Preparation Of Trastuzumab-bearing Paclitaxel-loaded Lipid Nanoparticles And Preliminary Study Of Targeting

Breast cancer has an increasing incidence year by year,which becomes the biggest threat to women's health.Paclitaxel(PTX)as first-line treatment drug of breast cancer,its use alone or in association with other drugs have showed a good effect.But the non-selective toxic effects cause serious systemic side effects.In tumor biological and targeted therapy,tumor markers are used as target site and drugs are selectively delivered to the tumor site without affecting the normal cells.HER2 is recognized as an important marker of breast cancer.HER2-targeted monoclonal antibody trastuzumab(Tmab)has been approved by the FDA as the first-line drug.Tmab in combination with PTX has improved therapeutic efficacy in breast cancer,but the targeting of PTX is still limited.Although antibody-drug conjugate(ADC)shows a strong targeting ability,the construction of attachment has high technical barriers and the position and number of conjugates is hard to control.Lipid nanoparticles as a novel drug delivery system,have feature of good biocompatibility,low toxicity and outstanding sustained-and controlled-release.Tmab has been used as a targeting ligand in combination with lipid nanoparticles,which achieves slow release,targeting and a relatively simple preparation process.In existing ligand conjugation strategy,Tmab is attached to the surface of the carrier through the chemical bond.Chemical reaction may cause the inactivation of monoclonal antibody and non-specific site may appear in antigen recognition area.How to maximize the biological activity of antibody has become the key to the application of antibody as a targeting ligand.In this paper,polyethyleneimines(PEIs)and cationic phospholipid(DODMA)have been used to construct cationic nanoparticles(CNPs).CNPs combined with negatively charged Tmab by electrostatic interactions.In the early explorations,the structure and natural activity of trastuzumab were characterized.Results indicated that the moderate electrostatic forces reserved the immune activity of antibodies very well.The matrix prescription,antibody conjugation strategy and conjugation ratio were optimized to prepare targeted nanoparticles with high efficiency and low toxicity.A preliminary study on in vitro anti-tumor effect and tumor targeting were carried out.The main contents included the following parts:1.The preparation and evaluation of PTX-loaded PEI cationic nanoparticlesIn this chapter,four different kinds of PEIs(800-b PEI,2000-b PEI,25k-b PEI and 25k-l PEI)were used mixing with PLGA/Egg PC to prepare PEI cationic nanoparticles.The size and zeta potantial were chosen as an index.By optimizing the PLGA/Egg PC ratios,the PEI type and content,PLGA/Egg PC(60: 20)and 5% 25k-b PEI were selected.The particle size and zeta potential of the cationic nanoparticles were 249.9 ± 4.76 nm and 35.3 ± 2.3 m V,respectively.In contrast with free PEI,blank cationic nanoparticles had no obvious toxicity on A549 cells and MCF7 cells,and the cell viability was more than 90%.The stability results showed that the particle size and PDI of the cationic nanoparticles did not basically change in PBS(10 m M,p H 7.4)within 2 weeks.In the cytotoxicity and cell uptake experiments,the PTX-loaded nanoparticles prepared from 25k-b PEI had the highest cytotoxicity(P < 0.001).Its cell uptake efficiency was significantly higher than that of the other three PEIs(P < 0.01).2.The preparation and evaluation of Tmab-bearing PTX-loaded PEI cationic nanoparticlesIn this chapter,Tmab combined with PTX-loaded PEI cationic nanoparticles through electrostatic adsorption and chemical bond,respectively.The increase of size,the decrease of surface potential and double-layer core-shell structure observed by TEM demonstrated the successful conjugation of antibodies.The particle size and surface potential of the nanoparticles prepared by electrostatic adsorption were 280.7 ± 7.8 nm and 1.00 ± 0.73 m V,respectively.The optimal conjugation ratio between cationic nanoparticles and Tmab was 1: 1.The antibody binding efficiency detected from BCA method was 92.7 ± 0.6%.During 24 h,the dissociation percentage of Tmab in physiological condition was below 25%.In vitro release of nanoparticles emerged biphasic and sustained release mode.The burst amount was 28.6% within 6 h and the cumulative release rate was 71.2% at 120 h.In MTT assay,HER2-positive BT474 cells and HER2-negative MCF7 cells were chosen as the experimental objects.The cell proliferation inhibition of nanoparticles on BT474 cells was receptor-and time-dependent.At 72 h,the inhibition ratio of electrostatic adsorption and chemical cross-linking groups on BT474 cells was 53.3% and 34.9%,respectively.The nanoparticles were labeled with double fluorescence mode.Cationic nanoparticles was labeled by Rhodamine B(Rhod·B)and Tmab was labeled by FITC.In BT474 cells,the internalized fluorescence intensities of FTIC and Rhod·B from electrostatic adsorption groups were higher than that of chemical cross-linking groups(P < 0.001).In MCF7 cells,two groups displayed the same fluorescence intensities.Tmab did not dissociate in electrostatic adsorption groups during the process of cell uptake.3.The preparation and evaluation of Tmab-bearing PTX-loaded cationic lipid nanoparticlesIn this chapter,cationic phospholipid DODMA with lower cytotoxicity and better biocompatibility was employed instead of cationic polymer PEI mixing with PLGA to prepare cationic lipid nanoparticles,followed by combination with Tmab.To achieve quantitatively controlling the antibody density on nanoparticles surface,three different antibody conjugation strategies were investigated to prepare targeted lipid nanoparticles.(1)For the improved pre-conjugation strategy(Imp),Tmab was first connected with DSPE-PEG2000-Mal molecule,and then was conjugated to nanoparticles surface through electrostatic adsorption.(2)For the pre-conjugation strategy(Pre),Tmab was connected with DSPE-PEG2000-Mal molecule.Then the nanoparticles were prepared by physical blending of DSPE-PEG2000-Tmab and DODMA/PLGA.(3)For the pos-conjugation strategy(Pos),Tmab was conjugated to the surface of DSPE-PEG2000-Mal/DODMA/PLGA nanoparticles by chemical bonds.The antibody density analysis results showed that the total loading of Tmab were 17.8%,14.5% and 4.1% for Imp,Pre and Pos,respectively.Thereinto,the amount of Tmab on the surface of nanoparticles were 17.8%,6.12% and 4.1%,respectively.For Pre,8.38% of antibody was wasted inside the nanoparticles during the preparation.Pos showed low chemical reactivity.In Imp,the antibody loading and the dose were linearly fitted with a R2 value of 0.995.Results indicated it is feasible to quantitatively control Tmab amount on the surfaces of nanoparticle by Imp.In MTT assay,the toxic effect of nanoparticles on BT474 was receptor-,timeand dose-dependent.In Imp,the inhibition rate of BT474 cells increased by 7.3%(P < 0.001)at 48 h when the loading of Tmab was increased from 0 to 17.8%.The flexible mode of administration in Imp facilitated the synergistic effect between Tmab and PTX.The order of cytotoxicity of the nanoparticles prepared from the three strategies was Imp > Pre > Pos,which was higher than that of the cationic nanoparticles and free PTX.In BT474 cells,Imp had the highest intracellular fluorescence level,followed by Pre and Pos,which were 30.3,23.6 and 20.7,respectively.The intracellular fluorescence level of nanoparticles in MCF7 cells was lower than that of BT474 cells.Finally,the effects of endocytosis inhibitors on the endocytosis pathway of nanoparticles were investigated.The results showed that targeted nanoparticles entered into tumor cells mainly through clathrin-and HER2-mediated endocytosis.At present,the studies about conjugation strategy of targeted ligand at home and abroad are mainly focused on chemical modification of ligands.However,the activity of antibody is easy to be damaged,thereby affecting its targeting.Chemical bonds have high-intensity which will block receptor-ligand recognition processes and subsequent chemotherapy drug release.In this paper,Tmab was conjugated to the surface of the cationic nanoparticles through electrostatic force,which greatly improved the structural stability and biological activity of antibody.The targeting effect could be regulated by adjusting the amount of antibody on the surface of nanoparticles from Imp.Tmab-bearing PTX-loaded cationic lipid nanoparticles provided theoretical basis,method reference and technical reference for the development of targeted vectors.