CN113633769B - 一种载脂多糖复合纳米粒的制备方法及其应用 - Google Patents
一种载脂多糖复合纳米粒的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种载脂多糖复合纳米粒的制备方法及其应用,属于生物医学技术领域。脂多糖复合纳米粒具有稳定的外膜,外膜由聚乳酸‑羟基乙酸共聚物(PLGA)制成,纳米粒内包封有由液态氟碳(PFP),吲哚菁绿(ICG),脂多糖(LPS)和水组成的混合溶液;复合纳米粒中,吲哚菁绿的载荷量为10.18%±0.12%,脂多糖的载荷量为1.69%±0.03%。本发明的脂多糖复合纳米粒,通过在PLGA纳米粒的基础上,加入了免疫佐剂LPS,光敏剂ICG,氧气以及携氧的相变材料PFP,不仅提高了ICG在循环过程中的稳定性和半衰期,使纳米粒具备良好的双模态成像能力,还可联合光声动力诱导机体免疫反应产生,与LPS共同激活免疫系统,在肿瘤免疫治疗方面,具有推广应用价值。
Description
技术领域
本发明涉及生物医学技术领域,具体涉及一种载脂多糖复合纳米粒的制备方法及其应用。
背景技术
在过去的十年中,诊疗一体化已经成为一个蓬勃发展的癌症治疗研究领域,这使得诊断和治疗更加紧密的结合,使治疗方案更加个体化。光疗法(如光热疗法和光动力疗法)因其侵袭性小、副作用小、易控制等优点,在新兴的肿瘤治疗中显示出巨大的优势。声动力疗法(Sonodynamic therapy,SDT)是在光动力疗法(Photodynamic therapy,PDT)的基础上发展起来的一种新的肿瘤治疗方法。与PDT不同,PDT组织穿透性差,超声能深入组织内,并以肿瘤组织为靶点,介导超声波增敏剂的细胞毒性。光声联合疗法(Sono-Photodynamictherapy,SPDT)是一种能够协同发挥各自的优势和增强免疫保护功能的方法。
吲哚菁绿(ICG)是FDA批准的用于测定肝功能和肝血流量的试剂。此外,ICG作为一种光谱吸收峰在800nm左右的染料,可用于PDT及PTT治疗。ICG在光和声的介导下可产生ROS,ROS可诱导肿瘤细胞发生免疫原性细胞死亡(immunogenic cell death,ICD),同时释放受损的相关分子模式(Damaged-associated molecular patterns,DAMP),包括钙网蛋白(calreticulin,CRT)和释放高迁移率群盒1(high mobility group box 1,HMGB 1)。DAMP的释放会激活免疫系统,特别是通过诱导树突状细胞(DC)的成熟,DC最终迁移到淋巴结,抗原与幼稚T细胞的相互作用使它们成为分化的CTL(CD8+)。此外,当ICD发生时,诱导的死亡肿瘤细胞可释放肿瘤相关抗原。这些即将死亡的肿瘤细胞可以作为全细胞癌症疫苗,它可以诱导对所有释放的潜在肿瘤抗原的免疫。因此,ICG在光声的作用下可以在肿瘤部位产生“肿瘤疫苗”,克服肿瘤抗原异质性,这可能是获得高免疫原性肿瘤疫苗,并将其用于癌症免疫治疗的一种很有效和简便的方法。
脂多糖(LPS)是革兰氏阴性菌外膜的组成部分,也是Toll-like receptors 4(TLR4)的激动剂。TLR4在免疫系统的单核细胞(如DC和巨噬细胞)、淋巴细胞和脾细胞中有较高的表达,它还可对HMGB 1进行选择性识别。DC是最强大的抗原提呈细胞(antigenpresenting cells,APC),也是适应性免疫应答的关键介质。LPS和HMGB 1通过激活TLR 4影响DC表面共刺激分子的表达和控制抗原的摄取进而促进抗原的加工和呈递,这是诱导和调节适应性免疫的关键环节。然而,静脉注射LPS常常会引起全身的毒副作用,如发热、寒战、低血压和肝毒性等。其他给药方式,如皮下注射、肿瘤内注射、肿瘤周围注射等,虽然能减少全身的毒性反应,但LPS很难穿透肿瘤组织,发挥有效作用,且较大剂量的局部注射会造成正常组织的损伤。因此,我们设计了一种纳米大小的药物载体来将LPS从给药点运送到肿瘤部位,联合光声治疗,实现肿瘤部位的可控释药,从而减少全身毒性反应。此外,纳米载药平台可以提高包裹药物的溶解性和稳定性,促进跨细胞膜的运输,延长循环半衰期,以提高安全性和有效性,为疾病的早期诊断和特异性治疗奠定了基础。
发明内容
有鉴于此,本发明的目的在于提供一种载脂多糖复合纳米粒的制备方法及其应用。
经研究,本发明提供以下技术方案:
1、一种载脂多糖复合纳米粒,所述纳米粒具有稳定的外膜,所述外膜由聚乳酸-羟基乙酸共聚物(PLGA)制成,所述纳米粒内包封有混合溶液,所述混合溶液由液态氟碳(PFP),含吲哚菁绿(ICG)和脂多糖(LPS)的水溶液组成;所述纳米粒中,吲哚菁绿的载荷量为10.18%±0.12%,脂多糖的载荷量为1.69%±0.03%。
优选的,所述所述纳米粒为球型构造,所述纳米粒的粒径为195.24±11.56nm。
优选的,所述纳米粒的zeta电位为-38.45±0.59mV。
优选的,所述液态氟碳为全氟戊烷、全氟己烷中的至少一种。
2、上述载脂多糖复合纳米粒的制备方法,包括以下步骤:
将聚乳酸-羟基乙酸共聚物(PLGA)溶解于二氯甲烷中,得混合溶液;
向液态氟碳(PFP)中通入氧气5~10min,得载氧液态氟碳(PFP);
将载氧液态氟碳(PFP)加入脂多糖(LPS)和吲哚菁绿(ICG)的水溶液中,超声0.5~1min,再向其中通入氧气5~10min,得乳化液;所述聚乳酸-羟基乙酸共聚物(PLGA),二氯甲烷,液态氟碳(PFP),脂多糖(LPS)和吲哚菁绿(ICG),按g:L:L:L:g计为100~300:15~20:1~2:5~10:15~20;
将混合溶液与乳化液混合,超声乳化3~5min后,倒入聚乙烯醇(PVA)水溶液中,并超声1~3min,加入异丙醇水溶液,搅拌4~6h,然后离心3~8min,取沉淀,用水洗涤后再次离心,将所得沉淀物重悬于充氧水中,即可。
优选的,所述聚乳酸-羟基乙酸共聚物(PLGA),二氯甲烷,液态氟碳(PFP),脂多糖(LPS)和吲哚菁绿(ICG),按g:L:L:L:g计为100:15:2:5:15。
优选的,所述混合溶液,载氧液态氟碳,乳化液和沉淀物的制备步骤,均在低温和避光条件下进行。
优选的,所述聚乙烯醇(PVA)水溶液的用量为5mL,所述异丙醇水溶液的用量为10mL。
优选的,所述聚乙烯醇(PVA)水溶液中,聚乙烯醇的浓度为1%,所述异丙醇水溶液中,异丙醇的体积百分含量为2%。
3、上述载脂多糖复合纳米粒在激光和超声下作为光声敏剂,且在光声介导下治疗肿瘤的应用。。
本发明的有益效果在于:
1)本发明的载脂多糖复合纳米粒(oxygen-carried and lipopolysaccharide/indocyanine green-loaded nanoparticles,OLI_NPs),通过在PLGA纳米粒的基础上,加入了免疫佐剂LPS,光敏剂ICG,氧气以及携氧的相变材料PFP,构建了新型的具有诊疗一体的具有相变型的多功能纳米粒--携氧载脂多糖/吲哚菁绿液态氟碳纳米粒,其中ICG被适当波长的可见光照射时,可随时间推移使纳米粒内部温度迅速上升,PFP随着温度的进一步升高而发生相变,从而导致液体转化为气体,有利于超声(US)和光声(PA)成像;脂多糖(lipopolysaccharide,LPS)是革兰氏阴性菌外膜的组成部分,可被Toll样受体4(TLR4)选择性识别,该受体在免疫系统的单核细胞细胞(如树突状细胞、巨噬细胞)中高度表达,LPS通过激活免疫细胞,使其在肿瘤免疫治疗中具有良好效果。当联合光声辐照肿瘤部位时,可诱导肿瘤细胞发生免疫原性死亡,从而释放HMGB1,细胞表面表达CRT,破裂的纳米粒同时释放LPS,可募集DC在肿瘤部位聚集和成熟,使抗原递呈效率提高,达到有效激活免疫反应的目的。
2)本发明的载脂多糖复合纳米粒(OLI_NPs)的制备方法,简单易操作,且所用材料价格便宜,可行性高,制得的载脂多糖复合纳米粒可同时作为造影剂和增敏剂,在超声成像、光声成像以及肿瘤免疫治疗领域,具有推广应用价值。
附图说明
图1为本发明的载脂多糖复合纳米粒的透射电镜分析图;
图2为本发明的载脂多糖复合纳米粒的粒径分析结果图;
图3为本发明的载脂多糖复合纳米粒的光镜分析结果图;
图4为本发明的载脂多糖复合纳米粒的共聚焦显微镜分析结果图;
图5为本发明的载脂多糖复合纳米粒的紫外吸收光谱分析结果图;
图6为本发明的载脂多糖复合纳米粒的荧光光谱分析结果图;
图7为本发明的载脂多糖复合纳米粒的体外超声成像分析结果图;
图8为本发明的载脂多糖复合纳米粒的体外光声成像分析结果图;
图9为注射载脂多糖复合纳米粒前后的体内光声成像效果对比图;
图10为本发明的载脂多糖复合纳米粒与游离吲哚菁绿的体内光声成像对比分析结果图;
图11为治疗后各组肿瘤组织表达CRT和HMGB1的免疫组织化学染色图;
图12为治疗后各组肿瘤组织表达CRT的平均光密度图;
图13为治疗后各组肿瘤组织表达HMGB1的平均光密度图;
图14和图15为瘤内DC细胞成熟情况对比图;
图16和图17为小鼠肿瘤生长情况对比图。
具体实施方式
下面结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
本实施例中,载脂多糖复合纳米粒的制备方法,包括以下步骤:
1)提前称量10mg PLGA溶解于1.5ml二氯甲烷中,放入4℃冰箱备用;
2)称取1.5mg ICG溶于500μl(1mg/ml)的脂多糖水溶液中,放入4℃冰箱备用;
3)向PFP中缓慢充氧5min,取200μl含氧PFP与步骤2)中所得溶解液声振30s,再向其中充氧5min;
4)将步骤1)中所得混合溶液与步骤3)中所得乳化液相混合,使用超声声振仪乳化3min后,倒入5mL PVA水溶液(1%w/v)中,并声振3min,然后加入10mL异丙醇水溶液(2%v/v),并用磁力搅拌4h,以充分挥发二氯甲烷,最后进行高速离心5min,去除上清液,取沉淀,用双蒸水充分洗涤后再次离心,重复三次,将所得沉淀物重悬于2mL充氧双蒸水中,储存于4℃条件下备用。上述步骤均在低温和避光条件下进行。
经过测定,实施例1制得的吲哚菁绿复合纳米粒的粒径和电位分别为195.24±11.56nm和-38.45±0.59mV,ICG的包封率为84.86%±0.96%,LPS的包封率为42.16%±0.79%,吲哚菁绿的载荷量为10.18%±0.12%,脂多糖的载荷量为1.69%±0.03%。
检测分析
1)透射电镜分析
具体操作为:将实施例1制得的载脂多糖复合纳米粒用双蒸水稀释一定倍数后,进行透射电镜观察,结果如图1所示。
从图1中分析可知,吲哚菁绿复合纳米粒的表面光滑呈球形,大小约为195nm左右,外层由PLGA包裹,内部为PFP混合含ICG和LPS的水溶液组成,其中,密度较高的为PFP,较低者为水溶液。
2)粒径分析
具体操作为:用Malvern粒径仪测定实施例1制得的载脂多糖复合纳米粒的粒径,结果如图2所示。
从图2中分析可知,实施例1制得的载脂多糖复合纳米粒的粒径分布在150~350nm之间,且对称性良好,平均粒径为195.24nm。
3)光镜分析
具体操作为:将实施例1制得的载脂多糖复合纳米粒用双蒸水稀释一定倍数后,进行光镜分析,结果如图3所示。
从图3中分析可知,实施例1制得的吲哚菁绿复合纳米粒在光镜下,可见复合纳米粒大小均一,具有良好的分散性,用49℃高温加热纳米粒1min后,纳米粒明显增大,产生相变现象,从而证明了载脂多糖复合纳米粒为相变型纳米粒。
4)共聚焦显微镜分析
具体操作为:将实施例1制得的载脂多糖复合纳米粒在共聚焦显微镜下进行观察分析,结果如图4所示。
从图4中分析可知,在共聚焦显微镜下可观察到绿色光为LPS,红色光为ICG,二者合成光为黄色,证明ICG和LPS被成功包裹在纳米粒中。
5)紫外吸收光谱和荧光光谱分析
紫外吸收光谱的具体操作为:将实施例1制得的载脂多糖复合纳米粒和吲哚菁绿分别进行紫外吸收光谱分析,使二者初始紫外吸光度一致,每三天观察一次紫外吸光度,持续15天。
荧光光谱分析的具体操作为:将实施例1制得的载脂多糖复合纳米粒和吲哚菁绿分别进行荧光光谱分析,使二者初始荧光吸光度一致,每三天观察一次荧光值,持续15天。结果如图5、图6所示。
从图5和图6中综合分析可知,吲哚菁绿复合纳米粒(OLI_NPs)的紫外吸收峰和荧光发射峰与游离ICG溶液基本一致。表明在纳米粒制备过程中并未改变ICG的光学特性。图5中,检测OLI_NPs和游离ICG每三天在780nm的紫外吸光度,在15天观察期内,与OLI_NPs相比,游离ICG的紫外吸光度显著下降,下降了初始强度的70%,OLI_NPs中的ICG吸收强度下降了约初始强度的20%。图6中,检测OLI_NPs和游离ICG每三天的荧光吸光度,在15天观察期内,与OLI_NPs相比,游离ICG的荧光吸光度显著下降,下降了初始强度的74%,OLI_NPs中的ICG吸收强度下降了约初始强度的18%。结果证明了,无论是在紫外光,还是在荧光特性方面,OLI_NPs都大大提高了游离ICG的光学稳定性,说明纳米结构有助于改善游离ICG易发生淬灭、半衰期短等缺点。
6)体外双模态成像分析
具体操作为:将实施例1制得的载脂多糖复合纳米粒进行体外光声成像检测分析,在800ml去离子水中加入4g琼脂糖凝胶粉末,逐渐加热至液体澄清且无气泡,迅速将液体倒入模具盒中,插入1ml枪头或者200μl枪头,待模具凝固成块后放入4℃冰箱备用。在超声成像中,使用每孔大小为1ml的模具,分四组:①PBS组;②游离ICG组(ICG 160μg/ml,1ml);③Blank NPs组(PLGA 1.25mg/ml,1ml);④OLI_NPs组(ICG 160μg/ml,1ml)。在光声成像中,使用每孔大小为200μl的模具,分四组:①PBS组;②游离ICG组(ICG 160μg/ml,200μl);③Blank NPs组(PLGA 1.25mg/ml,200μl);④OLI_NPs组(ICG 160μg/ml,200μl)。结果如图7和图8所示。
图7为808nm激光(1.5w 5min)照射前后和低功率聚焦超声(LIFU,其设备参数如下:超声输出频率:650KHz±10%,第一通道治疗头焦距:28mm±15%;第二通道治疗头焦距:12.5mm±15%)作用后的超声成像图,图8为808nm激光照射前后和LIFU(3w 1min)作用后的光声成像。
从图7的超声成像图中分析可知,激光照射后的B模式和CEUS模式下,PBS组和游离ICG组(free ICG)中均没有成像增强。在用808nm激光照射5分钟后,Blank NPs组超声成像B模式信号明显增强。这种增长可能是由于近红外激光照射后轻微的热辐射而导致BlankNPs相变所致。而OLI_NPs组的超声信号B模式和CEUS下较其他组显著增强。通过激发ICG引起PFP温度升高发生相变,导致超声成像明显增强。从而证明了OLI_NPs可以通过近红外光照射相变并且能成为超声成像的造影剂。
从图8的光声成像图中分析可知,PBS组和Blank NPs组在808nm激光照射前后光声信号没有明显的变化。由于PBS和没有包载ICG的Blank NPs在近红外范围内没有光吸收特性,所以它们没有显示出光声成像增强的能力。而通过808nm激光照射后,OLI_NPs组的光声信号较其他组明显增强。从而证明了OLI_NPs可以通过近红外光照射相变并且能成为超声成像的造影剂,且OLI_NPs也能作为一种良好的光声成像增敏剂。
无论是在超声成像或者是光声成像中,在激光辐照后,OLI_NPs的信号均明显增强,此时接着用超声辐照纳米粒后,可发现信号明显减弱,说明纳米粒发生了破裂,这也为实现纳米粒在肿瘤部位的可控释药奠定了基础。
7)体内光声成像分析
具体操作为:将实施例1制得的载脂多糖复合纳米粒(ICG 638μg/mL,100μL)和游离的ICG(ICG 638μg/mL,100μL)对荷瘤小鼠尾部进行静脉注射,然后在不同时间点的肿瘤局部进行体内光声成像检测分析,结果如图11所示。
图9为注射载脂多糖复合纳米粒前后的光声成像效果对比图。从图9中分析可知,注射前,肿瘤部位无光声信号,注射纳米粒后2h,肿瘤部位逐渐出现光声信号,信号很弱;4h后光声信号达到最强,说明此时纳米粒数量达到最大值,在这个时间点为最佳成像及治疗点;随着时间推移,肿瘤部位纳米粒信号逐渐减弱。
图10为注射载脂多糖复合纳米粒和游离的ICG的光声成像效果对比图。从图10中分析可知,游离ICG无论是在注射前还是注射后,体内均无法观察到光声信号。而OLI_NPs在注射4h之后,可在肿瘤部位明显成像,经1.5w激光照射5min后,光声信号明显增强。再经3w超声作用1min后,光声信号显著降低,说明此时纳米粒发生了破裂,证明OLI_NPs不仅提高了ICG的光学稳定性及肿瘤区域聚集能力,还可通过超声及光声成像对肿瘤进行靶向显影及对光声治疗过程进行动态监控。
8)纳米粒联合光声治疗肿瘤,诱导ICD作用
具体操作为:在小鼠左侧背部皮下种瘤,细胞数为1×106,待肿瘤体积达到100mm3时,分四组:①生理盐水组(Control);②载脂多糖复合纳米粒组(OLI_NPs);③光声动力组(PSDT);④载脂多糖复合纳米粒联合光声动力组(OLI_NPs+PSDT)。治疗后24小时收集肿瘤组织进行CRT和HMGB1免疫组织化学染色,结果如图11所示;统计并分析各组CRT和HMGB1的平均光密度,结果如图12和图13所示。
从图11和图12中分析可知,Control组细胞表面虽然有阳性染色,但不明显,OLI_NPs组、PSDT组与Control组CRT染色结果相近,OLI_NPs+PSDT组出现细胞结构和组织结构严重破坏,细胞周围黄染明显,且黄染程度强于间质,其平均光密度值显著高于其余各组,提示纳米粒联合光声治疗可诱导CRT发生外翻。
从图11和图13中分析可知,HMGB1存在于正常细胞的胞核内,所以Control组细胞胞核染色呈阳性,但细胞周围无黄染,OLI_NPs组和PSDT组与Control组HMGB1染色结果相近,但OLI_NPs+PSDT组细胞间质呈均一黄染,其平均光密度值显著高于其余各组,提示纳米粒联合光声治疗可诱导HMGB1释放到细胞外。
9)纳米粒联合光声治疗肿瘤,诱导免疫反应
具体操作为:在小鼠左侧背部皮下种瘤,细胞数为1×106,三天后,于对侧种等量细胞数,待左侧肿瘤体积达到100mm3时,分四组:①生理盐水组(Control);②载脂多糖复合纳米粒组(OLI_NPs);③光声动力组(PSDT);④载脂多糖复合纳米粒联合光声动力组(OLI_NPs+PSDT)。当治疗后第7天,取左侧肿瘤,分析瘤内DC细胞成熟情况,结果如图14和图15所示;其余小鼠继续观察肿瘤生长情况,分析双侧肿瘤生长趋势,结果如图16和图17所示。
从图14和图15中分析可知,OLI_NPs+PSDT组中成熟DC占比最多,其他3组无差异,从而证明了载脂多糖复合纳米粒联合光声治疗可促进抗原递呈细胞成熟。从图16和图17中分析可知,载脂多糖复合纳米粒联合光声治疗不仅可使原位肿瘤生长受到抑制,还可明显抑制远位肿瘤生长,证明载脂多糖复合纳米粒联合光声治疗可有效刺激小鼠体内免疫系统,达到控制肿瘤生长的目的。
本发明制得的载脂多糖复合纳米粒为一种用于诊疗一体化的多功能纳米粒。本发明的载脂多糖复合纳米粒中,PFP的携氧作用为辅,主要体现在成像方面的作用,以及作为一种可以随温度升高从而相变的物质,与ICG协同作用,使得纳米粒粒径增大甚至破裂,达到增强成像效果以及杀伤肿瘤细胞的效果。吲哚菁绿(indocyanine green,ICG)是一种高度生物相容性的近红外染料,但是ICG在循环的不稳定性和半衰期短,在长时间近红外光辐照后易发生光降解,经过纳米结构的保护性包裹后,不仅解决了ICG容易发生萃灭的难题,提高了ICG的稳定性和半衰期,同时也与PFP协同增强了ICG的光声成像效果,使纳米粒具备对肿瘤进行靶向显影及对光声治疗过程进行动态监控的能力。
本发明的载脂多糖复合纳米粒中,通过加入的免疫佐剂LPS,可激活TLR4,通过影响抗原递呈细胞表面共刺激分子的表达和控制抗原摄取来促进抗原的加工和呈递,是启动和调节适应性免疫应答的关键介质。ICG的光动力作用可诱导肿瘤细胞发生免疫原性死亡,从而暴露肿瘤抗原CRT和HMGB1,与LPS共同募集DC在肿瘤部位聚集,从而促进抗原的加工和呈递,二者协同作用可有效激活免疫系统,达到有效抑制肿瘤生长的目的。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
1.一种载脂多糖复合纳米粒,其特征在于,所述纳米粒具有稳定的外膜,所述外膜由聚乳酸-羟基乙酸共聚物制成,所述纳米粒内包封有混合水溶液,所述混合水溶液由液态氟碳、吲哚菁绿、脂多糖和水组成;所述纳米粒中,吲哚菁绿的载荷量为10.18%±0.12%,脂多糖的载荷量为1.69%±0.03%。
2.根据权利要求1所述载脂多糖复合纳米粒,其特征在于,所述纳米粒为球型构造,所述纳米粒的粒径为195.24±11.56nm。
3.根据权利要求1所述载脂多糖复合纳米粒,其特征在于,所述纳米粒的zeta电位为-38.45±0.59mV。
4.根据权利要求1所述载脂多糖复合纳米粒,其特征在于,所述液态氟碳为全氟戊烷、全氟己烷中的至少一种。
5.权利要求1至权利要求4任一项所述载脂多糖复合纳米粒的制备方法,其特征在于,包括以下步骤:
将聚乳酸-羟基乙酸共聚物溶解于二氯甲烷中,得混合溶液;
向液态氟碳中通入氧气5~10min,得载氧液态氟碳;
将载氧液态氟碳加入含脂多糖和吲哚菁绿的水溶液中,超声0.5~1min,再向其中通入氧气5~10min,得乳化液;所述聚乳酸-羟基乙酸共聚物、二氯甲烷、液态氟碳、脂多糖和吲哚菁绿按g:L:L:L:g计为100~300:15~20:1~2:5~10:15~20;
将混合溶液与乳化液混合,超声乳化3~5min后,倒入聚乙烯醇水溶液中,并超声1~3min,加入异丙醇水溶液,搅拌4~6h,然后离心3~8min,取沉淀,用水洗涤后再次离心,将所得沉淀物重悬于充氧水中,即可。
6.根据权利要求5所述载脂多糖复合纳米粒的制备方法,其特征在于,所述聚乳酸-羟基乙酸共聚物、二氯甲烷、液态氟碳、脂多糖和吲哚菁绿按g:L:L:L:g计为100:15:2:5:15。
7.根据权利要求5所述载脂多糖复合纳米粒的制备方法,其特征在于,所述混合溶液、载氧液态氟碳、乳化液和沉淀物的制备步骤均在0~4℃和避光条件下进行。
8.根据权利要求5所述载脂多糖复合纳米粒的制备方法,其特征在于,所述聚乙烯醇水溶液的用量为5mL,所述异丙醇水溶液的用量为10mL。
9.根据权利要求8所述载脂多糖复合纳米粒的制备方法,其特征在于,所述聚乙烯醇水溶液中,聚乙烯醇的浓度为1%,所述异丙醇水溶液中,异丙醇的体积百分含量为2%。
10.权利要求1至权利要求4任一项所述载脂多糖复合纳米粒在制备在光声介导下治疗肿瘤的光声敏剂中的应用。
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