CN114432230B - 一种经皮递送脂质体治疗银屑病的微针及其制备方法 - Google Patents
一种经皮递送脂质体治疗银屑病的微针及其制备方法 Download PDFInfo
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Abstract
本发明属于药物制剂领域,具体涉及一种经皮递送脂质体治疗银屑病的微针及其制备方法。所述制备方法首先通过逆向蒸发法制备了载药脂质体,并且将其制备成MTX脂质体冻干粉末,继而将粉末载入微针空穴中。本发明中的微针通过两步法制备,将脂质体冻干粉装载于可溶性微针的空穴中,经皮递送药物到达皮肤并提高药物在炎症部位的滞留时间。制得的微针机械性良好,可以突破角质层的屏障,并可以直接附于银屑病病变附近,提高药物疗效,且达到无痛微创的效果。本发明方法提高了药物的经皮渗透能力,降低药物毒性,提高生物相容性,实现缓慢释药充分发挥药效,同时减少副作用。
Description
技术领域
本发明属于药物制剂领域,具体涉及一种经皮递送脂质体治疗银屑病的微针及其制备方法。
背景技术
银屑病是一种T细胞介导的疾病,通常是由于自身免疫系统信号处理错误而引起的,表现在皮肤或关节中。银屑病的组织病理学表现为表皮增生、角质形成细胞分化障碍、明显的炎症浸润和血管形成增加,这严重影响患者生活质量。因此,通常使用小分子药物(甲氨蝶呤MTX,环孢素A,视黄酸等)和生物制剂(依那西普,阿达木单抗,赛妥珠单抗等)来治疗银屑病。但是靶向生物制剂高昂的价格使患者望而却步,导致依从性低,难以为继。
甲氨蝶呤MTX仍是目前治疗银屑病的一线药物,其价格合理,治疗效果明显,在临床上广泛使用。但是MTX的全身给药会导致许多副作用,口服MTX存在胃肠道刺激,肝脏首过效应,且生物利用率低、患者依从性差等问题,从而影响药效。约70%-80%患者患有轻度的银屑病,可以通过局部治疗的方法加以控制。局部治疗银屑病能够较大程度发挥药效,且减少副作用,以实现安全有效的治疗效果。
纳米技术的发展进步,使改善局部药物治疗效果和减少药物副作用成为可能。脂质体是磷脂分子在水中定向排列形成的疏水链向内、亲水头基向外的双分子层闭合囊泡。脂质体可控制药物释放、降低药物毒性、增强药物稳定性、实现药物定向释放,且生物相容性良好、成本较低,常用于局部或经皮给药中,可有效治疗皮肤病。除此之外,脂质体可在皮肤中形成储库,促进药物持续释放;磷脂分子扩散到角质层的脂质覆盖物中,作为渗透促进剂。
MTX的局部给药是有效避免首过效应和全身毒性的有效途径。皮肤是身体最大、最容易接触到的器官,是一种潜在的全身给药途径,具有重要的免疫功能。皮肤富含抗原呈递细胞(APC),尤其是在表皮和真皮中,皮肤中的常驻APC主动迁移到引流淋巴结,与T细胞和B细胞相互作用,从而连结了皮肤和整个免疫系统。因此经皮免疫具有得天独厚的优势。微针作为一种有前途的微创透皮给药系统,逐渐进入人们视野。微针长度通常为25-2000μm,这使其能够穿透皮肤,但不能到达真皮层的神经组织,从而减轻疼痛,提高患者舒适度并确保安全。常用于制备微针的材料有金属、硅、玻璃、镍、钛或可生物降解的聚合物等。微针的类型多样,主要有固体微针、包衣微针、中空微针和可溶性微针等。其中,制备可溶性微针常用的基质材料有透明质酸钠(HA)、羧甲基纤维素钠(CMC-Na)、聚乙烯醇(PVA)、聚乙烯基吡咯烷酮(PVP)、甲基乙烯基醚-马来酸酐(PMVEMA)(Gantrez AN-)、甲基乙烯基醚-马来酸(PMVEMAH)或低分子量糖等。
同时,查阅国内外文献,虽有释放银屑病治疗药物的微针贴片相关专利(申请号202010809588.0)报道,但其中选用难溶性聚合物,容易导致在人体内进行富集等从而造成毒副作用,且并未有负载甲氨蝶呤脂质体可溶性微针的相关文献报道。一种治疗银屑病关节炎的可溶性微针(申请号CN201811590175.7)中,其24h的药物皮肤滞留量仍然较低,从而影响治疗效果。申请号CN200910083494.3中采用金属、硅、玻璃制备微针,硅胶制作柔性背衬,材料的生物相容性不佳,难以避免微针断裂于皮肤中难以代谢出体外,而引起潜在危险。
综上所述,开发一种既能稳定、有效地负载MTX,又具有良好的机械性能和与皮肤的贴合度,增强MTX在皮肤的滞留时间的治疗银屑病的微针,是本领域技术人员急需解决的技术难题。
发明内容
本发明的目的在于解决现有技术中常用的银屑病治疗药物甲氨蝶呤MTX的缺陷和副作用,解决针对银屑病皮肤表面鳞屑、增厚等导致微针难以刺入的问题,提供一种经皮递送MTX脂质体治疗银屑病的可溶性柔性背衬微针。通过可溶性柔性背衬微针经皮递送MTX脂质体,避免了口服MTX引起的胃肠道副作用和肝脏首过效应,避免了注射MTX引起的疼痛、患者依从性差、频繁给药等问题,并实现MTX在银屑病炎症部位的富集,减少系统性副作用,提高药效,而微针针头机械性能良好,可刺入增厚皮肤,背衬柔软易于贴合鳞屑皮肤。
本发明解决其技术问题所采用的技术方案是:
本发明的目的之一是提供一种经皮递送MTX脂质体治疗银屑病的微针的制备方法,所述方法是通过两步离心法制备微针,将MTX脂质体冻干粉末装载于微针的空穴部位,得到柔性背衬的可溶性微针;具体包括以下步骤:
S1.将甲基乙烯基醚-马来酸(PMVE MAH)和水混匀,得到溶液A;将溶液A置于微针模具中,离心脱气后形成微针针头,刮去多余的针头材料,将带有微针针头的微针模具置于真空干燥器中干燥6-12h,除去材料中的气泡;
S2.干燥后,向微针针头处形成的空穴内加入MTX脂质体冻干粉末,继而加入背衬材料B,降速离心30min,形成微针的背衬层,转置于恒温恒湿箱中干燥并脱膜,得到具有柔性背衬的可溶性微针。
作为优选,所述步骤S1中溶液A为200mg/mL的PMVE MAH溶液。
作为优选,所述步骤S1中通过四维旋转混合仪或恒温振荡箱将甲基乙烯基醚-马来酸和水混匀。
作为优选,所述步骤S1中微针模具为聚二甲基硅氧烷(PDMS)模具。
作为优选,所述步骤S1中,离心的转速为2500-4000r/min,离心时间为20-60min;更优选的离心的转速为3500r/min,离心时间为30min。
作为优选,所述步骤S2中背衬材料B为硫酸软骨素、聚乳酸、聚乙醇酸、聚乙烯醇、丝素蛋白、羧甲基纤维素、羟丙基纤维素、透明质酸、聚乙烯吡咯烷酮、壳聚糖、麦芽糖和葡聚糖中的一种或多种混合材料;更优选的,背衬材料B为羟丙基纤维素HPC和可溶性壳聚糖WCS的混合溶液,其中羟丙基纤维素HPC和可溶性壳聚糖WCS的质量比为4:1;更优选的,以混合溶液体积计,HPC的浓度为20-40mg/mL,WCS的浓度为10mg/mL;更优选,HPC的浓度为40mg/mL,WCS的浓度为10mg/mL。
作为优选,所述步骤S2中,降速离心是指将微针模具依次在3000、2500、2000、1500、1000、500rpm的转速下水平离心各5min,降速离心有利于得到平整且厚度均匀的背衬材料,进一步增强与皮肤的贴合度。
作为优选,所述步骤S2中,恒温恒湿箱设置的温度为30℃,湿度为40%。
作为优选,如上所述的制备方法制成6×6的可溶性微针贴片,微针针头高度为500μm,针头为圆锥体。
作为优选,所述MTX脂质体冻干粉末的制备方法,包括以下步骤:
1)采用逆向蒸发法制备脂质体:将磷脂、胆固醇溶解在氯仿中,得到A溶液;用磷酸盐缓冲液PBS溶解MTX,得到B溶液,加至上述A溶液中,用细胞破碎仪冰浴超声5min(功率150W),形成w/o型乳剂,减压旋转去除有机溶剂,得半固态胶状物;
2)继续加入磷酸盐缓冲液PBS,减压旋蒸,除去残留有机溶剂,通过冰浴超声5min(功率150W),获得均匀的载药脂质体溶液;
3)脂质体溶液经透析(3500Da,48h),除去游离的MTX,在富集的脂质体溶液中加入冻干保护剂海藻糖,混合均匀,置入-20℃冰箱预冻12h后放入真空冷冻干燥机冷冻干燥48h,得到MTX脂质体冻干粉末,将获得的样品封装后放入-20℃冰箱保存。
作为优选,所述磷脂为蛋黄卵磷脂(EPC)、大豆磷脂、二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰磷脂酰胆碱(DSPC)、油酰基卵磷脂(DOPC)、氢化大豆卵磷脂(HSPC)、二油酰磷脂酰乙醇胺(DOPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二硬脂酰基磷脂酰乙醇胺(DSPE)或脑磷脂中的至少一种。
作为优选,所述步骤1)中磷脂为EPC。
作为优选,所述步骤2)中载药脂质体溶液中磷脂、胆固醇、MTX的浓度分别为5、2.5、1mg/mL。
作为优选,所述步骤1)中氯仿与PBS体积比为2-5:1,更优选为3:1。
作为优选,所述步骤1)和步骤2)中PBS的pH为6.0-8.0,更优选为6.5。
作为优选,所述步骤3)中冻干保护剂海藻糖投料量以载药脂质体溶液记为浓度为2mg/mL。
本发明的目的之二是提供一种通过上述制备方法制得的通过经皮递送脂质体来治疗银屑病的可溶性微针。
本发明的目的之三是提供一种上述的经皮递送MTX脂质体治疗银屑病的微针在银屑病中的应用。
与现有技术相比,本发明的有益效果是:
(1)通过微针治疗的手段实现银屑病的局部治疗,避免了口服MTX的胃肠道副作用和肝脏首过效应,避免了注射MTX引起的疼痛、患者依从性差、频繁给药等问题;
(2)通过脂质体载药方式,具有一定的缓释效果,可以延缓药物在体内的速释,使药物载炎症部位富集,从而提高治疗效果,且完全生物无害;
(3)通过控制微针干燥过程中的温度与湿度,节省制备时间,微针制作过程简单,同时对微针的制备材料进行选择,制得的微针机械性能良好、针头硬度好、可有效刺入增厚的皮内、不易造成脆裂,背衬柔软更贴合鳞屑皮肤,适于银屑病皮肤的透皮给药;
(4)将化学促渗技术(脂质体)与物理促渗剂技术(微针)相结合,提高经皮递药的效率和利用率,提高了药物在皮肤的储存时间,实现缓慢释药充分发挥药效。
附图说明
图1为本发明制备的柔性背衬微针整体图像;
图2为本发明的柔性背衬载药微针的扫描电镜图;
图3为MTX脂质体的TEM图(标尺200μm);
图4为MTX和MTX脂质体的体外释放曲线(mean±SD,n=3);
图5为不同制剂的MTX体外累积透皮渗透量(mean±SD,n=9);
图6为透皮实验24h时皮肤中的MTX沉积量(mean±SD,n=3);
图7为第8天时小鼠模型背部皮肤的银屑病样图;
图8为第8天时小鼠模型背部皮肤的PASI总分(mean±SD,n=6);
图9为第8天时小鼠模型背部皮肤的H&E染色图(标尺50μm);
图10为第8天时小鼠模型背部皮肤的免疫组化(Ki67)图(标尺100μm)。
具体实施方式
下面通过具体实施例,并结合附图,对本发明的技术方案作进一步的具体说明。
实施例1:MTX脂质体的制备
(1)采用逆向蒸发法制备脂质体:
将30mg蛋黄卵磷脂(EPC)、15mg胆固醇(Chol)溶解在9mL氯仿中,作为油相。用3mLPH为6.5的PBS溶解6mgMTX,作为水相,加至上述油相中,冰浴探头超声(功率150W)5min,形成w/o型乳剂,30℃下减压旋转30min蒸去有机溶剂,得半固态胶状物。继续加入3mLPBS,30℃下减压旋蒸5min,除去残留有机溶剂。最后再于细胞破碎仪中,冰浴探头超声(功率150W)5min,获得均匀的载药脂质体溶液。
(2)MTX脂质体冻干粉末制备:
脂质体溶液经透析(3500Da,48h)后,除去游离的药物MTX,将冻干保护剂(海藻糖)按照2mg/mL的量加入富集的脂质体溶液中,吹打混合均匀,置入-20℃冰箱冷冻过夜12h后放入真空冷冻干燥机冷冻干燥48h,得到MTX脂质体冻干粉末,将获得的样品放入-20℃冰箱保存。
实施例2:MTX脂质体的表征
(1)脂质体粒径、PDI、zeta电位的测量:
用DLS粒径仪测定实施例1中制备的脂质体在25℃时的粒径大小、PDI和zeta电位。将MTX脂质体溶液加入粒径杯中,平行测三次。测试时记录平均流体力学直径、多分散性指数(PDI)两种指标,加入电位测定电极,使电极片完全浸没于待测溶液,平行测三次,测试时记录脂质体的zeta电位,进行分析。
精密吸取1mL脂质体于100mL试管中,加入5mL破乳剂(氯仿/甲醇=1:1(V/V)),混合均匀,超声30~40min,使其破乳均匀,静置后分层取上层溶液稀释,用紫外分光光度计在303nm处测定其吸光度值,根据MTX的标准曲线计算其浓度,记为c1;
精密吸取1mL脂质体于50kDa的超滤管中,在8000r/min的超速离心机中离心30min,获得游离的MTX溶液,稀释后用紫外分光光度计在303nm处测定其吸光度值,根据MTX的标准曲线计算其浓度,记为c2。
包封率(EE%)根据式1计算:
表1 MTX脂质体的表征结果
由表1可知,制备的MTX粒径在200nm以下,且均一性较好。中国药典规定脂质体包封率不得低于80%,本实施例制备的MTX脂质体符合中国药典规定。
(2)透射电子显微镜(TEM)表征:
将MTX脂质体待测样品滴到电子显微镜的特殊铜网上,用滤纸除去多余的液体,自然干燥后,通过透射电子显微镜(TEM)观察样品形态,见图3。
图3中为TEM拍摄的脂质体显微图,从图中可以看出,脂质体数量较多,呈圆形囊泡状,粒径在200nm内。
(3)体外释放实验:
体外释放过程选用pH7.4的PBS作为释放介质。分别将5mLMTX和MTX脂质体转入截留分子量为14KDa的透析袋中,将透析袋完全浸没于装有100mL释放介质的烧杯中。将烧杯放入全温振荡培养箱内,温度设置为37℃,转速为100rad/min,进行体外释放。在开始释放后的0、0.5、1、2、4、6、8、12、24h后分别取样1mL用于释放率计算,同时补加同等体积的37℃新鲜介质。将取出的释放介质置于酶标仪中,设定激发波长为303nm,进行药物含量测定,计算各时间点累计释放百分率,结果如图4。
从图4中可以看出,当释放4h时,MTX已释放89.73%,而在9h时,MTX脂质体才释放90.74%的总投量,由此可见,脂质体的双层膜可以有效组织MTX的外泄,脂质体载体形式能延缓MTX的释放。
实施例3:空白可溶性微针的制备
微针模具由聚二甲基硅氧烷PDMS制成,由此可制得图1所示6×6圆锥形MNs(单根MNs底端直径130μm,尖端直径12μm,针长500μm)。
可溶性微针按以下步骤制得:称取甲基乙烯基醚-马来酸PMVE MAH,加纯化水溶解,于四维旋转混合仪上混匀,得到PMVE MAH溶液,呈微黄色。将PMVE MAH置于聚二甲基硅氧烷PDMS制成的微针模具中,3500rad/min离心30min后形成针头;刮去多余的针头材料PMVE MAH,将模具置于室温下的真空干燥器中,负压干燥12h后,HPC和WCS的混合背衬材料,降速离心30min(在3000、2500、2000、1500、1000、500rpm的转速下水平离心各5min),形成微针的背衬层。将微针转置于温度为30℃、湿度为40%的恒温恒湿箱中,放置8h后干燥完全,制备成空白微针。
实施例4:空白可溶性微针的表征
(1)微针的临界屈曲力
利用质构仪的压缩模式考察MNs的力学性能,检测MNs的临界屈曲力。MNs与探头轴向平行,针尖朝上,设置质构仪测试过程中的横坐标为压缩距离(mm),纵坐标为力(N),通过所得的力与位移的关系,得到MNs在断裂前的临界屈曲力。
(2)微针针头弯折率实验
为测试MNs在运输途中机械性能破坏程度,设计MNs针头弯折率实验,将制备好的MNs剪成3×3大小的阵列,放入10mL离心管内,将10mL离心管置于恒温震荡箱,设置转速100rad/min震荡1h后取出,查看MNs针头弯折情况并计算针头弯折率,计算公式如下所示。
(3)微针穿刺率实验
将制备的微针刺入小鼠背部皮肤,1min后揭开,用亚甲基蓝染色后,除去多余染液,记录孔洞数,计算皮肤穿刺率,值越高,机械性能越好。穿刺率的计算公式如下:穿刺率=(孔洞数/可溶性微针针体数)*100%。
(4)微针背衬最大弯曲角实验
将微针置于不同弯曲程度的平滑的曲面,曲面的两条切线所相交的角的补角度数为该检测台的弯曲角。检测台的弯曲角(10-180°)依次增大,直至微针与某角度的检测台贴合后,产生裂痕或断裂,则弯曲角为该处方下微针的最大弯曲角。通过微针背衬的最大弯曲角来考察其背衬与皮肤的贴合度,弯曲角越大,说明背衬越柔软,与皮肤的贴合度也越好。
表2不同配方的微针机械性能考察结果
表2为不同配方下制备而成的微针机械性能考察结果。PMVE MAH浓度为100mg/mL时,由于浓度过低,微针难以成型,因此分别考察针头浓度为200、300、400mg/mL的PMVEMAH;壳聚糖生物相容性良好,具有优良的抑菌、保湿、刺激细胞再生及修饰皮肤等功能,且机械性能良好,是用于制备微针的理想材料。但由于单一的WCS材料制备的微针在干燥时易皱缩,严重影响其成型性,且制成的WCS微针易吸湿,需要严格控制保存条件。为了解决上述问题,在WCS材料的基础上,加入HPC,共同作为微针背衬材料。混合背衬材料WCS和HPC,成膜时坚韧并有弹性,且不易吸湿,适合用于柔性背衬的制备。WCS赋予背衬材料更大的硬度,但是WCS:HPC的比例较高时(>1:1(w/w)),仍会引起微针的皱缩。因此,固定WCS的添加量为100mg/mL,改变HPC和WCS的比例分别为2:1、3:1、4:1、5:1(w/w)进行背衬比例的考察。根据临界屈曲力、针头弯折率、微针穿刺率、背衬弯曲角等因素综合考虑,选择了微针针头机械性能良好,但是背衬柔软更易贴合皮肤的最优处方为:200mg/mL的PMVE MAH作为针尖材料、浓度为40mg/mL的HPC和10mg/mL的WCS的混合溶液作为背衬材料。
实施例5:载MTX脂质体微针的制备
称取PMVE MAH,加纯化水溶解,于四维旋转混合仪上混匀,得到浓度200mg/mLPMVEMAH溶液。将PMVE MAH置于PDMS模具中,3500rpm离心30min后形成针头;刮去多余的PMVEMAH,将模具置于室温下的真空干燥器中,干燥6-12h后,在模具表面加入约1000μgMTX脂质体冻干粉,填入空穴,继而加入40mg/mL的HPC和10mg/mL的WCS的混合背衬材料,降速离心30min(在3000、2500、2000、1500、1000、500rpm的转速下水平离心各5min),形成微针的背衬层。将微针转置于温度为30℃、湿度为40%的恒温恒湿箱中,放置8h后干燥完全,制备成载药微针。载药微针的扫描电镜图见图2。
实施例6:药物的皮肤驻留实验
断颈处死Balb/c小鼠后,剃净其毛发,取其背部皮肤,用脱脂棉擦除皮下组织,并用生理盐水清洗,用透皮扩散仪对MTXMN、MTX-LipoMN、MTX水凝胶、MTX-Lipo水凝胶进行透皮扩散实验,其中每种制剂均载MTX约100μg。
采用PMVE MAH和聚乙二醇PEG-10000作为原料进行交联,制备水凝胶材料,制备过程如下:将200mg/mL PMVEMAH在纯化水中溶解后,剧烈搅拌,将其加热并保持在95-100℃。冷却后,按照50mg/mL加入PEG-10000,并分别加入药物制剂MTX或MTX-Lipo,混合均匀。将混合物缓慢倒入模具,使混合物均匀分布,在室温下干燥48h。80℃下固化24h以诱导PMVEMA和PEG之间的化学交联,分别制得载MTX和MTX-Lipo的水凝胶。
将MTXMN、MTX-LipoMN、MTX水凝胶、MTX-Lipo水凝胶置于皮肤中心。然后将皮肤牢固地安装在接收器隔室上。为了模拟体内条件,7.2mL的pH7.4PBS作为接收液(600rpm,,32±1℃)。在预定的时间点(0、1、2、3、6、9、12、24、36、60、72h)取出样品溶液(每种600μL),并将600μL的新鲜PBS添加到接收介质中,以确保体积保持恒定。样品通过0.2μm的过滤器过滤,并在303nm紫外波长下测定吸光度,根据公式计算MTX的累积释放百分比,结果如图5所示。
式中Q为t时间内单位面积累积经皮渗透量:Cn和Ci分别为第n次和第(n-1)次取样时接收液的浓度;A为有效渗透面积;V为接收池内溶液的体积;Vi为每次取样体积。
经过24h的透皮实验研究后,评估MTX在皮肤中的分布。为了测量皮肤中的药物浓度,用剪刀将皮肤切碎,浸泡在2mL提取溶剂(甲醇:10mM PBS=50:50v/v)中,放入6孔板中,持续摇晃24小时(100rpm)。样品通过0.2μm的过滤器过滤,并在303nm紫外波长下测定吸光度,计算MTX的皮肤驻留量,结果为图6,其中,*P<0.05,**P<0.01,***P<0.001,****P<0.0001。
图5中可知,MTX MN(MTX微针)透皮扩散的速率最快,当微针刺穿皮肤后,可直接将药物递送进入接受液中;而MTX-Lipo MN(MTX脂质体微针)透皮扩散效率紧随其后,可能是因为脂质体的载药形式使MTX释放速率降低;相比微针给药形式,水凝胶载体的透皮扩散量明显较低,这说明传统水凝胶形式的透皮效率十分有限,在透皮实验进行72h时,渗透量仍较低,且水凝胶制备过程中需要较高的温度(80℃)进行交联,对药物的活性可能会产生影响;但是脂质体的载体形式在一定程度上促进了水凝胶载药的透皮渗透量,可能是因为MTX为亲水性药物,穿透角质层阻碍的MTX较少,但脂质体的双层膜具有柔韧性和流动性,并作为渗透增强剂,使载药脂质体与皮肤更易融合并穿透皮肤。
图6为透皮扩散实验进行24h时,测量皮肤内MTX的沉积量所得的实验结果。MTX-Lipo MN组不仅透皮扩散量最多,其MTX在皮肤中的沉积量也最高,脂质体显着增加了MTX在皮肤中的积累(P<0.01)。
实施例6:可溶性微针对银屑病的治疗效果
将Balb/c小鼠随机分为6组,每组6只。适应性喂养一周后,用剃毛机剃除小鼠背部的毛发,除了对照组之外,其他各组每天以25mg/cm2施用咪喹莫特IMQ乳膏一次,持续7天,以在IMQ乳膏处理的皮肤区域上建立银屑病炎症。在造模后的第3、5、7天进行给药治疗,其中MTX制剂给药组的每次给药量为20μg/20g小鼠体重。分组情况如下:
(1)正常对照组
(2)模型组
(3)空白微针给药组
(4)MTX微针给药组
(5)MTX脂质体微针给药组
(6)MTX灌胃组
临床常用银屑病面积和严重程度指数PASI评估银屑病皮肤的严重程度,为红斑,鳞屑和皮肤增厚三部分得分的总和(0-12)。在第8天,对小鼠模型处皮肤进行红斑,鳞屑和增厚的评分(0分,无明显病变;1分,轻度;2分,中度;3分,明显;4分,非常明显)。用于评估银屑病的严重程度。将小鼠脱颈处死后,解剖取其银屑病造模处皮肤进行H&E染色和免疫组化分析(Ki67),实验结果分别如图7、8、9、10所示。
图7为第8天时小鼠模型背部皮肤的银屑病样图,图8则为其PASI总分。正常组小鼠皮肤呈暗红色,十分光滑,而经IMQ造模后的实验组PASI总分均显著性增加(P<0.05),但是经MTX-Lipo微针经皮治疗后,其鳞屑、增厚明显缓解(P<0.0001),效果优于单纯的MTX微针给药和口服给药。由此可见,MTX-Lipo微针经皮治疗可以高效地将药物递送至炎症部位,发挥药效。
图9为第8天时小鼠模型背部皮肤的H&E染色图,正常组的表皮(深色区域)厚度约为18μm,而模型组则增厚至正常皮肤的5倍左右,给予MTX制剂的三组表皮增厚情况有所减轻,但存在突出的棘层和角质层的脱落剥离情况。
Ki67可以识别增殖细胞中存在的核抗原,由此反映角质形成细胞的增殖率。图10为第8天时小鼠模型背部皮肤的免疫组化(Ki67)图,表皮的整体情况与H&E染色图相似,但是可以明显看出,基底层细胞增殖明显增多,且因基底细胞的不断增殖,形成棘层,突出下延至真皮层。
综上所述,柔性背衬的微针可以很好地与小鼠银屑病皮肤贴合,针头刺入皮肤后,药物进入体内,脂质体包封MTX后可延缓MTX的释放,增加MTX-Lipo在皮肤中的驻留量,从而发挥MTX的细胞毒性作用,抑制促炎细胞因子生成,抑制角质形成细胞合成DNA并诱导细胞凋亡,最终实现抑制银屑病的作用。
以上所述的实施例只是本发明的较佳方案,并非对本发明作任何形式上的限制,在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
Claims (9)
1.一种经皮递送MTX脂质体治疗银屑病的微针的制备方法,其特征在于,所述方法是通过两步离心法制备微针,将甲氨蝶呤MTX脂质体冻干粉末装载于微针的空穴部位,得到柔性背衬的可溶性微针;具体包括以下步骤:
S1.将甲基乙烯基醚-马来酸和水混匀,得到溶液A;将溶液A置于微针模具中,离心脱气后形成微针针头,置于真空干燥器中干燥6-12h;
S2.干燥后,向微针针头处形成的空穴内加入MTX脂质体冻干粉末,并加入背衬材料B,降速离心30min,转置于恒温恒湿箱中干燥并脱膜,得到柔性背衬的可溶性微针;
其中,所述MTX脂质体冻干粉末的制备方法,包括以下步骤:
1)采用逆向蒸发法制备脂质体:将磷脂、胆固醇溶解在氯仿中,得到A溶液;用磷酸盐缓冲液PBS溶解MTX,得到B溶液,加至上述A溶液中,用细胞破碎仪冰浴超声5min,形成w/o型乳剂,减压旋转去除有机溶剂,得半固态胶状物;
2)继续加入磷酸盐缓冲液PBS,减压旋蒸,除去残留有机溶剂,通过冰浴超声5min,获得载药脂质体溶液;
3)载药脂质体溶液经透析除去游离的MTX,加入冻干保护剂海藻糖,混合均匀,置入-20℃冰箱预冻12h后放入真空冷冻干燥机冷冻干燥48h,得到MTX脂质体冻干粉末。
2. 根据权利要求1所述一种经皮递送MTX脂质体治疗银屑病的微针的制备方法,其特征在于,所述步骤S1中溶液A为200mg/mL的PMVE MAH溶液。
3.根据权利要求1所述一种经皮递送MTX脂质体治疗银屑病的微针的制备方法,其特征在于,所述步骤S1中,离心的转速为2500-4000r/min,离心时间为20-60min。
4.根据权利要求1所述一种经皮递送MTX脂质体治疗银屑病的微针的制备方法,其特征在于,所述步骤S2中背衬材料B为硫酸软骨素、聚乳酸、聚乙醇酸、聚乙烯醇、丝素蛋白、羧甲基纤维素、羟丙基纤维素、透明质酸、聚乙烯吡咯烷酮、壳聚糖、麦芽糖和葡聚糖中的一种或多种混合材料。
5.根据权利要求4所述一种经皮递送MTX脂质体治疗银屑病的微针的制备方法,其特征在于,所述步骤S2中背衬材料B为羟丙基纤维素和可溶性壳聚糖的混合溶液,羟丙基纤维素和可溶性壳聚糖的质量比为4:1。
6.根据权利要求1所述一种经皮递送MTX脂质体治疗银屑病的微针的制备方法,其特征在于,所述步骤S2中,降速离心是指将微针模具依次在3000、2500、2000、1500、1000、500rpm的转速下水平离心各5min。
7.根据权利要求1所述一种经皮递送MTX脂质体治疗银屑病的微针的制备方法,其特征在于,所述步骤S2中,恒温恒湿箱设置的温度为30℃,湿度为40%。
8.根据权利要求1所述一种经皮递送MTX脂质体治疗银屑病的微针的制备方法,其特征在于,所述步骤2)中载药脂质体溶液中磷脂、胆固醇、MTX的浓度分别为5、2.5、1mg/mL;所述步骤3)中冻干保护剂海藻糖投料量以载药脂质体溶液记浓度为2mg/mL。
9.一种经皮递送MTX脂质体治疗银屑病的微针,其特征在于,所述微针是由权利要求1-8任意所述一种经皮递送MTX脂质体治疗银屑病的微针的制备方法制备得到。
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