Optimizing Gold Nanoparticles for Combination Therapy: Development of Hydrophobic Nanomedical Devices with Gemcitabine and Ascorbyl Palmitate

Optimizing Gold Nanoparticles for Combination Therapy: Development of Hydrophobic Nanomedical Devices with Gemcitabine and Ascorbyl Palmitate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Optimizing Gold Nanoparticles for Combination Therapy: Development of Hydrophobic Nanomedical Devices with Gemcitabine and Ascorbyl Palmitate Havva Rezaei, Mostafa Shourian This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7240807/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Breast cancer remains a significant global health challenge, often treated with gemcitabine hydrochloride (GEM). However, GEM's short half-life and poor sustained release can lead to severe side effects, including myelosuppression and nephrotoxicity. This study presents a novel nano-drug delivery system using gold nanoparticles (AuNPs) optimized with ascorbyl palmitate (AsP) to enhance GEM stability and efficacy. AuNPs were modified via single-phase emulsification to form a nanoemulsion coated with a hydrophobic AsP layer, improving tumor targeting through the enhanced permeability and retention (EPR) effect. Two formulations were developed: Au-GEM-AsP-COV (prodrug, 128.5 nm, -18.3 mV, 89.5% encapsulation efficiency) and Au-GEM-AsP-Phys (106 nm, -15.9 mV, 87% encapsulation efficiency). The Au-GEM-AsP-COV formulation demonstrated superior hydrophobicity, sustained release, and enhanced cytotoxicity (IC50 of 0.44 µg/mL) in the 4T1 cell line, significantly outperforming free GEM and modified Au-GEM formulations. Notably, it exhibited six months of accelerated stability, attributed to amide bond formation in the functionalized AuNP matrix. The study highlights the synergistic effects of AsP in enhancing the therapeutic efficacy of Au-GEM-based formulations, supporting its role as a key component in combination therapy. This research lays the foundation for future development of hydrophobic nanomedical devices combining GEM and AsP for therapeutic and diagnostic applications in nanomedicine. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Cancer Physical sciences/Chemistry Biological sciences/Drug discovery Physical sciences/Materials science Physical sciences/Nanoscience and technology Gold Nanoparticles Ascorbyl Palmitate Nanoemulsion Gemcitabine Breast cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cancer is a deadly disease characterized by uncontrolled cell proliferation and the spread of abnormal cells through invasion or metastasis 1 . Traditional cancer treatments include surgery, radiotherapy, and chemotherapy 2 . However, these treatments have significant side effects and limited efficacy, prompting researchers to explore new, effective targeted delivery systems based on nanochemistry platforms for the active targeting delivery of anticancer drugs, either alone or in combination with other effective active pharmaceutical ingredients (APIs) 3 – 6 . These novel drug delivery systems also affect pharmacokinetics and the ADME processes (absorption, distribution, metabolism, and excretion) 6 , 7 . Gemcitabine (GEM), a first-line chemotherapy for pancreatic cancer, is a nucleoside analog antimetabolite with proven antitumor activity and tolerability in non-small cell lung, ovarian, and metastatic breast cancers. However, its clinical utility is limited by rapid metabolism, resulting in a short plasma half-life (8–17 minutes) and systemic toxicity due to high dose (1000–1250 mg/m²) requirements for therapeutic levels. Additionally, after a few months, cells develop chemoresistance. Multiple clinical and experimental investigations have demonstrated that a combination or co-administration of other drugs as chemotherapies with GEM leads to superior therapeutic benefits 8 . Natural products have significantly contributed to anticancer research, as most clinically used anticancer drugs originate from natural sources 2 . Interest is growing in natural antioxidants like Vitamin C (L-ascorbic acid, ascorbate, VC), a water-soluble vitamin that scavenges free radicals and prevents DNA damage 9 – 11 . At pharmacologic concentrations, ascorbate undergoes oxidation via ascorbate radical, generating cytotoxic hydrogen peroxide (H₂O₂) through Fenton chemistry 8 , 11 – 14 . Several clinical trials have explored ascorbate's synergistic effects with cancer chemotherapeutics 8 , 15 . Michael Graham Espey et al. reported that GEM–ascorbate combinations in mice with pancreatic tumor xenografts enhanced growth inhibition versus GEM alone 2 . Daniel A. Monti et al., in phase I studies, observed increased toxicity with intravenous ascorbic acid combined with GEM and erlotinib in 14 metastatic stage IV pancreatic cancer patients, suggesting a longer phase II trial 3 . Joseph J. Cullen reviewed a Phase 2 trial (PACMAN 2.1) of high-dose ascorbate with nab-paclitaxel and GEM 4 . Ascorbyl palmitate (AsP), a key derivative of ascorbic acid, offers greater stability and functions as an antioxidant with antitumor activity via its antiproliferative effect 6 , 11 , 16 . However, combination therapies often cause severe systemic toxicity. Thus, developing a co-loaded drug delivery system with AsP and GEM is an attractive strategy to enhance anticancer treatment efficiency, improve stability and bioavailability, enable tumor-specific delivery, and minimize chemotherapy-related side effects. Mohamed El-Far et al. developed stable AsP-loaded Pluronic (F-127 or F-108) nano micelles to enhance AsP solubility and bioavailability using lower doses, reducing side effects compared to native AsP 11 . Min Zhou et al. designed AsP-based solid lipid nanoparticles combined with paclitaxel (AsP/PTX-SLNs) to maximize AsP’s therapeutic efficacy 15 . Mohamed El-Far et al. indicated the superiority of AsP-loaded Pluronic nanoparticles as a promising anticancer agent over native AsP, demonstrating a fantastic synergistic anticancer effect in combination with melatonin as a potential therapy against EAC-bearing mice 6 . Although AsP is more stable than vitamin C, its poor release capacity and water insolubility limit its bioavailability and therapeutic efficacy 15 , 17 . Thus, incorporating it into nanoparticle carriers can enhance circulation time and tumor accumulation via the enhanced permeability and retention (EPR) effect 18 , 19 . Recent studies show that nanocarriers with neutral, zwitterionic, or negative surface charge adsorb less protein, circulate longer, and internalize better than positively charged ones, leading to improved tumor distribution for similarly sized particles 16 , 18 – 21 . Nanoparticles sized 30–200 nm enhance cell uptake via increased surface area and membrane wrapping, effectively accumulating in tumors 22 , 23 . Overall, designing an optimal nanoparticle requires balancing drug-loading capacity, immune response, circulation time, and cellular uptake. Among many platforms, gold nanoparticles (AuNPs) stand out due to their physicochemical versatility, biocompatibility, and ease of surface modification 24 . Santiago et al. modified AuNP surfaces with GEM and folate/transferrin ligands to create a targeted controlled-release nanocarrier, reducing side effects and improving efficacy 14 . Developing a water-in-oil (W/O) emulsion using ultrasonic homogenizers, which mix two immiscible phases with a surfactant, can enable negatively charged surface modification of AuNPs’ hydrophilic surface by incorporating AsP 24 , 25 , 26 . Additionally, AuNPs can stabilize other drug carriers, such as liposomes, enhancing delivery efficiency 14 . Wang et al. demonstrated that nanoparticles adsorb phospholipids and induce gelation at the liposome surface. With 25% of the lipid surface occupied by nanoparticles, nanoparticle-modified liposomes showed no significant leakage over 50 days 27 . Yang et al. used AuNPs to stabilize oil-in-water emulsion droplets (< 100 nm), forming net negatively charged droplets. Positively charged AuNPs electrostatically bound to them, bridging strong repulsion and enhancing emulsion stability. The interaction between the AuNP-emulsion and AuNP-transferrin further improved droplet stability 28 . Surface charge polarity and density greatly influence immune clearance and cellular uptake of intravenously administered nanocarriers, affecting their delivery efficiency to target sites 29 . This study developed a gold nanocarrier for gemcitabine (GEM) with tunable surface properties within an ascorbyl palmitate (AsP) matrix. Gold nanoparticles (AuNPs) were synthesized with optimal size and surface charge, demonstrating stability in AsP presence. A novel nano-drug delivery system was created to enhance interaction between GEM and AuNPs using both physical (Au-GEM-AsP-Phys) and covalent (Au-GEM-AsP-COV) matrices. Chemical characterization via UV-visible spectroscopy, FT-IR, and in vitro assays confirmed effective binding and six-month accelerated stability of the covalent formulation. The MTT assay results revealed a significant reduction in IC 50 values, for the covalent formulation and the physical formulation, compared to the modified Au-GEM without AsP. These findings highlight the synergistic effect of AsP in enhancing therapeutic efficacy, attributed to its hydrophobicity and inherent anticancer properties. This research lays the groundwork for developing hydrophobic nanomedical devices that integrate GEM and AsP for dual applications in therapy and diagnostics. Future investigations will focus on evaluating in vivo toxicity and pharmacokinetics in relevant cancer models. Materials and Methods 2.1 Materials Tetra chloroauric acid (HAuCl 4 .2H 2 O), ascorbyl palmitate (AsP) (C 22 H 38 O 7 ), 11-mercaptoundecanoic acid (MUA) (HS(CH 2 ) 10 CO 2 H), 11-mercapto-1-undecanol (MU) (HS(CH 2 ) 11 OH), and 2-(3,5-diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole (MTT) (C 18 H 16 N 5 S + ) were purchased from Sigma-Aldrich. Gemcitabine hydrochloride (GEM) (C 9 H 11 F 2 N 3 O 4 ·HCl) was purchased from Shilpa. N-Hydroxy succinimide (NHS) (C 4 H 5 NO 3 ), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (C 8 H 17 N 3 ), tri-sodium Citrate (CIT) (C 6 H 5 Na 3 O 7 ), sodium dihydrogen phosphate (NaH 2 PO 4 ), sodium hydroxide (NaOH), hydrochloric acid (HCl), Tween 20 (C 26 H 50 O 10 ), dimethyl sulfoxide (DMSO) ((CH 3 )2SO), D-mannitol (C 6 H 14 O 6 ), propylene glycol (MPG) (C 3 H 8 O 2 ), polyethylene glycol 300 (PEG) (H(OCH 2 CH 2 ) n OH), absolute ethanol (ETOH) (C 2 H 6 O), and acetone ((CH 3 ) 2 CO) were obtained from Merck. The 4T1 cell line and acetonitrile (ACN) (CH 3 CN) were purchased from Zista Gene and Romil, respectively. All reagents were of analytical grade and doubly distilled water was used for the preparation of all solutions. 2.2 Synthesis of AuNPs in AsP matrix and Optimization So far, gold nanoparticle suspensions have been produced using sodium nitrate salt to achieve particles approximately 20 nanometers in size 30 . Additionally, the synthesis of gold nanoparticles using other reducing agents, such as ascorbic acid (Vitamin C), has also been reported 31 . In this study, we synthesized AuNPs using ascorbyl palmitate AsP as the reducing agent, emphasizing its potential to enhance the hydrophobicity of the nanoparticles due to its amphiphilic nature. For a comprehensive evaluation, we also synthesized AuNPs using CIT as the reducing agent, as it is a widely established method in nanoparticle synthesis, producing hydrophilic AuNPs. CIT not only acts as a reducing agent but also stabilizes the nanoparticles through the formation of a negatively charged surface, leading to good dispersibility in aqueous environments. By comparing the two, we aimed to highlight the potential of AsP-functionalized AuNPs to improve the enhanced permeability and retention (EPR) effect, which is crucial for drug delivery applications. The rationale for including CIT in this study is rooted in its historical significance and reliability in nanoparticle synthesis, ensuring a standard benchmark for evaluating the novel AsP method 30 . Before preparing the solutions, nitrogen was injected into 200 mL of water for 30 minutes to remove oxygen. This oxygen-free water was then used to prepare stock solutions from gold salt and AsP. To achieve stable AuNPs, five experimental sets were conducted at five different concentrations of the reducing solution AsP and hydrophobic AuNPs were prepared using the following synthetic route: Briefly, 10 µL of HAuCl 4 (100 mgmL − 1 ) was added to 9 mL of oxygen-free water at 70°C while mixing at 100 rpm. After 2 minutes, a specified volume of the 1 mgmL − 1 AsP stock solution was added dropwise with stirring at 150 rpm. After 20 minutes, the heater was turned off, and after 30 minutes, the shaker was also turned off, allowing the nanoparticles to gradually reach room temperature. The resulting nanoparticles from each experiment were analyzed for size, zeta potential, and UV-visible absorption spectrum to characterize their physicochemical properties. Additionally, the optimal formulation underwent MTT analysis on the 4T1 cell line to study the cytotoxic effects of the produced nanoparticles. A Transmission Electron Microscopy (TEM) image of the optimal sample was taken to examine morphology and determine size. Stability studies on the optimal sample, including changes in size, PDI, and visual appearance of the solution, were conducted at 0, 3, and 6 months at a temperature of 25°C ± 2°C and 60% ± 5% RH. 2.3 Gold metallic Nanocarrier containing GEM loaded in AsP matrix Loading the AuNPs containing GEM in an AsP matrix to leverage synergistic effects was achieved using nano emulsification technique. This process involved forming a nanoemulsion through micellization of the aqueous phase in the oil/surfactant (Oil/S) phase, followed by organic solvent evaporation and nanoparticle formation. The result was stable nanoparticles with higher concentrations of AsP, controllable size, and increased hydrophobicity 32 . 2.3.1 Innovation of the oil/surfactant (Oil/S) phase: Dispersing AsP in the PEG-MPG matrix To enhance the solubility of AsP, we used the placebo matrix consisting of polyethylene glycol 300 (PEG) and propylene glycol (MPG) in an ethanolic solution with a pH of 7.5, adjusted using ethanolic sodium hydroxide. This biocompatible matrix was incorporated into a new formulation, Au-GEM-AsP-Phys, with 70% matrix as the oil phase and 30% Tween 20 as a surfactant and cosolvent. A summary of Au-GEM-AsP-Phys nanoemulsion formation has shown in Fig. 1 A. For the aqueous phase, 500 µL of GEM stock solution (10 mgmL − 1 ) was mixed with 3.5 mL of optimized AuNPs (0.1 mgmL − 1 ) and stirred for 20 minutes. The aqueous phase was then grafted onto the AsP matrix (Oil/S phase) through emulsification, with dropwise addition and stirring at 300 rpm for 15 minutes, followed by homogenization at 12,000 rpm for 5 minutes. The nanoemulsion of Au-GEM-AsP-Phys was formed using sonication at 50 watts (20 seconds on, 10 seconds off) for 13 minutes, then shaken at 1,000 rpm for 15 minutes. Organic solvent evaporation was performed using a rotary evaporator at 30°C for two hours (Hrs). To separate any non-emulsified materials, aggregates and removal of excess surfactant, centrifugation has been performed at 12,000 rpm for 15 minutes at 15°C.The resulting Au-GEM-AsP-Phys nanoparticle solution was stored at 2–8°C and characterized using UV–Visible spectroscopy, size and zeta potential analysis, FT-IR spectroscopy, HPLC for encapsulation efficiency (EE) and assay, and cytotoxicity evaluation. Au-GEM-AsP-Phys Formulation stability was assessed by monitoring appearance, size distribution, PDI, and assay at 0, 3, and 6 months under storage conditions of 25°C ± 2°C and 60% ± 5% RH. 2.3.2 Surface Activation and Amide bond promotion As mentioned, the most significant drawback of GEM in its injectable form is its instability and short half-life due to enzymatic deactivation via its amine groups. Therefore, blocking this active site can be a solution to enhance its stability. To achieve this, the development of an amide bond between this amine group and the carboxylic group on the surface-modified gold nanoparticles has been advocated. Figure 1 B illustrates the activation of the AuNPs surface followed by the encapsulation of GEM within the AuNPs. Surface modification of gold nanoparticles was achieved through thiolation using 11-mercaptoundecanoic acid (MUA) and 11-mercaptoundecanol (MU). This process involved activating the carboxyl group on the surface of the nanoparticles using EDC/NHS chemistry. For this reason, to a suspension of optimized AuNPs (10 mL), 10 mL phosphate buffer with pH 8 and 50 µL Tween 20 were added and stirred at room temperature for 30 minutes. A 10 mL solution of 2 mM MUA/MU in an 8:1 volumetric ratio was then prepared and added to the nanoparticle suspension, which was stirred at room temperature for 12 Hrs. The MUA/MU-modified suspension was centrifuged at 14,000 rpm for 30 minutes at 4°C to remove the supernatant. The separated samples were washed first with phosphate buffer at pH 8 and then twice with phosphate buffer at pH 7, followed by centrifugation at 14,000 rpm for 30 minutes at 4°C to eliminate excess MUA/MU and Tween 20. The resulting precipitate was resuspended in MES buffer (pH 5.5) and centrifuged at 14,000 rpm for 30 minutes at 4°C. Then, 20 mL of a solution containing 10 mM EDC and 20 mM NHS was added, and the mixture was stirred at room temperature for 15 minutes using an incubator shaker, followed by centrifugation at 24,000 rpm for 10 minutes at 4°C 33 – 36 . To the carboxyl-activated AuNPs, 500 µL of GEM (10 mgmL - 1 ) was added, and the volume was adjusted to 2 mL using a phosphate buffer at pH 7.4. This Au-GEM suspension was stirred at room temperature for 20 minutes and then centrifuged at 24,000 rpm for 10 minutes at 4°C. The amount of free GEM in the supernatant was quantified. Subsequently, 5mL of mannitol (6.4 mgmL - 1 ) was added to the precipitate. The sample underwent freeze-drying under a pressure of 100 Pa for 48 Hrs, including a freezing stage at -40°C for 20 Hrs and primary drying at -25°C for 4 Hrs, followed by secondary drying for an additional 4 Hrs. Finally, the chamber temperature was raised to -5°C for final product freezing. The label claim of GEM in resulted Au-GEM powder was evaluated, along with its size and zeta potential 37 , 38 . 2.3.3 Au-GEM-AsP- COV nanoemulsion preparation Figure 1 C illustrates the single-phase emulsification process, where GEM-loaded modified gold nanoparticles are encapsulated within the AsP matrix, leading to the Au-AsP-GEM-COV nanoemulsion formation. The preparation of the Au-GEM-AsP-COV nanoemulsion involved dissolving AsP in acetone (12.5 mgmL − 1 ), mixing this oil phase with Tween 20 in a 70:30 ratio, and combining it with an aqueous phase reconstituted from freeze-dried Au-GEM powder in 4mL sodium phosphate buffer (pH 7.4). The aqueous phase was added dropwise to the oil/surfactant mixture, stirred at 300 rpm for 15 minutes, and homogenized at 12,000 rpm for 5 minutes. The mixture was then sonicated at 50 watts for 13 minutes (20 seconds on, 10 seconds off). Following sonication, the sample was stirred with an incubator shaker for 15 minutes, and the solvent was evaporated at 30°C for two Hrs. Centrifuge has been conducted at 12,000 rpm for 15 minutes at 15°C for separation and purification. The resulting Au-GEM-AsP-COV nanoparticles were characterized for drug loading, release profile, toxicity against 4T1 cell line. Also, the stability of the Au-GEM-AsP-COV formulation was evaluated by observing its appearance, size distribution, PDI, and assay over a period of 0, 3, and 6 months under storage conditions maintained at 25°C ± 2°C and 60% ± 5% RH. Additionally, the hydrophobic properties of the AuNPs, the modified Au-GEM, and the Au-GEM-AsP-COV were compared with each other. Results 3.1 Synthesis and Optimization of AuNPs in AsP matrix: Comparative Physico-Chemical Characterization with Au-Citrate (Au-CIT) NPs Due to the limited aqueous solubility of AsP, a fatty acid ester, optimizing its concentration was crucial for AuNP synthesis. Figure 2 A(a) presents five AuNP formulations with varying initial AsP concentrations. Their UV-Vis spectra were compared with a CIT-based sample (Au-CIT) containing 0.27 mg/mL CIT (Fig. 2 A(b)). The spectra revealed a wavelength shift from 520 nm to 560 nm and a color change from ruby red to purple, indicating increased nanoparticle size. Figure 2 B(a) shows these samples after a 6-month stability study at 5 ± 3°C. Unlike others, samples 1 and 2 remained clear and stable. The UV-Vis spectrum of sample 2 after 6 months, compared to Au-CIT, is shown in Fig. 2 B(b). Sample 2, maintaining stable absorbance similar to its initial spectrum and closely resembling Au-CIT with superior appearance stability (Fig. 2 B), was identified as the optimal formulation 39 . DLS results for the optimal formulation at initial, 3, and 6 months are illustrated in Fig. 3 A, showing an initial nanoparticle size of 90.9 nm. The size of nanoparticles remained consistent over 3 and 6 months. Surface charge measurements of the optimized formulation revealed a stable zeta potential of -1.1 mV initially, which slightly changed to -1.3 mV and − 1.6 mV at 3 and 6 months, respectively, likely due to the negatively charged AsP molecules used in the reduction process (Fig. 3 B). TEM images of the optimized formulation 2 (Fig. 3 C) at 100 nm and 200 nm scales show gold nanoparticles with a hydrodynamic diameter of 90.9 nanometers and an actual size of 78 nanometers. 3.2 Comparative Physico-Chemical Characterization of GEM in Au-ASP Matrix: Au-GEM, Au-GEM-AsP-Phys and Au-GEM-AsP-COV The encapsulation efficiency (%EE) of GEM was determined by HPLC analysis for each formulation. UV-Vis absorption spectra were obtained for all relevant materials: AuNPs, AsP, GEM, Au-GEM, and the final formulations Au-GEM-AsP-Phys and Au-GEM-AsP-COV (Fig. 4 A). The optimized AuNPs (sample 2), stabilized by AsP, exhibited a plasmon absorption band at 559 nm (intensity: 0.61), along with a secondary AsP-associated band at 236 nm (intensity: 1.07). Following modification with GEM, the plasmon band shifted to 562 nm with a reduced intensity of 0.30, and additional bands appeared at 235 nm and 270 nm, corresponding to AsP and GEM, respectively. The Au-GEM-AsP-Phys formulation displayed a further red shift to 571 nm (intensity: 0.42), with overlapping bands in the 240–280 nm range. In contrast, the covalently modified Au-GEM-AsP-COV formulation exhibited a shift to 568 nm, with a lower intensity of 0.27. The UV-Vis absorption shifts and intensity changes for the AuNP-based formulations are summarized in Table 1 . The particle size for the physically modified nanocarrier (Au-GEM-AsP-Phys) was 106.0 nm (Fig. 4 B(a)), and for the covalently modified nanocarrier (Au-GEM-AsP-COV) it was 125.8 nm (Fig. 4 B(b)). Zeta potential measurements showed a surface charge of -15.9 mV for the physical nanocarrier and − 18.3 mV for the covalent nanocarrier (Fig. 4 C(a–b)). Table 1 Summary of UV-Vis Absorption Shifts and Intensity Changes for AuNP-Based Formulations Sample Wavelength (nm) Intensity Description Shifts/Change AuNPs (Fig. 4 A-Yellow) 559 0.61 Plasmon absorption band of colloidal gold nanoparticles with a size of 90 nm, stabilized by AsP. No shift/change 236 1.07 Corresponding to AsP group. No shift/change GEM (Fig. 4 A-Dark blue) 270 0.71 Two distinct absorption bands; GEM sample used in the reaction. No shift/change 238 0.54 Au-GEM (Fig. 4 A- Green) 562 0.36 Plasmon band of gold nanoparticles after GEM binding. Shifted from 559 nm (intensity decrease from 0.61 to 0.30). 270 0.33 GEM drug bond on the nanoparticle surface. No shift/change 235 0.15 Overlapping bands of AsP (during synthesis) and GEM. No shift/change Au-GEM-AsP-Phys (Fig. 4 A- Purple) 571 0.42 Plasmon band of gold nanoparticles in the physical formulation. Shifted from 559 nm to 571 nm (intensity decreased from 0.61 to 0.42). 240–280 High Overlapping bands of AsP and GEM. No shift/change Au-GEM-AsP-COV (Fig. 4 A- Light blue) 568 0.27 Plasmon band of gold nanoparticles in the covalent formulation. Shifted from 559 nm to 568 nm (intensity decreased from 0.61 to 0.27, indicating stronger bonding). 240–280 High Overlapping bands of AsP and GEM. No shift/change To evaluate the surface hydrophobicity of the Au-GEM-AsP-COV nanocarrier versus optimized AuNPs, contact angle measurements were conducted. As shown in Fig. 5 A, the contact angle of bare AuNPs was 44°. After surface modification with MUA/MU and conjugation with GEM via amide bond formation, the contact angle decreased to 37° (Fig. 5 B), indicating increased hydrophilicity due to introduced polar carboxyl and amine groups. Following encapsulation of modified Au-GEM into the hydrophobic AsP matrix, the contact angle increased to 58° (Fig. 5 C). This consistent change across three independent measurements confirms increased surface hydrophobicity 40 . To confirm molecular interactions and amide bond formation, FT-IR spectra were recorded for Au-GEM, Au-GEM-AsP-Phys, and Au-GEM-AsP-COV at room temperature (Fig. 5 D). All spectra show characteristic amide bands, confirming successful conjugation. Notably, Au-GEM-AsP-COV (Fig. 5 D(b)) exhibits the most intense amide bands, indicating greater amide bond formation than Au-GEM-AsP-Phys (Fig. 5 D(a)) and modified Au-GEM (Fig. 5 D(c)). 3.3 Stability studies and optimal formulation To assess the stability and shelf-life potential of the Au-GEM-AsP-Phys and Au-GEM-AsP-COV formulations, accelerated stability tests were conducted following ICH Guideline Q1A (R2) standards 41 . The formulations were stored at 25 ± 2°C and 60 ± 5% RH for six months, with evaluations performed at 0, 3, and 6 months. As AuNP-based systems are typically stored long-term at 5 ± 3°C to prevent agglomeration, these elevated conditions were selected to simulate long-term behavior. The parameters monitored included visual appearance, particle size, polydispersity index (PDI), and drug content (assay), with summary data presented in Table 2 . Both formulations retained a clear and light purple appearance throughout the study, indicating no visible signs of instability. Particle size and PDI values showed minor fluctuations over time, with no significant aggregation or changes in distribution. Notably, changes were observed in drug content between the two formulations, with the Au-GEM-AsP-COV showing a more consistent assay profile over the testing period compared to Au-GEM-AsP-Phys. Table 2 Results of stability studies conducted on Au-GEM-AsP-Phys and Au-GEM-AsP-COV Product Name: Au-GEM-AsP-Phys 0.1mgmL − 1 Au, 1mgmL − 1 GEM, 2.5mgmL − 1 AsP Packing: Vial 6R, Clear Batch No.: Au-Phys − 22001 Mfg. Date: 06.2022 Stability Study Conditions Temperature (̊C): 25 ̊C ± 2 ̊C Relative Humidity: 60% ± 5% Test Initial M3 M6 Appearance Clear and Light Purple in color Clear and Light Purple in color Clear and Light Purple in color Assay (%) Acceptance Criteria: Initial ± 5% 87.00% 80.67% 77.72% Size (nm) 106.0 nm 115.4 nm 110.6 nm PDI 0.535 0.432 0.712 Product Name: Au-GEM-AsP-COV 0.1mgmL − 1 Au, 1mgmL − 1 GEM, 2.5mgmL − 1 AsP Packing: Vial 6R, Clear Batch No.: Au-COV-22001 Mfg. Date: 06.2022 Stability Study Conditions Temperature (̊C): 25 ̊C ± 2 ̊C Relative Humidity: 60% ± 5% Test Initial M3 M6 Appearance Clear and Light Purple in color Clear and Light Purple in color Clear and Light Purple in color Assay (%) Acceptance Criteria: Initial ± 5% 89.50% 84.78% 85.76% Size 125.8 nm 131.8 nm 134.0 nm PDI 0.664 0.704 0.701 3.4 In Vitro Release profile evaluation of Au-GEM-AsP-COV formulation To evaluate the role of the AsP matrix in enhancing hydrophobicity and achieving controlled drug release, the in vitro release profile of the Au-GEM-AsP-COV formulation was examined. As shown in Fig. 6 A(a), GEM release exhibited a time-dependent pattern, with cumulative release reaching 93.18% ± 1.68% at 72 hours, indicating sustained release behavior. Additionally, Fig. 6 A(b) presents the HPLC chromatograms of GEM at multiple time points, ranging from 5 minutes to 96 hours, further confirming the gradual release of the drug over time. 3.5 Evaluation of Cell Viability and Toxicity in 4T1 Cell Line: Comparing Au-GEM-AsP-Phys, Modified Au-GEM, and Au-GEM-AsP-COV with Free GEM To assess the cytotoxicity and therapeutic efficacy of the final formulations, MTT assays were performed on the 4T1 murine breast cancer cell line across a concentration range of 1–100 µg/mL over 48 hours. The study compared Au-GEM-AsP-COV and Au-GEM-AsP-Phys formulations against free GEM and other control groups. As shown in Fig. 6 B, the Au-GEM-AsP-COV formulation (Group B): 0.44 µg/mL demonstrated significantly lower cell viability compared to both the physical formulation (0.51 µg/mL) and the free drug (Group C): 0.89 µg/mL across all tested concentrations. Discussion In this study, ascorbyl palmitate (AsP), a hydrophobic derivative of vitamin C, was introduced as a reductant for AuNP formation, as well as a stabilizer and surface-modifying agent to enhance the physicochemical stability and encapsulation efficiency of GEM-loaded AuNPs. Results demonstrate that AsP provides steric stabilization to the nanoparticles and contributes to forming a hydrophobic matrix that more effectively retains GEM. Figure 2 A(a) shows five AuNP formulations immediately after synthesis. The corresponding UV-Vis spectra (Fig. 2 A(b)) for samples 1 to 5 revealed a redshift in the surface plasmon resonance (SPR) band from 520 nm to 560 nm in the presence of AsP, compared to the sodium citrate method 30 , 31 . This redshift, increasing with AsP concentration, indicates particle growth and successful surface coating. However, samples with excessive AsP (> 0.05 mg/mL) showed particle aggregation and reduced colloidal stability, consistent with reports that high surfactant levels induce interparticle bridging and destabilization. Sample 2 (0.05 mg/mL AsP) exhibited optimal stability, with consistent color, low turbidity (Fig. 2 B(a)), and minimal λmax shift over six-month storage (Fig. 2 B(b)). Further characterization using DLS and zeta potential analysis (Fig. 3 A, B) confirmed that Sample 2 initially had a uniform particle size of approximately 90.9 nm, low polydispersity, and a zeta potential of − 1.1 mV, indicating both electrostatic and steric stabilization. After 3 months, the particle size slightly increased to 101.2 nm and the zeta potential to − 1.3 mV, while at 6 months, the size decreased to 93.6 nm and the zeta potential shifted to − 1.6 mV. These minor fluctuations remained within acceptable ranges, suggesting good long-term colloidal stability. The findings were further supported by TEM images (Fig. 3 C), which consistently showed monodisperse, spherical particles, an ideal morphology for enhanced cellular uptake and biodistribution. Following the successful synthesis and functionalization of AuNPs, GEM was efficiently loaded onto the nanoparticle surface using two distinct strategies: physical adsorption and covalent attachment. The covalent approach, which involved surface modification with thiol-containing ligands followed by amide bond formation, improved drug stability compared to physical adsorption method. This improvement reflects the superior stability and interaction strength of covalent bonding, which minimizes premature drug release and enhances delivery potential 33 , 40 . The UV–Vis absorption spectra provided critical insights into the surface modifications and interaction behavior of the various nanoparticle formulations (Fig. 4 A, Table 1 ) 39 . The optimized colloidal gold nanoparticles (AuNPs), stabilized with AsP, exhibited a characteristic surface plasmon resonance (SPR) band at 559 nm with an intensity of 0.61, along with a secondary peak at 236 nm attributed to AsP. Upon conjugation with GEM to form Au-GEM, the SPR band red-shifted slightly to 562 nm, accompanied by a marked intensity decrease to 0.36, indicating surface functionalization and altered electronic environments. Additional absorption bands at 270 nm and 235 nm corresponded to GEM and AsP, confirming successful drug loading. Further modification in the physically adsorbed system (Au-GEM-AsP-Phys) caused a more pronounced red shift to 571 nm with an intensity of 0.42 (Fig. 4 A), suggesting effective but less compact surface interactions. In contrast, the covalently modified formulation (Au-GEM-AsP-COV) exhibited a shift to 568 nm and a significantly lower intensity of 0.27, likely reflecting a more compact and uniform surface coating due to stronger covalent bonding, which can dampen SPR oscillations. The overlapping absorption bands in the 240–280 nm region in both final formulations indicate the co-presence of AsP and GEM, supporting the multifunctional surface architecture. Overall, the observed spectral shifts and intensity changes confirm successful stepwise functionalization and reveal distinct surface environments between the covalent and physical systems. The surface engineering strategy employed in the covalently modified nanocarrier (Au-GEM-AsP-COV) resulted in a distinct enhancement in colloidal stability and particle uniformity. As shown in Fig. 4 B(b), the covalent formulation exhibited a hydrodynamic diameter of 125.8 nm, slightly larger than the physically adsorbed counterpart (Au-GEM-AsP-Phys), which measured 106.0 nm (Fig. 4 B(a)). Both nanocarriers were larger than the unmodified optimized gold nanoparticles (90.9 nm, sample 2), indicating successful surface functionalization. This increase in size for the covalent system likely reflects the additional surface layers formed via amide bond formation, contributing to structural robustness. Furthermore, zeta potential analysis demonstrated that the covalently modified nanocarriers possessed a more negative surface charge (–18.3 mV) compared to the physically adsorbed system (–15.9 mV), as shown in Fig. 4 C(a–b). This more negative potential is indicative of improved electrostatic repulsion and, consequently, greater colloidal stability. In contrast, the optimized AuNPs showed a near-neutral charge (–1.1 mV), underscoring the significance of surface modification in enhancing both stability and dispersion behavior 21 , 23 . Collectively, these findings highlight the superior physicochemical characteristics of the covalently engineered nanocarrier system, which are essential for maintaining long-term stability and preventing aggregation under physiological conditions. The contact angle measurements demonstrate the sequential surface modifications and their influence on nanoparticle hydrophobicity. As shown in Fig. 5 A and 5 B, the contact angle decreased from 44° to 37° following functionalization with MUA/MU and GEM, indicating enhanced hydrophilicity. This shift can be attributed to the introduction of polar functional groups, carboxylic acids and hydrophilic amines on the nanoparticle surface. In contrast, the subsequent increase in contact angle to 58° (Fig. 5 C) after encapsulation within the AsP matrix (Au-GEM-AsP-COV) reflects the addition of a hydrophobic layer 40 . This change confirms the successful entrapment of the hydrophilic-modified Au-GEM within a fatty acid ester-based AsP matrix, thereby altering the surface properties toward greater hydrophobicity. These findings validate the dual surface engineering approach for tailoring nanoparticle interactions with aqueous environments, which may have significant implications for biodistribution and cellular uptake in therapeutic applications. The FT-IR spectra (Fig. 5 D) confirm the successful formation of amide bonds during the surface modification and encapsulation steps of the nanoparticle formulations. Characteristic amide I and amide II bands, observed at approximately 1645–1646 cm⁻¹ and 1552–1553 cm⁻¹, respectively appear in all samples, indicating covalent attachment of GEM via amide bond formation. These bands correspond to C = O stretching (amide I) and N–H bending (amide II) vibrations, reflecting the conjugation of the carboxyl-functionalized gold nanoparticle surface with the amine groups of GEM 42 . Notably, the Au-GEM-AsP-COV formulation (Fig. 5 D(b)) exhibits the most intense peaks (~ 95% at 1646 cm⁻¹ and ~ 50% at 1552 cm⁻¹), compared to the physically loaded Au-GEM-AsP-Phys (Fig. 5 D(a); ~85% and ~ 35%) and the modified Au-GEM nanoparticles (Fig. 5 D(c); ~30% at 1627 cm⁻¹ and 1567 cm⁻¹). The higher intensity of amide bands in the covalently encapsulated formulation suggests a greater extent of bond formation, likely resulting from dual contributions of both the nanoparticle surface and the AsP matrix. These findings support the enhanced chemical integration of GEM in the Au-GEM-AsP-COV system and underscore the efficiency of the covalent encapsulation strategy. The accelerated stability study results summarized in Table 2 highlight the superior stability of the covalent Au-GEM-AsP-COV formulation compared to the physically loaded Au-GEM-AsP-Phys. Both formulations maintained their clear and light purple appearance throughout the six-month period, indicating good physical stability under accelerated conditions (25 ± 2°C, 60 ± 5% RH) 41 . Particle size and PDI values showed minor fluctuations but remained consistent overall, with no significant aggregation detected for either formulation. However, the drug assay data in Table 2 reveal distinct differences in chemical stability. The Au-GEM-AsP-COV formulation preserved GEM content within the acceptable ± 5% range over 6 months (initial: 89.5%, 3 months: 84.78%, 6 months: 85.76%), whereas the physically loaded Au-GEM-AsP-Phys formulation exhibited a substantial decline in drug content (initial: 87.0%, 3 months: 80.67%, 6 months: 77.72%), which exceeded the acceptable threshold, showing losses of over 6% and 9% at 3 and 6 months, respectively, indicating drug loss or degradation over time. These findings confirm that covalent conjugation of GEM to the nanoparticle surface, coupled with lyophilization, effectively reduces drug degradation and loss during storage, thereby enhancing the long-term stability of the nanocarrier system. As illustrated in Fig. 6 A(a), the Au-GEM-AsP-COV formulation demonstrated a sustained, time-dependent drug release pattern, achieving approximately 93.18% ± 1.68% cumulative release over 72 Hrs. This behavior is attributed to the covalent attachment of GEM to the gold nanoparticle surface and encapsulation within the AsP matrix, enhancing hydrophobicity and modulating diffusion. HPLC chromatograms in Fig. 6 A(b) confirm GEM’s gradual release from as early as 5 minutes to up to 96 hours. This sustained release is significant given GEM’s clinical limitations, its short plasma half-life (8-17minutes) due to rapid deamination by cytidine deaminase in blood and liver 8 . The controlled release observed with Au-GEM-AsP-COV may prolong systemic availability, protect GEM from enzymatic degradation, and maintain therapeutic levels longer. By minimizing the rapid peak-trough fluctuations typical of free GEM, this nanocarrier system could enhance efficacy and reduce dose-related toxicity. Overall, the release profile reinforces the nanocarrier’s potential to address GEM’s pharmacokinetic challenges and improve its clinical performance in cancer therapy 24 , 29 , 43 . These physicochemical advantages translated into improved biological activity. The covalently attached GEM-AuNPs exhibited significantly greater cytotoxicity against 4T1 breast cancer cells, with an IC₅₀ of 0.44 µg/mL (Fig. 6 B), which reinforces the potential of the AsP-based nanocarrier platform for the development of long-acting injectable formulations with enhanced therapeutic efficacy. The enhanced cytotoxic performance of the Au-GEM-AsP-COV formulation can be attributed to its sustained and controlled drug release, as previously demonstrated in the in vitro release study, where it achieved 93.18% ± 1.68% release over 72 Hrs. The slow and steady drug release allowed for prolonged drug availability and greater cellular uptake, resulting in a lower IC₅₀ value compared to both the free GEM (0.89 µg/mL) and the physical formulation (0.51 µg/mL). Moreover, the more negative surface charge of the covalent formulation (–18.3 mV compared to − 15.9 mV for the physical one) likely enhanced cellular internalization through electrostatic interactions, resulting in more effective intracellular drug delivery. The amide bond formation in the covalent formulation also provided additional chemical stability for GEM, minimizing premature degradation and enhancing efficacy. Collectively, these findings highlight the superior performance of the Au-GEM-AsP-COV formulation in targeted cancer therapy, supporting its potential for further preclinical development. Conclusion This study successfully demonstrated the multifunctional role of ascorbyl palmitate (AsP) in the synthesis and surface engineering of gold nanoparticles (AuNPs) for enhanced gemcitabine (GEM) delivery. AsP functioned not only as a reductant and stabilizer but also as a hydrophobic matrix component, contributing to improved nanoparticle stability and drug encapsulation. Among the various formulations tested, the covalently modified Au-GEM-AsP-COV system exhibited superior physicochemical characteristics, including optimal particle size, enhanced colloidal stability, more negative surface charge, and effective surface functionalization. These features contributed to significantly improved drug loading, controlled and sustained release over 72 Hrs, and reduced drug degradation over a six-month stability study. Critically, the Au-GEM-AsP-COV formulation demonstrated enhanced biological efficacy, achieving a lower IC₅₀ against 4T1 breast cancer cells compared to both free GEM and physically loaded formulations. The combined steric and electrostatic stabilization, along with the dual surface engineering strategy, enabled better control over GEM release, prolonged systemic availability, and greater cellular uptake. Altogether, these findings support the promise of AsP-based covalently engineered AuNPs as a robust platform for the development of long-acting, stable, and efficacious injectable chemotherapeutic nanocarriers. Declarations Conflict of interest: Not applicable. Ethical approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Funding This research did not receive any grant from funding agencies Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Author Contribution Havva Rezaei: Writing – original draft, Validation, Investigation, Formal analysis, Data curation, Validation, Methodology, Conceptualization, Visualization, Investigation, Formal analysis. Mostafa Shourian: Writing – review & editing, Supervision, Resources, Project administration, Data availability statement. Acknowledgement The authors appreciate Marzieh Ramezanpour, Department of Biology, Faculty of Science, University of Guilan, P.O.Box 4193833697, Rasht, Iran. Data Availability Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Heinemann, V. Role of Gemcitabine in the Treatment of Advanced and Metastatic Breast Cancer. Oncology 64 , 191–206 (2003). Espey, M. G. et al.' Pharmacologic ascorbate synergizes with gemcitabine in preclinical models of pancreatic cancer. Free Radic Biol. Med. 50 , 1610–1619 (2011). Monti, D. A. et al.'. Phase I Evaluation of Intravenous Ascorbic Acid in Combination with Gemcitabine and Erlotinib in Patients with Metastatic Pancreatic Cancer. PLoS ONE . 7 , 1–7 (2012). Cullen, J. J. A Phase 2 Trial of High-dose Ascorbate for Pancreatic Cancer (PACMAN 2.1). University of Iowa. 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Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 10 Oct, 2025 Reviews received at journal 01 Sep, 2025 Reviews received at journal 23 Aug, 2025 Reviewers agreed at journal 21 Aug, 2025 Reviewers agreed at journal 21 Aug, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers invited by journal 19 Aug, 2025 Editor assigned by journal 05 Aug, 2025 Editor invited by journal 05 Aug, 2025 Submission checks completed at journal 04 Aug, 2025 First submitted to journal 03 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7240807","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":504456517,"identity":"826154d0-66dd-434f-bb43-091c1269f4c6","order_by":0,"name":"Havva Rezaei","email":"","orcid":"","institution":"University of Guilan","correspondingAuthor":false,"prefix":"","firstName":"Havva","middleName":"","lastName":"Rezaei","suffix":""},{"id":504456519,"identity":"dcf717d1-cc35-4c0c-a744-136f3a42385e","order_by":1,"name":"Mostafa Shourian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDACCRBhwMDAD6QOPIAKHiBKi2QDUGUC8VpAukDKEnCrQwD52c3HpCsK7PKMz68xPJBQc4eBv/0A4+EKPFoM7hxLkzxjkFxsduONwYGEY88YJM4kMBw8g0+LRI6ZZIMBc+K2G2eAWtgOMzDcYGA42IDPYTPAWuoTN88Aafl3mEGekBaGG2AthxM38PcYHEhsO8xgQEiLwY20ZMsGg+OJM26wFRxI7DvMY3gmsYGAw5IP3mz4U53Y339484cP3w7LyR0/fPgjXofBgUQCmOJhYGAkTgMwxRwgUuEoGAWjYBSMOAAA35NWbMVsKxIAAAAASUVORK5CYII=","orcid":"","institution":"University of Guilan","correspondingAuthor":true,"prefix":"","firstName":"Mostafa","middleName":"","lastName":"Shourian","suffix":""}],"badges":[],"createdAt":"2025-07-29 08:23:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7240807/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7240807/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-32204-6","type":"published","date":"2025-12-26T15:58:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90045862,"identity":"e42d1124-0f68-4972-acee-732f0289dbcc","added_by":"auto","created_at":"2025-08-27 18:14:55","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":232340,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic of the AU-GEM-AsP-Phys nanoemulsion formation based on physical interaction, (B) Surface activation and amide bond formation, (C) single-phase emulsification process, showing the encapsulation of GEM-loaded modified AuNPs within the AsP matrix to Au-GEM-AsP-COV\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7240807/v1/03fe137645325ab5304e6169.jpeg"},{"id":90045860,"identity":"09207f5b-cc72-4b2c-87ff-1c5ca3b6a6d5","added_by":"auto","created_at":"2025-08-27 18:14:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":227786,"visible":true,"origin":"","legend":"\u003cp\u003eA(a) UV-Visible absorption spectra of five synthesized samples compared to the sample synthesized in the presence of CIT salt, A(b) Absorption wavelength shift corresponding to the increase in nanoparticle size in optimized formulation 2 and Au-CIT, B(a) Five formulation at initial - Temperature: 5 ̊C ± 3 ̊C, B(b) Synthesized samples after 6 months of long-term stability-Temperature: 5 ̊C ± 3 ̊C.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7240807/v1/a4c54d8958f719dc792ef890.jpeg"},{"id":90046712,"identity":"dd221f24-79e2-471b-9d0f-3d4f8618d2c9","added_by":"auto","created_at":"2025-08-27 18:31:00","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":514484,"visible":true,"origin":"","legend":"\u003cp\u003e(A) DLS results of synthesized samples with optimized formulation 2 at (a) initial, (b) 3 month and (c) 6 months of long-term stability, (B) Surface charge studies of the optimized formulation 2 at (a) initial, (b) 3 month and (c) 6 months of long-term stability, (C) (a) and (b) TEM images of optimized AuNPs at two magnifications: 100 nm and 200 nm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7240807/v1/e720c7bdf2342b49e689b20a.jpeg"},{"id":90046226,"identity":"c4488391-5153-4c6b-bd24-59e49e0827a5","added_by":"auto","created_at":"2025-08-27 18:22:55","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":465738,"visible":true,"origin":"","legend":"\u003cp\u003echaracterization studies including (A) Comparative UV-Vis spectra of different groups, AsP, GEM, AuNPs, Au-GEM, Au-GEM-AsP-COV, and Au-GEM-AsP-Phys (B) DLS results and (C) Zeta potential studies on (a) Au-GEM-AsP-Phys and (b) Au-GEM-AsP-COV\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7240807/v1/2ee004db71b3200618f44033.jpeg"},{"id":90046228,"identity":"a83bbac5-c74d-4021-9640-a1ac4a100133","added_by":"auto","created_at":"2025-08-27 18:22:55","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":253455,"visible":true,"origin":"","legend":"\u003cp\u003eSurface angle investigation as a hydrophobicity criterion for (A) AuNPs, (B) Au-GEM, (C) Au-GEM-AsP-COV, with three replicate runs, and (D) FT-IR spectra of (a) Au-GEM-AsP-Phys, (b) Au-GEM-AsP-COV, and (c) Au-GEM\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7240807/v1/553fa71373a0475d76bde820.jpeg"},{"id":90045859,"identity":"c416c7bf-8920-43de-bedd-d98d81d2c3e5","added_by":"auto","created_at":"2025-08-27 18:14:55","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":206249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eGEM drug release: (a) Cumulative release profile of GEM from the Au-GEM-AsP-COV formulation to examine release behavior in a simulated buffer environment at 37°C, pH 6.8 over a period of 96 Hrs, (b) HPLC chromatograms from the GEM release study of the Au-GEM-AsP-COV formulation, (B) Comparative Cell Viability Chart: (A) AuNPs, (B) Au-GEM-AsP-COV, (C) Free GEM, (G) Free AsP, (E) Modified Au-GEM, (F) Au-GEM-AsP-Phys on 4T1 cell line viability after 48 Hrs. The results are analyzed using the standard error of the mean with 3 replicates (n=3) and are presented as mean ± SD. The significance levels are indicated as follows: **** (p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7240807/v1/9e2b9d53ea32628fa8a28298.jpeg"},{"id":99172406,"identity":"cddb333d-cf69-42d7-b4b3-2c7ec91efc9c","added_by":"auto","created_at":"2025-12-29 16:08:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3067951,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7240807/v1/d789cbc0-a586-4646-97c7-97a37b813801.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimizing Gold Nanoparticles for Combination Therapy: Development of Hydrophobic Nanomedical Devices with Gemcitabine and Ascorbyl Palmitate","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer is a deadly disease characterized by uncontrolled cell proliferation and the spread of abnormal cells through invasion or metastasis \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Traditional cancer treatments include surgery, radiotherapy, and chemotherapy \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, these treatments have significant side effects and limited efficacy, prompting researchers to explore new, effective targeted delivery systems based on nanochemistry platforms for the active targeting delivery of anticancer drugs, either alone or in combination with other effective active pharmaceutical ingredients (APIs) \u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These novel drug delivery systems also affect pharmacokinetics and the ADME processes (absorption, distribution, metabolism, and excretion) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGemcitabine (GEM), a first-line chemotherapy for pancreatic cancer, is a nucleoside analog antimetabolite with proven antitumor activity and tolerability in non-small cell lung, ovarian, and metastatic breast cancers. However, its clinical utility is limited by rapid metabolism, resulting in a short plasma half-life (8\u0026ndash;17 minutes) and systemic toxicity due to high dose (1000\u0026ndash;1250 mg/m\u0026sup2;) requirements for therapeutic levels. Additionally, after a few months, cells develop chemoresistance. Multiple clinical and experimental investigations have demonstrated that a combination or co-administration of other drugs as chemotherapies with GEM leads to superior therapeutic benefits \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNatural products have significantly contributed to anticancer research, as most clinically used anticancer drugs originate from natural sources \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Interest is growing in natural antioxidants like Vitamin C (L-ascorbic acid, ascorbate, VC), a water-soluble vitamin that scavenges free radicals and prevents DNA damage \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. At pharmacologic concentrations, ascorbate undergoes oxidation via ascorbate radical, generating cytotoxic hydrogen peroxide (H₂O₂) through Fenton chemistry \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Several clinical trials have explored ascorbate's synergistic effects with cancer chemotherapeutics \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Michael Graham Espey et al. reported that GEM\u0026ndash;ascorbate combinations in mice with pancreatic tumor xenografts enhanced growth inhibition versus GEM alone \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Daniel A. Monti et al., in phase I studies, observed increased toxicity with intravenous ascorbic acid combined with GEM and erlotinib in 14 metastatic stage IV pancreatic cancer patients, suggesting a longer phase II trial \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Joseph J. Cullen reviewed a Phase 2 trial (PACMAN 2.1) of high-dose ascorbate with nab-paclitaxel and GEM \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAscorbyl palmitate (AsP), a key derivative of ascorbic acid, offers greater stability and functions as an antioxidant with antitumor activity via its antiproliferative effect \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, combination therapies often cause severe systemic toxicity. Thus, developing a co-loaded drug delivery system with AsP and GEM is an attractive strategy to enhance anticancer treatment efficiency, improve stability and bioavailability, enable tumor-specific delivery, and minimize chemotherapy-related side effects. Mohamed El-Far et al. developed stable AsP-loaded Pluronic (F-127 or F-108) nano micelles to enhance AsP solubility and bioavailability using lower doses, reducing side effects compared to native AsP \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Min Zhou et al. designed AsP-based solid lipid nanoparticles combined with paclitaxel (AsP/PTX-SLNs) to maximize AsP\u0026rsquo;s therapeutic efficacy \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Mohamed El-Far et al. indicated the superiority of AsP-loaded Pluronic nanoparticles as a promising anticancer agent over native AsP, demonstrating a fantastic synergistic anticancer effect in combination with melatonin as a potential therapy against EAC-bearing mice \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough AsP is more stable than vitamin C, its poor release capacity and water insolubility limit its bioavailability and therapeutic efficacy \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Thus, incorporating it into nanoparticle carriers can enhance circulation time and tumor accumulation via the enhanced permeability and retention (EPR) effect \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Recent studies show that nanocarriers with neutral, zwitterionic, or negative surface charge adsorb less protein, circulate longer, and internalize better than positively charged ones, leading to improved tumor distribution for similarly sized particles \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Nanoparticles sized 30\u0026ndash;200 nm enhance cell uptake via increased surface area and membrane wrapping, effectively accumulating in tumors \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Overall, designing an optimal nanoparticle requires balancing drug-loading capacity, immune response, circulation time, and cellular uptake. Among many platforms, gold nanoparticles (AuNPs) stand out due to their physicochemical versatility, biocompatibility, and ease of surface modification \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Santiago et al. modified AuNP surfaces with GEM and folate/transferrin ligands to create a targeted controlled-release nanocarrier, reducing side effects and improving efficacy \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDeveloping a water-in-oil (W/O) emulsion using ultrasonic homogenizers, which mix two immiscible phases with a surfactant, can enable negatively charged surface modification of AuNPs\u0026rsquo; hydrophilic surface by incorporating AsP \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, AuNPs can stabilize other drug carriers, such as liposomes, enhancing delivery efficiency \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Wang et al. demonstrated that nanoparticles adsorb phospholipids and induce gelation at the liposome surface. With 25% of the lipid surface occupied by nanoparticles, nanoparticle-modified liposomes showed no significant leakage over 50 days \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Yang et al. used AuNPs to stabilize oil-in-water emulsion droplets (\u0026lt;\u0026thinsp;100 nm), forming net negatively charged droplets. Positively charged AuNPs electrostatically bound to them, bridging strong repulsion and enhancing emulsion stability. The interaction between the AuNP-emulsion and AuNP-transferrin further improved droplet stability \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSurface charge polarity and density greatly influence immune clearance and cellular uptake of intravenously administered nanocarriers, affecting their delivery efficiency to target sites \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. This study developed a gold nanocarrier for gemcitabine (GEM) with tunable surface properties within an ascorbyl palmitate (AsP) matrix. Gold nanoparticles (AuNPs) were synthesized with optimal size and surface charge, demonstrating stability in AsP presence. A novel nano-drug delivery system was created to enhance interaction between GEM and AuNPs using both physical (Au-GEM-AsP-Phys) and covalent (Au-GEM-AsP-COV) matrices. Chemical characterization via UV-visible spectroscopy, FT-IR, and in vitro assays confirmed effective binding and six-month accelerated stability of the covalent formulation.\u003c/p\u003e\u003cp\u003eThe MTT assay results revealed a significant reduction in IC\u003csub\u003e50\u003c/sub\u003e values, for the covalent formulation and the physical formulation, compared to the modified Au-GEM without AsP. These findings highlight the synergistic effect of AsP in enhancing therapeutic efficacy, attributed to its hydrophobicity and inherent anticancer properties. This research lays the groundwork for developing hydrophobic nanomedical devices that integrate GEM and AsP for dual applications in therapy and diagnostics. Future investigations will focus on evaluating in vivo toxicity and pharmacokinetics in relevant cancer models.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eTetra chloroauric acid (HAuCl\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO), ascorbyl palmitate (AsP) (C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e38\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e), 11-mercaptoundecanoic acid (MUA) (HS(CH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e10\u003c/sub\u003eCO\u003csub\u003e2\u003c/sub\u003eH), 11-mercapto-1-undecanol (MU) (HS(CH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e11\u003c/sub\u003eOH), and 2-(3,5-diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole (MTT) (C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eS\u003csup\u003e+\u003c/sup\u003e) were purchased from Sigma-Aldrich. Gemcitabine hydrochloride (GEM) (C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u0026middot;HCl) was purchased from Shilpa. N-Hydroxy succinimide (NHS) (C\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e), tri-sodium Citrate (CIT) (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e), sodium dihydrogen phosphate (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), sodium hydroxide (NaOH), hydrochloric acid (HCl), Tween 20 (C\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e50\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e), dimethyl sulfoxide (DMSO) ((CH\u003csub\u003e3\u003c/sub\u003e)2SO), D-mannitol (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e), propylene glycol (MPG) (C\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), polyethylene glycol 300 (PEG) (H(OCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003en\u003c/sub\u003eOH), absolute ethanol (ETOH) (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO), and acetone ((CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eCO) were obtained from Merck. The 4T1 cell line and acetonitrile (ACN) (CH\u003csub\u003e3\u003c/sub\u003eCN) were purchased from Zista Gene and Romil, respectively. All reagents were of analytical grade and doubly distilled water was used for the preparation of all solutions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Synthesis of AuNPs in AsP matrix and Optimization\u003c/h2\u003e\u003cp\u003eSo far, gold nanoparticle suspensions have been produced using sodium nitrate salt to achieve particles approximately 20 nanometers in size \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Additionally, the synthesis of gold nanoparticles using other reducing agents, such as ascorbic acid (Vitamin C), has also been reported \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In this study, we synthesized AuNPs using ascorbyl palmitate AsP as the reducing agent, emphasizing its potential to enhance the hydrophobicity of the nanoparticles due to its amphiphilic nature. For a comprehensive evaluation, we also synthesized AuNPs using CIT as the reducing agent, as it is a widely established method in nanoparticle synthesis, producing hydrophilic AuNPs. CIT not only acts as a reducing agent but also stabilizes the nanoparticles through the formation of a negatively charged surface, leading to good dispersibility in aqueous environments. By comparing the two, we aimed to highlight the potential of AsP-functionalized AuNPs to improve the enhanced permeability and retention (EPR) effect, which is crucial for drug delivery applications. The rationale for including CIT in this study is rooted in its historical significance and reliability in nanoparticle synthesis, ensuring a standard benchmark for evaluating the novel AsP method \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBefore preparing the solutions, nitrogen was injected into 200 mL of water for 30 minutes to remove oxygen. This oxygen-free water was then used to prepare stock solutions from gold salt and AsP. To achieve stable AuNPs, five experimental sets were conducted at five different concentrations of the reducing solution AsP and hydrophobic AuNPs were prepared using the following synthetic route:\u003c/p\u003e\u003cp\u003eBriefly, 10 \u0026micro;L of HAuCl\u003csub\u003e4\u003c/sub\u003e (100 mgmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added to 9 mL of oxygen-free water at 70\u0026deg;C while mixing at 100 rpm. After 2 minutes, a specified volume of the 1 mgmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e AsP stock solution was added dropwise with stirring at 150 rpm. After 20 minutes, the heater was turned off, and after 30 minutes, the shaker was also turned off, allowing the nanoparticles to gradually reach room temperature.\u003c/p\u003e\u003cp\u003eThe resulting nanoparticles from each experiment were analyzed for size, zeta potential, and UV-visible absorption spectrum to characterize their physicochemical properties. Additionally, the optimal formulation underwent MTT analysis on the 4T1 cell line to study the cytotoxic effects of the produced nanoparticles. A Transmission Electron Microscopy (TEM) image of the optimal sample was taken to examine morphology and determine size. Stability studies on the optimal sample, including changes in size, PDI, and visual appearance of the solution, were conducted at 0, 3, and 6 months at a temperature of 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60% \u0026plusmn; 5% RH.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Gold metallic Nanocarrier containing GEM loaded in AsP matrix\u003c/h2\u003e\u003cp\u003eLoading the AuNPs containing GEM in an AsP matrix to leverage synergistic effects was achieved using nano emulsification technique. This process involved forming a nanoemulsion through micellization of the aqueous phase in the oil/surfactant (Oil/S) phase, followed by organic solvent evaporation and nanoparticle formation. The result was stable nanoparticles with higher concentrations of AsP, controllable size, and increased hydrophobicity \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Innovation of the oil/surfactant (Oil/S) phase: Dispersing AsP in the PEG-MPG matrix\u003c/h2\u003e\u003cp\u003eTo enhance the solubility of AsP, we used the placebo matrix consisting of polyethylene glycol 300 (PEG) and propylene glycol (MPG) in an ethanolic solution with a pH of 7.5, adjusted using ethanolic sodium hydroxide. This biocompatible matrix was incorporated into a new formulation, Au-GEM-AsP-Phys, with 70% matrix as the oil phase and 30% Tween 20 as a surfactant and cosolvent. A summary of Au-GEM-AsP-Phys nanoemulsion formation has shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. For the aqueous phase, 500 \u0026micro;L of GEM stock solution (10 mgmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was mixed with 3.5 mL of optimized AuNPs (0.1 mgmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and stirred for 20 minutes. The aqueous phase was then grafted onto the AsP matrix (Oil/S phase) through emulsification, with dropwise addition and stirring at 300 rpm for 15 minutes, followed by homogenization at 12,000 rpm for 5 minutes. The nanoemulsion of Au-GEM-AsP-Phys was formed using sonication at 50 watts (20 seconds on, 10 seconds off) for 13 minutes, then shaken at 1,000 rpm for 15 minutes. Organic solvent evaporation was performed using a rotary evaporator at 30\u0026deg;C for two hours (Hrs). To separate any non-emulsified materials, aggregates and removal of excess surfactant, centrifugation has been performed at 12,000 rpm for 15 minutes at 15\u0026deg;C.The resulting Au-GEM-AsP-Phys nanoparticle solution was stored at 2\u0026ndash;8\u0026deg;C and characterized using UV\u0026ndash;Visible spectroscopy, size and zeta potential analysis, FT-IR spectroscopy, HPLC for encapsulation efficiency (EE) and assay, and cytotoxicity evaluation. Au-GEM-AsP-Phys Formulation stability was assessed by monitoring appearance, size distribution, PDI, and assay at 0, 3, and 6 months under storage conditions of 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60% \u0026plusmn; 5% RH.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Surface Activation and Amide bond promotion\u003c/h2\u003e\u003cp\u003eAs mentioned, the most significant drawback of GEM in its injectable form is its instability and short half-life due to enzymatic deactivation via its amine groups. Therefore, blocking this active site can be a solution to enhance its stability. To achieve this, the development of an amide bond between this amine group and the carboxylic group on the surface-modified gold nanoparticles has been advocated. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB illustrates the activation of the AuNPs surface followed by the encapsulation of GEM within the AuNPs. Surface modification of gold nanoparticles was achieved through thiolation using 11-mercaptoundecanoic acid (MUA) and 11-mercaptoundecanol (MU). This process involved activating the carboxyl group on the surface of the nanoparticles using EDC/NHS chemistry. For this reason, to a suspension of optimized AuNPs (10 mL), 10 mL phosphate buffer with pH 8 and 50 \u0026micro;L Tween 20 were added and stirred at room temperature for 30 minutes. A 10 mL solution of 2 mM MUA/MU in an 8:1 volumetric ratio was then prepared and added to the nanoparticle suspension, which was stirred at room temperature for 12 Hrs. The MUA/MU-modified suspension was centrifuged at 14,000 rpm for 30 minutes at 4\u0026deg;C to remove the supernatant. The separated samples were washed first with phosphate buffer at pH 8 and then twice with phosphate buffer at pH 7, followed by centrifugation at 14,000 rpm for 30 minutes at 4\u0026deg;C to eliminate excess MUA/MU and Tween 20. The resulting precipitate was resuspended in MES buffer (pH 5.5) and centrifuged at 14,000 rpm for 30 minutes at 4\u0026deg;C. Then, 20 mL of a solution containing 10 mM EDC and 20 mM NHS was added, and the mixture was stirred at room temperature for 15 minutes using an incubator shaker, followed by centrifugation at 24,000 rpm for 10 minutes at 4\u0026deg;C \u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. To the carboxyl-activated AuNPs, 500 \u0026micro;L of GEM (10 mgmL\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) was added, and the volume was adjusted to 2 mL using a phosphate buffer at pH 7.4. This Au-GEM suspension was stirred at room temperature for 20 minutes and then centrifuged at 24,000 rpm for 10 minutes at 4\u0026deg;C. The amount of free GEM in the supernatant was quantified. Subsequently, 5mL of mannitol (6.4 mgmL\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) was added to the precipitate. The sample underwent freeze-drying under a pressure of 100 Pa for 48 Hrs, including a freezing stage at -40\u0026deg;C for 20 Hrs and primary drying at -25\u0026deg;C for 4 Hrs, followed by secondary drying for an additional 4 Hrs. Finally, the chamber temperature was raised to -5\u0026deg;C for final product freezing. The label claim of GEM in resulted Au-GEM powder was evaluated, along with its size and zeta potential \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Au-GEM-AsP- COV nanoemulsion preparation\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC illustrates the single-phase emulsification process, where GEM-loaded modified gold nanoparticles are encapsulated within the AsP matrix, leading to the Au-AsP-GEM-COV nanoemulsion formation. The preparation of the Au-GEM-AsP-COV nanoemulsion involved dissolving AsP in acetone (12.5 mgmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), mixing this oil phase with Tween 20 in a 70:30 ratio, and combining it with an aqueous phase reconstituted from freeze-dried Au-GEM powder in 4mL sodium phosphate buffer (pH 7.4). The aqueous phase was added dropwise to the oil/surfactant mixture, stirred at 300 rpm for 15 minutes, and homogenized at 12,000 rpm for 5 minutes. The mixture was then sonicated at 50 watts for 13 minutes (20 seconds on, 10 seconds off). Following sonication, the sample was stirred with an incubator shaker for 15 minutes, and the solvent was evaporated at 30\u0026deg;C for two Hrs. Centrifuge has been conducted at 12,000 rpm for 15 minutes at 15\u0026deg;C for separation and purification. The resulting Au-GEM-AsP-COV nanoparticles were characterized for drug loading, release profile, toxicity against 4T1 cell line. Also, the stability of the Au-GEM-AsP-COV formulation was evaluated by observing its appearance, size distribution, PDI, and assay over a period of 0, 3, and 6 months under storage conditions maintained at 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60% \u0026plusmn; 5% RH. Additionally, the hydrophobic properties of the AuNPs, the modified Au-GEM, and the Au-GEM-AsP-COV were compared with each other.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Synthesis and Optimization of AuNPs in AsP matrix: Comparative Physico-Chemical Characterization with Au-Citrate (Au-CIT) NPs\u003c/h2\u003e\u003cp\u003eDue to the limited aqueous solubility of AsP, a fatty acid ester, optimizing its concentration was crucial for AuNP synthesis. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA(a) presents five AuNP formulations with varying initial AsP concentrations. Their UV-Vis spectra were compared with a CIT-based sample (Au-CIT) containing 0.27 mg/mL CIT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA(b)). The spectra revealed a wavelength shift from 520 nm to 560 nm and a color change from ruby red to purple, indicating increased nanoparticle size. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB(a) shows these samples after a 6-month stability study at 5\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C. Unlike others, samples 1 and 2 remained clear and stable. The UV-Vis spectrum of sample 2 after 6 months, compared to Au-CIT, is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB(b). Sample 2, maintaining stable absorbance similar to its initial spectrum and closely resembling Au-CIT with superior appearance stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), was identified as the optimal formulation \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDLS results for the optimal formulation at initial, 3, and 6 months are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, showing an initial nanoparticle size of 90.9 nm. The size of nanoparticles remained consistent over 3 and 6 months. Surface charge measurements of the optimized formulation revealed a stable zeta potential of -1.1 mV initially, which slightly changed to -1.3 mV and \u0026minus;\u0026thinsp;1.6 mV at 3 and 6 months, respectively, likely due to the negatively charged AsP molecules used in the reduction process (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTEM images of the optimized formulation 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) at 100 nm and 200 nm scales show gold nanoparticles with a hydrodynamic diameter of 90.9 nanometers and an actual size of 78 nanometers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Comparative Physico-Chemical Characterization of GEM in Au-ASP Matrix: Au-GEM, Au-GEM-AsP-Phys and Au-GEM-AsP-COV\u003c/h2\u003e\u003cp\u003eThe encapsulation efficiency (%EE) of GEM was determined by HPLC analysis for each formulation.\u003c/p\u003e\u003cp\u003eUV-Vis absorption spectra were obtained for all relevant materials: AuNPs, AsP, GEM, Au-GEM, and the final formulations Au-GEM-AsP-Phys and Au-GEM-AsP-COV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The optimized AuNPs (sample 2), stabilized by AsP, exhibited a plasmon absorption band at 559 nm (intensity: 0.61), along with a secondary AsP-associated band at 236 nm (intensity: 1.07). Following modification with GEM, the plasmon band shifted to 562 nm with a reduced intensity of 0.30, and additional bands appeared at 235 nm and 270 nm, corresponding to AsP and GEM, respectively. The Au-GEM-AsP-Phys formulation displayed a further red shift to 571 nm (intensity: 0.42), with overlapping bands in the 240\u0026ndash;280 nm range. In contrast, the covalently modified Au-GEM-AsP-COV formulation exhibited a shift to 568 nm, with a lower intensity of 0.27. The UV-Vis absorption shifts and intensity changes for the AuNP-based formulations are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe particle size for the physically modified nanocarrier (Au-GEM-AsP-Phys) was 106.0 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB(a)), and for the covalently modified nanocarrier (Au-GEM-AsP-COV) it was 125.8 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB(b)). Zeta potential measurements showed a surface charge of -15.9 mV for the physical nanocarrier and \u0026minus;\u0026thinsp;18.3 mV for the covalent nanocarrier (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC(a\u0026ndash;b)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of UV-Vis Absorption Shifts and Intensity Changes for AuNP-Based Formulations\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWavelength (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIntensity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eShifts/Change\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAuNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-Yellow)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e559\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePlasmon absorption band of colloidal gold nanoparticles with a size of 90 nm, stabilized by AsP.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo shift/change\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e236\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCorresponding to AsP group.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo shift/change\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-Dark blue)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e270\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTwo distinct absorption bands; GEM sample used in the reaction.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNo shift/change\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e238\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAu-GEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA- Green)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePlasmon band of gold nanoparticles after GEM binding.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eShifted from 559 nm (intensity decrease from 0.61 to 0.30).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e270\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGEM drug bond on the nanoparticle surface.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo shift/change\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e235\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOverlapping bands of AsP (during synthesis) and GEM.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo shift/change\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAu-GEM-AsP-Phys (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA- Purple)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e571\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePlasmon band of gold nanoparticles in the physical formulation.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eShifted from 559 nm to 571 nm (intensity decreased from 0.61 to 0.42).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e240\u0026ndash;280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOverlapping bands of AsP and GEM.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo shift/change\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAu-GEM-AsP-COV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA- Light blue)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e568\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePlasmon band of gold nanoparticles in the covalent formulation.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eShifted from 559 nm to 568 nm (intensity decreased from 0.61 to 0.27, indicating stronger bonding).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e240\u0026ndash;280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOverlapping bands of AsP and GEM.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo shift/change\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the surface hydrophobicity of the Au-GEM-AsP-COV nanocarrier versus optimized AuNPs, contact angle measurements were conducted. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the contact angle of bare AuNPs was 44\u0026deg;. After surface modification with MUA/MU and conjugation with GEM via amide bond formation, the contact angle decreased to 37\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating increased hydrophilicity due to introduced polar carboxyl and amine groups. Following encapsulation of modified Au-GEM into the hydrophobic AsP matrix, the contact angle increased to 58\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This consistent change across three independent measurements confirms increased surface hydrophobicity \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. To confirm molecular interactions and amide bond formation, FT-IR spectra were recorded for Au-GEM, Au-GEM-AsP-Phys, and Au-GEM-AsP-COV at room temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). All spectra show characteristic amide bands, confirming successful conjugation. Notably, Au-GEM-AsP-COV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD(b)) exhibits the most intense amide bands, indicating greater amide bond formation than Au-GEM-AsP-Phys (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD(a)) and modified Au-GEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD(c)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Stability studies and optimal formulation\u003c/h2\u003e\u003cp\u003eTo assess the stability and shelf-life potential of the Au-GEM-AsP-Phys and Au-GEM-AsP-COV formulations, accelerated stability tests were conducted following ICH Guideline Q1A (R2) standards \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The formulations were stored at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH for six months, with evaluations performed at 0, 3, and 6 months. As AuNP-based systems are typically stored long-term at 5\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C to prevent agglomeration, these elevated conditions were selected to simulate long-term behavior. The parameters monitored included visual appearance, particle size, polydispersity index (PDI), and drug content (assay), with summary data presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eBoth formulations retained a clear and light purple appearance throughout the study, indicating no visible signs of instability. Particle size and PDI values showed minor fluctuations over time, with no significant aggregation or changes in distribution. Notably, changes were observed in drug content between the two formulations, with the Au-GEM-AsP-COV showing a more consistent assay profile over the testing period compared to Au-GEM-AsP-Phys.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of stability studies conducted on Au-GEM-AsP-Phys and Au-GEM-AsP-COV\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProduct Name: Au-GEM-AsP-Phys 0.1mgmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Au, 1mgmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GEM, 2.5mgmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e AsP\u003c/p\u003e\u003cp\u003ePacking: Vial 6R, Clear\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBatch No.: Au-Phys \u0026minus;\u0026thinsp;22001\u003c/p\u003e\u003cp\u003eMfg. Date: 06.2022\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eStability Study Conditions\u003c/p\u003e\u003cp\u003eTemperature (̊C): 25 ̊C\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ̊C\u003c/p\u003e\u003cp\u003eRelative Humidity: 60% \u0026plusmn; 5%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInitial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eM6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAppearance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eClear and Light Purple in color\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eClear and Light Purple in color\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eClear and Light Purple in color\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAssay (%)\u003c/p\u003e\u003cp\u003eAcceptance Criteria: Initial\u0026thinsp;\u0026plusmn;\u0026thinsp;5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e87.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e80.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e77.72%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSize (nm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e106.0 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e115.4 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e110.6 nm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.535\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.432\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.712\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eProduct Name: Au-GEM-AsP-COV 0.1mgmL\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eAu, 1mgmL\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eGEM, 2.5mgmL\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eAsP\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePacking: Vial 6R, Clear\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBatch No.: Au-COV-22001\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMfg. Date: 06.2022\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eStability Study Conditions\u003c/p\u003e\u003cp\u003eTemperature (̊C): 25 ̊C\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ̊C\u003c/p\u003e\u003cp\u003eRelative Humidity: 60% \u0026plusmn; 5%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInitial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eM6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAppearance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eClear and Light Purple in color\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eClear and Light Purple in color\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eClear and Light Purple in color\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAssay (%)\u003c/p\u003e\u003cp\u003eAcceptance Criteria: Initial\u0026thinsp;\u0026plusmn;\u0026thinsp;5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.50%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e84.78%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e85.76%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSize\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e125.8 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e131.8 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e134.0 nm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.664\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.704\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.701\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 In Vitro Release profile evaluation of Au-GEM-AsP-COV formulation\u003c/h2\u003e\u003cp\u003eTo evaluate the role of the AsP matrix in enhancing hydrophobicity and achieving controlled drug release, the in vitro release profile of the Au-GEM-AsP-COV formulation was examined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA(a), GEM release exhibited a time-dependent pattern, with cumulative release reaching 93.18% \u0026plusmn; 1.68% at 72 hours, indicating sustained release behavior. Additionally, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA(b) presents the HPLC chromatograms of GEM at multiple time points, ranging from 5 minutes to 96 hours, further confirming the gradual release of the drug over time.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5 Evaluation of Cell Viability and Toxicity in 4T1 Cell Line: Comparing Au-GEM-AsP-Phys, Modified Au-GEM, and Au-GEM-AsP-COV with Free GEM\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the cytotoxicity and therapeutic efficacy of the final formulations, MTT assays were performed on the 4T1 murine breast cancer cell line across a concentration range of 1\u0026ndash;100 \u0026micro;g/mL over 48 hours. The study compared Au-GEM-AsP-COV and Au-GEM-AsP-Phys formulations against free GEM and other control groups. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, the Au-GEM-AsP-COV formulation (Group B): 0.44 \u0026micro;g/mL demonstrated significantly lower cell viability compared to both the physical formulation (0.51 \u0026micro;g/mL) and the free drug (Group C): 0.89 \u0026micro;g/mL across all tested concentrations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, ascorbyl palmitate (AsP), a hydrophobic derivative of vitamin C, was introduced as a reductant for AuNP formation, as well as a stabilizer and surface-modifying agent to enhance the physicochemical stability and encapsulation efficiency of GEM-loaded AuNPs. Results demonstrate that AsP provides steric stabilization to the nanoparticles and contributes to forming a hydrophobic matrix that more effectively retains GEM.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA(a) shows five AuNP formulations immediately after synthesis. The corresponding UV-Vis spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA(b)) for samples 1 to 5 revealed a redshift in the surface plasmon resonance (SPR) band from 520 nm to 560 nm in the presence of AsP, compared to the sodium citrate method \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. This redshift, increasing with AsP concentration, indicates particle growth and successful surface coating. However, samples with excessive AsP (\u0026gt;\u0026thinsp;0.05 mg/mL) showed particle aggregation and reduced colloidal stability, consistent with reports that high surfactant levels induce interparticle bridging and destabilization. Sample 2 (0.05 mg/mL AsP) exhibited optimal stability, with consistent color, low turbidity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB(a)), and minimal λmax shift over six-month storage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB(b)).\u003c/p\u003e\u003cp\u003eFurther characterization using DLS and zeta potential analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B) confirmed that Sample 2 initially had a uniform particle size of approximately 90.9 nm, low polydispersity, and a zeta potential of \u0026minus;\u0026thinsp;1.1 mV, indicating both electrostatic and steric stabilization. After 3 months, the particle size slightly increased to 101.2 nm and the zeta potential to \u0026minus;\u0026thinsp;1.3 mV, while at 6 months, the size decreased to 93.6 nm and the zeta potential shifted to \u0026minus;\u0026thinsp;1.6 mV. These minor fluctuations remained within acceptable ranges, suggesting good long-term colloidal stability. The findings were further supported by TEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), which consistently showed monodisperse, spherical particles, an ideal morphology for enhanced cellular uptake and biodistribution.\u003c/p\u003e\u003cp\u003eFollowing the successful synthesis and functionalization of AuNPs, GEM was efficiently loaded onto the nanoparticle surface using two distinct strategies: physical adsorption and covalent attachment. The covalent approach, which involved surface modification with thiol-containing ligands followed by amide bond formation, improved drug stability compared to physical adsorption method. This improvement reflects the superior stability and interaction strength of covalent bonding, which minimizes premature drug release and enhances delivery potential \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe UV\u0026ndash;Vis absorption spectra provided critical insights into the surface modifications and interaction behavior of the various nanoparticle formulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The optimized colloidal gold nanoparticles (AuNPs), stabilized with AsP, exhibited a characteristic surface plasmon resonance (SPR) band at 559 nm with an intensity of 0.61, along with a secondary peak at 236 nm attributed to AsP. Upon conjugation with GEM to form Au-GEM, the SPR band red-shifted slightly to 562 nm, accompanied by a marked intensity decrease to 0.36, indicating surface functionalization and altered electronic environments. Additional absorption bands at 270 nm and 235 nm corresponded to GEM and AsP, confirming successful drug loading. Further modification in the physically adsorbed system (Au-GEM-AsP-Phys) caused a more pronounced red shift to 571 nm with an intensity of 0.42 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), suggesting effective but less compact surface interactions. In contrast, the covalently modified formulation (Au-GEM-AsP-COV) exhibited a shift to 568 nm and a significantly lower intensity of 0.27, likely reflecting a more compact and uniform surface coating due to stronger covalent bonding, which can dampen SPR oscillations. The overlapping absorption bands in the 240\u0026ndash;280 nm region in both final formulations indicate the co-presence of AsP and GEM, supporting the multifunctional surface architecture. Overall, the observed spectral shifts and intensity changes confirm successful stepwise functionalization and reveal distinct surface environments between the covalent and physical systems.\u003c/p\u003e\u003cp\u003eThe surface engineering strategy employed in the covalently modified nanocarrier (Au-GEM-AsP-COV) resulted in a distinct enhancement in colloidal stability and particle uniformity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB(b), the covalent formulation exhibited a hydrodynamic diameter of 125.8 nm, slightly larger than the physically adsorbed counterpart (Au-GEM-AsP-Phys), which measured 106.0 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB(a)). Both nanocarriers were larger than the unmodified optimized gold nanoparticles (90.9 nm, sample 2), indicating successful surface functionalization. This increase in size for the covalent system likely reflects the additional surface layers formed via amide bond formation, contributing to structural robustness. Furthermore, zeta potential analysis demonstrated that the covalently modified nanocarriers possessed a more negative surface charge (\u0026ndash;18.3 mV) compared to the physically adsorbed system (\u0026ndash;15.9 mV), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC(a\u0026ndash;b). This more negative potential is indicative of improved electrostatic repulsion and, consequently, greater colloidal stability. In contrast, the optimized AuNPs showed a near-neutral charge (\u0026ndash;1.1 mV), underscoring the significance of surface modification in enhancing both stability and dispersion behavior \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Collectively, these findings highlight the superior physicochemical characteristics of the covalently engineered nanocarrier system, which are essential for maintaining long-term stability and preventing aggregation under physiological conditions.\u003c/p\u003e\u003cp\u003eThe contact angle measurements demonstrate the sequential surface modifications and their influence on nanoparticle hydrophobicity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the contact angle decreased from 44\u0026deg; to 37\u0026deg; following functionalization with MUA/MU and GEM, indicating enhanced hydrophilicity. This shift can be attributed to the introduction of polar functional groups, carboxylic acids and hydrophilic amines on the nanoparticle surface. In contrast, the subsequent increase in contact angle to 58\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) after encapsulation within the AsP matrix (Au-GEM-AsP-COV) reflects the addition of a hydrophobic layer \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This change confirms the successful entrapment of the hydrophilic-modified Au-GEM within a fatty acid ester-based AsP matrix, thereby altering the surface properties toward greater hydrophobicity. These findings validate the dual surface engineering approach for tailoring nanoparticle interactions with aqueous environments, which may have significant implications for biodistribution and cellular uptake in therapeutic applications.\u003c/p\u003e\u003cp\u003eThe FT-IR spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) confirm the successful formation of amide bonds during the surface modification and encapsulation steps of the nanoparticle formulations. Characteristic amide I and amide II bands, observed at approximately 1645\u0026ndash;1646 cm⁻\u0026sup1; and 1552\u0026ndash;1553 cm⁻\u0026sup1;, respectively appear in all samples, indicating covalent attachment of GEM via amide bond formation. These bands correspond to C\u0026thinsp;=\u0026thinsp;O stretching (amide I) and N\u0026ndash;H bending (amide II) vibrations, reflecting the conjugation of the carboxyl-functionalized gold nanoparticle surface with the amine groups of GEM \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Notably, the Au-GEM-AsP-COV formulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD(b)) exhibits the most intense peaks (~\u0026thinsp;95% at 1646 cm⁻\u0026sup1; and ~\u0026thinsp;50% at 1552 cm⁻\u0026sup1;), compared to the physically loaded Au-GEM-AsP-Phys (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD(a); ~85% and ~\u0026thinsp;35%) and the modified Au-GEM nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD(c); ~30% at 1627 cm⁻\u0026sup1; and 1567 cm⁻\u0026sup1;). The higher intensity of amide bands in the covalently encapsulated formulation suggests a greater extent of bond formation, likely resulting from dual contributions of both the nanoparticle surface and the AsP matrix. These findings support the enhanced chemical integration of GEM in the Au-GEM-AsP-COV system and underscore the efficiency of the covalent encapsulation strategy.\u003c/p\u003e\u003cp\u003eThe accelerated stability study results summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e highlight the superior stability of the covalent Au-GEM-AsP-COV formulation compared to the physically loaded Au-GEM-AsP-Phys. Both formulations maintained their clear and light purple appearance throughout the six-month period, indicating good physical stability under accelerated conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH) \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Particle size and PDI values showed minor fluctuations but remained consistent overall, with no significant aggregation detected for either formulation. However, the drug assay data in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e reveal distinct differences in chemical stability. The Au-GEM-AsP-COV formulation preserved GEM content within the acceptable\u0026thinsp;\u0026plusmn;\u0026thinsp;5% range over 6 months (initial: 89.5%, 3 months: 84.78%, 6 months: 85.76%), whereas the physically loaded Au-GEM-AsP-Phys formulation exhibited a substantial decline in drug content (initial: 87.0%, 3 months: 80.67%, 6 months: 77.72%), which exceeded the acceptable threshold, showing losses of over 6% and 9% at 3 and 6 months, respectively, indicating drug loss or degradation over time. These findings confirm that covalent conjugation of GEM to the nanoparticle surface, coupled with lyophilization, effectively reduces drug degradation and loss during storage, thereby enhancing the long-term stability of the nanocarrier system.\u003c/p\u003e\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA(a), the Au-GEM-AsP-COV formulation demonstrated a sustained, time-dependent drug release pattern, achieving approximately 93.18% \u0026plusmn; 1.68% cumulative release over 72 Hrs. This behavior is attributed to the covalent attachment of GEM to the gold nanoparticle surface and encapsulation within the AsP matrix, enhancing hydrophobicity and modulating diffusion. HPLC chromatograms in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA(b) confirm GEM\u0026rsquo;s gradual release from as early as 5 minutes to up to 96 hours. This sustained release is significant given GEM\u0026rsquo;s clinical limitations, its short plasma half-life (8-17minutes) due to rapid deamination by cytidine deaminase in blood and liver \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The controlled release observed with Au-GEM-AsP-COV may prolong systemic availability, protect GEM from enzymatic degradation, and maintain therapeutic levels longer. By minimizing the rapid peak-trough fluctuations typical of free GEM, this nanocarrier system could enhance efficacy and reduce dose-related toxicity. Overall, the release profile reinforces the nanocarrier\u0026rsquo;s potential to address GEM\u0026rsquo;s pharmacokinetic challenges and improve its clinical performance in cancer therapy \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThese physicochemical advantages translated into improved biological activity. The covalently attached GEM-AuNPs exhibited significantly greater cytotoxicity against 4T1 breast cancer cells, with an IC₅₀ of 0.44 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), which reinforces the potential of the AsP-based nanocarrier platform for the development of long-acting injectable formulations with enhanced therapeutic efficacy. The enhanced cytotoxic performance of the Au-GEM-AsP-COV formulation can be attributed to its sustained and controlled drug release, as previously demonstrated in the in vitro release study, where it achieved 93.18% \u0026plusmn; 1.68% release over 72 Hrs. The slow and steady drug release allowed for prolonged drug availability and greater cellular uptake, resulting in a lower IC₅₀ value compared to both the free GEM (0.89 \u0026micro;g/mL) and the physical formulation (0.51 \u0026micro;g/mL). Moreover, the more negative surface charge of the covalent formulation (\u0026ndash;18.3 mV compared to \u0026minus;\u0026thinsp;15.9 mV for the physical one) likely enhanced cellular internalization through electrostatic interactions, resulting in more effective intracellular drug delivery. The amide bond formation in the covalent formulation also provided additional chemical stability for GEM, minimizing premature degradation and enhancing efficacy. Collectively, these findings highlight the superior performance of the Au-GEM-AsP-COV formulation in targeted cancer therapy, supporting its potential for further preclinical development.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully demonstrated the multifunctional role of ascorbyl palmitate (AsP) in the synthesis and surface engineering of gold nanoparticles (AuNPs) for enhanced gemcitabine (GEM) delivery. AsP functioned not only as a reductant and stabilizer but also as a hydrophobic matrix component, contributing to improved nanoparticle stability and drug encapsulation. Among the various formulations tested, the covalently modified Au-GEM-AsP-COV system exhibited superior physicochemical characteristics, including optimal particle size, enhanced colloidal stability, more negative surface charge, and effective surface functionalization. These features contributed to significantly improved drug loading, controlled and sustained release over 72 Hrs, and reduced drug degradation over a six-month stability study. Critically, the Au-GEM-AsP-COV formulation demonstrated enhanced biological efficacy, achieving a lower IC₅₀ against 4T1 breast cancer cells compared to both free GEM and physically loaded formulations. The combined steric and electrostatic stabilization, along with the dual surface engineering strategy, enabled better control over GEM release, prolonged systemic availability, and greater cellular uptake. Altogether, these findings support the promise of AsP-based covalently engineered AuNPs as a robust platform for the development of long-acting, stable, and efficacious injectable chemotherapeutic nanocarriers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research did not receive any grant from funding agencies\u003c/p\u003e\u003cp\u003eAvailability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHavva Rezaei: Writing \u0026ndash; original draft, Validation, Investigation, Formal analysis, Data curation, Validation, Methodology, Conceptualization, Visualization, Investigation, Formal analysis. Mostafa Shourian: Writing \u0026ndash; review \u0026amp; editing, Supervision, Resources, Project administration, Data availability statement.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors appreciate Marzieh Ramezanpour, Department of Biology, Faculty of Science, University of Guilan, P.O.Box 4193833697, Rasht, Iran.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAvailability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHeinemann, V. Role of Gemcitabine in the Treatment of Advanced and Metastatic Breast Cancer. \u003cem\u003eOncology\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 191\u0026ndash;206 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEspey, M. G. et al.' 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Two birds with one stone: triple negative breast cancer therapy by PtCo bimetallic nanozyme coated with gemcitabine\u0026ndash;hyaluronic acid\u0026ndash;polyethylene glycol. \u003cem\u003eCancer Nanotechnol\u003c/em\u003e. \u003cb\u003e14\u003c/b\u003e, 1\u0026ndash;21 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gold Nanoparticles, Ascorbyl Palmitate, Nanoemulsion, Gemcitabine, Breast cancer","lastPublishedDoi":"10.21203/rs.3.rs-7240807/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7240807/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreast cancer remains a significant global health challenge, often treated with gemcitabine hydrochloride (GEM). However, GEM's short half-life and poor sustained release can lead to severe side effects, including myelosuppression and nephrotoxicity. This study presents a novel nano-drug delivery system using gold nanoparticles (AuNPs) optimized with ascorbyl palmitate (AsP) to enhance GEM stability and efficacy. AuNPs were modified via single-phase emulsification to form a nanoemulsion coated with a hydrophobic AsP layer, improving tumor targeting through the enhanced permeability and retention (EPR) effect. Two formulations were developed: Au-GEM-AsP-COV (prodrug, 128.5 nm, -18.3 mV, 89.5% encapsulation efficiency) and Au-GEM-AsP-Phys (106 nm, -15.9 mV, 87% encapsulation efficiency). The Au-GEM-AsP-COV formulation demonstrated superior hydrophobicity, sustained release, and enhanced cytotoxicity (IC50 of 0.44 \u0026micro;g/mL) in the 4T1 cell line, significantly outperforming free GEM and modified Au-GEM formulations. Notably, it exhibited six months of accelerated stability, attributed to amide bond formation in the functionalized AuNP matrix. The study highlights the synergistic effects of AsP in enhancing the therapeutic efficacy of Au-GEM-based formulations, supporting its role as a key component in combination therapy. This research lays the foundation for future development of hydrophobic nanomedical devices combining GEM and AsP for therapeutic and diagnostic applications in nanomedicine.\u003c/p\u003e","manuscriptTitle":"Optimizing Gold Nanoparticles for Combination Therapy: Development of Hydrophobic Nanomedical Devices with Gemcitabine and Ascorbyl Palmitate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 18:14:50","doi":"10.21203/rs.3.rs-7240807/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-10T04:18:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-02T02:32:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-23T04:42:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83838783696335458893036116644908480553","date":"2025-08-21T16:10:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114349959448584958505089664850541245747","date":"2025-08-21T12:58:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73428485854473670270528864750765995015","date":"2025-08-19T10:13:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-19T07:42:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-05T09:12:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-05T09:06:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-04T05:26:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-03T05:54:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8d89f532-0b80-41b2-b167-fe530b79f287","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53597244,"name":"Biological sciences/Biochemistry"},{"id":53597246,"name":"Biological sciences/Biotechnology"},{"id":53597247,"name":"Biological sciences/Cancer"},{"id":53597249,"name":"Physical sciences/Chemistry"},{"id":53597250,"name":"Biological sciences/Drug discovery"},{"id":53597251,"name":"Physical sciences/Materials science"},{"id":53597252,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2025-12-29T16:03:21+00:00","versionOfRecord":{"articleIdentity":"rs-7240807","link":"https://doi.org/10.1038/s41598-025-32204-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-26 15:58:31","publishedOnDateReadable":"December 26th, 2025"},"versionCreatedAt":"2025-08-27 18:14:50","video":"","vorDoi":"10.1038/s41598-025-32204-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-32204-6","workflowStages":[]},"version":"v1","identity":"rs-7240807","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7240807","identity":"rs-7240807","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}