Nanocarrier-targeted Drug Delivery System Based On Thermosensitive Liposome And Low Density Lipoprotein Model

Background Active targeting drug delivery system has been widely concerned by many countries in tumor treatments.Although the paper of this research direction has increased exponentially,there is no listed products.Based on analysis of physical chemistry and biology principles,through thinking about differential diffusion ability of molecules and nanoparticles in aqueous medium,as well as in vivo targeting process of nanoparticles,we fund some theoretical misunderstanding of active targeting drug delivery system fundamentally,and revealed defects of the active nano carrier targeting that sometimes modification could not improve tumor targeting efficiency according to the original design intention of nano carriers.The EPR effect may only be applicable to drug molecules and nanoparticles with sufficient diffusion capability.Objective To improve the tumor targeting efficiency of nano carriers and the druggability,we focused on the environment depended specificity responsing targeting drug release nanoparticles(with thermosensitive liposomes as model),and active targeting nanoparticles with strong diffusion ability(with low density lipoprotein as a model).Both of them are most feasible targeting strategy.This study included the preparation of vincristine and adriamycin thermosensitive liposomes(VD-TSL)and vincristine artificially low density lipoprotein(VCR-a LDL),the evaluation of a series physical and chemical properties in vitro and in vivo,in vivo imaging,and pharmacodynamics research,which indicated these two kinds of targeting strategies could significantly improve nano carrier targeting efficiency.The contents and methods The first part: firstly,we established synchronization content analysis of VCR and DOX with HPLC detection method,and conducted a series of method validation,then prepared blank liposomes,and synchronous loaded 2 drugs using p H gradient active method,then formed VD-TSL.The appearance,particle size,zeta potential,morphology,encapsulation efficiency,thermal sensitivity,stability and phospholipid analysis were used as the main evaluation indexes.MTT method was used to determine the cytotoxicity in vitro,and the uptaken of fluorescent labeled thermosensitive liposomes combined with hyperthermia conditions was observed by confocal laser scan microscopy.Later,we established xenograft tumor model in nude mice,combining with local hyperthermia,we studied the in vivo distribution,pharmacodynamics and toxicity of VD-TSL in vivo.The second part: Firstly,through formulation optimization,we prepared 100 nm and 35 n M VCR-a LDL using thin film dispersion one step loading method.Then followed by particle size,zeta potential,morphology,encapsulation efficiency and stability of a series of physical and chemical evaluation.To compare the dispersal ability of two different particle size of VCR-a LDL,we carried out the MTT monolayer cell toxicity studies,MTT inverted diffusion cell toxicity studies,tumor cake and tumor ball model penetration ability studies using confocal observation.Then the xenograft tumor model of nude mice was established,we carried out the 3D in vivo imaging,tissue distribution and pharmacodynamics evaluation of VCR-a LDL.Results The first part: through the optimization of formulation,the prepared VD-TSL average particle size was about 90 nm,zeta potential was about zero;under transmission electron microscope,liposomes had round appearance and uniform particle size distribution.The encapsulation efficiency of VD-TSL could reach more than 95%,and the reproducibility of preparation process for different batch sample preparations was excellent.Furthermore,the long-term test showed that VD-TSL was stable within 6 months under the condition of-20℃.In vitro evaluation and cell uptaken experiments indicated that VCR and DOX have synergistic effect,and hyperthermia could promote the entrance of TSL to tumor cells at the same time,namely,TSL loaded with drugs and local hyperthermia had synergistic effect.The in vivo imaging of nude mouse xenograft model showed that TSL together with local hyperthermia of tumor tissue could significantly improve the targeting ability to tumor.The pharmacodynamics experiment results showed that VD-TSL combined with local hyperthermia of tumor had significant tumor growth inhibitory effect,the inhibition rate could reach more than 70%.At the same time,the mice of VD-TSL group ate and acted more active than the injection group,and the body weight of VDTSL group mice declined the smallest.It could be concluded that VD-TSL reduced systemic toxicity in nude mice.The second part: We prepared two kinds of artificial low density lipoprotein(a LDL)with different particle size,both of them had fat soluble core and hydrophilic shell structure.The middle particle size of them were 100 nm and 35 nm,respectively,and they had normal distribution,as well as near zero zeta potential.Under transmission electron microscope,we could see that a LDL had round appearance,uniform particle size distribution,and obvious core-shell structure.No matter 100 nm or 35 nm VCR-a LDL,the encapsulation efficiency could reach more than 95%,and the stability was very good.In MTT cytotoxicity experiments,the cell inhibition rate of two kinds of VCR-a LDL had no significant difference,but during the MTT diffusion inverted cell toxicity research,the diffusion capacity of 35 nm VCR-a LDL was better than 100 nm VCR-a LDL,then the inhibitory rate of cell proliferation was stronger.Through laser scanning confocal microscope,we fund that although the permeability of 35 nm fluorescence labeled a LDL was weaker than fluorescence molecular,it much stronger than 100 nm ones.Compared with the Injection group and 100 nm VCR-a LDL group,35 nm group VCR-a LDL had the strongest targeting tumor retention effect in the 3D imaging experiments.In pharmacodynamics experiments,the inhibition rate of 35 nm VCR-a LDL group mice was the strongest,while the body weight loss of them was the smallest,and the systemic toxicity was the least.Conclusion VD-TSL and VCR-a LDL were successfully prepared in this study.These two methods had different tumor targeting drug delivery principles,and had excellent in vitro physical and chemical properties,as well as in vivo tumor tissue targeting.Besides these,the process was stable and reproducible.VD-TSL combined the synergistic effect of VCR and DOX,as well as the synergistic effect of drug loaded thermosensitive liposome and hyperthermia effect,which was a dual control synergistic effect of the nano carrier.For VCR-a LDL,a LDL itself is active targeting nano carrier,through targeting effect study of 100 nm and 35 n M a LDL,indicating decreasing particle size could improve the diffusion ability of nano carrier,with sufficient diffusion capacity,there was sufficient passive targeting ability to make full use of the EPR effect,then played tumor targeting role better.