Document Type : Research Paper
Authors
Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran
Abstract
Keywords
INTRODUCTION
Gastric ulcers for a long time thought that factors such as eating spicy foods, stress, smoking and poor eating habits, and taking certain medications were the only causes of gastric ulcers until scientists discovered a bacterium in the gastric [1-4]. Researchers found that this bacterium was present in the stomach of many people called this bacterium “Helicobacter pylori. Gastric or peptic ulcer is the damage done to the lining of the stomach or small intestine [5-7]. This complication occurs when the protective mucosa inside the stomach becomes ineffective. The stomach produces strong acids to help digest food and to fight germs. As the mucosa loses its normal function, stomach acid can damage stomach tissue and lead to stomach ulcers [8-11]. Based on the available statistics, it is estimated that one in ten people may develop this disease during their lifetime. Treatment may vary depending on the cause of the wound in the patient.
Most of the wounds can be treated with simple and basic treatments, only in rare cases surgery may be require in which prompt and timely treatment of gastric ulcer is very important [12-16]. Therefore, the use of drug-containing hydrogels can be effective. Gelatin is one of the biopolymers on the market that is used in medical applications due to its excellent compatibility, insecurity and very wide biodegradability [17-19]. Collagen-derived gelatin is used in clinics for wound dressing, pharmacy, and adhesives because of its compatibility, biodegradability, and hydrogel properties, as well as its formability and cost-effectiveness. However, unlike synthetic polymers, which are generally non-ionic, gelatin is a type of polyelectrolyte polymer that has a large number of ionizing groups.
In addition, strong hydrogen bonding leads to the formation of a 3D macromolecular network, which drastically reduces the mobility of gelatin chains [20-24]. Magnetic nanoparticles (MNPs) in nanoscale have different properties. Some elements or compounds, such as iron or iron oxides have magnetic properties that retain these properties in the nanoscale [25-39]. MNPs consist of a magnetic core and an ultra-magnetic outer shell in which the outer shell enhances the magnetic properties of the nanoparticles. The enhanced magnetic properties of MNPs allow for highly sensitive detection as well as reduced nanoparticle aggregation [40-45]. Magnetic nanoparticles are divided into three groups: ferromagnetic, paramagnetic and diamagnetic [46-51]. The property of diamagnetism is apparently found in all types of materials [52-54]. The present study aims to make magnetic nanoparticles and load drugs on gelatin hydrogel.
MATERIALS AND METHODS
Dissolve 10 g of COSO4.H2O and 1 g of boric acid in 25 ml of deionized water. Place the cathode and anode of the electrolysis device in solution until MNPs are synthesized. The electrolysis process is performed at room temperature. After synthesis, the nanoparticles should be washed several times with distilled water. Pour the nanoparticles into a plate and place in the oven to dry completely. Then, mix the gelatin powder in 100 cc of deionized water. Three different amounts of gelatin are considered to find the optimal hydrogel. Finally, after the necessary experiments to investigate the magnetic properties, the synthesized nanoparticles are placed in a hydrogel by fusion method. According to the fusion method, first a magnetic fluid to avoid mass and oxidation by scattering of MNPs within the phase. Then, the mixture is combined with a hydrogel solution, and by crosslinking, the MNPs are encapsulated within the hydrogel structure.
Using UV, the crosslink process is performed for the hydrogel. Scanning electron microscope (SEM) is used for photographing surfaces and morphology of the hydrogel. X-ray diffraction (XRD) is a method for studying the microstructure of crystalline materials for shape and size of the building unit. The basis of the vibrating-sample magnetometer (VSM) is the Faraday induction law, which by vibrating the sample and applying a magnetic field to it, creates an inductive current in the coils embedded in the device. By transferring this inductive current to the computer connected to the device and displaying the waste loop, the magnetization of the sample can be measured.
Water absorption test is used and for this purpose, first the samples are weighed and after immersion in phosphate buffer saline (PBS), their weight eas measured again and the absorption rate may be obtained from Equation 1.
Weight loss = W-W0/W0 *100
In order to evaluate the stability and measure the rate of degradation in the external environment, the hydrolytic biodegradability test is used. At the end of every 24 hours, the dry weight of the compound was measured. Using the UV-Vis test, first the concentration of the drug is determined and then the concentration of the drug in the samples is examined. The test for measuring antibacterial properties is determined according to the relevant standard. Heterogeneous expression of genes in cancer cells that lead to diversity is called cellular parasitism. Cancer stem cells are cells that have the ability to self-regenerate and can differentiate into adult cancer cells. Cancer stem cells are similar to normal stem cells are very similar to normal stem cells in terms of self-renewal and metabolic properties. However, differences have been found between cancer and normal stem cells. Heat treatment methods can be divided into two main groups based on the technique used to generate heat: magnetic hyperthermia and heat optical heat treatment methods [55-58]. Metal spinelite ferrite nanoparticles have the general relationship MFe2O4 that M can be metals such as Zn, Fe, Mg, Co, Ni, and Mn.
These applications of cobalt ferrite nanoparticles are due to their ability to distribute cationically among subnets in tetrahedral and octahedral sites [59-62]. Cobalt ferrite nanoparticles are widely regarded for their interesting properties such as magneto-crystalline anisotropy, high inductance, medium saturation magnetism, high chemical stability, abrasion resistance and electrical insulation. The magnetic properties of the material depend on the size, shape and purity of the nanoparticles. Among the various ferrites, ferrites on ZnFe2O4 and cobalt ferrites on CoFe2O4 have been studied more because they are ferrites usually with normal spinel and inverted spinel, respectively [50-62]. The Co-Fe cobalt-ferrite composite ferrites are very interesting for research due to their interesting and completely different properties of ZnFe2O4 ferrite and CoFe2O4 cobalt ferrite. Magnetic properties of cobalt spinelite ferrite structure can be changed by cationic substitution to a composite structure. Based on new studies, replacement of Zn2+ with CO2 in cobalt nanoparticles of Co1-x Znx Fe2O4 has shown that properties such as chemical stability, high corrosion resistance, magneto-crystalline anisotropy, and magneto-optics have been improved. Thermotherapy is one of the treatment methods for cancer cells in which the temperature of cancer cells is raised to a temperature between 41°C and 45°C. In thermotherapy, a ferromagnetic or superparamagnetic compound is directed to the site of a cancer cell and then, by applying an external magnetic field, converts the strength of the external magnetic field into heat energy by applying several para or ferromagnetic particle mechanisms.
Zhang et al. [39] described TiO2/ZnCO2O4 nanorods a strategy to achieve highly sensitive and selective detection of formaldehyde (HCHO) and triethylamine (TEA) using porous ZnCO2O4 nanorods used with TiO2 as a marking agent Data. TiO2/ZnCo2O4 nanorods were prepared by molding method, in which solid ZnC2O4/CoC2O4 nanorods were pre-synthesized using a deposition method at room temperature and then immersed as a model of porous TiO2 nanorods. The ZnCO2O4 is used in C10H14O5Ti ethanol solution (before calcination) [35]. Accordingly, CoNWs-GO-PEGDOX demonstrates the satisfactory effect of killing cancer cells by chemotherapy in vitro. The present work provides a comprehensive overview of the potential of CoNWs-GO-PEG to serve as an antitumor agent in synergistic chemotherapeutic chemotherapy.
Heat treatment method that does not have the above defects has been used in the synthesis of various ferrites. In this research, researchers have synthesized cobalt ferrite nanoparticles on CoxZn1-xFe2O4 for (1, 0.8, 0.6, 0.4, 0.2 and x = 0) using heat treatment method and replacement effects of Co2+ with Zn2+ has been investigated for these nanoparticles. Initial anodizing was performed in 10% sulfuric acid solution and at 20 volts DC at 12°C for different times for sections A and B, 10 minutes and 30 minutes, respectively. To remove the surface oxide layer, a solution of 1% phosphoric acid was used at ambient temperature for 20 minutes. It should be noted that this solution may only remove the oxide layer created on the outer surface of the aluminum and may not damage the aluminum substrate.
Synthesis of nanoparticles
4 g of cobalt sulfate (4% wt) and add to 100 cc of deionized water was used in this study. After completion of the solution, the composition is removed from the electrolysis process. Electrolysis uses two electrodes made of aluminum and iron. After the electrolysis process, the obtained particles are poured into a falcon and used for better separation from the centrifuge for 10 minutes at a speed of 6000 rpm. After the particles inside the Falcon settle, the samples were washed several times with deionized water. Pour the particles into the plate and put the plate in the oven at 30°C for 24 hours to dry the water completely.
Gelatin hydrogels were prepared in different concentrations of 5, 10, 15 and 20 wt% using distilled water. Pantoprazole has been used for the combined treatment of gastrointestinal wound. In this article, metronidazole is used in addition to panaprazole. The nanoparticles made are mixed in the prepared solutions. In this study, mix 1 cc of gelatin hydrogel with 0.5 g of MNPs. The crosslinking process was performed on magnetic hydrogels using UV rays.
Table 1 shows the amount of material used for each magnetic hydrogel. Fig. 1 shows the schematic application of gelatin hydrogel/cobalt magnetic nanoparticles containing penaprazole and magnetic conduction in the extracorporeal phase. Fig. 2 shows the preparation technique for synthesize the gelatin hydrogel/cobalt magnetic nanoparticles after electrolysis.
Morphological study
Scanning electron microscope (SEM) analysis
Scanning electron microscopy (SEM: AIS2100) was used to evaluate the surface of MNPs and magnetic hydrogels. First, the samples were coated with gold and then imaging was performed and the nanoparticle size was measured using Image-J software. It may be possible to identify the constituent elements of the sample in semi-quantitative form. At least 5 mg of particle sample is required to analyze Energy dispersive spectroscopy (EDS) whicht may be calculated semi-quantitatively and the energy spectrum may be presented.
X-ray diffraction (XRD) analysis
To study the crystal structure and phase composition of the samples, X-ray diffraction (XRD) analysis was performed. X-ray wavelength is 1.54 A, copper anode and nickel filter. The stop time in each step was one second, the step size was 0.02 degrees and the sweeping range (2θ) was selected between 10 and 80° and the patterns were identified using references.
Vibrating-sample magnetometer (VSM)
Magnetometers measure the magnetization of a sample of material with different dimensions in different ways and under different conditions of temperature, magnetic field and sample orientation. The basis of the VSM magnetometer is the Faraday induction law, which, by vibrating the sample and applying a magnetic field to it, creates an inductive current in the coils embedded in the device that is proportional to the sample’s magnetism. This device is able to measure the magnetic properties of materials in powder and liquid state and thin layer up to 1.5 Tesla field.
Inflation evaluation
To evaluate the amount of water absorption on coated and uncoated scaffolds. The scaffolds sample placed in a PBS. Swelling was performed for all hydrogels containing MNPs. In order to evaluate the rate of degradation in the external environment. For this purpose, first the dry weighed scaffold was placed in PBS for 7 days. Every 24 hours a sample was taken out of solution and dried on filter paper and the weight of the sample was measured. The percentage of biodegradability of scaffold is obtained from the following equation.
Biodegradability = [(W-W0)/W0] × 100
Where W0 is the dry weight and W is the weight of the immersed scaffold. Swelling was performed for all hydrogels containing MNPs.
Antibacterial behaviour
One of the methods for measuring the antibacterial properties of a substance is the method of determining the diameter of the growth inhibition zone. The test specimens are then transferred to the culture medium. After storing the plates in the incubator for 24 hours at a temperature of 37°C, the plates are examined under light and the non-growth area is measured. Antibiogram classifies bacteria into susceptible, semi-susceptible or resistant groups based on qualitative results. Therefore, this method is a basic tool for determining the type of resistance phenotype among the tested bacteria. Gram-positive Staphylococcus bacteria are completely dispersed by Hooke into petri dishes containing solid LB medium based on McFarland concentration. Using disk fusion method, necessary studies have been performed for S. aureus (gram positive) and E. coli (gram negative) bacteria. These discs were completely sterilized by UV rays for 12 hours. Bacteria were completely dispersed by Hooke based on McFarland concentration into petri dishes containing solid LB medium. Finally, the plates were transferred to an incubator at 37°C and stored for 24 hours. The halo created around the specimens reflects the antibacterial properties of the scaffold.
Drug release analysis
The initial concentration of Pantoprazole and metronidazole is 2 ppm. To draw a standard curve, the drugs were diluted with PBS and prepared in 4 concentrations. Then, the absorbance of the samples was measured by UV spectrophotometer. To prepare the dialysis bag for the release test, the dialysis bag was placed in a solution containing 2 wt% baking soda and 0.05 wt% EDTA for 30 minutes. It was washed twice with distilled water and again placed in boiling distilled water. Insert 4 mg of magnetic hydrogel into the dialysis bag and close the top of the bag with another clip. 30 ml of PBS solution was poured into the human dialysis bag containing the samples. Sampling was performed at different intervals and the same amount of new solution was used instead. The samples were then placed in a UV spectrophotometer and a graph was plotted by calculating the release percentage of the drug released from the hydrogel.
Tensile strength analysis
The data obtained in this test are used to determine the mechanical properties of the material. Tensile strength or ultimate tensile strength (UTS) is the maximum tensile stress that the specimen can withstand and the ratio of the maximum force (load) to the initial cross-section of the specimen is defined. The slope of the linear part of the stress-strain curve is the elastic modulus. The samples were glued to the paper frame according to ISO-6824 standard with dimensions of 10.60 mm and were subjected to tensile strength test and compared. The paper frame was placed between the two jaws of the traction machine at a distance of 3 cm with an applied force of 10 N, a traction speed of 10 mm/min, a humidity of 30%.
RESULTS AND DISSCUSION
Fig. 3 (a-b) shows the morphological examination using a SEM image for nanoparticles synthesized in this study. Fig. 3(a) shows the particles obtained by block A and Fig. 3(b) is for the particles obtained by block B. As can be seen in the obtained images, the difference in the anodizing parameters has caused the particles to have slight changes in terms of morphology.
Fig. 4 shows a magnetic hydrogel after the blending process. Fig. 4 shows the light microscope image of a magnetic hydrogel. Fig. 4 indicates the MNPs are evenly distributed throughout the gelatin hydrogel. Fig. 5 (a) shows the XRD analysis of samples A and B. The XRD pattern is very wide for samples A and B obtained by anodizing aluminum parts. The taller and narrower the spikes, the more crystalline the material, and the wider and shorter the material, the closer it is to the amorphous form. The anodizing process has resulted in rod nanoparticles being structurally irregular. The obtained peaks represent cobalt sulfate, calcium phosphate hydrate (CaPO3(OH)2H2O) and boric acid (H3BO3). Sobhani et al. [47] study showed that CoSO4H2O has a monoclinic phase (space group C2 = c) by fixed network n = 0.695, b = 0.757 nm and c = 0.762 with card number 2104-70 = JCPDS It is [45]. Card numbers 0293-011-00 and 1455-072-01 are related to CaPs hydrate and cobalt sulfate, respectively. The peaks related to calcium phosphate hydrate for sample A are equal to 2θ = 29.2° and 2θ = 44.92° and for sample B are equal to 28.24° = 2θ and θ2 = 44.56 °. The cobalt sulfate peaks for samples A and B are around θ = 44.62 and θ2 = 45.3, respectively. Peaks of 10/48 (sample A) and 56/48 (sample B) are related to boric acid, which is consistent with the studies of Liu et al. [46].
Fig. 5 (b) shows the FTIR spectrum of the as-prepared sample. In this spectrum, absorption band from 3500 cm-1 to 2350 cm-1 is the features of stretching vibration of OH which indicates the availability of H2O in the hydrogel. In addition, strong absorption bands at 790, 1080, 1414 cm-1 and 1632 cm−1 which may be assigned vibration mode of ring structure of SiO2 tetrahedra.
Fig. 6 shows the presence of elements in samples A and B using EDS analysis. Accordingly, the EDS graph shows the rates of available chemical elements such as cobalt (Co), oxygen (O), carbon (C), iron (Fe), silicon (Si) and sulfur (S). Figs. 6 shows the presence of elements in particles A and B, which for cobalt, oxygen, carbon, iron, silicon and sulfur in particles A are 67.15, 14.92, 4.48, and 77, respectively. Also, the mentioned values for B particles are equal to 79.67, 9.02, 0.78, 3.52, 1.37 and 5.64 percent, respectively. The highest percentage of weight of cobalt is related to sample B.
Fig. 6 (b-bc) shows the results of the Map micrograph. The distribution of iron and cobalt elements in samples A and B is evenly distributed and uniform is established in different areas of the sample. Metals with semi-solid orbitals such as pure iron and cobalt have magnetic properties in which this group of metal solids is called ferromagnetic material [48]. The amount of drug loading in the hydrogel and the amount of drug release from them were measured by UV spectroscopy. The magnetic behavior of samples A and B can be measured using a VSM device by plotting the residual curve. The diagram of the magnetic effect of the synthesized particles, at ambient temperature and in the fields of 10,000 to 10,000 overs, is shown in Fig. 7. The results at this stage indicate the initial explosive release in the range of 30-50% of the loaded drugs. For the ferromagnetic properties of particles, the dispersion of iron and cobalt elements is of particular importance. Map micrograph examination shows the proper dispersion of both elements in both materials as shown in Fig. 7.
Fig. 8 shows the percentage of drug release from the hydrogel in the PBS. The area under the peak was calculated for all concentrations at 203 nm and finally the standard drug concentration curve was plotted in terms of UV uptake. The line diagram equation of standard drugs was obtained 0.0001 + x = 0.001 with R2 = 1 as shown in Fig. 8. As can be seen, the magnetization of sample A, 11.83 emu/g was obtained, which did not decrease or increase significantly compared to sample B, 11.61 emu/g.
Based on VSM analysis, the superparamagnetic structure in sample B is confirmed. The absence of a residual ring in the VSM diagram indicates superparamagnetic behavior and the magnetic property of magnetite is very sensitive to the structure of the sample and the superparamagnetic property occurs when the particles are small enough that thermal fluctuations can cause magnetic changes as shown in Fig. 9.
The absence of a residual loop in the VSM profile is an important criterion required for the superparamagnetic behavior of nanoparticles [50-55]. Drug release occurred in five stages with different slopes as shown in Fig. 10. In stage (d) up to 24 hours about 70% of the release has occurred. In the last step, the slope of the graph is almost fixed and the drug concentration inside and outside the films is the same. The results of the drug release test indicate release in the form of control infiltration. The study of swelling and water absorption properties in the hydrogel network is essential in various applications. In this study, the effect of increasing gelatin on hydrogel on swelling was investigated. Fig. 11 demonstrate the amount of gelatin increases and then the swelling rate increased too. The sample with 20% gelatin showed the highest swelling up to 233.33%. By far the lowest amount of gelatin is the percentage of swelling up to 44.74%. As the amount of gelatin increases in the first hour, more water is absorbed into the hydrogels.
Scaffolds with 5% and 10% were destroyed after 5 hours. Also, 15% and 20%G scaffolds were destroyed after 6 and 7 hours. This release rate is due to the degradation of surface abrasion of the hydrogel along with the release of drugs adhering to the sample surface as shown in Fig. 12. The slope of the release chart is reduced at this stage. However, at this stage, an increase in the release percentage of up to 60% is observed, which is due to the occurrence of the penetration phenomenon inside the hydrogel and the continued degradation of the polymer. In the third stage (c), which occurs in the range of 15 hours to 20 hours after the start of the test, the slope of the graph is almost constant.
Destruction for sample with 20% reinforcement in scaffold after 6 hours is 83.33%. The demolition time of the specimens is consistent with studies by Wiwatwongwana et al. [51]. They stated that compositing gelatin with another polymer such as carboxy methyl cellulose increases the degradation time in the hydrogel. Because the hydrogel is structurally stronger, but the pure gelatin hydrogel is degraded within 9 hours [51].
Fig. 13 (a-b) investigation of antibacterial properties of 20% G-NP sample containing metronidazole and penaprazole.
The mechanical properties of biological materials are critical among the parameters because they have a significant impact on cell behavior. The vascular system of the skin undergoes intermittent cyclic deformation, showing elastic modulus in the range of 25.2-130 MPa depending on the physiological anatomy, location and function of blood vessels. Therefore, the mechanical properties of scaffolds with different concentrations of gelatin have been investigated using tensile test and the results are according to Table 3.
Fig. 14 shows a diagram of the mechanical properties of 15% -NP and 20% -NP samples. As can be seen from the diagram, the amount of scaffold tension has also increased with increasing gelatin amount. As shown in Table 3, the percentage of elongation for the 20% G-NP sample is 44.98%. With decreasing gelatin amount, the percentage of elongation has decreased and reached to 31.89%.
CONCLUSION
Treatment of gastric wound is one of the most important challenges facing medical knowledge and drug delivery. Treatment of gastric wound with magnetic hydrogel can be directed to the target tissue by magnetic particles and with continuous release which leads to faster healing process. In this study, magnetic nanoparticles have been prepared using electrolysis process. The solution is prepared by cobalt (II) sulfate and boric acid. Using the anodizing process, porosities were created on the surface of the components. Modifications have been made to the anodizing process for both templates to identify the best parameter. It can be stated that placing the mold in a solution of sulfuric acid for 30 minutes is optimal. Gelatin hydrogels are prepared and the desired drugs are added to the solution. Using the blending technique, the optimal magnetic nanoparticles were loaded into the hydrogel. Morphological studies have shown that the anodizing process affects the morphology of the deposited particles. Microstructural studies using XRD pattern show peaks related to calcium phosphate, hydrate, cobalt sulfate, and boric acid. The EDS micrograph showed the highest amount of trace elements for cobalt and iron. The scattering of two elements with magnetic properties such as iron and cobalt is well shown in the map. VSM test studies have shown that nanoparticles obtained from anodized mold have superparamagnetic properties for a longer period of time. Drug release occurred in about 70% within 48 hours. The swelling test for the sample containing 20% gelatin showed the highest absorption rate up to 233%. Most detoration occurred after 6 hours for sample containing 20% gelatin in hydrogel. The pores created around the magnetic hydrogel was an evidence of the antibacterial property of the sample. Mechanical properties using tensile test have shown that with increasing gelatin amount, the elongation has increased from 31% to 44%.
ACKNOWLEDGEMENTS
The authors would like to extend their gratitude for the support provided by department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.