| Osteomyelitis is a refractory condition that may occur after bone surgery, potentially leading to amputation or even death; treatment often requires multiple surgical interventions and local or systemic antibiotic therapy. It involves the persistent bacterial infections which are commonly caused by gram-positive coccus. In the internal environment of bone, antibiotics are difficult to reach the efficient level. In some cases the bacterial proliferation could form biofilms which have a strong protective effect on the bacteria, and the drug-resistant plasmid can be easily transmitted and exchanged among the bacteria. As a result, in osteomyelitis growth of bacteria almost always leads to a significant decrease in susceptibility to anti-microbial agents compared with cultures grown in suspension. Therefore, seeking for effective treatment of osteomyelitis by novel drug delivery technologies is needed.Beta-tricalcium phosphate ((3-TCP) is a successfully used biodegradable scaffold for bone substitute with good biocompatibility. β-TCP also has the ability to induce bone formation. For its biodegradable and porous properties, calcium polyphosphate scaffolds can be used as carriers of antibiotics for treatment of bacteria-related osteomyelitis. They may result in a high level of drug release and obviate the need for removal; they are gradually replaced by ingrowing tissue. Furthermore, secondary release of the antibiotic may occur during the degradation phase of the carrier, this could increase the anti-microbial efficacy compared to non-biodegradable carriers.Liposome is a biocompatible and biodegradable drug carrier for sustained and targeted delivery of various drugs including antibiotics. It can modify the biopharmaceutical characters such as absorption and distribution of the drug. It has been proved to be an effective way to enhance the efficacy of antibiotics and to reduce their undesirable effects by incorporation of anti-microbial agents into liposomes. Liposomes have been found to be of use in the delivery of antibiotics to biofilms of various bacteria immobilized on solid supports. Liposomal systems have been widely studied, either to target antibiotics to the surface of bacterial biofilms, or by virtue of their property of being taken up cells of the reticuloendothelial system, to target antibiotics towards intracellular bacteria.Gentamicin sulfate (GS) is a kind of aminoglycoside antibiotic and is the typical treatment for osteomyelitis caused by Gram-positive bacteria. The efficacy of aminoglycosides depends on their ability to reach specific bacterial targets in their active form without enzymatic attack. High dosages of aminoglycosides potentially carry drug toxicity risks. Liposomal encapsulation of GS is a hopeful strategy that helps to increase the therapeutic index of the antibiotics by increasing the concentration of the antibiotics at the site of infection and prolong the drug release time, as well as reducing the toxicity.In this study we have investigated the effectiveness of delivery of GS by liposome combined β-TCP scaffold (LCS), with the aim of inhibiting the bacterial infection in the early stage of post-operative period which is a major cause of the refractory osteomyelitis.The study was divided into three parts:(1) Preparation and characterization of gentamicin sulfate liposomes.(2) Preparation of liposome combined β-TCP scaffold (LCS).(3) Evaluation of anti-biofilm activity in vitro.Main methods1. Preparation and characterization of gentamicin sulfate liposomes.A mixture of DPPC and cholesterol in the molar ratio of3:1was dissolved in a mixed solvent composed of methanol and chloroform (volume ratio1:4). The lipid solution was dried in a round-bottom flask using a rotatory evaporator, at40℃under reduced pressure, to form a dry lipid film. The lipid film was dried in vacuum for12h. Afterwards, the lipid film was hydrated with a2%(w/v) GS solution at55℃under magnetic stirring to form a homogeneous suspension. The suspension was applied to a probe-type sonicator for homogenization and reduction of the particle size, under different sonicating power and time. For separation of the unencapsulated GS, liposome was ultracentrifugated under60,000g and4℃for2h. The precipitate was re-suspended in an appropriate amount of deionized water. The encapsulation efficiency (EE) of liposome was calculated by determining the unencapsulated GS concentration in the supernatant after ultracentrifugation using an AxSym System, and compared with the initial GS amount added to the formulation. The particle size of GS liposomes prepared above was analyzed in a Nano-ZS zeta sizer at25℃. The liposomes were diluted with distilled water prior to analysis, and each measurement was carried out in triplicate.2. Preparation of liposome combined P-TCP scaffold(LCS).Medical grade P-TCP scaffold (0.5mm granules) was weighted and immersed in GS liposome sample and sonicated in a bath-type sonicator for20min, and further maintained in reduced pressure (0.09MPa) for another20min to allow the liposome permeating into the porous scaffold. Afterwards, samples were transferred into glass bottles and freezedried. For determination of the GS content in LCS, the granules were ground in1%Triton X-100solution and then sonicated for10min to completely release the liposomal GS. The resulting suspension was centrifuged at4000rpm for10min, and the GS content in the supernatant was determined by AxSym System.3.Scanning electron microscopy.The morphology of LCS was examined by scanning electron microscopy (SEM). The freeze-dried scaffolds were fractured, then attached to metal stubs and coated with gold under vacuum. The morphology was observed by a scanning electron microscope.4. In vitro release studyFor evaluating the release of liposomal GS from LCS,0.2g of the granules was introduced into a polyethylene tube with1.0mL of PBS solution (pH7.4) as release medium and kept in a shaking water bath at37℃,30rpm. At designated time intervals, the release medium was withdrawn and replaced with an equal volume of fresh medium. The withdrawn medium was ultracentrifugated under60,000g and4℃for2h. Afterwards the GS content in the supernatant was analyzed by AxSym System. The sediment was added to1.0mL of1%Triton X-100solution and sonicated for10min for liposome rupturing, and then the liposomal GS content was analyzed.5. Bacteriology and biofilm preparationStaphylococcus aureus biofilm was incubated in vitro according to a previously reported method. A strain of Staphylococcus aureus was planted to blood agar plates (composed of brain heart infusion, bacterial agar, defibrinated horse blood, and water). After incubation at37℃overnight, the bacteria were then transferred from the solid agar plate to Falcon tubes containing a nutrient broth (made by mixing brain heart infusion (3.7 g) and yeast extract powder (0.3g) in100mL of water) using a sterile disposable plastic loop. The Falcon tubes were left overnight on an agitator at37℃. The tubes were then centrifuged at2000rpm for10min. The supernatant was removed and the pellet washed with20mL sterile PBS. The process was repeated three times. The bacterial suspension was then diluted in PBS in order to give an optical density (OD) of0.5at550nm. Aliquots (200μL) of the above bacterial suspension were planted into a96-well flat-bottomed microtitre plate and incubated at room temperature for18h. Prior to use the supernatant was removed and the wells were washed three times with PBS.6.Evaluation of anti-biofilm activityLiposomal GS released from LCS were applied to the biofilms to assess its anti-biofilm activity. Briefly, after in vitro release for a certain interval as described earlier, the release medium was withdrawn, ultracentrifugated, and re-suspended to form an appropriate concentration, and aliquots of200μL were incubated with the biofilms for1h and then removed. As control, biofilms were exposed to GS aqueous solution and blank PBS, respectively, at identical overall drug concentrations. After exposure to test samples, the wells were washed with PBS three times to remove the free drug and the bacteria were re-dispersed in liquid growth medium to give an OD value of0.1at550nm. The OD of the bacteria suspension was measured by a DIAS Dynex plate reader under630nm at intervals of30min for an overall period of24h. The time necessary to reach the OD value of0.5(OD0.5) was recorded.7. Observation of biofilm formation with confocal laser scanning microscope.After the slides were cultured in6-well plates bacteria form a biofilm on the coverslip, the GS liposomes composite scaffold β-TCP, free gentamicin and PBS were used24hours after the intervention, cultured for48hours, using FITC-ConA and PI stain biofilm, and then confocal laser scanning microscopy (CLSM) was employed to observe biofilm formation.8.Statistical analysisExperiments were run in triplicate per sample and the data were expressed as means±standard deviation (SD). Statistical analysis was using one-way analysis of variance (ANOVA), using a statistical software SPSS11.0. p-values<0.05were considered to be significant. Main results1. Characterization of gentamicin sulfate liposomeThe particle size and EE of GS liposomes (GL) prepared is shown in Table1. Liposomes with particle size varying from-100nm to5μm were prepared for combination with β-TCP scaffold. The derivation of liposome particle size was obtained under different sonicating conditions, as are shown in Table1. The average particle size of liposome decreased when the sonicating power raised or sonicating time prolonged. The EE of liposome slightly decreased with the decrease in particle size, because the inner aqueous phase of liposomes had a decrease in volume after particle size reduction and thereby the drug loading was reduced. In order to obtain an equivalent drug content, the volume of liposome samples were adjusted using distilled water.2. Characterization of LCSLiposomes with different particle size (samples GL1-5) were combined with P-TCP scaffold to form LCS. The GS content in LCS with different liposome particle size is shown in Figure1. The GS liposome formula GL1-5correspond to LCS1-5, respectively. GS content reached the highest level in the case of LCS3,-7.05mg/g, while GL1resulted in the lowest GS level in LCS1,～4.95mg/g.3. The SEM morphology of LCS before and after combination with GS liposome.Before combination, β-TCP scaffold had a relative smooth surface with micropores of～100μm. In a more magnified view, the specific morphology of the surface was demonstrated. There were ultra-fine pores on the surface of the scaffold. After loading with GS liposome, the morphology of surface of the scaffold changed. It can be observed that liposome particles are adsorbed on the surface of scaffold.4. In vitro GS release from LCSThe release of GS form LCS was mainly in the form of liposome, rather than release as free drug. The cumulative release of free GS was only-5%in24h. This suggests that LCS has the potential to release and transport liposome encapsulated drug to target tissue.For liposomal GS, in the first hour the release profiles of various formulations were similarly in a fast release pattern. Afterward, the release decelerated and showed differences among groups. For comparison of release kinetics of the formulations, the time-release plots of liposomal GS from LCS were fitted by release models.According to correlation coefficient (R2), the release profiles of LCS with different liposome particle size all fitted best to Ritger-Peppas model. For the slab system, the release constant k has the limiting values of0.50in the case of Fickian diffusion. Therefore, the general release of liposomal GS from LCS can be regarded as mostly related to Fickian diffusion. The release constant k had a descending trend with the increase of liposomal particle size.In order to optimize the modeling and give a better understanding to the release mechanism, we selected two representative fractions from the release profile:from0.5-2h and from2-6h, in which two intervals over80%of the drug was released. They were fitted by Ritger-Peppas model respectively, and the results were displayed. In the segmental fitting, both of the two fractions from the release profiles complied with Ritger-Peppas model for each formulation. However, the trend of release constant k was diverse from the case of the gross fitting. In the interval of0.5-2h, the release constant k of the five formulations were similar, and two of them (LCS2and3) exceeded the limiting values of0.50for Fickian diffusion.4. Anti-biofilm activity of LCS evaluationBiofilm matrix polysaccharides Labeled with FITC-ConA, biofilm bacteria,with propidium iodide (PI),the biofilms were observed with the laser scanning confocal microscope. The results showed that compared with the control group, the liposomal gentamicin sulfate composite scaffold can significantly inhibit the formation of biofilm structure and it is effective in killing the bacteria colonization.In order to simulate the different stages of liposomal GS release from LCS, the GS content was ranging from2.5-800μg/mL in the antibiofilm study, and was compared with equivalent free GS solution. Figure7shows the effect of LCS3, which had the highest combination level with liposome GS, on the regrowth of bacteria of the biofilm. Even in the lowest GS concentration, LCS displayed a significant antibacterial activity compared with the control (p<0.01).ConclusionThe porous (3-TCP scaffolds were successfully combined with liposomes containing GS of different particle size. SEM morphology confirmed the complex structure and the binding rate of liposome was high enough to provide a potential drug delivery. The drug release from LCS can be recognized as an initial high dose of liposomal GS from the matrix and a further sustained release of free GS from the liposome, respectively, which is an ideal release pattern for treatment and prevention of post-operative osteomyelitis. The kinetics of the in vitro release showed identical characteristics among formulations and time segments, which is helpful for further understanding of this kind of release system. In the bacterial study, GS-LCS showed notable anti-biofilm activity compared with the free drug, and influences of liposomal particle size were also observed. The further potential of LCS system for anti-biofilm aimed drug delivery and its in vivo evaluation will be investigated in our undergoing studies. |
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