Covalent Grafting of Hyaluronic Acid on Drug Nanocrystals - Application in Mucosal Delivery

Covalent Grafting of Hyaluronic Acid on Drug Nanocrystals - Application in Mucosal 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 Research Article Covalent Grafting of Hyaluronic Acid on Drug Nanocrystals - Application in Mucosal Delivery Marta G. Fuster, Octavio E. Fandiño, Akhil Ramesh, Jonnathan A. Coulter, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7030744/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract Nanocrystals (NCs) represent an advanced drug delivery platform due to their nearly 100% drug loading capacity, enhanced solubility, and improved tissue penetration. The surface-rich structure NCs enable chemical modifications, with covalent grafting emerging as a superior strategy to impart stability and targeted functional groups. In this study, we explore the development of curcumin nanocrystals (CUR-NCs) covalently grafted with hyaluronic acid (HA). To enhance mucosal targeting and permeation, an innovative surface functionalization strategy was employed: hyaluronic acid (HA) was chemically grafted onto the Chitosan (CS) NCs through EDC/NHS-mediated chemistry. This surface conjugation was confirmed through FTIR and 1 H-NMR analyses, validating the successful formation of HA-CS-CUR-NCs. The resulting nanoparticles exhibited an average particle size of 110 nm, remaining within the ideal range for mucosal delivery. Importantly, cytotoxicity assays on THP1 monocytes and NIH/3T3 fibroblasts revealed that the HA-CS-CUR-NCs possessed excellent biocompatibility properties. Ex vivo mucosal deposition studies using neonatal porcine tissue demonstrated significantly improved mucopenetration with HA-functionalized NCs, achieving 37.3 ± 26% drug deposition after 24 hours, compared to only 1.81 ± 1% for non-functionalized CUR-NCs. These findings position HA-CS-CUR-NCs as a promising platform for advanced mucosal drug delivery, combining nanoscale precision with bioresponsive surface chemistry to enhance therapeutic outcomes. Curcumin nanosuspension targeted nanocrystals surface decoration in vitro uptake ex vivo mucopenetration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Nanocrystals (NCs) have emerged as a very promising drug delivery platform due to their near 100% drug loading capacity [ 1 ]. NCs are crystalline particles typically ranging in size from 10–500 nm, minimally stabilized by surfactants or polymers. Compared to conventional nanoparticles, which often require significant amounts of excipients or carriers, NCs offer improved dissolution rates and higher saturation solubility, making them particularly attractive for high-dose drug delivery with improved pharmacokinetics [ 2 ]. A major advantage of NCs lies in their surface accessibility, which allows for tailored functionalisation strategies. Among these, covalent grafting of functional groups on the NC surface stand out as the preferred method to facilitate targeting, muco-penetration [ 3 ], and controlled release [ 4 ]. Additionally, covalent grafting offers superior stability compared to physical adsorption or electrostatic interactions, ensuring durable surface modification under physiological conditions, a crucial aspect for clinical translation[ 5 – 7 ]. One of the most pressing challenges in drug delivery is overcoming mucosal barriers such as those found in the gastrointestinal, pulmonary, and nasal systems. The dense and viscous nature of mucus restricts the penetration and retention of therapeutic agents [ 8 ]. Efficient mucopenetration is critical for achieving localized drug delivery and enhancing systemic absorption. NCs, owing to their small size and high diffusion rates, exhibit superior muco-penetrative capabilities compared to larger drug carriers [ 9 – 11 ]. However, this activity can be further enhanced by modifying their surface chemistry [ 12 ]. In this context, covalent grafting with hydrophilic and biocompatible polymers such us hyaluronic acid (HA) offer a promising route to enhance mucus diffusion. HA is a naturally occurring glycosaminoglycan found in the extracellular matrix, characterized by its hydrophilicity, biodegradability, and excellent biocompatibility [ 13 ]. The covalent attachment of HA to NC surfaces can facilitate targeted delivery, while minimizing off-target effects and premature clearance [ 14 ]. Furthermore, the hydrophilic nature of HA reduces particle aggregation, which facilitates uniform and efficient diffusion through dense mucus layers. This is especially beneficial in nanopharmaceuticals, where uniform distribution and controlled drug delivery are critical for improving therapeutic outcomes and reducing side effects [ 15 ]. To exemplify the potential of this approach, curcumin (CUR) was selected as a model compound. CUR is a natural polyphenolic compound extracted from the rhizome of Curcuma longa , has attracted attention in biomedical research due to its broad spectrum of pharmacological activities [ 16 ]. CUR exhibits potent anti-inflammatory, antioxidant, antimicrobial, and anticancer properties, making it a promising candidate for the treatment of various chronic diseases, including cancer, arthritis, and neurodegenerative disorders [ 17 ]. Despite these benefits, clinical use of CUR remains low due to its poor water solubility (approximately 11 ng/mL in aqueous solutions), low chemical stability, and rapid systemic elimination. These challenging characteristics ultimately lead to suboptimal bioavailability and limited therapeutic efficacy [ 18 ]. NCs formulations has been proposed as an effective strategy to improve the solubility and pharmacokinetic profile of CUR [ 19 ]. Several studies have already shown that HA-modified nanoparticles can significantly enhance drug uptake and therapeutic efficacy. For instance, HA-based systems, such as solid lipid nanoparticles and polymeric nanogels, offer superior drug encapsulation, enhanced stability, and targeted release capabilities [ 20 ]. These advantages are particularly beneficial for treating diseases that require localized drug delivery across barriers, like ocular conditions or mucus-rich respiratory pathways [ 21 ]​. In addition, the hydrophilic HA coating reduces particle adhesion, enhancing mobility and preventing rapid clearance. Such properties make HA-modified nanoparticles promising for delivering drugs to hard-to-reach tissues, like tumors or inflamed sites, while minimizing systemic side effects. Improved mucoadhesion is also achieved through the natural affinity of HA for mucus glycoproteins, facilitating prolonged retention of polymeric nanoparticles on mucosal surfaces and enhancing drug absorption [ 22 ]. Several studies have demonstrated the benefits of HA in enhancing drug delivery systems by improving stability, bioavailability, and targeted therapeutic efficacy. Ji et al. [ 23 ] developed HA-coated CUR-NCs (HA-CUR-NCs) for breast-cancer therapy. The HA coating improved the bioavailability of CUR, prolonging circulation time and enhanced uptake in CD44 receptor-overexpressing cancer cells. In vivo , NCs exhibited superior anticancer effects with reduced side effects, demonstrating their potential as a targeted breast cancer treatment. Zerrillo et al. [ 24 ] designed a HA-decorated poly(lactide-co-glycolide) (PLGA) nanoparticles for osteoarthritis therapy. The HA modification improved binding to CD44 receptors on chondrocytes and enhanced cartilage tissue targeting, while conferring no direct cytotoxicity. Non-invasive imaging confirmed efficient nanoparticle accumulation, highlighting their suitability for targeted osteoarthritis therapies. Seo et al. [ 25 ] created a multilayered doxorubicin (DOX) and HA cellulose NCs (CNCs) for CD44-targeted delivery. This formulation improved drug penetration, ROS production, and cancer cell death in lung adenocarcinoma models. In vivo imaging confirmed enhanced tumor accumulation, demonstrating the potential for targeted chemotherapy. Given the superior drug loading capability of NCs and the benefits of stable surface functionalisation, this study focuses on the covalent grafting of HA onto CUR-NCs. The aim is to systematically synthesize, characterize, and optimize HA-grafted CUR-NCs, evaluating their impact on mucopenetration, bioavailability, and therapeutic potential. This work provides a foundation for the development of NCs-based drug delivery systems designed to leverage the synergistic advantages of high drug loading, covalent surface engineering, and targeted mucosal delivery. 2. Materials and methods 2.1. Materials All chemicals and materials used for this study were of analytical grade. Curcumin (CUR- CAS RN 458-37-7) was purchased from Tokyo Chemical Industries (London, UK), Poloxamer 188 (POL 188) from BASF chemical company (Ludwigshafen, Germany) and Poloxamer (POL 407). Chemicals such as chitosan (CS- LMW – 50-190KDa, Deacetylated chitin, Poly(D-glucosamine) and N-Hydroxysuccinimide (NHS- CAS 6066-82-6) were purchased from Sigma-Aldrich (Poole, Dorset, UK). 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC- CAS 25952-53-8) was obtained from EMD Millipore Corp (USA), sodium hyaluronate from Kewpie Corporation (Shibuya-Ku, Tokyo). For the milling process, YTZP Yttria-stabilized zirconia beads of 0.15 and 0.5 mm were purchased from Chemco (Guangfu, China). Parafilm M® was purchased from Bemis Company Inc. (Neenah, USA). Ultrapure water was obtained from a water purification system, Elga Purelab DV 25, Veolia Water Systems (Ireland). All other chemicals used were of analytical grade. 2.2. Methods 2.2.1. Optimisation of CUR-NCs Effect of surfactant type CUR-NCs were prepared by slightly modifying an already published media milling technique [ 1 ] wherein, 100 mg of CUR was sandwiched between 4.5 mL of zirconia beads and magnetic bars (25 x 8 mm) in a 10 mL vial. 5 mL of varying strengths (0.5%, 1%, 1.5% and 2% w/v) of P188 and P407 solutions were added. The vials were then completely covered using tin foil and agitated at 1200 rpm for 24 hours. The resultant CUR-NCs were filtered from the milling media using a 300-mesh sieve. 20 µL of the resultant filtrate was diluted with 3 mL HPLC water in a cuvette and analysed for PS, PDI and ZP. Effect of chitosan concentration A similar media milling technique was employed to prepare CS-CUR-NCs. Briefly, 100 mg of CUR, 4.5 mL of zirconia beads (0.1–0.2 mm), and two magnetic bars (25 × 8 mm) were measured and added to the milling system. Varying amounts of POL 188/POL 407 and CS 0.5% w/v solution were added, as summarized in Table 1 . The milling process was carried out at 1200 rpm for 24 hours. Afterward, NCs were filtered and characterized as previously described. Table 1 Optimization parameters for CS-CUR-NCs: poloxamer and chitosan volumes used. POL 188/POL 407 (% w/v) Final volume POL 188/POL 407 (mL) Final volume of 0.5% CS (mL) 0.5 4.75 0.25 1 4.5 0.5 1.5 4 1 2.2.2. Functionalisation of CS-CUR-NCs with hyaluronic acid To functionalize CS-CUR-NCs, HA was activated using a solution of EDC and NHS under stirring at 300 rpm for 5 minutes, then agitated for 60 minutes to form the HA-EDC-NHS intermediate. The molar ratios of both EDC:HA and NHS:HA were approximately 66:1, ensuring efficient activation of the HA carboxyl groups. Activated HA was then added dropwise to a suspension of CS-CUR-NCs and stirred at 300 rpm for 16 hours at room temperature. The resulting NCs were collected by centrifugation at 342 g for 15 minutes, before freeze-drying for 25 hours. The final freeze-dried HA-CS-CUR-NCs pellet was analysed using various physicochemical characterization techniques 2.2.3. Particle size, polydispersity index and zeta potential A NanoBrook Omni Dynamic Light Scattering (DLS) analyser (Brookhaven Instruments Corp., Holtsville, NY, USA) was used to determine particle size, polydispersity index (PDI), and zeta potential of the samples. For the measurements, 20 µL of the prepared NCs were diluted with 3 mL of distilled water in measurement cuvettes. All samples were measured in triplicate, and the results are reported as mean ± standard deviation (SD, n = 3). 2.2.4. Characterization Physicochemical characterization For the physicochemical characterization samples of CUR-NCs, CS-CUR-NCs, HA-CS-CUR-NCs and PM were analysed using a PerkinElmer Spectrum Two FTIR spectrometer (PerkinElmer Inc., USA). Wavelengths ranging from 4000 cm − 1 to 500 cm − 1 were subjected to analysis, averaging 64 scans per spectrum. All major bands obtained were recorded using OMNIC™ Spectra Software (Thermo Fisher Scientific Inc., Waltham, USA). Thermal characterization, including differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), was carried out using a Q100 DSC (TA Instruments, Bellingham, WA). Scans were conducted from 20°C to 350°C with a heating rate of 10°C/min under a constant flow of nitrogen (10 mL/min). TGA experiments were carried out between 25ºC and 350ºC with a heating rate of 10 ºC/min and a nitrogen flow of 10 mL/min, using a Q500 instrument (TA Instruments, New Castle, DE, USA). RM5 Confocal Raman Microscope (Edinburgh Instruments Ltd, Livingston, UK) was used to analyse the Raman spectra of the obtained NCs. Approximately 2 mg of sample were taken on a glass slide. The grating was set at 300 gr/mm, the laser power at 50% and laser wavelength at 785 nm. The spectrum was analysed within the range 0–3200 Raman shift/ cm − 1 at 5 accumulations each with exposure time 1.5 s. Signals were recorded and analysed via the Ramacle® Software (Edinburgh Instruments Ltd, Livingston, UK). Finally, the assessment of ligand attachment was evaluated using proton nuclear magnetic resonance (NMR) 1 H NMR. Approximately 8 mg of the FD samples were dissolved in 400 µL of DMSO (Dimethyl sulfoxide-d6), then transferred to 7” NMR sample tubes (Wilmad Economy, NJ, USA). The Bruker Ultrashield 400 spectrometer (Bruker, Leipzig, Germany) was operated at 400 MHz to record the NMR spectra. MestReNova 6.0.2© (Mestrelab Research, Santiago de Compostela, Spain) was used to process the spectra obtained. The observed chemical shifts relative to tetramethylsilane were reported in ppm ( δ ). Microscopy and imaging techniques Advanced microscopy imaging techniques were employed to characterise CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs. NCs were first desiccated to dryness, then deposited onto adhesive carbon tape, and examined using a TM3030 microscope (Hitachi, Krefeld, Germany). A Tabletop TM 3030 scanning electron microscope (SEM) (Hitachi, Tokyo, Japan) was utilised to reveal the topological features of NCs, providing detailed surface morphology data. 2.2.5. HPLC quantification The detection and quantification of CUR were performed by using HPLC-UV following the protocol reported before [ 27 ]. A ZORBAX Eclipse XDB-C18 column (50 × 4.6 mm internal diameter; 1.8 µm particle size) was selected as the stationary phase. The mobile phase was composed of 80:20% (v/v) ACN and water (0.1% phosphoric acid). The maxima absorption (λ max) was fixed at 425 nm. The injection volume was set as 20 µL, and the flow rate was 0.5 mL/min. The linearity of the method was explored between 0.1 and 50 µg/mL (r 2 = 0.9999). The limits of detection and quantification were 0.27 and 0.81 µg/mL, respectively. Determination of CUR content in the formulated nanosuspensions To determine the CUR content in the obtained formulations, 20 µL of the resulting nanosuspension was dissolved in 980 µL of acetonitrile (ACN). The resulting solution was centrifuged at 16,602 g for 15 minutes and subsequently filtered using a 0.22 µm filter (Millipore, USA) for subsequent quantification by high-performance liquid chromatography (HPLC). 2.2.6. In vitro release study A dialysis membrane technique was used to evaluate in vitro release profiles comparing pure CUR drug, a physical mixture (PM) of each nanoformulation, and the NCs. The release medium was a phosphate buffer at pH 6.4 (simulating saliva), supplemented with 2% w/v Tween 80 and 0.5% ascorbic acid to prevent degradation of the drug[ 28 ]. After confirming sink conditions, samples containing approximately 3 mg of CUR were added to preactivated (PBS for 1 h) dialysis membrane bags (molecular weight cut-off: 12,000–14,000 Da; Spectra-Por, Spectrum Medical Industries, Los Angeles, CA, USA). The bags were sealed with plastic clips and incubated in an ISF 7100 orbital shaker (Jeio Tech, MA, USA) at 37°C and 100 rpm. At predetermined time intervals (0 h, 1 h, 3 h, 6 h, 9 h, 1 day, 2 days, 3 days, 7 days, 14 days and 28 days), 1 mL of release media was withdrawn and replaced with fresh buffer. CUR content was quantified using HPLC (section 2.2.5). All experiments were conducted in triplicate, with results expressed as mean ± standard deviation (n = 3). 2.2.7. Cell culture experiments THP1 human monocytic and NIH/3T3 cell line fibroblast cell line from a mouse were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and stored according to the supplier’s guidelines. THP1 cells were kept in Roswell Park Memorial Institute medium (RPMI) and NIH/3T3 in Dulbecco’s modified Eagle medium (without phenol red) supplemented with 10% (v/v) heat inactivated foetal bovine serum. Cells were maintained in T75 culture flasks, in a humidified environment with 5% CO 2 /95% air at 37°C. When reaching 80% confluences, cells were detached using 0.25% trypsin-EDTA, then either seeded for experiment or passaged to maintain stocks. Viability assay The cytotoxic effects of CUR-NCs, CS-CUR-NCs, and HA-CS-CUR-NCs on NIH/3T3 cells were tested using Alamar Blue® assay (Thermo Fisher Scientific, Waltham, MA, USA). Cells were seeded in 96-well plates at a concentration of 5 × 10 4 cells/well. Twenty- four hours post seeding cells were treated with fresh medium (negative control) or various concentrations of NCs containing media for 24 h. Post treatment, excess NCs was replaced with complete media containing 10% Alamar blue solution for 4 h. Fluorescence levels, positivity correlating with cell viable cell number was measured using a FLUOstar Omega (BMG LABTECHGmbH, Freiburg, Germany) microplate spectrophotometer at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Data points were collected over at least three independent experiments. NCs cellular uptake by flow cytometry 1 × 10 4 cells were seeded into individual wells on a 6-well plate, then incubated at 37°C for 24 h to allow attachment. The culture medium was replaced by a fresh medium containing NCs at a concentration equivalent to 120 µg/mL of CUR for 24h. Following incubation, NCs were removed by washing in PBS (x3), cells detached using 0.25% trypsin-EDTA, then fixed in 4% paraformaldehyde for 20 min. Cellular uptake was assessed by quantifying the associated fluorescence using a Becton − Dickinson FACSCalibur flow cytometer (New Jersey, United States). Cell-associated fluorescence was quantified using a Becton − Dickinson FACSCalibur flow cytometer (New Jersey, United States). The intrinsic fluorescence of CUR was used to monitor cellular uptake [ 29 ]. Untreated cells were used as controls to determine background autofluorescence. 2.2.8. Ex vivo mucosal deposition As detailed below, the ex vivo mucosal deposition studies of NCs were performed in excised buccal neonatal porcine mucosa. The mucosa was cleaned with pH 7.4 buffer and gently cut with a sterile scalpel. Then, following a previously published methodology, each mucosa section was placed in a weigh boat containing tissue paper soaked in buffer [ 30 ]. 3D printed resin rings with an inner diameter of 6 mm and a height of 5 mm were attached to the mucosa with glue using manual force for 30 s. Inside the ring, 30 µL of CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs were added. The system was then sealed with Parafilm M® to prevent evaporation, then placed in an incubator at 37ºC. After 24 h, the rings were removed, the mucosa was rinsed with 1 mL pH 7.4 mucus buffer, and the excess formulation was wiped with a clean tissue paper wetted with mucus buffer. Subsequently, a 6 mm diameter punch biopsy was used to obtain a circular section of mucosa from which drug was extracted. This experiment was carried out in triplicate. Each circular section of mucosa was placed in a 2-mL Eppendorf tube with two stainless steel beads (0.5 cm diameter, Qiagen, Hilden, Germany) and 500 µL of purified water. Tubes were then placed in a TissueLyser® LT (Qiagen, Hilden, Germany) and processed for 10 min at 50 Hz to hydrate the mucosa. Next, 1 mL of acetonitrile was added to each tube, and the process was repeated for another 10 min at 50 Hz to extract the drug. The homogenized tissue was then centrifuged at 14,462 g for 10 min (Sigma microtube centrifuge SciQuip Ltd, Shropshire, UK), and the CUR content in the supernatant quantified by HPLC using the method detailed in Section 2.14. Skin without treatment was using as a control. 2.2.9. Statistical analysis Statistical analysis was performed using GraphPad Prism© software (version 8.0, GraphPad Software Inc., San Diego, California, USA). One-way ANOVA followed by Tukey’s post-hoc test was performed to compare particle size and surface charge among CUR, CUR-CS, and CUR-CS-HA NCs formulations. 3. Results and Discussion 3.1. Optimisation of NCs 3.1.1. Stabilizer selection study The concentration and choice of stabilizer used to synthesize CUR-NCs by media milling were compared using particle size, polydispersity index (PDI) and zeta potential. Both POL 188 and POL 407, were tested at four concentrations (0.5% w/v, 1% w/v, 1.5% w/v and 2% w/v). The results obtained regarding particle size showed no significant differences across any of the studied POL concentrations, nor when comparing the two types of poloxamer used (POL 188 and POL 407). For example, at a concentration of 1.5% w/v, both POL 188 and POL 407 displayed comparable particle sizes (90.81 ± 1.0 nm and 92.94 ± 0.09 nm, respectively) and similar PDI values (0.264 and 0.249, respectively). In contrast, at the highest concentration (2% w/v), particle sizes (85.43 ± 1.01 nm for POL 188 and 95.42 ± 0.68 nm for POL 407) and zeta potential values (0.13 ± 1.78 mV for POL 188 and − 0.09 ± 1.36 mV for POL 407) were not altered, as seen in Fig. 1 A. In terms of zeta potential (Fig. 1 B), a distinct trend was observed; as the concentration of POL increased, the absolute value of the zeta potential becoming less negative charged regardless of the type of POL used. This indicates that concentration, rather than the specific type of POL influenced the reduction in zeta potential. For instance, the zeta potential shifted from − 10.94 ± 1.28 mV (0.5% POL 188) and − 7.44 ± 1.84 mV (0.5% POL 407) to near-neutral values at the 2% concentration (0.13 ± 1.78 mV and − 0.09 ± 1.36 mV, respectively). Such a substantial reduction in zeta potential values, approaching neutrality, suggests particle instability and the potential for aggregation. This aligns with literature findings, which report that nanoparticles with zeta potential values near zero are prone to agglomeration due to insufficient electrostatic repulsion [ 31 ]. As the zeta potential moves closer to neutral, electrostatic stabilisation diminishes, leading to destabilisation of the particle suspension. Considering this, particles synthesised with the highest concentration of POL were deemed unsuitable for further investigation. Despite a relatively stable PDI (0.227 for POL 188 and 0.208 POL 407), the observed particle size and significantly reduced zeta potential render these formulations unfit as stabilisers. 3.1.2. Chitosan coated CUR-NCs Based on the results obtained from the stabiliser selection study (3.1.1), three concentrations (0.5%, 1% and 1.5% w/v) of both POL 188 and POL 407 were chosen to prepare six different CUR-NCs with 0.5% w/v CS solution. From Fig. 1 C,D, it can be observed that the particle size of the NCs decreases significantly (p < 0.0001) with the addition of CS as the percentage of POL increases. For instance, in the case of POL 188, the particle size reduces from 117.06 ± 1.27 nm to 79.79 ± 0.46 nm. A similar trend is observed for POL 407, showing a decrease from 193.00 ± 0.72 nm at lower POL concentrations to an optimal size of 74.85 ± 1.53 nm at 1.5% w/v. While both formulations demonstrated promising results for further experimentation, POL 407 was ultimately selected due to its superior final characteristics, including a lower PDI of 0.261 and a positive zeta potential of 9.53 ± 2.93 mV. 3.1.2.1. Stability study data for CS-CUR-NCs Stability studies for the optimised CS-CUR-NCs were conducted at 4°C over a 28-day period. The particle size results remained consistently below 80 nm across the full study period. Similarly, PDI values (Fig. 1 E dots) remained below 0.3, indicating a stable size distribution. Zeta potential measurements (Fig. 1 F) displayed minor fluctuations over time but consistently remained positive. 3.2. Functionalised NCs with hyaluronic acid (HA-CS-CUR-NCs). 3.2.1. Dynamic light scattering (DLS) The data obtained from the particle size and zeta potential analysis study are presented in Fig. 1 G, F. Comparing across samples it was evident that CS was successfully conjugated forming the CS-CUR-NCs sample, as the zeta potential following CS conjugation switched from negative (-4.62 ± 0.74 mV) to positive (10.02 ± 1.93 mV). This shift is consistent with the presence of numerous amino groups along the CS backbone, which confer a positive surface charge and facilitate subsequent derivatization. A slight decrease in the particle size from 92.94 ± 0.09 nm to 75.47 ± 1.19 nm was also observed. These changes in NCs physicochemical properties following surface conjugation are attributed to electrostatic interactions imparted by CS, increasing repulsive forces[ 32 ]. Importantly, PDI values also remained consistently below 0.3 for the three samples. 3.2.2. ATR-FTIR spectroscopy ATR-FTIR spectroscopy was performed to confirm the successful coating of HA onto the surface of CS-CUR-NCs. The spectra obtained (Fig. 2 ) revealed key characteristic bands for each analysed sample. A broad band between 3000 cm⁻¹ and 3500 cm⁻¹ indicated the presence of the COOH group, suggesting the successful incorporation of HA. Additionally, distinct peaks were identified for CS (amine stretching at 3355 cm⁻¹, amide I stretch at 1652 cm⁻¹), POL 407 (polyethylene oxide band at 1100 cm⁻¹, polypropylene stretch at 2897 cm⁻¹), and CUR (OH stretch at 3525 cm⁻¹, C = C at 1603 cm⁻¹, C = O at 1650 cm⁻¹). A comparison between the before purified sample and the purified sample (Fig. 2 A) highlighted the presence of an amide band at 1715 cm⁻¹, indicating a successful EDC-NHS coupling reaction. This band is associated with the formation of a urea byproduct, which serves as further confirmation of the functionalization process. After purification, all major characteristic peaks were still present; however, the intensity of the amide band at 1715 cm⁻¹ was notably reduced, suggesting that a significant portion of the urea byproduct had been removed [ 33 ]. While theoretically, a strong amide band (between 1650 cm⁻¹ and 1750 cm⁻¹) would still be expected even after purification, the presence of a small shoulder band at 1715 cm⁻¹ suggests that HA remains attached to the CS-CUR-NCs. The observed reduction in absorbance intensity may be attributed to factors affecting the efficiency of the EDC-NHS coupling reaction, including variations in pH, lower reagent concentrations, or temperature fluctuations during synthesis. These parameters could influence both the reaction yield and the intensity of the bands observed in the spectra[ 32 ]. Taken together, these results suggest the successful formation of an amide bond, indicating that covalent coupling successfully occurred. Nevertheless, further characterization techniques, including 1 H-NMR analysis, were used to confirm the structural modification, ensuring the reliability of the findings. 3.2.3. 1 H-NMR analysis 1 H-NMR analysis was conducted to complement the DLS and FTIR data, looking to gain deeper understanding on the molecular interactions and structural integrity of the NCs after surface modification. Three samples CUR-NCs, CS-CUR-NCs, and HA-CS-CUR-NCs were analysed, revealing various proton chemical shifts, as shown in Fig. 2 B. A strong peak at 2.5 ppm was observed in all spectra, corresponding to DMSO used in sample preparation. In CS-CUR-NCs spectrum, an upfield peak between 0.5 and 2.5 ppm was detected, suggesting the presence of an R-NH₂ functional group from CS [ 34 ]. However, this peak was absent in the spectrum of HA-CS-CUR-NCs (Fig. 2 B), indicating that the amine groups had reacted, confirming the functionalization of CS with HA. This conclusion was further supported by the appearance of two distinct peaks between 9.0 and 10.0 ppm in the HA-CS-CUR-NCs spectrum, which, based on literature, correspond to R-COOH and R-CO-NHR groups [ 35 ]. These shifts provide strong evidence of the successful attachment of HA onto CS-CUR-NCs via the EDC-NHS coupling reaction. Additionally, all three samples exhibited peaks between 6.0 and 8.0 ppm, corresponding to the aromatic protons of CUR [ 36 ], indicating that the core nanocrystal structure remained intact throughout the functionalization process. The overall spectral changes confirm the successful conjugation of HA onto CS-CUR-NCs and validate the efficiency of the EDC-NHS coupling chemistry. 3.2.4. Thermal analysis DSC thermograms were recorded for CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs. The results (Fig. 3 ) displayed fusion peaks for POL 407 (~ 50°C) and CUR (~ 180°C) across all samples, confirming their presence. In CS-CUR-NCs (Fig. 3 A), a broad peak around 101°C was observed, indicative of loss of water [ 37 ]. The thermogram for HA-CS-CUR-NCs showed two additional peaks, one endothermic and one exothermic, alongside the fusion peaks common to all samples. The broad endothermic peak at ~ 97°C and the exothermic peak at ~ 250°C suggest the presence of HA. However, since CS also exhibits an endothermic peak near 100°C, the possibility of peak overlap between CS and HA was considered. This risk is mitigated by the presence of an exothermic peak at ~ 250°C, a feature unique to HA, further supporting successful incorporation. Thermogravimetric Analysis (TGA) of CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs were analysed to assess their thermal behaviour. As shown in the Fig. 3 B, the thermograms of CUR and CS-NCs formulations exhibited a relatively stable mass up to approximately 230°C, after which an initial mass loss of approximately 8% was observed, indicating the onset of decomposition. This behaviour is typical for the individual components, with the weight loss likely attributed to the degradation of CUR and CS [ 38 ]. In contrast, the HA-CS-CUR-NCs formulation showed a more pronounced and earlier onset of mass loss, beginning around 190°C. This suggests that HA in the formulation undergoes decomposition at a lower temperature compared to CS and CUR, which is consistent with the known thermal behaviour of HA [ 39 ]. The faster degradation observed for HA-CS-CUR-NCs could be due to the specific characteristics of HA, which can be more thermally labile than CS. Despite this, all formulations remained thermally stable up to their respective temperatures, indicating that the NCs formation process via media milling did not affect the thermal stability of the active ingredients. These results suggest that the incorporation of HA does not significantly compromise the overall thermal stability of the formulation but may influence its decomposition dynamics. 3.2.5. Microscopy images SEM and TEM were used to characterise the morphology of CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs. SEM analysis (Fig. 4 A) of CUR-NCs revealed spherical structures, likely due to the use of POL as a surfactant [ 40 ]. In the case of CS-CUR-NCs, SEM images exhibited platelet-like formations, potentially attributable to CS deposition, despite the continued presence of POL as a stabiliser. This is consistent with prior studies reporting morphological alterations in nanoparticles following CS coating, where polymeric layers can influence surface roughness and structure [ 41 ]. Upon chemical modification with HA, the observed platelets appeared larger, which could be explained by the centrifugation step necessary for the conjugation reaction. This process may lead to the partial removal of POL, thereby exposing or restructuring the CS. However, it is important to note that these visible structures do not directly represent the NCs themselves. During sample drying, aggregation of the stabiliser occurs, forming a matrix or in which the NCs are embedded. Due to the limited resolution of SEM, typically in the micrometre scale, individual NCs are extremely difficult to visualise within this matrix. For this reason, TEM was employed to gain more detailed insight into the nanostructures (Fig. 4 B). CUR-NCs were observed as spherical. In CS-CUR-NCs samples, phase contrast TEM revealed a distinguishable outer layer, suggestive of CS adhesion via electrostatic interactions. Finally, the TEM analysis of HA-CS-CUR-NCs demonstrated the presence of a well-defined additional layer surrounding the NCs, indicative of successful conjugation with HA. These observations confirm that the chemical modification was effective and that TEM is a valuable tool for visualising the added HA layer, providing direct evidence of the surface functionalisation achieved in this study. 3.2.6. In vitro release profiles and mucosal deposition The in vitro release profiles of CUR, physical mixtures (PMs), and NCs are shown in Fig. 5 A. As expected, pure CUR exhibited a slow dissolution rate, with only 2.92 ± 1.74% of the drug released by the end of the study. The physical mixtures showed no significant improvement over the pure drug, with CUR PM, CS PM, and HA PM reaching 2.49 ± 1.45%, 1.96 ± 1.07%, and 2.29 ± 1.66% drug release, respectively. These results indicate that the presence of the compounds alone did not significantly enhance the dissolution rate of CUR. In contrast, the CUR-NCs demonstrated a markedly improved dissolution profile, with approximately 55.11 ± 5.20% of the drug released after 28 days. Similarly, CS-CUR-NCs reached 57.89 ± 5.82%, while HA-CS-CUR-NCs achieved 48.35 ± 6.02% release. These findings highlight two key points; first, that the PMs serve as appropriate controls, confirming that the observed enhancement in drug release is attributable to the NCs formulation rather than to the individual components alone; and second, that the nanoformulations are both effective and efficient in increasing the dissolution rate. The increased dissolution rate and saturation solubility observed with NCs can be attributed an enlarged surface area, which plays a critical role in dissolution kinetics, described by the Noyes–Whitney equation [ 42 ]. Moreover, the sustained drug release profile is a promising characteristic of these systems. It is hypothesized that, despite natural clearance mechanisms at the mucosal level, NCs may remain accumulated in the mucosa, contributing to prolonged drug availability. 3.2.6.1. Ex vivo mucosal deposition The results of the mucosal deposition assay on neonatal porcine mucosa were obtained as follows. Patches were cut into 5 mm diameter discs, with the drug content measured for each formulation. The drug content for CUR-NCs was 18.01 ± 0.11 mg/mL, for CS-CUR-NCs it was 18.54 ± 1.41 mg/mL, and for HA-CS-CUR-NCs it was 2.21 ± 1.14 mg/mL. After 24 hours of contact between the mucosa and the nanosuspension, the drug deposition was quantified. The results showed that 1.81 ± 1% of the drug from CUR-NCs was deposited in the mucosa, while 23.7 ± 5% of the drug from CS-CUR-NCs and 37.3 ± 26% from HA-CS-CUR-NCs were deposited, with the latter showing a significantly higher deposition compared to the CUR-NCs formulation (Fig. 5 B). The poor penetration of unmodified CUR-NCs may result from their limited ability to interact with mucus components or from rapid clearance by the mucus layer. Moreover, in immune-cell-rich environments such as mucosal tissues, it is plausible that a fraction of nanoparticles may be internalised by resident macrophages, thereby reducing the number of particles reaching deeper tissue layers. This hypothesis is supported by previous studies demonstrating that macrophages contribute significantly to nanoparticle sequestration and clearance within mucosal barriers [ 43 ]. In contrast, the enhanced deposition observed with the CS-CUR-NCs and HA-CS-CUR-NCs formulations can be attributed to the incorporation of CS and HA, which are known to improve the permeation and adhesion of nanoparticles to mucosal surfaces. CS, a natural polymer with a positive charge, enhances the interaction with the negatively charged cell membranes, facilitating the penetration of drug-loaded nanoparticles through the mucosa [ 44 ]. Additionally, HA, which exhibits mucoadhesive properties, can increase the residence time of NCs at the mucosal surface, promoting higher drug absorption. These findings are consistent with the literature on nanoparticle-based drug delivery systems, where smaller particle sizes enhanced mucoadhesion lead to improved drug permeation across biological membranes [ 45 , 46 ]. Therefore, the improved deposition achieved with HA-CS-CUR-NCs reflects not only enhanced mucoadhesion, but also an ability to circumvent immune clearance mechanisms to a certain extent, highlighting their potential for mucosal drug delivery applications. 3.2.7. In vitro cell culture assays Cell viability was evaluated using the Alamar Blue assay in THP1 monocytes and NIH/3T3 fibroblasts, testing CUR-NCs, CS-CUR-NCs, and HA-CS-CUR-NCs. While these in vitro models do not represent the mucosal epithelium directly, they provide valuable insights into cytocompatibility, immune cell interaction, and general NCs behaviour at the cellular level. THP1 monocytes are relevant due to the presence of innate immune cells within mucosal tissues, which play a role in NCs clearance, inflammation, and antigen sampling. NIH/3T3 fibroblasts, although not directly involved in NCs uptake at the mucosal interface, reside in the underlying connective tissue and are crucial for evaluating potential subepithelial toxicity and inflammatory signalling [ 47 ]. These two models thus complement the mucopenetration studies by providing mechanistic understanding of how surface modifications affect cell–NCs interactions. The results showed in Fig. 6 A demonstrate that for THP1 cells, none of the NCs tested exhibited direct cytotoxic effects, with viability remaining at ~ 100%. This finding suggests CUR-NCs would not negatively impact circulating monocytes, immune cells that often form the first line of defence against pathogens. Similarly, CUR-NCs and HA surface modified CUR-NCs exhibited no cytotoxicity in NIH/3T3 fibroblasts (Fig. 6 B). However, CS-coated NCs without HA modification (CS-CUR-NCs) exhibited a slight increase in sensitivity, with a trend towards a dose-dependent cytotoxic response. This behaviour has been previously reported in similar studies, where HA surface modification was shown to improve bioavailability and biocompatibility [ 48 ]. To further investigate the interaction between NCs and relevant cell types, cellular uptake was assessed 24h post-treatment using flow cytometry (Fig. 6 C). In THP1 cells, uptake was uniformly high compared to the control for all NCs tested 77.7% for CUR-NCs, 74.4% for CS-CUR-NCs, and 72.8% for HA-CS-CUR-NCs, suggesting that these cells internalise NCs effectively regardless of surface modification. This behaviour is consistent with the high phagocytic capacity of THP1 cells [ 49 ] and confirms that surface engineering does not impair internalisation in professional phagocytes. In contrast, NIH/3T3 fibroblasts exhibited a surface modification-dependent uptake profile: internalisation increased by 16.2% for CUR-NCs, 17% for CS-CUR-NCs, and 29.7% for HA-CS-CUR-NCs relative to control. The enhanced uptake observed for HA-CS-CUR-NCs may be attributed to receptor-mediated endocytosis via CD44, which is overexpressed in many cell types and known to mediate HA binding and internalisation [ 50 , 51 ]. The non-cytotoxic nature of these NCs ensures that their internalization does not adversely affect the viability, supporting their suitability for biomedical applications. Finally, the present work advances the field by replacing weak adsorption with covalent grafting of hyaluronic acid onto a CS-coated CUR-NCs core. This “graft-to” strategy locks the HA chains in place, conferring long-lived steric repulsion that is resistant to dilution or competitive displacement. More importantly, HA provides a bioresponsive motif; owing to its reported affinity for CD44 receptors [ 52 ], it may facilitate receptor-mediated interactions with mucosal and epithelial cells, while its hydrophilicity and negative charge create a hydration shell that minimises muco-adhesion and facilitates deep diffusion[ 53 ]. Compared to conventional PEGylated or poloxamer-coated systems, the HA–CS conjugate offers dual functionality, improved colloidal stability and selective tissue engagement [ 54 ]. These effects, together with the observed increases in mucosal deposition and cellular uptake, underscore the importance of fine-tuned surface properties in enhancing drug delivery performance. Moreover, considering that macrophages can actively internalise NCs and impact mucopenetration, the HA-CS-CUR-NCs system appears to at least partially overcome this clearance mechanism. These insights pave the way for the translational advancement of HA-functionalised NCs as next-generation drug delivery systems, particularly in applications where mucosal, transdermal, or transmucosal penetration is required. 4. Conclusion This work underscores the potential of HA-functionalised CS-coated NCs as a versatile and rationally engineered platform for mucosal drug delivery. Beyond achieving covalent HA grafting and demonstrating improved ex vivo mucopenetration, the system embodies a strategic convergence of NCs-based drug solubilization with stable, biointeractive surface design. The modularity of this approach opens the door to its application with a wide range of poorly soluble drugs and therapeutic biomolecules, particularly in indications where mucosal targeting and barrier traversal are critical. Looking ahead, in vivo validation of pharmacokinetics, biodistribution, and therapeutic performance will be key to assessing clinical translatability. Altogether, the findings presented here offer a promising step toward next-generation nanomedicine platforms that not only overcome physicochemical formulation hurdles but also engage biological systems with precision and purpose. Declarations Consent for publication The authors consent for publication of this article. Availability of data and materials Data will be made available upon request. Declaration of competing interests The authors have no conflict of interest in this work. Funding This work was supported by the Engineering and Physical Sciences Research Council (grant EP/Y001486/1). Author’s contribution MGF : Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. OF: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data curation, Writing – review & editing, Visualization. AR : Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data curation. JAC: Resources, Supervision. AJP: Conceptualization, Resources, Writing – review & editing, Supervision, Project administration. Acknowledgements The authors are thankful to the Engineering and Physical Sciences Research Council for the financial support. Availability of data and materials Data will be made available upon request. References Müller RH, Gohla S, Keck CM. 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Mura S, Nicolas J, Couvreur P. Stimuli-responsive nanocarriers for drug delivery. Nat Mater. 2013;12:991–1003. https://doi.org/10.1038/nmat3776 . Supplementary Files floatimage1.png Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major Revisions Needed 01 Oct, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers invited by journal 14 Jul, 2025 Editor assigned by journal 14 Jul, 2025 First submitted to journal 03 Jul, 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. 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Coulter","email":"","orcid":"","institution":"Queen's University Belfast","correspondingAuthor":false,"prefix":"","firstName":"Jonnathan","middleName":"A.","lastName":"Coulter","suffix":""},{"id":485354404,"identity":"3d6613fb-b1e4-4386-8f54-bd208a2329ce","order_by":4,"name":"Alejandro J. Paredes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYPACZgYG9gYQCQJsxGrhOUCyFokEIrXwN/Ae/FxRYS0nP/PxNukCBjt5Bom0BLxaJA7wJUueOZNubHA7rUx6BkOyYYNE2gH81hzgMZBsbDucuEE6x0yah4E5gUEivQGvDvkDPMY/G/8drp8/8wxISz1hLQYHeMwkGxsOJzDc4AFpATIIOczwMF+aZcOxdMMNZ9KKrWcYHDds43mWgFeL3PHewzcbaqzl5dsPb7xdUFEtz8+eZoBXCwMzD8KRYERERKJoGQWjYBSMglGABQAAtC08XTME0lsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0414-8972","institution":"Queen's University Belfast","correspondingAuthor":true,"prefix":"","firstName":"Alejandro","middleName":"J.","lastName":"Paredes","suffix":""}],"badges":[],"createdAt":"2025-07-02 15:22:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7030744/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7030744/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87268694,"identity":"18dc5ac0-e7aa-4c02-9956-42874480165b","added_by":"auto","created_at":"2025-07-22 08:11:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":299806,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size (bars) and PDI (circles) measured by intensity distribution in DLS and zeta potential for stabiliser selection studies (\u003cstrong\u003eA, B\u003c/strong\u003e), chitosan optimised studies (\u003cstrong\u003eC, D\u003c/strong\u003e), stability studies (\u003cstrong\u003eE, F\u003c/strong\u003e) and functionalised NCs (\u003cstrong\u003eG, F\u003c/strong\u003e). Data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test; significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7030744/v1/f83709348b95ea793179d693.png"},{"id":87268689,"identity":"b6c24463-bca6-4ebd-9e24-d04fb6cc12ce","added_by":"auto","created_at":"2025-07-22 08:11:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":443578,"visible":true,"origin":"","legend":"\u003cp\u003eATR-FTIR (\u003cstrong\u003eA\u003c/strong\u003e) and \u003csup\u003e1\u003c/sup\u003eH-NMR (\u003cstrong\u003eB\u003c/strong\u003e) spectra obtained for CUR-NCs, CS-CUR-NCs\u003cstrong\u003e \u003c/strong\u003eand HA-CS-CUR-NCs. Characteristic peaks were highlighted.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7030744/v1/3f4464cd1745eb1712fa2980.png"},{"id":87272446,"identity":"036c892b-8394-4531-8f68-6c19a3f898e0","added_by":"auto","created_at":"2025-07-22 08:27:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":322256,"visible":true,"origin":"","legend":"\u003cp\u003eThermal analysis of CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs. Figure \u003cstrong\u003e(A)\u003c/strong\u003e, correspond to DSC and figure \u003cstrong\u003e(B) \u003c/strong\u003eto TGA show the analyses of the samples for the temperature range used. Characteristic peaks were highlighted.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7030744/v1/9db0a4e1a21e720d1676770f.png"},{"id":87268692,"identity":"59aaee93-d594-4848-8e70-042a55d79647","added_by":"auto","created_at":"2025-07-22 08:11:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":590986,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs at scale bar of 300 µm \u003cstrong\u003e(A)\u003c/strong\u003e and transmission electron microscopy \u003cstrong\u003e(B) \u003c/strong\u003eimages of CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7030744/v1/a4aed5446d36af928301fe89.png"},{"id":87270388,"identity":"0090f18a-dbaf-4de1-8e20-e98b08610750","added_by":"auto","created_at":"2025-07-22 08:19:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":178033,"visible":true,"origin":"","legend":"\u003cp\u003eRelease study (\u003cstrong\u003eA\u003c/strong\u003e) and mucosal deposition assay (\u003cstrong\u003eB\u003c/strong\u003e). Data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test; significance is indicated as follows: ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7030744/v1/b14d0a22d7533c4472966e95.png"},{"id":87270394,"identity":"16d8300e-7042-4fb2-9c19-d4e0a31b188b","added_by":"auto","created_at":"2025-07-22 08:19:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":321805,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effect on THP1\u003cstrong\u003e (A)\u003c/strong\u003e and NIH/3T3 \u003cstrong\u003e(B)\u003c/strong\u003e and quantification by flow cytometry of THP1 and NIH/3T3 \u003cstrong\u003e(C)\u003c/strong\u003e. Data are presented as mean ± SD from three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7030744/v1/b055d1c704692ee15f114a21.png"},{"id":87273991,"identity":"70b7cae2-699e-4ed5-a574-5cdf58162620","added_by":"auto","created_at":"2025-07-22 08:43:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3092684,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7030744/v1/e492a806-35be-41df-a2bc-a4539ab5b33b.pdf"},{"id":87270386,"identity":"7bed812f-3624-477a-939c-2aa051c8e06e","added_by":"auto","created_at":"2025-07-22 08:19:12","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":307489,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7030744/v1/ec155b5359f0cc9a7cfe6f9a.png"}],"financialInterests":"","formattedTitle":"Covalent Grafting of Hyaluronic Acid on Drug Nanocrystals - Application in Mucosal Delivery","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanocrystals (NCs) have emerged as a very promising drug delivery platform due to their near 100% drug loading capacity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. NCs are crystalline particles typically ranging in size from 10\u0026ndash;500 nm, minimally stabilized by surfactants or polymers. Compared to conventional nanoparticles, which often require significant amounts of excipients or carriers, NCs offer improved dissolution rates and higher saturation solubility, making them particularly attractive for high-dose drug delivery with improved pharmacokinetics [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A major advantage of NCs lies in their surface accessibility, which allows for tailored functionalisation strategies. Among these, covalent grafting of functional groups on the NC surface stand out as the preferred method to facilitate targeting, muco-penetration [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and controlled release [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Additionally, covalent grafting offers superior stability compared to physical adsorption or electrostatic interactions, ensuring durable surface modification under physiological conditions, a crucial aspect for clinical translation[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. One of the most pressing challenges in drug delivery is overcoming mucosal barriers such as those found in the gastrointestinal, pulmonary, and nasal systems. The dense and viscous nature of mucus restricts the penetration and retention of therapeutic agents [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Efficient mucopenetration is critical for achieving localized drug delivery and enhancing systemic absorption. NCs, owing to their small size and high diffusion rates, exhibit superior muco-penetrative capabilities compared to larger drug carriers [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, this activity can be further enhanced by modifying their surface chemistry [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this context, covalent grafting with hydrophilic and biocompatible polymers such us hyaluronic acid (HA) offer a promising route to enhance mucus diffusion. HA is a naturally occurring glycosaminoglycan found in the extracellular matrix, characterized by its hydrophilicity, biodegradability, and excellent biocompatibility [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The covalent attachment of HA to NC surfaces can facilitate targeted delivery, while minimizing off-target effects and premature clearance [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, the hydrophilic nature of HA reduces particle aggregation, which facilitates uniform and efficient diffusion through dense mucus layers. This is especially beneficial in nanopharmaceuticals, where uniform distribution and controlled drug delivery are critical for improving therapeutic outcomes and reducing side effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo exemplify the potential of this approach, curcumin (CUR) was selected as a model compound. CUR is a natural polyphenolic compound extracted from the rhizome of \u003cem\u003eCurcuma longa\u003c/em\u003e, has attracted attention in biomedical research due to its broad spectrum of pharmacological activities [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. CUR exhibits potent anti-inflammatory, antioxidant, antimicrobial, and anticancer properties, making it a promising candidate for the treatment of various chronic diseases, including cancer, arthritis, and neurodegenerative disorders [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Despite these benefits, clinical use of CUR remains low due to its poor water solubility (approximately 11 ng/mL in aqueous solutions), low chemical stability, and rapid systemic elimination. These challenging characteristics ultimately lead to suboptimal bioavailability and limited therapeutic efficacy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. NCs formulations has been proposed as an effective strategy to improve the solubility and pharmacokinetic profile of CUR [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Several studies have already shown that HA-modified nanoparticles can significantly enhance drug uptake and therapeutic efficacy. For instance, HA-based systems, such as solid lipid nanoparticles and polymeric nanogels, offer superior drug encapsulation, enhanced stability, and targeted release capabilities [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These advantages are particularly beneficial for treating diseases that require localized drug delivery across barriers, like ocular conditions or mucus-rich respiratory pathways [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]​. In addition, the hydrophilic HA coating reduces particle adhesion, enhancing mobility and preventing rapid clearance. Such properties make HA-modified nanoparticles promising for delivering drugs to hard-to-reach tissues, like tumors or inflamed sites, while minimizing systemic side effects. Improved mucoadhesion is also achieved through the natural affinity of HA for mucus glycoproteins, facilitating prolonged retention of polymeric nanoparticles on mucosal surfaces and enhancing drug absorption [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Several studies have demonstrated the benefits of HA in enhancing drug delivery systems by improving stability, bioavailability, and targeted therapeutic efficacy. Ji \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] developed HA-coated CUR-NCs (HA-CUR-NCs) for breast-cancer therapy. The HA coating improved the bioavailability of CUR, prolonging circulation time and enhanced uptake in CD44 receptor-overexpressing cancer cells. \u003cem\u003eIn vivo\u003c/em\u003e, NCs exhibited superior anticancer effects with reduced side effects, demonstrating their potential as a targeted breast cancer treatment. Zerrillo \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] designed a HA-decorated poly(lactide-co-glycolide) (PLGA) nanoparticles for osteoarthritis therapy. The HA modification improved binding to CD44 receptors on chondrocytes and enhanced cartilage tissue targeting, while conferring no direct cytotoxicity. Non-invasive imaging confirmed efficient nanoparticle accumulation, highlighting their suitability for targeted osteoarthritis therapies. Seo \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] created a multilayered doxorubicin (DOX) and HA cellulose NCs (CNCs) for CD44-targeted delivery. This formulation improved drug penetration, ROS production, and cancer cell death in lung adenocarcinoma models. \u003cem\u003eIn vivo\u003c/em\u003e imaging confirmed enhanced tumor accumulation, demonstrating the potential for targeted chemotherapy.\u003c/p\u003e\u003cp\u003eGiven the superior drug loading capability of NCs and the benefits of stable surface functionalisation, this study focuses on the covalent grafting of HA onto CUR-NCs. The aim is to systematically synthesize, characterize, and optimize HA-grafted CUR-NCs, evaluating their impact on mucopenetration, bioavailability, and therapeutic potential. This work provides a foundation for the development of NCs-based drug delivery systems designed to leverage the synergistic advantages of high drug loading, covalent surface engineering, and targeted mucosal delivery.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eAll chemicals and materials used for this study were of analytical grade. Curcumin (CUR- CAS RN 458-37-7) was purchased from Tokyo Chemical Industries (London, UK), Poloxamer 188 (POL 188) from BASF chemical company (Ludwigshafen, Germany) and Poloxamer (POL 407). Chemicals such as chitosan (CS- LMW \u0026ndash; 50-190KDa, Deacetylated chitin, Poly(D-glucosamine) and N-Hydroxysuccinimide (NHS- CAS 6066-82-6) were purchased from Sigma-Aldrich (Poole, Dorset, UK). 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC- CAS 25952-53-8) was obtained from EMD Millipore Corp (USA), sodium hyaluronate from Kewpie Corporation (Shibuya-Ku, Tokyo). For the milling process, YTZP Yttria-stabilized zirconia beads of 0.15 and 0.5 mm were purchased from Chemco (Guangfu, China). Parafilm M\u0026reg; was purchased from Bemis Company Inc. (Neenah, USA). Ultrapure water was obtained from a water purification system, Elga Purelab DV 25, Veolia Water Systems (Ireland). All other chemicals used were of analytical grade.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Methods\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Optimisation of CUR-NCs\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eEffect of surfactant type\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eCUR-NCs were prepared by slightly modifying an already published media milling technique [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] wherein, 100 mg of CUR was sandwiched between 4.5 mL of zirconia beads and magnetic bars (25 x 8 mm) in a 10 mL vial. 5 mL of varying strengths (0.5%, 1%, 1.5% and 2% w/v) of P188 and P407 solutions were added. The vials were then completely covered using tin foil and agitated at 1200 rpm for 24 hours. The resultant CUR-NCs were filtered from the milling media using a 300-mesh sieve. 20 \u0026micro;L of the resultant filtrate was diluted with 3 mL HPLC water in a cuvette and analysed for PS, PDI and ZP.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eEffect of chitosan concentration\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eA similar media milling technique was employed to prepare CS-CUR-NCs. Briefly, 100 mg of CUR, 4.5 mL of zirconia beads (0.1\u0026ndash;0.2 mm), and two magnetic bars (25 \u0026times; 8 mm) were measured and added to the milling system. Varying amounts of POL 188/POL 407 and CS 0.5% w/v solution were added, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The milling process was carried out at 1200 rpm for 24 hours. Afterward, NCs were filtered and characterized as previously described.\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\u003eOptimization parameters for CS-CUR-NCs: poloxamer and chitosan volumes used.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePOL 188/POL 407 (% w/v)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFinal volume POL 188/POL 407 (mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFinal volume of 0.5% CS (mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Functionalisation of CS-CUR-NCs with hyaluronic acid\u003c/h2\u003e\u003cp\u003eTo functionalize CS-CUR-NCs, HA was activated using a solution of EDC and NHS under stirring at 300 rpm for 5 minutes, then agitated for 60 minutes to form the HA-EDC-NHS intermediate. The molar ratios of both EDC:HA and NHS:HA were approximately 66:1, ensuring efficient activation of the HA carboxyl groups. Activated HA was then added dropwise to a suspension of CS-CUR-NCs and stirred at 300 rpm for 16 hours at room temperature. The resulting NCs were collected by centrifugation at 342 g for 15 minutes, before freeze-drying for 25 hours. The final freeze-dried HA-CS-CUR-NCs pellet was analysed using various physicochemical characterization techniques\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. Particle size, polydispersity index and zeta potential\u003c/h2\u003e\u003cp\u003eA NanoBrook Omni Dynamic Light Scattering (DLS) analyser (Brookhaven Instruments Corp., Holtsville, NY, USA) was used to determine particle size, polydispersity index (PDI), and zeta potential of the samples. For the measurements, 20 \u0026micro;L of the prepared NCs were diluted with 3 mL of distilled water in measurement cuvettes. All samples were measured in triplicate, and the results are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD, n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4. Characterization\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003ePhysicochemical characterization\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eFor the physicochemical characterization samples of CUR-NCs, CS-CUR-NCs, HA-CS-CUR-NCs and PM were analysed using a PerkinElmer Spectrum Two FTIR spectrometer (PerkinElmer Inc., USA). Wavelengths ranging from 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were subjected to analysis, averaging 64 scans per spectrum. All major bands obtained were recorded using OMNIC\u0026trade; Spectra Software (Thermo Fisher Scientific Inc., Waltham, USA). Thermal characterization, including differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), was carried out using a Q100 DSC (TA Instruments, Bellingham, WA). Scans were conducted from 20\u0026deg;C to 350\u0026deg;C with a heating rate of 10\u0026deg;C/min under a constant flow of nitrogen (10 mL/min). TGA experiments were carried out between 25\u0026ordm;C and 350\u0026ordm;C with a heating rate of 10 \u0026ordm;C/min and a nitrogen flow of 10 mL/min, using a Q500 instrument (TA Instruments, New Castle, DE, USA). RM5 Confocal Raman Microscope (Edinburgh Instruments Ltd, Livingston, UK) was used to analyse the Raman spectra of the obtained NCs. Approximately 2 mg of sample were taken on a glass slide. The grating was set at 300 gr/mm, the laser power at 50% and laser wavelength at 785 nm. The spectrum was analysed within the range 0\u0026ndash;3200 Raman shift/ cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 5 accumulations each with exposure time 1.5 s. Signals were recorded and analysed via the Ramacle\u0026reg; Software (Edinburgh Instruments Ltd, Livingston, UK). Finally, the assessment of ligand attachment was evaluated using proton nuclear magnetic resonance (NMR) \u003csup\u003e1\u003c/sup\u003eH NMR. Approximately 8 mg of the FD samples were dissolved in 400 \u0026micro;L of DMSO (Dimethyl sulfoxide-d6), then transferred to 7\u0026rdquo; NMR sample tubes (Wilmad Economy, NJ, USA). The Bruker Ultrashield 400 spectrometer (Bruker, Leipzig, Germany) was operated at 400 MHz to record the NMR spectra. MestReNova 6.0.2\u0026copy; (Mestrelab Research, Santiago de Compostela, Spain) was used to process the spectra obtained. The observed chemical shifts relative to tetramethylsilane were reported in ppm (\u003cem\u003eδ\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eMicroscopy and imaging techniques\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eAdvanced microscopy imaging techniques were employed to characterise CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs. NCs were first desiccated to dryness, then deposited onto adhesive carbon tape, and examined using a TM3030 microscope (Hitachi, Krefeld, Germany). A Tabletop TM 3030 scanning electron microscope (SEM) (Hitachi, Tokyo, Japan) was utilised to reveal the topological features of NCs, providing detailed surface morphology data.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5. HPLC quantification\u003c/h2\u003e\u003cp\u003eThe detection and quantification of CUR were performed by using HPLC-UV following the protocol reported before [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A ZORBAX Eclipse XDB-C18 column (50 \u0026times; 4.6 mm internal diameter; 1.8 \u0026micro;m particle size) was selected as the stationary phase. The mobile phase was composed of 80:20% (v/v) ACN and water (0.1% phosphoric acid). The maxima absorption (λ max) was fixed at 425 nm. The injection volume was set as 20 \u0026micro;L, and the flow rate was 0.5 mL/min. The linearity of the method was explored between 0.1 and 50 \u0026micro;g/mL (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9999). The limits of detection and quantification were 0.27 and 0.81 \u0026micro;g/mL, respectively.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eDetermination of CUR content in the formulated nanosuspensions\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eTo determine the CUR content in the obtained formulations, 20 \u0026micro;L of the resulting nanosuspension was dissolved in 980 \u0026micro;L of acetonitrile (ACN). The resulting solution was centrifuged at 16,602 g for 15 minutes and subsequently filtered using a 0.22 \u0026micro;m filter (Millipore, USA) for subsequent quantification by high-performance liquid chromatography (HPLC).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6. \u003cem\u003eIn vitro\u003c/em\u003e release study\u003c/h2\u003e\u003cp\u003eA dialysis membrane technique was used to evaluate \u003cem\u003ein vitro\u003c/em\u003e release profiles comparing pure CUR drug, a physical mixture (PM) of each nanoformulation, and the NCs. The release medium was a phosphate buffer at pH 6.4 (simulating saliva), supplemented with 2% w/v Tween 80 and 0.5% ascorbic acid to prevent degradation of the drug[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. After confirming sink conditions, samples containing approximately 3 mg of CUR were added to preactivated (PBS for 1 h) dialysis membrane bags (molecular weight cut-off: 12,000\u0026ndash;14,000 Da; Spectra-Por, Spectrum Medical Industries, Los Angeles, CA, USA). The bags were sealed with plastic clips and incubated in an ISF 7100 orbital shaker (Jeio Tech, MA, USA) at 37\u0026deg;C and 100 rpm. At predetermined time intervals (0 h, 1 h, 3 h, 6 h, 9 h, 1 day, 2 days, 3 days, 7 days, 14 days and 28 days), 1 mL of release media was withdrawn and replaced with fresh buffer. CUR content was quantified using HPLC (section 2.2.5). All experiments were conducted in triplicate, with results expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7. Cell culture experiments\u003c/h2\u003e\u003cp\u003eTHP1 human monocytic and NIH/3T3 cell line fibroblast cell line from a mouse were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and stored according to the supplier\u0026rsquo;s guidelines. THP1 cells were kept in Roswell Park Memorial Institute medium (RPMI) and NIH/3T3 in Dulbecco\u0026rsquo;s modified Eagle medium (without phenol red) supplemented with 10% (v/v) heat inactivated foetal bovine serum. Cells were maintained in T75 culture flasks, in a humidified environment with 5% CO\u003csub\u003e2\u003c/sub\u003e/95% air at 37\u0026deg;C. When reaching 80% confluences, cells were detached using 0.25% trypsin-EDTA, then either seeded for experiment or passaged to maintain stocks.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eViability assay\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThe cytotoxic effects of CUR-NCs, CS-CUR-NCs, and HA-CS-CUR-NCs on NIH/3T3 cells were tested using Alamar Blue\u0026reg; assay (Thermo Fisher Scientific, Waltham, MA, USA). Cells were seeded in 96-well plates at a concentration of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well. Twenty- four hours post seeding cells were treated with fresh medium (negative control) or various concentrations of NCs containing media for 24 h. Post treatment, excess NCs was replaced with complete media containing 10% Alamar blue solution for 4 h. Fluorescence levels, positivity correlating with cell viable cell number was measured using a FLUOstar Omega (BMG LABTECHGmbH, Freiburg, Germany) microplate spectrophotometer at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Data points were collected over at least three independent experiments.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eNCs cellular uptake by flow cytometry\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were seeded into individual wells on a 6-well plate, then incubated at 37\u0026deg;C for 24 h to allow attachment. The culture medium was replaced by a fresh medium containing NCs at a concentration equivalent to 120 \u0026micro;g/mL of CUR for 24h. Following incubation, NCs were removed by washing in PBS (x3), cells detached using 0.25% trypsin-EDTA, then fixed in 4% paraformaldehyde for 20 min. Cellular uptake was assessed by quantifying the associated fluorescence using a Becton\u0026thinsp;\u0026minus;\u0026thinsp;Dickinson FACSCalibur flow cytometer (New Jersey, United States). Cell-associated fluorescence was quantified using a Becton\u0026thinsp;\u0026minus;\u0026thinsp;Dickinson FACSCalibur flow cytometer (New Jersey, United States). The intrinsic fluorescence of CUR was used to monitor cellular uptake [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Untreated cells were used as controls to determine background autofluorescence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.2.8. \u003cem\u003eEx vivo\u003c/em\u003e mucosal deposition\u003c/h2\u003e\u003cp\u003eAs detailed below, the \u003cem\u003eex vivo\u003c/em\u003e mucosal deposition studies of NCs were performed in excised buccal neonatal porcine mucosa. The mucosa was cleaned with pH 7.4 buffer and gently cut with a sterile scalpel. Then, following a previously published methodology, each mucosa section was placed in a weigh boat containing tissue paper soaked in buffer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. 3D printed resin rings with an inner diameter of 6 mm and a height of 5 mm were attached to the mucosa with glue using manual force for 30 s. Inside the ring, 30 \u0026micro;L of CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs were added. The system was then sealed with Parafilm M\u0026reg; to prevent evaporation, then placed in an incubator at 37\u0026ordm;C. After 24 h, the rings were removed, the mucosa was rinsed with 1 mL pH 7.4 mucus buffer, and the excess formulation was wiped with a clean tissue paper wetted with mucus buffer. Subsequently, a 6 mm diameter punch biopsy was used to obtain a circular section of mucosa from which drug was extracted. This experiment was carried out in triplicate. Each circular section of mucosa was placed in a 2-mL Eppendorf tube with two stainless steel beads (0.5 cm diameter, Qiagen, Hilden, Germany) and 500 \u0026micro;L of purified water. Tubes were then placed in a TissueLyser\u0026reg; LT (Qiagen, Hilden, Germany) and processed for 10 min at 50 Hz to hydrate the mucosa. Next, 1 mL of acetonitrile was added to each tube, and the process was repeated for another 10 min at 50 Hz to extract the drug. The homogenized tissue was then centrifuged at 14,462 g for 10 min (Sigma microtube centrifuge SciQuip Ltd, Shropshire, UK), and the CUR content in the supernatant quantified by HPLC using the method detailed in Section 2.14. Skin without treatment was using as a control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.2.9. Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using GraphPad Prism\u0026copy; software (version 8.0, GraphPad Software Inc., San Diego, California, USA). One-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test was performed to compare particle size and surface charge among CUR, CUR-CS, and CUR-CS-HA NCs formulations.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Optimisation of NCs\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1. Stabilizer selection study\u003c/h2\u003e\u003cp\u003eThe concentration and choice of stabilizer used to synthesize CUR-NCs by media milling were compared using particle size, polydispersity index (PDI) and zeta potential. Both POL 188 and POL 407, were tested at four concentrations (0.5% w/v, 1% w/v, 1.5% w/v and 2% w/v). The results obtained regarding particle size showed no significant differences across any of the studied POL concentrations, nor when comparing the two types of poloxamer used (POL 188 and POL 407). For example, at a concentration of 1.5% w/v, both POL 188 and POL 407 displayed comparable particle sizes (90.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 nm and 92.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 nm, respectively) and similar PDI values (0.264 and 0.249, respectively). In contrast, at the highest concentration (2% w/v), particle sizes (85.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01 nm for POL 188 and 95.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 nm for POL 407) and zeta potential values (0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78 mV for POL 188 and \u0026minus;\u0026thinsp;0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 mV for POL 407) were not altered, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. In terms of zeta potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), a distinct trend was observed; as the concentration of POL increased, the absolute value of the zeta potential becoming less negative charged regardless of the type of POL used. This indicates that concentration, rather than the specific type of POL influenced the reduction in zeta potential. For instance, the zeta potential shifted from \u0026minus;\u0026thinsp;10.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28 mV (0.5% POL 188) and \u0026minus;\u0026thinsp;7.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84 mV (0.5% POL 407) to near-neutral values at the 2% concentration (0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78 mV and \u0026minus;\u0026thinsp;0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 mV, respectively). Such a substantial reduction in zeta potential values, approaching neutrality, suggests particle instability and the potential for aggregation. This aligns with literature findings, which report that nanoparticles with zeta potential values near zero are prone to agglomeration due to insufficient electrostatic repulsion [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. As the zeta potential moves closer to neutral, electrostatic stabilisation diminishes, leading to destabilisation of the particle suspension. Considering this, particles synthesised with the highest concentration of POL were deemed unsuitable for further investigation. Despite a relatively stable PDI (0.227 for POL 188 and 0.208 POL 407), the observed particle size and significantly reduced zeta potential render these formulations unfit as stabilisers.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2. Chitosan coated CUR-NCs\u003c/h2\u003e\u003cp\u003eBased on the results obtained from the stabiliser selection study (3.1.1), three concentrations (0.5%, 1% and 1.5% w/v) of both POL 188 and POL 407 were chosen to prepare six different CUR-NCs with 0.5% w/v CS solution. From Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC,D, it can be observed that the particle size of the NCs decreases significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with the addition of CS as the percentage of POL increases. For instance, in the case of POL 188, the particle size reduces from 117.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27 nm to 79.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 nm. A similar trend is observed for POL 407, showing a decrease from 193.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72 nm at lower POL concentrations to an optimal size of 74.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 nm at 1.5% w/v. While both formulations demonstrated promising results for further experimentation, POL 407 was ultimately selected due to its superior final characteristics, including a lower PDI of 0.261 and a positive zeta potential of 9.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93 mV.\u003c/p\u003e\u003cdiv id=\"Sec18\" class=\"Section4\"\u003e\u003ch2\u003e3.1.2.1. Stability study data for CS-CUR-NCs\u003c/h2\u003e\u003cp\u003eStability studies for the optimised CS-CUR-NCs were conducted at 4\u0026deg;C over a 28-day period. The particle size results remained consistently below 80 nm across the full study period. Similarly, PDI values (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE dots) remained below 0.3, indicating a stable size distribution. Zeta potential measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) displayed minor fluctuations over time but consistently remained positive.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Functionalised NCs with hyaluronic acid (HA-CS-CUR-NCs).\u003c/h2\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1. Dynamic light scattering (DLS)\u003c/h2\u003e\u003cp\u003eThe data obtained from the particle size and zeta potential analysis study are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, F. Comparing across samples it was evident that CS was successfully conjugated forming the CS-CUR-NCs sample, as the zeta potential following CS conjugation switched from negative (-4.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74 mV) to positive (10.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93 mV). This shift is consistent with the presence of numerous amino groups along the CS backbone, which confer a positive surface charge and facilitate subsequent derivatization. A slight decrease in the particle size from 92.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 nm to 75.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 nm was also observed. These changes in NCs physicochemical properties following surface conjugation are attributed to electrostatic interactions imparted by CS, increasing repulsive forces[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Importantly, PDI values also remained consistently below 0.3 for the three samples.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2. ATR-FTIR spectroscopy\u003c/h2\u003e\u003cp\u003eATR-FTIR spectroscopy was performed to confirm the successful coating of HA onto the surface of CS-CUR-NCs. The spectra obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed key characteristic bands for each analysed sample. A broad band between 3000 cm⁻\u0026sup1; and 3500 cm⁻\u0026sup1; indicated the presence of the COOH group, suggesting the successful incorporation of HA. Additionally, distinct peaks were identified for CS (amine stretching at 3355 cm⁻\u0026sup1;, amide I stretch at 1652 cm⁻\u0026sup1;), POL 407 (polyethylene oxide band at 1100 cm⁻\u0026sup1;, polypropylene stretch at 2897 cm⁻\u0026sup1;), and CUR (OH stretch at 3525 cm⁻\u0026sup1;, C\u0026thinsp;=\u0026thinsp;C at 1603 cm⁻\u0026sup1;, C\u0026thinsp;=\u0026thinsp;O at 1650 cm⁻\u0026sup1;). A comparison between the before purified sample and the purified sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) highlighted the presence of an amide band at 1715 cm⁻\u0026sup1;, indicating a successful EDC-NHS coupling reaction. This band is associated with the formation of a urea byproduct, which serves as further confirmation of the functionalization process. After purification, all major characteristic peaks were still present; however, the intensity of the amide band at 1715 cm⁻\u0026sup1; was notably reduced, suggesting that a significant portion of the urea byproduct had been removed [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. While theoretically, a strong amide band (between 1650 cm⁻\u0026sup1; and 1750 cm⁻\u0026sup1;) would still be expected even after purification, the presence of a small shoulder band at 1715 cm⁻\u0026sup1; suggests that HA remains attached to the CS-CUR-NCs. The observed reduction in absorbance intensity may be attributed to factors affecting the efficiency of the EDC-NHS coupling reaction, including variations in pH, lower reagent concentrations, or temperature fluctuations during synthesis. These parameters could influence both the reaction yield and the intensity of the bands observed in the spectra[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Taken together, these results suggest the successful formation of an amide bond, indicating that covalent coupling successfully occurred. Nevertheless, further characterization techniques, including \u003csup\u003e1\u003c/sup\u003eH-NMR analysis, were used to confirm the structural modification, ensuring the reliability of the findings.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3. \u003csup\u003e1\u003c/sup\u003eH-NMR analysis\u003c/h2\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-NMR analysis was conducted to complement the DLS and FTIR data, looking to gain deeper understanding on the molecular interactions and structural integrity of the NCs after surface modification. Three samples CUR-NCs, CS-CUR-NCs, and HA-CS-CUR-NCs were analysed, revealing various proton chemical shifts, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. A strong peak at 2.5 ppm was observed in all spectra, corresponding to DMSO used in sample preparation. In CS-CUR-NCs spectrum, an upfield peak between 0.5 and 2.5 ppm was detected, suggesting the presence of an R-NH₂ functional group from CS [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, this peak was absent in the spectrum of HA-CS-CUR-NCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating that the amine groups had reacted, confirming the functionalization of CS with HA. This conclusion was further supported by the appearance of two distinct peaks between 9.0 and 10.0 ppm in the HA-CS-CUR-NCs spectrum, which, based on literature, correspond to R-COOH and R-CO-NHR groups [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These shifts provide strong evidence of the successful attachment of HA onto CS-CUR-NCs via the EDC-NHS coupling reaction. Additionally, all three samples exhibited peaks between 6.0 and 8.0 ppm, corresponding to the aromatic protons of CUR [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], indicating that the core nanocrystal structure remained intact throughout the functionalization process. The overall spectral changes confirm the successful conjugation of HA onto CS-CUR-NCs and validate the efficiency of the EDC-NHS coupling chemistry.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4. Thermal analysis\u003c/h2\u003e\u003cp\u003eDSC thermograms were recorded for CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) displayed fusion peaks for POL 407 (~\u0026thinsp;50\u0026deg;C) and CUR (~\u0026thinsp;180\u0026deg;C) across all samples, confirming their presence. In CS-CUR-NCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), a broad peak around 101\u0026deg;C was observed, indicative of loss of water [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The thermogram for HA-CS-CUR-NCs showed two additional peaks, one endothermic and one exothermic, alongside the fusion peaks common to all samples. The broad endothermic peak at ~\u0026thinsp;97\u0026deg;C and the exothermic peak at ~\u0026thinsp;250\u0026deg;C suggest the presence of HA. However, since CS also exhibits an endothermic peak near 100\u0026deg;C, the possibility of peak overlap between CS and HA was considered. This risk is mitigated by the presence of an exothermic peak at ~\u0026thinsp;250\u0026deg;C, a feature unique to HA, further supporting successful incorporation.\u003c/p\u003e\u003cp\u003eThermogravimetric Analysis (TGA) of CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs were analysed to assess their thermal behaviour. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, the thermograms of CUR and CS-NCs formulations exhibited a relatively stable mass up to approximately 230\u0026deg;C, after which an initial mass loss of approximately 8% was observed, indicating the onset of decomposition. This behaviour is typical for the individual components, with the weight loss likely attributed to the degradation of CUR and CS [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In contrast, the HA-CS-CUR-NCs formulation showed a more pronounced and earlier onset of mass loss, beginning around 190\u0026deg;C. This suggests that HA in the formulation undergoes decomposition at a lower temperature compared to CS and CUR, which is consistent with the known thermal behaviour of HA [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The faster degradation observed for HA-CS-CUR-NCs could be due to the specific characteristics of HA, which can be more thermally labile than CS. Despite this, all formulations remained thermally stable up to their respective temperatures, indicating that the NCs formation process via media milling did not affect the thermal stability of the active ingredients. These results suggest that the incorporation of HA does not significantly compromise the overall thermal stability of the formulation but may influence its decomposition dynamics.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e3.2.5. Microscopy images\u003c/h2\u003e\u003cp\u003eSEM and TEM were used to characterise the morphology of CUR-NCs, CS-CUR-NCs and HA-CS-CUR-NCs. SEM analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) of CUR-NCs revealed spherical structures, likely due to the use of POL as a surfactant [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In the case of CS-CUR-NCs, SEM images exhibited platelet-like formations, potentially attributable to CS deposition, despite the continued presence of POL as a stabiliser. This is consistent with prior studies reporting morphological alterations in nanoparticles following CS coating, where polymeric layers can influence surface roughness and structure [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Upon chemical modification with HA, the observed platelets appeared larger, which could be explained by the centrifugation step necessary for the conjugation reaction. This process may lead to the partial removal of POL, thereby exposing or restructuring the CS. However, it is important to note that these visible structures do not directly represent the NCs themselves. During sample drying, aggregation of the stabiliser occurs, forming a matrix or in which the NCs are embedded. Due to the limited resolution of SEM, typically in the micrometre scale, individual NCs are extremely difficult to visualise within this matrix. For this reason, TEM was employed to gain more detailed insight into the nanostructures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). CUR-NCs were observed as spherical. In CS-CUR-NCs samples, phase contrast TEM revealed a distinguishable outer layer, suggestive of CS adhesion via electrostatic interactions. Finally, the TEM analysis of HA-CS-CUR-NCs demonstrated the presence of a well-defined additional layer surrounding the NCs, indicative of successful conjugation with HA. These observations confirm that the chemical modification was effective and that TEM is a valuable tool for visualising the added HA layer, providing direct evidence of the surface functionalisation achieved in this study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e3.2.6. In vitro release profiles and mucosal deposition\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e release profiles of CUR, physical mixtures (PMs), and NCs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. As expected, pure CUR exhibited a slow dissolution rate, with only 2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74% of the drug released by the end of the study. The physical mixtures showed no significant improvement over the pure drug, with CUR PM, CS PM, and HA PM reaching 2.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45%, 1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07%, and 2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66% drug release, respectively. These results indicate that the presence of the compounds alone did not significantly enhance the dissolution rate of CUR. In contrast, the CUR-NCs demonstrated a markedly improved dissolution profile, with approximately 55.11\u0026thinsp;\u0026plusmn;\u0026thinsp;5.20% of the drug released after 28 days. Similarly, CS-CUR-NCs reached 57.89\u0026thinsp;\u0026plusmn;\u0026thinsp;5.82%, while HA-CS-CUR-NCs achieved 48.35\u0026thinsp;\u0026plusmn;\u0026thinsp;6.02% release. These findings highlight two key points; first, that the PMs serve as appropriate controls, confirming that the observed enhancement in drug release is attributable to the NCs formulation rather than to the individual components alone; and second, that the nanoformulations are both effective and efficient in increasing the dissolution rate. The increased dissolution rate and saturation solubility observed with NCs can be attributed an enlarged surface area, which plays a critical role in dissolution kinetics, described by the Noyes\u0026ndash;Whitney equation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Moreover, the sustained drug release profile is a promising characteristic of these systems. It is hypothesized that, despite natural clearance mechanisms at the mucosal level, NCs may remain accumulated in the mucosa, contributing to prolonged drug availability.\u003c/p\u003e\u003cdiv id=\"Sec26\" class=\"Section4\"\u003e\u003ch2\u003e3.2.6.1. Ex vivo mucosal deposition\u003c/h2\u003e\u003cp\u003eThe results of the mucosal deposition assay on neonatal porcine mucosa were obtained as follows. Patches were cut into 5 mm diameter discs, with the drug content measured for each formulation. The drug content for CUR-NCs was 18.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 mg/mL, for CS-CUR-NCs it was 18.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 mg/mL, and for HA-CS-CUR-NCs it was 2.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 mg/mL. After 24 hours of contact between the mucosa and the nanosuspension, the drug deposition was quantified. The results showed that 1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1% of the drug from CUR-NCs was deposited in the mucosa, while 23.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5% of the drug from CS-CUR-NCs and 37.3\u0026thinsp;\u0026plusmn;\u0026thinsp;26% from HA-CS-CUR-NCs were deposited, with the latter showing a significantly higher deposition compared to the CUR-NCs formulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The poor penetration of unmodified CUR-NCs may result from their limited ability to interact with mucus components or from rapid clearance by the mucus layer. Moreover, in immune-cell-rich environments such as mucosal tissues, it is plausible that a fraction of nanoparticles may be internalised by resident macrophages, thereby reducing the number of particles reaching deeper tissue layers. This hypothesis is supported by previous studies demonstrating that macrophages contribute significantly to nanoparticle sequestration and clearance within mucosal barriers [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In contrast, the enhanced deposition observed with the CS-CUR-NCs and HA-CS-CUR-NCs formulations can be attributed to the incorporation of CS and HA, which are known to improve the permeation and adhesion of nanoparticles to mucosal surfaces. CS, a natural polymer with a positive charge, enhances the interaction with the negatively charged cell membranes, facilitating the penetration of drug-loaded nanoparticles through the mucosa [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Additionally, HA, which exhibits mucoadhesive properties, can increase the residence time of NCs at the mucosal surface, promoting higher drug absorption. These findings are consistent with the literature on nanoparticle-based drug delivery systems, where smaller particle sizes enhanced mucoadhesion lead to improved drug permeation across biological membranes [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Therefore, the improved deposition achieved with HA-CS-CUR-NCs reflects not only enhanced mucoadhesion, but also an ability to circumvent immune clearance mechanisms to a certain extent, highlighting their potential for mucosal drug delivery applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e3.2.7. In vitro cell culture assays\u003c/h2\u003e\u003cp\u003eCell viability was evaluated using the Alamar Blue assay in THP1 monocytes and NIH/3T3 fibroblasts, testing CUR-NCs, CS-CUR-NCs, and HA-CS-CUR-NCs. While these \u003cem\u003ein vitro\u003c/em\u003e models do not represent the mucosal epithelium directly, they provide valuable insights into cytocompatibility, immune cell interaction, and general NCs behaviour at the cellular level. THP1 monocytes are relevant due to the presence of innate immune cells within mucosal tissues, which play a role in NCs clearance, inflammation, and antigen sampling. NIH/3T3 fibroblasts, although not directly involved in NCs uptake at the mucosal interface, reside in the underlying connective tissue and are crucial for evaluating potential subepithelial toxicity and inflammatory signalling [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. These two models thus complement the mucopenetration studies by providing mechanistic understanding of how surface modifications affect cell\u0026ndash;NCs interactions. The results showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA demonstrate that for THP1 cells, none of the NCs tested exhibited direct cytotoxic effects, with viability remaining at ~\u0026thinsp;100%. This finding suggests CUR-NCs would not negatively impact circulating monocytes, immune cells that often form the first line of defence against pathogens. Similarly, CUR-NCs and HA surface modified CUR-NCs exhibited no cytotoxicity in NIH/3T3 fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). However, CS-coated NCs without HA modification (CS-CUR-NCs) exhibited a slight increase in sensitivity, with a trend towards a dose-dependent cytotoxic response. This behaviour has been previously reported in similar studies, where HA surface modification was shown to improve bioavailability and biocompatibility [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. To further investigate the interaction between NCs and relevant cell types, cellular uptake was assessed 24h post-treatment using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In THP1 cells, uptake was uniformly high compared to the control for all NCs tested 77.7% for CUR-NCs, 74.4% for CS-CUR-NCs, and 72.8% for HA-CS-CUR-NCs, suggesting that these cells internalise NCs effectively regardless of surface modification. This behaviour is consistent with the high phagocytic capacity of THP1 cells [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and confirms that surface engineering does not impair internalisation in professional phagocytes. In contrast, NIH/3T3 fibroblasts exhibited a surface modification-dependent uptake profile: internalisation increased by 16.2% for CUR-NCs, 17% for CS-CUR-NCs, and 29.7% for HA-CS-CUR-NCs relative to control. The enhanced uptake observed for HA-CS-CUR-NCs may be attributed to receptor-mediated endocytosis via CD44, which is overexpressed in many cell types and known to mediate HA binding and internalisation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The non-cytotoxic nature of these NCs ensures that their internalization does not adversely affect the viability, supporting their suitability for biomedical applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFinally, the present work advances the field by replacing weak adsorption with covalent grafting of hyaluronic acid onto a CS-coated CUR-NCs core. This \u0026ldquo;graft-to\u0026rdquo; strategy locks the HA chains in place, conferring long-lived steric repulsion that is resistant to dilution or competitive displacement. More importantly, HA provides a bioresponsive motif; owing to its reported affinity for CD44 receptors [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], it may facilitate receptor-mediated interactions with mucosal and epithelial cells, while its hydrophilicity and negative charge create a hydration shell that minimises muco-adhesion and facilitates deep diffusion[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Compared to conventional PEGylated or poloxamer-coated systems, the HA\u0026ndash;CS conjugate offers dual functionality, improved colloidal stability and selective tissue engagement [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These effects, together with the observed increases in mucosal deposition and cellular uptake, underscore the importance of fine-tuned surface properties in enhancing drug delivery performance. Moreover, considering that macrophages can actively internalise NCs and impact mucopenetration, the HA-CS-CUR-NCs system appears to at least partially overcome this clearance mechanism. These insights pave the way for the translational advancement of HA-functionalised NCs as next-generation drug delivery systems, particularly in applications where mucosal, transdermal, or transmucosal penetration is required.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis work underscores the potential of HA-functionalised CS-coated NCs as a versatile and rationally engineered platform for mucosal drug delivery. Beyond achieving covalent HA grafting and demonstrating improved \u003cem\u003eex vivo\u003c/em\u003e mucopenetration, the system embodies a strategic convergence of NCs-based drug solubilization with stable, biointeractive surface design. The modularity of this approach opens the door to its application with a wide range of poorly soluble drugs and therapeutic biomolecules, particularly in indications where mucosal targeting and barrier traversal are critical. Looking ahead, \u003cem\u003ein vivo\u003c/em\u003e validation of pharmacokinetics, biodistribution, and therapeutic performance will be key to assessing clinical translatability. Altogether, the findings presented here offer a promising step toward next-generation nanomedicine platforms that not only overcome physicochemical formulation hurdles but also engage biological systems with precision and purpose.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors consent for publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Engineering and Physical Sciences Research Council (grant EP/Y001486/1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMGF\u003c/strong\u003e: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data curation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Visualization. \u003cstrong\u003eOF: \u003c/strong\u003eConceptualization, Methodology, Validation, Formal Analysis, Investigation, Data curation, Writing \u0026ndash; review \u0026amp; editing, Visualization. \u003cstrong\u003eAR\u003c/strong\u003e: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data curation. \u003cstrong\u003eJAC: \u003c/strong\u003eResources, Supervision. \u003cstrong\u003eAJP:\u003c/strong\u003e Conceptualization, Resources, Writing \u0026ndash; review \u0026amp; editing, Supervision, Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to the Engineering and Physical Sciences Research Council for the financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eM\u0026uuml;ller RH, Gohla S, Keck CM. 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Stimuli-responsive nanocarriers for drug delivery. Nat Mater. 2013;12:991\u0026ndash;1003. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmat3776\u003c/span\u003e\u003cspan address=\"10.1038/nmat3776\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Curcumin, nanosuspension, targeted nanocrystals, surface decoration, in vitro uptake, ex vivo mucopenetration","lastPublishedDoi":"10.21203/rs.3.rs-7030744/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7030744/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanocrystals (NCs) represent an advanced drug delivery platform due to their nearly 100% drug loading capacity, enhanced solubility, and improved tissue penetration. The surface-rich structure NCs enable chemical modifications, with covalent grafting emerging as a superior strategy to impart stability and targeted functional groups. In this study, we explore the development of curcumin nanocrystals (CUR-NCs) covalently grafted with hyaluronic acid (HA). To enhance mucosal targeting and permeation, an innovative surface functionalization strategy was employed: hyaluronic acid (HA) was chemically grafted onto the Chitosan (CS) NCs through EDC/NHS-mediated chemistry. This surface conjugation was confirmed through FTIR and \u003csup\u003e1\u003c/sup\u003eH-NMR analyses, validating the successful formation of HA-CS-CUR-NCs. The resulting nanoparticles exhibited an average particle size of 110 nm, remaining within the ideal range for mucosal delivery. Importantly, cytotoxicity assays on THP1 monocytes and NIH/3T3 fibroblasts revealed that the HA-CS-CUR-NCs possessed excellent biocompatibility properties. \u003cem\u003eEx vivo\u003c/em\u003e mucosal deposition studies using neonatal porcine tissue demonstrated significantly improved mucopenetration with HA-functionalized NCs, achieving 37.3 ± 26% drug deposition after 24 hours, compared to only 1.81 ± 1% for non-functionalized CUR-NCs. These findings position HA-CS-CUR-NCs as a promising platform for advanced mucosal drug delivery, combining nanoscale precision with bioresponsive surface chemistry to enhance therapeutic outcomes.\u003c/p\u003e","manuscriptTitle":"Covalent Grafting of Hyaluronic Acid on Drug Nanocrystals - Application in Mucosal Delivery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 08:11:07","doi":"10.21203/rs.3.rs-7030744/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-10-01T11:26:54+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-13T09:49:18+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T19:23:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-14T14:17:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2025-07-03T04:34:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8a626d1f-7b85-4afa-9ceb-69587de88312","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-10-01T15:28:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 08:11:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7030744","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7030744","identity":"rs-7030744","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}