Development of ascorbyl palmitate based hydrophobic gold nanoparticles as a nanocarrier system for gemcitabine delivery

Abstract

Breast cancer remains a major global health challenge, commonly treated with gemcitabine hydrochloride (GEM). However, GEM’s short half-life and rapid metabolism limit its therapeutic efficacy. This study introduces a novel nano-drug delivery system based on gold nanoparticles (AuNPs) modified with ascorbyl palmitate (AsP) to improve GEM stability and performance. AuNPs were surface-modified via single-phase emulsification to form a hydrophobic AsP-coated nanoemulsion. Two formulations were prepared: Au-GEM-AsP-MOD (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-MOD system exhibited superior hydrophobicity, controlled GEM release (93% over 72 h), and enhanced cytotoxicity (IC₅₀ = 0.44 µg/mL) in 4T1 breast cancer cells compared with free GEM and physically adsorbed formulations. Furthermore, it maintained stability for six months under stress conditions (25 ± 2 °C, 60 ± 5% RH), indicating robustness and extended shelf life due to effective surface modification. 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.

Introduction

Cancer is a deadly disease characterized by uncontrolled cell proliferation and the spread of abnormal cells through invasion or metastasis¹. Traditional cancer treatments include surgery, radiotherapy, and chemotherapy². 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 min) 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 benefits⁸.

Natural products have significantly contributed to anticancer research, as most clinically used anticancer drugs originate from natural sources². 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 alone². 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 trial³. Cullen reviewed a Phase 2 trial (PACMAN 2.1) of high-dose ascorbate with nab-paclitaxel and GEM⁴.

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 AsP¹¹. Min Zhou et al. designed AsP-based solid lipid nanoparticles combined with paclitaxel (AsP/PTX-SLNs) to maximize AsP’s therapeutic efficacy¹⁵. 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 mice⁶.

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 modification²⁴. Santiago et al. modified AuNP surfaces with GEM and folate/transferrin ligands to create a targeted controlled-release nanocarrier, reducing side effects and improving efficacy¹⁴.

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^24–26. Additionally, AuNPs can stabilize other drug carriers, such as liposomes, enhancing delivery efficiency¹⁴. 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 days²⁷. 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 stability²⁸.

Surface charge polarity and density greatly influence immune clearance and cellular uptake of intravenously administered nanocarriers, affecting their delivery efficiency to target sites²⁹. In this study, we developed and characterized a gold nanoparticle–based delivery platform for gemcitabine (GEM) within an ascorbyl palmitate (AsP) matrix to improve hydrophobicity, drug stability, and therapeutic efficacy. Two formulations were prepared and compared: a physically encapsulated system (Au-GEM-AsP-Phys) produced by a water-in-oil emulsification method, and a chemically activated, surface-modified system (Au-GEM-AsP-MOD) prepared by EDC/NHS activation of AuNP surfaces followed by GEM association. We characterized both formulations with respect to particle size, surface charge, drug loading, in vitro release, stability under stress conditions, and cytotoxicity in 4T1 cells, to determine how AsP encapsulation and surface modification affect physicochemical properties and anticancer performance.

Materials and methods

Materials

Tetra chloroauric acid (HAuCl₄.2H₂O), ascorbyl palmitate (AsP) (C₂₂H₃₈O₇), 11-mercaptoundecanoic acid (MUA) (HS(CH₂)₁₀CO₂H), 11-mercapto-1-undecanol (MU) (HS(CH₂)₁₁OH), and 2-(3,5-diphenyltetrazol-2-ium-2-yl)−4,5-dimethyl-1,3-thiazole (MTT) (C₁₈H₁₆N₅S⁺) were purchased from Sigma-Aldrich. Gemcitabine hydrochloride (GEM) (C₉H₁₁F₂N₃O₄·HCl) was purchased from Shilpa. N-Hydroxy succinimide (NHS) (C₄H₅NO₃), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (C₈H₁₇N₃), tri-sodium Citrate (CIT) (C₆H₅Na₃O₇), sodium dihydrogen phosphate (NaH₂PO₄), sodium hydroxide (NaOH), hydrochloric acid (HCl), Tween 20 (C₂₆H₅₀O₁₀), dimethyl sulfoxide (DMSO) ((CH₃)2SO), D-mannitol (C₆H₁₄O₆), propylene glycol (MPG) (C₃H₈O₂), polyethylene glycol 300 (PEG) (H(OCH₂CH₂)_nOH), absolute ethanol (ETOH) (C₂H₆O), and acetone ((CH₃)₂CO) were obtained from Merck. The 4T1 cell line and acetonitrile (ACN) (CH₃CN) 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.

Synthesis of AuNPs in asp matrix and optimization

Oxygen-free Milli-Q water was prepared by nitrogen purging 200 mL of water for 30 min prior to nanoparticle synthesis. An AsP stock solution (1.0 mgmL⁻¹) was prepared by dissolving ascorbyl palmitate (AsP) in absolute ethanol with heating at 70 °C and vortexing until a clear solution formed. The AsP stock was maintained at 70 °C during nanoparticle synthesis.

A gold precursor solution (HAuCl₄, 100 mgmL⁻¹) was used as received. For each synthesis, 9.0 mL of oxygen-free water was equilibrated at 70 °C under magnetic stirring (100 rpm). To this, 10 µL of HAuCl₄ (100 mgmL⁻¹) was added and stirred for 2 min. The AsP stock solution was then added dropwise at the specified volume while stirring at 150 rpm. The reaction mixture was maintained at 70 °C for 20 min, after which the heat was turned off and the suspension was allowed to cool to room temperature under gentle agitation.

Five different AsP concentrations were evaluated to optimize nanoparticle size distribution and colloidal stability. The resulting AuNPs were analyzed for UV–visible absorption (200–800 nm), hydrodynamic diameter, and zeta potential. The optimized formulation was further examined by transmission electron microscopy (TEM) for morphology and core size. Stability was monitored at 0, 3, and 6 months under stress conditions (25 ± 2 °C, 60 ± 5% RH) by measuring particle size, PDI, and visual appearance.

Gold metallic nanocarrier containing GEM loaded in asp matrix

Gemcitabine-loaded gold nanoparticles were incorporated into an AsP-based matrix using a nano emulsification technique to achieve stable, hydrophobic nanoformulations with controlled particle size and enhanced colloidal stability. The 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 formation³⁰. Two formulations were prepared: Au-GEM-AsP-Phys, obtained by the physical incorporation of GEM-loaded AuNPs into the AsP matrix, and Au-GEM-AsP-MOD, prepared by incorporating GEM-loaded AuNPs into AsP using a modified encapsulation approach.

Preparation of oil/surfactant phase and formation of Au-GEM-AsP-Phys nanoemulsion

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. 1A. 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 min. 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 min, followed by homogenization at 12,000 rpm for 5 min. The nanoemulsion of Au-GEM-AsP-Phys was formed using sonication at 50 watts (20 s on, 10 s off) for 13 min, then shaken at 1,000 rpm for 15 min. 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 min at 15 °C.The resulting Au-GEM-AsP-Phys nanoparticle solution was stored at 2–8 °C.

Fig. 1.

(A) Schematic of Au-GEM-AsP-Phys nanoemulsion formation based on physical incorporation, (B) surface treatment of AuNPs and GEM loading, (C) single-phase emulsification process showing encapsulation of GEM-loaded AuNPs within the AsP matrix to form Au-GEM-AsP-MOD.

Surface treatment of gold nanoparticles

Figure 1B illustrates the activation of the AuNPs surface followed by the encapsulation of GEM within the AuNPs. Optimized AuNP suspension (10 mL) was mixed with 10 mL phosphate buffer (pH 8.0) containing Tween 20 (50 µL) and stirred at room temperature for 30 min. A solution of 11-mercaptoundecanoic acid (MUA) and 11-mercaptoundecanol (MU) (2 mM, 8:1 v/v) was added, and the reaction mixture was stirred at room temperature for 12 h. The suspension was centrifuged (14,000 rpm, 30 min, 4 °C) and washed sequentially with phosphate buffer (pH 8 and pH 7).

The precipitate was resuspended in MES buffer (pH 5.5) and activated by adding 20 mL of EDC (10 mM)/NHS (20 mM) solution, followed by incubation at room temperature for 15 min with gentle shaking. Activated nanoparticles were collected by centrifugation (24,000 rpm, 10 min, 4 °C)^31–34..

To the activated AuNPs, 500 µL of GEM solution (10 mg mL⁻¹) was added and the total volume adjusted to 2 mL using phosphate buffer (pH 7.4). The mixture was stirred at room temperature for 20 min and centrifuged (24 000 rpm, 10 min, 4 °C). GEM content in the supernatant was determined to calculate conjugation efficiency. The pellet was combined with 5 mL of mannitol solution (6.4 mg mL⁻¹) and lyophilized under 100 Pa for 48 h (freezing − 40 °C/20 h; primary drying − 25 °C/4 h; secondary drying − 5 °C/4 h)^35,36. The final activated Au-GEM was stored at 2–8 °C.

Au-GEM-AsP-MOD nanoemulsion preparation

Figure 1C illustrates the single-phase emulsification process, where GEM-loaded modified gold nanoparticles are encapsulated within the AsP matrix, leading to the Au-AsP-GEM-MOD nanoemulsion formation. GEM-conjugated AuNPs (from Sect. Surface Treatment of Gold Nanoparticles) were encapsulated within an AsP matrix using a single-phase emulsification method. AsP was dissolved in acetone (12.5 mg mL⁻¹) and mixed with Tween 20 in a 70:30 ratio to form the oil/surfactant phase. Freeze-dried Au-GEM powder was reconstituted in 4 mL sodium-phosphate buffer (pH 7.4) to serve as the aqueous phase.

The aqueous phase was added dropwise to the oil/surfactant mixture under stirring (300 rpm, 15 min), followed by homogenization (12 000 rpm, 5 min) and probe sonication (50 W, 20 s on/10 s off cycles, 13 min). The emulsion was stirred (1000 rpm, 15 min) and the solvent evaporated at 30 °C for 2 h. The dispersion was centrifuged (12 000 rpm, 15 min, 15 °C) to remove residual aggregates. The resulting Au-GEM-AsP-MOD formulation was stored at 2–8 °C.

Results

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 2A(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 mgmL^{− 1} CIT (Fig. 2A(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 2B(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. 2B(b). Sample 2, maintaining stable absorbance similar to its initial spectrum and closely resembling Au-CIT with superior appearance stability (Fig. 2B), was identified as the optimal formulation³⁷.

Fig. 2.

A(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.

DLS results for the optimal formulation at initial, 3, and 6 months are illustrated in Fig. 3A, 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. 3B).

Fig. 3.

(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.

The morphology and size of the optimized formulation 2 was analyzed using Transmission Electron Microscopy (TEM, Philips CM30, Netherlands) operating at an accelerating voltage of 100 kV. A dilute aqueous dispersion (0.05 mg/mL) was sonicated for 2 min, and a 10 µL drop was placed on a carbon-coated copper grid (300 mesh) and air-dried for 10–15 min before imaging. TEM images of the optimized formulation 2 (Fig. 3C) 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.

Comparative physico-chemical characterization of GEM in Au-ASP matrix: Au-GEM, Au-GEM-AsP-Phys and Au-GEM-AsP-MOD

UV–Vis absorption spectra were recorded for all relevant materials, including AuNPs, AsP, GEM, Au-GEM, and the final formulations Au-GEM-AsP-Phys and Au-GEM-AsP-MOD (Fig. 4A). The optimized AuNPs (sample 2), stabilized with AsP, exhibited a characteristic plasmon absorption band at 559 nm (intensity: 0.61), along with a secondary band at 236 nm associated with AsP (intensity: 1.07). Following surface modification with GEM, the plasmon band shifted slightly to 562 nm with reduced intensity (0.30), and additional bands appeared at 235 nm and 270 nm, corresponding to AsP and GEM, respectively. The Au-GEM-AsP-Phys formulation showed a further red shift to 571 nm (intensity: 0.42) with overlapping bands in the 240–280 nm region. In comparison, the Au-GEM-AsP-MOD formulation exhibited a plasmon shift to 568 nm and a lower intensity of 0.27, consistent with surface modification and altered local electronic environments. The UV–Vis absorption shifts and intensity changes are summarized in Table 1.

Fig. 4.

characterization studies including (A) Comparative UV-Vis spectra of different groups, AsP, GEM, AuNPs, Au-GEM, Au-GEM-AsP-MOD, and Au-GEM-AsP-Phys (B) DLS results and (C) Zeta potential studies on (a) Au-GEM-AsP-Phys and (b) Au-GEM-AsP-MOD.

Table 1.

Summary of UV-Vis absorption shifts and intensity changes for AuNP-Based Formulations.

Sample	Wavelength (nm)	Intensity	Description	Shifts/Change
AuNPs (Fig. 4A-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. 4A-Dark blue)	270	0.71	Two distinct absorption bands; GEM sample used in the reaction.	No shift/change
	238	0.54
Au-GEM (Fig. 4A-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. 4A-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-MOD (Fig. 4A-Light blue)	568	0.27	Plasmon band of gold nanoparticles in the surface-modified formulation.	Shifted from 559 nm to 568 nm (intensity decreased from 0.61 to 0.27), consistent with surface modification and altered electronic environment.
	240–280	High	Overlapping bands of AsP and GEM.	No shift/change

Dynamic light scattering analysis showed that the hydrodynamic diameter of the physically modified nanocarrier (Au-GEM-AsP-Phys) was 106.0 nm, whereas the surface-modified Au-GEM-AsP-MOD measured 125.8 nm (Fig. 4B). Zeta potential measurements indicated a more negative surface charge for the surface-modified formulation (–18.3 mV) compared to the physically modified system (–15.9 mV), reflecting enhanced electrostatic stabilization (Fig. 4C(a–b)).

To evaluate the surface hydrophobicity of the Au-GEM-AsP-MOD nanocarrier versus optimized AuNPs, contact angle measurements were conducted. As shown in Fig. 5A, the contact angle of bare AuNPs was 44°. After surface modification with MUA/MU and conjugation with GEM, the contact angle decreased to 37° (Fig. 5B), 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. 5C). This consistent change across three independent measurements confirms increased surface hydrophobicity³⁸. To examine molecular interactions, FT-IR spectra were recorded for Au-GEM, Au-GEM-AsP-Phys, and Au-GEM-AsP-MOD at room temperature (Fig. 5D). All spectra show characteristic amide bands, consistent with interactions between GEM and the nanoparticle surface. Notably, Au-GEM-AsP-MOD (Fig. 5D(b)) exhibits the most intense amide bands compared to Au-GEM-AsP-Phys (Fig. 5D(a)) and surface-modified Au-GEM (Fig. 5D(c)), suggesting a higher degree of surface-associated amide interactions in this formulation.

Fig. 5.

Surface angle investigation as a hydrophobicity criterion for (A) AuNPs, (B) Au-GEM, (C) Au-GEM-AsP-MOD, with three replicate runs, and (D) FT-IR spectra of (a) Au-GEM-AsP-Phys, (b) Au-GEM-AsP-MOD, and (c) Au-GEM.

Stability studies and optimal formulation

To evaluate stability and shelf-life potential of the Au-GEM-AsP-Phys and Au-GEM-AsP-MOD formulations, stress condition studies were conducted at 25 ± 2 °C/60 ± 5% RH. These tests were designed to assess formulation robustness relative to the intended refrigerated storage (5 ± 3 °C)³⁹. The formulations were stored at 25 ± 2 °C and 60 ± 5% RH for six months, with evaluations performed at 0, 3, and 6 months. Although AuNP-based systems are typically stored at 5 ± 3 °C to minimize agglomeration, these elevated conditions were chosen to accelerate stability testing and simulate long-term behavior. Stability parameters included visual appearance, particle size, polydispersity index (PDI), and drug content, with % assay determined by HPLC analysis. The results are summarized in Table 2.

Table 2.

Results of stability studies conducted on Au-GEM-AsP-Phys and Au-GEM-AsP-MOD.

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 − 22,001 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-MOD 0.1mgmL− 1 Au, 1mgmL− 1 GEM, 2.5mgmL− 1 AsP Packing: Vial 6R, Clear	Batch No.: Au-MOD-22,001 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

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-MOD showing a more consistent assay profile over the testing period compared to Au-GEM-AsP-Phys.

In vitro release profile evaluation of Au-GEM-AsP-MOD formulation

To evaluate the role of the AsP matrix and surface modification in enhancing hydrophobicity and achieving controlled drug release, the in vitro release profile of the Au-GEM-AsP-MOD formulation was examined. As shown in Fig. 6A(a), GEM release exhibited a sustained, time-dependent pattern, reaching a cumulative release of 93.18 ± 1.68% after 72 h. The study was performed in phosphate buffer (pH 6.8) at 37 ± 0.5 °C under continuous shaking (100 rpm). GEM concentration in the release medium was quantified by HPLC, and cumulative release was calculated relative to the initial drug loading. All experiments were conducted in triplicate (n = 3). Additionally, Fig. 6A(b) presents the HPLC chromatograms of GEM at multiple time points, ranging from 5 min to 96 h, further confirming the gradual and controlled release behavior of the formulation.

Fig. 6.

(A) GEM drug release: (a) Cumulative release profile of GEM from the Au-GEM-AsP-MOD formulation to examine release behavior in a simulated buffer environment at 37 °C, pH 6.8 over a period of 96 h, (b) HPLC chromatograms from the GEM release study of the Au-GEM-AsP-MOD formulation, (B) Comparative Cell Viability Chart: (A) AuNPs, (B) Au-GEM-AsP-MOD, (C) Free GEM, (G) Free AsP, (E) Modified Au-GEM, (F) Au-GEM-AsP-Phys on 4T1 cell line viability after 48 h. 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 < 0.0001).

Evaluation of cell viability and toxicity in 4T1 cell line: comparing Au-GEM-AsP-Phys, modified Au-GEM, and Au-GEM-AsP-MOD 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 h. All formulations were prepared and tested at equivalent GEM concentrations, calculated from their encapsulation efficiency values determined by HPLC. The final treatment concentrations (1–100 µg/mL GEM equivalent) were identical for all samples, including the free GEM control. The results demonstrate that surface modification of AuNPs with AsP (Au-GEM-AsP-MOD) enhances GEM delivery and cytotoxic efficacy compared to both physical adsorption (Au-GEM-AsP-Phys) and free drug exposure. The study compared Au-GEM-AsP-MOD and Au-GEM-AsP-Phys formulations against free GEM and other control groups. As shown in Fig. 6B, the Au-GEM-AsP-MOD formulation (Group B; IC₅₀ = 0.44 µg/mL) demonstrated significantly lower cell viability compared to both the physical formulation (IC₅₀ = 0.51 µg/mL) and the free drug (Group C; IC₅₀ = 0.89 µg/mL).

Discussion

Gold nanoparticles (AuNPs) have traditionally been synthesized using various reducing agents, including sodium citrate, which typically yields particles of approximately 20 nm in size³⁰. Other common approaches utilize reducing agents such as ascorbic acid (vitamin C), which provide good control over particle formation and stability⁴⁰. In the present study, we employed ascorbyl palmitate (AsP), an amphiphilic derivative of vitamin C, as a reducing and stabilizing agent to synthesize AuNPs. This approach was intended to enhance nanoparticle hydrophobicity and improve their interfacial stability due to AsP’s dual hydrophilic–hydrophobic character.

Furthermore, AsP was introduced not only as a reductant but also as a surface-modifying agent to enhance the stability and encapsulation efficiency of GEM-loaded AuNPs. A dual formulation strategy was therefore designed to clarify the distinct stabilization mechanisms provided by (i) hydrophobic encapsulation within an AsP matrix and (ii) surface modification of GEM-loaded AuNPs. As shown in Fig. 1A, the Au-GEM-AsP-Phys formulation was prepared via a modified water-in-oil (W/O) emulsification method, in which GEM-loaded AuNPs were dispersed in a PEG/MPG–Tween 20 continuous phase containing AsP⁴¹. Although GEM is hydrophilic and prone to leakage, the amphiphilic nature of AsP promoted the formation of a semi-hydrophobic interfacial layer that reduced diffusion and improved drug retention. This configuration was deliberately chosen to assess AsP’s stabilizing contribution despite the expected reduction in entrapment efficiency. In contrast, the Au-GEM-AsP-MOD formulation (Figs. 1B–C) involved surface modification of AuNPs with GEM followed by AsP coating, which minimized drug loss and enhanced long-term stability. The comparative analysis of both systems revealed that physical encapsulation and surface modification act through complementary mechanisms to improve nanoparticle stability, drug-loading capacity, and controlled release under stress conditions.

Figure 2A(a) shows five AuNP formulations immediately after synthesis. The corresponding UV-Vis spectra (Fig. 2A(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^30,40. 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. 2B(a)), and minimal λmax shift over six-month storage (Fig. 2B(b)).

Further characterization using DLS and zeta potential analysis (Fig. 3A, 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. TEM images (Fig. 3C) revealed predominantly spherical nanoparticles with a relatively uniform size distribution. Occasional particle clustering is likely an artifact of solvent evaporation during grid preparation rather than true aggregation⁴². This interpretation is supported by DLS and zeta potential measurements (Fig. 3A), which displayed a single, narrow size distribution and stable colloidal behavior over six months, confirming that the AuNPs remained well-dispersed in solution. Following the successful synthesis and functionalization of AuNPs, GEM was efficiently loaded onto the nanoparticle surface using two distinct strategies: physical adsorption and surface modification. The surface modification approach, which involved functionalizing the AuNP surface with thiol-containing ligands and subsequent chemical activation, provided improved drug stability compared to the physical adsorption method. This improvement can be attributed to stronger surface interactions achieved through controlled modification, which likely minimized premature drug release and enhanced the overall delivery potential of the nanoparticles^31,38.

The UV–Vis absorption spectra provided critical insights into the surface modifications and interaction behavior of the various nanoparticle formulations (Fig. 4A; Table 1)³⁷. 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 loading with GEM to form Au-GEM, the SPR band red-shifted slightly to 562 nm, accompanied by a decrease in intensity to 0.36, indicating successful surface functionalization and altered electronic environments. Additional absorption bands at 270 nm and 235 nm corresponded to GEM and AsP, respectively, confirming drug incorporation.

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. 4A), suggesting effective but less compact surface interactions. In contrast, the surface-modified formulation (Au-GEM-AsP-MOD) exhibited a shift to 568 nm and a lower intensity of 0.27, likely reflecting a more compact and uniform surface coating due to stronger interfacial interactions, 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 variations confirm successful stepwise surface modification and reveal distinct interfacial environments between the two systems.

The surface engineering strategy employed in the surface-modified nanocarrier (Au-GEM-AsP-MOD) resulted in a distinct enhancement in colloidal stability and particle uniformity. As shown in Fig. 4B(b), the surface-modified 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. 4B(a)). Both nanocarriers were larger than the unmodified optimized gold nanoparticles (90.9 nm, sample 2), confirming successful surface functionalization. The size increase observed for the surface-modified system likely reflects the presence of additional surface layers formed during the modification process, contributing to improved structural integrity. Furthermore, zeta potential analysis revealed that the surface-modified nanocarriers possessed a more negative surface charge (–18.3 mV) compared to the physically adsorbed system (–15.9 mV), as shown in Fig. 4C(a–b). This increase in negative potential suggests stronger electrostatic repulsion between particles, thereby enhancing colloidal stability. In contrast, the optimized AuNPs exhibited a near-neutral charge (–1.1 mV), emphasizing the importance of surface modification in improving both dispersion and stability^21,23. Collectively, these findings highlight the superior physicochemical characteristics of the surface-modified nanocarrier system, which are essential for maintaining long-term stability and preventing aggregation under physiological stress conditions.

The contact angle measurements further confirmed the sequential surface modifications and their effect on nanoparticle hydrophobicity. As shown in Fig. 5A and B, the contact angle decreased from 44° to 37° following surface modification with MUA/MU and GEM, indicating enhanced hydrophilicity. This change can be attributed to the introduction of polar functional groups, such as carboxylic acids and hydrophilic amines, on the nanoparticle surface. In contrast, the subsequent increase in contact angle to 58° (Fig. 5C) after encapsulation within the AsP matrix (Au-GEM-AsP-MOD) reflects the formation of a hydrophobic surface layer, confirming the successful assembly of AsP around the modified nanoparticles³⁸. This change confirms the successful entrapment of the hydrophilic surface-modified Au-GEM within the fatty acid ester-based AsP matrix, thereby shifting the surface characteristics toward greater hydrophobicity. These results validate the dual surface engineering approach as an effective means to tailor nanoparticle interactions with aqueous environments, enhancing their stability and tunability for potential therapeutic applications.

The FT-IR spectra (Fig. 5D) provide evidence of amide-related interactions 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, consistent with surface modification involving interactions between the carboxyl-functionalized gold nanoparticle surface and the amine groups of GEM⁴³. Notably, the Au-GEM-AsP-MOD formulation (Fig. 5D(b)) exhibited the most intense amide-related peaks (~ 95% at 1646 cm⁻¹ and ~ 50% at 1552 cm⁻¹) compared to the physically loaded Au-GEM-AsP-Phys (Fig. 5D(a); ~85% and ~ 35%) and the surface-modified Au-GEM nanoparticles (Fig. 5D(c); ~30% at 1627 cm⁻¹ and 1567 cm⁻¹). The higher intensity of these bands in the Au-GEM-AsP-MOD formulation suggests a greater extent of surface-associated amide interactions, likely reflecting contributions from both the nanoparticle surface and the AsP matrix. These results indicate enhanced molecular association of GEM within the Au-GEM-AsP-MOD system and highlight the effectiveness of the surface modification and encapsulation strategy in achieving stable and uniform nanoparticle functionalization.

The stability results summarized in Table 2 highlight the superior robustness Au-GEM-AsP-MOD formulation compared to the physically loaded Au-GEM-AsP-Phys formulation. Both formulations maintained their clear and light purple appearance over six months, demonstrating good physical stability under stress conditions above the intended refrigerated storage (5 ± 3 °C, 25 ± 2 °C, 60 ± 5% RH)³⁹. Particle size and PDI values showed minor fluctuations but remained consistent overall, with no significant aggregation observed for either formulation. However, the drug assay data in Table 2 reveal distinct differences in chemical stability. The Au-GEM-AsP-MOD 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%). These losses exceeded the acceptable threshold, showing reductions of over 6% and 9% at 3 and 6 months, respectively, indicating gradual drug degradation or diffusion over time. These findings confirm that surface modification of GEM on the nanoparticle surface, coupled with lyophilization, effectively minimizes drug degradation and loss during storage, thereby enhancing the long-term physicochemical stability of the nanocarrier system.

As illustrated in Fig. 6A(a), the Au-GEM-AsP-MOD formulation exhibited a biphasic, sustained drug release pattern, achieving approximately 93.18 ± 1.68% cumulative release over 72 h. The rapid phase (~ 1.5 → 87% within 24 h) corresponds to GEM molecules adsorbed on or near the nanoparticle surface or located in hydrophilic regions of the AsP nanoemulsion, which readily diffuse into the release medium. The slower phase (~ 87 → 94.5% over 24–96 h) arises from GEM molecules entrapped within the hydrophobic AsP matrix or strongly associated with the surface-modified AuNPs, resulting in gradual, diffusion-controlled release. A similar biphasic release trend was reported by Emamzadeh et al. for polymer-coated gold nanoshells loaded with gemcitabine, confirming that diffusion through a polymeric matrix governs the sustained release of GEM molecules⁴⁴.

HPLC chromatograms in Fig. 6A(b) further confirm GEM’s gradual release from as early as 5 min up to 96 h. This prolonged and controlled release is particularly valuable considering GEM’s short plasma half-life (8–17 min) due to rapid deamination by cytidine deaminase in blood and liver⁸. The sustained release observed with Au-GEM-AsP-MOD may therefore extend systemic availability, protect GEM from enzymatic degradation, and maintain therapeutic concentrations for a longer duration. By minimizing the peak–trough fluctuations typical of free GEM, this nanocarrier system could enhance therapeutic efficacy while reducing dose-related toxicity. Overall, the release profile supports the potential of the Au-GEM-AsP-MOD nanocarrier to overcome GEM’s pharmacokinetic limitations and improve its clinical performance in cancer therapy^24,29,45.

These physicochemical advantages translated into improved biological activity. The surface-modified GEM-AuNPs (Au-GEM-AsP-MOD) exhibited significantly greater cytotoxicity against 4T1 breast cancer cells, with an IC₅₀ of 0.44 µg/mL (Fig. 6B), demonstrating the potential of the AsP-based nanocarrier platform for developing long-acting injectable formulations with enhanced therapeutic efficacy. The improved cytotoxic performance of the Au-GEM-AsP-MOD formulation can be attributed to its sustained and controlled drug release, as shown in the in vitro release study, where it achieved 93.18% ± 1.68% release over 72 h. This slow and steady release allowed for prolonged drug availability and greater cellular uptake, resulting in a lower IC₅₀ compared to both free GEM (0.89 µg/mL) and the physically loaded formulation (0.51 µg/mL).

Additionally, the more negative surface charge of the surface-modified formulation (–18.3 mV versus − 15.9 mV for the physical system) likely enhanced cellular internalization through electrostatic interactions, contributing to more efficient intracellular drug delivery. The surface modification in the Au-GEM-AsP-MOD system may also have contributed to improved chemical stability of GEM, reducing premature degradation and supporting sustained efficacy. Collectively, these findings highlight the enhanced performance of the Au-GEM-AsP-MOD formulation in cancer cell targeting, supporting its potential for further preclinical evaluation.

Conclusion

This study demonstrated the multifunctional role of ascorbyl palmitate (AsP) in the synthesis and surface engineering of gold nanoparticles (AuNPs) for gemcitabine (GEM) delivery. AsP served as a reductant, stabilizer, and hydrophobic matrix component, contributing to improved nanoparticle stability and drug encapsulation. Among the formulations tested, the surface-modified Au-GEM-AsP-MOD system exhibited the most favorable physicochemical characteristics, including optimal particle size, enhanced colloidal stability, more negative surface charge, and effective surface modification. These features contributed to improved drug loading, controlled and sustained release, and reduced drug degradation during a six-month stability study. In vitro evaluation showed that the Au-GEM-AsP-MOD formulation achieved a lower IC₅₀ against 4T1 breast cancer cells compared to free GEM and physically loaded formulations, reflecting sustained drug release, improved chemical stability, and likely enhanced cellular uptake. The combined steric and electrostatic stabilization, together with the dual surface engineering strategy, enabled better control over GEM release and prolonged drug availability. Overall, these findings highlight the potential of AsP-based surface-modified AuNPs as a robust platform for developing long-acting, stable, and efficacious injectable chemotherapeutic nanocarriers, providing a foundation for future mechanistic and preclinical studies.

Author contributions

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.

Funding

This research did not receive any grant from funding agencies.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.