Construction Of Brain Multi-targeting Biomimetic Nanoparticle And Its Anti-glioma Effect And Mechanism

Glioma is the most common tumor of central nerve system which occurred in the neuro derm.The World Health Organization data shows that the glioma is the second tumor killer among the young and middle-aged under 35.About 180~600 thousand young people died of glioma.Glioma is highly aggressive and it's not easy to separate it from the normal tissue leading to the poor surgical effects and prognosis.Postoperative radiotherapy and chemotherapy are very important.But chemotherapy drugs are difficult to penetrate into the tumor tissue to kill the tumor cells due to the physiological barrier of blood brain barrier.Nano drug delivery system is more and more widely used in the field of tumor therapy becasuse it can reduce the side effects of drugs and increase the accumulation of drugs in the tumor.The application of nano drug delivery system in safe and effective target treatment is becoming a trend in tumor therapy including glioma.Most materials of nanoparticles are systhetic and still have toxicity to the human body.The biomimetic nano drug delivery system is based on the natural particles.It has lower toxicity and higher targeting ability through the simulation of natural partices and other pathogens.The lipoprotein based nano drug delivery system is a member of the biomimetic nano system and it is biocompatible,biodegradable,and nonimmunogenic leading to good therapeutic effect.Cancer stem cells(CSCs)are a group of cells with the self-renewing and multi differentiation ability.CSCs are related with the aggressive growth,recurrence and drug resistance of tumor.The existence of glioma stem cells(GSCs)makes the glioma resistant to traditional radiotherapy and chemotherapy and maintains self-renewal state and proliferation inducing the recurrence of glioma.The treatment targeting GSCs is of great significance.It's reported that salinomycin(SAL)can kill various kinds of cancer stem cells including glioma stem cells selectively.SAL can also kill the tumor cells.We prepare the targeting biomimetic nano drug delivery system to pass the BBB,kill the glioma cells and inhibit the GSCs derived endothelial cells to realize the glioma multi-targeting.In the first part,two glioma cell lines U87 and SHG-44 were cultured and glioma spheres were isolated from them by the serum-free suspension culture method.The two glioma spheres were identified by the immunofluorescence method.The expression of the two surface markers CD133 and nestin could be observed through the confocal lazer scanning microscopy.The positive rate of CD133 investigated by the flow cytometry method in SHG-44 TS and U87 TS is 41.48% and 17.3%.The GSCs could be used in the following study.Next the cytotoxicity of SAL to glioma cells and GSCs was also investigated and the results showed that SAL had a direct cytotoxic effect on the glioma cells and GSCs.The GSCs were more sensitive to SAL.In the second part,low density lipoprotein(LDL)was separated from human blood and indentified by Fourier Transform Infrared Spectroscopy(FT-IR).The protein concentration measured by BCA method was 1.67mg/mL.The brain targeting peptide Angiopep-2 was conjugated with LDL to prepare LDL-Angiopep-2(LDL-Ang).SAL loaded LDL-Ang was prepared by the core-loading method called LDL-SAL-Ang.The formulation was optimized and prescription with the drug/carrier ratio of 20% was determined.The encapsulation efficiency was 35.63±2.65% and the drug loading was 5.83±0.7%.The particle size and zeta potential of the prepared nanoparticles was 22.95±1.58 nm and-5.98±0.20 mV which showed the uniform size and stable potential.The structure of the nanoparticles was observed by transmission electron microscope(TEM)and the sphere-like nanoparticles with clear boundary.In the third part,in vitro anti-tumor effect including glioma cells and GSCs of nanoparticles was investigated.The uptake of the fluorescent probe DiI loaded nanoparticles in glioma cells and GSCs was investigatedby the confocal lazer scanning microscopy and the flow cytometry method.The results revealed that LDL-SAL-DiI-Ang caused a higher accumulation in glioma cells and GSCswith significance in statistics compared to LDL-SAL-DiI.Special competitive inhibition experiment demonstrated thatforeign special LDL inhibited the binding of the LDL-Angwith glioma cells and GSCs,it could be verified that thecore loading of SAL and surface connection of Angiopep-2 did not affect the binding of nanoparticles to LDLR.The cytotoxicity of LDL and LDL-Ang was proved to be very low by the CCK-8 method.LDL-SAL-Ang showed the most cytotoxicity and can induce the most cell apoptosis,LDL-SAL second and SAL last in glioma cells and GSCs.Next the effect of nanoparticles on the formation of tumorsphere was investigated.The tumorsphere formation of LDL-SAL-Ang group was very small and loose which was the worst.The endothelial cells differentiating from glioma stem cellswere cultured in hypoxia.Immunofluorescence assay was used to identify and the tube forming ability was investigated.The results showed that the endothelial cells can be used in the study.The next experiments verified that LDL-SAL-Ang had a certain inhibition effect on the tube formation and migration of the cells.In the fourth part,the properties of brain transport was investigated.The uptake of the fluorescent probe DiI loaded nanoparticles in the human brain microvessel endothelial cells(HBMEC)-the main forming cells of BBB was investigated.The uptake intensity increased with time.Next the transwell chambers were used to evaluate permeability of the nanoparticles in HBMEC monolayer cells.The permeability of LDL-DiI-Ang was higher than LDL-DiI.HBMEC were seeded in the inserts and the glioma cells or GSCs were seeded on the bottom of the plate to evaluate the nanoparticle uptake penetrating a single layer of cells.DiR loaded nanoparticles including LDL-DiR and LDL-DiR-Ang were prepared.Next the particle size and zeta potential was measured.The brain stereotactic apparatus was used to establish glioma-bearing nude mice by injecting human glioma cell suspension into the caudate nucleus of nude mice.The living state of the nude mice was observed.The brain tissues were taken for paraffin pathological section and HE staining to verify the successful preparation of the model.The distribution of DiR loaded nanoparticles in mice was investigated by using small animal in vivo imaging system.The brain-targeting ability of LDL-DiR-Ang was better than that of LDL-DiR.The distribution of LDL-DiI-Ang was also observed by the freezing sectional method.The brain-targrting ability of LDL-DiI-Ang was better than that of LDL-DiI.ICR mice were administrated with the blank LDL and LDL-Ang by intraveous injection for a week continuously.CD68 staining of different tissues was used to evaluate the toxicity of blank LDL and LDL-Ang in vivo.The results showed that the blank LDL and LDL-Ang had low toxicity on tissue.In the fifth part,the study of pharmacokinetics and tissue distribution of drug loaded nanoparticles in vivo was taken.ICR mice were divided into three groups including SAL,LDL-SAL and LDL-SAL-Ang group.The changes of plasma concentration of SAL in different groups were observed.The results showed that LDL-SAL and LDL-SAL-Ang had a certain sustained release effect,and the distribution of LDL-SAL-Ang in brain tissue was higher than the other two groups.In the sixth part,the anti-tumor activity of drug loaded nanoparticles in vivo was investigated.First,the glioma-bearing nude mice model was established.The combination of nanoparticles with first-line anti glioma drug(TMZ)was used and 8 different treatment groups were set including Saline group,TMZ group,SAL group,LDL-SAL group,LDL-SAL-Ang group,the combination of SAL and TMZ group,the combination of LDL-SAL and TMZgroup and the combination of LDL-SAL-Ang and TMZ group.Cell apoptosis and the formation of vascular endothelial cells in glioma was evaluated by immunohistochemistry section.The survival status and weight change of the remaining mice in each group were observed and recorded.LDL-SAL-Ang can inhibit the apoptosis of glioma and angiogenesis,improve the survival state of the mice and slow down the weight loss.The combination of LDL-SAL-Ang and TMZ showed the best treatment in the 8 groups.Above all,LDL-SAL-Ang had strong anti-tumor activity and brain targeting ability,at the same time it inhibited the tube number and migration of endothelial cells differentiating from glioma stem cells.It also had muti-target annti-glioma effect which provided a new idea for glioma chemotherapy.