Research On Functional Bioprotein Molecules Nanoparticles

Objective:To Research the bioprotein molecules on drug delivery system: 1. we chose insulin as therapeutic bioprotein drug to study the synthesize of insulin conjugating a cell penetrating peptide(CPP), the preparation of oral insulin nanoparticles(NPs), and their regulation effects on blood glucose level(BGL) of diabetic rats; 2. we chose bovine serum albumin(BSA) as carrier bioprotein material to study the preparation of BSA NPs encapsulated sunitinib and curcumin, using combined drug therapy and nanoparticles technology to study the abilities and mechanisms of sunitinib and curcumin on inhibiting tumor cell proliferation. Methods:1. Insulin was chosen as therapeutic bioprotein drug: 1) the insulin-polyethylene glycol-low molecular weight protamine(Ins-PEG-LMWP) conjugate was synthesized. NPs encapsulated insulin or Ins-PEG-LMWP were prepared by ethyl acetate-polyvinyl acetate(EA-PVA) multiple emulsions method, considering N-trimethyl chitosan chloride(TMC) coated polylactic-co-glycolic acid(PLGA) as polymeric carrier material. The basic characteristics and drug release of NPs in simulated gastrointestinal fluids were measured. 2) Insulin/Ins-PEG-LMWP NPs were orally administered in diabetic rats, and the BGL was monitored.2. BSA was chosen as carrier bioprotein material: 1) The BSA NPs were prepared by optimizing the concentrations of BSA and drugs(sunitinib and curcumin), p H and processing time. Transmission electron microscopy(TEM) was used to estimate the formation and size of BSA NPs. Stability and drug release of NPs in vitro were measured. 2) The inhibition effects of drugs and BSA NPs on proliferation of cancer cells MCF-7 was detected by MTT assay. Uptakes of drugs and mechanisms of drugs induced anti-proliferation were observed by confocal laser scanning microscope(CLSM) and flow cytometry. 3) Drugs were injected at caudal vein to the tumor-burdened mice. The weights of mice and volumes of tumors were monitored. Results:1. Insulin was chosen as therapeutic bioprotein drug: 1) The particle size of oral insulin/Ins-PEG-LMWP NPs was measured as 246.9 ± 4.7 nm, zeta-potential was-42.2 ± 4.3 m V and encapsulation efficiency(EE) of insulin/Ins-PEG-LMWP was 45.4 ± 2.3%. The NPs behaved as two-phase drug release which appeared first quick back slow trend in simulated gastrointestinal fluids and there still 40% of drugs were encapsulated in NPs after 6 hours' release. 2) The BGL was decreased to 70% of initial value in 2 h and 50% in 12 h, leading to a high pharmacological bioavailability of Ins-PEG-LMWP NPs(17.98 ± 5.62%) and of insulin NPs(11.24 ± 3.92%) compared to subcutaneous injection.2. BSA was chosen as carrier bioprotein material: 1) The optimal BSA NPs, of which the average size was about 103 nm and the EE of sunitnib and curcumin was both more than 90%, were prepared when the concentrations of BSA, sunitinib and curcumin was 20 mg/m L, 125 ?g/m L, 250 ?g/m L, p H was 7 and the processing time was 4 h. Well-behaved stability and drug release in vitro were revealed. 2) MTT assay showed higher proliferation inhibition in combination drugs compared to single drug, and BSA NPs best. The more uptakes were monitored in combination drugs, and BSA NPs showed no adverse effect on uptakes of drugs. Unapparent effects of sunitinib and curcumin on promoting apoptosis of tumor cells existed. 3) In vivo experiments of drugs on tumor-burdened mice revealed the better anticancer ability of sunitinib combined curcumin but more toxicity, compared to single drug and BSA NPs loaded sunitinib combined curcumin showed the best anticancer effect and less toxicity. Conclusions:1. 1) TMC coated PLGA NPs encapsulated insulin/insulin-PEG-LMWP acted as a well-behaved drug delivery system for releasing drugs in gastrointestinal tract and regulating BGL in vivo. 2) Insulin-PEG-LMWP showed better regulation effect.2. 1) Sunitinib combined curcumin exhibited better anticancer proliferation ability compared to the single sunitinib or curcumin but more toxicity. 2) BSA NPs loaded drugs showed the best anticancer proliferation ability by EPR effect and long circulation of BSA. 3) No apparent effect on apoptosis was induced by sunitinib and curcumin, whereas possibility of blocking cell circulation was prompted.