Sialic acid-receptor targeted Epirubicin and Naringin-loaded sialic acid-conjugated silk fibroin nanoparticles for enhanced lung cancer treatment

Sialic acid-receptor targeted Epirubicin and Naringin-loaded sialic acid-conjugated silk fibroin nanoparticles for enhanced lung cancer treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sialic acid-receptor targeted Epirubicin and Naringin-loaded sialic acid-conjugated silk fibroin nanoparticles for enhanced lung cancer treatment Selvaraj Kunjiappan, Murugesan Sankaranarayanan, Parasuraman Pavadai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7970614/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Apr, 2026 Read the published version in Journal of Polymers and the Environment → Version 1 posted 20 You are reading this latest preprint version Abstract Lack of specificity, high burden of toxicity, and low bioavailability are the significant hurdles of conventional chemotherapies. Upregulated sialic acid receptors on the plasma membrane of lung cancer cells could be promising drug delivery targets for effective lung cancer treatment. In this view, the present study aimed to fabricate sialic acid (SA)-conjugated epirubicin (Epi) and naringin (NA)-loaded silk fibroin (SF) nanoparticles (SA-Epi-NA-SF-NPs) for selective delivery and enhanced lung cancer treatment. SF protein was initially extracted from silk cocoons, and the SA-conjugated SF was synthesized using simple EDC-conjugation chemistry. Later, the desolvation cross-linking technique was used to fabricate SA-Epi-NA-SF-NPs by encapsulating Epi and NA into an SA-conjugated SF. Various characterization methods were employed to confirm the physicochemical properties of SA-Epi-NA-SF-NPs. The fabricated SA-Epi-NA-SF-NPs ranged in size from 100 to 400 nm and had a spherical, crystalline nature. Epi and NA had encapsulation efficiency and loading capacity of 83 ± 1.5%, 80 ± 12%, 8.34 ± 0.9%, and 8.16 ± 0.3% into SA-conjugated SF, respectively. Drug release was substantially higher at pH 5.4 (84.46 ± 1.29% Epi and 70.99 ± 1.56% NA) than at pH 7.4. The cytotoxic potential of SA-Epi-NA-SF-NPs against A549 cells could diminish the viable number of cells after 24 h of treatment, and 13.16 µg×mL − 1 was observed as an IC 50 . The higher intracellular accumulation of Epi and NA in A549 cells targets mitochondria and the nucleus and causes apoptosis. Based on these outcomes, SA-Epi-NA-SF-NPs could have high therapeutic potential for lung cancer treatment, specifically targeting sialic acid receptors on A549 cells. A549 cells Apoptosis Drug delivery Sialic acid receptor Silk fibroin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Significant progress has been made in developing innovative therapeutic medicines and cancer detection and treatment methods. However, cancer remains a life-threatening condition, with the number of cases continually increasing. In terms of incidence and mortality, lung cancer ranks as the second leading cause of cancer worldwide, and the overall survival rate remains extremely poor [ 1 ]. Lung cancer was the most commonly diagnosed malignancy, making up about one out of every eight malignancies globally and over 2.5 million new cases, according to the GLOBOCAN 2022 report [ 2 ]. Cigarette smoking has been shown to raise the risk of acquiring lung cancer [ 3 ]. However, familial disease clustering and segregation analyses suggest that genetic predisposition may potentially play a role in the development of lung cancer [ 4 ]. Lung cancer can currently be treated with surgery, chemotherapy, radiation, laser therapy, and combinations of these [ 5 ]. Chemotherapy is the best, cost-effective, and widely accepted active treatment option for lung cancer [ 6 ]. Epirubicin (4'-epidoxorubicin), an Adriamycin analogue used to treat lung cancer, is one of the most often prescribed drugs [ 7 ]. At the same time, Epirubicin is non-specific in targeting cancer cells and has substantial side effects, which limit its use [ 8 ]. As a result, it is recommended to combine it with other medicines to reduce its dosage while maintaining its potency. Many studies have recommended that combination therapy has a high potential for treating lung cancer [ 9 – 12 ]. Chemotherapy coupled with plant-derived compounds considerably decreases chemotherapy's adverse effects and increases its anticancer potency [ 13 ]. Some natural chemicals may respond to conventional cytotoxic therapy, raise the medication's effective concentration, reverse drug resistance, improve the combined action of both administered drugs, or be cytotoxic to cancer cells [ 14 , 15 ]. Patients tolerate many plant-derived compounds well and do not experience severe adverse effects, even at large doses [ 16 ]. The interplay of conventional chemotherapeutics and natural chemicals brings up new avenues for cancer research and treatment possibilities. In this view, Naringin combined with Epirubicin may reduce its toxicity and enhance its efficacy. Naringin (4′,5,7-Trihydroxyflavanone-7-rhamnoglucoside) is a plant-derived compound found in grapes, oranges, and Kino [ 17 ]. Numerous studies have shown that naringin reduces cell proliferation, migration, and invasion while increasing apoptosis of cancer cells in in vitro and in vivo cancer models, indicating significant anticancer effects on various human cancers, including lung cancer [ 17 – 20 ]. Unfortunately, epirubicin not only targets malignant cells but also healthy cells, which is one of the reasons malignant cells become resistant to active therapy [ 21 ]. Furthermore, non-selective targeting, low aqueous solubility, and non-targetable drug distribution resulted in low bioavailability at the target site and drug resistance [ 22 ]. As a result, effective drug delivery systems are required to deliver drugs selectively, overcome drug resistance, and improve bioavailability at the target site [ 23 ]. In this scenario, various protein nanocarrier systems employ the most auspicious ways of delivering anticancer drugs into cancer cells [ 24 ]. Protein nanocarriers are nano-sized particles that carry drugs and deliver intracellularly into cancer cells [ 25 ]. Protein nanocarriers have numerous advantages, including biocompatibility, biodegradability, non-immunogenicity, loading a wide range of pharmaceuticals, excellent endocytosis efficiency, and controlled release of loaded entities [ 26 ]. Protein nanocarriers can increase blood plasma circulation and aggressively target malignant cells [ 27 , 28 ]. The surface of these nano-sized carrier systems can be easily modified by targeting moieties to improve cancer therapy [ 29 ]. Because of its biocompatibility, silk fibroin protein (SF) is one of the proteins most often employed for nanocarrier production in various biomedical applications [ 30 ]. Nanoparticles (NPs) were made using SF obtained from the cocoons of the silkworm Bombyx mori [ 31 ]. Silk fibroin nanoparticles (SF-NPs) efficiently deliver enzymes, nuclear materials, genetic materials, antibodies, and drugs into the target site [ 32 ]. SF contains numerous active amino, carboxyl and thiol groups that aid in conjugating different macromolecules [ 33 ]. Attaching or conjugating targeting molecules to SF addresses the nanoparticles' fundamental weaknesses by enhancing their selectivity for cell surface adhesion [ 33 ]. The ligands attached to nanoparticles easily bind to naturally upregulated receptor molecules on cancer cell surfaces and regulate bidirectional flux. Loaded drugs are also activated intracellularly following ligand cleavage [ 34 ]. The versatile SF-NPs can dynamically target lung cancer cells by altering the amino groups on their surfaces with ligands that bind to overexpressed surface receptors [ 24 ]. Combined with the SF-NPs, the ligand quickly recognizes the naturally overexpressed receptors on cancer cell surfaces [ 35 ]. Bidirectional flow occurs following ligand-receptor contact on the plasma membrane surface of cancer cells [ 36 ]. Designed nanoparticles are coupled with ligands for specific drug delivery to cancer cells, primarily targeting mitochondria and the nucleus. Cancer cells require abundant nutrients, including folic acid, amino acids, vitamins, and carbohydrates, to support aberrant development and uncontrolled cell multiplication [ 37 , 38 ]. Sialic acids (SAs) are nine-carbon carboxylated monosaccharides in cell surface glycoproteins and glycolipids. They tightly attach to the sialic acid receptor [ 39 ]. In recent years, sialic acid-binding proteins have been discovered, notably sialic acid-binding immunoglobulin-like lectins (Siglecs), also identified as sialic acid adhesins, which play an essential role in macrophages' pro-inflammatory responses [ 40 ]. Some studies have discovered that Siglecs are highly expressed on the surface of tumor-associated macrophages in mammals. Most Siglecs are endocytic receptors, allowing cytotoxic drugs or immunological modulators to be delivered to target cells by targeting Siglecs. These findings suggest that changing SAs on carriers to increase the formulation targeting is a promising cancer treatment strategy [ 41 – 44 ]. It is a critical component of both stealth and targeting ligands. The overall goal of this study was to develop a new drug delivery system with SAs-conjugated dual drugs (Epi and NA)-loaded SF-NPs for targeting sialic acid receptors to effectively deliver the loaded entities into lung cancer cells (A549 cells) and evaluate their anticancer potency. 2. Experimental section 2.1. Materials and Methods The silk cocoon of Bombyx mori was sourced from Mulberry farms, Hosur, Krishnagiri District, Tamil Nadu, India. Epirubicin (Epi), sialic acid ( N -acetylneuraminic acid) (SA), naringin (NA), lithium bromide, sodium carbonate, N -hydroxysuccinimide (NHS), N -(3-dimethyl aminopropyl)- N’ -ethyl carbodiimide hydrochloride (EDC) are procured from Sigma Aldrich, and Sisco Research Laboratories (SRL) Pvt. Ltd., Mumbai, India. Cell culture medium, staining, and other reagents were obtained from Gibco Laboratories, Thermo Fisher Scientific, and Sigma Aldrich Ltd., Mumbai, India. 2.2. Silk fibroin (SF) protein Dried Bombyx mori silk cocoons were fragmented into small pieces and degummed using a 0.02M sodium carbonate aqueous solution at 98°C for 30 min, with agitation. The whole mass was repeatedly washed with Milli-Q water to eliminate the adhesive sericin protein. After comprehensive washing with Milli-Q water and subsequent air-drying, the resultant dried product exhibited a luminous, white, cotton-like look. A silk fibroin solution was prepared by dissolving 10 g of degummed silk in a 9.30M lithium bromide solution at 60°C for 4 h. The silk fibroin solution underwent dialysis using a dialysis membrane-150 (molecular cutoff 12,400) against deionized water for three days, with water replacement every 6 h to remove lithium bromide. Post-dialysis, the silk fibroin solution underwent centrifugation at 5–10°C and 9000 rpm for 20 min, resulting in 5% fibre concentration in a 100% volume. The concentrated solutions were stored at 4°C for subsequent analysis [ 45 ]. FTIR and NMR analyses validated the chemical characterization of the extracted silk fibroin protein. Attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy (Cary 630 FTIR Spectrometer, Agilent Technologies, USA) was used to analyze the functional groups of silk fibroin protein. The sample was analyzed in reflection mode, with results reported as the average of 64 repeated scans ranging from 4000 to 400 cm − 1 . 1 H-NMR spectroscopy was assessed to examine the type and number of hydrogens present in the proteins. 2.3. Fabrication of sialic acid-conjugated Epirubicin and Naringin-loaded Silk fibroin Nanoparticles (SA-Epi-Na-SF-NPs) 2.3.1. Synthesis of Sialic acid-conjugated silk fibroin (SA-SF) The sialic acid-conjugated silk fibroin nanocarrier was synthesized by simple EDC-conjugation chemistry. Figure 1 describes the sialic acid-conjugated silk fibroin synthesis. Briefly, 81.04 mg of sialic acid ( N -acetylneuraminic acid) and 61.01 mg of EDC were solubilized in 10 mL of Milli-Q water while magnetically stirring. Following 10–15 min of stirring, 60.31 mg of N -hydroxysuccinimide was incorporated into the mixture above, and the reaction mixture was agitated for 1–2 h. The reaction mixture was subsequently filtered to eliminate O-acylisourea as a byproduct, resulting in a filtrate composed of sialic acid- N -hydroxy succinimide. Subsequently, 200 mg of silk fibroin protein, pre-dissolved in 5 mL of acetate buffer (pH 4.7), was combined with sialic acid- N -hydroxy succinimide and agitated overnight. The sialic acid-conjugated silk fibroin (SA-SF) mixture was subjected to dialysis using a dialysis membrane-150 against Milli-Q water to remove unreacted sialic acid. The structural and functional characteristics of the synthesized SA-SF nanocarrier were validated using FTIR and NMR spectroscopy. The 1 H-NMR spectrum was acquired using a Bruker 600 MHz AVANCE NMR spectrometer equipped with a TCI CryoProbe, with DMSO as the solvent and tetramethylsilane (TMS) as the internal standard. 2.3.2. Fabrication of Epirubicin and Naringin-loaded SA-SF nanoparticles (SA-Epi-NA-SF-NPs) The newly synthesized SA-SF nanocarrier (1 g) was pre-dissolved in 2 mL DMSO within 50 mL of carbonate/bicarbonate buffer (0.1 M; pH 10.0) and agitated using a magnetic stirrer. Subsequently, a 5 mg mixture of Epi (3 mg) and NA (2 mg) in 5 mL of ethanol, prepared via sonication, was incrementally added to the SA-SF solution along with 0.1 mL of glutaraldehyde as a cross-linking agent. This mixture was incubated with continuous magnetic stirring for approximately 48 h to achieve a clear solution. The nanoparticles were created by gradually encapsulating the hydrophobic drugs epirubicin and naringin. Subsequently, to exclude non-encapsulated medicines, the mixture underwent centrifugation for 5 min at 1500 rpm and was rinsed three times with deionized water. The synthesized SA-Epi-NA-SF-NPs were lyophilized and preserved for further biological uses. The drug loading and encapsulation efficiency were calculated throughout the formulation process. 2.3.3. Formulation of Epirubicin and Naringin-loaded silk fibroin nanoparticles (Epi-NA-SF NPs) Epi-NA-SF NPs were generated for comparative analysis. Epi-NA-SF NPs were produced with a modified desolvation cross-linking technique derived from coacervation. In summary, 2.5 mg of the compounds (Epi at 1.5 mg and NA at 1 mg) were solubilized by sonication in 3 mL of ethanol. The drug-infused solution was gradually incorporated into silk fibroin protein (1 g), which had been pre-dissolved in 2 mL of cell culture-grade DMSO within 50 mL of 0.1 M carbonate/bicarbonate buffer (pH 10.0) and glutaraldehyde (0.1 mL) as a cross-linking agent and was subjected to continuous magnetic stirring for approximately 48 h to achieve a transparent solution. The NPs were created by gradually encapsulating the hydrophobic medicines (Epi and NA) within silk fibroin protein. Subsequently, to exclude non-encapsulated Epi and NA, the mixture underwent centrifugation for 5 min at 1500 rpm and was rinsed with deionized water three times. The solution was redispersed in 3.0 mL of deionized water. 2.4. Characterization studies of SA-Epi-NA-SF-NPs 2.4.1. ATR-FTIR spectroscopy analysis Fourier Transform Infrared (FT-IR) spectral data of the Epi, NA, Sialic acid, Epi-NA-SF-NPs, and SA-Epi-NA-SF-NPs were recorded by ATR-FTIR using a Cary 630 FTIR Spectrometer, Agilent Technologies, USA. All spectra were acquired with 64 scans per spectrum in the 4000 − 600 cm − 1 range at a resolution of 4 cm − 1 . A background spectrum was captured before each measurement, with the same number of scans as the test sample. The spectra of the samples were acquired by positioning approximately 2 mg of the test sample atop the ATR crystal. The crystal was cleansed with isopropanol between each sample analysis and permitted to dry before the subsequent measurement. A pressure gauge applies force to the sample to ensure consistent contact between the sample and the crystal, facilitating optimal optical contact. The spectral bands 600–1800 and 2600–3800 cm − 1 indicated contributions from the biological component of the samples and were utilized for subsequent investigation [ 46 ]. 2.4.2. Dynamic Light Scattering (DLS) analysis The mean hydrodynamic diameter (Z-average), polydispersity index (PDI), and zeta potential of lyophilized Epi-NA-SF-NPs and SA-Epi-NA-SF-NPs were assessed via dynamic light scattering (DLS) utilizing a Zetasizer Nano ZS apparatus (Malvern Instruments Ltd., Worcestershire, UK). All measurements were conducted in Milli-Q water at 25°C and an angle of 173° to the source. Before the experiments, each sample underwent sonication for 3 min at 30% amplitude using 15-second ON/OFF intervals; the concentration of both NPs was 0.66 mg×mL − 1 . Measurements were conducted in triplicate, and data were presented as mean ± standard deviation (SD) [ 47 ]. 2.4.3. X-ray diffraction ( XRD) analysis The XRD pattern was obtained using a Bruker D8 ADVANCE ECO XRD system with an SSD160 1-D Detector to ascertain the physical characteristics of Epi-NA-SF NPs and SA-Epi-NA-SF-NPs. The x-ray diffractometer functioned under the following parameters: utilizing Cu Kα 1 radiation (λ = 0.1542) in a 2θ configuration at 20 keV and 30 mA current [ 48 ]. 2.4.4. X-ray photoelectron spectroscopy (XPS) analysis The presence of elements on the surface of SA-Epi-NA-SF-NPs was analyzed from 0 to 800 eV using high-resolution X-ray photoelectron spectroscopy (XPS; Thermo Fisher Scientific, East Grinstead, UK) with an Al/Ka X-ray source (hv = 1486.6 eV). The energy resolution was established at 0.1 eV with a pass energy of 1486.4 eV. The XPS spectra were examined utilizing CASA XPS software, which fitted the background using the Shirley function, removed it, and finally fitted the XPS peak with Gaussian-Lorentzian line shape functions [ 49 ]. 2.4.5. Field Emission Scanning Electron Microscopy ( FESEM) analysis The morphological characteristics of Epi-NA-SF-NPs and SA-Epi-NA-SF-NPs were evaluated using FESEM. For the experiment, 1 mg of freeze-dried Epi-NA-SF-NPs and SA-Epi-NA-SF-NPs were dissolved in 1 mL of Milli-Q water, and 2 µL of the resultant mixture was applied to a clean glass surface. Upon drying the solution, a thin layer of gold coating was applied to mitigate electrostatic charge during scanning electron microscopy. The research utilized a Thermo Fisher Scientific APREO 2SEM, FESEM [ 50 ]. 2.4.6. High-resolution transmission electron microscopy (HRTEM) analysis The dimensions and morphology of SA-Epi-NA-SF-NPs were examined via high-resolution transmission electron microscopy (HRTEM) utilizing a JEOL model 2100 equipment at an acceleration voltage of 200 kV, with lyophilized SA-Epi-NA-SF-NPs distributed in 1 mL of Milli-Q water. A few drops of redispersed SA-Epi-NA-SF NPs were deposited on a carbon-coated copper grid and air-dried to reveal the morphologically relevant aspects of the formed SA-Epi-NA-SF-NPs [ 51 ]. 2.5. Drug release studies The quantity of drug released in vitro from SA-Epi-NA-SF-NPs at various time intervals was assessed as follows: 4 mg of dual drug-encapsulated nanoparticles were combined with 20 mL of acetate buffer (pH 5.4) and phosphate-buffered saline (PBS) (pH 7.4), and agitated at 120 rpm at 37°C. At specified intervals, 2 mL was collected and centrifuged at 9000 rpm for 10 min. The concentration of the pharmaceuticals in the supernatant was quantified by UV-Visible spectrometry at 435 nm for Epi and 285 nm for NA. The drug release percentage was quantified as the ratio of the predicted quantity of medication released at different time intervals to the initial amount contained within the nanoparticles. All experiments were conducted in triplicate, and the average value was reported [ 52 ]. 2.6. Drug release kinetics studies Linear kinetic models were utilized to analyze the release data to determine the specific drug release kinetic profile and process [ 53 ]. The standard kinetic models for linear terms are as follows: The models employed include zero-order (cumulative drug release percentage versus time), first-order (logarithm of drug retention percentage versus time), Higuchi (cumulative drug release percentage versus the square root of time), Korsmeyer-Peppas (logarithm of drug released versus logarithm of time), and Hixson-Crowell (cube root of the amount of drug remaining in the dosage form versus time). In vitro drug release data were collected and analyzed to ascertain the kinetic process utilizing DD Solver 1.0, a Microsoft Excel add-in. The drug release kinetics of nanoparticle formulations are governed by the diffusional exponent "n." The drug release and diffusion mechanism demonstrates a non-Fickian or erosion process when n equals 0.45 and 0.89. For n = 0.45, the release mechanism is Fickian, whereas for n = 0.89, it corresponds to Case II transport. If drug release and diffusion are negligible, 'n' values and kinetic data lack significance. 2.7. Anticancer studies 2.7.1. Cytotoxicity assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining assay was employed to assess the cytotoxic effects of SA-Epi-NA-SF-NPs on lung cancer cells (A549 cells) and fibroblast cells (L929 cells) [ 54 ]. The cells were cultivated in 96-well plates at a density of 1×10 4 cells per well in a medium comprising 1% penicillin-streptomycin and 10% FBS, incubated at 37°C with 5% CO 2 for 24 h. The seeded cells were subjected to various concentrations of SA-Epi-NA-SF-NPs (10, 20, 50, 100, 150, 200 µg×mL − 1 ), 100 µg×mL − 1 of Epi-NA-SF NPs, 10 µg×mL − 1 of Epi, and 100 µg×mL − 1 of NA. Following 24 h of incubation, the medium in each well was substituted with 100 µL of MTT solution (1 mg×mL − 1 ) and incubated for 4 h at 37°C in a 5% CO 2 atmosphere. Living cells generated formazan, which was solubilized with 100 µL of isopropanol and quantified at 570 nm with a Biotek Epoch microplate spectrophotometer (Agilent Technologies, USA). The cytotoxicity was assessed by comparing the absorbance of treated cells to that of untreated cells, which served as a control. Every experiment was conducted a minimum of three times. 2.7.2. Cell migration study An in vitro scratch experiment was conducted to evaluate the efficacy of SA-Epi-NA-SF-NPs on cell-cell interactions and migration [ 55 ]. A549 cells were cultivated on a 6-well culture plate at a density of 1.5 × 10 4 cells per well. Upon achieving 80–90% confluency, a scratch was executed with a pointed instrument, such as a 10 µL pipette tip. The cells were subsequently washed with PBS to eliminate debris and treated with 7.5 µg×mL − 1 SA-Epi-NA-SF-NPs (½ IC 50 concentration), and pictures of control and experimental wells were captured at 0, 24, 48, and 72 h. To ascertain the % change in wound diameter for all formulations, the wound distance was randomly measured at various points for each scratch in an individual well plate, and the average of these independent measurements was computed. The migration rate of A549 cells was determined using the following formula: Cell migration rate (%) = (wound area at the 0 h-wound area at 24, 48, or 72 h)/wound area at 0 h×100 2.7.3. Detection of apoptosis 2.7.3.1. Acridine orange (AO)/Ethidium Bromide (EtBr) assay SA-Epi-NA-SF-NPs, subjected to AO/EtBr dual labeling, were examined via fluorescence microscopy to assess their apoptotic effects on A549 cells [ 56 ]. A549 cancer cells were plated in a 12-well dish at a density of 3 × 10 5 cells per well and incubated for 24 h. Following 24 h, the cells were administered an IC 50 concentration of SA-Epi-NA-SF NPs and cultured for 24 h. The treated cells were subsequently rinsed with 1× PBS buffer and stained with a dual fluorescent solution comprising 10 µL of AO (10 mg×mL − 1 ) and EtBr (10 mg×mL − 1 ), followed by a 30 min incubation period. Following incubation, unbound dyes were rinsed with 1x PBS buffer. The morphology of apoptotic cells was analyzed using a fluorescence microscope, and typical areas were documented at 40× magnification. The dual AO/EB staining technique was conducted a minimum of three times. 2.7.3.2. Calcein-Acetyoxymethyl (AM) cytotoxicity assay The calcein AM staining experiment evaluated the cytotoxicity of SA-Epi-NA-SF-NPs [ 57 ]. Cells were inoculated in 12-well plates at a density of 3 × 10 5 cells per well overnight and subsequently treated with the IC 50 concentration of SA-Epi-NA-SF-NPs diluted in DMEM media. Following a 24-h incubation at 37°C, 50 µL of 0.25 µM Calcein AM was introduced to the control-treated well and incubated for 30 min. Following incubation, unbound dyes were rinsed with 1x PBS buffer. The morphology of apoptotic cells was examined microscopically, with representative areas documented at 40× magnification. The calcein AM staining technique was performed a minimum of three times. 2.7.3.3. Hoechst assay The Hoechst 33342 staining experiment identified the induction of apoptosis following treatment with SA-Epi-NA-SF-NPs [ 58 ]. A549 cells were plated at a density of 3 × 10 5 cells per well in a 12-well plate and incubated for 24 h to facilitate cell adherence. After incubation, cells were administered the IC 50 concentration of SA-Epi-NA-SF-NPs, followed by an additional 24-h incubation period. Cells were stained with 1 µg × mL − 1 Hoechst 33342 (Invitrogen, Carlsbad, CA) for 1 min to counterstain the nuclei. After staining, the cells were rinsed and resuspended in 1x PBS before examination with an EVOS M 5000 imaging system (Thermo Fisher Scientific Inc., USA). 2.7.4. Mitochondrial targeting A549 cells were cultured on a 12-well plate at a density of 3 × 10 5 cells per well. Following 12 h of attachment, cells were exposed to the IC 50 concentration of SA-Epi-NA-SF-NPs for an additional 12 h. After 12 h, mitochondria were labelled with 1 µM MitoTracker Red; the cells were subsequently washed and resuspended in 1x PBS, then scanned using an EVOS M 5000 imaging system (Thermo Fisher Scientific Inc., USA) [ 59 ]. 2.8. Statistical Analysis Statistical outcomes from each experiment were presented as mean ± standard deviation for three independent repetitions. The distinction between the control and test samples was assessed utilizing the Student’s t-test unless specified otherwise in the legends. 3. Results 3.1. Silk fibroin (SF) SF protein was effectively isolated from silk cocoons, and its structure was validated using ATR-FTIR and proton NMR spectroscopy. The observed ATR-FTIR spectra (Fig. 2 (a)) displayed significant peaks at 3291 cm − 1 (amide A band), 3063 cm − 1 (N-H stretching), 2937 cm − 1 (C-H stretching), 2363 cm − 1 (O = C = O stretching), 1625 cm − 1 (amide I band), 1513 cm − 1 (amide II band), 1438 cm − 1 (O-H bending), 1222 cm − 1 (amide III band), and 1162 cm − 1 (C-O stretching). The ¹H NMR spectrum of SF protein (Fig. 2 (b)) verifies the prevalent amino acids as tyrosine (Tyr), glycine (Gly), serine (Ser), and alanine (Ala) because these amino acids constitute the backbone structure of silk fibroin protein. The proton signals from alanine methyl groups (-CH₃) range from 0.8 to 1.5 ppm, and the glycine and alanine β-protons (-CH₂) resonate between 2.0 and 2.5 ppm in the spectrum. The peaks within a 3.5–4.5 ppm area detect the α-protons (-CH) of tyrosine, glycine, serine, and alanine, thus confirming their place in the peptide backbone. The fibroin structure contains robust peptide linkages because the broad peaks at 8.0 ppm represent amide (-NH) protons. The spectral data confirm that glycine and alanine dominate the silk structure because these amino acids stabilize β-sheet conformations that deliver silk fibroin's impressive mechanical properties, including endurance and flexibility. 3.2. Sialic acid-conjugated silk fibroin nanocarrier ATR-FTIR and NMR spectral analysis confirmed sialic acid conjugation with silk fibroin protein protein. ATR-FTIR spectrum (Fig. 3 (a)) of synthesized sialic acid-conjugated silk fibroin protein nanocarrier displays peaks at 3270 cm − 1 (amide band), 3083 cm − 1 (N-H stretching), 2375 cm − 1 (N = C = O bonding), 1625 cm − 1 (C = O bond), 1541 cm − 1 (N-O bonding), 1229 cm − 1 (C-H stretching), and 1062 cm − 1 (C-O- bending). The sialic acid-conjugated silk fibroin FTIR spectrum displayed two significant absorption peaks at 1625 cm − 1 (C = O bond) and 1541 cm − 1 (N-O bonding). These absorption peaks indicate a carboxylic acid group of sialic acid linked with an amino group of silk fibroin protein. Further, the NMR spectrum (Fig. 3 (b)) of Sialic acid conjugated with silk fibroin protein peaks, representing silk fibroin and sialic acid molecules, to verify their effective linkage. The spectrum of the protein displays peaks that correspond to both glycine (Gly) and alanine (Ala) residues found in silk fibroin, with β-carbon (-CH₂) and methyl (-CH₃) protons appearing between 1.0 and 2.5 ppm. The broad peaks within the 7.5–8.5 ppm range confirm the existence of amide (-NH) protons, which verify the presence of the peptide backbone. The successful silk fibroin glycosylation can be verified through peaks between 3.0–4.5 ppm that indicate hydroxyl (-OH) and anomeric protons of sialic acid. The signals observed between 8.02 ppm confirm the conjugation through the presence of the carboxyl (-COO⁻) group. The spectral data confirm that glycine and alanine dominate the silk structure because these amino acids stabilize β-sheet conformations that deliver silk fibroin's impressive mechanical properties, including endurance and flexibility. 3.3. Fabrication of SA-Epi-NA-SF-NPs and Epi-NA-SF-NPs Epirubicin and naringin-loaded sialic acid-conjugated silk fibroin nanoparticles were successfully synthesized using glutaraldehyde as a cross-linking agent. Dual medicines were gradually encapsulated into a sialic acid-conjugated silk fibroin nanocarrier, resulting in a transparent solution. The faint red appears after adding dual medicines to the sialic acid-conjugated silk fibroin nanocarrier. Later, the pale red faded to a reddish yellow, indicating that dual medicines had been encapsulated inside sialic acid-conjugated silk fibroin nanocarriers and produced nanoparticles. Epi and NA were efficiently encapsulated and loaded into sialic acid-conjugated silk fibroin nanocarriers at 84 ± 0.2%, 82 ± 12%, and 8.23 ± 0.7%, 8.12 ± 13%, respectively. Similarly, Epi and NA-loaded silk fibroin nanoparticles were prepared and compared to SA-Epi-NA-SF-NPs. The desolvation cross-linking method was used to create Epi-NA-SF-NPs. The encapsulation efficiency and loading capacity of Epi and NA in SF protein were 83 ± 1.5%, 80 ± 12%, 8.34 ± 0.9%, and 8.16 ± 0.3%, respectively. 3.4. Characterization studies of SA-Epi-NA-SF-NPs 3.4.1. ATR-FTIR spectroscopy analysis FTIR spectral analysis was used to confirm the encapsulation of Epirubicin and Naringin in the SA-SF nanocarrier. The FTIR spectrums of epirubicin (Fig. 4 (a)), naringin (Fig. 4 (b)), Sialic acid (Fig. 4 (c)), Epi-NA-SF NPs (Fig. 4 (d)), and SA-Epi-NA-SF-NPs (Fig. 4 (e)) was presented. The observed IR spectra of SA-Epi-NA-SF-NPs displayed peaks at 3291 cm − 1 and 3083 cm − 1, indicating N-H stretching vibrations, 2937 cm − 1 indicating C-H stretching, 1625 cm − 1 indicating C = O bond, 1520 cm − 1 indicating Polyphenol skeletal (aromatic), 1437 cm − 1 O-H bending, 1229 cm − 1 CH 2 vibrations, 1062 cm − 1 indicating C-O stretching vibrations, and 833 cm − 1 indicating C-H bending vibrations of the aromatic ring. The SA-Epi-NA-SF-NPs spectra displayed multiple typical peaks similar to the Epirubicin, Naringin, silk fibroin protein, SA-silk fibroin protein, and Epi-NA-SF NPs spectra, with no shifts. The strength of peaks for Epi and NA was significantly lowered. This could be attributed to the hydrophilic environment and the encapsulating action of the cross-linking agent. The results confirmed the encapsulation of Epi and NA in SA-silk fibroin protein. 3.4.2. Dynamic Light Scattering (DLS) analysis DLS measurements were used to determine the intensity-weighted mean diameter (z-average) and zeta potential of SA-Epi-NA-SF-NPs and Epi-NA-SF-NPs, as shown in Figs. 4 (f), (g), (h), and (i), respectively. The mean diameter of SA-Epi-NA-SF-NPs was 659.2 nm, while Epi-NA-SF-NPs measured 413.1 nm. The zeta potential and polydispersity index of SA-Epi-NA-SF-NPs were 0.0238 mV and + 0.015, while Epi-NA-SF-NPs were − 1.13 mV and 0.0192, respectively. 3.4.3. X-ray diffraction ( XRD) analysis The physical nature of the produced NPs was validated by XRD analysis. The X-ray diffraction (XRD) spectrums of Epi-NA-SF NPs (Fig. 4 (j)) and SA-Epi-NA-SF-NPs (Fig. 4 (k)). The XRD spectra of SA-Epi-NA-SF-NPs revealed peaks at 2Ɵ conditions (22.5°, 28.74°, 33.74°, 48.32°, 57.08°, and 78.7°), demonstrating the crystalline character. The XRD spectra of Epi-NA-SF-NPs displayed large peaks at 2Ɵ condition at 20.8°, demonstrating their amorphous character. 3.4.4. X-ray photoelectron spectroscopy (XPS) analysis In Fig. 4 (l), XPS data of C1s, O1s, and N1s obtained as SA-Epi-NA-SF-NPs, the C1s signal shows that -C-C-/-C = C- group of sialic acid or silk fibroin protein of NPs at 290 eV, and the O1s peak at 547 eV would arise due to -C-O-H of silk fibroin protein or O = C- bonds of sialic acid. The N1s signal at 402 eV would come from the silk fibroin protein's C-N bond. 3.4.5. Field Emission Scanning Electron Microscopy ( FESEM) analysis The FESEM images (Figs. 5 (a), (b) and (c)) of the formed Epi-NA-SF NPs showed consistent 100–200 nm spherical forms with smooth surfaces. Further, the FESEM scans of SA-Epi-NA-SF-NPs indicated spherical particles ranging in size from 100 to 400 nm (Figs. 5 (d), (e) and (f)). 3.4.6. High-resolution transmission electron microscopy (HRTEM) analysis The smooth surface of Epi-NA-SF NPs may help to ensure the prolonged release of the loaded medications. SA-Epi-NA-SF NPs range in size from 100 to 300 nm, according to TEM images in Figs. 5 (g), (h), and (i); Epi and NA loading increased the size of the nanoparticles on conjugated sialic acid. 3.5. Drug release and release kinetics The release of epirubicin and naringin from SA-Epi-NA-SF-NPs was evaluated at 37 ± 1°C and 120 rpm using physiological buffer systems (acetate buffer pH 5.4 and phosphate buffer pH 7.4). A UV-Visible spectrophotometer was used to estimate the amount of Epirubicin and Naringin released from SA-Epi-NA-SF-NPs at predefined time intervals, and the results were expressed as a percentage of the total drug release. The encapsulated Epirubicin and Naringin from SA-conjugated NPs were released gradually and under control. SA-Epi-NA-SF-NPs were employed to release Epirubicin and Naringin for up to 40 h for each buffered system, with the highest percentage of the drug. As shown in Fig. 9 , epirubicin and naringin were released faster in the acetate buffer system at pH 5.4 than in the phosphate buffer at pH 7.4. Within 8 h, 26.6 ± 0.735% of Epirubicin and 23.50 ± 0.865% of Naringin were released at pH 7.4 of phosphate buffer, while 26.40 ± 0.611% of Epirubicin and 22.04 ± 0.867% of Naringin were released at pH 5.4 of acetate buffer. The release was steady and controlled. After 35 h, SA-Epi-NA-SF-NPs released a constant amount of Epi and NA (84.46 ± 1.29% and 70.99 ± 1.56%) at pH 5.4 acetate buffer and 7.4 phosphate buffer (81.83 ± 0.63% Epi and 68.75 ± 0.31% NA). The results showed that Epi and NA were released more significantly and efficiently from SA-Epi-NA-SF-NPs at pH 5.4 than at neutral pH 7.4. The dual drug release data were fitted into five basic kinetic models, as shown in Table 1 , to understand the drug release mechanism of SA-Epi-NA-SF-NPs better. The models were zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell. Each kinetic model had its release rate constant and regression coefficient (r 2 ) determined. The final r 2 value is closer to one, indicating a better fit or link between the two components. The first-order model r 2 values of pH 7.4, 0.9799 to 0.8712 (Epi), and 0.9774 to 0.8836 (NA), as well as pH 5.4, 0.9855 to 0.8894 (Epi), and 0.9786 to 0.8764 (NA), were greater than those of zero-order kinetics, according to the kinetic model analysis. The Higuchi model has kinetic r 2 values of 0.9817 (Epi), 0.9795 (NA) at pH 5.4, and 0.9617 (Epi), 0.9651 (NA) at pH 7.4. Because these numbers are so close to one, the models are expected to fit the data well. The Higuchi model was the most successful kinetic model, as evidenced by plots with high linearity and r 2 values ranging from 0.9817 (Epi), 0.9795 (NA) at pH 5.4 to 0.9617 (Epi), 0.9651 (NA), which predominantly suggested the diffusion process. The release kinetics of Epirubicin and Naringin were designed to produce Fickian diffusion. The diffusion mechanism predominantly controls Epi and NA release from SA-Epi-NA-SF-NPs and exhibits Fickian diffusion behavior. According to this kinetic model, first-order kinetics best describe the manufactured SA-Epi-NA-SF-NPs. Thus, the kinetic model mechanism depicts the nanoparticles' homogeneous disintegration and controlled release. Table 1 Drug-release kinetics profile of Epirubicin and Naringin from SA-Epi-NA-SF NPs Model Parameter Epirubicin and Naringin from SA-Epi-NA-SF NPs pH 5.4 pH 7.4 Epi NA Epi NA Zero order F = K 0 ×t K 0 0.032 0.027 0.033 0.028 r 2 adjusted 0.8712 0.8836 0.8894 0.8764 AIC 157.1008 148.9293 157.2013 152.7947 First order F = 100× [1-Exp (-k 1 ×t)] K 1 0.001 0.000 0.001 0.000 r 2 adjusted 0.9799 0.9774 0.9855 0.9786 AIC 119.9511 116.1182 116.5326 117.7297 Higuchi model F = K H ×t 1/2 K h 1.504 1.259 1.548 1.321 r 2 adjusted 0.9817 0.9795 0.9617 0.9651 AIC 118.0838 114.2066 135.9847 127.5186 Korsmeyer-Peppas model F = kKP×t n kKP 1.280 0.711 0.695 0.683 r 2 adjusted 0.9863 0.9850 0.9715 0.9717 n 0.523 0.576 0.606 0.587 AIC 114.0347 108.8952 131.0335 124.2223 Hixon-Crowell model F = 100×[1-(1-kHC×t) 3 ] kHC 0.000 0.000 0.000 0.000 r 2 adjusted 0.9670 0.9593 0.9794 0.9611 AIC 129.8932 127.9032 123.6243 129.6726 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. 3.6. Anticancer activity of SA-Epi-NA-SF-NPs 3.6.1. Cytotoxicity Cytotoxicity experiments revealed that epirubicin, naringin, Epi-NA-SF-NPs, and various concentrations of SA-Epi-NA-SF-NPs reduced the cellular viability of A549 cells in a concentration-dependent manner after 24 h of treatment. At the same time, SA-Epi-NA-SF-NPs had no cytotoxic effects on L929 cells (Fig. 7 (f)-7(g). Figure 7 shows the proportion of viable A549 cells after 24 h of treatment with IC 50 concentration of SA-Epi-NA-SF-NPs (Fig. 7 (b)), 10 µg×mL − 1 epirubicin (Fig. 7 (c)), 100 µg×mL − 1 naringin (Fig. 7 (d)), 100 µg×mL − 1 Epi-NA-SF-NPs (Fig. 7 (e)) and L929 cells with IC 50 concentration of SA-Epi-NA-SF-NPs (Fig. 7 (g)). Compared to epirubicin, naringin, and Epi-NA-SF-NPs, the observed cytotoxicity assay revealed that SA-Epi-NA-SF-NPs dramatically reduced the cellular viability of A549 cells. A 100 µg×mL − 1 of SA-Epi-NA-SF-NPs showed 42.82 ± 3.44% cellular viability. Similarly, 100 µg×mL − 1 of NA, 10 µg/mL of Epi, and 100 µg×mL − 1 of Epi-NA-SF-NPs increased A549 cell viability by 65.83 ± 2.44%, 53.08 ± 3.26%, and 71.07 ± 2.12%, respectively. Furthermore, the results showed that the treated SA-Epi-NA-SF-NPs considerably reduced cell proliferation compared to the control (Fig. 7 (a)). Figure 7 (h) showed that inhibition of SA-Epi-NA-SF-NPs was concentration-dependent. The IC 50 concentration of SA-Epi-NA-SF-NPs (13.16 µg×mL − 1 ) was selected for future investigation. 3.6.2. Scratch assay As seen in Figs. 8 (a)-8(d), the rate of cell migration was significantly reduced in the SA-Epi-NA-SF-NPs-treated cells. The migration rates of A549 cells in the absence of SA-Epi-NA-SF-NPs at 24, 48, and 72 h were 57.14%, 73.47%, and 80.61%, respectively. Meanwhile, cell migration rates (Figs. 8 (e)-8(h)) in the presence of ½ IC 50 concentration (7.5 µg×mL − 1 ) of SA-Epi-NA-SF NPs were 12.22%, 55.56%, and 57.78% at 24, 48, and 72 h, respectively. 3.6.3. AO/EtBr staining Apoptosis and necrosis were analyzed qualitatively using AO/EtBr dual staining. As seen in Fig. 9 (b), the IC 50 concentration of the SA-Epi-NA-SF-NPs-treated group had a large population of reddish cells, indicating cell death. Furthermore, cells treated with the IC 50 concentration of SA-Epi-NA-SF-NPs showed cell membrane blebbing and chromatin condensation, which are linked with early death (Fig. 9 (c)). Furthermore, reddish cells show AO binding to denatured DNA during late apoptosis. In comparison, untreated cells were green and had a normal shape (Fig. 12(a)). This finding suggests that the A549 cells were exposed to higher epirubicin and naringin concentrations. 3.6.4. Calcein AM staining To corroborate SA-Epi-NA-SF-NPs cytotoxicity on A549 cancer cells, Calcein AM staining (Live/Dead cell assay) was performed to verify cell death due to SA-Epi-NA-SF-NPs. A 24-h exposure to the IC 50 concentration of SA-Epi-NA-SF-NPs depleted A549 cells, resulting in severe and positive cytotoxicity (Fig. 9 (e) and (f)). There was no cell death in drug-free A549 cells (Fig. 9 (d)). Altogether, these results confirm the results of the MTT and AO/EtBr staining assays. 3.6.5. Hoechst staining The nuclear alterations caused by SA-Epi-NA-SF-NPs in A549 cells were evaluated using Hoechst 33342 staining. Fluorescence pictures revealed that cells treated with SA-Epi-NA-SF-NPs at the IC 50 concentration had constricted chromatin, fragmented nuclei, and intense blue fluorescence, indicating the production of apoptotic bodies (Fig. 9 (h) and (i)). In contrast, untreated cells seemed normal, with spherical nuclei and mild blue fluorescence (Fig. 9 (g)). These findings significantly indicate the successful induction of apoptosis in A549 cells by SA-Epi-NA-SF-NPs treatment. 3.6.6. Effect of SA-Epi-NA-SF NPs on A549 cells' mitochondrial morphology Another potential cytotoxic effect mechanism investigated was mitochondrial damage. Mitochondria were stained with MitoTracker Red after 24 h of exposure to the IC 50 concentration of SA-Epi-NA-SF-NPs. Compared to the control (Fig. 9 (j)), the SA-Epi-NA-SF NPs damaged the morphology and dispersion of mitochondria in A459 cells (Fig. 9 (k) and (l)). 4. Discussion In cancer treatment, the synergistic combination of conventional chemotherapy with compounds originating from plants is thought to be a viable way to get around unwanted toxicity and drug resistance. By creating and utilizing a biocompatible co-delivery system that can hold two or more drugs, transport and deliver them to the intended locations, the synergistic therapeutic effect of the drugs can be further improved [ 60 ]. According to earlier research, polymeric nanocarriers that may co-deliver hydrophobic medicinal compounds demonstrated improved anticancer efficacy and enhanced bioavailability [ 61 , 62 ]. Protein and polypeptide nanocarriers are being extensively studied for the site-specific delivery of therapeutic drugs into cancer cells [ 63 ]. Moreover, protein-based nanocarrier systems are biocompatible, nontoxic, and biodegradable [ 64 ]. To improve drug delivery to the tumor microenvironment while minimizing damage to healthy tissues, protein nanocarriers contain several amino and carboxyl groups that are readily linked to targeted ligands that recognize particular receptors overexpressed on the plasma membrane of cancer cells [ 65 ]. Doxorubicin-loaded magnetic silk fibroin nanoparticles are a nanoscale drug delivery device for chemotherapy in multidrug-resistant malignancies facilitated by a magnetic field [ 66 ]. The present study provides significant evidence for using sialic acid receptor-targeted epirubicin and naringin-loaded sialic acid conjugated-silk fibroin nanoparticles for enhanced anticancer activities against lung cancer cells. Around 100 µg×mL − 1 of naringin, 10 µg×mL − 1 of epirubicin, and 100 µg×mL − 1 of Epi-NA-SF-NPs reduced A549 cell viability by 65.83 ± 2.44%, 53.08 ± 3.26%, and 71.07 ± 2.12%, respectively. While sialic acid-conjugated Epi-NA-SF-NPs (100 µg/mL) significantly reduced cellular viability by 42.82 ± 3.44%. At the same time, no cytotoxic effects are observed in L929 cells. The potential cytotoxic effects of SA-Epi-NA-SF-NPs might be that SA conjugation is recognized, targets overexpressed sialic acid receptors, and intracellularly delivers loaded drugs into lung cancer cells rather than healthy cells. Silk protein, especially silk fibroin (SF), is a promising drug delivery vehicle owing to its exceptional biocompatibility, biodegradability, minimal immunological reaction, and unique characteristics that facilitate its integration with diverse therapeutic agents [ 67 ]. SF matrices have been found to deliver anticancer drugs, enzymes, and antibodies effectively [ 68 ]. The current study uses silk fibroin as a drug-delivery carrier that creates nanoparticles that effectively co-deliver two drugs (epirubicin and naringin) into cancer cells. Further, the surface of the SF nanoparticles was altered with sialic acid to target overexpressed sialic acid receptors. This method is especially promising for lung cancer treatment because of the distinctive "hypersialylation" in lung cancer cells [ 69 ]. The biological membrane of tumor cells contains abundant sialic acid, which is mimicked to target its receptors actively [ 70 ]. Sialic acids are primarily present at the terminal extremities of glycoproteins and glycolipids, and they serve essential functions in cellular communication and function [ 71 ]. Before dual drug-loaded SF nanoparticles was fabricated, sialic acid-conjugated silk fibroin nanocarriers was produced utilizing standard EDC-conjugation chemistry. FTIR and NMR spectra show that the carboxylic acid group of sialic acid is linked to an amino group of SF protein. These sialic acid-conjugated nanoparticles can revolutionize lung cancer treatment by boosting bioavailability, decreasing hepatic first-pass metabolism, facilitating drug endocytosis, and modifying molecular pathways of cell signalling at a specific site [ 72 ]. Active targeting involves recognizing overexpressed receptors while imitating the abundant sugars on cancer cells [ 73 ]. Treatment of cancer cells with a therapeutic drug embedded in a natural protein polymeric assembly, which is fully equipped with a sugar mimetic, protects against degradation, modulates the release profile, and increases therapeutic efficacy with a lower frequency of delivery [ 74 ]. Following active recognition, the attached biopolymer is fabricated into nanoparticles, which passively treat cancer cells via enhanced permeability and retention (EPR) [ 75 ]. The efficacy of EPR-based tumor tropic accumulation is also highly controlled by nanoparticle physicochemical parameters (size, shape, surface features, and biocompatibility), as well as the physiological characteristics of the tumor and its microenvironment [ 76 ]. The drug's and polymer's structure, composition, and interaction significantly influence the drug release rate and mechanism in vitro [ 77 ]. The generated SA-Epi-NA-SF-NPs were assessed based on their morphological characteristics, including size, shape, physical state, surface chemical compositions, and zeta potential, utilizing XRD, particle size analyzer, XPS, FE-SEM, and HR-TEM techniques. The X-ray diffraction revealed that the synthesized SA-Epi-NA-SF-NPs exhibited a crystalline structure. Drug nanocrystals typically consist of core drug particles and a few stabilizers, resulting in a high % loading capacity of about 100%. Additionally, it may be feasible to eradicate the detrimental side effects of the encapsulating/solubilizing excipients. The dimensions and morphology of drug-loaded nanoparticles are critical determinants of their anticancer efficacy, as they profoundly influence cellular uptake, tumor accumulation, circulation time, and drug release, thereby impacting treatment effectiveness. Numerous studies indicate that the optimal size of nanoparticles for EPR-mediated tumor targeting ranges from 50 to 200 nm in diameter [ 78 – 81 ]. Moreover, unlike spherical nanoparticles, those exhibiting low surface curvature and high aspect ratios, such as rod, discoidal, or worm-shaped nanoparticles, demonstrate enhanced phagocytosis resistance, extending their circulation duration and facilitating tumour development [ 82 ]. Upon examination by FE-SEM and HR-TEM, the synthesized SA-Epi-NA-SF-NPs exhibited a mainly spherical morphology, with particle sizes ranging from 100 to 400 nm, a finding corroborated by DLS analysis. Moreover, spherically shaped nanoparticles exhibited the greatest internalization within cancer cells compared to nanoparticles of varying geometries. Likewise, spherical nanoparticles provide a greater capacity for medication encapsulation, hence diminishing cancer cell survival [ 83 ]. Nanoparticle-mediated delivery methods were primarily utilized in cancer treatment. Zeta potential is an essential tool for evaluating the stability of nanoparticles in a colloidal condition [ 84 ]. Dispersed nanoparticles are likely to resist one another if their zeta potential is strong, whether positive or negative [ 85 ]. The surface charge of nanoparticles significantly influences their capacity to adhere to cell membranes. The synthesized SA-Epi-NA-SF-NPs displayed a zeta potential of + 0.015 mV, indicating enhanced stability. In vitro drug release studies were evaluated for the formulated nanoparticles' quality, safety, and efficacy. They are also used to assess formulation characteristics and production processes. These studies provide indirect measurements of drug availability in the early stages of development. The current study was performed to release epirubicin and naringin from SA-Epi-NA-SF-NPs at two different pH buffer solutions (i) Check if epirubicin and naringin have been properly encapsulated (ii) Examine the kinetics and mechanism of dual drugs (Epi and NA) released from SA-Epi-NA-SF-NPs (iii) Determine the best pH for maximum epirubicin and naringin release from SA-Epi-NA-SF-NPs. The observed results demonstrated that the highest contents of drugs (Epi and NA) were released in the acetic pH medium (pH 5.4). Further in-depth in vivo studies were required to confirm the same. Table 1 displays the observed release data from the five drug release kinetics models that release Epi and NA from SA-Epi-NA-SF-NPs. Based on the results above, it was determined that SA-Epi-NA-SF NPs released 84.46 ± 1.29% of Epi and 79.99 ± 1.6% of NA in 35 h after an acidic (pH 5.4) medium. Compared to healthy tissues, solid tumor tissues have a somewhat higher pH (between 5 and 6.5). This work found that the SA-Epi-NA-SF-NPs in a pH 5.4 medium smoothly release naringin and epirubicin, simulating a tumor microenvironment. Therefore, the fabricated SA-Epi-NA-SF-NPs may be a significant anticancer system. The cytotoxic potential of formulated SA-Epi-NA-SF-NPs was assessed against lung cancer cells (A549 cells) and mouse fibroblast cells (L929 cells) via MTT assay. Freshly formulated SA-Epi-NA-SF-NPs showed significant cytotoxic efficacy against A549 cells, and the observed IC 50 value was 13.16 µg/mL. At the same time, no cytotoxicity was observed against fibroblast cells (L929 cells); 200 µg/mL of SA-Epi-NA-SF-NPs displayed 73.05 ± 3.26% cell viability observed after 24 h exposure. The results of this study were further compared with 10 µg×mL − 1 Epirubicin, 100 µg×mL − 1 Naringin, and 100 µg/mL Epi-NA-SF-NPs, showing 53.08 ± 3.26%, 65.83 ± 2.44%, and 71.07 ± 2.12% cytotoxicity against A549 cells, respectively. Around 100 µg×mL − 1 of fabricated SA-Epi-NA-SF-NPs significantly inhibited (42.82 ± 3.44%) of A549 cells. This potential cytotoxic effect might be sialic acid conjugation in the dual drug-loaded silk fibroin nanoparticles, which deliver drugs into cancer cells. Similarly, Shunyao Zhu and colleagues explored a novel approach that involved the creation of sialic acid (SA)-modified liposomes, known as CA-DOX-SAL, that encapsulated both chlorogenic acid (CA) and doxorubicin (DOX) for the treatment of tumor, as part of immunochemotherapy. The direct cytotoxic effect of DOX on tumor cells was strategically employed. Simultaneously, CA targeted tumor-associated macrophages (TAMs) within the tumor microenvironment, playing a critical role in phenotypic reversal. Introducing SA modification to the liposomes was a necessary enhancement, significantly increasing cellular uptake and enabling superior drug accumulation within tumors in vivo contexts [ 86 ]. Specific signalling molecules initiate apoptosis that is activated by the mitochondrial-mediated intrinsic pathway. When the permeability of the mitochondrial membrane changes, cytochrome c is released from the mitochondria into the cytosol [ 87 ]. If the apoptotic signal is received, caspase-9 is the first to be activated, followed by caspase-3, -6, and − 7. B-cell Lymphoma (Bcl) family proteins and results ultimately regulate this process in the cleavage of apoptosis-related protein substrates [ 88 ]. Thus, the double-staining (AO/EB) assay has been used to evaluate the impact of SA-Epi-NA-SF-NPs on apoptotic/cell death in the MCF-7 cell lines. Around 13.16 µg×mL − 1 (IC 50 concentration) of SA-Epi-NA-SF-NPs leads to apoptotic cell death. Similarly, MCF-7 cells treated with CDK-4/6 inhibitor-loaded 4-carboxyphenyl boronic acid-linked pH-sensitive chitosan lecithin nanoparticles displayed typical apoptotic nuclei morphological alterations, such as nuclear condensation, enhanced brightness, and nuclear crinkling [ 89 ]. The effect of SA-Epi-NA-SF-NPs on the migration rate of A549 cell lines was evaluated after 24, 48, and 72 h. Compared to the control group, cells exposed to ½ IC 50 exhibited a notable reduction in wound closure rates, recorded at 57.78% after 72 h, whereas the control group achieved 80.61%. This discovery indicates that the produced SA-Epi-NA-SF-NPs display significant anti-migration properties. SA-Epi-NA-SF NPs may augment cellular internalization and facilitate the delivery of therapeutics nearer to the intracellular site of action. Additionally, by fluorescence, SA-Epi-NA-SF-NPs labelled with Hoechst and Mito tracker Red were employed to examine cancer cell uptake and the intercellular distribution of the nucleus and mitochondria. The observed data demonstrate blue fluorescence from Hoechst's nuclear stain and red fluorescence from the Mitotracker Red stain. These findings indicate that SA, NPs and medicines may effectively eradicate cancer cells. SA-Epi-NA-SF-NPs illustrated the localization of encapsulated drugs into the nucleus and mitochondria of the treated cells. Nonetheless, additional research is required to elucidate the precise mechanism of toxicity of the drug-loaded nanoparticles on cancer cells. Nevertheless, a comprehensive study is needed to explain the mechanism of SA-Epi-NA-SF-NPs concerning cellular uptake, intracellular trafficking, and the cytotoxic mechanism of the dual medicines administered by this system. 5. Conclusions In conclusion, a combination therapy of epirubicin and naringin-loaded sialic acid-conjugated silk fibroin nanoparticles was successfully developed for site-specific delivery into lung cancer cells. Combining naringin with epirubicin improves therapeutic efficacy by minimizing toxicity, synergizing, reversing resistance, and lowering the dose of epirubicin because of the poor solubility and lack of targeting efficacy of naringin and epirubicin encapsulated in silk fibroin protein nanoparticles. The sialic acid-conjugated silk fibroin nanocarrier effectively encapsulated epirubicin and naringin, with encapsulation efficiency and loading capacity of 83 ± 1.5% (Epi), 80 ± 12% (NA), and 8.34 ± 0.9% (Epi), 8.16 ± 0.3% (NA), respectively. The surface of the manufactured nanoparticles was modified with sialic acid to target malignant cells for medication delivery via increased sialic acid receptors. The drug release behaviour revealed that SA-Epi-NA-SF-NPs exhibited an extended and controlled release pattern. The largest concentrations of both medicines were released after 35 h at pH 5.4, compared to pH 7.4. FTIR, XRD, XPS, particle size analyzer, FE-SEM, and HR-TEM evaluated the physicochemical properties of the manufactured SA-Epi-NA-SF NP. SA-Epi-NA-SF NPs were monodispersed, 100–400 nm spherically shaped, and contained crystalline particles. The surface of SA-Epi-NA-SF-NPs was validated by the chemical composition of their C1, O1, and N1s. SA-Epi-NA-SF-NPs showed substantial cytotoxicity against A549 cell lines, inducing apoptosis at 13.16 µg/mL compared to Epi-NA-SF-NPs, free-epirubicin, and naringin. SA-Epi-NA-SF-NPs could transport epirubicin and naringin to A549 cells by recognizing overexpressed sialic acid receptors on their plasma membrane and facilitating endocytosis. The potential benefits of SA-Epi-NA-SF-NPs have opened up a new path for the safe and targeted killing of lung cancer cells by improving anticancer efficacy. Furthermore, in vivo animal investigations will be needed to further understand the molecular pathways driving cancer cell death caused by SA-Epi-NA-SF-NPs. Declarations Contribution statement Selvaraj Kunjiappan : Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation, Supervision. Murugesan Sankaranarayanan : Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. Parasuraman Pavadai : Writing – original draft, Methodology, Investigation, Formal analysis. Ethics declaration The authors declare no competing interests Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment We want to extend our heartfelt gratitude to the management and administration of Alliance University, Anekal, Bengaluru, India, for all the essential support. References Kratzer TB, Bandi P, Freedman ND, Smith RA, Travis WD, Jemal A, Siegel RL (2024) Lung cancer statistics, 2023. Cancer 130(8):1330–1348. https://doi.org/10.1002/cncr.35128 Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Sialic acid-conjugated silk fibroin protein nanocarrier (insert structure)\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7970614/v1/e3a658bdc65716cd63aad994.jpg"},{"id":96248809,"identity":"85529f95-acb9-431c-8008-eb419758cc84","added_by":"auto","created_at":"2025-11-19 07:29:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2213317,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of epirubicin (a), naringin(b), Sialic acid(c), Epi-NA-SF-NPs (d) and SA-Epi-NA-SF-NPs (e); DLS measurement of Z-average particle size of Epi-NA-SF-NPs (f) and Z-average particle size of SA-Epi-NA-SF-NPs (g), zeta potential of Epi-NA-SF-NPs (h) and zeta potential of SA-Epi-NA-SF-NPs (i); X-ray diffraction (XRD) pattern of Epi-NA-SF-NPs (j) and SA-Epi-NA-SF-NPs (k); XPS spectrum of SA-Epi-NA-SF-NPs\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7970614/v1/f553b302b023eb27ac977a34.jpg"},{"id":96108969,"identity":"b7e8a39e-2ad6-446e-a92a-e5da9707cedb","added_by":"auto","created_at":"2025-11-17 16:49:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9192686,"visible":true,"origin":"","legend":"\u003cp\u003eDetailed morphological size and shape analysis of SA-Epi-NA-SF-NPs. 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Control cells at 0 (a), 24 (b), 48 (c) and 72 (d) hours’ time intervals, and treated cells at 0 (e), 24 (f), 48 (g) and 72 (h) hours’ time intervals\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7970614/v1/951d4d14eb3d9bbb872a8fa1.jpg"},{"id":96108998,"identity":"44ae234f-95e9-4583-9e95-18be25add561","added_by":"auto","created_at":"2025-11-17 16:49:10","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":17207352,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of apoptotic morphological variation by double staining (AO/EtBr) control cells (untreated) (a), A549 cells treated with SA-Epi-NA-SF NPs for 24 h (b) and merged (c). Apoptotic effects of SA-Epi-NA-SF NPs measured by Calcine AM staining in A549 cancer cells, control cells (untreated) (d), treated cells (e) and merged (f). Hoechst 33342-stained image of A549 cancer cells. Control cells (g), treatment with SA-Epi-NA-SF-NPs (h) and merged (i). Chromatin fragmentation is shown with arrows. Effects of SA-Epi-NA-SF-NPs on mitochondria targeting in A549 cancer cells, control cells (untreated) (j), damaged mitochondria by Mito tracker Red stain on treated cells (k) and merged (l)\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7970614/v1/e64d0b4210204603fa8d0698.jpg"},{"id":106808835,"identity":"855a6426-3480-4bda-b1ec-7c87078d060d","added_by":"auto","created_at":"2026-04-13 16:03:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":51831102,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7970614/v1/9475fc80-9267-47e8-b42a-d1b2cbb09b10.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sialic acid-receptor targeted Epirubicin and Naringin-loaded sialic acid-conjugated silk fibroin nanoparticles for enhanced lung cancer treatment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSignificant progress has been made in developing innovative therapeutic medicines and cancer detection and treatment methods. However, cancer remains a life-threatening condition, with the number of cases continually increasing. In terms of incidence and mortality, lung cancer ranks as the second leading cause of cancer worldwide, and the overall survival rate remains extremely poor [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Lung cancer was the most commonly diagnosed malignancy, making up about one out of every eight malignancies globally and over 2.5\u0026nbsp;million new cases, according to the GLOBOCAN 2022 report [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Cigarette smoking has been shown to raise the risk of acquiring lung cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, familial disease clustering and segregation analyses suggest that genetic predisposition may potentially play a role in the development of lung cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Lung cancer can currently be treated with surgery, chemotherapy, radiation, laser therapy, and combinations of these [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Chemotherapy is the best, cost-effective, and widely accepted active treatment option for lung cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Epirubicin (4'-epidoxorubicin), an Adriamycin analogue used to treat lung cancer, is one of the most often prescribed drugs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. At the same time, Epirubicin is non-specific in targeting cancer cells and has substantial side effects, which limit its use [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As a result, it is recommended to combine it with other medicines to reduce its dosage while maintaining its potency.\u003c/p\u003e\u003cp\u003eMany studies have recommended that combination therapy has a high potential for treating lung cancer [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Chemotherapy coupled with plant-derived compounds considerably decreases chemotherapy's adverse effects and increases its anticancer potency [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Some natural chemicals may respond to conventional cytotoxic therapy, raise the medication's effective concentration, reverse drug resistance, improve the combined action of both administered drugs, or be cytotoxic to cancer cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Patients tolerate many plant-derived compounds well and do not experience severe adverse effects, even at large doses [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The interplay of conventional chemotherapeutics and natural chemicals brings up new avenues for cancer research and treatment possibilities. In this view, Naringin combined with Epirubicin may reduce its toxicity and enhance its efficacy. Naringin (4\u0026prime;,5,7-Trihydroxyflavanone-7-rhamnoglucoside) is a plant-derived compound found in grapes, oranges, and Kino [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Numerous studies have shown that naringin reduces cell proliferation, migration, and invasion while increasing apoptosis of cancer cells in \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e cancer models, indicating significant anticancer effects on various human cancers, including lung cancer [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Unfortunately, epirubicin not only targets malignant cells but also healthy cells, which is one of the reasons malignant cells become resistant to active therapy [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, non-selective targeting, low aqueous solubility, and non-targetable drug distribution resulted in low bioavailability at the target site and drug resistance [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As a result, effective drug delivery systems are required to deliver drugs selectively, overcome drug resistance, and improve bioavailability at the target site [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this scenario, various protein nanocarrier systems employ the most auspicious ways of delivering anticancer drugs into cancer cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Protein nanocarriers are nano-sized particles that carry drugs and deliver intracellularly into cancer cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Protein nanocarriers have numerous advantages, including biocompatibility, biodegradability, non-immunogenicity, loading a wide range of pharmaceuticals, excellent endocytosis efficiency, and controlled release of loaded entities [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Protein nanocarriers can increase blood plasma circulation and aggressively target malignant cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The surface of these nano-sized carrier systems can be easily modified by targeting moieties to improve cancer therapy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Because of its biocompatibility, silk fibroin protein (SF) is one of the proteins most often employed for nanocarrier production in various biomedical applications [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Nanoparticles (NPs) were made using SF obtained from the cocoons of the silkworm \u003cem\u003eBombyx mori\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Silk fibroin nanoparticles (SF-NPs) efficiently deliver enzymes, nuclear materials, genetic materials, antibodies, and drugs into the target site [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. SF contains numerous active amino, carboxyl and thiol groups that aid in conjugating different macromolecules [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Attaching or conjugating targeting molecules to SF addresses the nanoparticles' fundamental weaknesses by enhancing their selectivity for cell surface adhesion [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The ligands attached to nanoparticles easily bind to naturally upregulated receptor molecules on cancer cell surfaces and regulate bidirectional flux. Loaded drugs are also activated intracellularly following ligand cleavage [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe versatile SF-NPs can dynamically target lung cancer cells by altering the amino groups on their surfaces with ligands that bind to overexpressed surface receptors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Combined with the SF-NPs, the ligand quickly recognizes the naturally overexpressed receptors on cancer cell surfaces [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Bidirectional flow occurs following ligand-receptor contact on the plasma membrane surface of cancer cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Designed nanoparticles are coupled with ligands for specific drug delivery to cancer cells, primarily targeting mitochondria and the nucleus. Cancer cells require abundant nutrients, including folic acid, amino acids, vitamins, and carbohydrates, to support aberrant development and uncontrolled cell multiplication [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Sialic acids (SAs) are nine-carbon carboxylated monosaccharides in cell surface glycoproteins and glycolipids. They tightly attach to the sialic acid receptor [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In recent years, sialic acid-binding proteins have been discovered, notably sialic acid-binding immunoglobulin-like lectins (Siglecs), also identified as sialic acid adhesins, which play an essential role in macrophages' pro-inflammatory responses [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Some studies have discovered that Siglecs are highly expressed on the surface of tumor-associated macrophages in mammals. Most Siglecs are endocytic receptors, allowing cytotoxic drugs or immunological modulators to be delivered to target cells by targeting Siglecs. These findings suggest that changing SAs on carriers to increase the formulation targeting is a promising cancer treatment strategy [\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It is a critical component of both stealth and targeting ligands. The overall goal of this study was to develop a new drug delivery system with SAs-conjugated dual drugs (Epi and NA)-loaded SF-NPs for targeting sialic acid receptors to effectively deliver the loaded entities into lung cancer cells (A549 cells) and evaluate their anticancer potency.\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials and Methods\u003c/h2\u003e\u003cp\u003eThe silk cocoon of \u003cem\u003eBombyx mori\u003c/em\u003e was sourced from Mulberry farms, Hosur, Krishnagiri District, Tamil Nadu, India. Epirubicin (Epi), sialic acid (\u003cem\u003eN\u003c/em\u003e-acetylneuraminic acid) (SA), naringin (NA), lithium bromide, sodium carbonate, \u003cem\u003eN\u003c/em\u003e-hydroxysuccinimide (NHS), \u003cem\u003eN\u003c/em\u003e-(3-dimethyl aminopropyl)-\u003cem\u003eN\u0026rsquo;\u003c/em\u003e-ethyl carbodiimide hydrochloride (EDC) are procured from Sigma Aldrich, and Sisco Research Laboratories (SRL) Pvt. Ltd., Mumbai, India. Cell culture medium, staining, and other reagents were obtained from Gibco Laboratories, Thermo Fisher Scientific, and Sigma Aldrich Ltd., Mumbai, India.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Silk fibroin (SF) protein\u003c/h2\u003e\u003cp\u003eDried \u003cem\u003eBombyx mori\u003c/em\u003e silk cocoons were fragmented into small pieces and degummed using a 0.02M sodium carbonate aqueous solution at 98\u0026deg;C for 30 min, with agitation. The whole mass was repeatedly washed with Milli-Q water to eliminate the adhesive sericin protein. After comprehensive washing with Milli-Q water and subsequent air-drying, the resultant dried product exhibited a luminous, white, cotton-like look. A silk fibroin solution was prepared by dissolving 10 g of degummed silk in a 9.30M lithium bromide solution at 60\u0026deg;C for 4 h. The silk fibroin solution underwent dialysis using a dialysis membrane-150 (molecular cutoff 12,400) against deionized water for three days, with water replacement every 6 h to remove lithium bromide. Post-dialysis, the silk fibroin solution underwent centrifugation at 5\u0026ndash;10\u0026deg;C and 9000 rpm for 20 min, resulting in 5% fibre concentration in a 100% volume. The concentrated solutions were stored at 4\u0026deg;C for subsequent analysis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. FTIR and NMR analyses validated the chemical characterization of the extracted silk fibroin protein. Attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy (Cary 630 FTIR Spectrometer, Agilent Technologies, USA) was used to analyze the functional groups of silk fibroin protein. The sample was analyzed in reflection mode, with results reported as the average of 64 repeated scans ranging from 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. \u003csup\u003e1\u003c/sup\u003eH-NMR spectroscopy was assessed to examine the type and number of hydrogens present in the proteins.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Fabrication of sialic acid-conjugated Epirubicin and Naringin-loaded Silk fibroin Nanoparticles (SA-Epi-Na-SF-NPs)\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Synthesis of Sialic acid-conjugated silk fibroin (SA-SF)\u003c/h2\u003e\u003cp\u003eThe sialic acid-conjugated silk fibroin nanocarrier was synthesized by simple EDC-conjugation chemistry. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e describes the sialic acid-conjugated silk fibroin synthesis. Briefly, 81.04 mg of sialic acid (\u003cem\u003eN\u003c/em\u003e-acetylneuraminic acid) and 61.01 mg of EDC were solubilized in 10 mL of Milli-Q water while magnetically stirring. Following 10\u0026ndash;15 min of stirring, 60.31 mg of \u003cem\u003eN\u003c/em\u003e-hydroxysuccinimide was incorporated into the mixture above, and the reaction mixture was agitated for 1\u0026ndash;2 h. The reaction mixture was subsequently filtered to eliminate O-acylisourea as a byproduct, resulting in a filtrate composed of sialic acid-\u003cem\u003eN\u003c/em\u003e-hydroxy succinimide. Subsequently, 200 mg of silk fibroin protein, pre-dissolved in 5 mL of acetate buffer (pH 4.7), was combined with sialic acid-\u003cem\u003eN\u003c/em\u003e-hydroxy succinimide and agitated overnight. The sialic acid-conjugated silk fibroin (SA-SF) mixture was subjected to dialysis using a dialysis membrane-150 against Milli-Q water to remove unreacted sialic acid. The structural and functional characteristics of the synthesized SA-SF nanocarrier were validated using FTIR and NMR spectroscopy. The \u003csup\u003e1\u003c/sup\u003eH-NMR spectrum was acquired using a Bruker 600 MHz AVANCE NMR spectrometer equipped with a TCI CryoProbe, with DMSO as the solvent and tetramethylsilane (TMS) as the internal standard.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Fabrication of Epirubicin and Naringin-loaded SA-SF nanoparticles (SA-Epi-NA-SF-NPs)\u003c/h2\u003e\u003cp\u003eThe newly synthesized SA-SF nanocarrier (1 g) was pre-dissolved in 2 mL DMSO within 50 mL of carbonate/bicarbonate buffer (0.1 M; pH 10.0) and agitated using a magnetic stirrer. Subsequently, a 5 mg mixture of Epi (3 mg) and NA (2 mg) in 5 mL of ethanol, prepared via sonication, was incrementally added to the SA-SF solution along with 0.1 mL of glutaraldehyde as a cross-linking agent. This mixture was incubated with continuous magnetic stirring for approximately 48 h to achieve a clear solution. The nanoparticles were created by gradually encapsulating the hydrophobic drugs epirubicin and naringin. Subsequently, to exclude non-encapsulated medicines, the mixture underwent centrifugation for 5 min at 1500 rpm and was rinsed three times with deionized water. The synthesized SA-Epi-NA-SF-NPs were lyophilized and preserved for further biological uses. The drug loading and encapsulation efficiency were calculated throughout the formulation process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3. \u003cb\u003eFormulation of Epirubicin and Naringin-loaded silk fibroin nanoparticles (Epi-NA-SF NPs)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eEpi-NA-SF NPs were generated for comparative analysis. Epi-NA-SF NPs were produced with a modified desolvation cross-linking technique derived from coacervation. In summary, 2.5 mg of the compounds (Epi at 1.5 mg and NA at 1 mg) were solubilized by sonication in 3 mL of ethanol. The drug-infused solution was gradually incorporated into silk fibroin protein (1 g), which had been pre-dissolved in 2 mL of cell culture-grade DMSO within 50 mL of 0.1 M carbonate/bicarbonate buffer (pH 10.0) and glutaraldehyde (0.1 mL) as a cross-linking agent and was subjected to continuous magnetic stirring for approximately 48 h to achieve a transparent solution. The NPs were created by gradually encapsulating the hydrophobic medicines (Epi and NA) within silk fibroin protein. Subsequently, to exclude non-encapsulated Epi and NA, the mixture underwent centrifugation for 5 min at 1500 rpm and was rinsed with deionized water three times. The solution was redispersed in 3.0 mL of deionized water.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Characterization studies of SA-Epi-NA-SF-NPs\u003c/h2\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. ATR-FTIR spectroscopy analysis\u003c/h2\u003e\u003cp\u003eFourier Transform Infrared (FT-IR) spectral data of the Epi, NA, Sialic acid, Epi-NA-SF-NPs, and SA-Epi-NA-SF-NPs were recorded by ATR-FTIR using a Cary 630 FTIR Spectrometer, Agilent Technologies, USA. All spectra were acquired with 64 scans per spectrum in the 4000\u0026thinsp;\u0026minus;\u0026thinsp;600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range at a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A background spectrum was captured before each measurement, with the same number of scans as the test sample. The spectra of the samples were acquired by positioning approximately 2 mg of the test sample atop the ATR crystal. The crystal was cleansed with isopropanol between each sample analysis and permitted to dry before the subsequent measurement. A pressure gauge applies force to the sample to ensure consistent contact between the sample and the crystal, facilitating optimal optical contact. The spectral bands 600\u0026ndash;1800 and 2600\u0026ndash;3800 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicated contributions from the biological component of the samples and were utilized for subsequent investigation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. Dynamic Light Scattering (DLS) analysis\u003c/h2\u003e\u003cp\u003eThe mean hydrodynamic diameter (Z-average), polydispersity index (PDI), and zeta potential of lyophilized Epi-NA-SF-NPs and SA-Epi-NA-SF-NPs were assessed via dynamic light scattering (DLS) utilizing a Zetasizer Nano ZS apparatus (Malvern Instruments Ltd., Worcestershire, UK). All measurements were conducted in Milli-Q water at 25\u0026deg;C and an angle of 173\u0026deg; to the source. Before the experiments, each sample underwent sonication for 3 min at 30% amplitude using 15-second ON/OFF intervals; the concentration of both NPs was 0.66 mg\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Measurements were conducted in triplicate, and data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3. \u003cb\u003eX-ray diffraction\u003c/b\u003e (\u003cb\u003eXRD) analysis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe XRD pattern was obtained using a Bruker D8 ADVANCE ECO XRD system with an SSD160 1-D Detector to ascertain the physical characteristics of Epi-NA-SF NPs and SA-Epi-NA-SF-NPs. The x-ray diffractometer functioned under the following parameters: utilizing Cu Kα 1 radiation (λ\u0026thinsp;=\u0026thinsp;0.1542) in a 2θ configuration at 20 keV and 30 mA current [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.4.4. X-ray photoelectron spectroscopy (XPS) analysis\u003c/h2\u003e\u003cp\u003eThe presence of elements on the surface of SA-Epi-NA-SF-NPs was analyzed from 0 to 800 eV using high-resolution X-ray photoelectron spectroscopy (XPS; Thermo Fisher Scientific, East Grinstead, UK) with an Al/Ka X-ray source (hv\u0026thinsp;=\u0026thinsp;1486.6 eV). The energy resolution was established at 0.1 eV with a pass energy of 1486.4 eV. The XPS spectra were examined utilizing CASA XPS software, which fitted the background using the Shirley function, removed it, and finally fitted the XPS peak with Gaussian-Lorentzian line shape functions [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.4.5. \u003cb\u003eField Emission Scanning Electron Microscopy\u003c/b\u003e (\u003cb\u003eFESEM) analysis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe morphological characteristics of Epi-NA-SF-NPs and SA-Epi-NA-SF-NPs were evaluated using FESEM. For the experiment, 1 mg of freeze-dried Epi-NA-SF-NPs and SA-Epi-NA-SF-NPs were dissolved in 1 mL of Milli-Q water, and 2 \u0026micro;L of the resultant mixture was applied to a clean glass surface. Upon drying the solution, a thin layer of gold coating was applied to mitigate electrostatic charge during scanning electron microscopy. The research utilized a Thermo Fisher Scientific APREO 2SEM, FESEM [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.4.6. High-resolution transmission electron microscopy (HRTEM) analysis\u003c/h2\u003e\u003cp\u003eThe dimensions and morphology of SA-Epi-NA-SF-NPs were examined via high-resolution transmission electron microscopy (HRTEM) utilizing a JEOL model 2100 equipment at an acceleration voltage of 200 kV, with lyophilized SA-Epi-NA-SF-NPs distributed in 1 mL of Milli-Q water. A few drops of redispersed SA-Epi-NA-SF NPs were deposited on a carbon-coated copper grid and air-dried to reveal the morphologically relevant aspects of the formed SA-Epi-NA-SF-NPs [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Drug release studies\u003c/h2\u003e\u003cp\u003eThe quantity of drug released \u003cem\u003ein vitro\u003c/em\u003e from SA-Epi-NA-SF-NPs at various time intervals was assessed as follows: 4 mg of dual drug-encapsulated nanoparticles were combined with 20 mL of acetate buffer (pH 5.4) and phosphate-buffered saline (PBS) (pH 7.4), and agitated at 120 rpm at 37\u0026deg;C. At specified intervals, 2 mL was collected and centrifuged at 9000 rpm for 10 min. The concentration of the pharmaceuticals in the supernatant was quantified by UV-Visible spectrometry at 435 nm for Epi and 285 nm for NA. The drug release percentage was quantified as the ratio of the predicted quantity of medication released at different time intervals to the initial amount contained within the nanoparticles. All experiments were conducted in triplicate, and the average value was reported [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Drug release kinetics studies\u003c/h2\u003e\u003cp\u003eLinear kinetic models were utilized to analyze the release data to determine the specific drug release kinetic profile and process [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The standard kinetic models for linear terms are as follows: The models employed include zero-order (cumulative drug release percentage versus time), first-order (logarithm of drug retention percentage versus time), Higuchi (cumulative drug release percentage versus the square root of time), Korsmeyer-Peppas (logarithm of drug released versus logarithm of time), and Hixson-Crowell (cube root of the amount of drug remaining in the dosage form versus time). \u003cem\u003eIn vitro\u003c/em\u003e drug release data were collected and analyzed to ascertain the kinetic process utilizing DD Solver 1.0, a Microsoft Excel add-in. The drug release kinetics of nanoparticle formulations are governed by the diffusional exponent \"n.\" The drug release and diffusion mechanism demonstrates a non-Fickian or erosion process when n equals 0.45 and 0.89. For n\u0026thinsp;=\u0026thinsp;0.45, the release mechanism is Fickian, whereas for n\u0026thinsp;=\u0026thinsp;0.89, it corresponds to Case II transport. If drug release and diffusion are negligible, 'n' values and kinetic data lack significance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Anticancer studies\u003c/h2\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e2.7.1. Cytotoxicity assay\u003c/h2\u003e\u003cp\u003eThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining assay was employed to assess the cytotoxic effects of SA-Epi-NA-SF-NPs on lung cancer cells (A549 cells) and fibroblast cells (L929 cells) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The cells were cultivated in 96-well plates at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well in a medium comprising 1% penicillin-streptomycin and 10% FBS, incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h. The seeded cells were subjected to various concentrations of SA-Epi-NA-SF-NPs (10, 20, 50, 100, 150, 200 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Epi-NA-SF NPs, 10 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Epi, and 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of NA. Following 24 h of incubation, the medium in each well was substituted with 100 \u0026micro;L of MTT solution (1 mg\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and incubated for 4 h at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Living cells generated formazan, which was solubilized with 100 \u0026micro;L of isopropanol and quantified at 570 nm with a Biotek Epoch microplate spectrophotometer (Agilent Technologies, USA). The cytotoxicity was assessed by comparing the absorbance of treated cells to that of untreated cells, which served as a control. Every experiment was conducted a minimum of three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e2.7.2. Cell migration study\u003c/h2\u003e\u003cp\u003eAn \u003cem\u003ein vitro\u003c/em\u003e scratch experiment was conducted to evaluate the efficacy of SA-Epi-NA-SF-NPs on cell-cell interactions and migration [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. A549 cells were cultivated on a 6-well culture plate at a density of 1.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well. Upon achieving 80\u0026ndash;90% confluency, a scratch was executed with a pointed instrument, such as a 10 \u0026micro;L pipette tip. The cells were subsequently washed with PBS to eliminate debris and treated with 7.5 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e SA-Epi-NA-SF-NPs (\u0026frac12; IC\u003csub\u003e50\u003c/sub\u003e concentration), and pictures of control and experimental wells were captured at 0, 24, 48, and 72 h. To ascertain the % change in wound diameter for all formulations, the wound distance was randomly measured at various points for each scratch in an individual well plate, and the average of these independent measurements was computed. The migration rate of A549 cells was determined using the following formula:\u003c/p\u003e\u003cp\u003eCell migration rate (%) = (wound area at the 0 h-wound area at 24, 48, or 72 h)/wound area at 0 h\u0026times;100\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e2.7.3. Detection of apoptosis\u003c/h2\u003e\u003cdiv id=\"Sec22\" class=\"Section4\"\u003e\u003ch2\u003e2.7.3.1. Acridine orange (AO)/Ethidium Bromide (EtBr) assay\u003c/h2\u003e\u003cp\u003eSA-Epi-NA-SF-NPs, subjected to AO/EtBr dual labeling, were examined via fluorescence microscopy to assess their apoptotic effects on A549 cells [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. A549 cancer cells were plated in a 12-well dish at a density of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated for 24 h. Following 24 h, the cells were administered an IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF NPs and cultured for 24 h. The treated cells were subsequently rinsed with 1\u0026times; PBS buffer and stained with a dual fluorescent solution comprising 10 \u0026micro;L of AO (10 mg\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and EtBr (10 mg\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), followed by a 30 min incubation period. Following incubation, unbound dyes were rinsed with 1x PBS buffer. The morphology of apoptotic cells was analyzed using a fluorescence microscope, and typical areas were documented at 40\u0026times; magnification. The dual AO/EB staining technique was conducted a minimum of three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section4\"\u003e\u003ch2\u003e2.7.3.2. Calcein-Acetyoxymethyl (AM) cytotoxicity assay\u003c/h2\u003e\u003cp\u003eThe calcein AM staining experiment evaluated the cytotoxicity of SA-Epi-NA-SF-NPs [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Cells were inoculated in 12-well plates at a density of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well overnight and subsequently treated with the IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF-NPs diluted in DMEM media. Following a 24-h incubation at 37\u0026deg;C, 50 \u0026micro;L of 0.25 \u0026micro;M Calcein AM was introduced to the control-treated well and incubated for 30 min. Following incubation, unbound dyes were rinsed with 1x PBS buffer. The morphology of apoptotic cells was examined microscopically, with representative areas documented at 40\u0026times; magnification. The calcein AM staining technique was performed a minimum of three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section4\"\u003e\u003ch2\u003e2.7.3.3. Hoechst assay\u003c/h2\u003e\u003cp\u003eThe Hoechst 33342 staining experiment identified the induction of apoptosis following treatment with SA-Epi-NA-SF-NPs [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. A549 cells were plated at a density of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well in a 12-well plate and incubated for 24 h to facilitate cell adherence. After incubation, cells were administered the IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF-NPs, followed by an additional 24-h incubation period. Cells were stained with 1 \u0026micro;g \u0026times; mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Hoechst 33342 (Invitrogen, Carlsbad, CA) for 1 min to counterstain the nuclei. After staining, the cells were rinsed and resuspended in 1x PBS before examination with an EVOS M 5000 imaging system (Thermo Fisher Scientific Inc., USA).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e2.7.4. Mitochondrial targeting\u003c/h2\u003e\u003cp\u003eA549 cells were cultured on a 12-well plate at a density of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. Following 12 h of attachment, cells were exposed to the IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF-NPs for an additional 12 h. After 12 h, mitochondria were labelled with 1 \u0026micro;M MitoTracker Red; the cells were subsequently washed and resuspended in 1x PBS, then scanned using an EVOS M 5000 imaging system (Thermo Fisher Scientific Inc., USA) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical outcomes from each experiment were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation for three independent repetitions. The distinction between the control and test samples was assessed utilizing the Student\u0026rsquo;s t-test unless specified otherwise in the legends.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Silk fibroin (SF)\u003c/h2\u003e\u003cp\u003eSF protein was effectively isolated from silk cocoons, and its structure was validated using ATR-FTIR and proton NMR spectroscopy. The observed ATR-FTIR spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a)) displayed significant peaks at 3291 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (amide A band), 3063 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 2937 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C-H stretching), 2363 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (O\u0026thinsp;=\u0026thinsp;C\u0026thinsp;=\u0026thinsp;O stretching), 1625 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (amide I band), 1513 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (amide II band), 1438 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (O-H bending), 1222 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (amide III band), and 1162 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C-O stretching). The \u0026sup1;H NMR spectrum of SF protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b)) verifies the prevalent amino acids as tyrosine (Tyr), glycine (Gly), serine (Ser), and alanine (Ala) because these amino acids constitute the backbone structure of silk fibroin protein. The proton signals from alanine methyl groups (-CH₃) range from 0.8 to 1.5 ppm, and the glycine and alanine β-protons (-CH₂) resonate between 2.0 and 2.5 ppm in the spectrum. The peaks within a 3.5\u0026ndash;4.5 ppm area detect the α-protons (-CH) of tyrosine, glycine, serine, and alanine, thus confirming their place in the peptide backbone. The fibroin structure contains robust peptide linkages because the broad peaks at 8.0 ppm represent amide (-NH) protons. The spectral data confirm that glycine and alanine dominate the silk structure because these amino acids stabilize β-sheet conformations that deliver silk fibroin's impressive mechanical properties, including endurance and flexibility.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Sialic acid-conjugated silk fibroin nanocarrier\u003c/h2\u003e\u003cp\u003eATR-FTIR and NMR spectral analysis confirmed sialic acid conjugation with silk fibroin protein protein. ATR-FTIR spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)) of synthesized sialic acid-conjugated silk fibroin protein nanocarrier displays peaks at 3270 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (amide band), 3083 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 2375 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N\u0026thinsp;=\u0026thinsp;C\u0026thinsp;=\u0026thinsp;O bonding), 1625 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O bond), 1541 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-O bonding), 1229 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C-H stretching), and 1062 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C-O- bending). The sialic acid-conjugated silk fibroin FTIR spectrum displayed two significant absorption peaks at 1625 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O bond) and 1541 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-O bonding). These absorption peaks indicate a carboxylic acid group of sialic acid linked with an amino group of silk fibroin protein. Further, the NMR spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b)) of Sialic acid conjugated with silk fibroin protein peaks, representing silk fibroin and sialic acid molecules, to verify their effective linkage. The spectrum of the protein displays peaks that correspond to both glycine (Gly) and alanine (Ala) residues found in silk fibroin, with β-carbon (-CH₂) and methyl (-CH₃) protons appearing between 1.0 and 2.5 ppm. The broad peaks within the 7.5\u0026ndash;8.5 ppm range confirm the existence of amide (-NH) protons, which verify the presence of the peptide backbone. The successful silk fibroin glycosylation can be verified through peaks between 3.0\u0026ndash;4.5 ppm that indicate hydroxyl (-OH) and anomeric protons of sialic acid. The signals observed between 8.02 ppm confirm the conjugation through the presence of the carboxyl (-COO⁻) group. The spectral data confirm that glycine and alanine dominate the silk structure because these amino acids stabilize β-sheet conformations that deliver silk fibroin's impressive mechanical properties, including endurance and flexibility.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Fabrication of SA-Epi-NA-SF-NPs and Epi-NA-SF-NPs\u003c/h2\u003e\u003cp\u003eEpirubicin and naringin-loaded sialic acid-conjugated silk fibroin nanoparticles were successfully synthesized using glutaraldehyde as a cross-linking agent. Dual medicines were gradually encapsulated into a sialic acid-conjugated silk fibroin nanocarrier, resulting in a transparent solution. The faint red appears after adding dual medicines to the sialic acid-conjugated silk fibroin nanocarrier. Later, the pale red faded to a reddish yellow, indicating that dual medicines had been encapsulated inside sialic acid-conjugated silk fibroin nanocarriers and produced nanoparticles. Epi and NA were efficiently encapsulated and loaded into sialic acid-conjugated silk fibroin nanocarriers at 84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2%, 82\u0026thinsp;\u0026plusmn;\u0026thinsp;12%, and 8.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%, 8.12\u0026thinsp;\u0026plusmn;\u0026thinsp;13%, respectively. Similarly, Epi and NA-loaded silk fibroin nanoparticles were prepared and compared to SA-Epi-NA-SF-NPs. The desolvation cross-linking method was used to create Epi-NA-SF-NPs. The encapsulation efficiency and loading capacity of Epi and NA in SF protein were 83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%, 80\u0026thinsp;\u0026plusmn;\u0026thinsp;12%, 8.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9%, and 8.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Characterization studies of SA-Epi-NA-SF-NPs\u003c/h2\u003e\u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1. ATR-FTIR spectroscopy analysis\u003c/h2\u003e\u003cp\u003eFTIR spectral analysis was used to confirm the encapsulation of Epirubicin and Naringin in the SA-SF nanocarrier. The FTIR spectrums of epirubicin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a)), naringin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b)), Sialic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(c)), Epi-NA-SF NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d)), and SA-Epi-NA-SF-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(e)) was presented. The observed IR spectra of SA-Epi-NA-SF-NPs displayed peaks at 3291 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3083 cm\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e indicating N-H stretching vibrations, 2937 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicating C-H stretching, 1625 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicating C\u0026thinsp;=\u0026thinsp;O bond, 1520 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicating Polyphenol skeletal (aromatic), 1437 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e O-H bending, 1229 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CH\u003csub\u003e2\u003c/sub\u003e vibrations, 1062 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicating C-O stretching vibrations, and 833 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicating C-H bending vibrations of the aromatic ring. The SA-Epi-NA-SF-NPs spectra displayed multiple typical peaks similar to the Epirubicin, Naringin, silk fibroin protein, SA-silk fibroin protein, and Epi-NA-SF NPs spectra, with no shifts. The strength of peaks for Epi and NA was significantly lowered. This could be attributed to the hydrophilic environment and the encapsulating action of the cross-linking agent. The results confirmed the encapsulation of Epi and NA in SA-silk fibroin protein.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2. Dynamic Light Scattering (DLS) analysis\u003c/h2\u003e\u003cp\u003eDLS measurements were used to determine the intensity-weighted mean diameter (z-average) and zeta potential of SA-Epi-NA-SF-NPs and Epi-NA-SF-NPs, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(f), (g), (h), and (i), respectively. The mean diameter of SA-Epi-NA-SF-NPs was 659.2 nm, while Epi-NA-SF-NPs measured 413.1 nm. The zeta potential and polydispersity index of SA-Epi-NA-SF-NPs were 0.0238 mV and +\u0026thinsp;0.015, while Epi-NA-SF-NPs were \u0026minus;\u0026thinsp;1.13 mV and 0.0192, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003e3.4.3. \u003cb\u003eX-ray diffraction\u003c/b\u003e (\u003cb\u003eXRD) analysis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe physical nature of the produced NPs was validated by XRD analysis. The X-ray diffraction (XRD) spectrums of Epi-NA-SF NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(j)) and SA-Epi-NA-SF-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(k)). The XRD spectra of SA-Epi-NA-SF-NPs revealed peaks at 2Ɵ conditions (22.5\u0026deg;, 28.74\u0026deg;, 33.74\u0026deg;, 48.32\u0026deg;, 57.08\u0026deg;, and 78.7\u0026deg;), demonstrating the crystalline character. The XRD spectra of Epi-NA-SF-NPs displayed large peaks at 2Ɵ condition at 20.8\u0026deg;, demonstrating their amorphous character.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section3\"\u003e\u003ch2\u003e3.4.4. X-ray photoelectron spectroscopy (XPS) analysis\u003c/h2\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (l), XPS data of C1s, O1s, and N1s obtained as SA-Epi-NA-SF-NPs, the C1s signal shows that -C-C-/-C\u0026thinsp;=\u0026thinsp;C- group of sialic acid or silk fibroin protein of NPs at 290 eV, and the O1s peak at 547 eV would arise due to -C-O-H of silk fibroin protein or O\u0026thinsp;=\u0026thinsp;C- bonds of sialic acid. The N1s signal at 402 eV would come from the silk fibroin protein's C-N bond.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec36\" class=\"Section3\"\u003e\u003ch2\u003e3.4.5. \u003cb\u003eField Emission Scanning Electron Microscopy\u003c/b\u003e (\u003cb\u003eFESEM) analysis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe FESEM images (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a), (b) and (c)) of the formed Epi-NA-SF NPs showed consistent 100\u0026ndash;200 nm spherical forms with smooth surfaces. Further, the FESEM scans of SA-Epi-NA-SF-NPs indicated spherical particles ranging in size from 100 to 400 nm (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(d), (e) and (f)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section3\"\u003e\u003ch2\u003e3.4.6. High-resolution transmission electron microscopy (HRTEM) analysis\u003c/h2\u003e\u003cp\u003eThe smooth surface of Epi-NA-SF NPs may help to ensure the prolonged release of the loaded medications. SA-Epi-NA-SF NPs range in size from 100 to 300 nm, according to TEM images in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (g), (h), and (i); Epi and NA loading increased the size of the nanoparticles on conjugated sialic acid.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec38\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Drug release and release kinetics\u003c/h2\u003e\u003cp\u003eThe release of epirubicin and naringin from SA-Epi-NA-SF-NPs was evaluated at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 120 rpm using physiological buffer systems (acetate buffer pH 5.4 and phosphate buffer pH 7.4). A UV-Visible spectrophotometer was used to estimate the amount of Epirubicin and Naringin released from SA-Epi-NA-SF-NPs at predefined time intervals, and the results were expressed as a percentage of the total drug release. The encapsulated Epirubicin and Naringin from SA-conjugated NPs were released gradually and under control. SA-Epi-NA-SF-NPs were employed to release Epirubicin and Naringin for up to 40 h for each buffered system, with the highest percentage of the drug. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e, epirubicin and naringin were released faster in the acetate buffer system at pH 5.4 than in the phosphate buffer at pH 7.4. Within 8 h, 26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.735% of Epirubicin and 23.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.865% of Naringin were released at pH 7.4 of phosphate buffer, while 26.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.611% of Epirubicin and 22.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.867% of Naringin were released at pH 5.4 of acetate buffer. The release was steady and controlled. After 35 h, SA-Epi-NA-SF-NPs released a constant amount of Epi and NA (84.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29% and 70.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56%) at pH 5.4 acetate buffer and 7.4 phosphate buffer (81.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63% Epi and 68.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31% NA). The results showed that Epi and NA were released more significantly and efficiently from SA-Epi-NA-SF-NPs at pH 5.4 than at neutral pH 7.4. The dual drug release data were fitted into five basic kinetic models, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, to understand the drug release mechanism of SA-Epi-NA-SF-NPs better. The models were zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell. Each kinetic model had its release rate constant and regression coefficient (r\u003csup\u003e2\u003c/sup\u003e) determined. The final r\u003csup\u003e2\u003c/sup\u003e value is closer to one, indicating a better fit or link between the two components. The first-order model r\u003csup\u003e2\u003c/sup\u003e values of pH 7.4, 0.9799 to 0.8712 (Epi), and 0.9774 to 0.8836 (NA), as well as pH 5.4, 0.9855 to 0.8894 (Epi), and 0.9786 to 0.8764 (NA), were greater than those of zero-order kinetics, according to the kinetic model analysis. The Higuchi model has kinetic r\u003csup\u003e2\u003c/sup\u003e values of 0.9817 (Epi), 0.9795 (NA) at pH 5.4, and 0.9617 (Epi), 0.9651 (NA) at pH 7.4. Because these numbers are so close to one, the models are expected to fit the data well. The Higuchi model was the most successful kinetic model, as evidenced by plots with high linearity and r\u003csup\u003e2\u003c/sup\u003e values ranging from 0.9817 (Epi), 0.9795 (NA) at pH 5.4 to 0.9617 (Epi), 0.9651 (NA), which predominantly suggested the diffusion process. The release kinetics of Epirubicin and Naringin were designed to produce Fickian diffusion. The diffusion mechanism predominantly controls Epi and NA release from SA-Epi-NA-SF-NPs and exhibits Fickian diffusion behavior. According to this kinetic model, first-order kinetics best describe the manufactured SA-Epi-NA-SF-NPs. Thus, the kinetic model mechanism depicts the nanoparticles' homogeneous disintegration and controlled release.\u003c/p\u003e\u003cp\u003e\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 Epirubicin and Naringin from SA-Epi-NA-SF NPs\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eModel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eEpirubicin and Naringin from SA-Epi-NA-SF NPs\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003epH 5.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003epH 7.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEpi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEpi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\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\u003e0.032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.033\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.028\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\u003e0.8712\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.8836\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.8894\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.8764\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\u003e157.1008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e148.9293\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e157.2013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e152.7947\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\u003e0.001\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\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\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\u003e0.9799\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.9774\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9855\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9786\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAIC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e119.9511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e116.1182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e116.5326\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e117.7297\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\u003e1.504\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.259\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.548\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.321\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\u003e0.9817\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.9795\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9617\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9651\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\u003e118.0838\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e114.2066\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e135.9847\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e127.5186\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\u003e1.280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.711\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.695\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.683\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\u003e0.9863\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.9850\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9715\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9717\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\u003e0.523\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.576\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.606\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.587\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\u003e114.0347\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e108.8952\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e131.0335\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e124.2223\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\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\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\u003e0.9670\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.9593\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9794\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9611\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAIC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e129.8932\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e127.9032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e123.6243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e129.6726\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\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=\"Sec39\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Anticancer activity of SA-Epi-NA-SF-NPs\u003c/h2\u003e\u003cdiv id=\"Sec40\" class=\"Section3\"\u003e\u003ch2\u003e3.6.1. Cytotoxicity\u003c/h2\u003e\u003cp\u003eCytotoxicity experiments revealed that epirubicin, naringin, Epi-NA-SF-NPs, and various concentrations of SA-Epi-NA-SF-NPs reduced the cellular viability of A549 cells in a concentration-dependent manner after 24 h of treatment. At the same time, SA-Epi-NA-SF-NPs had no cytotoxic effects on L929 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(f)-7(g). Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the proportion of viable A549 cells after 24 h of treatment with IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(b)), 10 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e epirubicin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(c)), 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e naringin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(d)), 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Epi-NA-SF-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(e)) and L929 cells with IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(g)). Compared to epirubicin, naringin, and Epi-NA-SF-NPs, the observed cytotoxicity assay revealed that SA-Epi-NA-SF-NPs dramatically reduced the cellular viability of A549 cells. A 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of SA-Epi-NA-SF-NPs showed 42.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44% cellular viability. Similarly, 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of NA, 10 \u0026micro;g/mL of Epi, and 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Epi-NA-SF-NPs increased A549 cell viability by 65.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44%, 53.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26%, and 71.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12%, respectively. Furthermore, the results showed that the treated SA-Epi-NA-SF-NPs considerably reduced cell proliferation compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(a)). Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(h) showed that inhibition of SA-Epi-NA-SF-NPs was concentration-dependent. The IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF-NPs (13.16 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was selected for future investigation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec41\" class=\"Section3\"\u003e\u003ch2\u003e3.6.2. Scratch assay\u003c/h2\u003e\u003cp\u003eAs seen in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e (a)-8(d), the rate of cell migration was significantly reduced in the SA-Epi-NA-SF-NPs-treated cells. The migration rates of A549 cells in the absence of SA-Epi-NA-SF-NPs at 24, 48, and 72 h were 57.14%, 73.47%, and 80.61%, respectively. Meanwhile, cell migration rates (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e(e)-8(h)) in the presence of \u0026frac12; IC\u003csub\u003e50\u003c/sub\u003e concentration (7.5 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of SA-Epi-NA-SF NPs were 12.22%, 55.56%, and 57.78% at 24, 48, and 72 h, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec42\" class=\"Section3\"\u003e\u003ch2\u003e3.6.3. AO/EtBr staining\u003c/h2\u003e\u003cp\u003eApoptosis and necrosis were analyzed qualitatively using AO/EtBr dual staining. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e(b), the IC\u003csub\u003e50\u003c/sub\u003e concentration of the SA-Epi-NA-SF-NPs-treated group had a large population of reddish cells, indicating cell death. Furthermore, cells treated with the IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF-NPs showed cell membrane blebbing and chromatin condensation, which are linked with early death (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e(c)). Furthermore, reddish cells show AO binding to denatured DNA during late apoptosis. In comparison, untreated cells were green and had a normal shape (Fig.\u0026nbsp;12(a)). This finding suggests that the A549 cells were exposed to higher epirubicin and naringin concentrations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec43\" class=\"Section3\"\u003e\u003ch2\u003e3.6.4. Calcein AM staining\u003c/h2\u003e\u003cp\u003eTo corroborate SA-Epi-NA-SF-NPs cytotoxicity on A549 cancer cells, Calcein AM staining (Live/Dead cell assay) was performed to verify cell death due to SA-Epi-NA-SF-NPs. A 24-h exposure to the IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF-NPs depleted A549 cells, resulting in severe and positive cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e (e) and (f)). There was no cell death in drug-free A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e (d)). Altogether, these results confirm the results of the MTT and AO/EtBr staining assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec44\" class=\"Section3\"\u003e\u003ch2\u003e3.6.5. Hoechst staining\u003c/h2\u003e\u003cp\u003eThe nuclear alterations caused by SA-Epi-NA-SF-NPs in A549 cells were evaluated using Hoechst 33342 staining. Fluorescence pictures revealed that cells treated with SA-Epi-NA-SF-NPs at the IC\u003csub\u003e50\u003c/sub\u003e concentration had constricted chromatin, fragmented nuclei, and intense blue fluorescence, indicating the production of apoptotic bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e(h) and (i)). In contrast, untreated cells seemed normal, with spherical nuclei and mild blue fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e(g)). These findings significantly indicate the successful induction of apoptosis in A549 cells by SA-Epi-NA-SF-NPs treatment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec45\" class=\"Section3\"\u003e\u003ch2\u003e3.6.6. Effect of SA-Epi-NA-SF NPs on A549 cells' mitochondrial morphology\u003c/h2\u003e\u003cp\u003eAnother potential cytotoxic effect mechanism investigated was mitochondrial damage. Mitochondria were stained with MitoTracker Red after 24 h of exposure to the IC\u003csub\u003e50\u003c/sub\u003e concentration of SA-Epi-NA-SF-NPs. Compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e(j)), the SA-Epi-NA-SF NPs damaged the morphology and dispersion of mitochondria in A459 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e(k) and (l)).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn cancer treatment, the synergistic combination of conventional chemotherapy with compounds originating from plants is thought to be a viable way to get around unwanted toxicity and drug resistance. By creating and utilizing a biocompatible co-delivery system that can hold two or more drugs, transport and deliver them to the intended locations, the synergistic therapeutic effect of the drugs can be further improved [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. According to earlier research, polymeric nanocarriers that may co-deliver hydrophobic medicinal compounds demonstrated improved anticancer efficacy and enhanced bioavailability [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Protein and polypeptide nanocarriers are being extensively studied for the site-specific delivery of therapeutic drugs into cancer cells [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Moreover, protein-based nanocarrier systems are biocompatible, nontoxic, and biodegradable [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. To improve drug delivery to the tumor microenvironment while minimizing damage to healthy tissues, protein nanocarriers contain several amino and carboxyl groups that are readily linked to targeted ligands that recognize particular receptors overexpressed on the plasma membrane of cancer cells [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Doxorubicin-loaded magnetic silk fibroin nanoparticles are a nanoscale drug delivery device for chemotherapy in multidrug-resistant malignancies facilitated by a magnetic field [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The present study provides significant evidence for using sialic acid receptor-targeted epirubicin and naringin-loaded sialic acid conjugated-silk fibroin nanoparticles for enhanced anticancer activities against lung cancer cells. Around 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of naringin, 10 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of epirubicin, and 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Epi-NA-SF-NPs reduced A549 cell viability by 65.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44%, 53.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26%, and 71.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12%, respectively. While sialic acid-conjugated Epi-NA-SF-NPs (100 \u0026micro;g/mL) significantly reduced cellular viability by 42.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44%. At the same time, no cytotoxic effects are observed in L929 cells. The potential cytotoxic effects of SA-Epi-NA-SF-NPs might be that SA conjugation is recognized, targets overexpressed sialic acid receptors, and intracellularly delivers loaded drugs into lung cancer cells rather than healthy cells.\u003c/p\u003e\u003cp\u003eSilk protein, especially silk fibroin (SF), is a promising drug delivery vehicle owing to its exceptional biocompatibility, biodegradability, minimal immunological reaction, and unique characteristics that facilitate its integration with diverse therapeutic agents [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. SF matrices have been found to deliver anticancer drugs, enzymes, and antibodies effectively [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The current study uses silk fibroin as a drug-delivery carrier that creates nanoparticles that effectively co-deliver two drugs (epirubicin and naringin) into cancer cells. Further, the surface of the SF nanoparticles was altered with sialic acid to target overexpressed sialic acid receptors. This method is especially promising for lung cancer treatment because of the distinctive \"hypersialylation\" in lung cancer cells [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The biological membrane of tumor cells contains abundant sialic acid, which is mimicked to target its receptors actively [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Sialic acids are primarily present at the terminal extremities of glycoproteins and glycolipids, and they serve essential functions in cellular communication and function [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Before dual drug-loaded SF nanoparticles was fabricated, sialic acid-conjugated silk fibroin nanocarriers was produced utilizing standard EDC-conjugation chemistry. FTIR and NMR spectra show that the carboxylic acid group of sialic acid is linked to an amino group of SF protein. These sialic acid-conjugated nanoparticles can revolutionize lung cancer treatment by boosting bioavailability, decreasing hepatic first-pass metabolism, facilitating drug endocytosis, and modifying molecular pathways of cell signalling at a specific site [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Active targeting involves recognizing overexpressed receptors while imitating the abundant sugars on cancer cells [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Treatment of cancer cells with a therapeutic drug embedded in a natural protein polymeric assembly, which is fully equipped with a sugar mimetic, protects against degradation, modulates the release profile, and increases therapeutic efficacy with a lower frequency of delivery [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Following active recognition, the attached biopolymer is fabricated into nanoparticles, which passively treat cancer cells via enhanced permeability and retention (EPR) [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe efficacy of EPR-based tumor tropic accumulation is also highly controlled by nanoparticle physicochemical parameters (size, shape, surface features, and biocompatibility), as well as the physiological characteristics of the tumor and its microenvironment [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. The drug's and polymer's structure, composition, and interaction significantly influence the drug release rate and mechanism \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The generated SA-Epi-NA-SF-NPs were assessed based on their morphological characteristics, including size, shape, physical state, surface chemical compositions, and zeta potential, utilizing XRD, particle size analyzer, XPS, FE-SEM, and HR-TEM techniques. The X-ray diffraction revealed that the synthesized SA-Epi-NA-SF-NPs exhibited a crystalline structure. Drug nanocrystals typically consist of core drug particles and a few stabilizers, resulting in a high % loading capacity of about 100%. Additionally, it may be feasible to eradicate the detrimental side effects of the encapsulating/solubilizing excipients. The dimensions and morphology of drug-loaded nanoparticles are critical determinants of their anticancer efficacy, as they profoundly influence cellular uptake, tumor accumulation, circulation time, and drug release, thereby impacting treatment effectiveness. Numerous studies indicate that the optimal size of nanoparticles for EPR-mediated tumor targeting ranges from 50 to 200 nm in diameter [\u003cspan additionalcitationids=\"CR79 CR80\" citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Moreover, unlike spherical nanoparticles, those exhibiting low surface curvature and high aspect ratios, such as rod, discoidal, or worm-shaped nanoparticles, demonstrate enhanced phagocytosis resistance, extending their circulation duration and facilitating tumour development [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Upon examination by FE-SEM and HR-TEM, the synthesized SA-Epi-NA-SF-NPs exhibited a mainly spherical morphology, with particle sizes ranging from 100 to 400 nm, a finding corroborated by DLS analysis. Moreover, spherically shaped nanoparticles exhibited the greatest internalization within cancer cells compared to nanoparticles of varying geometries. Likewise, spherical nanoparticles provide a greater capacity for medication encapsulation, hence diminishing cancer cell survival [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Nanoparticle-mediated delivery methods were primarily utilized in cancer treatment. Zeta potential is an essential tool for evaluating the stability of nanoparticles in a colloidal condition [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Dispersed nanoparticles are likely to resist one another if their zeta potential is strong, whether positive or negative [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. The surface charge of nanoparticles significantly influences their capacity to adhere to cell membranes. The synthesized SA-Epi-NA-SF-NPs displayed a zeta potential of +\u0026thinsp;0.015 mV, indicating enhanced stability. \u003cem\u003eIn vitro\u003c/em\u003e drug release studies were evaluated for the formulated nanoparticles' quality, safety, and efficacy. They are also used to assess formulation characteristics and production processes. These studies provide indirect measurements of drug availability in the early stages of development. The current study was performed to release epirubicin and naringin from SA-Epi-NA-SF-NPs at two different pH buffer solutions (i) Check if epirubicin and naringin have been properly encapsulated (ii) Examine the kinetics and mechanism of dual drugs (Epi and NA) released from SA-Epi-NA-SF-NPs (iii) Determine the best pH for maximum epirubicin and naringin release from SA-Epi-NA-SF-NPs. The observed results demonstrated that the highest contents of drugs (Epi and NA) were released in the acetic pH medium (pH 5.4). Further in-depth \u003cem\u003ein vivo\u003c/em\u003e studies were required to confirm the same. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays the observed release data from the five drug release kinetics models that release Epi and NA from SA-Epi-NA-SF-NPs. Based on the results above, it was determined that SA-Epi-NA-SF NPs released 84.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29% of Epi and 79.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% of NA in 35 h after an acidic (pH 5.4) medium. Compared to healthy tissues, solid tumor tissues have a somewhat higher pH (between 5 and 6.5). This work found that the SA-Epi-NA-SF-NPs in a pH 5.4 medium smoothly release naringin and epirubicin, simulating a tumor microenvironment. Therefore, the fabricated SA-Epi-NA-SF-NPs may be a significant anticancer system.\u003c/p\u003e\u003cp\u003eThe cytotoxic potential of formulated SA-Epi-NA-SF-NPs was assessed against lung cancer cells (A549 cells) and mouse fibroblast cells (L929 cells) via MTT assay. Freshly formulated SA-Epi-NA-SF-NPs showed significant cytotoxic efficacy against A549 cells, and the observed IC\u003csub\u003e50\u003c/sub\u003e value was 13.16 \u0026micro;g/mL. At the same time, no cytotoxicity was observed against fibroblast cells (L929 cells); 200 \u0026micro;g/mL of SA-Epi-NA-SF-NPs displayed 73.05\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26% cell viability observed after 24 h exposure. The results of this study were further compared with 10 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Epirubicin, 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Naringin, and 100 \u0026micro;g/mL Epi-NA-SF-NPs, showing 53.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26%, 65.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44%, and 71.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12% cytotoxicity against A549 cells, respectively. Around 100 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of fabricated SA-Epi-NA-SF-NPs significantly inhibited (42.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44%) of A549 cells. This potential cytotoxic effect might be sialic acid conjugation in the dual drug-loaded silk fibroin nanoparticles, which deliver drugs into cancer cells. Similarly, Shunyao Zhu and colleagues explored a novel approach that involved the creation of sialic acid (SA)-modified liposomes, known as CA-DOX-SAL, that encapsulated both chlorogenic acid (CA) and doxorubicin (DOX) for the treatment of tumor, as part of immunochemotherapy. The direct cytotoxic effect of DOX on tumor cells was strategically employed. Simultaneously, CA targeted tumor-associated macrophages (TAMs) within the tumor microenvironment, playing a critical role in phenotypic reversal. Introducing SA modification to the liposomes was a necessary enhancement, significantly increasing cellular uptake and enabling superior drug accumulation within tumors \u003cem\u003ein vivo\u003c/em\u003e contexts [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. Specific signalling molecules initiate apoptosis that is activated by the mitochondrial-mediated intrinsic pathway. When the permeability of the mitochondrial membrane changes, cytochrome c is released from the mitochondria into the cytosol [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. If the apoptotic signal is received, caspase-9 is the first to be activated, followed by caspase-3, -6, and \u0026minus;\u0026thinsp;7. B-cell Lymphoma (Bcl) family proteins and results ultimately regulate this process in the cleavage of apoptosis-related protein substrates [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. Thus, the double-staining (AO/EB) assay has been used to evaluate the impact of SA-Epi-NA-SF-NPs on apoptotic/cell death in the MCF-7 cell lines. Around 13.16 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (IC\u003csub\u003e50\u003c/sub\u003e concentration) of SA-Epi-NA-SF-NPs leads to apoptotic cell death. Similarly, MCF-7 cells treated with CDK-4/6 inhibitor-loaded 4-carboxyphenyl boronic acid-linked pH-sensitive chitosan lecithin nanoparticles displayed typical apoptotic nuclei morphological alterations, such as nuclear condensation, enhanced brightness, and nuclear crinkling [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. The effect of SA-Epi-NA-SF-NPs on the migration rate of A549 cell lines was evaluated after 24, 48, and 72 h. Compared to the control group, cells exposed to \u0026frac12; IC\u003csub\u003e50\u003c/sub\u003e exhibited a notable reduction in wound closure rates, recorded at 57.78% after 72 h, whereas the control group achieved 80.61%. This discovery indicates that the produced SA-Epi-NA-SF-NPs display significant anti-migration properties. SA-Epi-NA-SF NPs may augment cellular internalization and facilitate the delivery of therapeutics nearer to the intracellular site of action. Additionally, by fluorescence, SA-Epi-NA-SF-NPs labelled with Hoechst and Mito tracker Red were employed to examine cancer cell uptake and the intercellular distribution of the nucleus and mitochondria. The observed data demonstrate blue fluorescence from Hoechst's nuclear stain and red fluorescence from the Mitotracker Red stain. These findings indicate that SA, NPs and medicines may effectively eradicate cancer cells. SA-Epi-NA-SF-NPs illustrated the localization of encapsulated drugs into the nucleus and mitochondria of the treated cells. Nonetheless, additional research is required to elucidate the precise mechanism of toxicity of the drug-loaded nanoparticles on cancer cells. Nevertheless, a comprehensive study is needed to explain the mechanism of SA-Epi-NA-SF-NPs concerning cellular uptake, intracellular trafficking, and the cytotoxic mechanism of the dual medicines administered by this system.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, a combination therapy of epirubicin and naringin-loaded sialic acid-conjugated silk fibroin nanoparticles was successfully developed for site-specific delivery into lung cancer cells. Combining naringin with epirubicin improves therapeutic efficacy by minimizing toxicity, synergizing, reversing resistance, and lowering the dose of epirubicin because of the poor solubility and lack of targeting efficacy of naringin and epirubicin encapsulated in silk fibroin protein nanoparticles. The sialic acid-conjugated silk fibroin nanocarrier effectively encapsulated epirubicin and naringin, with encapsulation efficiency and loading capacity of 83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5% (Epi), 80\u0026thinsp;\u0026plusmn;\u0026thinsp;12% (NA), and 8.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9% (Epi), 8.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% (NA), respectively. The surface of the manufactured nanoparticles was modified with sialic acid to target malignant cells for medication delivery via increased sialic acid receptors. The drug release behaviour revealed that SA-Epi-NA-SF-NPs exhibited an extended and controlled release pattern. The largest concentrations of both medicines were released after 35 h at pH 5.4, compared to pH 7.4. FTIR, XRD, XPS, particle size analyzer, FE-SEM, and HR-TEM evaluated the physicochemical properties of the manufactured SA-Epi-NA-SF NP. SA-Epi-NA-SF NPs were monodispersed, 100\u0026ndash;400 nm spherically shaped, and contained crystalline particles. The surface of SA-Epi-NA-SF-NPs was validated by the chemical composition of their C1, O1, and N1s. SA-Epi-NA-SF-NPs showed substantial cytotoxicity against A549 cell lines, inducing apoptosis at 13.16 \u0026micro;g/mL compared to Epi-NA-SF-NPs, free-epirubicin, and naringin. SA-Epi-NA-SF-NPs could transport epirubicin and naringin to A549 cells by recognizing overexpressed sialic acid receptors on their plasma membrane and facilitating endocytosis. The potential benefits of SA-Epi-NA-SF-NPs have opened up a new path for the safe and targeted killing of lung cancer cells by improving anticancer efficacy. Furthermore, \u003cem\u003ein vivo\u003c/em\u003e animal investigations will be needed to further understand the molecular pathways driving cancer cell death caused by SA-Epi-NA-SF-NPs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelvaraj Kunjiappan\u003c/strong\u003e: Writing \u0026ndash; original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation, Supervision. \u003cstrong\u003eMurugesan Sankaranarayanan\u003c/strong\u003e: Writing \u0026ndash; original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. \u003cstrong\u003eParasuraman Pavadai\u003c/strong\u003e: Writing \u0026ndash; original draft, Methodology, Investigation, Formal analysis. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to extend our heartfelt gratitude to the management and administration of Alliance University, Anekal, Bengaluru, India, for all the essential support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKratzer TB, Bandi P, Freedman ND, Smith RA, Travis WD, Jemal A, Siegel RL (2024) Lung cancer statistics, 2023. 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Int J Biol Macromol 258:128821. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.diamond.2025.112001\u003c/span\u003e\u003cspan address=\"10.1016/j.diamond.2025.112001\" 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":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"A549 cells, Apoptosis, Drug delivery, Sialic acid receptor, Silk fibroin","lastPublishedDoi":"10.21203/rs.3.rs-7970614/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7970614/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLack of specificity, high burden of toxicity, and low bioavailability are the significant hurdles of conventional chemotherapies. Upregulated sialic acid receptors on the plasma membrane of lung cancer cells could be promising drug delivery targets for effective lung cancer treatment. In this view, the present study aimed to fabricate sialic acid (SA)-conjugated epirubicin (Epi) and naringin (NA)-loaded silk fibroin (SF) nanoparticles (SA-Epi-NA-SF-NPs) for selective delivery and enhanced lung cancer treatment. SF protein was initially extracted from silk cocoons, and the SA-conjugated SF was synthesized using simple EDC-conjugation chemistry. Later, the desolvation cross-linking technique was used to fabricate SA-Epi-NA-SF-NPs by encapsulating Epi and NA into an SA-conjugated SF. Various characterization methods were employed to confirm the physicochemical properties of SA-Epi-NA-SF-NPs. The fabricated SA-Epi-NA-SF-NPs ranged in size from 100 to 400 nm and had a spherical, crystalline nature. Epi and NA had encapsulation efficiency and loading capacity of 83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%, 80\u0026thinsp;\u0026plusmn;\u0026thinsp;12%, 8.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9%, and 8.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% into SA-conjugated SF, respectively. Drug release was substantially higher at pH 5.4 (84.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29% Epi and 70.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56% NA) than at pH 7.4. The cytotoxic potential of SA-Epi-NA-SF-NPs against A549 cells could diminish the viable number of cells after 24 h of treatment, and 13.16 \u0026micro;g\u0026times;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was observed as an IC\u003csub\u003e50\u003c/sub\u003e. The higher intracellular accumulation of Epi and NA in A549 cells targets mitochondria and the nucleus and causes apoptosis. Based on these outcomes, SA-Epi-NA-SF-NPs could have high therapeutic potential for lung cancer treatment, specifically targeting sialic acid receptors on A549 cells.\u003c/p\u003e","manuscriptTitle":"Sialic acid-receptor targeted Epirubicin and Naringin-loaded sialic acid-conjugated silk fibroin nanoparticles for enhanced lung cancer treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 16:49:04","doi":"10.21203/rs.3.rs-7970614/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-08T14:27:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T11:13:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-28T03:21:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-17T02:57:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-16T10:04:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298961888108013145915902924016738729498","date":"2025-11-09T16:22:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-09T07:30:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247849647781152778446662619490861779830","date":"2025-11-08T12:29:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52414092410097954140041182188094192938","date":"2025-11-07T02:50:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T17:46:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216139517640724196042610931429818059190","date":"2025-11-06T17:09:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104758015105441149424966490970822558144","date":"2025-11-06T15:02:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43840973092028674328597953407237629852","date":"2025-11-06T13:15:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T13:11:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18554155046355526319721873753743031765","date":"2025-11-06T12:58:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232479917318133414334970692285732180427","date":"2025-11-06T12:43:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-06T12:24:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-29T18:52:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-29T18:52:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Polymers and the Environment","date":"2025-10-28T07:29:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3492e6fd-e881-485e-a376-d9119a7c804b","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:00:58+00:00","versionOfRecord":{"articleIdentity":"rs-7970614","link":"https://doi.org/10.1007/s10924-026-03820-8","journal":{"identity":"journal-of-polymers-and-the-environment","isVorOnly":false,"title":"Journal of Polymers and the Environment"},"publishedOn":"2026-04-06 15:58:15","publishedOnDateReadable":"April 6th, 2026"},"versionCreatedAt":"2025-11-17 16:49:04","video":"","vorDoi":"10.1007/s10924-026-03820-8","vorDoiUrl":"https://doi.org/10.1007/s10924-026-03820-8","workflowStages":[]},"version":"v1","identity":"rs-7970614","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7970614","identity":"rs-7970614","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}