Research On The Drug Delivery System Targeted To Gliomas By RDP-modified Long-circulting Curcumin-loaded Liposomes

Malignant gliomas（MG）are the most common cancers in the adult central nervous system and have the worst prognosis in primary brain tumors.It is difficult for MG to be completely removed by surgery and this poses a great threat to the human health because of high recurrence rate,short survival time and poor prognosis.For this reason,there is an urgent need for developing a kind of more effective therapeutic with fewer side effects.Thus,natural compounds or derivatives with multifunctional and multi-target characteristics have the potential to be developed as the best drugs for MG.In recent years,curcumin has received extensive attention because of its none of toxicity in high dose to human body and extensive and precise biological activity and pharmacological effects.Studies have shown that curcumin can inhibit the carcinogenesis of a variety of cell lines,and has a greater research value as a potential anticancer agent for the treatment of gliomas.And owing to some natural characteristics of curcumin which lead to its lower bioavailability and lack of pharmacokinetics,the application of curcumin in the treatment of GM is limited to some degree.An effective method of delivering therapic agents to tumor sites and improving their bioavailability have been a major challenge in current biomedical research.In recent decades,nano-carriers and nanotechnology have rapidly developed in the field of medical imaging and targeted-drug delivery.As a kind of drug delivery system,liposomes have a great advantage in the transmission of antineoplastic drugs.In addition,the cycle time of the liposomes is further increased by the modification of surface-bound hydrophilic molecules such as polyethylene glycol（PEG）which can target tumor cells passively by enhanced permeability and retention（EPR）effects.Although numerous studies have been conducted on targeted liposomes,there have been few studies on brain targeting liposomes.The blood-brain barrier（BBB）prevents the treatment of drugs from entering the central nervous system（CNS）.Clinically,failure of CNS drug delivery was mainly due to the lack of appropriate targeted drug delivery systems（TDDS）.Rabies virus has a high degree of neurotoxicity and a peptide derived from rabies virus glycoprotein（RDP）appears as a targeting ligand for drug delivery to the brain and has been shown to be expected as an effective route for brain-targeted therapy by a non-invasive approach to through BBB.It can be seen that the development of anti-brain tumor preparation,which is safe and effective and can target the tumor site,is of great significance in the treatment of malignant glioma.The purpose of this study is to develop safe and highly effective anti-brain tumor preparations,which apply RDP’s active tumor-targeted drug delivery approach and EPR effect of long-circulating liposomes for the treatment of malignant gliomas.In the first chapter,the research background and significance are briefly introduced,and then we summarize the research development of RDP targeting sequence.Subsequently,in the main part of the experiment,this research is composed of three interrelated chapters.（1）ThepreparationandcharacterizationofRDP-modifiedlong-circulating liposome-encapsulated curcumin（RDP-Lip/CUR）intended for targeted drug delivery.（2）Evaluation of targetability and efficacy of RDP-Lip/CUR in vitro.（3）Evaluation of targetability and efficacy of RDP-Lip/CUR in vivo.The specific contents and results are as follows.In Chapter 2,the RDP peptide was synthesized by solid phase synthesis method.Then,RDP-PEG2000-DSPE was synthesized by the reaction of the activated group malephthalamide in NHS-PEG2000-DSPE with N-terminal free amino group of RDP peptide.The synthesis was proved to be successful according to the determination identified by high-performance liquid chromatography（HPLC）method and MALDI-TOF-MS analysis.Subsequently,long-circulating PEGylated liposome-encapsulated curcumin（mPEG-Lip/CUR）and RDP-modified liposome-encapsulated curcumin（RDP-Lip/CUR）were prepared by the thin-film dispersion method and a design optimization process.mPEG-Lip/CUR and RDP-Lip/CUR had average particle sizes 98.3nm and 102.7nm,coefficients of dispersion 0.205and 0.239,zeta potentials-32.5mV and-30.8mV,encapsulation efficiencies 89.56%and88.72%,respectively.Two methods,transmission electron microscopy（TEM）and atomic force microscopy（AFM）,were used to observe the morphologies of liposomes,which indicated a homogeneous size and sphere shape distribution.The in vitro release assays results demonstrated that the modified RDP peptide on the prepared liposomes had no effect on the release of CUR.After 1 month at 4 degrees,the liposomes could maintain excellent stability.After the successfull synthesis,cell targeting and in vitro antitumor effect of curcumin-loaded liposomes were investigated.Neural human malignant glioma（U87）and non-neural human lung adenocarcinoma（A549）cell lines were chosen in the comparation of targetability.The cellular uptake of RDP-Lip/CUR and mPEG-Lip/CUR by U87 and A549 cells was investigated by fluorescence microscopy.The results showed that in the U87 cells,the fluorescence intensity of the RDP-Lip/CUR group was significantly higher than that of the mPEG-Lip/CUR group and was close to that of the free curcumin group,but there was no significant difference between the RDP-Lip/CUR group and the mPEG-Lip/CUR group in A549 cells.In addition,the result of fluorescence colocalization showed that compared to mPEG-Lip/CUR,RDP-Lip/CUR entered into the U87 cell nucleus more quickly.All these results indicated that RDP-Lip/CUR had good neural cell targeting which derived from RDP.The RDP-Lip/CUR was mainly uptaken by cells through a clathrin mediated endocytosis with time,concentration and temperature dependence,which can be inhibited by GABA ligands.Next,the cytotoxicity of the blank liposomes and the in vitro antitumor activity of the curcumin-loaded liposomes was analyzed by the inhibition of cell proliferation assays（CCK8）.The results showed that compared with PEGylated blank liposomes,RDP-modified blank liposomes were less cytotoxic.The cytotoxicity of RDP-Lip/CUR(IC₅₀=16)was higher than that of mPEG-Lip/CUR(IC₅₀=30),and was close to that of free curcumin in U87 cells.While,in A549,the difference of cytotoxicity of RDP-Lip/CUR(IC₅₀=32.5)and mPEG-Lip/CUR(IC₅₀=36.8)was not significant.The above points further demonstrated RDP-Lip/CUR had excellent activity to resist of glioma.Then,the influence of curcumin-loaded liposomes on the apoptosis and changes in cell cycle distribution were checked with flow cytometry.In addition,Western Blot was used to detect the expression of apoptosis-related proteins were detected by Western blotting.The results indicated that RDP-Lip/CUR could up-regulate pro-apoptotic protein（Bax and cleaved caspase3）expression,down-regulate anti-apoptotic protein（Bcl-2）expression,induce U87 cells apoptosis and block cell cycle in G2/M phase.The efficacy of RDP-Lip/CUR was similar to that of free curcumin（P>0.05）,while compared with mPEG-Lip/CUR,there was significant improvement（P<0.01）,indicating the effectiveness of RDP-Lip/CUR for anti-glioma therapy on the human glioma in vitro.Finally,the targetability and efficacy of RDP-Lip/CUR should be evaluated in vivo.the U87 tumor model of nude mice was established by brain stereotaxic device with a 100%tumor formation rate,100%mortality rate within 2 weeks after operation and 23 days average survival time.The model could be used as a good brain tumor model to evaluate the therapeutic efficacy.Subsequently,with saline as the control group,the anti-tumor efficacy of free curcumin,mPEG-Lip/CUR and RDP-Lip/CUR were examined and compared based on the fluorescence distribution of isolated organs by fluorescence imaging system after 4h intravenous injection with tumor model and normal mice.It was found that RDP-Lip/CUR could enter brain cells largely and be enriched in the glioma site by tumor EPR effect.mPEG-Lip/CUR,by contrast,performed poorly in the brain and no brain distribution in free curcumin groups（only distributed in the gallbladder）.These results demonstrated the excellent brain glioma targeting and long-circulating effects of RDP-Lip/CUR.Then,the targetability and therapeutic effects of RDP-Lip/CUR were valued and compared with other groups through a variety of approaches,including measurement of tumor size by magnetic resonance imaging（MRI）,observation of the degree of malignancy by the HE staining,detection of brain glioma apoptosis by TUNEL assays,investigation of the expression of tumor-associated proteins by immunohistochemistry and comparison of the survival time of tumor model mice.The results of MRI and HE staining showed that the RDP-Lip/CUR group had the highest brain tumor inhibition rate and lowest degree of malignancy,respectively.The results of immunohistochemistry showed that the expressions of Bax and cleaved caspase 3 positive cells in RDP-Lip/CUR group were significantly higher than those in other three groups,while the expressions of Bcl-2 and Ki67 positive cells were the lowest,which further demonstrated that RDP-Lip/CUR promoted the apoptosis of brain tumor cells most.Survival results showed that the median survival time of mice in RDP-Lip/CUR group 30 days was significantly higher than that of the other three groups,which was nearly 36%higher than that of the saline group（22 days）.The above results showed that RDP-Lip/CUR had a good anti-glioma effect in vivo.Finally,RDP-Lip/CUR was testified to be a good safe base on the results of pathology and blood and serum biochemical indicators.In summary,this study designed and prepared a brain-targeted DDS,that is,RDP peptide-modified long-circulating liposome-encapsulated curcumin and construct a good body model which can be used for glioma efficacy evaluation.The system,by means of the active brain-targeted drug delivery strategy of RDP and passive targeted strategy of liposomes,can effectively improve the brain tumor targeting and anti-glioma therapeutic efficacy and has the nature of safety in vivo.RDP-Lip/CUR is considered to have a good application prospect in the comprehensive treatment of GM.