Study on Cellular Penetrating Peptide and Transferrin Co-modified Liposomes Delivery Doxorubicin System for Anti-glioma

Author:Wang Zuo

Supervisor:zuo li rong

Database:Doctor

Degree Year:2019

Download:34

Pages:127

Size:5581K

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Glioma is characterized by rapid growth,high recurrence rate and strong invasiveness.Chemotherapy drugs are widely used in the course control of glioma,but due to the presence of the blood-brain barrier(BBB),most of the chemotherapy drugs are difficult to penetrate BBB for brain delivery,and drugs for cancer cells may not be selective,they have a certain toxicity to normal cells,these factors limit the effect of chemotherapy drugs on brain glioma.Liposomes have the advantages of no immunogenicity,low toxicity,biodegradability and high biocompatibility,etc.The surface of liposomes contains abundant functional groups,which can improve the efficacy of liposomes in the treatment of tumors after modfied.Cell penetrating peptides(CPPs)are kinds of short peptides could improve drugs or biological macromolecules to cross the blood-brain barrier such as the intestinal wall,retina,neurons and other biological barriers to deliver.Octapolyarginine(R8)is a classic CPPs.It is characterized by strong cell membrane penetration and cationic properties and it is capable of hydrogen bonding with negative cell surface residues to cross the cell membrane under the action of membrane potential.In addition,R8 is rich in amino groups,which can be modified by bonding with hydrophobic groups(such as fatty acids,oleic acids,etc.)through amide bonds.Studies have shown that transferrin receptor(TfRs)in cerebral microvascular endothelial cells and glioma cells overexpressing,transferrin(Tf)is a kind of glycoprotein,Tf as brain targeting ligands with TfRs with strong affinity,Tf modified liposome give brain tumor targeting ability,delivering chemotherapy drugs into the brain to achieve the purpose of targeted therapy of cerebral glioma.Based on the above research background,this paper mainly focused on the establishment of doxorubicin(DOX)analytical methodology,formulation optimization of CPPs modified liposomes,characterization of liposomes properties,preparation of CPPs and Tf co-modified liposomes,and research on anti-glioma activity.Specific research results are as follows:1.Establishment of doxorubicin assay methodology:(1)High performance liquid chromatography(HPLC)was used to study the precision,recovery and stability of DOX assay method,and to establish in vitro assay methodology.(2)An in vivo analysis method for determination of DOX content in brain tissues by UPLC/MS/MS was established and the accuracy,precision,recovery,matrix effect and stability of the method were evaluated.The results showed that HPLC method could accurately and precisely analyze the content of DOX in liposomes in vitro,and the liposome materials in liposomes had no obvious interference to the detection.The method meets the requirements of doxorubicin in vitro quantitative analysis.UPLC/MS/MS analysis method can accurately and precisely analyze the content of DOX in brain tissue,and the analysis method can eliminate the interference of biological matrix in brain tissue on DOX detection.The UPLC/MS/MS analytical methodology meets the requirements of DOX in vivo quantitative analysis.2.Prescription optimization of CPPs modified liposome membrane:ethanol injection method combined with the ammonium sulfate gradient method active drug preparation of CPPs modified DOX liposomes(R8PLP),using particle size,the encapsulation efficiency as evaluation indexes to optimize ratio of cationic lipids/OA-R8/ePC/cholesterol/DSPE-PEG2000 in R8PLP,kinds of cationic phospholipids,concentration of ammonium sulfate and volume ratio of lipid phase/ammonium sulfate solution.The best conditions for the preparation of R8PLP are DOTAP as cationic phospholipids,DOTAP/OA-R8/ePC/Chol/DSPE-PEG=20/25/18/32/3,aliphatic/aqueous phase=1/10,ammonium sulfate concentration of250μM,stirring speed of 300 rpm,injection rate of 5.5 mL/min to prepare R8PLP.DOX active drug loading condition:the R8PLP drug loading was completed with a lipid ratio of 1:10,a pH value of 7.4 and a temperature of 45℃for 30 mins.3.Characterization of liposomes modified by CPPs:firstly,OA-R8 modified liposomes used for DOX delivery to the brain were prepared,and then the physicochemical properties of the liposomes were characterized and also evaluated the anti-tumor activity and cell uptake of liposomes in vitro.The surface morphology of R8PLP was spherical,with smooth surface,good dispersibility and uniform particle size of about 100 nm.R8PLP of zeta potential of+12.1 mV;R8PLP was stable in 10.0%serum,PBS or deionized water at 37℃and 4℃for 48 h.The lyophilized R8PLP was stable in 10.0%serum,PBS or deionized water at 37℃and 4℃for 48 h.DOX,LP,PLP,R8LP and R8PL showed significant anti-tumor activity in U87 and GL261 cells,and the anti-tumor activity was time dependent.After 24 h of administration,the activity of OA-R8 modified liposomes(R8LP,R8PL)was significantly higher than that of adriamycin,LP and PLP,suggesting that OA-R8 modified liposomes could improve the anti-tumor activity of DOX in U87 and GL261cells.Finally,the blood-brain barrier passability of OA-R8 modified liposomes(R8PLP)was evaluated:compared with PLP,R8PLP had a significant accumulation effect in brain tissue,and the AUC0.5-12h area under the curve when R8PLP was treated in brain was 2.4 times that of PLP.However,the half-life of R8PLP(t1/2)was similar to that of PLP(3.47 vs 3.94 h),suggesting that OA-R8 modified liposomes had better BBB penetration and brain tissue accumulation.4.CPPs and Tf modified liposomes for the targeted treatment of glioma:this chapter on the basis of preparation of Tf-R8PLP,discussed the Tf content on the liposome targeting property and anti-tumor effect,the preparation of CPPs and Tf were modified(Tf-R8PLP),and particle size of liposomes,stability in the serum,encapsulation efficiency and evaluate the drug release in vitro performance.In addition,in vitro cell uptake of Tf-R8PLP was analyzed.The glioma model of nude mice was established,and the in vivo anti-tumor efficacy of Tf-R8PLP was evaluated.After Tf-R8PLP treatment,the distribution of the drug in tissues in nude mice and the pathology of organs in nude mice including brain,heart,heart,liver,spleen,lung and kidney were evaluated.The best preparation condition of Tf-R8PLP was 1/100 mole ratio of R8PLP/Tf-DSPE-PEG.Tf-R8PLP particle size of 128.64 nm,ζ-potential was+6.81 mV,Tf concentration is 16.15μg/mL;Tf-R8PLP has spherical structure and uniform particle size,and the stability of Tf-R8PLP in serum is good.Both R8PLP and Tf-R8PLP showed controlled release behavior.Tf-R8PLP had the highest tumor growth inhibition rate,and the in vivo anti-tumor activity of Tf-R8PLP was significantly higher than that of R8PLP and DOX.The survival time of mice in Tf-R8PLP group(25 days)was longer than that in normal saline group(20 days),DOX group(22 days)and R8PLP group(24 days),and Tf-R8PLP had significant effect on the treatment of glioma.