Targeted delivery of thymoquinone-encapsulated polyethyleneimine/poly (lactic acid) nanoparticles into breast cancer cells

Targeted delivery of thymoquinone-encapsulated polyethyleneimine/poly (lactic acid) nanoparticles into breast cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeted delivery of thymoquinone-encapsulated polyethyleneimine/poly (lactic acid) nanoparticles into breast cancer cells Jeganpandi Senthamarai Pandi, Parasuraman Pavadai, Ewa Babkiewicz, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6858817/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Breast cancer is the most often diagnosed cancer globally, affecting elderly women predominantly. Thymoquinone (TQ) is a potential anticancer agent; however, its pharmacological applications are restricted by inadequate aqueous solubility and bioavailability. Our study aimed to develop TQ-encapsulated polyethyleneimine/poly(lactic acid) nanoparticles (TQ-PEI/PLA-NPs) to enhance TQ delivery into breast cancer cells. The solvent evaporation-emulsification method was used to synthesize TQ-PEI/PLA-NPs, and their physicochemical characteristics were examined. TQ-PEI/PLA-NPs had a crystalline structure, a zeta potential of + 1 mV, and a spherical shape with a diameter of 80–90 nm. The encapsulation efficiency was 85% (w/w), while the drug loading capacity was 9.34% (w/w). The release rate of TQ from TQ-PEI/PLA-NPs was marginally elevated at pH 5.8 (81.21 ± 0.87%) compared to pH 3.5 and 7.2. The cytotoxicity of TQ-PEI/PLA-NPs was examined in MCF-7 cells. After 24 h of treatment, the MCF-7 cell counts decreased with an IC 50 of 21.99 µg/mL. The elevated intracellular accumulation of TQ in MCF-7 cells resulted in cell death, as evidenced by AO/EBr staining, mitochondrial transmembrane potential assay and Caspase-3 and − 9 studies. The observed MCF-7 cell death attributed to TQ was induced by increased reactive oxygen species (ROS) and impairment of mitochondrial membrane potential. The ROS potentially damaged the mitochondrial membrane and DNA, and further studies supported the induction of apoptosis. Our results indicated that TQ-PEI/PLA-NPs, which cause potent cytotoxicity to breast cancer cells, as evidenced by the decreased MCF-7 cell counts, may exhibit significant therapeutic potential for breast cancer therapies. Thymoquinone breast cancer cells polymeric nanoparticles apoptosis drug release Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Breast cancer is the most often diagnosed malignancy worldwide, with approximately 2.3 million new cases and 685,000 fatalities among women in 2020 [ 1 ]. It accounts for 14% of all cancer diagnoses [ 2 ]. While the majority of breast cancer incidence and deaths occur in those over the age of 50, a significant number of cases in Africa are diagnosed in younger individuals [ 3 ]. Incidence rates vary significantly across different regions, with the highest rates reported in Australia/New Zealand, Western Europe, and Northern America [ 4 ]. It is reported that every four minutes, an Indian woman is diagnosed with breast cancer. In India, breast cancer is becoming more common in both rural and urban settings [ 5 ]. According to the SURVCAN-3 study (Cancer Survival in Countries in Transition), the global median 3-year breast cancer survival rate is 84% in 2023 [ 6 ]. Of those instances, 68% occurred in India, which had a 5-year breast cancer survival rate of 66.1% based on the CONCORD-3 study, which was conducted between 2010 and 2014 [ 7 ]. Genetic, environmental, hormonal, and sedentary lifestyle factors all contribute to the etiology of breast cancer [ 8 ]. Researchers have found several risk factors that may elevate an individual's likelihood of acquiring breast cancer, despite the precise etiology of the disease remaining unidentified. High-fat junk food, excessive alcohol consumption and cigarette smoking, the use of medicines such as hormonal contraceptives and exogenous female hormones (menopausal hormone therapy), inactivity, and a poorly maintained body mass index (BMI) are common risk factors [ 9 ]. Most of the time, early cancer detection allows successful treatment, leading to a better prognosis and lower death rate. However, the prevalence of breast cancer has been rising annually [ 10 ]. The intricate molecular mechanisms of breast cancer present a considerable challenge; nonetheless, the diagnostic and therapeutic approaches now employed are inadequate in preventing disease development and minimizing toxicity. Current approaches, including surgical removal of the cancer tissue, laser and radiation therapy, immunotherapy, endocrine (hormone) therapy, personalized medicine, chemotherapy and combination therapy, are used to treat breast cancers, although chemotherapy appears more effective [ 11 ]. However, patients under chemotherapy suffer from significant toxicity linked to the treatment [ 12 ]. As a result, scientists look for safer and more potent anticancer drugs from natural resources. Since plants and their derivatives have played a crucial role in the development of effective anticancer medications [ 13 ]. For example, paclitaxel from Taxus brevifolia [ 14 ] irinotecan and camptothecin from Camptotheca acuminata [ 15 ], curcumin from Curcuma longa [ 16 ] vincristine and vinblastine from Vinca rosea [ 17 ], and etoposide and podophyllotoxin from the mandrake plant [ 18 ] have proven effective in treating breast cancer. Thymoquinone (TQ) is the primary active molecule present in the oil of black cumin seed ( Nigella sativa) [ 19 ]. Chemically, it is a monoterpene and consists of a benzoquinone moiety with antioxidant and anticancer properties [ 20 ]. Researchers have demonstrated the potential of TQ for the treatment of various cancer types, including breast cancer. By downregulating Bcl-2 and c-FLIP in renal cell cancer, it dramatically increases apoptosis. In human RCC 769-P and 786-O cell lines, it also suppresses metastasis and the epithelial-mesenchymal transition (EMT) by altering the LKB1/AMPK pathway [ 21 ]. It also reduces renal cell carcinoma by inducing autophagy through the AMPK/mTOR signalling pathway [ 22 ]. However, poor aqueous solubility restricts TQ's therapeutic applications [ 23 ]. This limitation can be mitigated by developing an innovative nano-carrier technology for targeted drug delivery to disease locations. Polymeric nanoparticles have been selected for this study because of their distinctive properties, including enhanced drug loading capacity, prolonged stability in blood circulation, biodegradability, and efficient drug delivery. Most researchers use biodegradable polymers, such as poly(lactic acid) (PLA), poly(caprolactone) (PCL), and poly(lactic-co-glycolic acid) (PLGA), which have been thoroughly tested for biocompatibility, for making polymeric nanoparticles [ 24 ]. Through matrix degradation or diffusion through the polymer matrix, these polymers offer sustained or extended drug release [ 25 ]. According to reports, polyethyleneimine (PEI)-coated nanoparticles showed enhanced cellular uptake and function as efficient drug delivery vehicles [ 26 ]. It is anticipated that PEI's higher positive charge will promote nanoparticle aggregation inside negatively charged cancer cells [ 27 ]. PEI-coated PLA nanoparticles (PEI/PLA-NPs) were developed to improve the therapeutic effectiveness and bioavailability of thymoquinone. Research by Connelly et al. demonstrated the use of TQ as an NF-κB inhibitor in breast cancer cells (PYG/L129) derived from PyVT mice crossed with NGL reporter mice, treated with breast cancer from PYG/L129 cells obtained from PyVT [ 28 ]. Similarly, Woo et al. investigated the anti-tumor effects of TQ in various cell types, including three distinct breast cancer cell lines (MCF-7, MDA-MB-231, and BT-474). Their findings suggest that the anti-tumor effect of TQ may be mediated through modulation of the PPAR-γ activation pathway [ 29 ]. These studies support the potential application of TQ as a breast cancer therapeutic. Since the poor aqueous solubility of TQ may limit its efficacy, we hypothesize that PEI's positive charge allows it to interact electrostatically with negatively charged biological molecules, such as proteins or nucleic acids, to promote cellular uptake. Hence, the current study aimed to overcome the limitations of TQ by encapsulating it in polymeric nanoparticles using polyethyleneimine/poly (lactic acid). We aimed to encapsulate TQ with PLA and modify the NP surface with positively charged PEI. The positive charge of PEI allows it to bind electrostatically with negatively charged biological molecules, including proteins or nucleic acids, enhancing cellular uptake and facilitating the effective transport of TQ into MCF-7 cells for breast cancer treatment. Experimental section Chemicals Thymoquinone (TQ), poly (lactic acid) (PLA), polyethyleneimine (PEI, Mw 25 kDa, branched) and Tween-80 were sourced from Sigma-Aldrich, USA. Dulbecco's Modified Eagle Medium (DMEM), Fetal bovine serum (FBS), and Penicillin-streptomycin were obtained from Gibco, USA. The Michigan Cancer Foundation 7 (MCF-7) cell line was received from the National Centre for Cell Science (NCCS), Pune, India. MCF-7 cells were passaged every 2–3 days upon achieving 70–80% confluence, utilizing 0.25% trypsin-EDTA for detachment, and subsequently resuspended in new medium. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2',7'-dichlorofluorescin diacetate (DCFDA) were purchased from Sigma-Aldrich, USA. 4',6-diamidino-2-phenylindole (DAPI), Rhodamine-123 were acquired from Thermo Scientific, USA. Caspase-Glo® 3 and 9 reagents were purchased from Promega, USA. All the other reagents and solvents used were of analytical grade and purchased from Merck, India. Milli-Q water was used for the entire experiment. Formulation of thymoquinone-encapsulated PEI/PLA nanoparticles The solvent evaporation-emulsification method was followed to generate PEI/PLA-NPs encapsulated with TQ [ 30 ]. In short, 5 mL of DMSO was used to dissolve 100 mg of PLA and 5 mg of TQ, and the mixture was sonicated for 30 min. In 50 mL of water, 0.5% (w/v) PEI and 1% Tween-80 were dissolved to form the aqueous phase. After 20 min of constant stirring in the magnetic stirrer, the organic phase was introduced to the aqueous phase drop by drop. It was then instantly homogenized for 3 min at 14,000 rpm. After being separated, washed, and freeze-dried (stored at -20°C overnight), the TQ-encapsulated PEI/PLA NPs were lyophilized for 48 h at -80°C using a freeze dryer (Martin Christ, Gefriertrocknungsanlagen GmbH). For further analysis, the prepared TQ-PEI/PLA-NPs were used. Encapsulation efficiency and loading capacity of TQ The encapsulation efficiency (EE%) and loading capacity (LC%) of TQ in PEI/PLA-NPs were evaluated using the method described in our earlier research [ 31 ]. 100 mg of TQ-PEI/PLA-NPs were centrifuged at 15000 rpm, and the supernatant was measured at UV-Visible spectrophotometer at 254 nm (UV-1800, Shimadzu, Japan). The TQ encapsulation efficiency (EE%) and loading capacity (LC%) were calculated using the formula given below: Percentage encapsulation efficiency (EE%) = \(\:\frac{\text{T}\text{Q}\text{a}\text{d}\text{d}\text{e}\text{d}\:-\:\text{T}\text{Q}\text{m}\text{e}\text{a}\text{s}\text{u}\text{r}\text{e}\text{d}}{\text{T}\text{Q}\text{a}\text{d}\text{d}\text{e}\text{d}}\:\) × 100 1 Percentage loading capacity (LC%) = \(\:\frac{\left(\text{T}\text{Q}\right)\text{a}\text{d}\text{d}\text{e}\text{d}\:-\:\left(\text{T}\text{Q}\right)\text{m}\text{e}\text{a}\text{s}\text{u}\text{r}\text{e}\text{d}}{Weight\:of\:the\:nanoparticles}\) × 100 2 In-vitro drug release The drug release profile of TQ from TQ-PEI/PLA-NPs was evaluated by the membrane dialysis method using 0.01 M acetate buffer at pH 3.5 and 0.01 M phosphate buffers at pH 5.8 and 7.2 [ 32 ]. Briefly, 50 mg of TQ-PEI/PLA-NPs was placed in a dialysis bag (3500 Da, HiMedia Laboratories, India), kept in a buffer solution, placed on a dissolution apparatus at 150 rpm, and maintained at 37°C. About 1mL of sample was taken out and replaced with fresh buffers. The drug content was estimated using a UV-Visible spectrophotometer at 254 nm. The experiments were performed thrice, and drug release (% DRC) was estimated using the below formula Percentage drug release = \(\:\frac{\text{A}0-\text{A}1}{\text{A}0}\) × 100 3 where, A 0 = absorbance of the control, and A 1 = absorbance of the sample. The release kinetics, such as zero-order (cumulative% drug release vs. time), log% drug remaining vs. time), Higuchi (cumulative% drug release vs. square root of time), Korsmeyer-Peppas (log drug release vs. log time), and Hixon-Crowell models, were applied to determine TQ release kinetics. Regression coefficients (r 2 ) and release rate constant values were generated for every kinetic model [ 33 ]. Stability studies In vitro stability was evaluated in physiological solution (10% NaCl containing 0.5% BSA), buffers like acetate buffer (pH 3.5 and 5.5), phosphate buffer (pH 7.2 and 9.0), and simulated gastric juice [ 34 ]. Briefly, equal volumes of physiological solution, buffers and simulated gastric juice were mixed with freshly prepared TQ-PEI/PLA-NPs and stored at room temperature. After 72 h, the absorbance was recorded using a UV-visible spectrophotometer at 100–500 nm. Characterization of TQ-PEI/PLA-NPs The formulated TQ-PEI/PLA-NPS were subjected to various physicochemical characterization. The functional groups were determined using FTIR spectroscopy (IR Tracer-100, Shimadzu, Japan). The IR spectra were obtained in the 4000 − 400 cm − 1 transmission range at a resolution of 4 cm − 1 . ZetaSizer (ZS Nano, Malvern Instruments, UK) was used to measure the particle size and size distribution of TQ-PEI/PLA-NPS. Using X-ray diffraction (XRD) analysis, the physical characteristics of TQ-PEI/PLA-NPS were investigated (BRUKER D8 Advance, Gmbh, Germany). The detector operates with Cu Kα 1 radiation (λ = 0.154060 nm) in a 2θ degree arrangement at a voltage of 40 kV and current power of 30 mA. The elemental composition of TQ-PEI/PLA-NPs was examined using high-resolution X-ray photoelectron spectroscopy (XPS; Thermo Scientific, UK) equipped with an Al/Ka X-ray source (hv = 1486.6 eV) with a surface between 0 and 1400 eV. Spectra were processed using XPS (Casa Software Ltd., Teignmouth, UK). The morphology of TQ-PEI/PLA-NPs was assessed using Field Emission Scanning Electron Microscopy (JEOL JSM6700 FESEM). Approximately 2 mg of freeze-dried TQ-PEI/PLA-NPs were reconstituted with distilled water, and the mixture was applied to a glass surface. A gold coating layer was applied over the mixture and dried to prevent electrostatic charge during the experiment. Molecular docking The protein data bank ( https://www.rcsb.org/structure/6SFO ) provided a three-dimensional X-ray structure of MAPK14 with the bound inhibitor SR-318 (PDB ID: 6SFO, Homo sapiens, resolution: 1.75 Å) of the target protein [ 35 ]. Later, the Swiss-PDB Viewer v4.1.0 tool was used to reinsert the missing residues into the final protein. Using the BIOVIA Discovery Studio Visualizer version 4.0, the precise amino acids of the active site were identified, and the protein structure was visually shown. Chem Sketch was used to create a three-dimensional structure for thymoquinone and doxorubicin (standard). Auto Dock Vina program was used to calculate the binding energies of different ligand and protein combinations. Discovery Studio Client 4.0 was used for the docking inquiry. The binding affinity energies of the complex were computed by considering the ligand conformation at the active binding site as well as the root mean square deviation (RMSD) between the initial and subsequent structures. Molecular dynamics simulation MD simulation was performed on the target molecule TQ-MAPK14 complex to assess the stability and interactions between the ligand and protein. An analysis was conducted on the binding mechanisms of potential lead candidates in relation to the protein targets they were designed to target [ 36 ]. The MD simulations were conducted on a Linux-based computer using the Desmond module and the Maestro simulation environment, both developed by Schrodinger, Inc., USA (utilizing the Academic Version, V3.6 algorithm). The intricate protein-ligand interaction was evaluated using the TIP3P water model [ 37 ]. The energy computations in the simulation utilized the OPLS-4 (Optimal Potentials for Liquid Simulation) force field, with recording intervals of 50 picoseconds. A molecular dynamics simulation was performed at three predetermined intervals, each lasting 100 nanoseconds. For a thorough examination of the outcomes derived from the MD simulation, we employed the Desmond module. The application of the Simulation Interaction Diagram greatly enhanced the quality of this analysis. The stability of ligand-protein complexes was assessed using several quantitative measures, such as root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and study of protein-ligand interactions found in simulated trajectories. Anticancer activity Cytotoxicity assay Cell viability using the MTT assay was performed by the method in Ellappan et al. [ 38 ]. Briefly, MCF-7 cell lines were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin at 37°C overnight in a humidified atmosphere of 5% CO 2 and 95% air in a CO 2 incubator. Cells were seeded at a density of 1×10 5 cells per well in a 96-well plate and allowed to incubate overnight. After 24 h of incubation, old medium was replaced with fresh medium containing TQ-PEI/PLA-NPs (100, 50, 25, 12.5, 6.25, 3.125 µg/mL), doxorubicin (0.25 µM) and TQ (100 µg/mL) in MCF-7 cells and incubated for another 24 h. To this, 200 µL of 0.5% MTT solution was added and the plate was placed in the dark at 37°C for 4 h to facilitate formazan crystal formation. After dissolving the formazan crystals in 100 µL of DMSO, the absorbance was measured by a microplate reader (Thermo Scientific, USA) at 570 nm. Cell viability was calculated from the following Eq. (4): Percentage cell viability = \(\:\frac{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}}{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\) × 100 4 Determination of apoptosis by AO/EB staining assay Cellular apoptosis was evaluated by AO/EB staining assay [ 39 ]. Briefly, MCF-7 cells were grown in a 96-well plate at 1×10 5 cells per well and allowed to incubate overnight. After 24 h of incubation, the cells were treated with 400 µL of TQ-PEI/PLA-NPs in DMEM at 37°C with 5% CO 2 for 24 h. Following treatment, the cells were washed twice with PBS and then incubated with a staining solution containing 1 µg/mL of AO and 1 µg/mL of EB for 15 min at 37°C in a dark. After incubation, the staining solution was removed, and the cells were washed with PBS. The cells were then observed under an inverted fluorescent microscope (Olympus IX71, Japan) at 450–490 nm. Approximately 300 cells were counted and scored for viable (green fluorescence) or dead cells (red fluorescence). The percentage of apoptotic and necrotic cells was calculated. DAPI staining DAPI (4',6-diamidino-2-phenylindole) staining was used to visualize nuclear abnormalities, including nuclear size, DNA damage, nuclear contour irregularities, and hyperchromatic, which can hinder cell growth [ 40 ]. MCF-7 cells were grown in a 24-well plate in a CO 2 incubator and treated with an IC 50 concentration of TQ-PEI/PLA-NPs for 24 h. After incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde. The fixed cells were stained with 20µL of DAPI (0.5 µg/mL) at room temperature in the dark. The nuclear morphology of the cells was examined by fluorescence microscopy (Olympus IX71, Japan). Evaluation of mitochondrial transmembrane potential (ΔΨm) The mitochondrial transmembrane potential (ΔΨm) was examined using the Rhodamine-123 staining method described by Zorova et al. [ 41 ]. Briefly, 1×10 5 MCF-7 cells were grown in a 6-well plate in a CO 2 incubator and treated with an IC 50 concentration of TQ-PEI/PLA-NPs for 24 h. Then, cells were washed with PBS and fixed with 4% paraformaldehyde. After fixing, the cells were stained with Rhodamine-123 for 30 min in a CO 2 incubator. Subsequently, the fluorescence intensity was measured at 480 and 530 nm using a fluorescence spectrophotometer (Spectramax M2, Molecular Devices, USA). Measurement of oxidative stress as reactive oxygen species (ROS) levels The intracellular generation of ROS was measured by the DCFH-DA method described by Zulueta et al., 1997 [ 42 ]. Approximately, 1×10 5 cells/well MCF-7 cells were grown in a 24-well plate in a CO 2 incubator. The cells were treated with the IC 50 concentration of TQ-PEI/PLA-NPs for 24 h. The cells were washed with PBS and incubated with 5 mM DCFH-DA for 30 min. Using a fluorescence spectrophotometer (Spectramax M2, Molecular Devices, USA), the excitation/ emission wavelength (485/520 nm) was recorded. Untreated cells are used as controls. Results are expressed as fold change compared to the control. Quantification of Caspase-3 and − 9 activities The caspase enzyme activity was evaluated using Caspase-3 and 9 assay kits (Caspase-Glo®, Promega) [ 43 ]. Briefly, 1×10 5 MCF-7 cells were grown in a 96-well plate in a CO2 incubator for 24 h. The IC 50 concentration of TQ-PEI/PLA-NPS was added to the cells and incubated overnight. Equal volumes of reagents and cell culture supernatants (100 µL) were mixed for 45 min, and samples were read at excitation /emission (400/505 nm) using a fluorescence spectrophotometer (Spectramax M2, Molecular Devices, USA). Statistical analysis The data are presented as mean ± SD of three distinct studies. * p < 0.05 is statistically significant compared to the control group. We used SPSS 20.0 software for the data analysis. Data analysis was performed using one-way ANOVA and Dunnett's multiple comparison tests. Results Encapsulation efficiency of TQ-PEI/PLA-NPs The solvent evaporation-emulsification technique successfully formulated thymoquinone-encapsulated polyethyleneimine/poly (lactic acid) nanoparticles. The encapsulation of TQ into PEI/PLA NPs was confirmed by measuring the absorption at various time intervals. Figure 1 shows the encapsulation of TQ into NPs. The TQ-encapsulation in PEI/PLA NPs exhibited a remarkable encapsulation efficiency of 85% (w/w), accompanied by an active drug loading capacity of 9.34% (w/w). The excellent drug loading capacity of nanocarriers is beneficial for cancer targeting applications. In vitro drug release of TQ-PEI/PLA-NPs The in vitro drug release study was performed in acetate buffer (pH 3.5) and phosphate-buffered saline (PBS) solution of pH 5.8 and pH 7.2 at 37°C. As seen in Fig. 2 , TQ-PEI/PLA-NPs exhibited a controlled release of TQ throughout the study period. Approximately, 33% (acetate buffer pH 3.5), 36% (PBS pH 5.8) and 32% (PBS pH 7.2) of TQ were released within 24 h of the study period while 76% (acetate buffer pH 3.5), 81% (PBS pH 5.8) and 73% (PBS pH 7.2) of TQ were released from PEI/PLA NPs at the end of 72 h. Drug release kinetics of TQ-PEI/PLA-NPs The results of the in vitro drug-release kinetics are summarised in Table 1 . We observed that the drug release profile of the TQ-PEI/PLA-NPs confirms the Higuchi model, with graphs showing high linearity and r 2 values in the range of 0.9889 (pH 3.5), 0.9898 (pH 5.8), and 0.9910 (pH 7.2), which mostly suggested the diffusion process. Furthermore, the observed release kinetics data demonstrated the non-Fickian behaviour of the TQ-PEI/PLA NPs formulation. The diffusion exponent (n) was shown to be larger than 0.69 (pH 7.2, or 0.697), indicating the involvement of the Case II transport drug-release mechanism. The Hixson–Crowell model's high r 2 values (0.9836) indicated a constant drug release and uniform dissolution, but TQ release was mostly sustained at pH 3.5, pH 5.8, and pH 7.2, where a fixed amount of drug-release pattern was seen. Table 1 Drug-release kinetics profile of thymoquinone from TQ-PEI/PLA-NPs. Model Parameter TQ-PEI/PLA-NPs pH 3.5 pH 5.8 pH7.2 Zero order F = K 0 ×t K 0 0.017 0.018 0.017 r 2 adjusted 0.9674 0.9570 0.9527 AIC 221.6597 233.2721 231.5051 First order F = 100× [1-Exp (-k 1 ×t)] K 1 0.000 0.000 0.000 r 2 adjusted 0.9824 0.9851 0.9865 AIC 201.2854 198.2880 190.1099 Higuchi model F = K H ×t 1/2 K h 0.982 1.036 0.969 r 2 adjusted 0.9442 0.9550 0.9601 AIC 239.3882 234.8125 225.9246 Korsmeyer-Peppas model F = kKP×t n kKP 0.125 0.185 0.198 r 2 adjusted 0.9889 0.9898 0.9910 n 0.756 0.714 0.697 AIC 187.0580 186.7438 177.8272 Hixon-Crowell model F = 100×[1-(1-kHC×t) 3 ] kHC 0.000 0.000 0.00 r 2 adjusted 0.9844 0.9847 0.9830 AIC 197.3270 199.1586 197.7952 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. Stability studies of TQ-PEI/PLA-NPs A key consideration in the formulation of therapeutics for the efficient treatment of diseases is the stability of the nanoparticles. The stability of the newly prepared TQ-PEI/PLA NPs is shown in Fig. 3 in a range of physiological solutions, including simulated gastric juice, buffer solutions (pH 7.2 and 9.0), acetate buffer (pH 3.5 and 5.5), and 0.5% BSA. TQ-PEI/PLA NPs exhibited a maximum absorbance of 305–315 nm in the physiological medium and buffer solutions. After 72 h of incubation in buffer solutions and physiological media, there is minimal change in the λ max . Characterization of TQ-PEI/PLA-NPs The FTIR spectra of TQ and TQ-PEI/PLA NPs are shown in Fig. 4 (a) and 4(b), respectively. The FTIR spectra of TQ exhibited significant peaks at wavenumbers of 3249 cm − 1 and 2971 − 2868 cm − 1 , which can be classified as stretching vibrations of the isopropyl and CH 3 groups. The peaks seen at 1639 cm − 1 , 1461 to 1354 cm − 1 , and 1127 cm − 1 correspond to the presence of the distinct C = O group, aromatic C-H stretching, and C = O group, respectively. The FTIR spectra of TQ-PEI/PLA NPs showed peaks at 3282 cm − 1 and 2924 − 2846 cm − 1 , corresponding to the stretching vibrations of the -OH and -CH 3 groups. The additional peaks observed were 1756 cm − 1 corresponding to C = O stretching vibration, 1645 cm − 1 corresponding to -C-O stretching, 1555 cm − 1 to 1401 cm − 1 according to CH 3 antisymmetric bending, 1313 cm − 1 corresponding to symmetric bending of tertiary carbon in the isopropyl group, and 1088 − 913 cm − 1 corresponding to CH 3 rocking. The presence of these identifiable peaks in the TQ and TQ-PEI/PLA NPs formulation suggests interaction between the drug and polymer matrices. The X-ray diffraction pattern of TQ-PEI/PLA NPs is depicted in Fig. 4 (c). The prominent peaks observed at 2θ = 20° and 41° suggest that the NPs have a crystalline structure. The TQ-encapsulated PEI/PLA NPs had a particle size of 80–90 nm (PDI approximately 0.2) (Fig. 4 (d)) and a zeta potential of + 1 mV (Fig. 4 (e)). High-resolution XPS analysis was conducted to investigate the surface chemical composition of formed TQ-PEI/PLA NPs (Fig. 4 (f)). The XPS data of O1s, N1s, and C1s obtained from TQ-PEI/PLA NPs in Fig. 4 (f) shows that the O1s peak at 528 eV corresponds to the = O-C––O–C-H bond of poly(lactic acid), the N1s signal at 402 eV corresponds to the C-NH 2 bond of polyethyleneimine, and the C1s signal indicates the presence of the -CH-CH 3 /-C-C- group of poly(lactic acid) in the NPs at 296 eV. FESEM pictures of TQ-encapsulated PEI/PLA nanoparticles showed the presence of uniform, monodisperse, spherical nanoparticles with a smooth surface and a diameter of around 100 nm, enclosed with TQ (Figs. 4 (g)-(j)). Molecular docking The binding energy of TQ, SR-318 and DOX to the target protein MAPK14 was quantified to be -7.8, -9.3 and − 8.5 kcal/mol, respectively. The molecular docking studies indicated that the binding energy of TQ is close to Doxorubicin. Figures 5 (a) and 5(d) demonstrate the formation of one hydrogen bonding (106A THR (2.03 Å)) and six hydrophobic contacts (38A VAL (3.74Å), 38A VAL (3.44Å), 51A ALA (3.84Å), 53A LYS (3.74Å), 53A LYS (3.55Å), and 167A LEU (3.91Å)) between the TQ and MAPK14 protein molecules. SR-318, formed 12 hydrophobic interactions (53A LYS (3.68Å), 67A ARG (3.49Å), 71A GLU (3.84Å), 71A GLU (3.72Å), 74A LEU (3.36Å), 84A ILE (3.70Å), 84A ILE (3.91Å), 106A THR (3.84Å), 167A LEU (3.65Å), 167A LEU (3.80Å), and 169A PHE (3.90Å), two hydrogen bonds 71A GLU (2.46Å) and 168A ASP (3.29Å), and one salt bridge 71A GLU (4.57Å) with the MAPK14 protein, as depicted in Figs. 5 (b) and 5(e). DOX, the standard drug, established four hydrogen bonding connections (67A ARG, 70A ARG, 149A ARG, and 170A GLY) and two hydrophobic interactions (75A LEU, 3.67Å, and 141A ILE, 3.57Å) with the MAPK14 protein, as depicted in Figs. 5 (c) and 5(f). Molecular dynamics simulation of TQ-PEI/PLA-NPs Molecular dynamics (MD) simulation was conducted to examine the stability and intermolecular bonding of the TQ-MAPK14 complex. The MD trajectory movements of the target TQ-MAPK14 complex exhibited RMSD values ranging from 2.8 Å to 3.6 Å (Fig. 6 (a)). RMSD fluctuations varied between 1.2 and 2.6 Å, suggesting stability. The amino acids’ binding to the major functional groups of the ligands did not result in any notable changes in the RMSF analysis (Fig. 6 (b)). The darker lines (Fig. 6 (c)) indicate ongoing involvement with the target. Figure 6 (d) illustrates the sequential arrangement of hydrogen bonds, hydrophobic interactions, ionic interactions, and water bridges in every amino acid residue. The protein-ligand contacts of the TQ-MAPK14 protein complex showed that the amino acid residue ALA 564 (89%) contributed the highest interaction with TQ (Fig. 6 (e)). Based on the molecular docking and dynamics studies, TQ strongly bind to the protein MAPK14, and the protein-ligand complex is highly stable. Cytotoxicity of TQ-PEI/PLA-NPs The cytotoxicity of TQ-PEI/PLA NPs, doxorubicin and free-TQ was tested against MCF-7 cells. TQ-PEI/PLA NPs exhibited a dose-dependent and time-dependent cytotoxic effect in MCF-7 cancer cells. TQ-PEI/PLA-NPs at 100 µg/mL showed 54.4 ± 1.86% cellular viability in MCF-7 cells with an IC 50 of 21.99 µg/mL after 24 h, Untreated control cells and those treated with 21.99 µg/mL TQ-PEI/PLA-NPs, produced morphological changes in MCF-7 cells are presented in Fig. 7 (a)). Figures 7 (b) and 7(c). Effect of TQ-PEI/PLA-NPs on apoptotic cells As shown in Fig. 7 (d), the control cells stained green and underwent no modifications. On the other hand, TQ-PEI/PLA-NPs-treated cells (Fig. 7 (e)) were stained orange, indicating the presence of apoptotic cells. The treated cells also show nuclear disintegration, shrinkage, and membrane blebbing. This result points to a substantial build-up of TQ inside the MCF-7 cells. Effect of TQ-PEI/PLA-NPs on nuclear morphology As shown in Fig. 7 (g), significant changes were observed in the chromatin structure after TQ-PEI/PLA-NPs treatment, compared to the untreated control (Figure (f)). The control cells reveal characteristic spherical nuclei with a normal blue tint, but the treated cells exhibit a vibrant colour, abnormal nuclei, and compressed chromatin with irregular cell morphology. Mitochondrial transmembrane potential damage of TQ-PEI/PLA-NPs As shown in Figs. 7 (h) and (i), treatment with TQ-PEI/PLA-NPs of MCF-7 cells at 21.99 µg/mL resulted in a significant decrease in mitochondrial membrane potential. Furthermore, the TQ-PEI/PLA nanoparticles induced depolarization of the mitochondrial membrane. ROS generation of TQ-PEI/PLA-NPs Figures 7 (j) and 7(k) show that the untreated MCF-7 cells exhibited reduced ROS production and delayed apoptosis initiation. The present study demonstrated that TQ-PEI/PLA NPs can stimulate the production of ROS in cancer cells (Fig. 7 (l)) using the DCFH-DA staining technique. Caspase-3 and − 9 activities of TQ-PEI/PLA-NPs Activation of Caspases-3 and − 9 by external stimuli marks them as the terminal phase inducers of programmed cell death in cancer cells. The activities of caspase-3 (Fig. 7 (m)) and caspase-9 (Fig. 7 (n)) were increased in cells exposed to 21.99 µg/mL TQ-PEI/PLA-NPs compared to control cells (untreated). Discussion Cancer is still one of the world’s leading causes of death and a significant health concern. The survival rate for cancer patients is relatively low, even with the availability of multiple cancer therapies. Searching and developing novel therapeutic compounds for treating and eradicating cancer cells is challenging, expensive, and time-consuming. Even though chemotherapy has dramatically improved the overall survival rate of patients, it still shows a variety of psychological and physical side effects that lower their quality of life. Therefore, in the fight against neoplasia, natural compounds are seen to provide a promising substitute for several chemotherapy treatments. In the last 30 years, about 74% of newly authorized anticancer agents have been derived from natural sources or were inspired by natural products [ 44 ]. TQ is a natural compound believed to be a potential antioxidant molecule, which can be combined with anticancer therapies to improve its efficacy and reduce toxicity [ 45 ]. Meanwhile, TQ alone has substantial anticancer activity against various types of cancers [ 46 ]. At the same time, TQ use remains limited in the research community because of its insolubility in aqueous medium and inability to reach the disease site [ 47 ]. However, TQ shows potential cytotoxic activity against breast cancer cells in vitro studies [ 48 ]. Therefore, TQ-PEI/PLA-NPs were formulated to increase the bioavailability of TQ at the disease site. For this, the nanoparticles are functionalized with a synthetic cationic polymer (PEI), a proven inducer of endosomal escape[ 49 ]. Meanwhile, PLA provides a biodegradable and biocompatible core for drug encapsulation polymeric nanocarrier system to improve solubility, stability, and controlled release of the encapsulated medicine [ 50 ]. Nevertheless, multiple things require discussion. Thymoquinone (TQ), which is isolated from Nigella sativa or black seeds, has many advantages, including anti-cancer efficacy [ 51 ]. TQ proved to be an anti-cancer agent against TNBC (triple negative breast cancer) through various mechanisms and modulation of the tumor micro-environment [ 52 ]. Long-term treatment with a low dose of TQ may inhibit breast cancer cell growth and prevent cancer cell growth in the sub-G1 phase. TQ induces apoptosis and regulates the expression of genes that promote and inhibit apoptosis [ 53 ]. Additionally, it has been demonstrated to reduce metastasis, ERK1/2, and PI3K activity, as well as the phosphorylation of NF-B and IKK [ 54 ]. However, TQ has a low bioavailability at the target site due to its high hydrophobicity and poor solubility in aqueous solutions [ 55 ]. TQ is also susceptible to environmental factors like light and temperature [ 56 ]. TQ has been encapsulated into various nanoparticles to improve its solubility and efficacy [ 57 ]. Nanoparticles afford controlled release of loaded drugs, at the right place and the right time. Regardless of their solubility, they enhance the bioavailability and cellular uptake of the encapsulated TQ. Nanoparticles can be used to transform sensitive compounds into stable substances. For instance, TQ loaded in a nanostructured lipid carrier significantly increased the anticancer activity in 4T1 tumour-bearing mice in a murine breast cancer model [ 58 ]. On the other hand, TQ was also incorporated into solid lipid nanoparticles (SLNs) and showed antidepressant-like effects in rats. TQ also prevents cervical cancer by delaying cell invasion and ROS-mediated death, as demonstrated by its loading into mesoporous silica nanoparticles. The physicochemical features of nanoparticulate systems, particularly their shape, size and surface charge, influence their physical stability, interactions with biological systems, release rates of encapsulated compounds, and interactions with target malignant cells. The drug-loaded nanoparticles exhibited particle sizes below 250 nm, with narrow size distributions and low polydispersity indices, which also play a critical role in influencing anticancer efficacy. In the present study, SEM examination revealed that the nanoparticles exhibited a spherical morphology with a smooth surface texture. Entrapment efficiency and loading capacity of thymoquinone in the developed nanoparticles were found to be 85% w/w and 9.34% w/w, respectively. The release of thymoquinone from the polymer matrix exhibited a biphasic pattern with an initial burst release during the first 24 h, followed by slower and sustained release of the drug. The slow and steady release of the drug ensures maximum efficacy over time. Similar patterns have been previously observed for the release of TQ from the polymeric systems. The cytotoxic profile of formulated TQ-PEI/PLA NPs was assessed using an MTT assay. The formulated TQ-PEI/PLA NPs demonstrated significant cytotoxicity in MCF-7 cells at 100 µg/ mL, showing 54.52 ± 1.01% of cellular viability. Similarly, 50 µM of TQ-loaded liposomes has more cytotoxic effects against MCF-7 and MCF-10 cells than 50 µM TQ [ 59 ]. The potential cytotoxic effects of TQ might be controlled by the release of TQ by polymeric carriers. Controlled release from polymeric carriers can result in elevated cytotoxic effects in the cancer microenvironment relative to free drug, attributable to the augmented drug concentration and targeted delivery, rather than an increase in the drug's intrinsic toxicity. The initiation of apoptosis may be a vital mechanism to impede breast cancer cell proliferation. Cancer cells may utilize many biological mechanisms to inhibit apoptosis and acquire resistance to chemotherapeutic or cytotoxic agents, including the production of antiapoptotic proteins or the downregulation or mutation of tumor suppressor genes. Apoptosis and associated cellular death mechanisms significantly impact cancer cell proliferation, making them viable targets for many cancer therapies [ 60 ]. Emerging research indicates that natural products play a crucial role in breast cancer treatment by inhibiting cell death. Selective fluorescent DNA-binding dyes, such as acridine orange and ethidium bromide, are used for morphological investigation due to their simplicity, precision, and speed. These tests eliminate the cell fixation stage, thereby avoiding several potential artifacts [ 61 ]. Thus, a DNA-specific AO/EBr double-staining assay was performed in this study to assess the apoptotic efficacy of the synthesized TQ-encapsulated PEI/PLA nanoparticles. TQ-PEI/PLA nanoparticles treated cells appeared to undergo nuclear disintegration, shrinkage, and membrane blebbing. Similarly, PLGA-PEI-EPI-PTX NPs induce cell membrane blebbing and chromatin condensation, indicating early apoptosis in cervical carcinoma cells (HeLa) [ 62 ]. Increased intracellular concentrations of reactive oxygen species (ROS) cause damage to proteins, nucleic acids, lipids, membranes, and organelles, potentially initiating cell death mechanisms such as apoptosis. Specific chemotherapies induce programmed cell death (apoptosis) in cancer cells by increasing Reactive Oxygen Species (ROS) levels. Apoptosis produced by ROS can transpire through various mechanisms, including mitochondrial dysfunction and the activation of apoptotic pathways. Different chemotherapy drugs increase intracellular reactive oxygen species (ROS) levels and can modify the redox equilibrium of cancer cells. TQ-PEI/PLA nanoparticles delivered to MCF-7 cells demonstrated elevated levels of reactive oxygen species compared to untreated cells. Reactive oxygen species (ROS) buildup can impair the respiratory chain, resulting in mitochondrial malfunction and perhaps activating a p53-mediated intrinsic apoptotic pathway. This route entails releasing mitochondrial components such as cytochrome c, which activate caspase enzymes and ultimately result in cell death. The efflux of caspase-3 and − 9, especially from the mitochondria, is a critical event in the intrinsic pathway of apoptosis, a programmed cell death mechanism that may result in the cancer cell death. When cells experience stress or damage, the mitochondrial membrane may become permeable, permitting cytochrome to escape into the cytoplasm. The released cytochrome c subsequently engages with Apaf-1 to construct the apoptosome, which activates caspase 9, leading to the activation of caspase 3, the "executioner" caspase that initiates cellular apoptosis [ 63 ]. The drop in mitochondrial membrane potential marks the commencement of the mitochondrial apoptotic process. The collapse of the mitochondrial membrane potential and the disturbance of the electron transport chain gradient leading to depolarization of the mitochondrial membrane. The preservation of an electron transport chain may elucidate the alteration in mitochondrial polarization noted following chemical exposure [ 64 ]. This work demonstrated that TQ-PEI/PLA nanoparticles induced significantly increased levels of ROS, caspase-3 and − 9, along with a reduction in mitochondrial membrane potential, in MCF-7 cells compared to untreated MCF-7 cells. Pt-coated Au nanoparticles (Pt-Au NPs; 27 ± 20 nm) exhibited increased uptake and cytotoxicity, along with enhanced levels of ROS, NO, caspase 9, and caspase 3, as well as a reduction in mitochondrial membrane potential in human breast cancer MCF-7 cells compared to non-cancerous human cells (HUVE). Gholinejad and colleagues have documented a comparable observation about TiO2 NP-induced cell death in HUVE cells [ 65 ]. Cadmium selenide/zinc sulfide quantum dot nanoparticles have been documented to elicit pyroptosis in hepatic L02 cells, mediated through mitochondrial reactive oxygen species induction and mitochondrial membrane potential loss [ 66 ]. The efficacy of apoptotic cell death was meticulously assessed using nuclear morphological alterations utilizing a DAPI assay. Apoptosis in mammalian cancer cells is frequently associated with specific morphological and physiological alterations, such as membrane blebbing, phosphatidylserine externalization, chromatin condensation, nuclear fragmentation, and DNA degradation, initially into large fragments and subsequently into small nucleosome fragments [ 67 ]. Conclusion The present study successfully formulated TQ-PEI/PLA nanoparticles via the solvent evaporation-emulsification method. TQ-PEI/PLA nanoparticles showed potential anticancer effectiveness against breast cancer cell lines (MCF-7) when compared to free TQ. The lead compound TQ exhibited a notable binding affinity for a breast cancer target (MAPK14). The significant anticancer activity of TQ may stem from improved distribution of encapsulated TQ into cancer cells, aided by certain polymers. The nanoparticles were synthesized with a synthetic cationic polymer (polyethylenimine (PEI), noted for its capacity to facilitate endosomal escape, whilst PLA provides a biodegradable and biocompatible core for drug encapsulation. TQ was well encapsulated into PEI/PLA nanoparticles, achieving an encapsulation efficiency of 85% w/w and a loading capacity of 9.34% w/w. The drug release profile demonstrated that TQ-PEI/PLA nanoparticles exhibited a sustained and regulated release mechanism. The physicochemical properties of the synthesized TQ-PEI/PLA nanoparticles were validated using FTIR, XRD, XPS, a particle size analyzer, and SEM. The synthesized TQ-PEI/PLA nanoparticles exhibited the ability to suppress the growth of MCF-7 cells and induce apoptosis. Moreover, in vivo animal investigations are crucial for elucidating the molecular pathways governing malignant cell death triggered by TQ encapsulated in PEI/PLA nanoparticles. Declarations Acknowledgements The authors are grateful to the management of Kalasalingam Academy of Research and Education, Krishnankoil, India, for research fellowships and for utilizing research facilities. Author Contributions SK supervision, fund acquisition, and project administration, resources, writing review and editing; JS, PP, EB, TP, MS, RM, PM, SK writing-original draft, formal analysis, investigation; SK, JS conceptualization, writing, investigation and editing. All authors have read and agreed to the published version of the manuscript. Funding Selvaraj Kunjiappan gratefully acknowledges the Management of Kalasalingam Academy of Research and Education for the Seed Money Grant (KARE/VC/R&D/SMPG/2021–2022/1). The University Research Fellowship provided by the management of Kalasalingam Academy of Research and Education to Mr. Jeganpandi Senthamarai Pandi is gratefully acknowledged. Data Availability All data generated or analyzed during this study are included in this manuscript. Conflict of interest The authors declare that they have no conflict of interest. Ethical Approval Ethical approval was not required for this research. Consent to Participate Not applicable. 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Microb Ecol 36:181–192. https://doi.org/10.1007/s002489900105 Hemananthan E (2020) In vitro studies to analyze the stability and bioavailability of thymoquinone encapsulated in the developed nanocarrier. J Dispers Sci Technol 41:243–256. https://doi.org/10.1080/01932691.2018.1564672 Sze OY (2019) Toxicity And Anti-Breast Cancer Properties Of Thymoquinone-Loaded Nanostructured Lipid Carrier In Mice, PhD thesis, Universiti Putra, Malaysia Sohrabi B, Qadbeigi M, Sabouni F, Hamta A (2024) Thymoquinone nanoparticle induces apoptosis and cell migration retardation through modulating of SUMOylation process genes in breast cancer cell line. Iran J Biotechnol 22:e3676. https://doi.org/10.30498/ijb.2024.390400.3676 Reed JC (2003) Apoptosis-targeted therapies for cancer Cancer cell 3 17–22 https://doi.org/10.1016/s1535-6108(02)00241-6 Debroy A, Yadav M, Dhawan R, Dey S, George N (2022) DNA dyes: toxicity, remediation strategies and alternatives Folia Microbiol 67 555 – 71 https://doi.org/10.1007/s12223-022-00963-8 Sharma N, Kumari RM, Gupta N, Syed A, Bahkali AH, Nimesh S (2020) Poly-(lactic-co-glycolic) acid nanoparticles for synergistic delivery of epirubicin and paclitaxel to human lung cancer cells Molecules 25 4243 https://doi.org/10.3390/molecules25184243 Stegh AH, Peter ME (2001) Apoptosis and caspases. Cardiol Clin 19:13–29. https://doi.org/10.1016/s0733-8651(05)70192-2 Ahmad M, Wolberg A, Kahwaji CI (2018) Biochemistry, electron transport chain Gholinejad Z, Ansari MHK, Rasmi Y (2019) Titanium dioxide nanoparticles induce endothelial cell apoptosis via cell membrane oxidative damage and p38, PI3K/Akt, NF-κB signaling pathways modulation. J Trace Elem Med Biol 54:27–35. https://doi.org/10.1016/j.jtemb.2019.03.008 Akhtar MJ, Ahamed M, Alhadlaq HA, Alshamsan A (2017) Mechanism of ROS scavenging and antioxidant signalling by redox metallic and fullerene nanomaterials: Potential implications in ROS associated degenerative disorders. Biochim Biophys Acta Gen Subj 1861:802–813. https://doi.org/10.1016/j.bbagen.2017.01.018 Mustafa M, Ahmad R, Tantry IQ, Ahmad W, Siddiqui S, Alam M, Abbas K, Hassan MI, Habib S, Islam S (2024) Apoptosis: a comprehensive overview of signaling pathways, morphological changes, and physiological significance and therapeutic implications Cells 13 1838 https://doi.org/10.3390/cells13221838 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6858817","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472899892,"identity":"04481495-4626-4ab8-8000-d23d355eb760","order_by":0,"name":"Jeganpandi Senthamarai Pandi","email":"","orcid":"","institution":"Kalasalingam Academy of Research and Education","correspondingAuthor":false,"prefix":"","firstName":"Jeganpandi","middleName":"Senthamarai","lastName":"Pandi","suffix":""},{"id":472899893,"identity":"d4913057-deb8-4979-845f-6811c339eba9","order_by":1,"name":"Parasuraman Pavadai","email":"","orcid":"","institution":"M.S. Ramaiah University of Applied Sciences","correspondingAuthor":false,"prefix":"","firstName":"Parasuraman","middleName":"","lastName":"Pavadai","suffix":""},{"id":472899894,"identity":"b5875819-a02a-4f40-af05-09bd33163919","order_by":2,"name":"Ewa Babkiewicz","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Babkiewicz","suffix":""},{"id":472899895,"identity":"5e23e214-2345-4869-974b-4ef6186e49ee","order_by":3,"name":"Theivendren Panneerselvam","email":"","orcid":"","institution":"Vels Institute of Science, Technology, and Advanced Studies","correspondingAuthor":false,"prefix":"","firstName":"Theivendren","middleName":"","lastName":"Panneerselvam","suffix":""},{"id":472899898,"identity":"443a7517-eecc-45c3-b7df-f44e1be4b785","order_by":4,"name":"Piotr Maszczyk","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Piotr","middleName":"","lastName":"Maszczyk","suffix":""},{"id":472899899,"identity":"fdd6959c-31a1-4eba-8908-a69fd39ad687","order_by":5,"name":"Murugesan Sankaranarayanan","email":"","orcid":"","institution":"Birla Institute of Technology and Science Pilani","correspondingAuthor":false,"prefix":"","firstName":"Murugesan","middleName":"","lastName":"Sankaranarayanan","suffix":""},{"id":472899901,"identity":"230d52d5-3bb9-405b-8f80-38e78022a8e0","order_by":6,"name":"Ramar Mohan","email":"","orcid":"","institution":"UConn School of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Ramar","middleName":"","lastName":"Mohan","suffix":""},{"id":472899905,"identity":"6344bb08-cd33-4630-98ac-644f99422228","order_by":7,"name":"Selvaraj Kunjiappan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3RuwrCMBiG4T8E6vJL1wiCtxBx9XArLbkHFwdbCsmiuLp5C4KrQ0pBl+LsWCk4OQhOgoOth7XRTTDv0EDoAx8EwGb7yWjw+ALSBLLHDQkMhLyJI8D7hgAgdl7EUEuFkl3X/RpgeuG+hJYbUJlVEZ7GsjE5Cgr16corSHuuieKVhPkRR00puPWVLghZApGsctjiELVvelwQzEsyMBLYkzBHnRTDJrQc5hsJT/0wb+otdXDT4d6OiXliGqYSHZ/0SLgocnYednszpY7Vw14J53my8lU/q//hfzabzfaP3QFDyUMJHWUdHAAAAABJRU5ErkJggg==","orcid":"","institution":"Kalasalingam Academy of Research and Education","correspondingAuthor":true,"prefix":"","firstName":"Selvaraj","middleName":"","lastName":"Kunjiappan","suffix":""}],"badges":[],"createdAt":"2025-06-10 04:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6858817/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6858817/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85004442,"identity":"964b3b8a-6eb1-4868-9282-51ebe3833538","added_by":"auto","created_at":"2025-06-19 20:11:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53213,"visible":true,"origin":"","legend":"\u003cp\u003eEncapsulation efficiency of Thymoquinone (TQ) at various time points.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6858817/v1/86f3a72aaa17c0c1f45dd55f.jpg"},{"id":85004448,"identity":"80ddaa34-3d2f-41ed-a68a-7a0802577b19","added_by":"auto","created_at":"2025-06-19 20:11:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46838,"visible":true,"origin":"","legend":"\u003cp\u003eDrug release of TQ at various buffers and time points.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6858817/v1/b3d48f99ed133981049bfc66.jpg"},{"id":85004444,"identity":"fb36f686-20d3-4dcd-970a-9e49df4acf71","added_by":"auto","created_at":"2025-06-19 20:11:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43545,"visible":true,"origin":"","legend":"\u003cp\u003eStability of TQ-PEI/PLA-NPs in various physiological solutions, buffers and simulated gastric juice.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6858817/v1/aed8a2a4a98eeab05c318a65.jpg"},{"id":85004590,"identity":"ea7fd35b-cf89-4733-b53b-65864db2da4f","added_by":"auto","created_at":"2025-06-19 20:27:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":83804,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of TQ (a), TQ-PEI/PLA-NPs (b), XRD spectrum of TQ-PEI/PLA-NPs (c), particle size analyzer of TQ-PEI/PLA-NPs (d), zeta potential of TQ-PEI/PLA-NPs (e), XPS spectrum of TQ-PEI/PLA-NPs (f), and FESEM images of TQ-PEI/PLA-NPs (g)-(j).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6858817/v1/c8a45e9a0504a17046c90c36.jpg"},{"id":85004591,"identity":"d9f8feb2-6215-481a-97a0-9c688e469c66","added_by":"auto","created_at":"2025-06-19 20:27:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":62410,"visible":true,"origin":"","legend":"\u003cp\u003eDisplayed 3D and 2D interaction between the TQ and MAPK14 (a) 7(d), 3D 2D interaction between the SR-318 and MAPK14 \u0026nbsp;(b) and (e), and 3D and 2D interaction between the Doxorubicin and MAPK14 (c) and (f).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6858817/v1/ffc8598a3d100dfcd068036d.jpg"},{"id":85004450,"identity":"2a8f7b53-f21d-4e9f-ab69-2af83ecfbb9d","added_by":"auto","created_at":"2025-06-19 20:11:51","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":45726,"visible":true,"origin":"","legend":"\u003cp\u003eRMSD plot for 100 ns MD simulation of TQ-MAPK14 protein docked complex (a);\u003c/p\u003e\n\u003cp\u003eRMSF plot of TQ for characterizing changes in the ligand atom positions (b); TQ-MAPK14 protein contacts timeline representation (c); Percentage of amino acid and water-mediated interactions contribution in MD simulation with TQ (d), and TQ contacts with respect to the amino acids in the target protein MAPK14 (e).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6858817/v1/63deeedf37de3b249ead8850.jpg"},{"id":85004462,"identity":"c25a0cc6-50da-4f97-871d-a451b952f37a","added_by":"auto","created_at":"2025-06-19 20:11:51","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":141764,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic activity of human breast adenoma cancer (MCF-7) cells using different concentrations of TQ, TQ-PEI/PLA-NPs and doxorubicin after 24 h treatment. The percentage of apoptotic cells increased dose-dependently. Values are mean ± standard deviation of triplicate measurements (p\u0026lt; 0.05) (a). Morphology of the control cells (b) and treated cells (c) observed using a phase-contrast microscope. Measurement of apoptosis by double staining (AO/EB) control cells (untreated) (d), MCF-7 cells treated with TQ-PEI/PLA-NPs for 24 h (e). DAPI-stained images of MCF-7 cancer cells. Control cells (f) and treatment with TQ-PEI/PLA-NPs (g). Effects of TQ-PEI/PLA-NPs on mitochondrial transmembrane potential in MCF-7 cancer cells, control cells (untreated) (h), loss of mitochondrial transmembrane potential on treated cells (i). ROS generation was measured as relative fluorescence intensity using a fluorescence microscope; an image of untreated control cells was taken (j), and an increase in ROS was found in TQ-PEI/PLA-NPs-treated cells (k). Results expressed as mean± standard deviation of triplicate measurements (p\u0026lt; 0.05) (l). Activity of Caspase-3 (m) and −9 (n) of 21.99 µg/mL of TQ-PEI/PLA-NPs against MCF-7 cells.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6858817/v1/5aa990e2186071b93517efa9.jpg"},{"id":88975791,"identity":"21ff49a6-6392-478b-ac81-750a3900e9c9","added_by":"auto","created_at":"2025-08-13 10:23:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1631411,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6858817/v1/81b80033-52ec-4af9-b226-911007a2346f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeted delivery of thymoquinone-encapsulated polyethyleneimine/poly (lactic acid) nanoparticles into breast cancer cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the most often diagnosed malignancy worldwide, with approximately 2.3\u0026nbsp;million new cases and 685,000 fatalities among women in 2020 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It accounts for 14% of all cancer diagnoses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While the majority of breast cancer incidence and deaths occur in those over the age of 50, a significant number of cases in Africa are diagnosed in younger individuals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Incidence rates vary significantly across different regions, with the highest rates reported in Australia/New Zealand, Western Europe, and Northern America [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It is reported that every four minutes, an Indian woman is diagnosed with breast cancer. In India, breast cancer is becoming more common in both rural and urban settings [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. According to the SURVCAN-3 study (Cancer Survival in Countries in Transition), the global median 3-year breast cancer survival rate is 84% in 2023 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Of those instances, 68% occurred in India, which had a 5-year breast cancer survival rate of 66.1% based on the CONCORD-3 study, which was conducted between 2010 and 2014 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Genetic, environmental, hormonal, and sedentary lifestyle factors all contribute to the etiology of breast cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Researchers have found several risk factors that may elevate an individual's likelihood of acquiring breast cancer, despite the precise etiology of the disease remaining unidentified. High-fat junk food, excessive alcohol consumption and cigarette smoking, the use of medicines such as hormonal contraceptives and exogenous female hormones (menopausal hormone therapy), inactivity, and a poorly maintained body mass index (BMI) are common risk factors [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Most of the time, early cancer detection allows successful treatment, leading to a better prognosis and lower death rate. However, the prevalence of breast cancer has been rising annually [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The intricate molecular mechanisms of breast cancer present a considerable challenge; nonetheless, the diagnostic and therapeutic approaches now employed are inadequate in preventing disease development and minimizing toxicity.\u003c/p\u003e \u003cp\u003eCurrent approaches, including surgical removal of the cancer tissue, laser and radiation therapy, immunotherapy, endocrine (hormone) therapy, personalized medicine, chemotherapy and combination therapy, are used to treat breast cancers, although chemotherapy appears more effective [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, patients under chemotherapy suffer from significant toxicity linked to the treatment [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As a result, scientists look for safer and more potent anticancer drugs from natural resources. Since plants and their derivatives have played a crucial role in the development of effective anticancer medications [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For example, paclitaxel from \u003cem\u003eTaxus brevifolia\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] irinotecan and camptothecin from \u003cem\u003eCamptotheca acuminata\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], curcumin from \u003cem\u003eCurcuma longa\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] vincristine and vinblastine from \u003cem\u003eVinca rosea\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and etoposide and podophyllotoxin from the mandrake plant [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] have proven effective in treating breast cancer. Thymoquinone (TQ) is the primary active molecule present in the oil of black cumin seed (\u003cem\u003eNigella sativa)\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Chemically, it is a monoterpene and consists of a benzoquinone moiety with antioxidant and anticancer properties [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Researchers have demonstrated the potential of TQ for the treatment of various cancer types, including breast cancer. By downregulating Bcl-2 and c-FLIP in renal cell cancer, it dramatically increases apoptosis. In human RCC 769-P and 786-O cell lines, it also suppresses metastasis and the epithelial-mesenchymal transition (EMT) by altering the LKB1/AMPK pathway [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It also reduces renal cell carcinoma by inducing autophagy through the AMPK/mTOR signalling pathway [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, poor aqueous solubility restricts TQ's therapeutic applications [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This limitation can be mitigated by developing an innovative nano-carrier technology for targeted drug delivery to disease locations.\u003c/p\u003e \u003cp\u003ePolymeric nanoparticles have been selected for this study because of their distinctive properties, including enhanced drug loading capacity, prolonged stability in blood circulation, biodegradability, and efficient drug delivery. Most researchers use biodegradable polymers, such as poly(lactic acid) (PLA), poly(caprolactone) (PCL), and poly(lactic-co-glycolic acid) (PLGA), which have been thoroughly tested for biocompatibility, for making polymeric nanoparticles [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Through matrix degradation or diffusion through the polymer matrix, these polymers offer sustained or extended drug release [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. According to reports, polyethyleneimine (PEI)-coated nanoparticles showed enhanced cellular uptake and function as efficient drug delivery vehicles [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It is anticipated that PEI's higher positive charge will promote nanoparticle aggregation inside negatively charged cancer cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. PEI-coated PLA nanoparticles (PEI/PLA-NPs) were developed to improve the therapeutic effectiveness and bioavailability of thymoquinone. Research by Connelly et al. demonstrated the use of TQ as an NF-κB inhibitor in breast cancer cells (PYG/L129) derived from PyVT mice crossed with NGL reporter mice, treated with breast cancer from PYG/L129 cells obtained from PyVT [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Similarly, Woo et al. investigated the anti-tumor effects of TQ in various cell types, including three distinct breast cancer cell lines (MCF-7, MDA-MB-231, and BT-474). Their findings suggest that the anti-tumor effect of TQ may be mediated through modulation of the PPAR-γ activation pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These studies support the potential application of TQ as a breast cancer therapeutic. Since the poor aqueous solubility of TQ may limit its efficacy, we hypothesize that PEI's positive charge allows it to interact electrostatically with negatively charged biological molecules, such as proteins or nucleic acids, to promote cellular uptake. Hence, the current study aimed to overcome the limitations of TQ by encapsulating it in polymeric nanoparticles using polyethyleneimine/poly (lactic acid). We aimed to encapsulate TQ with PLA and modify the NP surface with positively charged PEI. The positive charge of PEI allows it to bind electrostatically with negatively charged biological molecules, including proteins or nucleic acids, enhancing cellular uptake and facilitating the effective transport of TQ into MCF-7 cells for breast cancer treatment.\u003c/p\u003e"},{"header":"Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eThymoquinone (TQ), poly (lactic acid) (PLA), polyethyleneimine (PEI, Mw 25 kDa, branched) and Tween-80 were sourced from Sigma-Aldrich, USA. Dulbecco's Modified Eagle Medium (DMEM), Fetal bovine serum (FBS), and Penicillin-streptomycin were obtained from Gibco, USA. The Michigan Cancer Foundation 7 (MCF-7) cell line was received from the National Centre for Cell Science (NCCS), Pune, India. MCF-7 cells were passaged every 2\u0026ndash;3 days upon achieving 70\u0026ndash;80% confluence, utilizing 0.25% trypsin-EDTA for detachment, and subsequently resuspended in new medium. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2',7'-dichlorofluorescin diacetate (DCFDA) were purchased from Sigma-Aldrich, USA. 4',6-diamidino-2-phenylindole (DAPI), Rhodamine-123 were acquired from Thermo Scientific, USA. Caspase-Glo\u0026reg; 3 and 9 reagents were purchased from Promega, USA. All the other reagents and solvents used were of analytical grade and purchased from Merck, India. Milli-Q water was used for the entire experiment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFormulation of thymoquinone-encapsulated PEI/PLA nanoparticles\u003c/h3\u003e\n\u003cp\u003eThe solvent evaporation-emulsification method was followed to generate PEI/PLA-NPs encapsulated with TQ [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In short, 5 mL of DMSO was used to dissolve 100 mg of PLA and 5 mg of TQ, and the mixture was sonicated for 30 min. In 50 mL of water, 0.5% (w/v) PEI and 1% Tween-80 were dissolved to form the aqueous phase. After 20 min of constant stirring in the magnetic stirrer, the organic phase was introduced to the aqueous phase drop by drop. It was then instantly homogenized for 3 min at 14,000 rpm. After being separated, washed, and freeze-dried (stored at -20\u0026deg;C overnight), the TQ-encapsulated PEI/PLA NPs were lyophilized for 48 h at -80\u0026deg;C using a freeze dryer (Martin Christ, Gefriertrocknungsanlagen GmbH). For further analysis, the prepared TQ-PEI/PLA-NPs were used.\u003c/p\u003e\n\u003ch3\u003eEncapsulation efficiency and loading capacity of TQ\u003c/h3\u003e\n\u003cp\u003eThe encapsulation efficiency (EE%) and loading capacity (LC%) of TQ in PEI/PLA-NPs were evaluated using the method described in our earlier research [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. 100 mg of TQ-PEI/PLA-NPs were centrifuged at 15000 rpm, and the supernatant was measured at UV-Visible spectrophotometer at 254 nm (UV-1800, Shimadzu, Japan). The TQ encapsulation efficiency (EE%) and loading capacity (LC%) were calculated using the formula given below:\u003c/p\u003e \u003cp\u003ePercentage encapsulation efficiency (EE%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{T}\\text{Q}\\text{a}\\text{d}\\text{d}\\text{e}\\text{d}\\:-\\:\\text{T}\\text{Q}\\text{m}\\text{e}\\text{a}\\text{s}\\text{u}\\text{r}\\text{e}\\text{d}}{\\text{T}\\text{Q}\\text{a}\\text{d}\\text{d}\\text{e}\\text{d}}\\:\\)\u003c/span\u003e\u003c/span\u003e\u0026times; 100 1\u003c/p\u003e \u003cp\u003ePercentage loading capacity (LC%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\left(\\text{T}\\text{Q}\\right)\\text{a}\\text{d}\\text{d}\\text{e}\\text{d}\\:-\\:\\left(\\text{T}\\text{Q}\\right)\\text{m}\\text{e}\\text{a}\\text{s}\\text{u}\\text{r}\\text{e}\\text{d}}{Weight\\:of\\:the\\:nanoparticles}\\)\u003c/span\u003e\u003c/span\u003e \u0026times; 100 2\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-vitro\u003c/b\u003e \u003cb\u003edrug release\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe drug release profile of TQ from TQ-PEI/PLA-NPs was evaluated by the membrane dialysis method using 0.01 M acetate buffer at pH 3.5 and 0.01 M phosphate buffers at pH 5.8 and 7.2 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Briefly, 50 mg of TQ-PEI/PLA-NPs was placed in a dialysis bag (3500 Da, HiMedia Laboratories, India), kept in a buffer solution, placed on a dissolution apparatus at 150 rpm, and maintained at 37\u0026deg;C. About 1mL of sample was taken out and replaced with fresh buffers. The drug content was estimated using a UV-Visible spectrophotometer at 254 nm. The experiments were performed thrice, and drug release (% DRC) was estimated using the below formula\u003c/p\u003e \u003cp\u003ePercentage drug release = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}0-\\text{A}1}{\\text{A}0}\\)\u003c/span\u003e\u003c/span\u003e \u0026times; 100 3\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 \u003cp\u003eThe release kinetics, such as zero-order (cumulative% drug release vs. time), log% drug remaining vs. time), Higuchi (cumulative% drug release vs. square root of time), Korsmeyer-Peppas (log drug release vs. log time), and Hixon-Crowell models, were applied to determine TQ release kinetics. Regression coefficients (r\u003csup\u003e2\u003c/sup\u003e) and release rate constant values were generated for every kinetic model [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eStability studies\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e stability was evaluated in physiological solution (10% NaCl containing 0.5% BSA), buffers like acetate buffer (pH 3.5 and 5.5), phosphate buffer (pH 7.2 and 9.0), and simulated gastric juice [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Briefly, equal volumes of physiological solution, buffers and simulated gastric juice were mixed with freshly prepared TQ-PEI/PLA-NPs and stored at room temperature. After 72 h, the absorbance was recorded using a UV-visible spectrophotometer at 100\u0026ndash;500 nm.\u003c/p\u003e\n\u003ch3\u003eCharacterization of TQ-PEI/PLA-NPs\u003c/h3\u003e\n\u003cp\u003eThe formulated TQ-PEI/PLA-NPS were subjected to various physicochemical characterization. The functional groups were determined using FTIR spectroscopy (IR Tracer-100, Shimadzu, Japan). The IR spectra were obtained in the 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e transmission range at a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. ZetaSizer (ZS Nano, Malvern Instruments, UK) was used to measure the particle size and size distribution of TQ-PEI/PLA-NPS. Using X-ray diffraction (XRD) analysis, the physical characteristics of TQ-PEI/PLA-NPS were investigated (BRUKER D8 Advance, Gmbh, Germany). The detector operates with Cu Kα 1 radiation (λ\u0026thinsp;=\u0026thinsp;0.154060 nm) in a 2θ degree arrangement at a voltage of 40 kV and current power of 30 mA. The elemental composition of TQ-PEI/PLA-NPs was examined using high-resolution X-ray photoelectron spectroscopy (XPS; Thermo Scientific, UK) equipped with an Al/Ka X-ray source (hv\u0026thinsp;=\u0026thinsp;1486.6 eV) with a surface between 0 and 1400 eV. Spectra were processed using XPS (Casa Software Ltd., Teignmouth, UK). The morphology of TQ-PEI/PLA-NPs was assessed using Field Emission Scanning Electron Microscopy (JEOL JSM6700 FESEM). Approximately 2 mg of freeze-dried TQ-PEI/PLA-NPs were reconstituted with distilled water, and the mixture was applied to a glass surface. A gold coating layer was applied over the mixture and dried to prevent electrostatic charge during the experiment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eThe protein data bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/structure/6SFO\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/structure/6SFO\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) provided a three-dimensional X-ray structure of MAPK14 with the bound inhibitor SR-318 (PDB ID: 6SFO, Homo sapiens, resolution: 1.75 \u0026Aring;) of the target protein [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Later, the Swiss-PDB Viewer v4.1.0 tool was used to reinsert the missing residues into the final protein. Using the BIOVIA Discovery Studio Visualizer version 4.0, the precise amino acids of the active site were identified, and the protein structure was visually shown. Chem Sketch was used to create a three-dimensional structure for thymoquinone and doxorubicin (standard). Auto Dock Vina program was used to calculate the binding energies of different ligand and protein combinations. Discovery Studio Client 4.0 was used for the docking inquiry. The binding affinity energies of the complex were computed by considering the ligand conformation at the active binding site as well as the root mean square deviation (RMSD) between the initial and subsequent structures.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular dynamics simulation\u003c/h3\u003e\n\u003cp\u003eMD simulation was performed on the target molecule TQ-MAPK14 complex to assess the stability and interactions between the ligand and protein. An analysis was conducted on the binding mechanisms of potential lead candidates in relation to the protein targets they were designed to target [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The MD simulations were conducted on a Linux-based computer using the Desmond module and the Maestro simulation environment, both developed by Schrodinger, Inc., USA (utilizing the Academic Version, V3.6 algorithm). The intricate protein-ligand interaction was evaluated using the TIP3P water model [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The energy computations in the simulation utilized the OPLS-4 (Optimal Potentials for Liquid Simulation) force field, with recording intervals of 50 picoseconds. A molecular dynamics simulation was performed at three predetermined intervals, each lasting 100 nanoseconds. For a thorough examination of the outcomes derived from the MD simulation, we employed the Desmond module. The application of the Simulation Interaction Diagram greatly enhanced the quality of this analysis. The stability of ligand-protein complexes was assessed using several quantitative measures, such as root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and study of protein-ligand interactions found in simulated trajectories.\u003c/p\u003e\n\u003ch3\u003eAnticancer activity\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity assay\u003c/h2\u003e \u003cp\u003eCell viability using the MTT assay was performed by the method in Ellappan et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Briefly, MCF-7 cell lines were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin at 37\u0026deg;C overnight in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air in a CO\u003csub\u003e2\u003c/sub\u003e incubator. Cells were seeded at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well in a 96-well plate and allowed to incubate overnight. After 24 h of incubation, old medium was replaced with fresh medium containing TQ-PEI/PLA-NPs (100, 50, 25, 12.5, 6.25, 3.125 \u0026micro;g/mL), doxorubicin (0.25 \u0026micro;M) and TQ (100 \u0026micro;g/mL) in MCF-7 cells and incubated for another 24 h. To this, 200 \u0026micro;L of 0.5% MTT solution was added and the plate was placed in the dark at 37\u0026deg;C for 4 h to facilitate formazan crystal formation. After dissolving the formazan crystals in 100 \u0026micro;L of DMSO, the absorbance was measured by a microplate reader (Thermo Scientific, USA) at 570 nm. Cell viability was calculated from the following Eq.\u0026nbsp;(4):\u003c/p\u003e \u003cp\u003ePercentage cell viability = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d}}{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\)\u003c/span\u003e\u003c/span\u003e \u0026times; 100 4\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of apoptosis by AO/EB staining assay\u003c/h2\u003e \u003cp\u003eCellular apoptosis was evaluated by AO/EB staining assay [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Briefly, MCF-7 cells were grown in a 96-well plate at 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and allowed to incubate overnight. After 24 h of incubation, the cells were treated with 400 \u0026micro;L of TQ-PEI/PLA-NPs in DMEM at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h. Following treatment, the cells were washed twice with PBS and then incubated with a staining solution containing 1 \u0026micro;g/mL of AO and 1 \u0026micro;g/mL of EB for 15 min at 37\u0026deg;C in a dark. After incubation, the staining solution was removed, and the cells were washed with PBS. The cells were then observed under an inverted fluorescent microscope (Olympus IX71, Japan) at 450\u0026ndash;490 nm. Approximately 300 cells were counted and scored for viable (green fluorescence) or dead cells (red fluorescence). The percentage of apoptotic and necrotic cells was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDAPI staining\u003c/h2\u003e \u003cp\u003eDAPI (4',6-diamidino-2-phenylindole) staining was used to visualize nuclear abnormalities, including nuclear size, DNA damage, nuclear contour irregularities, and hyperchromatic, which can hinder cell growth [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. MCF-7 cells were grown in a 24-well plate in a CO\u003csub\u003e2\u003c/sub\u003e incubator and treated with an IC\u003csub\u003e50\u003c/sub\u003e concentration of TQ-PEI/PLA-NPs for 24 h. After incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde. The fixed cells were stained with 20\u0026micro;L of DAPI (0.5 \u0026micro;g/mL) at room temperature in the dark. The nuclear morphology of the cells was examined by fluorescence microscopy (Olympus IX71, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of mitochondrial transmembrane potential (ΔΨm)\u003c/h2\u003e \u003cp\u003eThe mitochondrial transmembrane potential (ΔΨm) was examined using the Rhodamine-123 staining method described by Zorova et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Briefly, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e MCF-7 cells were grown in a 6-well plate in a CO\u003csub\u003e2\u003c/sub\u003e incubator and treated with an IC\u003csub\u003e50\u003c/sub\u003e concentration of TQ-PEI/PLA-NPs for 24 h. Then, cells were washed with PBS and fixed with 4% paraformaldehyde. After fixing, the cells were stained with Rhodamine-123 for 30 min in a CO\u003csub\u003e2\u003c/sub\u003e incubator. Subsequently, the fluorescence intensity was measured at 480 and 530 nm using a fluorescence spectrophotometer (Spectramax M2, Molecular Devices, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of oxidative stress as reactive oxygen species (ROS) levels\u003c/h2\u003e \u003cp\u003eThe intracellular generation of ROS was measured by the DCFH-DA method described by Zulueta et al., 1997 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Approximately, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well MCF-7 cells were grown in a 24-well plate in a CO\u003csub\u003e2\u003c/sub\u003e incubator. The cells were treated with the IC\u003csub\u003e50\u003c/sub\u003e concentration of TQ-PEI/PLA-NPs for 24 h. The cells were washed with PBS and incubated with 5 mM DCFH-DA for 30 min. Using a fluorescence spectrophotometer (Spectramax M2, Molecular Devices, USA), the excitation/ emission wavelength (485/520 nm) was recorded. Untreated cells are used as controls. Results are expressed as fold change compared to the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of Caspase-3 and \u0026minus;\u0026thinsp;9 activities\u003c/h2\u003e \u003cp\u003eThe caspase enzyme activity was evaluated using Caspase-3 and 9 assay kits (Caspase-Glo\u0026reg;, Promega) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Briefly, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e MCF-7 cells were grown in a 96-well plate in a CO2 incubator for 24 h. The IC\u003csub\u003e50\u003c/sub\u003e concentration of TQ-PEI/PLA-NPS was added to the cells and incubated overnight. Equal volumes of reagents and cell culture supernatants (100 \u0026micro;L) were mixed for 45 min, and samples were read at excitation /emission (400/505 nm) using a fluorescence spectrophotometer (Spectramax M2, Molecular Devices, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three distinct studies. *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is statistically significant compared to the control group. We used SPSS 20.0 software for the data analysis. Data analysis was performed using one-way ANOVA and Dunnett's multiple comparison tests.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEncapsulation efficiency of TQ-PEI/PLA-NPs\u003c/h2\u003e \u003cp\u003eThe solvent evaporation-emulsification technique successfully formulated thymoquinone-encapsulated polyethyleneimine/poly (lactic acid) nanoparticles. The encapsulation of TQ into PEI/PLA NPs was confirmed by measuring the absorption at various time intervals. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the encapsulation of TQ into NPs. The TQ-encapsulation in PEI/PLA NPs exhibited a remarkable encapsulation efficiency of 85% (w/w), accompanied by an active drug loading capacity of 9.34% (w/w). The excellent drug loading capacity of nanocarriers is beneficial for cancer targeting applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003edrug release of TQ-PEI/PLA-NPs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e drug release study was performed in acetate buffer (pH 3.5) and phosphate-buffered saline (PBS) solution of pH 5.8 and pH 7.2 at 37\u0026deg;C. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, TQ-PEI/PLA-NPs exhibited a controlled release of TQ throughout the study period. Approximately, 33% (acetate buffer pH 3.5), 36% (PBS pH 5.8) and 32% (PBS pH 7.2) of TQ were released within 24 h of the study period while 76% (acetate buffer pH 3.5), 81% (PBS pH 5.8) and 73% (PBS pH 7.2) of TQ were released from PEI/PLA NPs at the end of 72 h.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDrug release kinetics of TQ-PEI/PLA-NPs\u003c/h2\u003e \u003cp\u003eThe results of the in vitro drug-release kinetics are summarised in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. We observed that the drug release profile of the TQ-PEI/PLA-NPs confirms the Higuchi model, with graphs showing high linearity and r\u003csup\u003e2\u003c/sup\u003e values in the range of 0.9889 (pH 3.5), 0.9898 (pH 5.8), and 0.9910 (pH 7.2), which mostly suggested the diffusion process. Furthermore, the observed release kinetics data demonstrated the non-Fickian behaviour of the TQ-PEI/PLA NPs formulation. The diffusion exponent (n) was shown to be larger than 0.69 (pH 7.2, or 0.697), indicating the involvement of the Case II transport drug-release mechanism. The Hixson\u0026ndash;Crowell model's high r\u003csup\u003e2\u003c/sup\u003e values (0.9836) indicated a constant drug release and uniform dissolution, but TQ release was mostly sustained at pH 3.5, pH 5.8, and pH 7.2, where a fixed amount of drug-release pattern was seen.\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 thymoquinone from TQ-PEI/PLA-NPs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \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=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eTQ-PEI/PLA-NPs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epH 3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epH 5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH7.2\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\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.017\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\u003e0.9674\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9527\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\u003e221.6597\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e233.2721\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e231.5051\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 (-k\u003csub\u003e1\u003c/sub\u003e\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\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.000\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\u003e0.9824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9865\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\u003e201.2854\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e198.2880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e190.1099\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;K\u003csub\u003eH\u003c/sub\u003e\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\u003e0.982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.969\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\u003e0.9442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9550\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9601\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\u003e239.3882\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e234.8125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e225.9246\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\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.198\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\u003e0.9889\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9898\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9910\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\u003e0.756\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.697\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\u003e187.0580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e186.7438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e177.8272\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\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\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\u003e0.9844\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9830\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\u003e197.3270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e199.1586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e197.7952\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\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 \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStability studies of TQ-PEI/PLA-NPs\u003c/h2\u003e \u003cp\u003eA key consideration in the formulation of therapeutics for the efficient treatment of diseases is the stability of the nanoparticles. The stability of the newly prepared TQ-PEI/PLA NPs is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e in a range of physiological solutions, including simulated gastric juice, buffer solutions (pH 7.2 and 9.0), acetate buffer (pH 3.5 and 5.5), and 0.5% BSA. TQ-PEI/PLA NPs exhibited a maximum absorbance of 305\u0026ndash;315 nm in the physiological medium and buffer solutions. After 72 h of incubation in buffer solutions and physiological media, there is minimal change in the λ\u003csub\u003emax\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of TQ-PEI/PLA-NPs\u003c/h2\u003e \u003cp\u003eThe FTIR spectra of TQ and TQ-PEI/PLA NPs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a) and 4(b), respectively. The FTIR spectra of TQ exhibited significant peaks at wavenumbers of 3249 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2971\u0026thinsp;\u0026minus;\u0026thinsp;2868 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which can be classified as stretching vibrations of the isopropyl and CH\u003csub\u003e3\u003c/sub\u003e groups. The peaks seen at 1639 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1461 to 1354 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1127 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to the presence of the distinct C\u0026thinsp;=\u0026thinsp;O group, aromatic C-H stretching, and C\u0026thinsp;=\u0026thinsp;O group, respectively. The FTIR spectra of TQ-PEI/PLA NPs showed peaks at 3282 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2924\u0026thinsp;\u0026minus;\u0026thinsp;2846 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to the stretching vibrations of the -OH and -CH\u003csub\u003e3\u003c/sub\u003e groups. The additional peaks observed were 1756 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to C\u0026thinsp;=\u0026thinsp;O stretching vibration, 1645 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to -C-O stretching, 1555 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1401 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e according to CH\u003csub\u003e3\u003c/sub\u003e antisymmetric bending, 1313 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to symmetric bending of tertiary carbon in the isopropyl group, and 1088\u0026thinsp;\u0026minus;\u0026thinsp;913 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to CH\u003csub\u003e3\u003c/sub\u003e rocking. The presence of these identifiable peaks in the TQ and TQ-PEI/PLA NPs formulation suggests interaction between the drug and polymer matrices.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe X-ray diffraction pattern of TQ-PEI/PLA NPs is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(c). The prominent peaks observed at 2θ\u0026thinsp;=\u0026thinsp;20\u0026deg; and 41\u0026deg; suggest that the NPs have a crystalline structure. The TQ-encapsulated PEI/PLA NPs had a particle size of 80\u0026ndash;90 nm (PDI approximately 0.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (d)) and a zeta potential of +\u0026thinsp;1 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(e)). High-resolution XPS analysis was conducted to investigate the surface chemical composition of formed TQ-PEI/PLA NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(f)). The XPS data of O1s, N1s, and C1s obtained from TQ-PEI/PLA NPs in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(f) shows that the O1s peak at 528 eV corresponds to the =\u0026thinsp;O-C\u0026ndash;\u0026ndash;O\u0026ndash;C-H bond of poly(lactic acid), the N1s signal at 402 eV corresponds to the C-NH\u003csub\u003e2\u003c/sub\u003e bond of polyethyleneimine, and the C1s signal indicates the presence of the -CH-CH\u003csub\u003e3\u003c/sub\u003e/-C-C- group of poly(lactic acid) in the NPs at 296 eV. FESEM pictures of TQ-encapsulated PEI/PLA nanoparticles showed the presence of uniform, monodisperse, spherical nanoparticles with a smooth surface and a diameter of around 100 nm, enclosed with TQ (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (g)-(j)).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eThe binding energy of TQ, SR-318 and DOX to the target protein MAPK14 was quantified to be -7.8, -9.3 and \u0026minus;\u0026thinsp;8.5 kcal/mol, respectively. The molecular docking studies indicated that the binding energy of TQ is close to Doxorubicin. Figures\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a) and 5(d) demonstrate the formation of one hydrogen bonding (106A THR (2.03 \u0026Aring;)) and six hydrophobic contacts (38A VAL (3.74\u0026Aring;), 38A VAL (3.44\u0026Aring;), 51A ALA (3.84\u0026Aring;), 53A LYS (3.74\u0026Aring;), 53A LYS (3.55\u0026Aring;), and 167A LEU (3.91\u0026Aring;)) between the TQ and MAPK14 protein molecules. SR-318, formed 12 hydrophobic interactions (53A LYS (3.68\u0026Aring;), 67A ARG (3.49\u0026Aring;), 71A GLU (3.84\u0026Aring;), 71A GLU (3.72\u0026Aring;), 74A LEU (3.36\u0026Aring;), 84A ILE (3.70\u0026Aring;), 84A ILE (3.91\u0026Aring;), 106A THR (3.84\u0026Aring;), 167A LEU (3.65\u0026Aring;), 167A LEU (3.80\u0026Aring;), and 169A PHE (3.90\u0026Aring;), two hydrogen bonds 71A GLU (2.46\u0026Aring;) and 168A ASP (3.29\u0026Aring;), and one salt bridge 71A GLU (4.57\u0026Aring;) with the MAPK14 protein, as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b) and 5(e). DOX, the standard drug, established four hydrogen bonding connections (67A ARG, 70A ARG, 149A ARG, and 170A GLY) and two hydrophobic interactions (75A LEU, 3.67\u0026Aring;, and 141A ILE, 3.57\u0026Aring;) with the MAPK14 protein, as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(c) and 5(f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamics simulation of TQ-PEI/PLA-NPs\u003c/h2\u003e \u003cp\u003eMolecular dynamics (MD) simulation was conducted to examine the stability and intermolecular bonding of the TQ-MAPK14 complex. The MD trajectory movements of the target TQ-MAPK14 complex exhibited RMSD values ranging from 2.8 \u0026Aring; to 3.6 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(a)). RMSD fluctuations varied between 1.2 and 2.6 \u0026Aring;, suggesting stability. The amino acids\u0026rsquo; binding to the major functional groups of the ligands did not result in any notable changes in the RMSF analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(b)). The darker lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(c)) indicate ongoing involvement with the target. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(d) illustrates the sequential arrangement of hydrogen bonds, hydrophobic interactions, ionic interactions, and water bridges in every amino acid residue. The protein-ligand contacts of the TQ-MAPK14 protein complex showed that the amino acid residue ALA 564 (89%) contributed the highest interaction with TQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(e)). Based on the molecular docking and dynamics studies, TQ strongly bind to the protein MAPK14, and the protein-ligand complex is highly stable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eCytotoxicity of TQ-PEI/PLA-NPs\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of TQ-PEI/PLA NPs, doxorubicin and free-TQ was tested against MCF-7 cells. TQ-PEI/PLA NPs exhibited a dose-dependent and time-dependent cytotoxic effect in MCF-7 cancer cells. TQ-PEI/PLA-NPs at 100 \u0026micro;g/mL showed 54.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86% cellular viability in MCF-7 cells with an IC\u003csub\u003e50\u003c/sub\u003e of 21.99 \u0026micro;g/mL after 24 h, Untreated control cells and those treated with 21.99 \u0026micro;g/mL TQ-PEI/PLA-NPs, produced morphological changes in MCF-7 cells are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(a)). Figures\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(b) and 7(c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eEffect of TQ-PEI/PLA-NPs on apoptotic cells\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(d), the control cells stained green and underwent no modifications. On the other hand, TQ-PEI/PLA-NPs-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(e)) were stained orange, indicating the presence of apoptotic cells. The treated cells also show nuclear disintegration, shrinkage, and membrane blebbing. This result points to a substantial build-up of TQ inside the MCF-7 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eEffect of TQ-PEI/PLA-NPs on nuclear morphology\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(g), significant changes were observed in the chromatin structure after TQ-PEI/PLA-NPs treatment, compared to the untreated control (Figure (f)). The control cells reveal characteristic spherical nuclei with a normal blue tint, but the treated cells exhibit a vibrant colour, abnormal nuclei, and compressed chromatin with irregular cell morphology.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial transmembrane potential damage of TQ-PEI/PLA-NPs\u003c/h2\u003e \u003cp\u003eAs shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (h) and (i), treatment with TQ-PEI/PLA-NPs of MCF-7 cells at 21.99 \u0026micro;g/mL resulted in a significant decrease in mitochondrial membrane potential. Furthermore, the TQ-PEI/PLA nanoparticles induced depolarization of the mitochondrial membrane.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eROS generation of TQ-PEI/PLA-NPs\u003c/h2\u003e \u003cp\u003eFigures \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(j) and 7(k) show that the untreated MCF-7 cells exhibited reduced ROS production and delayed apoptosis initiation. The present study demonstrated that TQ-PEI/PLA NPs can stimulate the production of ROS in cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(l)) using the DCFH-DA staining technique.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCaspase-3 and − 9 activities of TQ-PEI/PLA-NPs\u003c/h3\u003e\n\u003cp\u003eActivation of Caspases-3 and \u0026minus;\u0026thinsp;9 by external stimuli marks them as the terminal phase inducers of programmed cell death in cancer cells. The activities of caspase-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(m)) and caspase-9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(n)) were increased in cells exposed to 21.99 \u0026micro;g/mL TQ-PEI/PLA-NPs compared to control cells (untreated).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancer is still one of the world\u0026rsquo;s leading causes of death and a significant health concern. The survival rate for cancer patients is relatively low, even with the availability of multiple cancer therapies. Searching and developing novel therapeutic compounds for treating and eradicating cancer cells is challenging, expensive, and time-consuming. Even though chemotherapy has dramatically improved the overall survival rate of patients, it still shows a variety of psychological and physical side effects that lower their quality of life. Therefore, in the fight against neoplasia, natural compounds are seen to provide a promising substitute for several chemotherapy treatments. In the last 30 years, about 74% of newly authorized anticancer agents have been derived from natural sources or were inspired by natural products [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. TQ is a natural compound believed to be a potential antioxidant molecule, which can be combined with anticancer therapies to improve its efficacy and reduce toxicity [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Meanwhile, TQ alone has substantial anticancer activity against various types of cancers [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. At the same time, TQ use remains limited in the research community because of its insolubility in aqueous medium and inability to reach the disease site [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, TQ shows potential cytotoxic activity against breast cancer cells \u003cem\u003ein vitro\u003c/em\u003e studies [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Therefore, TQ-PEI/PLA-NPs were formulated to increase the bioavailability of TQ at the disease site. For this, the nanoparticles are functionalized with a synthetic cationic polymer (PEI), a proven inducer of endosomal escape[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Meanwhile, PLA provides a biodegradable and biocompatible core for drug encapsulation polymeric nanocarrier system to improve solubility, stability, and controlled release of the encapsulated medicine [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Nevertheless, multiple things require discussion.\u003c/p\u003e \u003cp\u003eThymoquinone (TQ), which is isolated from \u003cem\u003eNigella sativa\u003c/em\u003e or black seeds, has many advantages, including anti-cancer efficacy [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. TQ proved to be an anti-cancer agent against TNBC (triple negative breast cancer) through various mechanisms and modulation of the tumor micro-environment [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Long-term treatment with a low dose of TQ may inhibit breast cancer cell growth and prevent cancer cell growth in the sub-G1 phase. TQ induces apoptosis and regulates the expression of genes that promote and inhibit apoptosis [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Additionally, it has been demonstrated to reduce metastasis, ERK1/2, and PI3K activity, as well as the phosphorylation of NF-B and IKK [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, TQ has a low bioavailability at the target site due to its high hydrophobicity and poor solubility in aqueous solutions [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. TQ is also susceptible to environmental factors like light and temperature [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. TQ has been encapsulated into various nanoparticles to improve its solubility and efficacy [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Nanoparticles afford controlled release of loaded drugs, at the right place and the right time. Regardless of their solubility, they enhance the bioavailability and cellular uptake of the encapsulated TQ. Nanoparticles can be used to transform sensitive compounds into stable substances. For instance, TQ loaded in a nanostructured lipid carrier significantly increased the anticancer activity in 4T1 tumour-bearing mice in a murine breast cancer model [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. On the other hand, TQ was also incorporated into solid lipid nanoparticles (SLNs) and showed antidepressant-like effects in rats. TQ also prevents cervical cancer by delaying cell invasion and ROS-mediated death, as demonstrated by its loading into mesoporous silica nanoparticles.\u003c/p\u003e \u003cp\u003eThe physicochemical features of nanoparticulate systems, particularly their shape, size and surface charge, influence their physical stability, interactions with biological systems, release rates of encapsulated compounds, and interactions with target malignant cells. The drug-loaded nanoparticles exhibited particle sizes below 250 nm, with narrow size distributions and low polydispersity indices, which also play a critical role in influencing anticancer efficacy. In the present study, SEM examination revealed that the nanoparticles exhibited a spherical morphology with a smooth surface texture. Entrapment efficiency and loading capacity of thymoquinone in the developed nanoparticles were found to be 85% w/w and 9.34% w/w, respectively. The release of thymoquinone from the polymer matrix exhibited a biphasic pattern with an initial burst release during the first 24 h, followed by slower and sustained release of the drug. The slow and steady release of the drug ensures maximum efficacy over time. Similar patterns have been previously observed for the release of TQ from the polymeric systems.\u003c/p\u003e \u003cp\u003eThe cytotoxic profile of formulated TQ-PEI/PLA NPs was assessed using an MTT assay. The formulated TQ-PEI/PLA NPs demonstrated significant cytotoxicity in MCF-7 cells at 100 \u0026micro;g/ mL, showing 54.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01% of cellular viability. Similarly, 50 \u0026micro;M of TQ-loaded liposomes has more cytotoxic effects against MCF-7 and MCF-10 cells than 50 \u0026micro;M TQ [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The potential cytotoxic effects of TQ might be controlled by the release of TQ by polymeric carriers. Controlled release from polymeric carriers can result in elevated cytotoxic effects in the cancer microenvironment relative to free drug, attributable to the augmented drug concentration and targeted delivery, rather than an increase in the drug's intrinsic toxicity. The initiation of apoptosis may be a vital mechanism to impede breast cancer cell proliferation. Cancer cells may utilize many biological mechanisms to inhibit apoptosis and acquire resistance to chemotherapeutic or cytotoxic agents, including the production of antiapoptotic proteins or the downregulation or mutation of tumor suppressor genes. Apoptosis and associated cellular death mechanisms significantly impact cancer cell proliferation, making them viable targets for many cancer therapies [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Emerging research indicates that natural products play a crucial role in breast cancer treatment by inhibiting cell death. Selective fluorescent DNA-binding dyes, such as acridine orange and ethidium bromide, are used for morphological investigation due to their simplicity, precision, and speed. These tests eliminate the cell fixation stage, thereby avoiding several potential artifacts [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Thus, a DNA-specific AO/EBr double-staining assay was performed in this study to assess the apoptotic efficacy of the synthesized TQ-encapsulated PEI/PLA nanoparticles. TQ-PEI/PLA nanoparticles treated cells appeared to undergo nuclear disintegration, shrinkage, and membrane blebbing. Similarly, PLGA-PEI-EPI-PTX NPs induce cell membrane blebbing and chromatin condensation, indicating early apoptosis in cervical carcinoma cells (HeLa) [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIncreased intracellular concentrations of reactive oxygen species (ROS) cause damage to proteins, nucleic acids, lipids, membranes, and organelles, potentially initiating cell death mechanisms such as apoptosis. Specific chemotherapies induce programmed cell death (apoptosis) in cancer cells by increasing Reactive Oxygen Species (ROS) levels. Apoptosis produced by ROS can transpire through various mechanisms, including mitochondrial dysfunction and the activation of apoptotic pathways. Different chemotherapy drugs increase intracellular reactive oxygen species (ROS) levels and can modify the redox equilibrium of cancer cells. TQ-PEI/PLA nanoparticles delivered to MCF-7 cells demonstrated elevated levels of reactive oxygen species compared to untreated cells. Reactive oxygen species (ROS) buildup can impair the respiratory chain, resulting in mitochondrial malfunction and perhaps activating a p53-mediated intrinsic apoptotic pathway. This route entails releasing mitochondrial components such as cytochrome c, which activate caspase enzymes and ultimately result in cell death. The efflux of caspase-3 and \u0026minus;\u0026thinsp;9, especially from the mitochondria, is a critical event in the intrinsic pathway of apoptosis, a programmed cell death mechanism that may result in the cancer cell death. When cells experience stress or damage, the mitochondrial membrane may become permeable, permitting cytochrome to escape into the cytoplasm. The released cytochrome c subsequently engages with Apaf-1 to construct the apoptosome, which activates caspase 9, leading to the activation of caspase 3, the \"executioner\" caspase that initiates cellular apoptosis [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The drop in mitochondrial membrane potential marks the commencement of the mitochondrial apoptotic process. The collapse of the mitochondrial membrane potential and the disturbance of the electron transport chain gradient leading to depolarization of the mitochondrial membrane. The preservation of an electron transport chain may elucidate the alteration in mitochondrial polarization noted following chemical exposure [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. This work demonstrated that TQ-PEI/PLA nanoparticles induced significantly increased levels of ROS, caspase-3 and \u0026minus;\u0026thinsp;9, along with a reduction in mitochondrial membrane potential, in MCF-7 cells compared to untreated MCF-7 cells. Pt-coated Au nanoparticles (Pt-Au NPs; 27\u0026thinsp;\u0026plusmn;\u0026thinsp;20 nm) exhibited increased uptake and cytotoxicity, along with enhanced levels of ROS, NO, caspase 9, and caspase 3, as well as a reduction in mitochondrial membrane potential in human breast cancer MCF-7 cells compared to non-cancerous human cells (HUVE). Gholinejad and colleagues have documented a comparable observation about TiO2 NP-induced cell death in HUVE cells [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Cadmium selenide/zinc sulfide quantum dot nanoparticles have been documented to elicit pyroptosis in hepatic L02 cells, mediated through mitochondrial reactive oxygen species induction and mitochondrial membrane potential loss [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The efficacy of apoptotic cell death was meticulously assessed using nuclear morphological alterations utilizing a DAPI assay. Apoptosis in mammalian cancer cells is frequently associated with specific morphological and physiological alterations, such as membrane blebbing, phosphatidylserine externalization, chromatin condensation, nuclear fragmentation, and DNA degradation, initially into large fragments and subsequently into small nucleosome fragments [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study successfully formulated TQ-PEI/PLA nanoparticles via the solvent evaporation-emulsification method. TQ-PEI/PLA nanoparticles showed potential anticancer effectiveness against breast cancer cell lines (MCF-7) when compared to free TQ. The lead compound TQ exhibited a notable binding affinity for a breast cancer target (MAPK14). The significant anticancer activity of TQ may stem from improved distribution of encapsulated TQ into cancer cells, aided by certain polymers. The nanoparticles were synthesized with a synthetic cationic polymer (polyethylenimine (PEI), noted for its capacity to facilitate endosomal escape, whilst PLA provides a biodegradable and biocompatible core for drug encapsulation. TQ was well encapsulated into PEI/PLA nanoparticles, achieving an encapsulation efficiency of 85% w/w and a loading capacity of 9.34% w/w. The drug release profile demonstrated that TQ-PEI/PLA nanoparticles exhibited a sustained and regulated release mechanism. The physicochemical properties of the synthesized TQ-PEI/PLA nanoparticles were validated using FTIR, XRD, XPS, a particle size analyzer, and SEM. The synthesized TQ-PEI/PLA nanoparticles exhibited the ability to suppress the growth of MCF-7 cells and induce apoptosis. Moreover, in vivo animal investigations are crucial for elucidating the molecular pathways governing malignant cell death triggered by TQ encapsulated in PEI/PLA nanoparticles.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the management of Kalasalingam Academy of Research and Education, Krishnankoil, India, for research fellowships and for utilizing research facilities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSK supervision, fund acquisition, and project administration, resources, writing review and editing; JS, PP, EB, TP, MS, RM, PM, SK writing-original draft, formal analysis, investigation; SK, JS 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\u003eSelvaraj Kunjiappan gratefully acknowledges the Management of Kalasalingam Academy of Research and Education for the Seed Money Grant (KARE/VC/R\u0026amp;D/SMPG/2021\u0026ndash;2022/1). The University Research Fellowship provided by the management of Kalasalingam Academy of Research and Education to Mr. Jeganpandi Senthamarai Pandi is gratefully acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this manuscript.\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 interest.\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 consent for publication\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArnold M, Morgan E, Rumgay H, Mafra A, Singh D, Laversanne M, Vignat J, Gralow JR, Cardoso F, Siesling S (2022) Current and future burden of breast cancer: Global statistics for 2020 and 2040 \u003cem\u003eThe Breast\u003c/em\u003e 66 15\u0026ndash;23 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.breast.2022.08.010\u003c/span\u003e\u003cspan address=\"10.1016/j.breast.2022.08.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad A (2019) Breast cancer statistics: recent trends \u003cem\u003eBreast cancer metastasis and drug resistance: challenges and progress\u003c/em\u003e 1\u0026ndash;7 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-030-20301-6_1\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-20301-6_1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSopik V (2021) International variation in breast cancer incidence and mortality in young women. 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Biochim Biophys Acta Gen Subj 1861:802\u0026ndash;813. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbagen.2017.01.018\u003c/span\u003e\u003cspan address=\"10.1016/j.bbagen.2017.01.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMustafa M, Ahmad R, Tantry IQ, Ahmad W, Siddiqui S, Alam M, Abbas K, Hassan MI, Habib S, Islam S (2024) Apoptosis: a comprehensive overview of signaling pathways, morphological changes, and physiological significance and therapeutic implications \u003cem\u003eCells\u003c/em\u003e 13 1838 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells13221838\u003c/span\u003e\u003cspan address=\"10.3390/cells13221838\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Thymoquinone, breast cancer cells, polymeric nanoparticles, apoptosis, drug release","lastPublishedDoi":"10.21203/rs.3.rs-6858817/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6858817/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreast cancer is the most often diagnosed cancer globally, affecting elderly women predominantly. Thymoquinone (TQ) is a potential anticancer agent; however, its pharmacological applications are restricted by inadequate aqueous solubility and bioavailability. Our study aimed to develop TQ-encapsulated polyethyleneimine/poly(lactic acid) nanoparticles (TQ-PEI/PLA-NPs) to enhance TQ delivery into breast cancer cells. The solvent evaporation-emulsification method was used to synthesize TQ-PEI/PLA-NPs, and their physicochemical characteristics were examined. TQ-PEI/PLA-NPs had a crystalline structure, a zeta potential of +\u0026thinsp;1 mV, and a spherical shape with a diameter of 80\u0026ndash;90 nm. The encapsulation efficiency was 85% (w/w), while the drug loading capacity was 9.34% (w/w). The release rate of TQ from TQ-PEI/PLA-NPs was marginally elevated at pH 5.8 (81.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87%) compared to pH 3.5 and 7.2. The cytotoxicity of TQ-PEI/PLA-NPs was examined in MCF-7 cells. After 24 h of treatment, the MCF-7 cell counts decreased with an IC\u003csub\u003e50\u003c/sub\u003e of 21.99 \u0026micro;g/mL. The elevated intracellular accumulation of TQ in MCF-7 cells resulted in cell death, as evidenced by AO/EBr staining, mitochondrial transmembrane potential assay and Caspase-3 and \u0026minus;\u0026thinsp;9 studies. The observed MCF-7 cell death attributed to TQ was induced by increased reactive oxygen species (ROS) and impairment of mitochondrial membrane potential. The ROS potentially damaged the mitochondrial membrane and DNA, and further studies supported the induction of apoptosis. Our results indicated that TQ-PEI/PLA-NPs, which cause potent cytotoxicity to breast cancer cells, as evidenced by the decreased MCF-7 cell counts, may exhibit significant therapeutic potential for breast cancer therapies.\u003c/p\u003e","manuscriptTitle":"Targeted delivery of thymoquinone-encapsulated polyethyleneimine/poly (lactic acid) nanoparticles into breast cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-19 20:11:46","doi":"10.21203/rs.3.rs-6858817/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"221f0cea-1a07-485c-8032-4fe330ec1e4e","owner":[],"postedDate":"June 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-16T14:38:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-19 20:11:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6858817","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6858817","identity":"rs-6858817","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}