摘要
制备盐酸米诺环素微球贮库,并评价其体外缓释效果及理化性质。以聚乳酸-羟基乙酸共聚物[poly (lactic-co-glycolic acid),PLGA] 为原材料,采用静电喷雾的方法制备得到盐酸米诺环素微球,并通过偏振光显微镜以及扫描电镜对微球形态大小进行表征,将其与蔗糖乙酸酯异丁酸酯(sucrose acetate isobutyrate,SAIB)原位贮库以1∶10的比例混合形成盐酸米诺环素微球贮库,并考察其体外释放性能及孔隙率变化。结果表明,盐酸米诺环素微球表面光滑,直径(5.294±1.222)μm。盐酸米诺环素微球与SAIB混合形成盐酸米诺环素微球贮库后,盐酸米诺环素的突释大幅度降低,在第1天从60%降至3.27%并持续释放至第42天。同时结果表明该贮库在0~15 d孔隙率由(12.53±0.43)%迅速增加到(32.53±0.43)%,15~45 d孔隙率由(32.53±0.43)%缓慢增加到(33.81±0.54)%。本研究制备的盐酸米诺环素微球贮库制备工艺简单、释药性能良好,有望成为盐酸米诺环素给药的一种有效途径。
慢性牙周炎是最常见的口腔疾病之一,其导致的持续牙槽骨丢失将引起牙齿松
蔗糖醋酸盐异丁酸酯(sucrose acetate isobutyrate,SAIB)贮库是一种很有前景的注射型缓释系
聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA,n(LA)∶n(GA)=75∶25,济南岱港生物材料有限公司;相对分子质量80 kD];盐酸米诺环素标准品、蔗糖醋酸盐异丁酸酯(SAIB,相对分子质量846.91,1.146 g/mL)(美国Sigma公司)。其他试剂均为市售分析纯。
将PLGA 溶于氯仿,得到质量分数为8%的PLGA溶液,然后将盐酸米诺环素(相对于PLGA的质量分数为0%,2%)加入PLGA溶液中,用磁力搅拌器恒温搅拌2 h充分混合。在室温下,将所配置的溶液分别装入21号不锈钢钝性针头的5 mL注射器,针头与高压直流电源的正极相连,与针头相距20 cm的接收滚筒上接负极,滚筒上盖着铝箔纸,作为微球的接收器,制备得到空白微球(M)以及盐酸米诺环素微球(minocycline hydrochloride microsphere,MH-M)。制备微球的电喷参数如下:8% PLGA溶液在14 kV的高压电场中,以0.25 mL/h 的推注速度,相对湿度控制在31%~33%,温度控制在21~23 ℃。铝箔纸上覆盖载玻片收集微球,然后置于倒置相差显微镜下观察其形态。电喷结束后,铝箔纸置于恒温箱中干燥48 h,挥发残留的有机溶剂,收集到的微球转移至小玻璃瓶于-20 ℃保存。
收集到微球的铝箔纸剪成1 cm×1 cm,固定在样品台上,旋转蒸镀仪喷金,使用扫描电镜(SEM)进行观察;载玻片收集到的微球在偏振光显微镜下观察,用ImageJ软件测量微球直径并计数(每个样本超过100个),结果以表示;微球直径的单分散性即粒径分布一致性,用变异系数CV(%),CV越低表明微球单分散性即粒径分布一致性越佳。
针对包封率测定,用超速离心法测定微球表面游离MH的含量。称取微球5 mg,精确到0.1 mg,溶于PBS溶液1 mL中,混匀形成样品溶液,转移到1.5 mL超速离心管中,然后在13 000 r/min离心10 min。提取上清液,利用酶标仪在350 nm波长处分析。重复3次,计算包封率。
针对载药量的测定,通过将微球5 mg溶解在无水乙醇1 mL中,超声处理10 min后直至样品中聚合物完全溶解,然后在15 000 r/min离心20 min。最后提取上清液并利用酶标仪在350 nm波长处分析,从而来测定球体中的MH含量。重复3次,计算载药量。2.2.4 盐酸米诺环素微球的药物分配 盐酸米诺环素属于第二代半合成的四环素族药物,具有在一定条件下能够被激发出绿色荧光的特性。因此,将电喷微球样品置于玻片上,在激光扫描共聚焦显微镜下,使用375 nm的激发波长进行观察。
将SAIB与EtOH混合得到透明的SAIB-EtOH溶液(SAIB质量分数为80%)。使用前,将盐酸米诺环素0.1 mg分散在上述SAIB-EtOH系统中以产生盐酸米诺环素/SAIB(MH-SAIB)贮库。类似地,将盐酸米诺环素微球5 mg在SAIB-EtOH溶液50 mg中旋转5 min均匀混合,以获得盐酸米诺环素微球/SAIB(MH-M-SAIB)混合贮库(约含盐酸米诺环素0.1 mg)。
取盐酸米诺环素微球5 mg(约含盐酸米诺环素0.1 mg)以及MH-M-SAIB(约含盐酸米诺环素0.1 mg) 50 mg分别加入含有PBS溶液1 mL的1.5 mL EP管(pH 7.4, 0.02% NaN3)。所有的样本置于摇床中恒温37 ℃水浴。于定点时间取样,将各样本溶液进行13 000 r/min离心10 min后取上清液,采用酶标仪测量药物吸收度。所有的实验重复3次。
体外降解率是通过测定微球在PBS中浸泡过程中的失重进行测
将SAIB溶液注入水相,外水迅速扩散到贮库中,而溶剂则从基质中扩散到水相中。这导致富水区域的多孔网络的形成,周围是一个富含SAIB的系统。孔隙率是通过测试扩散到贮库的液体的体积来确定的。将约分析样品50 mg注入PBS 1.5 mL中,并置于37 ℃恒温箱中。在特定时间(2,8,15,30,45 d)取PBS。然后,贮库被冻干以除去吸收的液体。通过测量冻干前后的水位差,可以方便地确定贮库吸收的液体量。因此,孔隙率可用
(1) |
式中:p为孔隙率,W1为SAIB溶液的质量,W2为吸收液体后贮库的质量,W3为贮库冻干后的质量,c为SAIB溶液中SAIB的浓度,ρ1为液体的密度,ρ2为SAIB的密度。每种溶液的试验重复3次。

Figure 1 Characterization of minocycline hydrochloride microspheres(MH-M)
A: Optical microscope image; B: SEM image; C: Contact angle; D:Diameter distribution
接触角测试是材料亲水性能表征的重要指标之一,经测量微球接触角为(95.98 ± 0.67)°(

Figure 2 Laser confocal microscopy of MH-M
盐酸米诺环素微球的体外释放曲线如

Figure 3 Release curves of MH-M(A) and sucrose acetate isobutyrate(SAIB)-based depot MH-SAIB and MH-M-SAIB (B) ()
MH-M-SAIB和MH-SAIB贮库在PBS培养基中的体外药物释放曲线如
aSlope represented the drug release rate according to the Ritger-Peppas equation
局部应用抗生素治疗牙周炎的成功主要依赖于抗菌药物的持续释
已知PLGA降解遵循伪一级动力
aSlope represented the degradation release rate according to the hydrolysis kinetic equation

Figure 4 Degradation image of MH-M and blank microspheres(M)()
当微球分散在基质丰富的相并扩散到PBS缓冲液中时,贮库显示出不同的孔隙率(代表不同的扩散速率)。

Figure 5 Porosity of MH-M-SAIB and MH-SAIB ()
本实验通过静电喷雾的方法,成功制备表面光滑,尺寸分布均匀的盐酸米诺环素PLGA微球,并将其与SAIB混合成功制备盐酸米诺环素微球贮库。同时,研究结果表明,盐酸米诺环素微球贮库能显著降低药物的突释,达到持续稳定的长期释放,为后续进一步研究盐酸米诺环素微球贮库的成骨效果及抑菌实验奠定了基础。
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