{"paper_id":"1a9b74b6-ac11-4880-8387-cc0f1a160d17","body_text":"Folate receptor-targeted Camptothecin-loaded PLGA-Glutenin nanoparticles for effective breast cancer treatment | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Folate receptor-targeted Camptothecin-loaded PLGA-Glutenin nanoparticles for effective breast cancer treatment Raja Rajeswari Rajeshkumar, Theivendren Panneerselvam, Parasuraman Pavadai, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4513460/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Sep, 2024 Read the published version in Journal of Polymers and the Environment → Version 1 posted 10 You are reading this latest preprint version Abstract The combination of natural and synthetic polymers for nanomedicine development had many advantages, including less toxicity, biocompatibility, prolonged circulation, higher stability, and ease of surface modification. Here, a novel folic acid-conjugated Camptothecin-loaded-poly (lactic-co-glycolic) acid-glutenin nanoparticles (FA-CPT-PLGA-Glu NPs) was fabricated to treat breast cancer. FA-CPT-PLGA-Glu NPs target breast cancer cells via upregulated folate receptors and delivered their toxic payloads without disrupting healthy cells. First, CPT-loaded PLGA NPs were created using a modified emulsification/evaporation technique. Second, Glu-based CPT-PLGA NPs were synthesized using a layer-by-layer assembly, and their physiochemical properties were validated. CPT encapsulation efficiency and loading capacity into PLGA-Glu NPs were 74.95 ± 1.34% and 4.78 ± 1.08%, respectively. CPT-PLGA-Glu NPs exhibited sustained and controlled release of loaded-CPT from NPs, and the highest content was released in an acidic environment (pH 5.3), which will be advantageous for cancer treatment. Later, FA-CPT-PLGA-Glu NPs were synthesized by simple conjugation chemistry. The fabricated FA-CPT-PLGA-Glu NPs were around 100 nm in size, with a spherical form and crystalline nature. FA-CPT-PLGA-Glu NPs show strong cytotoxicity activity, and its IC 50 value was 16.33 µg × mL − 1 against breast cancer cell line (MCF-7). This folate-receptor-targeted NPs are more effectively internalized into MCF-7 cells, causing ROS generation, cell growth inhibition, and apoptosis. The activity of caspase-3 and − 9 causes MCF-7 cells apoptosis by internalized CPT. Further, internalized CPT induces potential loss of mitochondrial transmembrane and damages the nuclear integrity of the cancer cells. These results showed that the FA-CPT-PLGA-Glu NPs target upregulated folate receptors on the surface of MCF-7 cells. Camptothecin MCF-7 cells folate-receptor apoptosis cell death Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cancer is one of the deadliest non-communicable diseases (NCDs), which accounts for 9% of total deaths (63%) in India [ 1 ]. Particularly in India, breast and cervical cancers are majorly afflicted in women populations [ 2 ]. According to GLOBOCAN 2020, in India, breast cancer recorded 13.5% (178361) of all cancer cases and 10.6% (90408) of all deaths, with a cumulative risk of 2.81 million [ 3 ]. Women populations are the most vulnerable victims of breast cancer, but rare cases can also occur in men; in 2016, about 98.1% of 118000 total women breast cancer cases in India [ 4 ]. The reason for the occurrence of breast cancer might be sedentary lifestyle behaviors like being physically inactive, improperly-maintained body mass index (BMI), excessive alcohol consumption with cigarette smoking, consuming junk foods with high fats, and medicines such as exogenous female hormones (menopausal hormone therapy, and hormonal contraceptives) [ 5 , 6 ]. At the same time, mutations in the BRCA1 or BRCA2 gene, checkpoint kinase-2 (CHEK-2), and breast cancer-connected single-nucleotide polymorphisms (SNPs) also caused breast cancer [ 7 ]. Present today, surgical removal, radiation & laser therapy, hormonal therapy, chemotherapy, combination therapy, and personalized medicine are the major treatment strategies for breast cancer [ 8 ]. Among the various treatment strategies, chemotherapy was one of the most promising and effective treatment options for breast cancer [ 9 ]. Nowadays multiple classes of chemotherapeutic agents are used for the treatment of breast cancer, such as anti-metabolites (methotrexate) [ 10 ], alkylating agents (Cyclophosphamide) [ 11 ], anthracyclines (doxorubicin) [ 12 ], hormones (tamoxifen) [ 13 ], monoclonal antibodies (trastuzumab, pertuzumab) [ 14 ], mitotic inhibitors (paclitaxel, docetaxel) [ 15 ], topoisomerase inhibitors (etoposide, irinotecan) [ 16 ], and tubulin inhibitors (vinblastine) [ 17 ]. Cyclophosphamide (alkylating agent) and doxorubicin (anthracyclines) for four cycles, followed by paclitaxel (taxanes) for four cycles, were the common protocol for adjuvant chemotherapeutic treatment[ 18 ]. The mode of action of these compounds, such as cyclophosphamide, causes breakage of DNA [ 19 ], doxorubicin acts in DNA intercalation, inhibiting the macromolecular synthesis [ 20 ], and paclitaxel induces cell cycle arrest and apoptosis by binding to microtubules preventing their disassembly [ 21 ]. Anthracyclines and taxanes are primarily used to treat breast cancer. However, increased usage of these agents at an early phase of breast cancer frequently produces tumor drug-resistant against these treatments by the time the disease reoccurs, thus lowering the number of treatment choices for metastatic cancer [ 22 ]. Moreover, even when these drugs are used in metastatic cancer, treatment failure occurs usually, resulting in reduced 5-year survival rates for patients with metastatic breast cancer[ 23 ]. Drug resistance is a major issue for using chemotherapies during cancer treatment stages. Also, numerous side effects such as cytopenia, alopecia, fatigue, neurocognitive dysfunction, chemotherapy-induced peripheral neuropathy, and muscle pain occurred in the treatment of breast cancer [ 24 ]. After six months of treatment, patients experience chronic effects such as sterility, early menopause, psychological impacts, second cancer, and cardiomyopathy [ 25 ]. Naturally derived compounds are less toxic and have fewer side effects than synthetic chemotherapies [ 26 ]. Numerous natural compounds, such as resveratrol, vincristine, vinblastine, curcumin, capsaicin, piperine, etc., are already established for breast cancer treatment therapy[ 27 , 28 ]. Camptothecin (CPT) is a hydrophobic bioactive molecule, a plant-derived alkaloid extracted from the stem wood of Camptotheca acuminata [ 29 ]. The antineoplastic activity of the CPT makes it an efficient antioxidant and anti-cancer property for various types of cancers [ 30 ]. The mode of action of CPT was to induce apoptosis by inhibiting the enzyme topoisomerase-1 involved in the S-phase of the cell cycle [ 31 ]. Apart from the various advantages of this molecule, some shortcomings are there such as low solubility, rapid metabolism, lower stability, non-selectivity, low bioavailability, and biocompatibility are the major concerns of using this drug [ 32 ]. The drawbacks can be overcome by devising a novel nano-engineered carrier system to deliver loaded drugs to the disease sites. Hydrophobic drugs delivered through nanoparticulate systems have become a major backbone of cancer drug research. Among the most prevalent nanoparticulate systems, polymeric nanoparticles have several key advantages that provide a wide range of delivery platforms. However, polymeric nanoparticles have certain limitations, including using toxic solvents during manufacturing, polymer breakdown, drug leakage outside the sick tissue, and polymer cytotoxicity. Combining naturally obtained protein polymers with synthetic polymer is a hybrid nanocarrier for drug delivery applications, an upcoming field. It is much more efficient than a single nanocarrier system [ 33 ]. Synthetic polymeric nanocarriers are toxic and sometimes cause tissue damage; polymer-protein hybrid nanoparticles are considerably less harmful than individual nanocarriers [ 34 ]. The key factor in nanoparticle development is their drug-loading capacity, typically less than 2% for most approved drugs. Polymeric nanoparticles have a loading capacity lower than 5% [ 35 ]. Recent research reported combining PLGA-chitosan nanoparticles shows higher encapsulation efficiency and lower drug-loading capacity [ 36 ]. PLGA is an FDA-approved poly (lactic-co-glycolic) acid copolymer for drug encapsulation [ 37 ]. It is biodegradable, biocompatible, non-toxic, durable, easy to surface modification, stable, and ideal for controllable drug delivery. However, it shows higher cytotoxicity [ 38 ]. Thus, PLGA combined with protein reduces its toxicity. Protein is a natural polymer isolated from plants and animal sources [ 39 ]. Plant protein is more significant in drug delivery than animal protein since it is safe, economically cheap, easy to scale up, and renewable [ 40 ]. It is also biodegradable, biocompatible, non-antigenic, and reported as GRAS (Generally Recognized as Safe) [ 41 ]. The possibility of disease occurrence was minimal in plant-based protein unless animal protein causes diseases like bovine spongiform encephalitis (mad cow disease) [ 42 ]. Plant-based protein is hydrophobic; thus, there is no need for crosslinkers. It is highly stable, and it releases the drug in a controlled manner [ 43 ]. There are numerous plant proteins, such as collagen, Albumin, and cellulose, but these plant-based proteins lack stability in aqueous conditions [ 44 ]. Glutenin is a wheat protein that is highly stable in aqueous conditions and various physiological conditions [ 45 ]. PLGA-Glutenin (PLGA-Glu) hybrid nanoparticles have not yet been reported previously. It is highly anionic/cationic, enabling it to load various drugs effectively. PLGA-Glu nanocarrier system improves the pharmacodynamic and pharmacokinetic properties of the encapsulated drug [ 46 ]. Targeted delivery of drugs into the disease site/cancer cells is crucial in drug delivery carrier systems [ 47 ]. Cancer cells often upregulate many receptors on their surface for rapid proliferation and survival. These receptors are recognized as effective targets for drug intervention. A technique involves delivering toxic payloads to destroy cancer cells that express high quantities of certain receptors; one such receptor is the folate receptor (FR) [ 48 ]. FR is a membrane-bound glycoprotein that reduces folates (such as 5-methyltetrahydrofolate folic acid) and helps in the proliferation of cancer cells [ 49 ]. FR is upregulated in various types of cancers, including breast, ovarian, endometrial, mesothelioma, and lung [ 50 ]. It was upregulated 35–68% in triple-negative breast cancer and 20% in tissue samples of non-malignant breast cancer [ 49 ]. This is the main reason for considering FR as a valid anti-cancer drug target. Its expression is limited to non-malignant tissue and thus does not affect normal healthy cells [ 51 ]. Conjugating folic acid on the surface of the Glu-PLGA carrier to selectively deliver Camptothecin into cancer cells by entering the cell through FR by endocytosis [ 52 ]. In this work, we proposed a novel drug delivery system called the folic acid- conjugated Camptothecin-loaded glutenin-PLGA nanoparticles (FA-CPT-PLGA-Glu-NPs). This delivery system is capable of efficiently delivering Camptothecin to cancer cells. Furthermore, FA-CPT-PLGA-Glu was characterized by XRD, FTIR, particle size, and HRTEM. The cytotoxic effect of FA-CPT-PLGA-Glu nanoparticle was analyzed against MCF-7. Experimental section Chemicals Camptothecin and copolymer, poly(lactic-co-glycolic) acid were obtained from Sigma Aldrich, Bangaluru., India. MES (2-(N-morpholino) ethanesulfonic acid) buffer, Polyvinyl alcohol (PVA), and Dichloromethane (DCM) were received from HiMedia Laboratories, Pvt. Ltd., Mumbai, India. Folic acid (97.95%) was obtained as a gift from Alkem Laboratories, Mumbai, Maharashtra, India. All other remaining chemicals, reagents, and solvents were procured from Thermo Fisher Scientific Ltd., Mumbai, India. Cancer cell line MCF-7 cells (human adenoma breast cancer cells) were obtained from NCCS, Pune, Maharashtra, India, and maintained in RPMI1640 medium (pH 7.2–7.4) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% mixture of penicillin/streptomycin, & amphotericin B and incubated at 37°C in a humidified atmosphere with 5% CO 2 . Glutenin extraction As reported in our previous work, glutenin was extracted from wheat flour. The FTIR and 13 C-NMR spectrums were used to confirm glutenin before it was used in the nanoparticle formulation[ 53 ]. Formulation of PLGA-Camptothecin-loaded Nanoparticles (PLGA-CPT NPs) A slightly changed emulsification/evaporation method was applied to formulate camptothecin-loaded PLGA nanoparticles. Briefly, PVA (0.05g, w/w) was dissolved in deionized water to create an aqueous phase, which was then adjusted to a pH of 5.0 using MES buffer (50 mM). In the organic phase, 0.025 g of PLGA was mixed in 3 mL of dichloromethane and 3 mg of CPT in 2 mL of ethanol. The resulting combination (organic phase) was slowly added to the aqueous phase while stirring continuously with a magnetic stirrer. During the stirring process, the entire system was kept in an ice bath to prevent the oil phase from evaporating, and the mixture was magnetically swirled overnight. Nanoparticles were purified by centrifugation three times with a high-speed centrifuge. Layer-by-layer assembly of Glu-PLGA NPs loaded with Camptothecin Glutenin-composed CPT-loaded PLGA NPs were fabricated using a layer-by-layer assembly technique[ 54 ]. The freshly prepared PLGA nanoparticles (1 mg × mL -1 ) were distributed in the glutenin solution (2 mg diluted in 5 mL of acetic acid) by rotating and oscillating until the necessary layers were created. Next, a 0.5 mL of a 2% aqueous glutaraldehyde solution was introduced to facilitate cross-linking of the layers. Following centrifugation, a 30 mM sodium borohydride solution was employed to stop the cross-linking reaction after 30 min. Subsequently, the sample was rinsed three times with deionized water. The glutenin-layered particles were incubated with organic solvents, specifically tetrahydrofuran (THF), to remove the cores. The use of sonication facilitated this process. Determination of encapsulation efficiency of CPT-loaded- Glu-PLGA NPs A UV-visible spectrophotometer measured the drug encapsulation efficiency of the CPT-loaded into Glu-PLGA NPs at 225 nm. CPT-loaded-Glu-PLGA NPs were centrifuged at 5000 rpm, and the supernatant was analyzed by UV-visible spectrophotometer at 225 nm. The quantity of CPT was determined by plotting a graph with a standard graph of CPT. Further, loading capacity (%W/W) and encapsulation efficiency were determined using the below equations (1) and (2), respectively. Loading capacity (%) = [(CPT)added−(CPT)measured/Nanoparticle weight] × 100 Encapsulation efficiency (%) = [(CPT)added−(CPT)measured/(CPT)added] × 100 In vitro drug release study The release of CPT from CPT-PLGA-Glu NPs was measured using the dynamic membrane dialysis method in acetate buffer (pH 5.3, 0.01M) and phosphate buffer (pH 7.2, 0.01M) solutions. Drug release research was done to ascertain the solubility of the drug in buffer solutions, which served as the external medium. In summary, CPT-Glu-PLGA NPs (30 mg × mL -1 ) were enclosed in a dialysis membrane bag with a molecular weight cut-off of 3500 Da. The bag was then immersed in a buffer solution and placed on a shaker at a temperature of 37 ± 1.00°C and a speed of 150 rpm. At specific time intervals, samples were taken out and substituted with new media. The supernatant obtained from the collection was mixed with ethanol, and the quantity of the medicine (CPT) released from the NPs was measured at 225 nm using UV-visible spectrophotometry at certain time intervals. The formula below was employed to compute the percentage of medication release. % Drug release = (A 0 -A 1 ) × 100/A 0 where A 0 = absorbance of the control, and A 1 = absorbance of the sample Drug release kinetics The drug release kinetics and diffusion mechanisms of CPT-Glu-PLGA NPs were studied using five basic kinetic models: zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell models[ 55 ]. The Drug Distribution (DD Solver) 1.0 software, a Microsoft Excel plug-in tool, is utilized to assemble and analyze in vitro drug release data to determine the mechanism of drug release kinetics. The model with the lowest AIC (Akaike information criterion) value is the most suitable for predicting drug release. The exponent 'n' significantly influences the drug release kinetics. For values of 'n' less than 0.45, the release follows a Quasi-fickian behavior. When 'n' equals 0.45, it exhibits Fickian diffusion. When 'n' falls between 0.45 and 0.89, the diffusion is classified as non-fickian. For values of 'n' greater than 1, it is considered. Fabrication of folic acid-conjugated CPT-loaded-Glu-PLGA NPs (FA-CPT-PLGA-Glu NPs) Synthesis of N-hydroxysuccinimide ester of FA (FA-NHS) This procedure is performed in the dark to prevent the structural deprivation of folic acid[ 56 ]. Shortly, 1.05 g of FA was solubilized in 25 mL of DMSO. At the same time, 0.44 g of N-hydroxy succinimide, 1.05 g of N, N- dicyclohexylcarbodiimide, and 0.25 mL of triethyl amine were added. This mixture was kept in a water bath at 40°C for 48 h. After 48 h, it was filtered to remove the by-product of dicyclohexylurea. The synthesized FA-NHS was freeze-dried, which was confirmed by NMR and FTIR. FA-CPT-PLGA-Glu NPs The FA-CPT- PLGA-Glu NPs were fabricated by conjugating the carboxylic acid group of FA into an amino group of glutenin in PLGA-Glu-loaded CPT NPs (Fig. 1 ). Concisely, 5 mg of CPT- PLGA-Glu NPs dissolved in carbonate/bicarbonate buffer (pH 10.0; 3 mL; 0.1 M). DMSO solubilized FA-NHS (1 mg × mL -1 ) was added to the CPT-PLGA-Glu NPs solution and kept in a magnetic stirrer for 2–3 h. DMSO was removed by membrane dialysis against a phosphate buffer solution of pH 7.4 to remove unreacted FA-NHS. The FA-CPT-PLGA-Glu NPs were centrifuged for 30 min at 21000 rpm and then re-dispersed in deionized water (4.0 mL). Characterization studies of FA-CPT-PLGA-Glu NPs The X-ray diffractometer was used to evaluate the physical characteristics of the FA-CPT- PLGA-Glu NPs. The morphological features of the FA-CPT- PLGA-Glu NPs were assessed by high resolution transmission electron microscopy (HRTEM). Average particle size was measured by dynamic light scattering (DLS) analysis. Stability study of FA-CPT-PLGA-Glu NPs A stability study was performed under various physiological mediums, such as 5% NaCl, 0.5% BSA, acetate buffer (pH 5.3), and phosphate buffer (pH 7.0 and 9.0). 1 mL of FA-CPT-PLGA-Glu NPs was added to 9 mL of each physiological medium, and it was monitored for consecutive days. Then, the absorbance was measured in UV-visible spectroscopy between 200 to 600 nm. In silico molecular modelling Protein Preparation: The Research Collaboratory for Structural Bioinformatics manages the PDB database, which lists Caspase-3 as 3DEH at https://www.rcsb.org/ . X-ray crystallography is used to determine protein crystal structures. The protein is pre-processed using CHARMM-GUI to remove water molecules, ligands, and missing residues[ 57 ]. Ligand Preparation: The Camptothecin, Doxorubicin, and 3DEH cocrystal were docked with the Protein Data Bank Caspase-3 protein (3DEH). The free PubChem database ( https://pubchem.ncbi.nlm.nih.gov/ ) provided the ‘.sdp’ files. The chemicals and created cocrystals were processed using BIOVIA Discovery Studio Visualizer to prepare the cluster for analysis [ 1 – 5 ]. Docking Protocol: Intermediate steps AutoDock Vina-POAP for Virtual Screening converted proteins and ligands into ‘pdbqt’ files. A setup document includes protein, ligand, and grid box configuration metrics. The AutoDock Vina-POAP virtually screened the protein, revealing polar hydrogens, solvation parameters, and fragmental volumes. AutoGrid designed the grid map's grid box, with the grid centre at x (-46.8), y (15.02), and z (21.9). A scoring grid based on the ligand's structure was created to speed up computation. If proteins and ligands are stiff, AutoDock Vina docks them via iterated local search global optimization. The results with the lowest binding free energy were analyzed, while those with a positional root mean square deviation above 1.0A were discarded. Through the BIOVIA Discovery Studio Visualizer, the amino acid interaction was evaluated from the highest binding affinity or lowest binding site[ 58 ]. Identification of the binding site: Proteins need a unique binding site to hook onto molecules and a primary mechanism to ensure bioactive chemicals have adequate sites for chemical reactions. These proteins and enzymes need precise ligand or bioactive engagement sites to interact with the desired enzymes. PrankWeb ( https://prankweb.cz/ ) has been used to find every active target material binding site. The receptor grid was built from the protein's active site using AutoDock Vina-POAP for virtual screening[ 59 ]. Molecular dynamics We used molecular dynamics (MD) simulations to assess protein-ligand stability and variability. The major goal was to study how therapeutic drugs bind to target proteins. These simulations used Schrodinger Desmond module and the Maestro simulation platform on Linux. The TIP3 water model and cuboidal border constraint modified complex protein-ligand interactions. The system was neutralized with 0.15 M Na + and Cl - salts. MD simulations used the NPT ensemble to maintain 1.01325 bar pressure and 300 K temperature. OPLS-4 force field energy assessments were performed during simulations with 50 picosecond data gathering. One macro-level simulation lasted 100 nanoseconds. Schrödinger's Desmond module helped analyze simulation data. The study was improved using a simulated interaction diagram method to evaluate the ligand-protein complex's stability using RMSD, RMSF, and protein-ligand interaction analysis across simulated paths [ 60 ]. In vitro cytotoxicity assay The cytotoxic ability of FA-CPT-PLGA-Glu NPs was estimated against breast cancer cells (MCF-7) via colour change of yellow MTT (3,4,5-dimethyl thiazolyl-2-2-5-diaphenyl tetrazolium bromide) salt into purple formazan crystals. The change due to mitochondrial enzymes in metabolically active cells will affect MTT [ 61 ]. Briefly, the cells are cultured in a 96-well microplate and maintained at 37 ºC in a humidified environment with 5% CO 2 for 24h. After 24 h of initial incubation, the cells were expose with various concentrations of FA-CPT-PLGA-Glu NPs (100, 50, 25, 12.5, 6.25, 3125 µg × mL -1 ), 100 µg × mL -1 of CPT, and 0.25 µM of Doxorubicin for 24 h. After 24 h of exposure, the media was discarded, and cells were rinsed with phosphate buffer solution. Next, 20 µL of MTT staining (5 g × L -1 ) solution was added to the serum-free DMEM medium and incubated in a 5% CO 2 environment at 37 ºC for another 4 h. Then, the medium was removed, and 150 µL of DMSO was added to dissolve formazan crystals produced when tetrazolium MTT was converted. While it dissolves in DMSO, the purple colour varies based on viability. Absorbance was taken at 570 nm in an ELISA reader by which cytotoxicity was evaluated. The below-given equation was used to measure the % cellular viability: % Viability = At /Ac ×100 At and Ac are the mean absorbance of FA-CPT-PLGA-Glu NPs treated and control cells, respectively (n = 5, where n is the number of independent experiments). Evaluation of apoptosis by AO/EBr staining The MCF-7 cells were cultured in a 24-well plate in a CO 2 incubator overnight. After incubation, it was treated with IC 50 concentration of FA-CPT-PLGA-Glu NPs for 48 h. The cells were rinsed three times with ice-cold phosphate-buffered solution and treated with DNA binding strains 10 µL of acridine orange (AO) and ethidium bromide (EBr) (100 µg × mL -1 , each). AO invades the nuclei and makes them green, while EBr invades only cytoplasmic membrane integrity, which is lost to non-viable cells and stains the nuclei red, indicating apoptosis. Each experiment was repeated at least three times[ 62 ]. Measurement of mitochondrial transmembrane potential Rhodamine-123 (Rh-123) staining dye indicates potential damage to the mitochondrial membrane by penetrating the cell membrane. Mitochondrial function is important for predicting healthy cells; this assay will evaluate mitochondrial dysfunction. The cultured MCF-7 cells were treated with IC 50 concentration of FA-CPT-PLGA-Glu NPs for 24 h. After 24 h treatment, cells were harvested, stained with Rh-123 dye, and kept for 30 min. Later, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Morphological changes and membrane permeability were evaluated by fluorescence microscopy. The untreated cells were used as a control[ 63 ]. Measurement of nucleus-induced apoptosis by DAPI assay DAPI (4',6-diamidino-2-phenylindole) is a blue fluorescent DNA staining dye; it evaluates how far the drug affects the nucleus of the cell line[ 64 ]. A healthy cell has an intact cell membrane and does not allow the dye to pass through; thus, it stains dead cells effectively. The MCF-7 cells were grown on the 24-well plate and incubated in a CO 2 incubator at 37 ºC for 24 h. IC 50 concentration of FA-CPT-PLGA-Glu NPs was added and kept for 48 h. After 48h, the treated cells were washed with PBS buffer, and 4% paraformaldehyde was added to fix them for 30 min. The cells were incubated for 5 min with 20 µL DAPI (0.5 µg × mL -1 ) at room temperature and examined under a fluorescence microscope. Quantification of Caspase-3 and − 9 activities The quantification of caspase activity was performed using Caspase-3 and − 9 assay kits (Caspase-Glo® 3 and 9 reagents, Promega)[ 65 ]. In summary, 1×10 5 MCF-7 cells per well were cultured in a 96-well plate. The cell plate was placed in a humidified incubator at 37°C with 5% CO 2 for 24 h. The 96-well plate with the IC 50 concentration of FA-CPT-PLGA-Glu NPs and the untreated control cells were left to reach a stable state at room temperature. Each well of a 96-well plate (including both the test well and control) was supplemented with 100 µL of Caspase-Glo® 3 or 9 reagent, along with 100 µL of culture media. The plate was sealed, and the contents were agitated for 30 seconds at 500 revolutions per minute. The optical density was quantified using an ELISA reader at a wavelength of 405 nm following a 30 min incubation of the plate at room temperature. Analysis of intercellular reactive oxygen species (ROS) levels 2′,7′-dichlorofuorescein diacetate (DCFH-DA) was used to measure intracellular ROS levels[ 66 ]. MCF-7 cells were cultured for 24 h and treated with IC 5 0 concentration of FA-CPT-PLGA-Glu NPs. Treated cells were washed twice with PBS before labelling with 10 M DCFH-DA. DCFH-DA is a non-fluorescent probe that will be converted into fluorescent dichlorofluorescein (DCF) when oxidized by ROS. Fluorescence intensity was measured using a fluorescence spectrophotometer. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay The tunnel assay, also known as the Tdt-dUTP nick end labelling test, employs the Tdt enzyme and fluorescently tagged nucleotide to measure the number of apoptotic cells accurately[ 67 ]. MCF-7 cells were incubated for 24 h in a 5% carbon dioxide environment at 37°C. After a 24-h period, the cells were exposed to a concentration of FA-CPT-PLGA-Glu NPs that is equivalent to the IC 50 value. The cells were then incubated at 37°C for an additional 24 h. The sample is immersed in a 0.5 mL solution of PBS containing 4.5 mL of ice-cold 1% formaldehyde within a polypropylene tube. The tube is then placed in ice for a duration of 5 min. After 5 min, the substance is subjected to centrifugation at a speed of 1500 rpm for 5 min. The cellular sediment was gathered and resuspended in 5 mL of phosphate-buffered saline (PBS) and subjected to centrifugation at 1200 rpm for 3 min. Using a Pasteur pipette, move the cell suspension to a tube with 4.5 mL of ice-cold 70% ethanol. The ethanol was extracted and subsequently reconstituted in 0.5 mL of PBS. The sample was transferred to a volume of 50 µL of the Apo-Direct TUNEL Assay Kit and incubated for 40 min at 37°C. Subsequently, 1.5 mL of washing buffer was introduced and incubated for 1 h. Subsequently, 1 mL of propidium iodide staining solution was introduced and incubated for an additional 30 min at room temperature without light. The resulting mixture was then examined using Flow cytometry (BD FACS Calibur-Becton Dickinson, CytExpert v 1.2.11.0) to ascertain the presence of cells exhibiting DNA damage. The outcome is quantified as the proportion of apoptosis, calculated by adding the percentage of healthy cells to the percentage of cells with damaged DNA. Stability studies Stability in physiological medium and buffer solutions was one of the key parameters for the nano-formulation of an efficient drug delivery system. Figure 6 illustrates the stability of the freshly formulated FA-CPT-PLGA-Glu NPs in the physiological mediums (5% NaCl and 0.5% BSA) buffer solution of acetate buffer (pH 5.3) and PBS (pH: 7.4 and 9). The λmax of FA-CPT-PLGA-Glu NPs in the physiological medium and buffer solutions was 340 nm. There is no change in the λmax for 72 h incubation in physiological medium and buffer solutions. In silico molecular modelling Camptothecin, Doxorubicin, and Co-crystal molecular docking studies against Caspase-3 protein (PDB: 3DEH) to treat breast cancer. In addition to Co-crystal, Camptothecin, and Doxorubicin demonstrated high amino acid interaction and binding affinity for 3DEH Caspase-3 protein. The top-scoring medicines are Camptothecin, Doxorubicin, and 3DEH Co-crystal, with binding energies of -11.3, -9.1, and − 6.6 kcal × mol -1 , respectively, from the internal database. Amino acids Thr166(C), Ocs163(C), Gly122(A), and Doxorubicin Thr62(A) and His121(A) interacted most with Cocrystal. Figure 7 illustrate the 2D and 3D models of Camptothecin, Doxorubicin, co-crystal binding interactions. Molecular dynamics (MD) The protein-ligand complex's RMSD and RMSF were calculated. Additionally, 3DEH Camptothecin, 3DEH Doxorubicin, and 3DEH Co-crystal protein-ligand complexes were studied to determine their interaction patterns (supplementary Figs. 1–3). We found that protein-ligand contacts and interaction signatures significantly affect the complex by examining amino acid residue interactions and RMSD and RMSF computations. The MD simulation of Fig. 4 – 6 dissipated the range of interactions displayed by 3DEH Camptothecin complex from 0.21 to 3.33 in protein and 2.26 to 3.83 in ligand, 3DEH Doxorubicin complex from 0.17 to 4.47 in protein and 0.17 to 4.47 in ligand, and 3DEH Co-crystal complex from 0.16 to 6.32 in protein and 0.23 to 6.86 in ligand. Camptothecin, Doxorubicin, and Co-crystal protein-ligand complexes had consistent RMSF values during molecular dynamic modeling. During the MD simulation, the fluctuation curves showed that Camptothecin, Doxorubicin, and 3DEH Co-crystal had constant amino acid interactions, this compound had no fluctuation, and the residual index of RMSF had excellent stability. The protein-ligand interactions and RMSD complex show that amino acids interact directly with Camptothecin, Doxorubicin, and 3DEH Co-crystal. The MD simulation interaction and Camptothecin, Doxorubicin, and 3DEH Co-crystal binding contact confirmed the His121 amino acid interaction. Camptothecin, Doxorubicin, and Co-crystal docking research shows that His121 and Tyr204 interact similarly to the MD simulation. It demonstrates rigid and flexible connections in Camptothecin and Doxorubicin. Cytotoxicity Based on the results of in silico molecular modeling studies, we determined the cytotoxic potential of formulated FA-CPT-PLGA-Glu NPs against MCF-7 cells using the MTT assay. Figure 8 (a) shows the cytotoxic potential of different doses of FA-CPT-PLGA-Glu NPs, 200 µg CPT, and 0.25 µM Doxorubicin. After 24 hours of treatment, FA-CPT-PLGA-Glu NPs reduced cellular viability concentration-dependent. FA-CPT-PLGA-Glu NPs had an IC 50 value of 16.33 µg × mL -1 after 24 h of incubation with the cell line (n = 3). Cells treated with 3.125 µg × mL − 1 FA-CPT-PLGA-Glu NPs showed approximately 89% viability, while those incubated with 100 µg × mL − 1 CPT showed around 42% cell viability. Figures 8 (b) and (c) show how incubation with 16.33 µg × mL − 1 FA-CPT-PLGA-Glu NPs altered the shape of MCF-7 cells. Acridine Orange-Ethidium Bromide (AO/EBr) apoptosis analyses The AO/EBr fluorescent microscopic staining technique was employed to observe the morphological alterations in MCF-7 cells. The AO/EBr staining method can be used to distinguish between cells undergoing apoptosis and those that are in a normal state. Figures 8 (d) and (e) depict the untreated control and the exposure to FA-CPT-PLGA-Glu NPs at a concentration of 16.33 µg × mL -1 for 48 h. The illustration demonstrates that the control cells did not undergo any changes and remained green after staining. In contrast, the treated cells exhibited a colour change (orange), suggesting the presence of apoptotic cells. Furthermore, the treated cells exhibit membrane blebbing, shrinkage, and nuclear disintegration. This outcome suggests a significant accumulation of CPT within the MCF-7 cells. Mitochondrial transmembrane potential loss The decrease in mitochondrial membrane potential in MCF-7 cells incubated with FA-CPT-PLGA-Glu NPs was quantified using rhodamine-123 staining. A notable reduction in mitochondrial membrane potential was detected in MCF-7 cells following exposure to a dose of 16.33 µg × mL − 1 (IC 50 concentration) of FA-CPT-PLGA-Glu NPs, as shown in Figs. 8 (f) and (g). Further, the FA-CPT-Glu-PLGA NPs caused the mitochondrial membrane to depolarize. The findings of our study showed that the FA-CPT-PLGA-Glu NPs could enter the mitochondrial membrane through endocytosis and cause cell death by reducing the mitochondrial membrane potential. Nuclear Morphology The impact of FA-CPT-PLGA-Glu NPs on nuclear alterations was examined by DAPI staining technique. Figures 8 (h) and (i) demonstrates notable modifications in the structure of the chromatin nuclear material following DAPI labeling of FA-CPT-PLGA-Glu NPs-treated cells for 24 h compared to the untreated control. The control cells exhibit typical spherical nuclei with a normal blue hue, while the treated cells display a vivid colour, aberrant nuclei, and condensed chromatin with uneven cell shape. Caspase-3 and − 9 Activities The apoptosis was further authenticated by measuring the production levels of caspase-3 and − 9 in FA-CPT-PLGA-Glu NPs-treated MCF-7 cells over the untreated control group. Caspases 3 and 9 are the terminal phase inducers of programmed cell death in cancer cells when activated by external stimuli. The caspase-3 (Fig. 8 (j)) and − 9 (Fig. 8 (k)) activities were twofold enhanced in cells exposed to 16.33 µg × mL − 1 (IC 50 conc.) FA-CPT-PLGA-Glu NPs and extract compared to control (untreated) cells. ROS production The fluorescent microscopic observations determined intracellular reactive oxygen species (ROS) production in MCF-7 cells treated with FA-CPT-PLGA-Glu NPs (16.33 µg × mL − 1 ). The untreated MCF-7 cells demonstrated lower ROS generation (Figs. 8 (l) and 14(m)), the onset of apoptosis in the MCF-7 cells. The present work observed that FA-CPT-PLGA-Glu NPs can induce ROS generation in cancer cells (Fig. 8 (n)) using the DCFH-DA staining method. TUNEL Assay The TdT-mediated dUTP nick end labeling (TUNEL) test was used to evaluate nuclear DNA fragmentation (apoptosis) by flow cytometry. This investigation was conducted when the development of cancer cells was reduced through the activation of apoptosis. The flow cytometry analysis of MCF-7 cells is shown in Figs. 8 (o) and (p), depicting the cells' state before (Fig. 8 (o)) and after (Fig. 8 (p)) being treated with FA-CPT-PLGA-Glu NPs at a concentration that inhibits 50% of cell growth (IC 50 ) for a duration of 24 h. After being treated with a concentration of 16.33 µg × mL − 1 of FA-CPT-PLGA-Glu NPs, 38.6% of cells with DNA damage were observed, which was substantially higher than the percentage of DNA-damaged cells in the untreated group (9.46%). Results Glutenin FTIR and 13 C-NMR confirmed the structure of the extracted glutenin from wheat flour. The purified glutenin is observed at highly conserved sites and is involved in intrachain with disulfide (SS) linkages. FTIR and NMR spectral data are presented in our previously published article[ 53 ]. CPT-loaded PLGA NPs CPT-loaded PLGA nanoparticles were initially formulated using a modified emulsification /evaporation technique. The active drug (CPT) was slowly encapsulated into the copolymer PLGA; the encapsulation was confirmed by a UV-visible spectrophotometer (Fig. 2 ). From the UV-visible spectra, the absorption peak of camptothecin slowly disappeared, and a PLGA peak appeared. This peak change confirmed camptothecin encapsulation into PLGA. Layer-by-layer formulation of CPT-loaded PLGA-Glu NPs Layer-by-layer assembly of glutenin on the surface of CPT-loaded PLDA NPs was confirmed by FTIR spectroscopy. Figure 3 presents the FTIR spectra of CPT (a), CPT-loaded PLGA-Glu NPs (b), and glutenin (c). In the CPT spectrum, 3431 cm -1 represents the -OH stretch of hydrogen bonding, 3253 cm -1 represents the aromatic ring, 2976 cm -1 denotes -CH 3 (alkane stretch), 2364 cm -1 represents C = C stretching, and 1745 cm -1 represents -C = O group, and 1597 represents the amide group. CPT-loaded PLGA NPs spectrum showed 3333 cm -1 indicates the -OH stretching group of Glutenin, CPT and PLGA, 1583 cm -1 indicates the amide group from Glutenin and CPT, 1416 cm -1 denotes -CH 3 (alkane stretching) from CPT, Glu, and PLGA. These peaks indicated that CPT was confirmed to be encapsulated in the PLGA-Glu NPs. CPT encapsulation efficiency and loading capacity were observed at 74.95 ± 1.34% and 4.78 ± 1.08%, respectively. Drug release Various parameters, including polymer type and contents of drug-polymer interactions, can influence the drug release patterns of NPs. In vitro , drug release tests were conducted at 37 ± 1.0°C using a dialysis membrane, acetate buffer at pH 5.3, and phosphate buffer at pH 7.2. The release of CPT from CPT-loaded PLGA-Glu NPs occurred in two stages. Figure 4 shows the percentage of drug release from CPT-loaded PLGA-Glu NPs. PLGA-Glu NPs released CPT quickly, with burst release content of 23.47 ± 0.986% and 20.867 ± 1.1% at pH 5.3 and pH 7.2 within 8 h, respectively. CPT-loaded Glu-PLGA NPs produced the most CPT (75.517 ± 1.04% at pH 5.3 and 70.557 ± 0.86% at pH 7.2) within 28 h. Drug release kinetics Further, the above drug-release data was used to evaluate the CPT-release kinetics from the CPT-PLGA-Glu NPs. Five simple models were fitted to the acquired data, including zero-order, first-order, Higuchi, Korsmeyer–Peppas, and Hixson–Crowell. Table 1 summarizes the findings of the in vitro drug-release kinetics. The release rate constant of each kinetic model was predicted using the generated regression coefficient (r 2 ) values. In general, significantly fitting the model, the r 2 value is near 1. Here, 0.9847 to 0.9275 (pH 5.3) and 0.9846 to 0.9368 (pH 7.2) were the values of the first-order kinetics regression coefficient (r 2 ). These values were greater than those of the zero-order kinetics. Since these numbers are near 1, the models are regarded as good fits. On the other hand, the diffusion exponent (n) values in the Korsmeyer–Peppas model showed smaller variation, from 0.720 (pH 5.3) to 0.724 (pH 7.2). With plots displaying strong linearity and r 2 values in the range of 0.9344 (pH 5.3) and 0.9312 (pH 7.2), which mostly suggested the diffusion process, the Higuchi model was the most effective kinetic model. This shows that the formulation has been uniformly dissolved and released under control. Furthermore, the observed release kinetics data showed the CPT- PLGA-Glu NPs formulation non-Fickian behavior. Furthermore, because the diffusion exponent (n) was greater than 0.7 (pH 7.2, i.e., 0.724), it was demonstrated that the super case II transport drug-release mechanism was implicated. High r 2 values (0.9836) in the Hixson–Crowell model suggested uniform dissolution of the CPT- PLGA-Glu NPs and a consistent drug release. On the other hand, CPT release from the CPT- PLGA-Glu NPs was primarily maintained at a fixed amount of drug-release pattern at pH 5.3 and pH 7.2. Table 1 Drug-release kinetics profile of Camptothecin (CPT) from CPT-Glu-PLGA NPs. Model Parameter CPT from CPT-Glu-PLGA NPs pH 5.3 pH 7.2 Zero order F = K 0 ×t K 0 0.039 0.037 r 2 adjusted 0.9375 0.9368 AIC 147.4014 145.5374 First order F = 100× [1-Exp (-k1×t)] K 1 0.001 0.001 r 2 adjusted 0.9847 0.9846 AIC 117.8070 115.9276 Higuchi model F = KH×t 1/2 K h 1.516 1.433 r 2 adjusted 0.9344 0.9312 AIC 148.4185 147.3251 Korsmeyer-Peppas model F = kKP×t n kKP 0.310 0.283 r 2 adjusted 0.9758 0.9726 n 0.720 0.724 AIC 129.4737 128.9280 Hixon-Crowell model F = 100×[1-(1-kHC×t) 3 ] kHC 0.00 0.00 r 2 adjusted 0.9836 0.9815 AIC 119.3100 119.7788 Where, AIC = Akaike information criterion, F = fraction of drug release in time t, K 0 = apparent rate constant of zero order release constant, K 1 = first order release constant, K H =Higuchi constant, kKP = Korsmeyer-Peppas rate constant, kHC = Hixon-Crowell constant, n = diffusional exponent. And r 2 = Squared correlation coefficient. FA-CPT-Glu-PLGA NPs UV-visible and FTIR spectroscopy initially synthesized and confirmed FA-NHS (N-hydroxysuccinimide ester of Folic acid). The UV-visible spectrum of FA-NHS was presented in our previously published article. FA and FA-NHS FT-IR spectra are displayed in Figure 3 (d) and 3(e). The FA-NHS spectrum shows two notable absorption peaks at 1666 cm − 1 due to a N–O bond and 1688 cm − 1 due to a C = O bond. These absorption peaks indicate that FA has been structurally transferred into FA-NHS (Fig. 3 (e)). Folic acid-conjugated CPT-loaded PLGA-Glu NPs were formulated by simple conjugation chemistry with FA-NHS and CPT-loaded PLGA-Glu NPs. The carboxylic group of FA-NHS was linked with the amino group of Glutenin from CPT-Glu-PLGA NPs by adding a carbonate/bicarbonate buffer. Figure 3 (f) presents the FTIR spectrum of FA-CPT-PLGA-Glu NPs. As can be seen, the FTIR spectra of FA-CPT-PLGA-Glu NPs, 3458 cm -1 , indicate that the NH group from folic acid conjugation, 1937 cm -1 and 1666 cm -1 , indicate PLGA ester and acid bond absorption peaks. 1583 cm -1 and 1416 cm -1 indicate nitro compounds and -C-H bending stretching from glutenin protein. Characterization of FA-CPT-PLGA-Glu NPs Figure 5 (a) depicts the X-ray diffraction (XRD) spectrum of FA-CPT-PLGA-Glu NPs. The XRD pattern displayed stronger peaks at 2θ conditions (19°, 30°, 34°, 41°, 46°, and 57°), indicating their crystalline character. DLS analysis (Fig. 5 (b)) confirmed their average particle sizes between 10 to 100 nm. Further, HRTEM pictures (Figs. 5 (c)-(e)) demonstrate that the NPs had a 60 to 100 nm diameter and were spherical. Stability studies Stability in physiological medium and buffer solutions was one of the key parameters for the nano-formulation of an efficient drug delivery system. Figure 6 illustrates the stability of the freshly formulated FA-CPT-PLGA-Glu NPs in the physiological mediums (5% NaCl and 0.5% BSA) buffer solution of acetate buffer (pH 5.3) and PBS (pH: 7.4 and 9). The λmax of FA-CPT-PLGA-Glu NPs in the physiological medium and buffer solutions was 340 nm. There is no change in the λmax for 72 h incubation in physiological medium and buffer solutions. In silico molecular modelling Camptothecin, Doxorubicin, and Co-crystal molecular docking studies against Caspase-3 protein (PDB: 3DEH) to treat breast cancer. In addition to Co-crystal, Camptothecin, and Doxorubicin demonstrated high amino acid interaction and binding affinity for 3DEH Caspase-3 protein. The top-scoring medicines are Camptothecin, Doxorubicin, and 3DEH Co-crystal, with binding energies of -11.3, -9.1, and -6.6 kcal × mol -1 , respectively, from the internal database. Amino acids Thr166(C), Ocs163(C), Gly122(A), and Doxorubicin Thr62(A) and His121(A) interacted most with Cocrystal. Figure 7 illustrate the 2D and 3D models of Camptothecin, Doxorubicin, co-crystal binding interactions. Molecular dynamics (MD) The protein-ligand complex's RMSD and RMSF were calculated. Additionally, 3DEH Camptothecin, 3DEH Doxorubicin, and 3DEH Co-crystal protein-ligand complexes were studied to determine their interaction patterns (supplementary Figures 1-3). We found that protein-ligand contacts and interaction signatures significantly affect the complex by examining amino acid residue interactions and RMSD and RMSF computations. The MD simulation of Figure 4-6 dissipated the range of interactions displayed by 3DEH Camptothecin complex from 0.21 to 3.33 in protein and 2.26 to 3.83 in ligand, 3DEH Doxorubicin complex from 0.17 to 4.47 in protein and 0.17 to 4.47 in ligand, and 3DEH Co-crystal complex from 0.16 to 6.32 in protein and 0.23 to 6.86 in ligand. Camptothecin, Doxorubicin, and Co-crystal protein-ligand complexes had consistent RMSF values during molecular dynamic modeling. During the MD simulation, the fluctuation curves showed that Camptothecin, Doxorubicin, and 3DEH Co-crystal had constant amino acid interactions, this compound had no fluctuation, and the residual index of RMSF had excellent stability. The protein-ligand interactions and RMSD complex show that amino acids interact directly with Camptothecin, Doxorubicin, and 3DEH Co-crystal. The MD simulation interaction and Camptothecin, Doxorubicin, and 3DEH Co-crystal binding contact confirmed the His121 amino acid interaction. Camptothecin, Doxorubicin, and Co-crystal docking research shows that His121 and Tyr204 interact similarly to the MD simulation. It demonstrates rigid and flexible connections in Camptothecin and Doxorubicin. Cytotoxicity Based on the results of in silico molecular modeling studies, we determined the cytotoxic potential of formulated FA-CPT-PLGA-Glu NPs against MCF-7 cells using the MTT assay. Figure 8(a) shows the cytotoxic potential of different doses of FA-CPT-PLGA-Glu NPs, 200 µg CPT, and 0.25 µM Doxorubicin. After 24 hours of treatment, FA-CPT-PLGA-Glu NPs reduced cellular viability concentration-dependent. FA-CPT-PLGA-Glu NPs had an IC 50 value of 16.33 µg × mL -1 after 24 h of incubation with the cell line (n = 3). Cells treated with 3.125 μg × mL −1 FA-CPT-PLGA-Glu NPs showed approximately 89% viability, while those incubated with 100 μg × mL −1 CPT showed around 42% cell viability. Figures 8(b) and (c) show how incubation with 16.33 μg × mL −1 FA-CPT-PLGA-Glu NPs altered the shape of MCF-7 cells. Acridine Orange-Ethidium Bromide (AO/EBr) apoptosis analyses The AO/EBr fluorescent microscopic staining technique was employed to observe the morphological alterations in MCF-7 cells. The AO/EBr staining method can be used to distinguish between cells undergoing apoptosis and those that are in a normal state. Figures 8 (d) and (e) depict the untreated control and the exposure to FA-CPT-PLGA-Glu NPs at a concentration of 16.33 μg × mL -1 for 48 h. The illustration demonstrates that the control cells did not undergo any changes and remained green after staining. In contrast, the treated cells exhibited a colour change (orange), suggesting the presence of apoptotic cells. Furthermore, the treated cells exhibit membrane blebbing, shrinkage, and nuclear disintegration. This outcome suggests a significant accumulation of CPT within the MCF-7 cells. Mitochondrial transmembrane potential loss The decrease in mitochondrial membrane potential in MCF-7 cells incubated with FA-CPT-PLGA-Glu NPs was quantified using rhodamine-123 staining. A notable reduction in mitochondrial membrane potential was detected in MCF-7 cells following exposure to a dose of 16.33 μg × mL −1 (IC 50 concentration) of FA-CPT-PLGA-Glu NPs, as shown in Figures 8(f) and (g). Further, the FA-CPT-Glu-PLGA NPs caused the mitochondrial membrane to depolarize. The findings of our study showed that the FA-CPT-PLGA-Glu NPs could enter the mitochondrial membrane through endocytosis and cause cell death by reducing the mitochondrial membrane potential. Nuclear Morphology The impact of FA-CPT-PLGA-Glu NPs on nuclear alterations was examined by DAPI staining technique. Figures 8(h) and (i) demonstrates notable modifications in the structure of the chromatin nuclear material following DAPI labeling of FA-CPT-PLGA-Glu NPs-treated cells for 24 h compared to the untreated control. The control cells exhibit typical spherical nuclei with a normal blue hue, while the treated cells display a vivid colour, aberrant nuclei, and condensed chromatin with uneven cell shape. Caspase-3 and -9 Activities The apoptosis was further authenticated by measuring the production levels of caspase-3 and -9 in FA-CPT-PLGA-Glu NPs-treated MCF-7 cells over the untreated control group. Caspases 3 and 9 are the terminal phase inducers of programmed cell death in cancer cells when activated by external stimuli. The caspase-3 (Figure 8(j)) and -9 (Figure 8(k)) activities were twofold enhanced in cells exposed to 16.33 μg × mL −1 (IC 50 conc.) FA-CPT-PLGA-Glu NPs and extract compared to control (untreated) cells. ROS production The fluorescent microscopic observations determined intracellular reactive oxygen species (ROS) production in MCF-7 cells treated with FA-CPT-PLGA-Glu NPs (16.33 μg × mL −1 ). The untreated MCF-7 cells demonstrated lower ROS generation (Figures 8(l) and 14(m)), the onset of apoptosis in the MCF-7 cells. The present work observed that FA-CPT-PLGA-Glu NPs can induce ROS generation in cancer cells (Figure 8(n)) using the DCFH-DA staining method. TUNEL Assay The TdT-mediated dUTP nick end labeling (TUNEL) test was used to evaluate nuclear DNA fragmentation (apoptosis) by flow cytometry. This investigation was conducted when the development of cancer cells was reduced through the activation of apoptosis. The flow cytometry analysis of MCF-7 cells is shown in Figures 8 (o) and (p), depicting the cells' state before (Figure 8(o)) and after (Figure 8(p)) being treated with FA-CPT-PLGA-Glu NPs at a concentration that inhibits 50% of cell growth (IC 50 ) for a duration of 24 h. After being treated with a concentration of 16.33 μg × mL −1 of FA-CPT-PLGA-Glu NPs, 38.6% of cells with DNA damage were observed, which was substantially higher than the percentage of DNA-damaged cells in the untreated group (9.46 %). Discussion Camptothecin was previously thought to be an antioxidant molecule that might be used to combine anticancer and antidiabetic medicines[ 68 ]. Later, there was evidence of its anticancer efficacy in treating leukaemia and colon cancers. CPT has potent anticancer action by targeting DNA topoisomerase I [ 69 ]. However, CPT has three major limitations: (1). it is sparingly soluble in water, and its clinical applications are through intravenous administration (2). the biologically active form of CPT (lactone) rapidly undergoes a ring-opening reaction and converts to the biologically inactive form (carboxylate) under neutral conditions, and (3). it is unstable in human plasma and highly toxic[ 70 ]. To address these concerns, this study formulated CPT-loaded with folic acid-conjugated PLGA-glutenin nanoparticles (FA-CPT-PLGA-Glu NPs) to deliver CPT into cancer cells while preserving normal healthy cells. Polymeric nanoparticles efficiently deliver the loaded toxic payload into cancer cells because they are easily adjustable, and their morphology can be easily modified[ 71 ]. The selection of polymers for the development of nanoparticles is one of the key parameters for drug delivery applications. Currently, various types of biodegradable polymers (both natural and synthetic) are commonly used to develop nanoparticles, particularly for cancer drug delivery[ 72 ]. Interestingly, pharmaceutical research uses polymer-based nanoparticles to reduce drug toxicity and adverse effects, but until recently, it was not realized that carrier systems themselves could cause hazards to patients[ 73 ]. While developing a treatment approach using nanomedicine, the goal should be to minimize the toxic effects on the patient[ 74 ]. Synthetic polymers have many disadvantages, including toxicity, poor biocompatibility, and high cost of the production process. Natural polymers (derived from plants, animals, and bacteria) are less toxic, biocompatible, biodegradable, and easily available; at the same time, some disadvantages also occurred using as drug carrier material, such as structurally more complex and high degree of variability in materials from animal sources[ 75 ]. A combination of natural and synthetic polymers can be better compatible with higher stability, prolonged circulation, and controlled release behaviors of loaded drugs with less toxicity[ 76 ]. This study used a combination of glutenin and PLGA to formulate nanoparticles. PLGA has been widely used to encapsulate various anticancer agents, offering strong biodegradability, minimum systemic toxicity, and enhanced bioavailability of loaded drugs. Glutenin is isolated from wheat flour and comprises two polypeptide chains (gliadin and glutenin) and soluble carbohydrates. However, gliadin was extracted from gluten protein with 70% ethanol, and glutenin is soluble in acetic acid. In contrast to gliadin, glutenin was highly biocompatible and promotes fibroblast and osteoblast adhesion and proliferation more effectively than films composed of PLGA. Glutenin structure consists of multimeric aggregates of high and low molecular mass subunits linked together by disulfide links [ 53 ]. CPT-loaded PLGA NPs were formulated using a modified emulsification/evaporation method. Further, glutenin coated on the surface of CPT-loaded PLGA NPs via a layer-by-layer assembled technique. Furthermore, simple conjugation chemistry successfully formulated PLGA-Glu NPs conjugated with FA. The amino group of glutenin was conjugated with the carboxylic acid group of FA. The FA conjugation allowed for easy recognition and attachment of upregulated folate receptors on the surface of cancer cells. This facilitated the effective delivery of the loaded drugs into the cancer cells[ 77 ]. Because the folate receptor is a biomarker for many tumors, it is highly expressed in specific malignant cells such as breast, ovarian, lung, kidney, brain, and colon cancer cells. In addition, the cellular absorption of the drug is increased by folate-conjugated NPs through endocytosis. Our previous study demonstrated that retinoic acid-encapsulated folic acid-conjugated glutenin NPs (FA-RA-Glu NPs) effectively delivered the loaded RA into MCF-7 cells and significantly reduced the number of viable cells, and induced apoptosis. The cellular uptake study showed that the FA-RA-Glu NPs had facilitated endocytosis and delivered RA into MCF-7 cells[ 46 ]. The physiochemical features of nanoparticles, such as their size and shape, play a major role in determining their effectiveness and delivering drugs to the tumor site [ 78 ]. The X-ray diffraction analysis examined the formulated FA-CPT-PLGA-Glu NPs possess a crystalline structure, which enables a regulated and extended release of the loaded drug into the cancer cells. The molecular dimension and configuration of nanoparticles (NPs) are key factors that exert a substantial influence on regulating the circulation and biodistribution of therapeutic nanoparticles. When observed using transmission electron microscopy (TEM), the FA-CPT-PLGA-Glu NPs appeared predominantly spherical with a diameter of around 100 nm, consistent with the findings of the dynamic light scattering (DLS) investigation. The optimal particle size for effective distribution into solid tumors should be 10–200 nm. Furthermore, nanoparticles with a spherical shape demonstrated the highest level of internalization into cancer cells compared to nanoparticles of different shapes[ 79 ]. Additionally, the NPs with a spherical shape can encapsulate the highest drug content, leading to a decrease in the quantity of cancer cells. This is a significant utilization of nanoparticle-based drug delivery systems in cancer therapy. When formulating a targeted drug delivery system, it is necessary to consider drug encapsulation efficiency and loading capacity. The higher encapsulation efficiency with lesser nanoparticle loading capacity allows for more efficient drug delivery at the disease site. In the present study, the CPT encapsulation efficiency and loading capacity of PLGA-Glu NPs were 74.95 ± 1.34% and 4.78 ± 1.08%, respectively. Nanoparticles increase anticancer drugs' bioavailability and therapeutic efficacy while ensuring preferential accumulation at the targeted site. Here, we observed in vitro CPT release from CPT-PLGA-Glu NPs at various pH levels in the buffer solution to confirm the maximum content of CPT loaded. Investigate the CPT release kinetics and mechanism from FA-conjugated Glu-PLGA NPs. Determine the optimal conditions (pH) for the highest release of CPT-PLGA-Glu NPs, which is required for further in vivo studies. Our investigation revealed a consistent and gradual release of the drug during the studies, suggesting a sustained and controlled release of the loaded drug. This can be attributed to drug diffusion and matrix erosion technique mechanisms. Further, we found that the highest amount of CPT was released from CPT-PLGA-Glu NPs in a pH 5.3 acetate buffer solution, which simulated a cancerous environment. Similarly, our previous study demonstrated that the formulated glucose-conjugated CPT-loaded glutenin nanoparticles released their toxic payload of CPT in the acidic environment at pH 5.3. The primary goal of a targeted drug delivery system is to prolong circulation, locate at the disease site, target, and ensure a safe drug interaction with diseased tissue/cells. The results show that FA-CPT-PLGA-Glu NPs effectively reduced the viability of MCF-7 cells. This significant cytotoxicity of FA-CPT-PLGA-Glu NPs against MCF-7 cells is attributed to the highest CPT content that enters the cells. The higher cellular internalization of CPT-PLGA-Glu NPs leads to a higher cellular uptake of the entrapped therapeutic agent. The cellular uptake of FA-CPT-PLGA-Glu NPs may be influenced by the process of folate receptor-mediated endocytosis. The IC 50 value was calculated, representing the minimum concentration of a substance needed to induce 50% cell death in cancer cells within a specific period. The IC 50 concentration of FA-CPT-PLGA-Glu NPs against MCF-7 cells was 16.33 µg × mL − 1 . The MCF-7 cell line demonstrates a significant folate receptor positivity, with the folate receptor being abundantly upregulated on its surface. As the concentration increased, the viability of the cells reduced, indicating that the cytotoxicity of the drug depends on its concentration. Similarly, the cytotoxicity of α-mangosteen (AM), α-mangosteen-loaded folate-conjugated chitosan nanoparticles-high molecular weight (AM-F-CS-HMW NPs), and α-mangosteen-loaded folate-conjugated chitosan nanoparticles-high molecular weight (AM-F-CS-LMW NPs) against MCF-7 cells and found IC50 values of 8.47 ± 0.49, 5.3 ± 0.01, and 4.70 ± 0.11 µg × mL -1 , respectively. These results confirm the improved cytotoxicity of α-mangostin in MCF-7 cells when delivered via folate-conjugated chitosan NPs[ 80 ]. Most of the anticancer agents stimulate the process of apoptosis induction and associated cell death networks to eradicate malignant cells. Apoptosis is a crucial process in both carcinogenesis and cancer treatment. The FA-CPT-PLGA-Glu NPs effectively triggered cell death in MCF-7 cells, as demonstrated by the AO/EtBr double staining experiment. The cells treated with 16.33 µg × mL -1 of FA-CPT-PLGA-Glu NPs exhibited symptoms of cell death, which are typically characterized by distinct morphological alterations, including round and irregular forms, condensed nuclei, deformed membranes, and the presence of apoptotic bodies. Cancer cell cycle arrest, senescence, and apoptosis can be induced by the overproduction of intracellular reactive oxygen species (ROS). Our study showed FA-CPT-PLGA-Glu NPs activated ROS generation by the 2ʹ,7ʹ-Dichlorofluorescin Diacetate (DCFH-DA) method. 16.33 µg × mL -1 of FA-CPT-PLGA-Glu NPs treated cells showed increased levels of ROS. This might be interpreted as the proteasome inhibition in MCF-7 cells leading to the degradation of many proteins, disrupting redox equilibrium, and raising ROS levels. The buildup of ROS disrupts the respiratory chain and may activate the p53-mediated intrinsic apoptotic mechanism. Furthermore, phosphorylation and ubiquitination of cell cycle proteins disrupt the redox regulation of cell cycle progression, resulting in abnormal cell proliferation and cell death. The assessment of apoptosis was further validated by treating MCF-7 cells with 16.33 µg × mL − 1 of FA-CPT-PLGA-Glu NPs, which resulted in the release of cytochrome C and a decrease in mitochondrial membrane potential. Intrinsic apoptosis is linked to the integrity of the mitochondrial membrane damage. When a cell receives a death signal, the receptor's C terminal sequence becomes active. These target the outer membrane layer of the mitochondria and cause permeabilization, ultimately resulting in cell death. The caspase cascade is responsible for cell death after the cytochrome C release[ 81 ]. Targeted suppression of caspase-3 and − 9 enables the effective liberation of cytochrome C, impeding alterations in mitochondrial structure and generating reactive oxygen species (ROS). In addition, the effectiveness of apoptotic cell death was closely observed by examining changes in the morphology of the nucleus using the DAPI assay. Apoptosis in cancer cells of mammals is frequently accompanied by specific morphological and physiological alterations, such as membrane blebbing, phosphoserine externalization, chromatin condensation, nuclear fragmentation, and degradation of DNA initially into large fragments and subsequently into small nucleosomal fragments. The FA-CPT-PLGA-Glu NPs induced apoptosis in MCF-7 cells, as evidenced by nuclear fragmentation, cell shrinkage, and nucleus marginalization. The TUNEL assay detected apoptotic cells that undergo substantial DNA destruction during the late stages of apoptosis. This technique utilizes the autonomous labeling ability of terminal deoxynucleotidyl transferase to mark the blunt ends of double-stranded DNA breaks without requiring a template. Conclusion In this study, FA-CPT-PLGA-Glu NPs were successfully fabricated with the help of wheat glutenin protein and synthetic polymer PLGA, which is biocompatible and observed to deliver the loaded CPT into cancer cells effectively. The FA surface modification of NPs is easy to recognize and covalently binds to the overexpressed folate receptor on the plasma membrane of breast cancer cells and enters inside the cell via endocytosis. The released active molecule CPT from NPs targets caspase-3 and − 9 to induce cell death mechanism via overproduction of ROS. Due to the poor pharmacokinetics and toxic properties of CPT, it is converted into nanoparticles for targeting cancer cells without disturbing normal healthy cells. Fabricated FA-CPT-PLGA-Glu NPs were observed to be crystalline, spherical shaped, and around 100 nm in size. Active molecule, the CPT encapsulation efficiency and loading capacity of PLGA-Glu NPs were 74.95 ± 1.34% and 4.78 ± 1.08%, respectively. Further, the drug release behavior of CPT-PLGA-Glu NPs displayed a prolonged and controlled release pattern. Released CPT from NPs reduced cellular viability and inhibited caspase-3 and − 9 activity to cause cell death/apoptosis via increasing ROS generation, damaging mitochondrial membrane potential, and altering the morphology of MCF-7 nuclei. The findings suggested that the unique FA-CPT-PLGA-Glu NPs formulation showed strong antitumor activity. However, more extensive in vivo research is required to assess its clinical applicability fully. Declarations Acknowledgements RRR thanks to Kalasalingam Academy of Research and Education for the university research fellowship. Author Contributions SK supervision, project administration, funding acquisition, resources, writing review & editing; RRR, TP, PP, SRKP, ASKK writing-original draft, formal analysis, investigation; MS, SJK conceptualization, writing, investigation and editing. All authors have read and agreed to the published version of the manuscript. Funding SK gratefully acknowledge the Management of Kalasalingam Academy of Research and Education for Seed Money Grant (KARE/VC/R&D/SMPG/2021–2022/1). Data Availability All data generated or analysed during this study are included in this manuscript. Declarations Conflict of interest The authors declare that they have no conflict of interests. Ethical Approval Ethical approval was not required for this research. Consent to Participate Not applicable. Consent for Publication The authors give the consent for publication. 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Supplementary Files SupplementaryFigure1.jpg SupplementaryFigure2.jpg SupplementaryFigure3.jpg Cite Share Download PDF Status: Published Journal Publication published 03 Sep, 2024 Read the published version in Journal of Polymers and the Environment → Version 1 posted Editorial decision: Revision requested 07 Jul, 2024 Reviews received at journal 06 Jul, 2024 Reviews received at journal 05 Jul, 2024 Reviewers agreed at journal 26 Jun, 2024 Reviewers agreed at journal 26 Jun, 2024 Reviewers agreed at journal 07 Jun, 2024 Reviewers invited by journal 06 Jun, 2024 Submission checks completed at journal 01 Jun, 2024 Editor assigned by journal 01 Jun, 2024 First submitted to journal 01 Jun, 2024 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. 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Data are represented as mean ± standard deviation (n=3).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4513460/v1/144d3b0b26cd89fd3cc09e5a.jpg\"},{\"id\":58391310,\"identity\":\"e56f6512-34f5-483a-ba17-930b7fdca73a\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 20:44:55\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2524026,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eXRD pattern of FA-CPT-PLGA-Glu NPs (a), Particle distribution of FA-CPT-PLGA-Glu NPs (b), and HRTEM images of FA-CPT-PLGA-Glu NPs (c)-(e).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4513460/v1/e2e7c62f9ee93b4cb6a96cbc.jpg\"},{\"id\":58391012,\"identity\":\"7062ecf9-937e-48a5-9986-e67b37f86256\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 20:36:55\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":188629,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe \\u003cem\\u003ein vitro\\u003c/em\\u003e stability studies of FA-CPT-PLGA-Glu NPs in different physiological media, pH buffer systems. A stable peak appeared for 5% NaCl, 0.5% BSA, pH 5.3 acetate buffer and PBSs (pH: 7.4 and 9.0) at 340 nm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4513460/v1/107851cb3a6e6ed07eae989c.jpg\"},{\"id\":58391311,\"identity\":\"d395800b-d511-4414-afc7-e5d5198a7163\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 20:44:55\",\"extension\":\"jpg\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":5677869,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDepicted the interaction between the compound Camptothecin-Caspase-3 protein (a\\u0026amp; b), Doxorubicin- Caspase-3 protein (c \\u0026amp; d) and cocrystal (Isoquinoline-1,3,4-trione)-Caspase-3 protein complex (e \\u0026amp; f). The left side shows the 3D and the right side representing the 2D complex of the drug–caspase-3 protein interaction.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure7.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4513460/v1/703da6e85025e98cf77735ef.jpg\"},{\"id\":58391015,\"identity\":\"1706b8bb-c075-4532-80d0-8706885c57e3\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 20:36:55\",\"extension\":\"jpg\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3428488,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAssessment of cytotoxic and apoptotic effects of FA-CPT-PLGA-Glu NPs on MCF-7 cells. Cytotoxic activity of human adenoma breast cancer (MCF-7) cells using different concentrations of FA-CPT-PLGA-Glu NPs after 24 h treatment. The percentage of apoptotic cells increased dose-dependently (a). Morphology of the control cells (b) and treated cells (c) was observed using a phase-contrast microscope. Values are mean ± standard deviation of triplicate measurements (p\\u0026lt;0.05). Apoptotic morphological variations of MCF-7 cells were identified with AO/EB staining and observed using a fluorescence microscope. Control cells (d); 16.33 μg × mL\\u003csup\\u003e−1\\u003c/sup\\u003e of FA-CPT-PLGA-Glu NPs for 48 h (e). Effect of FA-CPT-PLGA-Glu NPs on mitochondrial transmembrane potential in MCF-7 cancer cells. Control cells (f); notable loss of mitochondrial transmembrane potential in treated cells (g). DAPI-stained image of MCF-7 cancer cells. Control cells (h) and treatment with FA-CPT-PLGA-Glu NPs (i). Chromatin fragmentation is shown with arrows. Activity of Caspase-3(j) and -9 (k) of 16.33 μg × mL\\u003csup\\u003e−1\\u003c/sup\\u003e of FA-CPT-PLGA-Glu NPs against MCF-7 cells. Effect of 16.33 μg × mL\\u003csup\\u003e−1\\u003c/sup\\u003e of FA-CPT-PLGA-Glu NPs on ROS generation (percentage of control) in MCF-7 cancer cells. ROS generation measured as relative fluorescence intensity using a fluorescence microscope (l); Image of untreated control cells (m); Results expressed as mean ± standard deviation of triplicate measurements (p \\u0026lt; 0.05) (n). Flow cytometry analysis of MCF-7 cells before (o) and after (p) treatment with 16.33 μg × mL\\u003csup\\u003e-1\\u003c/sup\\u003e of FA-CPT-PLGA-Glu NPs for 24 h.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure8.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4513460/v1/ca801d095ebcc92ec4944714.jpg\"},{\"id\":64185840,\"identity\":\"346584b5-3230-420e-837a-230b67a38a32\",\"added_by\":\"auto\",\"created_at\":\"2024-09-09 16:22:19\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":15783795,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4513460/v1/c37d773a-baae-4edf-a724-c0d3e371d061.pdf\"},{\"id\":58391019,\"identity\":\"09ac6e35-c00b-4c7f-996d-b8f6f28b6e42\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 20:36:56\",\"extension\":\"jpg\",\"order_by\":11,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":3408086,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryFigure1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4513460/v1/3e4c1ac32b6be60b8694fbd3.jpg\"},{\"id\":58391016,\"identity\":\"e9bd866a-2696-40a2-9542-70aa37a368fe\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 20:36:55\",\"extension\":\"jpg\",\"order_by\":12,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":3338782,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryFigure2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4513460/v1/32e40ffdf143166521c0e777.jpg\"},{\"id\":58391017,\"identity\":\"eeaa2847-039f-4f23-9b6e-67362d3d50d4\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 20:36:55\",\"extension\":\"jpg\",\"order_by\":13,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":3123290,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryFigure3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4513460/v1/78efe75ca97bcc57e7595332.jpg\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Folate receptor-targeted Camptothecin-loaded PLGA-Glutenin nanoparticles for effective breast cancer treatment\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eCancer is one of the deadliest non-communicable diseases (NCDs), which accounts for 9% of total deaths (63%) in India [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. Particularly in India, breast and cervical cancers are majorly afflicted in women populations [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. According to GLOBOCAN 2020, in India, breast cancer recorded 13.5% (178361) of all cancer cases and 10.6% (90408) of all deaths, with a cumulative risk of 2.81\\u0026nbsp;million [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Women populations are the most vulnerable victims of breast cancer, but rare cases can also occur in men; in 2016, about 98.1% of 118000 total women breast cancer cases in India [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. The reason for the occurrence of breast cancer might be sedentary lifestyle behaviors like being physically inactive, improperly-maintained body mass index (BMI), excessive alcohol consumption with cigarette smoking, consuming junk foods with high fats, and medicines such as exogenous female hormones (menopausal hormone therapy, and hormonal contraceptives) [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. At the same time, mutations in the BRCA1 or BRCA2 gene, checkpoint kinase-2 (CHEK-2), and breast cancer-connected single-nucleotide polymorphisms (SNPs) also caused breast cancer [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003ePresent today, surgical removal, radiation \\u0026amp; laser therapy, hormonal therapy, chemotherapy, combination therapy, and personalized medicine are the major treatment strategies for breast cancer [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Among the various treatment strategies, chemotherapy was one of the most promising and effective treatment options for breast cancer [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Nowadays multiple classes of chemotherapeutic agents are used for the treatment of breast cancer, such as anti-metabolites (methotrexate) [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e], alkylating agents (Cyclophosphamide) [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e], anthracyclines (doxorubicin) [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e], hormones (tamoxifen) [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e], monoclonal antibodies (trastuzumab, pertuzumab) [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e], mitotic inhibitors (paclitaxel, docetaxel) [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e], topoisomerase inhibitors (etoposide, irinotecan) [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e], and tubulin inhibitors (vinblastine) [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Cyclophosphamide (alkylating agent) and doxorubicin (anthracyclines) for four cycles, followed by paclitaxel (taxanes) for four cycles, were the common protocol for adjuvant chemotherapeutic treatment[\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. The mode of action of these compounds, such as cyclophosphamide, causes breakage of DNA [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e], doxorubicin acts in DNA intercalation, inhibiting the macromolecular synthesis [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e], and paclitaxel induces cell cycle arrest and apoptosis by binding to microtubules preventing their disassembly [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Anthracyclines and taxanes are primarily used to treat breast cancer. However, increased usage of these agents at an early phase of breast cancer frequently produces tumor drug-resistant against these treatments by the time the disease reoccurs, thus lowering the number of treatment choices for metastatic cancer [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Moreover, even when these drugs are used in metastatic cancer, treatment failure occurs usually, resulting in reduced 5-year survival rates for patients with metastatic breast cancer[\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eDrug resistance is a major issue for using chemotherapies during cancer treatment stages. Also, numerous side effects such as cytopenia, alopecia, fatigue, neurocognitive dysfunction, chemotherapy-induced peripheral neuropathy, and muscle pain occurred in the treatment of breast cancer [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. After six months of treatment, patients experience chronic effects such as sterility, early menopause, psychological impacts, second cancer, and cardiomyopathy [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Naturally derived compounds are less toxic and have fewer side effects than synthetic chemotherapies [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Numerous natural compounds, such as resveratrol, vincristine, vinblastine, curcumin, capsaicin, piperine, etc., are already established for breast cancer treatment therapy[\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Camptothecin (CPT) is a hydrophobic bioactive molecule, a plant-derived alkaloid extracted from the stem wood of \\u003cem\\u003eCamptotheca acuminata\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. The antineoplastic activity of the CPT makes it an efficient antioxidant and anti-cancer property for various types of cancers [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. The mode of action of CPT was to induce apoptosis by inhibiting the enzyme topoisomerase-1 involved in the S-phase of the cell cycle [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. Apart from the various advantages of this molecule, some shortcomings are there such as low solubility, rapid metabolism, lower stability, non-selectivity, low bioavailability, and biocompatibility are the major concerns of using this drug [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. The drawbacks can be overcome by devising a novel nano-engineered carrier system to deliver loaded drugs to the disease sites. Hydrophobic drugs delivered through nanoparticulate systems have become a major backbone of cancer drug research.\\u003c/p\\u003e \\u003cp\\u003eAmong the most prevalent nanoparticulate systems, polymeric nanoparticles have several key advantages that provide a wide range of delivery platforms. However, polymeric nanoparticles have certain limitations, including using toxic solvents during manufacturing, polymer breakdown, drug leakage outside the sick tissue, and polymer cytotoxicity. Combining naturally obtained protein polymers with synthetic polymer is a hybrid nanocarrier for drug delivery applications, an upcoming field. It is much more efficient than a single nanocarrier system [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. Synthetic polymeric nanocarriers are toxic and sometimes cause tissue damage; polymer-protein hybrid nanoparticles are considerably less harmful than individual nanocarriers [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. The key factor in nanoparticle development is their drug-loading capacity, typically less than 2% for most approved drugs. Polymeric nanoparticles have a loading capacity lower than 5% [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. Recent research reported combining PLGA-chitosan nanoparticles shows higher encapsulation efficiency and lower drug-loading capacity [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. PLGA is an FDA-approved poly (lactic-co-glycolic) acid copolymer for drug encapsulation [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. It is biodegradable, biocompatible, non-toxic, durable, easy to surface modification, stable, and ideal for controllable drug delivery. However, it shows higher cytotoxicity [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. Thus, PLGA combined with protein reduces its toxicity. Protein is a natural polymer isolated from plants and animal sources [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]. Plant protein is more significant in drug delivery than animal protein since it is safe, economically cheap, easy to scale up, and renewable [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. It is also biodegradable, biocompatible, non-antigenic, and reported as GRAS (Generally Recognized as Safe) [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. The possibility of disease occurrence was minimal in plant-based protein unless animal protein causes diseases like bovine spongiform encephalitis (mad cow disease) [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. Plant-based protein is hydrophobic; thus, there is no need for crosslinkers. It is highly stable, and it releases the drug in a controlled manner [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. There are numerous plant proteins, such as collagen, Albumin, and cellulose, but these plant-based proteins lack stability in aqueous conditions [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. Glutenin is a wheat protein that is highly stable in aqueous conditions and various physiological conditions [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. PLGA-Glutenin (PLGA-Glu) hybrid nanoparticles have not yet been reported previously. It is highly anionic/cationic, enabling it to load various drugs effectively. PLGA-Glu nanocarrier system improves the pharmacodynamic and pharmacokinetic properties of the encapsulated drug [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTargeted delivery of drugs into the disease site/cancer cells is crucial in drug delivery carrier systems [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. Cancer cells often upregulate many receptors on their surface for rapid proliferation and survival. These receptors are recognized as effective targets for drug intervention. A technique involves delivering toxic payloads to destroy cancer cells that express high quantities of certain receptors; one such receptor is the folate receptor (FR) [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. FR is a membrane-bound glycoprotein that reduces folates (such as 5-methyltetrahydrofolate folic acid) and helps in the proliferation of cancer cells [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e]. FR is upregulated in various types of cancers, including breast, ovarian, endometrial, mesothelioma, and lung [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. It was upregulated 35\\u0026ndash;68% in triple-negative breast cancer and 20% in tissue samples of non-malignant breast cancer [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e]. This is the main reason for considering FR as a valid anti-cancer drug target. Its expression is limited to non-malignant tissue and thus does not affect normal healthy cells [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. Conjugating folic acid on the surface of the Glu-PLGA carrier to selectively deliver Camptothecin into cancer cells by entering the cell through FR by endocytosis [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. In this work, we proposed a novel drug delivery system called the folic acid- conjugated Camptothecin-loaded glutenin-PLGA nanoparticles (FA-CPT-PLGA-Glu-NPs). This delivery system is capable of efficiently delivering Camptothecin to cancer cells. Furthermore, FA-CPT-PLGA-Glu was characterized by XRD, FTIR, particle size, and HRTEM. The cytotoxic effect of FA-CPT-PLGA-Glu nanoparticle was analyzed against MCF-7.\\u003c/p\\u003e\"},{\"header\":\"Experimental section\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eChemicals\\u003c/h2\\u003e \\u003cp\\u003eCamptothecin and copolymer, poly(lactic-co-glycolic) acid were obtained from Sigma Aldrich, Bangaluru., India. MES (2-(N-morpholino) ethanesulfonic acid) buffer, Polyvinyl alcohol (PVA), and Dichloromethane (DCM) were received from HiMedia Laboratories, Pvt. Ltd., Mumbai, India. Folic acid (97.95%) was obtained as a gift from Alkem Laboratories, Mumbai, Maharashtra, India. All other remaining chemicals, reagents, and solvents were procured from Thermo Fisher Scientific Ltd., Mumbai, India.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCancer cell line\\u003c/h2\\u003e \\u003cp\\u003eMCF-7 cells (human adenoma breast cancer cells) were obtained from NCCS, Pune, Maharashtra, India, and maintained in RPMI1640 medium (pH 7.2\\u0026ndash;7.4) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% mixture of penicillin/streptomycin, \\u0026amp; amphotericin B and incubated at 37\\u0026deg;C in a humidified atmosphere with 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGlutenin extraction\\u003c/h2\\u003e \\u003cp\\u003eAs reported in our previous work, glutenin was extracted from wheat flour. The FTIR and \\u003csup\\u003e13\\u003c/sup\\u003eC-NMR spectrums were used to confirm glutenin before it was used in the nanoparticle formulation[\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFormulation of PLGA-Camptothecin-loaded Nanoparticles (PLGA-CPT NPs)\\u003c/h2\\u003e \\u003cp\\u003eA slightly changed emulsification/evaporation method was applied to formulate camptothecin-loaded PLGA nanoparticles. Briefly, PVA (0.05g, w/w) was dissolved in deionized water to create an aqueous phase, which was then adjusted to a pH of 5.0 using MES buffer (50 mM). In the organic phase, 0.025 g of PLGA was mixed in 3 mL of dichloromethane and 3 mg of CPT in 2 mL of ethanol. The resulting combination (organic phase) was slowly added to the aqueous phase while stirring continuously with a magnetic stirrer. During the stirring process, the entire system was kept in an ice bath to prevent the oil phase from evaporating, and the mixture was magnetically swirled overnight. Nanoparticles were purified by centrifugation three times with a high-speed centrifuge.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eLayer-by-layer assembly of Glu-PLGA NPs loaded with Camptothecin\\u003c/h2\\u003e \\u003cp\\u003eGlutenin-composed CPT-loaded PLGA NPs were fabricated using a layer-by-layer assembly technique[\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. The freshly prepared PLGA nanoparticles (1 mg \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e) were distributed in the glutenin solution (2 mg diluted in 5 mL of acetic acid) by rotating and oscillating until the necessary layers were created. Next, a 0.5 mL of a 2% aqueous glutaraldehyde solution was introduced to facilitate cross-linking of the layers. Following centrifugation, a 30 mM sodium borohydride solution was employed to stop the cross-linking reaction after 30 min. Subsequently, the sample was rinsed three times with deionized water. The glutenin-layered particles were incubated with organic solvents, specifically tetrahydrofuran (THF), to remove the cores. The use of sonication facilitated this process.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDetermination of encapsulation efficiency of CPT-loaded- Glu-PLGA NPs\\u003c/h2\\u003e \\u003cp\\u003eA UV-visible spectrophotometer measured the drug encapsulation efficiency of the CPT-loaded into Glu-PLGA NPs at 225 nm. CPT-loaded-Glu-PLGA NPs were centrifuged at 5000 rpm, and the supernatant was analyzed by UV-visible spectrophotometer at 225 nm. The quantity of CPT was determined by plotting a graph with a standard graph of CPT. Further, loading capacity (%W/W) and encapsulation efficiency were determined using the below equations (1) and (2), respectively.\\u003c/p\\u003e \\u003cp\\u003eLoading capacity (%) = [(CPT)added\\u0026minus;(CPT)measured/Nanoparticle weight] \\u0026times; 100\\u003c/p\\u003e \\u003cp\\u003eEncapsulation efficiency (%) = [(CPT)added\\u0026minus;(CPT)measured/(CPT)added] \\u0026times; 100\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eIn vitro\\u003c/b\\u003e \\u003cb\\u003edrug release study\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe release of CPT from CPT-PLGA-Glu NPs was measured using the dynamic membrane dialysis method in acetate buffer (pH 5.3, 0.01M) and phosphate buffer (pH 7.2, 0.01M) solutions. Drug release research was done to ascertain the solubility of the drug in buffer solutions, which served as the external medium. In summary, CPT-Glu-PLGA NPs (30 mg \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e) were enclosed in a dialysis membrane bag with a molecular weight cut-off of 3500 Da. The bag was then immersed in a buffer solution and placed on a shaker at a temperature of 37\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.00\\u0026deg;C and a speed of 150 rpm. At specific time intervals, samples were taken out and substituted with new media. The supernatant obtained from the collection was mixed with ethanol, and the quantity of the medicine (CPT) released from the NPs was measured at 225 nm using UV-visible spectrophotometry at certain time intervals. The formula below was employed to compute the percentage of medication release.\\u003c/p\\u003e \\u003cp\\u003e% Drug release = (A\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003csub\\u003e1\\u003c/sub\\u003e) \\u0026times; 100/A\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003cp\\u003ewhere A\\u003csub\\u003e0\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;absorbance of the control, and A\\u003csub\\u003e1\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;absorbance of the sample\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDrug release kinetics\\u003c/h2\\u003e \\u003cp\\u003eThe drug release kinetics and diffusion mechanisms of CPT-Glu-PLGA NPs were studied using five basic kinetic models: zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell models[\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. The Drug Distribution (DD Solver) 1.0 software, a Microsoft Excel plug-in tool, is utilized to assemble and analyze \\u003cem\\u003ein vitro\\u003c/em\\u003e drug release data to determine the mechanism of drug release kinetics. The model with the lowest AIC (Akaike information criterion) value is the most suitable for predicting drug release. The exponent 'n' significantly influences the drug release kinetics. For values of 'n' less than 0.45, the release follows a Quasi-fickian behavior. When 'n' equals 0.45, it exhibits Fickian diffusion. When 'n' falls between 0.45 and 0.89, the diffusion is classified as non-fickian. For values of 'n' greater than 1, it is considered.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFabrication of folic acid-conjugated CPT-loaded-Glu-PLGA NPs (FA-CPT-PLGA-Glu NPs)\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eSynthesis of N-hydroxysuccinimide ester of FA (FA-NHS)\\u003c/h2\\u003e \\u003cp\\u003eThis procedure is performed in the dark to prevent the structural deprivation of folic acid[\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e]. Shortly, 1.05 g of FA was solubilized in 25 mL of DMSO. At the same time, 0.44 g of N-hydroxy succinimide, 1.05 g of N, N- dicyclohexylcarbodiimide, and 0.25 mL of triethyl amine were added. This mixture was kept in a water bath at 40\\u0026deg;C for 48 h. After 48 h, it was filtered to remove the by-product of dicyclohexylurea. The synthesized FA-NHS was freeze-dried, which was confirmed by NMR and FTIR.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFA-CPT-PLGA-Glu NPs\\u003c/h2\\u003e \\u003cp\\u003eThe FA-CPT- PLGA-Glu NPs were fabricated by conjugating the carboxylic acid group of FA into an amino group of glutenin in PLGA-Glu-loaded CPT NPs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Concisely, 5 mg of CPT- PLGA-Glu NPs dissolved in carbonate/bicarbonate buffer (pH 10.0; 3 mL; 0.1 M). DMSO solubilized FA-NHS (1 mg \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e) was added to the CPT-PLGA-Glu NPs solution and kept in a magnetic stirrer for 2\\u0026ndash;3 h. DMSO was removed by membrane dialysis against a phosphate buffer solution of pH 7.4 to remove unreacted FA-NHS. The FA-CPT-PLGA-Glu NPs were centrifuged for 30 min at 21000 rpm and then re-dispersed in deionized water (4.0 mL).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCharacterization studies of FA-CPT-PLGA-Glu NPs\\u003c/h2\\u003e \\u003cp\\u003eThe X-ray diffractometer was used to evaluate the physical characteristics of the FA-CPT- PLGA-Glu NPs. The morphological features of the FA-CPT- PLGA-Glu NPs were assessed by high resolution transmission electron microscopy (HRTEM). Average particle size was measured by dynamic light scattering (DLS) analysis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStability study of FA-CPT-PLGA-Glu NPs\\u003c/h2\\u003e \\u003cp\\u003eA stability study was performed under various physiological mediums, such as 5% NaCl, 0.5% BSA, acetate buffer (pH 5.3), and phosphate buffer (pH 7.0 and 9.0). 1 mL of FA-CPT-PLGA-Glu NPs was added to 9 mL of each physiological medium, and it was monitored for consecutive days. Then, the absorbance was measured in UV-visible spectroscopy between 200 to 600 nm.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eIn silico\\u003c/b\\u003e \\u003cb\\u003emolecular modelling\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eProtein Preparation: The Research Collaboratory for Structural Bioinformatics manages the PDB database, which lists Caspase-3 as 3DEH at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.rcsb.org/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.rcsb.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. X-ray crystallography is used to determine protein crystal structures. The protein is pre-processed using CHARMM-GUI to remove water molecules, ligands, and missing residues[\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e]. Ligand Preparation: The Camptothecin, Doxorubicin, and 3DEH cocrystal were docked with the Protein Data Bank Caspase-3 protein (3DEH). The free PubChem database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://pubchem.ncbi.nlm.nih.gov/\\u003c/span\\u003e\\u003cspan address=\\\"https://pubchem.ncbi.nlm.nih.gov/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) provided the \\u0026lsquo;.sdp\\u0026rsquo; files. The chemicals and created cocrystals were processed using BIOVIA Discovery Studio Visualizer to prepare the cluster for analysis [\\u003cspan additionalcitationids=\\\"CR2 CR3 CR4\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Docking Protocol: Intermediate steps AutoDock Vina-POAP for Virtual Screening converted proteins and ligands into \\u0026lsquo;pdbqt\\u0026rsquo; files. A setup document includes protein, ligand, and grid box configuration metrics. The AutoDock Vina-POAP virtually screened the protein, revealing polar hydrogens, solvation parameters, and fragmental volumes. AutoGrid designed the grid map's grid box, with the grid centre at x (-46.8), y (15.02), and z (21.9). A scoring grid based on the ligand's structure was created to speed up computation. If proteins and ligands are stiff, AutoDock Vina docks them via iterated local search global optimization. The results with the lowest binding free energy were analyzed, while those with a positional root mean square deviation above 1.0A were discarded. Through the BIOVIA Discovery Studio Visualizer, the amino acid interaction was evaluated from the highest binding affinity or lowest binding site[\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e]. Identification of the binding site: Proteins need a unique binding site to hook onto molecules and a primary mechanism to ensure bioactive chemicals have adequate sites for chemical reactions. These proteins and enzymes need precise ligand or bioactive engagement sites to interact with the desired enzymes. PrankWeb (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://prankweb.cz/\\u003c/span\\u003e\\u003cspan address=\\\"https://prankweb.cz/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) has been used to find every active target material binding site. The receptor grid was built from the protein's active site using AutoDock Vina-POAP for virtual screening[\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMolecular dynamics\\u003c/h2\\u003e \\u003cp\\u003eWe used molecular dynamics (MD) simulations to assess protein-ligand stability and variability. The major goal was to study how therapeutic drugs bind to target proteins. These simulations used Schrodinger Desmond module and the Maestro simulation platform on Linux. The TIP3 water model and cuboidal border constraint modified complex protein-ligand interactions. The system was neutralized with 0.15 M Na\\u0026thinsp;+\\u0026thinsp;and Cl\\u003csup\\u003e-\\u003c/sup\\u003e salts. MD simulations used the NPT ensemble to maintain 1.01325 bar pressure and 300 K temperature. OPLS-4 force field energy assessments were performed during simulations with 50 picosecond data gathering. One macro-level simulation lasted 100 nanoseconds. Schr\\u0026ouml;dinger's Desmond module helped analyze simulation data. The study was improved using a simulated interaction diagram method to evaluate the ligand-protein complex's stability using RMSD, RMSF, and protein-ligand interaction analysis across simulated paths [\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eIn vitro\\u003c/b\\u003e \\u003cb\\u003ecytotoxicity assay\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe cytotoxic ability of FA-CPT-PLGA-Glu NPs was estimated against breast cancer cells (MCF-7) via colour change of yellow MTT (3,4,5-dimethyl thiazolyl-2-2-5-diaphenyl tetrazolium bromide) salt into purple formazan crystals. The change due to mitochondrial enzymes in metabolically active cells will affect MTT [\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e]. Briefly, the cells are cultured in a 96-well microplate and maintained at 37 \\u0026ordm;C in a humidified environment with 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e for 24h. After 24 h of initial incubation, the cells were expose with various concentrations of FA-CPT-PLGA-Glu NPs (100, 50, 25, 12.5, 6.25, 3125 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e), 100 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e of CPT, and 0.25 \\u0026micro;M of Doxorubicin for 24 h. After 24 h of exposure, the media was discarded, and cells were rinsed with phosphate buffer solution. Next, 20 \\u0026micro;L of MTT staining (5 g \\u0026times; L\\u003csup\\u003e-1\\u003c/sup\\u003e) solution was added to the serum-free DMEM medium and incubated in a 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e environment at 37 \\u0026ordm;C for another 4 h. Then, the medium was removed, and 150 \\u0026micro;L of DMSO was added to dissolve formazan crystals produced when tetrazolium MTT was converted. While it dissolves in DMSO, the purple colour varies based on viability. Absorbance was taken at 570 nm in an ELISA reader by which cytotoxicity was evaluated. The below-given equation was used to measure the % cellular viability:\\u003c/p\\u003e \\u003cp\\u003e% Viability\\u0026thinsp;=\\u0026thinsp;At /Ac \\u0026times;100\\u003c/p\\u003e \\u003cp\\u003eAt and Ac are the mean absorbance of FA-CPT-PLGA-Glu NPs treated and control cells, respectively (n\\u0026thinsp;=\\u0026thinsp;5, where n is the number of independent experiments).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEvaluation of apoptosis by AO/EBr staining\\u003c/h2\\u003e \\u003cp\\u003eThe MCF-7 cells were cultured in a 24-well plate in a CO\\u003csub\\u003e2\\u003c/sub\\u003e incubator overnight. After incubation, it was treated with IC\\u003csub\\u003e50\\u003c/sub\\u003e concentration of FA-CPT-PLGA-Glu NPs for 48 h. The cells were rinsed three times with ice-cold phosphate-buffered solution and treated with DNA binding strains 10 \\u0026micro;L of acridine orange (AO) and ethidium bromide (EBr) (100 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e, each). AO invades the nuclei and makes them green, while EBr invades only cytoplasmic membrane integrity, which is lost to non-viable cells and stains the nuclei red, indicating apoptosis. Each experiment was repeated at least three times[\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMeasurement of mitochondrial transmembrane potential\\u003c/h2\\u003e \\u003cp\\u003eRhodamine-123 (Rh-123) staining dye indicates potential damage to the mitochondrial membrane by penetrating the cell membrane. Mitochondrial function is important for predicting healthy cells; this assay will evaluate mitochondrial dysfunction. The cultured MCF-7 cells were treated with IC\\u003csub\\u003e50\\u003c/sub\\u003e concentration of FA-CPT-PLGA-Glu NPs for 24 h. After 24 h treatment, cells were harvested, stained with Rh-123 dye, and kept for 30 min. Later, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Morphological changes and membrane permeability were evaluated by fluorescence microscopy. The untreated cells were used as a control[\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMeasurement of nucleus-induced apoptosis by DAPI assay\\u003c/h2\\u003e \\u003cp\\u003eDAPI (4',6-diamidino-2-phenylindole) is a blue fluorescent DNA staining dye; it evaluates how far the drug affects the nucleus of the cell line[\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e]. A healthy cell has an intact cell membrane and does not allow the dye to pass through; thus, it stains dead cells effectively. The MCF-7 cells were grown on the 24-well plate and incubated in a CO\\u003csub\\u003e2\\u003c/sub\\u003e incubator at 37 \\u0026ordm;C for 24 h. IC\\u003csub\\u003e50\\u003c/sub\\u003e concentration of FA-CPT-PLGA-Glu NPs was added and kept for 48 h. After 48h, the treated cells were washed with PBS buffer, and 4% paraformaldehyde was added to fix them for 30 min. The cells were incubated for 5 min with 20 \\u0026micro;L DAPI (0.5 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e) at room temperature and examined under a fluorescence microscope.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eQuantification of Caspase-3 and \\u0026minus;\\u0026thinsp;9 activities\\u003c/h2\\u003e \\u003cp\\u003eThe quantification of caspase activity was performed using Caspase-3 and \\u0026minus;\\u0026thinsp;9 assay kits (Caspase-Glo\\u0026reg; 3 and 9 reagents, Promega)[\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e]. In summary, 1\\u0026times;10\\u003csup\\u003e5\\u003c/sup\\u003e MCF-7 cells per well were cultured in a 96-well plate. The cell plate was placed in a humidified incubator at 37\\u0026deg;C with 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e for 24 h. The 96-well plate with the IC\\u003csub\\u003e50\\u003c/sub\\u003e concentration of FA-CPT-PLGA-Glu NPs and the untreated control cells were left to reach a stable state at room temperature. Each well of a 96-well plate (including both the test well and control) was supplemented with 100 \\u0026micro;L of Caspase-Glo\\u0026reg; 3 or 9 reagent, along with 100 \\u0026micro;L of culture media. The plate was sealed, and the contents were agitated for 30 seconds at 500 revolutions per minute. The optical density was quantified using an ELISA reader at a wavelength of 405 nm following a 30 min incubation of the plate at room temperature.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnalysis of intercellular reactive oxygen species (ROS) levels\\u003c/h2\\u003e \\u003cp\\u003e2\\u0026prime;,7\\u0026prime;-dichlorofuorescein diacetate (DCFH-DA) was used to measure intracellular ROS levels[\\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e]. MCF-7 cells were cultured for 24 h and treated with IC\\u003csub\\u003e5\\u003cb\\u003e0\\u003c/b\\u003e\\u003c/sub\\u003e concentration of FA-CPT-PLGA-Glu NPs. Treated cells were washed twice with PBS before labelling with 10 M DCFH-DA. DCFH-DA is a non-fluorescent probe that will be converted into fluorescent dichlorofluorescein (DCF) when oxidized by ROS. Fluorescence intensity was measured using a fluorescence spectrophotometer.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTerminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay\\u003c/h2\\u003e \\u003cp\\u003eThe tunnel assay, also known as the Tdt-dUTP nick end labelling test, employs the Tdt enzyme and fluorescently tagged nucleotide to measure the number of apoptotic cells accurately[\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e]. MCF-7 cells were incubated for 24 h in a 5% carbon dioxide environment at 37\\u0026deg;C. After a 24-h period, the cells were exposed to a concentration of FA-CPT-PLGA-Glu NPs that is equivalent to the IC\\u003csub\\u003e50\\u003c/sub\\u003e value. The cells were then incubated at 37\\u0026deg;C for an additional 24 h. The sample is immersed in a 0.5 mL solution of PBS containing 4.5 mL of ice-cold 1% formaldehyde within a polypropylene tube. The tube is then placed in ice for a duration of 5 min. After 5 min, the substance is subjected to centrifugation at a speed of 1500 rpm for 5 min. The cellular sediment was gathered and resuspended in 5 mL of phosphate-buffered saline (PBS) and subjected to centrifugation at 1200 rpm for 3 min. Using a Pasteur pipette, move the cell suspension to a tube with 4.5 mL of ice-cold 70% ethanol. The ethanol was extracted and subsequently reconstituted in 0.5 mL of PBS. The sample was transferred to a volume of 50 \\u0026micro;L of the Apo-Direct TUNEL Assay Kit and incubated for 40 min at 37\\u0026deg;C. Subsequently, 1.5 mL of washing buffer was introduced and incubated for 1 h. Subsequently, 1 mL of propidium iodide staining solution was introduced and incubated for an additional 30 min at room temperature without light. The resulting mixture was then examined using Flow cytometry (BD FACS Calibur-Becton Dickinson, CytExpert v 1.2.11.0) to ascertain the presence of cells exhibiting DNA damage. The outcome is quantified as the proportion of apoptosis, calculated by adding the percentage of healthy cells to the percentage of cells with damaged DNA.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eStability studies\\u003c/h3\\u003e\\n\\u003cp\\u003eStability in physiological medium and buffer solutions was one of the key parameters for the nano-formulation of an efficient drug delivery system. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e illustrates the stability of the freshly formulated FA-CPT-PLGA-Glu NPs in the physiological mediums (5% NaCl and 0.5% BSA) buffer solution of acetate buffer (pH 5.3) and PBS (pH: 7.4 and 9). The λmax of FA-CPT-PLGA-Glu NPs in the physiological medium and buffer solutions was 340 nm. There is no change in the λmax for 72 h incubation in physiological medium and buffer solutions.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eIn silico\\u003c/b\\u003e \\u003cb\\u003emolecular modelling\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eCamptothecin, Doxorubicin, and Co-crystal molecular docking studies against Caspase-3 protein (PDB: 3DEH) to treat breast cancer. In addition to Co-crystal, Camptothecin, and Doxorubicin demonstrated high amino acid interaction and binding affinity for 3DEH Caspase-3 protein. The top-scoring medicines are Camptothecin, Doxorubicin, and 3DEH Co-crystal, with binding energies of -11.3, -9.1, and \\u0026minus;\\u0026thinsp;6.6 kcal \\u0026times; mol\\u003csup\\u003e-1\\u003c/sup\\u003e, respectively, from the internal database. Amino acids Thr166(C), Ocs163(C), Gly122(A), and Doxorubicin Thr62(A) and His121(A) interacted most with Cocrystal. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e illustrate the 2D and 3D models of Camptothecin, Doxorubicin, co-crystal binding interactions.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec31\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMolecular dynamics (MD)\\u003c/h2\\u003e \\u003cp\\u003eThe protein-ligand complex's RMSD and RMSF were calculated. Additionally, 3DEH Camptothecin, 3DEH Doxorubicin, and 3DEH Co-crystal protein-ligand complexes were studied to determine their interaction patterns (supplementary Figs.\\u0026nbsp;1\\u0026ndash;3). We found that protein-ligand contacts and interaction signatures significantly affect the complex by examining amino acid residue interactions and RMSD and RMSF computations. The MD simulation of Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e\\u0026ndash;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e dissipated the range of interactions displayed by 3DEH Camptothecin complex from 0.21 to 3.33 in protein and 2.26 to 3.83 in ligand, 3DEH Doxorubicin complex from 0.17 to 4.47 in protein and 0.17 to 4.47 in ligand, and 3DEH Co-crystal complex from 0.16 to 6.32 in protein and 0.23 to 6.86 in ligand. Camptothecin, Doxorubicin, and Co-crystal protein-ligand complexes had consistent RMSF values during molecular dynamic modeling. During the MD simulation, the fluctuation curves showed that Camptothecin, Doxorubicin, and 3DEH Co-crystal had constant amino acid interactions, this compound had no fluctuation, and the residual index of RMSF had excellent stability. The protein-ligand interactions and RMSD complex show that amino acids interact directly with Camptothecin, Doxorubicin, and 3DEH Co-crystal. The MD simulation interaction and Camptothecin, Doxorubicin, and 3DEH Co-crystal binding contact confirmed the His121 amino acid interaction. Camptothecin, Doxorubicin, and Co-crystal docking research shows that His121 and Tyr204 interact similarly to the MD simulation. It demonstrates rigid and flexible connections in Camptothecin and Doxorubicin.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec32\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCytotoxicity\\u003c/h2\\u003e \\u003cp\\u003eBased on the results of \\u003cem\\u003ein silico\\u003c/em\\u003e molecular modeling studies, we determined the cytotoxic potential of formulated FA-CPT-PLGA-Glu NPs against MCF-7 cells using the MTT assay. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(a) shows the cytotoxic potential of different doses of FA-CPT-PLGA-Glu NPs, 200 \\u0026micro;g CPT, and 0.25 \\u0026micro;M Doxorubicin. After 24 hours of treatment, FA-CPT-PLGA-Glu NPs reduced cellular viability concentration-dependent. FA-CPT-PLGA-Glu NPs had an IC\\u003csub\\u003e50\\u003c/sub\\u003e value of 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e after 24 h of incubation with the cell line (n\\u0026thinsp;=\\u0026thinsp;3). Cells treated with 3.125 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FA-CPT-PLGA-Glu NPs showed approximately 89% viability, while those incubated with 100 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e CPT showed around 42% cell viability. Figures\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(b) and (c) show how incubation with 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FA-CPT-PLGA-Glu NPs altered the shape of MCF-7 cells.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec33\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eAcridine Orange-Ethidium Bromide (AO/EBr) apoptosis analyses\\u003c/h2\\u003e \\u003cp\\u003eThe AO/EBr fluorescent microscopic staining technique was employed to observe the morphological alterations in MCF-7 cells. The AO/EBr staining method can be used to distinguish between cells undergoing apoptosis and those that are in a normal state. Figures\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e (d) and (e) depict the untreated control and the exposure to FA-CPT-PLGA-Glu NPs at a concentration of 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e for 48 h. The illustration demonstrates that the control cells did not undergo any changes and remained green after staining. In contrast, the treated cells exhibited a colour change (orange), suggesting the presence of apoptotic cells. Furthermore, the treated cells exhibit membrane blebbing, shrinkage, and nuclear disintegration. This outcome suggests a significant accumulation of CPT within the MCF-7 cells.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec34\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eMitochondrial transmembrane potential loss\\u003c/h2\\u003e \\u003cp\\u003eThe decrease in mitochondrial membrane potential in MCF-7 cells incubated with FA-CPT-PLGA-Glu NPs was quantified using rhodamine-123 staining. A notable reduction in mitochondrial membrane potential was detected in MCF-7 cells following exposure to a dose of 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e (IC\\u003csub\\u003e50\\u003c/sub\\u003e concentration) of FA-CPT-PLGA-Glu NPs, as shown in Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(f) and (g). Further, the FA-CPT-Glu-PLGA NPs caused the mitochondrial membrane to depolarize. The findings of our study showed that the FA-CPT-PLGA-Glu NPs could enter the mitochondrial membrane through endocytosis and cause cell death by reducing the mitochondrial membrane potential.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eNuclear Morphology\\u003c/h3\\u003e\\n\\u003cp\\u003eThe impact of FA-CPT-PLGA-Glu NPs on nuclear alterations was examined by DAPI staining technique. Figures\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(h) and (i) demonstrates notable modifications in the structure of the chromatin nuclear material following DAPI labeling of FA-CPT-PLGA-Glu NPs-treated cells for 24 h compared to the untreated control. The control cells exhibit typical spherical nuclei with a normal blue hue, while the treated cells display a vivid colour, aberrant nuclei, and condensed chromatin with uneven cell shape.\\u003c/p\\u003e\\n\\u003ch3\\u003eCaspase-3 and − 9 Activities\\u003c/h3\\u003e\\n\\u003cp\\u003eThe apoptosis was further authenticated by measuring the production levels of caspase-3 and \\u0026minus;\\u0026thinsp;9 in FA-CPT-PLGA-Glu NPs-treated MCF-7 cells over the untreated control group. Caspases 3 and 9 are the terminal phase inducers of programmed cell death in cancer cells when activated by external stimuli. The caspase-3 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(j)) and \\u0026minus;\\u0026thinsp;9 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(k)) activities were twofold enhanced in cells exposed to 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e (IC\\u003csub\\u003e50\\u003c/sub\\u003e conc.) FA-CPT-PLGA-Glu NPs and extract compared to control (untreated) cells.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec37\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eROS production\\u003c/h2\\u003e \\u003cp\\u003eThe fluorescent microscopic observations determined intracellular reactive oxygen species (ROS) production in MCF-7 cells treated with FA-CPT-PLGA-Glu NPs (16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e). The untreated MCF-7 cells demonstrated lower ROS generation (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(l) and 14(m)), the onset of apoptosis in the MCF-7 cells. The present work observed that FA-CPT-PLGA-Glu NPs can induce ROS generation in cancer cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(n)) using the DCFH-DA staining method.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec38\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eTUNEL Assay\\u003c/h2\\u003e \\u003cp\\u003eThe TdT-mediated dUTP nick end labeling (TUNEL) test was used to evaluate nuclear DNA fragmentation (apoptosis) by flow cytometry. This investigation was conducted when the development of cancer cells was reduced through the activation of apoptosis. The flow cytometry analysis of MCF-7 cells is shown in Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e (o) and (p), depicting the cells' state before (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(o)) and after (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e(p)) being treated with FA-CPT-PLGA-Glu NPs at a concentration that inhibits 50% of cell growth (IC\\u003csub\\u003e50\\u003c/sub\\u003e) for a duration of 24 h. After being treated with a concentration of 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e of FA-CPT-PLGA-Glu NPs, 38.6% of cells with DNA damage were observed, which was substantially higher than the percentage of DNA-damaged cells in the untreated group (9.46%).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\" \\u003cdiv id=\\\"Sec22\\\" type=\\\"Results\\\" class=\\\"Section2\\\"\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eGlutenin\\u003c/h2\\u003e \\u003cp\\u003eFTIR and \\u003csup\\u003e13\\u003c/sup\\u003eC-NMR confirmed the structure of the extracted glutenin from wheat flour. The purified glutenin is observed at highly conserved sites and is involved in intrachain with disulfide (SS) linkages. FTIR and NMR spectral data are presented in our previously published article[\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCPT-loaded PLGA NPs\\u003c/h2\\u003e \\u003cp\\u003eCPT-loaded PLGA nanoparticles were initially formulated using a modified emulsification /evaporation technique. The active drug (CPT) was slowly encapsulated into the copolymer PLGA; the encapsulation was confirmed by a UV-visible spectrophotometer (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). From the UV-visible spectra, the absorption peak of camptothecin slowly disappeared, and a PLGA peak appeared. This peak change confirmed camptothecin encapsulation into PLGA.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec25\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eLayer-by-layer formulation of CPT-loaded PLGA-Glu NPs\\u003c/h2\\u003e \\u003cp\\u003eLayer-by-layer assembly of glutenin on the surface of CPT-loaded PLDA NPs was confirmed by FTIR spectroscopy. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e presents the FTIR spectra of CPT (a), CPT-loaded PLGA-Glu NPs (b), and glutenin (c). In the CPT spectrum, 3431 cm\\u003csup\\u003e-1\\u003c/sup\\u003e represents the -OH stretch of hydrogen bonding, 3253 cm\\u003csup\\u003e-1\\u003c/sup\\u003e represents the aromatic ring, 2976 cm\\u003csup\\u003e-1\\u003c/sup\\u003e denotes -CH\\u003csub\\u003e3\\u003c/sub\\u003e (alkane stretch), 2364 cm\\u003csup\\u003e-1\\u003c/sup\\u003e represents C\\u0026thinsp;=\\u0026thinsp;C stretching, and 1745 cm\\u003csup\\u003e-1\\u003c/sup\\u003e represents -C\\u0026thinsp;=\\u0026thinsp;O group, and 1597 represents the amide group. CPT-loaded PLGA NPs spectrum showed 3333 cm\\u003csup\\u003e-1\\u003c/sup\\u003e indicates the -OH stretching group of Glutenin, CPT and PLGA, 1583 cm\\u003csup\\u003e-1\\u003c/sup\\u003e indicates the amide group from Glutenin and CPT, 1416 cm\\u003csup\\u003e-1\\u003c/sup\\u003e denotes -CH\\u003csub\\u003e3\\u003c/sub\\u003e (alkane stretching) from CPT, Glu, and PLGA. These peaks indicated that CPT was confirmed to be encapsulated in the PLGA-Glu NPs. CPT encapsulation efficiency and loading capacity were observed at 74.95\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.34% and 4.78\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.08%, respectively.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec26\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eDrug release\\u003c/h2\\u003e \\u003cp\\u003eVarious parameters, including polymer type and contents of drug-polymer interactions, can influence the drug release patterns of NPs. \\u003cem\\u003eIn vitro\\u003c/em\\u003e, drug release tests were conducted at 37\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.0\\u0026deg;C using a dialysis membrane, acetate buffer at pH 5.3, and phosphate buffer at pH 7.2. The release of CPT from CPT-loaded PLGA-Glu NPs occurred in two stages. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e shows the percentage of drug release from CPT-loaded PLGA-Glu NPs. PLGA-Glu NPs released CPT quickly, with burst release content of 23.47\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.986% and 20.867\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.1% at pH 5.3 and pH 7.2 within 8 h, respectively. CPT-loaded Glu-PLGA NPs produced the most CPT (75.517\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.04% at pH 5.3 and 70.557\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.86% at pH 7.2) within 28 h.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec27\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eDrug release kinetics\\u003c/h2\\u003e \\u003cp\\u003eFurther, the above drug-release data was used to evaluate the CPT-release kinetics from the CPT-PLGA-Glu NPs. Five simple models were fitted to the acquired data, including zero-order, first-order, Higuchi, Korsmeyer\\u0026ndash;Peppas, and Hixson\\u0026ndash;Crowell. Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e summarizes the findings of the \\u003cem\\u003ein vitro\\u003c/em\\u003e drug-release kinetics. The release rate constant of each kinetic model was predicted using the generated regression coefficient (r\\u003csup\\u003e2\\u003c/sup\\u003e) values. In general, significantly fitting the model, the r\\u003csup\\u003e2\\u003c/sup\\u003e value is near 1. Here, 0.9847 to 0.9275 (pH 5.3) and 0.9846 to 0.9368 (pH 7.2) were the values of the first-order kinetics regression coefficient (r\\u003csup\\u003e2\\u003c/sup\\u003e). These values were greater than those of the zero-order kinetics. Since these numbers are near 1, the models are regarded as good fits. On the other hand, the diffusion exponent (n) values in the Korsmeyer\\u0026ndash;Peppas model showed smaller variation, from 0.720 (pH 5.3) to 0.724 (pH 7.2). With plots displaying strong linearity and r\\u003csup\\u003e2\\u003c/sup\\u003e values in the range of 0.9344 (pH 5.3) and 0.9312 (pH 7.2), which mostly suggested the diffusion process, the Higuchi model was the most effective kinetic model. This shows that the formulation has been uniformly dissolved and released under control. Furthermore, the observed release kinetics data showed the CPT- PLGA-Glu NPs formulation non-Fickian behavior. Furthermore, because the diffusion exponent (n) was greater than 0.7 (pH 7.2, i.e., 0.724), it was demonstrated that the super case II transport drug-release mechanism was implicated. High r\\u003csup\\u003e2\\u003c/sup\\u003e values (0.9836) in the Hixson\\u0026ndash;Crowell model suggested uniform dissolution of the CPT- PLGA-Glu NPs and a consistent drug release. On the other hand, CPT release from the CPT- PLGA-Glu NPs was primarily maintained at a fixed amount of drug-release pattern at pH 5.3 and pH 7.2.\\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\\u003eDrug-release kinetics profile of Camptothecin (CPT) from CPT-Glu-PLGA NPs.\\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 \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eModel\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eParameter\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c4\\\" namest=\\\"c3\\\"\\u003e \\u003cp\\u003eCPT from CPT-Glu-PLGA NPs\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003epH 5.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003epH 7.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eZero order F\\u0026thinsp;=\\u0026thinsp;K\\u003csub\\u003e0\\u003c/sub\\u003e\\u0026times;t\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eK\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.039\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.037\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003er\\u003csup\\u003e2\\u003c/sup\\u003e adjusted\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.9375\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.9368\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eAIC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e147.4014\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e145.5374\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eFirst order\\u003c/p\\u003e \\u003cp\\u003eF\\u0026thinsp;=\\u0026thinsp;100\\u0026times; [1-Exp (-k1\\u0026times;t)]\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eK\\u003csub\\u003e1\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.001\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.001\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003er\\u003csup\\u003e2\\u003c/sup\\u003e adjusted\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.9847\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.9846\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eAIC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e117.8070\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e115.9276\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eHiguchi model F\\u0026thinsp;=\\u0026thinsp;KH\\u0026times;t\\u003csup\\u003e1/2\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eK\\u003csub\\u003eh\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.516\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.433\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003er\\u003csup\\u003e2\\u003c/sup\\u003e adjusted\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.9344\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.9312\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eAIC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e148.4185\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e147.3251\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"3\\\" rowspan=\\\"4\\\"\\u003e \\u003cp\\u003eKorsmeyer-Peppas model\\u003c/p\\u003e \\u003cp\\u003eF\\u0026thinsp;=\\u0026thinsp;kKP\\u0026times;t\\u003csup\\u003en\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ekKP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.310\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.283\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003er\\u003csup\\u003e2\\u003c/sup\\u003e adjusted\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.9758\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.9726\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003en\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.720\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.724\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eAIC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e129.4737\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e128.9280\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eHixon-Crowell model F\\u0026thinsp;=\\u0026thinsp;100\\u0026times;[1-(1-kHC\\u0026times;t)\\u003csup\\u003e3\\u003c/sup\\u003e]\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ekHC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.00\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.00\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003er\\u003csup\\u003e2\\u003c/sup\\u003e adjusted\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.9836\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.9815\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eAIC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e119.3100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e119.7788\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003ctfoot\\u003e \\u003ctr\\u003e\\u003ctd colspan=\\\"4\\\"\\u003eWhere, AIC\\u0026thinsp;=\\u0026thinsp;Akaike information criterion, F\\u0026thinsp;=\\u0026thinsp;fraction of drug release in time t, K\\u003csub\\u003e0\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;apparent rate constant of zero order release constant, K\\u003csub\\u003e1\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;first order release constant, K\\u003csub\\u003eH\\u003c/sub\\u003e =Higuchi constant, kKP\\u0026thinsp;=\\u0026thinsp;Korsmeyer-Peppas rate constant, kHC\\u0026thinsp;=\\u0026thinsp;Hixon-Crowell constant, n\\u0026thinsp;=\\u0026thinsp;diffusional exponent. And r\\u003csup\\u003e2\\u003c/sup\\u003e\\u0026thinsp;=\\u0026thinsp;Squared correlation coefficient.\\u003c/td\\u003e\\u003c/tr\\u003e \\u003c/tfoot\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec28\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFA-CPT-Glu-PLGA NPs\\u003c/h2\\u003e \\u003cp\\u003eUV-visible and FTIR spectroscopy initially synthesized and confirmed FA-NHS (N-hydroxysuccinimide ester of Folic acid). The UV-visible spectrum of FA-NHS was presented in our previously published article. FA and FA-NHS FT-IR spectra are displayed in\\u003c/p\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e(d) and 3(e). The FA-NHS spectrum shows two notable absorption peaks at 1666 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e due to a N\\u0026ndash;O bond and 1688 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e due to a C\\u0026thinsp;=\\u0026thinsp;O bond. These absorption peaks indicate that FA has been structurally transferred into FA-NHS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e(e)). Folic acid-conjugated CPT-loaded PLGA-Glu NPs were formulated by simple conjugation chemistry with FA-NHS and CPT-loaded PLGA-Glu NPs. The carboxylic group of FA-NHS was linked with the amino group of Glutenin from CPT-Glu-PLGA NPs by adding a carbonate/bicarbonate buffer. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e(f) presents the FTIR spectrum of FA-CPT-PLGA-Glu NPs. As can be seen, the FTIR spectra of FA-CPT-PLGA-Glu NPs, 3458 cm\\u003csup\\u003e-1\\u003c/sup\\u003e, indicate that the NH group from folic acid conjugation, 1937 cm\\u003csup\\u003e-1\\u003c/sup\\u003e and 1666 cm\\u003csup\\u003e-1\\u003c/sup\\u003e, indicate PLGA ester and acid bond absorption peaks. 1583 cm\\u003csup\\u003e-1\\u003c/sup\\u003e and 1416 cm\\u003csup\\u003e-1\\u003c/sup\\u003e indicate nitro compounds and -C-H bending stretching from glutenin protein.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec29\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCharacterization of FA-CPT-PLGA-Glu NPs\\u003c/h2\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e(a) depicts the X-ray diffraction (XRD) spectrum of FA-CPT-PLGA-Glu NPs. The XRD pattern displayed stronger peaks at 2θ conditions (19\\u0026deg;, 30\\u0026deg;, 34\\u0026deg;, 41\\u0026deg;, 46\\u0026deg;, and 57\\u0026deg;), indicating their crystalline character. DLS analysis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e(b)) confirmed their average particle sizes between 10 to 100 nm. Further, HRTEM pictures (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e(c)-(e)) demonstrate that the NPs had a 60 to 100 nm diameter and were spherical.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStability studies\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eStability in physiological medium and buffer solutions was one of the key parameters for the nano-formulation of an efficient drug delivery system.\\u0026nbsp;Figure 6\\u0026nbsp;illustrates the stability of the freshly formulated FA-CPT-PLGA-Glu NPs in the physiological mediums (5% NaCl and 0.5% BSA) buffer solution of acetate buffer (pH 5.3) and PBS (pH: 7.4 and 9). The \\u0026lambda;max of FA-CPT-PLGA-Glu NPs in the physiological medium and buffer solutions was 340 nm. There is no change in the \\u0026lambda;max for 72 h incubation in physiological medium and buffer solutions.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cstrong\\u003eIn silico\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e\\u0026nbsp;molecular modelling\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCamptothecin, Doxorubicin, and Co-crystal molecular docking studies against Caspase-3 protein (PDB: 3DEH) to treat breast cancer. In addition to Co-crystal, Camptothecin, and Doxorubicin demonstrated high amino acid interaction and binding affinity for 3DEH Caspase-3 protein. The top-scoring medicines are Camptothecin, Doxorubicin, and 3DEH Co-crystal, with binding energies of -11.3, -9.1, and -6.6 kcal \\u0026times; mol\\u003csup\\u003e-1\\u003c/sup\\u003e, respectively, from the internal database. Amino acids Thr166(C), Ocs163(C), Gly122(A), and Doxorubicin Thr62(A) and His121(A) interacted most with Cocrystal.\\u0026nbsp;Figure 7\\u0026nbsp;illustrate the 2D and 3D models of Camptothecin, Doxorubicin, co-crystal binding interactions.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMolecular dynamics (MD)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe protein-ligand complex\\u0026apos;s RMSD and RMSF were calculated. Additionally, 3DEH Camptothecin, 3DEH Doxorubicin, and 3DEH Co-crystal protein-ligand complexes were studied to determine their interaction patterns (supplementary Figures 1-3). We found that protein-ligand contacts and interaction signatures significantly affect the complex by examining amino acid residue interactions and RMSD and RMSF computations. The MD simulation of Figure 4-6 dissipated the range of interactions displayed by 3DEH Camptothecin complex from 0.21 to 3.33 in protein and 2.26 to 3.83 in ligand, 3DEH Doxorubicin complex from 0.17 to 4.47 in protein and 0.17 to 4.47 in ligand, and 3DEH Co-crystal complex from 0.16 to 6.32 in protein and 0.23 to 6.86 in ligand. Camptothecin, Doxorubicin, and Co-crystal protein-ligand complexes had consistent RMSF values during molecular dynamic modeling. During the MD simulation, the fluctuation curves showed that Camptothecin, Doxorubicin, and 3DEH Co-crystal had constant amino acid interactions, this compound had no fluctuation, and the residual index of RMSF had excellent stability. The protein-ligand interactions and RMSD complex show that amino acids interact directly with Camptothecin, Doxorubicin, and 3DEH Co-crystal. The MD simulation interaction and Camptothecin, Doxorubicin, and 3DEH Co-crystal binding contact confirmed the His121 amino acid interaction. Camptothecin, Doxorubicin, and Co-crystal docking research shows that His121 and Tyr204 interact similarly to the MD simulation. It demonstrates rigid and flexible connections in Camptothecin and Doxorubicin.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCytotoxicity\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBased on the results of \\u003cem\\u003ein silico\\u003c/em\\u003e molecular modeling studies, we determined the cytotoxic potential of formulated FA-CPT-PLGA-Glu NPs against MCF-7 cells using the MTT assay.\\u0026nbsp;Figure 8(a)\\u0026nbsp;shows the cytotoxic potential of different doses of FA-CPT-PLGA-Glu NPs, 200 \\u0026micro;g CPT, and 0.25 \\u0026micro;M Doxorubicin. After 24 hours of treatment, FA-CPT-PLGA-Glu NPs reduced cellular viability concentration-dependent. FA-CPT-PLGA-Glu NPs had an IC\\u003csub\\u003e50\\u003c/sub\\u003e value of 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e after 24 h of incubation with the cell line (n = 3). Cells treated with 3.125 \\u0026mu;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e FA-CPT-PLGA-Glu NPs showed approximately 89% viability, while those incubated with 100 \\u0026mu;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e CPT showed around 42% cell viability.\\u0026nbsp;Figures 8(b) and (c)\\u0026nbsp;show how incubation with 16.33 \\u0026mu;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e FA-CPT-PLGA-Glu NPs altered the shape of MCF-7 cells.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcridine Orange-Ethidium Bromide (AO/EBr) apoptosis analyses\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe AO/EBr fluorescent microscopic staining technique was employed to observe the morphological alterations in MCF-7 cells. The AO/EBr staining method can be used to distinguish between cells undergoing apoptosis and those that are in a normal state. Figures 8 (d) and (e)\\u0026nbsp;depict the untreated control and the exposure to FA-CPT-PLGA-Glu NPs at a concentration of 16.33 \\u0026mu;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e for 48 h. The illustration demonstrates that the control cells did not undergo any changes and remained green after staining. In contrast, the treated cells exhibited a colour change (orange), suggesting the presence of apoptotic cells. Furthermore, the treated cells exhibit membrane blebbing, shrinkage, and nuclear disintegration. This outcome suggests a significant accumulation of CPT within the MCF-7 cells.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMitochondrial transmembrane potential loss\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe decrease in mitochondrial membrane potential in MCF-7 cells incubated with FA-CPT-PLGA-Glu NPs was quantified using rhodamine-123 staining. A notable reduction in mitochondrial membrane potential was detected in MCF-7 cells following exposure to a dose of 16.33 \\u0026mu;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e (IC\\u003csub\\u003e50\\u003c/sub\\u003e concentration) of FA-CPT-PLGA-Glu NPs, as shown in\\u0026nbsp;Figures 8(f) and (g). Further, the FA-CPT-Glu-PLGA NPs caused the mitochondrial membrane to depolarize. The findings of our study showed that the FA-CPT-PLGA-Glu NPs could enter the mitochondrial membrane through endocytosis and cause cell death by reducing the mitochondrial membrane potential.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eNuclear Morphology\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe impact of FA-CPT-PLGA-Glu NPs on nuclear alterations was examined by DAPI staining technique.\\u0026nbsp;Figures 8(h) and (i)\\u0026nbsp;demonstrates notable modifications in the structure of the chromatin nuclear material following DAPI labeling of FA-CPT-PLGA-Glu NPs-treated cells for 24 h compared to the untreated control. The control cells exhibit typical spherical nuclei with a normal blue hue, while the treated cells display a vivid colour, aberrant nuclei, and condensed chromatin with uneven cell shape.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCaspase-3 and -9 Activities\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe apoptosis was further authenticated by measuring the production levels of caspase-3 and -9 in FA-CPT-PLGA-Glu NPs-treated MCF-7 cells over the untreated control group. Caspases 3 and 9 are the terminal phase inducers of programmed cell death in cancer cells when activated by external stimuli. The caspase-3 (Figure 8(j)) and -9 (Figure 8(k))\\u0026nbsp;activities were twofold enhanced in cells exposed to 16.33 \\u0026mu;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e (IC\\u003csub\\u003e50\\u003c/sub\\u003e conc.) FA-CPT-PLGA-Glu NPs and extract compared to control (untreated) cells.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eROS production\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe fluorescent microscopic observations determined intracellular reactive oxygen species (ROS) production in MCF-7 cells treated with FA-CPT-PLGA-Glu NPs (16.33 \\u0026mu;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e). The untreated MCF-7 cells demonstrated lower ROS generation (Figures 8(l) and 14(m)), the onset of apoptosis in the MCF-7 cells. The present work observed that FA-CPT-PLGA-Glu NPs can induce ROS generation in cancer cells (Figure 8(n)) using the DCFH-DA staining method.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTUNEL Assay\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe TdT-mediated dUTP nick end labeling (TUNEL) test was used to evaluate nuclear DNA fragmentation (apoptosis) by flow cytometry. This investigation was conducted when the development of cancer cells was reduced through the activation of apoptosis. The flow cytometry analysis of MCF-7 cells is shown in\\u0026nbsp;Figures 8 (o) and (p), depicting the cells\\u0026apos; state before (Figure 8(o)) and after (Figure 8(p)) being treated with FA-CPT-PLGA-Glu NPs at a concentration that inhibits 50% of cell growth (IC\\u003csub\\u003e50\\u003c/sub\\u003e) for a duration of 24 h. After being treated with a concentration of 16.33 \\u0026mu;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;1\\u0026nbsp;\\u003c/sup\\u003eof FA-CPT-PLGA-Glu NPs, 38.6% of cells with DNA damage were observed, which was substantially higher than the percentage of DNA-damaged cells in the untreated group (9.46 %).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eCamptothecin was previously thought to be an antioxidant molecule that might be used to combine anticancer and antidiabetic medicines[\\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e68\\u003c/span\\u003e]. Later, there was evidence of its anticancer efficacy in treating leukaemia and colon cancers. CPT has potent anticancer action by targeting DNA topoisomerase I [\\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e69\\u003c/span\\u003e]. However, CPT has three major limitations: (1). it is sparingly soluble in water, and its clinical applications are through intravenous administration (2). the biologically active form of CPT (lactone) rapidly undergoes a ring-opening reaction and converts to the biologically inactive form (carboxylate) under neutral conditions, and (3). it is unstable in human plasma and highly toxic[\\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e70\\u003c/span\\u003e]. To address these concerns, this study formulated CPT-loaded with folic acid-conjugated PLGA-glutenin nanoparticles (FA-CPT-PLGA-Glu NPs) to deliver CPT into cancer cells while preserving normal healthy cells.\\u003c/p\\u003e \\u003cp\\u003ePolymeric nanoparticles efficiently deliver the loaded toxic payload into cancer cells because they are easily adjustable, and their morphology can be easily modified[\\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e71\\u003c/span\\u003e]. The selection of polymers for the development of nanoparticles is one of the key parameters for drug delivery applications. Currently, various types of biodegradable polymers (both natural and synthetic) are commonly used to develop nanoparticles, particularly for cancer drug delivery[\\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e72\\u003c/span\\u003e]. Interestingly, pharmaceutical research uses polymer-based nanoparticles to reduce drug toxicity and adverse effects, but until recently, it was not realized that carrier systems themselves could cause hazards to patients[\\u003cspan citationid=\\\"CR73\\\" class=\\\"CitationRef\\\"\\u003e73\\u003c/span\\u003e]. While developing a treatment approach using nanomedicine, the goal should be to minimize the toxic effects on the patient[\\u003cspan citationid=\\\"CR74\\\" class=\\\"CitationRef\\\"\\u003e74\\u003c/span\\u003e]. Synthetic polymers have many disadvantages, including toxicity, poor biocompatibility, and high cost of the production process. Natural polymers (derived from plants, animals, and bacteria) are less toxic, biocompatible, biodegradable, and easily available; at the same time, some disadvantages also occurred using as drug carrier material, such as structurally more complex and high degree of variability in materials from animal sources[\\u003cspan citationid=\\\"CR75\\\" class=\\\"CitationRef\\\"\\u003e75\\u003c/span\\u003e]. A combination of natural and synthetic polymers can be better compatible with higher stability, prolonged circulation, and controlled release behaviors of loaded drugs with less toxicity[\\u003cspan citationid=\\\"CR76\\\" class=\\\"CitationRef\\\"\\u003e76\\u003c/span\\u003e]. This study used a combination of glutenin and PLGA to formulate nanoparticles. PLGA has been widely used to encapsulate various anticancer agents, offering strong biodegradability, minimum systemic toxicity, and enhanced bioavailability of loaded drugs. Glutenin is isolated from wheat flour and comprises two polypeptide chains (gliadin and glutenin) and soluble carbohydrates. However, gliadin was extracted from gluten protein with 70% ethanol, and glutenin is soluble in acetic acid.\\u003c/p\\u003e \\u003cp\\u003eIn contrast to gliadin, glutenin was highly biocompatible and promotes fibroblast and osteoblast adhesion and proliferation more effectively than films composed of PLGA. Glutenin structure consists of multimeric aggregates of high and low molecular mass subunits linked together by disulfide links [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. CPT-loaded PLGA NPs were formulated using a modified emulsification/evaporation method. Further, glutenin coated on the surface of CPT-loaded PLGA NPs via a layer-by-layer assembled technique. Furthermore, simple conjugation chemistry successfully formulated PLGA-Glu NPs conjugated with FA. The amino group of glutenin was conjugated with the carboxylic acid group of FA. The FA conjugation allowed for easy recognition and attachment of upregulated folate receptors on the surface of cancer cells. This facilitated the effective delivery of the loaded drugs into the cancer cells[\\u003cspan citationid=\\\"CR77\\\" class=\\\"CitationRef\\\"\\u003e77\\u003c/span\\u003e]. Because the folate receptor is a biomarker for many tumors, it is highly expressed in specific malignant cells such as breast, ovarian, lung, kidney, brain, and colon cancer cells. In addition, the cellular absorption of the drug is increased by folate-conjugated NPs through endocytosis. Our previous study demonstrated that retinoic acid-encapsulated folic acid-conjugated glutenin NPs (FA-RA-Glu NPs) effectively delivered the loaded RA into MCF-7 cells and significantly reduced the number of viable cells, and induced apoptosis. The cellular uptake study showed that the FA-RA-Glu NPs had facilitated endocytosis and delivered RA into MCF-7 cells[\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe physiochemical features of nanoparticles, such as their size and shape, play a major role in determining their effectiveness and delivering drugs to the tumor site [\\u003cspan citationid=\\\"CR78\\\" class=\\\"CitationRef\\\"\\u003e78\\u003c/span\\u003e]. The X-ray diffraction analysis examined the formulated FA-CPT-PLGA-Glu NPs possess a crystalline structure, which enables a regulated and extended release of the loaded drug into the cancer cells. The molecular dimension and configuration of nanoparticles (NPs) are key factors that exert a substantial influence on regulating the circulation and biodistribution of therapeutic nanoparticles. When observed using transmission electron microscopy (TEM), the FA-CPT-PLGA-Glu NPs appeared predominantly spherical with a diameter of around 100 nm, consistent with the findings of the dynamic light scattering (DLS) investigation. The optimal particle size for effective distribution into solid tumors should be 10\\u0026ndash;200 nm. Furthermore, nanoparticles with a spherical shape demonstrated the highest level of internalization into cancer cells compared to nanoparticles of different shapes[\\u003cspan citationid=\\\"CR79\\\" class=\\\"CitationRef\\\"\\u003e79\\u003c/span\\u003e]. Additionally, the NPs with a spherical shape can encapsulate the highest drug content, leading to a decrease in the quantity of cancer cells. This is a significant utilization of nanoparticle-based drug delivery systems in cancer therapy.\\u003c/p\\u003e \\u003cp\\u003eWhen formulating a targeted drug delivery system, it is necessary to consider drug encapsulation efficiency and loading capacity. The higher encapsulation efficiency with lesser nanoparticle loading capacity allows for more efficient drug delivery at the disease site. In the present study, the CPT encapsulation efficiency and loading capacity of PLGA-Glu NPs were 74.95\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.34% and 4.78\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.08%, respectively. Nanoparticles increase anticancer drugs' bioavailability and therapeutic efficacy while ensuring preferential accumulation at the targeted site. Here, we observed \\u003cem\\u003ein vitro\\u003c/em\\u003e CPT release from CPT-PLGA-Glu NPs at various pH levels in the buffer solution to confirm the maximum content of CPT loaded. Investigate the CPT release kinetics and mechanism from FA-conjugated Glu-PLGA NPs. Determine the optimal conditions (pH) for the highest release of CPT-PLGA-Glu NPs, which is required for further in vivo studies. Our investigation revealed a consistent and gradual release of the drug during the studies, suggesting a sustained and controlled release of the loaded drug. This can be attributed to drug diffusion and matrix erosion technique mechanisms. Further, we found that the highest amount of CPT was released from CPT-PLGA-Glu NPs in a pH 5.3 acetate buffer solution, which simulated a cancerous environment. Similarly, our previous study demonstrated that the formulated glucose-conjugated CPT-loaded glutenin nanoparticles released their toxic payload of CPT in the acidic environment at pH 5.3.\\u003c/p\\u003e \\u003cp\\u003eThe primary goal of a targeted drug delivery system is to prolong circulation, locate at the disease site, target, and ensure a safe drug interaction with diseased tissue/cells. The results show that FA-CPT-PLGA-Glu NPs effectively reduced the viability of MCF-7 cells. This significant cytotoxicity of FA-CPT-PLGA-Glu NPs against MCF-7 cells is attributed to the highest CPT content that enters the cells. The higher cellular internalization of CPT-PLGA-Glu NPs leads to a higher cellular uptake of the entrapped therapeutic agent. The cellular uptake of FA-CPT-PLGA-Glu NPs may be influenced by the process of folate receptor-mediated endocytosis. The IC\\u003csub\\u003e50\\u003c/sub\\u003e value was calculated, representing the minimum concentration of a substance needed to induce 50% cell death in cancer cells within a specific period. The IC\\u003csub\\u003e50\\u003c/sub\\u003e concentration of FA-CPT-PLGA-Glu NPs against MCF-7 cells was 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. The MCF-7 cell line demonstrates a significant folate receptor positivity, with the folate receptor being abundantly upregulated on its surface. As the concentration increased, the viability of the cells reduced, indicating that the cytotoxicity of the drug depends on its concentration. Similarly, the cytotoxicity of α-mangosteen (AM), α-mangosteen-loaded folate-conjugated chitosan nanoparticles-high molecular weight (AM-F-CS-HMW NPs), and α-mangosteen-loaded folate-conjugated chitosan nanoparticles-high molecular weight (AM-F-CS-LMW NPs) against MCF-7 cells and found IC50 values of 8.47\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.49, 5.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.01, and 4.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.11 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e, respectively. These results confirm the improved cytotoxicity of α-mangostin in MCF-7 cells when delivered via folate-conjugated chitosan NPs[\\u003cspan citationid=\\\"CR80\\\" class=\\\"CitationRef\\\"\\u003e80\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eMost of the anticancer agents stimulate the process of apoptosis induction and associated cell death networks to eradicate malignant cells. Apoptosis is a crucial process in both carcinogenesis and cancer treatment. The FA-CPT-PLGA-Glu NPs effectively triggered cell death in MCF-7 cells, as demonstrated by the AO/EtBr double staining experiment. The cells treated with 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e of FA-CPT-PLGA-Glu NPs exhibited symptoms of cell death, which are typically characterized by distinct morphological alterations, including round and irregular forms, condensed nuclei, deformed membranes, and the presence of apoptotic bodies. Cancer cell cycle arrest, senescence, and apoptosis can be induced by the overproduction of intracellular reactive oxygen species (ROS). Our study showed FA-CPT-PLGA-Glu NPs activated ROS generation by the 2ʹ,7ʹ-Dichlorofluorescin Diacetate (DCFH-DA) method. 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e-1\\u003c/sup\\u003e of FA-CPT-PLGA-Glu NPs treated cells showed increased levels of ROS. This might be interpreted as the proteasome inhibition in MCF-7 cells leading to the degradation of many proteins, disrupting redox equilibrium, and raising ROS levels. The buildup of ROS disrupts the respiratory chain and may activate the p53-mediated intrinsic apoptotic mechanism. Furthermore, phosphorylation and ubiquitination of cell cycle proteins disrupt the redox regulation of cell cycle progression, resulting in abnormal cell proliferation and cell death. The assessment of apoptosis was further validated by treating MCF-7 cells with 16.33 \\u0026micro;g \\u0026times; mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e of FA-CPT-PLGA-Glu NPs, which resulted in the release of cytochrome C and a decrease in mitochondrial membrane potential. Intrinsic apoptosis is linked to the integrity of the mitochondrial membrane damage. When a cell receives a death signal, the receptor's C terminal sequence becomes active. These target the outer membrane layer of the mitochondria and cause permeabilization, ultimately resulting in cell death. The caspase cascade is responsible for cell death after the cytochrome C release[\\u003cspan citationid=\\\"CR81\\\" class=\\\"CitationRef\\\"\\u003e81\\u003c/span\\u003e]. Targeted suppression of caspase-3 and \\u0026minus;\\u0026thinsp;9 enables the effective liberation of cytochrome C, impeding alterations in mitochondrial structure and generating reactive oxygen species (ROS). In addition, the effectiveness of apoptotic cell death was closely observed by examining changes in the morphology of the nucleus using the DAPI assay. Apoptosis in cancer cells of mammals is frequently accompanied by specific morphological and physiological alterations, such as membrane blebbing, phosphoserine externalization, chromatin condensation, nuclear fragmentation, and degradation of DNA initially into large fragments and subsequently into small nucleosomal fragments. The FA-CPT-PLGA-Glu NPs induced apoptosis in MCF-7 cells, as evidenced by nuclear fragmentation, cell shrinkage, and nucleus marginalization. The TUNEL assay detected apoptotic cells that undergo substantial DNA destruction during the late stages of apoptosis. This technique utilizes the autonomous labeling ability of terminal deoxynucleotidyl transferase to mark the blunt ends of double-stranded DNA breaks without requiring a template.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eIn this study, FA-CPT-PLGA-Glu NPs were successfully fabricated with the help of wheat glutenin protein and synthetic polymer PLGA, which is biocompatible and observed to deliver the loaded CPT into cancer cells effectively. The FA surface modification of NPs is easy to recognize and covalently binds to the overexpressed folate receptor on the plasma membrane of breast cancer cells and enters inside the cell via endocytosis. The released active molecule CPT from NPs targets caspase-3 and \\u0026minus;\\u0026thinsp;9 to induce cell death mechanism via overproduction of ROS. Due to the poor pharmacokinetics and toxic properties of CPT, it is converted into nanoparticles for targeting cancer cells without disturbing normal healthy cells. Fabricated FA-CPT-PLGA-Glu NPs were observed to be crystalline, spherical shaped, and around 100 nm in size. Active molecule, the CPT encapsulation efficiency and loading capacity of PLGA-Glu NPs were 74.95\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.34% and 4.78\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.08%, respectively. Further, the drug release behavior of CPT-PLGA-Glu NPs displayed a prolonged and controlled release pattern. Released CPT from NPs reduced cellular viability and inhibited caspase-3 and \\u0026minus;\\u0026thinsp;9 activity to cause cell death/apoptosis via increasing ROS generation, damaging mitochondrial membrane potential, and altering the morphology of MCF-7 nuclei. The findings suggested that the unique FA-CPT-PLGA-Glu NPs formulation showed strong antitumor activity. However, more extensive \\u003cem\\u003ein vivo\\u003c/em\\u003e research is required to assess its clinical applicability fully.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRRR thanks to Kalasalingam Academy of Research and Education for the university research fellowship.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSK supervision, project administration, funding acquisition, resources, writing review \\u0026amp; editing; RRR, TP, PP, SRKP, ASKK writing-original draft, formal analysis, investigation; MS, SJK conceptualization, writing, investigation and editing. All authors have read and agreed to the published version of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSK gratefully acknowledge the Management of Kalasalingam Academy of Research and Education for Seed Money Grant (KARE/VC/R\\u0026amp;D/SMPG/2021\\u0026ndash;2022/1).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll data generated or analysed during this study are included in this manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclarations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no conflict of interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical Approval\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eEthical approval was not required for this research.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent to Participate\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for Publication\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors give the consent for publication.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eMehrotra R, Yadav K (2022) Breast cancer in India: Present scenario and the challenges ahead. 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Pharmaceuticals 3(4):839\\u0026ndash;915\\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\":\"info@researchsquare.com\",\"identity\":\"journal-of-polymers-and-the-environment\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jooe\",\"sideBox\":\"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)\",\"snPcode\":\"10924\",\"submissionUrl\":\"https://submission.nature.com/new-submission/10924/3\",\"title\":\"Journal of Polymers and the Environment\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Camptothecin, MCF-7 cells, folate-receptor, apoptosis, cell death\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4513460/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4513460/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe combination of natural and synthetic polymers for nanomedicine development had many advantages, including less toxicity, biocompatibility, prolonged circulation, higher stability, and ease of surface modification. Here, a novel folic acid-conjugated Camptothecin-loaded-poly (lactic-co-glycolic) acid-glutenin nanoparticles (FA-CPT-PLGA-Glu NPs) was fabricated to treat breast cancer. FA-CPT-PLGA-Glu NPs target breast cancer cells via upregulated folate receptors and delivered their toxic payloads without disrupting healthy cells. First, CPT-loaded PLGA NPs were created using a modified emulsification/evaporation technique. Second, Glu-based CPT-PLGA NPs were synthesized using a layer-by-layer assembly, and their physiochemical properties were validated. CPT encapsulation efficiency and loading capacity into PLGA-Glu NPs were 74.95 ± 1.34% and 4.78 ± 1.08%, respectively. CPT-PLGA-Glu NPs exhibited sustained and controlled release of loaded-CPT from NPs, and the highest content was released in an acidic environment (pH 5.3), which will be advantageous for cancer treatment. Later, FA-CPT-PLGA-Glu NPs were synthesized by simple conjugation chemistry. The fabricated FA-CPT-PLGA-Glu NPs were around 100 nm in size, with a spherical form and crystalline nature. FA-CPT-PLGA-Glu NPs show strong cytotoxicity activity, and its IC\\u003csub\\u003e50\\u003c/sub\\u003e value was 16.33 µg × mL\\u003csup\\u003e− 1\\u003c/sup\\u003e against breast cancer cell line (MCF-7). This folate-receptor-targeted NPs are more effectively internalized into MCF-7 cells, causing ROS generation, cell growth inhibition, and apoptosis. The activity of caspase-3 and − 9 causes MCF-7 cells apoptosis by internalized CPT. Further, internalized CPT induces potential loss of mitochondrial transmembrane and damages the nuclear integrity of the cancer cells. These results showed that the FA-CPT-PLGA-Glu NPs target upregulated folate receptors on the surface of MCF-7 cells.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Folate receptor-targeted Camptothecin-loaded PLGA-Glutenin nanoparticles for effective breast cancer treatment\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-06-14 20:36:50\",\"doi\":\"10.21203/rs.3.rs-4513460/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-07-07T13:02:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-06T14:44:56+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-05T09:11:06+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"310371373444736815866645944516905648482\",\"date\":\"2024-06-27T01:30:41+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"275290522780593218691444918489398635555\",\"date\":\"2024-06-27T00:34:55+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"155630107573190491013096451962239489186\",\"date\":\"2024-06-07T10:20:15+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-06-06T16:03:24+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-06-01T14:06:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-06-01T14:06:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Journal of Polymers and the Environment\",\"date\":\"2024-06-01T11:44:35+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-polymers-and-the-environment\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jooe\",\"sideBox\":\"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)\",\"snPcode\":\"10924\",\"submissionUrl\":\"https://submission.nature.com/new-submission/10924/3\",\"title\":\"Journal of Polymers and the Environment\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"732d45a8-690f-48e0-89a3-873a5d6c0397\",\"owner\":[],\"postedDate\":\"June 14th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-09-09T16:11:47+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4513460\",\"link\":\"https://doi.org/10.1007/s10924-024-03391-6\",\"journal\":{\"identity\":\"journal-of-polymers-and-the-environment\",\"isVorOnly\":false,\"title\":\"Journal of Polymers and the Environment\"},\"publishedOn\":\"2024-09-03 16:05:18\",\"publishedOnDateReadable\":\"September 3rd, 2024\"},\"versionCreatedAt\":\"2024-06-14 20:36:50\",\"video\":\"\",\"vorDoi\":\"10.1007/s10924-024-03391-6\",\"vorDoiUrl\":\"https://doi.org/10.1007/s10924-024-03391-6\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4513460\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4513460\",\"identity\":\"rs-4513460\",\"version\":[\"v1\"]},\"buildId\":\"qQ7_6M8ijIrYJ9CiyUnPg\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}