Study On The Preparation Of Low Molecular Weight Heparins PH-sensitive Reversed Polymer Nanoparticles And The Bioavailability In Rats

Objective:This thesis has established a vitro analysis method for low molecular weight heparin(LMWH) and investigated the stability of low molecular weight heparin in artificial gastric juice and intestinal juice. The preparation of LMWH p H-sensitive reversed polymer nanoparticles and its freeze-dried production have been studied, thiolated chitosan and O-carboxymethyl chitosan as carriers and LMWH as model medicine. It has also investigated the release characteristic in vitro and relative bioavailability in vivo of p H-sensitive TCS/O-CMC reversed polymer nanoparticles. Methods:(1)Establishing a vitro analysis method for LMWH and investigating the stability of low molecular weight heparin in artificial gastric juice and intestinal juiceThe content of LMWH was determined using the azure A colorimetric-ultraviolet spectrophotometry method. The stability of LMWH in artificial gastric juice and intestinal juice was observed by determine the content of LMWH in different time.(2)The preparation and characterization of low molecular weight heparin p H-sensitive TCS/O-CMC reversed polymer nanoparticlesLMWH p H-sensitive TCS/O-CMC reversed polymer nanoparticles have been prepared by ionic crosslinking method, using the reverse of thiolated chitosan and O-carboxymethyl chitosan as carriers, LMWH as model medicine and hydroxypropyl methyl cellulose phthalate ester(HP-55) as ionic crosslinking agent. Single factor experiment and orthogonal design have been applied to optimize the technology of preparation. Particles size, polydispersity index(PDI) and zeta potential were detected by Malvin particle size analyzer. The morphology of p H-sensitive reversed polymer nanoparticles has been observed by transmission electron microscope.In addition, loading capacity, encapsulation capability of the p H-sensitive reversed polymer nanoparticles have been analyzed by azure A colorimetric-ultraviolet spectrophotometry method.(3)The preparation and characterization of LMWH p H-sensitive TCS/O-CMC reversed polymer nanoparticles freeze-dried production.LMWH p H-sensitive reversed polymer nanoparticles freeze-dried products have been prepared by using respectively glucose, mannitol and glucose/mannitol as cryoprotectant. The effects of the cryoprotectant on the appearance, particle size, PDI and zeta potential of the nanoparticles are studied. The morphology of p H-sensitive reversed polymer nanoparticles is observed by transmission electron microscope.(4)Investigating release characteristic in vitro of p H-sensitive TCS/O-CMC reversed polymer nanoparticles.Dynamic dialysis system has been adopted to study the drug release from p H-sensitive reversed polymer nanoparticles in vitro in p H 1.0, p H 6.8 and simulated gastrointestinal p H gradient conditions.(5)Bioavailability of LMWH p H-sensitive TCS/O-CMC reversed polymer nanoparticles in rat plasma.A single oral dose of LMWH injection, LMWH solution, p H-sensitive TCS nanoparticles and p H-sensitive TCS/O-CMC reversed polymer nanoparticles have been respectively given to rats. Plasma concentrations are assayed by chromogenic substrate method afterwards. The pharmacokinetic parameters and bioavailability have been calculated by Win Nonlin program. Results:(1) Curve drawn through the work, getting in the 0.102 ~ 1.224 IU·m L-1 range of linear regression equation:ΔA = 0.3867C3- 1.2837C2 + 1.5597C- 0.0586, and correlation coefficient r = 0.9978. Drug percentage of LMWH has been reduced by 15% and 23% in the artificial gastric juice and artificial intestinal juice after 4 hours.(2) The morphology of p H-sensitive TCS/O-CMC reversed polymer nanoparticles have been observed by transmission electron microscopy. The p H-sensitive reversed polymer nanoparticles are spherical in shape and uniformly distributed, with particle size(332.0 ± 7.1) nm, PDI(0.279 ± 0.01), Zeta potential(28.1 ± 0.24) m V, drug loading capacity(12.04 ± 0.07) IU·mg-1, encapsulation efficiency(96.6 ± 0.72)%.(3) 6% glucose and 0.5% mannitol(volume ratio of 2:1) are chosen as cryoprotectant, which has a good effect of freeze-drying. LMWH p H-sensitive reversed polymer nanoparticles freeze-dried products are prepared in particle size(326.2 ± 4.0) nm, PDI(0.294 ± 0.04) and Zeta potential(32.3 ± 1.2) m V. They keep spherical in shape and relatively uniform as observed by transmission electron microscopy.(4) The release rate of LMWH within 24 hours from p H-sensitive reversed polymer nanoparticles are in the following order:(17.33 ± 0.04)%,(39.25 ± 0.01)% and(34.11 ± 2.51)% in 0.1 mol·L-1 HCl solution(p H 1.0), in PBS(p H 6.8) and in solution of gastrointestinal gradient p H.(5)The pharmacokinetic parameters of three formulations are as follows: Cmax are(0.103 ± 0.0254)、(0.425 ± 0.0463) and(0.522 ± 0.0421) IU·ml-1. AUC 0�'∞ are(0.477 ± 0.3475)、(4.710 ± 1.0090) and(7.961 ± 2.0369) IU·ml-1·h. The relative bioavailability are 2.10%、20.70% and 34.98%.There is significant difference of relative bioavailability between the three groups(P < 0.05). Conclusions:(1)The results have indicated that the azure A colorimetric-ultraviolet spectrophotometry method is accurate, convenient and could be used as in vitro assay of LMWH. LMWH is not stable in artificial gastric juice but relatively stable in the artificial intestinal juice, which is consistent with the literature and can be adopted for the further experiment.(2) The results of single factor investigation and orthogonal design have also showed that the preparation process of p H-sensitive reversed polymer nanoparticles is reasonable and feasible.(3) Mannitol/glucose can be a kind of good cryoprotector so that the p H-sensitive reversed polymer nanoparticles freeze-dried products are kept in a small size with a better appearance.(4) The preparation of p H-sensitive reversed polymer nanoparticles in simulated gastric conditions can slow down drug release but in intestinal juice it will increase the release of p H sensitive in vitro release characteristics.(5) Relative bioavailability of drug could be improved by p H-sensitive TCS/O-CMC reversed polymer nanoparticles, which therefore lays the foundation for the development of low molecular weight heparin nanoparticle drug delivery system.