IRGD Peptide Modified Co-loaded Carboplatin And Ce6 Liposome Mediated Sonodynamic Therapy To Inhibit Glioma

Glioma Cancer is the most common primary tumor in the nervous system.It has a high degree of malignancy and infiltrating growth.It is difficult to completely eliminate by operation.It is easy to damage the central nervous system by radiotherapy,and It is not easy for chemotherapeutic drugs to reach in brain tumor tissues.Moreover,the distribution of drugs in the body does not wipe out tumor cells and normal cellsselectively at the same time and it is difficult to treat with.Glioblastoma stem cells are a small part of gliomas that can cause tumorigenesis and maintain tumor growth.Under appropriate conditions,it can differentiate into new tumor cells and thus is considered as the origin of tumorigenesis,recurrence,and metastasis.Therefore,killing cancer stem cells is an important part of determining the efficacy of cancer,but so far all radiotherapy and chemotherapy methods are unable to kill cancer stem cells.Taking the limitations of these treatment methods into account,we have adopted another treatment method---Sonodynamic Tharapy(SDT).Sonodynamic Tharapy(SDT)is a new method proposed by Japanese scholar named Umemura S et al.in 1989 for the use of ultrasound synergistic sonosensitizers to kill tumor cells.There is a strong pnetration ability on biological tissue by the use of ultrasound in Sonodynamic Tharapy(SDT),in particular,focused ultrasound can focus on deep tissue by using sound energy non-invasively and activate the sonosensitizers to produce tumor tissue killing.The choice of sonosensitizers is the key of SDT.In this study,dihydroporphine e6,or Ce6,was chosen as one of the chlorophyll-degrading derivatives.The advantage is that there are a strong acoustic response capability,non-toxicity,high tumor tissue selectivity and high elimination rates of non-neoplastic tissue.In the selection of anticancer drugs,Carboplatin(CBP),an anticancer drug,was selected in this study.The target is not only DNA,but also inhibits the synthesis of RNA and protein.It is a kind of non-phase-specific anticancer drug.However,long-term high-dose administration will produce neurotoxicity and various adverse reactions.The emergence of bio-nanotechnology can provide solutions to the problems faced by toxicity of CBP.Liposome is used as an drug dilivery system with anti-cancer drug to enter target cells,which can significantly reduce the toxicity and side effects of chemotherapeutic drugs while treat tumors effectively.On the other hand,it is known from the literature that integrin ? v ? 3 is highly expressed on the surface of gliomas and other tumor cells,while iRGD-peptide is a short peptide sequence which can specifically bind to integrin ? v ? 3 and composed of arginine,glycine,aspartic acid.It is the recognition site for integrins and their ligand proteins.The major topic of our study is a targeted drug delivery system(tDDS)-targeted liposomes.Under ultrasound conditions,iRGD-peptides modified targeted liposomes contained the sensitizers Ce6 and the anticancer drug CBP.The drug was released at a specific time and under specific conditions by ultrasonic response,and the target was explored in terms of anticancer effect.Killing effect of liposomes on brain gliomas.This study mainly focus on the synthesis of target-loaded liposomes,the controlled release of drugs,the cytotoxicity in vitro and the evaluation of toxic effects of glioma microspheres.Our study will be discussed from the following four aspects:Part I:Preparation,characterization and drug release of iRGD-peptide modified CBP and Ce6 co-loaded liposomes.To study the selection of a standard preparation of PEGylated liposomes as a drug loading platform for the acoustic sensitizer Ce6 and the anticancer drug CBP.Ce6 is loaded into the liposome hydrophobic lipid bilayer and the water-soluble anticancer drug CBP is encapsulated within the hydrophilic core of the liposome.The membrane hydration method was used to prepare carboplatin-liposomes(CBP-L),dihydroporphine e6-liposomes(Ce6-L),carboplatin and Ce6 co-loaded liposomes(CBP-Ce6-L)and the iRGD-peptide modified carboplatin and Ce6 co-loaded liposomes(iRGD-CBP-Ce6-L).Transmission electron microscopy was used to observe its appearance characteristics;Malvern Nano-ZS90 dynamic light scattering particle size analyzer was used to determine the particle sizes,potential and poly dispersion index of different liposomes.The results showed that the preparation of liposomes by membrane hydration method is simple and the results are stable and easy to repeat.Through the study,it was found that the four liposomes were equally distributed,with a particle size of 100 nm-140 nm,a moderate distribution ranges,and a small amount of negative charges,effectively overcoming the problem of strong side effects in the clinical application of the anticancer drug carboplatin.Part II:Anti-tumor effect of iRGD-peptide modified CBP and Ce6 co-targeted liposomes combined with ultrasound on human glioma cells(U87).Using human glioma U87 cells as a model,MTT method,AnnexinV/PI and other experimental techniques were used to investigate the cytotoxic effects of different liposomes combined with ultrasound treatment.PI staining and flow cytometry were used to detect the DNA damage of different liposomes combined with ultrasound treatment.Flow cytometry combined with fluorescence microscopy were used to analysis the uptake of iRGD-peptide modified CBP and Ce6 co-targeted liposomes by U87 cells at different time points.The experimental results showed that there were the most cytotoxic and the strongest DNA damage on U87cells processed through iRGD-peptide modified CBP and Ce6 co-targeted liposomes.The uptake of targeted co-loaded liposomes also increased over time by U87 cells.Part ?:Anti-tumor Effects of iRGD-Peptide Modified CBP and Ce6 co-targeted liposome combined with ultrasound on Rat Glioma Cells(C6).Using rat glioma C6 cells as a model,MTT method,AnnexinV/PI and other experimental techniques were used to investigate the cytotoxic effects of different liposomes combined with ultrasound treatment.PI staining and flow cytometry were used to detect the DNA damage of different liposomes combined with ultrasound treatment.Flow cytometry combined with fluorescence microscopy were used to analysis the uptake of iRGD peptide modified CBP and Ce6 co-targeted liposomes by C6 cells at different time points.The experimental results showed that there were the most cytotoxic and the strongest DNA damage on C6 cells processed through iRGD-peptide modified CBP and Ce6 co-targeted liposomes.The uptake of targeted co-loaded liposomes also increased over time by C6 cells.Part IV:Anti-tumor effect of iRGD-peptide modified CBP and Ce6 co-targeted liposome combined with ultrasound on C6 rat glioma microspheres.Rat glioma cells(C6)were inoculated in serum-free medium[17]supplemented with growth factors and changed the culture medium[18]after four to five days after glioma microspheres[19]formation.After three to four weeks,the diameter of the microspheres can reach to 200?300um.When the serum was added to it,the cells turned to differentiation and adherent growth[20].When grown to the appropriate sizes,MTT assay,AnnexinV/PI and other experimental techniques were used to investigate the cytotoxic effects of different liposomes combined with ultrasound treatments.Flow cytometry was used to detect the uptake of iRGD-peptide modified CBP and Ce6 co-targeted liposome by C6 glioma microspheres over time.The targeted effect of the liposomes was determined by using a laser confocal microscope to photograph the penetration of the targeted co-loaded liposomes into the C6 glioma microspheres before and after sonication.