Synthesis of chitosan-folic acid (Chitosan-FA)
The results of chitosan as well as chitosan attached to folic acid are shown in Figure. 1 (image A and B). The results showed that there was a significant difference in the morphology of free chitosan and chitosan attached to folic acid. The results show that free chitosan has a spindle-shaped and a relatively uniform distribution. The size of nanoparticles obtained from 100 kDa chitosan was about 20-40 nm. The morphology of chitosan-derived nanoparticles and chitosan-folic acid nanoparticles were significantly different as well. The nanoparticles made from chitosan-folic acid exhibited high self-aggregation and cluster formation.
Confirmation of chitosan-folic acid synthesis using TGA
Thermal Gravimetry Analysis (TGA) is a method that records the weight changes of materials as a function of time or temperature in a controlled environment. In this method, heat is applied to the material at a specific temperature and in one environment. This method is used to identify and determine the amount of volatiles. In the range of 300 to 400° C, the TGA diagram generated from chitosan showed only one weight loss phase, as shown in Figure. 3, image (C). It shows that chitosan destruction is straightforward and just requires one step. In addition, the TGA device was used to record the weight variations of folic acid in a constant environment and at different temperatures. The results showed that weight loss was detected in folic acid at temperatures lower 200° C, which is likely resulted from loss of water molecules in folic acid. At temperatures over 200° C, folic acid degradation was more severe, with about 50% of its weight being reduced between 250 and 350° C. At temperatures ranging from 350 to 600° C, the third stage of weight loss was observed. In general, the process of folic acid weight loss was observed in three stages of 100 to 200°C, 250 to 350°C, and 350 to 600°C with increasing temperature at the same atmospheric pressure (Figure. 1, image C).
Unlike chitosan, the weight loss trend in chitosan-folic acid nanoparticles included 3 to 4 stages of weight reduction. At temperatures ranging from 120 to 250° C., about 5% of the weight was reduced. In addition, in the temperature range of 250 to 350° C, the second weight loss phase was observed, in which more than 50% of the weight was reduced. In the temperature range above 400° C, another weight loss was observed. Considering that the weight loss process in chitosan-folic acid nanoparticles is somewhat in the middle of the weight loss process of chitosan and folic acid, it can be concluded that the synthesis of chitosan-folic acid nanoparticles has been done correctly (Figure. 1, image C).
Confirmation of chitosan-folic acid synthesis
Because the binding of folic acid to chitosan causes many changes in the amount of chitosan absorption, UV spectroscopy of chitosan-folic acid nanoparticles was Figure 1, image D shows that the binding of folic acid to chitosan increases absorption in the 290 nm range, whereas, chitosan uptake was very low in this range.
Confirmation of chitosan-folic acid nanoparticle synthesis using FTIR spectroscopy
The FT-IR spectra of the synthesized chitosan-folic acid nanoparticles are shown in Figure. 1, image E. The peaks observed in the range of 3320 are associated with the O-H and N-H functional groups in chitosan. Furthermore, the peaks in the 2830-2900 range are linked to the C-H functional groups in chitosan. Also, the peak in the 1621 range is related to the amide I in chitosan, whereas the peak in the 1530 range demonstrates N-H binding of the amide groups and primary amine group’s acrylates in chitosan. Furthermore, the O-H and C-H bonds of the chitosan circular structure are visible in the 1386 and 1322 ranges, respectively, and 1010 range indicates C-O binding in chitosan.
The bands observed in pure folic acid in the range of 3421 to 3600 pertain to the hydroxyl (OH) group of glutamic acid and the NH-group of the pteridine ring. Also, The C=O bond in folic acid is related to the strong band observed in the 1730 range, and the NH-bond in folic acid is related to the band observed in the 1607 range. The intensity of bonds related to C=O binding related to the amide I group in the range of 1010, as well as bands related to C-N bonds with N-H related to amide II in chitosan, decreased after folic acid binding. The hydrophilic interaction between C = O, C-N, and N-H in amide I and amide II causes the strength of peaks related to folic acid and chitosan bonds to decrease. The FTIR spectra of chitosan showed no bands in the range of 2000 to 2200 (Figure 1, image E). ). However, after binding of chitosan to folic acid in this range, bands corresponding to folic acid and chitosan binding were seen. The above results are in agreement with the findings of (Pinto et al. 2021).
Evaluating the ability of PLA-PEG/Chitosan-FA and DNA nanoparticles to neutralize negative DNA charges
The results of the ability of PLA-PEG/Chitosan-FA nanoparticles to neutralize DNA negative charge showed that the speed of DNA movement in an agarose gel was gradually lowered by increasing the chitosan-folic acid ratio in PLA-PEG/Chitosan-FA/DNA nanoparticles compared to control DNA (uncoated DNA). DNA fully ceased moving at a PLA-PEG to chitosan ratio of 30 to 15, indicating that PLA-PEG/Chitosan-FA/DNA nanoparticles may neutralize DNA's negative charge (Figure 2).
Investigating the size and zeta potential characteristics of PLA-PEG/Chitosan-FA/DNA nanoparticles by DLS device
Increasing the amount of chitosan-folic acid in PLA-PEG/Chitosan-FA/DNA nanoparticles increased the amount of zeta-positive potential. Also, the results showed that the PLA-PEG/Chitosan-FA/DNA nanoparticles with the lowest percentage of chitosan-folic acid had the lowest zeta potential, whereas PLA-PEG/Chitosan-FA/DNA nanoparticles with the highest percentage of chitosan-folic acid had the highest zeta potential. On the other hand, the effect of increasing the percentage of chitosan-folic acid in PLA-PEG/Chitosan-FA/DNA nanoparticles on the size of nanoparticles was due to other factors studied by DLS. The results showed that despite the zeta potential, there was no link between increasing the percentage of chitosan-folic acid in PLA-PEG/Chitosan-FA/DNA nanoparticles and particle size. In addition, PLA-PEG/Chitosan-FA/DNA nanoparticles with the lowest percentage of chitosan-folic acid had the highest particle size. On the other hand, increasing the percentage of chitosan-folic acid in PLA-PEG/Chitosan-FA/DNA nanoparticles did not result in the same direct reduction in nanoparticle size. The size of PLA-PEG/Chitosan-FA/DNA nanoparticles increased again when the ratio of chitosan-folic acid to PLA-PEG was increased from 30:6 to 30:30 (ratio is not %) (Figure 3, B).
The DNA encapsulation efficiency is shown in Figure 3 (A). According to the results, the PLA-PEG/Chitosan-FA/DNA nanoparticles with the highest chitosan-folic acid ratio (63.82%) had the maximum DNA encapsulation effectiveness. In general, increasing the chitosan-folic acid ratio and DNA loading by PLA-PEG/Chitosan-FA nanoparticles had a direct and significant association. As a result, increasing the chitosan-folic acid ratio in PLA-PEG/Chitosan-FA nanoparticles increased the percentage of DNA loading. The neutralization of DNA negative charge by interaction with amino groups in chitosan could be one of the reasons for the increase in DNA encapsulation efficiency at high chitosan-folic acid ratios because of the negative charge in the DNA phosphate group as well as the carboxyl group in the PLA polymer.
Toxicity of PLA-PEG/Chitosan-FA/DNA nanoparticles
The cytotoxicity of some nanocarriers is a major obstacle to gene delivery systems. Therefore, determining the nanocarriers cytotoxicity is critical (Amani et al. 2019). The comparison of the mean results of the toxicity test of PLA-PEG/Chitosan-FA/DNA nanoparticles by the MTT test showed that the nanoparticles used in this study had very low toxicity (Figure 3, C). So, after treatment with PLA-PEG/Chitosan/DNA nanoparticles at a concentration of 1 mg/ml and PLA-PEG/Chitosan-FA/DNA nanoparticles containing a w/w ratio of 30: 30, (PLA-PEG: Chitosan-FA), MCF-7 cells showed the lowest percentage of viability (87.64%). As a result, increasing the chitosan-folic acid to PLA-PEG ratio in PLA-PEG/Chitosan-FA/DNA nanoparticles had no effect on MCF-7 cell mortality.
DNA release pattern from PLA-PEG/Chitosan-FA/ DNA nanoparticles
The DNA release pattern from PLA-PEG/Chitosan-FA/DNA nanoparticles is shown in Figure 3 (D). The results showed that the DNA release pattern from PLA-PEG/Chitosan-FA/DNA nanoparticles was explosive at first, then gradually decreased. More than 60% of the total amount of DNA released during the 28 days was released in the first three days. With increasing the amount of chitosan-folic acid in PLA-PEG/Chitosan-FA/DNA nanoparticles, DNA release increases significantly. The release rate of DNA was 63.74% after three days of incubation of PLA-PEG/Chitosan-FA/DNA nanoparticles in PBS buffer containing a 1: 30 ratio of PLA-PEG to chitosan-folic acid. However, after three days of incubation in PBS buffer, the amount of DNA released from PLA-PEG/Chitosan-FA/DNA nanoparticles containing equal ratios of PLA-PEG to chitosan-folic acid was only 82.16%. The results of DNA release from different nanoparticles used in this study showed that more than 60% of the total DNA released was released explosively in the first three days after nanoparticles were treated with PBS buffer.
MCF-7 cells transformation using PLA-PEG/Chitosan-FA/DNA nanoparticles
Fluorescence microscope image results showed that in some MCF-7 cells treated with PLA-PEG/Chitosan-FA/DNA nanoparticles, green fluorescence was observed (Figure 4, A, B, C and D). This demonstrated the ability of PLA-PEG/Chitosan-FA/DNA nanoparticles to transmit and release DNA within MCF-7 cells. However, the control DNA, was unable to transfer to MCF-7 cells. An electrostatic repulsion is formed between DNA and the cell membrane due to the negative charge in DNA and the cell membrane due to the presence of phosphorus groups in the structure of DNA, as well as the phospholipid membrane of cells, which inhibits DNA from penetrating into the cell. In addition, the appropriate structure and large size of DNA are other factors in the inability of control DNA to transmit to the cell. The results also indicated that increasing the ratio of chitosan-folic acid to PLA-PEG in PLA-PEG/Chitosan-FA/DNA nanoparticles increased gene transfer efficiency in a linear and significant manner. In a way, the efficacy of GFP gene transfer to MCF-7 cells was improved by increasing the ratio of chitosan-folic acid to PLA-PEG in PLA-PEG/Chitosan-FA/DNA nanoparticles. Cells treated with PLA-PEG/Chitosan-FA/DNA nanoparticles comprising equal proportions of PLA-PEG and chitosan-folic acid had the highest gene expression (38.22%). According to the results, the PLA-PEG/Chitosan-FA/DNA nanoparticles with the lowest chitosan-folic acid to PLA-PEG ratio (30: 2 PLA-PEG: Chitosan-FA) had the lowest gene transfer efficiency (34/18%). Gene expression efficiencies were 23.2% and 36.5% in PLA-PEG/Chitosan-FA/DNA nanoparticles having 30:6 and 30:15 (PLA-PEG: Chitosan-FA) ratios, respectively (Figure 4, E).