Synthesis, characterization, and molecular docking analysis of a novel nanocarrier for gene therapy in renal cell carcinoma

Synthesis, characterization, and molecular docking analysis of a novel nanocarrier for gene therapy in renal cell carcinoma | 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 Synthesis, characterization, and molecular docking analysis of a novel nanocarrier for gene therapy in renal cell carcinoma Yalda Asnaashari Kahnouji, Elaheh Mosaddegh, Fatemeh Tabibzadeh, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8882182/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 Today, nanotechnology has emerged as a promising approach in biomedical fields such as diagnosis and treatment of disease. Gene therapy is a technique that can treat a deficiency by sending a gene into the targeted cell instead of using drugs and surgery and can cause the least harm to humans. The combination of gene therapy techniques and nanotechnology opens a new way to improve clinical outcomes. The present study aimed to develop a high-potential carrier for gene delivery by synthesizing Eggshell/Citric Acid/Chitosan nanocomposites using the co-precipitation method. The FTIR spectrum confirmed the chemical structure of the prepared nanocomposite. TEM analysis revealed plate-like structures with an average diameter of 300–400 nm and DLS analysis showed the average hydrodynamic size of the synthesized product in the range of 700–4000 nm. In silico molecular docking using the MVD method in Schrodinger software was done and predicted favorable interactions between synthesized nanocomposite and enzyme F218V AtRCCR with − 137.39 KJmol − 1 for inhibition the active site of F218V AtRCCR, serving this nanocomposite as an appropriate carrier for gene delivery and potential product for in vivo studies to renal cell carcinoma treatment. Nanocomposites molecular docking gene therapy biocompatible materials chitosan Renal cell carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction These days, Nanotechnology provides new opportunities for researchers in various fields of science and technology. Nanomaterials can have disparate applications, due to their small size in the range of 1-1000 nm, high stability, proper conductivity, high carrier capacity, and optical properties [ 1 , 2 ]. They can simplify major improvements in the detection, diagnosis, and treatment of human cancers [ 3 ]. Additionally, nanomaterials are actively developed for in vivo applications, biomolecular profiling of cancer biomarkers, and targeted gene and drug delivery [ 3 , 4 ]. Generally, drug delivery systems can be classified into five recognized generations [ 5 ], presented in Table 1 . The first generation includes medications like tablets, capsules, etc. the second generation comprises repeat action and prolonged action, the third generation consists of the osmotically and swelling-controlled system, the fourth generation includes the targeted drug delivery system and the emerging fifth generation refers to gene therapy, nanorobots, etc [ 5 , 6 ]. Table 1 The generations of drug delivery systems Generations of Drug Delivery Systems First Generation Second Generation Third Generation Fourth Generation Fifth Generation Tablets and Capsules Repeat action tablet Osmotically controlled system Targeted drug delivery Gene therapy Suspension Prolong action tablet Swelling-controlled system Modulated drug delivery Nanorobots Emulsion Enteric-coated tablet Diffusion controlled system Self-regulated drug delivery Long-term delivery system Among all the methods, gene therapy is a special technique that uses therapeutic genes to prevent or cure disease [ 7 , 8 ]. This technique can treat the deficiency by sending a gene to the target cell instead of using drugs and surgery and may cause the least harm to humans [ 7 – 9 ]. These days, the interaction of small molecules with DNA has received much attention. It is the subject of interest in many research fields such as biochemistry, medicinal chemistry, life science, cancer therapy, etc [ 10 , 11 ]. Because these interactions can be the basis of many intracellular processes and induce predictable changes in DNA transcriptions and replications [ 12 , 13 ], these predictions can be used in the study of cell death, cell proliferation, gene mutations, cancer causes, and treatment [ 4 , 12 – 14 ]. DNA-binding compounds may have a wide spectrum of latent anticancer, antivirus, or carcinogenic effects [ 12 , 13 , 15 , 16 ], making it the target molecule for many drugs especially antitumor and anticancer drugs [ 17 , 18 ]. The covalent binding between the drug and DNA is irreversible and causes cell death due to the complete inhibition of DNA processes. Drugs with the non-covalent mode of interaction such as groove binders and intercalators are less cytotoxic than DNA-covalent agents and are reversible [ 19 , 20 ]. Intercalation involves π-π interactions between the planar aromatic moiety of the complex and the stacked aromatic planes of the nitrogen bases of DNA without breaking up the hydrogen bonds between the DNA bases [ 21 ]. An intercalator can insert between DNA base pairs deeply causing a change in the nucleotide structure and perturbation of DNA replication and transcription [ 22 ]. Examining the interactions of compounds with DNA can be used to design more effective drugs [ 12 , 15 ], study the changes in DNA structure due to reactions with different compounds [ 15 ], and even study protein-nucleic acid structures. Most of the available drugs cause a lot of damage to the healthy cells of the body and the cells of the target tissue [ 3 , 23 ]. These side effects limit the use of these drugs and significantly reduce their effectiveness in controlling the disease [ 15 , 23 ]. Nowadays, with the emergence of drug resistance and serious side effects, various chemotherapy drugs for cancer have become life-threatening and a major cause of death all over the world [ 3 , 14 – 16 , 23 , 24 ]. Since the free DNA can be rapidly degraded by nucleases in the blood nucleases and also due to the negative charge of the naked DNA and cell membrane, the repulsive electrostatic interactions between them can limit the entry of DNA into the cells [ 25 – 27 ]. In general, there are two types of vectors for gene transfer: viral and nonviral methods [ 27 ]. Viral vectors have major disadvantages such as difficulty in production and quality control, and low safety due to toxicity and immune responses [ 27 – 30 ]. Non-viral vectors have several advantages over viral vectors, such as improved safety, high gene loading capacity, simple preparation, and no immunogenicity [ 27 , 31 , 32 ]. Thus, the viral gene delivery systems have three main components, such as a plasmid-based gene expression system to control the function of a gene in the target cell, a gene encoding a therapeutic protein, and a gene delivery system that controls the delivery of the gene expression plasmid to a specific location within the body [ 33 – 37 ]. Renal cell carcinoma (RCC) is one of the top 10 most prevalent cancers globally, encompassing a diverse range of tumors originating from renal tubular epithelial cells [ 38 , 39 ]. Over the past 20 years, significant developments in the histopathological and molecular characterization of RCC have resulted in substantial changes to the disease's classification [ 39 , 40 ]. Clear cell RCC (ccRCC) [ 41 ], papillary RCC (pRCC) [ 42 ], and Chromophore RCC (chRCC) [ 43 ] are the major subtypes with ≥ 5% incidence. The remaining subtypes have a combined incidence of less than 1% making them extremely unusual. [ 44 ], and a tumor is classed as unclassified RCC (uRCC; ~4% overall incidence) [ 45 ] Suppose it does not match any subtype in the diagnostic criteria. The bulk of kidney cancer deaths are caused by the most prevalent subtype, ccRCC. [ 40 ]. Tumors with non-clear cell histology have been categorized as "nccRCC" for clinical trial viability due to the prevalence of clear cell histology in metastatic illness (83–88%) [ 46 , 47 ]. Additionally, the overt complexity of intra- and inter-tumor heterogeneity in ccRCC has been shown by recent cancer genomic studies. [ 48 , 49 ], which may be a factor in the varied clinical outcomes seen. Treatment options for localized RCC include ablation, which involves using heat or cold to destroy the cancerous tissue, and partial or radical nephrectomy, which involves removing the kidney. [ 50 ], or active surveillance, which involves routine radiographic examinations to track the development of tumors [ 51 ]. About 30% of patients with ccRCC with localized illness later develop metastases, which require systemic therapy and are associated with significant mortality even with nephrectomy performed to be curative. Although targeted treatments against the mTOR and VEGF pathways have been established, most patients eventually experience progression despite variable responses to treatment [ 40 ]. Recently, various nanomaterials including ceramic, iron oxide, and titanium oxide nanoparticles and polymeric nanomaterials have had wide applications in the clinical and biomedical fields [ 52 – 54 ]. Eggshell (ES) may be used as a new biocompatible material that is capable of displaying many applications in various fields [ 55 , 56 ] including drug delivery and biomedical purposes [ 57 ], the likelihood of bone replacement [ 55 ], as a vigorous catalyst [ 56 , 58 , 59 ], polymer industries, production of various nanocomposites [ 60 ], etc. This substance has a mesoporous structure in the nanoscale [ 56 , 59 , 61 ] and can enhance its properties and applications in collaborating with other materials. The eggshell waste consists of 90% carbonate and has better properties in an exceedingly composite compared to mineral carbonate [ 60 ]. Chitosan (Cs) is one of the most commonly used substances to generate valuable compounds [ 62 ]. Chitosan is a possible polymer created from chitin under alkaline conditions [ 61 ], and this substrate is the most abundant aminopolysaccharide [ 61 – 63 ]. The chemical and electrochemical binding of chitosan to metal ions leads to the stabilization of the compounds [ 64 , 65 ]. Due to chitosan's high biocompatibility, low toxicity, biodegradability, antibacterial properties, etc., this material is used in medicine such as tissue engineering, and drug and gene delivery [ 64 , 66 ]. It should be mentioned that chitosan can provide a strong binding with plasmids to adequately protect nucleic acids from nuclease degradation in the blood [ 4 , 67 ]. Today, computational methods play a fundamental role in various fields [ 68 , 69 ]. Molecular docking is one of these methods based on bioinformatics that has been an important technique for drug discovery [ 70 ]. During the docking process, two key steps are crucial: 1) recognizing the structure of the ligand and characterization of its location and orientation within designated sites, and 2) assessing the strength of the binding interaction. [ 70 , 71 ]. In molecular docking simulations, the focus is on predicting the non-covalent binding modes of small molecules (ligands) within the active site cavities of the target macromolecules (receptors), such as proteins or DNA [ 70 , 72 , 73 ]. Molecular docking typically employs two main strategies. One uses computer simulations to assess how well the ligand fits and interacts with the target receptor by calculating its energy levels in various positions. The second approach employs a technique to analyze the degree of shape complementarity between the ligands and the target molecule's binding pocket. [ 70 , 74 ]. The aim of this procedure is the production of a stable structure with enhanced specificity and potential efficacy leading to the development of more effective drugs. [ 75 ]. In recent years, extensive research has been conducted to explore improved sufficient structures for drug and gene delivery. Render et al. synthesized eggshell-derived calcium carbonate (CaCO 3 ) nanoparticles to develop the enteric drug delivery system. [ 76 ]. Iravani Kashkouli et al. synthesized the Fe 3 O 4 /chitosan biopolymer as a novel nanocarrier for targeted gene delivery and it was a highly efficient gene carrier with potential applications in cancer therapy. [ 4 ]. Jayasree et al. synthesized carbonated calcium-deficient hydroxyapatite (ECCDHA) nanoparticles from eggshell wastes using a simple method as a potential candidate as a carrier system for drug delivery. [ 77 ]. Iravani Kashkouli et al. investigated the synthesis of a novel functionalized chitosan nanocarrier as an efficient gene delivery system. [ 66 ]. Muthu et al. synthesized mesoporous carbonated hydroxyapatite using eggshell waste for developing biomedical applications like drug/protein delivery, bone fillers, and tissue engineering [ 78 ]. Atabaki et al. synthesized Fe 3 O 4 /chitosan nanocomposite with potential applications in drug delivery systems [ 79 ]. So, in the current study, eggshell/Citric Acid/Chitosan (ES/CA/Cs) nanocomposites were synthesized using the mild co-precipitation method, this composite has the potential properties for biomedical applications due to the presence of organic carbonate in eggshell and also chitosan biopolymer [ 59 , 66 , 76 ]. According to the previously mentioned literature, both eggshell and chitosan could have a strong potential carrier system in one combination for drug/gene delivery. The structure, shape, and size of the product nanoparticles were confirmed and evaluated by FTIR, TEM, and DLS analysis. The binding of the ES/Citric Acid/Chitosan nanocomposites with enzyme F218V AtRCCR was modeled by molecular docking using the MVD method by Schrödinger software and the in silico studies showed that this product could be used for in vivo studies, as suitable nanocarrier in renal cell carcinoma treatment. Scheme 1 presents a schematic structure of synthesized nanocomposite. 2. Experimental Section 2.1. Materials and Instrument The topological structure was performed by transmission electron microscopy (TEM) using a CM120 device (Philips, France) prepared on copper grids and operated at 31X-680KX and for one hour at 20°C. Fourier transforms infrared (FTIR) analysis was performed on the sample by the KBr technique (Bruker, Germany). The hydrodynamic size of the structure was performed by dynamic light scattering (DLS) using a Nano DS Dual scattering device (CILAS, France). Molecular docking studies were performed using the MVD method by Schrödinger software on a Corei-5 system (Schrödinger, United States). All chemical materials used in this research were prepared of analytical grade and purchased from Merck (Germany). 2.2. Synthesis of Eggshell/Citric Acid /Chitosan To synthesize the ES/Citric Acid/Chitosan nanocomposites, 0.5 g eggshell powder was dissolved in 20 ml water and was sonicated for 30 min and stirred then 1 g Citric Acid was added to it to bring the pH to 2.5 and then was stirred for 24 hours at room temperature. The next day 0.1 g chitosan was dissolved in 25 ml water and added to the solution drop by drop, again let stir for 24 hours. Finally, the resulting solution was washed and stored for further study. 2.3. Molecular Ducking After downloading the 3D structure of the enzyme and the compounds from the PDB and PubChem databases respectively (Fig. 1 (a, b)), they are minimized in terms of energy and structure using Chem3D. Molecular docking is performed using the MVD method on a Core-i5 computer to improve the interaction between the enzyme active site and composite structure. The components of the synthesized nanocomposite and the enzyme molecules were optimized and selected as inputs in MVD software. Then the molecular cavities of the enzyme were analyzed to determine the best-desired cavity (up to 5 holes were identified). Docking was done as moldock score and using the moldock Sean algorithm, the number of interactions was 10 and it was placed in the center of the active site as X: 3.34, Y: 2.87, Z: -1.46. After docking using MVD, it was used Schrödinger software to show the connection of the amino acid with the active site of F218V AtRCCR. 3. Results and discussion The novel ES/CA/Cs nanocarrier was successfully prepared via the co-precipitation method. Firstly, the CA was added to the eggshell solution at room temperature. After 24 hours of stirring, chitosan solution was added dropwise to the solution, and again after 2 hours of stirring, the final nanocomposites were made. Eventually, the shape, size, structure, and molecular docking of synthesized nanocomposites were studied. 3.1. Transmission Electron Microscopy Figure 2 indicates the transmission electron microscopy (TEM) that presents the topological structure and average particle size of ES/CA/Cs nanocomposites. Figure 2 (a-d) shows the plate-like form of particles on a scale of 70, 100, 200, and 500 nm respectively. The presence of CA and chitosan biopolymer forms the eggshell nanoparticles as smooth and flat plates shown in Fig. 2 (a, b). Additionally, chitosan biopolymer and CA play a major role in the well-dispersity and stability of synthesized nanocomposites, making the solution suitable for further work (Fig. 2 (d)). So, the average particle size of synthesized nanocomposite derived from TEM analysis is about 300–400 nm and the strong stability of the solution with this size is extraordinary. 3.2. FTIR analysis Fourier transform infrared spectroscopy (FTIR) spectrum using the KBr method in the range of 500–4000 cm − 1 is illustrated in Fig. 3 . According to this figure, the spectrum reveals several broad bands between 3327 and 3456 cm − 1 due to the overlapping stretching vibration of OH groups of citric acid and chitosan with N-H groups of chitosan [ 1 , 2 , 4 , 80 , 81 ]. The peak at 1557 cm − 1 can be assigned to the bending vibration of -N-H bands in chitosan [ 83 , 84 ]. The stretching vibration of the C = O group in the CA structure appeared at above 1700 cm − 1 [ 1 , 80 , 81 ]. The bands at 599 and 837 cm − 1 are referred to as the Citric Acid [ 81 , 82 ], and the bands at about 870, 1074 also 2938 cm − 1 are referred to as the CaCO 3 structure in the sample [ 56 , 59 , 83 ]. 3.3. DLS analysis The ES/CA/Cs nanocomposite was synthesized by the co-precipitation method, washed, and prepared for DLS analysis. As expected, the washed particles were polydispersed, and their hydrodynamic sizes ranged from the minimum diameter of 449 nm, where the majority of them were found within the range of 700–4000 nm (Fig. 4 ). So accordingly, it could be concluded that during the synthesis of nanocomposites and after washing them, the obvious layers of water molecules were formed around the particles due to the mesoporous nature of nano eggshells with the strong hydrogen bonding. So the hydrodynamic sizes of particles shifted to larger sizes. 3.4. Ducking Results The study of docking of some compounds on the active site of F218V AtRCCR (Fig. 5 ) indicates that amino acids such as Lys 147, Glu 262, Ile 266, Ser 145, and Val 259 are in bond with the active site of F218V AtRCCR (Fig. 6 ). The results show that the synthesized compound is capable of inhibiting the active site of F218V AtRCCR for in-silico studies. The amount of this inhibition energy varies from − 137.39 KJmol − 1 . The connection energy determines the connection between the compound and the enzyme's active site. If this number is lower, it indicates a strong bonding and the compound reveals strong and effective interaction with the enzyme. Conclusion This study aimed to synthesize sufficient nanocomposite as a carrier for in vivo studies. The main challenge was the binding interaction of this product with enzyme F218V AtRCCR. So, in this study, the ES/CA/Cs nanocarrier was successfully synthesized using the co-precipitation method, and the structure was confirmed through FTIR analysis. The average particle size was measured at approximately 300–400 nm, with a hydrodynamic size ranging from 700 to 4000 nm, respectively. Molecular docking was employed to evaluate the interaction between the synthesized nanocomposite and F218V AtRCCR. The theoretical in silico results revealed a favorable interaction between the product and F218V AtRCCR with a -137.39 KJmol − 1 energy for inhibition of the active site of F218V AtRCCR, serving it as an adequate carrier for gene delivery with potential applications for in vivo studies to renal cell carcinoma. Declarations Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. Funding Declaration The authors received no financial support for the research, Authorship, or publication of this article. Consent to Publish Declaration: not applicable Consent to Participate Declaration: not applicable Ethics Declaration: not applicable Author Contribution Y. Asnaashari Kahnouji wrote the original draft and was responsible for methodology, investigation and data analysis, E. 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Render D, Samuel T, King H, Vig M, Jeelani S, Babu RJ, Rangari V. (2016) Biomaterial-derived calcium carbonate nanoparticles for enteric drug delivery. J Nanomater 2016:3170248. https://doi.org/10.1155/2016/3170248 Jayasree R, Madhumathi K, Rana D, Ramalingam M, Nankar RP, Doble M, Kumar T. Development of egg shell derived carbonated apatite nanocarrier system for drug delivery. J Nanosci Nanotechnol. 2018;18:2318–24. https://doi.org/10.1166/jnn.2018.14377 . Muthu D, Kumar GS, Gowri M, Prasath M, Viswabaskaran V, Kattimani V, Girija E. Rapid synthesis of eggshell derived hydroxyapatite with nanoscale characteristics for biomedical applications. Ceram Int. 2022;48:1326–39. https://doi.org/10.1016/j.ceramint.2021.09.217 . Atabaki H. Synthesis of iron oxide magnetic nanoparticles and chitosan biopolymer in aqueous solutions. Inorg Chem Commun. 2024;162:112161. https://doi.org/10.1016/j.inoche.2024.112161 . Singh D, Gautam RK, Kumar R, Shukla BK, Shankar V, Krishna V. Citric acid coated magnetic nanoparticles: synthesis, characterization and application in removal of Cd (II) ions from aqueous solution. Water Process Eng. 2014;4:233–41. https://doi.org/10.1016/j.jwpe.2014.10.005 . Dheybm MA, Abdul Aziz A, Jameel MS, Noqta OA, Khaniabadi PM, Mehrdel B. Simple rapid stabilization method through citric acid modification for magnetite nanoparticles. Sci Rep. 2020;10:10793. https://doi.org/10.1038/s41598-020-67869-8 . Pimpang P, Sumang R, Choopun S. Effect of concentration of citric acid on size and optical properties of fluorescence graphene quantum dots prepared by tuning carbonization degree. Chiang Mai J Sci. 2018;45:2005. Mosaddegh E, Torkzadeh-Mahani M, Hassankhani A. Synthesis and characterization of acetamidotetrazole-grafted magnetic chitosan biopolymer as a novel non-virus vector for targeted gene delivery into HECK-293T cells. Iran J Chem Chem Eng. 2024;43:1302–13. https://doi.org/10.30492/ijcce.2024.1973567.5719 . Mosaddegh E, Torkzadeh-Mahani M. Synthesis and characterization of biocompatible chitosan/aminotetrazol nanoparticles as a novel nanocarier for gene delivery. Modares J Biotechnol. 2021;12:123–36. Tizo MS, Blanco LAV, Cagas ACQ, Cruz BRBD, Encoy JC, Gunting JV, Arazo RO, Mabayo VIF. Efficiency of calcium carbonate from eggshells as an adsorbent for cadmium removal in aqueous solution. Sustain Environ Res. 2018;28:326–32. https://doi.org/10.1016/j.serj.2018.09.002 . Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files floatimage1.png Scheme 1 The chemical structure of ES/CA/Cs nanocomposite 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. 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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-8882182","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601053192,"identity":"4ad7d211-0aff-41cb-a7b6-cbe3b0a46e81","order_by":0,"name":"Yalda Asnaashari Kahnouji","email":"","orcid":"","institution":"Graduate University of Advanced Technology","correspondingAuthor":false,"prefix":"","firstName":"Yalda","middleName":"Asnaashari","lastName":"Kahnouji","suffix":""},{"id":601053193,"identity":"ad8ce3a8-291c-4e96-946f-8e92323ce0aa","order_by":1,"name":"Elaheh 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Enzyme and the (b) ES/CA/Cs structure\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8882182/v1/821d57a3b7c0f277aaef7ff6.png"},{"id":104204431,"identity":"f78d0b46-e31c-4c11-80bc-c73bba980704","added_by":"auto","created_at":"2026-03-09 06:33:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2426947,"visible":true,"origin":"","legend":"\u003cp\u003eTEM image of synthesized ES/CA/Cs nanocomposites\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8882182/v1/97535158b8d7fdaea1cab83a.png"},{"id":104204430,"identity":"d1f717d5-0eca-4a99-8fb2-b1e18bc0c564","added_by":"auto","created_at":"2026-03-09 06:33:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41661,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR analysis of synthesized ES/CA/Cs nanocomposites\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8882182/v1/874436553f4137d1a2d7acbf.png"},{"id":104403939,"identity":"aa89cdd3-4f58-44ec-ada2-87f5cf7c887a","added_by":"auto","created_at":"2026-03-11 12:19:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24163,"visible":true,"origin":"","legend":"\u003cp\u003eDLS analysis of synthesized ES/CA/Cs nanocomposite\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8882182/v1/78feb1959becd2f9930b42c9.png"},{"id":104204434,"identity":"382d1665-cfd4-47ed-8246-0f89f924b684","added_by":"auto","created_at":"2026-03-09 06:33:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":224750,"visible":true,"origin":"","legend":"\u003cp\u003eThe active site of Enzyme F218V AtRCCR\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8882182/v1/e7ff7fbf8d63e7b3e3dfad57.png"},{"id":104204435,"identity":"c5641781-d8af-4711-9b03-f05dcf34e04f","added_by":"auto","created_at":"2026-03-09 06:33:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":267217,"visible":true,"origin":"","legend":"\u003cp\u003eThe interaction of amino acids with the active site of enzyme F218V AtRCCR (Blue and red dotted lines indicate the hydrogen bonding and electrostatic interactions respectively)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8882182/v1/4fce6f52f0c8c19b34019c55.png"},{"id":107522177,"identity":"5677f4d3-0413-4b03-b881-4055d19ba155","added_by":"auto","created_at":"2026-04-22 09:13:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3391288,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8882182/v1/8e5d7b7c-5b63-40b0-bf14-c71ff6227f30.pdf"},{"id":104204429,"identity":"1348be7b-684e-4765-85fe-5551f2dc8a06","added_by":"auto","created_at":"2026-03-09 06:33:34","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":128386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e The chemical structure of ES/CA/Cs nanocomposite\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8882182/v1/d5e1feef7a617b3d50cfe552.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis, characterization, and molecular docking analysis of a novel nanocarrier for gene therapy in renal cell carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThese days, Nanotechnology provides new opportunities for researchers in various fields of science and technology. Nanomaterials can have disparate applications, due to their small size in the range of 1-1000 nm, high stability, proper conductivity, high carrier capacity, and optical properties [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. They can simplify major improvements in the detection, diagnosis, and treatment of human cancers [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, nanomaterials are actively developed for in vivo applications, biomolecular profiling of cancer biomarkers, and targeted gene and drug delivery [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGenerally, drug delivery systems can be classified into five recognized generations [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The first generation includes medications like tablets, capsules, etc. the second generation comprises repeat action and prolonged action, the third generation consists of the osmotically and swelling-controlled system, the fourth generation includes the targeted drug delivery system and the emerging fifth generation refers to gene therapy, nanorobots, etc [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe generations of drug delivery systems\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eGenerations of Drug Delivery Systems\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFirst Generation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSecond Generation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eThird Generation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eFourth Generation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eFifth Generation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTablets and Capsules\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRepeat action tablet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOsmotically controlled system\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTargeted drug delivery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGene therapy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuspension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProlong action tablet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSwelling-controlled system\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModulated drug delivery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNanorobots\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmulsion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnteric-coated tablet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiffusion controlled system\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSelf-regulated drug delivery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLong-term delivery system\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAmong all the methods, gene therapy is a special technique that uses therapeutic genes to prevent or cure disease [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This technique can treat the deficiency by sending a gene to the target cell instead of using drugs and surgery and may cause the least harm to humans [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These days, the interaction of small molecules with DNA has received much attention. It is the subject of interest in many research fields such as biochemistry, medicinal chemistry, life science, cancer therapy, etc [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Because these interactions can be the basis of many intracellular processes and induce predictable changes in DNA transcriptions and replications [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], these predictions can be used in the study of cell death, cell proliferation, gene mutations, cancer causes, and treatment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. DNA-binding compounds may have a wide spectrum of latent anticancer, antivirus, or carcinogenic effects [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], making it the target molecule for many drugs especially antitumor and anticancer drugs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The covalent binding between the drug and DNA is irreversible and causes cell death due to the complete inhibition of DNA processes. Drugs with the non-covalent mode of interaction such as groove binders and intercalators are less cytotoxic than DNA-covalent agents and are reversible [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Intercalation involves π-π interactions between the planar aromatic moiety of the complex and the stacked aromatic planes of the nitrogen bases of DNA without breaking up the hydrogen bonds between the DNA bases [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. An intercalator can insert between DNA base pairs deeply causing a change in the nucleotide structure and perturbation of DNA replication and transcription [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Examining the interactions of compounds with DNA can be used to design more effective drugs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], study the changes in DNA structure due to reactions with different compounds [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and even study protein-nucleic acid structures. Most of the available drugs cause a lot of damage to the healthy cells of the body and the cells of the target tissue [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These side effects limit the use of these drugs and significantly reduce their effectiveness in controlling the disease [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Nowadays, with the emergence of drug resistance and serious side effects, various chemotherapy drugs for cancer have become life-threatening and a major cause of death all over the world [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince the free DNA can be rapidly degraded by nucleases in the blood nucleases and also due to the negative charge of the naked DNA and cell membrane, the repulsive electrostatic interactions between them can limit the entry of DNA into the cells [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In general, there are two types of vectors for gene transfer: viral and nonviral methods [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Viral vectors have major disadvantages such as difficulty in production and quality control, and low safety due to toxicity and immune responses [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Non-viral vectors have several advantages over viral vectors, such as improved safety, high gene loading capacity, simple preparation, and no immunogenicity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, the viral gene delivery systems have three main components, such as a plasmid-based gene expression system to control the function of a gene in the target cell, a gene encoding a therapeutic protein, and a gene delivery system that controls the delivery of the gene expression plasmid to a specific location within the body [\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRenal cell carcinoma (RCC) is one of the top 10 most prevalent cancers globally, encompassing a diverse range of tumors originating from renal tubular epithelial cells [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Over the past 20 years, significant developments in the histopathological and molecular characterization of RCC have resulted in substantial changes to the disease's classification [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Clear cell RCC (ccRCC) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], papillary RCC (pRCC) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and Chromophore RCC (chRCC) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] are the major subtypes with \u0026ge;\u0026thinsp;5% incidence. The remaining subtypes have a combined incidence of less than 1% making them extremely unusual. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and a tumor is classed as unclassified RCC (uRCC; ~4% overall incidence) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] Suppose it does not match any subtype in the diagnostic criteria. The bulk of kidney cancer deaths are caused by the most prevalent subtype, ccRCC. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Tumors with non-clear cell histology have been categorized as \"nccRCC\" for clinical trial viability due to the prevalence of clear cell histology in metastatic illness (83\u0026ndash;88%) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Additionally, the overt complexity of intra- and inter-tumor heterogeneity in ccRCC has been shown by recent cancer genomic studies. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], which may be a factor in the varied clinical outcomes seen. Treatment options for localized RCC include ablation, which involves using heat or cold to destroy the cancerous tissue, and partial or radical nephrectomy, which involves removing the kidney. [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], or active surveillance, which involves routine radiographic examinations to track the development of tumors [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. About 30% of patients with ccRCC with localized illness later develop metastases, which require systemic therapy and are associated with significant mortality even with nephrectomy performed to be curative. Although targeted treatments against the mTOR and VEGF pathways have been established, most patients eventually experience progression despite variable responses to treatment [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, various nanomaterials including ceramic, iron oxide, and titanium oxide nanoparticles and polymeric nanomaterials have had wide applications in the clinical and biomedical fields [\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Eggshell (ES) may be used as a new biocompatible material that is capable of displaying many applications in various fields [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] including drug delivery and biomedical purposes [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], the likelihood of bone replacement [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], as a vigorous catalyst [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], polymer industries, production of various nanocomposites [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], etc. This substance has a mesoporous structure in the nanoscale [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] and can enhance its properties and applications in collaborating with other materials. The eggshell waste consists of 90% carbonate and has better properties in an exceedingly composite compared to mineral carbonate [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Chitosan (Cs) is one of the most commonly used substances to generate valuable compounds [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Chitosan is a possible polymer created from chitin under alkaline conditions [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], and this substrate is the most abundant aminopolysaccharide [\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The chemical and electrochemical binding of chitosan to metal ions leads to the stabilization of the compounds [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Due to chitosan's high biocompatibility, low toxicity, biodegradability, antibacterial properties, etc., this material is used in medicine such as tissue engineering, and drug and gene delivery [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. It should be mentioned that chitosan can provide a strong binding with plasmids to adequately protect nucleic acids from nuclease degradation in the blood [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eToday, computational methods play a fundamental role in various fields [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Molecular docking is one of these methods based on bioinformatics that has been an important technique for drug discovery [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. During the docking process, two key steps are crucial: 1) recognizing the structure of the ligand and characterization of its location and orientation within designated sites, and 2) assessing the strength of the binding interaction. [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. In molecular docking simulations, the focus is on predicting the non-covalent binding modes of small molecules (ligands) within the active site cavities of the target macromolecules (receptors), such as proteins or DNA [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Molecular docking typically employs two main strategies. One uses computer simulations to assess how well the ligand fits and interacts with the target receptor by calculating its energy levels in various positions. The second approach employs a technique to analyze the degree of shape complementarity between the ligands and the target molecule's binding pocket. [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. The aim of this procedure is the production of a stable structure with enhanced specificity and potential efficacy leading to the development of more effective drugs. [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, extensive research has been conducted to explore improved sufficient structures for drug and gene delivery. Render et al. synthesized eggshell-derived calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) nanoparticles to develop the enteric drug delivery system. [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Iravani Kashkouli et al. synthesized the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/chitosan biopolymer as a novel nanocarrier for targeted gene delivery and it was a highly efficient gene carrier with potential applications in cancer therapy. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Jayasree et al. synthesized carbonated calcium-deficient hydroxyapatite (ECCDHA) nanoparticles from eggshell wastes using a simple method as a potential candidate as a carrier system for drug delivery. [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Iravani Kashkouli et al. investigated the synthesis of a novel functionalized chitosan nanocarrier as an efficient gene delivery system. [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Muthu et al. synthesized mesoporous carbonated hydroxyapatite using eggshell waste for developing biomedical applications like drug/protein delivery, bone fillers, and tissue engineering [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Atabaki et al. synthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/chitosan nanocomposite with potential applications in drug delivery systems [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSo, in the current study, eggshell/Citric Acid/Chitosan (ES/CA/Cs) nanocomposites were synthesized using the mild co-precipitation method, this composite has the potential properties for biomedical applications due to the presence of organic carbonate in eggshell and also chitosan biopolymer [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. According to the previously mentioned literature, both eggshell and chitosan could have a strong potential carrier system in one combination for drug/gene delivery. The structure, shape, and size of the product nanoparticles were confirmed and evaluated by FTIR, TEM, and DLS analysis. The binding of the ES/Citric Acid/Chitosan nanocomposites with enzyme F218V AtRCCR was modeled by molecular docking using the MVD method by Schr\u0026ouml;dinger software and the in silico studies showed that this product could be used for in vivo studies, as suitable nanocarrier in renal cell carcinoma treatment. Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents a schematic structure of synthesized nanocomposite.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.1. Materials and Instrument\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe topological structure was performed by transmission electron microscopy (TEM) using a CM120 device (Philips, France) prepared on copper grids and operated at 31X-680KX and for one hour at 20\u0026deg;C. Fourier transforms infrared (FTIR) analysis was performed on the sample by the KBr technique (Bruker, Germany). The hydrodynamic size of the structure was performed by dynamic light scattering (DLS) using a Nano DS Dual scattering device (CILAS, France). Molecular docking studies were performed using the MVD method by Schr\u0026ouml;dinger software on a Corei-5 system (Schr\u0026ouml;dinger, United States). All chemical materials used in this research were prepared of analytical grade and purchased from Merck (Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis of Eggshell/Citric Acid /Chitosan\u003c/h2\u003e \u003cp\u003eTo synthesize the ES/Citric Acid/Chitosan nanocomposites, 0.5 g eggshell powder was dissolved in 20 ml water and was sonicated for 30 min and stirred then 1 g Citric Acid was added to it to bring the pH to 2.5 and then was stirred for 24 hours at room temperature. The next day 0.1 g chitosan was dissolved in 25 ml water and added to the solution drop by drop, again let stir for 24 hours. Finally, the resulting solution was washed and stored for further study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Molecular Ducking\u003c/h2\u003e \u003cp\u003eAfter downloading the 3D structure of the enzyme and the compounds from the PDB and PubChem databases respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a, b)), they are minimized in terms of energy and structure using Chem3D. Molecular docking is performed using the MVD method on a Core-i5 computer to improve the interaction between the enzyme active site and composite structure. The components of the synthesized nanocomposite and the enzyme molecules were optimized and selected as inputs in MVD software. Then the molecular cavities of the enzyme were analyzed to determine the best-desired cavity (up to 5 holes were identified). Docking was done as moldock score and using the moldock Sean algorithm, the number of interactions was 10 and it was placed in the center of the active site as X: 3.34, Y: 2.87, Z: -1.46. After docking using MVD, it was used Schr\u0026ouml;dinger software to show the connection of the amino acid with the active site of F218V AtRCCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eThe novel ES/CA/Cs nanocarrier was successfully prepared via the co-precipitation method. Firstly, the CA was added to the eggshell solution at room temperature. After 24 hours of stirring, chitosan solution was added dropwise to the solution, and again after 2 hours of stirring, the final nanocomposites were made. Eventually, the shape, size, structure, and molecular docking of synthesized nanocomposites were studied.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Transmission Electron Microscopy\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicates the transmission electron microscopy (TEM) that presents the topological structure and average particle size of ES/CA/Cs nanocomposites. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a-d) shows the plate-like form of particles on a scale of 70, 100, 200, and 500 nm respectively. The presence of CA and chitosan biopolymer forms the eggshell nanoparticles as smooth and flat plates shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a, b). Additionally, chitosan biopolymer and CA play a major role in the well-dispersity and stability of synthesized nanocomposites, making the solution suitable for further work (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (d)). So, the average particle size of synthesized nanocomposite derived from TEM analysis is about 300\u0026ndash;400 nm and the strong stability of the solution with this size is extraordinary.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2. FTIR analysis\u003c/h2\u003e \u003cp\u003eFourier transform infrared spectroscopy (FTIR) spectrum using the KBr method in the range of 500\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. According to this figure, the spectrum reveals several broad bands between 3327 and 3456 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e due to the overlapping stretching vibration of OH groups of citric acid and chitosan with N-H groups of chitosan [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. The peak at 1557 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be assigned to the bending vibration of -N-H bands in chitosan [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. The stretching vibration of the C\u0026thinsp;=\u0026thinsp;O group in the CA structure appeared at above 1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. The bands at 599 and 837 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are referred to as the Citric Acid [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e], and the bands at about 870, 1074 also 2938 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are referred to as the CaCO\u003csub\u003e3\u003c/sub\u003e structure in the sample [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3. DLS analysis\u003c/h2\u003e \u003cp\u003eThe ES/CA/Cs nanocomposite was synthesized by the co-precipitation method, washed, and prepared for DLS analysis. As expected, the washed particles were polydispersed, and their hydrodynamic sizes ranged from the minimum diameter of 449 nm, where the majority of them were found within the range of 700\u0026ndash;4000 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). So accordingly, it could be concluded that during the synthesis of nanocomposites and after washing them, the obvious layers of water molecules were formed around the particles due to the mesoporous nature of nano eggshells with the strong hydrogen bonding. So the hydrodynamic sizes of particles shifted to larger sizes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Ducking Results\u003c/h2\u003e \u003cp\u003eThe study of docking of some compounds on the active site of F218V AtRCCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) indicates that amino acids such as Lys 147, Glu 262, Ile 266, Ser 145, and Val 259 are in bond with the active site of F218V AtRCCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results show that the synthesized compound is capable of inhibiting the active site of F218V AtRCCR for in-silico studies. The amount of this inhibition energy varies from \u0026minus;\u0026thinsp;137.39 KJmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The connection energy determines the connection between the compound and the enzyme's active site. If this number is lower, it indicates a strong bonding and the compound reveals strong and effective interaction with the enzyme.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study aimed to synthesize sufficient nanocomposite as a carrier for in vivo studies. The main challenge was the binding interaction of this product with enzyme F218V AtRCCR. So, in this study, the ES/CA/Cs nanocarrier was successfully synthesized using the co-precipitation method, and the structure was confirmed through FTIR analysis. The average particle size was measured at approximately 300\u0026ndash;400 nm, with a hydrodynamic size ranging from 700 to 4000 nm, respectively. Molecular docking was employed to evaluate the interaction between the synthesized nanocomposite and F218V AtRCCR. The theoretical in silico results revealed a favorable interaction between the product and F218V AtRCCR with a -137.39 KJmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e energy for inhibition of the active site of F218V AtRCCR, serving it as an adequate carrier for gene delivery with potential applications for in vivo studies to renal cell carcinoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no financial support for the research, Authorship, or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish Declaration:\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate Declaration:\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declaration:\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY. Asnaashari Kahnouji wrote the original draft and was responsible for methodology, investigation and data analysis, E. Mosaddegh supervised the project, conceptualized the study and contributed to manuscript revision. F. Tabibzadeh assisted with the investigation and data analysis. M. Salarvand was responsible for simulation and data analysis. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data generated during the current study, including experimental synthesis and characterization of the compound as well as molecular docking simulations, are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMikelashvili V, Kekutia S, Markhulia J, Saneblidze L, Maisuradze N, Kriechbaum M, Alm\u0026aacute;sy L. 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Sustain Environ Res. 2018;28:326\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.serj.2018.09.002\u003c/span\u003e\u003cspan address=\"10.1016/j.serj.2018.09.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\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":"Nanocomposites, molecular docking, gene therapy, biocompatible materials, chitosan, Renal cell carcinoma","lastPublishedDoi":"10.21203/rs.3.rs-8882182/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8882182/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eToday, nanotechnology has emerged as a promising approach in biomedical fields such as diagnosis and treatment of disease. Gene therapy is a technique that can treat a deficiency by sending a gene into the targeted cell instead of using drugs and surgery and can cause the least harm to humans. The combination of gene therapy techniques and nanotechnology opens a new way to improve clinical outcomes. The present study aimed to develop a high-potential carrier for gene delivery by synthesizing Eggshell/Citric Acid/Chitosan nanocomposites using the co-precipitation method. The FTIR spectrum confirmed the chemical structure of the prepared nanocomposite. TEM analysis revealed plate-like structures with an average diameter of 300\u0026ndash;400 nm and DLS analysis showed the average hydrodynamic size of the synthesized product in the range of 700\u0026ndash;4000 nm. In silico molecular docking using the MVD method in Schrodinger software was done and predicted favorable interactions between synthesized nanocomposite and enzyme F218V AtRCCR with \u0026minus;\u0026thinsp;137.39 KJmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for inhibition the active site of F218V AtRCCR, serving this nanocomposite as an appropriate carrier for gene delivery and potential product for in vivo studies to renal cell carcinoma treatment.\u003c/p\u003e","manuscriptTitle":"Synthesis, characterization, and molecular docking analysis of a novel nanocarrier for gene therapy in renal cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 06:33:29","doi":"10.21203/rs.3.rs-8882182/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":"4e7925c9-f2f4-4ac0-bc1c-2e09d7a91ab0","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T09:09:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 06:33:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8882182","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8882182","identity":"rs-8882182","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}