Thymoquinone-Loaded Lipid Nanocapsules Improve Anticancer Efficacy Following Intraperitoneal Administration in a CT26 Xenograft Model of Colorectal Cancer

Thymoquinone-Loaded Lipid Nanocapsules Improve Anticancer Efficacy Following Intraperitoneal Administration in a CT26 Xenograft Model of Colorectal Cancer | 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 Thymoquinone-Loaded Lipid Nanocapsules Improve Anticancer Efficacy Following Intraperitoneal Administration in a CT26 Xenograft Model of Colorectal Cancer Mouna SELMI, Maram SELMI, Abir SALEK, Fedia JABER, Haifa MESSAI, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8239390/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Colorectal cancer (CRC) remains a major cause of cancer-related mortality worldwide, with limited treatment options at advanced stages. Thymoquinone (TQ), a natural bioactive compound derived from Nigella sativa , has demonstrated significant anticancer potential but is limited by poor aqueous solubility and low bioavailability. In this study, TQ was successfully encapsulated into lipid nanocapsules (LNCs) to overcome these limitations and enhance its anticancer efficacy after intraperitoneal administration. The LNCs-TQ exhibited a mean diameter of ~60 nm with a high encapsulation efficiency exceeding 85%. Their antiproliferative activity was evaluated in vitro against murine colorectal carcinoma CT26 cells, showing that TQ significantly reduced cell viability compared to free TQ (IC₅₀ : 5 µM vs. 15 µM). In vivo , TQ-LNCs has significantly suppressed tumor growth in a CT26 subcutaneous xenograft mouse model, with a tumor growth inhibition rate of up to 85%. Comet assay and histopathological analysis confirmed enhanced DNA damage and extensive tumor cell necrosis. These results suggest that LNCs-TQ improved the delivery and potency of TQ, making it a promising adjuvant strategy in colorectal cancer therapy. Thymoquinone Lipid nanocapsules Colorectal cancer Drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cancer remains a global health challenge and one of the leading causes of mortality worldwide. The burden of cancer is projected to increase significantly, with estimates suggesting 35 million new cancer cases worldwide by 2050 ( 1 ). Colorectal cancer (CRC) ranks among the most prevalent malignancies worldwide, with over 1.9 million new cases and approximately 935.000 deaths reported in 2020 alone ( 2 ). Current treatment strategies for CRC rely heavily on surgery, radiotherapy, and chemotherapy. Unfortunately, these conventional medical treatments can cause complications or even side effects that are ofen very severe for the patient including systemic toxicity, non-specific distribution, and the emergence of chemoresistance ( 3 – 5 ). Advances in nanotechnology have generated hopes for innovative cancer therapies that may be more effective, better targeted, and better tolerated ( 6 , 7 ). Biomedical nanotechnology, which involves the manipulation of materials at the nanoscale for biological applications, offers promising opportunities to improve cancer diagnosis, therapy, and tissue regeneration through highly targeted interventions. Nanoparticles (NPs) exhibit improved physical properties compared to their original material, including increased surface area, reactivity, and greater sensitivity. Their tiny size and high chemical reactivity enhance cellular uptake and interactions with biomolecules and tissues, facilitating their selective accumulation in tumor sites through crossing biological membranes ( 8 – 10 ), particularly the vascular endothelium, and reaching tumors through the enhanced permeation retention (EPR) mechanism ( 11 , 12 ). This selective targeting reduces systemic toxicity and enhances therapeutic efficacy compared to traditional treatments. Thus, the development of nanoparticle-based drug delivery systems can improve drug therapeutic efficacy while reducing the doses administered and the adverse effects of chemotherapeutic drugs on patients ( 13 , 14 ). Recently, the integration of phytochemicals into conventional therapies has emerged as a promising strategy to enhance treatment efficacy while reducing adverse effects. Thymoquinone (TQ), the major bioactive component of Nigella sativa seeds, has attracted considerable interest for its anticancer properties, including pro-apoptotic, anti-proliferative, anti-inflammatory, and antioxidant activities ( 15 – 17 ). Preclinical studies have demonstrated that TQ can inhibit tumor growth and modulate key signaling pathways involved in cancer progression ( 18 – 22 ). However, its poor aqueous solubility, low oral bioavailability, and rapid metabolic degradation severely limit its clinical application. To overcome these drawbacks, nanotechnology-based delivery systems have been developed to encapsulate TQ within controlled-release carriers. These nanovectors ; including liposomes, micelle nanoparticles, and chitosan nanoparticles ( 23 – 25 ). These naocarriers aimed to enhance drug biodistribution, minimize systemic toxicity, and improve antitumor efficacy ( 23 – 25 ). However, their use is often limited by low drug loading capacity and instability in biological environments, which can lead to rapid drug degradation before reaching the tumor site ( 23 – 25 ). Consequently, developing a biocompatible nanocarrier that can effectively address TQ’s pharmacological and toxicological limitations remains a critical challenge. As selective nanomaterials, Lipid nanocapsules (LNCs) appear as innovative and promising drug delivery system. These biocompatible nano-materials exhibit a great capacity in the encapsulations of anticancer drugs ( 26 – 28 ). LNCs have recently emerged as valuable tools in biomedical research. Our findings demonstrate that their unique tubular shape enhances cellular internalization, which can significantly improve targeted drug delivery. Notably, we reported for the first time that TiNTs achieve a high TQ loading capacity (8 mg/g of LNCs) combined with substantial entrapment efficiency (up to 85%), supporting controlled drug release and elevated cellular uptake of TQ-loaded TiNTs ( 29 – 31 ). Moreover, in vivo anticancer efficacy was validated through intratumoral administration in mice at doses of 5 mg/kg over 15 days without any adverse effects ( 31 ). These nanostructured materials possess distinctive advantages for drug encapsulation and sustained release, making them strong candidates for next-generation nanomedicine platforms ( 29 – 31 ). The work is structured around two different strands. The first focuses on the efficay of TQ-loaded LNCs on cell viability of CCR cells. The second examines the anti-tumor efficiency of two galenic forms of Thymoquinone : the free drug versus the drug encapsulated in lipid nanocapsules after intraperitonel administration into a murine subcutaneous xenograft model of CRC. This study seeks to contribute to the development of safer and more efficient nanomedicine approaches for colorectal and other solid tumors, with a particular focus on systemic drug delivery routes. 2. Materials and Methods 2.1. Materials Thymoquinone (99%) and DMSO have been purchased from Sigma–Aldrich (Saint-Quentin Fallavier, France). Captex® 8000 was provided by Abitec Corp (Saint-Quentin Fallavier, France). Lipoid® S100 and Kolliphor® HS15 were supplied from Lipoid Gmbh (Steinhausen, Switzerland) and BASF (Ludwigshafen, Germany), respectively. Sodium chloride was purchased from Prolabo VWR International (Fontenay-sous-Bois, France). All the other reactants, including HPLC grade acetic acid, acetonitrile and methanol have been purchased from Fisher Scientific (Loughborough, United Kingdom). Deionized water was produced using a Milli-Q system (Millipore). 2.2. Preparation of TQ-Loaded Lipid Nanocapsules (TQ-NLCs) Lipid nanocapsules loading Thymoquinone (TQ-LNCs) were prepared using the phase inversion temperature (PIT) method as previously described ( 30 , 31 ). 10 mg of TQ was firstly solubilized within Captex® 8000 at a final concentration of 6.66% w/w under magnetic stirring. Then, Captex®-TQ (13.12% w/w) and Lipoid® S100 (0.73% w/w) were heated to 75°C under magnetic stirring. Kolliphor® HS15 (10.94% w/w), NaCl (0.8% w/w), and water (19.73% w/w) were subquently added. Three heat-cool cycles were applied (65–90°C). The formulation was rapidly diluted with cold purified water (2°C, 54.68% w/w) at the inversion temperature (70°C) to induce nanocapsule formation at the last cycle. The final dispersion was gently stirred for 5 min and filtered through a 0.2 µm membrane filter (Merck KGaA, Darmstadt, Germany). Unloaded LNCs were prepared in a similar manner as describe above without addition of TQ. Suspensions were stored at 4°C until further use. 2.3. Physicochemical Characterization: size, PDI and zeta potential Nanoparticle mean size, polydispersity index (PDI), and zeta potential were measured using Malvern Zetasizer Nano ZS setup (Malvern Instruments S.A., UK). Unloaded LNCs or TQ-LNCs were diluted 1:400 (v/v) in deionized water and analyzed in triplicate at room temperature after equilibration for 2 min. The data were obtained with the average of three measurements. 2.3. Drug loading and Encapsulation Efficiency The amount of encapsulated TQ was quantified by HPLC-UV (Waters system) using a BEH C18 column (250 × 4.6 mm, 5 µm). The mobile phase composition consisted of phase A (2% acetic acid in water, pH = 2.5) and a phase B (methanol) and the separation of TQ was optimized using an isocratic mode (A:B 25:75 (v/v). The flow rate was fixed at 1.5 mL.min − 1 and the run time was 7 min. The retention time of TQ was 4.8 min. The column was operated at 25°C and TQ was monitored and quantified at 254 nm. Calibration curve was prepared from a stock solution of TQ in methanol at 10 mg.mL − 1 ) followed by appropriate dilutions to form concentrations ranging from 5 to 50 µg.mL − 1 . Samples of filtrated TQ-LNCs were diluted by 200 in methanol before injection of 10 µL. The analytical method was developed and validated as described previously with some modifications ( 30 ). The LNCs drug loading capacity (%) was calculated as the amount of total entrapped TQ divided by the total nanoparticles weight. Then the drug loading efficiency (%) was calculated as the ratio of the amount of TQ in the nanoparticles to the total amount of TQ applied in formulation of the nanoparticles. 2.4. Cell Culture and Viability Assays CT26 murine adenocarcinoma cell lines were provided by the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in RPMI-1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 1% L-glutamine, and 1% penicillin/streptomycin (Sigma Aldrich, St. Quentin Fallavier, France). Cells were maintained in culture in a humidified incubator at 37˚C and 5% CO 2 . For the viability assessment, Cells were seeded in 96-well plates (5×10³ cells/well) and treated for 24 h with increasing concentrations (0–40 µM) of free TQ dissolved in 0.1% DMSO (Fisher Scientific), LNCs-TQ and empty LNCs. Untreated cells served as controls and were set as 100% viability for normalization. Following treatment, cells were washed with PBS, fixed with glutaraldehyde (Fisher Scientific), and stained with crystal violet solution (Fisher Scientific). After rinsing and drying, violet crystal was solubilized in acetic acid (Fisher Scientific), and absorbance was measured at 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was calculated as the percentage of absorbance relative to control wells. 2.5. Animal Model of subcutaneously colorectal cancer xenograft Animal care procedures were conducted in conformity with the legislation for the protection of animals used for scientific purposes provided by the relevant Tunisian law and European Union Directive (Tunisian Legislative Decree 2009–2200 and 2010/63/EU) and the International Guiding Principles for the Biomedical Research involving animals (Council for the International Organizations of Medical Sciences, CH). Animals were subjected to experimental protocols approved by the Animal Ethics Committee of the Institute of Biotechnology of Monastir (Authorization number: CERSVS/ISBM 009/2022). Eighteen male BALB/c mice, 5 weeks old, were obtained from Pasteur Institute (Tunisia). All animals were maintained in an air-conditioned room (22–25° C) with 12 h light/dark cycle. Food and water were available throughout the experimental period. After 7 days of acclimatization, all mice were subcutaneously injected with CT26 murine colorectal cancer cells (2×10 6 cells/mouse, suspended in 100 µL of PBS) in the right flank to form a solid tumor. 2.6. Animal treatment One week after CT26 cells inoculation, when the tumor size grew to an average of 100 mm 3 , animals were randomly divided into three groups of six mice (n = 6) each depending on the experimental treatment. In the TQ-treated group, TQ was solubilized in 0.1% of DMSO in PBS administrated intraperitionnally at a dose of 10 mg.kg − 1 body weight of TQ. This treatment was repeated every 48 h for two weeks as described ( 31 ). Similarly, a TQ-LNCs group received TQ-LNCs at the equivalent dose of 10 mg.kg − 1 body weight of TQ. Further, control group was treated in the same manner, by intraperitionnally injection of 100 µL of PBS with 0.1% of DMSO. In all treated groups, the application site was washed twice with NaCl 0.9% and betadine before and after intraperitionnally injection to avoid local site infection. During the two weeks following the treatment, the tumor nodule volume were measured every 2 days using a caliper. The tumor volumes were calculated using the following formula : V = L × W 2 × 0.523 (where V is the volume, L is the length and W is the width) ( 31 ). After two weeks, all animals were anesthetized under isoflurane and sacrificed. Solid tumor from each mouse were collected, washed with cooled NaCl 0.9%, weighed then stored under − 20°C until analysis. At the end of experiment, the tumor inhibitory rate (TIR) was calculated using the following equation : TIR (%) = [(Tumor Volume Control - Tumor Volume Treated ) / Tumor Volume Control ] ×100 ​ 2.7. Comet assay : genotoxicity assessment The genotoxicity effect of free TQ versus TQ-LNCs was assessed by the quantification of DNA damage induced in tumor cells using the comet assay in neutral and alkali conditions as reported previously ( 31 ). Briefly, 10 mg of solid tumor were accurately weighed and then homogenized with 1mL of PBS. After shaking for 5 min, the homogenate was mixed gently with low melting agarose (1.2%) for cell adhesion then placed on comet slides for 10 min at 4°C. Subsequently, cells were incubated in lysis buffer (pH = 10) for 1 h at 4°C in darkness. Next, electrophoresis was performed under neutral condition at 25 V and 300 mA for 15 min then embedded cells were fixed with ethanol (70%). At the end, comet slides were stained with diluted Ethidium Bromide (0.02 mg. mL − 1 ) then observed by fluorescence microscopy (Olympus B×51 TRF, USA) using a FITC filter. Finally, the DNA damage percentage was determined according to Eke and Celik 2016, using the following formula: Genotoxicity (%) = (Total DNA Damage of sample - Total DNA Damage of control/ Total DNA Damage of control) x 100. 2.8. Histology For histological evaluation, collected organs and solid tumors were immediately fixed in 10% buffered neutral formalin and embedded in a paraffin wax. Sections of 5 µm were cut from each sample and stained with hematoxylin-eosin as reported previously ( 31 ). 2.9. Satistical Analyses Data are expressed as mean ± standard deviation (SD). Statistical significance was assessed using one-way and two-way ANOVA followed by Tukey’s post hoc test using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). P-values ≤ 0.05 were considered significant (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001). 3. Results 3.1. Physicochemical Characterization of Thymoquinone Loaded LNCs (TQ-LNCs) Lipid nanocapsules loading Thymoquinone (LNCs-TQ) were prepared via the phase inversion temperature (PIT) method ( 30 ). The freshly prepared TQ-LNCs dispersion appeared macroscopically homogeneous, translucent, and yellowish in color ( Fig. 1 ) . Dynamic light scattering analysis revealed a mean hydrodynamic diameter of 57.47 ± 3.75 for unloaded LNCs to 58.34 ± 3.7 for TQ-LNCs indicating no significant difference even when TQ is loaded within nanoparticles ( Table 1 ) . Likewise, unloaded LNCs and TQ-LNCs aqueous suspensions showed homogeneous monodisperse nanoparticles system according to the polydispersity index which corresponded to 0.04 ± 0.01 and 0.05 ± 0.02 for unloaded and TQ-loaded nanoparticles, respectively. Moreover, a similar neutral zeta potential (∼ 3.8 mV) was measured for both formulations indicating that TQ loading did not influence the surface charge of the nanoparticles. Finally, a nanoparticles drug loading up to 8.41 ± 0.56 mg.mL − 1 of TQ per LNCs suspension was obtained corresponding to a high drug loading efficiency of 89.13 ± 4.93%. Table 1 Mean particle size, polydispersity index (PDI) and zeta potential values of unloaded LNCs and TQ-LNCs. Drug loading capacity and efficiency of LNCs after filtration (n = 6, mean ± SD). Size (nm) PDI Zeta potential (mV) Drug loading capacity (%) Drug loading efficiency (%) LNCs-vides 55.37 ± 2.35 0.04 ± 0.01 -3.57 ± 0.47 - - LNCs-TQ 56.42 ± 2.12 0.05 ± 0.02 -3.8 ± 1.41 8.41 ± 0.56 89.13 ± 4.45% 3.2. Free TQ vs TQ loaded LNCs Decreased Colorectal Cancer Cells Viability CT26 cells were adopted to assess the cytotoxicity of TQ and its lipid nanocapsule formulation (TQ-LNCs). CRC cells were exposed to increasing concentrations (0–40µM) of each agent for 24 h. Cell viability was subsequently assesed using crystal violet staining. As Shown in Fig. 2 the treatment with TQ significantly reduced CT26 cell viability in a dose-dependent manner, exhibiting IC50 values of 15 µM ( Fig. 2 ) . Notably, the IC50 value for TQ was approximately threefold lower following encapsulation in LNCs, decreasing from 15 µM for free TQ to 5 µM for TQ loaded LNCs ( Fig. 2 ) . This marked reduction strongly suggests that lipid nanocapsules substantially facilitated the cellular internalization of TQ, thereby enhancing its cytotoxic effects. Although, treatment with unloaded LNCs maintained cell viability above 80%. This may be attributed to the higher cellular uptake of TQ-loaded LNCs followed by the faster TQ release as a consequence of the intracellular degradation conditions. 3.3. Antitumor Activity of TQ versus TQ-LNCs in subcutaneous tumor xenograft model after intraperitoneal administration The antitumoral activity of TQ-LNCs was tested on xenograft model of murine colorectal cancer developed by the injection of CT26 murine colorectal carcinoma cells in the flank of BALB/c mice. Animal growth and tumor size development were monitored during the peroid of treatment following TQ-LNCs intraperitoneal administration. Animal growth showed that The mice treated with free TQ and TQ-LNCs had a significant high relative weight compared to the control group (Figure S1) confrming the absence of toxic effects due to TQ-LNCs, as we reported for the TQ-loaded LNCs after the intratumoal injection of 5 mg kg − 1 in mice bearing CRC tumor ( 31 ). Moreover, tumors growth was controlled for 15 days following the intraperitoneal injection of TQ at 10 mg. kg − 1 in solution or encapsulated in LNCs. For control group (treated with PBS) the average tumor volume increased exponentially from 94.29 ± 13.53 mm³ to 1786.08 ± 275.93 mm³ at day 15 ( Fig. 3 ) . After administration of free TQ three times per week, tumor volume significantly reduced to an average of 1093.29 ± 103.23 mm³ corresponding to a substantial tumor growth inhibition by 38.81% (p < 0.01) compared to the control group. Moreover, a more drastic tumor regression has been occurred after TQ-LNCs mice intraperitoneal treatment resulting in lower tumor volume reaching 415.81 mm 3 ± 80.63 mm³ indicating a tumor growth inhibition by 76.77% (p < 0.01) compared to control group. Overall, our results demonstrated that TQ-LNCs markedly enhance the antitumor activity of TQ compared with the free drug at an equivalent dose (10 mg.Kg − 1 ). Moreover, representative tumor photographs ( Fig. 4 A ) revealed a clear reduction in tumor size from control to TQ, with the smallest tumor observed in the TQ-LNCs–treated group, suggessing a substantial decrease in tumor burden and may indicate reduced viability relative to both control and free TQ. Quantitatively, tumor inhibition rates ( Fig. 4 B ) results revealed that the control group exhibits a very low tumor inhibition rate, confirming progressive tumor growth in the absence of treatment. Allthough, Free TQ at 10 mg/kg produces a moderate inhibition reaching 63.89% ± 3.4, whereas TQ-LNCs at the same dose achieve a high inhibition rate reaching 86.83% ± 9.3, showing a statistically significant difference compared with both the control and free TQ groups ( p < 0.01). 3.4. Genotoxicity effect of TQ versus TQ-LNCs Genotoxicity measurement was approved using the comet assay to analyze DNA damage and tail parameters in cells isolated from solid tumor of control group and mice treated with free TQ and TQ-LNCs at 10 mg.kg − 1 after intraperitoneal administration. The treatment with free TQ and TQ-LNCs ( Fig. 5 ) showed a high positive response in the comet assay, indicating maximum of genotoxicity with relative tail moment values of 16.6 ± 1.3 and 39.2 ± 3.6 (p < 0.01), respectively. This effect is higher (up to 13-fold) than the control group treated with PBS 3.4 ± 1.8 (p < 0.01). Moreover, the relative tail moment value in TQ-LCNs treated group is almost 2.4-fold (p < 0.01) higher than that of free TQ treated group evidencing an enhanced DNA damage in cancer cells upon LNCs mediated drug delivery. 3.5. Histopathological examination Histopathological examonation of the tumor sections revealed a significant reduction of viable cells in TQ and TQ-LNCs groups compared with control group ( Fig. 6 ) . Interestingly, TQ and its nano formulation revealed the presence of necrotic areas in tumor sections evidencing the antitumor potential of TQ and TQ-loaded LNCs. Additionally, histology analyses of the major organs of the treated groups including the liver, spleen, lungs, kidneys, heart, and brain, were similar to that of the organs of the control groups (Figure S2) confirming the absence of any systemic toxicity. Discussion This study aimed to evaluate the antitumor activity of TQ-LNCs against CRC model following an intaperitoneal administration of a dose of (10 mg.Kg -1 ) of TQ to mice. Prior to the animal’s treatment, our study was conducted initially to formulate and characterize TQ loaded LNCs with a maximum of encapsulation efficiency acccording to Selmi et al described method ( 30 , 31 ). LNCs showed a high drug loading efficiency close to 90% and a great drug loading capacity around to 8.4%. Furthermore, both of the unloaded and TQ loading LNCs suspensions were characterized for their size distribution and surface charge which plays a crucial role in cellular uptake within tumoral cells, prior to the in vivo experiments. The obtained results revealed that TQ-LNCs have been successfully formulated with a mean diameter above 60 nm, a relatively neutral zeta potential charge (-3.8 mV) and with a narrow distribution (PDI < 0.2). As shown also in our experiment, no significant changes were recorded in the hydrodynamic diameter of LNCs due to TQ loading compared to that of the unloaded LNCs. These physicochemical propreties are promising for a high tumor cells uptake since nanoscale size (150–200 nm). While a size less then 100 nm decrease the recognize of phagocytic system and consequently the destruction of nanocarriers ( 8 – 10 ). Numerous studies confirm that nanoparticles with near-neutral surface charge exhibit superior tumor accumulation and reduced opsonization by serum proteins, extending circulation time. This stealth-like property minimizes nonspecific interactions, facilitating direct membrane traversal or energy-independent internalization pathways without reliance on receptor-mediated endocytosis.​ Consequently, the physicochemical profile of TQ-LNCs—optimal size and neutral zeta potential—renders them highly suitable for effective cancer therapy by enhancing targeted drug delivery and cellular penetration ( 29 , 32 ). Moreover the TQ-loaded LNCs was tested for their cytotoxicity effect on murine CRC cells, CT26. The biocompatibility of LNCs was investigated on CT26 cells at the higher concentration 40 µM. The IC50 values of CT26 cells after 24 h incubation with free TQ and TQ-loaded-LNCs are shown in Fig. 2 . It can be found that the IC50 value of the TQ loaded LNCs for CT26 cells was 5 µM, which was a three fold less than TQ alone (15 µM) after 24 h incubation. It is concluded that the TQ-loaded nanoparticles exhibited significantly higher cytotoxicity for CT26 cells owing to the sustained drug release manner. The prolonged circulation time of nanoparticles is one of the most important parameters which must be improved to obtain efficient drug delivery. In this regard, several studies were focused on surface functionalization of nanoparticles using stabilizing agents such as polyethyleneglycol (PEG), dextran, or chitosan. These coating agents obviously prevented the nanoparicle aggregation and showing a better colloidal stability in biological media and long-time residence in the bloodstream circulation ( 33 – 35 ). PEG chains at the surface of our formulated nanocarrier conferring a steric stabilization and make them a potential candidate as a dug delivery system of TQ. Beyond nanoparticle size and surface functionalization, abnormalities in the tumor microenvironment significantly enhance nanocarrier retention through the enhanced permeability and retention (EPR) effect ( 11 , 12 ). Tumor-associated defective angiogenesis results in blood vessels with large fenestrations (150–200 nm), far wider than those in normal tissue, which allows nanoparticles smaller than 200 nm to extravasate more easily into the tumor interstitium. Additionally, the impaired lymphatic drainage common in tumors, combined with rapid cancer cell proliferation, not only promotes accumulation of nanoparticles but also facilitates the sustained release of their therapeutic payloads within tumor tissue, as observed for various nanoparticle systems ( 33 – 35 ). On the other hand, the antitumor efficacy of TQ-LNCs has been investigated on mice bearing the colorectal cancer CT26 cell line following intraperitoneal injection of a dose of TQ-LNCs (at 10 mg kg − 1 body weight). Fifteen days following the treatment, mice treated with TQ-LNCs showed a more drastic tumor-growth inhibition of up to 86% compared with control group without any body-weight loss, evidencing their anticancer efficacy. In our previous study, our results revealed that intratumoral administration of TQ-LNCs (5 mg/kg) yielded to 86% tumor growth inhibition in subcutaneous CT26 colorectal cancer xenografts ( 31 ). This inhibtion of tumor rate was signficantly higher than TQ alone, without body weight loss or organ toxicity. Histopathology revealed extensive necrosis and reduced viable cells, attributed to enhanced TQ internalization, P-gp inhibition by shell components (Kolliphor ® HS15). Elevated tumor cytokines (GM-CSF, TNF-α) indicated immune modulation, aligning with TQ's genotoxic effects across cancers ( 31 ).​ Despite shifting to systemic intraperitoneal administration (10 mg/kg), TQ-LNCs elicited even similar antitumor activity to intratumoral dosing, overcoming free TQ's bioavailability limitations noted previously without LNCs ( 31 ). This enhancement stems from extended circulation time, EPR-driven accumulation, and endocytosis-mediated uptake, with neutral charge minimizing protein opsonization. Such route flexibility addresses intratumoral injection challenges like incomplete coverage in heterogeneous tumors.​ According to the histopathological assessment, cell necrotic areas increased significantly in tumor tissues of mice treated with TQ-LNCs compared to those treated with the control group. Meanwhile treatment with free TQ induces less necrosis cells as compared to TQ-LNCs, suggesting lower pharmacological effectiveness probably due to its lipophilicity, which limits its affinity for the tumoral microenvironment and favored its rapid degradation and systemic elimination. The ameliorated anticancer activity was also observed with improved the drug delivery into the tumor cells, and thus equivalent TQ anticancer activity could be obtained via systemic administation garanting lower drug concentrations. This can be explained by long circulation time of TQ in blood stream and then the enhanced absorption of TQ-LNCs in tumor via EPR mechanisms. Once in tumor microenvirment, Thus, LNCs could enhance the resorption of TQ within tumor cells via endocytosis mechanisms ( 29 ), improving its cytotoxic effect. These results are in accordance with the previous findings ( 31 ). Collectively, TQ-LNCs appear to form a better delivery system than free TQ in colorectal cancer treatment without observable toxicity to the other organs, thus improving the efficacy and the therapeutic index of the drug. This proves how the design of TQ-LNCs has gained increasing interest as a means of improving the treatment of neoplastic diseases. Regarding the toxicity, all studied parameters, including the healthy behavior of animals and absence of toxicity in the kidneys, spleen, and liver, lung, heart and brain confirm the biocompatibility of TQ-LNCs. These results clearly confirm the interest in the use of these non-toxic LNCs as a nanocarrier for drug delivery for biomedical applications, notably in cancer therapy. Collectively, these results demonstrate that LNCs encapsulation substantially improves TQ’s bioavailability by prolonging its systemic circulation and reducing clearance, which could translate into enhanced therapeutic efficacy and reduced dosing frequency. This enhancement highlights the potential of TQ carriers to optimize the delivery and performance of TQ after intraperitoneal administration with prolonging the circulation time of encapsulated drugs by protecting them from rapid metabolism and clearance ( 36 – 38 ). Conslusion LNCs, which combine exceptional physicochemical propreties and high drug encapsulation capacity, are a promising safe and efficient drug delivery system. Upon intraperitoneal administration, LNCs loaded with TQ were able to drastically enhance anticancer efficay of TQ, and minimizing associated tissue toxicity and avoids histological damage. These results open fascinating perspectives for the safe and efficient drug delivery system using biocompatible nanomaterials like LNCs. Declarations Informed Consent Statement Not applicable. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Institutional Review Board Statement: Not applicable. Author Contribution M.S., F.J. H.M and M.S. performed the experiments and analyzed the data; M.S., A.S., supervised experiments. M.S wrote the manuscript. A.T. supervised HPLC experiments. M.L. supervised characterization experiments of LNCs. L.C.G supervised the overall project. All authors have read and agreed to the published version of the manuscript. Acknowledgement The work was supported by the Tunisian Ministry of Higher Education and Scientific Research (TMHESR). 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Baati T, Kefi BB, Aouane A, Njim L, Chaspoul F, Heresanu V, et al. Biocompatible titanate nanotubes with high loading capacity of genistein: Cytotoxicity study and anti-migratory effect on U87-MG cancer cell lines. RSC Adv. 2016;6(103):101688–96. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":147312,"visible":true,"origin":"","legend":"\u003cp\u003e(A, B) Images of the LNCs suspensions were taken by Selmi et al. (2024) : unloaded lipid nanocapsules (A) and Thymoquinone-loaded lipid nanocapsules (B) prepared by the phase inversion temperature (PIT) method. The prepared TQ-LNCs dispersion appears homogeneous, translucent, and yellowish in color.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8239390/v1/9302f8f5264de63346a8905f.png"},{"id":97982232,"identity":"18913a0d-55f1-4f3a-9b25-6886c9e7d9f6","added_by":"auto","created_at":"2025-12-11 13:03:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50287,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of murine CRC cells (CT26) determined by crystal violet staining after 24h of incubation with differents concentrations of TQ/ TQ-LNCs ranging from 0 to 40 µM. Each data point corresponds to mean values of three experiments (Results shown are representative curves from three independent experiments).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8239390/v1/f2dc27c4f8b35560ed2b9faf.png"},{"id":97982234,"identity":"d2643428-b5d3-4edd-bf48-ce762e071c06","added_by":"auto","created_at":"2025-12-11 13:03:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75415,"visible":true,"origin":"","legend":"\u003cp\u003eTumor growth curves in CT26 tumor-bearing mice treated with free Thymoquinone (TQ) or TQ-loaded lipid nanocapsules (TQ-LNCs). Tumor volumes were measured every two days after the start of treatment. Data are presented as mean ± SD (n = 6). Both TQ and TQ-LNCs significantly suppressed tumor growth compared with the untreated control group, with TQ-LNCs showing the strongest inhibition throughout the study period (p \u0026lt; 0.01 vs. control and free TQ).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8239390/v1/a40698387b6eb05d414fc74c.png"},{"id":97982239,"identity":"29ce17e4-0594-4cb8-9704-fa90a18e11a9","added_by":"auto","created_at":"2025-12-11 13:03:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63139,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo antitumor effect after intraperitoneal treatment with : PBS as Control, TQ and TQ loaded LNCs at 10 mg.kg\u003csup\u003e-1\u003c/sup\u003e body weight on a colorectal cancer subcutaneous model (n= 6 mice/group). (A) Macroscopic effects on mice tumor at days 15 (B) Tumor inhibitory rate. (a : P\u0026lt;0.01 vs control\u0026nbsp;group ; b : P\u0026lt;0.01 vs TQ group).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8239390/v1/610063abf632e2aa0fc133bf.png"},{"id":98424744,"identity":"4efccddc-4040-4feb-af51-417fa94df942","added_by":"auto","created_at":"2025-12-17 16:33:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":181274,"visible":true,"origin":"","legend":"\u003cp\u003eComet images of cells isolated from mice tumors showing comet tail formation. Cells were stained with Ethidium Bromide (0.02 mg.mL\u003csup\u003e-1\u003c/sup\u003e) then observed by epifluorescence microscopy using a FITC filter. Magnification = 400 ×, scale bar = 200 μm. DNA damage in comet cell contains a tail and a head like a comet; apoptotic cells have a large tail and a small head. Effect on total DNA damage assessed through the alkaline comet assay in tumor cells. \u0026nbsp;(a : P\u0026lt;0.01 vs control\u0026nbsp;group ; b : P\u0026lt;0.01 vs TQ group).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8239390/v1/9078fd96ebea7656c419dde7.png"},{"id":98424847,"identity":"0812a40b-7487-4334-9bad-8988f6a16204","added_by":"auto","created_at":"2025-12-17 16:33:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1768200,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathology assessments of tumor masses in colorectal cancer subcutaneous model. \u003cstrong\u003eN\u003c/strong\u003e refers to the necrotic area in tumor tissues after intraperitoneal treatment with PBS (Control), free TQ and TQ-LNCs with a dose of 10 mg.kg\u003csup\u003e-1\u003c/sup\u003e of TQ.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8239390/v1/8d0f0f2a1ccca95e146b1ad9.png"},{"id":98443955,"identity":"4a263f0d-f5b4-4ba4-ba50-7c416e8309b6","added_by":"auto","created_at":"2025-12-17 17:14:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3050476,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8239390/v1/e2098119-837d-49ec-bc99-dc2431798d0b.pdf"},{"id":97982233,"identity":"f88f21c0-9120-42c1-a320-14d6d0235bcb","added_by":"auto","created_at":"2025-12-11 13:03:17","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":164165,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8239390/v1/f5ba18d7eb12ecb0596331a6.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Thymoquinone-Loaded Lipid Nanocapsules Improve Anticancer Efficacy Following Intraperitoneal Administration in a CT26 Xenograft Model of Colorectal Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer remains a global health challenge and one of the leading causes of mortality worldwide. The burden of cancer is projected to increase significantly, with estimates suggesting 35\u0026nbsp;million new cancer cases worldwide by 2050 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Colorectal cancer (CRC) ranks among the most prevalent malignancies worldwide, with over 1.9\u0026nbsp;million new cases and approximately 935.000 deaths reported in 2020 alone (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Current treatment strategies for CRC rely heavily on surgery, radiotherapy, and chemotherapy. Unfortunately, these conventional medical treatments can cause complications or even side effects that are ofen very severe for the patient including systemic toxicity, non-specific distribution, and the emergence of chemoresistance (\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAdvances in nanotechnology have generated hopes for innovative cancer therapies that may be more effective, better targeted, and better tolerated (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Biomedical nanotechnology, which involves the manipulation of materials at the nanoscale for biological applications, offers promising opportunities to improve cancer diagnosis, therapy, and tissue regeneration through highly targeted interventions. Nanoparticles (NPs) exhibit improved physical properties compared to their original material, including increased surface area, reactivity, and greater sensitivity. Their tiny size and high chemical reactivity enhance cellular uptake and interactions with biomolecules and tissues, facilitating their selective accumulation in tumor sites through crossing biological membranes (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), particularly the vascular endothelium, and reaching tumors through the enhanced permeation retention (EPR) mechanism (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). This selective targeting reduces systemic toxicity and enhances therapeutic efficacy compared to traditional treatments. Thus, the development of nanoparticle-based drug delivery systems can improve drug therapeutic efficacy while reducing the doses administered and the adverse effects of chemotherapeutic drugs on patients (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecently, the integration of phytochemicals into conventional therapies has emerged as a promising strategy to enhance treatment efficacy while reducing adverse effects. Thymoquinone (TQ), the major bioactive component of \u003cem\u003eNigella sativa\u003c/em\u003e seeds, has attracted considerable interest for its anticancer properties, including pro-apoptotic, anti-proliferative, anti-inflammatory, and antioxidant activities (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Preclinical studies have demonstrated that TQ can inhibit tumor growth and modulate key signaling pathways involved in cancer progression (\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). However, its poor aqueous solubility, low oral bioavailability, and rapid metabolic degradation severely limit its clinical application.\u003c/p\u003e\u003cp\u003eTo overcome these drawbacks, nanotechnology-based delivery systems have been developed to encapsulate TQ within controlled-release carriers. These nanovectors ; including liposomes, micelle nanoparticles, and chitosan nanoparticles (\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). These naocarriers aimed to enhance drug biodistribution, minimize systemic toxicity, and improve antitumor efficacy (\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, their use is often limited by low drug loading capacity and instability in biological environments, which can lead to rapid drug degradation before reaching the tumor site (\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Consequently, developing a biocompatible nanocarrier that can effectively address TQ\u0026rsquo;s pharmacological and toxicological limitations remains a critical challenge.\u003c/p\u003e\u003cp\u003eAs selective nanomaterials, Lipid nanocapsules (LNCs) appear as innovative and promising drug delivery system. These biocompatible nano-materials exhibit a great capacity in the encapsulations of anticancer drugs (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). LNCs have recently emerged as valuable tools in biomedical research. Our findings demonstrate that their unique tubular shape enhances cellular internalization, which can significantly improve targeted drug delivery. Notably, we reported for the first time that TiNTs achieve a high TQ loading capacity (8 mg/g of LNCs) combined with substantial entrapment efficiency (up to 85%), supporting controlled drug release and elevated cellular uptake of TQ-loaded TiNTs (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Moreover, \u003cem\u003ein vivo\u003c/em\u003e anticancer efficacy was validated through intratumoral administration in mice at doses of 5 mg/kg over 15 days without any adverse effects (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). These nanostructured materials possess distinctive advantages for drug encapsulation and sustained release, making them strong candidates for next-generation nanomedicine platforms (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe work is structured around two different strands. The first focuses on the efficay of TQ-loaded LNCs on cell viability of CCR cells. The second examines the anti-tumor efficiency of two galenic forms of Thymoquinone : the free drug versus the drug encapsulated in lipid nanocapsules after intraperitonel administration into a murine subcutaneous xenograft model of CRC. This study seeks to contribute to the development of safer and more efficient nanomedicine approaches for colorectal and other solid tumors, with a particular focus on systemic drug delivery routes.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eThymoquinone (99%) and DMSO have been purchased from Sigma\u0026ndash;Aldrich (Saint-Quentin Fallavier, France). Captex\u0026reg; 8000 was provided by Abitec Corp (Saint-Quentin Fallavier, France). Lipoid\u0026reg; S100 and Kolliphor\u0026reg; HS15 were supplied from Lipoid Gmbh (Steinhausen, Switzerland) and BASF (Ludwigshafen, Germany), respectively. Sodium chloride was purchased from Prolabo VWR International (Fontenay-sous-Bois, France). All the other reactants, including HPLC grade acetic acid, acetonitrile and methanol have been purchased from Fisher Scientific (Loughborough, United Kingdom). Deionized water was produced using a Milli-Q system (Millipore).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Preparation of TQ-Loaded Lipid Nanocapsules (TQ-NLCs)\u003c/h2\u003e\u003cp\u003eLipid nanocapsules loading Thymoquinone (TQ-LNCs) were prepared using the phase inversion temperature (PIT) method as previously described (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). 10 mg of TQ was firstly solubilized within Captex\u0026reg; 8000 at a final concentration of 6.66% w/w under magnetic stirring. Then, Captex\u0026reg;-TQ (13.12% w/w) and Lipoid\u0026reg; S100 (0.73% w/w) were heated to 75\u0026deg;C under magnetic stirring. Kolliphor\u0026reg; HS15 (10.94% w/w), NaCl (0.8% w/w), and water (19.73% w/w) were subquently added. Three heat-cool cycles were applied (65\u0026ndash;90\u0026deg;C). The formulation was rapidly diluted with cold purified water (2\u0026deg;C, 54.68% w/w) at the inversion temperature (70\u0026deg;C) to induce nanocapsule formation at the last cycle. The final dispersion was gently stirred for 5 min and filtered through a 0.2 \u0026micro;m membrane filter (Merck KGaA, Darmstadt, Germany). Unloaded LNCs were prepared in a similar manner as describe above without addition of TQ. Suspensions were stored at 4\u0026deg;C until further use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Physicochemical Characterization: size, PDI and zeta potential\u003c/h2\u003e\u003cp\u003eNanoparticle mean size, polydispersity index (PDI), and zeta potential were measured using Malvern Zetasizer Nano ZS setup (Malvern Instruments S.A., UK). Unloaded LNCs or TQ-LNCs were diluted 1:400 (v/v) in deionized water and analyzed in triplicate at room temperature after equilibration for 2 min. The data were obtained with the average of three measurements.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Drug loading and Encapsulation Efficiency\u003c/h2\u003e\u003cp\u003eThe amount of encapsulated TQ was quantified by HPLC-UV (Waters system) using a BEH C18 column (250 \u0026times; 4.6 mm, 5 \u0026micro;m). The mobile phase composition consisted of phase A (2% acetic acid in water, pH\u0026thinsp;=\u0026thinsp;2.5) and a phase B (methanol) and the separation of TQ was optimized using an isocratic mode (A:B 25:75 (v/v). The flow rate was fixed at 1.5 mL.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the run time was 7 min. The retention time of TQ was 4.8 min. The column was operated at 25\u0026deg;C and TQ was monitored and quantified at 254 nm. Calibration curve was prepared from a stock solution of TQ in methanol at 10 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) followed by appropriate dilutions to form concentrations ranging from 5 to 50 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Samples of filtrated TQ-LNCs were diluted by 200 in methanol before injection of 10 \u0026micro;L. The analytical method was developed and validated as described previously with some modifications (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The LNCs drug loading capacity (%) was calculated as the amount of total entrapped TQ divided by the total nanoparticles weight. Then the drug loading efficiency (%) was calculated as the ratio of the amount of TQ in the nanoparticles to the total amount of TQ applied in formulation of the nanoparticles.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Cell Culture and Viability Assays\u003c/h2\u003e\u003cp\u003eCT26 murine adenocarcinoma cell lines were provided by the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in RPMI-1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 1% L-glutamine, and 1% penicillin/streptomycin (Sigma Aldrich, St. Quentin Fallavier, France). Cells were maintained in culture in a humidified incubator at 37˚C and 5% CO\u003csub\u003e2\u003c/sub\u003e. For the viability assessment, Cells were seeded in 96-well plates (5\u0026times;10\u0026sup3; cells/well) and treated for 24 h with increasing concentrations (0\u0026ndash;40 \u0026micro;M) of free TQ dissolved in 0.1% DMSO (Fisher Scientific), LNCs-TQ and empty LNCs. Untreated cells served as controls and were set as 100% viability for normalization. Following treatment, cells were washed with PBS, fixed with glutaraldehyde (Fisher Scientific), and stained with crystal violet solution (Fisher Scientific). After rinsing and drying, violet crystal was solubilized in acetic acid (Fisher Scientific), and absorbance was measured at 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was calculated as the percentage of absorbance relative to control wells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Animal Model of subcutaneously colorectal cancer xenograft\u003c/h2\u003e\u003cp\u003e Animal care procedures were conducted in conformity with the legislation for the protection of animals used for scientific purposes provided by the relevant Tunisian law and European Union Directive (Tunisian Legislative Decree 2009\u0026ndash;2200 and 2010/63/EU) and the International Guiding Principles for the Biomedical Research involving animals (Council for the International Organizations of Medical Sciences, CH). Animals were subjected to experimental protocols approved by the Animal Ethics Committee of the Institute of Biotechnology of Monastir (Authorization number: CERSVS/ISBM 009/2022). Eighteen male BALB/c mice, 5 weeks old, were obtained from Pasteur Institute (Tunisia). All animals were maintained in an air-conditioned room (22\u0026ndash;25\u0026deg; C) with 12 h light/dark cycle. Food and water were available throughout the experimental period. After 7 days of acclimatization, all mice were subcutaneously injected with CT26 murine colorectal cancer cells (2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mouse, suspended in 100 \u0026micro;L of PBS) in the right flank to form a solid tumor.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Animal treatment\u003c/h2\u003e\u003cp\u003eOne week after CT26 cells inoculation, when the tumor size grew to an average of 100 mm\u003csup\u003e3\u003c/sup\u003e, animals were randomly divided into three groups of six mice (n\u0026thinsp;=\u0026thinsp;6) each depending on the experimental treatment. In the TQ-treated group, TQ was solubilized in 0.1% of DMSO in PBS administrated intraperitionnally at a dose of 10 mg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e body weight of TQ. This treatment was repeated every 48 h for two weeks as described (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Similarly, a TQ-LNCs group received TQ-LNCs at the equivalent dose of 10 mg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e body weight of TQ. Further, control group was treated in the same manner, by intraperitionnally injection of 100 \u0026micro;L of PBS with 0.1% of DMSO. In all treated groups, the application site was washed twice with NaCl 0.9% and betadine before and after intraperitionnally injection to avoid local site infection.\u003c/p\u003e\u003cp\u003eDuring the two weeks following the treatment, the tumor nodule volume were measured every 2 days using a caliper. The tumor volumes were calculated using the following formula :\u003c/p\u003e\u003cp\u003e\u003cb\u003eV\u0026thinsp;=\u0026thinsp;L \u0026times; W\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e\u0026times; 0.523\u003c/b\u003e\u003c/p\u003e\u003cp\u003e(where V is the volume, L is the length and W is the width) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). After two weeks, all animals were anesthetized under isoflurane and sacrificed. Solid tumor from each mouse were collected, washed with cooled NaCl 0.9%, weighed then stored under \u0026minus;\u0026thinsp;20\u0026deg;C until analysis. At the end of experiment, the tumor inhibitory rate (TIR) was calculated using the following equation :\u003c/p\u003e\u003cp\u003e\u003cb\u003eTIR (%) = [(Tumor Volume\u003c/b\u003e \u003csub\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Tumor Volume\u003c/b\u003e \u003csub\u003e\u003cb\u003eTreated\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e) / Tumor Volume\u003c/b\u003e \u003csub\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e] \u0026times;100\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e​\u003c/b\u003e\u003cb\u003e2.7. Comet assay : genotoxicity assessment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe genotoxicity effect of free TQ versus TQ-LNCs was assessed by the quantification of DNA damage induced in tumor cells using the comet assay in neutral and alkali conditions as reported previously (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Briefly, 10 mg of solid tumor were accurately weighed and then homogenized with 1mL of PBS. After shaking for 5 min, the homogenate was mixed gently with low melting agarose (1.2%) for cell adhesion then placed on comet slides for 10 min at 4\u0026deg;C. Subsequently, cells were incubated in lysis buffer (pH\u0026thinsp;=\u0026thinsp;10) for 1 h at 4\u0026deg;C in darkness. Next, electrophoresis was performed under neutral condition at 25 V and 300 mA for 15 min then embedded cells were fixed with ethanol (70%). At the end, comet slides were stained with diluted Ethidium Bromide (0.02 mg. mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) then observed by fluorescence microscopy (Olympus B\u0026times;51 TRF, USA) using a FITC filter. Finally, the DNA damage percentage was determined according to Eke and Celik 2016, using the following formula: \u003cb\u003eGenotoxicity (%) = (Total DNA Damage of sample - Total DNA Damage of control/ Total DNA Damage of control) x 100.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Histology\u003c/h2\u003e\u003cp\u003eFor histological evaluation, collected organs and solid tumors were immediately fixed in 10% buffered neutral formalin and embedded in a paraffin wax. Sections of 5 \u0026micro;m were cut from each sample and stained with hematoxylin-eosin as reported previously (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Satistical Analyses\u003c/h2\u003e\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was assessed using one-way and two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). P-values\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered significant (* p\u0026thinsp;\u0026le;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026le;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026le;\u0026thinsp;0.001 and **** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Physicochemical Characterization of Thymoquinone Loaded LNCs (TQ-LNCs)\u003c/h2\u003e\u003cp\u003eLipid nanocapsules loading Thymoquinone (LNCs-TQ) were prepared via the phase inversion temperature (PIT) method (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The freshly prepared TQ-LNCs dispersion appeared macroscopically homogeneous, translucent, and yellowish in color \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Dynamic light scattering analysis revealed a mean hydrodynamic diameter of 57.47\u0026thinsp;\u0026plusmn;\u0026thinsp;3.75 for unloaded LNCs to 58.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 for TQ-LNCs indicating no significant difference even when TQ is loaded within nanoparticles \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Likewise, unloaded LNCs and TQ-LNCs aqueous suspensions showed homogeneous monodisperse nanoparticles system according to the polydispersity index which corresponded to 0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 for unloaded and TQ-loaded nanoparticles, respectively. Moreover, a similar neutral zeta potential (\u0026sim; 3.8 mV) was measured for both formulations indicating that TQ loading did not influence the surface charge of the nanoparticles. Finally, a nanoparticles drug loading up to 8.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of TQ per LNCs suspension was obtained corresponding to a high drug loading efficiency of 89.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean particle size, polydispersity index (PDI) and zeta potential values of unloaded LNCs and TQ-LNCs. Drug loading capacity and efficiency of LNCs after filtration (n\u0026thinsp;=\u0026thinsp;6, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSize (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZeta potential (mV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDrug loading capacity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDrug loading efficiency (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLNCs-vides\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e55.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e-3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLNCs-TQ\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e56.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e-3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e8.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e89.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.45%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Free TQ vs TQ loaded LNCs Decreased Colorectal Cancer Cells Viability\u003c/h2\u003e\u003cp\u003eCT26 cells were adopted to assess the cytotoxicity of TQ and its lipid nanocapsule formulation (TQ-LNCs). CRC cells were exposed to increasing concentrations (0\u0026ndash;40\u0026micro;M) of each agent for 24 h. Cell viability was subsequently assesed using crystal violet staining. As Shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e the treatment with TQ significantly reduced CT26 cell viability in a dose-dependent manner, exhibiting IC50 values of 15 \u0026micro;M \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Notably, the IC50 value for TQ was approximately threefold lower following encapsulation in LNCs, decreasing from 15 \u0026micro;M for free TQ to 5 \u0026micro;M for TQ loaded LNCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. This marked reduction strongly suggests that lipid nanocapsules substantially facilitated the cellular internalization of TQ, thereby enhancing its cytotoxic effects. Although, treatment with unloaded LNCs maintained cell viability above 80%. This may be attributed to the higher cellular uptake of TQ-loaded LNCs followed by the faster TQ release as a consequence of the intracellular degradation conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Antitumor Activity of TQ versus TQ-LNCs in subcutaneous tumor xenograft model after intraperitoneal administration\u003c/h2\u003e\u003cp\u003eThe antitumoral activity of TQ-LNCs was tested on xenograft model of murine colorectal cancer developed by the injection of CT26 murine colorectal carcinoma cells in the flank of BALB/c mice. Animal growth and tumor size development were monitored during the peroid of treatment following TQ-LNCs intraperitoneal administration. Animal growth showed that The mice treated with free TQ and TQ-LNCs had a significant high relative weight compared to the control group (Figure S1) confrming the absence of toxic effects due to TQ-LNCs, as we reported for the TQ-loaded LNCs after the intratumoal injection of 5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in mice bearing CRC tumor (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Moreover, tumors growth was controlled for 15 days following the intraperitoneal injection of TQ at 10 mg. kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in solution or encapsulated in LNCs. For control group (treated with PBS) the average tumor volume increased exponentially from 94.29\u0026thinsp;\u0026plusmn;\u0026thinsp;13.53 mm\u0026sup3; to 1786.08\u0026thinsp;\u0026plusmn;\u0026thinsp;275.93 mm\u0026sup3; at day 15 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. After administration of free TQ three times per week, tumor volume significantly reduced to an average of 1093.29\u0026thinsp;\u0026plusmn;\u0026thinsp;103.23 mm\u0026sup3; corresponding to a substantial tumor growth inhibition by 38.81% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the control group. Moreover, a more drastic tumor regression has been occurred after TQ-LNCs mice intraperitoneal treatment resulting in lower tumor volume reaching 415.81 mm\u003csup\u003e3\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;80.63 mm\u0026sup3; indicating a tumor growth inhibition by 76.77% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOverall, our results demonstrated that TQ-LNCs markedly enhance the antitumor activity of TQ compared with the free drug at an equivalent dose (10 mg.Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Moreover, representative tumor photographs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e revealed a clear reduction in tumor size from control to TQ, with the smallest tumor observed in the TQ-LNCs\u0026ndash;treated group, suggessing a substantial decrease in tumor burden and may indicate reduced viability relative to both control and free TQ. Quantitatively, tumor inhibition rates \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e results revealed that the control group exhibits a very low tumor inhibition rate, confirming progressive tumor growth in the absence of treatment. Allthough, Free TQ at 10 mg/kg produces a moderate inhibition reaching 63.89% \u0026plusmn; 3.4, whereas TQ-LNCs at the same dose achieve a high inhibition rate reaching 86.83% \u0026plusmn; 9.3, showing a statistically significant difference compared with both the control and free TQ groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Genotoxicity effect of TQ versus TQ-LNCs\u003c/h2\u003e\u003cp\u003eGenotoxicity measurement was approved using the comet assay to analyze DNA damage and tail parameters in cells isolated from solid tumor of control group and mice treated with free TQ and TQ-LNCs at 10 mg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after intraperitoneal administration. The treatment with free TQ and TQ-LNCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e showed a high positive response in the comet assay, indicating maximum of genotoxicity with relative tail moment values of 16.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 and 39.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), respectively. This effect is higher (up to 13-fold) than the control group treated with PBS 3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Moreover, the relative tail moment value in TQ-LCNs treated group is almost 2.4-fold (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) higher than that of free TQ treated group evidencing an enhanced DNA damage in cancer cells upon LNCs mediated drug delivery.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Histopathological examination\u003c/h2\u003e\u003cp\u003eHistopathological examonation of the tumor sections revealed a significant reduction of viable cells in TQ and TQ-LNCs groups compared with control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Interestingly, TQ and its nano formulation revealed the presence of necrotic areas in tumor sections evidencing the antitumor potential of TQ and TQ-loaded LNCs. Additionally, histology analyses of the major organs of the treated groups including the liver, spleen, lungs, kidneys, heart, and brain, were similar to that of the organs of the control groups \u003cb\u003e(Figure S2)\u003c/b\u003e confirming the absence of any systemic toxicity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study aimed to evaluate the antitumor activity of TQ-LNCs against CRC model following an intaperitoneal administration of a dose of (10 mg.Kg\u003csup\u003e-1\u003c/sup\u003e) of TQ to mice. Prior to the animal\u0026rsquo;s treatment, our study was conducted initially to formulate and characterize TQ loaded LNCs with a maximum of encapsulation efficiency acccording to Selmi et \u003cem\u003eal\u003c/em\u003e described method (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). LNCs showed a high drug loading efficiency close to 90% and a great drug loading capacity around to 8.4%. Furthermore, both of the unloaded and TQ loading LNCs suspensions were characterized for their size distribution and surface charge which plays a crucial role in cellular uptake within tumoral cells, prior to the \u003cem\u003ein vivo\u003c/em\u003e experiments. The obtained results revealed that TQ-LNCs have been successfully formulated with a mean diameter above 60 nm, a relatively neutral zeta potential charge (-3.8 mV) and with a narrow distribution (PDI\u0026thinsp;\u0026lt;\u0026thinsp;0.2). As shown also in our experiment, no significant changes were recorded in the hydrodynamic diameter of LNCs due to TQ loading compared to that of the unloaded LNCs. These physicochemical propreties are promising for a high tumor cells uptake since nanoscale size (150\u0026ndash;200 nm). While a size less then 100 nm decrease the recognize of phagocytic system and consequently the destruction of nanocarriers (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Numerous studies confirm that nanoparticles with near-neutral surface charge exhibit superior tumor accumulation and reduced opsonization by serum proteins, extending circulation time. This stealth-like property minimizes nonspecific interactions, facilitating direct membrane traversal or energy-independent internalization pathways without reliance on receptor-mediated endocytosis.​ Consequently, the physicochemical profile of TQ-LNCs\u0026mdash;optimal size and neutral zeta potential\u0026mdash;renders them highly suitable for effective cancer therapy by enhancing targeted drug delivery and cellular penetration (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Moreover the TQ-loaded LNCs was tested for their cytotoxicity effect on murine CRC cells, CT26. The biocompatibility of LNCs was investigated on CT26 cells at the higher concentration 40 \u0026micro;M. The IC50 values of CT26 cells after 24 h incubation with free TQ and TQ-loaded-LNCs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. It can be found that the IC50 value of the TQ loaded LNCs for CT26 cells was 5 \u0026micro;M, which was a three fold less than TQ alone (15 \u0026micro;M) after 24 h incubation. It is concluded that the TQ-loaded nanoparticles exhibited significantly higher cytotoxicity for CT26 cells owing to the sustained drug release manner. The prolonged circulation time of nanoparticles is one of the most important parameters which must be improved to obtain efficient drug delivery. In this regard, several studies were focused on surface functionalization of nanoparticles using stabilizing agents such as polyethyleneglycol (PEG), dextran, or chitosan. These coating agents obviously prevented the nanoparicle aggregation and showing a better colloidal stability in biological media and long-time residence in the bloodstream circulation (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). PEG chains at the surface of our formulated nanocarrier conferring a steric stabilization and make them a potential candidate as a dug delivery system of TQ. Beyond nanoparticle size and surface functionalization, abnormalities in the tumor microenvironment significantly enhance nanocarrier retention through the enhanced permeability and retention (EPR) effect (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Tumor-associated defective angiogenesis results in blood vessels with large fenestrations (150\u0026ndash;200 nm), far wider than those in normal tissue, which allows nanoparticles smaller than 200 nm to extravasate more easily into the tumor interstitium. Additionally, the impaired lymphatic drainage common in tumors, combined with rapid cancer cell proliferation, not only promotes accumulation of nanoparticles but also facilitates the sustained release of their therapeutic payloads within tumor tissue, as observed for various nanoparticle systems (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). On the other hand, the antitumor efficacy of TQ-LNCs has been investigated on mice bearing the colorectal cancer CT26 cell line following intraperitoneal injection of a dose of TQ-LNCs (at 10 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e body weight). Fifteen days following the treatment, mice treated with TQ-LNCs showed a more drastic tumor-growth inhibition of up to 86% compared with control group without any body-weight loss, evidencing their anticancer efficacy. In our previous study, our results revealed that intratumoral administration of TQ-LNCs (5 mg/kg) yielded to 86% tumor growth inhibition in subcutaneous CT26 colorectal cancer xenografts (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). This inhibtion of tumor rate was signficantly higher than TQ alone, without body weight loss or organ toxicity. Histopathology revealed extensive necrosis and reduced viable cells, attributed to enhanced TQ internalization, P-gp inhibition by shell components (Kolliphor\u003csup\u003e\u0026reg;\u003c/sup\u003e HS15). Elevated tumor cytokines (GM-CSF, TNF-α) indicated immune modulation, aligning with TQ's genotoxic effects across cancers (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).​ Despite shifting to systemic intraperitoneal administration (10 mg/kg), TQ-LNCs elicited even similar antitumor activity to intratumoral dosing, overcoming free TQ's bioavailability limitations noted previously without LNCs (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). This enhancement stems from extended circulation time, EPR-driven accumulation, and endocytosis-mediated uptake, with neutral charge minimizing protein opsonization. Such route flexibility addresses intratumoral injection challenges like incomplete coverage in heterogeneous tumors.​ According to the histopathological assessment, cell necrotic areas increased significantly in tumor tissues of mice treated with TQ-LNCs compared to those treated with the control group. Meanwhile treatment with free TQ induces less necrosis cells as compared to TQ-LNCs, suggesting lower pharmacological effectiveness probably due to its lipophilicity, which limits its affinity for the tumoral microenvironment and favored its rapid degradation and systemic elimination. The ameliorated anticancer activity was also observed with improved the drug delivery into the tumor cells, and thus equivalent TQ anticancer activity could be obtained via systemic administation garanting lower drug concentrations. This can be explained by long circulation time of TQ in blood stream and then the enhanced absorption of TQ-LNCs in tumor via EPR mechanisms. Once in tumor microenvirment, Thus, LNCs could enhance the resorption of TQ within tumor cells via endocytosis mechanisms (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), improving its cytotoxic effect. These results are in accordance with the previous findings (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Collectively, TQ-LNCs appear to form a better delivery system than free TQ in colorectal cancer treatment without observable toxicity to the other organs, thus improving the efficacy and the therapeutic index of the drug. This proves how the design of TQ-LNCs has gained increasing interest as a means of improving the treatment of neoplastic diseases. Regarding the toxicity, all studied parameters, including the healthy behavior of animals and absence of toxicity in the kidneys, spleen, and liver, lung, heart and brain confirm the biocompatibility of TQ-LNCs. These results clearly confirm the interest in the use of these non-toxic LNCs as a nanocarrier for drug delivery for biomedical applications, notably in cancer therapy. Collectively, these results demonstrate that LNCs encapsulation substantially improves TQ\u0026rsquo;s bioavailability by prolonging its systemic circulation and reducing clearance, which could translate into enhanced therapeutic efficacy and reduced dosing frequency. This enhancement highlights the potential of TQ carriers to optimize the delivery and performance of TQ after intraperitoneal administration with prolonging the circulation time of encapsulated drugs by protecting them from rapid metabolism and clearance (\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conslusion","content":"\u003cp\u003eLNCs, which combine exceptional physicochemical propreties and high drug encapsulation capacity, are a promising safe and efficient drug delivery system. Upon intraperitoneal administration, LNCs loaded with TQ were able to drastically enhance anticancer efficay of TQ, and minimizing associated tissue toxicity and avoids histological damage. These results open fascinating perspectives for the safe and efficient drug delivery system using biocompatible nanomaterials like LNCs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eInformed Consent Statement\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Institutional Review Board Statement: Not applicable.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.S., F.J. H.M and M.S. performed the experiments and analyzed the data; M.S., A.S., supervised experiments. M.S wrote the manuscript. A.T. supervised HPLC experiments. M.L. supervised characterization experiments of LNCs. L.C.G supervised the overall project. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe work was supported by the Tunisian Ministry of Higher Education and Scientific Research (TMHESR).\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e\u003cp\u003eThe authors declare that all data supporting the findings of this study are available within the article\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGlobal cancer burden growing. amidst mounting need for services [Internet]. [cit\u0026eacute; 12 oct 2025]. 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RSC Adv. 2016;6(103):101688\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Thymoquinone, Lipid nanocapsules, Colorectal cancer, Drug delivery","lastPublishedDoi":"10.21203/rs.3.rs-8239390/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8239390/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) remains a major cause of cancer-related mortality worldwide, with limited treatment options at advanced stages. Thymoquinone (TQ), a natural bioactive compound derived from \u003cem\u003eNigella sativa\u003c/em\u003e, has demonstrated significant anticancer potential but is limited by poor aqueous solubility and low bioavailability. In this study, TQ was successfully encapsulated into lipid nanocapsules (LNCs) to overcome these limitations and enhance its anticancer efficacy after intraperitoneal administration. The LNCs-TQ exhibited a mean diameter of ~60 nm with a high encapsulation efficiency exceeding 85%. Their antiproliferative activity was evaluated \u003cem\u003ein vitro\u003c/em\u003e against murine colorectal carcinoma CT26 cells, showing that TQ significantly reduced cell viability compared to free TQ (IC₅₀ : 5 µM vs. 15 µM). \u003cem\u003eIn vivo\u003c/em\u003e, TQ-LNCs has significantly suppressed tumor growth in a CT26 subcutaneous xenograft mouse model, with a tumor growth inhibition rate of up to 85%. Comet assay and histopathological analysis confirmed enhanced DNA damage and extensive tumor cell necrosis. These results suggest that LNCs-TQ improved the delivery and potency of TQ, making it a promising adjuvant strategy in colorectal cancer therapy.\u003c/p\u003e","manuscriptTitle":"Thymoquinone-Loaded Lipid Nanocapsules Improve Anticancer Efficacy Following Intraperitoneal Administration in a CT26 Xenograft Model of Colorectal Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 13:03:12","doi":"10.21203/rs.3.rs-8239390/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af574931-39da-4d4a-895c-caca47b190fb","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-12T13:39:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 13:03:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8239390","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8239390","identity":"rs-8239390","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}