CN112316138A - 一种pcp靶向修饰的黑磷纳米粒子及其制备方法和应用 - Google Patents
一种pcp靶向修饰的黑磷纳米粒子及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种PCP靶向修饰的黑磷纳米粒子,包含如下重量份的组分:黑磷10~60份、聚乙二醇5~25份、PCP多肽1~20份、阿霉素1~10份。本发明针对目前前列腺癌骨转移治疗手段的局限性,设计了一种联合治疗的纳米体系BP@PEG‑PCP/DOX,以黑磷纳米片为基础,聚乙二醇修饰增强其生理稳定性,PCP肽增强纳米体系靶向前列腺癌细胞的能力,高效负载阿霉素至肿瘤部位进行缓释;同时,由于黑磷纳米片具有优异光热性能,使得该靶向纳米诊疗平台同时具备靶向、药物/光热联合治疗功能,用于前列腺癌骨转移的精准、高效治疗,为前列腺癌骨转移的临床治疗提供新的解决方案和科学依据。
Description
技术领域
本发明涉及一种PCP靶向修饰的黑磷纳米粒子及其制备方法和应用,属于生物医学工程材料技术领域。
背景技术
前列腺癌是十分常见的恶性肿瘤,在西方男性中是最常见的恶性肿瘤类型。随着前列腺癌的进展,其会逐渐向直肠、膀胱、骨骼、淋巴结、肺和肝脏转移。其中,骨转移是影响侵袭性前列腺癌患者生活质量,导致前列腺癌患者死亡的主要原因之一。前列腺根治性切除术不适用于前列腺癌骨转移的治疗,因此,前列腺骨转移的临床治疗方式主要包括放疗、激素治疗和化疗。然而,放射治疗可能引起性功能障碍。单一激素疗法对晚期前列腺癌尤其是前列腺癌骨转移效果不显著,并可使前列腺癌逐渐发展为去势抵抗型前列腺癌。而且,传统化疗会引起患者不容忽视的全身毒副作用。考虑到这些治疗方式的缺点,有研究将金属、聚合物、水凝胶、陶瓷、脂质载体等纳米级药物传递系统应用于抗肿瘤药物的传递中,从而延长药物的血液循环时间,提高药物在肿瘤内积聚,进而达到更高效的抗肿瘤效果。
近来,新发现的黑磷成为了二维纳米材料的研究热点,引起了生物医学界的广泛关注,为纳米药物输送系统带来了新的契机。黑磷是一种非金属层状半导体,能隙变化从0.3ev(堆叠)到2.0ev(单层)不等,吸收带横跨整个紫外到近红外区域。黑磷由折叠的层状磷片通过微弱的范德华力结合而成,通常可以被剥离成多层甚至单层的超薄二维纳米片。由于黑磷独特的电子结构,研究发现黑磷可作为高效的光敏剂,能产生大量单线态氧,应用于光动力治疗。此外,黑磷的吸收谱横跨整个紫外到近红外区域,具有良好的光热转换效率,在光热治疗(PTT)领域具有潜在的应用前景。初步研究表明,黑磷作为一种无机材料,具有极佳的生物相容性,在各类细胞中尚未发现生物毒性。尤其是尺寸厚度比较小的黑磷纳米片,很容易与氧气和水发生反应,最终降解为无毒的磷酸盐和膦酸酯,磷是人体的组成元素之一,这些降解的磷氧化物与机体具有很好的相容性,对人体无害。由于黑磷遇水和氧气容易氧化降解,一些研究报道将黑磷表面修饰上聚乙二醇或TiL4,从而能延缓降低黑磷的氧化。
阿霉素又称多柔比星,是一种蒽环类抗肿瘤抗生素,对急性白血病、淋巴瘤、乳腺瘤、甲状腺瘤、肺癌、前列腺癌等实体瘤有效。但是由于其对心脏及神经等正常组织细胞有严重的毒副作用,加上用药剂量大,且能到达肿瘤病灶发挥抗肿瘤的药量很低,因此降低了其生物利用度,限制了其临床应用。因此,研究者们试图解决毒副作用大的问题,比如通过调整给药方案,研制心脏毒性低的阿霉素类似物、联合用药以及改变药物剂型,但最终研究者们普遍认为只有通过改变剂型,尤其是改良为靶向制剂是最为有效的解决办法。
靶向给药系统是指药物载体通过局部或者全身血液循环而使药物选择性地富集定位于靶组织、靶器官、靶细胞或者细胞内,在该靶部位发挥治疗作用的药物递送系统。纳米载体表面可以功能化达到主动靶向的效果,从而提高药物在肿瘤内部的聚集,这可以通过在纳米材料表面标记靶标的特定部分或配体实现。这样的配体被公认为是在特定类型的肿瘤细胞表面是过度表达。前列腺特异性膜抗原(PSMA)是一种分子量为100KDa跨膜蛋白,在人前列腺癌细胞表面以及多种实体瘤肿瘤相关的血管系统中均有高度表达,在前列腺癌的靶向治疗中呈现良好的应用前景。Romanov等人对PSMA表达阳性的LNCaP细胞受体的特异性配体,他们发现前列腺癌靶向肽(DPRATPGS序列)对LNCaP细胞表面具有良好的亲和性;Zitzmann等应用噬菌体表面展示技术制备前列腺癌靶向肽,得到靶向肽DUP-1;Kim利用此衍生肽(Ac-CFRPNRAQDYNTN)合成PEI-PEG-PCP聚合物,用于传递VEGF siRNA,结果显示此复合物显著抑制前列腺PC-3细胞的血管内皮细胞的生长。基于以上考虑,本研究试图利用PCP作为靶向肽用于制备靶向药物递送系统。前列腺癌靶向多肽(Cys-Asp-Pro-Arg-Ala-Thr-Pro-Gly-Ser,简写为PCP)由九种氨基酸通过肽键相连而成的多肽,可与PSMA表达阳性的前列腺癌细胞特异性结合,从而使含有PCP的纳米载体可以靶向性进入前列腺癌细胞。
基于以上文献资料调研,结合发明人多年来对于纳米载体的研究,在本发明中,拟采用生物相容性好、生物可降解的黑磷纳米片(BP NSs),利用PEG修饰提高其稳定性,得到BP@PEG并用其与靶向多肽PCP相结合,负载阿霉素。构建一种靶向的纳米联合治疗平台用于前列腺癌骨转移的精准、高效治疗。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种PCP靶向修饰的黑磷纳米粒子及其制备方法和应用,该PCP靶向修饰的黑磷纳米粒子用于前列腺癌骨转移的精准、高效治疗,为前列腺癌骨转移的临床治疗提供新的解决方案和科学依据。
为实现上述目的,本发明采取的技术方案为:一种PCP靶向修饰的黑磷纳米粒子,所述纳米粒子包含如下重量份的组分:黑磷10~60份、聚乙二醇5~25份、PCP多肽1~20份、阿霉素1~10份。
本发明采用液相剥离法合成黑磷纳米片,由于其优异的光学性质,使得纳米载体具有光声成像的功能;其次利用PEG修饰,用于提高黑磷纳米片的生物相容性;PCP多肽促进了纳米材料在肿瘤细胞的富集。该纳米材料提高了前列腺癌骨转移的治疗效果,为前列腺癌骨转移的高效联合治疗提供了新思路。
作为本发明所述纳米粒子的优选实施方式,所述纳米粒子包含如下重量份的组分:黑磷10份、聚乙二醇15份、PCP多肽5份、阿霉素4.1份。
第二方面,本发明提供了上述纳米粒子的制备方法,所述制备方法包括以下步骤:
(1)黑磷纳米片的制备
将黑磷分散在纯水中,通入氩气,冰浴下超声,然后离心,收集上清液;将上清液放入离心管中,离心以除去水,得到纯黑磷纳米片;将纯黑磷纳米片悬浮在PBS中,保存待用;
(2)聚乙二醇修饰的黑磷纳米片的合成
将聚乙二醇分散在步骤(1)所得的溶液中,超声,搅拌反应,离心以去除多余的聚乙二醇,并水洗,得到聚乙二醇修饰的黑磷纳米片;将聚乙二醇修饰的黑磷纳米片悬浮于PBS中,保存待用;
(3)靶向纳米载体的合成
将PCP多肽溶于二甲基亚砜中,加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺搅拌反应;并将上述溶液逐滴加入到步骤(2)所得的溶液中,室温搅拌反应过夜;反应结束,离心并水洗,得到靶向纳米载体;
(4)阿霉素的负载
将阿霉素溶于纯水中,将其逐滴加入到步骤所得的(3)溶液中,并用NaOH调节pH至7.4,避光搅拌过夜,离心,水洗后,得到PCP靶向修饰的黑磷纳米粒子。
作为本发明所述制备方法的优选实施方式,所述步骤(1)中,黑磷与纯水的质量体积比为(10~60)mg:(5~100)mL。
作为本发明所述制备方法的优选实施方式,所述步骤(1)中,超声时间为10~20h,超声强度为100~600W,离心速率为500~2000rpm,离心时间为5~20min,第二次离心速率为1000~8000rpm,第二次离心时间为10~30min。
优选地,所述步骤(1)通过液相剥离法制备黑磷纳米片(BP NSs):将一定量的黑磷(BP)分散在纯水中,通入几分钟氩气以消除溶解氧分子,减少剥离过程中的氧化。然后在冰浴条件下对混合溶液进行超声处理。将所得棕色分散液离心以去除大块的BP,并收集含有BP NSs的上清液。然后,将收集的含BP NSs的上清液放入离心管中,并在4℃条件下,离心以除去水。将获得的纯BP NSs重新悬浮在PBS中,并在4℃下储存,以便进一步使用。
优选地,所述黑磷的用量为10~60mg,优选为30mg;所述蒸馏水在反应体系中的体积为5~100mL,优选为55mL;所述超声时间为10~20h,优选是15h;所述的超声强度为100~600W,优选为300W;所述离心速率为500~2000rpm,优选是1000rpm;所述离心时间为5~20min,优选为10min;所述第二次离心速率为1000~8000rpm,优选为4000rpm;所述第二次离心时间为10~30min,优选为30min。
作为本发明所述制备方法的优选实施方式,所述步骤(2)中,步骤(1)所得的溶液的浓度为50~300μg/mL。
作为本发明所述制备方法的优选实施方式,所述步骤(2)中,超声时间为10~40min,搅拌反应时间为2~6h,离心速率为2000~6000rpm。
优选地,所述步骤(2)为:将聚乙二醇分散在适量的BP NSs溶液中。超声,搅拌反应一段时间后,将所得混合物在离心管中,并在4℃条件下,离心以去除多余的PEG,并水洗两次;将聚乙二醇修饰的黑磷纳米片(BP@PEG)样品重新悬浮于PBS中,并最终储存于4℃下,以备进一步使用。
优选地,所述PEG的质量为5~25mg,优选为20mg;所述黑磷纳米片的浓度为50~300μg/mL,优选为150μg/mL;所述超声时间为10~40min,优选是20min;所述搅拌反应时间为2~6小时,优选为4小时;所述离心速率为2000~6000rpm,优选为4000rpm。
作为本发明所述制备方法的优选实施方式,所述步骤(3)中,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的摩尔比为1:(1~3),PCP多肽与二甲基亚砜的质量体积比为(1~20)mg:(1~10)mL。
作为本发明所述制备方法的优选实施方式,所述步骤(3)中,反应时间为2~6h,离心速率为1000~8000rpm。
优选地,所述步骤(3)为:将PCP多肽溶于一定量的DMSO中,将适量的EDC和NHS加入到PCP溶液中搅拌反应一段时间。并将上述逐滴加入到步骤(2)溶液中,室温搅拌反应过夜;反应结束,在4℃条件下,离心并水洗两次,得到靶向纳米载体(BP@PEG-PCP)。
优选地,所述PCP多肽的用量为1~20mg,优选为10mg;所述EDC与NHS的摩尔比为1:(1~3),优选为1:1.5;所述DMSO的用量为1~10mL,优选为6mL;所述反应时间为2~6h,优选为4h;所述步骤(2)材料的用量为10~20mL,优选为15mL;所述离心速率为1000~8000rpm,优选为4000rpm。
作为本发明所述制备方法的优选实施方式,所述步骤(4)中,阿霉素与纯水的质量体积比为(1~10)mg:(1~4)mL,反应时间为12~20h。
优选地,所述步骤(4)为:称量一定量的DOX溶于纯水中,将其逐滴加入到步骤(3)溶液中,并用NaOH调节pH至7.4,避光搅拌过夜,离心,水洗两次后,得到PCP靶向修饰的黑磷纳米粒子(BP@PEG-PCP/DOX)。
优选地,所述阿霉素的用量为1~10mg,优选为5mg;所述的纯水的用量为1~4mL,优选为2mL;所述的反应时间为12~20h,优选为16h;所述的步骤(3)材料用量为5~15mL,优选为10mL。
与现有技术相比,本发明的有益效果为:
(1)本发明针对目前前列腺癌骨转移治疗手段的局限性,设计了一种联合治疗的纳米体系BP@PEG-PCP/DOX,以黑磷纳米片(BP NSs)为基础,聚乙二醇修饰增强其生理稳定性,PCP肽增强纳米体系靶向前列腺癌细胞的能力,高效负载阿霉素至肿瘤部位进行缓释;同时,由于BP NSs具有优异光热性能,使得该靶向纳米诊疗平台同时具备靶向、药物/光热联合治疗功能,用于前列腺癌骨转移的精准、高效治疗,为前列腺癌骨转移的临床治疗提供新的解决方案和科学依据。
(2)本发明为前列腺癌骨转移的治疗提供了一种新思路,探究出具有靶向功能的纳米载体的制备方法,该纳米载体材料具有良好的生物相容性,能够递送化疗药物,并具有光热抑制肿瘤增殖的能力,提高了前列腺癌骨转移的治疗效果。本发明合成方法简单,所需的原材料易得,有望在生物医学工程材料领域得到广泛的应用。
附图说明
图1为BP NS和BP@PEG NS的透射电镜图,其中,A为BP NS的透射电镜图,B为BP@PEGNS的透射电镜图。
图2为DOX、BP、BP-DOX的紫外谱图。
图3为BP@PEG/DOX在不同PH环境下的DOX释放曲线图。
图4为不同浓度BP@PEG-PCP对PC-3的细胞毒性的统计图。
图5为对PC-3细胞依次进行BP@PEG、BP@PEG+NIR、BP@PEG-PCP/DOX、BP@PEG-PCP/DOX+NIR处理后的细胞存活率的统计图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。
实施例1 BP NSs的制备
通过液相剥离法制备BP NSs:将30mg BP溶于55mL纯水中,通入几分钟氩气以消除溶解氧分子,减少剥离过程中的氧化。然后在冰浴条件下对混合溶液进行超声处理15h(强度:300W,开/关循环:45s/15s)。将所得棕色分散液以1000rpm离心10min以去除大块的BP,并收集含有BP NSs的上清液。然后,将收集的含BP NSs的上清液放入离心管中,并在4℃条件下,以4000rpm离心30min以除去水。将获得的纯BP NSs重新悬浮在PBS中,并在4℃下储存以便进一步使用。
实施例2 PEG修饰的BP NSs的制备
将20mg PEG分散在30mL BP NSs溶液中,BP浓度为500μg/mL。超声20分钟,搅拌4h后,将所得混合物在离心管中,并在4℃条件下,以4000rpm离心30min去除多余的PEG,并水洗两次;将纯BP@PEG样品重新悬浮于PBS中,并最终储存于4℃下以备进一步使用。对其进行形貌表征如图1所示,从图1中可以看到,BP纳米片是分散的片状结构,横向尺寸约为200纳米,PEG的修饰对其形貌并未产生影响。
实施例3 BP@PEG-PCP纳米载体的制备
取10mg PCP肽溶于6mL DMSO中,将2mol EDC和NHS加入到PCP溶液中搅拌反应4h。并将上述溶液逐滴加入到15mL实施例2的溶液中,室温搅拌反应过夜。反应结束,在4℃条件下,以4000rpm离心30min去除多余的PCP多肽,并水洗两次,得到BP@PEG-PCP。
实施例4 BP@PEG-PCP/DOX复合纳米粒子的制备
称量5mg DOX溶于2mL纯水中,将其逐滴加入到10mL实施例3所得的溶液中,并用NaOH调节pH至7.4,避光搅拌16h,离心(4000rpm,30min),水洗两次后,将样品分散在水中待进一步使用。利用紫外光谱对BP@PEG-PCP/DOX进行表征,如图2所示,从紫外图谱可以看出DOX和BP-DOX在480nm处有明显的紫外吸收峰,单纯的BP无吸收峰,则BP-DOX有紫外吸收峰,是由于DOX的存在,表明DOX的成功负载。
该BP@PEG-PCP/DOX复合纳米粒子中,黑磷为10mg,阿霉素为4.1mg,聚乙二醇为15mg,PCP多肽为5mg。
实施例5 药物释放试验
为探究DOX的体外释放情况,将DOX置于不同pH的PBS体系中(PH=7.4或者PH=5.7)。首先取1mL BP@PEG/DOX离心,然后将其分别分散到1mL不同PH含有10%的吐温80的PBS中(PH=7.4或者PH=5.7),并置于37℃恒温摇床孵育。孵育不同的时间间隔(0.5、1、3、5、20、36、48和72h)后,离心并收集上清,然后再加入新鲜的不同pH的PBS继续振荡孵育。使用紫外分光光度计测试上清液在480nm处的吸光度,根据DOX的释放标准曲线,计算出释放的DOX量,然后计算累计释放量。结果如图3所示,在pH=5.7时释放速率比在pH=7.4时增加了2倍,这是由于pH=5.7时DOX的加速溶解,使得BP和DOX之间的静电作用减弱。由于酸性肿瘤微环境的存在,BP-DOX对DOX的依赖性释放有利于肿瘤内药物的释放。
实施例6 纳米载体的细胞毒性试验
利用CCK-8检测细胞活性的方法来评价BP@PEG-PCP对PC-3细胞的细胞毒性。具体操作步骤如下:首先将PC-3细胞以5000个/孔的密度接种于96孔板中,然后将其置于二氧化碳培养箱中培养贴壁过夜。随后,吸出原有的培养基,换上新鲜的含有不同浓度的BP@PEG-PCP的完全培养基,所选的BP@PEG-PCP的浓度范围为5-200μg/mL,每个浓度有5个平行。然后在培养箱中培养24h,培养后用PBS将细胞洗涤一次后并向各孔中加入100μL的新鲜培养基(含有10%CCK-8)。置于培养箱中孵育一段时间,最后使用酶标仪检测并记录在450nm波长处的吸光度,通过以下公式计算细胞存活率:细胞存活率(%)=(实验组吸光度-空白组吸光度)/(阴性对照组吸光度-空白组吸光度)×100%。
结果如图4所示,从图中可以看出,当BP-PEG纳米粒子浓度为5μg/mL时PC-3细胞的细胞存活率分别为106.46%,可见低浓度的黑磷纳米粒子并未影响细胞增殖。此外,随着黑磷纳米粒子浓度的逐渐增加,PC-3细胞的存活率稍有下降但依然保持着很高的存活率,当BP@PEG-PCP纳米粒子浓度为50μg/mL时,细胞的存活率依次为95.05%。当BP@PEG-PCP纳米粒子浓度高达200μg/mL时,细胞的细胞存活率分别为84.03%,可见即使黑磷纳米粒子很高,但细胞毒性仍然很低。综上所述,我们认为黑磷纳米粒子本身的生物毒性很低,对细胞增殖没有明显影响,生物相容性很好,是优良安全的纳米药物载体。
实施例7 体外抑制肿瘤细胞增殖试验
前面的研究已经表明黑磷具有良好的光热性能,在NIR近红外激光照射下温度能在短时间内急剧升高,这对癌细胞光热治疗具有重要的应用价值。盐酸阿霉素(DOX)是常用的抗肿瘤药物,对癌细胞具有强烈的毒性作用,阿霉分子嵌入DNA抑制癌细胞遗传物质核酸的合成从而杀死癌细胞。
为了探究黑磷纳米药物载体在抗癌药物和光热条件下的细胞毒性,本实验选用前列腺癌细胞PC-3,设立了4组实验对照,对每组细胞分别添加以下纳米粒子共培养:BP@PEG,BP@PEG+NIR,BP@PEG-PCP/DOX,BP@PEG-PCP/DOX+NIR,以PBS组作为对照,其中NIR代表用808nm NIR近红外激光照射处理,激光功率密度为1.5W/cm2,光照时间为5min,黑磷纳米粒子的浓度均为5、10、20、40和80μg/mL。各组细胞在同等条件下共培养24h后,用CCK-8法细胞增殖检测试剂盒检测细胞活性。
结果如图5所示,经BP@PEG,BP@PEG+NIR,BP@PEG-PCP/DOX和BP@PEG-PCP/DOX+NIR处理后的PC-3细胞的相对存活率相差很大。对比经BP@PEG和BP@PEG+NIR处理的细胞存活率可见,当BP浓度为80μg/mL时,BP@PEG+NIR组对癌细胞有很大的毒性,其细胞存活率下降了60.18%,这说明光热发挥了很好的杀死癌细胞的作用但是当BP的浓度较小时,光热效果不够明显,这可能是由于纳米粒子浓度过低,光照温度上升较低,不足以导致大量的细胞死亡。对比BP@PEG与BP@PEG-PCP/DOX组,随着BP浓度的增加,DOX的浓度增加,细胞存活率下降。对比BP@PEG与BP@PEG-PCP/DOX+NIR组的细胞存活率,表明药物与光热的联合作用能够显著抑制细胞的增殖。
实施例8
一种PCP靶向修饰的黑磷纳米粒子,其制备方法如下:
(1)将10mg BP溶于5mL纯水中,通入几分钟氩气以消除溶解氧分子,减少剥离过程中的氧化。然后在冰浴条件下对混合溶液进行超声处理10h(强度:100W,开/关循环:45s/15s)。将所得棕色分散液以500rpm离心5min以去除大块的BP,并收集含有BP NSs的上清液。然后,将收集的含BP NSs的上清液放入离心管中,并在4℃条件下,以1000rpm离心10min以除去水。将获得的纯BP NSs重新悬浮在PBS中,并在4℃下储存以便进一步使用。
(2)将5mg PEG分散在BP NSs溶液中,超声10分钟,搅拌2h后,将所得混合物在离心管中,并在4℃条件下,以2000rpm离心30min去除多余的PEG,并水洗两次;将纯BP@PEG样品重新悬浮于PBS中,并最终储存于4℃下以备进一步使用。
(3)取1mg PCP肽溶于1mL DMSO中,将2mol EDC和NHS加入到PCP溶液中搅拌反应4h。并将上述溶液逐滴加入到实施例2的溶液中,室温搅拌反应过夜。反应结束,在4℃条件下,以1000rpm离心30min去除多余的PCP多肽,并水洗两次,得到BP@PEG-PCP。
(4)称量1mg DOX溶于1mL纯水中,将其逐滴加入到实施例3所得的溶液中,并用NaOH调节pH至7.4,避光搅拌12h,离心(4000rpm,30min),水洗两次后,得PCP靶向修饰的黑磷纳米粒子。
实施例9
一种PCP靶向修饰的黑磷纳米粒子,其制备方法如下:
(1)将60mg BP溶于100mL纯水中,通入几分钟氩气以消除溶解氧分子,减少剥离过程中的氧化。然后在冰浴条件下对混合溶液进行超声处理20h(强度:600W,开/关循环:45s/15s)。将所得棕色分散液以2000rpm离心20min以去除大块的BP,并收集含有BP NSs的上清液。然后,将收集的含BP NSs的上清液放入离心管中,并在4℃条件下,以8000rpm离心30min以除去水。将获得的纯BP NSs重新悬浮在PBS中,并在4℃下储存以便进一步使用。
(2)将25mg PEG分散在BP NSs溶液中,超声40分钟,搅拌6h后,将所得混合物在离心管中,并在4℃条件下,以6000rpm离心30min去除多余的PEG,并水洗两次;将纯BP@PEG样品重新悬浮于PBS中,并最终储存于4℃下以备进一步使用。
(3)取20mg PCP肽溶于10mL DMSO中,将2mol EDC和NHS加入到PCP溶液中搅拌反应4h。并将上述溶液逐滴加入到实施例2的溶液中,室温搅拌反应过夜。反应结束,在4℃条件下,以8000rpm离心30min去除多余的PCP多肽,并水洗两次,得到BP@PEG-PCP。
(4)称量10mg DOX溶于4mL纯水中,将其逐滴加入到实施例3所得的溶液中,并用NaOH调节pH至7.4,避光搅拌20h,离心(4000rpm,30min),水洗两次后,得PCP靶向修饰的黑磷纳米粒子。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种PCP靶向修饰的黑磷纳米粒子,其特征在于,所述纳米粒子包含如下重量份的组分:黑磷10~60份、聚乙二醇5~25份、PCP多肽1~20份、阿霉素1~10份。
2.如权利要求1所述的纳米粒子,其特征在于,所述纳米粒子包含如下重量份的组分:黑磷10份、聚乙二醇15份、PCP多肽5份、阿霉素4.1份。
3.如权利要求1~2任一项所述的纳米粒子的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)黑磷纳米片的制备
将黑磷分散在纯水中,通入氩气,冰浴下超声,然后离心,收集上清液;将上清液放入离心管中,离心以除去水,得到纯黑磷纳米片;将纯黑磷纳米片悬浮在PBS中,保存待用;
(2)聚乙二醇修饰的黑磷纳米片的合成
将聚乙二醇分散在步骤(1)所得的溶液中,超声,搅拌反应,离心以去除多余的聚乙二醇,并水洗,得到聚乙二醇修饰的黑磷纳米片;将聚乙二醇修饰的黑磷纳米片悬浮于PBS中,保存待用;
(3)靶向纳米载体的合成
将PCP多肽溶于二甲基亚砜中,加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺搅拌反应;并将上述溶液逐滴加入到步骤(2)所得的溶液中,室温搅拌反应过夜;反应结束,离心并水洗,得到靶向纳米载体;
(4)阿霉素的负载
将阿霉素溶于纯水中,将其逐滴加入到步骤所得的(3)溶液中,并用NaOH调节pH至7.4,避光搅拌过夜,离心,水洗后,得到PCP靶向修饰的黑磷纳米粒子。
4.如权利要求3所述的制备方法,其特征在于,所述步骤(1)中,黑磷与纯水的质量体积比为(10~60)mg:(5~100)mL。
5.如权利要求3所述的制备方法,其特征在于,所述步骤(1)中,超声时间为10~20h,超声强度为100~600W,离心速率为500~2000rpm,离心时间为5~20min,第二次离心速率为1000~8000rpm,第二次离心时间为10~30min。
6.如权利要求3所述的制备方法,其特征在于,所述步骤(2)中,步骤(1)所得的溶液的浓度为50~300μg/mL。
7.如权利要求3所述的制备方法,其特征在于,所述步骤(2)中,超声时间为10~40min,搅拌反应时间为2~6h,离心速率为2000~6000rpm。
8.如权利要求3所述的制备方法,其特征在于,所述步骤(3)中,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的摩尔比为1:(1~3),PCP多肽与二甲基亚砜的质量体积比为(1~20)mg:(1~10)mL。
9.如权利要求3所述的制备方法,其特征在于,所述步骤(3)中,反应时间为2~6h,离心速率为1000~8000rpm。
10.如权利要求3所述的制备方法,其特征在于,所述步骤(4)中,阿霉素与纯水的质量体积比为(1~10)mg:(1~4)mL,反应时间为12~20h。
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