Noncovalent Complexation Of Amphotericin B With Poly(β-amino Ester)derivates For Treatment Of C. Neoformans Infection

Background:In recent years,the incidence of cryptococcal disease has increased dramatically in patients immunocompromised because of HIV infection,organ transplantation,or treatment with cytotoxic chemotherapy.Amphotericin B（AMB）is a polyene macrolide antifungal agent and the preferred drug for the treatment of cryptococcal infection.The clinical application of amphotericin B is limited by its poor solubility in aqueous medium and also by its renal toxicity in the host.Therefore,it is important to study how to increasing the water solubility of AMB and reducing its side effects by nano-drug delivery systems.Objective:With the development of the nanotechnology,drug delivery systems based on nanomaterials have exhibited great potential applications in the fields of drug delivery.The nanocarriers are able to increase the water solubility of hydrophobic drugs,change the distribution and pharmacokinetics,and improve the safety.In view of this,based on interactions between drugs and nanomaterials,we will fabricate a kind of nanocomplex delivery systems for AMB in order to overcome its poor water solubility and serious side effects.Methods and Results:The main content of this dissertation are described as below:1)Design and synthesis of MPEG-PLA-PAE copolymer;2)Preparation and characterization of AMB-loaded nanocomplex;3)In vitro and in vivo evaluation of AMB-loaded nanocomplex.1)Firstly,MPEG-PLA copolymers were synthesized by a ring-opening polymerization of _D,L-lactide using Sn（Oct）₂ as a catalyst and MPEG₂₀₀₀000 as a macroinitiator.Then,the MPEG-PLA-PAE was synthesized by Michael addition of TDP to MPEG-PLA acrylate and DDD.The polymer structure was characterized by¹H-NMR spectroscopy to confirm the synthesis of copolymers.From GPC analysis,the average molecular weight（Mn）of MPEG-PLA and MPEG-PLA-PAE were determined to be 4062 and 10736,respectively.Compared with MPEG-PLA,MPEG-PLA-PAE has its specific acidity-responsiveness.2)Based on the result of prescription design,an optimal formulation was successfully screened by single factor test.An emulsion-solvent evaporation method was used to prepare the AMB/MPEG-PLA-PAE complex,while AMB/MPEG-PLA micelles were relatively easy to fabricate by a thin-film hydration method.The average size and zeta potential of AMB/MPEG-PLA were 205.7±3.5 nm and 10.7±0.8 mV,respectively.Due to the potential interactions between AMB and PAE segments,the size of the AMB/MPEG-PLA-PAE was generally smaller（115.8±2.8 nm）than that of AMB/MPEG-PLA,and its zeta potential values increased to 35.1±1.2 mV.HPLC was used for drug analysis,which was performed with a C₁₈8 column（Inertsil ODS-SP C18,4.6×250 mm,5μm）（mobile phase:acetonitrile-0.02 mol/L EDTA·2Na（35:65,v/v）,flow rate:1.0 mL/min and UV detection at 386 nm）.At the same feed,with a weight ratio of 1:10 of the drug:polymer,the drug encapsulation efficiency of AMB/MPEG-PLA-PAE（95.6±1.2%）wasobviouslyhigherthanthatof AMB/MPEG-PLA（71.2±2.5%）,indicating that the PAE segments in polymers could enhance the drug-loading capacity.Moreover,the release rate of AMB from AMB/MPEG-PLA-PAE was obviously lower than that of AMB/MPEG-PLA.And the AMB-entrapped nanocomplex formation exhibited better serum and storage stability than its micellar formation.In the UV spectral analysis,AMB is highly aggregated in AMB/MPEG-PLA-PAE,mostly due to the close association of AMB to the PAE3)HPLC internal standard method for the determination of AMB in biosample has been established.The internal standard was 4-nitro-1-naphthylamine,which has good separation with AMB.And the chromatographic system provided good separation of the compound without no endogenous interference.The results of pharmacokinetics showed that the AMB-loaded nanocomplex had the faster clearance rate from blood than its micellar formation.Compared to AMB/MPEG-PLA,AMB/MPEG-PLA-PAE had a higher degree of accumulation in the liver,lung and spleen.Additionally,all AMB formulations exerted obvious antifungal activity against C.neoformans cells.Based on the results from in vitro assays,the AMB-loaded nanocomplex was used for further evaluation in the mice model infected with C.neoformans.As expected,AMB/MPEG-PLA-PAE reduced the C.neoformans burden in lungs,liver,and spleen more efficiently than AMB/MPEG-PLA and AMB.And no obvious side effects on hepatic and renal function were observed after injection of AMB-entrapped nanocomplex.Thus,AMB nanocomplex was able to reduce AMB acute toxicity and increase drug dose to some extent.Conclusion:In the current study,we developed a noncovalent complex for AMB delivery based on MPEG-PLA-PAE copolymers.Due to the electrostatic interactions between AMB and PAE blocks,entrapped AMB in the nanocomplex showed dramatically increased water solubility,high loading capacity,and enhanced stability.In vivo antifungal activity tested against C.neoformans in mice was found to improve the therapeutic benefits of AMB significantly when loaded within the nanocomplex.Moreover,no obvious side effects on liver and kidneys were observed after treatment with the AMB nanocomplex.Overall,our results indicate that we have designed a suitable nanocarrier for efficient delivery of AMB in the treatment of C.neoformans infection.