Preparation Process And Pharmacokinetics Study On Chitosan-modified Honokiol Liposomes

Honokiol(HNK)potent with extensive pharmacological activities and the possibility of being a patent medicine,and thus has great development potential.However,due to its poor water solubility and low bioavailability,its further development and utilization are greatly limited.In this thesis,we attempt to improve the solubility,oral bioavailability,and reduce the cytotoxicity of HNK through preparing of HNK liposomes.Then chitosan was used for modification of HNK liposomes to enhance the slowing release effect,light and thermal stability of this active ingredient.The research idea in this thesis is anticipated to provide a reference for designing of novel formulation for active ingredients from traditional Chinese medicine.HNK-Lip was prepared by thin film dispersion-ultrasonic method,and the optimal prescription of HNK-Lip was obtained by single-factor investigation and Box-Behnken response surface optimization.Then CS-HNK-Lip was prepared drop-in method,and to improve the storage stability of liposome,the CS-HNK-Lip solution was freeze-dried to finally obtain HNK-Lip and CS-HNK-Lip lyophilized products.The HNK-Lip and CS-HNKLip lyophilized products were subsequently to adequate quality evaluation tests and preliminary investigations on the in vitro pharmacodynamics and in vivo pharmacokinetics.Through single factor analysis and Box-Behnken response surface optimization,the formulation of HNK-Lip was determined as follows: the ratio of phospholipid to cholesterol,5:1,the ratio of HNK to lipid,6:60,and the content of TPGS,9%.The preparation process was as follows: the prescription amount of phospholipids,HNK and TPGS were weighed and dissolved in methanol-chloroform(1:2,v/v)mixed solution,shaking under ultrasound to fully dissolve them,and evaporation was conducted under reduced pressure at 40℃in a rotary evaporator to slowly remove organic reagents.Then pure water was added for hydrating at room temperature for 30 minutes and then shake fully until it is fully hydrated.After then probe ultrasonic was performed in ice water bath environment(Φ 3(British 1/8 "),300 W ultrasonic power for 6 min(ultrasonic 3 s,interval 3 s))to prepare HNK-Lip solution with pale blue opalescence.The diameter of the nanoparticles prepared under this condition is 151 nm,and the encapsulation efficiency is 90.1%.To improve the storage stability of liposomes,HNK-Lip was modified with chitosan.We determined to use chitosan with molecular weight of 100 k Da as the best material,and chitosan(100 k Da)sodium acetate buffer solution(p H 4.5)with concentration of 2% was prepared,and then the same volume of HNK-Lip suspension was injected into the chitosan solution dropwise under 400 rpm magnetic stirring.After adding,continue to stir for 1 hour,and leave it overnight to obtain CS-HNK-Lip with light blue opalescence.The diameter and charge of nanoparticles prepared under this condition was 183 nm and +12.2 m V,respectively.In order to prepare HNK Lip and CS-HNK Lip freeze-dried samples,the type and amount of freeze-dried protective agents for HNK Lip and CS-HNK Lip were determined to be 10%sucrose and 5% trehalose respectively.The appearance of HNK Lip and CS-HNK Lip lyophilized samples prepared under this condition were white or white loose powder with particle sizes of 161 nm and 360 nm,which could be completely dissolute in water within 5minutes.Transmission electron microscopy(TEM)results confirmed that CS-HNK-Lip was homogeneously dispersed sphere-like,with a dark black material chitosan wrapped around the outside.Differential calorimetric scanning(DSC)showed that HNK was encapsulated in the inner liposome.The in vitro release tests of HNK-Lip and CS-HNK-Lip in different p H media showed the slow release rate of HNK in preparations was significantly decreased,and HNK in CS-HNK-Lip showed better stability under light and high temperature compared with that of HNK-Lip.Results demonstrated the cytotoxicity of HNK in HNK-Lip and CS-HNK-Lip was significantly reduced in RAW 264.7,and intracellular uptake for HNK in these two preparations was also significantly increased.Single dose of 50 mg/kg HNK(equal amount of HNK for preparations)was administered by gavage for rats.Results showed the pharmacokinetic behaviors of HNK suspension,HNK-Lip,and CS-HNK-Lip were all consistent with the two-compartment model after oral administration to rats.the C max for HNK-Lip and CS-HNK-Lip groups were 1.48 and 1.44 times higher than that of the HNK suspension group,respectively.It was demonstrated that HNK-Lip and CS-HNK-Lip could enhance the absorption of HNK by oral administration in rats,and the mean retention time(MRT)of HNK-Lip and CS-HNK-Lip groups were 1.25 and 1.44 times higher than that of the HNK suspension HNK group,respectively,and it was deduced the significantly changed pharmacokinetic profiles for HNK in preparations might related to the fact that liposome system decreased phagocytosis by reticuloendothelial system.For HNK in CS-HNK-Lip,due to the unique adhesion properties of chitosan,the retention time of it was longer compared to that of HNK-Lip.Taken together,CS-HNK-Lip and its lyophilized products were successfully prepared in our experiments,which greatly improved the solubility,oral bioavailability and reduced cytotoxicity of HNK.At the same time,the liposome carried with slow release characteristic and good light and thermal stability.The follow-up research should focus on the connections between pharmacodynamics and pharmacokinetics in vivo to further optimize and improve the design for the preparations,and provide a more solid research foundation for pharmaceutical research and data accumulation for preparation design for the truly realization of clinical treatment services for HNK.