Lipid Nanocapsule-Chitosan and Iota-Carrageenan Hydrogel Composite for Sustained Hydrophobic Drug Delivery

Lipid Nanocapsule-Chitosan and Iota-Carrageenan Hydrogel Composite for Sustained Hydrophobic Drug Delivery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lipid Nanocapsule-Chitosan and Iota-Carrageenan Hydrogel Composite for Sustained Hydrophobic Drug Delivery Grady K. Mukubwa, Justin B. Safari, Zikhona N. Tetana, Caroline N. Jones, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7078136/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Hydrophobic drug delivery via oral or pulmonary routes presents significant challenges for clinical translation, particularly for poorly soluble antiviral drugs. Physiological barriers—such as enzymatic degradation, harsh pH, and rapid transit in the gastrointestinal tract, or mucociliary clearance and alveolar macrophage uptake in the lungs—can severely limit therapeutic efficacy. To address these challenges, we developed a novel lipid nanocapsule (LNC) and chitosan/iota-carrageenan hydrogel composite tailored for sustained delivery of hydrophobic antiviral agents. This composite system was designed to encapsulate and deliver Efavirenz (EFV) under simulated gastrointestinal conditions. EFV was first encapsulated in LNCs, which were subsequently embedded within a mucoadhesive hydrogel matrix to form the EFV-LNC hydrogel composite. The LNCs significantly enhanced EFV solubility compared to water alone (p < 0.0001), and droplet size was controlled (57.4 ± 0.5 nm). The hydrogel composite exhibited an optimized swelling ratio (~ 300 g water per 1 g hydrogel) and achieved an encapsulation efficiency of approximately 53%. Importantly, EFV release from the composite was significantly prolonged under various gastrointestinal pH conditions compared to the unformulated drug (p < 0.0001). Cytotoxicity assays confirmed the composite’s cytocompatibility, supporting its potential safety for future mucosal administration. These findings suggest that the LNC-hydrogel composite enhances solubility, enables controlled release, and may improve mucosal retention, supporting its utility as a versatile platform for oral and pulmonary delivery of hydrophobic antiviral drugs. Biological sciences/Biotechnology Biological sciences/Drug discovery Physical sciences/Materials science Health sciences/Medical research Lipid nanocapsules Hydrogel Synergistic Use Drug Delivery Chitosan Iotacarrageenan Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Efavirenz (EFV) is a widely prescribed first-line antiretroviral agent belonging to the class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), commonly utilized as part of combination therapy for the treatment of HIV infection 1 ​. However, it is a highly lipophilic Biopharmaceutics Classification System (BCS) Class II compound with extremely low aqueous solubility (~ 9 µg/mL at 25°C), resulting in dissolution-limited and variable oral absorption (oral bioavailability ~ 40–45%) 1 . This poor and erratic pharmacokinetic profile can lead to inconsistent drug exposure; indeed, genetic variability in EFV metabolism has been linked to subtherapeutic concentrations or doselimiting side effects 2 ​. These issues pose a significant challenge in maintaining effective and safe EFV plasma levels during longterm HIV treatment. This variability underscores the need for improved EFV formulations to achieve more reliable therapeutic outcomes. Approximately half of all approved antiviral drugs (including lopinavir, ritonavir, and remdesivir) also fall into BCS Class II and suffer from low aqueous solubility​ 3 . Inadequate dissolution in the gastrointestinal tract is a major factor limiting the efficacy of such oral antivirals, which has motivated the development of formulation strategies to enhance solubility and absorption. Lipidbased nanoformulations have demonstrated success in improving the oral absorption of BCS II antivirals; for example, ritonavir and saquinavir have been marketed in lipidbased formulations to increase their bioavailability​ 4 –8 . In the case of EFV, various nanocarrier approaches – such as solid lipid nanoparticles, nanoemulsions, and selfemulsifying drug delivery systems – have been explored to boost its dissolution rate and absorption, with encouraging outcomes in preclinical studies 1 , 9 –11 ​. These findings reinforce the potential of advanced delivery systems to overcome EFV’s biopharmaceutical limitations. Among lipidbased nanocarriers, lipid nanocapsules (LNCs) are an attractive option for enhancing EFV delivery. LNCs consist of a liquid lipid core surrounded by a surfactant shell, a structure that enables high encapsulation efficiency of hydrophobic drugs like EFV (log P ~ 4.6) 1 2–16 . The small (~ 20–100 nm) LNC particles disperse readily in gastrointestinal fluids, maintaining EFV in a solubilized state and facilitating its transport to absorptive sites. Encapsulating EFV in LNCs can also convert the drug into an amorphous form, therefore enhancing its apparent solubility 17 ​. Additionally, LNC formulations help protect the drug from degradation and can modulate its release, which may reduce presystemic metabolism and improve the fraction of EFV absorbed. While LNCs address the solubility issue, a complementary approach is needed to prolong EFV’s residence time in the gut for absorption as LNCs are exposed to rapid gastrointestinal clearance, enzymatic degradation, and premature drug release under harsh gastrointestinal conditions 18–20 . In this study, we incorporated EFV-containing lipid nanocapsules (LNCs) into a chitosan/iota-carrageenan hydrogel to create a mucoadhesive nanoparticle-in-hydrogel delivery system (Fig. 1 ). Chitosan, a cationic polysaccharide, is well known for its mucoadhesive properties, enabling formulations to adhere to the intestinal mucosa and extend drug residence time at the absorption site. ​ 21 . Iota-carrageenan, an anionic polysaccharide, interacts with chitosan to form a crosslinked hydrogel network capable of entrapping the nanocapsules. This biopolymer-based matrix anchors the EFV-containing LNCs in the gastrointestinal environment, limiting rapid transit and enabling sustained drug release. Such hybrid systems combine the solubilization advantages of nanocarriers with the localized retention properties of hydrogels, offering a synergistic strategy to enhance oral drug delivery.​ 22 , 23 . We demonstrated that encapsulating EFV in LNCs within a chitosan/iota-carrageenan hydrogel significantly improved key oral biopharmaceutical properties, including solubility, physicochemical stability, sustained release, and cytocompatibility. The LNCs enhanced EFV solubility and protected the drug from premature degradation, while the mucoadhesive hydrogel extended gastrointestinal residence and facilitated more complete absorption. The primary objective of this work was to develop and characterize the LNC-in-hydrogel formulation and to evaluate its potential to overcome EFV’s solubility and delivery limitations. This combined strategy addresses a major formulation challenge by simultaneously improving drug solubility and prolonging mucosal retention. If successful, it could lead to more consistent EFV therapy and serve as a platform for improving the oral delivery of other poorly soluble antiviral agents. MATERIAL AND METHODS This section was adapted from the author’s previously submitted dissertation [Mukubwa, G. K., 2022] 24 . Chemicals. Crude soybean lecithin granules were sourced from Health Connection Wholefoods (USA). As per manufacturer information, the granules predominantly consist of phosphatidylcholine, phosphatidylinositol, polyunsaturated and saturated fatty acids, glycaemic carbohydrates, and sodium. Medium-chain triglyceride (MCT) oil of high purity was acquired from Absolute Organix (Jeppestown, Johannesburg), while Labrafac Lipophile 1349 was kindly provided by Gattfossé (France). Polysorbate 80 (Tween 80) was obtained from Merck (Johannesburg, Gauteng, South Africa), and Efavirenz was generously supplied by Adcock Ingram Ltd. (Wadeville, Gauteng, South Africa) 24 . Additional reagents, including iota-carrageenan (iCar), low molecular weight chitosan (CS), acrylic acid (AA), N,N′-methylenebisacrylamide (MBA), and acrylamide (Aam), were procured from Sigma Aldrich (Darmstadt, Germany). Glacial acetic acid, sodium chloride, and acetone were purchased from Minema (Roodepoort, South Africa), and ammonium persulfate (APS) from Saarchem (Wadeville, South Africa). HPLC-grade acetonitrile was sourced from Merck (Germany), and ultrapure HPLC-grade water was prepared using a RephiLe Bioscience Direct Purefi Ultrapure RO Water System (Boston, MA, USA). Human cervical adenocarcinoma (HeLa) cell lines were obtained from ATCC® (Manassas, Virginia, USA) 24 . Instruments. High-performance liquid chromatography (HPLC) was carried out using an Agilent 1100 system comprising a quaternary pump (model G1311A), a degasser (G1322A), a diode array detector (G1315B), and a manual injection unit (G1328B), with separation achieved on a Phenomenex® Kinetex® C18 column (2.6 µm, 100 Å, 150 × 4.6 mm i.d.). Sample lyophilization was performed using a LABCONCO FreeZone® 6 Liter Benchtop Freeze Dryer (Kansas City, MO, USA) 24 . Infrared (IR) spectra were collected using a PerkinElmer Spectrum 100 FTIR spectrometer. Differential scanning calorimetry (DSC) was conducted on a TA Instruments DSC 250. Crystalline structure of the materials was characterized by X-ray diffraction (XRD) using a Bruker D2 Phaser (second generation, Bruker, Billerica, MA, USA). The mean hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the lipid nanocapsules (LNCs) were measured using a Zetasizer Nano ZEN 3600 (Malvern Instruments, Malvern, UK) 24 . Transmission electron microscopy (TEM) was conducted using a Zeiss Libra 120KV instrument (Oberkochen, Germany) to assess particle morphology. Elemental composition was determined via energy-dispersive X-ray spectroscopy (EDS) using an INCA PENTA FET system coupled with a VAGA TESCAM unit (Brno, Czech Republic). Surface features of the hydrogels were examined using a TESCAN VEGA scanning electron microscope (Brno, Czech Republic). Fluorescence imaging was performed with an Olympus BX60 fluorescence microscope (Japan) 24 . Preparation of Efavirenz based Lipid Nanocapsules (EFVLNCs). Efavirenz-loaded lipid nanocapsules were formulated using a phase inversion technique, as established in our earlier work 1 7 . In summary, the formulation consisted of a mixture of medium-chain triglyceride (MCT) oil, crude soybean lecithin, Tween 80, and aqueous sodium chloride solution (18.5:0.5 g, water:NaCl) in a weight ratio of 12:3:9:76, incorporating Efavirenz at a final concentration of 17.5 mg/mL. Specifically, a total of 25 g of the formulation was prepared by combining 3 g MCT oil, 0.75 g lecithin, 2.25 g Tween 80, and 19 g NaCl solution in a 250 mL round-bottom flask, followed by the addition of 1.35 g of Efavirenz. The mixture was stirred magnetically at 1300 rpm and heated to 95°C using an oil bath on a hot plate. A series of three controlled heating–cooling cycles between 70°C and 95°C were then performed, each lasting approximately 90 minutes, during which phase inversion was visually indicated by the appearance of a translucent emulsion. To induce nanocapsule formation, the emulsion was rapidly quenched by adding cold HPLC-grade water (1–4°C) in a volume three times greater than the initial aqueous phase during the final cycle. A water-cooled condenser was used throughout the procedure to prevent evaporation of volatile constituents. Design and Optimization of chitosangiotacarrageenangpoly (acrylamide co acrylic acid) hydrogel (CS/iCarp(Aam co AA)). An I-optimal mixture design was conducted using Design-Expert® software version 13 (Stat-Ease, Inc., Minneapolis, MN, USA) to investigate how the composition of the CS/iCar p(AAm-co-AA) hydrogel influences its swelling behavior. The formulation components included two polymeric materials (chitosan and iota-carrageenan), two monomers (acrylic acid and acrylamide), and one crosslinking agent (N,N′-methylenebisacrylamide). These were defined as independent variables, while the hydrogel’s swelling capacity served as the response variable. A polynomial model was fitted to predict the swelling performance based on variations in component proportions. The input variables and their respective percentage contributions, which together sum to 100%, are presented in Table 1 . Table 1 Ioptimal mixture design input variables Low Limit Constraint High Limit 3.000 ≤ A: IOTACARRAGEENAN ≤ 6.000 3.000 ≤ B: CHITOSAN ≤ 6.000 40.000 ≤ C: ACRYLIC ACID ≤ 53.000 40.000 ≤ D: ACRYLAMIDE ≤ 53.000 1.000 ≤ E: N , N METHYLENE BISACRYLAMIDE ≤ 3.000 A + B + C + D + E = 100.000 Model Optimization and Confirmation. To evaluate the predictive accuracy of the developed polynomial model, swelling capacity maximization was set as the sole optimization criterion. The software identified the most desirable formulation, which was synthesized in triplicate. Triplicate swelling measurements were then performed, and the resulting average was compared to the model’s 95% prediction interval (95% PI) to validate the model. where is the predicted value of the response, \(\:{t}_{\left(\frac{\propto\:}{2},\:residual\:df\right)}\) is the Student’s t critical value and \(\:{SE}_{pred}\) is the standard error of the prediction 24 . Synthesis of CS/iCarp(AamcoAA) hydrogel. The hydrogel synthesis protocol was adapted from the approach described by Rahmani et al. 25 , with modifications introduced to suit the present study. The hydrogels were fabricated via free radical precipitation graft copolymerization of acrylic acid (AA) and acrylamide (AAm) onto a chitosan–iota carrageenan backbone, using ammonium persulfate (APS) as the radical initiator. For each batch, a total of 500 mg of combined ingredients was prepared. Iota carrageenan was first dissolved in 10 mL of HPLC-grade water at 60°C (Mixture A), while chitosan was separately solubilized in 10 mL of 1% (w/v) acetic acid solution (Mixture B). Both solutions were stirred independently at 800 rpm for 1 hour to ensure uniform dispersion. Subsequently, Mixture B was added to Mixture A, followed by the incorporation of the monomers (acrylic acid and acrylamide) and the crosslinker (MBA) in specified amounts. The polymerization reaction was initiated by adding APS (ranging from 0.05 to 0.2 g) under a nitrogen atmosphere. After polymerization, the hydrogels were thoroughly washed with approximately 500 mL of acetone to remove unreacted residues, and then dried in an oven at 40°C 24 . Development of EFVLNCsCS/iCarp(AamcoAA) hydrogel composite. To develop the EFV LNCs CS/iCar p(AAm-co-AA) hydrogel composite, Efavirenz-loaded lipid nanocapsules were incorporated into the hydrogel matrix, followed by quantification of the entrapped drug. A customized D-optimal design with randomized experimental runs was constructed using Design-Expert® version 13.0 (Stat-Ease, Inc., Minneapolis, MN, USA) to model and optimize the encapsulation efficiency based on varying concentrations of EFV in the lipid nanocapsules. This design yielded 13 experimental runs with EFV concentrations ranging from 17.5 mg/mL to 87.5 mg/mL 24 . Encapsulation was performed using the swelling equilibrium technique as previously described 26 . In brief, a measured volume of EFV LNCs corresponding to the target EFV concentration was diluted in a 25 mL volumetric flask. This solution was then used to hydrate 50 mg of dried hydrogel for a duration of 24 hours at ambient temperature. After the incubation period, the swollen hydrogel was separated by filtration and rinsed with HPLC-grade water. The filtrate, containing unencapsulated EFV LNCs, was collected for analysis. A 150 µL aliquot of this filtrate was diluted in a 10 mL volumetric flask using a 40:60 acetonitrile–water mixture and analyzed by a reversed-phase HPLC method, as developed by Bienvenu et al. and validated in our previous study 17 , 27 . The encapsulation efficiency (EE%) was calculated using the following equation: \(\:\text{%EE=}\frac{\text{Mi-Mf}}{\text{Mi}}\text{×100}\) ( Eq. 2 ) Where %EE stands for encapsulation efficiency, Mf is the Efavirenz mass determined from the filtrate and Mi the initial mass of Efavirenz in the soaking solution 24 . Droplet size, polydispersity index and zeta potential of EFVLNCs 2 4 . Droplet size (DS) and polydispersity index (PDI) measurements were performed using a Malvern Nano ZS Zetasizer (Malvern Instruments, Worcester, UK), operated in dynamic light scattering (DLS) mode for DS/PDI and in laser Doppler anemometry (LDA) mode for zeta potential (ZP) analysis. Before measurement, 500 µL of the saturated nanocapsule suspension was diluted in 50 mL of HPLC-grade water. For DS and PDI assessments, samples were transferred into BRAND® disposable cuvettes (12.5 × 12.5 × 45 mm; BRAND GmbH + CO KG, Wertheim, Germany). Zeta potential determinations were conducted using a folded capillary cell. All measurements were carried out at 22°C with a backscatter detection angle of 173°. Swelling Ratio of CS/iCarp(AamcoAA) hydrogel 24 . The swelling ratio of the synthesized hydrogel, expressed as grams of water absorbed per gram of dry hydrogel (g/g), was determined using a filtration-based approach. A known quantity of dry hydrogel (50–100 mg), denoted as W₀, was placed into a beaker containing 40–80 mL of HPLC-grade water and allowed to swell over a 24-hour period at room temperature. Subsequently, the mass of a piece of filter paper pre-wetted with distilled water was recorded as W₁ and placed in a Büchner funnel. The swollen hydrogel was separated from the solution via vacuum filtration to eliminate excess surface water. The filter paper, now holding the swollen hydrogel, was carefully removed and weighed again to obtain the final mass (W₂). The swelling ratio (SR) was calculated using the equation below, and all measurements were conducted in triplicate for accuracy 28 : Fourier Transform InfraRed Spectroscopy (FTIR) 2 4 . Fourier-transform infrared (FTIR) spectroscopy was employed to compare the spectral profiles of the synthesized hydrogel and the EFV LNCs–loaded hydrogel composite with those of the individual raw materials, in order to detect potential modifications in functional group characteristics 2 9 . Spectra were acquired using a PerkinElmer Spectrum 100 FTIR spectrometer operating in attenuated total reflection (ATR) mode 3 0 . Each sample was analyzed in the spectral range of 650–4000 cm⁻¹, with a total of sixteen scans per run 3 1 . The measurements were performed on freeze-dried forms of both the plain hydrogel and the nanocapsule-loaded composite 3 0 . Thermal Gravimetric Analysis 24 . Thermogravimetric analysis (TGA) was conducted to evaluate the thermal stability and decomposition patterns of the CS/iCar p(AAm-co-AA) hydrogel and its EFV LNC–loaded composite, in comparison with the native polymers chitosan and iota-carrageenan 32 . The analysis was carried out under an inert nitrogen atmosphere (flow rate: 20 mL/min) using a PerkinElmer TGA 4000 system. For each run, 2–5 mg of sample was heated from 30°C to 600°C at a constant rate of 10°C/min. Powder Xray Diffraction 2 4 . Powder X-ray diffraction (XRD) was employed to investigate the phase behavior and crystallinity of the freeze-dried CS/iCar p(AAm-co-AA) hydrogel and its EFV LNC–incorporated composite, in comparison with the native polymers chitosan and iota-carrageenan. Crystallinity profiles were analyzed to assess structural differences, and statistical significance in crystallinity variations was evaluated using a one-way ANOVA test. Measurements were conducted using a Bruker D2 Phaser (Second Generation) diffractometer equipped with a LynxEye proportional counter detector, a nickel filter, and Cu Kα radiation (λ = 1.5404 Å). Scans were performed over a 2θ range of 10–60° at a scanning rate of 1°/min, with a fixed slit width of 6.0 mm. Scanning Electron Spectroscopy (SEM) and EnergyDispersive Xray Spectroscopy (EDS) 24 . Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were used to examine the surface morphology and elemental composition of the CS/iCar p(AAm-co-AA) hydrogel and its EFV LNC–loaded composite. The EDS analysis was performed using an INCA PENTA FET system coupled with a VAGA TESCAN unit. Prior to imaging, hydrogels were equilibrated in HPLC-grade water for 24 hours at ambient temperature to reach maximum swelling, then lyophilized using a LABCONCO FreeZone® 6 Liter Benchtop Freeze Dry System. The resulting dried samples were mounted onto aluminum stubs and coated with a thin layer of gold to enhance conductivity before SEM and EDS analysis. Transmission Electron Microscopy (TEM) 24 . Transmission electron microscopy (TEM) was employed to assess the morphology and structural features of the EFV LNCs–loaded CS/iCar p(AAm-co-AA) hydrogel composite at an accelerating voltage of 80 kV. To ensure optimal electron transparency and maintain sample integrity, the composite underwent a carefully controlled preparation protocol. The sample was first embedded in optimal cutting temperature (OCT) compound to provide structural support during sectioning. Ultra-thin sections were then obtained via cryo-ultramicrotomy. These sections were transferred onto 3.05 mm diameter copper grids coated with holey carbon film (FORMVAR/Carbon, 300 mesh; TAAB Laboratories Equipment Ltd., Aldermaston, Berks, UK). For contrast enhancement, the sections were stained with uranyl acetate. Prior to imaging, samples were thoroughly dried in a desiccator to eliminate residual moisture that could interfere with electron beam interaction. This method enabled high-resolution visualization of the hydrogel composite while preserving its native structural characteristics. Fluorescence Microscopy 24 . Fluorescence microscopy was employed to confirm the presence and distribution of lipid nanocapsules (LNCs) within the CS/iCar p(AAm-co-AA) hydrogel matrix. To enable visualization, the composite was stained using amaranth dye. Specifically, 500 µg of amaranth was added to 25 mL of LNCs suspension, and this dye-loaded solution was used to hydrate 50 g of the dried hydrogel, resulting in a stained LNC–hydrogel composite. The composite was subsequently freeze-dried prior to imaging. A small section of the dried sample was placed between two glass slides and mounted under the microscope for observation. Imaging was conducted using an Olympus BX60 fluorescence microscope (Japan), equipped with a U-MWU DM400 filter set for DAPI fluorescence, and images were captured at 10× magnification. Drug Release Studies 24 . The release behavior of Efavirenz (EFV), encapsulated within lipid nanocapsules and further embedded in the CS/iCar p(AAm-co-AA) hydrogel matrix, was evaluated using a 1% sodium lauryl sulfate (SLS) solution as the release medium. Experiments were conducted at two pH levels—7.0 (neutral) and 4.0 (acidic)—to simulate physiological and acidic conditions, respectively. A portion of hydrogel containing an equivalent of 3 mg of EFV was weighed and placed into a dialysis bag (cellulose membrane tubing, flat width 25 mm; Sigma Aldrich, St. Louis, MO, USA), which was then immersed in 25 mL of the release medium. The setup was maintained at 37°C with continuous agitation at 100 rpm. At predetermined intervals (0.5, 1, 1.5, 2, 4, 8, 12, 24, 48, 72, 96, 144, and 216 hours), 5 mL aliquots were withdrawn and replaced with an equal volume of fresh pre-warmed medium to maintain sink conditions. Each time point was analyzed in triplicate. The experiment involved two categorical variables—sampling time and formulation—and one continuous response variable, namely the cumulative percentage of EFV released. It was hypothesized that both the pH of the release medium and the composite nature of the drug delivery system would influence the release kinetics. Two-way ANOVA was employed to assess the statistical significance of these effects across different conditions. Cytotoxicity studies 24 . Cytotoxicity assessments were carried out using HeLa cells. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere with 5% CO₂. After a 24-hour incubation period, cells were harvested, resuspended in fresh medium, and seeded into a 96-well plate preloaded with various treatments: unloaded lipid nanocapsules (LNCs), Efavirenz-loaded LNCs (EFV LNCs), LNCs embedded within CS/iCar p(AAm-co-AA) hydrogel, and the fully integrated EFV LNCs–hydrogel composite. All treatments were prepared at a final concentration of 50 µg/mL. The plate was incubated for an additional 24 hours at 37°C and 5% CO₂ to allow interaction between the cells and the test formulations. Following treatment, resazurin reagent—diluted in culture medium according to manufacturer guidelines (0.1 to 0.5 mg/mL)—was added to each well to cover the cell monolayer. The plate was then returned to the incubator for 1 to 4 hours to allow viable cells to reduce resazurin to fluorescent resorufin. Fluorescence intensity was measured using a multi-well plate reader, with all readings performed in triplicate. Emetine, a known inducer of apoptosis, served as the positive control. Cell viability was calculated using untreated cells as the 100% viability reference, and results were expressed as mean ± standard deviation (SD) based on triplicate measurements. The viability calculation followed a previously reported method 33 . Statistical Analysis 24 . Prior to performing statistical comparisons, data were assessed for normality using the Shapiro–Wilk test. Based on the distribution of the data, appropriate statistical tests were applied. For normally distributed datasets, comparisons between two groups were made using Student’s t-test. When the data did not meet parametric assumptions, the Kruskal–Wallis test was employed as a non-parametric alternative to ANOVA. Post hoc analyses were conducted using Dunn’s test to identify significant pairwise differences, with p-values adjusted for multiple comparisons. Cytotoxicity data involving comparisons between treatment groups and the negative control were analyzed using Welch’s t-test (two-tailed, assuming unequal variances). A p-value of less than 0.05 was considered statistically significant, while results with p < 0.0001 were interpreted as highly significant. Data analysis, modeling, and visualization were carried out using a combination of software tools, including Design-Expert®, OriginPro version 9, GraphPad Prism, R, Python, and Microsoft Excel. RESULTS AND DISCUSSION EFV-lipid nanocapsule preparation and characterization. EFVLNCs were produced for encapsulation in a chitosan-grafted iota-carrageenan poly(acrylamide-co-acrylic acid) hydrogel (CS/iCar-p(Aam-co-AA)). Solubility tests showed that medium chain triglycerides oil (MCT oil) significantly solubilized Efavirenz compared to water (Ttest, p 0.5) and analyzed using a t-test, revealing with 95% confidence that the true difference in solubility means between the two dissolving media is between 266.9 and 281.4 mg/mL. Based on previous findings reported by Mukubwa et al. 17 , an optimized formulation of EFVLNCs was prepared using the phase inversion method 17 ,34 . The obtained EFVLNCs were characterized for their hydrodynamic diameter, polydispersity index (PDI) and zeta potential (ZP). The average values of these parameters were approximately 57.4 ± 0.5 nm, 0.148, and − 54.9 mV, respectively, consistent with results observed in the previous study 17 . MCT oil is effective for solubilizing the non-polar EFV molecule in water as it consists of a lipid-structured molecular tail that matches favorably with the hydrophobic moieties present on EFV. This property also makes MCT oil a prime candidate for EFV delivery enhancement. The hydrodynamic diameter of 57.4 nm, a low polydispersity index (0.148), as well as their zeta potential (-54.9 mV) suggests an effective, stable and homogenous nanoparticle system. These properties together indicate that LNCs are effective nanocarriers for drug delivery with enhanced stability and potential enhanced bioavailability 17 . Synthesis and Optimization of Swelling Capacity of CS/iCar-p(Aam-co-AA) Hydrogel The CS/iCar-p(AAm-co-AA) hydrogel was synthesized through free radical copolymerization, with formulation optimization and statistical evaluation performed using Design-Expert® software version 13.0 (Stat-Ease, Inc., Minneapolis, MN, USA). An I-optimal mixture design yielded 35 distinct formulations, each prepared according to the synthesis protocol described earlier. Table S1 summarizes the component ratios for each formulation alongside their corresponding swelling capacities, which were used as the response variable for optimization. Swelling capacity was expressed as a percentage, calculated by the ratio of water absorbed (g) to the dry hydrogel mass (g). The proposed reaction mechanism for hydrogel formation is depicted in Figures S2 and S3 . Multiple polynomial models were evaluated to fit the experimental data, with the linear model demonstrating superior performance. This model yielded consistently low standard errors (< 0.5σ, where σ denotes the estimated variability within the dataset) across the design space ( Figure S4 ). Table S2 presents a comparative analysis of model fits, where the linear model was automatically selected as optimal based on its lowest predicted residual error sum of squares (PRESS) value and a significantly small sequential p-value, both indicators of strong model predictive accuracy 11 . While swelling behavior was selected as the primary criterion for optimizing the CS/iCar-p(Aam-co-AA) hydrogel, given its direct relationship with drug absorption and controlled release in gastrointestinal conditions, rheological properties also critically influence the composite’s performance. Mechanical and rheological characteristics significantly impact hydrogel behavior within the dynamic gastrointestinal environment, affecting aspects such as mucoadhesion, retention time, and stability against physiological shear forces. Evaluating rheological parameters, including viscosity, elasticity, and shear-thinning behavior, is therefore crucial to predict the hydrogel’s performance under realistic gastrointestinal conditions 35 , 36 . Although detailed rheological characterization was beyond the scope of this study, future work should investigate these properties to optimize the formulation further, ensuring enhanced gastrointestinal retention, controlled drug release, and improved patient compliance in oral antiviral drug delivery applications. Varying the portion of polymers, monomer and crosslinkers either increased or decreased the swelling capacity. In this study, a linear regression model was developed to accurately predict the swelling ratio of hydrogels based on the proportions of iota-carrageenan, chitosan, acrylic acid, acrylamide, and N,N′-methylenebisacrylamide (MBA) used in the formulation. These five components served as the independent variables in the model. The resulting predictive equation for swelling behavior is given as: %SC = 0.556464 x A + -0.241395 x B + 0.0694467 x C + 0.0907205 X D + 0.0431736 X E ( Eq. 4 ). In the regression model, factors A through E represent the following components: iota-carrageenan (A), chitosan (B), acrylic acid (C), acrylamide (D), and N,N′-methylenebisacrylamide (E). This model is statistically significant (p = 0.0451), but it only accounts for a moderate portion of the variance in swelling ratio (adjusted R² = 0.1643). The insignificant lack-of-fit p-value (0.3168) means that this model is decent for predicting data without being an overfit and thus serves as a good reference to formulate hydrogels in the future 37 – 39 . The low R² value is likely to be due to variability in hydrogel synthesis; small differences during preparation can cause significant variations in swelling behavior 40 , 41 . While the model is linear and insubstantial in as much as mimicking more complex component interactions, it provides a useful approximation of swelling behavior within these experimental confines. More complex models could improve prediction, but this simple linear regression provides a nice balance between simplicity and accuracy for early-stage hydrogel development. The swelling ratios of the synthesized hydrogel formulations varied significantly, ranging from 2.84 to 434.43. This variation was directly influenced by the relative composition of the hydrogel constituents. An increase in iota-carrageenan content led to a marked enhancement in swelling capacity, whereas higher chitosan levels were associated with a reduction in water absorption (Fig. 2 ). Additionally, decreasing the proportions of acrylic acid, acrylamide, and MBA resulted in improved swelling performance. This trend is attributed to lower crosslinking density at reduced monomer and crosslinker concentrations, which facilitates greater expansion of the hydrogel network and allows more water uptake. Validation of the hydrogel composition model yielded a swelling ratio exceeding 300, meaning that over 300 g of water was absorbed per gram of hydrogel. To assess the predictive accuracy of the developed linear regression model, Design-Expert® software generated five optimized formulations based on the criterion of maximizing swelling capacity. Among these, the formulation with the highest desirability score was selected for experimental validation and tested in triplicate, as summarized in Table S3 . The optimized formulation consisted of 6% iota-carrageenan, 3% chitosan, 40% acrylic acid, 50% acrylamide, and 1% MBA, as also depicted in Fig. 3 a. The average swelling ratio observed experimentally fell within the 95% prediction interval, thereby confirming the model’s reliability. The close overlap of observed and predicted swelling ratio values for the 95% prediction interval (Table 2 ), confirms the effectiveness of our prediction model. This predictability indicates that the model can reasonably estimate swelling ratios despite inherent variability in hydrogel synthesis. This inherent variability is reflected in the wide prediction interval of the swelling ratios provided by the model (32.6 to 1407.1). Overall, this model can be used to predict different proportions of iCar, CS, AA, Aam and MBA to synthesize a hydrogel with desired properties. It also underlines the potential of our optimized hydrogel formulation in drug delivery systems where maximum swelling is required. This formulation was designated for further experiments, including LNCshydrogel composite preparation, EFVLNCshydrogel composite preparation and EFV release studies. Table 2 Observed swelling ratio mean against the prediction interval Response Mean 95% Prediction Predicted Observed 95% PI low 95% PI high Formulation 1 Swelling ratio 444.5 304.2 32.6 1407.1 CS/iCarp(AamcoAA) hydrogel composite entrapped LNCs containing over 50 mg/mL of EFV 24 . Following the synthesis of the CS/iCar p(AAm-co-AA) hydrogel, an Efavirenz-loaded LNC solution was incorporated to form the EFV LNCs–hydrogel composite. To investigate the impact of LNC drug concentration on encapsulation efficiency, thirteen different formulations were prepared based on a D-optimal (custom) randomized experimental design ( Fig. 3 ) . A clear trend emerged wherein encapsulation efficiency increased with rising EFV concentrations, reaching a broad maximum between 55 and 60 mg/mL before declining at higher concentrations. The experimental data were modeled using a reduced quadratic equation, as outlined below: %EE = 4.25921 + 1.4527t1×A+-0.0123934×A 2 ( Eq. 5 ) where A represents the concentration of Efavirenz (EFV) within the lipid nanocapsules (LNCs). To identify an optimal EFV LNC concentration for subsequent experiments and to evaluate the model’s predictive capability, encapsulation efficiency was set as the primary optimization target. Design-Expert® software generated a single optimal formulation, a concentration of 58 mg/mL EFV LNCs was recommended, corresponding to a predicted encapsulation efficiency of 46.83%. In practice, 50 mg of the optimized hydrogel were immersed in 25 mL of an EFV LNC solution containing 58.61 mg/mL of the drug. The experimentally determined encapsulation efficiency fell within the 95% prediction interval ( Table 3 ) , confirming the model’s validity for predictive applications. Table 3 Comparison of the experimentally observed mean swelling ratio with the 95% prediction interval generated by the regression model. Response Mean 95% Prediction Predicted % Observed % 95% PI low 95% PI high Hydrogel + 58 mg/mL EFVLNCs Encapsulation efficiency 46.83 53.04 28.03 65.63 The CS/iCar p(AAm-co-AA) hydrogel exhibited thermal stability above 200°C. Thermogravimetric analysis (TGA), conducted to compare the thermal behavior of the synthesized hydrogel with its individual components, revealed a series of distinct decomposition events corresponding to the chemical crosslinking process. The incorporation of acrylic acid and acrylamide into the polysaccharide network altered the degradation profile of the final hydrogel. As shown in Fig. 4 a, multiple weight loss stages were observed during thermal decomposition. Among the base polysaccharides, iota-carrageenan demonstrated a more rapid thermal degradation, with approximately 28% weight loss at 208°C, whereas chitosan showed only a 10% reduction at the same temperature. Figures 4 a and 4 b illustrate the unique decomposition pattern of the hydrogel, confirming its enhanced thermal resistance beyond 200°C. These findings support the successful chemical modification of the polysaccharide matrix, as reflected in its altered thermal characteristics. The CS/iCarp(AamcoAA) hydrogel demonstrated excellent thermal stability, exceeding 200°C, due to chemical crosslinking that improved its resistance to thermal degradation. The decomposition profiles in Figs. 4 a and 4 b illustrate that chitosan and iota-carrageenan undergo distinct degradation patterns, indicating their unique contributions to the composite’s thermal resilience. Such thermal stability is particularly advantageous for oral drug delivery formulations, as it ensures that manufacturing processes involving elevated temperatures (e.g., drying, tablet compression, sterilization) will not compromise hydrogel integrity 42 –44 . Additionally, these properties suggest the hydrogel’s suitability for other advanced biomedical applications, such as transdermal patches or implantable devices requiring sterilization via autoclaving 45 . These observations reinforce the importance of chemical crosslinking for obtaining hydrogels with robust thermal stability suitable for various biomedical applications, including oral dosage forms 46 . Chemical interactions between polymers, monomers, and the crosslinker resulted in shifts in wavenumbers observed in the FTIR spectra of the synthesized hydrogel. Fourier-transform infrared (FTIR) spectroscopy was utilized to examine potential chemical bonding and structural interactions within the material resulting from the synthesis of the CS/iCarp(AAmcoAA) hydrogel. Both chitosan (CS) and iota-carrageenan (iCar) contain hydroxyl groups that may interact with the carbon–carbon double bonds (–C = C–) present in the monomers and crosslinker. Additionally, interactions between the EFV-loaded lipid nanocapsules (EFV LNCs) and the hydrogel network were evaluated through spectral analysis. FTIR spectra of the synthesized materials were compared with those of the raw components (Figs. 4 c and 4 d). The hydrogel spectrum displayed a broad absorption band in the 3300–3500 cm⁻¹ range, corresponding to O–H and N–H stretching vibrations. Peaks between 1000 and 1200 cm⁻¹ were attributed to C–O stretching, while weaker bands observed in the 2000–2300 cm⁻¹ region, also present in CS and iCar, were likely overtones of C–O vibrations, possibly arising due to the thickness of the hydrogel or polysaccharide films. The prominent peak at 1657 cm⁻¹ confirmed the presence of C = O groups, consistent with the incorporation of acrylamide and acrylic acid. The FTIR spectrum of the EFV LNCs–loaded hydrogel composite retained the key absorption bands observed in the plain hydrogel. However, the 2000–2300 cm⁻¹ bands appeared diminished or absent, potentially due to differences in film thickness. Notably, additional bands in the 2950–2840 cm⁻¹ range, characteristic of aliphatic C–H stretching from saturated fatty acids, were evident in the composite, along with carbonyl peaks near 1750–1745 cm⁻¹, consistent with the presence of lipid-based nanocapsules. The wavenumber shifts that we observed in the FTIR spectra following CS/iCarp(AamcoAA) hydrogel and its EFV-LNCs composite synthesis demonstrates successful yield of a stable hydrogel network from chemical interactions among the polymers, monomers, and crosslinking agent. The acrylamide and acrylic acid grafting onto the polysaccharide backbone is confirmed by the presence of stretches like the OH and NH, coupled with C = O and CO vibrations, aligning with previous studies where similar spectral shifts have been demonstrated when polysaccharides are chemically modified to enhance their drug delivery potential 47,48 . For example, improvement of properties like the swelling ratio have been proven to be associated with the introduction of functional groups like amides and carboxylates in the hydrogel’s structure 49 . Moreover, upon EFVLNCs incorporation, the hydrogel structure was maintained, suggesting that the encapsulation process does not compromise the hydrogel’s integrity. On the other hand, the presence of CH bands which are characteristic of saturated fatty acids found in medium chain triglycerides oil (MCT oil), supports successful encapsulation of EFV-LNCs 50 . Overall, this new CS/iCarp(AamcoAA) hydrogel delivery system possesses potential practical applications in improving controlled drug delivery of hydrophobic drugs like EFV. The synthesized CS/iCar p(AAm-co-AA) hydrogel displayed a fibrous surface architecture, with elemental analysis revealing the presence of nitrogen and sulfur groups indicative of its polysaccharide components. Surface morphology and elemental composition were evaluated using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS), respectively. SEM micrographs (Fig. 5 a: EFV LNC–loaded hydrogel composite; Fig. 5 b: unloaded hydrogel) revealed a rough, fibrous network with abundant porous structures, which are potentially advantageous for drug loading and release 51 . The incorporation of EFV LNCs did not visibly alter the porous morphology of the hydrogel. EDS spectra further confirmed the elemental signatures of the hydrogel matrix. Both the composite (Fig. 5 c) and unloaded hydrogel (Fig. 5 d) exhibited characteristic peaks for nitrogen, originating from chitosan and sulfur, derived from iota-carrageenan, alongside common elements such as carbon and oxygen. Additionally, chlorine was detected exclusively in the EFV-loaded composite (Fig. 5 c), corresponding to the chemical structure of Efavirenz. These results indicate that both CS/iCar-p(Aam-co-AA) hydrogel and its EFV-LNCs composite exhibit porous and fibrous structures suitable for drug delivery. Although pore size was not determined, the previously observed correlation between the swelling ratio and the proportion of the hydrogel components implies that pore sizes also vary accordingly. For instance, increasing the amount of iota-carrageenan and decreasing the amount of chitosan resulted in higher swelling ratios, implying the presence of larger or more accessible pores, with the potential to affect drug loading and release. In the future, pore size should be measured and optimized to further understand its relationship with swelling ratio, drug loading and drug release 52 – 55 . The CS/iCarp(AamcoAA) hydrogel and EFVLNCsCS/iCarp(AamcoAA) hydrogel composite display relatively lower crystallinity compared to chitosan and iota-carrageenan. Powder X-ray diffraction (XRD) analysis was performed to evaluate changes in the crystalline structure following hydrogel formation and drug encapsulation. The data passed normality testing using the Shapiro–Wilk test (p > 0.05), and subsequent ANOVA analysis revealed that the crystallinity of both the plain hydrogel and the EFV-loaded composite was significantly lower than that of chitosan, EFV, and EFV-LNCs (p < 0.01), as shown in Fig. 5 e. These findings provide additional evidence of successful chemical crosslinking between the monomers and polysaccharides. Crosslinking typically disrupts the orderly molecular arrangement in a polymer matrix, thereby reducing its degree of crystallinity 56 . The crystallinity reduction of Efavirenz upon encapsulation, first from 89.75% for nonencapsulated EFV to 57.52% for EFV-LNCs, and further down to 7.13% for the EFVLNCsCS/iCarp(AamcoAA) hydrogel composite highlights the effectiveness of double encapsulation in influencing the drug’s polymorphism. Interestingly, CS/iCarp(Aam-co-AA) hydrogel’s crystallinity which was initially 7.12% was not affected by EFV-LNCs incorporation. This supports that CS/iCar-p(Aam-co-AA) hydrogel’s structural integrity was maintained. Decreasing EFV crystallinity is critical since lower crystallinity improves solubility and bioavailability, which are essential for effective drug delivery. Moreover, CS/iCar-p(Aam-co-AA) hydrogel structural integrity maintenance suggests that its matrix can entrap a drug without altering its inherent properties. Our previous Differential Calorimetric Analysis (DSC) of EFV-LNCs demonstrated the transformation of encapsulated EFV to an amorphous state, explaining the reduction of its crystallinity rather than mere masking it 17 . DSC analysis of the EFVLNCsCS/iCarp(AamcoAA) hydrogel composite compared to non-encapsulated EFV should be preconized to further demonstrate how the hydrogel matrix affected EFV polymorphism. When compared to Chitosan, CS/iCarp(AamcoAA) hydrogel crystallinity significantly decreased, and this could be accounted for by the introduction of a threedimensional network that limits the mobility of Chitosan resulting in a more amorphous structure 57 . The microstructural insights of EFVLNCsCS/iCarp(AamcoAA) hydrogel composite reveal generated entanglements of the hydrogel scaffold and tiny spherical particles of entrapped EFVLNCs. Transmission electron microscopy enabled the visualization of the microscopic structure of the composites. Figure 6 a highlights the EFV LNCs CS/iCar p(Aam-co-AA) hydrogel composite network, showing proposed entanglements at 200 nm. At 100 nm, small spherical particles are visible, indicating the presence of EFV LNCs. A TEM micrograph of EFVLNCs has previously been reported, exhibiting particles with sizes below 100 nm 1 7 . The presence of these particles confirms that the EFVLNCs particles are incorporated into the hydrogel matrix, and their distinct appearance suggests a specific and controlled distribution. This insight into the composite structure further supports the entrapment of EFVLNCs in the hydrogel scaffold. This entrapment could have important implications for drug delivery, where the controlled release of the encapsulated EFV is desired. EFVLNCs particles entrapped in the CS/iCarp(AamcoAA hydrogel scaffold The presence of the EFV-LNCs in CS/iCar p(Aam-co-AA) hydrogel scaffold was confirmed by using fluorescence microscopy since the LNCs lacked a heavy atom. Amaranth dye was used to stain the composite to increase the contrast between the incorporated LNCs and the rest of the constituents in the composite. The images obtained using the DAPI filter (Fig. 6 b ) , which excites at 358 nm and emits blue fluorescence at 461 nm, revealed a bright reddish color of the background corresponding to the outer hydrogel layer. Besides, the bright and distinct blue-color dots represent the clusters of LNCs. While Amaranth dye is weakly fluorescent, its association with the phospholipids in soybean lecithin of the LNCs likely enhanced the fluorescent signal. This is consistent with previous research on soybean oil and soy protein, where phospholipid interactions with some molecules resulted in amplified fluorescence under specific conditions 58 . The blue fluorescence not only evidenced the structural integrity of the EFV-LNCs-CS/iCar p(Aam-co-AA) hydrogel composite but also the success of the encapsulation procedure, making a huge potential for further application, including controlled release of the entrapped drug (EFV). LNCsCS/iCarp(AamcoAA) hydrogel composite achieves enhanced and sustained In vitro Release of EFV. The in vitro release study was conducted using a 1% sodium lauryl sulfate (SLS) solution adjusted to pH 7 and pH 4 to simulate gastrointestinal conditions, providing physiologically relevant environments for assessing drug release. The pH 7 condition represents the intestinal environment, while pH 4 condition simulates fed-state gastric conditions or the transitional environment between the stomach and proximal intestine, conditions frequently experienced after food intake, thereby enabling evaluation of the composite's performance throughout different regions of the gastrointestinal tract 59 – 61 . Figure 7 illustrates the comparative release profiles of free Efavirenz (non-encapsulated) and the EFV LNCs–CS/iCar p(AAm-co-AA) hydrogel composite under the tested conditions. As the data did not meet normality assumptions (Shapiro–Wilk test, p < 0.05 for all groups), the Kruskal–Wallis test was employed for non-parametric analysis of overall group differences. This was followed by Dunn’s post-hoc test for pairwise comparisons. The Kruskal–Wallis test identified statistically significant differences in the drug release behavior between free EFV and the hydrogel composite across multiple time points. Free EFV exhibited a significantly faster release, exceeding 75% in both pH conditions over the 9-days experimental period (p < 0.0001). Notably, the free EFV release profile demonstrated a burst increase after 72 hours (region between 50 ~ 100 hours as seen in Fig. 7 ), likely due to saturation of the dialysis bag, alterations in solution properties, and the lack of a diffusion barrier, all of which contribute to unrestricted drug diffusion. In other words, EFV saturated the dialysis bag and precipitated; once some precipitated drug gradually dissolved or the medium was refreshed, a sudden increase in diffusion occurred 62 . In contrast, the hydrogel formulation averted such bursts, maintaining a steadier, more linear release without spikes. Pairwise comparisons were examined using the Wilcoxon Signed-Rank Test. The release kinetics of Free EFV at pH 4 and pH 7 were not significantly different (W = 42.0, p = 0.807), suggesting that pH had minimal influence on free EFV diffusion dynamics. However, significant differences were observed between Free EFV at pH 4 vs. EFVLNCCS/iCar at pH 4 (W = 0.0, p = 0.0015) and Free EFV at pH 7 vs. EFVLNCCS/iCar at pH 7 (W = 0.0, p = 0.0015), confirming that encapsulation significantly reduced drug release rates under both pH conditions. Additional comparisons between Free EFV at pH 4 vs. EFVLNCCS/iCar at pH 7 (W = 0.0, p = 0.0015) and Free EFV at pH 7 vs. EFV-LNC-CS/iCar at pH 4 (W = 0.0, p = 0.0015) further reinforced the hydrogel’s ability to modulate drug release more effectively than the free drug, irrespective of pH. The composite’s sustained, slower release is governed by diffusion through multiple barriers and pH-responsive matrix behavior. EFV must traverse the lipid nanocapsule core and then the chitosan/iota-carrageenan hydrogel network, which slows down its mobility 63 . The hydrogel’s mesh size and swelling depend on pH – it stays dense at pH 4 (limiting EFV diffusion) and expands at pH 7 (allowing faster diffusion) 64 . Interestingly, a significant difference was detected within the EFV-LNC-CS/iCar hydrogel composite, with higher drug release at pH 7 compared to pH 4 (W = 0.0, p = 0.0077). This pH-dependent release profile suggests that the hydrogel matrix responds to environmental pH, releasing less drug under mildly acidic conditions. This means that there is minimal release in the stomach (pH 4) means most EFV remains protected from acidic degradation or irritation, and the dose is not wasted where little drug uptake occurs. Once the hydrogel reaches the intestine and encounters pH 7, it swells and rapidly releases EFV at the site of optimal absorption 65 . This targeted release maximizes drug bioavailability by delivering EFV where it can be efficiently absorbed, while the sustained release profile prolongs drug presence in the absorption window. Given that pH 4 simulates fed-state gastric conditions or the transitional environment between the stomach and proximal intestine, this characteristic could be advantageous for protecting the drug from premature release in the stomach and promoting more targeted and sustained drug release upon entering the neutral intestinal environment, ultimately enhancing oral bioavailability and therapeutic efficacy as already demonstrated in previous studies 66 , 67 . The hydrogel composite demonstrated sustained and controlled drug release over 72 hours without significant burst effects, in contrast to free EFV. The hydrogel’s porous structure effectively acted as a diffusion barrier, modulating EFV release through swelling and erosion under gastrointestinal conditions. This controlled release profile is advantageous for improving EFV’s oral bioavailability and reducing dosing frequency. LNCs enhance EFV’s solubility and stability, preventing precipitation and protecting the drug from premature degradation, while also facilitating absorption via mechanisms like lymphatic uptake 68 . The hydrogel, in turn, localizes and slowly releases the LNCs (and encapsulated EFV) in the GI tract. This dual system offers a synergistic effect that prevents the rapid clearance or burst release that might occur with LNCs alone and effectively marries the sustained release capacity of hydrogels with the absorption-enhancing benefits of nanoparticles 69 . Consequently, the composite facilitates prolonged drug retention and absorption compared to either component alone, highlighting its potential as an advanced oral delivery platform to enhance therapeutic efficacy and patient compliance for poorly soluble antiviral drugs. Hydrogel is biocompatible with HELA cells. Cytocompatibility is essential to ensure future clinical use of any newly developed product. For this reason, we performed cytotoxicity experiments by treating HeLa cells with lipid nanocapsules, CS/iCarp(AamcoAA) hydrogel, LNCsCS/iCarp(AamcoAA) hydrogel composite and EFVLNCsCS/iCarp(AamcoAA) hydrogel composite using a resazurinbased assay. HeLa cells were chosen due to their widespread use and well-characterized response in preliminary cytotoxicity studies. This allows for consistency and comparability with existing literature, providing a reliable initial assessment of biocompatibility 70–73 . The treatments were prepared in a 96 well plate at a concentration of 50 µg/mL. The plate was put in the incubator at 37°C with 5% CO 2 to allow the cells to grow and interact with the treatments for 24 hours. After incubation, the resazurin was used to fully cover each well of the 96‑well plate, which was then returned into the incubator. In this resazurin-based assay, viable cells with active metabolism reduce the non-fluorescent resazurin to the fluorescent compound resorufin. The resulting fluorescence intensity, measured after 1–4 hours of incubation, is directly proportional to the number of metabolically active cells. Cytotoxicity was expressed as percent viability, calculated as the ratio of fluorescence in wells with treated cells compared to wells containing untreated cells. The viability of HeLa cells treated with various formulations is illustrated in Fig. 8 . The LNCs-Hydrogel composite showed a viability of ~ 90% (t = -0.8552, p = 0.4825, ns), with no statistically significant difference from the negative control, suggesting cytocompatibility. At the tested concentration of 50 µg/mL, none of the treatments reduced cell viability by more than 50%, except the positive control Emetine, a well-established inducer of apoptosis. This observation indicates minimal cytotoxicity of LNCs formulations compared to no treatment. Compounds with IC50 concentrations above 10 µg/mL are deemed safe candidates to use clinically 74 – 79 . Among the formulations, LNCs exhibited the highest viability (~ 135%, t = 16.1692, p = 0.0038, **), indicating enhanced metabolic activity. EFVLNCs and EFVLNCs Hydrogel composite exhibited viabilities of ~ 65% (t = -30.7915, p = 0.0011, **) and ~ 85% (t = -6.7239, p = 0.0214, *), respectively, indicating that the incorporation of EFV reduced cell viability, as expected for an active pharmaceutical ingredient. Emetine, used as a positive control, dramatically reduced cell viability to approximately 10% (t = -250.7546, p < 0.0001, ****), confirming its known cytotoxicity. These findings suggest that LNCs and the new LNCsHydrogel composite are cytocompatible, and the observed decrease in viability upon EFV loading aligns with the drug’s expected biological activity. The elevated viability observed with LNCs may reflect increased metabolic activity, potentially facilitated by mediumchain triglycerides (MCTs) as an energy source, soy lecithin supporting membrane integrity, and Tween 80 alleviating cellular stress 8 0–84 . Additionally, the aqueous medium and salt content likely help maintain osmolarity and hydration, contributing to an optimal environment for cell survival. Additionally, the resazurin assay likely detected this metabolic boost as increased viability. These results suggest that lipid-based systems, such as LNCs and their composites, can support high cell viability, warranting further investigation into their invivo application. While preliminary cytotoxicity studies using HeLa cells have demonstrated promising biocompatibility of our formulations, follow-up experiments will be conducted on gastrointestinalrelevant cell lines to better represent oral conditions. Suitable cell lines for evaluating gastrointestinal cytotoxicity include Caco-2 (human intestinal epithelial cells), HT29-MTX (human colon carcinoma cells secreting mucus), and IEC-6 (normal rat small intestine epithelial cells) 8 5, 8 6 . These intestinal cell models are anticipated to provide critical insights into the safety, biocompatibility, and potential efficacy of our hydrogel- and lipid nanocapsulebased formulation for oral drug delivery, supporting further clinical translation. EFVLNCsCS/iCarp(AamcoAA) Hydrogel Composite as Oral Solid Dosage Form For future studies, the EFVLNCCS/iCarp(AamcoAA) hydrogel composite can be optimized into a solid oral dosage form, enhancing patient convenience and therapeutic efficacy. Lyophilization could transform the composite into a dry, easily administrable powder or tablet, maintaining the integrity of both lipid nanocapsules and hydrogel components 87–89 . Freeze-drying would preserve the internal structure of the composite, minimizing aggregation or premature drug release. Additionally, compression into oral tablets or encapsulation in capsules could facilitate dosage uniformity, ease patient administration, and maintain sustained release properties 90,91 . Although solid dosage form development was not performed in this study, this approach represents a promising direction for enhancing the clinical applicability of EFV-loaded hybrid composites, potentially improving the oral bioavailability and therapeutic consistency of hydrophobic antiviral drugs. CONCLUSION This study introduces an innovative lipid nanocapsule and hydrogel composite specifically developed for oral delivery of hydrophobic antiviral drugs. Biocompatible polysaccharides, chitosan (CS) and iotacarrageenan (iCar), were used to synthesize a porous hydrogel scaffold via free-radical copolymerization. The synthesized CS/iCarp(AamcoAA) hydrogel demonstrated high swelling capacity, facilitating effective encapsulation and sustained release of Efavirenzloaded lipid nanocapsules (LNCs). The composite improved EFV solubility and demonstrated controlled, pHresponsive release profiles under simulated gastrointestinal conditions. Furthermore, structural and cytotoxicity analyses confirmed the biocompatibility and safety of the developed formulation, suggesting favorable interactions within gastrointestinal environments. Future studies will focus on evaluating biocompatibility with relevant intestinal cell lines and optimizing mechanical and mucoadhesive properties. These next steps will be essential for translating this novel composite system into an effective clinical solution for not only orally delivering poorly soluble antiviral medications, but also for application in other mucosal tissues, such as vaginal drug delivery 92,93 , where hydrogels have been extensively employed as vehicles to deliver drugs embedded in nanoparticle, transdermal delivery and even pulmonary delivery 94 . Abbreviations LNCs – Lipid Nanocapsules CS - Chitosan iCar - Iota-Carrageenan EFV - Efavirenz SEM - Scanning Electron Microscopy EDS - Energy-Dispersive X-ray Spectroscopy FTIR - Fourier Transform Infrared Spectroscopy TGA - Thermal Gravimetric Analysis DS - Droplet Size PDI - Polydispersity Index ZP - Zeta Potential MBA - N,N'-Methylene Bisacrylamide AA - Acrylic Acid Aam - Acrylamide SC - Swelling Capacity SR - Swelling Ratio IC50 - Inhibitory Concentration 50% ANOVA - Analysis of Variance SLS - Sodium Lauryl Sulfate DSC - Differential Scanning Calorimetry Declarations Supporting information Solubility test : the T‑test in Figure S1 shows that Efavirenz was significantly soluble in MCT oil unlike in water. Hydrogel swelling capacity optimization : Table S1 lists all the hydrogels formulations prepared, with their corresponding swelling capacity values. Table S2 shows the results of the analysis of variance of models fitted to the swelling capacity. Potential mechanism of hydrogel formation : Figure S2 and Figure S3 respectively describe the formation of free radical sites on chitosan and iota‑carrageenan backbones under ammonium persulfate initiation, and the plausible reactional mechanism of CS/iCar‑p(Aam‑co‑AA) hydrogel formation. Standard error plot : Figure S4 presents the contour plot of the standard error at different points in the design space that shows small values of the standard error that ranged from 0.2 to 0.35, which is an indication that the means of the samples are a reliable reflection of the true population means. Swelling capacity prediction : Table S3 lists automatically generated hydrogel composition batches for model optimization and confirmation. AUTHOR INFORMATION Corresponding Authors *Email: [email protected] (C.N.J.) *Email: [email protected] (R.W.M.K.), *Email: [email protected] (G.K.M), ORCID Grady K. Mukubwa: 0000-0002-1678-4722 Justin B. Safari: 0000-0002-8940-9552 Zikhona N. Tetana: 0000-0002-0767-1056 Caroline N. Jones: 0000-0001-6003-9335 Roderick B. Walker: 0000-0003-2781-4154 Rui W.M. Krause: 0000-0001-6788-6449 Author contribution The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Project design and conceptualization, methodology, data collection, software, data analysis and interpretation, investigation, data curation, writing original draft preparation, writing review, editing and visualization: G.K.M.; methodology, data collection and analysis, writing review and editing: J.B.S.; methodology, data collection, writing review and editing: Z.N.T.; conceptualization, validation, writing review and editing, supervision, project administration, resources and funding acquisition: R.W.M.K., R.B.W and C.N.J. All authors have read and agreed to the published version of the manuscript. Notes The authors declare no competing financial interest. Data availability All data supporting the findings of this study are provided within the main article and its supplementary materials. Additional datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Acknowledgments We thank the Rhodes University Electron Beam Laboratory for EM facilities, especially Dr. D. van Niekerk and Mr. M. Randall for training, selecting experimental parameters, and guidance with sample preparation and data collection. Thanks to the Rhodes CCBR for bioassay data, the Edkins lab for confocal data, and the SMaLL-group for XRD, FTIR, and TGA facilities. This manuscript contains content adapted from the first author’s publicly available dissertation submitted to Rhodes University, archived at [http://vital.seals.ac.za:8080/vital/access/manager/Repository/vital:65665?site_name=GlobalView]. The material has been revised and expanded for the purpose of journal publication. Funding Declaration This research was funded by the NGO Förderverein Uni Kinshasa e.V. (fUNIKIN), ElseKroener-Fresenius Stiftung, and Holger-Poehlmann Foundation through the excellence scholarship program “Bourse d’Excellence Bringmann aux Universités Congolaises, BEBUC” ( [email protected] ; [email protected] ), and Rhodes University, South Africa, supported by the National Research Foundation, NRF (including Grant No: 141979). G.K.M and C.N.J received support from R35 GM133610/GM/NIGMS NIH HHS/United States. In memory of Prof. Rui W.M. Krause (1970–2024), a renowned figure in organic medicinal chemistry and nanomaterials, whose leadership and mentorship left an indelible mark on the field. His dedication to inspiring and guiding the next generation of scholars has empowered many to realize their full potential. References Gupta, S., Kesarla, R., Chotai, N., Misra, A. & Omri, A. Systematic Approach for the Formulation and Optimization of Solid Lipid Nanoparticles of Efavirenz by High Pressure Homogenization Using Design of Experiments for Brain Targeting and Enhanced Bioavailability. Biomed Res Int 2017 , 5984014 (2017). Langmia, I. M. et al. CYP2B6 Functional Variability in Drug Metabolism and Exposure Across Populations—Implication for Drug Safety, Dosing, and Individualized Therapy. Front Genet 12 , 692234 (2021). Chen, R. et al. Antiviral Drug Delivery System for Enhanced Bioactivity, Better Metabolism and Pharmacokinetic Characteristics. Int J Nanomedicine 16 , 4959 (2021). Arora, S., Chawla, A., Pawar, P. & Sharma, P. Novel drug delivery approaches on antiviral and antiretroviral agents. J Adv Pharm Technol Res 3 , 147–159 (2012). Lembo, D. & Cavalli, R. Nanoparticulate delivery systems for antiviral drugs. Antivir Chem Chemother 21 , 53–70 (2010). Cojocaru, F. D. et al. Nanomaterials designed for antiviral drug delivery transport across biological barriers. Pharmaceutics 12 , 1–34 (2020). Samimi, S., Maghsoudnia, N., Eftekhari, R. B. & Dorkoosh, F. Lipid-Based Nanoparticles for Drug Delivery Systems . Characterization and Biology of Nanomaterials for Drug Delivery: Nanoscience and Nanotechnology in Drug Delivery (Elsevier Inc., 2018). doi:10.1016/B978-0-12-814031-4.00003-9. Kalepu, S., Manthina, M. & Padavala, V. Oral lipid-based drug delivery systems – an overview. Acta Pharm Sin B 3 , 361–372 (2013). Kamble, R. N., Mehta, P. P. & Kumar, A. Efavirenz Self-Nano-Emulsifying Drug Delivery System: In Vitro and In Vivo Evaluation. AAPS PharmSciTech 17 , 1240–1247 (2016). Kotta, S., Khan, A. W., Ansari, S. H., Sharma, R. K. & Ali, J. Anti HIV nanoemulsion formulation: Optimization and in vitro–in vivo evaluation. Int J Pharm 462 , 129–134 (2014). Mazonde, P., Khamanga, S. M. M., Walker, R. B. & Krause, R. W. M. Design, optimization, manufacture and characterization of Efavirenz-loaded flaxseed oil nanoemulsions. Pharmaceutics 12 , 1–22 (2020). APA. PubChem Compound Summary for CID 64139, Efavirenz. National Center for Biotechnology Information https://pubchem.ncbi.nlm.nih.gov/compound/Efavirenz (2021). Persson, L. C., Porter, C. J. H., Charman, W. N. & Bergström, C. A. S. Computational prediction of drug solubility in lipid based formulation excipients. Pharm Res 30 , 3225–3237 (2013). Rane, S. S. & Anderson, B. D. What determines drug solubility in lipid vehicles: Is it predictable? Adv Drug Deliv Rev 60 , 638–656 (2008). Moura, R. P., Pacheco, C., Pêgo, A. P., des Rieux, A. & Sarmento, B. Lipid nanocapsules to enhance drug bioavailability to the central nervous system. Journal of Controlled Release 322 , 390–400 (2020). Heurtault, B., Saulnier, P., Proust, J.E., Benoit, J. P. Lipid nanocapsules, preparation process and use as medicine. 2 , (2000). Mukubwa, G. K., Safari, J. B. & Walker, R. B. Design , Manufacturing , Characterization and Evaluation of Lipid Nanocapsules to Enhance the Biopharmaceutical Properties of Efavirenz. (2022). Hua, S. Advances in Oral Drug Delivery for Regional Targeting in the Gastrointestinal Tract - Influence of Physiological, Pathophysiological and Pharmaceutical Factors. Front Pharmacol 11 , 524 (2020). Liu, J., Leng, P. & Liu, Y. Oral drug delivery with nanoparticles into the gastrointestinal mucosa. Fundam Clin Pharmacol 35 , 86–96 (2021). Suk, J. S., Xu, Q., Kim, N., Hanes, J. & Ensign, L. M. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev 99 , 28 (2015). Ways, T. M. M., Lau, W. M. & Khutoryanskiy, V. V. Chitosan and Its Derivatives for Application in Mucoadhesive Drug Delivery Systems. Polymers (Basel) 10 , 267 (2018). Azhar, S. N. A. S., Ashari, S. E., Zainuddin, N. & Hassan, M. Nanostructured Lipid Carriers-Hydrogels System for Drug Delivery: Nanohybrid Technology Perspective. Molecules 27 , 289 (2022). Desfrançois, C., Auzély, R. & Texier, I. Lipid nanoparticles and their hydrogel composites for drug delivery: A review. Pharmaceuticals 11 , (2018). Mukubwa, G. K. Design, synthesis, manufacture, characterization and evaluation of lipid nanocapsules in chitosan-iota-carrageenan based hydrogel scaffold as a potential anti-Covid-19 drug delivery system. (2022). Rahmani, Z., Ghaemy, M. & Olad, A. Preparation of nanogels based on kappa-carrageenan/chitosan and N-doped carbon dots: study of drug delivery behavior. Polymer Bulletin vol. 78 2709–2726 Preprint at https://doi.org/10.1007/s00289-020-03236-x (2021). Qi, X. et al. Salecan-Based pH-Sensitive Hydrogels for Insulin Delivery. Mol Pharm 14 , 431–440 (2017). Bienvenu, E., Hoffmann, K. J., Ashton, M. & Kayumba, P. C. A rapid and selective HPLC-UV method for the quantitation of efavirenz in plasma from patients on concurrent HIV/AIDS and tuberculosis treatments. Biomedical Chromatography 27 , 1554–1559 (2013). Safari, J. B., Bapolisi, A. M. & Krause, R. W. M. Development of pH-sensitive chitosan-g-poly (Acrylamide-co-acrylic acid) hydrogel for controlled drug delivery of tenofovir disoproxil fumarate. Polymers (Basel) 13 , (2021). Naeem, A., Yu, C., Zhu, W., Zang, Z. & Guan, Y. Study of Hydroxypropyl β-Cyclodextrin and Puerarin Inclusion Complexes Encapsulated in Sodium Alginate-Grafted 2-Acrylamido-2-Methyl-1-Propane Sulfonic Acid Hydrogels for Oral Controlled Drug Delivery. Gels 9 , 246 (2023). Magli, S. et al. Combined analytical approaches to standardize and characterize biomaterials formulations: Application to chitosan-gelatin cross-linked hydrogels. Biomolecules 11 , 683 (2021). H Mohd Yusoff, S. N. et al. Akademia Baru Studies on Thermal and Conductivity of Modified Natural Rubber. Journal of Advanced Research in Materials Science ISSN 12 , 2289–7992. Klein, R. S. et al. Iota-Carrageenan/Chitosan Nanoparticles via Coacervation: Achieving Stability for Tiny Particles. Nanomaterials 15 , 161 (2025). Gonçalves, R. C. R. et al. Bioimaging of Lysosomes with a BODIPY pH-Dependent Fluorescent Probe. Molecules 27 , (2022). Heurtault, B., Saulnier, P., Pech, B., Proust, J. E. & Benoit, J. P. A novel phase inversion-based process for the preparation of lipid nanocarriers. Pharm Res 19 , 875–880 (2002). Budai, L., Budai, M., Fülöpné Pápay, Z. E., Vilimi, Z. & Antal, I. Rheological Considerations of Pharmaceutical Formulations: Focus on Viscoelasticity. Gels 2023, Vol. 9, Page 469 9 , 469 (2023). Souza, A. B. de, Chaud, M. V., Alves, T. F., Souza, J. F. de & Santana, M. H. A. Hyaluronic Acid in the Intestinal Tract: Influence of Structure, Rheology, and Mucoadhesion on the Intestinal Uptake in Rats. Biomolecules 2020, Vol. 10, Page 1422 10 , 1422 (2020). Li, Z., Lu, D. & Gao, X. Optimization of mixture proportions by statistical experimental design using response surface method - A review. Journal of Building Engineering 36 , 102101 (2021). Aydar, A. Y. Utilization of Response Surface Methodology in Optimization of Extraction of Plant Materials. Statistical Approaches With Emphasis on Design of Experiments Applied to Chemical Processes (2018) doi:10.5772/INTECHOPEN.73690. Breig, S. J. M. & Luti, K. J. K. Response surface methodology: A review on its applications and challenges in microbial cultures. Mater Today Proc 42 , 2277–2284 (2021). Jastram, A., Lindner, T., Luebbert, C., Sadowski, G. & Kragl, U. Swelling and diffusion in polymerized ionic liquids-based hydrogels. Polymers (Basel) 13 , (2021). Kamoun, E. A. & Menzel, H. HES-HEMA nanocomposite polymer hydrogels: Swelling behavior and characterization. Journal of Polymer Research 19 , (2012). Ciolacu, D. E., Nicu, R. & Ciolacu, F. Cellulose-Based Hydrogels as Sustained Drug-Delivery Systems. Materials 2020, Vol. 13, Page 5270 13 , 5270 (2020). Lin, J., Jiao, G. & Kermanshahi-Pour, A. Algal Polysaccharides-Based Hydrogels: Extraction, Synthesis, Characterization, and Applications. Marine Drugs 2022, Vol. 20, Page 306 20 , 306 (2022). Kuo, C. C., Clark, S., Qin, H. & Shi, X. Development of a shelf-stable, gel-based delivery system for probiotics by encapsulation, 3D printing, and freeze-drying. LWT 157 , 113075 (2022). Swathi, H. P. et al. Effect of gamma sterilization on the properties of microneedle array transdermal patch system. Drug Dev Ind Pharm 46 , 606–620 (2020). Wang, H., Meng, Z., Zhao, C.-Y. & -, al. Biocompatibility evaluation for the developed hydrogel wound dressing – ISO-10993-11 standards – in vitro and in vivo study. Biomed Phys Eng Express 8 , 015010 (2021). Bashir, S., Teo, Y. Y., Naeem, S., Ramesh, S. & Ramesh, K. PH responsive N-succinyl chitosan/Poly (acrylamide-co-acrylic acid) hydrogels and in vitro release of 5-fluorouracil. PLoS One 12 , (2017). Thang, N. H., Chien, T. B. & Cuong, D. X. Polymer-Based Hydrogels Applied in Drug Delivery: An Overview. Gels vol. 9 Preprint at https://doi.org/10.3390/gels9070523 (2023). Li, B. et al. Preparation and properties of CS/P(AM-co-AA) composite hydrogels by frontal polymerization of ternary DES. RSC Adv 12 , 34724–34729 (2022). Krambeck, K. et al. Design and characterization of Nanostructured lipid carriers (NLC) and Nanostructured lipid carrier-based hydrogels containing Passiflora edulis seeds oil. Int J Pharm 600 , (2021). Hoare, T. R. & Kohane, D. S. Hydrogels in drug delivery: Progress and challenges. Polymer (Guildf) 49 , 1993–2007 (2008). Sonker, M. et al. Review of Recent Advances and Their Improvement in the Effectiveness of Hydrogel-Based Targeted Drug Delivery: A Hope for Treating Cancer. ACS Applied Bio Materials vol. 4 8080–8109 Preprint at https://doi.org/10.1021/acsabm.1c00857 (2021). Vigata, M., Meinert, C., Hutmacher, D. W. & Bock, N. Hydrogels as drug delivery systems: A review of current characterization and evaluation techniques. Pharmaceutics 12 , 1–45 (2020). Hameed, H., Faheem, S., Paiva-Santos, A. C., Sarwar, H. S. & Jamshaid, M. A Comprehensive Review of Hydrogel-Based Drug Delivery Systems: Classification, Properties, Recent Trends, and Applications. AAPS PharmSciTech vol. 25 Preprint at https://doi.org/10.1208/s12249-024-02786-x (2024). Zhang, W. et al. Factors affecting the properties of superabsorbent polymer hydrogels and methods to improve their performance: a review. Journal of Materials Science vol. 56 16223–16242 Preprint at https://doi.org/10.1007/s10853-021-06306-1 (2021). Chen, J., Garcia, E. S. & Zimmerman, S. C. Intramolecularly Cross-Linked Polymers: From Structure to Function with Applications as Artificial Antibodies and Artificial Enzymes. Acc Chem Res 53 , 1244–1256 (2020). Paajanen, A., Vaari, J. & Verho, T. Crystallization of cross-linked polyethylene by molecular dynamics simulation. Polymer (Guildf) 171 , 80–86 (2019). Liang, J.-H. Fluorescence Due to Interactions of Oxidizing Soybean Oil and Soy Proteins . Singh, J. & Nayak, P. pH-responsive polymers for drug delivery: Trends and opportunities. Journal of Polymer Science 61 , 2828–2850 (2023). Yoshida, T., Lai, T. C., Kwon, G. S. & Sako, K. pH- and ion-sensitive polymers for drug delivery. Expert Opin Drug Deliv 10 , 1497 (2013). Golla, V. M. et al. Separation, Identification, and Characterization of Transformation Products Related to Relugolix Upon Subjecting to In Vitro Gastrointestinal Fluids Utilizing Liquid Chromatography, Mass Spectrometry, and Nuclear Magnetic Resonance Spectroscopy. Sep Sci Plus 8 , e202400195 (2025). Abouelmagd, S. A., Sun, B., Chang, A. C., Ku, Y. J. & Yeo, Y. Release Kinetics Study of Poorly Water-Soluble Drugs from Nanoparticles: Are We Doing It Right? Mol Pharm 12 , 997 (2015). Lavrentev, F. V. et al. Diffusion-Limited Processes in Hydrogels with Chosen Applications from Drug Delivery to Electronic Components. Molecules 28 , 5931 (2023). Safari, J. B., Bapolisi, A. M. & Krause, R. W. M. Development of pH-Sensitive Chitosan-g-poly(acrylamide-co-acrylic acid) Hydrogel for Controlled Drug Delivery of Tenofovir Disoproxil Fumarate. Polymers (Basel) 13 , 3571 (2021). Vegad, U. et al. pH stimuli-responsive hydrogels from non-cellulosic biopolymers for drug delivery. Front Bioeng Biotechnol 11 , 1270364 (2023). Chiappetta, D. A., Hocht, C., Taira, C. & Sosnik, A. Efavirenz-loaded polymeric micelles for pediatric anti-HIV pharmacotherapy with significantly higher oral bioavailability [corrected]. Nanomedicine (Lond) 5 , 11–23 (2010). Almeida dos Santos, K., Maria Coelho de Carvalho Moreira, L., Lamartine Soares-Sobrinho, J. & Felts de La Roca Soares, M. New Horizons in Antiretroviral Drug Delivery Systems for HIV Management. Curr Med Chem 31 , (2024). Varshosaz, J., Taymouri, S., Jahanian-Najafabadi, A. & Alizadeh, A. Efavirenz oral delivery via lipid nanocapsules: formulation, optimisation, and ex‐vivo gut permeation study. IET Nanobiotechnol 12 , 795 (2018). Pitorre, M. et al. Polymer-free hydrogel made of lipid nanocapsules, as a local drug delivery platform. Mater Sci Eng C Mater Biol Appl 126 , (2021). Saleemi, M. A. et al. Toxicity of Carbon Nanotubes: Molecular Mechanisms, Signaling Cascades, and Remedies in Biomedical Applications. Chemical Research in Toxicology vol. 34 24–46 Preprint at https://doi.org/10.1021/acs.chemrestox.0c00172 (2021). Miura, N. & Shinohara, Y. Cytotoxic effect and apoptosis induction by silver nanoparticles in HeLa cells. Biochem Biophys Res Commun 390 , 733–737 (2009). Manna, S. K. et al. Single-walled carbon nanotube induces oxidative stress and activates nuclear transcription factor-κB in human keratinocytes. Nano Lett 5 , 1676–1684 (2005). Gravely, M. et al. Aggregation Reduces Subcellular Localization and Cytotoxicity of Single-Walled Carbon Nanotubes. ACS Appl Mater Interfaces 14 , 19168–19177 (2022). Happi, G. M. et al. Bioassay-Guided Isolation of Antiplasmodial Compounds from Hypericum lanceolatum Lam. (Hypericaceae) and Their Cytotoxicity and Molecular Docking. Biomed Res Int 2023 , 4693765 (2023). Tenorio-Borroto, E. et al. Model for high-throughput screening of drug immunotoxicity – Study of the anti-microbial G1 over peritoneal macrophages using flow cytometry. Eur J Med Chem 72 , 206–220 (2014). Mustafa, Y. F. Synthesis of novel 6-aminocoumarin derivatives as potential –biocompatible antimicrobial and anticancer agents. J Mol Struct 1320 , 139658 (2025). Nordin, M. L., Abdul Kadir, A., Zakaria, Z. A., Abdullah, R. & Abdullah, M. N. H. In vitro investigation of cytotoxic and antioxidative activities of Ardisia crispa against breast cancer cell lines, MCF-7 and MDA-MB-231. BMC Complement Altern Med 18 , (2018). Hughes, J. P., Rees, S. S., Kalindjian, S. B. & Philpott, K. L. Principles of early drug discovery. Br J Pharmacol 162 , 1239 (2011). Akinboye, E. S. & Bakare, O. Biological Activities of Emetine . The Open Natural Products Journal (2011). Amenta, F., Battineni, G., Traini, E. & Pallotta, G. Choline-containing phospholipids and treatment of adult-onset dementia disorders. in Diagnosis and Management in Dementia: The Neuroscience of Dementia, Volume 1 477–493 (Elsevier, 2020). doi:10.1016/B978-0-12-815854-8.00030-6. Onaolapo, M. C. et al. Lecithin and cardiovascular health: a comprehensive review. Egyptian Heart Journal vol. 76 Preprint at https://doi.org/10.1186/s43044-024-00523-0 (2024). St-Onge, M.-P. & Jones, P. J. H. Recent Advances in Nutritional Sciences Physiological Effects of Medium-Chain Triglycerides: Potential Agents in the Prevention of Obesity 1 . https://academic.oup.com/jn/article-abstract/132/3/329/4687297 (2018). van Gastel, N. et al. Induction of a Timed Metabolic Collapse to Overcome Cancer Chemoresistance. Cell Metab 32 , 391-403.e6 (2020). Thomas, D. D. et al. Effects of medium chain triglycerides supplementation on insulin sensitivity and beta cell function: A feasibility study. PLoS One 14 , (2019). Dosh, R. H., Jordan-Mahy, N., Sammon, C. & Le Maitre, C. L. Tissue Engineering Laboratory Models of the Small Intestine. Tissue Eng Part B Rev 24 , 98–111 (2018). Peng, C., Lu, W. & Fang, Y. An insight into the effect of food nanoparticles on the metabolism of intestinal cells. Curr Opin Food Sci 43 , 174–182 (2022). Sornkamnerd, S., Okajima, M. K. & Kaneko, T. Tough and Porous Hydrogels Prepared by Simple Lyophilization of LC Gels. ACS Omega 2 , 5304–5314 (2017). Mohite, P. et al. Lipid-based oral formulation in capsules to improve the delivery of poorly water-soluble drugs. Frontiers in Drug Delivery 3 , 1232012 (2023). Žigrayová, D., Mikušová, V. & Mikuš, P. Advances in Antiviral Delivery Systems and Chitosan-Based Polymeric and Nanoparticulate Antivirals and Antiviral Carriers. Viruses 2023, Vol. 15, Page 647 15 , 647 (2023). Siyawamwaya, M. Three dimensional printed controlled release tritherapeutic tablet (3D CRTT) for the delivery of anti-HIV drugs. Preprint at https://hdl.handle.net/10539/24794 (2017). Mabrouk, M. et al. Design of a novel crosslinked HEC-PAA porous hydrogel composite for dissolution rate and solubility enhancement of efavirenz. Int J Pharm 490 , 429–437 (2015). Carvalho, G. C. et al. Highlights in poloxamer-based drug delivery systems as strategy at local application for vaginal infections. Int J Pharm 602 , 120635 (2021). Thapa, R., Gurung, S., Parat, M. O., Parekh, H. S. & Pandey, P. Application of Sol–Gels for Treatment of Gynaecological Conditions—Physiological Perspectives and Emerging Concepts in Intravaginal Drug Delivery. Gels 2022, Vol. 8, Page 99 8 , 99 (2022). Vashist, A. et al. Recent trends on hydrogel based drug delivery systems for infectious diseases. Biomater Sci 4 , 1535–1553 (2016). Additional Declarations No competing interests reported. Supplementary Files MukubwaManuscriptSupportinginformationJuly2025.docx Cite Share Download PDF Status: Published Journal Publication published 27 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 02 Sep, 2025 Reviews received at journal 31 Aug, 2025 Reviewers agreed at journal 26 Aug, 2025 Reviews received at journal 20 Aug, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviewers invited by journal 12 Aug, 2025 Editor assigned by journal 12 Aug, 2025 Editor invited by journal 12 Aug, 2025 Submission checks completed at journal 02 Aug, 2025 First submitted to journal 02 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7078136","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":499824616,"identity":"5b90c86b-70d8-4b15-a395-a92d85603ef6","order_by":0,"name":"Grady K. 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Walker","email":"","orcid":"","institution":"Rhodes University","correspondingAuthor":false,"prefix":"","firstName":"Roderick","middleName":"B.","lastName":"Walker","suffix":""},{"id":499824621,"identity":"80531cbd-5db4-4e89-b0c4-6f296e758b36","order_by":5,"name":"Rui W.M. Krause","email":"","orcid":"","institution":"Rhodes University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"W.M.","lastName":"Krause","suffix":""}],"badges":[],"createdAt":"2025-07-08 21:53:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7078136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7078136/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-26390-6","type":"published","date":"2025-11-27T15:58:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89465860,"identity":"57178a51-5021-4f8e-8ced-99bc88f0fc28","added_by":"auto","created_at":"2025-08-20 08:34:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":471523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of lipid nanocapsules and chitosan hydrogel for enhanced delivery of hydrophobic drugs and antiviral efficacy. (a) \u003c/strong\u003eThe LNCs‑hydrogel composite is designed for potential oral delivery of hydrophobic antiviral drugs. This combination leverages the solubility enhancement properties of lipid nanocapsules and the three‑dimensional scaffold of the hydrogel, which facilitates better deposition of small particles in the gut regions and their sustained release. Created with BioRender.com. \u003cstrong\u003e(b)\u003c/strong\u003e Structure of synthesized chitosan and \u003cem\u003eiota\u003c/em\u003e‑Carrageenan crosslinked hydrogel; a detailed chemical reaction of its formation can be found in supporting material \u003cstrong\u003eFigure S3\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/23b279837444177dd436ac64.png"},{"id":89467010,"identity":"eff747eb-dcae-4580-8489-afb70ea26827","added_by":"auto","created_at":"2025-08-20 08:42:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2522531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControlling the swelling capacity of hydrogels by varying proportions of monomers, polymers, and crosslinker. (a)\u003c/strong\u003e A combination of high iota-carrageenan content with reduced chitosan levels significantly enhances the swelling behavior of the hydrogel; \u003cstrong\u003e(b)\u003c/strong\u003e Increasing iota-carrageenan while simultaneously lowering the amount of acrylic acid leads to greater swelling capacity; \u003cstrong\u003e(c)\u003c/strong\u003e A higher proportion of iota-carrageenan paired with a lower proportion of acrylamide results in increased water absorption; and \u003cstrong\u003e(d)\u003c/strong\u003e Swelling capacity also increases when iota-carrageenan content is elevated while the concentration of N,N′-methylenebisacrylamide (MBA) is reduced.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/9a60cae9bb45e1da93dc1c52.png"},{"id":89467005,"identity":"1f260049-e176-42eb-9c1b-df7e90e4de7e","added_by":"auto","created_at":"2025-08-20 08:42:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":730837,"visible":true,"origin":"","legend":"\u003cp\u003eOptimal swelling capacity of CS/iCar‑p(Aam‑\u003cem\u003eco\u003c/em\u003e‑AA) hydrogel and encapsulation efficiency of EVF-LNCs in CS/iCar‑p(Aam‑co‑AA) hydrogel. (a) Highest and optimal swelling capacity (%SC) with corresponding proportions. Of the starting materials used for the rest of the experiments, namely the encapsulation capacity and drug release study. (b) Eight different concentrations of encapsulated EFV in LNCs were evaluated to yield the optimized hybrid system with optimum EVF concentration. The curve shows that the encapsulation efficiency stops increasing after reaching concentrations between 46 – 55 mg/mL.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/1359e79c9c1b5d9764966107.png"},{"id":89465865,"identity":"22dc496b-f0c4-45a2-9c75-8b10ec8c7d1e","added_by":"auto","created_at":"2025-08-20 08:34:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":419688,"visible":true,"origin":"","legend":"\u003cp\u003eThermal gravimetric and fourier transform infrared spectroscopy analysis of hydrogels. (a) Thermogravimetric analysis (TGA) profiles showing thermal decomposition of CS/iCar p(AAm-co-AA) hydrogel, EFV LNCs–loaded CS/iCar p(AAm-co-AA) hydrogel composite, chitosan (CS), and iota-carrageenan (iCar), with weight loss plotted against temperature. (b) First-order derivative curves (DTG) of the TGA data illustrating the rate of decomposition. (c) FTIR spectra of the raw materials: chitosan (CS), iota-carrageenan (iCar), acrylic acid (AA), and acrylamide (AAm). (d) FTIR spectra of N,N′-methylenebisacrylamide (MBA), Efavirenz-loaded lipid nanocapsules (EFV LNCs), CS/iCar p(AAm-co-AA) hydrogel, and the EFV‑LNCs‑CS/iCar p(Aam‑co‑AA) hydrogel composite.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/ca36c22c8ac5669f802ddad8.png"},{"id":89467006,"identity":"942d2eac-5dcb-4a3b-acb8-4ad1d3493f22","added_by":"auto","created_at":"2025-08-20 08:42:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":745129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNitrogen and sulfur appeared on the surface of porous CS/iCar‑p(Aam‑co‑AA) hydrogel structure and\u003c/strong\u003e \u003cstrong\u003ecrystallinity significantly changes during hydrogel synthesis and addition of EFV. \u003c/strong\u003eScanning electron micrographs (scale bar: 50 µm) reveal the porous surface morphology of \u003cstrong\u003e(a)\u003c/strong\u003e the unloaded CS/iCar p(AAm-co-AA) hydrogel and \u003cstrong\u003e(b)\u003c/strong\u003e the EFV LNCs–loaded composite hydrogel. Energy-dispersive X-ray spectroscopy (EDS) spectra indicating the elemental composition of \u003cstrong\u003e(c)\u003c/strong\u003e the EFV‑LNCs‑CS/iCar p(AAm-co-AA) hydrogel composite and \u003cstrong\u003e(d) \u003c/strong\u003ethe unloaded hydrogel, confirming the presence of nitrogen, sulfur, carbon, oxygen, and chlorine attributed to Efavirenz. \u003cstrong\u003e(e)\u003c/strong\u003e Powder X-ray diffraction (XRD) patterns comparing the crystallinity profiles of the CS/iCar p(AAm-co-AA) hydrogel and its EFV LNCs composite with the raw materials—chitosan, iota-carrageenan, free Efavirenz (EFV), and EFV-LNCs—alongside their corresponding mean crystallinity values.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/be4ab7b6358d1c02ffbec9d8.png"},{"id":89468113,"identity":"c54e559a-6bce-48ea-96c6-198ce5c2b132","added_by":"auto","created_at":"2025-08-20 08:58:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":378608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEM and fluorescent microscopy analysis of EFV‑LNCs‑CS/iCar‑p(Aam‑co‑AA) hydrogel composite\u003c/strong\u003e. \u003cstrong\u003e(a) \u003c/strong\u003eTEM micrographs of EFV‑LNCs‑CS/iCar‑p(Aam‑co‑AA) hydrogel composite highlighting scaffold entanglements and tiny particles at 100 nm (left) and 200 nm (right), respectively; \u003cstrong\u003e(b) \u003c/strong\u003eMicrograph of LNCs‑CS/iCar‑p(Aam‑co‑AA) hydrogel composite visualized under fluorescence microscope using U-MWU DM400 filter (DAPI fluorescence) at 10x magnification, the fluorescent amaranth purple dyed dots being indicative of LNCs clusters embedded in hydrogel at 10x magnification.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/ff7163e0c06cb5aa276b5d08.png"},{"id":89465866,"identity":"5cdd4a4e-f876-4ea5-9f1a-65a8eca11d44","added_by":"auto","created_at":"2025-08-20 08:34:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":273123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEFV‑LNCs‑CS/iCar‑p(Aam‑co‑AA hydrogel composite\u003c/strong\u003e \u003cstrong\u003eprolongs release of drug at both pH 4 and 7. \u003c/strong\u003e\u0026nbsp;Cumulative release profiles of Efavirenz encapsulated in the hydrogel composite at pH 7 and pH 4, demonstrating prolonged release of the encapsulated EFV (red and green lines) compared to the non-encapsulated EFV (orange and blue lines). Wilcoxon signed-rank test revealed no significant difference between Free EFV at pH 4 and pH 7 (n.s., p = 0.807), while encapsulation significantly slowed release in both conditions (p ≤ 0.001). Furthermore, the hydrogel composite demonstrated pH-responsive release behavior, with significantly greater Efavirenz release observed at pH 7 compared to pH 4 (p = 0.0077). These results underscore the composite’s capacity for controlled and sustained drug delivery, highlighting its potential for targeted release under physiological conditions. n.s. = not significant; *** = p ≤ 0.001. Experiments were conducted in triplicate.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/404a8cafd77af6e8fa5c6393.png"},{"id":89465870,"identity":"b9671df7-b11e-47bb-994e-692fb1138510","added_by":"auto","created_at":"2025-08-20 08:34:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":230582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNew LNCs‑hydrogel composite is cytocompatible. \u003c/strong\u003eViability of HeLa cells treated with various formulations—LNCs [50 µg/mL], Hydrogel [50 µg/mL], LNCs-Hydrogel composite [50 µg/mL], EFV‑NCs, EFV‑LNCs Hydrogel composite, and Emetine [50µg/mL] (positive control)—compared to a Negative control (untreated cells), assessed using the resazurin assay. LNCs significantly increased viability (~135%, t = 16.1692, p = 0.0038, **), while Hydrogel (~85%, t = -10.1806, p = 0.0095, **), EFV-LNCs (~65%, t = -30.7915, p = 0.0011, **), and EFV-LNCs Hydrogel (~85%, t = -6.7239, p = 0.0214, *) significantly reduced viability. The LNCs-Hydrogel composite (~90%, t = -0.8552, p = 0.4825, ns) did not significantly differ from the Negative control. Data are presented as mean ± SD, and statistical significance is indicated by ns (not significant), * (p \u0026lt; 0.05), ** (p \u0026lt; 0.01), and **** (p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/8c83943c4e83771bd383b8de.png"},{"id":97178681,"identity":"647d2d3b-47e0-4e89-87c5-dca647c7bf28","added_by":"auto","created_at":"2025-12-01 16:12:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8162454,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/18d063a5-75a9-42e5-931f-93c132cf7d59.pdf"},{"id":89465863,"identity":"ac8786bd-07a7-43df-85a2-fe7a1d1cb520","added_by":"auto","created_at":"2025-08-20 08:34:02","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":399614,"visible":true,"origin":"","legend":"","description":"","filename":"MukubwaManuscriptSupportinginformationJuly2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7078136/v1/ca9312065df8d8d08929b27d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lipid Nanocapsule-Chitosan and Iota-Carrageenan Hydrogel Composite for Sustained Hydrophobic Drug Delivery","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEfavirenz (EFV) is a widely prescribed first-line antiretroviral agent belonging to the class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), commonly utilized as part of combination therapy for the treatment of HIV infection\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e​. However, it is a highly lipophilic Biopharmaceutics Classification System (BCS) Class II compound with extremely low aqueous solubility (~\u0026thinsp;9 \u0026micro;g/mL at 25\u0026deg;C), resulting in dissolution-limited and variable oral absorption (oral bioavailability\u0026thinsp;~\u0026thinsp;40\u0026ndash;45%)\u003csup\u003e1\u003c/sup\u003e. This poor and erratic pharmacokinetic profile can lead to inconsistent drug exposure; indeed, genetic variability in EFV metabolism has been linked to subtherapeutic concentrations or doselimiting side effects\u003csup\u003e2\u003c/sup\u003e​. These issues pose a significant challenge in maintaining effective and safe EFV plasma levels during longterm HIV treatment. This variability underscores the need for improved EFV formulations to achieve more reliable therapeutic outcomes. Approximately half of all approved antiviral drugs (including lopinavir, ritonavir, and remdesivir) also fall into BCS Class II and suffer from low aqueous solubility​\u003csup\u003e3\u003c/sup\u003e. Inadequate dissolution in the gastrointestinal tract is a major factor limiting the efficacy of such oral antivirals, which has motivated the development of formulation strategies to enhance solubility and absorption.\u003c/p\u003e\u003cp\u003eLipidbased nanoformulations have demonstrated success in improving the oral absorption of BCS II antivirals; for example, ritonavir and saquinavir have been marketed in lipidbased formulations to increase their bioavailability​\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;8\u003c/sup\u003e. In the case of EFV, various nanocarrier approaches \u0026ndash; such as solid lipid nanoparticles, nanoemulsions, and selfemulsifying drug delivery systems \u0026ndash; have been explored to boost its dissolution rate and absorption, with encouraging outcomes in preclinical studies\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;11\u003c/sup\u003e​. These findings reinforce the potential of advanced delivery systems to overcome EFV\u0026rsquo;s biopharmaceutical limitations. Among lipidbased nanocarriers, lipid nanocapsules (LNCs) are an attractive option for enhancing EFV delivery. LNCs consist of a liquid lipid core surrounded by a surfactant shell, a structure that enables high encapsulation efficiency of hydrophobic drugs like EFV (log P\u0026thinsp;~\u0026thinsp;4.6)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e2\u0026ndash;16\u003c/sup\u003e. The small (~\u0026thinsp;20\u0026ndash;100 nm) LNC particles disperse readily in gastrointestinal fluids, maintaining EFV in a solubilized state and facilitating its transport to absorptive sites. Encapsulating EFV in LNCs can also convert the drug into an amorphous form, therefore enhancing its apparent solubility\u003csup\u003e17\u003c/sup\u003e​. Additionally, LNC formulations help protect the drug from degradation and can modulate its release, which may reduce presystemic metabolism and improve the fraction of EFV absorbed. While LNCs address the solubility issue, a complementary approach is needed to prolong EFV\u0026rsquo;s residence time in the gut for absorption as LNCs are exposed to rapid gastrointestinal clearance, enzymatic degradation, and premature drug release under harsh gastrointestinal conditions\u003csup\u003e18\u0026ndash;20\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we incorporated EFV-containing lipid nanocapsules (LNCs) into a chitosan/iota-carrageenan hydrogel to create a mucoadhesive nanoparticle-in-hydrogel delivery system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Chitosan, a cationic polysaccharide, is well known for its mucoadhesive properties, enabling formulations to adhere to the intestinal mucosa and extend drug residence time at the absorption site. ​\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Iota-carrageenan, an anionic polysaccharide, interacts with chitosan to form a crosslinked hydrogel network capable of entrapping the nanocapsules. This biopolymer-based matrix anchors the EFV-containing LNCs in the gastrointestinal environment, limiting rapid transit and enabling sustained drug release. Such hybrid systems combine the solubilization advantages of nanocarriers with the localized retention properties of hydrogels, offering a synergistic strategy to enhance oral drug delivery.​\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. We demonstrated that encapsulating EFV in LNCs within a chitosan/iota-carrageenan hydrogel significantly improved key oral biopharmaceutical properties, including solubility, physicochemical stability, sustained release, and cytocompatibility. The LNCs enhanced EFV solubility and protected the drug from premature degradation, while the mucoadhesive hydrogel extended gastrointestinal residence and facilitated more complete absorption. The primary objective of this work was to develop and characterize the LNC-in-hydrogel formulation and to evaluate its potential to overcome EFV\u0026rsquo;s solubility and delivery limitations. This combined strategy addresses a major formulation challenge by simultaneously improving drug solubility and prolonging mucosal retention. If successful, it could lead to more consistent EFV therapy and serve as a platform for improving the oral delivery of other poorly soluble antiviral agents.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eThis section was adapted from the author\u0026rsquo;s previously submitted dissertation [Mukubwa, G. K., 2022]\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemicals.\u003c/b\u003e Crude soybean lecithin granules were sourced from Health Connection Wholefoods (USA). As per manufacturer information, the granules predominantly consist of phosphatidylcholine, phosphatidylinositol, polyunsaturated and saturated fatty acids, glycaemic carbohydrates, and sodium. Medium-chain triglyceride (MCT) oil of high purity was acquired from Absolute Organix (Jeppestown, Johannesburg), while Labrafac Lipophile 1349 was kindly provided by Gattfoss\u0026eacute; (France). Polysorbate 80 (Tween 80) was obtained from Merck (Johannesburg, Gauteng, South Africa), and Efavirenz was generously supplied by Adcock Ingram Ltd. (Wadeville, Gauteng, South Africa)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Additional reagents, including iota-carrageenan (iCar), low molecular weight chitosan (CS), acrylic acid (AA), N,N\u0026prime;-methylenebisacrylamide (MBA), and acrylamide (Aam), were procured from Sigma Aldrich (Darmstadt, Germany). Glacial acetic acid, sodium chloride, and acetone were purchased from Minema (Roodepoort, South Africa), and ammonium persulfate (APS) from Saarchem (Wadeville, South Africa). HPLC-grade acetonitrile was sourced from Merck (Germany), and ultrapure HPLC-grade water was prepared using a RephiLe Bioscience Direct Purefi Ultrapure RO Water System (Boston, MA, USA). Human cervical adenocarcinoma (HeLa) cell lines were obtained from ATCC\u0026reg; (Manassas, Virginia, USA)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eInstruments.\u003c/b\u003e High-performance liquid chromatography (HPLC) was carried out using an Agilent 1100 system comprising a quaternary pump (model G1311A), a degasser (G1322A), a diode array detector (G1315B), and a manual injection unit (G1328B), with separation achieved on a Phenomenex\u0026reg; Kinetex\u0026reg; C18 column (2.6 \u0026micro;m, 100 \u0026Aring;, 150 \u0026times; 4.6 mm i.d.). Sample lyophilization was performed using a LABCONCO FreeZone\u0026reg; 6 Liter Benchtop Freeze Dryer (Kansas City, MO, USA)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Infrared (IR) spectra were collected using a PerkinElmer Spectrum 100 FTIR spectrometer. Differential scanning calorimetry (DSC) was conducted on a TA Instruments DSC 250. Crystalline structure of the materials was characterized by X-ray diffraction (XRD) using a Bruker D2 Phaser (second generation, Bruker, Billerica, MA, USA). The mean hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the lipid nanocapsules (LNCs) were measured using a Zetasizer Nano ZEN 3600 (Malvern Instruments, Malvern, UK)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Transmission electron microscopy (TEM) was conducted using a Zeiss Libra 120KV instrument (Oberkochen, Germany) to assess particle morphology. Elemental composition was determined via energy-dispersive X-ray spectroscopy (EDS) using an INCA PENTA FET system coupled with a VAGA TESCAM unit (Brno, Czech Republic). Surface features of the hydrogels were examined using a TESCAN VEGA scanning electron microscope (Brno, Czech Republic). Fluorescence imaging was performed with an Olympus BX60 fluorescence microscope (Japan)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of Efavirenz based Lipid Nanocapsules (EFVLNCs).\u003c/b\u003e Efavirenz-loaded lipid nanocapsules were formulated using a phase inversion technique, as established in our earlier work\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e7\u003c/sup\u003e. In summary, the formulation consisted of a mixture of medium-chain triglyceride (MCT) oil, crude soybean lecithin, Tween 80, and aqueous sodium chloride solution (18.5:0.5 g, water:NaCl) in a weight ratio of 12:3:9:76, incorporating Efavirenz at a final concentration of 17.5 mg/mL. Specifically, a total of 25 g of the formulation was prepared by combining 3 g MCT oil, 0.75 g lecithin, 2.25 g Tween 80, and 19 g NaCl solution in a 250 mL round-bottom flask, followed by the addition of 1.35 g of Efavirenz. The mixture was stirred magnetically at 1300 rpm and heated to 95\u0026deg;C using an oil bath on a hot plate. A series of three controlled heating\u0026ndash;cooling cycles between 70\u0026deg;C and 95\u0026deg;C were then performed, each lasting approximately 90 minutes, during which phase inversion was visually indicated by the appearance of a translucent emulsion. To induce nanocapsule formation, the emulsion was rapidly quenched by adding cold HPLC-grade water (1\u0026ndash;4\u0026deg;C) in a volume three times greater than the initial aqueous phase during the final cycle. A water-cooled condenser was used throughout the procedure to prevent evaporation of volatile constituents.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDesign and Optimization of chitosangiotacarrageenangpoly (acrylamide\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003eacrylic acid) hydrogel (CS/iCarp(Aam\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003eAA)).\u003c/b\u003e An I-optimal mixture design was conducted using Design-Expert\u0026reg; software version 13 (Stat-Ease, Inc., Minneapolis, MN, USA) to investigate how the composition of the CS/iCar p(AAm-co-AA) hydrogel influences its swelling behavior. The formulation components included two polymeric materials (chitosan and iota-carrageenan), two monomers (acrylic acid and acrylamide), and one crosslinking agent (N,N\u0026prime;-methylenebisacrylamide). These were defined as independent variables, while the hydrogel\u0026rsquo;s swelling capacity served as the response variable. A polynomial model was fitted to predict the swelling performance based on variations in component proportions. The input variables and their respective percentage contributions, which together sum to 100%, are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIoptimal mixture design input variables\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow Limit\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConstraint\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHigh Limit\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eA: IOTACARRAGEENAN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB: CHITOSAN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eC: ACRYLIC ACID\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e53.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eD: ACRYLAMIDE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e53.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eE: \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003eMETHYLENE BISACRYLAMIDE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;E\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e=\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e100.000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eModel Optimization and Confirmation.\u003c/b\u003e To evaluate the predictive accuracy of the developed polynomial model, swelling capacity maximization was set as the sole optimization criterion. The software identified the most desirable formulation, which was synthesized in triplicate. Triplicate swelling measurements were then performed, and the resulting average was compared to the model\u0026rsquo;s 95% prediction interval (95% PI) to validate the model.\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"width: 241px; height: 27.4223px;\" width=\"241\" height=\"27.4223\"\u003e where \u003cimg src=\"data:image/png;base64,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\" style=\"width: 17px; height: 37.4px;\" width=\"17\" height=\"37.4\"\u003e is the predicted value of the response, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{t}_{\\left(\\frac{\\propto\\:}{2},\\:residual\\:df\\right)}\\)\u003c/span\u003e\u003c/span\u003e is the Student\u0026rsquo;s t critical value and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{SE}_{pred}\\)\u003c/span\u003e\u003c/span\u003e is the standard error of the prediction\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynthesis of CS/iCarp(AamcoAA) hydrogel.\u003c/b\u003e The hydrogel synthesis protocol was adapted from the approach described by Rahmani et al.\u003csup\u003e25\u003c/sup\u003e, with modifications introduced to suit the present study. The hydrogels were fabricated via free radical precipitation graft copolymerization of acrylic acid (AA) and acrylamide (AAm) onto a chitosan\u0026ndash;iota carrageenan backbone, using ammonium persulfate (APS) as the radical initiator. For each batch, a total of 500 mg of combined ingredients was prepared. Iota carrageenan was first dissolved in 10 mL of HPLC-grade water at 60\u0026deg;C (Mixture A), while chitosan was separately solubilized in 10 mL of 1% (w/v) acetic acid solution (Mixture B). Both solutions were stirred independently at 800 rpm for 1 hour to ensure uniform dispersion. Subsequently, Mixture B was added to Mixture A, followed by the incorporation of the monomers (acrylic acid and acrylamide) and the crosslinker (MBA) in specified amounts. The polymerization reaction was initiated by adding APS (ranging from 0.05 to 0.2 g) under a nitrogen atmosphere. After polymerization, the hydrogels were thoroughly washed with approximately 500 mL of acetone to remove unreacted residues, and then dried in an oven at 40\u0026deg;C\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDevelopment of EFVLNCsCS/iCarp(AamcoAA) hydrogel composite.\u003c/b\u003e To develop the EFV LNCs CS/iCar p(AAm-co-AA) hydrogel composite, Efavirenz-loaded lipid nanocapsules were incorporated into the hydrogel matrix, followed by quantification of the entrapped drug. A customized D-optimal design with randomized experimental runs was constructed using Design-Expert\u0026reg; version 13.0 (Stat-Ease, Inc., Minneapolis, MN, USA) to model and optimize the encapsulation efficiency based on varying concentrations of EFV in the lipid nanocapsules. This design yielded 13 experimental runs with EFV concentrations ranging from 17.5 mg/mL to 87.5 mg/mL\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEncapsulation was performed using the swelling equilibrium technique as previously described \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In brief, a measured volume of EFV LNCs corresponding to the target EFV concentration was diluted in a 25 mL volumetric flask. This solution was then used to hydrate 50 mg of dried hydrogel for a duration of 24 hours at ambient temperature. After the incubation period, the swollen hydrogel was separated by filtration and rinsed with HPLC-grade water. The filtrate, containing unencapsulated EFV LNCs, was collected for analysis. A 150 \u0026micro;L aliquot of this filtrate was diluted in a 10 mL volumetric flask using a 40:60 acetonitrile\u0026ndash;water mixture and analyzed by a reversed-phase HPLC method, as developed by Bienvenu et al. and validated in our previous study\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe encapsulation efficiency (EE%) was calculated using the following equation:\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{%EE=}\\frac{\\text{Mi-Mf}}{\\text{Mi}}\\text{\u0026times;100}\\)\u003c/span\u003e\u003c/span\u003e (\u003cb\u003eEq.\u0026nbsp;2\u003c/b\u003e)\u003c/p\u003e\u003cp\u003eWhere %EE stands for encapsulation efficiency, Mf is the Efavirenz mass determined from the filtrate and Mi the initial mass of Efavirenz in the soaking solution\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDroplet size, polydispersity index and zeta potential of EFVLNCs\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e4\u003c/sup\u003e. Droplet size (DS) and polydispersity index (PDI) measurements were performed using a Malvern Nano ZS Zetasizer (Malvern Instruments, Worcester, UK), operated in dynamic light scattering (DLS) mode for DS/PDI and in laser Doppler anemometry (LDA) mode for zeta potential (ZP) analysis. Before measurement, 500 \u0026micro;L of the saturated nanocapsule suspension was diluted in 50 mL of HPLC-grade water. For DS and PDI assessments, samples were transferred into BRAND\u0026reg; disposable cuvettes (12.5 \u0026times; 12.5 \u0026times; 45 mm; BRAND GmbH\u0026thinsp;+\u0026thinsp;CO KG, Wertheim, Germany). Zeta potential determinations were conducted using a folded capillary cell. All measurements were carried out at 22\u0026deg;C with a backscatter detection angle of 173\u0026deg;.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSwelling Ratio of CS/iCarp(AamcoAA) hydrogel\u003c/b\u003e\u003csup\u003e24\u003c/sup\u003e. The swelling ratio of the synthesized hydrogel, expressed as grams of water absorbed per gram of dry hydrogel (g/g), was determined using a filtration-based approach. A known quantity of dry hydrogel (50\u0026ndash;100 mg), denoted as W₀, was placed into a beaker containing 40\u0026ndash;80 mL of HPLC-grade water and allowed to swell over a 24-hour period at room temperature. Subsequently, the mass of a piece of filter paper pre-wetted with distilled water was recorded as W₁ and placed in a B\u0026uuml;chner funnel. The swollen hydrogel was separated from the solution via vacuum filtration to eliminate excess surface water. The filter paper, now holding the swollen hydrogel, was carefully removed and weighed again to obtain the final mass (W₂). The swelling ratio (SR) was calculated using the equation below, and all measurements were conducted in triplicate for accuracy\u003csup\u003e28\u003c/sup\u003e:\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"width: 142px; height: 33.4622px;\" width=\"142\" height=\"33.4622\"\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFourier Transform InfraRed Spectroscopy (FTIR)\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e4\u003c/sup\u003e. Fourier-transform infrared (FTIR) spectroscopy was employed to compare the spectral profiles of the synthesized hydrogel and the EFV LNCs\u0026ndash;loaded hydrogel composite with those of the individual raw materials, in order to detect potential modifications in functional group characteristics\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e9\u003c/sup\u003e. Spectra were acquired using a PerkinElmer Spectrum 100 FTIR spectrometer operating in attenuated total reflection (ATR) mode\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e0\u003c/sup\u003e. Each sample was analyzed in the spectral range of 650\u0026ndash;4000 cm⁻\u0026sup1;, with a total of sixteen scans per run\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e1\u003c/sup\u003e. The measurements were performed on freeze-dried forms of both the plain hydrogel and the nanocapsule-loaded composite \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e0\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThermal Gravimetric Analysis\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Thermogravimetric analysis (TGA) was conducted to evaluate the thermal stability and decomposition patterns of the CS/iCar p(AAm-co-AA) hydrogel and its EFV LNC\u0026ndash;loaded composite, in comparison with the native polymers chitosan and iota-carrageenan\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The analysis was carried out under an inert nitrogen atmosphere (flow rate: 20 mL/min) using a PerkinElmer TGA 4000 system. For each run, 2\u0026ndash;5 mg of sample was heated from 30\u0026deg;C to 600\u0026deg;C at a constant rate of 10\u0026deg;C/min.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePowder Xray Diffraction\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e4\u003c/sup\u003e. Powder X-ray diffraction (XRD) was employed to investigate the phase behavior and crystallinity of the freeze-dried CS/iCar p(AAm-co-AA) hydrogel and its EFV LNC\u0026ndash;incorporated composite, in comparison with the native polymers chitosan and iota-carrageenan. Crystallinity profiles were analyzed to assess structural differences, and statistical significance in crystallinity variations was evaluated using a one-way ANOVA test. Measurements were conducted using a Bruker D2 Phaser (Second Generation) diffractometer equipped with a LynxEye proportional counter detector, a nickel filter, and Cu Kα radiation (λ\u0026thinsp;=\u0026thinsp;1.5404 \u0026Aring;). Scans were performed over a 2θ range of 10\u0026ndash;60\u0026deg; at a scanning rate of 1\u0026deg;/min, with a fixed slit width of 6.0 mm.\u003c/p\u003e\u003cp\u003e\u003cb\u003eScanning Electron Spectroscopy (SEM) and EnergyDispersive Xray Spectroscopy (EDS)\u003c/b\u003e\u003csup\u003e24\u003c/sup\u003e. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were used to examine the surface morphology and elemental composition of the CS/iCar p(AAm-co-AA) hydrogel and its EFV LNC\u0026ndash;loaded composite. The EDS analysis was performed using an INCA PENTA FET system coupled with a VAGA TESCAN unit. Prior to imaging, hydrogels were equilibrated in HPLC-grade water for 24 hours at ambient temperature to reach maximum swelling, then lyophilized using a LABCONCO FreeZone\u0026reg; 6 Liter Benchtop Freeze Dry System. The resulting dried samples were mounted onto aluminum stubs and coated with a thin layer of gold to enhance conductivity before SEM and EDS analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransmission Electron Microscopy (TEM)\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Transmission electron microscopy (TEM) was employed to assess the morphology and structural features of the EFV LNCs\u0026ndash;loaded CS/iCar p(AAm-co-AA) hydrogel composite at an accelerating voltage of 80 kV. To ensure optimal electron transparency and maintain sample integrity, the composite underwent a carefully controlled preparation protocol. The sample was first embedded in optimal cutting temperature (OCT) compound to provide structural support during sectioning. Ultra-thin sections were then obtained via cryo-ultramicrotomy. These sections were transferred onto 3.05 mm diameter copper grids coated with holey carbon film (FORMVAR/Carbon, 300 mesh; TAAB Laboratories Equipment Ltd., Aldermaston, Berks, UK). For contrast enhancement, the sections were stained with uranyl acetate. Prior to imaging, samples were thoroughly dried in a desiccator to eliminate residual moisture that could interfere with electron beam interaction. This method enabled high-resolution visualization of the hydrogel composite while preserving its native structural characteristics.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFluorescence Microscopy\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Fluorescence microscopy was employed to confirm the presence and distribution of lipid nanocapsules (LNCs) within the CS/iCar p(AAm-co-AA) hydrogel matrix. To enable visualization, the composite was stained using amaranth dye. Specifically, 500 \u0026micro;g of amaranth was added to 25 mL of LNCs suspension, and this dye-loaded solution was used to hydrate 50 g of the dried hydrogel, resulting in a stained LNC\u0026ndash;hydrogel composite. The composite was subsequently freeze-dried prior to imaging. A small section of the dried sample was placed between two glass slides and mounted under the microscope for observation. Imaging was conducted using an Olympus BX60 fluorescence microscope (Japan), equipped with a U-MWU DM400 filter set for DAPI fluorescence, and images were captured at 10\u0026times; magnification.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDrug Release Studies\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The release behavior of Efavirenz (EFV), encapsulated within lipid nanocapsules and further embedded in the CS/iCar p(AAm-co-AA) hydrogel matrix, was evaluated using a 1% sodium lauryl sulfate (SLS) solution as the release medium. Experiments were conducted at two pH levels\u0026mdash;7.0 (neutral) and 4.0 (acidic)\u0026mdash;to simulate physiological and acidic conditions, respectively. A portion of hydrogel containing an equivalent of 3 mg of EFV was weighed and placed into a dialysis bag (cellulose membrane tubing, flat width 25 mm; Sigma Aldrich, St. Louis, MO, USA), which was then immersed in 25 mL of the release medium. The setup was maintained at 37\u0026deg;C with continuous agitation at 100 rpm. At predetermined intervals (0.5, 1, 1.5, 2, 4, 8, 12, 24, 48, 72, 96, 144, and 216 hours), 5 mL aliquots were withdrawn and replaced with an equal volume of fresh pre-warmed medium to maintain sink conditions. Each time point was analyzed in triplicate. The experiment involved two categorical variables\u0026mdash;sampling time and formulation\u0026mdash;and one continuous response variable, namely the cumulative percentage of EFV released. It was hypothesized that both the pH of the release medium and the composite nature of the drug delivery system would influence the release kinetics. Two-way ANOVA was employed to assess the statistical significance of these effects across different conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytotoxicity studies\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Cytotoxicity assessments were carried out using HeLa cells. The cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO₂. After a 24-hour incubation period, cells were harvested, resuspended in fresh medium, and seeded into a 96-well plate preloaded with various treatments: unloaded lipid nanocapsules (LNCs), Efavirenz-loaded LNCs (EFV LNCs), LNCs embedded within CS/iCar p(AAm-co-AA) hydrogel, and the fully integrated EFV LNCs\u0026ndash;hydrogel composite. All treatments were prepared at a final concentration of 50 \u0026micro;g/mL. The plate was incubated for an additional 24 hours at 37\u0026deg;C and 5% CO₂ to allow interaction between the cells and the test formulations. Following treatment, resazurin reagent\u0026mdash;diluted in culture medium according to manufacturer guidelines (0.1 to 0.5 mg/mL)\u0026mdash;was added to each well to cover the cell monolayer. The plate was then returned to the incubator for 1 to 4 hours to allow viable cells to reduce resazurin to fluorescent resorufin. Fluorescence intensity was measured using a multi-well plate reader, with all readings performed in triplicate. Emetine, a known inducer of apoptosis, served as the positive control. Cell viability was calculated using untreated cells as the 100% viability reference, and results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) based on triplicate measurements. The viability calculation followed a previously reported method\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cimg 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\" width=\"495\" height=\"82\"\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analysis\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Prior to performing statistical comparisons, data were assessed for normality using the Shapiro\u0026ndash;Wilk test. Based on the distribution of the data, appropriate statistical tests were applied. For normally distributed datasets, comparisons between two groups were made using Student\u0026rsquo;s t-test. When the data did not meet parametric assumptions, the Kruskal\u0026ndash;Wallis test was employed as a non-parametric alternative to ANOVA. Post hoc analyses were conducted using Dunn\u0026rsquo;s test to identify significant pairwise differences, with p-values adjusted for multiple comparisons. Cytotoxicity data involving comparisons between treatment groups and the negative control were analyzed using Welch\u0026rsquo;s t-test (two-tailed, assuming unequal variances). A p-value of less than 0.05 was considered statistically significant, while results with p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 were interpreted as highly significant. Data analysis, modeling, and visualization were carried out using a combination of software tools, including Design-Expert\u0026reg;, OriginPro version 9, GraphPad Prism, R, Python, and Microsoft Excel.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cb\u003eEFV-lipid nanocapsule preparation and characterization.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEFVLNCs were produced for encapsulation in a chitosan-grafted iota-carrageenan poly(acrylamide-co-acrylic acid) hydrogel (CS/iCar-p(Aam-co-AA)). Solubility tests showed that medium chain triglycerides oil (MCT oil) significantly solubilized Efavirenz compared to water (Ttest, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The data were normally distributed (Shapiro-Wilk test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.5) and analyzed using a t-test, revealing with 95% confidence that the true difference in solubility means between the two dissolving media is between 266.9 and 281.4 mg/mL. Based on previous findings reported by Mukubwa \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e17\u003c/sup\u003e, an optimized formulation of EFVLNCs was prepared using the phase inversion method\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,34\u003c/sup\u003e. The obtained EFVLNCs were characterized for their hydrodynamic diameter, polydispersity index (PDI) and zeta potential (ZP). The average values of these parameters were approximately 57.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nm, 0.148, and \u0026minus;\u0026thinsp;54.9 mV, respectively, consistent with results observed in the previous study\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMCT oil is effective for solubilizing the non-polar EFV molecule in water as it consists of a lipid-structured molecular tail that matches favorably with the hydrophobic moieties present on EFV. This property also makes MCT oil a prime candidate for EFV delivery enhancement. The hydrodynamic diameter of 57.4 nm, a low polydispersity index (0.148), as well as their zeta potential (-54.9 mV) suggests an effective, stable and homogenous nanoparticle system. These properties together indicate that LNCs are effective nanocarriers for drug delivery with enhanced stability and potential enhanced bioavailability\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynthesis and Optimization of Swelling Capacity of CS/iCar-p(Aam-co-AA) Hydrogel\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe CS/iCar-p(AAm-co-AA) hydrogel was synthesized through free radical copolymerization, with formulation optimization and statistical evaluation performed using Design-Expert\u0026reg; software version 13.0 (Stat-Ease, Inc., Minneapolis, MN, USA). An I-optimal mixture design yielded 35 distinct formulations, each prepared according to the synthesis protocol described earlier. \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e summarizes the component ratios for each formulation alongside their corresponding swelling capacities, which were used as the response variable for optimization. Swelling capacity was expressed as a percentage, calculated by the ratio of water absorbed (g) to the dry hydrogel mass (g). The proposed reaction mechanism for hydrogel formation is depicted in \u003cb\u003eFigures S2 and S3\u003c/b\u003e. Multiple polynomial models were evaluated to fit the experimental data, with the linear model demonstrating superior performance. This model yielded consistently low standard errors (\u0026lt;\u0026thinsp;0.5σ, where σ denotes the estimated variability within the dataset) across the design space (\u003cb\u003eFigure S4\u003c/b\u003e). \u003cb\u003eTable S2\u003c/b\u003e presents a comparative analysis of model fits, where the linear model was automatically selected as optimal based on its lowest predicted residual error sum of squares (PRESS) value and a significantly small sequential p-value, both indicators of strong model predictive accuracy\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhile swelling behavior was selected as the primary criterion for optimizing the CS/iCar-p(Aam-co-AA) hydrogel, given its direct relationship with drug absorption and controlled release in gastrointestinal conditions, rheological properties also critically influence the composite\u0026rsquo;s performance. Mechanical and rheological characteristics significantly impact hydrogel behavior within the dynamic gastrointestinal environment, affecting aspects such as mucoadhesion, retention time, and stability against physiological shear forces. Evaluating rheological parameters, including viscosity, elasticity, and shear-thinning behavior, is therefore crucial to predict the hydrogel\u0026rsquo;s performance under realistic gastrointestinal conditions\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Although detailed rheological characterization was beyond the scope of this study, future work should investigate these properties to optimize the formulation further, ensuring enhanced gastrointestinal retention, controlled drug release, and improved patient compliance in oral antiviral drug delivery applications.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVarying the portion of polymers, monomer and crosslinkers either increased or decreased the swelling capacity.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, a linear regression model was developed to accurately predict the swelling ratio of hydrogels based on the proportions of iota-carrageenan, chitosan, acrylic acid, acrylamide, and N,N\u0026prime;-methylenebisacrylamide (MBA) used in the formulation. These five components served as the independent variables in the model. The resulting predictive equation for swelling behavior is given as: \u003cb\u003e%SC\u003c/b\u003e\u0026thinsp;=\u0026thinsp;0.556464 x A + -0.241395 x B\u0026thinsp;+\u0026thinsp;0.0694467 x C\u0026thinsp;+\u0026thinsp;0.0907205 X D\u0026thinsp;+\u0026thinsp;0.0431736 X E (\u003cb\u003eEq.\u0026nbsp;4\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eIn the regression model, factors A through E represent the following components: iota-carrageenan (A), chitosan (B), acrylic acid (C), acrylamide (D), and N,N\u0026prime;-methylenebisacrylamide (E).\u003c/p\u003e\u003cp\u003eThis model is statistically significant (p\u0026thinsp;=\u0026thinsp;0.0451), but it only accounts for a moderate portion of the variance in swelling ratio (adjusted R\u0026sup2; = 0.1643). The insignificant lack-of-fit p-value (0.3168) means that this model is decent for predicting data without being an overfit and thus serves as a good reference to formulate hydrogels in the future\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe low R\u0026sup2; value is likely to be due to variability in hydrogel synthesis; small differences during preparation can cause significant variations in swelling behavior\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. While the model is linear and insubstantial in as much as mimicking more complex component interactions, it provides a useful approximation of swelling behavior within these experimental confines. More complex models could improve prediction, but this simple linear regression provides a nice balance between simplicity and accuracy for early-stage hydrogel development.\u003c/p\u003e\u003cp\u003eThe swelling ratios of the synthesized hydrogel formulations varied significantly, ranging from 2.84 to 434.43. This variation was directly influenced by the relative composition of the hydrogel constituents. An increase in iota-carrageenan content led to a marked enhancement in swelling capacity, whereas higher chitosan levels were associated with a reduction in water absorption (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, decreasing the proportions of acrylic acid, acrylamide, and MBA resulted in improved swelling performance. This trend is attributed to lower crosslinking density at reduced monomer and crosslinker concentrations, which facilitates greater expansion of the hydrogel network and allows more water uptake.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eValidation of the hydrogel composition model yielded a swelling ratio exceeding 300, meaning that over 300 g of water was absorbed per gram of hydrogel.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the predictive accuracy of the developed linear regression model, Design-Expert\u0026reg; software generated five optimized formulations based on the criterion of maximizing swelling capacity. Among these, the formulation with the highest desirability score was selected for experimental validation and tested in triplicate, as summarized in \u003cb\u003eTable S3\u003c/b\u003e. The optimized formulation consisted of 6% iota-carrageenan, 3% chitosan, 40% acrylic acid, 50% acrylamide, and 1% MBA, as also depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. The average swelling ratio observed experimentally fell within the 95% prediction interval, thereby confirming the model\u0026rsquo;s reliability.\u003c/p\u003e\u003cp\u003eThe close overlap of observed and predicted swelling ratio values for the 95% prediction interval (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), confirms the effectiveness of our prediction model. This predictability indicates that the model can reasonably estimate swelling ratios despite inherent variability in hydrogel synthesis. This inherent variability is reflected in the wide prediction interval of the swelling ratios provided by the model (32.6 to 1407.1). Overall, this model can be used to predict different proportions of iCar, CS, AA, Aam and MBA to synthesize a hydrogel with desired properties. It also underlines the potential of our optimized hydrogel formulation in drug delivery systems where maximum swelling is required. This formulation was designated for further experiments, including LNCshydrogel composite preparation, EFVLNCshydrogel composite preparation and EFV release studies.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eObserved swelling ratio mean against the prediction interval\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eResponse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eMean\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e95% Prediction\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePredicted\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eObserved\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e95% PI low\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95% PI high\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFormulation 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSwelling ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e444.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e304.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1407.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCS/iCarp(AamcoAA) hydrogel composite entrapped LNCs containing over 50 mg/mL of EFV\u003c/b\u003e\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFollowing the synthesis of the CS/iCar p(AAm-co-AA) hydrogel, an Efavirenz-loaded LNC solution was incorporated to form the EFV LNCs\u0026ndash;hydrogel composite. To investigate the impact of LNC drug concentration on encapsulation efficiency, thirteen different formulations were prepared based on a D-optimal (custom) randomized experimental design \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. A clear trend emerged wherein encapsulation efficiency increased with rising EFV concentrations, reaching a broad maximum between 55 and 60 mg/mL before declining at higher concentrations. The experimental data were modeled using a reduced quadratic equation, as outlined below:\u003c/p\u003e\u003cp\u003e\u003cb\u003e%EE\u003c/b\u003e\u0026thinsp;=\u0026thinsp;4.25921\u0026thinsp;+\u0026thinsp;1.4527t1\u0026times;A+-0.0123934\u0026times;A\u003csup\u003e2\u003c/sup\u003e (\u003cb\u003eEq.\u0026nbsp;5\u003c/b\u003e) where A represents the concentration of Efavirenz (EFV) within the lipid nanocapsules (LNCs).\u003c/p\u003e\u003cp\u003eTo identify an optimal EFV LNC concentration for subsequent experiments and to evaluate the model\u0026rsquo;s predictive capability, encapsulation efficiency was set as the primary optimization target. Design-Expert\u0026reg; software generated a single optimal formulation, a concentration of 58 mg/mL EFV LNCs was recommended, corresponding to a predicted encapsulation efficiency of 46.83%. In practice, 50 mg of the optimized hydrogel were immersed in 25 mL of an EFV LNC solution containing 58.61 mg/mL of the drug. The experimentally determined encapsulation efficiency fell within the 95% prediction interval \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, confirming the model\u0026rsquo;s validity for predictive applications.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of the experimentally observed mean swelling ratio with the 95% prediction interval generated by the regression model.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eResponse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eMean\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e95% Prediction\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePredicted %\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eObserved %\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e95% PI low\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95% PI high\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHydrogel\u0026thinsp;+\u0026thinsp;58 mg/mL\u003c/p\u003e\u003cp\u003eEFVLNCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eEncapsulation efficiency\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e46.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e53.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e28.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e65.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe CS/iCar p(AAm-co-AA) hydrogel exhibited thermal stability above 200\u0026deg;C.\u003c/b\u003e Thermogravimetric analysis (TGA), conducted to compare the thermal behavior of the synthesized hydrogel with its individual components, revealed a series of distinct decomposition events corresponding to the chemical crosslinking process. The incorporation of acrylic acid and acrylamide into the polysaccharide network altered the degradation profile of the final hydrogel. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, multiple weight loss stages were observed during thermal decomposition. Among the base polysaccharides, iota-carrageenan demonstrated a more rapid thermal degradation, with approximately 28% weight loss at 208\u0026deg;C, whereas chitosan showed only a 10% reduction at the same temperature. Figures\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb illustrate the unique decomposition pattern of the hydrogel, confirming its enhanced thermal resistance beyond 200\u0026deg;C. These findings support the successful chemical modification of the polysaccharide matrix, as reflected in its altered thermal characteristics.\u003c/p\u003e\u003cp\u003eThe CS/iCarp(AamcoAA) hydrogel demonstrated excellent thermal stability, exceeding 200\u0026deg;C, due to chemical crosslinking that improved its resistance to thermal degradation. The decomposition profiles in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb illustrate that chitosan and iota-carrageenan undergo distinct degradation patterns, indicating their unique contributions to the composite\u0026rsquo;s thermal resilience. Such thermal stability is particularly advantageous for oral drug delivery formulations, as it ensures that manufacturing processes involving elevated temperatures (e.g., drying, tablet compression, sterilization) will not compromise hydrogel integrity\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;44\u003c/sup\u003e. Additionally, these properties suggest the hydrogel\u0026rsquo;s suitability for other advanced biomedical applications, such as transdermal patches or implantable devices requiring sterilization via autoclaving\u003csup\u003e45\u003c/sup\u003e. These observations reinforce the importance of chemical crosslinking for obtaining hydrogels with robust thermal stability suitable for various biomedical applications, including oral dosage forms\u003csup\u003e46\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical interactions between polymers, monomers, and the crosslinker resulted in shifts in wavenumbers observed in the FTIR spectra of the synthesized hydrogel.\u003c/b\u003e Fourier-transform infrared (FTIR) spectroscopy was utilized to examine potential chemical bonding and structural interactions within the material resulting from the synthesis of the CS/iCarp(AAmcoAA) hydrogel. Both chitosan (CS) and iota-carrageenan (iCar) contain hydroxyl groups that may interact with the carbon\u0026ndash;carbon double bonds (\u0026ndash;C\u0026thinsp;=\u0026thinsp;C\u0026ndash;) present in the monomers and crosslinker. Additionally, interactions between the EFV-loaded lipid nanocapsules (EFV LNCs) and the hydrogel network were evaluated through spectral analysis. FTIR spectra of the synthesized materials were compared with those of the raw components (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The hydrogel spectrum displayed a broad absorption band in the 3300\u0026ndash;3500 cm⁻\u0026sup1; range, corresponding to O\u0026ndash;H and N\u0026ndash;H stretching vibrations. Peaks between 1000 and 1200 cm⁻\u0026sup1; were attributed to C\u0026ndash;O stretching, while weaker bands observed in the 2000\u0026ndash;2300 cm⁻\u0026sup1; region, also present in CS and iCar, were likely overtones of C\u0026ndash;O vibrations, possibly arising due to the thickness of the hydrogel or polysaccharide films. The prominent peak at 1657 cm⁻\u0026sup1; confirmed the presence of C\u0026thinsp;=\u0026thinsp;O groups, consistent with the incorporation of acrylamide and acrylic acid. The FTIR spectrum of the EFV LNCs\u0026ndash;loaded hydrogel composite retained the key absorption bands observed in the plain hydrogel. However, the 2000\u0026ndash;2300 cm⁻\u0026sup1; bands appeared diminished or absent, potentially due to differences in film thickness. Notably, additional bands in the 2950\u0026ndash;2840 cm⁻\u0026sup1; range, characteristic of aliphatic C\u0026ndash;H stretching from saturated fatty acids, were evident in the composite, along with carbonyl peaks near 1750\u0026ndash;1745 cm⁻\u0026sup1;, consistent with the presence of lipid-based nanocapsules.\u003c/p\u003e\u003cp\u003eThe wavenumber shifts that we observed in the FTIR spectra following CS/iCarp(AamcoAA) hydrogel and its EFV-LNCs composite synthesis demonstrates successful yield of a stable hydrogel network from chemical interactions among the polymers, monomers, and crosslinking agent. The acrylamide and acrylic acid grafting onto the polysaccharide backbone is confirmed by the presence of stretches like the OH and NH, coupled with C\u0026thinsp;=\u0026thinsp;O and CO vibrations, aligning with previous studies where similar spectral shifts have been demonstrated when polysaccharides are chemically modified to enhance their drug delivery potential\u003csup\u003e47,48\u003c/sup\u003e. For example, improvement of properties like the swelling ratio have been proven to be associated with the introduction of functional groups like amides and carboxylates in the hydrogel\u0026rsquo;s structure\u003csup\u003e49\u003c/sup\u003e. Moreover, upon EFVLNCs incorporation, the hydrogel structure was maintained, suggesting that the encapsulation process does not compromise the hydrogel\u0026rsquo;s integrity. On the other hand, the presence of CH bands which are characteristic of saturated fatty acids found in medium chain triglycerides oil (MCT oil), supports successful encapsulation of EFV-LNCs\u003csup\u003e50\u003c/sup\u003e. Overall, this new CS/iCarp(AamcoAA) hydrogel delivery system possesses potential practical applications in improving controlled drug delivery of hydrophobic drugs like EFV.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe synthesized CS/iCar p(AAm-co-AA) hydrogel displayed a fibrous surface architecture, with elemental analysis revealing the presence of nitrogen and sulfur groups indicative of its polysaccharide components.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSurface morphology and elemental composition were evaluated using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS), respectively. SEM micrographs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea: EFV LNC\u0026ndash;loaded hydrogel composite; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb: unloaded hydrogel) revealed a rough, fibrous network with abundant porous structures, which are potentially advantageous for drug loading and release\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The incorporation of EFV LNCs did not visibly alter the porous morphology of the hydrogel. EDS spectra further confirmed the elemental signatures of the hydrogel matrix. Both the composite (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) and unloaded hydrogel (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) exhibited characteristic peaks for nitrogen, originating from chitosan and sulfur, derived from iota-carrageenan, alongside common elements such as carbon and oxygen. Additionally, chlorine was detected exclusively in the EFV-loaded composite (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), corresponding to the chemical structure of Efavirenz.\u003c/p\u003e\u003cp\u003eThese results indicate that both CS/iCar-p(Aam-co-AA) hydrogel and its EFV-LNCs composite exhibit porous and fibrous structures suitable for drug delivery. Although pore size was not determined, the previously observed correlation between the swelling ratio and the proportion of the hydrogel components implies that pore sizes also vary accordingly. For instance, increasing the amount of iota-carrageenan and decreasing the amount of chitosan resulted in higher swelling ratios, implying the presence of larger or more accessible pores, with the potential to affect drug loading and release. In the future, pore size should be measured and optimized to further understand its relationship with swelling ratio, drug loading and drug release\u003csup\u003e\u003cspan additionalcitationids=\"CR53 CR54\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe CS/iCarp(AamcoAA) hydrogel and EFVLNCsCS/iCarp(AamcoAA) hydrogel composite display relatively lower crystallinity compared to chitosan and iota-carrageenan.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePowder X-ray diffraction (XRD) analysis was performed to evaluate changes in the crystalline structure following hydrogel formation and drug encapsulation. The data passed normality testing using the Shapiro\u0026ndash;Wilk test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and subsequent ANOVA analysis revealed that the crystallinity of both the plain hydrogel and the EFV-loaded composite was significantly lower than that of chitosan, EFV, and EFV-LNCs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee. These findings provide additional evidence of successful chemical crosslinking between the monomers and polysaccharides. Crosslinking typically disrupts the orderly molecular arrangement in a polymer matrix, thereby reducing its degree of crystallinity\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe crystallinity reduction of Efavirenz upon encapsulation, first from 89.75% for nonencapsulated EFV to 57.52% for EFV-LNCs, and further down to 7.13% for the EFVLNCsCS/iCarp(AamcoAA) hydrogel composite highlights the effectiveness of double encapsulation in influencing the drug\u0026rsquo;s polymorphism. Interestingly, CS/iCarp(Aam-co-AA) hydrogel\u0026rsquo;s crystallinity which was initially 7.12% was not affected by EFV-LNCs incorporation. This supports that CS/iCar-p(Aam-co-AA) hydrogel\u0026rsquo;s structural integrity was maintained. Decreasing EFV crystallinity is critical since lower crystallinity improves solubility and bioavailability, which are essential for effective drug delivery. Moreover, CS/iCar-p(Aam-co-AA) hydrogel structural integrity maintenance suggests that its matrix can entrap a drug without altering its inherent properties.\u003c/p\u003e\u003cp\u003eOur previous Differential Calorimetric Analysis (DSC) of EFV-LNCs demonstrated the transformation of encapsulated EFV to an amorphous state, explaining the reduction of its crystallinity rather than mere masking it\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. DSC analysis of the EFVLNCsCS/iCarp(AamcoAA) hydrogel composite compared to non-encapsulated EFV should be preconized to further demonstrate how the hydrogel matrix affected EFV polymorphism. When compared to Chitosan, CS/iCarp(AamcoAA) hydrogel crystallinity significantly decreased, and this could be accounted for by the introduction of a threedimensional network that limits the mobility of Chitosan resulting in a more amorphous structure\u003csup\u003e57\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe microstructural insights of EFVLNCsCS/iCarp(AamcoAA) hydrogel composite reveal generated entanglements of the hydrogel scaffold and tiny spherical particles of entrapped EFVLNCs.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTransmission electron microscopy enabled the visualization of the microscopic structure of the composites. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea highlights the EFV LNCs CS/iCar p(Aam-co-AA) hydrogel composite network, showing proposed entanglements at 200 nm. At 100 nm, small spherical particles are visible, indicating the presence of EFV LNCs. A TEM micrograph of EFVLNCs has previously been reported, exhibiting particles with sizes below 100 nm\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e7\u003c/sup\u003e. The presence of these particles confirms that the EFVLNCs particles are incorporated into the hydrogel matrix, and their distinct appearance suggests a specific and controlled distribution. This insight into the composite structure further supports the entrapment of EFVLNCs in the hydrogel scaffold. This entrapment could have important implications for drug delivery, where the controlled release of the encapsulated EFV is desired.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEFVLNCs particles entrapped in the CS/iCarp(AamcoAA hydrogel scaffold\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe presence of the EFV-LNCs in CS/iCar p(Aam-co-AA) hydrogel scaffold was confirmed by using fluorescence microscopy since the LNCs lacked a heavy atom. Amaranth dye was used to stain the composite to increase the contrast between the incorporated LNCs and the rest of the constituents in the composite. The images obtained using the DAPI filter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, which excites at 358 nm and emits blue fluorescence at 461 nm, revealed a bright reddish color of the background corresponding to the outer hydrogel layer. Besides, the bright and distinct blue-color dots represent the clusters of LNCs. While Amaranth dye is weakly fluorescent, its association with the phospholipids in soybean lecithin of the LNCs likely enhanced the fluorescent signal. This is consistent with previous research on soybean oil and soy protein, where phospholipid interactions with some molecules resulted in amplified fluorescence under specific conditions\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The blue fluorescence not only evidenced the structural integrity of the EFV-LNCs-CS/iCar p(Aam-co-AA) hydrogel composite but also the success of the encapsulation procedure, making a huge potential for further application, including controlled release of the entrapped drug (EFV).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLNCsCS/iCarp(AamcoAA) hydrogel composite achieves enhanced and sustained\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eRelease of EFV.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe in vitro release study was conducted using a 1% sodium lauryl sulfate (SLS) solution adjusted to pH 7 and pH 4 to simulate gastrointestinal conditions, providing physiologically relevant environments for assessing drug release. The pH 7 condition represents the intestinal environment, while pH 4 condition simulates fed-state gastric conditions or the transitional environment between the stomach and proximal intestine, conditions frequently experienced after food intake, thereby enabling evaluation of the composite's performance throughout different regions of the gastrointestinal tract\u003csup\u003e\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e illustrates the comparative release profiles of free Efavirenz (non-encapsulated) and the EFV LNCs\u0026ndash;CS/iCar p(AAm-co-AA) hydrogel composite under the tested conditions. As the data did not meet normality assumptions (Shapiro\u0026ndash;Wilk test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all groups), the Kruskal\u0026ndash;Wallis test was employed for non-parametric analysis of overall group differences. This was followed by Dunn\u0026rsquo;s post-hoc test for pairwise comparisons. The Kruskal\u0026ndash;Wallis test identified statistically significant differences in the drug release behavior between free EFV and the hydrogel composite across multiple time points. Free EFV exhibited a significantly faster release, exceeding 75% in both pH conditions over the 9-days experimental period (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Notably, the free EFV release profile demonstrated a burst increase after 72 hours (region between 50\u0026thinsp;~\u0026thinsp;100 hours as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), likely due to saturation of the dialysis bag, alterations in solution properties, and the lack of a diffusion barrier, all of which contribute to unrestricted drug diffusion. In other words, EFV saturated the dialysis bag and precipitated; once some precipitated drug gradually dissolved or the medium was refreshed, a sudden increase in diffusion occurred\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In contrast, the hydrogel formulation averted such bursts, maintaining a steadier, more linear release without spikes.\u003c/p\u003e\u003cp\u003ePairwise comparisons were examined using the Wilcoxon Signed-Rank Test. The release kinetics of Free EFV at pH 4 and pH 7 were not significantly different (W\u0026thinsp;=\u0026thinsp;42.0, p\u0026thinsp;=\u0026thinsp;0.807), suggesting that pH had minimal influence on free EFV diffusion dynamics. However, significant differences were observed between Free EFV at pH 4 vs. EFVLNCCS/iCar at pH 4 (W\u0026thinsp;=\u0026thinsp;0.0, p\u0026thinsp;=\u0026thinsp;0.0015) and Free EFV at pH 7 vs. EFVLNCCS/iCar at pH 7 (W\u0026thinsp;=\u0026thinsp;0.0, p\u0026thinsp;=\u0026thinsp;0.0015), confirming that encapsulation significantly reduced drug release rates under both pH conditions. Additional comparisons between Free EFV at pH 4 vs. EFVLNCCS/iCar at pH 7 (W\u0026thinsp;=\u0026thinsp;0.0, p\u0026thinsp;=\u0026thinsp;0.0015) and Free EFV at pH 7 vs. EFV-LNC-CS/iCar at pH 4 (W\u0026thinsp;=\u0026thinsp;0.0, p\u0026thinsp;=\u0026thinsp;0.0015) further reinforced the hydrogel\u0026rsquo;s ability to modulate drug release more effectively than the free drug, irrespective of pH. The composite\u0026rsquo;s sustained, slower release is governed by diffusion through multiple barriers and pH-responsive matrix behavior. EFV must traverse the lipid nanocapsule core and then the chitosan/iota-carrageenan hydrogel network, which slows down its mobility\u003csup\u003e63\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe hydrogel\u0026rsquo;s mesh size and swelling depend on pH \u0026ndash; it stays dense at pH 4 (limiting EFV diffusion) and expands at pH 7 (allowing faster diffusion)\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Interestingly, a significant difference was detected within the EFV-LNC-CS/iCar hydrogel composite, with higher drug release at pH 7 compared to pH 4 (W\u0026thinsp;=\u0026thinsp;0.0, p\u0026thinsp;=\u0026thinsp;0.0077). This pH-dependent release profile suggests that the hydrogel matrix responds to environmental pH, releasing less drug under mildly acidic conditions. This means that there is minimal release in the stomach (pH 4) means most EFV remains protected from acidic degradation or irritation, and the dose is not wasted where little drug uptake occurs. Once the hydrogel reaches the intestine and encounters pH 7, it swells and rapidly releases EFV at the site of optimal absorption\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. This targeted release maximizes drug bioavailability by delivering EFV where it can be efficiently absorbed, while the sustained release profile prolongs drug presence in the absorption window. Given that pH 4 simulates fed-state gastric conditions or the transitional environment between the stomach and proximal intestine, this characteristic could be advantageous for protecting the drug from premature release in the stomach and promoting more targeted and sustained drug release upon entering the neutral intestinal environment, ultimately enhancing oral bioavailability and therapeutic efficacy as already demonstrated in previous studies\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe hydrogel composite demonstrated sustained and controlled drug release over 72 hours without significant burst effects, in contrast to free EFV. The hydrogel\u0026rsquo;s porous structure effectively acted as a diffusion barrier, modulating EFV release through swelling and erosion under gastrointestinal conditions. This controlled release profile is advantageous for improving EFV\u0026rsquo;s oral bioavailability and reducing dosing frequency. LNCs enhance EFV\u0026rsquo;s solubility and stability, preventing precipitation and protecting the drug from premature degradation, while also facilitating absorption via mechanisms like lymphatic uptake\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. The hydrogel, in turn, localizes and slowly releases the LNCs (and encapsulated EFV) in the GI tract. This dual system offers a synergistic effect that prevents the rapid clearance or burst release that might occur with LNCs alone and effectively marries the sustained release capacity of hydrogels with the absorption-enhancing benefits of nanoparticles\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Consequently, the composite facilitates prolonged drug retention and absorption compared to either component alone, highlighting its potential as an advanced oral delivery platform to enhance therapeutic efficacy and patient compliance for poorly soluble antiviral drugs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHydrogel is biocompatible with HELA cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCytocompatibility is essential to ensure future clinical use of any newly developed product. For this reason, we performed cytotoxicity experiments by treating HeLa cells with lipid nanocapsules, CS/iCarp(AamcoAA) hydrogel, LNCsCS/iCarp(AamcoAA) hydrogel composite and EFVLNCsCS/iCarp(AamcoAA) hydrogel composite using a resazurinbased assay. HeLa cells were chosen due to their widespread use and well-characterized response in preliminary cytotoxicity studies. This allows for consistency and comparability with existing literature, providing a reliable initial assessment of biocompatibility\u003csup\u003e70\u0026ndash;73\u003c/sup\u003e. The treatments were prepared in a 96 well plate at a concentration of 50 \u0026micro;g/mL. The plate was put in the incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e to allow the cells to grow and interact with the treatments for 24 hours. After incubation, the resazurin was used to fully cover each well of the 96‑well plate, which was then returned into the incubator. In this resazurin-based assay, viable cells with active metabolism reduce the non-fluorescent resazurin to the fluorescent compound resorufin. The resulting fluorescence intensity, measured after 1\u0026ndash;4 hours of incubation, is directly proportional to the number of metabolically active cells. Cytotoxicity was expressed as percent viability, calculated as the ratio of fluorescence in wells with treated cells compared to wells containing untreated cells.\u003c/p\u003e\u003cp\u003eThe viability of HeLa cells treated with various formulations is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The LNCs-Hydrogel composite showed a viability of ~\u0026thinsp;90% (t = -0.8552, p\u0026thinsp;=\u0026thinsp;0.4825, ns), with no statistically significant difference from the negative control, suggesting cytocompatibility. At the tested concentration of 50 \u0026micro;g/mL, none of the treatments reduced cell viability by more than 50%, except the positive control Emetine, a well-established inducer of apoptosis. This observation indicates minimal cytotoxicity of LNCs formulations compared to no treatment. Compounds with IC50 concentrations above 10 \u0026micro;g/mL are deemed safe candidates to use clinically \u003csup\u003e\u003cspan additionalcitationids=\"CR75 CR76 CR77 CR78\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Among the formulations, LNCs exhibited the highest viability (~\u0026thinsp;135%, t\u0026thinsp;=\u0026thinsp;16.1692, p\u0026thinsp;=\u0026thinsp;0.0038, **), indicating enhanced metabolic activity. EFVLNCs and EFVLNCs Hydrogel composite exhibited viabilities of ~\u0026thinsp;65% (t = -30.7915, p\u0026thinsp;=\u0026thinsp;0.0011, **) and ~\u0026thinsp;85% (t = -6.7239, p\u0026thinsp;=\u0026thinsp;0.0214, *), respectively, indicating that the incorporation of EFV reduced cell viability, as expected for an active pharmaceutical ingredient. Emetine, used as a positive control, dramatically reduced cell viability to approximately 10% (t = -250.7546, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, ****), confirming its known cytotoxicity. These findings suggest that LNCs and the new LNCsHydrogel composite are cytocompatible, and the observed decrease in viability upon EFV loading aligns with the drug\u0026rsquo;s expected biological activity. The elevated viability observed with LNCs may reflect increased metabolic activity, potentially facilitated by mediumchain triglycerides (MCTs) as an energy source, soy lecithin supporting membrane integrity, and Tween 80 alleviating cellular stress\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e0\u0026ndash;84\u003c/sup\u003e. Additionally, the aqueous medium and salt content likely help maintain osmolarity and hydration, contributing to an optimal environment for cell survival. Additionally, the resazurin assay likely detected this metabolic boost as increased viability. These results suggest that lipid-based systems, such as LNCs and their composites, can support high cell viability, warranting further investigation into their \u003cem\u003einvivo\u003c/em\u003e application.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhile preliminary cytotoxicity studies using HeLa cells have demonstrated promising biocompatibility of our formulations, follow-up experiments will be conducted on gastrointestinalrelevant cell lines to better represent oral conditions. Suitable cell lines for evaluating gastrointestinal cytotoxicity include Caco-2 (human intestinal epithelial cells), HT29-MTX (human colon carcinoma cells secreting mucus), and IEC-6 (normal rat small intestine epithelial cells)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e5, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e6\u003c/sup\u003e. These intestinal cell models are anticipated to provide critical insights into the safety, biocompatibility, and potential efficacy of our hydrogel- and lipid nanocapsulebased formulation for oral drug delivery, supporting further clinical translation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEFVLNCsCS/iCarp(AamcoAA) Hydrogel Composite as Oral Solid Dosage Form\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor future studies, the EFVLNCCS/iCarp(AamcoAA) hydrogel composite can be optimized into a solid oral dosage form, enhancing patient convenience and therapeutic efficacy. Lyophilization could transform the composite into a dry, easily administrable powder or tablet, maintaining the integrity of both lipid nanocapsules and hydrogel components\u003csup\u003e87\u0026ndash;89\u003c/sup\u003e. Freeze-drying would preserve the internal structure of the composite, minimizing aggregation or premature drug release. Additionally, compression into oral tablets or encapsulation in capsules could facilitate dosage uniformity, ease patient administration, and maintain sustained release properties\u003csup\u003e90,91\u003c/sup\u003e. Although solid dosage form development was not performed in this study, this approach represents a promising direction for enhancing the clinical applicability of EFV-loaded hybrid composites, potentially improving the oral bioavailability and therapeutic consistency of hydrophobic antiviral drugs.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study introduces an innovative lipid nanocapsule and hydrogel composite specifically developed for oral delivery of hydrophobic antiviral drugs. Biocompatible polysaccharides, chitosan (CS) and iotacarrageenan (iCar), were used to synthesize a porous hydrogel scaffold via free-radical copolymerization. The synthesized CS/iCarp(AamcoAA) hydrogel demonstrated high swelling capacity, facilitating effective encapsulation and sustained release of Efavirenzloaded lipid nanocapsules (LNCs). The composite improved EFV solubility and demonstrated controlled, pHresponsive release profiles under simulated gastrointestinal conditions. Furthermore, structural and cytotoxicity analyses confirmed the biocompatibility and safety of the developed formulation, suggesting favorable interactions within gastrointestinal environments. Future studies will focus on evaluating biocompatibility with relevant intestinal cell lines and optimizing mechanical and mucoadhesive properties. These next steps will be essential for translating this novel composite system into an effective clinical solution for not only orally delivering poorly soluble antiviral medications, but also for application in other mucosal tissues, such as vaginal drug delivery\u003csup\u003e92,93\u003c/sup\u003e, where hydrogels have been extensively employed as vehicles to deliver drugs embedded in nanoparticle, transdermal delivery and even pulmonary delivery\u003csup\u003e94\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eLNCs \u0026ndash;\u0026nbsp;\u003c/strong\u003eLipid Nanocapsules\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCS -\u0026nbsp;\u003c/strong\u003eChitosan\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eiCar -\u0026nbsp;\u003c/strong\u003eIota-Carrageenan\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eEFV -\u0026nbsp;\u003c/strong\u003eEfavirenz\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSEM -\u0026nbsp;\u003c/strong\u003eScanning Electron Microscopy\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eEDS -\u0026nbsp;\u003c/strong\u003eEnergy-Dispersive X-ray Spectroscopy\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eFTIR -\u0026nbsp;\u003c/strong\u003eFourier Transform Infrared Spectroscopy\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eTGA -\u0026nbsp;\u003c/strong\u003eThermal Gravimetric Analysis\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDS -\u0026nbsp;\u003c/strong\u003eDroplet Size\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePDI -\u0026nbsp;\u003c/strong\u003ePolydispersity Index\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eZP -\u0026nbsp;\u003c/strong\u003eZeta Potential\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eMBA -\u0026nbsp;\u003c/strong\u003eN,N\u0026apos;-Methylene Bisacrylamide\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAA -\u0026nbsp;\u003c/strong\u003eAcrylic Acid\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAam -\u0026nbsp;\u003c/strong\u003eAcrylamide\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSC -\u0026nbsp;\u003c/strong\u003eSwelling Capacity\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSR -\u0026nbsp;\u003c/strong\u003eSwelling Ratio\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eIC50 -\u0026nbsp;\u003c/strong\u003eInhibitory Concentration 50%\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eANOVA -\u0026nbsp;\u003c/strong\u003eAnalysis of Variance\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSLS -\u0026nbsp;\u003c/strong\u003eSodium Lauryl Sulfate\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDSC -\u0026nbsp;\u003c/strong\u003eDifferential Scanning Calorimetry\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupporting information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSolubility test\u003c/strong\u003e: the T‑test in \u003cstrong\u003eFigure S1\u003c/strong\u003e shows that Efavirenz was significantly soluble in MCT oil unlike in water. \u003cstrong\u003eHydrogel swelling capacity optimization\u003c/strong\u003e: \u003cstrong\u003eTable S1\u003c/strong\u003e lists all the hydrogels formulations prepared, with their corresponding swelling capacity values. \u003cstrong\u003eTable S2\u003c/strong\u003e shows the results of the analysis of variance of models fitted to the swelling capacity. \u003cstrong\u003ePotential mechanism of hydrogel formation\u003c/strong\u003e: \u003cstrong\u003eFigure S2\u003c/strong\u003e and \u003cstrong\u003eFigure S3\u003c/strong\u003e respectively describe the formation of free radical sites on chitosan and iota‑carrageenan backbones under ammonium persulfate initiation, and the plausible reactional mechanism of CS/iCar‑p(Aam‑co‑AA) hydrogel formation. \u003cstrong\u003eStandard error plot\u003c/strong\u003e: \u003cstrong\u003eFigure S4\u003c/strong\u003e presents the contour plot of the standard error at different points in the design space that shows small values of the standard error that ranged from 0.2 to 0.35, which is an indication that the means of the samples are a reliable reflection of the true population means. \u003cstrong\u003eSwelling capacity prediction\u003c/strong\u003e: \u003cstrong\u003eTable S3\u003c/strong\u003e lists automatically generated hydrogel composition batches for model optimization and confirmation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e*Email: [email protected] (C.N.J.)\u003c/p\u003e\n\u003cp\u003e*Email: [email protected] (R.W.M.K.),\u003c/p\u003e\n\u003cp\u003e*Email: [email protected] (G.K.M),\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrady K. Mukubwa: 0000-0002-1678-4722\u003c/p\u003e\n\u003cp\u003eJustin B. Safari: 0000-0002-8940-9552\u003c/p\u003e\n\u003cp\u003eZikhona N. Tetana: 0000-0002-0767-1056\u003c/p\u003e\n\u003cp\u003eCaroline N. Jones: 0000-0001-6003-9335\u003c/p\u003e\n\u003cp\u003eRoderick B. Walker: 0000-0003-2781-4154\u003c/p\u003e\n\u003cp\u003eRui W.M. Krause: 0000-0001-6788-6449\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Project design and conceptualization, methodology, data collection, software, data analysis and interpretation, investigation, data curation, writing original draft preparation, writing review, editing and visualization: G.K.M.; methodology, data collection and analysis, writing review and editing: J.B.S.; methodology, data collection, writing review and editing: Z.N.T.; conceptualization, validation, writing review and editing, supervision, project administration, resources and funding acquisition: R.W.M.K., R.B.W and C.N.J. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are provided within the main article and its supplementary materials. Additional datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Rhodes University Electron Beam Laboratory for EM facilities, especially Dr. D. van Niekerk and Mr. M. Randall for training, selecting experimental parameters, and guidance with sample preparation and data collection. Thanks to the Rhodes CCBR for bioassay data, the Edkins lab for confocal data, and the SMaLL-group for XRD, FTIR, and TGA facilities. This manuscript contains content adapted from the first author\u0026rsquo;s publicly available dissertation submitted to Rhodes University, archived at [http://vital.seals.ac.za:8080/vital/access/manager/Repository/vital:65665?site_name=GlobalView]. The material has been revised and expanded for the purpose of journal publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the NGO F\u0026ouml;rderverein Uni Kinshasa e.V. (fUNIKIN), ElseKroener-Fresenius Stiftung, and Holger-Poehlmann Foundation through the excellence scholarship program \u0026ldquo;Bourse d\u0026rsquo;Excellence Bringmann aux Universit\u0026eacute;s Congolaises, BEBUC\u0026rdquo; ([email protected]; [email protected]), and Rhodes University, South Africa, supported by the National Research Foundation, NRF (including Grant No: 141979). G.K.M and C.N.J received support from R35 GM133610/GM/NIGMS NIH HHS/United States.\u003c/p\u003e\n\u003cp\u003eIn memory of Prof. Rui W.M. Krause (1970\u0026ndash;2024), a renowned figure in organic medicinal chemistry and nanomaterials, whose leadership and mentorship left an indelible mark on the field. His dedication to inspiring and guiding the next generation of scholars has empowered many to realize their full potential.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGupta, S., Kesarla, R., Chotai, N., Misra, A. \u0026amp; Omri, A. Systematic Approach for the Formulation and Optimization of Solid Lipid Nanoparticles of Efavirenz by High Pressure Homogenization Using Design of Experiments for Brain Targeting and Enhanced Bioavailability. \u003cem\u003eBiomed Res Int\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, 5984014 (2017).\u003c/li\u003e\n\u003cli\u003eLangmia, I. M. \u003cem\u003eet al.\u003c/em\u003e CYP2B6 Functional Variability in Drug Metabolism and Exposure Across Populations\u0026mdash;Implication for Drug Safety, Dosing, and Individualized Therapy. \u003cem\u003eFront Genet\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 692234 (2021).\u003c/li\u003e\n\u003cli\u003eChen, R. \u003cem\u003eet al.\u003c/em\u003e Antiviral Drug Delivery System for Enhanced Bioactivity, Better Metabolism and Pharmacokinetic Characteristics. \u003cem\u003eInt J Nanomedicine\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 4959 (2021).\u003c/li\u003e\n\u003cli\u003eArora, S., Chawla, A., Pawar, P. \u0026amp; Sharma, P. Novel drug delivery approaches on antiviral and antiretroviral agents. \u003cem\u003eJ Adv Pharm Technol Res\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 147\u0026ndash;159 (2012).\u003c/li\u003e\n\u003cli\u003eLembo, D. \u0026amp; Cavalli, R. Nanoparticulate delivery systems for antiviral drugs. \u003cem\u003eAntivir Chem Chemother\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 53\u0026ndash;70 (2010).\u003c/li\u003e\n\u003cli\u003eCojocaru, F. D. \u003cem\u003eet al.\u003c/em\u003e Nanomaterials designed for antiviral drug delivery transport across biological barriers. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1\u0026ndash;34 (2020).\u003c/li\u003e\n\u003cli\u003eSamimi, S., Maghsoudnia, N., Eftekhari, R. B. \u0026amp; Dorkoosh, F. \u003cem\u003eLipid-Based Nanoparticles for Drug Delivery Systems\u003c/em\u003e. \u003cem\u003eCharacterization and Biology of Nanomaterials for Drug Delivery: Nanoscience and Nanotechnology in Drug Delivery\u003c/em\u003e (Elsevier Inc., 2018). doi:10.1016/B978-0-12-814031-4.00003-9.\u003c/li\u003e\n\u003cli\u003eKalepu, S., Manthina, M. \u0026amp; Padavala, V. Oral lipid-based drug delivery systems \u0026ndash; an overview. \u003cem\u003eActa Pharm Sin B\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 361\u0026ndash;372 (2013).\u003c/li\u003e\n\u003cli\u003eKamble, R. N., Mehta, P. P. \u0026amp; Kumar, A. Efavirenz Self-Nano-Emulsifying Drug Delivery System: In Vitro and In Vivo Evaluation. \u003cem\u003eAAPS PharmSciTech\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1240\u0026ndash;1247 (2016).\u003c/li\u003e\n\u003cli\u003eKotta, S., Khan, A. W., Ansari, S. H., Sharma, R. K. \u0026amp; Ali, J. Anti HIV nanoemulsion formulation: Optimization and in vitro\u0026ndash;in vivo evaluation. \u003cem\u003eInt J Pharm\u003c/em\u003e \u003cstrong\u003e462\u003c/strong\u003e, 129\u0026ndash;134 (2014).\u003c/li\u003e\n\u003cli\u003eMazonde, P., Khamanga, S. M. M., Walker, R. B. \u0026amp; Krause, R. W. M. Design, optimization, manufacture and characterization of Efavirenz-loaded flaxseed oil nanoemulsions. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1\u0026ndash;22 (2020).\u003c/li\u003e\n\u003cli\u003eAPA. PubChem Compound Summary for CID 64139, Efavirenz. \u003cem\u003eNational Center for Biotechnology Information\u003c/em\u003e https://pubchem.ncbi.nlm.nih.gov/compound/Efavirenz (2021).\u003c/li\u003e\n\u003cli\u003ePersson, L. C., Porter, C. J. H., Charman, W. N. \u0026amp; Bergstr\u0026ouml;m, C. A. S. Computational prediction of drug solubility in lipid based formulation excipients. \u003cem\u003ePharm Res\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 3225\u0026ndash;3237 (2013).\u003c/li\u003e\n\u003cli\u003eRane, S. S. \u0026amp; Anderson, B. D. What determines drug solubility in lipid vehicles: Is it predictable? \u003cem\u003eAdv Drug Deliv Rev\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 638\u0026ndash;656 (2008).\u003c/li\u003e\n\u003cli\u003eMoura, R. P., Pacheco, C., P\u0026ecirc;go, A. P., des Rieux, A. \u0026amp; Sarmento, B. Lipid nanocapsules to enhance drug bioavailability to the central nervous system. \u003cem\u003eJournal of Controlled Release\u003c/em\u003e \u003cstrong\u003e322\u003c/strong\u003e, 390\u0026ndash;400 (2020).\u003c/li\u003e\n\u003cli\u003eHeurtault, B., Saulnier, P., Proust, J.E., Benoit, J. P. Lipid nanocapsules, preparation process and use as medicine. \u003cstrong\u003e2\u003c/strong\u003e, (2000).\u003c/li\u003e\n\u003cli\u003eMukubwa, G. K., Safari, J. B. \u0026amp; Walker, R. B. Design , Manufacturing , Characterization and Evaluation of Lipid Nanocapsules to Enhance the Biopharmaceutical Properties of Efavirenz. (2022).\u003c/li\u003e\n\u003cli\u003eHua, S. Advances in Oral Drug Delivery for Regional Targeting in the Gastrointestinal Tract - Influence of Physiological, Pathophysiological and Pharmaceutical Factors. \u003cem\u003eFront Pharmacol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 524 (2020).\u003c/li\u003e\n\u003cli\u003eLiu, J., Leng, P. \u0026amp; Liu, Y. Oral drug delivery with nanoparticles into the gastrointestinal mucosa. \u003cem\u003eFundam Clin Pharmacol\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 86\u0026ndash;96 (2021).\u003c/li\u003e\n\u003cli\u003eSuk, J. S., Xu, Q., Kim, N., Hanes, J. \u0026amp; Ensign, L. M. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. \u003cem\u003eAdv Drug Deliv Rev\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 28 (2015).\u003c/li\u003e\n\u003cli\u003eWays, T. M. M., Lau, W. M. \u0026amp; Khutoryanskiy, V. V. Chitosan and Its Derivatives for Application in Mucoadhesive Drug Delivery Systems. \u003cem\u003ePolymers (Basel)\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 267 (2018).\u003c/li\u003e\n\u003cli\u003eAzhar, S. N. A. S., Ashari, S. E., Zainuddin, N. \u0026amp; Hassan, M. Nanostructured Lipid Carriers-Hydrogels System for Drug Delivery: Nanohybrid Technology Perspective. \u003cem\u003eMolecules\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 289 (2022).\u003c/li\u003e\n\u003cli\u003eDesfran\u0026ccedil;ois, C., Auz\u0026eacute;ly, R. \u0026amp; Texier, I. Lipid nanoparticles and their hydrogel composites for drug delivery: A review. \u003cem\u003ePharmaceuticals\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003eMukubwa, G. K. Design, synthesis, manufacture, characterization and evaluation of lipid nanocapsules in chitosan-iota-carrageenan based hydrogel scaffold as a potential anti-Covid-19 drug delivery system. (2022).\u003c/li\u003e\n\u003cli\u003eRahmani, Z., Ghaemy, M. \u0026amp; Olad, A. Preparation of nanogels based on kappa-carrageenan/chitosan and N-doped carbon dots: study of drug delivery behavior. \u003cem\u003ePolymer Bulletin\u003c/em\u003e vol. 78 2709\u0026ndash;2726 Preprint at https://doi.org/10.1007/s00289-020-03236-x (2021).\u003c/li\u003e\n\u003cli\u003eQi, X. \u003cem\u003eet al.\u003c/em\u003e Salecan-Based pH-Sensitive Hydrogels for Insulin Delivery. \u003cem\u003eMol Pharm\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 431\u0026ndash;440 (2017).\u003c/li\u003e\n\u003cli\u003eBienvenu, E., Hoffmann, K. J., Ashton, M. \u0026amp; Kayumba, P. C. A rapid and selective HPLC-UV method for the quantitation of efavirenz in plasma from patients on concurrent HIV/AIDS and tuberculosis treatments. \u003cem\u003eBiomedical Chromatography\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1554\u0026ndash;1559 (2013).\u003c/li\u003e\n\u003cli\u003eSafari, J. B., Bapolisi, A. M. \u0026amp; Krause, R. W. M. Development of pH-sensitive chitosan-g-poly (Acrylamide-co-acrylic acid) hydrogel for controlled drug delivery of tenofovir disoproxil fumarate. \u003cem\u003ePolymers (Basel)\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eNaeem, A., Yu, C., Zhu, W., Zang, Z. \u0026amp; Guan, Y. Study of Hydroxypropyl \u0026beta;-Cyclodextrin and Puerarin Inclusion Complexes Encapsulated in Sodium Alginate-Grafted 2-Acrylamido-2-Methyl-1-Propane Sulfonic Acid Hydrogels for Oral Controlled Drug Delivery. \u003cem\u003eGels\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 246 (2023).\u003c/li\u003e\n\u003cli\u003eMagli, S. \u003cem\u003eet al.\u003c/em\u003e Combined analytical approaches to standardize and characterize biomaterials formulations: Application to chitosan-gelatin cross-linked hydrogels. \u003cem\u003eBiomolecules\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 683 (2021).\u003c/li\u003e\n\u003cli\u003eH Mohd Yusoff, S. N. \u003cem\u003eet al.\u003c/em\u003e Akademia Baru Studies on Thermal and Conductivity of Modified Natural Rubber. \u003cem\u003eJournal of Advanced Research in Materials Science ISSN\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2289\u0026ndash;7992.\u003c/li\u003e\n\u003cli\u003eKlein, R. S. \u003cem\u003eet al.\u003c/em\u003e Iota-Carrageenan/Chitosan Nanoparticles via Coacervation: Achieving Stability for Tiny Particles. \u003cem\u003eNanomaterials\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 161 (2025).\u003c/li\u003e\n\u003cli\u003eGon\u0026ccedil;alves, R. C. R. \u003cem\u003eet al.\u003c/em\u003e Bioimaging of Lysosomes with a BODIPY pH-Dependent Fluorescent Probe. \u003cem\u003eMolecules\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eHeurtault, B., Saulnier, P., Pech, B., Proust, J. E. \u0026amp; Benoit, J. P. A novel phase inversion-based process for the preparation of lipid nanocarriers. \u003cem\u003ePharm Res\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 875\u0026ndash;880 (2002).\u003c/li\u003e\n\u003cli\u003eBudai, L., Budai, M., F\u0026uuml;l\u0026ouml;pn\u0026eacute; P\u0026aacute;pay, Z. E., Vilimi, Z. \u0026amp; Antal, I. Rheological Considerations of Pharmaceutical Formulations: Focus on Viscoelasticity. \u003cem\u003eGels 2023, Vol. 9, Page 469\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 469 (2023).\u003c/li\u003e\n\u003cli\u003eSouza, A. B. de, Chaud, M. V., Alves, T. F., Souza, J. F. de \u0026amp; Santana, M. H. A. Hyaluronic Acid in the Intestinal Tract: Influence of Structure, Rheology, and Mucoadhesion on the Intestinal Uptake in Rats. \u003cem\u003eBiomolecules 2020, Vol. 10, Page 1422\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1422 (2020).\u003c/li\u003e\n\u003cli\u003eLi, Z., Lu, D. \u0026amp; Gao, X. Optimization of mixture proportions by statistical experimental design using response surface method - A review. \u003cem\u003eJournal of Building Engineering\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 102101 (2021).\u003c/li\u003e\n\u003cli\u003eAydar, A. Y. Utilization of Response Surface Methodology in Optimization of Extraction of Plant Materials. \u003cem\u003eStatistical Approaches With Emphasis on Design of Experiments Applied to Chemical Processes\u003c/em\u003e (2018) doi:10.5772/INTECHOPEN.73690.\u003c/li\u003e\n\u003cli\u003eBreig, S. J. M. \u0026amp; Luti, K. J. K. Response surface methodology: A review on its applications and challenges in microbial cultures. \u003cem\u003eMater Today Proc\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 2277\u0026ndash;2284 (2021).\u003c/li\u003e\n\u003cli\u003eJastram, A., Lindner, T., Luebbert, C., Sadowski, G. \u0026amp; Kragl, U. Swelling and diffusion in polymerized ionic liquids-based hydrogels. \u003cem\u003ePolymers (Basel)\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eKamoun, E. A. \u0026amp; Menzel, H. HES-HEMA nanocomposite polymer hydrogels: Swelling behavior and characterization. \u003cem\u003eJournal of Polymer Research\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, (2012).\u003c/li\u003e\n\u003cli\u003eCiolacu, D. E., Nicu, R. \u0026amp; Ciolacu, F. Cellulose-Based Hydrogels as Sustained Drug-Delivery Systems. \u003cem\u003eMaterials 2020, Vol. 13, Page 5270\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 5270 (2020).\u003c/li\u003e\n\u003cli\u003eLin, J., Jiao, G. \u0026amp; Kermanshahi-Pour, A. Algal Polysaccharides-Based Hydrogels: Extraction, Synthesis, Characterization, and Applications. \u003cem\u003eMarine Drugs 2022, Vol. 20, Page 306\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 306 (2022).\u003c/li\u003e\n\u003cli\u003eKuo, C. C., Clark, S., Qin, H. \u0026amp; Shi, X. Development of a shelf-stable, gel-based delivery system for probiotics by encapsulation, 3D printing, and freeze-drying. \u003cem\u003eLWT\u003c/em\u003e \u003cstrong\u003e157\u003c/strong\u003e, 113075 (2022).\u003c/li\u003e\n\u003cli\u003eSwathi, H. P. \u003cem\u003eet al.\u003c/em\u003e Effect of gamma sterilization on the properties of microneedle array transdermal patch system. \u003cem\u003eDrug Dev Ind Pharm\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 606\u0026ndash;620 (2020).\u003c/li\u003e\n\u003cli\u003eWang, H., Meng, Z., Zhao, C.-Y. \u0026amp; -, al. Biocompatibility evaluation for the developed hydrogel wound dressing \u0026ndash; ISO-10993-11 standards \u0026ndash; in vitro and in vivo study. \u003cem\u003eBiomed Phys Eng Express\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 015010 (2021).\u003c/li\u003e\n\u003cli\u003eBashir, S., Teo, Y. Y., Naeem, S., Ramesh, S. \u0026amp; Ramesh, K. PH responsive N-succinyl chitosan/Poly (acrylamide-co-acrylic acid) hydrogels and in vitro release of 5-fluorouracil. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, (2017).\u003c/li\u003e\n\u003cli\u003eThang, N. H., Chien, T. B. \u0026amp; Cuong, D. X. Polymer-Based Hydrogels Applied in Drug Delivery: An Overview. \u003cem\u003eGels\u003c/em\u003e vol. 9 Preprint at https://doi.org/10.3390/gels9070523 (2023).\u003c/li\u003e\n\u003cli\u003eLi, B. \u003cem\u003eet al.\u003c/em\u003e Preparation and properties of CS/P(AM-co-AA) composite hydrogels by frontal polymerization of ternary DES. \u003cem\u003eRSC Adv\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 34724\u0026ndash;34729 (2022).\u003c/li\u003e\n\u003cli\u003eKrambeck, K. \u003cem\u003eet al.\u003c/em\u003e Design and characterization of Nanostructured lipid carriers (NLC) and Nanostructured lipid carrier-based hydrogels containing Passiflora edulis seeds oil. \u003cem\u003eInt J Pharm\u003c/em\u003e \u003cstrong\u003e600\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eHoare, T. R. \u0026amp; Kohane, D. S. Hydrogels in drug delivery: Progress and challenges. \u003cem\u003ePolymer (Guildf)\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 1993\u0026ndash;2007 (2008).\u003c/li\u003e\n\u003cli\u003eSonker, M. \u003cem\u003eet al.\u003c/em\u003e Review of Recent Advances and Their Improvement in the Effectiveness of Hydrogel-Based Targeted Drug Delivery: A Hope for Treating Cancer. \u003cem\u003eACS Applied Bio Materials\u003c/em\u003e vol. 4 8080\u0026ndash;8109 Preprint at https://doi.org/10.1021/acsabm.1c00857 (2021).\u003c/li\u003e\n\u003cli\u003eVigata, M., Meinert, C., Hutmacher, D. W. \u0026amp; Bock, N. Hydrogels as drug delivery systems: A review of current characterization and evaluation techniques. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1\u0026ndash;45 (2020).\u003c/li\u003e\n\u003cli\u003eHameed, H., Faheem, S., Paiva-Santos, A. C., Sarwar, H. S. \u0026amp; Jamshaid, M. A Comprehensive Review of Hydrogel-Based Drug Delivery Systems: Classification, Properties, Recent Trends, and Applications. \u003cem\u003eAAPS PharmSciTech\u003c/em\u003e vol. 25 Preprint at https://doi.org/10.1208/s12249-024-02786-x (2024).\u003c/li\u003e\n\u003cli\u003eZhang, W. \u003cem\u003eet al.\u003c/em\u003e Factors affecting the properties of superabsorbent polymer hydrogels and methods to improve their performance: a review. \u003cem\u003eJournal of Materials Science\u003c/em\u003e vol. 56 16223\u0026ndash;16242 Preprint at https://doi.org/10.1007/s10853-021-06306-1 (2021).\u003c/li\u003e\n\u003cli\u003eChen, J., Garcia, E. S. \u0026amp; Zimmerman, S. C. Intramolecularly Cross-Linked Polymers: From Structure to Function with Applications as Artificial Antibodies and Artificial Enzymes. \u003cem\u003eAcc Chem Res\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 1244\u0026ndash;1256 (2020).\u003c/li\u003e\n\u003cli\u003ePaajanen, A., Vaari, J. \u0026amp; Verho, T. Crystallization of cross-linked polyethylene by molecular dynamics simulation. \u003cem\u003ePolymer (Guildf)\u003c/em\u003e \u003cstrong\u003e171\u003c/strong\u003e, 80\u0026ndash;86 (2019).\u003c/li\u003e\n\u003cli\u003eLiang, J.-H. \u003cem\u003eFluorescence Due to Interactions of Oxidizing Soybean Oil and Soy Proteins\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eSingh, J. \u0026amp; Nayak, P. pH-responsive polymers for drug delivery: Trends and opportunities. \u003cem\u003eJournal of Polymer Science\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 2828\u0026ndash;2850 (2023).\u003c/li\u003e\n\u003cli\u003eYoshida, T., Lai, T. C., Kwon, G. S. \u0026amp; Sako, K. pH- and ion-sensitive polymers for drug delivery. \u003cem\u003eExpert Opin Drug Deliv\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1497 (2013).\u003c/li\u003e\n\u003cli\u003eGolla, V. M. \u003cem\u003eet al.\u003c/em\u003e Separation, Identification, and Characterization of Transformation Products Related to Relugolix Upon Subjecting to In Vitro Gastrointestinal Fluids Utilizing Liquid Chromatography, Mass Spectrometry, and Nuclear Magnetic Resonance Spectroscopy. \u003cem\u003eSep Sci Plus\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e202400195 (2025).\u003c/li\u003e\n\u003cli\u003eAbouelmagd, S. A., Sun, B., Chang, A. C., Ku, Y. J. \u0026amp; Yeo, Y. Release Kinetics Study of Poorly Water-Soluble Drugs from Nanoparticles: Are We Doing It Right? \u003cem\u003eMol Pharm\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 997 (2015).\u003c/li\u003e\n\u003cli\u003eLavrentev, F. V. \u003cem\u003eet al.\u003c/em\u003e Diffusion-Limited Processes in Hydrogels with Chosen Applications from Drug Delivery to Electronic Components. \u003cem\u003eMolecules\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 5931 (2023).\u003c/li\u003e\n\u003cli\u003eSafari, J. B., Bapolisi, A. M. \u0026amp; Krause, R. W. M. Development of pH-Sensitive Chitosan-g-poly(acrylamide-co-acrylic acid) Hydrogel for Controlled Drug Delivery of Tenofovir Disoproxil Fumarate. \u003cem\u003ePolymers (Basel)\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 3571 (2021).\u003c/li\u003e\n\u003cli\u003eVegad, U. \u003cem\u003eet al.\u003c/em\u003e pH stimuli-responsive hydrogels from non-cellulosic biopolymers for drug delivery. \u003cem\u003eFront Bioeng Biotechnol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1270364 (2023).\u003c/li\u003e\n\u003cli\u003eChiappetta, D. A., Hocht, C., Taira, C. \u0026amp; Sosnik, A. Efavirenz-loaded polymeric micelles for pediatric anti-HIV pharmacotherapy with significantly higher oral bioavailability [corrected]. \u003cem\u003eNanomedicine (Lond)\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 11\u0026ndash;23 (2010).\u003c/li\u003e\n\u003cli\u003eAlmeida dos Santos, K., Maria Coelho de Carvalho Moreira, L., Lamartine Soares-Sobrinho, J. \u0026amp; Felts de La Roca Soares, M. New Horizons in Antiretroviral Drug Delivery Systems for HIV Management. \u003cem\u003eCurr Med Chem\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, (2024).\u003c/li\u003e\n\u003cli\u003eVarshosaz, J., Taymouri, S., Jahanian-Najafabadi, A. \u0026amp; Alizadeh, A. Efavirenz oral delivery via lipid nanocapsules: formulation, optimisation, and ex‐vivo gut permeation study. \u003cem\u003eIET Nanobiotechnol\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 795 (2018).\u003c/li\u003e\n\u003cli\u003ePitorre, M. \u003cem\u003eet al.\u003c/em\u003e Polymer-free hydrogel made of lipid nanocapsules, as a local drug delivery platform. \u003cem\u003eMater Sci Eng C Mater Biol Appl\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eSaleemi, M. A. \u003cem\u003eet al.\u003c/em\u003e Toxicity of Carbon Nanotubes: Molecular Mechanisms, Signaling Cascades, and Remedies in Biomedical Applications. \u003cem\u003eChemical Research in Toxicology\u003c/em\u003e vol. 34 24\u0026ndash;46 Preprint at https://doi.org/10.1021/acs.chemrestox.0c00172 (2021).\u003c/li\u003e\n\u003cli\u003eMiura, N. \u0026amp; Shinohara, Y. Cytotoxic effect and apoptosis induction by silver nanoparticles in HeLa cells. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e \u003cstrong\u003e390\u003c/strong\u003e, 733\u0026ndash;737 (2009).\u003c/li\u003e\n\u003cli\u003eManna, S. K. \u003cem\u003eet al.\u003c/em\u003e Single-walled carbon nanotube induces oxidative stress and activates nuclear transcription factor-\u0026kappa;B in human keratinocytes. \u003cem\u003eNano Lett\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1676\u0026ndash;1684 (2005).\u003c/li\u003e\n\u003cli\u003eGravely, M. \u003cem\u003eet al.\u003c/em\u003e Aggregation Reduces Subcellular Localization and Cytotoxicity of Single-Walled Carbon Nanotubes. \u003cem\u003eACS Appl Mater Interfaces\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 19168\u0026ndash;19177 (2022).\u003c/li\u003e\n\u003cli\u003eHappi, G. M. \u003cem\u003eet al.\u003c/em\u003e Bioassay-Guided Isolation of Antiplasmodial Compounds from Hypericum lanceolatum Lam. (Hypericaceae) and Their Cytotoxicity and Molecular Docking. \u003cem\u003eBiomed Res Int\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 4693765 (2023).\u003c/li\u003e\n\u003cli\u003eTenorio-Borroto, E. \u003cem\u003eet al.\u003c/em\u003e Model for high-throughput screening of drug immunotoxicity \u0026ndash; Study of the anti-microbial G1 over peritoneal macrophages using flow cytometry. \u003cem\u003eEur J Med Chem\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 206\u0026ndash;220 (2014).\u003c/li\u003e\n\u003cli\u003eMustafa, Y. F. Synthesis of novel 6-aminocoumarin derivatives as potential \u0026ndash;biocompatible antimicrobial and anticancer agents. \u003cem\u003eJ Mol Struct\u003c/em\u003e \u003cstrong\u003e1320\u003c/strong\u003e, 139658 (2025).\u003c/li\u003e\n\u003cli\u003eNordin, M. L., Abdul Kadir, A., Zakaria, Z. A., Abdullah, R. \u0026amp; Abdullah, M. N. H. In vitro investigation of cytotoxic and antioxidative activities of Ardisia crispa against breast cancer cell lines, MCF-7 and MDA-MB-231. \u003cem\u003eBMC Complement Altern Med\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003eHughes, J. P., Rees, S. S., Kalindjian, S. B. \u0026amp; Philpott, K. L. Principles of early drug discovery. \u003cem\u003eBr J Pharmacol\u003c/em\u003e \u003cstrong\u003e162\u003c/strong\u003e, 1239 (2011).\u003c/li\u003e\n\u003cli\u003eAkinboye, E. S. \u0026amp; Bakare, O. \u003cem\u003eBiological Activities of Emetine\u003c/em\u003e. \u003cem\u003eThe Open Natural Products Journal\u003c/em\u003e (2011).\u003c/li\u003e\n\u003cli\u003eAmenta, F., Battineni, G., Traini, E. \u0026amp; Pallotta, G. Choline-containing phospholipids and treatment of adult-onset dementia disorders. in \u003cem\u003eDiagnosis and Management in Dementia: The Neuroscience of Dementia, Volume 1\u003c/em\u003e 477\u0026ndash;493 (Elsevier, 2020). doi:10.1016/B978-0-12-815854-8.00030-6.\u003c/li\u003e\n\u003cli\u003eOnaolapo, M. C. \u003cem\u003eet al.\u003c/em\u003e Lecithin and cardiovascular health: a comprehensive review. \u003cem\u003eEgyptian Heart Journal\u003c/em\u003e vol. 76 Preprint at https://doi.org/10.1186/s43044-024-00523-0 (2024).\u003c/li\u003e\n\u003cli\u003eSt-Onge, M.-P. \u0026amp; Jones, P. J. H. \u003cem\u003eRecent Advances in Nutritional Sciences Physiological Effects of Medium-Chain Triglycerides: Potential Agents in the Prevention of Obesity 1\u003c/em\u003e. https://academic.oup.com/jn/article-abstract/132/3/329/4687297 (2018).\u003c/li\u003e\n\u003cli\u003evan Gastel, N. \u003cem\u003eet al.\u003c/em\u003e Induction of a Timed Metabolic Collapse to Overcome Cancer Chemoresistance. \u003cem\u003eCell Metab\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 391-403.e6 (2020).\u003c/li\u003e\n\u003cli\u003eThomas, D. D. \u003cem\u003eet al.\u003c/em\u003e Effects of medium chain triglycerides supplementation on insulin sensitivity and beta cell function: A feasibility study. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eDosh, R. H., Jordan-Mahy, N., Sammon, C. \u0026amp; Le Maitre, C. L. Tissue Engineering Laboratory Models of the Small Intestine. \u003cem\u003eTissue Eng Part B Rev\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 98\u0026ndash;111 (2018).\u003c/li\u003e\n\u003cli\u003ePeng, C., Lu, W. \u0026amp; Fang, Y. An insight into the effect of food nanoparticles on the metabolism of intestinal cells. \u003cem\u003eCurr Opin Food Sci\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 174\u0026ndash;182 (2022).\u003c/li\u003e\n\u003cli\u003eSornkamnerd, S., Okajima, M. K. \u0026amp; Kaneko, T. Tough and Porous Hydrogels Prepared by Simple Lyophilization of LC Gels. \u003cem\u003eACS Omega\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 5304\u0026ndash;5314 (2017).\u003c/li\u003e\n\u003cli\u003eMohite, P. \u003cem\u003eet al.\u003c/em\u003e Lipid-based oral formulation in capsules to improve the delivery of poorly water-soluble drugs. \u003cem\u003eFrontiers in Drug Delivery\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1232012 (2023).\u003c/li\u003e\n\u003cli\u003eŽigrayov\u0026aacute;, D., Miku\u0026scaron;ov\u0026aacute;, V. \u0026amp; Miku\u0026scaron;, P. Advances in Antiviral Delivery Systems and Chitosan-Based Polymeric and Nanoparticulate Antivirals and Antiviral Carriers. \u003cem\u003eViruses 2023, Vol. 15, Page 647\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 647 (2023).\u003c/li\u003e\n\u003cli\u003eSiyawamwaya, M. Three dimensional printed controlled release tritherapeutic tablet (3D CRTT) for the delivery of anti-HIV drugs. Preprint at https://hdl.handle.net/10539/24794 (2017).\u003c/li\u003e\n\u003cli\u003eMabrouk, M. \u003cem\u003eet al.\u003c/em\u003e Design of a novel crosslinked HEC-PAA porous hydrogel composite for dissolution rate and solubility enhancement of efavirenz. \u003cem\u003eInt J Pharm\u003c/em\u003e \u003cstrong\u003e490\u003c/strong\u003e, 429\u0026ndash;437 (2015).\u003c/li\u003e\n\u003cli\u003eCarvalho, G. C. \u003cem\u003eet al.\u003c/em\u003e Highlights in poloxamer-based drug delivery systems as strategy at local application for vaginal infections. \u003cem\u003eInt J Pharm\u003c/em\u003e \u003cstrong\u003e602\u003c/strong\u003e, 120635 (2021).\u003c/li\u003e\n\u003cli\u003eThapa, R., Gurung, S., Parat, M. O., Parekh, H. S. \u0026amp; Pandey, P. Application of Sol\u0026ndash;Gels for Treatment of Gynaecological Conditions\u0026mdash;Physiological Perspectives and Emerging Concepts in Intravaginal Drug Delivery. \u003cem\u003eGels 2022, Vol. 8, Page 99\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 99 (2022).\u003c/li\u003e\n\u003cli\u003eVashist, A. \u003cem\u003eet al.\u003c/em\u003e Recent trends on hydrogel based drug delivery systems for infectious diseases. \u003cem\u003eBiomater Sci\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1535\u0026ndash;1553 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lipid nanocapsules, Hydrogel, Synergistic Use, Drug Delivery, Chitosan, Iotacarrageenan","lastPublishedDoi":"10.21203/rs.3.rs-7078136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7078136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHydrophobic drug delivery via oral or pulmonary routes presents significant challenges for clinical translation, particularly for poorly soluble antiviral drugs. Physiological barriers\u0026mdash;such as enzymatic degradation, harsh pH, and rapid transit in the gastrointestinal tract, or mucociliary clearance and alveolar macrophage uptake in the lungs\u0026mdash;can severely limit therapeutic efficacy. To address these challenges, we developed a novel lipid nanocapsule (LNC) and chitosan/iota-carrageenan hydrogel composite tailored for sustained delivery of hydrophobic antiviral agents. This composite system was designed to encapsulate and deliver Efavirenz (EFV) under simulated gastrointestinal conditions. EFV was first encapsulated in LNCs, which were subsequently embedded within a mucoadhesive hydrogel matrix to form the EFV-LNC hydrogel composite. The LNCs significantly enhanced EFV solubility compared to water alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and droplet size was controlled (57.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nm). The hydrogel composite exhibited an optimized swelling ratio (~\u0026thinsp;300 g water per 1 g hydrogel) and achieved an encapsulation efficiency of approximately 53%. Importantly, EFV release from the composite was significantly prolonged under various gastrointestinal pH conditions compared to the unformulated drug (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Cytotoxicity assays confirmed the composite\u0026rsquo;s cytocompatibility, supporting its potential safety for future mucosal administration. These findings suggest that the LNC-hydrogel composite enhances solubility, enables controlled release, and may improve mucosal retention, supporting its utility as a versatile platform for oral and pulmonary delivery of hydrophobic antiviral drugs.\u003c/p\u003e","manuscriptTitle":"Lipid Nanocapsule-Chitosan and Iota-Carrageenan Hydrogel Composite for Sustained Hydrophobic Drug Delivery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 08:33:57","doi":"10.21203/rs.3.rs-7078136/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-02T20:37:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-31T07:27:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51894610592689588727930084115445452215","date":"2025-08-27T00:29:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T05:14:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"226088707205961462085085461829500351403","date":"2025-08-13T07:21:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313804569754450222503745320992124018543","date":"2025-08-12T16:00:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-12T14:59:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-12T14:56:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-12T12:46:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-02T04:26:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-02T04:21:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de3d8e57-35eb-4336-a1d2-0ede8220ce73","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53090290,"name":"Biological sciences/Biotechnology"},{"id":53090291,"name":"Biological sciences/Drug discovery"},{"id":53090292,"name":"Physical sciences/Materials science"},{"id":53090293,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-12-01T16:05:25+00:00","versionOfRecord":{"articleIdentity":"rs-7078136","link":"https://doi.org/10.1038/s41598-025-26390-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-27 15:58:43","publishedOnDateReadable":"November 27th, 2025"},"versionCreatedAt":"2025-08-20 08:33:57","video":"","vorDoi":"10.1038/s41598-025-26390-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-26390-6","workflowStages":[]},"version":"v1","identity":"rs-7078136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7078136","identity":"rs-7078136","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}