Smart biomaterials for enhancing cancer therapy by overcoming tumor hypoxia: a review.

Hypoxia is a distinctive feature of most solid tumors due to insufficient oxygen supply of the abnormal vasculature, which cannot work with the demands of the fast proliferation of cancer cells. One of the main obstacles to limiting the efficacy of cancer medicines is tumor hypoxia. Thus, oxygen is a vital parameter for controlling the efficacy of different types of cancer therapy, such as chemotherapy (CT), photodynamic therapy (PDT), photothermal therapy (PTT), immunotherapy (IT), and radiotherapy (RT). Numerous technologies have attracted much attention for enhancing oxygen distribution in humans and improving the efficacy of cancer treatment. Such technologies include treatment with hyperbaric oxygen therapy (HBO), delivering oxygen by polysaccharides (e.g., cellulose, gelatin, alginate, and silk) and other biocompatible synthetic polymers (e.g., PMMA, PLA, PVA, PVP and PCL), decreasing oxygen consumption, producing oxygen in situ in tumors, and using polymeric systems as oxygen carriers. Herein, this review provides an overview of the relationship between hypoxia in tumor cells and its role in the limitation of different cancer therapies alongside the numerous strategies for oxygen delivery using polysaccharides and other biomaterials as carriers and for oxygen generation.

Antitumor/anti-angiogenesis efficacy of epigallocatechin gallate nanoformulated with antioxidant in melanoma.

Aim: Epigallocatechin gallate (EGCG) derived from green tea has poor stability; therefore, to enhance its bioavailability and anticancer efficiency, we synthesized three different nanoformulations. We hypothesized that these three nanoformulations of EGCG (nano-EGCG) would enhance EGCG's stability and improve its anticancer and antiangiogenic activity against melanoma compared with free EGCG. Methods: We prepared nano-EGCG using a copolymerization method with the UV blocker ZnO and the antioxidants lycopene and olive oil. Results: The different nano-EGCG formulation exhibited improved EGCG stability and greater suppression of melanoma growth than free EGCG. Nanoformulation preparation methods efficiently prevented the loss of EGCG activity and are a favorable approach for the treatment of melanoma. Conclusion: Nano-EGCG formulations had enhanced stability and produced greater suppression of melanoma tumor growth and angiogenesis compared with free EGCG.

Edible Green Solvent for Optimized Catechins Extraction from Green Tea Leaves: Anti-Hypercholesterolemia.

Catechin polyphenols are the major bioactive ingredients in green tea with various human health benefits. Extraction of catechins from green tea (GTE) leaves at optimized standard conditions is still a challenging approach. An optimized, rapid, and economic extraction method is industrially needed. We hypothesized that certain extraction techniques in the presence of natural polymers and antioxidants might improve GTE catechin extraction yield and its biological activity. The effect of microwave (30-60 seconds irradiation in a typical kitchen microwave) assisted extraction (MAE) and ultrasonic assisted extraction (UAE) techniques were evaluated separately and in combination. To study the effect of the extraction solvent, nine edible green solvent combinations were investigated namely water, ascorbic acid, chitosan/ascorbic acid, carboxymethylcellulose /ascorbic acid, methylcellulose /ascorbic acid, chitosan/methylcellulose/ascorbic acid, methylcellulose, chitosan/acetic acid, and ethanol. The amounts of extracted catechins from green tea leaves were quantified with HPLC-UV. Data showed that the use of MAE & UAE technique was the optimal in producing a higher extraction yield of catechins. Chitosan/ascorbic acid was the optimized solvent with high extraction efficiencies of catechins. Studies in high fat diet fed animals demonstrated significant reduction of total cholesterol and LDL-C by GTE after 3 weeks of oral daily administration. In conclusion, efficient extraction, and stabilization of catechins from green tea leaves demonstrated a significant lowering of high fat diet-mediated elevation in blood cholesterol and LDL-C levels.

Correction: Karakus et al. Development of Triiodothyronine Polymeric Nanoparticles for Targeted Delivery in the Cardioprotection against Ischemic Insult. Biomedicines 2021, 9, 1713.

In the published manuscript [...].

Photochemoprevention of ultraviolet Beam Radiation-induced DNA damage in keratinocytes by topical delivery of nanoformulated Epigallocatechin-3-gallate.

Ultraviolet Beam (UVB) radiation is the main cause of skin cancer worldwide. Besides biocompatibility, the instability and limited skin permeability are the most challenging features of many effective photochemopreventive agents. (-)-Epigallocatechin-3-gallate (EGCG) is a natural polyphenolic compound extracted from Camellia sinensis that has been demonstrated to have antioxidant, anti-inflammatory, and anti-cancer properties. We evaluated the efficacy of three innovative EGCG nanoformulations in chemoprevention of UVB-induced DNA damage in keratinocytes. Results indicated that the EGCG nanoformulations reduced UVB-induced oxidative stress elevation and DNA damage. The nanoformulations also reduced the UVB-induced formation of pyrimidine and pyrimidone photoproducts in 2D human immortalized HaCaT keratinocytes and SKH-1 hairless mice through antioxidant effects and possibly through absorption of UVB radiation. In addition, EGCG nanoformulations inhibited UVB-induced chemokine/cytokine activation and promoted EGCG skin permeability and stability. Taken together, the results suggest the use of EGCG nanoformulations as potential natural chemopreventive agents during exposure to UVB radiation.

Silica-coated graphene compared to Si-CdSe/ZnS quantum dots: Toxicity, emission stability, and role of silica in the uptake process for imaging purposes.

Quantum dots (QDs) present a special type of nanocrystals (NCs) due to their unique optical and chemical properties. While cadmium-based QDs (Cd-QDs) have the most favorable physicochemical properties, their toxicity, instability in the aqueous phase, and loss of brightness at high temperature are some of the obstacles that prevent the wide use of Cd-QDs. Carbon-based QDs as graphene quantum dots (GQDs) represent a very promising biocompatible replacement. In the present work, we mainly focus on comparing the efficiency and uptake of GQDs and Cd-QDs for fluorescent imaging purposes and studying the effect of growing silica shell on the emission and the uptake of QDs inside living human and bacterial cells. Graphene and CdSe/ZnS QDs were prepared and encapsulated in silica to increase their emission and uptake by living cells. Moreover, we studied their photostability and cytotoxicity. The Prepared G-Si QDs showed good emission inside the cytoplasmic portion of the liver hepatocellular carcinoma cell line (HepG2) and Bacillus subtilis (B. subtilis), but they revealed lower photoluminescence (PL) intensity compared to Si-CdSe/ZnS NCs although G-Si QDs are advantageous in other aspects, i.e. possess lower toxicity and higher stability with temperature variations.

Tissue Distribution, Histopathological and Genotoxic Effects of Magnetite Nanoparticles on Ehrlich Solid Carcinoma.

Nanoparticles can potentially cause adverse effects on cellular and molecular level. The present study aimed to investigate the histopathological changes and DNA damage effects of magnetite nanoparticles (MNPs) on female albino mice model with Ehrlich solid carcinoma (ESC). Magnetite nanoparticles coated with L-ascorbic acid (size ~ 25.0 nm) were synthesized and characterized. Mice were treated with MNPs day by day, intraperitoneally (IP), intramuscularly (IM), or intratumorally (IT). Autopsy samples were taken from the solid tumor, thigh muscle, liver, kidney, lung, spleen, and brain for assessment of iron content, histopathological examination, and genotoxicity using comet assay. The liver, spleen, lung, and heart had significantly higher iron content in groups treated IP. On the other hand, tumor, muscles, and the liver had significantly higher iron content in groups treated IT. MNPs induced a significant DNA damage in IT treated ESC. While a significant DNA damage was detected in the liver of the IP treated group, but no significant DNA damage could be detected in the brain. Histopathological findings in ESC revealed a marked tumor necrosis, 50% in group injected IT but 40% in group injected IP and 20% only in untreated tumors. Other findings include inflammatory cell infiltration, dilatation, and congestion of blood vessels of different organs of treated groups in addition to appearance of metastatic cancer cells in the liver of non-treated tumor group. MNPs could have an antitumor effect but it is recommended to be injected intratumorally to be directed to the tumor tissues and reduce its adverse effects on healthy tissues.

Novel oral nano-hepatic targeted anti-PCSK9 in hypercholesterolemia.

Proprotein convertase subtilisin/kexin type 9 is a protease enzyme secreted by liver that downregulates hepatic low-density lipoprotein receptor (LDLR) by binding and chaperoning LDLR to lysosomes for degradation, causing hypercholesteremia. The development of anti-PCSK9 therapeutics attracted considerable attention for the management of cardiovascular disease risk. However, only subcutaneous injectable PCSK9 monoclonal antibodies have been FDA approved. Oral administration of small-molecule PCSK9 inhibitors has the potential to become a practical therapeutic option if achievable. In the present work, we used nanotechnological approaches to develop the first small oral molecule nano-hepatic targeted anti-PCSK9. Using high-throughput optimization and a series of evaluations, a stable water-dispersible 150-200 nm nano-encapsulated drug (named P-4) conjugated with hepatic targeting moiety was synthesized and characterized (named P-21). Pharmacodynamic (PD), pharmacokinetic (PK) and bioavailability studies were conducted using a high fat diet nutritionally induced hypercholesterolemia mouse model to evaluate the efficacy of P-21 as an anti-PCSK9 LDL-cholesterol lowering hepatic targeted nanodrug. The PD results demonstrate that P-21 in a dose-dependent manner is highly effective in lowering LDL-C by 50-90%. PK results show the maximum plasma concentration (Cmax) of P-4 was observed after 30 min of administration with 31% oral bioavailability and had a sustained longer half-life up to 24 h. In vivo safety studies in rats showed no apparent adverse effects, normal chemical biomarkers and normal histopathological findings in all P-21 treated groups at different escalating doses. Compared to the FDA-approved monoclonal antibodies, P-21 offers a more efficient, and practical treatment protocol for targeting uncontrolled hypercholesterolemia in reducing the risk of cardiovascular diseases. The present study introduced a nano-targeted drug delivery approaches for PCSK9/LDLR antagonist.

Development of Triiodothyronine Polymeric Nanoparticles for Targeted Delivery in the Cardioprotection against Ischemic Insult.

Ischemic heart disease is the main cause of death globally. Cardioprotection is the process whereby mechanisms that reduce myocardial damage, and activate protective factors, contribute to the preservation of the heart. Targeting these processes could be a new strategy in the treatment of post-ischemic heart failure (HF). Triiodothyronine (T3) and thyroxine (T4), which have multiple effects on the heart, prevent myocardial damage. This study describes the formulation, and characterization, of chemically modified polymeric nanoparticles incorporating T3, to target the thyroid hormone receptors. Modified T3 was conjugated to polylactide-co-glycolide (PLGA) to facilitate T3 delivery and restrict its nuclear translocation. Modified T3 and PLGA-T3 was characterized with 1H-NMR. The protective role of synthesized phosphocreatine (PCr) encapsulated PLGA-T3 nanoparticles (PLGA-T3/PCr NPs) and PLGA-T3 nanoparticles (PLGA-T3 NPs) in hypoxia-mediated cardiac cell insults was investigated. The results showed that PLGA-T3/PCr NPs represent a potentially new therapeutic agent for the control of tissue damage in cardiac ischemia and resuscitation.

Mercaptopurine-Loaded Sandwiched Tri-Layered Composed of Electrospun Polycaprolactone/Poly(Methyl Methacrylate) Nanofibrous Scaffolds as Anticancer Carrier with Antimicrobial and Antibiotic Features: Sandwich Configuration Nanofibers, Release Study and in vitro Bioevaluation Tests.

BACKGROUND: 6-Mercaptopurine (6-MP) is a potential anti-cancer agent which its therapeutic and limitation applicability due to its high toxicity. OBJECTIVE: Herein, 6-MP was loaded into tri-layered sandwich nanofibrous scaffold (the top layer composed of poly methyl methacrylate/polycaprolactone (PMMA/PCL), the middle layer was PCL/PMMA/6-MP, and the bottom layer was PCL/PMMA to improve its bioactivity, adjusting the release-sustainability and reduce its toxicity. METHODS: Electrospun tri-layered nanofibers composed of PCL/PMMA were utilized as nano-mats for controlling sustained drug release. Four groups of sandwich scaffold configurations were investigated with alteration of (PMMA: PCL) composition. RESULTS: The sandwich scaffold composed of 2%PCL/4%PMMA/1%6-MP showed the best miscibility, good homogeneity and produced the smoothest nanofibers and low crystallinity. All fabricated 6-MP-loaded-PCL/PMMA scaffolds exhibited antimicrobial properties on the bacterial and fungal organisms, where the cytotoxicity evaluation proved the safety of scaffolds on normal cells, even at high concentration. Scaffolds provided a sustained-drug release profile that was strongly dependent on (PCL: PMMA). As (PCL: PMMA) decreased, the sustained 6-MP release from PCL/PMMA scaffolds increased. Results established that ~18% and 20% of 6-MP were released after 23h from (4%PCL/4%PMMA/1%6-MP) and (2%PCL/4%PMMA/1%6-MP), respectively, where this release was maintained for more than 20 days. The anti-cancer activity of all fabricated scaffolds was also investigated using different cancerous cell lines (e.g., Caco-2, MDA, and HepG-2) results showed that 6-MP-loaded-nanofibrous mats have an anti-cancer effect, with a high selective index for breast cancer. We observed that viability of a cancer cell was dropped to about 10%, using nanofibers containing 2%PCL/4%PMMA/1%6-MP. CONCLUSION: Overall, the PCL: PMMA ratio and sandwich configuration imparts a tight control on long-term release profile and initial burst of 6-MP for anticancer treatment purposes.

Nanonutraceuticals: Anti-Cancer Activity and Improved Safety of Chemotherapy by Costunolide and Its Nanoformulation against Colon and Breast Cancer.

Costunolide (COS) is a sesquiterpene lactone with anticancer properties. The present study investigated the anticancer effects of COS against the human colon (HCT116) and breast (MDA-MB-231-Luc) cancer cell lines. Inhibition of cell lines viability and IC50 of COS were assessed via an MTT assay. Furthermore, the apoptotic rate was detected by assessment of Bcl2-associated X (Bax) and B-cell lymphoma 2 (Bcl2) protein levels by flow cytometry. Xenograft mice model of HCT116 and MDA-MB-231-Luc were carried out to determine the effect of COS and its nanoparticles (COS-NPs). The results demonstrated that COS inhibited the viability of HCT116 and MDA-MB-231-Luc cells, with a half maximal inhibitory concentration value (IC50) of 39.92 µM and 100.57 µM, respectively. COS significantly increased Bax and decreased Bcl2 levels in treated cells. COS and COS-NPs, in combination with doxorubicin (DOX), significantly decreased the tumor growth of HCT116 and MDA-MB-231-Luc implants in mice. Furthermore, oral administration of COS and COS-NPs significantly decreased the viable cells and increased necrotic/apoptotic cells of HCT116 and MDA-MB-231-Luc implants. Interestingly, both COS and COS-NPs protected the cardiac muscles against DOX's cardiotoxicity. The current results indicated the promising anticancer and cardiac muscles protection of COS and COS-NPs when administered with chemotherapy.

Using Zinc Oxide Nanoparticles to Improve the Color and Berry Quality of Table Grapes Cv. Crimson Seedless.

Producing high-quality table grapes is becoming a challenge in the warmer area of the world due to the global increase in temperature, which negatively affects anthocyanin biosynthesis and other fruit quality attributes. Nanotechnology is a growing field that can be a very useful tool to improve crop productivity and sustainability. The red color is one of the major fruit quality parameters that determine table grape marketability. This study aimed to investigate the role of the zinc element in improving the marketable characteristics of Crimson seedless (Vitis vinifera L.) table grape berries i.e., color, firmness, total soluble solids and sugars; besides its role in activating PAL and SOD enzymatic systems. Additionally, this paper investigated the additive advantages of zinc when applied in nanometric form. Five concentrations of zinc oxide nanoparticles, ZnO NPs (0, 25, 50, 100 and 250 ppm), were compared to zinc oxide in mineral form at a concentration of 250 ppm to investigate their effects on the marketable characteristics of Crimson seedless grape cultivar. The treatments were applied as foliar spray on three-year-old Crimson seedless vines grafted on Richter 110 rootstock grown in one of the major table grape production area in Egypt. The experiment was arranged in completely randomized block design and each vine was sprayed with five letters of the solution. The use of the lowest concentration (25 ppm) of ZnO NPs achieved the highest significant enzyme activity (PAL and SOD). Moreover, the T.S.S, sugars and anthocyanin content in berries increased significantly in association of decreasing acidity. On the other hand, the use of a 50 ppm concentration led to an increase in fruit firmness. Collectively, our data showed that 25 ppm of zinc nanoparticles improved PAL and SOD enzymes activity, improved red coloration in table grape and was more effective than the 250 ppm zinc oxide mineral form.

Thymoquinone and its nanoformulation attenuate colorectal and breast cancers and alleviate doxorubicin-induced cardiotoxicity.

Aim: To investigate the anti-cancer potential of thymoquinone (TQ) and TQ nanoparticles (TQ-NPs) and their protection against doxorubicin (DOX)-induced cardiotoxicity. Methods: TQ-NPs were prepared by double emulsion method and characterized. The efficacy of TQ and TQ-DOX was studied against HCT116 and MDA-MB-231-Luc cancer cell lines in vitro and in a xenograft tumor model. Results: TQ and TQ + DOX increased Bax levels in HCT116 cells and decreased Bcl2 levels in MDA-MB-231-Luc cells. In the xenograft model, the TQ-NPs, with an average size of 218 nm, in combination with DOX, significantly reduced tumor size. The combination of TQ or TQ-NPs with DOX significantly reduced DOX-induced cardiotoxicity. Conclusion: Data suggest the promising role of TQ and TQ-NPs alone and with DOX for anti-cancer and cardiac protection benefits.

Novel oxygen-generation from electrospun nanofibrous scaffolds with anticancer properties: synthesis of PMMA-conjugate PVP-H2O2 nanofibers, characterization, and in vitro bio-evaluation tests.

Released oxygen plays a critical role in reducing destructive tumor behavior. This study aims to utilize decomposed hydrogen peroxide as an oxygen source by conjugating it with polyvinylpyrrolidone (PVP). PVP-hydrogen peroxide complex (PHP) composed of different ratios of (PVP : H2O2) (0.5 : 1, 1 : 1, 1 : 1.5, 1 : 5, and 1 : 10) were successfully synthesized. PHP complex with a ratio of 1 : 1.5 was chosen as the optimized ratio, and it was incorporated into the polymethyl methacrylate (PMMA) nanofibrous scaffold via the electrospinning technique. Results have revealed that the PMMA-10% PHP complex provided a significant morphological structure of nanofibrous scaffolds. The mechanical properties of PMMA-10% PHP nanofibers showed the most suitable mechanical features such as Young's modulus, elongation-at-break (%), and maximum strength, in addition to the highest degree of swelling. All PHP complex scaffolds released oxygen in a sustained manner. However, the PMMA-10% PHP complex gave the highest concentration of released-oxygen with (∼8.9 mg L-1, after 2.5 h). PMMA-10% PHP nanofibers provided an ideal model for released-oxygen scaffold with anti-cancer effect and high selectivity for cancer cells, especially for breast cancer cells. Nanofibrous scaffolds with different composition revealed high cell viability for normal cells. Such outcomes support the suitability of using synthesized nanofibrous scaffolds as released-oxygen biomaterials to enhance cancer cells' sensitivity and maximize the treatment effect.

Spectroscopic and photostability study of water-soluble hypericin encapsulated with polyvinylpyrrolidone.

Hypericin has gained great attention as a powerful photosensitizing and fluorescent agent for photodynamic therapy (PDT) and fluorescence diagnosis (FD) of cancer. However, native hypericin is hydrophobic and nearly insoluble in aqueous media which hinders its photobiological activity. Herein, we demonstrate the encapsulation of hypericin and polyvinylpyrrolidone (hypericin@PVP) as an attractive class of water-soluble formula of hypericin with improved absorption and emission characteristics in water. The absorption and fluorescence properties of the water-soluble hypericin@PVP were studied at room temperature. Also, the photostability of the prepared hypericin@PVP was studied under visible light irradiation. The absorbance and emission measurements confirm the association and binding of hypericin and PVP with a binding constant (Kb) of 1.2 × 105 M-1. The interaction between hypericin and PVP in water could lead to the dissociation of aggregated hypericin into their monomeric state which is crucial for effective photobiological implementation in PDT and FD. Upon encapsulation with PVP, hypericin showed a significant increase in the fluorescence properties with an enhanced emission intensity of 300% at a PVP concentration of 1 × 10-4 M. Moreover, water-soluble hypericin@PVP demonstrated high photostability under visible light irradiation with an irradiance of 15 mW/cm2 and exposure time up to 150 min. This enhancement in the absorption, emission, and photostability of hypericin in water is related to the effects of encapsulation with PVP and the unique spectroscopic properties of the formulated hypericin@PVP.

Functionalized nano-targeted moieties in management of prostate cancer.

Multimodal properties of nanoparticles, such as simultaneously carrying drugs and/or diagnostic probes for site-specific delivery, make them excellent carriers for diagnosis and treatment of prostate cancer. Advantages are high permeability and selectivity to malignant cells to reduce systemic toxicity of chemotherapeutic drugs. Based on a review of current literature, the lack of efficient and highly specific prostate cancer cell targeting moieties is hindering successful in vivo prostate cancer-targeted drug delivery systems. Highly specific nano-targeting moieties as drug delivery vehicles might improve chemotherapeutic delivery via targeting to specific receptors expressed on the surface of prostate cancer cells. This review describes nano-targeting moieties for management of prostate cancer and its cancer stem cells. Descriptions of targeting moieties using anti-prostate-specific membrane antigen, aptamer, anti-cluster of differentiation 24/44, folic acid and other targeting strategies are highlighted. Current research results are promising and may yield development of next-generation nanoscale theragnostic targeted modalities for prostate cancer treatment.

IR-enhanced photothermal therapeutic effect of graphene magnetite nanocomposite on human liver cancer HepG2 cell model.

Background: Graphene magnetite nanocomposites (G/Fe3O4) exhibit light photothermal conversion upon enhancement by 808 nm IR laser excitation. We evaluated the cytotoxic and photothermal effects of G/Fe3O4 on a HepG2 human liver cancer cell model. Methods: Graphene nanosheets (rGO), magnetite nanoparticles (Fe3O4), and G/Fe3O4 were prepared by chemical methods and characterized using transmission electron microscopy, Raman spectroscopy, zeta analysis, and vibrating sample magnemeter. Dark and light cytotoxicity were screened with colorimetric Sulforhodamine B cell viability assay after 24 and 48 hours. DNA fragmentation and some apoptotic genes on a transcriptional RNA level expression were performed. All prepared nanomaterials were evaluated for their photothermal effect at concentrations of 10 and 50 µg/mL. The power density incident on the cells by 300 mW 808 IR diode laser was 0.597 W/cm2. Results: Treatment of HepG2 with 400 µg/mL of rGO, Fe3O4, and G/Fe3O4 showed alteration in cell morphology after 24 hours of cell treatment and revealed toxic effects on cellular DNA. Evaluation of the cytotoxic effects showed messenger RNA (mRNA) in ß-actin and Bax apoptotic genes, but no expression of mRNA of caspase-3 after 24 hours of cell exposure, suggesting the involvement of an intrinsic apoptotic caspase-independent pathway. A photothermal effect was observed for G/Fe3O4 after irradiation of the HepG2 cells. A marked decrease was found in cell viability when treated with 10 and 50 µg/mL G/Fe3O4 from 40% to 5% after 48 hours of cell treatment. Conclusion: Results indicate that G/Fe3O4 nanocomposite was effective at transformation of light into heat and is a promising candidate for cancer therapy.

Ascorbic acid prevents cellular uptake and improves biocompatibility of chitosan nanoparticles.

Chitosan nanoparticles have many applications, such as gene and drug delivery, due to their biocompatibility. Chitosan nanoparticles are currently produced by dissolution in acetic acid that affects the biocompatibility at acidic pH. Here, we synthesized and characterized chitosan (CS) and ascorbate chitosan (AsCS) nanoparticles and investigated their cytotoxic effects, internalization, and distribution in the human colon carcinoma cell line using confocal laser scanning microscopy (CLSM). The CS and AsCS nanoparticles were spherical with average particle sizes of 44±8.4nm and 87±13.6nm, respectively. CS nanoparticles were taken up by the cells and showed dose-dependent cytotoxicity. By contrast, AsCS nanoparticles were not internalized and showed no cytotoxicity. Therefore, AsCS nanoparticles are more biocompatible than CS nanoparticles and may be more suitable for extracellular drug delivery.

Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene.

BACKGROUND: Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body. AIM OF WORK: The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system. METHODS AND RESULTS: Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50). CONCLUSION: Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV.