Study On The Preparation Of Drug Loaded Nanoparticles With Bovine Serum Albumin Conjugated Biguanides And Its Anti-tumor Effect

Biguanides are commonly used antidiabetic drugs.In recent years,anti-cancer efficacy of biguanide drugs has been reported,and the biguanide in this class of drugs is considered a chemical group with potential anti-tumor effect.Furthermore,the functional group possess relative high affinity for nucleic acid phosphates and great cell penetrating ability through the plasma membrane contained a lipid bilayer.NDNB is a small chemical drug for the treatment of idiopathic pulmonary fibrosis,and as a kind of multi-target receptor tyrosine kinase inhibitor which can inhibits angiogenesis and tissue fibrosis.A large number of preclinical and clinical studies have shown that NDNB has a good anti-tumor effect.In this study,using dicyandiamide as the substrate,the intermediate with biguanide was synthesized which was conjugated to bovine serum albumin by the mixed anhydride method.Then,nano-carrier with the biguanide-conjugated the bovine serum albumin as material was prepared to carry NDNB as its model drug,evaluating its pharmacodynamics or pharmacokinetics in vitro and in vivo.The ultimate aim is to enhance the anti-tumor effect of NDNB by using conjugated nano-carrier.This paper includes six parts as follows:Part one: ReviewsIn this part,we mainly introduced the research progress on biguanide anti-tumor effect,the conjugated/modified albumin carrier and the anti-tumor effect of NDNB.the research frontiers to anti-tumor effect based-biguanides drugs and biguanides,protein conjugated/modified methods and ideas,physical and chemical properties,pharmacological effects and pharmacokinetic properties of NDNB were elucidated in order to lay the foundation for the subsequent studies of preparations.Part two: preformulation studiesIn this part,an ultraviolet(UV)spectrophotometric method was established as an analytical method for the detection of drug content in vitro.Determined by UV full wavelength scanning,the maximum absorption wavelength of NDNB was 385 nm which was used as the drug detection wavelength.The different concentration solution was used to establish standard curve.The experimental results ofmethodology validation showed that the method is stable,reliable and consistent with the methodology.Besides,the solubility of NDNB in the release medium was investigated.The results showed that the solubility of NDNB in water was high,and the solubility in the phosphate solution was very small.Addition of surfactant tween80 can increase its solubility in the phosphate solution.Part three: Preparation of biguanides-conjugated bovine serum albuminIn this part,1-(p-carboxyphenyl)biguanide(DP)hydrochloride was prepared by the reaction of dicyandiamide and p-aminobenzoic acid as the substrate.Bovine serum albumin(BSA)was conjugated with DP by the mixed anhydride method.The structure and purity of DP was characterized by thin-layer chromatography(TLC),infrared spectrometry(IR),proton nuclear magnetic resonance(1HNMR),mass spectrometry(MS),UV scanning and high performance liquid chromatography(HPLC).UV scanning and gel-electrophoresis were used to confirm and characterize the biguanides-conjugated BSA(DP-BSA).the conjugation ratio is analyzed and estimated using matrix-assisted desorption ionization-time of flight(MALDI-TOF MS)and UV-spectrophotometry.The optimal process and formulation was obtained by the single-factor test of the feed ratio,the reaction time and the reaction pH environment,in which DP-BSA prepared had a conjugation ratio 42.19.In addition,we also investigate the solubility and other properties of DP-BSA prepared.Part four: Preparation of drug-loaded nano-carrier and Releasing in vitroIn this part,nano-carrier with the biguanide-conjugated the bovine serum albumin as material was prepared to carry NDNB as its model drug.laser particle size analyzer,transmission electron microscopy(TEM)and UV-spectrophotometry were used to detect or characterize the particle size distribution,entrapment efficiency(EE)and drug content of drug-loaded nanoparticles(NDNB-DP-BSANPs)solution.The prescription and preparation process were optimized by the single-factor test with the particle size distribution,appearance,entrapment efficiency and drug content as indexes.The results showed that the average particle size of the nanoparticles prepared was 47.18 nm,and the polydispersity was 0.15,and the average drug concentration was 1.586mg/mL.the drug-loaded concentration was stable within 48 h.Besides,the UV spectrophotometry was used as the method detecting drug concentration in the release test in vitro.By dialysis method,the release of drug NDNB API solution and NDNB-DP-BSANPs solution in PBS(0.5% Tween80)solution was evaluated.The results of the release test in vitro showed that the drug loaded on nanoparticles released slowly.Part five: Pharmacokinetics in vivoIn this part,Establishing an HPLC method for the determination of the drug concentration in plasma,the pharmacokinetics in vivo was investigated on SD rat administrated by tail vein injection.The experimental results of methodology validation showed that the method had strong specificity,high precision,high recovery rate and good stability.The results of pharmacokinetic study showed that the pharmacokinetic process of NDNB in rats could be fitted to two compartment open models.Drug concentrations in the plasma at each time point of NDNB-DP-BSANPs group in SD rats were higher than that of NDNB API group.The half-life in vivo of NDNB API group was 2.526 h,while that of NDNB-DP-BSANPs group was 4.588 h.The average residence time of API group was 2.914 h,which was significantly lower than that of drug-loaded nanoparticles group(5.905h).The relative bioavailability of the nanoparticles was 162.96%.These results indicated that drug-loaded nanoparticles could increase the half-life and bioavailability of drugs.Part six: Pharmacodynamics study in vivo antitumor model and cell experimentIn this part,the antitumor effect of the biguanides conjugated-BSA nanoparticles and NDNB-loaded nanoparticles was investgated by tumor bearing animal model and cell experiment.Antitumor efficacy test and cell test results showed that biguanides conjugated-BSA nanoparticles had a certain inhibitory effect on tumor cells,and NDNB-loaded nanoparticles had a good tumor growth inhibition in both experiments.