Anticancer drug delivery system based on starch-coated zinc oxide nanoparticles for targeted delivery of gemcitabine

Abstract

One of the urgent needs of modern medical science is the development of drug carriers with good bioavailability and fewer side effects than existing drugs. In the present invention, zinc oxide nanoparticles were produced through a green synthesis method based on wild barberry extract and were coated and functionalized with starch polymer and folic acid to increase fluidity and targeted release, and then loaded with the anticancer drug gemcitabine. The physicochemical properties of the synthesized nanoparticles were investigated by ultraviolet-visible spectroscopy (UV-Vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). Subsequently, the drug release rate from the synthesized nanoparticles was studied in vitro. Also, their antibacterial, antioxidant, and anticancer activities were evaluated. The synthesized nanostructures were mainly spherical with an average size of less than 70 nm. In vitro drug release studies demonstrated sustained and controlled drug release from the synthesized nanoparticles. The results of MTT assay showed that gemcitabine loaded in synthetic nanostructures had higher biocompatibility and anticancer activity compared to free gemcitabine. Based on these findings, the synthesized nanostructures could be a promising candidate for use as a targeted drug delivery system to suppress cancer cells and reduce drug side effects.

Description

Description of the invention Title of the invention (as stated in the declaration) Anticancer drug delivery system based on starch-coated zinc oxide nanoparticles for targeted delivery of gemcitabine Technical background of the relevant invention This invention is aimed at developing biocompatible nanostructures related to the fields of nanomedicine, nanobiotechnology, and healthcare industries, which can be used as a targeted drug delivery system. Technical problem and stating the objectives of the invention In recent years, the application of nanotechnology to treat a wide range of cancers has received much attention. Many current chemotherapy methods are based on alkylating agents, which are often unable to produce a complete anticancer response. These methods also cannot distinguish between cancer and normal cells, resulting in systemic toxicity and undesirable side effects. One of these chemotherapy methods is the use of the drug gemcitabine, which is widely used to treat metastatic ovarian cancer. Gemcitabine has a short plasma half-life due to its small molecular weight and high hydrophilicity and is rapidly degraded to inactive compounds after administration. In addition, the administration of this drug is often associated with high toxicity and severe side effects. One of the most important strategies to increase the half-life, reduce the toxicity and side effects of antitumor drugs is to change the chemical structure of the compounds in them or to use targeted drug delivery systems.In this regard, biocompatible nanostructures are widely considered as a suitable option for the targeted delivery of drugs, genes, and proteins. Also, despite the expansion of antibiotics in terms of number and effectiveness, complications due to bacterial infections are still a major issue in the medical field. Many antibiotics can play a role in controlling pathogenic microorganisms, but most of them have short plasma half-lives and high side effects, which has made their continuous and excessive use a major challenge. Therefore, the development of efficient drug delivery systems for targeted drug delivery, improved efficacy, and reduced side effects in the treatment of infectious diseases has also received attention. Zinc oxide nanoparticles have attracted the attention of many researchers in the field of nanobiotechnology due to their unique properties and wide applications. These nanoparticles are used in various fields such as the design of drug delivery systems, catalysts and optical sensors, energy converters, the production of cosmetic products, and the development of antimicrobial drugs. Zinc oxide belongs to the group of metal oxides that act with photocatalytic and photo-oxidative capacity against chemical and biological species and inhibit malignant cells through the production of reactive oxygen species, including hydrogen peroxide and superoxide. Direct contact of these ions with cells will lead to disruption of mitochondrial function and cell death. Although zinc is considered relatively non-toxic, there is evidence that free zinc ions may cause the destruction of normal cells. Therefore, in order to eliminate its cytotoxic effect, zinc cations are conjugated with active ligands or zinc oxide nanoparticles are synthesized.The use of zinc nanoparticles with anticancer drugs significantly increases the cellular uptake of the target drug and causes the accumulation of the drug in the target cell. Zinc oxide nanoparticles also have good antimicrobial effects against a wide range of bacteria. The antimicrobial properties of zinc oxide nanoparticles are due to the increased association of nanoparticles with cells through electrostatic interaction between nanoparticles and the cell surface. There are several methods for preparing nanoparticles with different morphologies, among which the green synthesis method has been developed due to its environmental compatibility and the absence of the use of toxic and expensive chemicals. This method is based on the use of microorganisms and plant products such as extracts and is very economical and can be easily applied to large and industrial scales. Extracts are rich in plant secondary metabolites that selectively reduce metal ions and lead to the formation of metal nanoparticles. Wild barberry (Berberis integerrima) extract has various medicinal properties, including strengthening the nerves, treating cardiovascular diseases, reducing blood lipids, anti-inflammatory, anti-cancer and antimicrobial properties. In addition, in order to create more fluidity and targeted release, biodegradable polymers such as starch can be used to coat nanoparticles. Polymers such as starch and gelatin are considered the best choices for coating nanoparticles for drug delivery for various reasons such as compatibility with the physiological conditions of the body. Starch is a natural polymer that is biocompatible, biodegradable, non-toxic, available and inexpensive. Compared to natural starch, amino-modified starch mainly has improved physical properties such as greater solubility in water and helps in encapsulation and delivery of hydrophilic drugs to target sites. Folic acid can also be used as one of the most important ligands to produce targeted nanocarriers for cancer cells. These ligands can significantly increase the effectiveness of nanocarriers by binding to active receptors on cancer cells due to increased expression of the folate receptor on the surface of some cancer cells, including ovarian cancer. In view of the above, the objectives of this invention are as follows: Green synthesis of starch-coated zinc oxide nanoparticles by wild barberry plant extract through a facile and environmentally friendly process. Antibacterial and antioxidant activity of synthesized nanoparticles. Anticancer properties of synthesized nanoparticles against ovarian cancer cells. Increase the biocompatibility and half-life of gemcitabine. A description of the state of the prior art and the history of developments related to the claimed invention. So far, numerous studies have been conducted in the field of green synthesis of zinc oxide nanoparticles with diverse biological purposes and applications both domestically and internationally. However, a review of past studies and research shows that this is the first time that these nanoparticles have been synthesized using wild barberry extract and coated and functionalized with starch and folic acid for effective and targeted delivery of gemcitabine. The patent, registered number IN202241036346 in 2022, describes a method for the green synthesis of zinc oxide nanoparticles using Justicia Adhatoda leaf extract. The inventors describe the method as cost-effective and environmentally friendly. Patent No. CN114099697 filed in 2022 describes a drug delivery system based on organometallic porphyrin nanoparticles loaded with the drug gemcitabine, prepared using layer-by-layer coating. The results showed that the synthetic nanoparticles with a size of 60-100 nm increased the depth of drug penetration into tumors and relieved the immunosuppression state. In patent registration number 100572 in 2018, zinc oxide nanoparticles were synthesized using Hypnea musciformis marine macroalgae extract, which can replace chemical nanoparticles as an environmentally friendly compound and ultraviolet absorber in the cosmetic and health industries. In the patent registration number 91292 in 2016, zinc oxide nanoparticles were synthesized by green chemistry using an amino acid mineral biocomplex. Sabet Bashari et al. (2022) succeeded in synthesizing zinc oxide nanoparticles using the extract of Zataria multiflora. They investigated the antimicrobial, cytotoxic, and apoptotic effects of the nanoparticles on colon cancer cell lines. The results showed that nanoparticles with a size of 35-95 nm can be used as part of antitumor drug formulations. Mariadoss et al. (2020) synthesized copper oxide nanoparticles using Helianthus tuberosus extract, encapsulated them with starch polymer, and finally conjugated them with folic acid to facilitate targeted release of these nanoparticles into MDA-MB-231 cells. Cytotoxicity results proved that folic acid and starch enhance the penetration of nanoparticles into the cell through folate receptor-mediated endocytosis for the treatment of breast cancer. Tang et al. (2020) synthesized zinc oxide nanoparticles using Morus nigra extract and investigated its effect on human adenocarcinoma cell line. The synthesized nanoparticles showed good anticancer properties and increased ROS levels in human adenocarcinoma cells. Anitha et al. (2019) investigated the effect of chitosan-coated zinc oxide nanoparticles on breast cancer cells. The results of this study indicated that these nanoparticles had less toxicity on normal cells and higher lethality on cancer cells compared to the control group. Saneja et al. (2019) used a PLGA-PEG polymer nanoparticle system to encapsulate gemcitabine and betulinic acid to enhance chemotherapy response. Cytotoxicity studies on Panc1 cells showed higher efficacy of the combined nanoparticles compared to the pure drug. Santhoshkumar et al. (2017) synthesized zinc oxide nanoparticles with an average size of 37 nm using Passiflora caerulea leaf extract and investigated its inhibitory effect on a number of urinary tract infection pathogens. Their results confirmed the antibacterial effect of this nanoparticle. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention The steps for the green synthesis of starch-coated and folic acid-functionalized zinc oxide nanoparticles are given below, respectively: 1-Preparation of wild barberry plant extract First, the wild barberry fruit was separated from the rest of the plant parts and washed. Then it was placed in an oven at 40°C for 48 hours. Next, 20 grams of ground plant samples were mixed with 100 ml of 80% methanol and placed on a heater stirrer for 60 minutes at 60°C. Then, it was placed in a shaker at 37°C for 15 hours at 120 rpm and subsequently at room temperature for 5 hours. The resulting extract was centrifuged at 6000 rpm for 10 minutes and then filtered with Whatman paper. The obtained methanol extract was stored at refrigerator temperature for subsequent experiments. The total phenol content of wild barberry extract was measured according to the Folin-Ciocaltio method. 300 μL of the extract at a concentration of 1 mg / mL was mixed with 1500 μL of 1 M Folin reagent and 1200 μL of sodium carbonate, and the final volume was brought to 4000 μL by adding deionized water and kept at 25°C for 50 minutes. Then, the absorbance of the samples was measured by spectrophotometer at a wavelength of 765 nm. 80% methanol, folin and sodium carbonate were used as blanks of the device and gallic acid at concentrations of 0.3, 0.2, 0.1, 0.075, and 0.024 μg / mL was used to draw a standard curve. To calculate the amount of total flavonoids in the extract, 1500 μL of wild barberry extract at a concentration of 1 mg / mL was mixed with 1500 μL of aluminum chloride (20%) and kept in the dark for 40 minutes at 20°C. Then, the absorbance of the samples was measured by spectrophotometer at a wavelength of 415 nm and the amount of total flavonoids in the extract was reported using a quercetin standard curve. The antioxidant activity of the extract was evaluated by DPPH free radical scavenging and iron reduction methods. 2- Green synthesis of zinc oxide nanoparticles 1.78 g of zinc acetate was mixed with 80 ml of distilled water (on a stirrer). Then 20 ml of methanolic extract of wild barberry was added dropwise to it until the total volume of the solution reached 100 ml. After adjusting the pH, the resulting solution was placed on a heater stirrer at 80 °C for 4 hours at a speed of 120 rpm. The resulting solution was centrifuged for 15 minutes at 6000 rpm. The resulting precipitate was washed three times with deionized water and centrifuged for 15 minutes each time. The samples were dried for 24 hours by a freeze dryer (OPERON, made in South Korea) and finally stored at 4 °C. 3- Starch functionalization In order to modify and prepare the starch polymer, first 2 grams of starch powder was mixed with 1 M sodium hydroxide solution and placed in an ultrasonic device for 20 minutes and finally placed on a heater stirrer for 100 minutes at 100 ° C. Then 900 microliters of epichlorohydrin solution was added to this mixture and placed on a heater stirrer at 50 ° C overnight. After 24 hours, the pH of the solution was reduced from 13 to 7 with hydrochloric acid and then 4 milliliters of ethylene diamine were added to this mixture and placed on a heater stirrer for one night. 4- Synthesis of zinc oxide / starch / folic acid nanoparticles (ZNO NPs / ST / FA) To coat the zinc oxide nanoparticles, 0.6 g of functionalized starch and 0.3 g of nano zinc oxide were dissolved separately in 20 ml of distilled water and ultrasonicated for 10 minutes for better dissolution. Then, the starch solution was added dropwise to the nano zinc oxide and placed on a heater stirrer at room temperature overnight. After drying the nano zinc oxide-starch mixture using a freeze dryer, 0.7 g of this material was weighed and dissolved in 20 ml of sterile water. Next, 200 mg of folic acid was mixed with 40 ml of sterile water, 80 mg of carbodiimide hydrochloride (EDC) and 70 mg of N-hydroxysuccinimide (NHS) were added, and ultrasonicated for 30 minutes and then placed on a heater stirrer at room temperature for 1 hour. The zinc oxide / starch solution was gradually added to the above mixture and placed on a heater stirrer for 24 hours.In order to purify this compound and separate unbound folic acid, the resulting solution was poured into a 12 kDa dialysis bag and placed in an aqueous environment for 24 hours. 5- Binding of gemcitabine to nano-zinc oxide coated with starch and folic acid In order to attach gemcitabine to starch-coated zinc oxide nanoparticles functionalized with folic acid, 0.1 g of ZnO NPs / ST / FA was dissolved in 20 ml of deionized water. Then, 100 mg of EDC and 80 mg of NHS were added to this mixture and its pH was adjusted to 4.5-6.0. 0.2 g of gemcitabine was dissolved in 20 ml of deionized water and added gradually. The final mixture was placed on a heater stirrer at room temperature for 24 hours. Then, in order to separate unbound gemcitabine, the resulting solution was poured into a 12 kDa dialysis bag and placed in an aqueous environment for 24 hours. To determine the amount of drug loaded, the absorbance of the supernatant was measured at a wavelength of 268 nm and then calculated using a calibration curve and the following formula. EE % = [(Drug Total - Drug Filtered) ̸ (Drug Total)] × 100 6-Investigating drug release in vitro In order to investigate the release rate of gemcitabine from the starch-coated zinc oxide nanostructure, two buffer environments with pH = 4.7 and pH = 4.5 were used. Initially, 5 mg of each substance was dissolved in 2 ml of both buffers and placed in a 12 kDa dialysis bag. The dialysis bags were placed in environments containing 60 ml of buffer at 37°C and in a shaking incubator. Then, over three days at specific intervals (every hour on the first and second day and every three hours on the third day), 3 ml of the buffer around the dialysis bags was removed each time and its absorption was measured at a wavelength of 268 nm by an ultraviolet-visible (UV-Vis) spectrometer. A standard curve was also prepared using different concentrations of gemcitabine. 7-Ultraviolet-Visible Spectroscopy (UV-Vis) For this purpose, the synthesized zinc oxide nanoparticle powder was completely dissolved and homogenized in 3 mL of deionized water. Then, its absorption was recorded in the range of 200 to 800 nm using a spectrophotometer (Thermo Biomate, USA). 8-Fourier Transform Infrared Spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR) was used to identify organic compounds and functional groups in the extract, zinc oxide nanoparticles, and zinc oxide nanoparticles coated with starch and conjugated with gemcitabine. For this measurement, each of the samples was first converted into tablets using potassium bromide, and then the spectroscopy of these materials was examined in the frequency range of 400-4000 / cm using an instrument (AVATAR, Thermo, USA). 9-X-ray diffraction (XRD) patterns XRD or X-ray diffraction is a unique equipment for analyzing and determining the characteristics of crystals, including the detection of crystal phase, crystal grain size and shape, the distance between crystal layers, determining crystal orientation and position, measuring the percentage of crystallinity of the sample and its structure. The principles of XRD device design are based on irradiating X-rays to the sample at different angles and analyzing its diffraction or reflection pattern. In general, by comparing the obtained X-ray diffraction pattern with the standard diffraction pattern, the crystalline composition is identified. The Debye-Scherer equation is used to measure the crystal structure of synthesized nanoparticles. 10-Transmission electron microscope analysis One of the analytical tools for determining the structure and morphology of materials is the transmission electron microscope (TEM). This microscope can be used to study the structure of crystals, symmetry and orientation, and crystal defects. In this study, a Philips CM200 / FEG microscope was used to investigate the morphology of zinc oxide nanoparticles and zinc oxide nanoparticles coated with starch and conjugated with gemcitabine. The purity of the materials and the analysis of the elements present in the synthesized products were also examined by EDX analysis. 11- Investigating the antibacterial properties of wild barberry plant extract and coated and functionalized zinc oxide nanoparticles using the disk diffusion method For this purpose, concentrations of 1000, 500 and 250 μg / ml of the extract and concentrations of 70, 30, 10 and 5 μg / ml of coated zinc oxide nanoparticles were prepared. Then, 30 μl of bacterial suspension (Escherichia coli, Bacillus subtilis, Listeria monocytogenes, Salmonella typhimurium) was spread on a Petri dish containing nutrient agar medium and spread as a lawn culture using a sterile swab, and the surface of the Petri dish was disced at a certain distance. Then, the raw discs were impregnated with 20 μl of different concentrations of plant extract and ZNO NPs / ST, and the Petri dishes were placed in an incubator at 37°C for 24 hours. Finally, the diameter of the zone of no growth was measured using a caliper. 12-Measuring the anticancer properties of synthesized nanoparticles To investigate the anticancer activity of synthetic nanoparticles, the MTT colorimetric assay was used. In this method, first, by culturing the cell line in a flask and reaching the density of ovarian cancer (SKOV3) and normal (Vero) cell lines to 105, 104, 25×103, and 3×103, respectively, 100 μl of it was poured into the plate and placed in a CO2 incubator for 24 hours to eliminate the stress caused by trypsinization of the cells. After the cells adhered to the bottom of the plate, the culture medium was discarded and 100 μl of different concentrations of gemcitabine (GEM), zinc oxide nanoparticles (ZNO NPs), and functionalized zinc oxide nanoparticles loaded with gemcitabine (ZNO NPs / ST / FA-GEM) (5, 10, 20, 40, and 80 μg / ml) were added to the plate and the plates were placed in the incubator for 24 hours. Next, the supernatant was removed and 20 μl of MTT dye at a concentration of 5 μg / ml was added to the plate and incubated at 37°C in the dark for 4 hours.In the next step, the MTT dye was removed and the formazan crystal formed by the mitochondrial dehydrogenase enzyme of the living cell was dissolved in 50 μl of dimethyl sulfoxide (DMSO). Finally, the absorbance of the wells was read at a wavelength of 570 nm and the percentage of viability was obtained from the following formula: Cell viability (%) = × 100 Explanation of shapes, maps and diagrams Figure 1) shows the antioxidant activity of different concentrations of wild barberry extract. The results showed that with increasing extract concentration, the amount of DPPH free radical inhibition also increased. In such a way that the IC50 value for this extract was 20.2±0.8 μg / ml. The results also showed that this extract had a total phenol content of 46.9±0.7 mg gallic acid / g extract and a total flavonoid content of 5.6±0.01 mg quercetin / g extract, respectively. Figure 2) shows the UV-Vis spectrum obtained for the synthesized zinc oxide nanoparticles. The results showed a prominent absorption peak in the 360 ​​nm range, which confirms the synthesis of zinc oxide nanoparticles. Figure 3) shows a comparison of the FTIR spectra obtained from wild barberry extract (BI), zinc oxide nanoparticles (ZNO NPs), amino starch (Aminated ST), and starch-coated zinc oxide nanoparticles (ZNO NPs / ST). Figure 4) shows a comparison of the FTIR spectra of folic acid (FA), gemcitabine (GEM), zinc oxide nanoparticles coated with starch and folic acid (ZNO NPs / ST / FA), and zinc oxide nanoparticles loaded with gemcitabine (ZNO NPs / ST / FA-GEM). Figure 5) shows the X-ray diffraction pattern of gemcitabine (GEM), starch-coated zinc oxide nanoparticles (ZNO NPs / ST) and gemcitabine-loaded zinc oxide nanoparticles (ZNO NPs / ST / FA-GEM). The presence of characteristic peaks at diffraction angles of 32.15°, 34.5°, 36.7°, 47.75° and 63° indicates the crystalline and hexagonal structure of zinc oxide nanoparticles. The presence of a prominent peak at diffraction angle of 25° indicates the presence of amino-modified starch. The presence of a peak at diffraction angle of 27.3° indicates the presence of a crystalline structure of folic acid in the compound. Also, the presence of peaks at diffraction angles of 21° and 10° due to overlap with the XRD spectrum of gemcitabine drug probably indicates the presence of a crystalline structure of this drug in the final compound. Figure 6) shows the transmission electron microscope images of a) ZNO NPs / ST b) ZNO NPs c) ZNO NPs / ST / FA-GEM. Based on the images, the average size of the synthesized nanoparticles was observed to be 64 nm with a spherical structure and uniform size distribution. Figure 7) shows the in vitro release of gemcitabine from synthetic nanoparticles in two simulated buffer environments with pH=7.4 and pH=4.5. The results obtained from the release of gemcitabine after 44 hours indicated 86% drug release at pH=4.5 and 81% drug release at pH=4.7. The drug loading percentage was also calculated to be 70%. Figure 8) shows the antimicrobial activity of functionalized zinc oxide nanoparticles against four bacterial strains based on the average diameter of the inhibition zone obtained from the disk diffusion test. The highest inhibition zone diameter was recorded at a concentration of 70 μg / mL against the gram-negative bacterium Salmonella typhimurium (24 ± 2 mm) and the lowest inhibition zone was recorded at a concentration of 5 μg / mL against the gram-positive bacterium Bacillus subtilis (10 ± 1 mm). Figure 9) shows the comparison of the anticancer activity of ZNO NPs, ZNO NPs / ST / FA-GEM and GEM on the ovarian cancer cell line (SKOV3). The results indicated a decrease in the viability of the cancer cell line with increasing concentrations of all three compounds. So that the cytotoxicity rate at a concentration of 5 μg / mL for ZNO NPs, ZNO NPs / ST / FA-GEM and GEM in the cancer cell line was reported to be 5%, 17% and 10%, respectively, and at a concentration of 80 μg / mL it was reported to be 56%, 87% and 84%, respectively. The results also indicated a higher lethality of cancer cells by the ZNO NPs / ST / FA-GEM combination at all concentrations compared to free gemcitabine and zinc oxide nanoparticles. Figure 10) shows the comparison of anticancer activity of ZNO NPs, ZNO NPs / ST / FA-GEM and GEM on normal cell line. The viability of normal cell line at a concentration of 5 μg / ml was reported to be 97%, 95% and 95% for ZNO NPs, ZNO NPs / ST / FA-GEM and GEM respectively. However, the viability decreased with increasing concentration so that at a concentration of 80 μg / ml the viability of normal cell line was reported to be 75%, 64% and 51% respectively. The results of the lethality study of the mentioned compounds on normal cell line showed significant toxicity of free gemcitabine compared to zinc oxide nanoparticles loaded with gemcitabine, which indicates higher biocompatibility of synthetic nanoparticles. A clear and precise statement of the advantages of the claimed invention over prior inventions. 1- Using a biocompatible, safe, and easy method, without the use of toxic and expensive materials, for the design and green synthesis of zinc oxide nanoparticles based on wild barberry plant extract, which is capable of replacing commercial chemical samples in terms of quality and physicochemical properties. 2- Significant antioxidant activity of wild barberry extract due to the presence of phenolic and flavonoid bioactive compounds. 3- Uniform and highly suitable size distribution of synthetic nanoparticles for effective drug delivery to the surface of cancer cells. 4- Increasing fluidity and targeted release of synthesized nanoparticles through appropriate environmental binding with starch and folic acid. 5- The ability to maintain and release the drug continuously and for a relatively long time from the synthesized nanoparticles, without losing the performance and physico-chemical properties of the loaded drug. 6- The suitable antimicrobial activity of coated zinc oxide nanoparticles against the studied bacterial strains makes it a promising and suitable option to replace commercial antibiotics. 7- The effective and highly suitable anticancer activity of zinc oxide nanoparticles loaded with gemcitabine on ovarian cancer cell lines, as well as its very low lethal effect on normal cell lines compared to free gemcitabine, indicates their anticancer properties and high biocompatibility. Description of at least one implementation method for implementing the invention The synthetic method used in this invention has the potential to be industrialized due to its short production process, ease, and high biocompatibility and efficiency compared to other methods. For this purpose, after synthesizing zinc oxide nanoparticles loaded with gemcitabine, it can be transported to the desired location to suppress cancer cells so that the loaded drug can be released through controlled release. Also, due to the presence of folic acid, the synthesized nanoparticles act in a targeted manner and, with a size of approximately 64 nm, have the ability to be transported and penetrate tumor tissues with low pH. Explicit mention of the industrial application of the invention Due to the sustained release, high biocompatibility, and significant anticancer activity of the synthesized nanoparticles, it can be used as a potential anticancer drug for the effective and targeted treatment of cancer cells, especially ovarian cancer. Due to the high antimicrobial and antioxidant properties of the synthesized nanoparticles, they can be used as a safe protective and preservative agent in the packaging of food, beverages, as well as health and medical products.

Claims

Claims What is claimed: Claim 1) This invention relates to the design and synthesis of a targeted drug delivery system based on zinc oxide nanoparticles that can effectively and controllably release the drug gemcitabine for use as a potential anticancer compound. Claim 2) According to claim 1, zinc oxide nanoparticles with an average size of 64 nm, spherical structure, and uniform size distribution were synthesized. Claim 3) According to claim number 1, the anticancer drug gemcitabine was loaded onto starch-coated zinc oxide nanoparticles functionalized with folic acid through an optimized method, and its loading percentage was calculated to be 70%. Claim 4) According to claim 1, the drug delivery system exhibited a sustained and controlled release profile under simulated physiological conditions (pH=7.4), such that after 44 hours, more than 86% of the drug was released. Claim 5) According to claim number 1, effective and appropriate anticancer activity of the synthesized nanoparticles on ovarian cancer cell lines, as well as its very low lethal effect on normal cell lines compared to free gemcitabine, was recorded.

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