Development and Optimization of PEGylated Darolutamide-Loaded Liposomes for Treatment of Prostate Cancer: In vitro and In-vivo Characterization

Development and Optimization of PEGylated Darolutamide-Loaded Liposomes for Treatment of Prostate Cancer: In vitro and In-vivo Characterization | 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 Development and Optimization of PEGylated Darolutamide-Loaded Liposomes for Treatment of Prostate Cancer: In vitro and In-vivo Characterization Vinod Patil, Harshal Pawar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7203702/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Journal of Pharmaceutical Innovation → Version 1 posted You are reading this latest preprint version Abstract The present study aimed to develop a darolutamide (DRM) loaded PEGylated liposomal (DRML) to improve therapeutic efficacy in the treatment of prostate cancer. A 3² full factorial design was employed to evaluate the influence of Hydrogenated Soy Phosphatidylcholine (HSPC) and cholesterol concentrations on key formulation parameters, including particle size and entrapment efficiency (EE). The optimized PEGylated DRM liposomal formulation (DRML-9) was further characterized by transmission electron microscopy (TEM) for morphology, X-ray diffraction (XRD) for crystallinity, and differential scanning calorimetry (DSC) for thermal behavior. Among 13 liquid formulations (PDL–1 to PDL–13), PDL–9, comprising 15 mg HSPC, 35mg cholesterol, exhibited optimal characteristics, including particle size (87.8 ± 3.25) and higher entrapment (95.57 ± 1.67%). FTIR spectra confirmed drug-excipient compatibility, and DSC as well as XRD data indicated the amorphization and molecular dispersion of DRM in the liposomal formulation. The DRML-9 formulation exhibited superior drug release (71.56 ± 3.48%) at tumor pH compared to blood pH (16.35 ± 1.17%). DRML-9 significantly inhibited LNCaP prostate cancer cell proliferation (P < 0.001) and induced apoptosis as evidenced by Annexin-V/PI staining. Cell cycle study in DRML resulted in almost 2-fold increase in S-phase arrest compared to DRM alone. Pharmacokinetic evaluation demonstrated a 3.6-fold improvement in intravenous bioavailability of PEGylated DRML over pure DRM. Collectively, these findings establish PEGylated DRML-9 as a promising intravenous delivery platform for improving the therapeutic potential of darolutamide in treatment of prostate cancer. Darolutamide Liposomes Cancer Pharmacokinetic study Apoptosis and Cell cycle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background The global burden of cancer continues to rise at an alarming rate, with an urgent need for ideal therapeutic strategies that can effectively manage this complex disease. Prostate cancer (PC) ranks as the second most frequently diagnosed malignancy and the fifth leading cause of cancer-related deaths in men globally, with approximately 1.46 million new cases and 3.96 million fatalities reported in 2022 [1]. Alarmingly, projections indicate that by 2040, the global incidence of PC will rise to approximately 2.4 million cases, with associated deaths reaching 7.12 million, primarily driven by population aging and growth [2]. Therapeutic strategies for PC typically include surgery, radiotherapy, chemotherapy, and hormone therapy [3–5]. Despite advancements in these approaches, limitations persist. Radiotherapy, while effective, often suffers from low specificity, leading to damage of surrounding healthy tissues when higher doses are administered to achieve therapeutic efficacy. Such treatments may lead to undesirable side effects, including bone pain, fatigue, gastrointestinal issues, hematological toxicity, as well as reductions in platelet and white blood cell counts, primarily due to damage to the surrounding bone marrow [6]. Chemotherapy is associated with systemic side effects, including alopecia, mucositis, anorexia, gastrointestinal disturbances, and profound fatigue related to anemia [7]. Hormone therapies, aimed at reducing testosterone levels, often lead to significant side effects, including hot flashes, sexual dysfunction, fatigue, weight gain, muscle loss, bone demineralization, and gynecomastia [8]. Darolutamide (DRM) is a next-generation androgen receptor inhibitor approved for treating non-metastatic castration-resistant and metastatic hormone-sensitive prostate cancer [9]. However, the clinical utility of darolutamide is hampered by its poor aqueous solubility, incomplete oral absorption, and low bioavailability [10]. Furthermore, its use has been associated with adverse cardiac events, including arrhythmias, coronary artery disease, hypertension, and heart failure [11]. Liposomal drug delivery systems, comprising microscopic lipid-based vesicles, have emerged as a promising approach to overcome the limitations of conventional drug delivery. These systems utilize the amphiphilic, biocompatible, and self-assembling properties of lipids to encapsulate therapeutic agents, including hydrophobic small molecules, peptides, and nucleic acids, thus improving drug solubility, stability, and targeted delivery [12]. Liposomal formulations offer several advantages, including enhanced bioavailability, prolonged systemic circulation, reduced systemic toxicity, and controlled or site-specific drug release [13–14]. Notably, nanoparticulate drug delivery systems such as liposomes exhibit superior pharmacokinetics and pharmacodynamics, widening the therapeutic window and minimizing off-target toxicities [15–16]. Incorporation of functional lipids, such as phospholipids and cholesterol, has shown added benefits in improving liposome stability, drug entrapment efficiency, and reducing drug leakage, as demonstrated in our previous studies [17–18]. Cholesterol, in particular, enhances membrane stability, reduces haemolysis, improves entrapment efficiency, and regulates drug release kinetics, thereby enhancing the overall therapeutic potential of liposomes. Additionally, functionalizing liposomes with ligands like antibodies, peptides, or aptamers allows active targeting of prostate-specific membrane antigen (PSMA), which is highly overexpressed on prostate cancer cells, enhancing therapeutic specificity [19]. Liposomal encapsulation also protects hydrophobic drugs from premature degradation in the systemic circulation, promoting prolonged drug residence time and sustained therapeutic release [20]. Given these advantages, the present research aims to develop and optimize darolutamide-loaded liposomes (DRM-liposomes) to enhance their bioavailability and anticancer efficacy. A 3² full factorial design was utilized to systematically develop and optimize DRM-loaded liposomes. The optimized formulation underwent detailed characterization, including particle size, zeta potential, entrapment efficiency, and in vitro drug release. Additionally, it’s in vitro-anticancer efficacy was assessed through studies on apoptosis induction and cell cycle arrest, followed by in-vivo pharmacokinetic evaluation to determine its therapeutic potential for prostate cancer treatment Materials and Methods Materials Darolutamide has been acquired from Clearsynth Andheri West, Mumbai, India. Cholesterol and HSPC were procured from Sigma Aldrich India Pvt. Ltd. Mumbai, India. DSPE-PEG2000 was obtained from TCI Chemicals Japan. Methanol and Milli-Q water, were procured from Unique Biologicals Kolhapur, India. All other chemical, and reagents used in current research work, are of analytical grade. Cell Culture The NCCS (National Centre for Cell Science), located in Pune, India, provided human prostate cancer cell lines DU-145, PC-3, LNCaP. Cells have been cultivated in DMEM (Dulbecco's Modified Eagle's Medium), which has been improved with one percent penicillin-streptomycin as well as 10% FBS (fetal bovine serum). Cultures have been kept at 37°C with five percent CO₂ in a humidified incubator. Preparation and optimization of PEGylated Liposomes PEGylated Darolutamide-loaded liposomes (DRML) were formulated utilizing thin-film hydration method. Briefly, to accomplish full solubilization, 50 mg of DRM and liposomal components—namely, HSPC, cholesterol, and DSPE-PEG2000—were carefully weighed and dissolved in 15 mL of a methanol: chloroform mixture (1:2, v/v) using bath sonication. Organic solvents were later evaporated at 65 ± 2°C using rotary evaporator (BUCHI Rotavapor R-200, BUCHI India Private Ltd, Mumbai, India), leading to the formation of thin lipid layer on the inner surface of the round-bottom flask. The lipid film was additionally vacuum-dried for an entire night in order to remove any remaining solvents. Finally, dried lipid film has been hydrated with 40 mL of SWI (sterile water for injection) at 70 ± 2°C for fifteen minutes with gentle agitation to obtain multilamellar vesicles (MLVs). The MLVs were further subjected to probe sonication (LABMAN Pro-656) to reduce the vesicle size and produce small unilamellar vesicles (SUVs). Probe sonication was performed for five cycles of one minute each, with each cycle consisting of 12 sec. of sonication at 240 V and 0.6 A, followed by an 8-second pause [21–22]. 2.4. Experimental design A 3² full factorial design has been utilized for the systematic development and optimization of PEGylated DRML with Design Expert® software (Version 7.0.0, Stat-Ease Inc., USA). Two independent variables, X₁ (HSPC amount) and X₂ (cholesterol amount), were studied at three levels (-1, 0, + 1) to evaluate their effect on Y₁ (particle size) and Y₂ (entrapment efficiency, %EE). A total of 13 experimental runs were generated as per the design (Table 1 ), and all formulations have been evaluated for particle size and %EE. The responses have been fitted to various models (linear, quadratic, 2FI, cubic), the best-fit model has been selected depending on ANOVA results, considering R², adjusted R², predicted R², and p-values. Response surface plots, contour plots, and perturbation graphs were generated to visualize the effect of variables. The optimized formulation was identified using numerical and graphical optimization with a desirability function, targeting minimum particle size and maximum %EE. An overlay plot indicated the optimal region, and the optimized formulation was prepared to validate the model by comparing predicted and experimental responses. Table 1 Full factorial Design matrix summarizing the levels, factors, and responses of 13 runs for optimization of Darolutamide loaded liposomes. Batch code X 1 X 2 Y 1 Y 2 PDL–1 10 25 103.27 ± 2.25 83.8 ± 1.75 PDL − 2 10 15 92.24 ± 1.54 81.24 ± 1.67 PDL − 3 5 25 165.32 ± 4.25 72.58 ± 2.68 PDL − 4 15 15 58.12 ± 0.67 87.25 ± 2.68 PDL − 5 15 25 76.84 ± 2.19 91.10 ± 3.18 PDL − 6 10 25 102.25 ± 2.24 84.12 ± 1.18 PDL − 7 5 15 142.9 ± 3.47 68.25 ± 1.85 PDL − 8 10 25 101.25 ± 2.37 62.34 ± 1.67 PDL–9 15 35 87.8 ± 3.25 95.57 ± 1.67 PDL–10 10 35 126.57 ± 3.24 85.34 ± 1.58 PDL–11 10 25 101.98 ± .25 83.67 ± 1.98 PDL–12 10 25 102.12 ± 1.25 82.92 ± 1.27 PDL–13 5 35 183.12 ± 4.95 77.35 ± 2.27 Factor (Independent variables) Actual levels Low (-1) Medium (0) High (+ 1) Factor X1: Amount of HSPC (mg) 5 10 15 Factor X2: Amount of Cholesterol (mg) 15 25 35 Dependent variables or Responses Constraint Response 1: Particle size (Y1) (nm) Minimize Response 2: Entrapment efficiency (Y2) (%) Maximize Evaluation of Liposomes Containing DRM Particle size and zeta potential Particle size as well as zeta potential of optimized PEGylated DRML have been determined by utilizing a DLS (dynamic light scattering) technique with particle size analyzer (Malvern Zetasizer, Version 7.11, Malvern Instruments Ltd., United Kingdom). The analysis has been performed at controlled temperature of 25 ± 1°C, with cell drive voltage maintained at 150 mV. To prevent numerous scattering effects, the liposomal dispersion was suitably diluted with double-distilled water for every measurement. Three independent samples of the optimized formulation were analyzed to determine mean particle size and zeta potential. Results have been recorded as mean ± SD [23]. Entrapment efficiency : Entrapment efficiency (%EE) of PEGylated DRML has been determined utilizing an indirect centrifugation technique. Briefly, liposomal dispersion has been subjected to centrifugation at 10,000 rpm for one hour to separate unentrapped (free) drug present in the aqueous phase. After centrifugation, the supernatant containing the free drug was carefully collected and appropriately diluted with methanol. Concentration of free Darolutamide (DRM) in supernatant has been quantified utilizing UV-Visible spectrophotometer at 286 nm, employing methanol as the blank.[24] Surface Morphology Morphological characteristics of optimized PEGylated DRML have been examined by utilizing TEM (Transmission Electron Microscopy). After the extra fluid has been removed, 5 µL of liposomal dispersion has been carefully applied to a 300-mesh carbon-coated copper grid. Grid has been allowed to stand for a few minutes to enable sample adherence. Subsequently, the sample was air-dried and negatively stained with 1percent (w/v) uranyl acetate for 3 to 5 min. to improve contrast. After drying, grid was observed under Transmission Electron Microscope (JEOL-JEM 1400, USA) to visualize shape, size, structural integrity of liposomes [25]. In vitro drug release study Drug release profile of DRM has been examined at pH 7.4 (physiological pH), pH 5.8 (tumor microenvironment pH) using the dialysis bag method in a USP Type II (paddle) apparatus at 37 ± 2°C. Briefly, DRML dispersions containing an amount equivalent to 5 mg of drug have been placed into dialysis bags (LA390-10mt-HiMedia), securely sealed, attached to the paddle, and immersed individually into vessels containing 100 mL of PBS (phosphate buffer saline) at pH 7.4 or 5.8, each supplemented with 5% (v/v) methanol [30]. The media were continuously stirred at 100 rpm while being kept at 37°C. At predetermined time intervals (0, 1, 2, 4, 6, 12, 24 h), 3 mL samples were withdrawn and analyzed by utilizing UV-visible spectrophotometer at 286 nm. Fresh buffer has been added to the withdrawn volume in order to preserve sink conditions. The % of drug released was calculated and plotted as a function of time [26] Lyophilization of optimized PEGylated Liposomes The optimized PEGylated Darolutamide-loaded liposomes (DRML) were subjected to lyophilization to enhance their long-term stability. Prior to freeze-drying, trehalose was used as a cryoprotectant at varying weight ratios with respect to the lipid content to protect the liposomal structure during freeze-drying process. Under carefully monitored conditions, a liposomal dispersion including cryoprotectant was frozen and lyophilized utilizing Martin Christ Alpha 1–2 LD Plus freeze dryer (Martin Christ GmbH, Germany). Resulting lyophilized powder has been collected and stored in airtight containers for further characterization.[27] 6.4 Compatibility study: 2.11. Four Transform Infrared Analysis (FTIR) Bruker Alpha-II FTIR spectrophotometer has been used to record FTIR spectra of DRM, HSPC, Cholesterol, and PEGylated DRML along with liposomal formulation excipients in a range of 4000 to 650 cm − 1 frequency.[28] 2.12. Differential Scanning Calorimeter (DSC) Analysis DSC thermograms (DSC-60, Shimadzu, Japan) of DRM, HSPC, Cholesterol, and PEGylated DRML, along with liposomal formulation excipients, were performed to examine changes in their thermal behavior.[29] 2.13. Powder X-ray diffraction (P-XRD) The crystalline characteristics of DRM and the lyophilized PEGylated DRM-liposomal formulation were examined using P-XRD (Powder X-ray diffraction) with Philips diffractometer (Netherlands) operated at 40 kV or 30mA. Samples have been scanned over 2θ range of 10–80°, with step size of 0.020°, as well as scanning speed of one second per step, under ambient temperature conditions [30] In vitro anticancer activity Cytotoxicity MTT assay was utilized to assess cytotoxic potential of DRM and improved PEGylated DRML formulation against human prostate cancer cell lines (LNCaP, DU-145, PC-3). To enable adhesion, cells have been sown in 96-well plates or treated for the entire night at 37°C. Subsequently, cells have been exposed to varying concentrations of test compounds for 48 hours. After treatment, test solutions have been removed, and 100 µL of MTT solution (6 mg /10 mL in PBS) has been added to each well. Plates have been incubated for an additional 4 h under identical conditions. One hundred microliters of DMSO have been added to each well to dissolve formazan crystals that have been produced by metabolically active cells. Microplate reader has been utilized to detect absorbance at 570 nm, and dose-response curves have been used to calculate IC₅₀ values. [31] Apoptosis by Flow cytometer Cells have been seeded, and plates were subjected to heat at 37°C overnight. Cells have been incubated with 50µg/mL of DRM or optimized DRML for 24h. Later, cells were subjected to centrifugation (Remi-CM8 Plus, India) for about 5 min at 4°C. Following collection, the cell pellets (1 × 10¹/mL) were meticulously cleaned using 2 mL of 1X PBS. After setting the tubes on ice, 5µL of propidium iodide and 1µL of annexin-V fluorescein isothiocyanate were added. Tubes were then left to incubate for 15 min. In less than half an hour, tubes were examined by utilizing flow cytometer following the addition of 400 µL of cold buffer.[32] Apoptosis by DAPI Each well of 24-well plate has been seeded with 1 × 10⁴ cells, and cells were then allowed to adhere by being incubated in a CO₂ incubator for the entire night at 37°C. DRM and the optimized PEGylated DRML formulation were subsequently added to the cells at concentrations less than 50 µg/mL, and they were incubated for 24 h under the same circumstances. Following treatment, the cells underwent a 30-min. dark incubation period before being rinsed with PBS. Then, each well received 20 µL of DAPI staining solution (0.1 µg/mL). The proportion of apoptotic cells was determined by randomly observing and counting cells displaying characteristic apoptotic features under a fluorescence microscope [33] Cell cycle study After cells have been seeded in 24 wells of micro titer plate with flat bottoms containing a cover slip and kept for incubation in CO 2 at 37°C for duration of experiment, for 48h, cells added 10µL/mL of the developed formulation. After the cells have been incubated for appropriate time, they were centrifuged for 5 min. at a rate of 4°C. Supernatant has been removed by centrifugation, and cell pellets were subsequently mixed with ice-cold 1X binding buffer at concentration of 1x105/mL. After gently mixing cells, incubate them for 15 min. and then treat them with approximately 10µL of propidium iodide. The tubes should be placed on ice right away. In addition, a 1X binding buffer with a concentration of 400µL/mL has been thoroughly mixed and stirred before being subjected to analysis in a flow cytometer within the next half an hour.[34] 3. In-Vivo Pharmacokinetic Study Study protocol has been accepted by Institutional Animal Ethics Committee (IAEC/BiRD/Sangli/2024-25/13). Male Wistar albino rats (n = 9; 180 to 220g) were obtained from Crystal Biological Solutions, Pune, India. Animals had been housed under standard laboratory settings, having free water supply as well as food for two weeks prior to study. Rats had been divided at random into three groups such as Group I (Control): Received normal saline, Group II (Standard): Pure DRM administered 5mg/kg suspended in PEG-400 (40%), Ethanol (10%) and Sterile saline (50%) and Group III(Test): Administered PEGylated DRML-9 intravenously at a dose equal to 5mg/kg of DRM. Blood samples (500µL) had been gathered from retro-orbital plexus at predefined time intervals (0.5 to 24 h) post-dosing into heparinized tubes. Plasma has been separated using centrifugation at 700 rpm for 10 min, alongside kept until analysis at -20°C. DRM concentration in plasma has been determined utilizing a validated HPLC technique with a Shimadzu LC-2030C 3D Plus system, equipped with Shimpack GIST C18 column along with mobile phase delivered at 1mL/min. Pharmacokinetic parameters, including Cₘₐₓ, Tₘₐₓ, AUC₀–∞, AUMC₀–∞, and MRT, were calculated using non-compartmental analysis [35]. 2.6. Stability Study A stability study of the optimized PEGylated DRML was carried out. The LPs formulations containing 5% of sucrose as a cryoprotectant. The formulation was also stored at refrigeration (2–8°c). The prepared batch were tested for particle size and % EE after 1,2 and 3 months.[36]. Statistical Analysis Data are presented as mean ± standard deviation (SD). Statistical comparisons between groups were conducted using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Analyses were performed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). A p-value of < 0.05 was considered statistically significant. [36] Results and Discussion Fitting of data into the model : To select the most suitable model, experimental responses from the 13 formulations were analyzed using Design-Expert® software. Models were compared based on higher R², adjusted R², and predicted R² values, along with lower standard deviation (SD), coefficient of variation (% CV), and predicted residual sum of squares (PRESS) (Table 2 ). A lower PRESS value reflects better model predictability. Based on these criteria, the quadratic model best fit particle size data, while the linear model was optimal for entrapment efficiency (%EE). Table 2 Regression analysis results obtained for various responses Y 1 (particle size) and Y 2 (% entrapment efficiency) of darolutamide loaded liposomes for fitting to different models Models SD R 2 Adjusted R 2 Predicted R 2 PRESS CV (%) Remark Response (Y 1 ) Linear 2FI Quadratic Cubic 9.08 9.40 3.31 3.89 0.9438 0.9457 0.9948 0.9948 0.9326 0.9276 0.9910 0.9876 0.9004 0.8300 0.9649 0.4168 1459.75 2492.06 513.94 8547.98 8.17 8.47 2.98 3.50 Suggested Response (Y 2 ) Linear 2FI Quadratic Cubic 6.34 6.68 7.37 8.65 0.5960 0.5961 0.6170 0.6240 0.5152 0.4615 0.3434 0.0975 0.5076 0.4743 0.3308 -0.6026 489.51 522.61 665.22 1593.08 7.80 8.23 9.08 10.65 Suggested SD: standard deviation, R 2 : multiple correlation coefficient, 2FI: two factor interaction, PRESS: predicted residual sum of square, CV: coefficient of variation. Effect of independent variables on particle size (Y 1 ) of PEGylated DRM liposomes The particle size of PEGylated DRM liposomes formulations is displayed in Table 1 . Particle size has been found in the range of 87.8 ± 3.25 to 183.12 ± 4.95 nm. The following quadratic equation describes how the independent variables affect the particle size. Y 1 = + 103.60–44.76X 1 + 17.37X 2 -2.63 X 1 X 2 + 13.92X 1 2 + 2.25X 2 2 Where Y 1 is particle size, X 1 is the amount of HSPC, and X 2 is the amount of cholesterol. The equation indicates that amount of HSPC has a negative effect and amount of cholesterol has a positive effect on particle size. This demonstrates that particle size PEGylated DRM-loaded liposomes decreases with increase in the amount of HSPC, while it increases with increase in the amount of cholesterol added. High coefficient value for X₁ indicates that amount of HSPC exerts more pronounced influence on particle size of DRM-loaded liposomes in comparison to amount of cholesterol. The ANOVA results for particle size are displayed in Table 3 . Model's F-value of 266.53 suggests that model is highly significant, with only 0.01% probability that like high F-value could be attributed to random variation. A p-value (Prob > F) < 0.05 confirms statistical significance of model terms. In the present analysis, X₁, X₂, and interaction term X₁₂ were found to be significant contributors. Predicted R² value is 0.9649 demonstrates good agreement with Adjusted R² is 0.9910, reflecting model's robustness. Adequate Precision value, that reflects signal-to-noise ratio, was 55.30, exceeding the desirable threshold of 4, confirming an adequate signal. As a result, the created model can be trusted to explore and optimize the formulation design space (Table 2 ). Table 3 ANOVA results for various responses of darolutamide loaded liposomes Source Responses Y 1 (Particle Size) Y 2 (% Entrapment efficiency) F-value p-value Prob > F Adequacy precision F-value p-value Prob > F Adequacy precision Model 266.53 < 0.0001 55.301 7.38 0.0108 8.437 X 1 1098.86 < 0.0001 12.85 0.0050 X 2 165.49 < 0.0001 1.90 0.1977 X1X2 2.54 0.1551 X 1 2 48.92 0.0002 X 2 2 1.27 0.2963 X 1 , and X 2 are coded terms for independent variables; X 1 X 2 interaction terms; X 1 2 and X 2 2 are quadratic terms The influence of the independent variables on particle size is visually illustrated through 3D response surface plots (Fig. 1 (A)) and perturbation plots (Fig. 1 (B)). These graphical representations clearly show that increasing the amount of HSPC from 5 to 15 mg leads to a reduction in particle size, whereas increasing the amount of cholesterol from 15 to 35 mg results in an increase in particle size. Increasing the amount of HSPC provides more amphiphilic molecules that can self-assemble into smaller and more stable vesicles due to better packing and curvature stabilization. [37] Higher HSPC concentration enhances bilayer rigidity and reduces surface energy, promoting the formation of smaller unilamellar vesicles during sonication or extrusion. [38] As cholesterol content increases, it stabilizes the bilayer but also reduces its curvature, favoring the formation of larger and more rigid liposomes. [39] At high cholesterol levels, the bilayer becomes more condensed and thicker, resisting size reduction during mechanical processing. [40] The perturbation plot (Fig. 1 (B)) for particle size further validated these findings. The plot shows a steeper slope for factor A (HSPC) compared to factor B (cholesterol), indicating that the amount of HSPC has a more substantial impact on particle size than cholesterol. Effect of independent variables on % entrapment efficiency (Y 2 ) of PEGylated DRM loaded liposomes The % EE of DRM loaded liposomes formulations are shown in Table 1 . The % EE was found in the range of 62.34 ± 1.67 to 95.57 ± 1.67%. The effect of the independent variables on the % EE can be explained by the following linear equation. Y 2 = + 81.20 + 9.27X 1 + 3.57X 2 (2) Here, Y₂ represents the percentage entrapment efficiency (%EE), while X₁ and X₂ correspond to the amounts of HSPC and cholesterol, respectively. The equation suggests that both HSPC and cholesterol concentrations positively influence %EE, indicating that increasing either component leads to enhanced entrapment efficiency of the PEGylated DRM-loaded liposomes. The higher coefficient for X₁ reflects the greater impact of HSPC on %EE compared to cholesterol. The ANOVA results for %EE are shown in Table 3 . The model's F-value of 7.38 confirms its statistical significance, with only a 0.01% chance of such a result occurring randomly. A p-value below 0.05 indicates that the model terms, particularly X₁, are significant. The predicted R² (0.5076) shows acceptable alignment with the adjusted R² (0.5152), while the adequate precision ratio of 8.437 reflects a good signal-to-noise ratio (Table 2 ). These results confirm the suitability of the quadratic model for exploring the design space. The 3D response surface and perturbation plots illustrating the effect of independent variables on %EE are shown in Fig. 1 C. These plots illustrate that %EE increases with higher concentrations of HSPC (5 to 15 mg) and cholesterol (15 to 35 mg). Increasing HSPC concentration means more bilayer material is available to form liposomes, resulting in more drug can be accommodated either in the aqueous core (hydrophilic) or in the lipid bilayer (hydrophobic). [41] Cholesterol stabilizes the bilayer by filling the spaces between phospholipid tails. Especially for hydrophobic drugs, cholesterol increases bilayer thickness and hydrophobic volume, enhancing drug incorporation. [42] A perturbation plot is a useful tool to assess the influence of individual independent variables on the response. The perturbation plot for %EE (Fig. 1 D) further supports the trends observed in the 3D response surface plots. Optimization A numerical optimization approach was used to identify the optimized PEGylated DRM-loaded liposome formulation within the design space by setting constraints to minimize particle size and maximize entrapment efficiency (%EE). The software suggested an optimized formulation containing 15 mg of HSPC and 24.07 mg of cholesterol, with a desirability value of 1. The overlay plot illustrates the optimized formulation and its predicted response values (Fig. 1 E). The optimized formulation of PEGylated DRM-loaded liposomes was prepared and experimentally evaluated for the selected responses. The predicted and actual response values, along with their percentage prediction errors, are presented in Table 4 . The close agreement between these values confirms the success and reliability of the design and optimization process. The % prediction errors ranged from − 3.05 to 1.52%, indicating the robustness and validity of the QbD approach applied for optimizing PEGylated DRM-loaded liposomes. Table 4 Validation of optimized formulation Response Predicted value Observed value Prediction error (%) Particle size (nm) 71.40 73.58 − 3.05 EE (%) 90.13 88.76 1.52 Evaluation of DRM loaded Liposomes Particle size and Zeta Potential The optimized PEGylated liposomal formulation, DRML-9, displayed the smallest vesicle size among all tested formulations, with an average diameter of 87.8 ± 3.25 nm (Fig. 2 A). Nanoparticles within this size range are generally considered favorable for drug delivery, as they can facilitate enhanced permeability and retention (EPR) effects, promote cellular internalization, and improve biodistribution.[43] Smaller vesicles also increase the surface area-to-volume ratio, which enhances drug loading and interaction with biological membranes. Zeta potential analysis was specifically carried out for DRML-9 to assess its colloidal stability.[44] The observed zeta potential of − 30.4 mV (Fig. 2 B) indicates that the formulation possesses a sufficient negative surface charge to prevent particle aggregation through electrostatic repulsion. A zeta potential magnitude greater than ± 30 mV is typically regarded as indicative of good physical stability, which is essential for maintaining the uniformity and shelf life of nanoparticulate systems. The targeted characterization of DRML-9 in terms of particle size and surface charge highlights its promising physicochemical attributes, supporting its potential for efficient and stable therapeutic delivery.[45] Surface Morphology by TEM Transmission Electron Microscopy (TEM) analysis was conducted to confirm the formation of PEGylated DRM liposomes and to investigate their surface morphology and structural characteristics. The TEM images (Fig. 2 C) revealed the presence of spherical to nearly spherical unilamellar vesicles with moderate size distribution. The liposomes exhibited well-defined boundaries, indicating successful formation of intact bilayer structures. No significant aggregation or deformation was observed, suggesting good colloidal stability of the formulation. [46] The absence of aggregation is a positive indication of the colloidal stability of the PEGylated liposomes. PEGylation is known to impart steric stabilization to liposomes, reducing inter-vesicular interactions and preventing fusion or aggregation during storage or in biological environments [47]. Nanoscale and uniform size distribution visualized in the TEM micrographs suggests controlled vesicle formation, which is essential for predictable pharmacokinetic behavior and biodistribution. FTIR Figure 3 A presents the FTIR spectra of DRM, showing characteristic peaks corresponding to the functional groups present in its structure. These peaks include stretching vibrations at 3764.95 cm⁻¹ (-O-H -) alcohol, 3051.45 cm⁻¹ (C-H), 2229.34cm⁻¹ (Nitrile), 1646.82cm⁻¹ (C = N), 1440..69 cm⁻¹ (O-H) carboxylic acid, 1233.30cm⁻¹ (C-N), and (C = C alkene) at 990.63 cm⁻¹, which is attributed to the presence of four neighboring hydrogen bonds on the hetero-aromatic nucleus. These values closely match those observed in the standard DRM structure, confirming the integrity of the functional groups in the DRM. In contrast, the FTIR spectra of optimized PEGylated DRM liposomes (DRML-9) (Fig. 3 D) revealed similar stretching vibrations: 3740.84 cm⁻¹ (-O-H -) alcohol, 3116.11 cm⁻¹ (C-H), 2354.01cm⁻¹ (Nitrile), 1682.29cm⁻¹ (C = N), 1412.34 cm⁻¹ (O-H) carboxylic acid, 1225.81cm⁻¹ (C-N), and (C = C alkene) at 941.44 cm⁻¹,The FTIR analysis shows that the key functional peaks of DRM were retained in the optimized DRML formulation, suggesting that no significant chemical interaction occurred between DRM and the excipients used in the formulation. These results indicate that the drug and excipients are compatible, and the drug’s chemical integrity is preserved in the nanoemulsion formulation [53–54]. DSC Studies : Figure 4 represents the DSC thermograms of DRM, cholesterol, HSPC, and optimized PEGylated DRM liposomes (DRML-9). It is evident that the DSC thermogram of DRM (Fig. 4 A), cholesterol (Fig. 4 B), HSPC (Fig. 4 C) and DRML-9 (Fig. 4 D) exhibits a single sharp characteristic, endothermic melting peak at 169.07°C, 145.51°C, 207.38°C and 233.42°C which is in agreement with” that reported previously. The crystalline state of the DRM is specified by the sharp endotherm. The thermogram of the optimized PEGylated DRML revealed that DRM is molecularly dispersed in DRML; however, DRML did not exhibit the “characteristic endothermic peak from the crystalline DRM. The endothermic melting peak at the 233.42°C corresponds to either HSPC or Cholesterol”.[55] This shows that the DRM that was added to the liposomal formulation was not crystalline, suggesting that the DRM may have been molecularly dispersed within the liposomal matrix or that it may have transformed into an amorphous state. It also proposes that the DRM is entirely trapped within the liposomal lipid matrix.[56] XRD Studies : XRD studies were performed to investigate the physical state of DRM when loaded into liposomes, comparing it to pure DRM, cholesterol, HSPC and lyophilized DRML. The diffractogram of DRM (Fig. 5 A) exhibited characteristic intense reflections at specific diffraction angles, including 79.23, 13.78, 14.94, 16.35, 16.38, 16.42, 16.46, 16.48, 16.50, 16.53, 18.29, 22.75, and 24.90 (2θ), with corresponding intensity counts of 577, 423, 493, 1998,2376, 2155, 2286, 2150, 1932, 1755, 660, 995, and 975. These distinct peaks indicate the crystalline nature of DRM, confirming that it exists in its solid, crystalline form. In contrast, the X-ray diffraction pattern of DRML (Fig. 5 D) showed a significant reduction or complete disappearance of these characteristic crystalline peaks. This observation suggests that DRM has transformed its crystalline state to an amorphous or less crystalline form when incorporated into the liposomal formulation. The loss of sharp diffraction peaks is consistent with the DSC results, indicating a shift in the melting temperature and the disappearance of the crystalline DRM peak in the optimized PEGylated DRML formulation. This transformation is likely due to the dispersion of DRM within the liposomal lipid matrix of, leading to a more amorphous structure [57]. In vitro drug release study DRM exhibited poor, pH-independent release due to its low solubility, whereas PEGylated DRM liposomes showed sustained release at blood pH (7.4) and enhanced, pH-responsive release at tumor pH (5.8) [36]. To maintain sink conditions during the study, 5% (v/v) methanol was added to the release medium [48]. The DRM showed 24.35 ± 2.75% drug release in blood pH when compared to tumor pH showed 29.54 ± 2.62%. The developed PEGylated DRM liposomes exhibited significantly higher drug release of 71.56 ± 3.48% at the acidic tumor pH of 5.8, compared to 16.35 ± 1.17% at the physiological blood pH of 7.4, confirming their pH-sensitive behaviour. This characteristic is advantageous for targeted drug delivery within the tumor microenvironment. These findings further support the hypothesis that acidic conditions promote the destabilization of liposomes and other nanocarriers, resulting in enhanced drug release [49–50]. PEGylation improves the stability and systemic circulation of DRM liposomes by minimizing drug leakage at physiological pH. At tumor pH, the acidic environment can promote destabilization of PEGylated liposomes, resulting in a controlled, targeted burst release of DRM. Anticancer Activity Cytotoxicity Study It is vital to evaluate the cytotoxicity of DRM and optimized PEGylated DRML-9 for their potential application as anticancer agents. The viability of PC-3, LNCaP and DU-145 cells treated with 0.325 to 100 µg/mL exhibited concentration-dependent cytotoxicity. The viability of the PC-3, LNCaP and DU-145 cells was drastically reduced after treatment with DRM and optimized DRML-9 shown in Fig. 6 . DRM and optimized DRML-9 exhibited significant (p < 0.01) cytotoxicity against LNCaP cells (low IC 50 value 9.45 ± 0.87 µg/mL and 5.62 ± 0.38 µg/mL) for PC-3 (14.78 ± 1.12µg/mL and 10.27 ± 0.95µg/mL) and against DU-145 (20.18 ± 1.51µg/mL and 13.86 ± 1.27µg/mL) respectively after 48 h of incubation. DRM and DRML-9 exhibited considerable cytotoxicity against PC-3 cells, in contrast to LNCaP and DU-145 cells. As depicted in Fig. 5 A, the use of optimized DRML-9 results in a considerable (p < 0.05) suppression of cancer cell growth in comparison to DRM. The LNCaP cells were chosen for further examination due to their heightened susceptibility to optimized DRML-9 compared to other cells. Optimized DRML-9 has been shown to significantly enhance cytotoxicity in prostate cancer cell lines (LNCaP). DRML displays higher cellular uptake due to the nano-size range, which facilitates endocytosis-mediated internalization by cancer cells. Once internalized, liposomes provide a sustained release of the drug, ensuring a prolonged intracellular presence and a higher local concentration of the drug at the target site, leading to enhanced AR inhibition and apoptosis .Moreover, liposomes can protect DRM from enzymatic degradation, Improve bioavailability and pharmacokinetics, Allow passive targeting via the enhanced permeability and retention (EPR) effect in tumor tissues and Be modified with surface ligands such as PEGylation for active targeting to prostate cancer cells. Cytotoxicity conforms DRML liposomes have significantly lower IC₅₀ values compared to free DRM in LNCaP when compared to PC-3 and DU-145 prostate cancer cell lines, indicating superior cytotoxic potential.[51–52 ]. Apoptosis by Flow cytometer One of the ways that anticancer moieties may cause cell death in tumor tissue is by apoptosis. We evaluated the apoptosis-inducing capacity of DRM and optimized PEGylated DRML-9 in LNCaP cells using the Annexin V-FITC/PI staining method. In control sample Just 0.33, 0.41, and 0.16% of the cells were in early apoptotic, late apoptotic, and necrotic phases, respectively Fig. 7 A. When treated with DRM and optimized DRML-9 the % of live cells decreased from 99.10–83.9% (Fig. 7 B) and 73.7% (Fig. 7 C), respectively. DRML-treated cells exhibited that 16.04% of the cells were in the apoptotic state where 11.79% of the cells were in the apoptotic stage in DRM treated. Both DRM and optimized DRML-9 are significantly (p < 0.05) causing apoptosis, as compared to the untreated group. Similarly, as compared to DRM, DRML-9 exhibited a considerable (p < 0.05) ability to induce apoptosis, a form of cell death. Based on these results, DRML-9 treatment resulted in a much higher % of cells in the apoptotic state than DRM treatment and control. DRM, a potent second-generation androgen receptor (AR) antagonist, liposomal encapsulation significantly enhances its apoptotic potential in LNCaP cell lines compared to the pure DRM. This enhancement is attributed to improved cellular uptake, sustained intracellular release, and higher bioavailability at the tumor site. DRM-loaded liposomes induce apoptosis more effectively due to Enhanced Cellular Uptake due to nanoscale size (typically < 200 nm) and lipid composition of liposomes facilitate endocytosis-mediated internalization, ensuring higher intracellular DRM concentrations than pure DRM, which may enter cells via passive diffusion. [53] DRML provide sustained drug release, prolonging the intracellular exposure of cancer cells to DRM. This allows more effective suppression of AR signalling pathways, which are crucial for prostate cancer cell survival and resistance. DRML lead to increased mitochondrial membrane depolarization, cytochrome c release, and activation of caspase-9 and caspase-3, hallmarks of intrinsic apoptotic pathway activation. This effect is more pronounced than in cells treated with pure DRM. Studies have shown that liposomal formulations increase the Bax/Bcl-2 ratio, enhance cleaved PARP expression, and promote DNA fragmentation, further supporting their stronger apoptotic potential. DRML more effectively suppress Survivin and Bcl-2, proteins that inhibit apoptosis and are often upregulated in androgen-dependent prostate cancers. [54] Apoptosis by DAPI Following a 48-hour treatment with DRM and optimized PEGylated DRML-9, DAPI staining was used to detect the programmed cell death in LNCaP cells. Figure 7 D-F displays fluorescence microscopic images of cells that had not been treated and cells that had been treated with DRM and optimized DRML-9. The untreated cells, referred to as the negative control, exhibited normal intact nuclei with faint and uniform blue staining. In contrast, the groups treated with DRM (Fig. 7 E) and DRML-9 (Fig. 7 F) displayed small nuclei with intense chromatin condensation, nuclear fragmentation, and the formation of apoptotic bodies. The results indicate that both DRM and optimized DRML-9 induce apoptosis in LNCaP cells. Cells treated with DRM and optimized DRML-9 have fragmented nuclei and uneven margins close to the nuclei, which are signs of apoptosis. For apoptosis study, fluorescence microscopy with cell permeable nucleic acid dye, such as DAPI, is frequently employed to evaluate nuclear morphology. [55–56] These changes can be caused by cell permeability or the activation of the apoptotic-inducing enzyme. The literature also contains similar findings. [57–60] Cell Cycle Arrest The cell cycle is a highly regulated process that controls cell division in living organisms. Through specific checkpoints, it prevents uncontrolled cell proliferation by detecting DNA damage, promoting DNA repair, or triggering the elimination of potentially cancerous cells. Both DRM and optimized PEGylated DRML-9 induced permanent cell death specifically during the S phase [61]. In control cells (Fig. 8 A) (67.2%) cells are in sub G 1 phase (14.5%) cells are in G 0 /G 1 phase. In the S phase (7.90%) and the G2/M phase (4.25%), distinct populations of cells were also seen. DRM and optimized DRML-9 caused permanent cell death during the S phase [62–63]. Cells treatment with DRM (Fig. 8 B) and DRML-9 (Fig. 8 C) undergo a massive shift of cells from sub G1 to G0/G1 and S phase arrest of almost 18.3% and 45.7% and 8.77% and 15.6% of the cell population respectively. The therapy with DRML resulted in almost 2-fold increase in S phase arrest compared to DRM alone. Furthermore, the administration of DRM and optimized DRML-9 resulted in a partial decrease in the number of cells arrested in the G2/M phase of the cell cycle.[64] The encapsulation of DRM in liposomal carriers has been shown to significantly enhance its anti-proliferative activity in prostate cancer cells by inducing a more pronounced cell cycle arrest, especially at the G1 phase, compared to the free DRM. This enhancement is primarily due to improved drug delivery efficiency, cellular internalization, and sustained intracellular release, which collectively boost the suppression of androgen receptor (AR)-mediated signalling pathways involved in cell cycle progression. Pure DRM, as a potent AR antagonist, disrupts AR transcriptional activity and suppresses genes necessary for G1 to S phase transition, such as Cyclin D1, CDK4/6, and E2F1, leading to G1 phase arrest.[65] Enhanced Cell Cycle Arrest by DRML is mainly due to Improved Intracellular Accumulation: DRML enhance the intracellular concentration of DRM through endocytosis, facilitating more effective inhibition of AR-dependent gene expression required for cell cycle progression. The sustained release from DRML ensures prolonged exposure of cancer cells to DRM, allowing continuous inhibition of Cyclin D–CDK4/6 complex activity, which is essential for G1/S transition.[ ] Liposomes more effectively reduce the expression of proliferative markers like Ki-67 and PCNA, indicating a stronger blockade of mitotic entry. LNCaP cells treated with liposomal formulations often reveals a significantly higher percentage of cells in G0/G1 phase and a reduced S-phase population compared to cells treated with pure DRM. Liposomal delivery enhances the downregulation of AR-regulated genes such as CDC25A, Cyclin E, and Skp2, further reinforcing the G1 phase blockade.[66] In-Vivo Pharmacokinetic Study : In plasma RT (retention time) of DRM has been observed to be 8.8 min. Upon intravenous administration of DRM and PEGylated DRML-9 in concentration V/s time profile in plasma C max for DRML has been observed to be 8.33 ± 0.62 µ g /mL − 1 at 1 h while C max of plain DRM (7.99 ± 0.48 µ g mL − 1) at zero hours (T max ) plasma concentration was noted for both DRM and DRML-9 (Fig. 9 A). “AUC of DRML-9 was observed to be significantly greater ( P < 0.05 ) as compared to DRM (64.09 ± 2.67 µ g. h. mL − 1) than DRM (17.68 ± 1.32 µ g.h. mL − 1) correspondingly. The elimination rate constant (K), & elimination half-life (T 1/2 ) for DRML-9 and” DRM (0.06 ± 0.005 & 0.26 ± 0.031 µg/mL)/h ) and (7.94 ± 0.64, & 6.82 ± 0.53 h) correspondingly. MRT for DRML-9 & DRM has been observed to be 11.07 ± 0.98 & 6.19 ± 0.62 h respectively. Detailed results have been shown in Table 5 . Table 5 Pharmacokinetic Parameters of DRM after intravenous administration of DRM and optimized PEGylated DRM loaded liposomes (DRML-9) (5mg/kg) in rats) Parameter DRM DRML-9 T max (h) 0 0 C max (µg/ml) 7.99 ± 0.48 8.33 ± 0.62 AUC 0 − t (µg/ml*h) 17.68 ± 1.32 64.09 ± 2.67 AUC 0 − inf (µg/ml*h^2) 18.63 ± 1.43 73.37 ± 3.25 t 1/2 (h) 6.82 ± 0.53 7.94 ± 0.64 MRT (h) 6.19 ± 0.62 11.07 ± 0.98 CL ( µg/ml)/h 0.26 0.06 Relative Bioavailability 362.50 A significant increase of 3.63 times in bioavailability was seen when DRML-9 was administered intravenously compared to pure DRM. Liposomal drug delivery systems enhance bioavailability through several key mechanisms. The primary factor is the encapsulation of drugs within a liposomal matrix, which protects them from enzymatic degradation in the bloodstream, thereby improving drug stability and extending circulation time. [65–66] PEGylated liposomes further evade the reticuloendothelial system (RES), prolonging systemic circulation and enhancing bioavailability. [67] PEGylated liposomes can be engineered for passive or active targeting, increasing drug accumulation at the desired site of action while minimizing off-target distribution. [68–69] This selective delivery improves therapeutic efficacy and reduces adverse effects. Additionally, liposomes can encapsulate hydrophobic drugs within their lipid bilayer, enhancing aqueous solubility and systemic availability following intravenous administration. [70] By enabling controlled release and influencing biodistribution, liposomes reduce systemic toxicity and improve the therapeutic index of encapsulated drugs. Furthermore, liposomes can enhance cellular uptake through endocytosis or membrane fusion, particularly benefiting drugs with poor membrane permeability. [71–72] 3.5. Accelerated Stability Study There are no significant changes in the particle size and entrapment efficiency of optimized PEGylated DRML-9 after storage of 3 months at 2 to 8°C. The particle size and entrapment efficiency of optimized DRML after 1, 2, and 3 months of storage are depicted in Table 6 and Fig. 9 B. Table 6 Stability study of optimized PEGylated DRM loaded liposomes (DRML-9) at 2–8°C showing % EE and particle size Parameter 00 Days 30 Days 60 Days 90 Days EE (%) 95.84 ± 1.54 95.47 ± 1.62 95.14 ± 1.53 94.89 ± 1.37 Particle size (nm) 86.3 ± 2.67 86.7 ± 2.81 87.5 ± 2.32 87.8 ± 2.05 Conclusions In the present study, PEGylated Darolutamide-loaded liposomes (DRML) were successfully developed and optimized using a 3² factorial design. The optimized PEGylated DRML exhibited high entrapment efficiency, minimal vesicle size in the nanometer range, and good colloidal stability with a negative zeta potential. Notably, PEGylated DRML demonstrated a significant enhancement in the anticancer potential of Darolutamide against prostate cancer cells. Both in-vitro and in-vivo studies confirmed the improved therapeutic efficacy of PEGylated DRML in the treatment of prostate carcinoma. Overall, PEGylated DRML represents a promising strategy to enhance the therapeutic potential of Darolutamide. Declarations Funding No funding was received for this study. Author Contribution Vinod V. Patil: Investigation, Methodology, Writing – original draft, Validation, Conceptualization, Resources. Harshal Pawar: Methodology, Software, Writing – review, editing, Conceptualization, Supervision. Acknowledgement The authors are thankful to School of Pharmacy, Mansarovar Global University, Kolar Road, Bhopal 462042, Madhya Pradesh, India for providing required guidance and support for completion of this research work. Authors are also thankful to Gattefosse India and BASF India for providing gift samples of lipids and surfactants. We thank Diya Labs Mumbai for Characterization of formulations. The authors are also thankful to NCBI Pune for providing –cancer cell line, Maratha Mandal Dental College and research center Belagavi for anticancer activity, and Biocyte Sangli for bioanalytical work. References Schafer EJ, Laversanne M, Sung H, Soerjomataram I, Briganti A, Dahut W, Bray F, Jemal A. Recent patterns and trends in global prostate cancer incidence and mortality: an update. Eur Urol . 2025 Mar 1;87(3):302-13. doi:10.1016/j.eururo.2024.11.031. Podgoršek E, Mehra N, van Oort IM, Somford DM, Boerrigter E, van Erp NP. 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Subcutaneous liposomal delivery improves monoclonal antibody pharmacokinetics in vivo. Acta Biomater . 2025 Mar 15;195:522-35. doi:10.1016/j.actbio.2024.12.045. Additional Declarations No competing interests reported. Supplementary Files GA.jpg Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Journal of Pharmaceutical Innovation → 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. 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(E) The overlay plot displaying the optimized DRM loaded liposomes formulation in the design space and their predicted response values.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/deb2d903ff2a1368d49a71e9.jpg"},{"id":96794633,"identity":"4a551e88-96e1-445b-bc2c-3dfd31d37bd2","added_by":"auto","created_at":"2025-11-26 07:18:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8874598,"visible":true,"origin":"","legend":"\u003cp\u003eA) Globule size B) Zeta Potential and C)Surface Morphology of Optimized PEGylatedDRMloaded liposomes\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/4f6daef5b710f0583fa6c369.jpg"},{"id":96794627,"identity":"cc52eef7-23a1-4781-ae82-1f259ebe3458","added_by":"auto","created_at":"2025-11-26 07:18:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3380334,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of A) DRM B) Cholesterol C) HSPC D) Optimized DRML formulation.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/ae83d5bea8744dcf39bfd6a2.jpg"},{"id":96794628,"identity":"90c828fd-fcdf-4b86-805c-749dc40182d4","added_by":"auto","created_at":"2025-11-26 07:18:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1362503,"visible":true,"origin":"","legend":"\u003cp\u003eDSC thermogram of A) DRM B) Cholesterol C) HSPC D) Optimized DRML formulation.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/05b95196def99e52b5f240c5.jpg"},{"id":96916798,"identity":"5b60c856-5090-44aa-b6aa-2f8508ebb256","added_by":"auto","created_at":"2025-11-27 14:08:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2962858,"visible":true,"origin":"","legend":"\u003cp\u003eXRD diffractogram of A) DRM B) Cholesterol C) HSPC D) Optimized DRML formulation.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/80bd57e7665b523ad06e02ee.jpg"},{"id":96917236,"identity":"d5ee80e8-1854-4428-b85c-12260f1a21c9","added_by":"auto","created_at":"2025-11-27 14:09:25","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1777637,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Cytotoxicity of DRM and DRML on prostate cancer cell line (MTT assay). Data is expressed as mean ± SEM (n = 3). The significance difference indicated as *p \u0026lt; 0.05 as DRM compared to DRML.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/cb756b4147cc18e3d8efcca0.jpg"},{"id":96794657,"identity":"48c6cd7e-8489-4ca3-9455-e57299365cb9","added_by":"auto","created_at":"2025-11-26 07:18:09","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":189982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFlow cytometric\u003c/em\u003e assessment of apoptosis in (A) Untreated (B) DRM (C) DRML treated LNCaP cells for 12 h, at IC50 value; Apoptotic nuclear changes observed in D) Untreated E) DRM treated and F) DRML treated LNCaP cancer cells after 24 h of incubation. Circle represents chromatin condensation and nuclear shrinkage and square represents nuclear blebbing and nuclear fragmentation.\u003c/p\u003e","description":"","filename":"7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/49fd24a5240f08a5219620d0.jpeg"},{"id":96916354,"identity":"b949ba02-1578-4c99-bf78-de4f39d6d6ef","added_by":"auto","created_at":"2025-11-27 14:08:29","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":76036,"visible":true,"origin":"","legend":"\u003cp\u003eCell cycle arrest in (A) Untreated (UN) (B) DRM (G) DRML treated LNCaP cells after 12 h, at IC50 value.\u003c/p\u003e","description":"","filename":"8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/d3b4a55b77701a80091aa127.jpeg"},{"id":96917055,"identity":"ad31521e-3b5c-47b8-acae-c4cdea8a2dad","added_by":"auto","created_at":"2025-11-27 14:09:13","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1970307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eIn-vivo Pharmacokinetic study of DRM and DRML after 24 hours in albino wistar rats (B) Particle size of Optimized DRM-liposomal formulation after 3 months storage.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/637ce15c3fd455ede6fdb53e.jpg"},{"id":102785819,"identity":"4c6b0ce4-d5a8-4f2e-ac81-2fbc30259eb0","added_by":"auto","created_at":"2026-02-16 16:10:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27680618,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/acc3b57d-19ac-481a-baaa-48d0d8a4cce8.pdf"},{"id":96794626,"identity":"c996bc3c-65ab-4d05-84ea-9f3c94a6e013","added_by":"auto","created_at":"2025-11-26 07:18:08","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3133120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7203702/v1/acbd1c031e69dea15de36882.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Optimization of PEGylated Darolutamide-Loaded Liposomes for Treatment of Prostate Cancer: In vitro and In-vivo Characterization","fulltext":[{"header":"Background","content":"\u003cp\u003eThe global burden of cancer continues to rise at an alarming rate, with an urgent need for ideal therapeutic strategies that can effectively manage this complex disease. Prostate cancer (PC) ranks as the second most frequently diagnosed malignancy and the fifth leading cause of cancer-related deaths in men globally, with approximately 1.46\u0026nbsp;million new cases and 3.96\u0026nbsp;million fatalities reported in 2022 [1]. Alarmingly, projections indicate that by 2040, the global incidence of PC will rise to approximately 2.4\u0026nbsp;million cases, with associated deaths reaching 7.12\u0026nbsp;million, primarily driven by population aging and growth [2]. Therapeutic strategies for PC typically include surgery, radiotherapy, chemotherapy, and hormone therapy [3\u0026ndash;5]. Despite advancements in these approaches, limitations persist. Radiotherapy, while effective, often suffers from low specificity, leading to damage of surrounding healthy tissues when higher doses are administered to achieve therapeutic efficacy. Such treatments may lead to undesirable side effects, including bone pain, fatigue, gastrointestinal issues, hematological toxicity, as well as reductions in platelet and white blood cell counts, primarily due to damage to the surrounding bone marrow [6]. Chemotherapy is associated with systemic side effects, including alopecia, mucositis, anorexia, gastrointestinal disturbances, and profound fatigue related to anemia [7]. Hormone therapies, aimed at reducing testosterone levels, often lead to significant side effects, including hot flashes, sexual dysfunction, fatigue, weight gain, muscle loss, bone demineralization, and gynecomastia [8]. Darolutamide (DRM) is a next-generation androgen receptor inhibitor approved for treating non-metastatic castration-resistant and metastatic hormone-sensitive prostate cancer [9]. However, the clinical utility of darolutamide is hampered by its poor aqueous solubility, incomplete oral absorption, and low bioavailability [10]. Furthermore, its use has been associated with adverse cardiac events, including arrhythmias, coronary artery disease, hypertension, and heart failure [11]. Liposomal drug delivery systems, comprising microscopic lipid-based vesicles, have emerged as a promising approach to overcome the limitations of conventional drug delivery. These systems utilize the amphiphilic, biocompatible, and self-assembling properties of lipids to encapsulate therapeutic agents, including hydrophobic small molecules, peptides, and nucleic acids, thus improving drug solubility, stability, and targeted delivery [12]. Liposomal formulations offer several advantages, including enhanced bioavailability, prolonged systemic circulation, reduced systemic toxicity, and controlled or site-specific drug release [13\u0026ndash;14]. Notably, nanoparticulate drug delivery systems such as liposomes exhibit superior pharmacokinetics and pharmacodynamics, widening the therapeutic window and minimizing off-target toxicities [15\u0026ndash;16]. Incorporation of functional lipids, such as phospholipids and cholesterol, has shown added benefits in improving liposome stability, drug entrapment efficiency, and reducing drug leakage, as demonstrated in our previous studies [17\u0026ndash;18]. Cholesterol, in particular, enhances membrane stability, reduces haemolysis, improves entrapment efficiency, and regulates drug release kinetics, thereby enhancing the overall therapeutic potential of liposomes. Additionally, functionalizing liposomes with ligands like antibodies, peptides, or aptamers allows active targeting of prostate-specific membrane antigen (PSMA), which is highly overexpressed on prostate cancer cells, enhancing therapeutic specificity [19]. Liposomal encapsulation also protects hydrophobic drugs from premature degradation in the systemic circulation, promoting prolonged drug residence time and sustained therapeutic release [20]. Given these advantages, the present research aims to develop and optimize darolutamide-loaded liposomes (DRM-liposomes) to enhance their bioavailability and anticancer efficacy. A 3\u0026sup2; full factorial design was utilized to systematically develop and optimize DRM-loaded liposomes. The optimized formulation underwent detailed characterization, including particle size, zeta potential, entrapment efficiency, and in vitro drug release. Additionally, it\u0026rsquo;s in vitro-anticancer efficacy was assessed through studies on apoptosis induction and cell cycle arrest, followed by in-vivo pharmacokinetic evaluation to determine its therapeutic potential for prostate cancer treatment\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDarolutamide has been acquired from Clearsynth Andheri West, Mumbai, India. Cholesterol and HSPC were procured from Sigma Aldrich India Pvt. Ltd. Mumbai, India. DSPE-PEG2000 was obtained from TCI Chemicals Japan. Methanol and Milli-Q water, were procured from Unique Biologicals Kolhapur, India. All other chemical, and reagents used in current research work, are of analytical grade.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NCCS (National Centre for Cell Science), located in Pune, India, provided human prostate cancer cell lines DU-145, PC-3, LNCaP. Cells have been cultivated in DMEM (Dulbecco's Modified Eagle's Medium), which has been improved with one percent penicillin-streptomycin as well as 10% FBS (fetal bovine serum). Cultures have been kept at 37\u0026deg;C with five percent CO₂ in a humidified incubator.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation and optimization of PEGylated Liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePEGylated Darolutamide-loaded liposomes (DRML) were formulated utilizing thin-film hydration method. Briefly, to accomplish full solubilization, 50 mg of DRM and liposomal components\u0026mdash;namely, HSPC, cholesterol, and DSPE-PEG2000\u0026mdash;were carefully weighed and dissolved in 15 mL of a methanol: chloroform mixture (1:2, v/v) using bath sonication. Organic solvents were later evaporated at 65\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C using rotary evaporator (BUCHI Rotavapor R-200, BUCHI India Private Ltd, Mumbai, India), leading to the formation of thin lipid layer on the inner surface of the round-bottom flask. The lipid film was additionally vacuum-dried for an entire night in order to remove any remaining solvents. Finally, dried lipid film has been hydrated with 40 mL of SWI (sterile water for injection) at 70\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for fifteen minutes with gentle agitation to obtain multilamellar vesicles (MLVs). The MLVs were further subjected to probe sonication (LABMAN Pro-656) to reduce the vesicle size and produce small unilamellar vesicles (SUVs). Probe sonication was performed for five cycles of one minute each, with each cycle consisting of 12 sec. of sonication at 240 V and 0.6 A, followed by an 8-second pause [21\u0026ndash;22].\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4. Experimental design\u003c/h2\u003e\n\u003cp\u003eA 3\u0026sup2; full factorial design has been utilized for the systematic development and optimization of PEGylated DRML with Design Expert\u0026reg; software (Version 7.0.0, Stat-Ease Inc., USA). Two independent variables, X₁ (HSPC amount) and X₂ (cholesterol amount), were studied at three levels (-1, 0, +\u0026thinsp;1) to evaluate their effect on Y₁ (particle size) and Y₂ (entrapment efficiency, %EE). A total of 13 experimental runs were generated as per the design (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), and all formulations have been evaluated for particle size and %EE. The responses have been fitted to various models (linear, quadratic, 2FI, cubic), the best-fit model has been selected depending on ANOVA results, considering R\u0026sup2;, adjusted R\u0026sup2;, predicted R\u0026sup2;, and p-values. Response surface plots, contour plots, and perturbation graphs were generated to visualize the effect of variables. The optimized formulation was identified using numerical and graphical optimization with a desirability function, targeting minimum particle size and maximum %EE. An overlay plot indicated the optimal region, and the optimized formulation was prepared to validate the model by comparing predicted and experimental responses.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eFull factorial Design matrix summarizing the levels, factors, and responses of 13 runs for optimization of Darolutamide loaded liposomes.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBatch code\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eY\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eY\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL\u0026ndash;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e103.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL \u0026minus;\u0026thinsp;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL \u0026minus;\u0026thinsp;3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e165.32\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e72.58\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL \u0026minus;\u0026thinsp;4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e58.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e87.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL \u0026minus;\u0026thinsp;5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.10\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL \u0026minus;\u0026thinsp;6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e102.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e84.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL \u0026minus;\u0026thinsp;7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e142.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL \u0026minus;\u0026thinsp;8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e101.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL\u0026ndash;9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e87.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL\u0026ndash;10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e126.57\u0026thinsp;\u0026plusmn;\u0026thinsp;3.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e85.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL\u0026ndash;11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e101.98\u0026thinsp;\u0026plusmn;\u0026thinsp;.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL\u0026ndash;12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e102.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e82.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL\u0026ndash;13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e183.12\u0026thinsp;\u0026plusmn;\u0026thinsp;4.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\" rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eFactor (Independent variables)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eActual levels\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eLow (-1)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMedium (0)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHigh (+\u0026thinsp;1)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eFactor X1: Amount of HSPC (mg)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eFactor X2: Amount of Cholesterol (mg)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDependent variables or Responses\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eConstraint\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eResponse 1: Particle size (Y1) (nm)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMinimize\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eResponse 2: Entrapment efficiency (Y2) (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMaximize\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of Liposomes Containing DRM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticle size and zeta potential\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticle size as well as zeta potential of optimized PEGylated DRML have been determined by utilizing a DLS (dynamic light scattering) technique with particle size analyzer (Malvern Zetasizer, Version 7.11, Malvern Instruments Ltd., United Kingdom). The analysis has been performed at controlled temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, with cell drive voltage maintained at 150 mV. To prevent numerous scattering effects, the liposomal dispersion was suitably diluted with double-distilled water for every measurement. Three independent samples of the optimized formulation were analyzed to determine mean particle size and zeta potential. Results have been recorded as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD [23].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEntrapment efficiency\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eEntrapment efficiency (%EE) of PEGylated DRML has been determined utilizing an indirect centrifugation technique. Briefly, liposomal dispersion has been subjected to centrifugation at 10,000 rpm for one hour to separate unentrapped (free) drug present in the aqueous phase. After centrifugation, the supernatant containing the free drug was carefully collected and appropriately diluted with methanol. Concentration of free Darolutamide (DRM) in supernatant has been quantified utilizing UV-Visible spectrophotometer at 286 nm, employing methanol as the blank.[24]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurface Morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphological characteristics of optimized PEGylated DRML have been examined by utilizing TEM (Transmission Electron Microscopy). After the extra fluid has been removed, 5 \u0026micro;L of liposomal dispersion has been carefully applied to a 300-mesh carbon-coated copper grid. Grid has been allowed to stand for a few minutes to enable sample adherence. Subsequently, the sample was air-dried and negatively stained with 1percent (w/v) uranyl acetate for 3 to 5 min. to improve contrast. After drying, grid was observed under Transmission Electron Microscope (JEOL-JEM 1400, USA) to visualize shape, size, structural integrity of liposomes [25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003edrug release study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDrug release profile of DRM has been examined at pH 7.4 (physiological pH), pH 5.8 (tumor microenvironment pH) using the dialysis bag method in a USP Type II (paddle) apparatus at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Briefly, DRML dispersions containing an amount equivalent to 5 mg of drug have been placed into dialysis bags (LA390-10mt-HiMedia), securely sealed, attached to the paddle, and immersed individually into vessels containing 100 mL of PBS (phosphate buffer saline) at pH 7.4 or 5.8, each supplemented with 5% (v/v) methanol [30]. The media were continuously stirred at 100 rpm while being kept at 37\u0026deg;C. At predetermined time intervals (0, 1, 2, 4, 6, 12, 24 h), 3 mL samples were withdrawn and analyzed by utilizing UV-visible spectrophotometer at 286 nm. Fresh buffer has been added to the withdrawn volume in order to preserve sink conditions. The % of drug released was calculated and plotted as a function of time [26]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLyophilization of optimized PEGylated Liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimized PEGylated Darolutamide-loaded liposomes (DRML) were subjected to lyophilization to enhance their long-term stability. Prior to freeze-drying, trehalose was used as a cryoprotectant at varying weight ratios with respect to the lipid content to protect the liposomal structure during freeze-drying process. Under carefully monitored conditions, a liposomal dispersion including cryoprotectant was frozen and lyophilized utilizing Martin Christ Alpha 1\u0026ndash;2 LD Plus freeze dryer (Martin Christ GmbH, Germany). Resulting lyophilized powder has been collected and stored in airtight containers for further characterization.[27]\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e6.4 Compatibility study:\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003e2.11. Four Transform Infrared Analysis (FTIR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBruker Alpha-II FTIR spectrophotometer has been used to record FTIR spectra of DRM, HSPC, Cholesterol, and PEGylated DRML along with liposomal formulation excipients in a range of 4000 to 650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e frequency.[28]\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e2.12. Differential Scanning Calorimeter (DSC) Analysis\u003c/h2\u003e\n\u003cp\u003eDSC thermograms (DSC-60, Shimadzu, Japan) of DRM, HSPC, Cholesterol, and PEGylated DRML, along with liposomal formulation excipients, were performed to examine changes in their thermal behavior.[29]\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e2.13. Powder X-ray diffraction (P-XRD)\u003c/h2\u003e\n\u003cp\u003eThe crystalline characteristics of DRM and the lyophilized PEGylated DRM-liposomal formulation were examined using P-XRD (Powder X-ray diffraction) with Philips diffractometer (Netherlands) operated at 40 kV or 30mA. Samples have been scanned over 2\u0026theta; range of 10\u0026ndash;80\u0026deg;, with step size of 0.020\u0026deg;, as well as scanning speed of one second per step, under ambient temperature conditions [30]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro anticancer activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTT assay was utilized to assess cytotoxic potential of DRM and improved PEGylated DRML formulation against human prostate cancer cell lines (LNCaP, DU-145, PC-3). To enable adhesion, cells have been sown in 96-well plates or treated for the entire night at 37\u0026deg;C. Subsequently, cells have been exposed to varying concentrations of test compounds for 48 hours. After treatment, test solutions have been removed, and 100 \u0026micro;L of MTT solution (6 mg /10 mL in PBS) has been added to each well. Plates have been incubated for an additional 4 h under identical conditions. One hundred microliters of DMSO have been added to each well to dissolve formazan crystals that have been produced by metabolically active cells. Microplate reader has been utilized to detect absorbance at 570 nm, and dose-response curves have been used to calculate IC₅₀ values. [31]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis by Flow cytometer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells have been seeded, and plates were subjected to heat at 37\u0026deg;C overnight. Cells have been incubated with 50\u0026micro;g/mL of DRM or optimized DRML for 24h. Later, cells were subjected to centrifugation (Remi-CM8 Plus, India) for about 5 min at 4\u0026deg;C. Following collection, the cell pellets (1 \u0026times; 10\u0026sup1;/mL) were meticulously cleaned using 2 mL of 1X PBS. After setting the tubes on ice, 5\u0026micro;L of propidium iodide and 1\u0026micro;L of annexin-V fluorescein isothiocyanate were added. Tubes were then left to incubate for 15 min. In less than half an hour, tubes were examined by utilizing flow cytometer following the addition of 400 \u0026micro;L of cold buffer.[32]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis by DAPI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach well of 24-well plate has been seeded with 1 \u0026times; 10⁴ cells, and cells were then allowed to adhere by being incubated in a CO₂ incubator for the entire night at 37\u0026deg;C. DRM and the optimized PEGylated DRML formulation were subsequently added to the cells at concentrations less than 50 \u0026micro;g/mL, and they were incubated for 24 h under the same circumstances. Following treatment, the cells underwent a 30-min. dark incubation period before being rinsed with PBS. Then, each well received 20 \u0026micro;L of DAPI staining solution (0.1 \u0026micro;g/mL). The proportion of apoptotic cells was determined by randomly observing and counting cells displaying characteristic apoptotic features under a fluorescence microscope [33]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter cells have been seeded in 24 wells of micro titer plate with flat bottoms containing a cover slip and kept for incubation in CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for duration of experiment, for 48h, cells added 10\u0026micro;L/mL of the developed formulation. After the cells have been incubated for appropriate time, they were centrifuged for 5 min. at a rate of 4\u0026deg;C. Supernatant has been removed by centrifugation, and cell pellets were subsequently mixed with ice-cold 1X binding buffer at concentration of 1x105/mL. After gently mixing cells, incubate them for 15 min. and then treat them with approximately 10\u0026micro;L of propidium iodide. The tubes should be placed on ice right away. In addition, a 1X binding buffer with a concentration of 400\u0026micro;L/mL has been thoroughly mixed and stirred before being subjected to analysis in a flow cytometer within the next half an hour.[34]\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e3. In-Vivo Pharmacokinetic Study\u003c/h3\u003e\n\u003cp\u003eStudy protocol has been accepted by Institutional Animal Ethics Committee (IAEC/BiRD/Sangli/2024-25/13). Male Wistar albino rats (n\u0026thinsp;=\u0026thinsp;9; 180 to 220g) were obtained from Crystal Biological Solutions, Pune, India. Animals had been housed under standard laboratory settings, having free water supply as well as food for two weeks prior to study. Rats had been divided at random into three groups such as Group I (Control): Received normal saline, Group II (Standard): Pure DRM administered 5mg/kg suspended in PEG-400 (40%), Ethanol (10%) and Sterile saline (50%) and Group III(Test): Administered PEGylated DRML-9 intravenously at a dose equal to 5mg/kg of DRM. Blood samples (500\u0026micro;L) had been gathered from retro-orbital plexus at predefined time intervals (0.5 to 24 h) post-dosing into heparinized tubes. Plasma has been separated using centrifugation at 700 rpm for 10 min, alongside kept until analysis at -20\u0026deg;C. DRM concentration in plasma has been determined utilizing a validated HPLC technique with a Shimadzu LC-2030C 3D Plus system, equipped with Shimpack GIST C18 column along with mobile phase delivered at 1mL/min. Pharmacokinetic parameters, including Cₘₐₓ, Tₘₐₓ, AUC₀\u0026ndash;\u0026infin;, AUMC₀\u0026ndash;\u0026infin;, and MRT, were calculated using non-compartmental analysis [35].\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e2.6. Stability Study\u003c/h2\u003e\n\u003cp\u003eA stability study of the optimized PEGylated DRML was carried out. The LPs formulations containing 5% of sucrose as a cryoprotectant. The formulation was also stored at refrigeration (2\u0026ndash;8\u0026deg;c). The prepared batch were tested for particle size and % EE after 1,2 and 3 months.[36].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical comparisons between groups were conducted using one-way analysis of variance (ANOVA) followed by Dunnett\u0026rsquo;s multiple comparison test. Analyses were performed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. [36]\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eFitting of data into the model\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eTo select the most suitable model, experimental responses from the 13 formulations were analyzed using Design-Expert\u0026reg; software. Models were compared based on higher R\u0026sup2;, adjusted R\u0026sup2;, and predicted R\u0026sup2; values, along with lower standard deviation (SD), coefficient of variation (% CV), and predicted residual sum of squares (PRESS) (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). A lower PRESS value reflects better model predictability. Based on these criteria, the quadratic model best fit particle size data, while the linear model was optimal for entrapment efficiency (%EE).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRegression analysis results obtained for various responses Y\u003csub\u003e1\u003c/sub\u003e (particle size) and Y\u003csub\u003e2\u003c/sub\u003e (% entrapment efficiency) of darolutamide loaded liposomes for fitting to different models\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eModels\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAdjusted R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePredicted R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePRESS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCV (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRemark\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth colspan=\"8\" align=\"left\"\u003e\n \u003cp\u003eResponse (Y\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLinear\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2FI\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eQuadratic\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCubic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.08\u003c/p\u003e\n \u003cp\u003e9.40\u003c/p\u003e\n \u003cp\u003e3.31\u003c/p\u003e\n \u003cp\u003e3.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9438\u003c/p\u003e\n \u003cp\u003e0.9457\u003c/p\u003e\n \u003cp\u003e0.9948\u003c/p\u003e\n \u003cp\u003e0.9948\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9326\u003c/p\u003e\n \u003cp\u003e0.9276\u003c/p\u003e\n \u003cp\u003e0.9910\u003c/p\u003e\n \u003cp\u003e0.9876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9004\u003c/p\u003e\n \u003cp\u003e0.8300\u003c/p\u003e\n \u003cp\u003e0.9649\u003c/p\u003e\n \u003cp\u003e0.4168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1459.75\u003c/p\u003e\n \u003cp\u003e2492.06\u003c/p\u003e\n \u003cp\u003e513.94\u003c/p\u003e\n \u003cp\u003e8547.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.17\u003c/p\u003e\n \u003cp\u003e8.47\u003c/p\u003e\n \u003cp\u003e2.98\u003c/p\u003e\n \u003cp\u003e3.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSuggested\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eResponse (Y\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLinear\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2FI\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eQuadratic\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCubic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.34\u003c/p\u003e\n \u003cp\u003e6.68\u003c/p\u003e\n \u003cp\u003e7.37\u003c/p\u003e\n \u003cp\u003e8.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5960\u003c/p\u003e\n \u003cp\u003e0.5961\u003c/p\u003e\n \u003cp\u003e0.6170\u003c/p\u003e\n \u003cp\u003e0.6240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5152\u003c/p\u003e\n \u003cp\u003e0.4615\u003c/p\u003e\n \u003cp\u003e0.3434\u003c/p\u003e\n \u003cp\u003e0.0975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5076\u003c/p\u003e\n \u003cp\u003e0.4743\u003c/p\u003e\n \u003cp\u003e0.3308\u003c/p\u003e\n \u003cp\u003e-0.6026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e489.51\u003c/p\u003e\n \u003cp\u003e522.61\u003c/p\u003e\n \u003cp\u003e665.22\u003c/p\u003e\n \u003cp\u003e1593.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.80\u003c/p\u003e\n \u003cp\u003e8.23\u003c/p\u003e\n \u003cp\u003e9.08\u003c/p\u003e\n \u003cp\u003e10.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSuggested\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003eSD: standard deviation, R\u003csup\u003e2\u003c/sup\u003e: multiple correlation coefficient, 2FI: two factor interaction, PRESS: predicted residual sum of square, CV: coefficient of variation.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of independent variables on particle size (Y\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) of PEGylated DRM liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe particle size of PEGylated DRM liposomes formulations is displayed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Particle size has been found in the range of 87.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25 to 183.12\u0026thinsp;\u0026plusmn;\u0026thinsp;4.95 nm. The following quadratic equation describes how the independent variables affect the particle size.\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;103.60\u0026ndash;44.76X\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;17.37X\u003csub\u003e2\u003c/sub\u003e -2.63 X\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;13.92X\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;2.25X\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eWhere Y\u003csub\u003e1\u003c/sub\u003e is particle size, X\u003csub\u003e1\u003c/sub\u003e is the amount of HSPC, and X\u003csub\u003e2\u003c/sub\u003e is the amount of cholesterol. The equation indicates that amount of HSPC has a negative effect and amount of cholesterol has a positive effect on particle size. This demonstrates that particle size PEGylated DRM-loaded liposomes decreases with increase in the amount of HSPC, while it increases with increase in the amount of cholesterol added. High coefficient value for \u003cstrong\u003eX₁\u003c/strong\u003e indicates that amount of HSPC exerts more pronounced influence on particle size of DRM-loaded liposomes in comparison to amount of cholesterol. The ANOVA results for particle size are displayed in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Model\u0026apos;s F-value of 266.53 suggests that model is highly significant, with only 0.01% probability that like high F-value could be attributed to random variation. A p-value (Prob\u0026thinsp;\u0026gt;\u0026thinsp;F)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 confirms statistical significance of model terms. In the present analysis, X₁, X₂, and interaction term X₁₂ were found to be significant contributors. Predicted R\u0026sup2; value is 0.9649 demonstrates good agreement with Adjusted R\u0026sup2; is 0.9910, reflecting model\u0026apos;s robustness. Adequate Precision value, that reflects signal-to-noise ratio, was 55.30, exceeding the desirable threshold of 4, confirming an adequate signal. As a result, the created model can be trusted to explore and optimize the formulation design space (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eANOVA results for various responses of darolutamide loaded liposomes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" align=\"left\"\u003e\n \u003cp\u003eResponses\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eY\u003csub\u003e1\u003c/sub\u003e (Particle Size)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eY\u003csub\u003e2\u003c/sub\u003e (% Entrapment efficiency)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003cp\u003eProb\u0026thinsp;\u0026gt;\u0026thinsp;F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdequacy\u003c/p\u003e\n \u003cp\u003eprecision\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003cp\u003eProb\u0026thinsp;\u0026gt;\u0026thinsp;F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdequacy\u003c/p\u003e\n \u003cp\u003eprecision\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e266.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"6\" align=\"left\"\u003e\n \u003cp\u003e55.301\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"6\" align=\"left\"\u003e\n \u003cp\u003e8.437\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1098.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0050\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e165.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1977\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX1X2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1551\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2963\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eX\u003csub\u003e1\u003c/sub\u003e, and X\u003csub\u003e2\u003c/sub\u003e are coded terms for independent variables; X\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e interaction terms; X\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e and X\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e are quadratic terms\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe influence of the independent variables on particle size is visually illustrated through 3D response surface plots (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(A)) and perturbation plots (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(B)). These graphical representations clearly show that increasing the amount of HSPC from 5 to 15 mg leads to a reduction in particle size, whereas increasing the amount of cholesterol from 15 to 35 mg results in an increase in particle size.\u003c/p\u003e\n\u003cp\u003eIncreasing the amount of HSPC provides more amphiphilic molecules that can self-assemble into smaller and more stable vesicles due to better packing and curvature stabilization. [37] Higher HSPC concentration enhances bilayer rigidity and reduces surface energy, promoting the formation of smaller unilamellar vesicles during sonication or extrusion. [38] As cholesterol content increases, it stabilizes the bilayer but also reduces its curvature, favoring the formation of larger and more rigid liposomes. [39] At high cholesterol levels, the bilayer becomes more condensed and thicker, resisting size reduction during mechanical processing. [40] The perturbation plot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(B)) for particle size further validated these findings. The plot shows a steeper slope for factor A (HSPC) compared to factor B (cholesterol), indicating that the amount of HSPC has a more substantial impact on particle size than cholesterol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of independent variables on % entrapment efficiency (Y\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) of PEGylated DRM loaded liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe % EE of DRM loaded liposomes formulations are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The % EE was found in the range of 62.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67 to 95.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67%. The effect of the independent variables on the % EE can be explained by the following linear equation.\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;81.20\u0026thinsp;+\u0026thinsp;9.27X\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;3.57X\u003csub\u003e2\u003c/sub\u003e (2)\u003c/p\u003e\n\u003cp\u003eHere, \u003cstrong\u003eY₂\u003c/strong\u003e represents the percentage entrapment efficiency (%EE), while \u003cstrong\u003eX₁\u003c/strong\u003e and \u003cstrong\u003eX₂\u003c/strong\u003e correspond to the amounts of HSPC and cholesterol, respectively. The equation suggests that both HSPC and cholesterol concentrations positively influence %EE, indicating that increasing either component leads to enhanced entrapment efficiency of the PEGylated DRM-loaded liposomes. The higher coefficient for \u003cstrong\u003eX₁\u003c/strong\u003e reflects the greater impact of HSPC on %EE compared to cholesterol. The ANOVA results for %EE are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The model\u0026apos;s F-value of 7.38 confirms its statistical significance, with only a 0.01% chance of such a result occurring randomly. A p-value below 0.05 indicates that the model terms, particularly X₁, are significant. The predicted R\u0026sup2; (0.5076) shows acceptable alignment with the adjusted R\u0026sup2; (0.5152), while the adequate precision ratio of 8.437 reflects a good signal-to-noise ratio (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). These results confirm the suitability of the quadratic model for exploring the design space.\u003c/p\u003e\n\u003cp\u003eThe 3D response surface and perturbation plots illustrating the effect of independent variables on %EE are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC. These plots illustrate that %EE increases with higher concentrations of HSPC (5 to 15 mg) and cholesterol (15 to 35 mg). Increasing HSPC concentration means more bilayer material is available to form liposomes, resulting in more drug can be accommodated either in the aqueous core (hydrophilic) or in the lipid bilayer (hydrophobic). [41] Cholesterol stabilizes the bilayer by filling the spaces between phospholipid tails. Especially for hydrophobic drugs, cholesterol increases bilayer thickness and hydrophobic volume, enhancing drug incorporation. [42] A perturbation plot is a useful tool to assess the influence of individual independent variables on the response. The perturbation plot for %EE (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD) further supports the trends observed in the 3D response surface plots.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA numerical optimization approach was used to identify the optimized PEGylated DRM-loaded liposome formulation within the design space by setting constraints to minimize particle size and maximize entrapment efficiency (%EE). The software suggested an optimized formulation containing 15 mg of HSPC and 24.07 mg of cholesterol, with a desirability value of 1. The overlay plot illustrates the optimized formulation and its predicted response values (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). The optimized formulation of PEGylated DRM-loaded liposomes was prepared and experimentally evaluated for the selected responses. The predicted and actual response values, along with their percentage prediction errors, are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The close agreement between these values confirms the success and reliability of the design and optimization process. The % prediction errors ranged from \u0026minus;\u0026thinsp;3.05 to 1.52%, indicating the robustness and validity of the QbD approach applied for optimizing PEGylated DRM-loaded liposomes.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eValidation of optimized formulation\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResponse\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePredicted value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eObserved value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrediction error (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eParticle size (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026minus;\u0026thinsp;3.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEE (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of DRM loaded Liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticle size and Zeta Potential\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimized PEGylated liposomal formulation, DRML-9, displayed the smallest vesicle size among all tested formulations, with an average diameter of 87.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25 nm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Nanoparticles within this size range are generally considered favorable for drug delivery, as they can facilitate enhanced permeability and retention (EPR) effects, promote cellular internalization, and improve biodistribution.[43] Smaller vesicles also increase the surface area-to-volume ratio, which enhances drug loading and interaction with biological membranes. Zeta potential analysis was specifically carried out for DRML-9 to assess its colloidal stability.[44] The observed zeta potential of \u0026minus;\u0026thinsp;30.4 mV (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) indicates that the formulation possesses a sufficient negative surface charge to prevent particle aggregation through electrostatic repulsion. A zeta potential magnitude greater than \u0026plusmn;\u0026thinsp;30 mV is typically regarded as indicative of good physical stability, which is essential for maintaining the uniformity and shelf life of nanoparticulate systems. The targeted characterization of DRML-9 in terms of particle size and surface charge highlights its promising physicochemical attributes, supporting its potential for efficient and stable therapeutic delivery.[45]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurface Morphology by TEM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransmission Electron Microscopy (TEM) analysis was conducted to confirm the formation of PEGylated DRM liposomes and to investigate their surface morphology and structural characteristics. The TEM images (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC) revealed the presence of spherical to nearly spherical unilamellar vesicles with moderate size distribution. The liposomes exhibited well-defined boundaries, indicating successful formation of intact bilayer structures. No significant aggregation or deformation was observed, suggesting good colloidal stability of the formulation. [46] The absence of aggregation is a positive indication of the colloidal stability of the PEGylated liposomes. PEGylation is known to impart steric stabilization to liposomes, reducing inter-vesicular interactions and preventing fusion or aggregation during storage or in biological environments [47]. Nanoscale and uniform size distribution visualized in the TEM micrographs suggests controlled vesicle formation, which is essential for predictable pharmacokinetic behavior and biodistribution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTIR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA presents the FTIR spectra of DRM, showing characteristic peaks corresponding to the functional groups present in its structure. These peaks include stretching vibrations at 3764.95 cm⁻\u0026sup1; (-O-H -) alcohol, 3051.45 cm⁻\u0026sup1; (C-H), 2229.34cm⁻\u0026sup1; (Nitrile), 1646.82cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;N), 1440..69 cm⁻\u0026sup1; (O-H) carboxylic acid, 1233.30cm⁻\u0026sup1; (C-N), and (C\u0026thinsp;=\u0026thinsp;C alkene) at 990.63 cm⁻\u0026sup1;, which is attributed to the presence of four neighboring hydrogen bonds on the hetero-aromatic nucleus. These values closely match those observed in the standard DRM structure, confirming the integrity of the functional groups in the DRM. In contrast, the FTIR spectra of optimized PEGylated DRM liposomes (DRML-9) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD) revealed similar stretching vibrations: 3740.84 cm⁻\u0026sup1; (-O-H -) alcohol, 3116.11 cm⁻\u0026sup1; (C-H), 2354.01cm⁻\u0026sup1; (Nitrile), 1682.29cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;N), 1412.34 cm⁻\u0026sup1; (O-H) carboxylic acid, 1225.81cm⁻\u0026sup1; (C-N), and (C\u0026thinsp;=\u0026thinsp;C alkene) at 941.44 cm⁻\u0026sup1;,The FTIR analysis shows that the key functional peaks of DRM were retained in the optimized DRML formulation, suggesting that no significant chemical interaction occurred between DRM and the excipients used in the formulation. These results indicate that the drug and excipients are compatible, and the drug\u0026rsquo;s chemical integrity is preserved in the nanoemulsion formulation [53\u0026ndash;54].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDSC Studies\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e represents the DSC thermograms of DRM, cholesterol, HSPC, and optimized PEGylated DRM liposomes (DRML-9). It is evident that the DSC thermogram of DRM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), cholesterol (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), HSPC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC) and DRML-9 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD) exhibits a single sharp characteristic, endothermic melting peak at 169.07\u0026deg;C, 145.51\u0026deg;C, 207.38\u0026deg;C and 233.42\u0026deg;C which is in agreement with\u0026rdquo; that reported previously. The crystalline state of the DRM is specified by the sharp endotherm. The thermogram of the optimized PEGylated DRML revealed that DRM is molecularly dispersed in DRML; however, DRML did not exhibit the \u0026ldquo;characteristic endothermic peak from the crystalline DRM. The endothermic melting peak at the 233.42\u0026deg;C corresponds to either HSPC or Cholesterol\u0026rdquo;.[55] This shows that the DRM that was added to the liposomal formulation was not crystalline, suggesting that the DRM may have been molecularly dispersed within the liposomal matrix or that it may have transformed into an amorphous state. It also proposes that the DRM is entirely trapped within the liposomal lipid matrix.[56]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXRD Studies\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eXRD studies were performed to investigate the physical state of DRM when loaded into liposomes, comparing it to pure DRM, cholesterol, HSPC and lyophilized DRML. The diffractogram of DRM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA) exhibited characteristic intense reflections at specific diffraction angles, including 79.23, 13.78, 14.94, 16.35, 16.38, 16.42, 16.46, 16.48, 16.50, 16.53, 18.29, 22.75, and 24.90 (2\u0026theta;), with corresponding intensity counts of 577, 423, 493, 1998,2376, 2155, 2286, 2150, 1932, 1755, 660, 995, and 975. These distinct peaks indicate the crystalline nature of DRM, confirming that it exists in its solid, crystalline form. In contrast, the X-ray diffraction pattern of DRML (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD) showed a significant reduction or complete disappearance of these characteristic crystalline peaks. This observation suggests that DRM has transformed its crystalline state to an amorphous or less crystalline form when incorporated into the liposomal formulation. The loss of sharp diffraction peaks is consistent with the DSC results, indicating a shift in the melting temperature and the disappearance of the crystalline DRM peak in the optimized PEGylated DRML formulation. This transformation is likely due to the dispersion of DRM within the liposomal lipid matrix of, leading to a more amorphous structure [57].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003edrug release study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDRM exhibited poor, pH-independent release due to its low solubility, whereas PEGylated DRM liposomes showed sustained release at blood pH (7.4) and enhanced, pH-responsive release at tumor pH (5.8) [36]. To maintain sink conditions during the study, 5% (v/v) methanol was added to the release medium [48].\u003c/p\u003e\n\u003cp\u003eThe DRM showed 24.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.75% drug release in blood pH when compared to tumor pH showed 29.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62%. The developed PEGylated DRM liposomes exhibited significantly higher drug release of 71.56\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48% at the acidic tumor pH of 5.8, compared to 16.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17% at the physiological blood pH of 7.4, confirming their pH-sensitive behaviour. This characteristic is advantageous for targeted drug delivery within the tumor microenvironment. These findings further support the hypothesis that acidic conditions promote the destabilization of liposomes and other nanocarriers, resulting in enhanced drug release [49\u0026ndash;50].\u003c/p\u003e\n\u003cp\u003ePEGylation improves the stability and systemic circulation of DRM liposomes by minimizing drug leakage at physiological pH. At tumor pH, the acidic environment can promote destabilization of PEGylated liposomes, resulting in a controlled, targeted burst release of DRM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnticancer Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt is vital to evaluate the cytotoxicity of DRM and optimized PEGylated DRML-9 for their potential application as anticancer agents. The viability of PC-3, LNCaP and DU-145 cells treated with 0.325 to 100 \u0026micro;g/mL exhibited concentration-dependent cytotoxicity. The viability of the PC-3, LNCaP and DU-145 cells was drastically reduced after treatment with DRM and optimized DRML-9 shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eDRM and optimized DRML-9 exhibited significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) cytotoxicity against LNCaP cells (low IC\u003csub\u003e50\u003c/sub\u003e value 9.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 \u0026micro;g/mL and 5.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 \u0026micro;g/mL) for PC-3 (14.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u0026micro;g/mL and 10.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u0026micro;g/mL) and against DU-145 (20.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u0026micro;g/mL and 13.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u0026micro;g/mL) respectively after 48 h of incubation. DRM and DRML-9 exhibited considerable cytotoxicity against PC-3 cells, in contrast to LNCaP and DU-145 cells. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, the use of optimized DRML-9 results in a considerable (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) suppression of cancer cell growth in comparison to DRM. The LNCaP cells were chosen for further examination due to their heightened susceptibility to optimized DRML-9 compared to other cells.\u003c/p\u003e\n\u003cp\u003eOptimized DRML-9 has been shown to significantly enhance cytotoxicity in prostate cancer cell lines (LNCaP). DRML displays higher cellular uptake due to the nano-size range, which facilitates endocytosis-mediated internalization by cancer cells. Once internalized, liposomes provide a sustained release of the drug, ensuring a prolonged intracellular presence and a higher local concentration of the drug at the target site, leading to enhanced AR inhibition and apoptosis .Moreover, liposomes can protect DRM from enzymatic degradation, Improve bioavailability and pharmacokinetics, Allow passive targeting via the enhanced permeability and retention (EPR) effect in tumor tissues and Be modified with surface ligands such as PEGylation for active targeting to prostate cancer cells. Cytotoxicity conforms DRML liposomes have significantly lower IC₅₀ values compared to free DRM in LNCaP when compared to PC-3 and DU-145 prostate cancer cell lines, indicating superior cytotoxic potential.[51\u0026ndash;52 ].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis by Flow cytometer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne of the ways that anticancer moieties may cause cell death in tumor tissue is by apoptosis. We evaluated the apoptosis-inducing capacity of DRM and optimized PEGylated DRML-9 in LNCaP cells using the Annexin V-FITC/PI staining method. In control sample Just 0.33, 0.41, and 0.16% of the cells were in early apoptotic, late apoptotic, and necrotic phases, respectively Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA. When treated with DRM and optimized DRML-9 the % of live cells decreased from 99.10\u0026ndash;83.9% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB) and 73.7% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC), respectively.\u003c/p\u003e\n\u003cp\u003eDRML-treated cells exhibited that 16.04% of the cells were in the apoptotic state where 11.79% of the cells were in the apoptotic stage in DRM treated. Both DRM and optimized DRML-9 are significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) causing apoptosis, as compared to the untreated group. Similarly, as compared to DRM, DRML-9 exhibited a considerable (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) ability to induce apoptosis, a form of cell death. Based on these results, DRML-9 treatment resulted in a much higher % of cells in the apoptotic state than DRM treatment and control.\u003c/p\u003e\n\u003cp\u003eDRM, a potent second-generation androgen receptor (AR) antagonist, liposomal encapsulation significantly enhances its apoptotic potential in LNCaP cell lines compared to the pure DRM. This enhancement is attributed to improved cellular uptake, sustained intracellular release, and higher bioavailability at the tumor site. DRM-loaded liposomes induce apoptosis more effectively due to Enhanced Cellular Uptake due to nanoscale size (typically\u0026thinsp;\u0026lt;\u0026thinsp;200 nm) and lipid composition of liposomes facilitate endocytosis-mediated internalization, ensuring higher intracellular DRM concentrations than pure DRM, which may enter cells via passive diffusion. [53] DRML provide sustained drug release, prolonging the intracellular exposure of cancer cells to DRM. This allows more effective suppression of AR signalling pathways, which are crucial for prostate cancer cell survival and resistance. DRML lead to increased mitochondrial membrane depolarization, cytochrome c release, and activation of caspase-9 and caspase-3, hallmarks of intrinsic apoptotic pathway activation. This effect is more pronounced than in cells treated with pure DRM. Studies have shown that liposomal formulations increase the Bax/Bcl-2 ratio, enhance cleaved PARP expression, and promote DNA fragmentation, further supporting their stronger apoptotic potential. DRML more effectively suppress Survivin and Bcl-2, proteins that inhibit apoptosis and are often upregulated in androgen-dependent prostate cancers. [54]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis by DAPI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing a 48-hour treatment with DRM and optimized PEGylated DRML-9, DAPI staining was used to detect the programmed cell death in LNCaP cells. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD-F displays fluorescence microscopic images of cells that had not been treated and cells that had been treated with DRM and optimized DRML-9. The untreated cells, referred to as the negative control, exhibited normal intact nuclei with faint and uniform blue staining. In contrast, the groups treated with DRM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE) and DRML-9 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF) displayed small nuclei with intense chromatin condensation, nuclear fragmentation, and the formation of apoptotic bodies. The results indicate that both DRM and optimized DRML-9 induce apoptosis in LNCaP cells. Cells treated with DRM and optimized DRML-9 have fragmented nuclei and uneven margins close to the nuclei, which are signs of apoptosis. For apoptosis study, fluorescence microscopy with cell permeable nucleic acid dye, such as DAPI, is frequently employed to evaluate nuclear morphology. [55\u0026ndash;56] These changes can be caused by cell permeability or the activation of the apoptotic-inducing enzyme. The literature also contains similar findings. [57\u0026ndash;60]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Cycle Arrest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell cycle is a highly regulated process that controls cell division in living organisms. Through specific checkpoints, it prevents uncontrolled cell proliferation by detecting DNA damage, promoting DNA repair, or triggering the elimination of potentially cancerous cells. Both DRM and optimized PEGylated DRML-9 induced permanent cell death specifically during the S phase [61]. In control cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA) (67.2%) cells are in sub G\u003csub\u003e1\u003c/sub\u003e phase (14.5%) cells are in G\u003csub\u003e0\u003c/sub\u003e/G\u003csub\u003e1\u003c/sub\u003e phase. In the S phase (7.90%) and the G2/M phase (4.25%), distinct populations of cells were also seen. DRM and optimized DRML-9 caused permanent cell death during the S phase [62\u0026ndash;63]. Cells treatment with DRM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB) and DRML-9 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC) undergo a massive shift of cells from sub G1 to G0/G1 and S phase arrest of almost 18.3% and 45.7% and 8.77% and 15.6% of the cell population respectively. The therapy with DRML resulted in almost 2-fold increase in S phase arrest compared to DRM alone. Furthermore, the administration of DRM and optimized DRML-9 resulted in a partial decrease in the number of cells arrested in the G2/M phase of the cell cycle.[64]\u003c/p\u003e\n\u003cp\u003eThe encapsulation of DRM in liposomal carriers has been shown to significantly enhance its anti-proliferative activity in prostate cancer cells by inducing a more pronounced cell cycle arrest, especially at the G1 phase, compared to the free DRM. This enhancement is primarily due to improved drug delivery efficiency, cellular internalization, and sustained intracellular release, which collectively boost the suppression of androgen receptor (AR)-mediated signalling pathways involved in cell cycle progression. Pure DRM, as a potent AR antagonist, disrupts AR transcriptional activity and suppresses genes necessary for G1 to S phase transition, such as Cyclin D1, CDK4/6, and E2F1, leading to G1 phase arrest.[65] Enhanced Cell Cycle Arrest by DRML is mainly due to Improved Intracellular Accumulation:\u003c/p\u003e\n\u003cp\u003eDRML enhance the intracellular concentration of DRM through endocytosis, facilitating more effective inhibition of AR-dependent gene expression required for cell cycle progression.\u003c/p\u003e\n\u003cp\u003eThe sustained release from DRML ensures prolonged exposure of cancer cells to DRM, allowing continuous inhibition of Cyclin D\u0026ndash;CDK4/6 complex activity, which is essential for G1/S transition.[ ] Liposomes more effectively reduce the expression of proliferative markers like Ki-67 and PCNA, indicating a stronger blockade of mitotic entry. LNCaP cells treated with liposomal formulations often reveals a significantly higher percentage of cells in G0/G1 phase and a reduced S-phase population compared to cells treated with pure DRM. Liposomal delivery enhances the downregulation of AR-regulated genes such as CDC25A, Cyclin E, and Skp2, further reinforcing the G1 phase blockade.[66]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn-Vivo\u003c/strong\u003e \u003cstrong\u003ePharmacokinetic Study\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eIn plasma RT (retention time) of DRM has been observed to be 8.8 min. Upon intravenous administration of DRM and PEGylated DRML-9 in concentration V/s time profile in plasma C\u003csub\u003emax\u003c/sub\u003e for DRML has been observed to be 8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 \u003cstrong\u003e\u0026micro;\u003c/strong\u003eg /mL\u0026thinsp;\u0026minus;\u0026thinsp;1 at 1 h while C max of plain DRM (7.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 \u003cstrong\u003e\u0026micro;\u003c/strong\u003eg mL\u0026thinsp;\u0026minus;\u0026thinsp;1) at zero hours (T\u003csub\u003emax\u003c/sub\u003e) plasma concentration was noted for both DRM and DRML-9 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;AUC of DRML-9 was observed to be significantly greater (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) as compared to DRM (64.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67 \u003cstrong\u003e\u0026micro;\u003c/strong\u003eg. h. mL\u0026thinsp;\u0026minus;\u0026thinsp;1) than DRM (17.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32 \u003cstrong\u003e\u0026micro;\u003c/strong\u003eg.h. mL\u0026thinsp;\u0026minus;\u0026thinsp;1) correspondingly. The elimination rate constant (K), \u0026amp; elimination half-life (T\u003csub\u003e1/2\u003c/sub\u003e) for DRML-9 and\u0026rdquo; DRM (0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 \u0026amp; 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031 \u003cstrong\u003e\u0026micro;g/mL)/h\u003c/strong\u003e) and (7.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64, \u0026amp; 6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 h) correspondingly. MRT for DRML-9 \u0026amp; DRM has been observed to be 11.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 \u0026amp; 6.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 h respectively. Detailed results have been shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab9\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePharmacokinetic Parameters of DRM after intravenous administration of DRM and optimized PEGylated DRM loaded liposomes (DRML-9) (5mg/kg) in rats)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDRM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDRML-9\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003emax\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003e(h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e \u003csub\u003e\u003cstrong\u003emax\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003e(\u0026micro;g/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAUC\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003e(\u0026micro;g/ml*h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAUC\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;inf\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003e(\u0026micro;g/ml*h^2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003et\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1/2\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003e(h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMRT (h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCL ( \u0026micro;g/ml)/h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Bioavailability\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e362.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eA significant increase of 3.63 times in bioavailability was seen when DRML-9 was administered intravenously compared to pure DRM. Liposomal drug delivery systems enhance bioavailability through several key mechanisms. The primary factor is the encapsulation of drugs within a liposomal matrix, which protects them from enzymatic degradation in the bloodstream, thereby improving drug stability and extending circulation time. [65\u0026ndash;66] PEGylated liposomes further evade the reticuloendothelial system (RES), prolonging systemic circulation and enhancing bioavailability. [67]\u003c/p\u003e\n\u003cp\u003ePEGylated liposomes can be engineered for passive or active targeting, increasing drug accumulation at the desired site of action while minimizing off-target distribution. [68\u0026ndash;69] This selective delivery improves therapeutic efficacy and reduces adverse effects. Additionally, liposomes can encapsulate hydrophobic drugs within their lipid bilayer, enhancing aqueous solubility and systemic availability following intravenous administration. [70] By enabling controlled release and influencing biodistribution, liposomes reduce systemic toxicity and improve the therapeutic index of encapsulated drugs. Furthermore, liposomes can enhance cellular uptake through endocytosis or membrane fusion, particularly benefiting drugs with poor membrane permeability. [71\u0026ndash;72]\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Accelerated Stability Study\u003c/h2\u003e\n \u003cp\u003eThere are no significant changes in the particle size and entrapment efficiency of optimized PEGylated DRML-9 after storage of 3 months at 2 to 8\u0026deg;C. The particle size and entrapment efficiency of optimized DRML after 1, 2, and 3 months of storage are depicted in Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab11\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStability study of optimized PEGylated DRM loaded liposomes (DRML-9) at 2\u0026ndash;8\u0026deg;C showing % EE and particle size\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e00 Days\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e30 Days\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e60 Days\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e90 Days\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEE (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eParticle size (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, PEGylated Darolutamide-loaded liposomes (DRML) were successfully developed and optimized using a 3\u0026sup2; factorial design. The optimized PEGylated DRML exhibited high entrapment efficiency, minimal vesicle size in the nanometer range, and good colloidal stability with a negative zeta potential. Notably, PEGylated DRML demonstrated a significant enhancement in the anticancer potential of Darolutamide against prostate cancer cells. Both in-vitro and in-vivo studies confirmed the improved therapeutic efficacy of PEGylated DRML in the treatment of prostate carcinoma. Overall, PEGylated DRML represents a promising strategy to enhance the therapeutic potential of Darolutamide.\u003c/p\u003e"},{"header":"Declarations","content":"\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eNo funding was received for this study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eVinod V. Patil: Investigation, Methodology, Writing \u0026ndash; original draft, Validation, Conceptualization, Resources. Harshal Pawar: Methodology, Software, Writing \u0026ndash; review, editing, Conceptualization, Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are thankful to School of Pharmacy, Mansarovar Global University, Kolar Road, Bhopal 462042, Madhya Pradesh, India for providing required guidance and support for completion of this research work. Authors are also thankful to Gattefosse India and BASF India for providing gift samples of lipids and surfactants. We thank Diya Labs Mumbai for Characterization of formulations. The authors are also thankful to NCBI Pune for providing \u0026ndash;cancer cell line, Maratha Mandal Dental College and research center Belagavi for anticancer activity, and Biocyte Sangli for bioanalytical work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchafer EJ, Laversanne M, Sung H, Soerjomataram I, Briganti A, Dahut W, Bray F, Jemal A. 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A., Grillo, R., Swamy, M. K., Sharma, S., Habtemariam, S., \u0026amp; Shin, H. S. (2018). Nano-based drug delivery systems: Recent developments and future prospects. \u003cem\u003eJournal of Nanobiotechnology\u003c/em\u003e, 16, 71.\u003cbr /\u003e https://doi.org/10.1186/s12951-018-0392-8.\u003c/li\u003e\n\u003cli\u003eAllen, T. M., \u0026amp; Cullis, P. R. (2013). Liposomal drug delivery systems: From concept to clinical applications. \u003cem\u003eAdvanced Drug Delivery Reviews\u003c/em\u003e, 65(1), 36\u0026ndash;48.\u003cbr /\u003e https://doi.org/10.1016/j.addr.2012.09.037.\u003c/li\u003e\n\u003cli\u003eTorchilin, V. P. (2011). Multifunctional and stimuli-sensitive pharmaceutical nanocarriers. \u003cem\u003eEuropean Journal of Pharmaceutics and Biopharmaceutics\u003c/em\u003e, 77(3), 453\u0026ndash;463.https://doi.org/10.1016/j.ejpb.2010.12.006.\u003c/li\u003e\n\u003cli\u003eAkbarzadeh, A., Rezaei-Sadabady, R., Davaran, S., Joo, S. W., Zarghami, N., Hanifehpour, Y., Samiei, M., Kouhi, M., \u0026amp; Nejati-Koshki, K. (2013). Liposome: Classification, preparation, and applications. \u003cem\u003eNanoscale Research Letters\u003c/em\u003e, 8, 102.\u003cbr /\u003e https://doi.org/10.1186/1556-276X-8-102.\u003c/li\u003e\n\u003cli\u003eKarimi M, Aslanabadi A, Atkinson B, Hojabri M, Munawwar A, Zareidoodeji R, Ray K, Habibzadeh P, Parlayan HN, DeVico A, Heredia A. Subcutaneous liposomal delivery improves monoclonal antibody pharmacokinetics in vivo. \u003cem\u003eActa Biomater\u003c/em\u003e. 2025 Mar 15;195:522-35. doi:10.1016/j.actbio.2024.12.045.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"Darolutamide, Liposomes, Cancer, Pharmacokinetic study, Apoptosis, and Cell cycle","lastPublishedDoi":"10.21203/rs.3.rs-7203702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7203702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study aimed to develop a darolutamide (DRM) loaded PEGylated liposomal (DRML) to improve therapeutic efficacy in the treatment of prostate cancer. A 3\u0026sup2; full factorial design was employed to evaluate the influence of Hydrogenated Soy Phosphatidylcholine (HSPC) and cholesterol concentrations on key formulation parameters, including particle size and entrapment efficiency (EE). The optimized PEGylated DRM liposomal formulation (DRML-9) was further characterized by transmission electron microscopy (TEM) for morphology, X-ray diffraction (XRD) for crystallinity, and differential scanning calorimetry (DSC) for thermal behavior. Among 13 liquid formulations (PDL\u0026ndash;1 to PDL\u0026ndash;13), PDL\u0026ndash;9, comprising 15 mg HSPC, 35mg cholesterol, exhibited optimal characteristics, including particle size (87.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25) and higher entrapment (95.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67%). FTIR spectra confirmed drug-excipient compatibility, and DSC as well as XRD data indicated the amorphization and molecular dispersion of DRM in the liposomal formulation. The DRML-9 formulation exhibited superior drug release (71.56\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48%) at tumor pH compared to blood pH (16.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17%). DRML-9 significantly inhibited LNCaP prostate cancer cell proliferation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and induced apoptosis as evidenced by Annexin-V/PI staining. Cell cycle study in DRML resulted in almost 2-fold increase in S-phase arrest compared to DRM alone. Pharmacokinetic evaluation demonstrated a 3.6-fold improvement in intravenous bioavailability of PEGylated DRML over pure DRM. Collectively, these findings establish PEGylated DRML-9 as a promising intravenous delivery platform for improving the therapeutic potential of darolutamide in treatment of prostate cancer.\u003c/p\u003e","manuscriptTitle":"Development and Optimization of PEGylated Darolutamide-Loaded Liposomes for Treatment of Prostate Cancer: In vitro and In-vivo Characterization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 07:18:04","doi":"10.21203/rs.3.rs-7203702/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":"9c7784d8-9846-44f2-8a65-b3ae73dd793d","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:06:25+00:00","versionOfRecord":{"articleIdentity":"rs-7203702","link":"https://doi.org/10.1007/s12247-025-10352-6","journal":{"identity":"journal-of-pharmaceutical-innovation","isVorOnly":false,"title":"Journal of Pharmaceutical Innovation"},"publishedOn":"2026-02-13 15:59:07","publishedOnDateReadable":"February 13th, 2026"},"versionCreatedAt":"2025-11-26 07:18:04","video":"","vorDoi":"10.1007/s12247-025-10352-6","vorDoiUrl":"https://doi.org/10.1007/s12247-025-10352-6","workflowStages":[]},"version":"v1","identity":"rs-7203702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7203702","identity":"rs-7203702","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}