Anti-proliferative and Apoptotic Efficacy of Nano-PLGA encapsulated Quercetin Molecules by down-regulation of Akt in K-ras mutated NSCLC cell lines, A549 and H460

Anti-proliferative and Apoptotic Efficacy of Nano-PLGA encapsulated Quercetin Molecules by down-regulation of Akt in K-ras mutated NSCLC cell lines, A549 and H460 | 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 Anti-proliferative and Apoptotic Efficacy of Nano-PLGA encapsulated Quercetin Molecules by down-regulation of Akt in K-ras mutated NSCLC cell lines, A549 and H460 Avinaba Mukherjee, Sandip Ghosh, Sayak Ganguli, Biswarup Basu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4086530/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 To test if encapsulating hydrophobic flavonoids in nanoparticles could offer a new possibility in the therapeutics of non-small cell lung cancer (NSCLC), quercetin was encapsulated in PLGA nanoparticles by solvent displacement technique. The synthesised nanoparticles were then characterised by dynamic light scattering (DLS), Fourier transforms infrared spectroscopy (FTIR), and atomic force microscopy (AFM). The size of the nanoparticles with smooth surface topology was estimated at 110 nm. Treatment with nano-PLGA encapsulated quercetin (NPEQ) triggered the death of K-ras mutated NSCLC cells, A549 and H460, and showed 50% cell cytotoxicity in them at a dose of 406 ng/ml and 306 ng/ml, respectively. NPEQ was able to block uncontrolled cell proliferation by inducing concomitant destruction of BrdU activity and a lower incidence of cell migrations. Cell death was due to the induction of apoptosis rather than necrosis, as revealed by morphological alterations and phosphatidylserine externalisation induced by NPEQ. NPEQ also caused the arrest of A549 and H460 cells at the sub-G1 stage. NPEQ induced down-regulation of Akt, which is usually found to be hyperactive in NSCLC due to K-ras mutation. This indicates that NPEQ caused target-specific apoptotic and antiproliferative activity by targeting the downregulation of Akt. Further, when NPEQ was generated in the tumour-bearing mice model, it showed antitumor efficacy. Besides this, histological alteration of tissue architecture and modulation of an apoptotic marker protein in mice indicates the prospect and advantages of nanoparticulate quercetin delivery in therapeutic formulations against cancer. PLGA encapsulated nanoparticles Quercetin cell proliferation Akt apoptosis anti-proliferation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Among all the cancers, non-small cell lung cancer (NSCLC) is the most prevalent cancer of all types and has shown a great deal of drug resistance [ 1 ]. In 2020, worldwide, an estimated 2,206,771 people were diagnosed with lung cancer [ 2 ]. Regular synthetic drugs like- cisplatin, gefitinib, etc., have shown limited therapeutic efficacy, leading to normal cell cytotoxicity and limited bioavailability. The poor aqueous solubility of certain drugs, especially the flavonoids, reflects the limitations of target-based therapy in NSCLC [ 3 , 4 ]. Therefore, nano-encapsulation of drug molecules has now become a significant area of research that helps to increase its specificity towards malignant cells regardless of their poor aqueous solubility [ 5 , 6 ]. Quercetin is a major flavonoid found in fruits and vegetables and has been reported to show anticancer efficacy against lung cancer [ 7 , 8 , 9 ]. Unfortunately, due to its poor aqueous solubility, the effectiveness of quercetin has not been successfully elucidated in insignificant cancers. Among the RAS family of oncogenes, K-ras is a vital member, a collection of small guanosine triphosphate (GTP)-binding proteins that activate intracellular signalling pathways to regulate the proliferation of cells. Gain-of-function mutations that confer transforming capacity are frequently observed in K-ras, predominantly arising as single amino acid substitutions at residues G12, G13, or Q61. In NSCLC, K-ras mutations are highly prevalent (20–30%) and are associated with unfavourable clinical outcomes [ 10 , 11 ]. K-ras mutated NSCLC cells generally show constitutive activation of Akt. Therefore, target-based therapy in K-ras mutated NSCLC, using drugs that successfully block the downstream PI3Kor EGFR signal proteins, fails to work efficiently due to the constitutive activation of their upstream molecule Akt [ 12 , 13 ]. Therefore, we hypothesised that Akt could be a novel target in K-ras mutated NSCLC for drugs that successfully block their activity through direct interaction with the signal protein. In this work, our main objective was to decipher if quercetin in PLGA nanoparticles could sensitise K-ras mutated NSCLC cells by showing better efficacy at a minimal dose. Further, we also evaluated the role of drug-induced modulation of tumorigenesis in the mice model. This study would be advantageous in controlling the proliferation of those NSCLC cells that bear target-based therapeutic limitations because of K-ras mutation. 2. Materials and methods 2.1. Chemicals Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute medium (RPMI)-1640, fetal bovine serum (FBS), penicillin, streptomycin, neomycin (PSN) antibiotic, trypsin, and ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Grand Island, NY, USA). Tissue culture plastic wares were obtained from Tarsons, India. Quercetin was obtained from Himedia, India. MTT [3-(4, 5-Dimethyl-thiazol-2-yl)-2, S-diphenyltetrazolium bromide], propidium iodide, PLGA, and all secondary antibodies were purchased from Sigma Aldrich, USA. Santa Cruz Biotechnology Inc, USA purchased Annexin V-FITC, anti-Akt, anti-cleaved caspase3, and anti-GAPDH monoclonal antibodies. The primary antibodies of anti-BrdU were procured from Cell Signaling, USA. The anti-phospho-Akt antibody was procured from St John’s Laboratory, and the HRP-tagged secondary antibody was purchased from Cell Signaling Technology. Impact Novared was purchased from Vector Lab and used as a substrate for HRP. 2.2 Experimental animals An in-bred strain of Swiss albino mice, Mus musculus, weighing 30 ± 5 g at the initiation of the experiment, was reared and maintained in the animal house of the Chittaranjan National Cancer Institute (CNCI), Kolkata ((IAEC-1774-BB-3/2019/12). The animals were kept at an ambient temperature of 24 ± 1°C in 12-hour light and 12-hour dark cycles with food and water ad libitum. 2.2. Preparation of nanoparticles Quercetin-loaded PLGA nanoparticles were formulated using the solvent displacement technique [ 14 ]. Briefly, 10 mg quercetin was dissolved in 3 ml acetone, and then 50 mg PLGA was added to that mixture and dissolved completely by vigorous vortexing. This organic phase mixture was added drop-wise (0.5 ml/min) to 20 ml of an aqueous solution containing F68, the stabiliser (1% polyoxyethylene–polyoxypropylene;w/v). A laboratory magnetic stirrer then stirred the mixture until complete evaporation of the organic solvent. The redundant stabiliser was removed from it by centrifugation at 2500 g at 4 0 C for 30 min. After that, the pellet was re-suspended in Mili-Q water and washed thrice. Blank nanoparticles were also prepared in the same manner except for quercetin addition. 2.3. Particle size determination by Dynamic light scattering (DLS) The average size and size distribution of nano-PLGA encapsulated quercetin (NPEQ) was determined by DLS using a Zetasizer, Nano-ZS instrument (Malvern Instruments, Southborough, UK). Data were analysed in the automatic mode, and the measured size was presented as the average value of 20 runs, with triplicate measurements within each run. 2.4. Determination of zeta potential The Zeta potential of the NPEQ molecule was analysed by DLS using a Zetasizer Nano-ZS instrument (Malvern Instruments, Southborough, UK). 2.5. Atomic force microscopy (AFM) Samples were prepared for AFM imaging by placing a drop of nano-PLGA compound suspension on a freshly cleaved mica sheet and allowing it to dry in the air. The observation was recorded through AFM (Bruker AXS, Innova) imaging using AFM in amplitude and tapping modes. 2.6. Fourier transform infrared spectroscopy (FTIR) To ascertain whether the encapsulation procedure altered the characteristics of the polymer (PLGA) or the drug (quercetin) and whether interactions had occurred between the drug and the polymer after encapsulation (quercetin-loaded nano-PLGA), FTIR spectral study was performed by potassium bromide (KBr) technique. Mid-infrared (IR) spectral data (KBr, cm − 1 ) of quercetin-loaded nano-PLGA and blank nano-PLGA particles were recorded using Perkin Elmer, Spectrum One, FTIR spectrum instrument. 2.7. Molecular docking analysis of quercetin-PLGA and quercetin-Akt interaction The initial structure of the protein (7NH4) was downloaded from the protein data bank, and the prepare protein protocol of Discovery Studio was used to perform tasks such as inserting missing atoms in incomplete residues, standardising atom names, and protonate titratable residues using predicted pKs. The following steps were carried out sequentially: cleaning of protein, optimisation of side-chain conformation for residues with inserted atoms using the ChiRotor algorithm, removal of water molecules, prediction of titration site pKs, and protonation of the structure at the specified pH. The CDOCKER module of Discovery Studio performed the protein-ligand docking investigation. CDOCKER is a grid-based molecular docking method that employs a CHARMm-based molecular dynamics scheme to dock ligands into a receptor binding site. The ligands were allowed to flex with the rigidly held receptor during the refinement. High-temperature molecular dynamics generated random ligand conformations. This was followed by random rotations, refined by grid-based (GRID1) simulated annealing and forcefield minimisation. Each component's refined ligand poses were generated and analysed based on the lowest binding free energy, hydrogen bonds, and hydrophobic interactions. The top-scoring conformation identified through docking analyses was selected and used as a molecular dynamics (MD) simulation starting point. The ligand-receptor complex was subjected to the CHARMm36 force field and was then solvated in an orthorhombic box with Explicit Periodic Boundary conditions to avoid surface artefacts. This prevented the water molecules from diffusing away from the protein during the simulation and permitted a relatively small number of water molecules to act as a bulk solution. For studying the nature of interactions, LIGPLOT was used, where the docked complexes were uploaded to generate the relevant diagrams. 2.8. Cell lines Human K-ras mutated NSCLC cell lines, A549 and H460, were collected from the National Centre for Cell Science, Pune, India and were cultured in DMEM and RPMI-1640, respectively, with 10% heat-inactivated FBS and 1% antibiotic (PSN) at 37ºC with 5% CO 2 in a humidified incubator. Cells were harvested with 0.025% trypsin and 0.52 mM EDTA in phosphate buffer saline, plated at required cell numbers, and allowed to adhere for a minimum of 24 hours before treatment. 2.8. Cell viability assay A549 and H460 cells (1×10 2 cells per well) were seeded in 96 healthy plates and treated with NPEQ (10–100 ng/ml) for 24 hours. Cell viability was measured by MTT assay [ 15 ]. The concentration that reduced the cell viability by 50% was taken as an IC 50 dose and was used for further experiments. 2.9. Cellular morphological analysis Both the cell lines were seeded at the density 1×10 3, and NPEQ was treated in them for 12, 18, and 24 hours. After that, morphological analysis was done by an ordinary phase-contrast microscope (Leica, Wechsler, Germany). 2.10. Wound healing assay Cells were cultured in a 6-well plate to almost 100% confluence. A wound was generated at the centre of the well by scratching it with a plastic pipette tip. Cells were washed once with PBS. After NPEQ treatment with less than IC 50 dose for respective hours, the plate was placed under an inverted microscope (Axis cope plus 2, Zees, Germany), the reference point was matched, the photographed regions of the first image were aligned, and then the second image was obtained [ 16 ]. 2.11. BrdU incorporation assay A549 cells (1×10 3 cells per well) were dispensed in 96-well flat-bottom microtiter plates. After 24h of incubation, they were treated with BrdU solution and three different drug concentrations for 6, 12, and 24 hrs of exposure, respectively. BrdU incorporation was measured using a primary anti-BrdU antibody (1:500 dilutions) and its respective secondary antibody. After that, pap was used as a colour developer, and colour intensity was measured at 405 nm concerning blank. 2.12. Phosphatidyl externalisation assay after Annexin V-FITC/PI staining After 12, 18, and 24 hr of NPEQ induction, A549 and H460 cells were harvested after trypsinisation and were incubated in 100µl binding buffer (10mM HEPES, pH 7.4; 150 mM NaCl; 5mM KCl; 1 mM MgCl 2 and 1.8 mM CaCl 2 ) with FITC labeled Annexin V (100 ng/ml) and propidium iodide (50 µg/ml) at room temperature for 15 min in the dark and analysed using a FACS Calibur (BD Bioscience) taking minimum 10,000 cells in each sample [ 17 ]. 2.13. Cell cycle analysis after PI staining Briefly, 2 x 10 4 cells were seeded and treated with NPEQ at different times. Cells were then recovered, washed twice in cold PBS, and fixed in 70% chilled ethanol for one hour. Then, the cells were washed twice in PBS and incubated with RNase A (100 µg/ml) for 1 h at 37 0 C. After that, 50 µg/ml PI was added, and cells were incubated for 15 min in the dark and were analysed using a FACS Calibur flow cytometer (BD Bioscience). Ten thousand events were analysed for each sample. 2.14. Cell extract and protein isolation Cells (2 x 10 6 ) were plated in 90 mm culture dishes and were allowed to increase for 48 h. Following NPEQ treatment, A549 and H460 cells were collected and washed twice in ice-cold PBS. The total protein was isolated and stored at -20°C for further use. 2.15. Immunoblot analysis For immunoblot analysis, equal amounts (70 µg) of protein were loaded. Samples were denatured in 12% SDS-PAGE for Akt (1:1000). Separated proteins were transferred individually onto PVDF membranes and were probed with respective primary antibodies overnight at 4°C, followed by 1h incubation with ALKP-conjugated secondary antibody (1:500). Then they were developed using BCIP-NBT [ 18 ]. GAPDH (1:1000) was used as a loading control. Quantifying proteins was performed by densitometry using Image J software [ 19 ]. 2.16. Tumor cell transplantation and treatments Ehrlich Ascites Carcinoma (EAC), murine mammary adenocarcinoma cells were used as a tumour model. Five groups of healthy Swiss albino mice (n = 5), all from the age group, i.e. 3–5 weeks, were injected subcutaneously in the right leg flank with 5x10 6 cells in 100 µl PBS. All mice were divided into five groups, as vehicle-treated control, Quercetin at 25 mg/kg body weight (Q1), 50 mg/kg body weight (Q2), and NPEQ at 25 mg/kg body weight (NQ1), 50 mg/kg body weight (NQ2). After ten days, a palpable tumour appeared, and treatment started. All compounds were injected intraperitoneally (IP) with mentioned doses. Each group was treated on every alternate day, i.e. 1st day followed by 3rd day, and continued for up to 15 days. After 30 days, all animals were sacrificed, and tumours and other vital organs were dissected. 2.17. Acute toxicity studies and drug dose selection For sensitive toxicity testing, mice (n = 6) were fed 25, 50, 100, and 200 mg/kg body weight (bw) of NPEQ. After that, signs of mortality, clinical signs, and behavioural changes in mice for 24 h for any sign of acute toxicity. 2.18. Chronic toxicity analysis of liver and kidney tissue after Eosin Hematoxylin staining For the established toxicity assay, mice were treated with Quercetin and NPEQ at 25 mg/kg body weight and 50 mg/kg body weight for 30 days. After that, the histological structure of the liver and kidney were analysed after eosin and hematoxylin staining. Stained slides were mounted with DPX mounting media and observed under a bright field microscope. 2.19. In vivo tumour growth analysis Tumour size was measured every other day using a Vernier calliper to determine both the shortest diameter (A) and the longest diameter (B). Volume calculation was done using the formula V=(A2B)/2. Mice were sacrificed after 30 days after treatment started. The relative tumour volume (RTV) on day ‘n’ was calculated using RTV = TVn/TV0, where TVn is the Tumor volume on day n, and TV0 is the Tumor volume on day 0. The following formula calculated [ 20 ] tumour growth inhibition rate (TIR) TIR = {(1 - (mean volume of treated tumors)/mean volume of control tumors)} × 100% 2.20. Tumor tissue architecture analysis by Hematoxylin and Eosin staining Tumours of untreated and treated mice were fixed in 10% neutral buffered formalin for 24 hours. The histological structure of the tumour tissue was analysed after eosin and hematoxylin staining following the protocol discussed earlier. Stained slides were observed under a bright field microscope. 2.20. Protein expression analysis after Immunohistochemistry For immunohistochemistry staining, the tissue slide was made. The primary antibodies, cleaved caspase-3 (1:400) and anti-phospho-Akt antibody (1:100), were applied in a humidified chamber at 4 o C overnight. After washing, the HRP-tagged secondary antibody (1:800) was applied the next day for 30 minutes. Impact Novared was used as a substrate for HRP to generate a colour. Sections were then dehydrated and mounted with DPX mounting media. 2.21. Statistical analysis The values of the determined parameters were expressed as mean ± standard deviation (SD). The mean values from three independent experiments were included. Comparisons were performed using two-way ANOVA and an LSD post hoc test for multiple comparisons. The results were statistically significant at * p < 0.05, ** p < 0.01, *** p < 0.001. All analyses were performed using SPSS 16.0 software. 3. Results 3.1. Characterization of nano-PLGA encapsulated quercetin (NPEQ) The size of the synthesised nanoparticles was approximately 110 nm with a polydispersity index value of 0.186 (Fig. 1 A and 1 B). The zeta potential was found to be negative. AFM characterisation of quercetin-loaded PLGA nanoparticles revealed their smooth and bright surface topology (Fig. 1 C). FTIR analysis indicated that quercetin was efficiently loaded in PLGA molecules. The primary characteristic peaks of quercetin were present in free and PLGA-encapsulated quercetin, which was absent in only PLGA nanoparticles. The presence of distinct peaks of quercetin in PLGA-quercetin nano molecule is an indirect way of confirming the successful encapsulation of quercetin in PLGA nanoparticles (Fig. 1 D). 3.2. NPEQ interacts with PLGA In the case of the Quercetin - PLGA interaction, the detection of hydrogen bonds was not found. These two small molecules seemed to interact via van der Waals forces only. The stability of the complex was identified based on the free energy value of − 119.30 KCal/mol ( Fig. 2 ) . A weak interaction between quercetin and PLGA was found. When implied in a cell, this might help sustain the quercetin molecules' sustainable release. 3.3. NPEQ showed cytotoxicity on A549 and H460 cells NPEQ reduced the cell viability of A549 and H460 after 24 hrs of induction. The IC 50 value was determined to be 406 ng/ml and 347 ng/ml for A549 and H460, respectively (Fig. 3 A). On the other hand, free quercetin was earlier found to be cytotoxic against A549 and H460 [ 21 ] but with a higher dose of IC 50 value 66 µM (~ 19x10 6 ng/ml) for A549 and > 100 µM (~ 30x10 6 ng/ml) (Table 1 ). This shows that quercetin in nano-encapsulated forms is more precise and effective target-specific. Table 1 IC 50 values of free quercetin and NPEQ in A549 and H460 cell line Name of the compound Cell lines A549 H460 Free Quercetin ~ 19x10 6 ng/ml [ 21 ] > 30 x10 6 ng/ml [ 21 ] NPEQ 406 ng/ml 347 ng/ml 3.4. NPEQ-induced morphological alterations and blocked the proliferation of A549 and H460 cells Compared with the untreated ones, NPEQ-induced cell shrinkage and membrane blebbing after 12–24 hrs of exposure (Fig. 3 B). Uncontrolled cell proliferation activity was significantly inhibited time-dependent after NPEQ induction, as revealed by wound healing and BrdU incorporation assay (Fig. 3 C and Fig. 3 D). 3.5. Phosphatidylserine externalisation and cell cycle arrest after NPEQ induction An increased number of AnnexinV positive A549 and H460 cell populations was observed after NPEQ treatment, as compared to the untreated control (Fig. 4 A). Flow cytometric analysis also indicated a sub-G1 arrest in NPEQ-treated cells in a time-dependent manner (Fig. 4 B). Onset of this sub-G1 cell cycle arrest suggested that NPEQ was potent in inducing apoptosis in both A549 and H460 cells. 3.6. Modulation of Akt expression upon NPEQ treatment, possibly through Quercetin-Akt interaction From the immunoblot data, NPEQ was found to be effective in inhibiting Akt expression in both the NSCLC cell lines after 12–24 hrs of exposure, as compared with the untreated ones (Fig. 5 ). Down-regulation of Akt was more pronounced in both the cell lines 12 hrs onwards of NPEQ induction. As the quercetin downregulated the Akt expression after release in a sustainable manner from PLGA nanoencapsulation, we have studied whether it has any interactive activity with Akt. Through molecular docking analysis, Quercetin was found to interact with the protein molecule using a total of 52 noncovalent interactions, out of which a hydrogen bond was detected between the aspartate 293 and the first carbon of quercetin having a distance of 3.22 A 0 . Fifty-one non-bonded contacts (Van der Waals interactions) were identified ( Supplementary Table ). The overall interaction was stable, and a free energy of 236.53KCal/mol was obtained, indicating the stability of the complex (Fig. 6 ). 3.7. NPEQ toxicity assessment and dose selection Quercetin was relatively non-toxic to mice in vivo in free and nano-encapsulated form. No significant changes in mortality or abnormal clinical signs or symptoms were observed. The 100 mg/kg bw dose of NPEQ followed partial signs of behavioural change. Thus, we preferred to use 25 and 50 mg/kg body weight of the drug (free quercetin and NPEQ) as the optimum ones for the study. Furthermore, histological studies indicated that after quercetin treatment (free and nano-encapsulated form), liver and kidney tissue architecture remains unaltered compared to the untreated control (Fig. 7 ). This revealed that the selected drug dose is optimum and safe for further testing and analysis. On the other hand, nano-encapsulated quercetin recovered the tissue architecture (marked in red) compared with the free quercetin when applied. This reveals that this has the potential to reduce tumorigenesis. 3.8. Antitumor activity of the compounds in vivo Differential regression in tumour volume in different treatment groups is shown in dissected mice images (Fig. 8 ). Q1 and Q2 inhibited tumour growth by 19% and 46%, whereas NQ1 and NQ2 inhibited tumour growth by 38% and 72% (Fig. 8 ). 3.9. NPEQ-induced tissue structure alteration in tumour-bearing mice The excised tumours were sectioned and examined by H&E staining. Mean tumour apoptosis (%) was determined from several high-magnification images and apoptotic areas indicated by a red arrow (Fig. 8 ). The stained tumour histology section from the control group showed hematoxylin-stained, well-organised, dense tumour nuclei (Fig. 9 A). The NQ2 group showed an elevated level of necrotic areas (64%) with very few seats. Q1 and Q2 groups showed 30% and 39% apoptotic area in tumour tissue sections, whereas the NQ1 group showed 46% apoptotic area. This revealed the potential of NPEQ to recover the tissue architecture compared to free quercetin and the untreated control. 3.10. Modulation of cleaved caspase three and Akt activation The immunohistochemical assay revealed that compared with free quercetin treatment, NPEQ, in a dose-dependent manner, upregulated cleaved caspase 3 (Fig. 9 B) and downregulated p-Akt expression (Fig. 9 C). This signified quercetin in a PLGA nano-encapsulated form is a more potent antiproliferative and pro-apoptotic agent than free quercetin. 4. Discussion Designing and testing target-specific drug molecules on biological systems demands the formulation and usage of nanoparticles encapsulated in biodegradable polymers with better efficacy and higher bioavailability. We tried to examine the effectiveness of nano-encapsulated quercetin in inhibiting cell proliferation against K-ras mutated NSCLC cells. Furthermore, we have induced the formulation in tumour-bearing mice to evaluate its antitumorigenic effect. Experimental results indicated successful encapsulation of the hydrophobic flavonoid quercetin into PLGA nanoparticles. NPEQ showed an approximate size of 110 nm with an unimodal particle size distribution, as revealed by the low polydispersity index, which is typical of mono-dispersed systems. The negative zeta potential value confirmed that the NPEQ is highly stable without particle aggregation. AFM characterisation of NPEQ revealed their smooth and bright surface topology. This indicates the uniformity in dimensions of the formulated nanocapsules. FTIR spectral analysis showed that quercetin was efficiently loaded in PLGA molecules with no significant interaction, as similar characteristic peaks of quercetin were present in both free and NPEQ. Besides this, molecular docking analysis between quercetin and PLGA showed no hydrogen bond interaction and weak van der Waals interaction. This indicates that quercetin can quickly be released in the target cell after being incorporated in a nanoencapsulation form. After that, the synthesised NPEQ was examined for its anti-proliferative and apoptotic efficacy against two major K-ras mutated NSCLC cell lines, A549 and H460. Results revealed that NPEQ had significant inhibitory effects, with 406 ng/ml and 306 ng/ml as IC 50 values in A549 and H460, respectively. Time-dependent inhibitory effects of NPEQ revealed that proliferation of both the cell lines was inhibited, showing concomitant destruction of BrdU activity and lower incidence of cell migration. Besides this, NPEQ-mediated cell death was confirmed to be a consequence of apoptosis and not necrosis. After its exposure, effective phosphatidylserine externalisation and cell cycle arrest at sub-G1 stages were observed. Hence, we could infer that early-hour inhibition of NSCLC cell proliferation may eventually lead the cells to apoptosis. It has been demonstrated that Akt, a significant protein related to cell proliferation, remains constitutively active in A549 and H460 cells due to K-ras mutation [ 22 , 23 ]. Therefore, the inhibition of downstream PI3K alone is insufficient to treat K-ras mutated cancer and may not be sensitive to PI3K pathway inhibitors. Targeted knockdown of Akt in K-ras mutated NSCLC cell lines would provide critical confirmation of the role of Akt in tumorigenesis, resulting in both the suppression of tumour growth and sensitisation to PI3K inhibitors. Our results revealed that after the sustained release of the NPEQ, the drug molecules, the quercetin itself, could efficiently interact with Akt. Here, the strong interaction of the released quercetin and Akt may have caused the downregulation of Akt despite the K-ras mutation. A molecular docking study indicates that quercetin significantly interacts with Akt with 52 noncovalent interactions, including one hydrogen bond and 51 non-bonded contacts (Van der Waals interactions). This significant interaction might be a causative factor of the quercetin-induced downregulation of Akt in both A549 and H460 cell lines. However, the precise downregulation at such a lower dose would not have been possible in an accessible form of quercetin. Nanoencapsulation might help the target-specific sustained release of quercetin to exert its efficacy at a lower dose. Hence, our studies have shown that NPEQ mediated the down-regulation of Akt at 12–18 hrs of exposure in the A549 and H460 cell lines. The significant down-regulation of Akt was likely to play an initial cause in blocking cell proliferation, which presumably directed the cells towards apoptosis. On the other hand, in tumour-bearing mice, NPEQ showed better efficacy in lowering tumour growth and the tumorigenesis process. NPEQ also indicated tissue structure recovery in tumour-bearing mice in a dose-dependent manner. NPEQ at this particular dose has not shown any normal cell toxicity, indicating that the amount is safe and might be target-specific. Furthermore, immunohistochemistry analysis results showed an upregulation of cleaved caspase three and simultaneous downregulation in the activated form of Akt. This indicated the tissue apoptosis and uncontrolled growth retardation that may be beneficial for the mice’s bodies to recover from the tumorigenic condition. 5. Conclusion The above findings suggest that quercetin in a nanoparticulate form can effectively block uncontrolled NSCLC cell proliferation and trigger the apoptotic event. Furthermore, this NPEQ showed its antitumorigenic potential in mice bodies, better than the free quercetin. Thus, encapsulation would offer a new dimension in the target-based drug delivery system, especially in the treatment modality. Understanding how NPEQ regulates proliferation inhibition and cell death may contribute to novel therapeutic strategies in cancer research. Declarations Conflict of Interest None to declare Acknowledgments The authors sincerely thank Dr Sanjaya Mallick, ex-application scientist and COE manager, BD BioSciences, and associated with the Centre for Research in Nanoscience and Nanotechnology, University of Calcutta, for his help conducting FACS. Ethical Approval For animal studies, the animals were maintained in the animal facility of the Chittaranjan National Cancer Institute (CNCI), Kolkata, after the institutional animal ethical committee approved the protocol with Sanction No. IAEC-1774-BB-3/2019/12. Funding The research work was partially funded by the West Bengal Department of Science, Technology and Biotechnology, Sanction Order No.: 252(Sanc.)/STBT-11012(12)/2/2022- ST SEC. References Spaans JN, Goss GD. (2014). Drug resistance to molecular targeted therapy and its consequences for treatment decisions in non-small-cell lung cancer. Front Oncol , doi: 10.3389/fonc.2014.00190. International Agency for Research on Cancer. GLOBOCAN Lung Cancer Facts Sheet 2020. Kopustinskiene DM, Jakstas V, Sav ickas A, Bernatoniene J. (2020). Flavonoids as Anticancer Agents. Nutrients , 12(2), 457. Cai X, Fang Z, Dou J, Yu A, Zhai G. 2013. Bioavailability of quercetin: problems and promises, Curr Med Chem ., 20(20): 2572-2582. 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Efferth T, Giaisi M, Merling A, Krammer PH, Li-Weber M. (2007). Artesunate induces ROS-mediated apoptosis in doxorubicin-resistant T leukemia cells, PLoS ONE. , 2, e693. Sambrook J, Russell DW, Molecular Cloning, (2001) 3rd ed. Cold Spring Harbor Laboratory Press, New York. Schneider CA, Rasbandws WS, Eliceiri KW. (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 9(7): 671-675. Kaur H, Ghosh S, Kumar P, Basu B, Nagpal K. (2021) Ellagic acid-loaded, tween 80-coated, chitosan nanoparticles as a promising therapeutic approach against breast cancer: In-vitro and in-vivo study. Life Sci. , 284:119927. Mukherjee A, Khuda-Bukhsh AR. (2015). Quercetin Down-regulates IL-6/STAT-3 Signals to Induce Mitochondrial-mediated Apoptosis in a Non small- cell Lung-cancer Cell Line, A549. J Pharmacopuncture . 18(1):19-26. Rosell R, Monzo M., Molina F., Martinez E., Pifarre A., Moreno I., Mate J. L., de Anta J. M., Sanchez M., Font A. (1995). K-ras genotypes and prognosis in non-small cell lung cancer. Ann. Oncol. , 6: S15-S20. Brognard J, Clark AS, Yucheng Ni, Dennis PA. (2001). Akt/Protein Kinase B Is Constitutively Active in Non-Small Cell Lung Cancer Cells and Promotes Cellular Survival and Resistance to Chemotherapy and Radiation. Cancer Res. , 61(10): 3986-3997. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.docx 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-4086530","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280336326,"identity":"01521c29-1a1b-4428-a27b-deee22edd37a","order_by":0,"name":"Avinaba Mukherjee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYFACNjYQAWYeACF+ECuhgBQtkg0gLQYEtcDBAQaDAyAajxbdGWlpDz7u4ZPjZz9jeLig5o688fnViR8eGDDI84sdwKrF7EbaccMZz9iMJXtyDA7POPbMcNuNt5slgA4znDk7AYeW9DZpngNsiRsOpCUc5mE7zLjtxtkNIC0JBrfxaPlzgK1+//lnQC3/DttvnnF28w/8WtKOSTMcYEswkEg+cJi37XDiBv7ebfhtOfMsTbLnAJvhjBuPgVr6niXPuMG7zQJoAm6/HE8zk/hx4Jg8f39i82eeb3ds+/vPbr75o8JGnl8auxYoOIbElgCrlMCnHARqkNj8BwipHgWjYBSMghEGAD7qarOSlrweAAAAAElFTkSuQmCC","orcid":"","institution":"Charuchandra College, University of Calcutta","correspondingAuthor":true,"prefix":"","firstName":"Avinaba","middleName":"","lastName":"Mukherjee","suffix":""},{"id":280336327,"identity":"31a02ca9-c793-4ad5-b583-addb3741b260","order_by":1,"name":"Sandip Ghosh","email":"","orcid":"","institution":"Chittaranjan National Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Sandip","middleName":"","lastName":"Ghosh","suffix":""},{"id":280336328,"identity":"c627c6e1-557e-4087-98f1-94719a2ede85","order_by":2,"name":"Sayak Ganguli","email":"","orcid":"","institution":"St Xavier’s College","correspondingAuthor":false,"prefix":"","firstName":"Sayak","middleName":"","lastName":"Ganguli","suffix":""},{"id":280336329,"identity":"f22b8fa3-4063-4ad6-8bc9-8c8027e1e042","order_by":3,"name":"Biswarup Basu","email":"","orcid":"","institution":"Chittaranjan National Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Biswarup","middleName":"","lastName":"Basu","suffix":""}],"badges":[],"createdAt":"2024-03-12 16:56:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4086530/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4086530/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52985333,"identity":"f46560b1-a12b-4dad-bca1-a6aa7f470dae","added_by":"auto","created_at":"2024-03-19 11:04:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6857041,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the PLGA encapsulated quercetin nanoparticles by (A) and (B) DLS study. (C) The surface topology of NPEQ was analyzed by AFM image (2D) of the size and surface morphology of NC. (D) Interactions between quercetin and the polymer after encapsulation (quercetin-loaded nano-PLGA) were analyzed by FTIR analysis.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/419abee3b3c06046d26fe6cb.png"},{"id":52985337,"identity":"a4adfae3-f738-49fd-b865-f9393bb239fd","added_by":"auto","created_at":"2024-03-19 11:04:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8224373,"visible":true,"origin":"","legend":"\u003cp\u003eInteractions of Quercetin with PLGA. In Figure A the nature of the interaction of quercetin (green) is depicted with the structure of PLGA (Yellow). Figure B indicates the overlapping electrostatic surfaces of the two molecules displayed as a grid. Figure C identifies the nature of the interaction between the two small molecules as van der Waals contact (the chemical structure of Quercetin displayed in a combination of carbon and oxygen while unk1 represents PLGA).\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/a90fd325cd6aea2c897f7be3.png"},{"id":52985334,"identity":"d2fd8ea1-d29f-4eaf-8bdd-31af1b0e8356","added_by":"auto","created_at":"2024-03-19 11:04:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1004297,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of NPEQ on A549 and H460 cells by MTT assay. The values are represented as mean ± SD of three independent experiments with six replicates in each. Statistical significance was considered as \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001. B) Cell morphological analysis normal phase-contrast microscope after treatment with NPEQ for 12, 18, and 24 h. C) Wound healing assay of A549 and H460 respectively after NPEQ treatment for 12,18 and 24 h. Arrows indicate the migration of cells. D) BrdU incorporation assay after treatment with NPEQ for 12, 18, and 24 hrs along with the incorporation of BrdU. Incorporated BrdU was labelled with an anti-BrdU antibody and intensity was measured at 405nm using PNPP as a color developer. Each data was expressed as mean ± SD (N = 6). Statistical significance was considered as \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01 and \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/6e6d4d835f50d3540a9c541f.png"},{"id":52985336,"identity":"deca5080-c375-4e75-9719-e2745a550ea4","added_by":"auto","created_at":"2024-03-19 11:04:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1034755,"visible":true,"origin":"","legend":"\u003cp\u003eNPEQ induces apoptosis in A549 (A) AnnexinV-FITC FACS analysis where LR-lower quadrant, indicated early apoptotic cells and UR- the upper quadrant indicated late apoptosis. (B) Cell cycle analysis after PI staining. Percentages of Sub GO/G1 cell populations were indicated.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/a24c992ff964e2fe9082c78c.png"},{"id":52985339,"identity":"693e418c-a224-4fac-b28c-9d9cde4656fd","added_by":"auto","created_at":"2024-03-19 11:04:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182368,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot of Akt and band intensities were calculated and relative band intensity was calculated Statistical significance was considered as \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/e7d69d878a91b943eac35602.png"},{"id":52985341,"identity":"82408992-2513-46f0-9bba-f1f5ded8cc8a","added_by":"auto","created_at":"2024-03-19 11:04:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6254023,"visible":true,"origin":"","legend":"\u003cp\u003eInteractions of AKT with Quercetin. A. The binding site of quercetin (red) is depicted in the 3D structure of Akt (Green). B. It indicates the major residues that participate in this interaction with the hydrogen bond shown in green.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/ed55e3e3476476739dd836ad.png"},{"id":52985342,"identity":"d9bc4baf-8f51-4e53-851e-bbfdb24d8953","added_by":"auto","created_at":"2024-03-19 11:04:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":59539510,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin staining of liver and kidney (x100 magnification).\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/f53f70694ae8ac56f96f433a.png"},{"id":52985338,"identity":"540325c4-72b2-41f4-9f2b-4a9a3e963b29","added_by":"auto","created_at":"2024-03-19 11:04:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":492541,"visible":true,"origin":"","legend":"\u003cp\u003eTreatment schedule, (B) Changes in mean relative tumor volume in different treatment groups. Values indicate mean ± SD (n\u0026nbsp;= 6). \u003csup\u003e*\u003c/sup\u003eP\u0026nbsp;\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026nbsp;\u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003eP\u0026nbsp;\u0026lt; 0.001. (C) Sacrificed mice with tumors from different groups. Tumors are indicated with a red arrow.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/9f643a08aa9a57d3f60a4484.png"},{"id":52986705,"identity":"a278999c-ae81-4b29-a103-432c83960363","added_by":"auto","created_at":"2024-03-19 11:12:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":111421669,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining of tumor. (A) Representative photomicrograph of Cleaved Caspase-3 and p-Akt (scale bar 100 µm) (B) \u0026amp; (C) Cleaved Caspase-3 and p-Akt expression. Positive immunohistochemical reactions were determined by ImageJ. Values are expressed as mean ± SD. Significantly different compared with the control group at \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001. Significantly different compared with the EAC control group at \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/dd48213fa11296d7953cfaaf.png"},{"id":53067691,"identity":"55cc0b4f-cbe7-4d9b-98d8-8412c80ea4ba","added_by":"auto","created_at":"2024-03-20 08:17:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6725898,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/5ece12b2-0c10-444b-923d-dd9015f18141.pdf"},{"id":52985335,"identity":"f174abef-db11-4853-897c-50116f462a77","added_by":"auto","created_at":"2024-03-19 11:04:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":507270,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-4086530/v1/53ec7b5290a13ea1e9399c58.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-proliferative and Apoptotic Efficacy of Nano-PLGA encapsulated Quercetin Molecules by down-regulation of Akt in K-ras mutated NSCLC cell lines, A549 and H460","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAmong all the cancers, non-small cell lung cancer (NSCLC) is the most prevalent cancer of all types and has shown a great deal of drug resistance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In 2020, worldwide, an estimated 2,206,771 people were diagnosed with lung cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Regular synthetic drugs like- cisplatin, gefitinib, etc., have shown limited therapeutic efficacy, leading to normal cell cytotoxicity and limited bioavailability. The poor aqueous solubility of certain drugs, especially the flavonoids, reflects the limitations of target-based therapy in NSCLC [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, nano-encapsulation of drug molecules has now become a significant area of research that helps to increase its specificity towards malignant cells regardless of their poor aqueous solubility [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Quercetin is a major flavonoid found in fruits and vegetables and has been reported to show anticancer efficacy against lung cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Unfortunately, due to its poor aqueous solubility, the effectiveness of quercetin has not been successfully elucidated in insignificant cancers.\u003c/p\u003e \u003cp\u003eAmong the RAS family of oncogenes, K-ras is a vital member, a collection of small guanosine triphosphate (GTP)-binding proteins that activate intracellular signalling pathways to regulate the proliferation of cells. Gain-of-function mutations that confer transforming capacity are frequently observed in K-ras, predominantly arising as single amino acid substitutions at residues G12, G13, or Q61. In NSCLC, K-ras mutations are highly prevalent (20\u0026ndash;30%) and are associated with unfavourable clinical outcomes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. K-ras mutated NSCLC cells generally show constitutive activation of Akt. Therefore, target-based therapy in K-ras mutated NSCLC, using drugs that successfully block the downstream PI3Kor EGFR signal proteins, fails to work efficiently due to the constitutive activation of their upstream molecule Akt [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, we hypothesised that Akt could be a novel target in K-ras mutated NSCLC for drugs that successfully block their activity through direct interaction with the signal protein.\u003c/p\u003e \u003cp\u003eIn this work, our main objective was to decipher if quercetin in PLGA nanoparticles could sensitise K-ras mutated NSCLC cells by showing better efficacy at a minimal dose. Further, we also evaluated the role of drug-induced modulation of tumorigenesis in the mice model. This study would be advantageous in controlling the proliferation of those NSCLC cells that bear target-based therapeutic limitations because of K-ras mutation.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute medium (RPMI)-1640, fetal bovine serum (FBS), penicillin, streptomycin, neomycin (PSN) antibiotic, trypsin, and ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Grand Island, NY, USA). Tissue culture plastic wares were obtained from Tarsons, India. Quercetin was obtained from Himedia, India. MTT [3-(4, 5-Dimethyl-thiazol-2-yl)-2, S-diphenyltetrazolium bromide], propidium iodide, PLGA, and all secondary antibodies were purchased from Sigma Aldrich, USA. Santa Cruz Biotechnology Inc, USA purchased Annexin V-FITC, anti-Akt, anti-cleaved caspase3, and anti-GAPDH monoclonal antibodies. The primary antibodies of anti-BrdU were procured from Cell Signaling, USA. The anti-phospho-Akt antibody was procured from St John\u0026rsquo;s Laboratory, and the HRP-tagged secondary antibody was purchased from Cell Signaling Technology. Impact Novared was purchased from Vector Lab and used as a substrate for HRP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental animals\u003c/h2\u003e \u003cp\u003eAn in-bred strain of Swiss albino mice, Mus musculus, weighing 30\u0026thinsp;\u0026plusmn;\u0026thinsp;5 g at the initiation of the experiment, was reared and maintained in the animal house of the Chittaranjan National Cancer Institute (CNCI), Kolkata ((IAEC-1774-BB-3/2019/12). The animals were kept at an ambient temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C in 12-hour light and 12-hour dark cycles with food and water ad libitum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of nanoparticles\u003c/h2\u003e \u003cp\u003eQuercetin-loaded PLGA nanoparticles were formulated using the solvent displacement technique [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Briefly, 10 mg quercetin was dissolved in 3 ml acetone, and then 50 mg PLGA was added to that mixture and dissolved completely by vigorous vortexing. This organic phase mixture was added drop-wise (0.5 ml/min) to 20 ml of an aqueous solution containing F68, the stabiliser (1% polyoxyethylene\u0026ndash;polyoxypropylene;w/v). A laboratory magnetic stirrer then stirred the mixture until complete evaporation of the organic solvent. The redundant stabiliser was removed from it by centrifugation at 2500 g at 4\u003csup\u003e0\u003c/sup\u003eC for 30 min. After that, the pellet was re-suspended in Mili-Q water and washed thrice. Blank nanoparticles were also prepared in the same manner except for quercetin addition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Particle size determination by Dynamic light scattering (DLS)\u003c/h2\u003e \u003cp\u003eThe average size and size distribution of nano-PLGA encapsulated quercetin (NPEQ) was determined by DLS using a Zetasizer, Nano-ZS instrument (Malvern Instruments, Southborough, UK). Data were analysed in the automatic mode, and the measured size was presented as the average value of 20 runs, with triplicate measurements within each run.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Determination of zeta potential\u003c/h2\u003e \u003cp\u003eThe Zeta potential of the NPEQ molecule was analysed by DLS using a Zetasizer Nano-ZS instrument (Malvern Instruments, Southborough, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Atomic force microscopy (AFM)\u003c/h2\u003e \u003cp\u003eSamples were prepared for AFM imaging by placing a drop of nano-PLGA compound suspension on a freshly cleaved mica sheet and allowing it to dry in the air. The observation was recorded through AFM (Bruker AXS, Innova) imaging using AFM in amplitude and tapping modes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Fourier transform infrared spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eTo ascertain whether the encapsulation procedure altered the characteristics of the polymer (PLGA) or the drug (quercetin) and whether interactions had occurred between the drug and the polymer after encapsulation (quercetin-loaded nano-PLGA), FTIR spectral study was performed by potassium bromide (KBr) technique. Mid-infrared (IR) spectral data (KBr, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of quercetin-loaded nano-PLGA and blank nano-PLGA particles were recorded using Perkin Elmer, Spectrum One, FTIR spectrum instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Molecular docking analysis of quercetin-PLGA and quercetin-Akt interaction\u003c/h2\u003e \u003cp\u003eThe initial structure of the protein (7NH4) was downloaded from the protein data bank, and the prepare protein protocol of Discovery Studio was used to perform tasks such as inserting missing atoms in incomplete residues, standardising atom names, and protonate titratable residues using predicted pKs. The following steps were carried out sequentially: cleaning of protein, optimisation of side-chain conformation for residues with inserted atoms using the ChiRotor algorithm, removal of water molecules, prediction of titration site pKs, and protonation of the structure at the specified pH.\u003c/p\u003e \u003cp\u003eThe CDOCKER module of Discovery Studio performed the protein-ligand docking investigation. CDOCKER is a grid-based molecular docking method that employs a CHARMm-based molecular dynamics scheme to dock ligands into a receptor binding site. The ligands were allowed to flex with the rigidly held receptor during the refinement. High-temperature molecular dynamics generated random ligand conformations. This was followed by random rotations, refined by grid-based (GRID1) simulated annealing and forcefield minimisation. Each component's refined ligand poses were generated and analysed based on the lowest binding free energy, hydrogen bonds, and hydrophobic interactions.\u003c/p\u003e \u003cp\u003eThe top-scoring conformation identified through docking analyses was selected and used as a molecular dynamics (MD) simulation starting point. The ligand-receptor complex was subjected to the CHARMm36 force field and was then solvated in an orthorhombic box with Explicit Periodic Boundary conditions to avoid surface artefacts. This prevented the water molecules from diffusing away from the protein during the simulation and permitted a relatively small number of water molecules to act as a bulk solution. For studying the nature of interactions, LIGPLOT was used, where the docked complexes were uploaded to generate the relevant diagrams.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Cell lines\u003c/h2\u003e \u003cp\u003eHuman K-ras mutated NSCLC cell lines, A549 and H460, were collected from the National Centre for Cell Science, Pune, India and were cultured in DMEM and RPMI-1640, respectively, with 10% heat-inactivated FBS and 1% antibiotic (PSN) at 37\u0026ordm;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator. Cells were harvested with 0.025% trypsin and 0.52 mM EDTA in phosphate buffer saline, plated at required cell numbers, and allowed to adhere for a minimum of 24 hours before treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Cell viability assay\u003c/h2\u003e \u003cp\u003eA549 and H460 cells (1\u0026times;10\u003csup\u003e2\u003c/sup\u003e cells per well) were seeded in 96 healthy plates and treated with NPEQ (10\u0026ndash;100 ng/ml) for 24 hours. Cell viability was measured by MTT assay [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The concentration that reduced the cell viability by 50% was taken as an IC\u003csub\u003e50\u003c/sub\u003e dose and was used for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Cellular morphological analysis\u003c/h2\u003e \u003cp\u003eBoth the cell lines were seeded at the density 1\u0026times;10\u003csup\u003e3,\u003c/sup\u003e and NPEQ was treated in them for 12, 18, and 24 hours. After that, morphological analysis was done by an ordinary phase-contrast microscope (Leica, Wechsler, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Wound healing assay\u003c/h2\u003e \u003cp\u003eCells were cultured in a 6-well plate to almost 100% confluence. A wound was generated at the centre of the well by scratching it with a plastic pipette tip. Cells were washed once with PBS. After NPEQ treatment with less than IC\u003csub\u003e50\u003c/sub\u003e dose for respective hours, the plate was placed under an inverted microscope (Axis cope plus 2, Zees, Germany), the reference point was matched, the photographed regions of the first image were aligned, and then the second image was obtained [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.11. BrdU incorporation assay\u003c/h2\u003e \u003cp\u003eA549 cells (1\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well) were dispensed in 96-well flat-bottom microtiter plates. After 24h of incubation, they were treated with BrdU solution and three different drug concentrations for 6, 12, and 24 hrs of exposure, respectively. BrdU incorporation was measured using a primary anti-BrdU antibody (1:500 dilutions) and its respective secondary antibody. After that, pap was used as a colour developer, and colour intensity was measured at 405 nm concerning blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Phosphatidyl externalisation assay after Annexin V-FITC/PI staining\u003c/h2\u003e \u003cp\u003eAfter 12, 18, and 24 hr of NPEQ induction, A549 and H460 cells were harvested after trypsinisation and were incubated in 100\u0026micro;l binding buffer (10mM HEPES, pH 7.4; 150 mM NaCl; 5mM KCl; 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e and 1.8 mM CaCl\u003csub\u003e2\u003c/sub\u003e) with FITC labeled Annexin V (100 ng/ml) and propidium iodide (50 \u0026micro;g/ml) at room temperature for 15 min in the dark and analysed using a FACS Calibur (BD Bioscience) taking minimum 10,000 cells in each sample [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Cell cycle analysis after PI staining\u003c/h2\u003e \u003cp\u003eBriefly, 2 x 10\u003csup\u003e4\u003c/sup\u003e cells were seeded and treated with NPEQ at different times. Cells were then recovered, washed twice in cold PBS, and fixed in 70% chilled ethanol for one hour. Then, the cells were washed twice in PBS and incubated with RNase A (100 \u0026micro;g/ml) for 1 h at 37\u003csup\u003e0\u003c/sup\u003eC. After that, 50 \u0026micro;g/ml PI was added, and cells were incubated for 15 min in the dark and were analysed using a FACS Calibur flow cytometer (BD Bioscience). Ten thousand events were analysed for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Cell extract and protein isolation\u003c/h2\u003e \u003cp\u003eCells (2 x 10\u003csup\u003e6\u003c/sup\u003e) were plated in 90 mm culture dishes and were allowed to increase for 48 h. Following NPEQ treatment, A549 and H460 cells were collected and washed twice in ice-cold PBS. The total protein was isolated and stored at -20\u0026deg;C for further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Immunoblot analysis\u003c/h2\u003e \u003cp\u003eFor immunoblot analysis, equal amounts (70 \u0026micro;g) of protein were loaded. Samples were denatured in 12% SDS-PAGE for Akt (1:1000). Separated proteins were transferred individually onto PVDF membranes and were probed with respective primary antibodies overnight at 4\u0026deg;C, followed by 1h incubation with ALKP-conjugated secondary antibody (1:500). Then they were developed using BCIP-NBT [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. GAPDH (1:1000) was used as a loading control. Quantifying proteins was performed by densitometry using Image J software [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.16. Tumor cell transplantation and treatments\u003c/h2\u003e \u003cp\u003eEhrlich Ascites Carcinoma (EAC), murine mammary adenocarcinoma cells were used as a tumour model. Five groups of healthy Swiss albino mice (n\u0026thinsp;=\u0026thinsp;5), all from the age group, i.e. 3\u0026ndash;5 weeks, were injected subcutaneously in the right leg flank with 5x10\u003csup\u003e6\u003c/sup\u003e cells in 100 \u0026micro;l PBS. All mice were divided into five groups, as vehicle-treated control, Quercetin at 25 mg/kg body weight (Q1), 50 mg/kg body weight (Q2), and NPEQ at 25 mg/kg body weight (NQ1), 50 mg/kg body weight (NQ2). After ten days, a palpable tumour appeared, and treatment started. All compounds were injected intraperitoneally (IP) with mentioned doses. Each group was treated on every alternate day, i.e. 1st day followed by 3rd day, and continued for up to 15 days. After 30 days, all animals were sacrificed, and tumours and other vital organs were dissected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.17. Acute toxicity studies and drug dose selection\u003c/h2\u003e \u003cp\u003eFor sensitive toxicity testing, mice (n\u0026thinsp;=\u0026thinsp;6) were fed 25, 50, 100, and 200 mg/kg body weight (bw) of NPEQ. After that, signs of mortality, clinical signs, and behavioural changes in mice for 24 h for any sign of acute toxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.18. Chronic toxicity analysis of liver and kidney tissue after Eosin Hematoxylin staining\u003c/h2\u003e \u003cp\u003eFor the established toxicity assay, mice were treated with Quercetin and NPEQ at 25 mg/kg body weight and 50 mg/kg body weight for 30 days. After that, the histological structure of the liver and kidney were analysed after eosin and hematoxylin staining. Stained slides were mounted with DPX mounting media and observed under a bright field microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.19. In vivo tumour growth analysis\u003c/h2\u003e \u003cp\u003eTumour size was measured every other day using a Vernier calliper to determine both the shortest diameter (A) and the longest diameter (B). Volume calculation was done using the formula V=(A2B)/2. Mice were sacrificed after 30 days after treatment started. The relative tumour volume (RTV) on day \u0026lsquo;n\u0026rsquo; was calculated using RTV\u0026thinsp;=\u0026thinsp;TVn/TV0, where TVn is the Tumor volume on day n, and TV0 is the Tumor volume on day 0. The following formula calculated [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] tumour growth inhibition rate (TIR)\u003c/p\u003e \u003cp\u003eTIR = {(1 - (mean volume of treated tumors)/mean volume of control tumors)} \u0026times; 100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e2.20. Tumor tissue architecture analysis by Hematoxylin and Eosin staining\u003c/h2\u003e \u003cp\u003eTumours of untreated and treated mice were fixed in 10% neutral buffered formalin for 24 hours. The histological structure of the tumour tissue was analysed after eosin and hematoxylin staining following the protocol discussed earlier. Stained slides were observed under a bright field microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e2.20. Protein expression analysis after Immunohistochemistry\u003c/h2\u003e \u003cp\u003eFor immunohistochemistry staining, the tissue slide was made. The primary antibodies, cleaved caspase-3 (1:400) and anti-phospho-Akt antibody (1:100), were applied in a humidified chamber at 4\u003csup\u003eo\u003c/sup\u003eC overnight. After washing, the HRP-tagged secondary antibody (1:800) was applied the next day for 30 minutes. Impact Novared was used as a substrate for HRP to generate a colour. Sections were then dehydrated and mounted with DPX mounting media.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e2.21. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe values of the determined parameters were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The mean values from three independent experiments were included. Comparisons were performed using two-way ANOVA and an LSD post hoc test for multiple comparisons. The results were statistically significant at \u003csup\u003e*\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003csup\u003e***\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001. All analyses were performed using SPSS 16.0 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of nano-PLGA encapsulated quercetin (NPEQ)\u003c/h2\u003e \u003cp\u003eThe size of the synthesised nanoparticles was approximately 110 nm with a polydispersity index value of 0.186 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The zeta potential was found to be negative. AFM characterisation of quercetin-loaded PLGA nanoparticles revealed their smooth and bright surface topology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). FTIR analysis indicated that quercetin was efficiently loaded in PLGA molecules. The primary characteristic peaks of quercetin were present in free and PLGA-encapsulated quercetin, which was absent in only PLGA nanoparticles. The presence of distinct peaks of quercetin in PLGA-quercetin nano molecule is an indirect way of confirming the successful encapsulation of quercetin in PLGA nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.2. NPEQ interacts with PLGA\u003c/h2\u003e \u003cp\u003eIn the case of the Quercetin - PLGA interaction, the detection of hydrogen bonds was not found. These two small molecules seemed to interact via van der Waals forces only. The stability of the complex was identified based on the free energy value of \u0026minus;\u0026thinsp;119.30 KCal/mol \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. A weak interaction between quercetin and PLGA was found. When implied in a cell, this might help sustain the quercetin molecules' sustainable release.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.3. NPEQ showed cytotoxicity on A549 and H460 cells\u003c/h2\u003e \u003cp\u003eNPEQ reduced the cell viability of A549 and H460 after 24 hrs of induction. The IC\u003csub\u003e50\u003c/sub\u003e value was determined to be 406 ng/ml and 347 ng/ml for A549 and H460, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). On the other hand, free quercetin was earlier found to be cytotoxic against A549 and H460 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] but with a higher dose of IC\u003csub\u003e50\u003c/sub\u003e value 66 \u0026micro;M (~\u0026thinsp;19x10\u003csup\u003e6\u003c/sup\u003e ng/ml) for A549 and \u0026gt;\u0026thinsp;100 \u0026micro;M (~\u0026thinsp;30x10\u003csup\u003e6\u003c/sup\u003e ng/ml) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This shows that quercetin in nano-encapsulated forms is more precise and effective target-specific.\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\u003eIC\u003csub\u003e50\u003c/sub\u003e values of free quercetin and NPEQ in A549 and H460 cell line\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eName of the compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCell lines\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA549\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH460\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFree Quercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e~\u0026thinsp;19x10\u003csup\u003e6\u003c/sup\u003e ng/ml [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;30 x10\u003csup\u003e6\u003c/sup\u003e ng/ml [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNPEQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e406 ng/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e347 ng/ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.4. NPEQ-induced morphological alterations and blocked the proliferation of A549 and H460 cells\u003c/h2\u003e \u003cp\u003eCompared with the untreated ones, NPEQ-induced cell shrinkage and membrane blebbing after 12\u0026ndash;24 hrs of exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Uncontrolled cell proliferation activity was significantly inhibited time-dependent after NPEQ induction, as revealed by wound healing and BrdU incorporation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Phosphatidylserine externalisation and cell cycle arrest after NPEQ induction\u003c/h2\u003e \u003cp\u003eAn increased number of AnnexinV positive A549 and H460 cell populations was observed after NPEQ treatment, as compared to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Flow cytometric analysis also indicated a sub-G1 arrest in NPEQ-treated cells in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Onset of this sub-G1 cell cycle arrest suggested that NPEQ was potent in inducing apoptosis in both A549 and H460 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Modulation of Akt expression upon NPEQ treatment, possibly through Quercetin-Akt interaction\u003c/h2\u003e \u003cp\u003eFrom the immunoblot data, NPEQ was found to be effective in inhibiting Akt expression in both the NSCLC cell lines after 12\u0026ndash;24 hrs of exposure, as compared with the untreated ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Down-regulation of Akt was more pronounced in both the cell lines 12 hrs onwards of NPEQ induction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs the quercetin downregulated the Akt expression after release in a sustainable manner from PLGA nanoencapsulation, we have studied whether it has any interactive activity with Akt. Through molecular docking analysis, Quercetin was found to interact with the protein molecule using a total of 52 noncovalent interactions, out of which a hydrogen bond was detected between the aspartate 293 and the first carbon of quercetin having a distance of 3.22 A\u003csup\u003e0\u003c/sup\u003e. Fifty-one non-bonded contacts (Van der Waals interactions) were identified (\u003cb\u003eSupplementary Table\u003c/b\u003e). The overall interaction was stable, and a free energy of 236.53KCal/mol was obtained, indicating the stability of the complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e3.7. NPEQ toxicity assessment and dose selection\u003c/h2\u003e \u003cp\u003eQuercetin was relatively non-toxic to mice in vivo in free and nano-encapsulated form. No significant changes in mortality or abnormal clinical signs or symptoms were observed. The 100 mg/kg bw dose of NPEQ followed partial signs of behavioural change. Thus, we preferred to use 25 and 50 mg/kg body weight of the drug (free quercetin and NPEQ) as the optimum ones for the study. Furthermore, histological studies indicated that after quercetin treatment (free and nano-encapsulated form), liver and kidney tissue architecture remains unaltered compared to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This revealed that the selected drug dose is optimum and safe for further testing and analysis. On the other hand, nano-encapsulated quercetin recovered the tissue architecture (marked in red) compared with the free quercetin when applied. This reveals that this has the potential to reduce tumorigenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Antitumor activity of the compounds in vivo\u003c/h2\u003e \u003cp\u003eDifferential regression in tumour volume in different treatment groups is shown in dissected mice images (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Q1 and Q2 inhibited tumour growth by 19% and 46%, whereas NQ1 and NQ2 inhibited tumour growth by 38% and 72% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003e3.9. NPEQ-induced tissue structure alteration in tumour-bearing mice\u003c/h2\u003e \u003cp\u003eThe excised tumours were sectioned and examined by H\u0026amp;E staining. Mean tumour apoptosis (%) was determined from several high-magnification images and apoptotic areas indicated by a red arrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The stained tumour histology section from the control group showed hematoxylin-stained, well-organised, dense tumour nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). The NQ2 group showed an elevated level of necrotic areas (64%) with very few seats. Q1 and Q2 groups showed 30% and 39% apoptotic area in tumour tissue sections, whereas the NQ1 group showed 46% apoptotic area. This revealed the potential of NPEQ to recover the tissue architecture compared to free quercetin and the untreated control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003e3.10. Modulation of cleaved caspase three and Akt activation\u003c/h2\u003e \u003cp\u003eThe immunohistochemical assay revealed that compared with free quercetin treatment, NPEQ, in a dose-dependent manner, upregulated cleaved caspase 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB) and downregulated p-Akt expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). This signified quercetin in a PLGA nano-encapsulated form is a more potent antiproliferative and pro-apoptotic agent than free quercetin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDesigning and testing target-specific drug molecules on biological systems demands the formulation and usage of nanoparticles encapsulated in biodegradable polymers with better efficacy and higher bioavailability. We tried to examine the effectiveness of nano-encapsulated quercetin in inhibiting cell proliferation against K-ras mutated NSCLC cells. Furthermore, we have induced the formulation in tumour-bearing mice to evaluate its antitumorigenic effect. Experimental results indicated successful encapsulation of the hydrophobic flavonoid quercetin into PLGA nanoparticles. NPEQ showed an approximate size of 110 nm with an unimodal particle size distribution, as revealed by the low polydispersity index, which is typical of mono-dispersed systems. The negative zeta potential value confirmed that the NPEQ is highly stable without particle aggregation. AFM characterisation of NPEQ revealed their smooth and bright surface topology. This indicates the uniformity in dimensions of the formulated nanocapsules. FTIR spectral analysis showed that quercetin was efficiently loaded in PLGA molecules with no significant interaction, as similar characteristic peaks of quercetin were present in both free and NPEQ. Besides this, molecular docking analysis between quercetin and PLGA showed no hydrogen bond interaction and weak van der Waals interaction. This indicates that quercetin can quickly be released in the target cell after being incorporated in a nanoencapsulation form.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter that, the synthesised NPEQ was examined for its anti-proliferative and apoptotic efficacy against two major K-ras mutated NSCLC cell lines, A549 and H460. Results revealed that NPEQ had significant inhibitory effects, with 406 ng/ml and 306 ng/ml as IC\u003csub\u003e50\u003c/sub\u003e values in A549 and H460, respectively. Time-dependent inhibitory effects of NPEQ revealed that proliferation of both the cell lines was inhibited, showing concomitant destruction of BrdU activity and lower incidence of cell migration. Besides this, NPEQ-mediated cell death was confirmed to be a consequence of apoptosis and not necrosis. After its exposure, effective phosphatidylserine externalisation and cell cycle arrest at sub-G1 stages were observed. Hence, we could infer that early-hour inhibition of NSCLC cell proliferation may eventually lead the cells to apoptosis.\u003c/p\u003e \u003cp\u003eIt has been demonstrated that Akt, a significant protein related to cell proliferation, remains constitutively active in A549 and H460 cells due to K-ras mutation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, the inhibition of downstream PI3K alone is insufficient to treat K-ras mutated cancer and may not be sensitive to PI3K pathway inhibitors. Targeted knockdown of Akt in K-ras mutated NSCLC cell lines would provide critical confirmation of the role of Akt in tumorigenesis, resulting in both the suppression of tumour growth and sensitisation to PI3K inhibitors. Our results revealed that after the sustained release of the NPEQ, the drug molecules, the quercetin itself, could efficiently interact with Akt. Here, the strong interaction of the released quercetin and Akt may have caused the downregulation of Akt despite the K-ras mutation. A molecular docking study indicates that quercetin significantly interacts with Akt with 52 noncovalent interactions, including one hydrogen bond and 51 non-bonded contacts (Van der Waals interactions). This significant interaction might be a causative factor of the quercetin-induced downregulation of Akt in both A549 and H460 cell lines. However, the precise downregulation at such a lower dose would not have been possible in an accessible form of quercetin. Nanoencapsulation might help the target-specific sustained release of quercetin to exert its efficacy at a lower dose. Hence, our studies have shown that NPEQ mediated the down-regulation of Akt at 12\u0026ndash;18 hrs of exposure in the A549 and H460 cell lines. The significant down-regulation of Akt was likely to play an initial cause in blocking cell proliferation, which presumably directed the cells towards apoptosis.\u003c/p\u003e \u003cp\u003eOn the other hand, in tumour-bearing mice, NPEQ showed better efficacy in lowering tumour growth and the tumorigenesis process. NPEQ also indicated tissue structure recovery in tumour-bearing mice in a dose-dependent manner. NPEQ at this particular dose has not shown any normal cell toxicity, indicating that the amount is safe and might be target-specific. Furthermore, immunohistochemistry analysis results showed an upregulation of cleaved caspase three and simultaneous downregulation in the activated form of Akt. This indicated the tissue apoptosis and uncontrolled growth retardation that may be beneficial for the mice\u0026rsquo;s bodies to recover from the tumorigenic condition.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe above findings suggest that quercetin in a nanoparticulate form can effectively block uncontrolled NSCLC cell proliferation and trigger the apoptotic event. Furthermore, this NPEQ showed its antitumorigenic potential in mice bodies, better than the free quercetin. Thus, encapsulation would offer a new dimension in the target-based drug delivery system, especially in the treatment modality. Understanding how NPEQ regulates proliferation inhibition and cell death may contribute to novel therapeutic strategies in cancer research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone to declare\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank Dr Sanjaya Mallick, ex-application scientist and COE manager, BD BioSciences, and associated with the Centre for Research in Nanoscience and Nanotechnology, University of Calcutta, for his help conducting FACS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor animal studies, the animals were maintained in the animal facility of the Chittaranjan National Cancer Institute (CNCI), Kolkata, after the institutional animal ethical committee approved the protocol with Sanction No. IAEC-1774-BB-3/2019/12.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research work was partially funded by the West Bengal Department of Science, Technology and Biotechnology, Sanction Order No.: 252(Sanc.)/STBT-11012(12)/2/2022- ST SEC.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSpaans JN, Goss GD. 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Quercetin Down-regulates IL-6/STAT-3 Signals to Induce Mitochondrial-mediated Apoptosis in a Non small- cell Lung-cancer Cell Line, A549. \u003cem\u003eJ Pharmacopuncture\u003c/em\u003e. 18(1):19-26.\u003c/li\u003e\n\u003cli\u003eRosell R, Monzo M., Molina F., Martinez E., Pifarre A., Moreno I., Mate J. L., de Anta J. M., Sanchez M., Font A. (1995). K-ras genotypes and prognosis in non-small cell lung cancer. \u003cem\u003eAnn. Oncol.\u003c/em\u003e, 6: S15-S20.\u003c/li\u003e\n\u003cli\u003eBrognard J, Clark AS, Yucheng Ni, Dennis PA. (2001). Akt/Protein Kinase B Is Constitutively Active in Non-Small Cell Lung Cancer Cells and Promotes Cellular Survival and Resistance to Chemotherapy and Radiation. \u003cem\u003eCancer Res.\u003c/em\u003e, 61(10): 3986-3997.\u003c/li\u003e\n\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":"PLGA encapsulated nanoparticles, Quercetin, cell proliferation, Akt, apoptosis, anti-proliferation","lastPublishedDoi":"10.21203/rs.3.rs-4086530/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4086530/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo test if encapsulating hydrophobic flavonoids in nanoparticles could offer a new possibility in the therapeutics of non-small cell lung cancer (NSCLC), quercetin was encapsulated in PLGA nanoparticles by solvent displacement technique. The synthesised nanoparticles were then characterised by dynamic light scattering (DLS), Fourier transforms infrared spectroscopy (FTIR), and atomic force microscopy (AFM). The size of the nanoparticles with smooth surface topology was estimated at 110 nm. Treatment with nano-PLGA encapsulated quercetin (NPEQ) triggered the death of K-ras mutated NSCLC cells, A549 and H460, and showed 50% cell cytotoxicity in them at a dose of 406 ng/ml and 306 ng/ml, respectively. NPEQ was able to block uncontrolled cell proliferation by inducing concomitant destruction of BrdU activity and a lower incidence of cell migrations. Cell death was due to the induction of apoptosis rather than necrosis, as revealed by morphological alterations and phosphatidylserine externalisation induced by NPEQ. NPEQ also caused the arrest of A549 and H460 cells at the sub-G1 stage. NPEQ induced down-regulation of Akt, which is usually found to be hyperactive in NSCLC due to K-ras mutation. This indicates that NPEQ caused target-specific apoptotic and antiproliferative activity by targeting the downregulation of Akt. Further, when NPEQ was generated in the tumour-bearing mice model, it showed antitumor efficacy. Besides this, histological alteration of tissue architecture and modulation of an apoptotic marker protein in mice indicates the prospect and advantages of nanoparticulate quercetin delivery in therapeutic formulations against cancer.\u003c/p\u003e","manuscriptTitle":"Anti-proliferative and Apoptotic Efficacy of Nano-PLGA encapsulated Quercetin Molecules by down-regulation of Akt in K-ras mutated NSCLC cell lines, A549 and H460","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-19 11:04:42","doi":"10.21203/rs.3.rs-4086530/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":"107845f3-7dd3-4f3e-ae6c-c68ae34c4f0a","owner":[],"postedDate":"March 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-03T05:24:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-19 11:04:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4086530","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4086530","identity":"rs-4086530","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}