The Research And Pharmaprojects Of Colorectal Cancer Drug Based On HKP-miR-150

ObjectiveRNA interference(RNA interference, RNAi) technology offers us a new way for the treatment of colorectal cancer.This topic to a miRNA- 150(miR- 150) as the active pharmaceutical ingredient(API), Polymer with histidine-lysine polymer,(Histidine-Lysine Polymer, HKP) for the body into microRNA polypeptide injection drug carrier and used for specific target therapy for the research and development of colorectal cancer drug. MethodsLigand targeting carrier type mixed with microRNA reagent preparation.In this study, observe the HKP entrapment capacity to miRNA by 1% agarose gel electrophoresis to analysis the nitrogen and phosphorus suitable mass ratio of HKP and miR-150 by agarose gel electrophoresis.Particle size distribution of nanoparticles in aqueous suspension can be measured directly by laser granulometer, which using dynamic light scattering principle.Using dynamic light scattering principle to measuring quality of different proportion of HKP laser granulometer and microRNA forming nanometer complex particle size of the potential.RiboGreen RNA quantitation reagent is an ultra-sensitive nucleic acid fluorescent dyes. Thus, drew the standard curve between fluorescent activity and the miRNA concentration, and then according to the fluorescence value on the standard curve to obtain the corresponding concentration by calculating the entrapment efficiency.Using real-time PCR method for the import of target gene mRNA expression level in Lo Vo cells which is half quantitative analysis, to detect the mixture HKP- miR- 150 in vitro biological activities.LoVo cells were inoculated subcutaneously into nude mice to form subcutaneous transplantation tumor and Pharmacodynamic study on polypeptide formulation for targeted HKP-miR-150. The pharmaceutical formulation and other control group were injected into the tumor tissue for continuous treatment every other day, 1 OD mi RNA / 70 ul / mouse/ administration, a total of 9 times administration. The whole process took 18 days from the first administration till got all specimens. Observe and record nude mice subcutaneously transplanted tumor size, shape, growth status. Executed nude mice and the tumor tissue specimens of fluorescence quantitative PCR to detect the expression of tumor tissue specimens of mi R-150, PCNA(Proliferating Cell Nuclear Antigen, PCNA) method to detect the cancer cells proliferation and in TUNEL method to detect the cancer cells apoptosis.By cy-3fluorescent reagent for the HKP-miR-150 and without HKP in the package of miR-150 for qualitative analysis, observation of the mixture HKP-miR-150 in animal metabolism and distribution.Through the poisonous and toxic test in mice and cynomolgus monkey two animal species, and guinea pig skin allergy test to evaluation for the safety of HPK reagent. ResultsAfter agarose gel electrophoresis, it is failed to observe free miRNA in the mass ratio of 4: 1, that is, when mi RNA and HKP are mixed in this ratio or even higher, miRNA can be entrapped completely by HKP.Mixed HKP(API) with the oligo(mimics) in the different mass ratios: 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, to measure the partical size and potential of these nanoparticles. The data of all ratios(Table 2 and Table 3) showed the stable potential and particle size.With the increment of HKP(figure 5), the concentration of free miRNA was reducing, it means that the entrapment effect was increasing with obvious gradient, and the concentration of free mi RNA was extremely low in the ratio of 4: 1, so the ratio is suitable for further research.The complex of HKP(API) and miRNA(unmodified)(N/P ratio: 4: 1) was transfected into LOVO cell lines, and using real-time fluorescence quantification PCR in vitro to detect mi R-150 relative expression for its semi-quantitative analysis,and the result the result reach the ideal.Successfully established a nude mice subcutaneously transplanted tumor animal models.As the treatment continued 18 days, in addition to the miRNA-150 treatment group, whose tumor growth slowed remarkablely, other groups presented similar growth trend with the control group, and started from the seventh treatment, tumor growth significantly inhibited.Total RNA were extracted from tumor tissues, which were took out from euthanasia of mice in the end of treatment, to detect target gene expression level in tumor tissue by real-time fluorescent quantitative PCR. The results showed the target gene miR-150 expression of the treatment group(HKP-miR-150 mimics) was significantly higher than other group.After the treatment, it was detected that the PCNA(Proliferating Cell Nuclear Antigen) in tumor tissue of HKP-miR-150 mimics treatment group had significant difference from other control groups. PCNA got a substantial reduction in the tumor cell nucleus after miRNA150 / HKP formulation treatment, indicated tumor cell death.After the treatment, it was detected that HKP-miR-150 mimic treatment group had significant difference from other control groups by classic TUNEL method. TUNEL nuclei got a substantial increase in the tumor cell after HKP-miR-150 mimic formulation treatment, indicated the treatment induced programmed tumor cell death.Cy-3fluorescent qualitative analysis results showed that HKP-miR-150 in animals duration than miR- 150 in animals were significantly prolonged.Abdominal imaging results showed that after 24 hourscy-3-HKP-mi R-150 HKP in small intestine, colorectal, kidney, spleen, there is still a drug concentration. Safety evaluation results show that the application of HKP package carrier animals are not seen obvious systemic acute toxicity and long-termtoxicity and skin allergy reaction. HKP safe doses greater than 1080 ug/kg. ConclusionAfter agarose gel electrophoresis, it is failed to observe free miRNA in the mass ratio of 4: 1, that is, when mi RNA and HKP are mixed in this ratio or even higher, miRNA can be entrapped completely by HKP. And Pharmacodynamics experiment showed that the mixed preparation through within the tumor injection can induce tumor cell apoptosis, stylized effectively inhibiting tumor cell growth.HKP can serve as a kind of efficient and low toxicity of micrornas drug carrier, is applied to the mi RNA drug carrier in the body So HKP- miR- 150 in the study of the pharmaceutical preparations can be used as a new generation of 1.1 class to sustain colon cancer drug candidate innovation continue to drive towards the direction of the clinical trials.