Smart co-delivery of Erlotinib and Camptothecin using silica-coated gold nanorods functionalized with recombinant anti-BMP receptor type AI

Smart co-delivery of Erlotinib and Camptothecin using silica-coated gold nanorods functionalized with recombinant anti-BMP receptor type AI | 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 Smart co-delivery of Erlotinib and Camptothecin using silica-coated gold nanorods functionalized with recombinant anti-BMP receptor type AI Fatemeh Sabzalizadeh, Hamed Mirshekari, Nediljko Budisa, Khosro Khajeh, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6709389/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Non-small cell lung carcinoma is a particularly aggressive cancer with a poor outlook. Although Erlotinib (ELT) and Camptothecin (CPT) are commonly used together in chemotherapy, their effectiveness is limited when administered as free drugs. To enhance their efficacy, we developed a novel nanomedicine consisting of gold nanorods (Au-NRs) coated with a functionalized silica network to deliver both drugs simultaneously. This strategy aims to improve cancer cell targeting, suppress cell proliferation, and induce apoptosis. The nanomedicine was further engineered with a recombinant anti-BMP receptor AI (BMPR-AI) single-chain variable fragment (scFv) fused with maltose-binding protein for targeted delivery. Successful coating and functionalization were confirmed through various analyses, including HR-TEM, EDS/EDAX, zeta potential measurements, and FT-IR. The resulting CPT/ELT/scFv@Au-NR nanomedicine effectively targeted BMPR-AI-overexpressing cancer cells, significantly inhibiting cell growth and inducing apoptosis more efficiently than the free drugs. This promising approach exhibits enhanced cytotoxic effects and holds the potential for more effective chemotherapy and future advancements in cancer treatment. Non-small cell lung cancer BMPR-AI Erlotinib Camptothecin gold nanorods Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Lung cancer ranks among the most widespread cancers globally and is the leading cause of cancer-related deaths, with 1.8 million people succumbing to the disease in 2020, according to the World Health Organization. Despite significant progress in cancer research, diagnosis, and treatment, the five-year survival rate for lung cancer has only improved by five percent over the past 20 years. Sadly, many patients face a grim prognosis, often passing away within the first year of diagnosis [ 1 ]. Lung cancer is divided into two main types: small-cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). While NSCLC is more common and generally grows slower, SCLC, though less prevalent, tends to be more aggressive. More than half of NSCLC cases (55%) are diagnosed at advanced stages, and only 20% of patients respond favorably to chemotherapy [ 1 ]. Nano-drug delivery systems harness the power of nanotechnology to achieve controlled drug release, enhanced cellular absorption, prolonged drug stability within cells and the bloodstream, minimized side effects, improved accessibility, biocompatibility, targeted delivery, reduced dosage requirements, controllable pharmacokinetics, and traceability [ 2 ]. These systems utilize various carriers, including liposomes, micelles, polymers, polysaccharides, self-assembled peptides, dendrimers, silica-based nanoparticles, bioactive glasses, hydrogels, carbon-based nanoparticles, metal nanoparticles, exosomes, and gold nanostructures [ 3 , 4 ]. Among these, gold nanostructures are increasingly used in medical applications as carriers for antigen delivery, vaccination, gene therapy, and other therapeutic targets [ 5 ]. Camptothecin (CPT), a potent antitumor drug originally sourced from Camptotheca acuminata , a tree native to Tibet and China and used in traditional Chinese medicine, inhibits the enzyme topoisomerase I (Topo I). It forms irreversible covalent complexes with DNA during replication, leading to DNA strand breaks and subsequent apoptosis [ 6 , 7 ]. Erlotinib (ELT) is a small-molecule tyrosine kinase inhibitor approved by the FDA for treating NSCLC and metastatic pancreatic cancer, often in combination with gemcitabine (U.S. Food and Drug Administration). It selectively binds to the adenosine triphosphate (ATP) binding sites of the epidermal growth factor receptor (EGFR), reversibly inhibiting EGFR activation and blocking downstream signaling pathways. This action reduces cell proliferation, angiogenesis, and metastasis [ 8 , 9 ]. Bone morphogenetic proteins BMP2 and BMP4 are crucial for embryo development and lung growth. Although BMP signaling diminishes after lung formation, it can be reactivated during inflammation and in lung cancer, playing a significant role in tumor development [ 10 ]. BMP2 expression is elevated in 98% of NSCLC patients and is linked to tumor spread in various other cancers [ 11 ]. Therefore, targeting BMP signaling presents a promising approach for treating NSCLC. Extracellular antagonists such as germline and noggin bind to BMP2 and BMP4, preventing their interaction with receptors [ 12 , 13 ]. Several generations of BMP receptor inhibitors have been developed, with DMH1 showing a strong affinity for the ACVR-1 receptor. In 2014, Hao et al. demonstrated that DMH1 effectively reduced cell division, induced cell death, and decreased cell migration in NSCLC [ 14 ]. In 2018, Newman introduced a new inhibitor targeting BMP type I and II receptors in NSCLC, which lowers the expression of Id1, XIAP, and TAK1 genes [ 15 ]. Additionally, Browning highlighted in 2018 that inhibiting BMPR-IA aids in differentiating helper T cells from CD4 + T cells [ 16 ]. Monoclonal antibodies (mAbs) are widely recognized as the gold standard for targeting tumor cells due to their exceptional specificity. However, full-size mAbs have several drawbacks, including their large size, complexity, and issues with post-translational modifications, which can limit their ability to penetrate cancer cells effectively. Consequently, there has been a move towards using smaller antibody fragments such as Fab, scFv, and VHH [ 17 ]. Among these, single-chain variable fragments (scFv) stand out for their compact size, low immunogenicity, and cost-effectiveness. scFv fragments are created from recombinant molecules where the variable regions of light (VL) and heavy (VH) chains are combined into a single polypeptide connected by a flexible linker [ 18 ]. Although scFv expression requires an oxidizing environment—such as the endoplasmic reticulum in eukaryotic cells or the periplasm in bacteria—this does not detract from their potential [ 19 , 20 ]. The E. coli system is a popular choice for producing recombinant proteins in both industrial and research settings. However, generating proteins with disulfide bonds or post-translational modifications like glycosylation in bacteria can be challenging. While producing full-sized antibodies in bacteria is problematic due to the lack of necessary post-translational modifications, many antibody fragments can be successfully produced in the cytoplasm or periplasm of engineered strains [ 21 ]. To improve the solubility and yield of these proteins, fusion partners such as glutathione S-transferase (GST), maltose-binding protein (MBP), small ubiquitin-like modifier (SUMO), and thioredoxin (Trx) are frequently used, particularly for scFv fragments [ 22 ]. To overcome the challenges associated with E. coli cytoplasmic expression, the CyDisCo system was developed. This system co-expresses disulfide bond formation catalysts, like Erv1p, DsbB, or VKOR, and disulfide bond isomerization catalysts, such as DsbC or PDI. The CyDisCo system has effectively produced scFv and Fab fragments from known antibodies within the E. coli cytoplasm [ 23 , 24 ]. In this study, we developed an advanced drug delivery system specifically designed to target lung cancer cells. This system utilizes silica-coated gold nanorods (SiO₂@Au-NRs) modified with anti-BMP receptor AI scFv fragments (scFv). The scFv, fused with maltose-binding protein (MBP-scFv), is produced using a co-expression system in E. coli BL21 (DE3). To facilitate the loading of MBP-scFv, the SiO₂@Au-NRs are coated with aminated maltose (NH₂-maltose). Additionally, Camptothecin (CPT) is covalently attached to the surface, and Erlotinib (ELT) is incorporated into the SiO₂@Au-NRs and modified with MBP-scFv to create a sophisticated targeting system (Fig. 1 ). Our study assesses this system's toxicity, apoptotic effects, and cellular uptake using cell assays and fluorescence microscopy on the A549 and MRC-5 cell lines. Early results suggest that co-delivery of ELT and CPT significantly enhances their therapeutic effectiveness by inhibiting cell proliferation and inducing apoptosis. Material and methods Materials, strains, and cell lines Gold (III) chloride trihydrate (HAuCl₄), cetyltrimethylammonium bromide (CTAB), L-ascorbic acid, 3-(mercaptopropyl)trimethoxysilane (MPTMS), 3-(triethoxysilyl)propylamine (APTMS), sodium borohydride (NaBH₄), and silver nitrate (AgNO₃) were sourced from Sigma-Aldrich in Germany. All experiments were conducted using double-distilled water. Fermentas, Lithuania, supplied restriction enzymes and T4 ligase. The helper plasmid pMJS205 was generously provided by Professor Lloyd Ruddock under a signed Material Transfer Agreement (MTA). E. coli BL21 (DE3) was obtained from Invitrogen (Thermo Fisher Scientific), while the pMAL-c2X plasmid was acquired from Addgene (#75286). Dulbecco's Modified Eagle's Medium (DMEM) high glucose, along with trypsin-EDTA, penicillin/streptomycin, and heat-inactivated fetal bovine serum (FBS), were purchased from Gibco®, USA. The human lung adenocarcinoma cell line (A549) and the normal human lung cell line (MRC-5) were procured from the Department of Cell Bank at the Pasteur Institute of Iran. Additionally, Camptothecin and Erlotinib were purchased from Alfa Aesar. Synthesis of CTAB-functionalized gold nanorods (CTAB@Au-NRs) Gold nanorods were synthesized using a seed-mediated growth method [ 25 ]. First, 125 µL of 10 mM HAuCl₄ was mixed with 3.75 mL of 100 mM CTAB to prepare the gold seed solution. Then, 300 µL of 10 mM NaBH₄, freshly prepared on ice, was added to the mixture. The solution was vigorously stirred for two minutes, resulting in a yellow-brown seed solution. To remove excess NaBH₄, the solution was allowed to stand at room temperature in the dark for 2 hours. For the growth solution, 2 mL of 10 mM HAuCl 4 was mixed with 47 mL of 100 mM CTAB in a Falcon tube. Following this, 300 µL of 10 mM AgNO 3 was added to control the aspect ratio of the gold nanorods. After thorough stirring, 320 µL of freshly prepared 100 mM ascorbic acid solution was slowly introduced, causing the solution to transition from yellow-brown to colorless. Immediately afterward, 320 µL of the gold seed solution was rapidly added, and the mixture was gently stirred for 20 seconds. Over the next 15 minutes, the solution gradually changed color and stabilized. The nanorods were allowed to grow overnight at 25°C without further stirring. Preparation of Silica-functionalized Au-NRs (SiO@Au-NRs) The procedure began with sonicating 4 mL of the gold nanorod suspension in a water bath for two minutes at 90 W and 50–60 kHz. Following this, the suspension was centrifuged at 4,000 x g for 5 minutes to remove impurities, and the resulting supernatant, which contained the purified gold nanorods, was collected. To coat the gold nanorods, the method described by Mirshekari et al. (2024) was used. First, to remove CTAB and other impurities, the nanorods (Au-NRs) were centrifuged at 12,000 x g for 20 minutes. The purified nanorods were then resuspended in 5 mL of deionized water, and the pH was adjusted to 4.0 using 100 mM hydrochloric acid. This suspension was mixed at 400 x g for 20 minutes. Subsequently, 41 µL of 100 mM MPTMS ethanol solution was added, stirring the mixture for 3 hours at the same speed. Afterward, 62 µL of a 20% APTMS ethanol solution was introduced, and the suspension was stirred for one hour at 800 x g. The pH was then adjusted to 10.0 using 100 mM sodium hydroxide, and the mixture was stirred at 800 x g for an additional 20 minutes. The suspension was then left to rest at room temperature for 20 hours. Finally, the suspension was centrifuged at 10,000 x g for 10 minutes, and the gold nanorods were washed three times with absolute ethanol and three times with deionized water. Amination of maltose (NH-Maltose) To start, 0.004 moles (1.37 g) of maltose and 0.004 moles (0.31 g) of NH₄HCO₃ were added to 20 mL of an aqueous ammonium solution. This mixture was refluxed at 42°C for 36 hours until the volume was reduced by half. Subsequently, ammonium hydroxide was separated from the maltose using a freeze dryer, and the aminated maltose was dried as described by Zhou et al. (2012). Carboxylation of SiO@Au-NRs (COOH@Au-NRs) A 10 mL ethanol solution of Au-NRs (OD ~ 1.0) was treated with 100 mM of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 100 mM of N-hydroxy succinimide (NHS). After the reaction, 100 mM of succinic anhydride was added to the solution, which was then stirred for three hours at + 4°C. Following this, the nanorods were washed three times with water and three times with ethanol to remove unreacted chemicals. Preparation of maltose-coated gold nanorods 100 mM NH₂-Maltose was added to 10 mL of an aqueous solution containing activated COOH@Au-NRs (OD ~ 1.0). The mixture was stirred for one hour at + 4°C. Following this, the nanorods were washed three times with ethanol and then three times with water. Camptothecin-conjugation on carboxylated gold nanorods surface To covalently attach CPT to the gold nanorod surfaces, a reaction mixture was prepared with 1 milliliter of COOH@Au-NRs (optical density ~ 1.0), 1 micromole of CPT, 1 micromole of triethylamine (TEA), and 2 micromoles of N,N'-dicyclohexylcarbodiimide (DCC). This mixture was incubated at room temperature for 24 hours. To deactivate any remaining carboxyl groups, 100 mM NH₂-maltose was introduced and allowed to react for one hour. Afterward, the gold nanorods were thoroughly washed three times with ethanol, followed by three washes with water. Drug loading evaluation Erlotinib was loaded onto both Mal@Au-NRs and CPT/Mal@Au-NRs using an absorption technique. First, the nanorods, with an optical density at the local surface plasmon resonance (OD LSPR ) of approximately 0.58, were suspended in Tris-HCl buffer (pH 7.4). Various Erlotinib concentrations were introduced into the nanorod suspension at 20-minute intervals while the mixture was continuously rotated. Afterward, the mixture was centrifuged three times to remove any unbound Erlotinib. The concentration of unbound Erlotinib in the supernatant was measured using UV–visible absorption spectroscopy at 333 nm (Supplementary Fig. 1), based on a calibration curve for Erlotinib. The specific loading content and efficiency of Erlotinib were subsequently calculated using the equations provided below: $$\:\text{E}\text{n}\text{c}\text{a}\text{p}\text{s}\text{u}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{e}\text{f}\text{f}\text{i}\text{c}\text{i}\text{e}\text{n}\text{c}y\:\%\:=\:\frac{drug\:initial\:weigth\:\left(mg\right)\:-\:unloaded\:drug\:in\:aqueous\:phase\:\left(mg\right)}{drug\:initial\:weigth\:\left(mg\right)}\times\:100$$ $$\:Loading\:capacity\:=\:\frac{drug\:initial\:weigth\:\left(mg\right)\:-\:unloaded\:drug\:in\:aqueous\:phase\:\left(mg\right)}{gold\:nanorods\:weigth\:\left(mg\right)}$$ In vitro drug release at different pH values For the in vitro release test, nanorods were dispersed in 1 mL of either acetate buffer (pH 5.0) or phosphate buffer (pH 7.4), each placed in separate dialysis bags (MW cutoff = 3.5 kDa). These bags were then immersed in 10 mL of the corresponding buffer solutions. At regular intervals, 0.5 mL samples were taken from the release medium, and absorbance was measured at 333 nm for Erlotinib (ELT) and 368 nm for Camptothecin (CPT). Blank buffer solutions of equal volume were used for calibration. All experiments were performed in triplicate, with average results reported. Synthesis and cloning of the scFv gene General Biosystems company synthesized the codon-optimized sequence of the scFv fragment, which was derived from the anti-BMP receptor AI Fab (PDB: 3NH7) and cloned in the pUC57-Amp cloning vector. The sequence comprises the light (V L ) and heavy (V H ) variable chains of the Fab fragment, linked by (G 4 S) 3 , with a histidine sequence at the C-terminal. The synthesized sequence was inserted into the pMAL-c2X vector using Bam HI and Hind III restriction sites to produce the recombinant fusion protein of MBP-scfv. Overexpression and purification of the MBP-scFv fusion protein The pMAL-scFv vector and the helper plasmid pMJS205 were co-transformed into E. coli BL21(DE3) cells. The LB medium and the ZYM5052 autoinduction system were employed to express the MBP-scFv fusion protein. A single bacterial colony was grown in an LB medium with ampicillin and chloramphenicol at appropriate concentrations, incubating at 37°C for 16 hours. Expression of the recombinant MBP-scFv fusion protein was induced by adding IPTG at concentrations of 20, 50, and 80 µM to the LB medium, while 0.2% lactose was used in the ZYM5052 autoinduction medium. This process was carried out at 25°C with shaking at 280 rpm for 24 hours. After incubation, the bacterial cultures were harvested by centrifugation, and the resulting cell pellets were resuspended in lysis buffer containing 20 mM Tris (pH 8.0). To purify the overexpressed MBP-scFv protein efficiently, the soluble fraction of the cell extract was passed through a Ni-NTA agarose column. Following purification, the MBP-scFv fusion protein was dialyzed against 20 mM Tris (pH 8.0). The sample’s homogeneity was finally confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% acrylamide gels. Immobilization of MBP-scFv fusion protein on nanocarrier The MBP-scFv fusion protein was successfully immobilized onto the surface of Mal@Au-NRs by incubating 200 ng of the fusion protein with 2 ml of Mal@Au-NRs (OD ≈ 1) at 4°C for 24 hours. To assess the effectiveness of this immobilization, the nanorod solution was centrifuged, and the supernatant was analyzed through absorbance measurements at 280 nm. Colloidal stability investigation of CPT/ELT/scFv@Au-NRs An aliquot of 100 microliters of CPT/ELT/scFv@Au-NRs was suspended in 500 microliters of 20 mM Tris-base, pH 7.4. The colloidal stability of the CPT/ELT/scFv@Au-NRs was assessed at four time points: 0, 15, 30, and 60 minutes. Cytotoxicity evaluation and synergistic effect To evaluate the formulations' cytotoxicity under controlled conditions, A549 and MRC-5 cells were plated in 96-well plates at densities of 8 × 10³ and 12 × 10³ cells per well, respectively. Each well contained 100 µl of DMEM high-glucose medium enriched with 10% FBS and necessary antibiotics. The cells were incubated at 37°C with 5% CO₂ in a humidified environment. After 16 hours, free drugs and various nanoformulations were introduced to the wells, and the incubation continued for 24, 48, and 72 hours. To determine cell viability, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay was performed. Cells were first washed with PBS buffer, then treated with 100 µl of MTT solution (0.025 mg/ml) and incubated in the dark at 37°C for 4 hours. Afterward, the medium was discarded, and 100 µl of DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm with a reference at 630 nm using a microplate reader. The Combination Index (CI) was calculated following the Chou-Talalay method, and isobologram analysis was conducted to assess the synergistic effects of the treatments. Cellular uptake The fluorescent microscopy technique was used to examine the absorption and distribution of the formulations within cells. Cells were initially seeded in 48-well plates at a density of 15,000 cells per well and incubated at 37°C for 24 hours. Following this incubation period, the cells were exposed to CPT@Au-NRs, CPT/ELT@Au-NRs, and CPT/ELT/scFv@Au-NRs in fresh culture medium for 2 hours at 37°C with 5% CO₂. Rhodamine was introduced to each well at a concentration of 40 µg/ml. After this treatment, the cells were thoroughly washed three times with PBS and then analyzed using a fluorescent microscope. Apoptosis assay Cell apoptosis was evaluated using the Annexin V-FITC/PI double staining kit. Initially, 10 5 cells were plated into each well of a 6-well plate and incubated overnight at 37°C. The following day, the cells were exposed to free drugs and various formulations for a duration of 72 hours. After treatment, the cells were collected and resuspended in 400 µL of 1× reconstitution buffer. To each tube, 5 µL of Annexin V-FITC was added, and the samples were incubated for 15 minutes in the dark at 4°C. Following this, 10 µL of propidium iodide (PI) was introduced to each tube, which was then incubated for another 5 minutes in the dark. The evaluation of apoptosis was conducted within 30 minutes after staining. Cell-based ELISA analysis MRC-5 and A549 cell lines were seeded into 96-well plates and incubated overnight at 37°C to allow a monolayer of cells to form at the bottom of each well. After incubation, the culture medium was removed, and the cells were gently rinsed with PBS. They were then fixed with 4% formaldehyde in PBS for 20 minutes and washed three times with PBS. To neutralize any residual fixative, the cells were treated with 0.1 M glycine in PBS for 30 minutes. Next, the cells were exposed to a 3% (w/v) hydrogen peroxide solution in PBS for 5 minutes to minimize endogenous peroxidase activity. Following two 5-minute washes with PBS, the cells were incubated in a blocking solution (5% fetal bovine serum and 1% BSA in PBS) for 30 minutes. After blocking, the cells were treated with MBP-scFv fusion protein for 1 hour, followed by three washes with the blocking solution, each lasting 3–5 minutes. Subsequently, the cells were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, diluted 1:3000 in the blocking solution, for 1 hour. After four washes with PBS (each lasting 4–5 minutes), an HRP substrate reagent (3,3',5,5'-tetramethylbenzidine enzyme substrate; 150 µl per well) was added and incubated for 10 minutes. The reaction was halted with 1 M HCl, and the absorbance of the samples was measured at 450 nm using a microplate reader [ 26 ]. A positive control was included by immobilizing 100 µg/ml of the recombinant fusion protein in a blank well. Nanocarrier characterization We obtained the UV-visible scattering spectrum using a spectrophotometer. To visualize the SiO₂@Au-NRs, we employed high-resolution transmission electron microscopy (HR-TEM) with a Tec9G20 model. The size distribution of the gold nanorods (Au-NRs) and the thickness of the silicon shell surrounding them were measured by analyzing at least 100 particles and 100 shells with ImageJ software. Elemental composition was determined using EDAX or EDS, while the gold concentration was quantified through inductively coupled plasma optical emission spectroscopy (ICP-OES). The surface charge of the gold nanorods was evaluated with the SZ-100 nanoparticle system (Nano ZS90 Zetasizer, Malvern Panalytical, Malvern, UK). Fourier-transform infrared (FT-IR) spectroscopy was performed on dried samples within the 400–4000 cm⁻¹ range. Additionally, proton nuclear magnetic resonance (¹H-NMR) spectra were recorded using a 400 MHz NMR spectrometer (Bruker, AVANCE™ III HD 400 MHz). Statistics analysis The data in this study were analyzed using GraphPad Prism software, version 8.3.0. Comparisons between groups were conducted using either one-way ANOVA with Dunnett’s test or two-way ANOVA with the Tukey test. Statistical significance was defined by p-values less than 0.05. Combination index (CI) values were calculated using CompuSyn software, version 1.0. Results and discussion Synthesis and characterization of SiO 2 @Au-NRs CTAB is a crucial surfactant used in gold nanorods (Au-NRs) synthesis, but its significant cytotoxicity limits the use of CTAB-capped nanorods in medical applications. Additionally, CTAB molecules create a positive charge on the surface of Au-NRs by forming a double layer, further complicating their use in biological settings. Therefore, to facilitate the use of Au-NRs in medical applications, it is necessary to either replace CTAB or apply a coating to the nanorods [ 27 ]. The strong interaction between CTAB and the Au-NR surface, coupled with CTAB’s role in stabilizing colloidal suspensions, makes replacing it through ligand exchange a challenging task. Early research on coating Au-NRs with silica focused on using silane coupling agents or polymers to improve the affinity between the gold and silica surfaces [ 28 ]. High-resolution transmission electron microscopy (HR-TEM) images confirm that the gold nanorods are effectively coated with silica. The synthesized gold nanorods show distinct length and width dimensions (Fig. 2 a). The average thickness of the silica coating, as observed in HR-TEM images of SiO₂@Au-NRs, is 2.08 ± 0.08 nm (Fig. 2 b). These SiO₂@Au-NRs are relatively uniform in size and shape, with an average length of 33.63 ± 5.38 nm, as depicted in Fig. 2 c. HR-TEM images also reveal that the gold nanorods grow along the {100} plane with an interplanar distance of 0.196 nm (Supplementary Fig. 2a). The single-crystal nature of the gold nanorods is supported by their selected area electron diffraction (SAED) patterns, which display bright circular rings corresponding to the crystalline lattice planes of the gold nanoparticles (Supplementary Fig. 2b). The composition of SiO₂@Au-NRs was analyzed using Energy-dispersive X-ray spectroscopy (EDS), as shown in Supplementary Fig. 3a. This analysis revealed the presence of gold (Au) in the SiO₂@Au-NRs and confirmed the presence of oxygen, silicon, and nitrogen on the surface of the gold nanorods. Point-scan analysis detected the elements O, Si, and Au in the SiO₂@Au-NRs, but nitrogen was not observed (Supplementary Fig. 3b). These EDS/EDAX findings were consistent with the results from the HR-TEM analysis. To investigate bond formation and interactions within the structure, we utilized Fourier transform infrared spectroscopy (FT-IR). This technique allows us to trace shifts, formations, or disappearances of spectral bands to specific interactions. As depicted in Fig. 3 a, the peak at 1589 cm⁻¹ corresponds to the N-H bending vibration of the amine group attached to maltose. The broad band observed between 3100 and 3600 cm⁻¹ is associated with the O-H stretching vibrations from hydroxyl groups present in the nanorods, maltose, and water [ 29 , 30 ]. A peak at 1429 cm⁻¹ signifies O-H groups, while the band at 1074 cm⁻¹ indicates C-O stretching in secondary alcohol [ 31 ]. Additionally, the peak at 2935 cm⁻¹ is linked to C-H stretching in the sp³ carbon of the maltose ring [ 32 ]. The presence of CTAB was confirmed by two bands at 2915 and 2896 cm⁻¹ (Fig. 3 b) [ 33 ]. Bands at 1487 cm⁻¹, 1473 cm⁻¹, 1462 cm⁻¹, and 1431 cm⁻¹ correspond to the symmetric and asymmetric C-H shear vibrations of the CH₃-N⁺ group. Significant differences emerged when comparing the spectra of CTAB@Au-NRs and SiO₂@Au-NRs. The peak at 1030 cm⁻¹ represents the asymmetric stretching vibrations of Si-O-Si [ 34 ]. Si-O-Si aromatic symmetric bending and O-Si-O stretching were confirmed at 455 cm⁻¹ and 791 cm⁻¹, respectively [ 34 , 35 ]. The peak at 1112 cm⁻¹ further indicated Si-O-Si vibrations [ 36 ]. A new vibrational mode observed at approximately 1630 cm⁻¹ corresponds to the N-H bending vibration of the amine group in SiO₂@Au-NRs [ 35 ] and the stretching vibration of the carbonyl group (C = O) in carboxyl and amide bonds [ 37 ]. Lastly, the peak at 3425 cm⁻¹ reflects the presence of N-H bonds associated with the amine group of APTMS and the hydroxyl group of carboxyl groups [ 38 ]. The surface charge of gold nanorods was assessed under various conditions using a Tris buffer at pH 7.4. Nanorods synthesized in a 1.6% cationic surfactant environment had a surface charge of about + 43 millivolts, leading to electrostatic repulsion between the particles. After applying silane grafting, the surface charge of the nanorods varied according to the coverage and functional groups of the silane. The charge of the coated nanorods was measured at -1.93 mV, which is due to the presence of amine groups from APTMS. Further modification through carboxylation of SiO₂@Au-NRs reduced the positive surface charge to -5.98 mV. Gold nanorods are widely used in biomedical fields such as drug delivery, cell imaging, and cancer therapy because of their strong plasmon resonance properties. Coating these nanorods with SiO₂ shells improves their stability and biocompatibility. In 2001, Murphy and colleagues successfully encapsulated high aspect ratio gold nanorods with a thin (5–10 nm) silica shell using MPTMS. This choice was based on sulfur’s strong affinity for gold, which helps displace the tightly bound CTAB molecules [ 39 ]. Silica coatings offer several advantages: they enhance colloidal and thermal stability, increase the surface area of the nanorods while preserving the gold core's optical properties, and allow for precise control over porosity. Additionally, silica improves the biocompatibility of gold nanorods. Its reactive surface silanols facilitate efficient drug loading and enable the attachment of functional ligands or biomolecules for targeted delivery [ 28 ]. For example, Zhou et al. developed a dual-targeted chemo-photo thermal therapy system with gold nanorods coated in mesoporous silica. This system showed a high photothermal effect and pH- and NIR-triggered drug release, highlighting its potential as an anticancer treatment [ 40 ]. Similarly, Gao et al. engineered folate-functionalized gold nanorods, demonstrating high biocompatibility and effective tumor cell uptake [ 41 ]. Synthesis and characterization of CPT@Au-NRs The creation of CPT@Au-NRs involves establishing an ester bond between CPT's hydroxyl group and the carboxyl groups on the surface of COOH@Au-NRs (Fig. 1 a). We employed 1 H-NMR spectroscopy and synthesized methyl hydrogen succinate to react with the gold nanorod surface to validate this esterification reaction. Peaks at 2.6 ppm and 3.6 ppm in the spectrum confirm the presence of succinic anhydride and the methylation of one carboxyl group (Supplementary Fig. 4a). The 1 H-NMR spectrum for CPT-methyl hydrogen succinate reveals peaks at 1.2 ppm and 1.91 ppm, associated with the CPT drug [ 42 ], while peaks at 3.06 ppm and 4.2 ppm are linked to the methyl hydrogen succinate chain (Supplementary Fig. 4b). These findings confirm the esterification reaction with an efficiency of 52%. After CPT was incorporated onto the SiO 2 @Au-NRs, the surface charge decreased to -19.5 mV, demonstrating successful CPT binding to the gold nanorod surface. Following the addition of NH 2 -maltose to block any remaining active groups, the surface charge further shifted to -8.66 mV (Fig. 3 c). In vitro release assessment of ELT The study evaluated the efficiency of loading ELT onto CPT/Mal@Au-NRs and Mal@Au-NRs to determine how much drug could be incorporated before the nanorods began to aggregate. We found that the encapsulation efficiency of ELT was 78.7% in CPT/Mal@Au-NRs and 72.6% in Mal@Au-NRs. At the same conditions, the drug loading was 4.2% for CPT/Mal@Au-NRs and 4.0% for Mal@Au-NRs. To test the effectiveness of this smart release system, we examined how ELT was released from CPT/ELT@Au-NRs and ELT/Mal@Au-NRs in environments that mimic physiological conditions. After 24 hours, the gold nanorods released 76.7% and 70% of ELT in a fluid that simulates intracellular conditions (pH 5.0). In contrast, the release of ELT in a fluid mimicking extracellular conditions (pH 7.4) was 60.8% for CPT/ELT@Au-NRs and 65% for ELT/Mal@Au-NRs. The release profiles in these simulated environments showed significant statistical differences (Fig. 3 d), and no CPT release was detected. The literature indicates that nanoparticles are usually taken up by endocytic vesicles within cells. While body fluids and extracellular environments typically have a pH of around 7.4, the pH in intracellular late endosomes is about 5.0. Our Au-NRs system is engineered to release CPT and ELT primarily in the acidic environment of endocytic compartments, reducing diffusion into body fluids and extracellular spaces. The data suggest that ELT release from Au-NRs nanomedicine is limited during bloodstream circulation but accelerates once reaching the tumor site, where the acidic conditions trigger enhanced drug release. This pH-sensitive release mechanism underscores the innovative approach of our research, which improves targeted drug delivery to tumor cells, minimizes systemic side effects, and enhances therapeutic efficacy. Cloning, expression, and purification of MBP-scFv recombinant fusion protein We optimized the scFv coding sequence for expression in E. coli , which was synthesized by General Biosystems in Germany and then inserted into the pUC57 cloning vector. This gene was cloned into the expression vector pMAL-c2X using Bam HI and Hind III restriction enzymes (Fig. 4 a). Confirmation of successful integration into the pMAL-c2X vector was achieved through DNA sequencing. The construct underwent further processing with enzymatic digestion using Bam HI and Hind III (Fig. 4 a). The resulting MBP-scFv fusion protein features maltose-binding protein (MBP) linked to the N-terminus of the scFv sequence via an asparagine linker. For purification, a His6x tag was included at the C-terminus, allowing us to utilize Ni-NTA affinity chromatography. The expression of this recombinant protein is controlled by the tacI promoter. We co-transformed the pMAL-scFv construct along with the pMJS205 helper vector into E. coli BL21(DE3) cells. Protein expression was induced with IPTG at concentrations of 20, 50, and 80 µM in LB medium at 25°C and 280 rpm. Despite these IPTG concentrations, the yield of soluble protein was low (Supplementary Fig. S5). To improve expression, we switched to autoinduction medium ZYM5052 at 25°C and 280 rpm. This adjustment successfully produced a 70.7 kDa protein, induced by 0.2% lactose, in its soluble form (Fig. 4 b). While using a rich medium, the presence of two disulfide bonds in the scFv fragment, combined with the reductive environment of the bacterial cytoplasm, impeded proper protein folding and caused misfolded protein accumulation. To address this, the pMJS205 helper vector, which provides eukaryotic oxidase (Ervp1) and disulfide isomerase (PDI), was utilized to facilitate disulfide bond formation and correct protein folding, thereby enhancing the yield of soluble recombinant protein. Additionally, high aeration (280 rpm) and a temperature of 25°C further contributed to the increased protein yield. Purification of the protein was carried out using Ni-NTA agarose column chromatography, targeting the His6x tag at the C-terminus of the scFv fragment. We tested various imidazole concentrations for elution, finding that 250 mM imidazole was effective in eluting the MBP-scFv from the column. SDS-PAGE analysis using ImageJ confirmed that the MBP-scFv protein purity exceeded 95% (Fig. 4 c). The initial evaluation of the fusion protein function The study employed cell-based ELISA to evaluate how well the fusion protein binds to the BMP receptor AI on the cell surface. Cells were treated with three distinct concentrations of the fusion protein. The data revealed that as the concentration of the fusion protein increased, so did the binding to the cell surface, with a statistically significant difference observed compared to the control. Furthermore, at the highest concentration of the fusion protein, MRC-5 cells showed an absorbance at 450 nm that was almost identical to that of the control sample (See Fig. 5 ). Maintaining the colloidal stability of the nanomedicine We evaluated the stability of CPT/ELT/scFv@Au-NRs over a one-hour period using UV-visible spectroscopy. The results indicated that these nanorods retained approximately 76% stability in a buffer environment (see Supplementary Fig. 6). In a related study, Pavelka et al. (2021) demonstrated that gold nanorods coated with silica maintained high colloidal stability for a long time. In vitro cytotoxicity assessment The MRC-5 and A549 cell lines, representing normal and cancerous lung cells, respectively, were utilized to examine their responses to free drugs, the MBP-scFv fusion protein, and a targeted drug delivery system (Fig. 6 ). SiO₂@Au-NRs exhibited no toxicity in A549 cells at concentrations up to 120 pM, with a 96% survival rate after 72 hours of treatment (Fig. 6 a). Consequently, the concentration of SiO₂@Au-NRs in the drug-loaded samples was maintained below 120 pM during subsequent cell viability tests. Figure 6 b shows that the MBP-scFv fusion protein displayed significant time-dependent cytotoxicity after 72 hours at concentrations greater than 6.25 nM. IC 50 values for free ELT and CPT were determined in both A549 and MRC-5 cells over 24, 48, and 72 hours. The free drugs exhibited minimal cytotoxic effects on MRC-5 cells, with statistically significant effects only observed at high concentrations after 72 hours. Cell viability remained at 80% for CPT and 70% for ELT (Fig.s 6c and 6d). Fig.s 6e and 6f illustrate that both CPT (at concentrations < 25 nM) and ELT demonstrated concentration-dependent cytotoxicity in A549 cells. Additionally, CPT showed time-dependent cytotoxicity at concentrations above 25 nM. Given ELT’s known selectivity for lung cancer cells and the influence of proliferation rates on CPT performance, A549 cells displayed higher cytotoxicity compared to MRC-5 cells. Figure 7 highlights our main findings. We observed that the IC 50 for CPT@Au-NRs was higher than that for free CPT after 72 hours, indicating that CPT's toxicity decreases when it is loaded onto GNRs, likely due to the slower release of the drug. In contrast, ELT@Au-NRs demonstrated increased toxicity towards cells compared to free ELT, as evidenced by a lower IC 50 . Moreover, ELT/scFv@Au-NRs exhibited a significantly higher cytotoxic effect on the A549 cell line after 72 hours of treatment compared to non-targeted GNRs. However, no significant difference in cytotoxicity was noted between CPT@Au-NRs and CPT/scFv@ Au-NRs. Although CPT/ELT@Au-NRs increased cytotoxicity in A549 cells, the targeted versions did not show a significant advantage over the non-targeted ones. These results emphasize the potential of GNRs in drug delivery and their role in enhancing cytotoxic activity, although the full effectiveness of the targeting system needs to be confirmed through in vivo testing. Our results align with the study's goal of enhancing drug toxicity on A549 cells through GNRs loading and functionalization. Statistical analysis showed no significant differences in cytotoxicity between CPT@Au-NRs and CPT/scFv@Au-NRs, but notable effects were observed for ELT-loaded Au-NRs (see Fig. 7 ). Overall, the data indicate that cytotoxicity is influenced by both drug concentration and exposure time, with increased toxicity corresponding to higher drug concentrations and longer exposure. Additionally, functionalizing Au-NRs with an anti-BMPR-AI antibody sometimes enhanced their cytotoxic effects. Cells respond more acutely to free CPT compared to drug-loaded nanorods, which release the drug at a slower rate. Notably, the CPT/ELT/scFv@Au-NRs exhibited greater cell toxicity than free CPT, ELT, or CPT/ELT@Au-NRs. This heightened toxicity is likely due to the nanomedicine's functionalization with the MBP-scFv fusion protein, which improves targeting of BMPR-AI on A549 cells. The increased accessibility of hydrophobic drugs to cells may diminish the impact of the MBP-scFv fusion protein’s targeting capabilities. To assess the cytotoxic effects of CPT and ELT, we used the MTT assay to determine their IC 50 values at various molar ratios (Table 1 ). We then calculated the combination index (CI) to evaluate whether the drug combination had a synergistic, antagonistic, or additive effect. A CI value greater than 1 indicates antagonism, less than 1 suggests synergy, and a value of 1 reflects an additive effect [43{Mirzaeinia, 2022 #1253]. The results showed that the CPT and ELT combination had a more pronounced anticancer effect than either drug alone, as evidenced by fractional inhibition (Fa) values ranging from 0.10 to 0.95 (Table 1 ). This indicates that combining CPT and ELT is more effective at inhibiting cancer cell growth compared to using either drug individually. Table 1 Combination index values and the cytotoxic effects of combined CPT and ELT. Drug combination ratio CI Interpretation CPT + ELT (IC 50 :1/2IC 50 ) 0.49612 Synergism CPT + ELT (IC 50 :1/4IC 50 ) 0.73912 Synergism CPT + ELT (IC 50 :IC 50 ) 0.36487 Synergism CPT + ELT (1/2IC 50 :IC 50 ) 0.21218 Synergism CPT + ELT (1/4IC 50 :IC 50 ) 0.12403 Synergism Cellular uptake evaluation To evaluate cellular uptake effectiveness, we used an inverted fluorescence microscope. The results demonstrated that our formulations successfully delivered the drugs into the cells, with fluorescence significantly increasing after 120 minutes (see Fig. 8 ). CPT’s autofluorescence allowed us to track its entry into the cells directly with the microscope. The hydrophobic nature of the compounds facilitated their easy penetration into the cells in their free form. Notably, the fluorescence observed in cells treated with CPT/ELT/scFv@GNRs confirmed the effectiveness of our targeting strategy, as the nanomedicine was taken up through specific antibody-receptor interactions. Cells typically absorb mesoporous silica particles non-specifically via clathrin-coated vesicles due to their siliceous nature [ 44 ]. However, rod-shaped mesoporous silica particles interact with the cell membrane over a larger surface area, particularly along the length of the rods. This enhanced interaction significantly influences the rate and extent of cellular uptake compared to spherical nanoparticles [ 45 ]. Although passive targeting is less controlled and more variable in its effects on cell function, it is not as precise as targeted uptake. The MBP-scFv fusion protein, which targets BMPR-AI on the cell surface, was attached to the gold nanorods to enhance site-specific delivery. Analysis of cell apoptosis using flow cytometry Flow cytometry was employed to evaluate the combined cytotoxic effects of ELT and CPT treatments and their potential to enhance apoptosis in A549 cells. The study included treatments with PBS, free CPT, free ELT, CPT/scFv@Au-NRs, ELT/scFv@Au-NRs, and CPT/ELT/scFv@Au-NRs. Apoptosis was quantified using a dual-parameter dot plot generated through flow cytometry. To distinguish between early apoptosis, late apoptosis, and necrosis, we used Annexin V-FITC and PI double staining (see Fig. 9 ). The total percentage of apoptotic cells was calculated by summing early and late apoptotic cells (Annexin V-FITC positive). Figure 9 shows that 92.3% of untreated A549 cells remained viable after 72 hours. In contrast, treatment with free CPT or ELT resulted in increased apoptosis rates—67.7% for CPT and 68.2% for ELT. In cells treated with CPT/scFv@Au-NRs, 59.2% were in the early apoptotic stage, indicating that this treatment induced apoptosis more effectively than free CPT. Cells exposed to free ELT were predominantly in the late apoptosis phase (66.2%), while those treated with ELT/scFv@Au-NRs showed a predominance of early apoptosis (60.0%). This notable shift, likely due to the more controlled release of drugs from the nanocarriers compared to the free drugs, highlights the precision of our findings. Significantly, A549 cells treated with CPT/ELT/scFv@Au-NRs dramatically reduced viable cells to 4.3%, with 93.4% of cells undergoing early or late apoptosis. Conclusion This study presents a cutting-edge nanocarrier system using silica-coated gold nanorods (SiO₂@Au-NRs) for the targeted delivery of ELT and CPT. The gold nanorods were functionalized with recombinant anti-BMP receptor AI and loaded with either ELT, CPT, or both. The therapeutic effects were evaluated through cytotoxicity assays in A549 cells. The SiO₂@Au-NRs nanocarrier shows great promise for delivering multiple drugs and can be tailored with various scFv proteins fused to maltose-binding protein (MBP). Nanorods coated with the MBP-scFv fusion protein exhibited a high capacity for drug loading while maintaining their ability to target specific receptors. When ELT and CPT were combined in nanorods conjugated with anti-BMP receptor AI, cellular uptake in BMP receptor-overexpressing cells was significantly enhanced, leading to increased cytotoxicity. This innovative drug delivery system holds considerable potential as a cancer treatment strategy. However, further research is required to assess the stability of SiO₂@Au-NRs and their cytotoxic effects in animal models. Declarations Acknowledgments We gratefully appreciate the support from Shiraz University and Tarbiat Modarres University, which were instrumental in the completion of this project. Thanks to Prof. Lloyd Ruddock for kindly providing the pMJS205 plasmid through a Material Transfer Agreement. Authors’ contributions As the lead author, Fatemeh Sabzalizadeh played a central role in conducting the research, analyzing data, and writing the manuscript. Hamed Mirshakari contributed to laboratory work, while Hamid-Reza Karbalaei Heidari aided in idea development, data review, and manuscript editing. Nediljko Budisa participated in manuscript editing and data evaluation, and Khosro Khajeh supervised the research process. Funding This work was funded by the Iran National Science Foundation (INSF), grant number 98017210. Data availability statement The original contributions in this study are detailed in the article or Supplementary Material. For further inquiries, please contact the corresponding authors. Ethics approval and consent to participate :No primary studies with animals or human subjects have been conducted. Consent for publication: No studies with human subjects Competing interests: 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. References Zou B, Zhou XL, Lai SQ, Liu JC. 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Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010;70(2):440–6. Manzanares D, Ceña V. Endocytosis: The Nanoparticle and Submicron Nanocompounds Gateway into the Cell. Pharm 2020, 12(4). Babaei M, Abnous K, Taghdisi SM, Taghavi S, Saljooghi AS, Ramezani M, Alibolandi M. Targeted rod-shaped mesoporous silica nanoparticles for the co-delivery of camptothecin and survivin shRNA in to colon adenocarcinoma in vitro and in vivo. Eur J Pharm Biopharm. 2020;156:84–96. Supplementary Files GA.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6709389","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":465420267,"identity":"cabb2d35-b1e8-4041-a781-e354b9f18163","order_by":0,"name":"Fatemeh Sabzalizadeh","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Sabzalizadeh","suffix":""},{"id":465420268,"identity":"0540e1c0-dc49-4265-b9d9-49c0aeb17a69","order_by":1,"name":"Hamed Mirshekari","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Hamed","middleName":"","lastName":"Mirshekari","suffix":""},{"id":465420269,"identity":"c71e90e3-ef4f-4543-b9e7-55d909ed2c22","order_by":2,"name":"Nediljko Budisa","email":"","orcid":"","institution":"University of Manitoba","correspondingAuthor":false,"prefix":"","firstName":"Nediljko","middleName":"","lastName":"Budisa","suffix":""},{"id":465420270,"identity":"a219712e-2723-456e-b647-dfb5fec53cd8","order_by":3,"name":"Khosro Khajeh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACCQkGBgPGBhsgMwEmxthAjJY0ErUA1RxG1kIASM5uflDwccf5aH725GcfPvxhkOdvYG77gE+LtMwxA8OZZ27nzux5ZjxzZhuD4YwDjM0z8GmRk0gwMOZtu5274UaCMTNvAwPjBgbGZrwOk5NI/2D8t+0cUEv6Z+Y/fxjsCWqRlsgxMGZsOwDUkmPMzMDGkEhQi+SMnALD3rZkoF/eFDP2tkkkzzhMQIvEjfRtBj/b7HL72dM3M/z4Y2Pb397+GK8WIGAzQDaCgYGZkAagkgeE1YyCUTAKRsGIBgAxm0oltWpHIwAAAABJRU5ErkJggg==","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":true,"prefix":"","firstName":"Khosro","middleName":"","lastName":"Khajeh","suffix":""},{"id":465420271,"identity":"63582274-d3ed-4e98-85a3-7896a3f26dfa","order_by":4,"name":"Hamid-Reza Karbalaei-Heidari","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Hamid-Reza","middleName":"","lastName":"Karbalaei-Heidari","suffix":""}],"badges":[],"createdAt":"2025-05-20 15:44:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6709389/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6709389/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84050327,"identity":"84aa6bc1-8500-4a90-90d7-bcc353040931","added_by":"auto","created_at":"2025-06-06 08:20:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":274456,"visible":true,"origin":"","legend":"\u003cp\u003eThe schematic illustration depicts the step-by-step construction of CPT/Mal@Au-NRs nanomedicine (a), as well as how it actively targets BMPR-AI, inhibits growth, reduces proliferation, and induces apoptosis in cancer cells through the controlled release of anticancer drugs with minimal side effects (b).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/ef42de60bf2fcc85a7ae4e99.jpg"},{"id":84050325,"identity":"d09a1fa2-2a5d-4842-b0af-2614e2617804","added_by":"auto","created_at":"2025-06-06 08:20:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":118380,"visible":true,"origin":"","legend":"\u003cp\u003eThe initial characterization of SiO₂@Au-NRs includes a) an HR-TEM image of SiO₂@Au-NRs, b) an HR-TEM image of individual SiO₂@Au-NRs, and c) a size distribution histogram of SiO₂@Au-NRs.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/2abdacbde3b5e4a76c627366.jpg"},{"id":84050329,"identity":"1bc94f70-b39c-41c3-af56-22147421acfd","added_by":"auto","created_at":"2025-06-06 08:20:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":215178,"visible":true,"origin":"","legend":"\u003cp\u003eThe data include a) FT-IR spectra of maltose and aminated maltose; b) FT-IR curves of gold nanorods at various stages; c) zeta potential measurements; and d) cumulative percentage release of ELT at pH 5.0 and pH 7.4. The statistical significance is indicated by **** (p-value \u0026lt; 0.0001), calculated using a t-test.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/88dac8896a088f2de255cf5d.jpg"},{"id":84050337,"identity":"16f979d7-8980-43d8-be4f-50bdcede84bb","added_by":"auto","created_at":"2025-06-06 08:20:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":209916,"visible":true,"origin":"","legend":"\u003cp\u003eThe schematic illustration of the pUC57-scFv and pMAL-scFv constructs and the cloning confirmation on an agarose gel (a); MBP-scFv fusion protein expression in autoinduction media ZYM5052, with Line 1 and Line 2 representing total and soluble protein fractions, respectively (b); and MBP-scFv purification stages, with Line 1 showing soluble protein, Line 2 showing unbound proteins, Line 3 and Line 4 showing washed proteins with 50 mM imidazole, and Line 5 showing purified protein with 250 mM imidazole (c).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/c847e3fba79e7fd1ad74b917.jpg"},{"id":84050330,"identity":"4cf42e58-afc5-4b21-a20f-570b12a3c0eb","added_by":"auto","created_at":"2025-06-06 08:20:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":75114,"visible":true,"origin":"","legend":"\u003cp\u003eCell-based ELISA was conducted to evaluate the MBP-scFv fusion protein at various concentrations. MRC-5 cells and wells coated with MBP-scFv fusion protein served as negative and positive controls, respectively. Statistical significance was determined using a t-test, with *** indicating a p-value \u0026lt; 0.0002 and **** indicating a p-value \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/a71038983fa0d17386da5cc7.jpg"},{"id":84050333,"identity":"b2aa39d5-5d1e-4964-b374-b5a5769b5fff","added_by":"auto","created_at":"2025-06-06 08:20:10","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":429245,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability was assessed using the MTT assay.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/ee968664d9431d04c859e209.jpg"},{"id":84050338,"identity":"7aa47717-cb8f-4211-8075-b846573c9693","added_by":"auto","created_at":"2025-06-06 08:20:10","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":179209,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of IC\u003csub\u003e50\u003c/sub\u003e values for free drugs and nanoformulations after 72 hours. ** p-value\u0026lt;0.0021, *** p-value\u0026lt;0.0002, and **** p-value\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/16f995cf01cd2fd02acefeaf.jpg"},{"id":84051471,"identity":"e7018ce0-0a93-447c-9cf3-d2348e567771","added_by":"auto","created_at":"2025-06-06 08:28:10","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":345165,"visible":true,"origin":"","legend":"\u003cp\u003eCellular uptake of CPT@Au-NRs, CPT/ELT@Au-NRs, and CPT/ELT/scFv@Au-NRs in A549 cells after two hours of treatment.\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/8cf190769549e984a260429d.jpg"},{"id":84050331,"identity":"417d98a4-07c7-4be5-9174-f4a73d0414b4","added_by":"auto","created_at":"2025-06-06 08:20:10","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":264891,"visible":true,"origin":"","legend":"\u003cp\u003eA549 cell apoptosis evaluation using flow cytometry after staining with Annexin V/FITC-PI. Panels show effect of a) PBS, b) CPT, c) ELT, d) CPT/scFv@Au-NRs, e) ELT/scFv@Au-NRs, and f) CPT/ELT/scFv@Au-NRs.\u003c/p\u003e","description":"","filename":"Picture10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/b29c4e5bbe737ec67efb274d.jpg"},{"id":86107020,"identity":"7e1a3b37-8aa3-45c7-9f44-719cde7fadd9","added_by":"auto","created_at":"2025-07-06 16:49:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3257264,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/4eb92c6c-722d-4ba7-b9d9-1163f8d00588.pdf"},{"id":84050332,"identity":"13f5b161-4582-449f-bfe3-8ce400b8b6dc","added_by":"auto","created_at":"2025-06-06 08:20:10","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":621662,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-6709389/v1/45336fc088bb0ff8b8bb99e9.png"}],"financialInterests":"","formattedTitle":"Smart co-delivery of Erlotinib and Camptothecin using silica-coated gold nanorods functionalized with recombinant anti-BMP receptor type AI","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLung cancer ranks among the most widespread cancers globally and is the leading cause of cancer-related deaths, with 1.8\u0026nbsp;million people succumbing to the disease in 2020, according to the World Health Organization. Despite significant progress in cancer research, diagnosis, and treatment, the five-year survival rate for lung cancer has only improved by five percent over the past 20 years. Sadly, many patients face a grim prognosis, often passing away within the first year of diagnosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Lung cancer is divided into two main types: small-cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). While NSCLC is more common and generally grows slower, SCLC, though less prevalent, tends to be more aggressive. More than half of NSCLC cases (55%) are diagnosed at advanced stages, and only 20% of patients respond favorably to chemotherapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNano-drug delivery systems harness the power of nanotechnology to achieve controlled drug release, enhanced cellular absorption, prolonged drug stability within cells and the bloodstream, minimized side effects, improved accessibility, biocompatibility, targeted delivery, reduced dosage requirements, controllable pharmacokinetics, and traceability [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These systems utilize various carriers, including liposomes, micelles, polymers, polysaccharides, self-assembled peptides, dendrimers, silica-based nanoparticles, bioactive glasses, hydrogels, carbon-based nanoparticles, metal nanoparticles, exosomes, and gold nanostructures [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among these, gold nanostructures are increasingly used in medical applications as carriers for antigen delivery, vaccination, gene therapy, and other therapeutic targets [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCamptothecin (CPT), a potent antitumor drug originally sourced from \u003cem\u003eCamptotheca acuminata\u003c/em\u003e, a tree native to Tibet and China and used in traditional Chinese medicine, inhibits the enzyme topoisomerase I (Topo I). It forms irreversible covalent complexes with DNA during replication, leading to DNA strand breaks and subsequent apoptosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eErlotinib (ELT) is a small-molecule tyrosine kinase inhibitor approved by the FDA for treating NSCLC and metastatic pancreatic cancer, often in combination with gemcitabine (U.S. Food and Drug Administration). It selectively binds to the adenosine triphosphate (ATP) binding sites of the epidermal growth factor receptor (EGFR), reversibly inhibiting EGFR activation and blocking downstream signaling pathways. This action reduces cell proliferation, angiogenesis, and metastasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBone morphogenetic proteins BMP2 and BMP4 are crucial for embryo development and lung growth. Although BMP signaling diminishes after lung formation, it can be reactivated during inflammation and in lung cancer, playing a significant role in tumor development [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. BMP2 expression is elevated in 98% of NSCLC patients and is linked to tumor spread in various other cancers [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, targeting BMP signaling presents a promising approach for treating NSCLC.\u003c/p\u003e \u003cp\u003eExtracellular antagonists such as germline and noggin bind to BMP2 and BMP4, preventing their interaction with receptors [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Several generations of BMP receptor inhibitors have been developed, with DMH1 showing a strong affinity for the ACVR-1 receptor. In 2014, Hao et al. demonstrated that DMH1 effectively reduced cell division, induced cell death, and decreased cell migration in NSCLC [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In 2018, Newman introduced a new inhibitor targeting BMP type I and II receptors in NSCLC, which lowers the expression of Id1, XIAP, and TAK1 genes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, Browning highlighted in 2018 that inhibiting BMPR-IA aids in differentiating helper T cells from CD4\u0026thinsp;+\u0026thinsp;T cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eMonoclonal antibodies (mAbs) are widely recognized as the gold standard for targeting tumor cells due to their exceptional specificity. However, full-size mAbs have several drawbacks, including their large size, complexity, and issues with post-translational modifications, which can limit their ability to penetrate cancer cells effectively. Consequently, there has been a move towards using smaller antibody fragments such as Fab, scFv, and VHH [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Among these, single-chain variable fragments (scFv) stand out for their compact size, low immunogenicity, and cost-effectiveness. scFv fragments are created from recombinant molecules where the variable regions of light (VL) and heavy (VH) chains are combined into a single polypeptide connected by a flexible linker [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although scFv expression requires an oxidizing environment\u0026mdash;such as the endoplasmic reticulum in eukaryotic cells or the periplasm in bacteria\u0026mdash;this does not detract from their potential [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eE. coli\u003c/em\u003e system is a popular choice for producing recombinant proteins in both industrial and research settings. However, generating proteins with disulfide bonds or post-translational modifications like glycosylation in bacteria can be challenging. While producing full-sized antibodies in bacteria is problematic due to the lack of necessary post-translational modifications, many antibody fragments can be successfully produced in the cytoplasm or periplasm of engineered strains [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To improve the solubility and yield of these proteins, fusion partners such as glutathione S-transferase (GST), maltose-binding protein (MBP), small ubiquitin-like modifier (SUMO), and thioredoxin (Trx) are frequently used, particularly for scFv fragments [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To overcome the challenges associated with \u003cem\u003eE. coli\u003c/em\u003e cytoplasmic expression, the CyDisCo system was developed. This system co-expresses disulfide bond formation catalysts, like Erv1p, DsbB, or VKOR, and disulfide bond isomerization catalysts, such as DsbC or PDI. The CyDisCo system has effectively produced scFv and Fab fragments from known antibodies within the \u003cem\u003eE. coli\u003c/em\u003e cytoplasm [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we developed an advanced drug delivery system specifically designed to target lung cancer cells. This system utilizes silica-coated gold nanorods (SiO₂@Au-NRs) modified with anti-BMP receptor AI scFv fragments (scFv). The scFv, fused with maltose-binding protein (MBP-scFv), is produced using a co-expression system in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3). To facilitate the loading of MBP-scFv, the SiO₂@Au-NRs are coated with aminated maltose (NH₂-maltose). Additionally, Camptothecin (CPT) is covalently attached to the surface, and Erlotinib (ELT) is incorporated into the SiO₂@Au-NRs and modified with MBP-scFv to create a sophisticated targeting system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our study assesses this system's toxicity, apoptotic effects, and cellular uptake using cell assays and fluorescence microscopy on the A549 and MRC-5 cell lines. Early results suggest that co-delivery of ELT and CPT significantly enhances their therapeutic effectiveness by inhibiting cell proliferation and inducing apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials, strains, and cell lines\u003c/h2\u003e \u003cp\u003eGold (III) chloride trihydrate (HAuCl₄), cetyltrimethylammonium bromide (CTAB), L-ascorbic acid, 3-(mercaptopropyl)trimethoxysilane (MPTMS), 3-(triethoxysilyl)propylamine (APTMS), sodium borohydride (NaBH₄), and silver nitrate (AgNO₃) were sourced from Sigma-Aldrich in Germany. All experiments were conducted using double-distilled water. Fermentas, Lithuania, supplied restriction enzymes and T4 ligase. The helper plasmid pMJS205 was generously provided by Professor Lloyd Ruddock under a signed Material Transfer Agreement (MTA). \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) was obtained from Invitrogen (Thermo Fisher Scientific), while the pMAL-c2X plasmid was acquired from Addgene (#75286). Dulbecco's Modified Eagle's Medium (DMEM) high glucose, along with trypsin-EDTA, penicillin/streptomycin, and heat-inactivated fetal bovine serum (FBS), were purchased from Gibco\u0026reg;, USA. The human lung adenocarcinoma cell line (A549) and the normal human lung cell line (MRC-5) were procured from the Department of Cell Bank at the Pasteur Institute of Iran. Additionally, Camptothecin and Erlotinib were purchased from Alfa Aesar.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynthesis of CTAB-functionalized gold nanorods (CTAB@Au-NRs)\u003c/h3\u003e\n\u003cp\u003eGold nanorods were synthesized using a seed-mediated growth method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. First, 125 \u0026micro;L of 10 mM HAuCl₄ was mixed with 3.75 mL of 100 mM CTAB to prepare the gold seed solution. Then, 300 \u0026micro;L of 10 mM NaBH₄, freshly prepared on ice, was added to the mixture. The solution was vigorously stirred for two minutes, resulting in a yellow-brown seed solution. To remove excess NaBH₄, the solution was allowed to stand at room temperature in the dark for 2 hours.\u003c/p\u003e \u003cp\u003eFor the growth solution, 2 mL of 10 mM HAuCl\u003csub\u003e4\u003c/sub\u003e was mixed with 47 mL of 100 mM CTAB in a Falcon tube. Following this, 300 \u0026micro;L of 10 mM AgNO\u003csub\u003e3\u003c/sub\u003e was added to control the aspect ratio of the gold nanorods. After thorough stirring, 320 \u0026micro;L of freshly prepared 100 mM ascorbic acid solution was slowly introduced, causing the solution to transition from yellow-brown to colorless. Immediately afterward, 320 \u0026micro;L of the gold seed solution was rapidly added, and the mixture was gently stirred for 20 seconds. Over the next 15 minutes, the solution gradually changed color and stabilized. The nanorods were allowed to grow overnight at 25\u0026deg;C without further stirring.\u003c/p\u003e\n\u003ch3\u003ePreparation of Silica-functionalized Au-NRs (SiO@Au-NRs)\u003c/h3\u003e\n\u003cp\u003eThe procedure began with sonicating 4 mL of the gold nanorod suspension in a water bath for two minutes at 90 W and 50\u0026ndash;60 kHz. Following this, the suspension was centrifuged at 4,000 x g for 5 minutes to remove impurities, and the resulting supernatant, which contained the purified gold nanorods, was collected. To coat the gold nanorods, the method described by Mirshekari et al. (2024) was used. First, to remove CTAB and other impurities, the nanorods (Au-NRs) were centrifuged at 12,000 x g for 20 minutes. The purified nanorods were then resuspended in 5 mL of deionized water, and the pH was adjusted to 4.0 using 100 mM hydrochloric acid. This suspension was mixed at 400 x g for 20 minutes. Subsequently, 41 \u0026micro;L of 100 mM MPTMS ethanol solution was added, stirring the mixture for 3 hours at the same speed. Afterward, 62 \u0026micro;L of a 20% APTMS ethanol solution was introduced, and the suspension was stirred for one hour at 800 x g. The pH was then adjusted to 10.0 using 100 mM sodium hydroxide, and the mixture was stirred at 800 x g for an additional 20 minutes. The suspension was then left to rest at room temperature for 20 hours. Finally, the suspension was centrifuged at 10,000 x g for 10 minutes, and the gold nanorods were washed three times with absolute ethanol and three times with deionized water.\u003c/p\u003e\n\u003ch3\u003eAmination of maltose (NH-Maltose)\u003c/h3\u003e\n\u003cp\u003eTo start, 0.004 moles (1.37 g) of maltose and 0.004 moles (0.31 g) of NH₄HCO₃ were added to 20 mL of an aqueous ammonium solution. This mixture was refluxed at 42\u0026deg;C for 36 hours until the volume was reduced by half. Subsequently, ammonium hydroxide was separated from the maltose using a freeze dryer, and the aminated maltose was dried as described by Zhou et al. (2012).\u003c/p\u003e\n\u003ch3\u003eCarboxylation of SiO@Au-NRs (COOH@Au-NRs)\u003c/h3\u003e\n\u003cp\u003eA 10 mL ethanol solution of Au-NRs (OD\u0026thinsp;~\u0026thinsp;1.0) was treated with 100 mM of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 100 mM of N-hydroxy succinimide (NHS). After the reaction, 100 mM of succinic anhydride was added to the solution, which was then stirred for three hours at +\u0026thinsp;4\u0026deg;C. Following this, the nanorods were washed three times with water and three times with ethanol to remove unreacted chemicals.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of maltose-coated gold nanorods\u003c/h2\u003e \u003cp\u003e100 mM NH₂-Maltose was added to 10 mL of an aqueous solution containing activated COOH@Au-NRs (OD\u0026thinsp;~\u0026thinsp;1.0). The mixture was stirred for one hour at +\u0026thinsp;4\u0026deg;C. Following this, the nanorods were washed three times with ethanol and then three times with water.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCamptothecin-conjugation on carboxylated gold nanorods surface\u003c/h3\u003e\n\u003cp\u003eTo covalently attach CPT to the gold nanorod surfaces, a reaction mixture was prepared with 1 milliliter of COOH@Au-NRs (optical density\u0026thinsp;~\u0026thinsp;1.0), 1 micromole of CPT, 1 micromole of triethylamine (TEA), and 2 micromoles of N,N'-dicyclohexylcarbodiimide (DCC). This mixture was incubated at room temperature for 24 hours. To deactivate any remaining carboxyl groups, 100 mM NH₂-maltose was introduced and allowed to react for one hour. Afterward, the gold nanorods were thoroughly washed three times with ethanol, followed by three washes with water.\u003c/p\u003e\n\u003ch3\u003eDrug loading evaluation\u003c/h3\u003e\n\u003cp\u003eErlotinib was loaded onto both Mal@Au-NRs and CPT/Mal@Au-NRs using an absorption technique. First, the nanorods, with an optical density at the local surface plasmon resonance (OD\u003csub\u003eLSPR\u003c/sub\u003e) of approximately 0.58, were suspended in Tris-HCl buffer (pH 7.4). Various Erlotinib concentrations were introduced into the nanorod suspension at 20-minute intervals while the mixture was continuously rotated. Afterward, the mixture was centrifuged three times to remove any unbound Erlotinib. The concentration of unbound Erlotinib in the supernatant was measured using UV\u0026ndash;visible absorption spectroscopy at 333 nm (Supplementary Fig.\u0026nbsp;1), based on a calibration curve for Erlotinib. The specific loading content and efficiency of Erlotinib were subsequently calculated using the equations provided below:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{E}\\text{n}\\text{c}\\text{a}\\text{p}\\text{s}\\text{u}\\text{l}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\text{e}\\text{f}\\text{f}\\text{i}\\text{c}\\text{i}\\text{e}\\text{n}\\text{c}y\\:\\%\\:=\\:\\frac{drug\\:initial\\:weigth\\:\\left(mg\\right)\\:-\\:unloaded\\:drug\\:in\\:aqueous\\:phase\\:\\left(mg\\right)}{drug\\:initial\\:weigth\\:\\left(mg\\right)}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Loading\\:capacity\\:=\\:\\frac{drug\\:initial\\:weigth\\:\\left(mg\\right)\\:-\\:unloaded\\:drug\\:in\\:aqueous\\:phase\\:\\left(mg\\right)}{gold\\:nanorods\\:weigth\\:\\left(mg\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro drug release at different pH values\u003c/h2\u003e \u003cp\u003eFor the in vitro release test, nanorods were dispersed in 1 mL of either acetate buffer (pH 5.0) or phosphate buffer (pH 7.4), each placed in separate dialysis bags (MW cutoff\u0026thinsp;=\u0026thinsp;3.5 kDa). These bags were then immersed in 10 mL of the corresponding buffer solutions. At regular intervals, 0.5 mL samples were taken from the release medium, and absorbance was measured at 333 nm for Erlotinib (ELT) and 368 nm for Camptothecin (CPT). Blank buffer solutions of equal volume were used for calibration. All experiments were performed in triplicate, with average results reported.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and cloning of the scFv gene\u003c/h2\u003e \u003cp\u003eGeneral Biosystems company synthesized the codon-optimized sequence of the scFv fragment, which was derived from the anti-BMP receptor AI Fab (PDB: 3NH7) and cloned in the pUC57-Amp cloning vector. The sequence comprises the light (V\u003csub\u003eL\u003c/sub\u003e) and heavy (V\u003csub\u003eH\u003c/sub\u003e) variable chains of the Fab fragment, linked by (G\u003csub\u003e4\u003c/sub\u003eS)\u003csub\u003e3\u003c/sub\u003e, with a histidine sequence at the C-terminal. The synthesized sequence was inserted into the pMAL-c2X vector using \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eHind\u003c/em\u003eIII restriction sites to produce the recombinant fusion protein of MBP-scfv.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOverexpression and purification of the MBP-scFv fusion protein\u003c/h2\u003e \u003cp\u003eThe pMAL-scFv vector and the helper plasmid pMJS205 were co-transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cells. The LB medium and the ZYM5052 autoinduction system were employed to express the MBP-scFv fusion protein. A single bacterial colony was grown in an LB medium with ampicillin and chloramphenicol at appropriate concentrations, incubating at 37\u0026deg;C for 16 hours. Expression of the recombinant MBP-scFv fusion protein was induced by adding IPTG at concentrations of 20, 50, and 80 \u0026micro;M to the LB medium, while 0.2% lactose was used in the ZYM5052 autoinduction medium. This process was carried out at 25\u0026deg;C with shaking at 280 rpm for 24 hours. After incubation, the bacterial cultures were harvested by centrifugation, and the resulting cell pellets were resuspended in lysis buffer containing 20 mM Tris (pH 8.0). To purify the overexpressed MBP-scFv protein efficiently, the soluble fraction of the cell extract was passed through a Ni-NTA agarose column. Following purification, the MBP-scFv fusion protein was dialyzed against 20 mM Tris (pH 8.0). The sample\u0026rsquo;s homogeneity was finally confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% acrylamide gels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmobilization of MBP-scFv fusion protein on nanocarrier\u003c/h2\u003e \u003cp\u003eThe MBP-scFv fusion protein was successfully immobilized onto the surface of Mal@Au-NRs by incubating 200 ng of the fusion protein with 2 ml of Mal@Au-NRs (OD\u0026thinsp;\u0026asymp;\u0026thinsp;1) at 4\u0026deg;C for 24 hours. To assess the effectiveness of this immobilization, the nanorod solution was centrifuged, and the supernatant was analyzed through absorbance measurements at 280 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eColloidal stability investigation of CPT/ELT/scFv@Au-NRs\u003c/h2\u003e \u003cp\u003eAn aliquot of 100 microliters of CPT/ELT/scFv@Au-NRs was suspended in 500 microliters of 20 mM Tris-base, pH 7.4. The colloidal stability of the CPT/ELT/scFv@Au-NRs was assessed at four time points: 0, 15, 30, and 60 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity evaluation and synergistic effect\u003c/h2\u003e \u003cp\u003eTo evaluate the formulations' cytotoxicity under controlled conditions, A549 and MRC-5 cells were plated in 96-well plates at densities of 8 \u0026times; 10\u0026sup3; and 12 \u0026times; 10\u0026sup3; cells per well, respectively. Each well contained 100 \u0026micro;l of DMEM high-glucose medium enriched with 10% FBS and necessary antibiotics. The cells were incubated at 37\u0026deg;C with 5% CO₂ in a humidified environment. After 16 hours, free drugs and various nanoformulations were introduced to the wells, and the incubation continued for 24, 48, and 72 hours.\u003c/p\u003e \u003cp\u003eTo determine cell viability, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay was performed. Cells were first washed with PBS buffer, then treated with 100 \u0026micro;l of MTT solution (0.025 mg/ml) and incubated in the dark at 37\u0026deg;C for 4 hours. Afterward, the medium was discarded, and 100 \u0026micro;l of DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm with a reference at 630 nm using a microplate reader. The Combination Index (CI) was calculated following the Chou-Talalay method, and isobologram analysis was conducted to assess the synergistic effects of the treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCellular uptake\u003c/h2\u003e \u003cp\u003eThe fluorescent microscopy technique was used to examine the absorption and distribution of the formulations within cells. Cells were initially seeded in 48-well plates at a density of 15,000 cells per well and incubated at 37\u0026deg;C for 24 hours. Following this incubation period, the cells were exposed to CPT@Au-NRs, CPT/ELT@Au-NRs, and CPT/ELT/scFv@Au-NRs in fresh culture medium for 2 hours at 37\u0026deg;C with 5% CO₂. Rhodamine was introduced to each well at a concentration of 40 \u0026micro;g/ml. After this treatment, the cells were thoroughly washed three times with PBS and then analyzed using a fluorescent microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assay\u003c/h2\u003e \u003cp\u003eCell apoptosis was evaluated using the Annexin V-FITC/PI double staining kit. Initially, 10\u003csup\u003e5\u003c/sup\u003e cells were plated into each well of a 6-well plate and incubated overnight at 37\u0026deg;C. The following day, the cells were exposed to free drugs and various formulations for a duration of 72 hours. After treatment, the cells were collected and resuspended in 400 \u0026micro;L of 1\u0026times; reconstitution buffer. To each tube, 5 \u0026micro;L of Annexin V-FITC was added, and the samples were incubated for 15 minutes in the dark at 4\u0026deg;C. Following this, 10 \u0026micro;L of propidium iodide (PI) was introduced to each tube, which was then incubated for another 5 minutes in the dark. The evaluation of apoptosis was conducted within 30 minutes after staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCell-based ELISA analysis\u003c/h2\u003e \u003cp\u003eMRC-5 and A549 cell lines were seeded into 96-well plates and incubated overnight at 37\u0026deg;C to allow a monolayer of cells to form at the bottom of each well. After incubation, the culture medium was removed, and the cells were gently rinsed with PBS. They were then fixed with 4% formaldehyde in PBS for 20 minutes and washed three times with PBS. To neutralize any residual fixative, the cells were treated with 0.1 M glycine in PBS for 30 minutes.\u003c/p\u003e \u003cp\u003eNext, the cells were exposed to a 3% (w/v) hydrogen peroxide solution in PBS for 5 minutes to minimize endogenous peroxidase activity. Following two 5-minute washes with PBS, the cells were incubated in a blocking solution (5% fetal bovine serum and 1% BSA in PBS) for 30 minutes. After blocking, the cells were treated with MBP-scFv fusion protein for 1 hour, followed by three washes with the blocking solution, each lasting 3\u0026ndash;5 minutes.\u003c/p\u003e \u003cp\u003eSubsequently, the cells were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, diluted 1:3000 in the blocking solution, for 1 hour. After four washes with PBS (each lasting 4\u0026ndash;5 minutes), an HRP substrate reagent (3,3',5,5'-tetramethylbenzidine enzyme substrate; 150 \u0026micro;l per well) was added and incubated for 10 minutes. The reaction was halted with 1 M HCl, and the absorbance of the samples was measured at 450 nm using a microplate reader [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A positive control was included by immobilizing 100 \u0026micro;g/ml of the recombinant fusion protein in a blank well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eNanocarrier characterization\u003c/h2\u003e \u003cp\u003eWe obtained the UV-visible scattering spectrum using a spectrophotometer. To visualize the SiO₂@Au-NRs, we employed high-resolution transmission electron microscopy (HR-TEM) with a Tec9G20 model. The size distribution of the gold nanorods (Au-NRs) and the thickness of the silicon shell surrounding them were measured by analyzing at least 100 particles and 100 shells with ImageJ software. Elemental composition was determined using EDAX or EDS, while the gold concentration was quantified through inductively coupled plasma optical emission spectroscopy (ICP-OES). The surface charge of the gold nanorods was evaluated with the SZ-100 nanoparticle system (Nano ZS90 Zetasizer, Malvern Panalytical, Malvern, UK). Fourier-transform infrared (FT-IR) spectroscopy was performed on dried samples within the 400\u0026ndash;4000 cm⁻\u0026sup1; range. Additionally, proton nuclear magnetic resonance (\u0026sup1;H-NMR) spectra were recorded using a 400 MHz NMR spectrometer (Bruker, AVANCE\u0026trade; III HD 400 MHz).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistics analysis\u003c/h2\u003e \u003cp\u003eThe data in this study were analyzed using GraphPad Prism software, version 8.3.0. Comparisons between groups were conducted using either one-way ANOVA with Dunnett\u0026rsquo;s test or two-way ANOVA with the Tukey test. Statistical significance was defined by p-values less than 0.05. Combination index (CI) values were calculated using CompuSyn software, version 1.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and characterization of SiO\u003csub\u003e2\u003c/sub\u003e@Au-NRs\u003c/h2\u003e \u003cp\u003eCTAB is a crucial surfactant used in gold nanorods (Au-NRs) synthesis, but its significant cytotoxicity limits the use of CTAB-capped nanorods in medical applications. Additionally, CTAB molecules create a positive charge on the surface of Au-NRs by forming a double layer, further complicating their use in biological settings. Therefore, to facilitate the use of Au-NRs in medical applications, it is necessary to either replace CTAB or apply a coating to the nanorods [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The strong interaction between CTAB and the Au-NR surface, coupled with CTAB\u0026rsquo;s role in stabilizing colloidal suspensions, makes replacing it through ligand exchange a challenging task. Early research on coating Au-NRs with silica focused on using silane coupling agents or polymers to improve the affinity between the gold and silica surfaces [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHigh-resolution transmission electron microscopy (HR-TEM) images confirm that the gold nanorods are effectively coated with silica. The synthesized gold nanorods show distinct length and width dimensions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The average thickness of the silica coating, as observed in HR-TEM images of SiO₂@Au-NRs, is 2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These SiO₂@Au-NRs are relatively uniform in size and shape, with an average length of 33.63\u0026thinsp;\u0026plusmn;\u0026thinsp;5.38 nm, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec. HR-TEM images also reveal that the gold nanorods grow along the {100} plane with an interplanar distance of 0.196 nm (Supplementary Fig.\u0026nbsp;2a). The single-crystal nature of the gold nanorods is supported by their selected area electron diffraction (SAED) patterns, which display bright circular rings corresponding to the crystalline lattice planes of the gold nanoparticles (Supplementary Fig.\u0026nbsp;2b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe composition of SiO₂@Au-NRs was analyzed using Energy-dispersive X-ray spectroscopy (EDS), as shown in Supplementary Fig.\u0026nbsp;3a. This analysis revealed the presence of gold (Au) in the SiO₂@Au-NRs and confirmed the presence of oxygen, silicon, and nitrogen on the surface of the gold nanorods. Point-scan analysis detected the elements O, Si, and Au in the SiO₂@Au-NRs, but nitrogen was not observed (Supplementary Fig.\u0026nbsp;3b). These EDS/EDAX findings were consistent with the results from the HR-TEM analysis.\u003c/p\u003e \u003cp\u003eTo investigate bond formation and interactions within the structure, we utilized Fourier transform infrared spectroscopy (FT-IR). This technique allows us to trace shifts, formations, or disappearances of spectral bands to specific interactions. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the peak at 1589 cm⁻\u0026sup1; corresponds to the N-H bending vibration of the amine group attached to maltose. The broad band observed between 3100 and 3600 cm⁻\u0026sup1; is associated with the O-H stretching vibrations from hydroxyl groups present in the nanorods, maltose, and water [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A peak at 1429 cm⁻\u0026sup1; signifies O-H groups, while the band at 1074 cm⁻\u0026sup1; indicates C-O stretching in secondary alcohol [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, the peak at 2935 cm⁻\u0026sup1; is linked to C-H stretching in the sp\u0026sup3; carbon of the maltose ring [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe presence of CTAB was confirmed by two bands at 2915 and 2896 cm⁻\u0026sup1; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Bands at 1487 cm⁻\u0026sup1;, 1473 cm⁻\u0026sup1;, 1462 cm⁻\u0026sup1;, and 1431 cm⁻\u0026sup1; correspond to the symmetric and asymmetric C-H shear vibrations of the CH₃-N⁺ group. Significant differences emerged when comparing the spectra of CTAB@Au-NRs and SiO₂@Au-NRs. The peak at 1030 cm⁻\u0026sup1; represents the asymmetric stretching vibrations of Si-O-Si [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Si-O-Si aromatic symmetric bending and O-Si-O stretching were confirmed at 455 cm⁻\u0026sup1; and 791 cm⁻\u0026sup1;, respectively [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The peak at 1112 cm⁻\u0026sup1; further indicated Si-O-Si vibrations [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. A new vibrational mode observed at approximately 1630 cm⁻\u0026sup1; corresponds to the N-H bending vibration of the amine group in SiO₂@Au-NRs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and the stretching vibration of the carbonyl group (C\u0026thinsp;=\u0026thinsp;O) in carboxyl and amide bonds [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Lastly, the peak at 3425 cm⁻\u0026sup1; reflects the presence of N-H bonds associated with the amine group of APTMS and the hydroxyl group of carboxyl groups [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe surface charge of gold nanorods was assessed under various conditions using a Tris buffer at pH 7.4. Nanorods synthesized in a 1.6% cationic surfactant environment had a surface charge of about\u0026thinsp;+\u0026thinsp;43 millivolts, leading to electrostatic repulsion between the particles. After applying silane grafting, the surface charge of the nanorods varied according to the coverage and functional groups of the silane. The charge of the coated nanorods was measured at -1.93 mV, which is due to the presence of amine groups from APTMS. Further modification through carboxylation of SiO₂@Au-NRs reduced the positive surface charge to -5.98 mV.\u003c/p\u003e \u003cp\u003eGold nanorods are widely used in biomedical fields such as drug delivery, cell imaging, and cancer therapy because of their strong plasmon resonance properties. Coating these nanorods with SiO₂ shells improves their stability and biocompatibility. In 2001, Murphy and colleagues successfully encapsulated high aspect ratio gold nanorods with a thin (5\u0026ndash;10 nm) silica shell using MPTMS. This choice was based on sulfur\u0026rsquo;s strong affinity for gold, which helps displace the tightly bound CTAB molecules [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Silica coatings offer several advantages: they enhance colloidal and thermal stability, increase the surface area of the nanorods while preserving the gold core's optical properties, and allow for precise control over porosity. Additionally, silica improves the biocompatibility of gold nanorods. Its reactive surface silanols facilitate efficient drug loading and enable the attachment of functional ligands or biomolecules for targeted delivery [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For example, Zhou et al. developed a dual-targeted chemo-photo thermal therapy system with gold nanorods coated in mesoporous silica. This system showed a high photothermal effect and pH- and NIR-triggered drug release, highlighting its potential as an anticancer treatment [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Similarly, Gao et al. engineered folate-functionalized gold nanorods, demonstrating high biocompatibility and effective tumor cell uptake [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and characterization of CPT@Au-NRs\u003c/h2\u003e \u003cp\u003eThe creation of CPT@Au-NRs involves establishing an ester bond between CPT's hydroxyl group and the carboxyl groups on the surface of COOH@Au-NRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We employed \u003csup\u003e1\u003c/sup\u003eH-NMR spectroscopy and synthesized methyl hydrogen succinate to react with the gold nanorod surface to validate this esterification reaction. Peaks at 2.6 ppm and 3.6 ppm in the spectrum confirm the presence of succinic anhydride and the methylation of one carboxyl group (Supplementary Fig.\u0026nbsp;4a). The \u003csup\u003e1\u003c/sup\u003eH-NMR spectrum for CPT-methyl hydrogen succinate reveals peaks at 1.2 ppm and 1.91 ppm, associated with the CPT drug [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], while peaks at 3.06 ppm and 4.2 ppm are linked to the methyl hydrogen succinate chain (Supplementary Fig.\u0026nbsp;4b). These findings confirm the esterification reaction with an efficiency of 52%. After CPT was incorporated onto the SiO\u003csub\u003e2\u003c/sub\u003e@Au-NRs, the surface charge decreased to -19.5 mV, demonstrating successful CPT binding to the gold nanorod surface. Following the addition of NH\u003csub\u003e2\u003c/sub\u003e-maltose to block any remaining active groups, the surface charge further shifted to -8.66 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eIn vitro release assessment of ELT\u003c/h2\u003e \u003cp\u003eThe study evaluated the efficiency of loading ELT onto CPT/Mal@Au-NRs and Mal@Au-NRs to determine how much drug could be incorporated before the nanorods began to aggregate. We found that the encapsulation efficiency of ELT was 78.7% in CPT/Mal@Au-NRs and 72.6% in Mal@Au-NRs. At the same conditions, the drug loading was 4.2% for CPT/Mal@Au-NRs and 4.0% for Mal@Au-NRs.\u003c/p\u003e \u003cp\u003eTo test the effectiveness of this smart release system, we examined how ELT was released from CPT/ELT@Au-NRs and ELT/Mal@Au-NRs in environments that mimic physiological conditions. After 24 hours, the gold nanorods released 76.7% and 70% of ELT in a fluid that simulates intracellular conditions (pH 5.0). In contrast, the release of ELT in a fluid mimicking extracellular conditions (pH 7.4) was 60.8% for CPT/ELT@Au-NRs and 65% for ELT/Mal@Au-NRs. The release profiles in these simulated environments showed significant statistical differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), and no CPT release was detected.\u003c/p\u003e \u003cp\u003eThe literature indicates that nanoparticles are usually taken up by endocytic vesicles within cells. While body fluids and extracellular environments typically have a pH of around 7.4, the pH in intracellular late endosomes is about 5.0. Our Au-NRs system is engineered to release CPT and ELT primarily in the acidic environment of endocytic compartments, reducing diffusion into body fluids and extracellular spaces. The data suggest that ELT release from Au-NRs nanomedicine is limited during bloodstream circulation but accelerates once reaching the tumor site, where the acidic conditions trigger enhanced drug release. This pH-sensitive release mechanism underscores the innovative approach of our research, which improves targeted drug delivery to tumor cells, minimizes systemic side effects, and enhances therapeutic efficacy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eCloning, expression, and purification of MBP-scFv recombinant fusion protein\u003c/h2\u003e \u003cp\u003eWe optimized the scFv coding sequence for expression in \u003cem\u003eE. coli\u003c/em\u003e, which was synthesized by General Biosystems in Germany and then inserted into the pUC57 cloning vector. This gene was cloned into the expression vector pMAL-c2X using \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eHind\u003c/em\u003eIII restriction enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Confirmation of successful integration into the pMAL-c2X vector was achieved through DNA sequencing. The construct underwent further processing with enzymatic digestion using \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eHind\u003c/em\u003eIII (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe resulting MBP-scFv fusion protein features maltose-binding protein (MBP) linked to the N-terminus of the scFv sequence via an asparagine linker. For purification, a His6x tag was included at the C-terminus, allowing us to utilize Ni-NTA affinity chromatography. The expression of this recombinant protein is controlled by the tacI promoter.\u003c/p\u003e \u003cp\u003eWe co-transformed the pMAL-scFv construct along with the pMJS205 helper vector into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cells. Protein expression was induced with IPTG at concentrations of 20, 50, and 80 \u0026micro;M in LB medium at 25\u0026deg;C and 280 rpm. Despite these IPTG concentrations, the yield of soluble protein was low (Supplementary Fig. S5). To improve expression, we switched to autoinduction medium ZYM5052 at 25\u0026deg;C and 280 rpm. This adjustment successfully produced a 70.7 kDa protein, induced by 0.2% lactose, in its soluble form (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eWhile using a rich medium, the presence of two disulfide bonds in the scFv fragment, combined with the reductive environment of the bacterial cytoplasm, impeded proper protein folding and caused misfolded protein accumulation. To address this, the pMJS205 helper vector, which provides eukaryotic oxidase (Ervp1) and disulfide isomerase (PDI), was utilized to facilitate disulfide bond formation and correct protein folding, thereby enhancing the yield of soluble recombinant protein. Additionally, high aeration (280 rpm) and a temperature of 25\u0026deg;C further contributed to the increased protein yield.\u003c/p\u003e \u003cp\u003ePurification of the protein was carried out using Ni-NTA agarose column chromatography, targeting the His6x tag at the C-terminus of the scFv fragment. We tested various imidazole concentrations for elution, finding that 250 mM imidazole was effective in eluting the MBP-scFv from the column. SDS-PAGE analysis using ImageJ confirmed that the MBP-scFv protein purity exceeded 95% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eThe initial evaluation of the fusion protein function\u003c/h2\u003e \u003cp\u003eThe study employed cell-based ELISA to evaluate how well the fusion protein binds to the BMP receptor AI on the cell surface. Cells were treated with three distinct concentrations of the fusion protein. The data revealed that as the concentration of the fusion protein increased, so did the binding to the cell surface, with a statistically significant difference observed compared to the control. Furthermore, at the highest concentration of the fusion protein, MRC-5 cells showed an absorbance at 450 nm that was almost identical to that of the control sample (See Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eMaintaining the colloidal stability of the nanomedicine\u003c/h2\u003e \u003cp\u003eWe evaluated the stability of CPT/ELT/scFv@Au-NRs over a one-hour period using UV-visible spectroscopy. The results indicated that these nanorods retained approximately 76% stability in a buffer environment (see Supplementary Fig.\u0026nbsp;6). In a related study, Pavelka et al. (2021) demonstrated that gold nanorods coated with silica maintained high colloidal stability for a long time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro cytotoxicity assessment\u003c/h2\u003e \u003cp\u003eThe MRC-5 and A549 cell lines, representing normal and cancerous lung cells, respectively, were utilized to examine their responses to free drugs, the MBP-scFv fusion protein, and a targeted drug delivery system (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). SiO₂@Au-NRs exhibited no toxicity in A549 cells at concentrations up to 120 pM, with a 96% survival rate after 72 hours of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Consequently, the concentration of SiO₂@Au-NRs in the drug-loaded samples was maintained below 120 pM during subsequent cell viability tests. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb shows that the MBP-scFv fusion protein displayed significant time-dependent cytotoxicity after 72 hours at concentrations greater than 6.25 nM.\u003c/p\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e values for free ELT and CPT were determined in both A549 and MRC-5 cells over 24, 48, and 72 hours. The free drugs exhibited minimal cytotoxic effects on MRC-5 cells, with statistically significant effects only observed at high concentrations after 72 hours. Cell viability remained at 80% for CPT and 70% for ELT (Fig.s 6c and 6d). Fig.s 6e and 6f illustrate that both CPT (at concentrations\u0026thinsp;\u0026lt;\u0026thinsp;25 nM) and ELT demonstrated concentration-dependent cytotoxicity in A549 cells. Additionally, CPT showed time-dependent cytotoxicity at concentrations above 25 nM. Given ELT\u0026rsquo;s known selectivity for lung cancer cells and the influence of proliferation rates on CPT performance, A549 cells displayed higher cytotoxicity compared to MRC-5 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e highlights our main findings. We observed that the IC\u003csub\u003e50\u003c/sub\u003e for CPT@Au-NRs was higher than that for free CPT after 72 hours, indicating that CPT's toxicity decreases when it is loaded onto GNRs, likely due to the slower release of the drug. In contrast, ELT@Au-NRs demonstrated increased toxicity towards cells compared to free ELT, as evidenced by a lower IC\u003csub\u003e50\u003c/sub\u003e. Moreover, ELT/scFv@Au-NRs exhibited a significantly higher cytotoxic effect on the A549 cell line after 72 hours of treatment compared to non-targeted GNRs. However, no significant difference in cytotoxicity was noted between CPT@Au-NRs and CPT/scFv@ Au-NRs. Although CPT/ELT@Au-NRs increased cytotoxicity in A549 cells, the targeted versions did not show a significant advantage over the non-targeted ones. These results emphasize the potential of GNRs in drug delivery and their role in enhancing cytotoxic activity, although the full effectiveness of the targeting system needs to be confirmed through in vivo testing.\u003c/p\u003e \u003cp\u003eOur results align with the study's goal of enhancing drug toxicity on A549 cells through GNRs loading and functionalization. Statistical analysis showed no significant differences in cytotoxicity between CPT@Au-NRs and CPT/scFv@Au-NRs, but notable effects were observed for ELT-loaded Au-NRs (see Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Overall, the data indicate that cytotoxicity is influenced by both drug concentration and exposure time, with increased toxicity corresponding to higher drug concentrations and longer exposure. Additionally, functionalizing Au-NRs with an anti-BMPR-AI antibody sometimes enhanced their cytotoxic effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCells respond more acutely to free CPT compared to drug-loaded nanorods, which release the drug at a slower rate. Notably, the CPT/ELT/scFv@Au-NRs exhibited greater cell toxicity than free CPT, ELT, or CPT/ELT@Au-NRs. This heightened toxicity is likely due to the nanomedicine's functionalization with the MBP-scFv fusion protein, which improves targeting of BMPR-AI on A549 cells. The increased accessibility of hydrophobic drugs to cells may diminish the impact of the MBP-scFv fusion protein\u0026rsquo;s targeting capabilities.\u003c/p\u003e \u003cp\u003eTo assess the cytotoxic effects of CPT and ELT, we used the MTT assay to determine their IC\u003csub\u003e50\u003c/sub\u003e values at various molar ratios (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We then calculated the combination index (CI) to evaluate whether the drug combination had a synergistic, antagonistic, or additive effect. A CI value greater than 1 indicates antagonism, less than 1 suggests synergy, and a value of 1 reflects an additive effect [43{Mirzaeinia, 2022 #1253]. The results showed that the CPT and ELT combination had a more pronounced anticancer effect than either drug alone, as evidenced by fractional inhibition (Fa) values ranging from 0.10 to 0.95 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This indicates that combining CPT and ELT is more effective at inhibiting cancer cell growth compared to using either drug individually.\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\u003eCombination index values and the cytotoxic effects of combined CPT and ELT.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug combination ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInterpretation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPT\u0026thinsp;+\u0026thinsp;ELT (IC\u003csub\u003e50\u003c/sub\u003e:1/2IC\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.49612\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPT\u0026thinsp;+\u0026thinsp;ELT (IC\u003csub\u003e50\u003c/sub\u003e:1/4IC\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.73912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPT\u0026thinsp;+\u0026thinsp;ELT (IC\u003csub\u003e50\u003c/sub\u003e:IC\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.36487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPT\u0026thinsp;+\u0026thinsp;ELT (1/2IC\u003csub\u003e50\u003c/sub\u003e:IC\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.21218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPT\u0026thinsp;+\u0026thinsp;ELT (1/4IC\u003csub\u003e50\u003c/sub\u003e:IC\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12403\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCellular uptake evaluation\u003c/h3\u003e\n\u003cp\u003eTo evaluate cellular uptake effectiveness, we used an inverted fluorescence microscope. The results demonstrated that our formulations successfully delivered the drugs into the cells, with fluorescence significantly increasing after 120 minutes (see Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). CPT\u0026rsquo;s autofluorescence allowed us to track its entry into the cells directly with the microscope. The hydrophobic nature of the compounds facilitated their easy penetration into the cells in their free form. Notably, the fluorescence observed in cells treated with CPT/ELT/scFv@GNRs confirmed the effectiveness of our targeting strategy, as the nanomedicine was taken up through specific antibody-receptor interactions.\u003c/p\u003e \u003cp\u003eCells typically absorb mesoporous silica particles non-specifically via clathrin-coated vesicles due to their siliceous nature [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, rod-shaped mesoporous silica particles interact with the cell membrane over a larger surface area, particularly along the length of the rods. This enhanced interaction significantly influences the rate and extent of cellular uptake compared to spherical nanoparticles [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Although passive targeting is less controlled and more variable in its effects on cell function, it is not as precise as targeted uptake. The MBP-scFv fusion protein, which targets BMPR-AI on the cell surface, was attached to the gold nanorods to enhance site-specific delivery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of cell apoptosis using flow cytometry\u003c/h2\u003e \u003cp\u003eFlow cytometry was employed to evaluate the combined cytotoxic effects of ELT and CPT treatments and their potential to enhance apoptosis in A549 cells. The study included treatments with PBS, free CPT, free ELT, CPT/scFv@Au-NRs, ELT/scFv@Au-NRs, and CPT/ELT/scFv@Au-NRs. Apoptosis was quantified using a dual-parameter dot plot generated through flow cytometry.\u003c/p\u003e \u003cp\u003eTo distinguish between early apoptosis, late apoptosis, and necrosis, we used Annexin V-FITC and PI double staining (see Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The total percentage of apoptotic cells was calculated by summing early and late apoptotic cells (Annexin V-FITC positive). Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e shows that 92.3% of untreated A549 cells remained viable after 72 hours. In contrast, treatment with free CPT or ELT resulted in increased apoptosis rates\u0026mdash;67.7% for CPT and 68.2% for ELT.\u003c/p\u003e \u003cp\u003eIn cells treated with CPT/scFv@Au-NRs, 59.2% were in the early apoptotic stage, indicating that this treatment induced apoptosis more effectively than free CPT. Cells exposed to free ELT were predominantly in the late apoptosis phase (66.2%), while those treated with ELT/scFv@Au-NRs showed a predominance of early apoptosis (60.0%). This notable shift, likely due to the more controlled release of drugs from the nanocarriers compared to the free drugs, highlights the precision of our findings. Significantly, A549 cells treated with CPT/ELT/scFv@Au-NRs dramatically reduced viable cells to 4.3%, with 93.4% of cells undergoing early or late apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents a cutting-edge nanocarrier system using silica-coated gold nanorods (SiO₂@Au-NRs) for the targeted delivery of ELT and CPT. The gold nanorods were functionalized with recombinant anti-BMP receptor AI and loaded with either ELT, CPT, or both. The therapeutic effects were evaluated through cytotoxicity assays in A549 cells. The SiO₂@Au-NRs nanocarrier shows great promise for delivering multiple drugs and can be tailored with various scFv proteins fused to maltose-binding protein (MBP). Nanorods coated with the MBP-scFv fusion protein exhibited a high capacity for drug loading while maintaining their ability to target specific receptors. When ELT and CPT were combined in nanorods conjugated with anti-BMP receptor AI, cellular uptake in BMP receptor-overexpressing cells was significantly enhanced, leading to increased cytotoxicity. This innovative drug delivery system holds considerable potential as a cancer treatment strategy. However, further research is required to assess the stability of SiO₂@Au-NRs and their cytotoxic effects in animal models.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully appreciate the support from Shiraz University and Tarbiat Modarres University, which were instrumental in the completion of this project. Thanks to Prof. Lloyd Ruddock for kindly providing the pMJS205 plasmid through a Material Transfer Agreement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs the lead author, Fatemeh Sabzalizadeh played a central role in conducting the research, analyzing data, and writing the manuscript. Hamed Mirshakari contributed to laboratory work, while Hamid-Reza Karbalaei Heidari aided in idea development, data review, and manuscript editing. Nediljko Budisa participated in manuscript editing and data evaluation, and Khosro Khajeh supervised the research process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Iran National Science Foundation (INSF), grant number 98017210.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions in this study are detailed in the article or Supplementary Material. For further inquiries, please contact the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e:No primary studies with animals or human subjects have been conducted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003eNo studies with human subjects\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\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.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZou B, Zhou XL, Lai SQ, Liu JC. Notch signaling and non-small cell lung cancer. Oncol Lett. 2018;15(3):3415\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmirmahani N, Mahmoodi NO, Galangash MM, Ghavidast A. 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Eur J Pharm Biopharm. 2020;156:84\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Non-small cell lung cancer, BMPR-AI, Erlotinib, Camptothecin, gold nanorods","lastPublishedDoi":"10.21203/rs.3.rs-6709389/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6709389/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNon-small cell lung carcinoma is a particularly aggressive cancer with a poor outlook. Although Erlotinib (ELT) and Camptothecin (CPT) are commonly used together in chemotherapy, their effectiveness is limited when administered as free drugs. To enhance their efficacy, we developed a novel nanomedicine consisting of gold nanorods (Au-NRs) coated with a functionalized silica network to deliver both drugs simultaneously. This strategy aims to improve cancer cell targeting, suppress cell proliferation, and induce apoptosis. The nanomedicine was further engineered with a recombinant anti-BMP receptor AI (BMPR-AI) single-chain variable fragment (scFv) fused with maltose-binding protein for targeted delivery. Successful coating and functionalization were confirmed through various analyses, including HR-TEM, EDS/EDAX, zeta potential measurements, and FT-IR. The resulting CPT/ELT/scFv@Au-NR nanomedicine effectively targeted BMPR-AI-overexpressing cancer cells, significantly inhibiting cell growth and inducing apoptosis more efficiently than the free drugs. This promising approach exhibits enhanced cytotoxic effects and holds the potential for more effective chemotherapy and future advancements in cancer treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Smart co-delivery of Erlotinib and Camptothecin using silica-coated gold nanorods functionalized with recombinant anti-BMP receptor type AI","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-06 08:20:06","doi":"10.21203/rs.3.rs-6709389/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc646b73-9997-48c0-8642-be619fb2b0cf","owner":[],"postedDate":"June 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-06T16:41:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-06 08:20:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6709389","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6709389","identity":"rs-6709389","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}