Recombinant Hirudin -2 (rhv2), Intranasal Administration Of Chitosan Nanoparticles

To improve the bio-availability of rHV2, a peptide, after nasal administration furthest, we have chosen chitosan as the envelopment material and prepared the rHV2 chitosan nanoparticles which have the function of bio-adhesive and then researched the influence of chitosan nanoparticles on nasal absorption of rHV2. Firstly, the preparation technique and formulation of rHV2 nanoparticles were researched, and then the physical-chemical characterization and the release in vitro were studied. Secondly, the cilia toxicity of the nanoparticles was evaluated and the freeze-dried power of the rHV2 nanoparticles was prepared. Finally, the penetrability of rHV2 nanoparticles through the rabbit nasal mucous membrane in vitro was researched. The pharmacokinetic and pharmacodynamic studies on the formulation were also carried out in rats.Part one: Literature ReviewSummarize the new developments of nasal delivery system of the peptides and protein; the survey on nanoparticles for nasal delivery research; the medical research progress of rHV2.Part two: Experiment Studies1. The preparation of rHV2 chitosan nanoparticlesTaking efficiency as the index, the single factor and orthogonal design were used to optimize the preparative technique and formulation of rHV2 chitosan nanoparticles. Experiment results proved the selected preparation technique and formulation of rHV2 chitosan nanoparticles were stable and practicable. The efficiency could reach above 70%.2. The characterization of rHV2 chitosan nanoparticlesThe morphological, particle size distribution and zeta potential were determined. Results showed that the particle size distribution was symmetrical, the mean size of rHV2 chitosan nanoparticles was about 200nm and the zeta potential was above 30 mV which implied the possibility of stable existence of the nanoparticles suspension.3. In vitro release studiesThe method of ultracentrifugation was used in rHV2 chitosan nanoparticles release studies in vitro. Results displayed that rHV2 released from the nanoparticles slowly and the amount of release in the first two hour was less than 40 %.4. Ciliotoxicity evaluationThe ciliotoxicity of rHV2 was evaluated with in situ toad palate model. The results showed that ciliary movement was affected weakly by rHV2 chitosan nanoparticles for nasal administration which proved the safety of this formulation.5. Preparation of freeze-dried powerWe have chosen A, B, C and D as cryoprotector, optimized the preparative technique and the formulation of rHV2 chitosan nanoparticles freeze-dried power and investigated the effect of the illumination and high temperature factors on the freeze-dried power.6. The penetrability of rHV2 nanoparticles through the rabbit nasal mucous membrane in vitroThe experiments of penetrability in vitro were studied in using diffusion cell technique. We took the rabbit nasal mucous membranes as the permeability barriers and determined the amount of rHV2 across the barriers, finally calculated the permeability coefficient. The permeability coefficient of chitosan nanoparticles was 5 times more than that of the salt solution of rHV2. It was indicated that chitosan nanoparticles was more effective in increasing the permeability of rHV2.7. Pharmacokinetic in vivoCompared to subcutaneous administration of rHV2 salt solution, the relative bioavailabilities (Fr) of rHV2 salt solution nasal administration and rHV2 chitosan nanoparticles nasal administration were determined and the pharmacokinetic parameters were calculated by the software 3P87. The results indicated that chitosan nanoparticles showed obvious sustain-released attribute in vivo and it could improve the Fr of rHV2 nasal administration greatly. The Fr of the rHV2 chitosan nanoparticles could approach 30%.8. Pharmacodynamic studiesPharmacodynamic studies on the rHV2 chitosan nanoparticles after nasal administration were carried out in normal rats, taking APTT as the index and the subcutaneous administration of rHV2 salt solution as control groups. Significant effects of anticoagulation were investigated in normal rats after nasal administration of rHV2 chitosan nanoparticles. Compared to the control groups, chitosan nanoparticles showed the function of sustain-release. Part three: Analysis and Discussion The results of the experiments are summed up, analyzed and discussed.