Killer Peptide Loaded Nanoparticles: Characterization And Anticryptcoccal Efficiacy Evaluation

[Backgroud]Cryptococcosis is a systemic disease caused by Cryptococcus neoformans.In recent years,there has been a dramatic rise in the incidence of cryptococcosis,especially in immunocompromised patients,such as AIDS people,or those who received organ or marrow transplantation.It has been proved that the Cryptococcus neoformans is an facultative intracellular pathogen which can evade the host immune response and under certain circumstances survive in a dormant state until reactivation occurs upon sustained immunodeficiency.This special parasitism probably contributes to the poor outcome of therapy and common relapse.Killer peptide(sequence AKVTMTCSAS) is a water-soluble synthetic decapeptide, which was engineered on the basis of the sequenced KTscFv gene.Killer peptide(KP) has been proved to target onβ1,3-glucan in the cell wall of yeast and not lethal for mammalian cells.In previous work,KP demonstrated a strong fungicidal activity against C. neoformans,Acanthamoeba castellanii,Paracoccidioides brasiliensis, fluconazole-susceptible and-resistant C.albicans strains.Because of this,it probably will be a promising antifungal agent.Traditional anti-cryptococcus agents mainly include amphotericin B and some azole derivatives such as Fluconazole.Amphotericin B is effective but toxic,what more important is that several C.neoformans strains have shown strong resistance to treatments with AmB and azoles as well.Therefore,the recent emergence of new resistant strains requires the development of more effective antifungal agents,possibly with different mechanism of action,in order to avoid the classical mode of fungal resistance.This is the case of novel peptides that have been taken into account as potential new antifungal agents because of their particular interaction with fungal biological membranesMoreover,to eradicate the pathogen adopting intracellular parasitism,particulate system has been proposed for intracellular delivery of antimicrobial agents,it targets the intracellular sites of infection,thereby helps to improve drug activity against intracellular disease involving the mononuclear phagocyte system.So far,some conventional colloidal drug delivery systems,such as liposomes and nanoparticles,have been developed and tested for intracellular pathogen eradication,they ended almost exclusively in the mononuclear phagocyte system(MPS) and provided high local concentration of pharmacotherapeutic agents,thereby improved the antimicrobial efficiacy.[Objective]In this article,we used Poly(D,L-Lactide-Coglycolide)(PLGA) nanoparticles(NPs) for delivery of the KP against systemic C.neoformans infection in mice.It is anticipated that the carder would enhance KP's efficiacy in treatment of systemic murine cryptococcosis[Method]In present study,we adopted a modified double-emulsion method to prepare the nanoparticle,investigated the antifungal activity of killer peptide entrapped in PLGA naoparticles in a murine model.Mice infected with C.neoformans were treated with free killer peptide,as well as killer peptide loaded in PLGA nanoparticles.The anticryptococcal efficacy was assessed by continued survival and colony-forming units(cfu) in vital organs of the treated mice.The toxicity of the KP-PLGA-NP preparation was also evaluated by the means of blood test.[Result]In the first part,we optimized the parameters to acquire the most suitable agent, among all the process parameters,the concentration of PVA plays the most important role in affecting the characteristics of the nanopartiles,the release test in vitro of two preparations(1%,5%PVA) were performed over 12 days and resulted sufficiently sustained release with more than 50%of the peptide released.We demonstrated that by selecting proper conditions it is possible to obtain KP loaded NPs with suitable amount of entrapped peptide,shape,size,and good in vitro sustained release pattern.In the second part,we tested the anticryptococcal efficacy of the sythentic killer peptide by colony counting method.As expected,it manifested a strong anticryptococcal activity in vitro against the ATCC 32609 strain in a dose-dependent manner.In the third part,Killer peptide entrapped in the PLGA nanoparticles showed better efficacy compared with the free killer peptide against C.neoformans systemic infection in BALB/c mice.Mice treated with killer peptide loaded PLGA nanoparticles showed significant higher survival and with less fungal burden in liver,lung and brain of treated mice.We speculate that both of the uptake by macrophages and activation of the MPS may be of great importance for the therapeutic efficiacy of PLGA naonopaticle loaded with KP to treat intracellular infection.Thus we can suppose that KP loaded NPs could represent a new effective therapeutic system for Cryptococcus neoformans infectionIn the final part,the toxicity of several preparations was also evaluated.The results of biochemical analysis disclosed KP-PLGA-NP retained in nano-particles to be atoxic to kidney,liver,as well as hematopoietic system.[Conclusion]we demonstrated that by selecting proper conditions it is possible to obtain KP loaded NPs with suitable amount of entrapped peptide,shape,size,and good in vitro sustained release pattern.Both of the uptake by macrophages and activation of MPS may be of great importance for the therapeutic efficiacy of PLGA naonopaticle loaded with KP to treat intracellular infection.Thus we can suppose that KP loaded NPs could represent a new effective therapeutic system for Cryptococcus neoformans infection...