| Osteomyelitis is a progressive infection of bone, resulting in inflammatory destructionor necrosis of the bone, and may progress to a chronic and persistent state. Staphylococcusaureus (S. aureus) is the most commonly isolated organism from patients with chronicalosteomyelitis. S. aureus would evolve multiple drug resistance rapidly by forming biofilm.Osteomyelitis is a persistent type of biofilm infection known to be refractory to surgicalintervention and antibiotic therapy. Antibiotic-loaded scaffolds have been attemptedextensively in the treatment of osteomyelitis. However, the effectiveness of antibiotic-loadedscaffolds is limited. Furthermore, high concentration of some antibiotics at infection sites canaffect the regeneration and metabolism of bone by interfering with the proliferation of humanosteoblasts and endothelial cells. Therefore, it is necessary to develope a new strategy fortargeting antimicrobial agents for the treatment of S. aureus biofilm infections.According to the results of some previous reseaches, antibiotic-impregnated cationicliposomes have been shown to reduce the congregation of microorganisms. Furthermore,antibiotic-impregnated liposomes have a strong affinity for biofilms and increase theconcentration of antibiotics at the surface of biofilms. The results of our researchdemonstrated that cationic liposomal ceftazidime could inhibit the formation of S. aureusbiofilm more effectively than free ceftazidime. However, the rapid removal of liposomalantibiotics by the reticuloendothelial system of liver Kupffer cells and spleen macrophageslimits the use of intravenously injected liposomal drugs. Therefore, incorporating liposomalantibiotics into a suitable artificial bone scaffold and setting it at the area of infection mayallow for the selective increase of liposomal antibiotic concentrations at the site of bone infections, allowing more effective treatment of bone infections. This has been one of thefocus in the area of chronic osteomyelitis researches.A number of scaffolds, such as beads,ceramics and kinds of bone cements, have beenused extensively in researches and clinic. However, a high temperature and pressure areneeded for the preparation of beads or ceramics. Therefore, for beads, and ceramic, drugs orliposomes can be incorporated into scaffolds only by the process of soaking and vacuumevaporation or by centrifugalization. These means are difficult to distribute liposomes intoporous of the scaffolds uniformly, and difficult to control the drug dose and to use in clinic.For acrylic bone cement, organic solvents must be used in the preparation procedure, organicsolvents can damage the microstructure of liposomes. Furthermore acrylic bone cementcannot be biodegradation and need be taken out by operation after a period of time, whichincrease injury to the patient and dangerous of infections. In our early reseach, we found thatwhen we tried to incorporate liposomal antibiotics into calcium phosphate cement (CPC),the scaffold is difficult to form a certain shape and cannot be used in forth research. Up tonow, it is nearly impossible to find a scaffold, which can carry drug and promot boneregeneration in the defection space simultaneously. In recent years, nano-hydroxyapatite/beta-tricalcium phosphate ceramics (n-HA/β-TCP) have been used as a carrier scaffold,with its good biocompatibility,biodegradation and bioactivity, which have been favoured bysome researcher. For this reason, we select n-HA/β-TCP as our experimental scaffold at last.In the present report, we had prepared cationic liposomal ceftazidime (CLC) andstuded their properties. Then we also had prepared nano-hydroxyapatite/beta-tricalciumphosphate scaffolds loaded with cationic liposomal ceftazidime (CLCS), and investigated therelease properties of CLC and CLCS in simulated body fluid (SBF). The anti-biofilmactivity of CLC and CLCS was also evaluated initially..The study includes three parts. Part one: Preparation and Properties of CationicLiposomal ceftazidime. Part two: Nano-hydroxyapatite/beta-tricalcium phosphatescaffolds loaded with cationic liposomal ceftazidime(CLCS): preparation and in vitro drugrelease behavior. Part three: Nano-hydroxyapatite/beta-tricalcium phosphate scaffoldsloaded with cationic liposomal ceftazidime(CLCS): activity against Staphylococcus aureusbiofilms in vitro.Main experiments are shown below: Part one: Preparation and Properties of Cationic Liposomal CeftazidimeMethods:1CLC was prepared by modified reverse phase evaporation method.2The concentrations of ceftazidime in the liposomes were determined by HPLC at254nm with the mobile phase of triethylamineglacial acetic acid buffer solution.3Methods of the separation of CLC from the free ceftazidime.4The optimum lipid composition was screened by orthogonal design.5CLC,shape was observed in transmission electron microscopy (TEM), and itsparticle size and zeta potential were measured with laser scattering method.Main results:The optimized lipid composition used for preparing liposomes was composed of soyaphosphatidylcholine (SPC), stearamide (SA) and cholesterol (Chol) at the molar ratios of7:1:1, lipid and drug at weight ratio of5:1and organic phase:aqueous phase3:1(v/v).Themean values of particle size, zeta potential and entrapment efficiency of the CLC studied were161.5±5.37nm,60.60±5.24mV,6.90±0.07and16.57±0.13%, respectively. The CLC wasstable at the store condition of4℃or-80℃, which appeared steady with leak ratio below5%.Part two: Nano-hydroxyapatite/beta-tricalcium phosphate scaffolds loaded withcationic liposomal ceftazidime: preparation and in vitro drug release behaviorMethods:1Preparation of Nano-hydroxyapatite/beta-tricalcium phosphate scaffolds by a methodof high temperature sinter.2By the process of soaking and vacuum evaporation, cationic liposomal ceftazidime(CLC) is incorporated successfully with nano-hydroxyapatite/beta-tricalcium phosphatescaffolds to form a novel complex drug carrier (CLCS).3The kinetics of free ceftazidime and CLC release from scaffolds were examined invitro.Main results:Electron microscope images indicated that the liposomes were well preserved in thescaffolds, and that it was the CLC rather than free ceftazidime releasing from the scaffolds.The test results and statistical analyses showed that the release behavior of both the free ceftazidime and CLC from scaffolds was based on a dissolve/diffusion Fickian process.Part three: Nano-hydroxyapatite/beta-tricalcium phosphate scaffolds loadedwith cationic liposomal ceftazidime: activity against Staphylococcus aureus biofilmsMethods:1The minimal inhibitory concentration (MIC) of samples to Staphylococcus aureus wasdetermined by a broth microdilution assay.2The anti-biofilm activity of CLCS was studied using a biofilm regrowth assay.Main results:The MIC of free ceftazidime and CLC against Staphylococcus aureus were6.0μg/mland4.25μg/ml, respectively; and against Penicillin-resistant Staphylococcus aureus were8.0μg/ml and3.16μg/ml, respectively. CLC could inhibit the formation of both Staphylococcusaureus biofilms and Penicillin-resistant Staphylococcus aureus biofilms more effectively thanfree ceftazidime (P＜0.05). CLCS was more effective at inhibiting the formation of S. aureusbiofilms and can release CLC from scaffolds persistently.Conclusions:1The modified reverse phase evaporation method was a simple, reproducible method toprepare CLC, and with a high entrapment efficiency.2The porous n-HA/β-TCP scaffolds were successfully combined with both liposomescontaining ceftazidime and free ceftazidime. The test results and statistical analyses showedthat the release behavior of both the free ceftazidime and CLC from scaffolds was based on adissolve/diffusion Fickian process.3Both CLC and CLCS could be more effective at inhibiting the formation of S. aureusbiofilms in vitro..4CLCS is a novel and promising vehicle for the controlled delivery of liposomalantibiotics for the treatment of osteomyelitis caused by S. aureus biofilm infection.
|
PDF Full Text Request