Better Treatment And Diagnosis Biology Essay

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200 mg of pre-synthesized manganese oxide nanoparticles stabilized with oleic acid was dispersed in 1000 of CHCl3. This dispersion was added to 0.5 g CTAB in 10 mL Millipore Milli-Q water in a round bottom flask which was kept stirring for 2 h and then at 60oC for 1 h to remove chloroform. To this solution 10% Pluronics-123 was added slowly and stirred for 30 min. To this dispersion appropriate amounts of 2 N HCl and NH4F was added and again stirred for another 30 min. TEOS was added to the above mixture which was allowed to stir for 24 h at room temperature and then at 80oC for another 24 h without stirring. Solvent extraction procedure was carried out using a mixture of ethanol and HCl for 6 h under stirring. The procedure was carried out for 3 times and then vacuum dried at 50oC overnight. Thus obtained powder sample was characterized. Briefly, 0.1 g of the mesoporous sample (MA-SBA/MO-SBA) was taken in a round bottom flask containing 20 mL of toluene and kept stirring for 30 min. To this dispersion, 0.02 mL of APTES was added and kept overnight under reflex condition at 80oC with stirring. Modified mesoporous sample was washed several times with toluene and water to remove unreacted APTES and vacuum dried at 80oC. Amine functionalized sample was characterized and stored till further use. Folic acid tagging was done to attain targeting capability to the mesoporous sample. Click chemistry was employed to tag folic acid to the amine functionalized mesoporous sample. To 16.4 mg of N-hydroxyl succinimide (NHS) solution, 7.2 mg of EDC was added along with excess of folic acid. The above mixture was kept in dark for 2 h to activate carboxylic group of folic acid. 50 mg of amine functionalized mesoporous sample (MA-SBA/MO-SBA) was added to the mixture and kept overnight for tagging to takes place. Tagged sample was washed several times with PBS to remove unreacted components and then stored in PBS until further use. 5-fluoro Uracil is highly soluble in water. A stock solution of 2 mg per 1 mL was prepared. 10 mg of the as synthesized MA-SBA/MO-SBA was taken in a glass vial with 2 mL millipore water in it. Nanoparticles were dispersed using bath sonication for 2 min. Later, to the dispersion, a mL of the stock solution of 5-fluoro Uracil was added. The entire solution was make-up to 3 mL using millipore water and bath sonicated for 5min and then allowed to stir overnight at room temperature. Nanoparticles were separated using centrifugation and lyophilized for later use. Estimation of 5-fluoro Uracil loading was done using UV spectrophotometer at 265 nm. Encapsulation efficiency was estimated using equation: Procedure was developed somewhere else [28] was adopted here with some modification. Briefly, 5ml of fresh blood collected from a healthy adult was taken into a test tube containing 10 µL of 0.5 M EDTA solution. The blood sample was centrifuged and supernatant was discarded. To the sample was diluted using freshly prepared phosphate buffer saline. From this, 50 µL cell suspension containing RBCs was treated with mesoporous samples. The samples were sealed using paraffin film and then incubated for 2 h at 37°C. Later, 40 µL of 25% glutaraldehyde was added to each sample to stabilize and kept at 4°C for 4 h. Stabilized samples were further centrifuged at 1000 rpm for 10 min and precipitates were made into dispersion using PBS and a drop of these solutions was imaged using Leica DM EP microscopy (Leica microsystem, India) fitted with a digital camera. Morphology and particle size was studied using field emission scanning electron microscopy (FE-SEM) (JSM 6701F, JEOL, Japan) and field emission transmission electron microscopy (FE-TEM) (JEM 2100F, JEOL, Japan). Dried sample was used for FE-SEM with platinum coating over it. Ethanol dispersions of nanoparticles were used for FE-TEM imaging. Aggregate particle size and zeta potential was determined using dynamic light scattering (DLS) using zetasizer (Nano-ZS, MALVERN Instruments, USA) at room temperature. Appropriate dilutions of aqueous dispersions of nanoparticles where used for DLS studies. Data collected were obtained after 3 readings, each reading comprising 12 runs. Ultra sonication was performed where ever it was necessary. Diffraction studies were done using a powder X-ray Diffractometer (D8 FOCUS, Bruker AXS, Germany) in the range of 10 to 60° in steps of 0.01° at a scan rate of 1 step/sec. CuK α radiation (λ = 1.5406 Å) was employed to irradiate the dried sample taken for analysis with no further sample preparation. X-ray fluorescence spectroscopy (S8 TIGER, Bruker AXS, India) and Energy dispersive X-ray spectroscopy was employed to know the presence of manganese oxides in synthesized samples. Standard sampling procedures were followed during the experimental studies. Brunauer-Emmet-Teller (BET) model and Barett-Joyner-Halenda (BJH) model was used to estimate the surface area, total pore volume, and the pore size respectively. Analysis was carried out using Physisorption analyzer (ASAP 2020 - Physisorption analyzer, micromeritics®, India). Degassing was done at 350oC and 200oC for MA-SBA and MO-SBA respectively. Less temperature was used to avoid desorption of chemisorbed oleate on MO nanoparticles in MO-SBA. FT-IR spectrometer (Spectrum 100, Perkin Elmer, USA) was employed to know the presence or absence of various functionalities in the samples during the synthesis procedure. KBr pellets loaded with dried samples were used for spectroscopic analysis. Measurements were performed in the 400-4000 cm⁻¹ range with an interval of 4 cm⁻¹, by averaging the 12 scans obtained for each sample. Data was processed using Origin 8 software, MS Excel and presented as mean ± standard error of the mean (S.E.M.). Sol-gel science and technology reached its advanced stage to produce some highly sophisticated network structures. One such includes mesoporous silica (eg. MCM-41, SBA-15). Here, for the first time, we developed manganese oxide doped SBA-15 spherical nanoparticles by applying some basic concepts in sol-gel science and technology. MA-SBA synthesis was carried out like any common methods of SBA-15 synthesis (schematically represented in fig.1). During the synthesis procedure, the addition of manganese (II) acetate tetra hydrate in the acidic environment (2N HCL) helped in the formation of manganese dihydroxide (Mn (OH)2). As it was known, during the condensation step of the sol-gel synthesis of mesoporous silica, monomers condense to form polymeric networks along a guiding support (SDA).

In general:

Or

In presence of Mn (OH)2:

Or

There is every possibility for the above reactions to takes place which results in the presence of manganese as a part of internal structure in mesoporous silica. However, there is also a probability that manganese (II) acetate salt might get trapped in the mesopores which on calcination results in the formation of manganese oxide. Further the presence of acidic medium accelerates the formation of manganese hydroxide and acetic acid.

Under normal conditions this results in formation of numerous oxides of manganese preferably manganese dioxide (MnO2) and hausmannite (Mn3O4). Overall, manganese will be present within the internal structure and also the pores of mesoporous silica. Synthesis and growth mechanism was depicted in the schematic diagram (fig. 1).

With respect to MO-SBA, it was more of a composite where different materials get deposited one over the other based on secondary forces and finally silica was grown over the guiding structure. Oleate coated silica was further stabilized with CTAB to disperse it in water. Further, P-123 was added to get SBA-15 and NH4F helps in maintaining the spherical shape. Solvent extraction was a crucial process in this method as calcination, if done results in removal of oleate coating around the MnO which can potentiate the formation of other oxides of manganese (MnO2, Mn3O4 etc). So, solvent extraction was performed. In this case, manganese oxide exists in the pores but not in the internal structure of silica. Various steps involved in the synthesis of MO-SBA were depicted schematically in figure 2. Particle size of the mesoporous nanoparticles was studied using electron microscopy. Nanoparticles were spherical shaped as expected due to the addition of NH4F. MA-SBA particles were found to be in the range of 20 nm to 40 nm as shown in the figure 3. Dynamic light scattering results showed the presences of aggregates having a mean size of 148.7±28 nm (Table 1). Table 1 shows the particle size and surface of the nanoparticles. The surface charge showed the nanoparticles can form a stable dispersion. After amine functionalization of the nanoparticles the size increases as expected due to the deposition of silica and the amine groups attract more water which resulted in high hydrodynamic radius in both MA-SBA-NH2 and MO-SBA-NH2. Transmission Electron micrographs of the MA-SBA revealed the porous nature of the MA-SBA (fig. 4). Inset in the TEM micrograph of figure 4 revealed the presence of manganese oxide in the nanoparticles, pointed using red arrows in the figure. Manganese oxide nanoparticles stabilized with oleic acid (MO) synthesized using the procedure reported by us [58] was checked for its particle size using scanning electron microscope. Micrographs of these MO nanoparticles revealed an aggregate size of 25 nm to 40 nm (fig. 5). Aggregation of MO nanoparticles may be due to interlocking of oleate carbon chains of adjacent particles via hydrophobic forces. MO nanoparticles internalized SBA-15 SEM micrographs had a particle size ranging from 40 nm to 70 nm (fig. 6 (A)) and the zeta sizer showed the aggregate mean size around 238±9 nm (Table 1). The increase in the particle size of MO-SBA can be attributed to the use of pre-synthesized manganese oxide nanoparticles. Since prepared nanoparticles were in the range of 10 nm (single particle) to 40 nm (aggregate), growing of silica over the surfactant stabilized nanoparticles will invariably result in larger size. TEM micrographs revealed the internal porous structure of MO-SBA shown in figure 6 (B) and (C). Although the pores were not very clearly visible, the porous nature which was further confirmed by N2 absorption-desorption studies. The presence of dark patches similar to TEM micrographs of MA-SBA can be attributed to the presence of manganese oxide nanoparticles (fig. 4). N2 absorption and desorption studies were carried out to know the internal structure, porosity of the mesoporous nanoparticles. This is important as the technique clearly elucidates various parameters that define the internal structure of the mesoporous material. Moreover, the internal structure directs the type of application. With high surface area and porosity, the material can be used for catalytic as well as drug delivery applications. MA-SBA with its manganese oxide in the internal pores, seen in TEM micrographs (fig. 4), had very high surface area of 454.33 m2/g. Similarly, MO-SBA had high surface area, 397.90 m2/g. Relatively less surface area of MO-SBA than MA-SBA might be due to the presence of MO in the pores which was may be filled partially in the pores of MA-SBA (present in both walls and pores). Graphs representing the BET and BJT isotherms were depicted in the figure 7. Various parameters defining the surface area and porosity of the nanoparticles were tabulated in table 2. XRD of MA-SBA and MO-SBA were depicted in figure 8. Both MA-SBA and MO-SBA diffractograms showed a broad peak from 2θ values, 12o to 25o come from silica portion of the nanomaterial. In case of MA-SBA, the presence of peaks in and around 30o attributed to Mn2O3 which was in consent with literature data [59] and JCPDS Card No. 24-0734. MO-SBA showed diffraction peaks specifically at 30o and 42o which was in consent with XRD data obtained for MO nanoparticles in this paper, literature [29] and JCPDS Card No. 07-0230. Once again the presence of manganese in the form of manganese oxide was confirmed.

Compositional studies of the MA-SBA and MO-SBA were carried out using X-ray fluorescence (XRF) technique and Energy Dispersive X-ray Spectroscopy (EDS) respectively. XRF results for MA-SBA (Table 3) showed the presence of 3 % of manganese which is important and sufficient as excess of manganese content might result in toxicity; manganese has a proven history of causing neurodegenerative diseases [30]. Figure 9 shows the EDS of MO-SBA with presence of silica in higher amounts and manganese oxide. Short peaks around silica peak representing the presence of platinum which comes from the platinum coating used to avoid charging effect as the sample is non-conducting. FT-IR studies were carried out to study the fundamental vibrations and associated rotational vibrations which form a signature of chemical entities. Vibrational spectra of various samples of MA-SBA, MO-SBA at various stages of synthesis was showed in the table 4. 5-fluoro Uracil loading and release kinetics were done to analyze the efficiency of the nanocarrier for drug delivery application. Encapsulation efficiency of the drug on to the MA-SBA and MO-SBA carrier was found to be ~92 % and ~91 % respectively. This high encapsulation efficiency can be attributed to the mesoporous structure of the nanocarrier and also its high hydrophilic nature. As the drug, 5-fluoro Uracil was highly hydrophilic with its two -C=O groups, two -N-H groups and one -F group. Intense hydrogen bonding between the -OH groups of silica and the highly electronegative atoms (-F, -O and -N) of the drug might result in this high efficiency. Mesoporous silica as such results in a sustained release formulation for longer periods of time, as the hydrogen bonding remains quite strong the release of the drug could become very slow which was proven in the release studies. This resulted in release of only ~20 % after one week. The release was biphasic in both MA-SBA and MO-SBA as expected and showed sustained release characteristics. As the release rate was infinitesimally slow in both MA-SBA and MO-SBA (figure 10) the nanoparticles on entering the cells will dump a massive dose of drug which will be highly effective in cancer therapy. As the drug used here was an anti-cancerous agent, initial release of drug before reaching the target site was detrimental to normal cells. Moreover, these release characteristics can be very useful in case of highly resistant tumors where a constant drug supply was needed to kill them. Overall, the release system was very good in holding lot of drug owing to its porous nature and the choice of the drug was crucial in achieving this prolonged sustained release. First contact for any intra venous formulation was blood and its components. To test the compatibility of the nanocarrier system, we performed haemocompatibility studies. Studies were carried on MA-SBA and MO-SBA along with a control. From figure 11 one can clearly see that the red blood cells were intact and no damage was done. Moreover there is no deposition of nanoparticles during the treatment period, revealing the non-toxic behaviour of the nanocarrier system. Overall, the nanocarrier system was found to be non-toxic to the blood. Manganese oxide doped mesoporous SBA was synthesized, characterized and successfully presented as a potential drug delivery agent. During the synthesis and characterization of the material some important findings were made and based on those conclusions were drawn. Synthesized MA-SBA revealed the formation of manganese oxide inside the mesoporous silica which was in sizes below nanometer range. This shows that the mesoporous material had pores less than nanometer range and it was successful in confining the size of the manganese oxide formed. High surface area and spherical shape of the synthesized MA-SBA and MO-SBA supported the ability of synthesis procedures and mechanism elucidated. Although micrographs showed the presence of nanoparticles below 100 nm, but the nanoparticles exist in the form of aggregates which was revealed by dynamic scattering experiments. During the synthesis process of MA-SBA, the precursors were used in 1:1 ratio. But XRF results revealed that the doping of manganese was low which was a decent ~3 %. This showed that some of the manganese precursor might get washed way during vacuum filtration which was done before calcination. In case of MO-SBA, they too exist as aggregates but the size was still in required range. EDS spectra showed a strong peak of Mn-O which comes from MO nanoparticles entrapped in MO-SBA. Further the presence of metal-oxide was confirmed by FT-IR studies. Post-synthesis functionalization of mesoporous samples resulted in increased aggregate size which was expected. High porous nature of the silica material was further ascertained by high encapsulation efficiency of drug, 5-fluoro Uracil. Drug release rates were very slow and showed biphasic sustained release kinetics. This slow release can be attributed to presence of high electronegative atoms in the drug which forms strong hydrogen bonding with the hydroxyl groups of the silica material. These release characteristics make this drug delivery system a suitable candidate for drug resistant tumors which need to be supplied with constant amount of drug. Toxicity evaluation was done. As high as 500 µg of sample concentration was used to treat red cells even then there was no deformation and membrane rupture. Overall, as synthesized manganese doped/entrapped mesoporous nanoparticles can be used in nanomedicine for application like drug delivery and imaging.

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