Role of Folate Functionalized Human Serum Albumin Nano-formulation for Bleomycin Delivery on Gene Expression in Gastric Cancer Cells

Role of Folate Functionalized Human Serum Albumin Nano-formulation for Bleomycin Delivery on Gene Expression in Gastric Cancer Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of Folate Functionalized Human Serum Albumin Nano-formulation for Bleomycin Delivery on Gene Expression in Gastric Cancer Cells Ali Hamad Abd Kelkaw, Ali Hussein F Alnasraui, Sharafaldin Al-Musawi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6742375/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 Nanoparticles (NPs) are considered an effective and accurate approach for targeted drug delivery. This research focuses on the modification of human serum albumin (HSA) using folic acid (FA) to enhance the targeted delivery of bleomycin (BLM) to gastric cancer cells. The structure and properties of FA-HSA-BLM formulated nanoparticles were effectively analyzed. The methods utilized included dynamic light scattering (DLS), scanning electron microscopy (SEM), atomic force microscopy (AFM), and ultraviolet-visible spectroscopy (UV-Vis). The rate of drug release was measured. The toxicity and viability percentages of various treatments, including BLM, FA-HSA, and FA-BLM-HAS, were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Molecular docking models were created to examine the interactions between the BLM drug and AKT1, Caspace3, NF1, and P21, aiming to identify potential binding sites. The results demonstrate that the drug release is pH-dependent, exhibits high loading efficiency, and possesses sustained release capabilities in this nano-formulation. The cytotoxic effects of FA-HSA-BLM nanoparticles on SNU-5 cell lines indicate extended anticancer activity. RT-PCR was employed to assess the expression levels of the Capase3, NF1, p21, and Akt1 genes in tumor cells. Expression levels of Caspase 3 and NF1 genes were elevated, while p21 and Akt1 gene expression levels were decreased. Tumor cells (SNU-5) and healthy cells (CCL-241) exhibit no response to bare FA-HSA nanoparticles. An analysis of the results confirmed that the formulated nanoparticles (FA-BLM-HAS) exhibit significant therapeutic activity against gastric cancer, as indicated by their cell toxicity profile and the induction of apoptosis. Human Serum Albumin Nanoparticle Gastric Cancer Bleomycin Molecular Docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction BLM is one of the most effective natural anticancer medications. It has been employed as a chemotherapeutic agent in the treatment of several cancers [ 1 , 2 ]. Due to its adverse effects, primarily pulmonary damage [ 3 ], BLM's therapeutic efficacy is restricted. If lower doses could be supplied closer to the target and confined, the use of this specific anticancer treatment might be increased [ 4 , 5 ]. To generate DNA double-strand breaks (DSBs), BLM must attach to Deoxyribonucleic Acid (DNA) [ 6 ]. Several types of cancer may be treated with bleomycin, including Hodgkin's lymphoma and non-lymphoma Hodgkin's as well as testicular cancer, breast cancer, and ovarian and cervical cancer. DNA lesions known as double-strand breaks (DSBs) are regarded as the most dangerous because of their impact on genomic stability and DNA integrity. Gastric tumors are known as stomach cancer because they grow from the stomach lining and spread to other parts of the body. One of the most common kinds of stomach cancer is stomach adenocarcinomas, subdivided into many subtypes. In addition to lymphomas and mesenchymal tumors, stomach cancers may occur frequently. Treatments for various types of cancer include chemotherapy drugs, radiation therapy, and surgery, all of which might harm or kill healthy cells in the therapeutic process. Several studies have been conducted to reduce the detrimental impact of cancer therapy on healthy cells. Recent years have seen an increase in the use of biocompatible nanomaterials in the biotechnological and biomedical fields, including medication delivery, cancer therapy and bioseparation. There are many applications of nanomaterial such as sensors, solar cell water cement, drug delivery and so on [ 7 – 11 ]. Molecular docking serves as a computational method to determine the binding affinity between the drug and its target. Consequently, employing in silico and in vitro screening methods may accelerate the identification of toxicity in the tested drugs or molecules, potentially eliminating the necessity for additional phases like in vivo and preclinical studies, particularly if the results from in silico and in vitro assessments are unfavorable [ 12 , 13 ]. In silico investigations that involve docking, studies require at least two essential components: a protein/drug database and a molecular docking program. Protein and drug databases are collections that include various protein and drug structures. Nanomaterials may be effective in reducing radiation treatment adverse effects in healthy cells around malignant ones. As a result of their nanoscale characteristics and improved retention and permeability, nanoparticles, particularly natural polymeric NPs, have received considerable interest [ 14 , 15 ]. Natural materials with high biocompatibility, like protein-NPs, may be employed as suitable drug carriers because of their unique characterizations, such as amphiphilicity, surface modification capability, shelf life and biodegradability [ 16 , 17 ]. Plasma proteins, such as HSA are the most abundant parts and assist in maintaining the body's normal osmotic pressure. The HSA is classified as a nontoxic, biocompatible, and biodegradable polymeric material that may have various applications. This molecule was made of amino acids linked together by peptide bonds, which allow for facilitating the drug loading and embedding processes. Drug stability and the capacity to enter cancer cells through cellular absorption make HSA an attractive platform for the fabrication of NPs [ 18 – 21 ]. FA receptors may be found on the surfaces of many tumor cells, including kidney, lung, brain, intestine, and uterine/cervix cancers [ 22 , 23 ]. However, in healthy cells, this protein is exceedingly rare to be seen [ 24 – 28 ]; This contrast may be utilized to target cancerous cells to optimize medication administration. In this study, we employed folate-NPs to increase the delivery of BLM medication to epithelial carcinoma cells (SNU-5) in an in vitro setting. The anticancer activity of the FA-HSA-BLM NPs was tested in gastric (SNU-5) and normal (CCL-241) cells, as well as their release of the BLM drug. When we treated SNU-5 cancer cells with BLM-containing FA-HSA nanoparticles, we also studied changes in cell proliferation and mRNA expression of Capase3 and NF1 gene and their antagonists p21 and Akt1. Experimental part 2.1. Materials All materials utilized in this work such as Bleomycin, HSA, MTT powder, DMSO, Dulbecco’s Eagle media medium, distilled deionized water (DDW), penicillin/streptomycin, Fetal bovine serum (FBS), and FA were obtained from Sigma-Aldrich Company, USA. The SNU-5 cancer and CCL-241 normal cell lines were purchased from ATCC, USA. 2.2. Preparation of FA-BLM-HSA NPs: The ethanol desolvation approach [ 18 ] was utilized in the preparation of FA-functionalized HSA NPs. First, 55 mg of HSA was mixed with 10 mM of NaCl (10 mL) at 25 o C while the solution was being stirred at (800) rpm. The solution was swirled continuously for 15 minutes, after which the pH was 8.5 using 1 M NaOH while stirring for another 5 minutes under continuous stirring, adding the ethanol to HSA after changing the HSA solution into turbid (1–2) mL, at which point the ethanol was removed. To create stable HSA NPs, 10% glutaraldehyde was added to the HSA solution as a cross-linking agent. NPs were separated from the solution by centrifuging it at 14000 rpm for 15 minutes, after which they were washed with DDW. The washed NPs were then suspended in an equivalent amount of PBS to complete the experiment. FA powder was dissolved in distal water after adding NaOH. Subsequently, this solution was added to the HSA. The FA molecules were bonded electrostatically to the HSA surface; The combined solution was shaken for 60 seconds at 25 o C. The drug-loaded FA-HSA nanocarrier was synthesized after adding BLM to 1 mL FA decorated HSA and allowed to stand under continual stirring for five hours, followed by dropwise addition of ethanol. 2.3. Characterization: The researchers employed a DLS (Nanopartica, Japan) to quantify the polydispersity, surface charge, and size of NPs in the study. The NPs scale, distribution, and charge were determined using passing them through a Zeta sizer with deionized water (1 mg/1 mL) (Malvern, UK). AFM (Nanoscopy Digital) and SEM (Philips XL30) instruments were used to analyze the morphological characteristics of the samples. 2.5. UV-vis spectrophotometer: While constructing nanocomposites, it is critical to use the right mix of pharmaceuticals and to combine target agents in the right way. Using UV-vis to corroborate the results, an investigation into the combination of FA, HSA molecules, and BLM on FA-BLM-HSA NPs was carried out. The absorption range was detected at different nm (200–700) nm using a UV spectrophotometer and the absorption spectrum of these NPs was detected at different intervals (200–750) nm. 2.6. Encapsulation rate: After centrifuging the NPs at 14000 rpm for 20 minutes with the use of a UV spectrophotometer, the amount of drug-loaded NPs was calculated and the amount of free BLM was determined. The following equation was used to determine the encapsulation efficiency of the material: \(\:Encapsulation\:efficiency\:\left(\%\right)\:=\frac{Total\:amount\:of\:drug\:‐\:Free\:drug}{Initial\:amount\:of\:drug}\times\:100\) Eq. (1) 2.7. BLM release study: To calculate the released BLM from the produced Nanosystem under the FA-BLM-HSA suspension 1 mL was put into a dialysis bag and submerged in 150 mL before incubating at 37°C while being shaken. 1 ml of FA-BLM-HSA solution was sampled, freeze-dried, and added to 2 ml methanol for further analysis and testing. The quantity of BLM released from the FA-BLM-HSA formulation was determined by fluorescence spectroscopy (Varian, USA) at 450 nm using a BIO UV spectrophotometer (Varian, USA). For the calculation of BLM drug release, the equation was used: calculated by the mentioned: \(\:R=\:\frac{V\:{\sum\:}_{i}^{n-1}{C}_{i}+\:{V}_{o}{C}_{n}}{{m}_{drug}}\) Eq. (2) V is the sample volume, Ci and Cn are BLM level, V 0 is initial drug volume, R is drug release (%), the times indicated by i and n, and the BLM in NPs mass was presented via m drug . 2.8. Molecular Docking The ligand and Fe 3 O 4 @Au-CU nanoparticles were subjected to molecular docking analysis utilizing the Auto Dock 1.5.7 software, employing BCL-XL and BAK genes. The crystal structures of AKT1 (8UW9), Caspace3 (1NMQ), NF1 (3PG7) and P21 (5BYD) were acquired from the Protein Data Bank ( www.rcsb.org ), which serves as a comprehensive resource for the storage and dissemination of three-dimensional biological macromolecular structural data. The water molecules surrounding the AKT1, Caspace3, NF1, and P21 complex were eliminated. The hydrogen atoms that are necessary for the structure, along with the Kollman charges, were included in the receptor structure. The Discovery Studio Visualizer and chimeraX were employed to visualize interactions [ 29 ]. AutoDock Tools provides diverse techniques for examining the outcomes of docking simulations, including conformational similarity, visualization of the binding site and its energy, intermolecular energy, and inhibition constant [ 12 , 30 ]. 2.9. Cell culture: ATCC provided the cell lines CCL-241, a human normal gastro intestine cell line, and SNU-5, a human gastric cancer cell line (UK). The cells were grown in DMEM media with 2 mM of glutamine and 10% FBS and incubated at 5% CO 2 environment and 37°C for the duration of the experiment. 2.10. MTT assay 96-well plates with 1 × 10 5 cells per well were seeded with CCL-241 and SNU-5 cells and overnighted to ensure the cells adhering to the wells. CCL-241 cells were used as a control. After that, the media were changed to include the desired dose of BLM, FA-HSA, and FA- BLM-HSA NPs, which were dissolved in a DMEM with a concentration of 10 to 60 M in the previous step. Following the completion of 24 and 48 hours of therapy. Following the addition of an MTT solution (5 mg/mL), the toxicity was investigated after 240 minutes of treatment with the MTT solution. The media were then withdrawn from each well, adding 100 L of DMSO to each well. In this study, the absorbance was measured using the reader (Tecan, Switzerland) at 570 and 690 nm, respectively, on a wavelength of 690 nm. 2.11. RT-PCR: The SNU-5 cells were treated with bare nanocarrier, BLM, and BLM-loaded nanocarrier, seeded with 10 5 cells/mL density and grown for 48 hours before being harvested. TRIzol (Invitrogen, UK) was used to completely extract the RNA from the cells after 48 hours of treatment. Complementary DNA (cDNA) synthesis was accomplished by the cDNA Synthesis Kit, which followed the manufacturer's instructions. After incubating at 42°C for 1 hour, the reaction was carried out via bio-rad t100 thermal cycler (USA) according to the protocol, which included heating at 75°C for 5 minutes after incubating at 25°C for 6 minutes. The real-time PCR method makes direct use of cDNA. A list of primers for endogenous genes and targets created using the primer express programme can be seen in Table 1. Applied Biosystems, USA, provided the cDNA and 0.5 mL of primers in addition to the SYBR Green-I dye in the amplification procedures. The concentration of the primers in the final amount of 20 mL was 100 nM. The following procedures were followed for doing real-time PCR: The analysis of the melting curve was applied to evaluate the success of the RT-PCR, which consisted of 50 cycles starting at 95°C for 10 min, followed by 15 s at 95°C and 1 min at 60°C. 2.12. Statistical evaluation The statistical evaluation was done by applying SPSS (V: 24), and data were presented as (mean ± SEM) by ANOVA and unpaired Student's t-test with *p < 0.05. Results and Discussion 3.1. Characterization of FA-HSA-BLM NPs Over the last several years, numerous studies have investigated HSA as a potential nanocarrier for delivering anticancer medicines. The use of ethanol as a solvent may be used to manufacture such NPs without the usage of organic solvents [ 31 – 33 ]. In this experiment, HSA was dispersed in water, and methanol was used as a dehydrating material after the pH was changed to an alkaline level. Compared to previous approaches, this method is considered simple and low-cost [ 34 – 37 ]. Using DLS findings from the generated FA-BLM-HSA has a diameter size of 167 nm, which was in accordance with prior research [ 38 ]. The polydispersity of the NPs was 0.012, which indicates that there is a limited variation in the NP size (Fig. 1 ), which suggests that the NPs have great stability and are suitable for a range of biological applications. The researchers also presented the camptothecin-loaded HAS-FA with about 230 nm [ 39 ], which they believe is consistent with previous findings [ 40 ]. The results obtained by SEM and AFM were consistent with the DLS data, indicating that FA- BLM-BSA has a spherical form (Fig. 1 ). According to the results, the Encapsulation rate of FA-BLM-HSA NPs was 78%. The stability experiment measured the dimensions and polydispersity of the FA-BLM-HSA nanoformulation (see Fig. 1 ). The microscopic examination of FA-BLM-HSA is shown in Figs. 2 A and B. 3.3. UV-vis spectroscopy The NPs are subjected to further UV-vis spectroscopy testing to determine if FA, HSA, and BLM have been successfully encapsulated. The results showed that the spectrum data specifics of the energy band gaps and optical transition could be used to demonstrate that the anticancer drug bonds to the FA-HSA. The UV-vis spectra of pure FA, HSA, and BLM were considerably different from those of their NP counterparts. Each of the three absorbance spectra showed an absorbance peak at 554, 343, and 565 nm, respectively. In contrast, both a combination of free medicines and FA-BLM-HSA NPs showed an absorbance peak at 570 nm that was wide (Fig. 3 ). 3.4. Release profile As shown in Fig. 4 , at 37°C, the BLM drug release was obtained in PBS and citrate buffer but not in the absence of either of these solutions. The quantity of BLM released from FA- BLM-HSA NPs was determined by measuring the fluorescence intensity emission at various pH levels. Using drug release curves, it was discovered that when the loading nanocarrier system (FA- BLM-HSA) was treated with a PBS (pH 7.4) rather than an acidic citrate buffer at pH 5.4, the release time from the nanocarrier system (FA- BLM-HSA) was slower. Also, the in vitro release of free BLM revealed a comparable release algorithm to that seen in the laboratory (pH 7.4–5.4). The findings of the curve analysis showed that BLM released at a quicker rate at pH 5.4 than at pH 7.4 [ 41 ]. 3.5. Docking study Molecular docking is a valuable computational model for examining the interaction between protein and ligand molecules. Estimating the binding affinity between substances' interaction with a protein target can be obtained from the docking score or the binding energy [ 42 ]. Molecular docking analysis has examined the interaction between BLM drug and the receptors of two proteins associated with gastric cancer, specifically AKT1, Caspace3, NF1 and P21. To establish a correlation between the empirical data gained from in vitro and the theoretical results provided by in-silico simulations. The negative binding energy value obtained for the complexes indicates the favourable association of BLM drug with the protein. The best dock AKT1 (8uw9) with binding energy − 8.3 kcal/mol (Fig. 5 A), has a binding pattern with fourteen hydrogen bonds with ASN53, ASN54, SER56, GLN59, TRP80, GLU114, ARG200, GLN203, SER205, THR211, LYS268, TYR272, THR291, and ASP292 with bond lengths of 3.29 Å, 3.37 Å, 3.32 Å, 2.84 Å, 2.00 Å, 3.02 Å, 2.44 Å, 3.62 Å, 2.37 Å, 2.45 Å, 2.14 Å, 2.55 Å, 3.00 Å, 2.60 Å respectively and hydrophobic interactions were established in (Fig. 5 B). The binding energy of the compound dock with the protein Caspace3 (1nmq) is computed as -7.3 kcal/mol (Fig. 6 A), and binding pattern with ten hydrogen bonds THR62, HIS121, TYR204, SER205, ARG207, ASN208, SER209, LYS242, PHE250, and SER251 with bond lengths of 2.18 Å,2.31 Å, 2.28 Å, 2.39 Å, 3.12 Å, 2.88 Å, 2.28 Å, 2.24 Å, 2.38 Å, 2.17 Å, and 2.46 Å respectively and hydrophobic interactions was established in (Fig. 6 B). The binding energy of the compound dock with the protein NF1 (3pg7) is computed as -8.1 kcal/mol (Fig. 7 A), and the binding pattern with seven hydrogen bonds THR1618, LEU1675, GLY1678, LYS1680, GLY1681, LYS1683, and ARG1684 with bond lengths of 2.418 Å,2.21 Å, 2.55 Å, 2.81 Å, 2.11 Å, 2.32 Å, and 2.07 Å respectively and hydrophobic interactions were established in (Fig. 7 B). The binding energy of the compound dock with the protein P21 (5bdy) is computed as -9.3 kcal/mol (Fig. 8A), and binding pattern with fifteen hydrogen bonds ASP27, LYS54, ASN57, ASN199, GLU202, THR235, GLY237, THR256, THR264, THR265, GLY266, ASP269, ASN295, and THR307 with bond lengths of 1.87 Å,1.95 Å, 2.79 Å, 1.86 Å, 3.05 Å, 2.64 Å, 3.41 Å, 3.19 Å, 2.27 Å, 3.55 Å, 2.28 Å, 3.00 Å, 1.82 Å, 1.87 Å, 3.17 Å respectively and hydrophobic interactions were established in (Fig. 8B). 3.6. MTT assay According to Fig. 9 , the MTT test was used to determine if the FA-BLM-HSA NPs were cytotoxic to the SNU-5 and CCL-241 cell lines, and the results showed that they were. The cancer cells were treated with both empty BLM and bare FA-BLM-HSA, and even at the maximum dose of 60 µM mL1, there was no evidence of damage to the cells. It was shown that more than 80% of the cells survived after 48 hours of incubation. This showed that the treatments, BLM without FA and BLM without HSA, were cytocompatible. It was shown that cancer cells had stronger inhibitory activity when treated with the FA-BLM-HSA NPs than when treated with free BLM or bare FA-BLM-HSA NPs alone. This was in contrast to when treated with free BLM or bare FA-BLM-HSA NPs alone. To estimate the IC 50 concentration, a dose-response curve fitting of the cell viability data was performed. The FA-BLM-HSA NPs had IC50 values of 28 and 13 µM for SNU-5 cells after 24 and 48 hours, respectively, when tested in vitro. This discovery might be explained by detecting more apoptotic cells following treatment with the FA-BLM-HSA NPs, as compared to necrotic cells after treatment with the NPs. Furthermore, it was not able to distinguish between these two forms of cell death just based on the increasing loss of integrity of the plasma membrane as an indication [ 43 ], which was previously reported. The use of NPs as anticancer drug capsules has been shown to increase the uptake of anticancer medications by cells and lysosomes, resulting in increased cytotoxic activity [ 44 – 46 ]. According to one prior study, boosting the production of intracellular ROS may be responsible, at least in part, for the lethal impact of anticancer medications by increasing their production. Apoptosis and altered cell proliferation have been linked to the incubation of cancer cells with NPs, which is regulated by variations in ROS generation levels [ 47 ]. 3.7. RT- PCR and gene expression: Activation of (Capase3 and NF1) and (p21 and Akt1) were studied in the present work. Researchers have discovered that the ratio of pro-apoptotic to anti-apoptotic proteins regulates the occurrence of cellular apoptosis. N Pilco-Ferreto published a paper describing the need for Bcl-2 expression in response to BLM. Following treatment with FA-BLM-HSA NPs in SNU-5 cells, we examined the expression of the genes mentioned above compared to FA-HSA and void BLM. We discovered that the FA-BLM-HSA reduced p21 and Akt1 expression after treatment in the control group, whereas the FA-HSA had no effect (untreated). Furthermore, FA-BLM-HSA significantly increased Capase3 and NF1 gene expression compared to their baseline levels (P < 0.01). (Fig. 10 ). Conclusions Following these results, we were able to effectively build a functional FA-BLM-HSA nanocarrier system that was capable of efficiently delivering large concentrations of BLM into SNU-5 cells while maintaining the great potential for cancer treatment. The FA-BLM-HSA NPs showed excellent cellular uptake, were able to overcome chemotherapeutic resistance and were able to induce apoptosis in SNU-5 cells. The conjugates formed by combining FA-HSA with BLM have excellent activity and pH-dependent drug release, and they are safe to use. Molecular docking results show that prepared ligands with BLM drug have better binding ability with biomolecules (AKT1, Caspace3, NF1 & P21). The presence of enhanced ROS concentrations and related mitochondrial membrane modifications may be responsible for events such as increased morphological apoptotic alterations and DNA damage, among other things. However, to standardize and establish an effective treatment regime, it is vital to explain the mechanism of action of FA-BLM-HSA NPs' anti-proliferative properties. The compound functions as a novel biocompatible anticancer medication containing BLM drug and has a minimal cellular toxicity impact compared with BLM drug alone. It has the potential to be a significant contribution to cancer therapy shortly. Declarations Acknowledgements The authors appreciatively acknowledge all support provided by the Al-Qasim Green University and Applied Sciences Department, University of Technology, Baghdad-Iraq. Author Contributions: All researchers collectively contributed to this research. Funding: This study was supported by the researchers' private account and was not supported by any other institution. Data Availability: The data used in the study are available upon request from the corresponding authors. Conflict of interest The authors declare no conflicts of interest References R. 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Essam, Assessing the Toxicity of Aluminum Oxide Nanoparticles (Al2O3 NPS) Prepared by Laser Ablation Technique on Blood Components, Journal of Applied Sciences and Nanotechnology. 3 (2023) 8–17. https://doi.org/10.53293/jasn.2022.4746.1142. S. Al-Musawi, S. Albukhaty, H. Al-Karagoly, F. Almalki, Design and synthesis of multi-functional superparamagnetic core-gold shell coated with chitosan and folate nanoparticles for targeted antitumor therapy, Nanomaterials. 11 (2021) 1–14. https://doi.org/10.3390/nano11010032. S. Al-Musawi, S. Ibraheem, S.A. Mahdi, S. Albukhaty, A.J. Haider, A.A. Kadhim, K.A. Kadhim, H.A. Kadhim, H. Al-Karagoly, Smart nanoformulation based on polymeric magnetic nanoparticles and vincristine drug: A novel therapy for apoptotic gene expression in tumors, Life. 11 (2021) 1–12. https://doi.org/10.3390/life11010071. S. Abdul Mahdi, A. Ali Kadhim, S. Albukhaty, S. Nikzad, A.J. Haider, S. Ibraheem, H. Ali Kadhim, S. Al-Musawi, Gene expression and apoptosis response in hepatocellular carcinoma cells induced by biocompatible polymer/magnetic nanoparticles containing 5-fluorouracil, Electronic Journal of Biotechnology. 52 (2021) 21–29. https://doi.org/10.1016/j.ejbt.2021.04.001. M. Al-Kinani, A. Haider, S. Al-Musawi, Study the Effect of Laser Wavelength on Polymeric Metallic Nanocarrier Synthesis for Curcumin Delivery in Prostate Cancer Therapy: In Vitro Study, Journal of Applied Sciences and Nanotechnology. 1 (2021) 43–50. https://doi.org/10.53293/jasn.2021.11023. Tables Tables are available in the Supplementary Files section. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1. The schematic presentation illustrates the employed folate-NPs to increase the delivery of BLM medication to epithelial carcinoma cells (SNU-5) in an in vitro setting. 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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-6742375","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":462658834,"identity":"42232381-d4a1-49d7-816c-259cde92524c","order_by":0,"name":"Ali Hamad Abd Kelkaw","email":"","orcid":"","institution":"University of Kerbala","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Hamad Abd","lastName":"Kelkaw","suffix":""},{"id":462658835,"identity":"fd30afbd-3575-471a-bdb4-a1d57572c8f6","order_by":1,"name":"Ali Hussein F Alnasraui","email":"","orcid":"","institution":"Al-Qasim Green 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and polydispersity of the molecule.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/3b90524018ceac5ffb0dce9a.png"},{"id":83573387,"identity":"2a589614-cc89-4d00-9e20-aa7114ce1a34","added_by":"auto","created_at":"2025-05-28 18:05:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":841360,"visible":true,"origin":"","legend":"\u003cp\u003e(A) SEM and (B) AFM image of FA-BLM-HSA\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/747426c1136ef9ce180a1710.png"},{"id":83573891,"identity":"eb7b4315-2a20-4282-a979-d2353f220eba","added_by":"auto","created_at":"2025-05-28 18:21:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":662890,"visible":true,"origin":"","legend":"\u003cp\u003eThe normalized UV-vis spectra for FA, bar BLM, HSA, FA-HSA and FA-HSA-BLM NP.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/a095bcef44783bfa12aa72b0.png"},{"id":83573392,"identity":"9cbb2db4-99fb-4925-a0fd-516329b2b142","added_by":"auto","created_at":"2025-05-28 18:05:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":450617,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro release profile of FA-BLM-HSA at pH 7.4 and pH 5.4 at 37°C.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/29ea4c9961a7b7409e1f5463.png"},{"id":83573890,"identity":"c9a7f3ab-8c96-4e81-922d-38a505ffb42e","added_by":"auto","created_at":"2025-05-28 18:21:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1660845,"visible":true,"origin":"","legend":"\u003cp\u003eStructural of Molecular docking analysis for BLM with AKT1 (8UW9) Protein-ligand complex with target compound in the binding site, (A) hydrogen bonds interaction with amino acid residues (B) Hydrophobic interaction\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/65f17fa584b633fb76482117.png"},{"id":83573397,"identity":"036be5a3-9eef-4a14-98e6-78b85aa3b8a7","added_by":"auto","created_at":"2025-05-28 18:05:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1536714,"visible":true,"origin":"","legend":"\u003cp\u003eStructural of Molecular docking analysis for BLM with Caspace3 (1NMQ) Protein-ligand complex with target compound in the binding site, (A) hydrogen bonds interaction with amino acid residues (B) Hydrophobic interaction\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/c127e6d283a051db14fcb204.png"},{"id":83573403,"identity":"bbd466ec-fc8e-40fb-b4fd-5a4b1b62c1bd","added_by":"auto","created_at":"2025-05-28 18:05:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1495669,"visible":true,"origin":"","legend":"\u003cp\u003eStructural of Molecular docking analysis for BLM with NF1 (3PG7) Protein-ligand complex with target compound in the binding site, (A) hydrogen bonds interaction with amino acid residues (B) Hydrophobic interaction\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/926a7a4da424b8676df3e43e.png"},{"id":83573405,"identity":"6c1ed17f-4221-4aa6-b991-68d5c02f77e9","added_by":"auto","created_at":"2025-05-28 18:05:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1466928,"visible":true,"origin":"","legend":"\u003cp\u003eStructural of Molecular docking analysis for BLM with P21 (5BDY) Protein-ligand complex with target compound in the binding site, (A) hydrogen bonds interactionwith amino acid residues (B) Hydrophobic interaction\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/3d54e1a12394fae2a138322e.png"},{"id":83573398,"identity":"7e038abc-180f-479f-a5b6-e9f5f844d56b","added_by":"auto","created_at":"2025-05-28 18:05:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":704847,"visible":true,"origin":"","legend":"\u003cp\u003eMTT Assay for FA-BLM-HSA nanoformulation at 24 h (A), 48 h (B) and void BLM (C) \u0026amp; FA-HSA (D) at 48 h on cancer cells (SNU-5) and normal cells (CCL-241).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/b63418da1da84faee351e483.png"},{"id":83573777,"identity":"310dc60e-7b37-4700-b8bf-9c868f768f3a","added_by":"auto","created_at":"2025-05-28 18:13:56","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":388810,"visible":true,"origin":"","legend":"\u003cp\u003eRT-PCR gene expression of SNU-5 cell with void BLM, FA-HSA and FA-BLM-HSA by ANOVA and Bonferroni post-test. Values were expressed as mean±SD ***P\u0026lt;0.001, ****P\u0026lt;0.0001 for showing the differences among the groups.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/d224fe290d1b10c192604c7d.png"},{"id":83707770,"identity":"3ad4d0e1-7b1f-4ee6-a94f-50d135f6387e","added_by":"auto","created_at":"2025-05-31 13:16:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15183412,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/ad445208-b440-48d4-a5e9-c6fc67cb412c.pdf"},{"id":83573773,"identity":"cc231544-21b2-4892-8f28-46d81366900a","added_by":"auto","created_at":"2025-05-28 18:13:56","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":842266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e The schematic presentation illustrates the employed folate-NPs to increase the delivery of BLM medication to epithelial carcinoma cells (SNU-5) in an in vitro setting.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/dba2a07597cf7a9806d96cfa.png"},{"id":83573386,"identity":"399fc890-09bc-4f30-a25a-42fbf7a81dfc","added_by":"auto","created_at":"2025-05-28 18:05:56","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30843,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6742375/v1/45b8650c98de5eaeeb91b4f4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of Folate Functionalized Human Serum Albumin Nano-formulation for Bleomycin Delivery on Gene Expression in Gastric Cancer Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBLM is one of the most effective natural anticancer medications. It has been employed as a chemotherapeutic agent in the treatment of several cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to its adverse effects, primarily pulmonary damage [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], BLM's therapeutic efficacy is restricted. If lower doses could be supplied closer to the target and confined, the use of this specific anticancer treatment might be increased [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To generate DNA double-strand breaks (DSBs), BLM must attach to Deoxyribonucleic Acid (DNA) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Several types of cancer may be treated with bleomycin, including Hodgkin's lymphoma and non-lymphoma Hodgkin's as well as testicular cancer, breast cancer, and ovarian and cervical cancer. DNA lesions known as double-strand breaks (DSBs) are regarded as the most dangerous because of their impact on genomic stability and DNA integrity. Gastric tumors are known as stomach cancer because they grow from the stomach lining and spread to other parts of the body. One of the most common kinds of stomach cancer is stomach adenocarcinomas, subdivided into many subtypes. In addition to lymphomas and mesenchymal tumors, stomach cancers may occur frequently. Treatments for various types of cancer include chemotherapy drugs, radiation therapy, and surgery, all of which might harm or kill healthy cells in the therapeutic process. Several studies have been conducted to reduce the detrimental impact of cancer therapy on healthy cells. Recent years have seen an increase in the use of biocompatible nanomaterials in the biotechnological and biomedical fields, including medication delivery, cancer therapy and bioseparation. There are many applications of nanomaterial such as sensors, solar cell water cement, drug delivery and so on [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Molecular docking serves as a computational method to determine the binding affinity between the drug and its target. Consequently, employing in silico and in vitro screening methods may accelerate the identification of toxicity in the tested drugs or molecules, potentially eliminating the necessity for additional phases like in vivo and preclinical studies, particularly if the results from in silico and in vitro assessments are unfavorable [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In silico investigations that involve docking, studies require at least two essential components: a protein/drug database and a molecular docking program. Protein and drug databases are collections that include various protein and drug structures. Nanomaterials may be effective in reducing radiation treatment adverse effects in healthy cells around malignant ones. As a result of their nanoscale characteristics and improved retention and permeability, nanoparticles, particularly natural polymeric NPs, have received considerable interest [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Natural materials with high biocompatibility, like protein-NPs, may be employed as suitable drug carriers because of their unique characterizations, such as amphiphilicity, surface modification capability, shelf life and biodegradability [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Plasma proteins, such as HSA are the most abundant parts and assist in maintaining the body's normal osmotic pressure. The HSA is classified as a nontoxic, biocompatible, and biodegradable polymeric material that may have various applications. This molecule was made of amino acids linked together by peptide bonds, which allow for facilitating the drug loading and embedding processes. Drug stability and the capacity to enter cancer cells through cellular absorption make HSA an attractive platform for the fabrication of NPs [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. FA receptors may be found on the surfaces of many tumor cells, including kidney, lung, brain, intestine, and uterine/cervix cancers [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, in healthy cells, this protein is exceedingly rare to be seen [\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]; This contrast may be utilized to target cancerous cells to optimize medication administration.\u003c/p\u003e \u003cp\u003eIn this study, we employed folate-NPs to increase the delivery of BLM medication to epithelial carcinoma cells (SNU-5) in an in vitro setting. The anticancer activity of the FA-HSA-BLM NPs was tested in gastric (SNU-5) and normal (CCL-241) cells, as well as their release of the BLM drug. When we treated SNU-5 cancer cells with BLM-containing FA-HSA nanoparticles, we also studied changes in cell proliferation and mRNA expression of Capase3 and NF1 gene and their antagonists p21 and Akt1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Experimental part","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eAll materials utilized in this work such as Bleomycin, HSA, MTT powder, DMSO, Dulbecco’s Eagle media medium, distilled deionized water (DDW), penicillin/streptomycin, Fetal bovine serum (FBS), and FA were obtained from Sigma-Aldrich Company, USA. The SNU-5 cancer and CCL-241 normal cell lines were purchased from ATCC, USA.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.2. Preparation of FA-BLM-HSA NPs:\u003c/h3\u003e\n\u003cp\u003eThe ethanol desolvation approach [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] was utilized in the preparation of FA-functionalized HSA NPs. First, 55 mg of HSA was mixed with 10 mM of NaCl (10 mL) at 25 \u003csup\u003eo\u003c/sup\u003eC while the solution was being stirred at (800) rpm. The solution was swirled continuously for 15 minutes, after which the pH was 8.5 using 1 M NaOH while stirring for another 5 minutes under continuous stirring, adding the ethanol to HSA after changing the HSA solution into turbid (1–2) mL, at which point the ethanol was removed. To create stable HSA NPs, 10% glutaraldehyde was added to the HSA solution as a cross-linking agent. NPs were separated from the solution by centrifuging it at 14000 rpm for 15 minutes, after which they were washed with DDW. The washed NPs were then suspended in an equivalent amount of PBS to complete the experiment. FA powder was dissolved in distal water after adding NaOH. Subsequently, this solution was added to the HSA. The FA molecules were bonded electrostatically to the HSA surface; The combined solution was shaken for 60 seconds at 25\u003csup\u003eo\u003c/sup\u003eC. The drug-loaded FA-HSA nanocarrier was synthesized after adding BLM to 1 mL FA decorated HSA and allowed to stand under continual stirring for five hours, followed by dropwise addition of ethanol.\u003c/p\u003e\n\u003ch3\u003e2.3. Characterization:\u003c/h3\u003e\n\u003cp\u003eThe researchers employed a DLS (Nanopartica, Japan) to quantify the polydispersity, surface charge, and size of NPs in the study. The NPs scale, distribution, and charge were determined using passing them through a Zeta sizer with deionized water (1 mg/1 mL) (Malvern, UK). AFM (Nanoscopy Digital) and SEM (Philips XL30) instruments were used to analyze the morphological characteristics of the samples.\u003c/p\u003e\n\u003ch3\u003e2.5. UV-vis spectrophotometer:\u003c/h3\u003e\n\u003cp\u003eWhile constructing nanocomposites, it is critical to use the right mix of pharmaceuticals and to combine target agents in the right way. Using UV-vis to corroborate the results, an investigation into the combination of FA, HSA molecules, and BLM on FA-BLM-HSA NPs was carried out. The absorption range was detected at different nm (200–700) nm using a UV spectrophotometer and the absorption spectrum of these NPs was detected at different intervals (200–750) nm.\u003c/p\u003e\n\u003ch3\u003e2.6. Encapsulation rate:\u003c/h3\u003e\n\u003cp\u003eAfter centrifuging the NPs at 14000 rpm for 20 minutes with the use of a UV spectrophotometer, the amount of drug-loaded NPs was calculated and the amount of free BLM was determined. The following equation was used to determine the encapsulation efficiency of the material:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:Encapsulation\\:efficiency\\:\\left(\\%\\right)\\:=\\frac{Total\\:amount\\:of\\:drug\\:‐\\:Free\\:drug}{Initial\\:amount\\:of\\:drug}\\times\\:100\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;(1)\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7. BLM release study:\u003c/h2\u003e \u003cp\u003eTo calculate the released BLM from the produced Nanosystem under the FA-BLM-HSA suspension 1 mL was put into a dialysis bag and submerged in 150 mL before incubating at 37°C while being shaken. 1 ml of FA-BLM-HSA solution was sampled, freeze-dried, and added to 2 ml methanol for further analysis and testing. The quantity of BLM released from the FA-BLM-HSA formulation was determined by fluorescence spectroscopy (Varian, USA) at 450 nm using a BIO UV spectrophotometer (Varian, USA). For the calculation of BLM drug release, the equation was used: calculated by the mentioned:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:R=\\:\\frac{V\\:{\\sum\\:}_{i}^{n-1}{C}_{i}+\\:{V}_{o}{C}_{n}}{{m}_{drug}}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;(2)\u003c/p\u003e \u003cp\u003eV is the sample volume, Ci and Cn are BLM level, V\u003csub\u003e0\u003c/sub\u003e is initial drug volume, R is drug release (%), the times indicated by i and n, and the BLM in NPs mass was presented via m\u003csub\u003edrug\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.8. Molecular Docking\u003c/h3\u003e\n\u003cp\u003eThe ligand and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@Au-CU nanoparticles were subjected to molecular docking analysis utilizing the Auto Dock 1.5.7 software, employing BCL-XL and BAK genes. The crystal structures of AKT1 (8UW9), Caspace3 (1NMQ), NF1 (3PG7) and P21 (5BYD) were acquired from the Protein Data Bank ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.rcsb.org\u003c/span\u003e\u003c/span\u003e ), which serves as a comprehensive resource for the storage and dissemination of three-dimensional biological macromolecular structural data. The water molecules surrounding the AKT1, Caspace3, NF1, and P21 complex were eliminated. The hydrogen atoms that are necessary for the structure, along with the Kollman charges, were included in the receptor structure. The Discovery Studio Visualizer and chimeraX were employed to visualize interactions [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. AutoDock Tools provides diverse techniques for examining the outcomes of docking simulations, including conformational similarity, visualization of the binding site and its energy, intermolecular energy, and inhibition constant [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003e2.9. Cell culture:\u003c/h3\u003e\n\u003cp\u003eATCC provided the cell lines CCL-241, a human normal gastro intestine cell line, and SNU-5, a human gastric cancer cell line (UK). The cells were grown in DMEM media with 2 mM of glutamine and 10% FBS and incubated at 5% CO\u003csub\u003e2\u003c/sub\u003e environment and 37°C for the duration of the experiment.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10. MTT assay\u003c/h2\u003e\n \u003cp\u003e96-well plates with 1 × 10\u003csup\u003e5\u003c/sup\u003e cells per well were seeded with CCL-241 and SNU-5 cells and overnighted to ensure the cells adhering to the wells. CCL-241 cells were used as a control. After that, the media were changed to include the desired dose of BLM, FA-HSA, and FA- BLM-HSA NPs, which were dissolved in a DMEM with a concentration of 10 to 60 M in the previous step. Following the completion of 24 and 48 hours of therapy. Following the addition of an MTT solution (5 mg/mL), the toxicity was investigated after 240 minutes of treatment with the MTT solution. The media were then withdrawn from each well, adding 100 L of DMSO to each well. In this study, the absorbance was measured using the reader (Tecan, Switzerland) at 570 and 690 nm, respectively, on a wavelength of 690 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11. RT-PCR:\u003c/h2\u003e\n \u003cp\u003eThe SNU-5 cells were treated with bare nanocarrier, BLM, and BLM-loaded nanocarrier, seeded with 10\u003csup\u003e5\u003c/sup\u003e cells/mL density and grown for 48 hours before being harvested. TRIzol (Invitrogen, UK) was used to completely extract the RNA from the cells after 48 hours of treatment. Complementary DNA (cDNA) synthesis was accomplished by the cDNA Synthesis Kit, which followed the manufacturer's instructions. After incubating at 42°C for 1 hour, the reaction was carried out via bio-rad t100 thermal cycler (USA) according to the protocol, which included heating at 75°C for 5 minutes after incubating at 25°C for 6 minutes. The real-time PCR method makes direct use of cDNA. A list of primers for endogenous genes and targets created using the primer express programme can be seen in Table 1. Applied Biosystems, USA, provided the cDNA and 0.5 mL of primers in addition to the SYBR Green-I dye in the amplification procedures. The concentration of the primers in the final amount of 20 mL was 100 nM. The following procedures were followed for doing real-time PCR: The analysis of the melting curve was applied to evaluate the success of the RT-PCR, which consisted of 50 cycles starting at 95°C for 10 min, followed by 15 s at 95°C and 1 min at 60°C.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e2.12. Statistical evaluation\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003cp\u003eThe statistical evaluation was done by applying SPSS (V: 24), and data were presented as (mean ± SEM) by ANOVA and unpaired Student's t-test with *p \u0026lt; 0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003ch2\u003e3.1. Characterization of FA-HSA-BLM NPs\u003c/h2\u003e\u003cp\u003eOver the last several years, numerous studies have investigated HSA as a potential nanocarrier for delivering anticancer medicines. The use of ethanol as a solvent may be used to manufacture such NPs without the usage of organic solvents [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this experiment, HSA was dispersed in water, and methanol was used as a dehydrating material after the pH was changed to an alkaline level. Compared to previous approaches, this method is considered simple and low-cost [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Using DLS findings from the generated FA-BLM-HSA has a diameter size of 167 nm, which was in accordance with prior research [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. The polydispersity of the NPs was 0.012, which indicates that there is a limited variation in the NP size (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), which suggests that the NPs have great stability and are suitable for a range of biological applications. The researchers also presented the camptothecin-loaded HAS-FA with about 230 nm [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e], which they believe is consistent with previous findings [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. The results obtained by SEM and AFM were consistent with the DLS data, indicating that FA- BLM-BSA has a spherical form (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). According to the results, the Encapsulation rate of FA-BLM-HSA NPs was 78%.\u003c/p\u003e\u003cp\u003eThe stability experiment measured the dimensions and polydispersity of the FA-BLM-HSA nanoformulation (see Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The microscopic examination of FA-BLM-HSA is shown in Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and B.\u003c/p\u003e\u003ch2\u003e3.3. UV-vis spectroscopy\u003c/h2\u003e\u003cp\u003eThe NPs are subjected to further UV-vis spectroscopy testing to determine if FA, HSA, and BLM have been successfully encapsulated. The results showed that the spectrum data specifics of the energy band gaps and optical transition could be used to demonstrate that the anticancer drug bonds to the FA-HSA. The UV-vis spectra of pure FA, HSA, and BLM were considerably different from those of their NP counterparts. Each of the three absorbance spectra showed an absorbance peak at 554, 343, and 565 nm, respectively. In contrast, both a combination of free medicines and FA-BLM-HSA NPs showed an absorbance peak at 570 nm that was wide (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003e3.4. Release profile\u003c/h2\u003e\u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, at 37°C, the BLM drug release was obtained in PBS and citrate buffer but not in the absence of either of these solutions. The quantity of BLM released from FA- BLM-HSA NPs was determined by measuring the fluorescence intensity emission at various pH levels. Using drug release curves, it was discovered that when the loading nanocarrier system (FA- BLM-HSA) was treated with a PBS (pH 7.4) rather than an acidic citrate buffer at pH 5.4, the release time from the nanocarrier system (FA- BLM-HSA) was slower. Also, the in vitro release of free BLM revealed a comparable release algorithm to that seen in the laboratory (pH 7.4–5.4). The findings of the curve analysis showed that BLM released at a quicker rate at pH 5.4 than at pH 7.4 [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e3.5. Docking study\u003c/h2\u003e\u003cp\u003eMolecular docking is a valuable computational model for examining the interaction between protein and ligand molecules. Estimating the binding affinity between substances' interaction with a protein target can be obtained from the docking score or the binding energy [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. Molecular docking analysis has examined the interaction between BLM drug and the receptors of two proteins associated with gastric cancer, specifically AKT1, Caspace3, NF1 and P21. To establish a correlation between the empirical data gained from in vitro and the theoretical results provided by in-silico simulations. The negative binding energy value obtained for the complexes indicates the favourable association of BLM drug with the protein. The best dock AKT1 (8uw9) with binding energy − 8.3 kcal/mol (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), has a binding pattern with fourteen hydrogen bonds with ASN53, ASN54, SER56, GLN59, TRP80, GLU114, ARG200, GLN203, SER205, THR211, LYS268, TYR272, THR291, and ASP292 with bond lengths of 3.29 Å, 3.37 Å, 3.32 Å, 2.84 Å, 2.00 Å, 3.02 Å, 2.44 Å, 3.62 Å, 2.37 Å, 2.45 Å, 2.14 Å, 2.55 Å, 3.00 Å, 2.60 Å respectively and hydrophobic interactions were established in (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). The binding energy of the compound dock with the protein Caspace3 (1nmq) is computed as -7.3 kcal/mol (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA), and binding pattern with ten hydrogen bonds THR62, HIS121, TYR204, SER205, ARG207, ASN208, SER209, LYS242, PHE250, and SER251 with bond lengths of 2.18 Å,2.31 Å, 2.28 Å, 2.39 Å, 3.12 Å, 2.88 Å, 2.28 Å, 2.24 Å, 2.38 Å, 2.17 Å, and 2.46 Å respectively and hydrophobic interactions was established in (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). The binding energy of the compound dock with the protein NF1 (3pg7) is computed as -8.1 kcal/mol (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA), and the binding pattern with seven hydrogen bonds THR1618, LEU1675, GLY1678, LYS1680, GLY1681, LYS1683, and ARG1684 with bond lengths of 2.418 Å,2.21 Å, 2.55 Å, 2.81 Å, 2.11 Å, 2.32 Å, and 2.07 Å respectively and hydrophobic interactions were established in (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). The binding energy of the compound dock with the protein P21 (5bdy) is computed as -9.3 kcal/mol (Fig. 8A), and binding pattern with fifteen hydrogen bonds ASP27, LYS54, ASN57, ASN199, GLU202, THR235, GLY237, THR256, THR264, THR265, GLY266, ASP269, ASN295, and THR307 with bond lengths of 1.87 Å,1.95 Å, 2.79 Å, 1.86 Å, 3.05 Å, 2.64 Å, 3.41 Å, 3.19 Å, 2.27 Å, 3.55 Å, 2.28 Å, 3.00 Å, 1.82 Å, 1.87 Å, 3.17 Å respectively and hydrophobic interactions were established in (Fig. 8B).\u003c/p\u003e\u003ch2\u003e3.6. MTT assay\u003c/h2\u003e\u003cp\u003eAccording to Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, the MTT test was used to determine if the FA-BLM-HSA NPs were cytotoxic to the SNU-5 and CCL-241 cell lines, and the results showed that they were. The cancer cells were treated with both empty BLM and bare FA-BLM-HSA, and even at the maximum dose of 60 µM mL1, there was no evidence of damage to the cells. It was shown that more than 80% of the cells survived after 48 hours of incubation. This showed that the treatments, BLM without FA and BLM without HSA, were cytocompatible. It was shown that cancer cells had stronger inhibitory activity when treated with the FA-BLM-HSA NPs than when treated with free BLM or bare FA-BLM-HSA NPs alone. This was in contrast to when treated with free BLM or bare FA-BLM-HSA NPs alone. To estimate the IC\u003csub\u003e50\u003c/sub\u003e concentration, a dose-response curve fitting of the cell viability data was performed. The FA-BLM-HSA NPs had IC50 values of 28 and 13 µM for SNU-5 cells after 24 and 48 hours, respectively, when tested in vitro.\u003c/p\u003e\u003cp\u003eThis discovery might be explained by detecting more apoptotic cells following treatment with the FA-BLM-HSA NPs, as compared to necrotic cells after treatment with the NPs. Furthermore, it was not able to distinguish between these two forms of cell death just based on the increasing loss of integrity of the plasma membrane as an indication [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e], which was previously reported. The use of NPs as anticancer drug capsules has been shown to increase the uptake of anticancer medications by cells and lysosomes, resulting in increased cytotoxic activity [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAccording to one prior study, boosting the production of intracellular ROS may be responsible, at least in part, for the lethal impact of anticancer medications by increasing their production. Apoptosis and altered cell proliferation have been linked to the incubation of cancer cells with NPs, which is regulated by variations in ROS generation levels [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e3.7. RT- PCR and gene expression:\u003c/h2\u003e\u003cp\u003eActivation of (Capase3 and NF1) and (p21 and Akt1) were studied in the present work. Researchers have discovered that the ratio of pro-apoptotic to anti-apoptotic proteins regulates the occurrence of cellular apoptosis. N Pilco-Ferreto published a paper describing the need for Bcl-2 expression in response to BLM. Following treatment with FA-BLM-HSA NPs in SNU-5 cells, we examined the expression of the genes mentioned above compared to FA-HSA and void BLM. We discovered that the FA-BLM-HSA reduced p21 and Akt1 expression after treatment in the control group, whereas the FA-HSA had no effect (untreated). Furthermore, FA-BLM-HSA significantly increased Capase3 and NF1 gene expression compared to their baseline levels (P \u0026lt; 0.01). (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eFollowing these results, we were able to effectively build a functional FA-BLM-HSA nanocarrier system that was capable of efficiently delivering large concentrations of BLM into SNU-5 cells while maintaining the great potential for cancer treatment. The FA-BLM-HSA NPs showed excellent cellular uptake, were able to overcome chemotherapeutic resistance and were able to induce apoptosis in SNU-5 cells. The conjugates formed by combining FA-HSA with BLM have excellent activity and pH-dependent drug release, and they are safe to use. Molecular docking results show that prepared ligands with BLM drug have better binding ability with biomolecules (AKT1, Caspace3, NF1 \u0026amp; P21). The presence of enhanced ROS concentrations and related mitochondrial membrane modifications may be responsible for events such as increased morphological apoptotic alterations and DNA damage, among other things. However, to standardize and establish an effective treatment regime, it is vital to explain the mechanism of action of FA-BLM-HSA NPs' anti-proliferative properties.\u003c/p\u003e\u003cp\u003eThe compound functions as a novel biocompatible anticancer medication containing BLM drug and has a minimal cellular toxicity impact compared with BLM drug alone. It has the potential to be a significant contribution to cancer therapy shortly.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors appreciatively acknowledge all support provided by the Al-Qasim Green University and Applied Sciences Department, University of Technology, Baghdad-Iraq.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e All researchers collectively contributed to this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was supported by the researchers\u0026apos; private account and was not supported by any other institution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e The data used in the study are available upon request from the corresponding authors.\u003c/p\u003e\n\u003cp\u003eConflict of interest The authors declare no conflicts of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eR. 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Al-Musawi, Study the Effect of Laser Wavelength on Polymeric Metallic Nanocarrier Synthesis for Curcumin Delivery in Prostate Cancer Therapy: In Vitro Study, Journal of Applied Sciences and Nanotechnology. 1 (2021) 43\u0026ndash;50. https://doi.org/10.53293/jasn.2021.11023.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\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":"Human Serum Albumin, Nanoparticle, Gastric Cancer, Bleomycin, Molecular Docking","lastPublishedDoi":"10.21203/rs.3.rs-6742375/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6742375/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanoparticles (NPs) are considered an effective and accurate approach for targeted drug delivery. This research focuses on the modification of human serum albumin (HSA) using folic acid (FA) to enhance the targeted delivery of bleomycin (BLM) to gastric cancer cells. The structure and properties of FA-HSA-BLM formulated nanoparticles were effectively analyzed. The methods utilized included dynamic light scattering (DLS), scanning electron microscopy (SEM), atomic force microscopy (AFM), and ultraviolet-visible spectroscopy (UV-Vis). The rate of drug release was measured. The toxicity and viability percentages of various treatments, including BLM, FA-HSA, and FA-BLM-HAS, were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Molecular docking models were created to examine the interactions between the BLM drug and AKT1, Caspace3, NF1, and P21, aiming to identify potential binding sites. The results demonstrate that the drug release is pH-dependent, exhibits high loading efficiency, and possesses sustained release capabilities in this nano-formulation. The cytotoxic effects of FA-HSA-BLM nanoparticles on SNU-5 cell lines indicate extended anticancer activity. RT-PCR was employed to assess the expression levels of the Capase3, NF1, p21, and Akt1 genes in tumor cells. Expression levels of Caspase 3 and NF1 genes were elevated, while p21 and Akt1 gene expression levels were decreased. Tumor cells (SNU-5) and healthy cells (CCL-241) exhibit no response to bare FA-HSA nanoparticles. An analysis of the results confirmed that the formulated nanoparticles (FA-BLM-HAS) exhibit significant therapeutic activity against gastric cancer, as indicated by their cell toxicity profile and the induction of apoptosis.\u003c/p\u003e","manuscriptTitle":"Role of Folate Functionalized Human Serum Albumin Nano-formulation for Bleomycin Delivery on Gene Expression in Gastric Cancer Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-28 18:05:51","doi":"10.21203/rs.3.rs-6742375/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":"3549a51a-b008-431a-ac46-4015a77323ef","owner":[],"postedDate":"May 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-31T13:08:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-28 18:05:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6742375","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6742375","identity":"rs-6742375","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}