Bactericidal Activity And Biosafety Of Chlorhexidine-loaded Silver-based Nanoantiseptics

Silver,as one of the antiseptics,has been used for thousand years and extensively studied worldwide for various applications,particularly in the food and biomedical areas owing to its highly efficient antibacterial effect and broad spectrum against bacteria with less drug-resistance development.Except for silver,Chlorhexidine(CHX)is a preferred antiseptic for skin and oral infection due to its rapid onset and broad-spectrum biocide effectiveness with less risk for the occurrence of an opportunistic infection.With the increasing challenges brought up by drug-resistant bacteria worldwide,a combination of antiseptics has become a common strategy for treating bacterial infections.Recent studies demonstrate that the combination of CHX and silver could engender a synergistic bactericidal effect and greatly improve the bactericidal efficacy.However,the clinical application of silver-CHX combination can be ineffective or limited as a result of off-targeted administration and side effects.Thus,it is necessary to design a targeted and biosecured drug carrier with high drug-loading rate and encapsulation efficiency.The carrier should offer the controlled-release of CHX and silver ions in response to the acidic microenvironment of bacterial infection,thereby exerting the maximal antibacterial effect with minimal side effects.To date,the increasing nanotechnology together with biomedicine have given rise to a novel interdiscipline,nanomedicine.It brings new opportunities for the development of highly efficient and safe drug delivery systems therefore currently being most promising in regard of medicine translation.Herein,our thesis aims at improving the efficiency and biosafety of the concomitant use of silver and CHX by taking the advantages of mesoporous silica nanoparticles(MSNs),including the superb biocompatibility,excellent loading capacity and easily surface modification properties.We fabricated an MSNs-based multi-functional nano-antiseptic,which was capable of co-loading CHX and silver with p H-triggered drug releasing behavior,and investigated its antibacterial effect,and evaluated its biocompatibility.In the second chapter,we prepared the uniform amino-functionalized MSNs according to a modified one-pot sol-gel method.The resulting spherical nanoparticles(NPs)with good dispersity had an average diameter of 105.3 nm and Zeta potential of +19.8 m V.The surface area,pore volume and average pore diameter of MSNs were determined to be 533.5 m2/g,1.23 cm3/g and 5.6 nm,respectively.In this regard,the growth of nanosilvers on the MSNs' surface was then achieved under ultrasonication by introducing silver ammonia complex cation([Ag(NH3)2]+)into the MSNs-NH2 suspension.A large amount of nanosilver was homogeneously distributed on the surface of MSNs,presenting in the form of Ag0.The so-called Ag-MSNs had an average diameter of 123.7 nm and Zeta potential of +37.5 m V.Compared to MSNs,the surface area,pore volume and average pore diameter of MSNs were decreased to 359.1 m2/g,0.91 cm3/g and 3.0 nm,respectively,holding as an ideal vector for loading small antiseptics.In the third chapter,we introduced carboxylate functional groups onto the pore surface of the Ag-MSNs for loading positively charged CHX through electrostatic interaction.The CHX-loaded,nanosilver-decorated MSNs(Ag-MSNs@CHX)with good dispersity and stability had an average diameter of 165.8 nm and Zeta potential of +8.2 m V.We demonstrated that more CHX and silver ions were released from Ag-MSNs@CHX into the acidic environment in comparison with the neutral media.The p H-responsive releasing properity of Ag-MSNs@CHX laid the foundation for its futher antibacterial activities.In the fourth chapter,we demonstrated that the corresponding minimum inhibition concentration(MIC)of Ag-MSNs@CHX at 24 h for Staphylococcus aureus and Escherichia coli was 25 ?g/m L and 12.5 ?g/m L,respectively.The minimum biocidal concentration(MBC)of Ag-MSNs@CHX was consistent with MIC in both bacteria,which indicated that Ag-MSNs@CHX possessed less antibacterial activity against Gram-positive bacteria than Gram-negative bacteria.We further demonstrated that the bacterial colonies formation of Ag-MSNs@CHX in both kinds of bacteria was much lower than that of materials containing equivalent concentration including CHX,Ag NO3,Ag-MSNs,or MSNs.Importantly,the combination of silver and CHX in the nano-antiseptic exhibited a significant synergistic effect(FIC index = 0.75 and 0.625)against S.aureus and E.coli,respectively.In the fifth chapter,we demonstrated that the cell viability of both NIH-3T3 cells and HUVEC remained more than 80% even at Ag-MSNs concentration of 50 ?g/m L,whereas MSNs exhibited no toxic effects at the same concentration.Importantly,Ag-MSNs@CHX significantly reduced the toxicity of silver ions and CHX.However,Ag-MSNs would cause hemolysis at high concentration(50 ?g/m L)while MSNs would not.In the oral toxicity studies,although there was slightly body weight loss in the mice of Ag-MSNs group,the hematological parameters,blood biochemistry,and organ histopathology at the end of day 30 did not significantly change when compared with that of the mice in MSNs group or normal group.These results suggested a good biocompability of MSNs and Ag-MSNs and their great potential in clincal application.Taken together,monodisperse MSNs nanospheres were successfully developed as an ideal carrier for co-delivery of CHX and nanosilver through a facile and environmental-friendly method in this thesis.The as-formed nanoantiseptics with high density and well-distributed silver nanodots exhibited a p H-responsive releasing manner of both CHX and silver ions simultaneously.Thus,Ag-MSNs@CHX exerted an excellent bactericidal effect on Gram-positive bacteria S.aureus and Gram-negative bacteria E.coli at a low concentration.Meanwhile,it possessed a good biocompatibility for somatic cells,blood cells in vitro and mice after 30 days of oral exposure in vivo.Importantly,the synergistic bactericidal effect of Ag-MSNs@CHX towards both S.aureus and E.coli were demonstrated.To this end,our findings lay the groundwork for exploiting Ag-MSNs@CHX as an economic and biosafe nanoantiseptic agent for the treatment of bacterial infections in the future.