1. Introduction

Gold nanoparticles (AuNPs) have garnered extensive attention for their potential applications in cancer therapy. Notably, they exhibit surface plasmon resonance (SPR) phenomenon, characterized by strong absorption and scattering interactions with light at specific wavelengths, making AuNPs ideal for bioimaging and sensor applications. Furthermore, the small size of AuNPs allows them to penetrate biological membranes and interact directly with biomolecules, playing a crucial role in cancer diagnosis and treatment . However, the stability and targeting efficiency of these nanoparticles within biological systems pose significant challenges for their clinical applications. The objective of this study is to construct a novel form of gold nanoparticles through surface modification techniques, thereby improving their biocompatibility and targeting ability, and enhancing their effectiveness and safety in cancer treatment.

The crux of this research involves exploring a new formulation of AuNPs, simultaneously modified with polyethylene glycol (PEG) and lipidized peptides (pepducins) on their surface to enhance stability and targeting toward cancer cells . PEGylation is a widely used strategy to improve nanoparticle stability and biocompatibility; whereas the incorporation of pepducins, a novel type of lipidized peptide, aims to enhance the cellular uptake of AuNPs and their potential for targeting cancer cells. This dual modification strategy promises to yield AuNPs with high stability and superior targeting capabilities, offering a new approach for cancer therapy.

The study delves into the application of nanotechnology in cancer treatment and the progress in AuNPs surface modification techniques. It then outlines the materials used, the synthesis and functionalization process of AuNPs, experimental methods, and statistical analysis. Subsequently, the physicochemical properties, stability, and internalization of the novel AuNPs in cancer cells are thoroughly analyzed. The study concludes with a summary, discussing its limitations and future research directions.

2. Literature Review

2.1. Surface Modification of Gold Nanoparticles

Gold nanoparticles (AuNPs) have made progress in medical research and applications due to their unique localized surface plasmon resonance (LSPR) effect and photothermal effect. However, ultrasmall luminescent gold nanoparticles lack corresponding surface chemical properties, and upon entering the bloodstream, they are typically enveloped by serum proteins in the blood, forming a protein corona. This leads to phagocytosis by the reticuloendothelial system (RES), severely affecting the nanoparticles’ targeted entry into tumor tissues. Nanoparticles engulfed in this manner tend to accumulate in the body, causing inflammation and other side effects at the accumulation sites, thus reducing their efficiency in passively targeting tumors. Therefore, relying solely on the enhanced permeability and retention (EPR) effect for passive targeting strategies lacks clinical applicability, necessitating the adoption of surface modification strategies. For instance, modifying AuNPs with polymers to prolong their circulation half-life enhances their stability in application environments . Researchers like Zoë Rachael Goddard have modified AuNPs with biocompatible molecules such as DNA, peptides, and antibodies to actively target gold nanoparticles, thereby improving their accumulation rate and photothermal therapy effects in tumor cells . Based on the interaction between polymer molecules and gold nanoparticles, Luo et al. used thiol-terminated amphiphilic block copolymers poly(2-(dimethylamino)ethyl methacrylate) (PDMA) and poly(ethylene oxide) (PEO) to synthesize hybrid core/shell gold nanoparticles, finding that crosslinking the PDMA inner shell connected to the gold core enhances colloidal stability . Professor Reznickova discovered that modifying AuNPs with thiol-ended polyethylene glycol (PEG-SH) imparts good biocompatibility to AuNPs and enables their application in biomedical detection both in vivo and in vitro .

2.2. Targeted Modification of Gold Nanoparticles

Surface PEGylation of nanocarriers is the primary method for enhancing carrier stability and prolonging blood circulation. However, it reduces the interaction between nanoparticles and the plasma membrane, impeding cellular internalization and endosomal escape. Additionally, chemotherapeutic and genetic drugs need to be transported into cells to exert their therapeutic effects fully. Active targeting, based on the Enhanced Permeability and Retention (EPR) effect of nanoparticles, involves attaching targeting ligands to the nanocarrier surface, allowing them to bind to specific receptors at tumor cell target sites. This targeting enables tumor cells to endocytose the polymeric nanocarriers more “selectively” at the tumor site. Torchilin and colleagues have linked ligands or transmembrane peptides to the ends of long-chain PEG or short-chain PEG, respectively. They found that in tumor microenvironments with high MMP enzyme expression, the long-chain PEG on the carrier’s surface responsively detaches, exposing the short-chain PEG with ligands or transmembrane peptides, thereby promoting cellular drug uptake . Among various targeting ligands, cell-penetrating peptides (CPPs) are the most commonly used. CPPs are small peptide molecules that can directly cross cell membranes and (or) nuclear membranes in a non-receptor dependent, non-classical endocytosis manner. Typically no longer than 30 amino acids, CPPs are rich in basic amino acids, guiding gold nanoparticles to rapidly penetrate negatively charged cell membranes. Dozens of CPPs have been discovered and synthesized, broadly categorized into three types: those derived from homologous structural domains, such as Pepducin; those from naturally occurring proteins in viruses or microbes, like TAT; and various artificially synthesized short peptides like polyarginine, MAP, Transportan, and signal sequence-based peptides like MTS . Covic and colleagues first reported the pepducin technology in their research. Pepducin involves attaching a lipid part (such as palmitoyl, myristoyl, or cholic acid) to a peptide targeting the cytoplasmic loops (C1, C2, or C3) or C-terminal tail (C4) of G-protein-coupled receptors (GPCRs). Pepducin can act as an agonist, antagonist, or activity modulator of GPCRs .

3. Research Method

3.1. Preparation of AuNPs-citrate

This section primarily describes the preparation of 15 nm gold nanoparticles modified with sodium citrate. Before starting the experiment, all glassware must be thoroughly cleaned with aqua regia, followed by rinsing with ultrapure water and drying overnight in an oven at 80℃. Initially, 400 ml of gold salt solution (0.01 w/v concentration) is prepared and transferred into a round-bottom flask. The flask is then placed in a heating mantle and heated under reflux condensation, with the entire process conducted in a fume hood for safety. The solution in the flask is stirred during heating. Upon boiling, 9 ml of a pre-prepared 1% w/v sodium citrate solution is added. As the reaction progresses, the solution color changes from transparent to deep blue/black, and finally to wine red. The reflux boiling is continued for 60 minutes. Afterwards, heating is stopped, and the solution is allowed to cool down to room temperature overnight. The next day, the prepared citrate-wrapped gold nanoparticles (AuNP-citrate) are bottled, labeled, and stored at 4°C for future use.

3.2. Preparation of AuNP-PEG with Different Coverage Rates

Different concentrations of PEG-SH solutions (2.14 µg/ml, 3.20 µg/ml, 4.27 µg/ml, and 5.34 µg/ml) are added to tubes containing AuNP-citrate and left to stand at room temperature for 30 minutes. Subsequently, these polyethylene glycol-modified (PEGylated) gold nanoparticle (Au) samples are centrifuged at 4℃ for 30 minutes at a relative centrifugal force (rcf) of 10,000. The supernatant with 90% unbound PEG is gently removed, and an equal volume of ultrapure distilled water is added to the centrifuged volume. This process yields AuNP-PEG formulations with different surface coverage rates, with PEG coverages of 10%, 15%, 20%, and 25%, respectively.

3.3. Preparation of the Novel Nanogold Particle AuX2R

Ensuring 100% surface coverage, 6.25 µg/ml of pepducin is added to the AuNP-PEG solution and left to stand at room temperature for half an hour. The samples are then centrifuged at 4℃ for 30 minutes at a relative centrifugal force of 10,000. After centrifugation, the samples are removed, and 90% of the supernatant is gently aspirated. The sample is then replaced with an equal volume of ultrapure distilled water to prepare AuX2R for experimental use.

3.4. Characterization and Stability Experiments of Gold Nanoparticles

Dynamic Light Scattering (DLS) technology was employed to characterize all samples using a Zetasizer instrument, determining their hydrodynamic particle size and Polydispersity Index (PDI). A volume of 50µl of the sample was placed in a cuvette, with each sample undergoing three readings at a measurement temperature of 25℃. Subsequently, long-term aqueous phase stability tests were conducted. Two batches of gold nanoparticle formulations were prepared and stored under room temperature and refrigeration at 4℃. Using DLS, the Z-average and PDI of the samples were measured immediately post-synthesis and then weekly for four weeks to assess their stability under the respective storage conditions.

The stability of the gold nanoparticles in saline solution and serum was tested. The synthesized gold nanoparticles were exposed to 0.15M NaCl solution and incubated in a 37°C constant temperature incubator for 24 and 48 hours before removal. Following the aforementioned centrifugation conditions, 90% of the supernatant was removed and replaced with ultrapure water. The Z-average and PDI of the samples were analyzed using DLS. The gold nanoparticle formulations were also exposed to serum containing 10% Fetal Bovine Serum (FBS) and 10% Bovine Calf Serum (BCS) and incubated at 37℃. Samples were taken out for testing after 24 and 48 hours. Samples were processed according to the described centrifugation procedure, with 90% of the supernatant removed and replaced with ultrapure water. After repeating this operation twice, the Z-average and PDI of the samples were measured again using DLS..

3.5. Cellular Internalization Experiments of Gold Nanoparticles

Approximately 40,000 cells were cultured on each well of a four-well slide. After 48 hours of cultivation, the cells were treated for 24 hours with solutions containing AuNP-citrate, AuNP-PEG, and AuX2R (concentration 100µg/ml). Cells were then washed thrice with PBS, fixed for 20 minutes with 4% formaldehyde, washed again with PBS, and encapsulated with a DAPI-containing fluorescent agent. Dark-field and hyperspectral imaging were performed using a Cytoviva microscope. Additionally, a six-well plate was prepared, with each well containing 80,000 cells, and treated in the same manner as the four-well plate. After 24 hours of treatment, the cells were washed with PBS, dissolved in aqua regia, and the gold content was determined using Atomic Absorption Spectroscopy (AAS).

4. Results and Disscussions

4.1. Stability of AuNPs Modified with Varying Concentrations of PEG

Experimental results indicate that at 25% PEG coverage, the dispersion of AuNPs did not differ significantly from the control group, suggesting that a higher proportion of PEG coverage can effectively maintain the stable dispersion of nanoparticles. However, when the PEG coverage was reduced to 10%, the Polydispersity Index (PDI) of AuNP-15% PEG exhibited a significant difference compared to the control group (P<0.05), with a 1.80-fold increase in PDI. This data implies that lower PEG coverage might lead to decreased particle stability.

Figure 1. PDI of AuNP-PEG at different ratios in physiological saline solution.

The tests on AuNP-PEG-pepducin (AuX2R) complexes at different PEG:pepducin ratios (25:75 and 15:85) demonstrated that both formulations exhibited good stability in saline solution after 24 hours, with no significant changes. This indicates that the introduction of pepducin does not negatively affect the stability of AuNPs.

Figure 2. PDI of AuX2R at different ratios in physiological saline solution.

4.2. Long-term Stability of AuNPs with Different Surface Modifications in Aqueous Solution

Experiments at room temperature and refrigerated conditions demonstrated that AuX2R exhibits excellent long-term stability in aqueous solution. Despite similar trends observed with AuNP-citrate and AuNP-PEG at room temperature storage, a statistically significant change in the Z-average value of AuX2R was noted in comparison to controls at week four (p<0.05), indicating superior long-term stability.

4.3. Stability of AuNPs with Different Surface Modifications in Saline Solution

After PEGylation of AuNP-citrate, AuNP-25% PEG demonstrated enhanced stability over 24 and 48 hours in saline solution compared to AuNP-citrate. Specifically, the Z-average diameter of unmodified AuNP-citrate increased nearly 62.66-fold after 24 hours in saline solution, whereas PEG-modified formulations did not show such a significant size increase.

4.4. Stability of AuNPs with Different Surface Modifications under Physiological Serum Conditions

AuNP-citrate was found to be highly unstable in saline solution, with particle size increasing almost 60-fold within 24 hours. However, after the addition of 10% FBS, the average particle size increased by 50% after 24 and 48 hours, from 20.52 nm to 44.56 nm and 44.03 nm, respectively, indicating poor stability of unmodified AuNP-citrate in simulated biological environments. AuNP-PEG and AuX2R demonstrated better stability. PEGylation significantly improved nanoparticle stability in serum. The molecular weight of PEG used was 5000, which enhanced nanoparticle stability while reducing serum protein adsorption and thus, protein corona formation. In experiments with AuNP-PEG and AuX2R, no significant difference in Z-average diameter was observed compared to controls after adding 10% FBS and 10% BCS.

4.5. Internalization of AuX2R in Prostate Cancer Cells

Results indicated a significant increase in intracellular concentration of AuX2R compared to AuNP-PEG. This data revealed the significant effect of AuX2R in enhancing AuNPs internalization, with concentrations nearly 250 times that of AuNP-PEG. Further analysis showed that the concentration of AuNPs in cancer cells treated with AuX2R was about 300 times higher than that with AuNP-PEG, proving that the addition of pepducin in AuX2R not only did not reduce the stability of AuNPs but also enhanced their targeting efficacy in prostate cancer cells.

Figure 11. Distribution of Different Formulations in Prostate Cancer Cells

Figure 12. Internalization of AuNPs in Prostate Cancer Cells In Vitro

5. Conclusions

This study primarily focuses on the development of a novel gold nanoparticle formulation, AuX2R, characterized by the conjugation of polyethylene glycol (PEG) and lipidized peptide (pepducin) on the surface of AuNPs. The innovative dual-surface modification aims to enhance the stability of nanoparticles while improving their targeting towards cancer cells. In this research, a detailed stability comparison was conducted for three different formulations of AuNPs (AuNP-citrate, AuNP-PEG, and AuX2R) in aqueous solution, physiological saline, and serum. The results showed that the addition of PEG significantly improved the stability of the nanoparticles, while the inclusion of pepducin did not have a negative impact on their stability.

Furthermore, this study explored the internalization of the nanoparticle formulations in prostate cancer cells using microscopy and atomic absorption spectroscopy (AAS). Experimental results indicated that compared to the other two formulations, AuX2R exhibited higher internalization in cancer cells, demonstrating stronger targeting and intracellular delivery efficiency.

Nevertheless, this study has certain limitations. Since all experiments were conducted in vitro, without in vivo applications, further validation is required to determine the specific behavior, safety, and efficacy of AuX2R within biological organisms. Additionally, research on the long-term toxicity and side effects of AuX2R in biological systems remains insufficient. Furthermore, the specificity of AuX2R’s targeting toward different cancer cell types and its ability to distinguish between healthy and cancerous cells also require further investigation. Lastly, challenges related to the large-scale production and quality control of AuX2R are important aspects for future research.

In summary, this study successfully synthesized a novel nanoparticle formulation, AuX2R, with dual functionality, demonstrating excellent stability and targeting capabilities. Future research efforts will focus on in vivo experiments, assessing long-term toxicity, improving targeting specificity, and exploring the potential for clinical applications, with the aim of developing AuX2R into an effective tool for cancer therapy.