The Construction Of The Fusion Protein Anti-EGFR-iRGD Decorated Red Blood Cell Membrane Derived Nano-carrier And Its Role In The Targeted Therapy Of Gastric Cancer

In worldwide,gastric cancer is one of the top five commonly diagnosed malignant tumor,which is also one of the top three leading cause of cancer related death.Nowadays,gastric cancer has caused a serious social health burden to the whole world.And for our country,China,which is located in the south-east Asia region,the high-risk areas of stomach cancer around the world,considering of the population size of our country,now we have a huge number of diagnosed gastric caner patient,and high proportion of all the diagnosed patients were diagnosed as advanced gastric cancer at the very beginning of diagnosis.All of these make gastric cancer a significant heavry burden for our whole society.Considering of all above,early diagnosis and systematic treatment of gastric cancer are needed very urgently in our country.At present,according to the NCCN diagnosis and treatment guidelines for primary gastric cancer,which is now using internationally,the main treatment strategies of gastric cancer remain as operation,chemotherapy and radiotherapy.And preoperative and postoperative chemotherapy is also an important treatment of gastric cancer.One of traitional chemodrug,which was proofed to be effective in gastric cancer is paclitaxel.Paclitaxel is now commonly used in treatment for gastric cancer.But side effects such as hypersensitive reaction,serverly suppression of bone marrow function,due to its chemical properties or formulate type,make its application in the clinic high risk and complicated.Researchers have been working hard on how to improve the formular form of paclitaxel,improve the curative effect and reduce the side effect.In recent years,with research progress in biomimic drug carrier feild,autologous biological membranes,including red blood cell membrane,white blood cell membrane,cancer cell membrance,et al were used to motified into carriers for different kinds of drugs,including traditional chemotherapy drug.And autologous biological membranes derived nanocarriers were proofed to have their unique advantages in application.Among them,the red blood cell membrane derived nano-carrier was confirmed that could protect the loaded drug to avoid the ABC phonomenno,which leads to fast clearance of drug from the body,and also could keep the drug in circulation for long time.And since everyone has plenty of red blood cells in our body system,it’s easy to get,makes red blood cells a ideal provider for drug delivery.Purpose:In this study,we use red blood cell membrane to manufacture RBC membrane nanoparticles for loading PTX(RBCm-PTX),then anti-EGFR-i RGD,a recombinant protein,firstly designed and purified by our team were used to decorate the RBCm nanoparticles(anti-EGFR-i RGD-RBCm-PTX).And further,the tumor targeting and killing ability of anti-EGFR-i RGD-RBCm-PTX were studied in vitro and in vivo,using human tumor cell line MKN45 and MKN45 subcutaneous tumor model.Methods:Red blood cells were separated,spined down from freshly collected human peripheral blood,then were washed by 0.09%salin.Red blood cells were ruputured using low permeable salin,to collect the ghost of the red blood cells.Then the collected ghosts of red blood cells were used to prepare red blood cell membrane vesicles.And later-on the vesicles were further used to wrap paclitaxel solution using liposome extrusion implements,to form PTX loaded red blood cell membrane nanoparticles(RBCm-PTX).After preparation,the RBCm-PTX was characterized by dynamic light scattering and transmission electron microscopy,and its stability in vitro was observed.The encapsulation rate,dosage and its release profile in vitro of RBCm-PTX were detected by HPLC.DSPE-PEG-Mal and anti-EGFR-i RGD were used to form DSPE-PEG-anti-EGFR-i RGD by Michael addition reaction,then DSPE-PEG-anti-EGFR-i RGD were inserted into bilayer lipid of red blood cell membrane by incubation at 37℃,to form anti-EGFR-i RGD-RBCm-PTX nanoparticles.MKN45 cell lines were involved in this study,MTT were carried out to test the antitumor effect of nanoparticles and PTX,and the antitumor effect of anti-EGFR-i RGD-RBCm-PTX was detected in vivo in MKN45 subcutaneous tumor animal model,weight of experimental animals,volum of subcutaneous tumor were monitored.Near infrared in vivo imaging system were used to study in vivo distribution and tumor targeting ability of anti-EGFR-i RGD-RBCm-PTX.Mian internal organs of experimental animal were harvested to evaluate biological safety of anti-EGFR-i RGD-RBCm-PTX in vivo.Results:1.RBCm-PTX nanoparticles were successfully prepared.Dynamic light scattering analysis showed that RBCm-PTX average particle size is about 168.7±2.2nm,dispersion index(PDI)0.200±0.002,the encapsulation rate is about 60.00%,drug loading efficiency about 34.07%.RBCm-PTX nanoparticles are spheroid,uniformly size under transmission electron microscopy(TEM),and could be stable in vitro for about 8 days.2.DSPE-PEG-anti-EGFR-i RGD were formed by Michael addition reaction of DSPE-PEG-Mal and anti-EGFR-i RGD.Protein electrophoresis showed that,a new coomassie bright blue colorable stripe was found in the gel,which was located above the anti-EGFR-i RGD.3.anti-EGFR-i RGD-RBCm-PTX nanoparticles were successfully prepared.Dynamic light scattering analysis showed that RBCm-PTX average particle size is about 171.7±4.7 nm,dispersion index(PDI)0.214±0.005. anti-EGFR-i RGD-RBCm-PTX nanoparticles are spheroid,uniformly size under transmission electron microscopy(TEM),and could be stable in vitro for about 8days.4.MTT assay results showed that,for MKN45,IC50 of PTX is 5.1 ng/m L in vitro,RBCm-PTX,anti-EGFR-i RGD-RBCm-PTX could also significantly inhibit proliferation of MKN45 cells in vitro,and the inhibitory effect were comparable to PTX,no statistical difference,P>0.05.5.in vivo distribution and tumor targeting study,subcutaneous tumor animal model was involved.Results showed that,about 2h after injection of drugs through tail vein,fluorescent signal could be detected in subcutaneous tumor site of anti-EGFR-i RGD-RBCm-PTX group,and the singal could still be detected 24 h,48h after injection.While,tumor-burdened animals of RBCm-PTX,co-administration of anti-EGFR-i RGD and RBCm-PTX group,no obviously fluorescent signal were detected at all the time point collected,2h,24 h,48 h after injection.And also,fluorescence signals were observed in heart,liver,spleen and kidney of experimental animals in all animal of anti-EGFR-i RGD-RBCm-PTX group,RBCm-PTX group,co-administration of anti-EGFR-i RGD and RBCm-PTX group.6.Results showed that,the weight of subcutaneous tumor was 0.78±0.24,0.46±0.17,0.33±0.09,0.24±0.05,0.13±0.07g for control group,PTX group,RBCm-PTX group,co-administration of anti-EGFR-i RGD and RBCm-PTX group,Anti-EGFR-i RGD-RBCm-PTX group.And the tumor volum of anti-EGFR-i RGD-RBCm-PTX group had accumulated389±72 mm~3,less than the control group(1006±145 mm~3),PTX group(793±73mm~3),RBCm-PTX group(673±52 mm~3),co-administration of anti-EGFR-i RGD and RBCm-PTX group(607±66 mm3),statistically significant(P<0.05).Compared with the control group,the subcutaneous tumor volume in each group decreased by 61%,21%,33%and 40%respectively.And the H&E staining of vital organs,including heart,liver,spleen,lung,kidney of experimental animal,showed no obvious tissue damage.Conclusion:In this study,the paclitaxel loaded erythrocyte membrane nanoparticles(RBCm-PTX)were successfully prepared,and anti-EGFR-i RGD were successfully motifed onto RBCm-PTX,named anti-EGFR-i RGD-RBCm-PTX.Results of this study showed that,RBCm-PTX and anti-EGFR-i RGD-RBCm-PTX nanoparticles are spheroid,uniformly size,and could be stable in vitro for about 8 days.And anti-EGFR-i RGD-RBCm-PTX could target subcutaneous tumor of MKN45 in vivo,could inhibit the growth of MKN45 cells in vitro and in vivo.Considering of its easy preparation,feasibility and and safety,anti-EGFR-i RGD-RBCm-PTX could be one of the candidater for potential clinical application.