| Protein drugs played a very important role in the diagnosis and treatment of various human diseases.However,due to its unique structure and property,the delivery of protein drugs has always been a major obstacle.Polymersomes are liposome-like nanoparticles composed of a lipophilic membrane and a hydrophilic inner cavity.As a novel nano-delivery system,they have gradually attracted people’s attention in recent years.Firstly,in this study,we synthesised amphiphilic polyphosphazene polymer with methoxyl poly（ethylene glycol）（mPEG2000）as hydrophilic side group,octadecylphosphoethanolamine（OPA）and bihexylphosphoethanolamine as hydrophobic side groups,abbreviated as PEOP and PEBP.A series of PEOPs were obtained via the graft ratio adjustment of mPEG and OPA.The structure of PEOPs were characterized by Fourier Translation Infrared Spectrum（FT-IR）,Nuclear Magnetic Resonance（NMR）and Gel Permeation Chromatography（GPC）.Then PEOP polymersomes were prepared by simple dialysis method with dimethyl formamide（DMF）as solvent.Accordding to the Transmission electron microscope（TEM）and confocal laser scanning microscope（CLSM）,PEOP polymer can form self-assemblies in water solution.When the weight ratio of PEG f PEG（w）reduced to53.5%-61.8%,the morphology of the self-assemblies changed from micelles to vesicles.Then,we investigate the feasibility of PEOP self-assemblies as an oral delivery carrier for insulin（INS）.We prepared by common insulin-loaded PEOP nanoparticles by dialysis method,found that whether the PEOP polymer formed micelles or vesicles can efficiently load insulin.The enhanced OPA graft ratio facilitated efficient encapsulation of drugs.Especially,the insulin loading content of PEOP-1 vesicles reached 8.07%and encapsulation efficiency was as high as 87.8%.2D1H-1H NOESY spectrum and FT-IR proved the existence of strong inter-molecular forces between the PEOP polymer and the protein drug,so that whether the PEOP polymer forms micelles or vesicles can efficiently load insulin.In vitro stability experiments,insulin was easily inactivated under conditions of strong acidity and trypsin,but when insulin was loaded into the PEOP self-assemblies.The PEOP self-assemblies can protect the encapsulated insulin well,avoid them to contact with enzymes and acids in the gastrointestinal tract,make them release slowly.The ability of PEOP vesicles in improving oral absorption in vitro was investigated,MDCK cell monolayer as in vitro GI tract model,cellular uptake and transport study as evaluation criterions.It was found that the rate of cell uptake was improved after insulin was encapsulated into PEOP-1 vesicles or PEOP-4 micelles according to flow cytometry.Papp values of INS/PEOP-1 vesicles and INS/PEOP-4 micelles were approximately 1.4-fold higher than that of free insulin,which illustrated that the carrier could promote the transport.Diabetic SD rat model was established to investigate the hypoglycemic effect of oral insulin-loaded PEOP nanoparticles,demonstrating that oral administration of INS/PEOP-1 vesicles can effectively reduce blood glucose levels in diabetic rats,and can avoid the phenomenon of hypoglycem.The results of pharmacokinetic studies showed that the relative bioavailability of PEOP-1 vesicles group was 3872%compared with free insulin,and the relative bioavailability of PEOP-4 micelles group was 1215%.The results show that PEOP-1 vesicles can effectively increase the oral bioavailability of insulin.In order to solve the problem of interferon-γ（IFN-γ）about short half-life and instability,we also used PEBP vesicles to load IFN-γ（IFN-γ/PEBP vesicles）,and conbined with 5-fluorouracil（5-Fu）to treat colon cancer.The relevant mechanism is that after 5-Fu enter tumor cells,it is decomposed into the active intermediate fluorodeoxyuridine monophosphate（FdUMP）in the presence of thymidine phosphorylase（TP）,and inhibits DNA replication and transcription of tumor cells,thereby inhibiting the growth of tumor cells and inducing apoptosis of tumor cells.After combined administration of 5-Fu and IFN-γ,when IFN-γis taken up into tumor cells,it can activate TP so that more 5-Fu is converted to FdUMP,thereby achieving better anti-tumor effect.The MTT experiment indicated that the combination of 5-Fu and IFN-γ/PEBP vesicles can effectively inhibit the proliferation of tumor cells.Cell apoptosis and DNA ladder experiments showed that the combination of 5-Fu and IFN-γ/PEBP vesicles could effectively induce tumor cell apoptosis.Cell cycle experiments show that the combination of 5-Fu and IFN-γ/PEBP vesicles can effectively reduce the proportion of tumor cells in the G2 phase,thereby inhibiting DNA replication and transcription of tumor cells.In vivo anti-tumor experiments show,The tumor inhibitory rates of intravenous injected free IFN-γat a dose of 0.5mg/kg,IFN-γ/PEBP vesicles at a dose of 0.5 mg/kg,intraperitoneal injected 5-Fu at a dose of 5 mg/kg,combination administration of 5-Fu at a dose of 5 mg/kg and free IFN-γat a dose of 0.5 mg/kg,and combination administration of 5-Fu at a dose of 5mg/kg and IFN-γ/PEBP vesicles at a dose of 0.5 mg/kg were 27.2%,21.9%,39.9%and 66.9%,respectively,indicated that combination of 5-Fu and IFN-γ/PEBP vesicles has significant antitumor effect.In conclusion,PEOP and PEBP polymer vesicles containing lipid-mimic groups can efficiently load protein drugs.In terms of hypoglycemia,drug carriers have good drug-loading and transmembrane transport capabilities,which can greatly improve the oral bioavailability of insulin.In the aspect of anti-tumor,it can improve the efficacy and reduce toxic and side effects.This study has laid a good foundation for the subsequent research. |
