Synthesis And Studies Of Starch Nanoparticles As Anti-cancer Drugs/DNA Carriers

Nanotechnology was been widely used in the field of medicine, especially in the preparation of nano-drug carrier. But the choice of material was usually focused on the man-made polymer, and few natural material of starch was applied. In this study anion starch nanoparticle (StNP) was prepared by nanotechnology, and a series of nanoparticles which can be used as gene carrier, tumor-targeting drug carrier, anti-cancer complex drug carrier and active drug carrier were obtained through physical, chemical and biological modifying. The biochemical characteristic and anti-cancer capability of these nanoparticles were discussed in vitro and in vivo deeply.1. Preparation of StNP and the properties researchSynthesis of nanoparticle: Anion starch nanoparticle was synthesized in water-in-oil microemulsion under room temperature, and soluble starch was used as raw materials and POCl3 as crosslinking agent.Determination of StNP: The average diameter of nanoparticles was 50nm detected by atomic force microscope (AFM), with uniform in size and well dispersed, and the Zeta electric potential value of StNP was -8.5mV. The particles swelled slightly in saline solution, but the surface characteristics did not change obviously. StNP could load arginine (Arg) largely, which takes positive charge in neutral aqueous solution. The kinetics of drug release showed that the release time of Arg from StNP was 48h, and the drug was loaded through routes of physical absorption as well as static combination.2. Synthesize of gene carrier poly-L-lysine (PLL)-StNP and gene transformation in breast cancer cell line MCF-7Synthesize of PLL-StNP: PLL-StNPs were prepared by linking poly-L-lysine (PLL) on the surface of StNPs. Observed under atomic force microscope, PLL-StNPs distributed evenly, and their size was about 50 nm. The Characteristics of PLL-StNPs are : (1) Conjugating DNA efficiently: When DNA was added to PLL-StNPs solution, gel electrophoresis illuminated that PLL-StNPs could integrate DNA effectively. DNA could be released from the DNA-PLL-StNPs compounds, and the bands of DNA in electrophoresis gel did not change.It means that PLL-StNPs didn't change the property of DNA. (2) Protecting DNA from DNase I cleavage and ultrasound damage: DNA-PLL-StNPs complexes were treated with ultrasound for different time. Gel electrophoresis showed that DNA still bound to PLL-StNPs and bands of released DNA did not change, indicating that PLL-StNPs could prevent DNA from ultrasound damage. After digestion of PLL-StNPs-DNA complexes with DNase I, the strips of eluted DNA remained the same. So, PLL-StNPs might prevent DNA not only from ultrasound damage but also from DNase I cleavage. (3) No cell toxicity: Blank nanoparticles were cultivated with MCF-7 cell,and the MTT assay showed that PLL-StNP was nontoxical to cells.Gene transfection in breast cancer cell line MCF-7: PLL-StNP loading plasmid DNA pEGFP, which comprised green fluorescence protein gene, could be transfected into MCF-7 cells, and green fluorescence protein was expressed efficiently in cells. The best transfection efficiences was with N/P = 15.3. Preparation of folate conjugated starch nanoparticles and the targeting function to liver cancer cellsSynthesis of drug-loading FA-PEG-StNP: Actived folate was conjugated with PEG- (NH2) 2, the production was then modified on the surface of StNP, and FA-PEG-StNP was obtained with diameter of 130nm. Characteristics of FA-PEG-StNP: (1) hydrophilicity of FA-PEG-StNP: FA-PEG- StNP and FA-StNP were injected into peritoneum of mice respectively, and the number of peritoneal macrophages induced by FA-PEG- StNP was half of that induced by FA-StNP group, and the results shows that the FA-PEG-StNP was more hydrophilical than FA-StNP. (2) drug Sustaining release function: Anticancer drug doxorubicin (DOX) could be conjugated to FA-PEG-StNPs with the saturating capacity of 28μg/mg, and could be sustaining released for 48h. (3) Targeting of FA-PEG-StNP to liver cancer cells: FITC-loading FA-PEG-StNP was co-cultivated with liver cancer cells and natural liver cells, and the fluorescence intensity in former cells was 3 times higher than that of later cells. FITC-loading FA-PEG-StNP and FITC-loading StNP were co-cultivated with liver cancer cells, and the fluorescence intensity of former NP was 3 times higher than that of later NP. These results indicated that the nanoparticle modified by FA had the targeting function to liver cancer cells.Inhibition effect of drug loaded FA-PEG/StNP on liver cancer cells BEL7404: DOX-loaded FA-PEG/StNP and DOX-loaded StNP were incubated with liver cancer cells BEL7404 respectively. The cell death ratio of the former was 2 times than that of the later, and the Lc50 of the former was about 30% of that of the later. These results suggested that more DOX could enter into the cancer cells because of the combination of folate on FA-PEG/StNP with the folate receptor on cancer cells, thus the growth of cancer cells was inhibited effectively.4. Synthesis and synergetic anti-tumor effect of drug-loaded Se-StNP Synthesis of drug-loaded Se-StNP: Drug-loaded Se-StNP with size of 70nm was obtained by covertion of red element Se on the surface of DOX-loaded StNP, which had obvious shell-core structure, and the surface charge was -13.9mv.Synergetic anti-tumor effect research: Drug-loaded Se-StNPs were incubated with liver cancer BEL7404 cell, and MTT assay was used to detect the cell growth-inhibition ratio. the result showed that the growth-inhibition ratio of Drug-loaded Se-StNPs was higher by 13% than the summation of growth-inhibition ratios of element Se and DOX-StNP. This indicated that the synergetic anti-tumor effect was achieved by using DOX-loaded Se-StNPs. The possible reasons for the synergetic anti-tumor effect of complex nanoparticles are the follows: the first is that the speed of drug-release was slowed down because of the protection of shell-core structure; the second is that element Se eliminated the oxidated material around cells, and inhibited the speed of tumor cell division. In addition the permeability of cell membrane was improved and was available for drugs's transporting into cell; the last reason is that the negative element Se increased the electronegation of cell membrane and the permeability of cell membrane was thus improved further.5. Synthesize of dialdehyde starch nanoparticle (DASNP) and its application as anticancer drug carrier in vivoSynthesis, determination and drug-loaded of DASNP: Hydrolyzing starch was oxidized by sodium periodate (NaIO4) to obtain dialdehyde starch (DAS), and DAS was crosslinked by POCl3 in water-in-oil micro-emulsion to obtain dialdehyde starch nanoparticles(DASNPs)under room temperature. The oxidation degree (OD) of prepared DASNPs was(40±5)%, and the diameter was in range of 80~100nm, with uniform in size and well dispersed. DASNPs could sustain resolve in solution with pH5~8. MTT assay showed that DASNPs had no cell toxicity in vitro. DASNPs suspension was injected in nude mice subcutaneously, and there was no inflammation reaction in the hypodermal tissue. It means that DASNPs is biocompatible and biosecurity, and can be used as drug carrier. Anticancer drug 5-Fu was combined to DASNPs through the imine bond, and the load of drug was 10%. The drug-loaded DASNPs could sustaining release 5-Fu for about 20h in pH5~7.Tumor cell analysis of drug-loaded DASNPs in vitro: When the concentration of drug-loaded DASNPs was 1mg/ml, the breast cancer MCF-7 cell growth inhibition ratio was 74.5%. Flow cytometry assay showed that drug-loaded DASNPs changed the cell cycle and more cells were blocked in S phase.Studies on tumor suppression of drug-loaded DASNPs in vivo: Drug-loaded DASNPs were injected subcutaneously in nude mice loaded solid tumor, and the tumor suppression was 75%, which was higher by 30% than that of single drug. H&E staining showed that there was obvious tissue cells apoptosis in tissue slices, and immunohistochemical studies showed that the expression of Bcl-2 decreased and the expression of Bax increased obviously. All these results indicated that drug-loaded DASNPs could suppress tumor growth effectively in vivo, and the suppressor was achieved by inducing tumor cell apoptosis.