دانشگاه اراک سلول و بافت 2228-7035 17 1 2026 03 21 Targeted Delivery of Curcumin to Hs-578T Cells Using Multifunctional PLA-PEG-Fe3O4 Nanoparticles Modified with Folic Acid and Glucose انتقال هدفمند کورکومین به سلول‌های Hs-578T به کمک نانوذرات چندمنظوره PLA-PEG-Fe3O4 اصلاح‌شده با فولیک‌اسید و گلوکز 33 58 731557 10.66224/JCT.17.1.33 FA هاشم یعقوبی گروه زیست شناسی ،واحد اردبیل، دانشگاه آزاد اسلامی، اردبیل، ایران 0000-0003-3449-3037 Journal Article 2025 10 01 Introduction: Cancer is one of the most significant diseases of the present century and ranks as the second leading cause of death worldwide. Its incidence, particularly breast cancer in women, is steadily increasing. This disease arises from uncontrolled cell proliferation and the ability of cancer cells to migrate to healthy tissues, leading to tumor formation and metastasis. The genetic and biological heterogeneity of cancer cells, especially in breast cancer, complicates effective treatment. Conventional therapies such as surgery, chemotherapy, and radiotherapy, although effective in reducing tumor size, are associated with limitations, including damage to healthy cells, drug resistance, and systemic side effects. Therefore, the development of targeted and innovative therapeutic strategies is a priority in cancer research. One promising approach is the use of nanoparticles for targeted drug delivery. Due to their small size, high surface-to-volume ratio, and modifiable surfaces, nanoparticles can efficiently carry drugs and deliver them to target cells. Controlled drug release from nanoparticles reduces uptake by healthy cells and minimizes systemic toxicity. Iron oxide nanoparticles (Fe3O4) possess unique magnetic and chemical properties that enable precise guidance to tumor sites and allow real-time monitoring of drug distribution via MRI. Surface modification with biocompatible polymers such as polyethylene glycol (PEG) enhances nanoparticle stability, prolongs systemic circulation, reduces immune clearance, and provides sites for conjugating targeting ligands such as folic acid and glucose. Polylactic acid (PLA), a biodegradable and biocompatible polymer, increases drug-loading capacity and enables sustained and controlled drug release. Combining PLA with PEG and targeting ligands creates multifunctional nanoparticles that are stable, biocompatible, and capable of selectively recognizing cancer cells while minimizing side effects on healthy tissues. Targeting ligands such as folic acid and glucose facilitate selective cellular uptake; folic acid binds to overexpressed receptors on many cancer cells, promoting intracellular drug delivery, while glucose exploits the high metabolic demand of cancer cells, improving the delivery of therapeutic agents or genes. Aims: Dual-ligand nanoparticles enable multi-pathway targeting, enhancing therapeutic efficacy and potentially overcoming drug resistance. In this study, multifunctional PLA-PEG nanoparticles functionalized with folic acid and glucose and incorporating Fe3O4 were developed for targeted drug delivery to triple-negative breast cancer cells (Hs-578T). This system combines magnetic guidance, biocompatibility, controlled drug release, and selective targeting, providing a promising platform for effective and safe breast cancer treatment with reduced side effects compared to conventional therapies. Materials and Methods: Various chemicals and reagents, including MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)), Ethanol, HCl, NaCl, KH2PO4, Chloroform, NH2-PEG-FA, NH2-PEG-Glu, NaOH, and paraformaldehyde were procured from commercial suppliers, while FBS, DMEM, and trypsin-EDTA were used for in vitro studies with Hs-578T breast cancer cells. Multifunctional nanoparticles PPF (PLA-PEG-FA), PPG (PLA-PEG-Glu), and PPGF (PLA-PEG-Glu/FA) were synthesized via the reaction of PLA-acrylate with the respective NH2-PEG derivatives in chloroform under mechanical stirring and mild heating, followed by purification through dialysis and freeze-drying. Methanolic extract of Silybum marianum was prepared by ultrasonication of powdered aerial parts in 80% methanol, followed by centrifugation and filtration. Fe3O4 nanoparticles were synthesized via co-precipitation of Fe (III) and Fe (II) salts under nitrogen, followed by gradual addition of Silybum marianum plant extract and NaOH, leading to nanoparticle formation. The particles were collected magnetically, washed, freeze-dried, and stored in dark, dry conditions. Targeted delivery of Curcumin (CUR) was achieved using folic acid and glucose ligands. CUR and Fe3O4-OA were incorporated into PPF, PPG, or PPGF matrices using sonication and emulsification in PVA solutions, followed by rotary evaporation to remove chloroform, washing, and filtration to remove unencapsulated drug and large particles. Structural and chemical composition were analyzed using 1H-NMR and FTIR spectroscopy. Morphology was observed via TEM, and average size and zeta potential were measured using dynamic light scattering (DLS). Drug encapsulation efficiency was determined spectrophotometrically by measuring unencapsulated CUR in the supernatant. CUR release kinetics were evaluated in two pH environments: pH=7.4 (normal cells), and pH=4.5 (tumor-like), with periodic sampling and replacement with fresh medium over three days. Cytotoxicity was assessed via the MTT assay, with IC50 values determined after treating Hs-578T cells with varying concentrations of CUR, nanoparticles with CUR, or blank nanoparticles. Apoptosis was analyzed using Annexin V/propidium iodide staining and flow cytometry to distinguish apoptotic and necrotic cells. All experiments were performed in triplicate. Data were analyzed using one-way ANOVA followed by Duncan’s test at a 5% significance level, and normality was verified with the Kolmogorov–Smirnov test. Results are reported as mean ± standard deviation. Results: Targeted drug delivery has emerged as a promising strategy for treating difficult-to-treat diseases, including cancer, by directing therapeutic agents specifically to diseased tissues while minimizing uptake by normal cells. Biocompatible and biodegradable polymers are essential for efficient delivery systems. PLA, a widely used biodegradable polymer, allows loading and controlled release of hydrophobic drugs like curcumin. However, PLA alone can aggregate in serum due to hydrophobic interactions, leading to immune recognition and clearance. To overcome this, PLA was conjugated with PEG, which enhances nanoparticle circulation time and provides a stealth effect, reducing recognition by the immune system. PEG also improves drug loading and stability, although it is not biodegradable and may accumulate over repeated doses. Magnetic Fe3O4 nanoparticles were incorporated to increase targeting efficiency, while folic acid and glucose ligands were used for selective recognition of cancer cells. The synthesized PLA-based nanoparticles demonstrated good biocompatibility with Hs-578T cells, showing no significant cytotoxicity. Drug encapsulation within PLA-PEG nanoparticles allowed controlled and sustained release of curcumin, which was particularly enhanced under acidic conditions mimicking the tumor microenvironment. Notably, nanoparticles containing both folic acid and PEG showed higher drug release compared to those with PEG alone, highlighting the role of folic acid in facilitating curcumin release. This pH-sensitive release profile minimizes drug exposure to normal tissues, potentially reducing side effects. Cytotoxicity assay revealed that curcumin-loaded nanoparticles significantly inhibited Hs-578T cell proliferation compared to free curcumin, with the dual-ligand nanoparticles exhibiting the lowest IC50 values. Flow cytometry analysis demonstrated that these nanoparticles primarily induced apoptosis rather than necrosis, suggesting selective activation of programmed cell death pathways. The enhanced apoptotic effect is likely due to increased cellular uptake and targeted delivery, which facilitates higher intracellular concentrations of curcumin. Discussion: Overall, the results indicate that PLA-PEG-based nanoparticles, functionalized with magnetic Fe3O4 and surface ligands, provide an effective platform for targeted curcumin delivery. These nanoparticles combine biocompatibility, controlled release, tumor-specific accumulation, and apoptosis induction, which collectively enhance therapeutic efficacy while minimizing systemic toxicity. The study underscores the potential of multifunctional nanocarriers as a promising approach for improving the effectiveness of anticancer therapies and offers a foundation for the further development of targeted, stimuli-responsive drug delivery systems. Conclusion: As a final conclusion of this study, it can be stated that the development of novel drug delivery systems based on PLA-PEG copolymer nanoparticles is a promising and strategic step toward overcoming the therapeutic challenges of intractable diseases such as cancer. This study clearly demonstrated that the design of such nanocarriers not only offers high biocompatibility and safety for normal cells, but also, by taking advantage of the camouflage effect caused by PEG, increases blood circulation time and prevents premature elimination by the immune system. The outstanding feature of this system is the ability to control and intelligently release the drug in response to specific stimuli of the tumor microenvironment, especially higher acidity, which allows for maximum drug delivery to the target tissue and, subsequently, a significant reduction in systemic side effects. In this study, the effect of adding targeting ligands such as folic acid and glucose on the accuracy and efficiency of these nanoparticles in recognizing and binding to cancer cells was also clearly observed. In addition, the results of this study showed that the designed nanoparticles were successfully able to increase the induction of cell apoptosis in Hs-578T cancer cells. These findings strongly support the superiority of this nanotechnology compared to conventional drug formulations, both in increasing therapeutic efficacy and reducing toxicity. Overall, it can be concluded that PLA-PEG-based smart nanoparticles, with their multimodal capabilities, have great potential to become a comprehensive and reliable approach in the next generation of targeted cancer therapies, paving the way for further studies and ultimately effective clinical applications. مقدمه و هدف سرطان پستان سه‌گانه منفی با تهاجم بالا و مقاومت دارویی مواجه است. هدف این مطالعه طراحی و تولید نانوحامل هوشمند چندمنظوره مبتنی بر کوپلیمر PLA-PEG برای انتقال هدفمند کورکومین به سلول‌های سرطانی پستان (Hs-578T) و بهبود اختصاصیت و کارایی درمانی بود. مواد و روش‌ها نانوذرات PLA-PEG با لیگاندهای اسید فولیک (FA) و گلوکز (Glu) برای هدف‌گیری سلولی و با اکسید آهن (Fe3O4) برای هدایت مغناطیسی و ایجاد پتانسیل درمان ترکیبی سنتز شدند. ساختار نانوذرات PPF (PLA-PEG-FA-Fe3O4)، PPG (PLA-PEG-Glu-Fe3O4) و PPGF (PLA-PEG-Glu-FA-Fe3O4) با استفاده از 1H-NMR و FTIR تأیید شد. مورفولوژی و اندازه نانوذرات پس از بارگذاری کورکومین (CUR) با TEM، SEM و DLS بررسی گردید. سینتیک رهایش دارو در شرایط مختلف pH مورد ارزیابی قرار گرفت و سمیت سلولی با آزمون MTT و فلوسایتومتری سنجیده شد. نتایج نانوذرات کروی با اندازه ۱۵۰ الی ۲۰۰ نانومتر و بدون تجمع مشاهده شدند. رهایش کورکومین وابسته به pH بود و در محیط اسیدی (8/4pH=) که مشابه ریزمحیط تومور است، افزایش قابل توجهی داشت. نانوذرات بدون دارو سمیت قابل ملاحظه‌ای نداشتند، در حالی که فرمولاسیون‌های بارگذاری شده با کورکومین فعالیت ضدسرطانی معناداری نشان دادند. بیشترین اثر سیتوتوکسیک متعلق به نانوذرات دارای هر دو لیگاند و اکسید آهن (PPGFFe/CUR) با کمترین IC50 (µg/mL ۱۶۱/۵۶) بود. نتایج حاصل از فلوسایتومتری نشان داد که این نانوذرات عمدتاً آپوپتوز را القا کرده و از نکروز جلوگیری می‌کنند. نتیجه‌گیری نانوحامل طراحی‌شده، قابلیت رهایش pH-حساس، سمیت پایین نانوحامل و اثر ضدسرطانی مؤثر را نشان داد و پتانسیل بالایی برای درمان ترکیبی سرطان پستان سه‌گانه منفی دارد.

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