Nano-hydroxyapatite/chitosan/konjac Glucomannan Scaffolds Loaded With Cationic Liposomal Vancomycin: Activity Against Staphylococcus Aureus Biofilms

Osteomyelitis is a persistent type of biofilm infection known to be refractory to surgical interventions and antibiotic therapy. Antibiotic-loaded scaffolds have been effective in the treatment of osteomyelitis. However, in some infections caused by gram-positive cocci biofilms, the effectiveness of antibiotic-loaded scaffolds is limited. Furthermore, high concentrations of some antibiotics at infection sites can delay the healing of fractures by interfering with the proliferation of human osteoblasts and endothelial cells. Therefore, the development of a new strategy for targeting antimicrobial agents for the treatment of bacterial biofilms is necessary.Antibiotic-impregnated liposomes have been shown to reduce the congregation of microorganisms. Furthermore, antibiotic-impregnated liposomes have a strong affinity for biofilms and increase the concentration of antibiotics at the surface of biofilms. Cationic liposomal vancomycin inhibits the formation of Staphylococcus aureus (S. aureus) biofilms more effectively than vancomycin alone at equivalent concentrations. However, the rapid removal of liposomal antibiotics by the reticuloendothelial system of liver Kupffer cells and spleen macrophages limits the use of intravenously injected liposomal drugs. Therefore, incorporating liposomal antibiotics into a suitable scaffold may allow for the selective increase of liposomal antibiotic concentrations at the site of bone infections, allowing more effective treatment of bone infections.A number of studies have focused on the use of scaffolds such as beads, ceramics or bone cement as most of these can carry antibiotics. However, a high temperature and pressure are needed for the preparation of beads or ceramics and organic solvents are used in the preparation of acrylic bone cement. These preparation conditions can damage the microstructure of liposomes. Therefore, liposomes cannot be incorporated into beads, ceramic or bone cement. n-HA/CS/KGM scaffolds are prepared at room temperature without the use of organic solvents, and that the n-HA content within the scaffold can be adjusted to be similar to that of natural bone. This scaffold also has good biocompatibility and bioactivity, making it an ideal candidate scaffold for use as a carrier of liposomal antibiotics. In the present report, we prepare cationic liposomal vancomycin (CLV) and study their properties and investigated the preparation and release properties of Nano-hydroxyapatite/chitosan/konjac glucomannan scaffolds loaded with cationic liposomal vancomycin (LLS) in simulated body fluid (SBF). We also determined the impact of varying the amounts of KGM and cationic liposomes on the release of liposomes from the scaffolds. The anti-biofilm activity of LLS was also evaluated.The study includes three parts. Part one: Preparation and Properties of Cationic Liposomal Vancomycin. Part two: Nano-hydroxyapatite/chitosan/konjac glucomannan scaffolds loaded with cationic liposomal vancomycin: preparation and in vitro drug release behavior. Part three: Nano-hydroxyapatite/chitosan/konjac glucomannan scaffolds loaded with cationic liposomal vancomycin: activity against Staphylococcus aureus biofilms.Main experiments show below:Part one: Preparation and Properties of Cationic Liposomal VancomycinMethods:1 CLV was prepared by modified reverse phase evaporation method,and separated from the suspention with Sephadex G-50 minicolumns.2 The optimum lipid composition was screened by orthogonal design.3 CLV, shape was observed in transmission electron microscopy (TEM), and its particle size and zeta potential were measured with laser scattering method.Main results:The optimized lipid composition used for preparing liposomes was composed of soya phosphatidylcholine (SPC), stearamide (SA) and cholesterol (Chol) at the molar ratios of 7:3:1. The mean values of particle size, zeta potential and entrapment efficiency of the CLV studied were 185.75±16.33 nm, 69.11±4.62 mV and 8.58±0.045 %, respectively. The CLV was stable at the store condition of 4?or -80?, which appeared steady with leak ratio below 5%.Part two: Nano-hydroxyapatite/chitosan/konjac glucomannan scaffolds loaded with cationic liposomal vancomycin: preparation and in vitro drug release behaviorMethods:1 Porous nano-hydroxyapatite/chitosan/konjac glucomannan (n-HA/CS/KGM) scaffolds were loaded with cationic liposomal vancomycin (CLV) to form a novel complex drug carrier (LLS).2 The kinetics of CLV release from LLS and the effect of the amount of konjac glucomannan (KGM) and CLV in LLS were examined in vitro.Main results:Electron microscope images indicated that the liposomes were well preserved in the scaffolds, and that it was the CLV rather than free vancomycin releasing from the scaffolds. The test results and statistical analyses showed that the weight percentage of KGM or the CLV in scaffolds could greatly influence the release behavior of scaffolds, and the release of CLV from scaffold was based on an anomalous mechanism, a swelling/diffusion process. The increase in the amount of KGM or the decrease in the amount of the CLV in the scaffolds could significantly reduce the release of CLV from the scaffolds.Part three: Nano-hydroxyapatite/chitosan/konjac glucomannan scaffolds loaded with cationic liposomal vancomycin: activity against Staphylococcus aureus biofilmsMethods:The minimal inhibitory concentration (MIC) of samples to Staphylococcus aureus was determined by a broth microdilution assay. The anti-biofilm activity of LLS was studied using a regrowth assay.Main results:The MIC of free vancomycin and CLV against Staphylococcus aureus were 1.0?g/ml and 0.6?g/ml, respectively. CLV could inhibit the formation of Staphylococcus aureus biofilms more effectively than free vancomycin (P<0.05). LLS was more effective at inhibiting the formation of S. aureus biofilms than the free drug-loaded scaffold at equivalent vancomycin doses, especially at low concentrations and short exposure times. The n-HA/CS/KGM scaffolds loaded with CLV could sustain the release of CLV and inhibit Staphylococcus aureus biofilms.Conclusions:1 The modified reverse phase evaporation method was a simple, reproducible method to prepare CLV2 The porous n-HA/CS/KGM scaffolds were successfully combined with liposomes containing vancomycin, resulting in sustained CLV release and inhibition of S. aureus biofilm formation.3 By adjusting the ratio of KGM/CLV (w/w), which significantly influenced CLV release rate, we successfully prepared customized scaffolds with different release rates capable of meeting diverse therapy requirements.4 LLS is a novel and promising vehicle for the controlled delivery of liposomal antibiotics for the treatment of osteomyelitis caused by biofilm infection.