Application of MNPs-IHSPN nanoparticles in really stabilization of biomolecules bio drugs In-vitro environment

All scientists are working on biomedicine and Nano-biotechnology. So, one of the most important issues is the study of magnetic nanoparticles (MNPs-IHSPN). The purpose of this project is to examine the approach of using a common buffer to determine the degree of stabilization and release of two molecules of drugs that have been analyzed In-vitro. The structures as a SEM and FT-IR instruments and were used in the amount of 25 mg. After the reaction with biomolecules, the absorption rate was about 60-80%. The release of biomolecules was done by a buffer PBS by spectrophotometer analysis. It is noteworthy that these biomolecules was tested in two forms of covalent and electrostatic bonding. EDX analysis and electrophoresis were used to stabilize the absorption rate in the electrostatic transplant and FT-IR analysis for fixation of covalent bonding. The overall result of this project is to use the common characteristics of some biomolecules for important drug exchange in the living environment of cells.


INTRODUCTION
Nanotechnology is a key element in understanding the nature of the coming decade.
Interdisciplinary research collaboration, special training and transfer of ideas from people in the industry; including the benefits of nanotechnology in the future.
Nanocatalysts nanotechnology industry had been made significant progress.
Nanocatalysts types are designed, among them, iron oxide nanoparticles (based on magnetic properties) had been many applications especially in the field of drug delivery, the proteins and biodrug (MTX biomolecule drug) are necessary. One of the most important and most widely used magnetic nanoparticles in which a variety of 3 materials to create their own unique characteristics compared to other Nanospecific applications screwed. These particles are applied in various branches. But the role of them in life_medicine and, as mentioned, are significant in terms of its delivery to the inherent magnetism gives them a lot of things, including facilitating spotter the delivery of these are very important [1].

Application and structure of magnetic nanoparticles
Over the past few years, efforts have been devoted to the magnetic Functionalized nanoparticles as the level of cover will gain significant benefits from it. However, there are many types of materials available in magnetic coatings Nanoparticles, such as metal oxides, metal, and plastic; Silica is still considered to be the best candidate surfaces Functionalization because it is highly stable against degradation.
In addition, the silica to improve the biocompatibility, hydrophobicity profile as well as the availability of high-level performance Group silanol (-SiOH) on the surface [2], that makes a promise Materials for a variety of biological applications. Silicacoated magnetic nanoparticles are used for various applications in recent years, such as separation of protein and enzyme immobilization [3,4].

TEOS-MPTES (3-mercaptopropyl triethoxy silane), SH-polymer
Although silica coating enhances the surface performance of magnetic nanoparticles, the surface can be modified to increase this performance as well as to increase the biomolecule uptake at the nanoparticle surface. For this purpose, another coating called mercapto (with a sulfur functional group derived from the green chemical composition of imidazole ion) was used which results showed that the size of the magnetic nanoparticles decreased after adding the mercapto functional group and The stability of the reaction is increased. Nowadays, chemicalorganic compounds known as green solvents without harmful effects on the 4 environment are used, as indicated by imidazole ion combining ionic liquids with two cationic and anionic components to provide stability and enhance the performance of nanoparticles used. It should be noted that in this complex, the imidazole ion (C 3 N 2 H 4 ) as a cationic portion and a variety of halides such as chloride and bromide ion as the anionic portion provided the complex composition for the mercapto (sulfur functional group) on the surface of the magnetic nanoparticles [5].

Magnetite
Methotrexate conjugated with large microparticles made of gelatin or polyglutaraldehyde improves methotrexate levels when interacting with tumor cells. This is called a targeted pharmacologist and its therapeutic efficacy has improved.
The size of the compound (the molecule conjugated to the microparticles) should be small, rather than the movement of the drug molecule in the flow of blood circulation (in the veins) to the target location is easy. Thus, nanoscale nanoparticles with small particle sizes are used at the nanoscale, for example, a drug substance (methotrexate) conjugate to the surface of the magnetite magnetic nanoparticles that are being studied [6]. Magnetic nanoparticles have been prioritized to Nano-compounds due to their stability, their small particle size, and their controllability by external magnetism for targeted drug delivery, and antibacterial properties [7][8]. There are many papers and researchers to stabilize biomolecules on magnetic nanoparticles. The main goal of this project is to cover the SiO 2 activation of Fe 3 O 4 magnetic nanoparticles for the stabilization of biomolecules (protein (BSA) (serum albumin)) and Fe 3 O 4 @SiO 2 /SH/NH 2 for the 5 stabilization of biomolecules methotrexate (MTX) drug molecule. In this study, they sought to stabilize and release them In-vitro according to the result, the basic point is the stabilization of our electrostatic bond (for protein biomolecules) and covalent bond (for MTX drug) between them (by electrophoresis and EDX analyses) at room temperature (25 o C) for protein and 37 o C for MTX (Figure 1).

Materials
All solvents and chemicals are purchased from commercial Suppliers. The structure Deionized water was used in each experiment.

Synthesis of silica-coated with Fe 3 O 4 magnetic nanoparticles
Chemical Co-precipitation also one of the easiest and most convenient methods of synthesis of magnetic nanoparticles with core/shell structure. So, in this way, sample container iron salts with amounts of 1 to 2 (1.5mg of FeCl 2 .4H 2 O and 3mg of Fe (NO 3 ) 3 .9H 2 O) were dissolved in distilled water. The reaction temperature was 25 o C and high-intensity spinning under inert nitrogen gas. After 3 hours to prevent additional oxidation and increasing the absorption of biomolecules for biological targets of 3 ml tetraethyl-orthosilicate was used. Finally, the yellowish-brown product was obtained in the same magnetic nanoparticles. In the read more, the solution was washed repeatedly with methanol and water and then dried in the oven, the powder was gathered.

Synthesis of Fe 3 O 4 @SiO 2 /SH/NH 2 magnetic nanoparticles
After synthesizing Fe 3 O 4 @SiO 2 /SH magnetic nanoparticles, weigh out 100 mg of same magnetic nanoparticles and dissolve in distilled water 4 ml to completely dissolve in water. In another part, it prepared 3 ml of the APTES solution (3aminopropyl tri-methoxy-silane), and then the magnetic nanoparticles Fe 3 O 4 @SiO 2 , After synthesizing Fe 3 O 4 @SiO 2 magnetic nanoparticles weigh out 100 mg of same magnetic nanoparticles and dissolve in double distilled water 4 ml to completely double dissolve in water. After complete dissolution of sample, place in reflex conditions at 100°C, and the amount 250 μl (CPTES) linker is added to the solution.
Then 24 hours, 250 μl of (MPTES) was added to the solution. On another 36 hr, 250 7 μl of N-methyl imidazole was added to remove chloride from the first linker. Which are completely dissolved (in water) in the case of a heater and powered by a magnet the solution was completely dissolved in a drop-drop of APTES solution to magnetic nanoparticles and placed in a non-temperature condition for one night.
Finally, we wash the magnetic nanoparticles Fe 3 O 4 @SiO 2 /SH/NH 2 prepared by distilled water twice ionized and placed in the oven. Finally, magnetic nanoparticles containing a yellowish-brown amine agent group was prepared.

Synthesis and characterization of magnetic nanoparticle coated with silica
In this section, we will try to use the aforementioned magnetic nanocomposite first to prepare its core / shell structure and then to evaluate it by highly specialized analyzers. For this purpose, various analytical devices are used which we have been able to record by the SEM analyzer, the orderly structure of the nanocatalyst with the active reactant surface. As it appears from the nanocatalyst formulation, it provides a suitable surface for conducting a variety of chemical-biological reactions with most biomolecular biomolecules. In the range of 1-100nm magnetic nanoparticles should be made, such as the size of the magnetic nanoparticles in chemical reactions and medical procedures are important. The structure of magnetic nanoparticles coated with silica by analytical SEM is shown in (Figure   2a).

The FT-IR spec)trum of Fe3O4@SiO2/SH/NH 2 (MNPS-IHSPN)
In order to show the bond between the functional groups to make the covalent 9 bond, FT-IR analysis is used, the range of frequencies is 500-4000 cm -1 , the absorption band for the 808 SiO 2 functional group and 1.222 cm -1 Si-O-Si. In the present magnetic nanocatalysis, the amount of silica coating at 879 and 694 and for mercapto (-SH functional group) was at 1.212 cm -1 and so, 2930-3390 cm -1 is about amino linker (-NH 2 functional group, which after the addition of sulfur functional group, the peak rate became narrower ie more regular. This would indicate an increase in the level of performance of the magnetic nanoparticles (Figure 2b).

Results of protein and MTX loaded onto magnetic nanoparticlesFe 3 O 4 @ (SiO 2 /SHor SiO 2 /SH/NH 2 ) by spectrophotometry
The purpose of this section is to investigate the absorption of drug biomolecules on the magnetic nanoparticle bed. Therefore, using the equation [7][8], we can examine the absorption rate. Under standard conditions, the amount of 30 micrograms per μl of the bio-molecule protein and drug are dissolved in 2 ml of sterile water, and then twice 25 milligrams of magnetic nanoparticles weigh in two separate dishes and on each One of the bio-molecule proteins and drug (60min, 36h) were solved separately.
The instant of dissolution of the two mixtures was continued at the instant of zero minutes to about an hour for each of the mixtures and summarized the obtained data. The results showed that the absorption rate of the protein in the first half hour was about 60% and the absorption rate of the drug on the nanoparticles were about 80%. The results obtained in the future did not change later, and the results showed that the absorption of nanoparticles in non-co-volcanic and covalent magnetic nanoparticles is approximately equal to one. After methotrexate has been stabilized on a surface of Fe 3 O 4 @SiO 2 /SH/NH 2 magnetic nanoparticles with different methods, we will now consider the methotrexate release rate. This is done with a phosphate buffer solution which is a mixture of 4 K 2 HPO 3 , Na 2 HPO 3 , KCl, and NaCl salts in a specified amount of distilled water, which is ready for use in the reaction after the autoclave. Using phosphate buffered saline, sodium hydroxide, and chloride acid adjust the pH to 8.8 to adjust the pH of the pH in the presence of phosphate buffer of methotrexate on the surface of nanoparticles and with sodium hydroxide and chloride. And the rate of release has been measured over a period of 2 hr to 72 hr at pH of 7.28-8.0. Looking at the data in the chart, it can be concluded that after 24 hr, the release of the methotrexate drug molecule from the surface of nanoparticles is about 80% at pH 7.8. With these results, can be said that is a full stabilization of MTX drug molecule on MNPs-IHSPN In-vitro. The results are shown in (Figure 3). All Error bars are selected as standard, indicating the accuracy of the data presented in the chart.

The effect of time in absorption
Reaction time for absorption, 1hr and 36hr in this test as the standard time for this research is very important. At the beginning of the reaction, patterns were to study the magnetic nanoparticles containing silica-protein-MTX absorption studies conducted showed that the uptake protein the concentration (30 μg/ml) and 10µg/ml for MTX, in The wavelength of 595nm for 30min (absorption wavelength protein) is 60% and the uptake MTX the concentration (10 μg/ml), in The wavelength of 304nm for 36h (absorption wavelength MTX) is 80%, and after 1hr and 36hr absorption be stable over time because the absorption process of protein and MTX was complete on the surface of magnetic nanoparticles. The absorption of biomolecules (MTX, protein) within a half-hour and twenty four-hour episodes were tested and the results were observed in (Figure 4). And according to the results, we find that over time gradually increased uptake, and it been stable after 1h for protein and 36h for MTX drug molecule.

Stability of magnetic nanoparticles in repeated use after recycling
The results of the magnetic nanoparticles with silica coating for MTX or BSA adsorptions were analyzed by spectrophotometric analysis over a period of 12-170 h for 10 periods for protein and 4 days for MTX, and the results showed that the efficiency of nanoparticles in the application again and again the stabilization of biomolecules, MNPs-MTX, BSA even decreased by 10 percent over the course of 15 percent. Magnetic nanoparticles are very important for sustainability under favorable reaction conditions and having the ability to re-use these magnetic nanoparticles. On the picture 6, the magnitude of this stability has been investigated in 7 days (the reaction process and optimal conditions are the same as 13 in the discussion and conclusion). The results are shown in (Figure 6).

The results of the absorption of protein by electrophoresis
In this section, protein is analyzed by the electrophoresis. Electrophoresis analysis is based on absorption at absorption times. Here, vertical electrophoresis to measure protein absorption. With respect to (Figure 7), it can be seen that the amount of stained specimens in the range of 0 to 60 mins, the absorption of protein on the magnetic nanoparticles have gradually dimmed, which this fading stain shows that biomolecules are absorbed on surface of magnetic nanoparticles.

Conclusion
In this project, the maximum capacity of magnetic nanoparticles (MNPs-IHSPN) with silica coating was used to stabilize and liberate biomolecules (MTX and protein). To do this, the MNPs were originally synthesized using a chemical co-precipitation method and their structure was identified with tools such as SEM and FT-IR. To do this, the amount of 25 mg of MNPs in 1 ml of solution of each biomolecules (protein, with an optimal concentration of 30 μg.ml -1 and MTX, with an optimal concentration of 10 µg.ml -1 ) at 25° C (room temperature) for protein and 37 o C for MTX in a water solvent for protein and water with DMSO for MTX in the specimens were then isolated for 60 minutes for the protein and 36 hour for MTX, after the reaction (these times are optimal) and tested in the spectrophotometer and UV-Vis apparatus to measure their absorbance. The results showed that the absorption rate a protein higher than 60% and 80% for MTX, which indicates a high percentage of absorption of biomolecule (protein or MTX) on the surface of magnetic nanoparticles, addition of biomolecule (protein or MTX) from the surface of the nanoparticles in the presence of external magnetic field and PBS buffer was shown to be more than 90%.
After the experiments, magnetic nanoparticles were extracted by external magnetic field. Overall results showed that biomolecules (MTX, protein) were almost completely stabilized on surface of magnetic nanoparticles. With the endured studies, this project can be a way of stabilizing biological and drug-mental molecules by MNPs-(MTX, protein) systems. Figure 1 The general process of MNPS-ISPN nanoparticles formation and its reaction with biomedical b Figure 1 The general process of MNPS-ISPN nanoparticles formation and its reaction with biomedical b 18 Figure 2 Left picture (figure 2b) is FT-IR analysis of nanoparticles (of up to down) and so, right picture  Results of EDX analysis. Up picture is for BSA protein and, down picture for MTX biodrug, left   Electrophoresis results to stabilize the amount of protein adsorbed on the surface of the nan Electrophoresis results to stabilize the amount of protein adsorbed on the surface of the nan