复方奥硝唑/培氟沙星PLGA缓释微球的制备及对慢性牙周炎的药效学研究
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摘要
研究背景
慢性牙周炎是最常见的感染性疾病之一，是造成牙齿缺失的最主要原因。它是由多种牙周致病菌混合感染引起的，在疾病的发生阶段以厌氧菌为主，进入疾病发展期后，兼性厌氧菌也参与其中。这些微生物可以通过释放蛋白水解酶以及有毒代谢产物直接破坏牙周组织，也可以通过刺激宿主细胞产生炎症相关因子来破坏牙周组织。因此，需要抗菌药物配合牙周手术治疗对这些牙周致病菌进行有效清除。
奥硝唑和培氟沙星分别对厌氧菌和兼性厌氧菌抑菌效果明显，两种药物联合用药，可发挥高效抑菌效果。但是长期口服抗生素会造成菌群失调、肝肾损伤等不良反应，而且全身用药无法高效维持局部的药物治疗浓度。因此，局部牙周组织用药是目前牙周病治疗学中的研究热点之一。局部用药需要一个适合的药物载体，乳酸羟基乙酸共聚物（PLGA）作为FDA认证通过的生物可降解高分子聚合物，具有良好的生物相容性，被广泛应用于药物、蛋白以及其他一些大分子载体材料的研发领域，这其中缓释微球由于具有适宜的释药性能以及相对简单的制备方式，是PLGA材料中具有良好应用前景的一种载体形式。
基于此，本研究选取PLGA作为载体材料，经过处方和工艺优化制备出适合于局部牙周组织注射用药的复方奥硝唑/培氟沙星PLGA缓释微球，以期该药物制剂作为牙周手术的辅助手段，在治疗慢性牙周炎方面能够发挥积极的作用。
材料和方法
1.复方奥硝唑/培氟沙星PLGA缓释微球的初步制备
复乳→溶剂挥发法制备复方奥硝唑/培氟沙星PLGA缓释微球并建立体外药物高效液相色谱分析检测方法，在此基础上以包封率作为主要考察指标，对制备装置选择、有机溶剂组合以及外水相PVA浓度进行初步改良。
2.复方奥硝唑/培氟沙星PLGA缓释微球处方与工艺优化
在对复乳→溶剂挥发法进行初步改良的基础上，以包封率作为主要考察指标，应用单因素试验对PLGA的分子量、浓度及PLA/PGA比例，药物与PLGA的质量比，油相中二氯甲烷/乙酸乙酯比，高速剪切机搅拌的转速和时间进行优化。之后再应用3因素3水平正交设计试验，对PLGA材料在有机溶剂中的浓度、有机溶剂中二氯甲烷/乙酸乙酯的比例、剪切速度这三个关键因素进一步进行优化。
3.复方奥硝唑/培氟沙星PLGA缓释微球的干燥与灭菌
针对微球“后优化”阶段中干燥与灭菌这两个重要环节进行研究，比较了低温冷冻干燥法和室温真空干燥法对微球粉体特性、表面形态、包封率以及“突释效应”产生的不同影响，选择出适合的干燥方式；从2、5、10、15kGy四种剂量中，通过微生物检查筛选60Co辐射灭菌的最佳辐射剂量。
4.优化后复方奥硝唑/培氟沙星PLGA缓释微球性能检测
在处方和工艺优化完成之后，对载药微球粉末的粉体学特性，载药微球表面形态学、包封率与载药量，载药微球混悬液pH值，载药微球体外释药情况进行检测，考察其是否适合于牙周组织局部用药。
5.复方奥硝唑/培氟沙星PLGA缓释微球对牙周膜成纤维细胞增殖、矿化的影响
采用组织块培养法培养人牙周膜成纤维细胞，对细胞进行形态学和免疫组化鉴定后，选用第4代细胞作为研究对象，用MTT法和碱性磷酸酶活性测定法考察了1～7d，50mg/ml的载药微球悬液、空白载药微球、5mg/ml的奥硝唑与培氟沙星纯药品组合对细胞增殖活性、细胞毒性以及矿化活性的影响，初步判断载药微球的细胞安全性。
6.复方奥硝唑/培氟沙星PLGA缓释微球体外抑菌实验
体外培养牙周主要致病厌氧菌（牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌、消化链球菌）和兼性厌氧菌（变形链球菌、金黄色葡萄球菌、表皮葡萄球菌）。并分别在1d、2d、3d、4d、5d、6d、8d、10d、12d、14d、16d、18d、20d、22d、24d收集缓释微球释放的药物，采用K-B药物扩散法对不同时间点的抑菌效果进行评价。
7.复方奥硝唑/培氟沙星PLGA缓释微球对大鼠慢性牙周炎的疗效评价
运用结扎法+牙龈沟区域接种牙龈卟啉单胞菌+高糖饮食来诱导大鼠右上颌第一磨牙慢性牙周炎，8周后，通过慢性牙周炎的各项基本临床指标（牙龈出血指数、菌斑指数、牙周袋探诊深度）和龈沟液中天冬氨酸转氨酶含量、牙槽骨丧失度以及牙周组织病理学检查，判断动物模型建立的成功性。在此基础上，以20d作为治疗周期，对奥硝唑全身用药、复方奥硝唑/培氟沙星全身用药、复方奥硝唑/培氟沙星缓释微球局部用药、市售牙康（甲硝唑棒）局部用药作为牙周手术辅助治疗手段的疗效进行对比评价。
结果
1.高效液相色谱分析中，奥硝唑和培氟沙星保留时间分别为10.299min和13.107min，二者分离情况良好，便于检测，两种药物的标准曲线在1～30μg/ml范围内线性关系良好。
2.微球制备方式优化结果：微球制备过程中，容器选择烧杯；搅拌装置选择高速剪切机，剪切时间选择1.5min；有机溶剂选择二氯甲烷/乙酸乙酯=1:2的混合物；PLGA分子量选择25000，PLGA在有机溶剂中的浓度选择200mg/ml，PLA/PGA选择75:25；药脂比选择1:10；外水相中PVA浓度选择3%。
3.低温冷冻干燥处理的微球粉末流动性好、无粘连，室温真空干燥的微球粉末则出现结块、粘连现象。室温真空干燥处理的微球，“突释效应”明显增加且包封率低。经过60Co-辐射灭菌的微球，2kGy辐射剂量达不到完全的灭菌效果，5、10、15kGy辐射剂量均能达到良好的灭菌效果，检测期内未出现任何微生物生长。
4.优化后制备的微球呈白色粉末，粉末的圆整度为35.99±1.03°，休止角为36.1±2.20°，堆密度为0.3804±0.0311g/ml。光镜与扫描电镜下微球成球性良好，光滑、粘连状态较少、分散性良好。微球粒径正态分布于14～30μm，均值为：21.60±2.41μm。奥硝唑和培氟沙星的包封率分别为68.15±0.40%和64.07±0.37%，载药量分别为6.18±0.15%和3.95±0.21%。微球在1%生理盐水、5%葡萄糖和纯水中的pH值分别为7.01±0.03、4.91±0.27、5.57±0.16，存在差异性。24h时，奥硝唑体外释放度达25%左右，培氟沙星达35%以上，存在“突释效应”；随时间推移，微球释药速率基本保持稳定。第20d，奥硝唑体外释放度达95%左右，培氟沙星达到97%以上。
5.体外培养的牙周膜成纤维细胞来自中胚层，状态良好。各时间点，不同处理组之间细胞的增殖活性未出现明显变化，载药微球组细胞毒性处于0级（绝对安全级别），不同处理组的碱性磷酸酶活性未出现明显变化。
6.缓释微球体外抑菌实验表明：整个检测时间段内药物制剂均显示出有效抑菌活性，第1d的抑菌效果最为明显，2～20d药物的抑菌效果维持稳定，20d之后抑菌活性明显下降。
7.慢性牙周炎动物模型诱导8周后，牙龈出血指数、菌斑指数、牙周袋探诊深度、龈沟液天冬氨酸转氨酶、牙槽骨丧失度与正常大鼠相比均有明显变化，实验区牙周组织病理学检测显示出明显的炎症细胞浸润、结合上皮根方迁移及牙骨质破坏。经20d治疗，与慢性牙周炎组相比，各治疗组牙周组织临床指标均显示出了明显的改善，组织病理学检查显示：各治疗组中炎症细胞的浸润程度均受到抑制，牙骨质获得一定程度的恢复，其中局部缓释微球组显示了最为显著的疗效。
结论
1.处方和工艺优化后，复乳→溶剂挥发法制备的复方奥硝唑/培氟沙星缓释微球流动性、成球性、粒径分布、包封率以及体外缓释性能均比较良好，适合于牙周组织局部注射用药。微球制备完成后，适合用低温冷冻干燥以及5kGy60Co辐射灭菌后保存。
2.载药微球没有对细胞的增殖和矿化活性产生明显影响，本剂型中的载体材料、药物及相关辅剂成分在此浓度下对细胞无毒性作用。
3.体外，载药微球在20d内对牙周主要致病菌有明显的抑菌效果。
4.本实验牙周炎动物模型的建立是成功的，本研究开发的局部复方奥硝唑/培氟沙星PLGA缓释微球作为牙周手术的辅助治疗手段，在治疗慢性牙周炎方面具有一定优势。
BACKGROUD
Periodontitis is one of the most common infections and is a major cause of tooth loss.It is a polymicrobial infection, and thus, the complex interaction among microorganismsmakes the disease a challenging one to understand and treat. These pathogens includeanaerobes, such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacteriumnudeatum and Peptostreptococcus anaerobiu, as well as facultative anaerobes, includingStaphylococcus aureus and Staphylococcus epidermidis, among others. Bacterial plaque isrecognized as the primary agent for the initiation and progression of periodontitis. Plaquemicroorganisms can damage the periodontium by releasing their proteolytic and noxiouswaste products and by stimulating the host cells to produce pro-inflammatory cytokines,inducing connective tissue and alveolar bone destruction.
Therefore, a successful treatment depends on the elimination or control of thepathogens together with a microbial shift toward a microbial population, which is typicallypresent in healthy individuals. Treatments include mechanical and drug therapy. Mechanicaltherapy involves nonsurgical scaling and root planing (SRP) and drug therapy is adjunctiveto SRP.
Drug therapy includes local and systemic administration. Although systemicadministration has some benefits, an inadequate concentration in the periodontium and ahigh plasma concentration may be associated with bacterial resistance and side effects. Animportant advantage of local administration is the higher therapeutic concentration in thelesion location, leaving residual parts unaffected.
Ornidazole is a member of the nitromidazoles and is widely used in the treatment ofthe oral disease, which have better activity against anaerobes than quinolones. Pefloxacinmesylate is a member of the quinolones, and these have better activity against facultative anaerobes than nitromidazoles. As previously mentioned, anaerobe is dominant microbialpopulation, but facultative anaerobe also plays a part in break down of deep periodontaltissues.
The use of PLGA as a biodegradable polymer in microsphere production is common inthe literature due to its attractive properties, including the availability of variousco-polymer compositions and molecular weights, which makes the manufacture ofmicrospheres with tailored characteristics accessible.
So, the aim of this study can be summarized as preparation of ornidazole/pefloxacin-loaded PLGA microspheres with optimized characteristics and in vivo evaluation of t thepharmacological effect on chronic periodontitis.
MATERIALS AND METHODS
1. Preliminary preparation of compound ornidazole/pefloxacin PLGA sustained releasemicrospheres
Double emulsion→solvent evaporation method was used to prepare the microspheres,high performance liquid chromatographic analysis (HPLC) was used to measure thedrug content. With the encapsulation efficiency as the main investigation index,preparation device, organic solvents combination, PVA concentration in external waterphase was preliminary optimized.
2. Prescription and process optimization of compound ornidazole/pefloxacin PLGAsustained release microspheres
On the basis of preliminary improvement, with the encapsulation efficiency as themain investigation index, preparation device, the molecular weight and concentration ofPLGA, proportion of PLA/PGA, the proportion of drugs/PLGA, the proportion ofmethylene chloride/ethyl acetate in oil phase, stirring speed and time of high-speed shearmachine were optimized through single factor experiment. And then, the concentration ofPLGA, the proportion of methylene chloride/ethyl acetate in oil phase, and the stirring timeof high-speed shear machine were futher optimized through orthogonal test on3factors3levels.
3. Drying and sterilization
Powder traits, surface morphology, encapsulation efficiency and burst release effect ofthe microspheres were investigated and compared under freezing drying and room temperature vacuum drying methods. Through the microbial inspection, the best radiationsterilization dose of60Co was screened from2,5,10,15kGy.
4. Performance testing of compound ornidazole/pefloxacin PLGA sustained releasemicrospheres after optimization
In order to determinate wheather pharmaceutical preparation is suitable for localadministration for periodontitis, the powder traits, surface morphology, encapsulationefficiency, pH value of suspension liquid drugs-release trait in vitro of the microsphereswere investigated after optimization.
5. Impacts of the microspheres in vitro on proliferation, mineralization of periodontalligamnent fibroblasts
Periodontal ligamnent fibroblasts were cultured by tissue culture method,cell wasidentificated through morphology and immunohistochemistry. In order to judge cell safetyof the Pharmaceutical preparation, proliferation activity, and cell toxicity andmineralization activity in50mg/ml microspheres, blank microspheres, and5mg/ml ofornidazole/pefloxacin in1~7d were investigated through MTT method and alkalinephosphatase activity determination method.
6. Antibacterial experiment of the microspheres in vitro
Anaerobes (Porphyromonas gingivalis, Prevotella intermedia, Fusobacteriumnudeatum and Peptostreptococcus anaerobiu), afacultative anaerobes (Staphylococcusaureus and Staphylococcus epidermidis) were cultured in vitro. Drugs released fromsustained release microspheres were collected at1d,2d,3d,4d,5d,6d,8d,10d,12d,14d,16d,18d,20d,22d and24d. K-B method was used to evaluate the antibacterial effect atdifferent time points.
7. Effects of the microspheres on experimental periodontitis in rats
Periodontitis was induced in rats by placing a thin-steel ligature around the upper firstmolars and inoculating them with Porphyromonas gingivalis381. After8weeks, SBI, PI,PPD, ABL, AST-GCF and histopathology were used to evaluate the success of the rat ECP.After the successful induction, effects of systemic ornidazole, systemic compoundornidazole, local sustained release microspheres and local YAKANG (metronidazole) onexperimental periodontitis in rats were evaluated.
RESULTS
1. In HPLC, the retention time of ornidazole and pefloxacin respectively is10.299minand13.107min, the separation in good condition. The linear relation between peak area andconcentration of drug was good in1~30μ g/ml range.
2. Optimization results: the container was beaker, the stirring device was high-speedshear machine, the stirring time was1.5min, methylene chloride/ethyl acetate was1:2,PLGA molecular weight was25000, the concentration of PLGA in organic solvent was200mg/ml, LA/GA was75:25, drug/PLGA was1:10, the concentration of PVA in externalwater phase was3%.
3. After freeze-drying processing the powder of microspheres was in good liquiditywithout adhesion, however, after room temperature vacuum drying processing the powderappeared agglomerate and adhesion phenomenon. After room temperature vacuum dryingprocessing, the burst release effect increased obviously and the encapsulation efficiency islow.2kGy60Co could not achieve sterilization thoroughly,5,10,15kGy60Co could allachieve sterilization thoroughly.
4. After optimization, color of powder was white, roundness degrees of powder were35.99±1.03°, repose angle of powder was36.1±2.20°, and bulk density of powder was0.3804±0.0311g/ml. Under Light and scanning electron microscopy, the microspheres weresmooth, in good balling index and in lesss adhesion state. After screening throughmolecular sieve, the particle sizes of microspheres were in normal distribution (14~30μm); the mean value was21.60±2.41μm. the encapsulation efficiency of ornidazole andpefloxacin was68.15±0.40%and64.07±0.37%respectively, drug loadings was6.18±0.15%and3.95±0.21%respectively. pH value of Microspheres in1%physiological saline,5%glucose and pure water was7.01±0.03、4.91±0.27、5.57±0.16respectively.At24h,ornidazole in vitro released about25%by degrees, pefloxacin released about35%. Theburst release effects existed. Over time, the drug release rate remained stable.At20d,ornidazole in vitro released about95%by degrees, pefloxacin released above97%.
5. Periodontal ligamnent fibroblasts cultured in vitro were from mesoderm and were ingood condition. At different time point, there was no obvious change between differenttreatment groups on cell proliferation, there was no cell toxicity in microspheres, and there wasno obvious change between different treatment groups on cell alkaline phosphatase activity.
6. The antibacterial experiment of sustained release microspheres in vitro showed thatduring the whole experiment time, the pharmaceutical preparation could have effectiveantibacterial activity; at the fist day, the bacteriostatic effect was the strongest; during2~20d,the bacteriostatic effect maintain stable; after20d the bacteriostatic effect decreasedobviously.
7. After8weeks’ induction, the periodontal health indicators in periodontitis rats, suchas SBI, PI and PPD, GCF-AST, ABL showed statistically significant differences comparedwith normal rats.Histopathology observation in ECP rats revealed inflammatory cellinfiltration, apical migration of the junctional epithelium, severe cementum destructionresembling a periodontitis lesion. The disorders of these indicators indicated the success ofperiodontitis mode. After20days treatment, compared with ECP, all the combinedtreatments produced beneficial changes in the clinical indicators.The histopathology of theperiodontium revealed a significant reduction in the numbers of inflammatory cellinfiltration as well as in the inhibition of destruction of cementum. Among these treatmentgroups, the Local sustained release microspheres gruop is most effective.
CONCLUSIONS
1. After optimization, the microspheres were smooth, in good balling index and inlesss adhesion state. The encapsulation efficiency, drug loadings and release activity in vitroof the microspheres were in good condition. The pharmaceutical preparation was suitablefor local injection treatment of periodontitis.
2. The microspheres exerted no obvious influence on the proliferation andmineralization of periodontal ligamnent fibroblasts. The pharmaceutical preparation exertedno toxicity on periodontal ligamnent fibroblasts.
3. The pharmaceutical preparation had obvious bacteriostatic effect to the mainpathogenic peiodontitis bacteria in20days in vitro.
4. A rat model of peiodontiti, which can provide an important basis for follow-uptreatment experiments, was successfully established. In the treatment of chronicperiodontitis, the pharmaceutical preparation (microspheres) was adjuvant to periodontalinstruments, has certain advantages.
慢性牙周炎是最常见的感染性疾病之一，是造成牙齿缺失的最主要原因。它是由多种牙周致病菌混合感染引起的，在疾病的发生阶段以厌氧菌为主，进入疾病发展期后，兼性厌氧菌也参与其中。这些微生物可以通过释放蛋白水解酶以及有毒代谢产物直接破坏牙周组织，也可以通过刺激宿主细胞产生炎症相关因子来破坏牙周组织。因此，需要抗菌药物配合牙周手术治疗对这些牙周致病菌进行有效清除。
奥硝唑和培氟沙星分别对厌氧菌和兼性厌氧菌抑菌效果明显，两种药物联合用药，可发挥高效抑菌效果。但是长期口服抗生素会造成菌群失调、肝肾损伤等不良反应，而且全身用药无法高效维持局部的药物治疗浓度。因此，局部牙周组织用药是目前牙周病治疗学中的研究热点之一。局部用药需要一个适合的药物载体，乳酸羟基乙酸共聚物（PLGA）作为FDA认证通过的生物可降解高分子聚合物，具有良好的生物相容性，被广泛应用于药物、蛋白以及其他一些大分子载体材料的研发领域，这其中缓释微球由于具有适宜的释药性能以及相对简单的制备方式，是PLGA材料中具有良好应用前景的一种载体形式。
基于此，本研究选取PLGA作为载体材料，经过处方和工艺优化制备出适合于局部牙周组织注射用药的复方奥硝唑/培氟沙星PLGA缓释微球，以期该药物制剂作为牙周手术的辅助手段，在治疗慢性牙周炎方面能够发挥积极的作用。
材料和方法
1.复方奥硝唑/培氟沙星PLGA缓释微球的初步制备
复乳→溶剂挥发法制备复方奥硝唑/培氟沙星PLGA缓释微球并建立体外药物高效液相色谱分析检测方法，在此基础上以包封率作为主要考察指标，对制备装置选择、有机溶剂组合以及外水相PVA浓度进行初步改良。
2.复方奥硝唑/培氟沙星PLGA缓释微球处方与工艺优化
在对复乳→溶剂挥发法进行初步改良的基础上，以包封率作为主要考察指标，应用单因素试验对PLGA的分子量、浓度及PLA/PGA比例，药物与PLGA的质量比，油相中二氯甲烷/乙酸乙酯比，高速剪切机搅拌的转速和时间进行优化。之后再应用3因素3水平正交设计试验，对PLGA材料在有机溶剂中的浓度、有机溶剂中二氯甲烷/乙酸乙酯的比例、剪切速度这三个关键因素进一步进行优化。
3.复方奥硝唑/培氟沙星PLGA缓释微球的干燥与灭菌
针对微球“后优化”阶段中干燥与灭菌这两个重要环节进行研究，比较了低温冷冻干燥法和室温真空干燥法对微球粉体特性、表面形态、包封率以及“突释效应”产生的不同影响，选择出适合的干燥方式；从2、5、10、15kGy四种剂量中，通过微生物检查筛选60Co辐射灭菌的最佳辐射剂量。
4.优化后复方奥硝唑/培氟沙星PLGA缓释微球性能检测
在处方和工艺优化完成之后，对载药微球粉末的粉体学特性，载药微球表面形态学、包封率与载药量，载药微球混悬液pH值，载药微球体外释药情况进行检测，考察其是否适合于牙周组织局部用药。
5.复方奥硝唑/培氟沙星PLGA缓释微球对牙周膜成纤维细胞增殖、矿化的影响
采用组织块培养法培养人牙周膜成纤维细胞，对细胞进行形态学和免疫组化鉴定后，选用第4代细胞作为研究对象，用MTT法和碱性磷酸酶活性测定法考察了1～7d，50mg/ml的载药微球悬液、空白载药微球、5mg/ml的奥硝唑与培氟沙星纯药品组合对细胞增殖活性、细胞毒性以及矿化活性的影响，初步判断载药微球的细胞安全性。
6.复方奥硝唑/培氟沙星PLGA缓释微球体外抑菌实验
体外培养牙周主要致病厌氧菌（牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌、消化链球菌）和兼性厌氧菌（变形链球菌、金黄色葡萄球菌、表皮葡萄球菌）。并分别在1d、2d、3d、4d、5d、6d、8d、10d、12d、14d、16d、18d、20d、22d、24d收集缓释微球释放的药物，采用K-B药物扩散法对不同时间点的抑菌效果进行评价。
7.复方奥硝唑/培氟沙星PLGA缓释微球对大鼠慢性牙周炎的疗效评价
运用结扎法+牙龈沟区域接种牙龈卟啉单胞菌+高糖饮食来诱导大鼠右上颌第一磨牙慢性牙周炎，8周后，通过慢性牙周炎的各项基本临床指标（牙龈出血指数、菌斑指数、牙周袋探诊深度）和龈沟液中天冬氨酸转氨酶含量、牙槽骨丧失度以及牙周组织病理学检查，判断动物模型建立的成功性。在此基础上，以20d作为治疗周期，对奥硝唑全身用药、复方奥硝唑/培氟沙星全身用药、复方奥硝唑/培氟沙星缓释微球局部用药、市售牙康（甲硝唑棒）局部用药作为牙周手术辅助治疗手段的疗效进行对比评价。
结果
1.高效液相色谱分析中，奥硝唑和培氟沙星保留时间分别为10.299min和13.107min，二者分离情况良好，便于检测，两种药物的标准曲线在1～30μg/ml范围内线性关系良好。
2.微球制备方式优化结果：微球制备过程中，容器选择烧杯；搅拌装置选择高速剪切机，剪切时间选择1.5min；有机溶剂选择二氯甲烷/乙酸乙酯=1:2的混合物；PLGA分子量选择25000，PLGA在有机溶剂中的浓度选择200mg/ml，PLA/PGA选择75:25；药脂比选择1:10；外水相中PVA浓度选择3%。
3.低温冷冻干燥处理的微球粉末流动性好、无粘连，室温真空干燥的微球粉末则出现结块、粘连现象。室温真空干燥处理的微球，“突释效应”明显增加且包封率低。经过60Co-辐射灭菌的微球，2kGy辐射剂量达不到完全的灭菌效果，5、10、15kGy辐射剂量均能达到良好的灭菌效果，检测期内未出现任何微生物生长。
4.优化后制备的微球呈白色粉末，粉末的圆整度为35.99±1.03°，休止角为36.1±2.20°，堆密度为0.3804±0.0311g/ml。光镜与扫描电镜下微球成球性良好，光滑、粘连状态较少、分散性良好。微球粒径正态分布于14～30μm，均值为：21.60±2.41μm。奥硝唑和培氟沙星的包封率分别为68.15±0.40%和64.07±0.37%，载药量分别为6.18±0.15%和3.95±0.21%。微球在1%生理盐水、5%葡萄糖和纯水中的pH值分别为7.01±0.03、4.91±0.27、5.57±0.16，存在差异性。24h时，奥硝唑体外释放度达25%左右，培氟沙星达35%以上，存在“突释效应”；随时间推移，微球释药速率基本保持稳定。第20d，奥硝唑体外释放度达95%左右，培氟沙星达到97%以上。
5.体外培养的牙周膜成纤维细胞来自中胚层，状态良好。各时间点，不同处理组之间细胞的增殖活性未出现明显变化，载药微球组细胞毒性处于0级（绝对安全级别），不同处理组的碱性磷酸酶活性未出现明显变化。
6.缓释微球体外抑菌实验表明：整个检测时间段内药物制剂均显示出有效抑菌活性，第1d的抑菌效果最为明显，2～20d药物的抑菌效果维持稳定，20d之后抑菌活性明显下降。
7.慢性牙周炎动物模型诱导8周后，牙龈出血指数、菌斑指数、牙周袋探诊深度、龈沟液天冬氨酸转氨酶、牙槽骨丧失度与正常大鼠相比均有明显变化，实验区牙周组织病理学检测显示出明显的炎症细胞浸润、结合上皮根方迁移及牙骨质破坏。经20d治疗，与慢性牙周炎组相比，各治疗组牙周组织临床指标均显示出了明显的改善，组织病理学检查显示：各治疗组中炎症细胞的浸润程度均受到抑制，牙骨质获得一定程度的恢复，其中局部缓释微球组显示了最为显著的疗效。
结论
1.处方和工艺优化后，复乳→溶剂挥发法制备的复方奥硝唑/培氟沙星缓释微球流动性、成球性、粒径分布、包封率以及体外缓释性能均比较良好，适合于牙周组织局部注射用药。微球制备完成后，适合用低温冷冻干燥以及5kGy60Co辐射灭菌后保存。
2.载药微球没有对细胞的增殖和矿化活性产生明显影响，本剂型中的载体材料、药物及相关辅剂成分在此浓度下对细胞无毒性作用。
3.体外，载药微球在20d内对牙周主要致病菌有明显的抑菌效果。
4.本实验牙周炎动物模型的建立是成功的，本研究开发的局部复方奥硝唑/培氟沙星PLGA缓释微球作为牙周手术的辅助治疗手段，在治疗慢性牙周炎方面具有一定优势。
BACKGROUD
Periodontitis is one of the most common infections and is a major cause of tooth loss.It is a polymicrobial infection, and thus, the complex interaction among microorganismsmakes the disease a challenging one to understand and treat. These pathogens includeanaerobes, such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacteriumnudeatum and Peptostreptococcus anaerobiu, as well as facultative anaerobes, includingStaphylococcus aureus and Staphylococcus epidermidis, among others. Bacterial plaque isrecognized as the primary agent for the initiation and progression of periodontitis. Plaquemicroorganisms can damage the periodontium by releasing their proteolytic and noxiouswaste products and by stimulating the host cells to produce pro-inflammatory cytokines,inducing connective tissue and alveolar bone destruction.
Therefore, a successful treatment depends on the elimination or control of thepathogens together with a microbial shift toward a microbial population, which is typicallypresent in healthy individuals. Treatments include mechanical and drug therapy. Mechanicaltherapy involves nonsurgical scaling and root planing (SRP) and drug therapy is adjunctiveto SRP.
Drug therapy includes local and systemic administration. Although systemicadministration has some benefits, an inadequate concentration in the periodontium and ahigh plasma concentration may be associated with bacterial resistance and side effects. Animportant advantage of local administration is the higher therapeutic concentration in thelesion location, leaving residual parts unaffected.
Ornidazole is a member of the nitromidazoles and is widely used in the treatment ofthe oral disease, which have better activity against anaerobes than quinolones. Pefloxacinmesylate is a member of the quinolones, and these have better activity against facultative anaerobes than nitromidazoles. As previously mentioned, anaerobe is dominant microbialpopulation, but facultative anaerobe also plays a part in break down of deep periodontaltissues.
The use of PLGA as a biodegradable polymer in microsphere production is common inthe literature due to its attractive properties, including the availability of variousco-polymer compositions and molecular weights, which makes the manufacture ofmicrospheres with tailored characteristics accessible.
So, the aim of this study can be summarized as preparation of ornidazole/pefloxacin-loaded PLGA microspheres with optimized characteristics and in vivo evaluation of t thepharmacological effect on chronic periodontitis.
MATERIALS AND METHODS
1. Preliminary preparation of compound ornidazole/pefloxacin PLGA sustained releasemicrospheres
Double emulsion→solvent evaporation method was used to prepare the microspheres,high performance liquid chromatographic analysis (HPLC) was used to measure thedrug content. With the encapsulation efficiency as the main investigation index,preparation device, organic solvents combination, PVA concentration in external waterphase was preliminary optimized.
2. Prescription and process optimization of compound ornidazole/pefloxacin PLGAsustained release microspheres
On the basis of preliminary improvement, with the encapsulation efficiency as themain investigation index, preparation device, the molecular weight and concentration ofPLGA, proportion of PLA/PGA, the proportion of drugs/PLGA, the proportion ofmethylene chloride/ethyl acetate in oil phase, stirring speed and time of high-speed shearmachine were optimized through single factor experiment. And then, the concentration ofPLGA, the proportion of methylene chloride/ethyl acetate in oil phase, and the stirring timeof high-speed shear machine were futher optimized through orthogonal test on3factors3levels.
3. Drying and sterilization
Powder traits, surface morphology, encapsulation efficiency and burst release effect ofthe microspheres were investigated and compared under freezing drying and room temperature vacuum drying methods. Through the microbial inspection, the best radiationsterilization dose of60Co was screened from2,5,10,15kGy.
4. Performance testing of compound ornidazole/pefloxacin PLGA sustained releasemicrospheres after optimization
In order to determinate wheather pharmaceutical preparation is suitable for localadministration for periodontitis, the powder traits, surface morphology, encapsulationefficiency, pH value of suspension liquid drugs-release trait in vitro of the microsphereswere investigated after optimization.
5. Impacts of the microspheres in vitro on proliferation, mineralization of periodontalligamnent fibroblasts
Periodontal ligamnent fibroblasts were cultured by tissue culture method,cell wasidentificated through morphology and immunohistochemistry. In order to judge cell safetyof the Pharmaceutical preparation, proliferation activity, and cell toxicity andmineralization activity in50mg/ml microspheres, blank microspheres, and5mg/ml ofornidazole/pefloxacin in1~7d were investigated through MTT method and alkalinephosphatase activity determination method.
6. Antibacterial experiment of the microspheres in vitro
Anaerobes (Porphyromonas gingivalis, Prevotella intermedia, Fusobacteriumnudeatum and Peptostreptococcus anaerobiu), afacultative anaerobes (Staphylococcusaureus and Staphylococcus epidermidis) were cultured in vitro. Drugs released fromsustained release microspheres were collected at1d,2d,3d,4d,5d,6d,8d,10d,12d,14d,16d,18d,20d,22d and24d. K-B method was used to evaluate the antibacterial effect atdifferent time points.
7. Effects of the microspheres on experimental periodontitis in rats
Periodontitis was induced in rats by placing a thin-steel ligature around the upper firstmolars and inoculating them with Porphyromonas gingivalis381. After8weeks, SBI, PI,PPD, ABL, AST-GCF and histopathology were used to evaluate the success of the rat ECP.After the successful induction, effects of systemic ornidazole, systemic compoundornidazole, local sustained release microspheres and local YAKANG (metronidazole) onexperimental periodontitis in rats were evaluated.
RESULTS
1. In HPLC, the retention time of ornidazole and pefloxacin respectively is10.299minand13.107min, the separation in good condition. The linear relation between peak area andconcentration of drug was good in1~30μ g/ml range.
2. Optimization results: the container was beaker, the stirring device was high-speedshear machine, the stirring time was1.5min, methylene chloride/ethyl acetate was1:2,PLGA molecular weight was25000, the concentration of PLGA in organic solvent was200mg/ml, LA/GA was75:25, drug/PLGA was1:10, the concentration of PVA in externalwater phase was3%.
3. After freeze-drying processing the powder of microspheres was in good liquiditywithout adhesion, however, after room temperature vacuum drying processing the powderappeared agglomerate and adhesion phenomenon. After room temperature vacuum dryingprocessing, the burst release effect increased obviously and the encapsulation efficiency islow.2kGy60Co could not achieve sterilization thoroughly,5,10,15kGy60Co could allachieve sterilization thoroughly.
4. After optimization, color of powder was white, roundness degrees of powder were35.99±1.03°, repose angle of powder was36.1±2.20°, and bulk density of powder was0.3804±0.0311g/ml. Under Light and scanning electron microscopy, the microspheres weresmooth, in good balling index and in lesss adhesion state. After screening throughmolecular sieve, the particle sizes of microspheres were in normal distribution (14~30μm); the mean value was21.60±2.41μm. the encapsulation efficiency of ornidazole andpefloxacin was68.15±0.40%and64.07±0.37%respectively, drug loadings was6.18±0.15%and3.95±0.21%respectively. pH value of Microspheres in1%physiological saline,5%glucose and pure water was7.01±0.03、4.91±0.27、5.57±0.16respectively.At24h,ornidazole in vitro released about25%by degrees, pefloxacin released about35%. Theburst release effects existed. Over time, the drug release rate remained stable.At20d,ornidazole in vitro released about95%by degrees, pefloxacin released above97%.
5. Periodontal ligamnent fibroblasts cultured in vitro were from mesoderm and were ingood condition. At different time point, there was no obvious change between differenttreatment groups on cell proliferation, there was no cell toxicity in microspheres, and there wasno obvious change between different treatment groups on cell alkaline phosphatase activity.
6. The antibacterial experiment of sustained release microspheres in vitro showed thatduring the whole experiment time, the pharmaceutical preparation could have effectiveantibacterial activity; at the fist day, the bacteriostatic effect was the strongest; during2~20d,the bacteriostatic effect maintain stable; after20d the bacteriostatic effect decreasedobviously.
7. After8weeks’ induction, the periodontal health indicators in periodontitis rats, suchas SBI, PI and PPD, GCF-AST, ABL showed statistically significant differences comparedwith normal rats.Histopathology observation in ECP rats revealed inflammatory cellinfiltration, apical migration of the junctional epithelium, severe cementum destructionresembling a periodontitis lesion. The disorders of these indicators indicated the success ofperiodontitis mode. After20days treatment, compared with ECP, all the combinedtreatments produced beneficial changes in the clinical indicators.The histopathology of theperiodontium revealed a significant reduction in the numbers of inflammatory cellinfiltration as well as in the inhibition of destruction of cementum. Among these treatmentgroups, the Local sustained release microspheres gruop is most effective.
CONCLUSIONS
1. After optimization, the microspheres were smooth, in good balling index and inlesss adhesion state. The encapsulation efficiency, drug loadings and release activity in vitroof the microspheres were in good condition. The pharmaceutical preparation was suitablefor local injection treatment of periodontitis.
2. The microspheres exerted no obvious influence on the proliferation andmineralization of periodontal ligamnent fibroblasts. The pharmaceutical preparation exertedno toxicity on periodontal ligamnent fibroblasts.
3. The pharmaceutical preparation had obvious bacteriostatic effect to the mainpathogenic peiodontitis bacteria in20days in vitro.
4. A rat model of peiodontiti, which can provide an important basis for follow-uptreatment experiments, was successfully established. In the treatment of chronicperiodontitis, the pharmaceutical preparation (microspheres) was adjuvant to periodontalinstruments, has certain advantages.
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