CN1177291A - 稳定的含脂质药物传递复合体及其生产方法 - Google Patents

稳定的含脂质药物传递复合体及其生产方法 Download PDF

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CN1177291A
CN1177291A CN96192287A CN96192287A CN1177291A CN 1177291 A CN1177291 A CN 1177291A CN 96192287 A CN96192287 A CN 96192287A CN 96192287 A CN96192287 A CN 96192287A CN 1177291 A CN1177291 A CN 1177291A
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高祥
黄礼夫
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Abstract

描述了新型稳定的、浓的、有生物活性和即可使用的含脂质药物传递复合体及其生产方法。生产的复合体的生物活性比得上按现有技术掺合方法配制的调和物,由本发明的方法生产的复合体比由掺合而形成的复合体组分浓50～500倍。文中描述的方法可供大规模生产适用于基因疗法和其它应用的含脂质药物传递系统。

Description

稳定的含脂质药物传递复合体及其生产方法
本发明涉及阳离子脂质及其作为载体而用于将核酸或如蛋白质的其它大分子转入细胞的用途。确切地说，本发明涉及含脂质的药物传递复合体，该复合体是稳定的、有生物活性、可被浓缩，本发明还涉及其生产方法。
随着将诸如蛋白质或核酸这样的大分子用作治疗剂的新治疗方式的发展，已形成了这样一种需求，即要求开发新颖而有效的方法以将这类大分子传递至其合适的细胞靶。可能需要这种新传递系统的治疗实例有：基于利用特异的多肽生长因子或专效基因来代替或补充缺乏的或缺损的基因的疗法。这类疗法的临床应用，不但依赖于新传递系统的功效，还依赖于基于这些系统的技术的安全性和容易性，该技术是指适于治疗调和物的大规模药学生产、贮存和配给方法。基因疗法已成为治疗各种遗传病的日益重要的方法。提供有效治疗和甚至治愈的可能性，业已刺激巨大的努力以将该技术用于尚无有效疗法的疾病。该领域内近期的进展已表明：基因疗法不仅对单一基因病的治疗，而且对如癌症的其它更复杂疾病的治疗有显著疗效。然而，要达到有效的基因治疗尚存在一个重要的障碍，即难于设计出将治疗用核酸传递到细胞靶的新颖而有效的方法。因此，用于传递外源基因至细胞和组织的理想载体，在核酸传递、安全使用、易于大量生产和具有实用作药物的足够的稳定性方面应该是高效的。
主要以阳离子脂质体为代表的非病毒性载体，基于下述理由已被认为是适用于基因疗法的一种载体。首先，脂质体介导的基因疗法所需的质粒DNA可被广泛、常规地大规模制备，且比如逆转录病毒的病毒性载体的使用更简便、危险性更小。其次，脂质体介导的基因传递可传递RNA或DNA，这就不同于逆转录病毒介导的基因传递。因此，DNA、RNA或寡核苷酸可被直接引入细胞中。此外，阳离子脂质体是无毒的、非免疫原性的，于是可在活体内重复使用，这已由阳离子脂质体在体内成功地传递基因至插有导管的血管(Nabel，E.G.，et al.(1990)Science，249：1285-1288)、肺上皮细胞(Brigham，K.L.，et al.(1989)Am.J.Respir.CellMol.Biol.，195-200，Stribling，R.，et al.(1992)Proc.Natl.Acad.Sci.U.S.A.89：11277-11281)和全身的其它用途(Zhu，N.，et al.(1993)Science.261：209-211，Philip，R.，et al.(1993)Science，261：209-211)所证实。
尽管各种阳离子脂质体调和物、包括可商购的阳离子脂质体试剂DOTMA/DOPE(N-1，-(2，3-二油酰氧基)丙基-N，N，N-三甲基氯化铵/二油酰基磷脂酰乙醇胺)是本技术中已知的(Felgner，P.L.et al.(1987) Proc.Natl.Acad.Sci.U.S.A，84：7413-7417)，但名为DC-Chol/DOPE(3βN-(N′，N′-二甲氨基乙烷)-氨基甲酰基胆甾醇)/(二油酰基磷脂酰乙醇胺)的阳离子脂质体调和物，已由体外研究证实(Gao，X.，and Huang，L.(1991)Biochem.Biophys.Res.Commun.179：280-285)较为无毒且比DOTMA/DOPE更有效。此外，在下述广泛的活体内研究中(Plautz，G.E.，et al.(1993) Proc.Natl.Acad.Sci.U.S.A.，90：4645-4649，Stewart，M.J.，et al.(1992)Hum.Gene Ther，3：267-275)其中DC-Chol/DOPE已被证明作为核酸传递系统既安全又有效，该调和物已获得the U.S.Food and Drug Administration(FDA)和theU.K.Medicines Control Agency(MCA)的许可，并且已被应用于两例不同的基因疗法临床试验中(Nabel，G.J.，etal.(1993)Proc.Natl.Acad.Sci.U.S.A.，90：11307-11311，Caplen，N.J.，et al.(1995)Nature Medicine，1：39-46)。
然而，有很多理由认为，将DC-ChOl/DOPE和现有的其它阳离子脂质体作为载体以传递核酸至细胞靶，在大规模的治疗应用上是不合适的。首先，在现有技术的掺合方法中，形成核酸脂质体复合体而采用的脂质体与核酸的比率会导致大直径复合体的形成，  因而稳定性相当低。所以，现用的包括DC-Chol/DOPE在内的阳离子脂质体调和物，没有一种被设计成稳定的、即可使用的核酸/脂质体复合体的药物调和物。掺合方法的该局限性要求使用者在每次使用前配制复合体，不便的是要求经专门训练的人员才行。此外，每次使用前用掺和法配制复合体会引起可能的剂量变化，这就会因受体可能过量用药或用药不足而妨碍对利用这些复合体来治疗的评价。
其次，在每次使用前配制核酸/阳离子脂质体复合体的掺和方法，要求采用稀核酸溶液(低于4μg/ml)和稀脂质体分散体(低于50μM)来配制核酸/脂质体复合体以减小形成大而活性低的凝聚物的可能性。该局限性使得未采用亚于最佳条件如减小脂质体的量(会引起核酸转移活性的降低)或增大脂质体的量(会导致毒性的增高)时，难于制备小的生物活性复合体。还有，复合体必须在稀浓度下配制，实际上是临床应用的重大缺点，尤其是当肿瘤内注射该复合体时，因为在各部位只能注入小体积的复合体(Nabel，G.J.，et al.(1993)Proc.Natl.Acad.Sci.U.S.A.，90：11307-11311).
因此，本发明的目的在于，提供稳定的、有生物活性、含脂质的药物传递复合体，它可被浓缩；并提供这类复合体的生产方法。
本发明提供生产含脂质药物传递复合体的方法，该复合体具有净正电荷和/或正电性表面。在整个本说明书和权利要求书中用到的“药物”是指任意分子实体，它可以是单体或低聚的，且当它与脂质或脂质和聚阳离子复合后，被施药给个体以向受体提供治疗效果。因此，具有整体净负电荷或负电性区的大分子，有望形成本发明的传递复合体。特别适于和本发明的复合体合用的大分子例如有：DNA、RNA、寡核苷酸或负电性蛋白质。然而，带正电荷的大分子(如大的阳离子蛋白质)也有望形成本发明的复合体，是通过将阳离子大分子先与阴离子脂质或聚合物、再与阳离子脂质复合。
本发明的复合体包括将待传递的药物与阳离子脂质体混合而形成的药物/脂质复合体，其中药物与脂质的比率使得形成的药物/脂质复合体带净正电荷；还包括将药物与阳离子脂质体和聚阳离子混合而形成的药物/脂质/聚阳离子复合体，其中药物与脂质与聚阳离子的比率使形成的药物/脂质/聚阳离子复合体带净正电荷。用于药物/脂质复合体的“净正电荷”是指脂质的正电荷多于药物。用于药物/脂质/聚阳离子复合体的“净正电荷”是指阳离子脂质和聚阳离子的正电荷多于药物的负电荷。但是，应理解，本发明也包括这样的药物/脂质以及药物/脂质/聚阳离子复合体，即不管复合体的净电荷是阳性、中性甚或是阴性，只需具有正电性的表面。复合体的正电性表面可通过本技术中已知方法由复合体在电场中的迁移而测知，例如通过测定ξ电位(Martin，A.，Swarick，J.，andCammarata，A.，Physical Parmacy & Physical Chemical Principlesin the Pharmaceutical Sciences，3rd ed.Lea andFebriger，Philadelphia，1983)；或者由复合体对细胞表面的结合亲和力而测知。与具有中性或负电性表面的复合体相比，呈正电性细胞表面的复合体对细胞表面具有更强的结合亲和力。
因此，本发明涉及生产这些药物/脂质和药物/脂质/聚阳离子复合体的方法，该方法包括将待传递的药物与阳离子脂质体混合并选择性地与聚阳离子混合，其比率使得所形成的复合体具有净正电荷和/或正电性表面。
在本发明的另一实施方案中，生产药物/脂质或药物/脂质/聚阳离子复合体的方法可进一步包括，在其生产之后于过量的游离组分(药物、脂质、聚阳离子)中纯化该复合体这一步骤。
本发明的药物/脂质和药物/脂质/聚阳离子复合体通常是稳定的、可在较高浓度下生产，而且长期贮存后仍保持生物活性。这类复合体在传递核酸、蛋白质和其它大分子至细胞和组织方面有实用性。
图1显示核酸/脂质体复合体的典型的大小分布(平均直径)，是由DC-Chol/DOPE(3∶2)脂质体和pRSVL质粒DNA(2μg)按所示的脂质与DNA的比率以掺合物形式制备的。
图2A和2B显示脂质体标示物3H-胆甾烯基十六烷基醚(○)和32P-DNA标示物(■)在蔗糖梯度级分中的分布。图2A和2B的蔗糖梯度中的各级分的位置如图2A的顶端所示。
图2A显示游离脂质体(10μmolDC-Chol/DOPE(2∶3)于2ml体积中)或游离DNA(50μg PRSVL DNA于2ml体积中)经蔗糖密度梯度在超速离心分离后，3H和32P标示物的分布。图2B显示DNA-脂质复合体(由20μmol DC-Chol/DOPE(2∶3)脂质体和0.4mg pRSVL DNA混合于2ml体积中而形成)经蔗糖密度梯度在超离心分离后，3H和32P标示物的分布。
图3显示DNA/脂质体掺合复合体(○)、DNA/脂质体/聚L-赖氨酸(PLL)掺合复合体(□)、DNA/脂质复合体(●)和DNA/脂质/PLL复合体(■)在CHO细胞中的转染活性。用于形成上述复合体的DC-Chol/DOPE脂质体，如图3的底部所示含不同mol％的DC-Chol。DNA/脂质(●)和DNA/脂质/PLL(■)复合体在被测定转染活性之前先通过蔗糖密度梯度纯化。转染活性如纵轴所示，以荧光素酶活性的相对光单位表示。
图4显示DNA/脂质体掺合复合体(○)和DNA/脂质体/PLL掺合复合体(□)的转染活性与DNA/脂质(●)和DNA/脂质/PLL复合体的转染活性的比较，是当它们通过蔗糖密度梯度纯化后在4℃下贮存达130天进行比较的。用于形成上述复合体的DC-Chol/DOPE脂质体含有如图4的底部所示的不同mol％的DC-Chol。转染活性如纵轴所示，以荧光素酶活性的相对光单位表示。
图5显示用DNA/脂质体掺合复合体(○)；DNA/脂质体/PLL掺合复合体(□)；DNA/脂质复合体(●)或DNA/脂质/PLL复合体(■)处理细胞36小时后，可从CHO细胞中提取的蛋白质的浓度。DNA/脂质和DNA/脂质/PLL复合体在被测定转染活性之前先通过蔗糖密度梯度纯化。用于形成上述复合体的DC-Chol/DOPE脂质体含有如图5的底部所示的不同mol％的DC-Chol。
图6显示从有卵巢肿瘤的小鼠制备的肿瘤提取物的CAT测定结果。在第0天时，将2×106个人卵巢癌细胞经皮下注入SCID小鼠。在第14天，将含有与以掺和物形式与DC-Chol脂质体(30nmol)复合的pUCCMVCAT DNA(含30 μ g大肠杆菌(E.coli)的氯霉素乙酰转移酶基因)的100μl溶液(第1和2列，两重复试样)，或者将相同量的DNA以纯化的复合体形式(从DNA/DC-Chol脂质体以1μg/25mmol的比率制得，第3和4列；两重复试样)直接注入肿瘤内。当转染48小时后，将小鼠杀死，测定含100μg蛋白质的肿瘤提取物的CAT活性。第5列给出标准的大肠杆菌CAT的阳性对照组CAT活性。
图7A-7C显示DNA/脂质掺合复合体和纯化过的和未纯化的DNA/脂质/PLL复合体在293细胞(图7A)、C3细胞(图7B)和BL6细胞(图7C)中的转染活性。转染活性如图7A-7C的纵轴所示，以荧光素酶活性的相对光单位表示。
本发明涉及含脂质的药物传递复合体，它在pH6.0-8.0时具有净正电荷和/或正电性表面。这些复合体包括阳离子脂质、药物，并选择地进一步包括聚阳离子。本发明进一步涉及生产这些复合体的方法，其中该方法可选择地包括从过量的、各成分中纯化这些调和物的步骤。至于生产本发明的药物/脂质复合体，包括纯化步骤是优选的实施方案。应明白，将纯化步骤应用于药物/脂质/聚阳离子复合体时，则纯化后呈不含多余组分的纯净态的这些复合体的回收率，低于纯化后药物/脂质复合体的回收率；因为经密度离心的蔗糖纯化之后含药物/脂质/聚阳离子复合体的峰比含药物/脂质复合体的峰宽；且因此与游离组份的峰重叠。
本发明的含脂质药物传递复合体是稳定的，可在较高浓度下生产，并在长期贮存后仍保持生物活性。生产这些复合体的方法基于两种带相反电荷的聚合物(例如负电性的核酸和正电性的脂质)之间的结合模式，其中通过利用过量的、呈阳离子脂质体或阳离子脂质体和聚阳离子的正电荷来中和药物的负电荷，从而避免形成大而不稳定的凝聚物。业已发现，当贮存于10％蔗糖缓冲溶液中达4个月之后，本发明的复合体仍可保持其初始直径和生物活性。
本发明的含脂质药物传递复合体中包含的“药物”可以是核酸、聚阴离子蛋白质、多糖和可直接与阳离子脂质复合的其它大分子。然而，阳离子药物(例如大的阳离子蛋白质)可直接与阴离子脂质复合，或者相继地先与阴离子脂质或聚合物复合、再与阳离子脂质复合。利用该方法，可由本发明的复合体将正电性的或电中性的药物传递至细胞。
为生产带净正电荷的药物/脂质和药物/脂质/聚阳离子复合体，脂质相对药物或者脂质和聚阳离子相对药物的过量正电荷，在复合体中总的脂质相对药物或者脂质和聚阳离子相对药物可高达约30倍正电荷超出量；优选是约2～10倍电荷超出量，且最优选是约2～6倍电荷超出量。表面带正电荷的复合体可具有表面电荷相对于药物过量的类似的优选范围。若要生产脂质的正电荷多于核酸的核酸/脂质复合体，为产生在pH6.0-8.0时脂质的正电荷多于核酸的核酸/脂质复合体而与1μg核酸混合的阳离子脂质体脂质的摩尔量范围可从约0.1nmol至约200nmol脂质，优选是约5nmol至约100nmol脂质，这取决于阳离子脂质体的正电荷含量。当然，如果药物是蛋白质，则与1μg负电性蛋白质混合的脂质的量，同上述与1μgDNA混合的脂质的量相比，前者至少应小10倍，因为蛋白质的电荷密度低于核酸。本领域普通技术人员易于理解，为了使脂质的正电荷多于药物，必须将不同摩尔量的阳离子脂质体与等当量药物混合，它取决于阳离子脂质体的正电荷含量。
若要生产具有净正电荷和/或正电性表面的药物/脂质/聚阳离子复合体，掺入聚阳离子可减少脂质的量，它必须与药物混合至如此程度即脂质的正电荷少于药物的负电荷。脂质的量的减少会降低含聚阳离子的调和物的毒性。配制核酸/脂质/聚阳离子复合体所用到的阳离子脂质体的摩尔量，其范围可以是每1μg核酸约0.1nmol至约200nmol脂质，更优选是每1μg核酸约1至约25nmol脂质，这取决于阳离子脂质体的正电荷含量。通常应明白，在生产本发明的核酸/脂质和核酸/脂质/聚阳离子复合体时，生产这些复合体所需的脂质体的摩尔量将随与脂质体混合的核酸浓度的增大而增加。
本领域普通技术人员易于理解，当纯化本发明的复合体时，就在混合之前，阳离子脂质体相对药物或者阳离子脂质体和聚阳离子相对药物的正电荷超出量，将大于纯化过的复合体中脂质相对药物或者脂质和聚阳离子相对药物的正电荷超出量，因为纯化步骤导致除去过量的游离脂质和/或游离聚阳离子。
为了阐述在pH6.0-8.0时如何确定阳离子脂质、药物和聚阳离子的电荷，而提供下列实例。假如待传递的药物是DNA，则可这样确定待传递的DNA的负电荷：将待混合的DNA的量，或复合体中DNA的量除以330，330是指单个的核苷酸的分子量，此时一个核苷酸等于一个负电荷。因此，1μgDNA的负电荷是3.3nmol。
至于10nmolDC-Chol/DOPE(2∶3)脂质体，脂质的有效电荷可计算如下：将总脂质体脂质(10nmol)的量乘以0.4(总脂质体脂质的40％是阳离子脂质DC-Chol)而得脂质体中4nmolDC-Chol。由于在pH6-8时，一分子DC-Chol有一个正电荷，则在混合时或复合体中，脂质体脂质的有效正电荷是4.0nmol。当然，本领域技术人员易于明白，在pH6-8.0时，其它阳离子脂质，每分子阳离子脂质的正电荷量可能少于或大于DC-Chol。
假如待混合而形成复合体的聚阳离子是聚L-赖氨酸(PLL)的溴盐，则混合时PLL的正电荷可由待混合的PLL的量除以207而得，207是指一个赖氨酰残基的分子量，此时一个赖氨酰残基等于一个正电荷。因此，1μgPLL的正电荷约为5.0nmol。若要计算所形成的复合体中赖氨酰残基贡献的正电荷，则考虑到反荷离子如果存在的话的重量，将复合体中存在的赖氨酸的量除以一个赖氨酰残基的分子量。
将上述计算法应用于本文表1中所示的数据(参见实施例3)，可说明在混合DNA和脂质体时，和纯化由DNA和脂质体混合而产生的复合体之后，如何计算正电荷与负电荷的比率。在实施例3的表1中，将0.4mgDNA和20μmol阳离子DC-Chol/DOPE脂质体混合而产生DNA/脂质复合体。至于DC-Chol/DOPE比率为4∶6的阳离子脂质体，求得脂质体脂质的正电荷含量为8000nmol，而有待与脂质体混合的0.4mgDNA的负电荷含量为1320nmol，都基于上文中给出的计算范例。因此，混合时正电荷与负电荷的比率是6.06(8000除以1320)。不过，一旦复合体被纯化后，该纯化过的复合体中脂质与DNA的比率为12.7nmol脂质/μgDNA，如表1所示(见“4：6行”)。12.7这一比率转化成正电荷与负电荷的比率为1.5，于是表明纯化作用可除去游离脂质体的过量正电荷。
也是在表1中，其中将4μmol脂质体(4：6DC-Chol/DOPE)和1mgPLL与0.4mg DNA混合而配制成DNA/脂质/PLL复合体，可如下求算混合时正电荷与负电荷的比率：基于上文中给出的计算范例，4μmol脂质体脂质贡献1600nmol正电荷，1mgPLL贡献5000nmol正电荷，而0.4mgDNA贡献1320nmol负电荷。所以，将脂质体、PLL与DNA混合时，正电荷与负电荷的比率为5。
本领域技术人员进一步理解，复合体的净电荷可这样确定：使用合适的分析技术如利用各组分的放射性同位素标记或采用元素分析法，通过测定复合体中DNA、脂质和如果存在时的聚阳离子含量来确定。一旦知道在给定的pH下复合体中各组分(DNA、脂质和如果存在时的聚阳离子)的量，就可考虑已知的或经分析测知的各级分的PK值来求算给定pH下复合体的大致的净电荷。
在一个优选的实施方案中，药物是核酸序列，优选是编码有治疗用途的基因产物的核酸序列。
在本发明的一个实施方案中，生产在pH6-8.0时带有净正电荷和/或正电性表面的核酸/脂质复合体的方法包括：将核酸与阳离子脂质体结合，其中核酸与脂质的比率使得形成的核酸/脂质复合体中脂质的正电荷多于核酸。
在另一实施方案中，可将核酸和阳离子脂质体与聚阳离子混合，其中核酸与脂质与聚阳离子的比率使得形成的核酸/脂质/聚阳离子复合体在pH6-8时，脂质和聚阳离子的正电荷多于核酸。
在一个优选的实施方案中，是这样生产核酸/脂质和核酸/脂质/聚阳离子复合体的：将核酸缓慢地加入脂质体或者脂质体和聚阳离子的溶液，并用搅拌棒混合而混合片刻。此外，可将脂质体或脂质体/聚阳离子混合物通过第一入口加到单一槽内，同时将核酸通过第二入口加到该槽内。然后同时用机械方法在同一槽中混合这些组分。
与药物或者与药物和聚阳离子混合而形成本发明的复合体的阳离子脂质体，可只含阳离子脂质或是含有结合有中性磷脂的阳离子脂质。合适的阳离子脂质包括但不局限于：1，2-双(油酰氧基)-3-(三甲铵基)丙烷(DOTAP)；N-1，-(2，3-二油酰氧基)丙基-N，N，N-三甲基氯化铵(DOTMA)或其它N-(N，N-1-二烷氧基)-烷基-N，N，N-三取代的铵表面活性剂；1，2-二油酰基-3-(4′-三甲铵基)丁酰-sn-甘油(DOBT)或胆甾醇(4′-三甲铵基)丁酸酯(ChOTB)， 中三甲铵基通过丁酰间隔臂连接到双链(对DOTB)或胆甾烯基(对ChOTB)上；/DORI(DL-1，2-二油酰基-3-二甲氨基丙基-β-羟乙铵)或DORIE(DL-1，2-O-二油酰-3-二甲氨基丙基-β-羟乙铵)(DORIE)或其类似物，如W093/03709中所述；1，2-二油酰-3-丁二酰-sn-甘油胆碱酯(DOSC)；胆甾醇半丁二酸酯(ChOSC)；脂多胺类如双十八烷基酰氨基甘氨酰精胺(DOGS)和双十六烷酰磷脂酰乙醇酰胺精胺(DPPES)或阳离子脂质如US PatentNumber5,283,185中所示，胆甾醇-3β-羧基-酰氨基-亚乙基碘化三甲铵，1-二甲氨基-3-三甲铵基-DL-2-丙基-胆甾醇羧化碘，胆甾醇-3β-羧基酰氨基亚乙胺，胆甾醇-3β-羟丁二酰胺-亚乙基碘化三甲铵，1-二甲氨基-3-三甲铵基-DL-2-丙基-胆甾醇-3β-羟丁二酸碘，2-(2-三甲铵基)-乙基甲氨基乙基-胆甾醇-3β-羟丁二酸碘，3βN-(N′，N′-二甲氨基乙烷)氨基甲酰基胆甾醇(DC-Chol)，和3β-N-(聚乙烯亚胺)-氨基甲酰基胆甾醇。
优选的阳离子脂质的实例包括：胆甾醇-3β-羧基酰氨基亚乙基碘化三甲铵，1-二甲氨基-3-三甲铵基-DL-2-丙基-胆甾醇羧化碘，胆甾醇-3β-羧基酰氨基亚乙胺，胆甾醇-3β-羟丁二酰胺亚乙基碘化三甲铵，1-二甲氨基-3-三甲铵基-DL-2-丙基-胆甾醇-3β-羟丁二酸碘，2-(2-三甲铵基)乙基甲氨基乙基-胆甾醇-3β-羟丁二酸碘，3βN-(N′，N′-二甲氨基乙烷)-氨基甲酰基-胆甾醇(DC-Chol)，和3βN-(聚乙烯亚胺)-氨基甲酰基胆甾醇。
由于本发明的复合体的一个特征是其贮存稳定性(即它们形成之后可长时间保持小直径和保留生物活性的能力)；本领域普通技术人员应理解，优选的阳离子脂质是其中的亲脂基和氨基之间的键在水溶液中稳定的脂质。虽然阳离子脂质中的这种键包括酰胺键、酯键、醚键和氨基甲酰基键，但优选的阳离子脂质具有氨基甲酰基键。具有氨基甲酰基键的优选的阳离子脂质实例是DC-Chol。本领域技术人员易于明白，含有多于一种阳离子脂质的脂质体可用于生产本发明的复合体。例如，业已公开了包括两种阳离子脂质的脂质体，即包括赖氨酰-磷脂酰乙醇胺和β-丙氨酰胆甾醇酯的脂质体(Brunette，E.et al.(1992)Nucl.Acids Res.，20：1151)。
应进一步明白，就适于与药物和选择性地与聚阳离子混合以形成本发明的复合体的阳离子脂质体来说，本发明的方法不只限于使用上述脂质，只要能产生阳离子脂质体，也可使用任何脂质成分。
因此，除了阳离子脂质，用来形成本发明的复合体的阳离子脂质体可包括除阳离子脂质之外的其它脂质。这些脂质包括但不限于：溶解脂例如溶血卵磷脂(1-油酰基溶血卵磷脂)，胆甾醇，或中性磷脂包括二油酰磷脂酰乙醇胺(DOPE)或二油酰卵磷脂(DOPC)。本发明的脂质复合体也可含有负电性的脂质和阳离子脂质，只要所形成的复合体净电荷为正电荷和/或复合体表面是正电性的即可。本发明的负电性脂质是这样的脂质，即该脂质包括至少一种在生理pH或其附近时带净负电荷的脂质，或是这些脂质的混合物。合适的负电性脂质包括磷脂酰甘油和磷脂酸或相似的磷脂类似物。
进一步设想，在形成本发明的复合体而用到的阳离子脂质体中，可改变脂质的比率以包括结合有胆甾醇或结合有溶解脂质或中性脂质混合物的大部分阳离子脂质。如果要将选定的阳离子脂质与另一脂质结合，则优选的脂质是中性磷脂，最优选是DOPE。
制备用来生产本发明的含脂药物传递复合体的脂质体的方法，是通常本领域普通技术人员已知的。有关脂质体制备方法的综述可参见：LiPosome Technology(CFC Press Ny 1984)；Liposomesby Ortro(Marcel Schher，1987)；Methods Biochem. Anol.33：337-462(1988)和U.S.Patent 5,283,185。这些方法包括冻融挤出和超声处理。单层脂质体(平均直径小于约200nm)和多层脂质体(平均直径大于约300nm)均可用作生产本发明的复合体的起始组分。
在用于生产本发明的药物/脂质复合体的阳离子脂质体中，存在于脂质体中的阳离子脂质占总脂质体脂质的约10～约100mol％，优选是约20～约80mol％，且最优选是约20～约60mol％。中性脂质若包括于脂质体中，则它存在的浓度为总脂质体脂质的约0～约90mol％，优选为约20～约80mol％，且最优选为40～80mol％。脂质体中若包括负电性脂质，则它存在的浓度占总脂质体脂质的约0mol％～约49mol％，优选为约0mol％～约40mol％。在一个优选的实施方案中，脂质体含有阳离子脂质和中性脂质，最好是DC-Chol和DOPE，其比为约2∶8～约6∶4。应进一步明白，本发明的复合体可含有修饰脂质、蛋白质、聚阳离子或受体配件，它们起导向因子的作用，将复合体引导至特定的组织或细胞类型。导向因子的实例包括但不限于：脱唾液酸糖蛋白、胰岛素、低密度脂蛋白(LDL)、叶酸和针对细胞表面分子的单克隆和多克隆抗体。可能的靶包括但不限于：肝、血细胞、内皮细胞和肿瘤细胞。
应进一步了解，本发明的复合体的正电荷不但受复合体的脂质组成的影响，还受形成药物/脂质复合体的溶液pH的影响。例如，提高pH(碱性更强)会逐渐中和阳离子脂质DC-Chol的叔胺的正电荷。在一个优选的实施方案中，本发明的复合体是在可使复合体带有净正电荷和/或具有正电性表面的pH下被制备和贮存的。优选的pH范围是pH6.0-8.0，最优选是pH7.0-7.8。
若欲将聚阳离子与核酸和阳离子脂质体混合，则聚阳离子可选自分子量约300～约200,000的有机聚阳离子。这些聚阳离子在pH7.0时还优选具有约3～约1000的化合价。聚阳离子可以是天然的或合成的氨基酸、肽、蛋白质、多胺、糖类和任何合成的阳离子聚合物。聚阳离子的非限制性实例包括：聚精氨酸、聚鸟氨酸、鱼精蛋白和聚赖氨酸、Polybrene(海美溴铵)、组蛋白、阳离子树枝状物(cationic dendrimer)、精氨、亚精氨和来自SV40大T抗原的合成多肽，它有多余的正电荷并代表核定位信号。优选的聚阳离子是聚L-赖氨酸(PLL)。在生产本发明的核酸/脂质/聚阳离子复合体时，固定聚阳离子与核酸的比率而改变脂质体的量。不过，本领域技术人员会认识到，聚阳离子与核酸的比率受有待与核酸和聚阳离子混合的脂质体的电荷密度的影响。例如，若脂质体的电荷密度因脂质体中脂质组成的变化而减小(例如降低脂质体中阳离子脂质与中性脂质的比率)，则可增加有待与核酸和脂质体混合的聚阳离子的量，以补偿因脂质体导致的正电荷的减少。但是，当用到聚阳离子时，优选是使用亚饱和量的聚阳离子(即不会将核酸的全部负电荷饱和的量)以便使阳离子脂质与核酸复合。这样，在本发明优选的一个实施方案中，甚至将聚阳离子与脂质和核酸混合时，也使用相对于核酸而言正电荷过多的脂质。可与1μg核酸和变化量的阳离子脂质体混合的聚阳离子的量，在本发明中为约0.01μg～约100μg聚阳离子/μg核酸，优选为约0.1μg～约10μg聚阳离子/μg核酸。
如果需要从过量的游离DNA、游离脂质体和游离聚阳离子中纯化核酸/脂质和核酸/脂质/聚阳离子复合体，则可采用离心法通过蔗糖密度梯度或适合于形成密度梯度的其它介质来进行纯化。然而应明白，也可运用其它纯化方法如色谱法、过滤、相分配、沉淀或吸附法。在优选的方法中，采用离心法通过蔗糖密度梯度来纯化。蔗糖梯度可在约0％蔗糖～约60％蔗糖范围，优选是约5％蔗糖～约30％蔗糖。配制蔗糖密度梯度的缓冲剂可以是适于贮存含复合体的级分的任何缓冲水溶液，且优选是适于将复合体施药至细胞和组织的缓冲剂。优选的缓冲剂是pH7.0～8.0 Hepes。
应理解，在本发明中，优选的核酸序列是可导引蛋白质表达的那些。这种序列可用惯常方法插入本领域技术人员知道的质粒表达载体内，再与阳离子脂质体或者脂质体和聚阳离子混合而形成本发明的含脂质药物传递复合体。与阳离子脂质体或与阳离子脂质体和聚阳离子混合的核酸的量可为约0.01μg～约10mg，优选是约为0.1μg～约1.0mg。应明白，当质粒表达载体中含有感兴趣的核酸时，则上述核酸的量是指含有感兴趣的核酸的质粒。
纯化本发明的核酸/脂质和核酸/脂质/聚阳离子复合体，是为了将含于所生成的复合体中的核酸和脂质浓缩约50倍～约500倍，使得复合体中所含的脂质含量高达约40μmol/ml，且核酸含量可高达约2mg/ml。
本发明的方法生产的复合体直径小于约400nm，优选小于约200nm，且更优选小于150nm。
用本发明的方法形成的复合体在4℃下贮存时可稳定长达约一年。复合体从蔗糖梯度中收集后可贮存于10％蔗糖溶液中，或者可将复合体冻干而在使用前重新溶于10％蔗糖溶液中。在一优选的实施方案中，是将复合体贮存于溶液中。本发明的复合体的稳定性可用专门的方法测定，以确定贮存一段时间后复合体的物理稳定性和生物活性。复合体的物理稳定性的测定，可用本领域普通技术人员所知的方法通过测定复合体的直径来进行，这些方法例如包括：电子显微镜、凝胶过滤色谱，或者采用准弹性光散射法(其中用附如实施例中所述的Coulter N4SD粒度计)。当贮存后复合体的直径与纯化时测定的复合体直径相比，前者增大不高于100$，优选是不高于50％，且最优选是不高于30％时，则认为贮存后复合体的物理稳定性“实质上未改变”。
用于测定复合体的生物活性的分析方法视复合体中所含的药物而定。例如，若药物是编码基因产物的核酸，则可在本领域普通技术人员采用的转染细胞用的转染条件下，通过用DNA和阳离子脂质体的掺和物体外处理细胞而测定生物活性。可被复合体转染的细胞包括那些可被DNA/脂质体掺合复合体转染的细胞。然后将贮存过的复合体的活性与经掺和制得的复合体的转染活性进行比较。如果药物是蛋白质，则可用适于该蛋白质的生物分析法来测定活性。
本领域技术人员应进一步明白，本发明的复合体可用作活体内基因疗法的载体。
利用本发明的复合体的治疗用调和物优选包括生理相容的缓冲液中的复合体，例如磷酸盐缓冲的盐水、等渗盐水或低离子强度的缓冲液如10％蔗糖于H2O(pH7.4-7.6)或于Hepes(pH7-8，更优选的pH为7.4-7.6)中。复合体可以气溶胶或液体溶液的形式施用于肿瘤内、静脉内、气管内、腹膜内和肌内。
文中引用的任何文献或专利均并入作参考。下述实施例阐述本发明的各方面，但决不是想限制其范围。
实施例
原料
DOPE购自Avanti Polar Lipid，Inc.(Alabaster，AL).pRSVL，这种在Rous肉瘤病毒长末端重复序列控制下编码荧光素酶基因的质粒(De Wet，J.R.et al.(1987)Mol.Cell.Biol.，7：725-737)在大肠杆菌中扩增并利用标准的CsCl-EtBr超离心法纯化(Sambrook，J.，Fritsch，E.F.and Maniatis，T.，Molecular Cloning：ALaboratory Manual(2d ed)Cold Spring Harbor LaboratoryPress：New York(1989))。所有的组织培养基均得自 GibcoBRL(Gaithersburg，MD)。人胚胎肾293细胞、CHO(中国仓鼠卵巢细胞)、BL6和BHK(幼仓鼠肾细胞)均得自American TypeCulture Collection(Rockville，MD)。小鼠肺细胞(MLC)系源自Balb/c小鼠的肺的初级培养细胞，由Dr.S.Kenned(Oak RidgeNational Laboratory，TN)而得。293、BL6、BHK和MLC细胞均用DMEM培养基培养，CHO细胞用F12培养基培养，而C3Hela细胞于RPMI-1640培养基中培养。所有培养基中均添加10％胎牛血清(Hyclone Laboratories，Inc.，Logan，UT)和100单位/ml青霉素和100μg/ml链霉素。聚L-赖氨酸氢溴化物(MW 3000和MW25，600)和其它化学药品均得自Sigma(St Louis，MI)。
DC-Chol按Gao和Huang(1991)的方法合成(Gao，X.，andHuang，L.(1991)Biochem.Biophys.Res.Commun.，179：280-285)，其纯化步骤中作如下改动：在反应后加入10ml己烷，并用10ml水提取混合物三次。收集有机相并在4℃下真空干燥。在加热下将所得固体溶于少量无水乙醇，并在0℃的乙腈中重结晶。  DC-Chol的纯度至少＞95％，是由TLC法和1H-NMR分析测得的，且产率约为 70％，这比Gao，X.和Huang，L.先前报道过的方法((1991)Biochem.Biophys.Res.Commun.，179：280-285)有显著改进。
方法
复合体的制备和纯化
在20mM总脂质浓度下的阳离子脂质体的制备为：按报道过的方法(Gao，X.，and Huang，L.(1991)Biochem.Biophys.Res.Commun.，179：280-285)，通过超声处理法用不同比率的DC-Chol和DOPE制备。基于定量目的包括有痕量的3H胆甾醇十六烷基醚(Amersham，Arlington Heights，IL)。这些脂质体的直径大小介于100～150nm，是利用Coulter N4SD粒度计(Coulter Electronics，Inc.，Hialeah，FL)通过准弹性光散射法测定的。除非另有说明，下述实施例中的DNA/脂质复合体是在典型的实验室规模制备的：将各实施例中所示剂量的、在体积为1ml的2mM Hepes缓冲液(pH7.6)中的游离DC-Chol/DOPE脂质体加入15×7.5聚苯乙烯培养管(Baxter，McGraw Pare，IL)，管中放入一颗微磁力搅拌子，并充分混合此溶液。然后按各实施例中所示的pRSVL DNA剂量，自贮液(0.2mg/ml，于2mM Hepes缓冲液中，pH7.6)中逐滴加入脂质体溶液，历时3分钟。为定量目的，采用缺口平移试剂盒(Promega，Madison，WI)和 32P dCTP(Amersham，ArlingtonHeights，IL)以32P标记的痕量的pRSVL被包括其中。
为制备纯的脂质/PLL/DNA复合体，将如各实施例中所示剂量的上述0.2mg/ml DNA溶液加入含有如各实施例中所示剂量的脂质体和PLL的1ml PLL/脂质体混合物中。将DNA/脂质复合体加注到由分别为0.5ml的5％、7.5％、10％和15％蔗糖(W/W)组成的蔗糖梯度顶层，并将DNA/脂质/PLL复合体加注到由分别为0.5ml的5％、10％、15％、20％、25％和30％蔗糖(W/W)组成的蔗糖梯度顶层。然后在4℃下通过100,000g下超离心从游离的脂质和PLL中纯化DNA/脂质和DNA/脂质/PLL复合体。离心后，将200μl各级分从管顶部至管底部取出。用闪烁计数器对来自各级分的等分试样分析3H和32p放射活性。收集并汇集含有32p峰值的级分。然后测定这些汇集的级分的粒度和转染活性。
体外转染分析
通过下述实施例中细胞的体外转染来分析上述复合体的生物活性。简言之，将生长于48孔板中的细胞与用0.5ml CHO-S-SFM(Gibco BRL)稀释的DNA/脂质复合体或者与按Gao和Huang(1991)(Gao，X.，and Huang，L.(1991)Biochem.Biophys.Res.Commun.，179：280-285)的方法制备的DNA/脂质体掺合复合体一起培养。至于在PLL存在下用DC-Chol脂质体转染pRSVLDNA，是将脂质体先与PLL混合、再与DNA复合而进行的。所有的转染作用均在37℃下进行4小时。转染后，在含10％胎牛血清的合适的培养基中将细胞进一步培养36小时。再以PBS洗涤细胞，并用由荧光素酶分析试剂盒(Promega，Madison，W1)提供的100μlIX溶胞缓冲液溶解。取4μl溶胞产物试样分析荧光素酶活性，其中用到来自重组的荧光素酶分析盒的100μl底物溶液与AutoLumat LB953光度计(Berthold，Germany)。各溶胞产物的蛋白质浓度，是按厂商的说明(Pierce，Rockford，IL)采用考马斯蓝染料方法来分析的。
实施例1
以DNA与脂质比率确定DNA/脂质复合体的大小
进行该实验以显示：由掺合而形成的DNA-脂质复合体的大小，随着与脂质体混合的DNA的比率的改变而变化。简言之，将pRSVL质粒DNA(2μg)与不同量的DC-CHOL/DOPE(3∶2)脂质体于pH7.6的2mM Hepes缓冲液中混合，终体积为500μl。在7分钟后，用Coulter N4SD激光散射粒度计在单一模式下操作以确定复合体的大小。
如图1中所示，当DNA过量时(脂质体与DNA的比率小于7)，不会形成大的凝聚物；但当脂质与DNA的比率为电中性时(～10)，则复合体的直径达极大值。此外，当脂质体与DNA的比率恒定于10nmol/μg时，则DNA和脂质体的浓度均增大时，复合体的大小也会增大，并最后形成沉淀。但是，当增大脂质体与DNA的比率时，复合体的大小逐渐降低直至恒定(250-300nm)，此时脂质体与DNA的比率高于25nmol脂质/μgDNA。该结果可能是由于DNA可能被过量的脂质体包覆，所以复合体之间不会发生凝聚。
基于图1中给出的数据，脂质/DNA复合体的制备如下：利用50nmol/Pμg的脂质体与DNA的比率，将200μg/ml的DNA溶液缓慢地加入过量的(10μmol)脂质体中而得。所形成的复合体大小约为250nm。如果将脂质体与DNA的比率改为25nmol/μg，则复合体的大小增大到大约350nm。利用25nmol/μg或50nmol/μg的比率形成的复合体似乎是物理稳定的，因为在4℃下贮存4周之久也未形成沉淀。
实施例2
DNA/脂质复合体的纯化
当将DNA与脂质体在1μg/50nmol的比率下混合后，则可看到过量的游离脂质体脂质与DNA/脂质复合体共存。由于过量的游离脂质对细胞有毒，所以进行实验以确定是否可以用密度梯度超离心法将游离脂质体脂质从DNA/脂质复合体中分离。简言之，游离脂质体(10μmol的DC-Chol/DOPE(2∶3)于2ml体积中)；游离DNA(50μgpRSVL于2ml体积中)和通过将20μmol DC-Chol/DOPE(2∶3)与0.4mg pRSVL质粒DNA(50nmol/μg)混合而形成的DNA/脂质复合体，分别在由各0.5ml的5％、7.5％、10％和15％蔗糖(W/W)组成的梯度上于4℃下以100,000g离心30分钟。再分别从管顶至管底采集200μl各级分，并分析DNA标示物(32P，■)和脂质标示物(3H，O)的分布。图2A和2B显示在蔗糖梯度上，游离脂质体脂质、游离DNA(图2A)和DNA/脂质复合体(图2B)的典型分离结果。图2B的结果显示：离心后，复合体在10％蔗糖层形成一条主带。比较起来，图2A显示：游离DNA或游离脂质体脂质的大部分放射活性分布在管的上半部，且并未进入蔗糖梯度。此外，尽管在图2B中3H的峰和32P的峰共存于第16号级分，仍有相当量的3H分布于级分1～10，表明过量的游离脂质体脂质已从DNA/脂质复合体中充分分离。
实施例3
纯化过的脂质/DNA和脂质/PLL/DNA复合体的物理稳定性
将20μmol各种DC-Chol/DOPE组成(参见表1)的脂质体与0.4mgpRSVL质粒DNA在1μg DNA/50nmol脂质的比率下混合，以形成DNA/脂质复合体。而脂质/PLL/DNA复合体的形成如下：将4μmol各种DC-Chol/DOPE组成的脂质体与1mg PLL(MW＝3000)和0.4mg pRSVL质粒DNA混合。然后通过如方法部分中所述的蔗糖梯度离心，将这两种复合体从游离脂质、游离DNA和游离PLL中纯化。收集并汇集峰级分。收集后立即(0天)或在4℃下贮存于10％蔗糖中达120天后分析所汇集的试样的直径。表1给出这些分析结果。
表1.纯化后的脂质/DNA和脂质/PLL/DNA预复合体的物理稳定性
脂质体组成物(DC-Chol/DOPE) PLL(μg/μgDNA) 大小(nm)0天    120天 纯化后的复合体脂质/DNA比率(nmol/μg) DNA的回收率(总数的％)
2∶8 0 168 280 23.2 51
3∶7 0 187 252 14.0 66
4∶6 0 175 195 12.7 73
5∶5 0 174 210 13.2 70
6∶4 0 198 232 10.1 69
2∶8 2.5 165 287 20.8 17
3∶7 2.5 99 101 19.2 22
4∶6 2，5 138 132 38.3 29
5∶5 2.5 184 178 22.4 27
表1中所示的数据显示：纯化后的脂质/DNA和脂质/PLL/DNA复合体在第0天时尺寸很小(小于200nm)，并且其尺寸不因贮存而急剧增大。再者，纯化后的复合体中DNA与脂质的比率介于10到23nmol脂质/μgDNA之间，这取决于所用的脂质体组成；且贮存120天后该比率不会改变。还可看出脂质体中DC-Chol的浓度与复合体中脂质含量之间的倒数关系，表明富含DC-Chol的脂质体比含更少DC-Chol的脂质体显示更强的DNA结合或电荷中和活性。最右一栏显示经蔗糖密度梯度纯化后，32P标记的DNA从DNA/脂质和DNA/脂质/PLL复合体中的回收率。结果表明，从不含PLL的复合体中DNA的回收率高于从含PLL的复合体中DNA的回收率。
实施例4
纯化后的复合体在各种细胞中的生物活性
由于通过脂质体与DNA在脂质对DNA的高比率下混合而形成的DNA/脂质复合体既小又长期稳定，所以可进行实验以比较这些复合体的转染活性与由掺合法配制的DNA/脂质体复合体的活性。
在一个实验中，将培养于48孔板中的CHO细胞用1μgpRSVL只和不同脂质组成的10nmol DC-Chol/DOPE脂质体形成的掺合物(○)或二者还与1μgPLL(MW＝3,000)形成的掺和物(□)处理4小时；或者将细胞用纯化过的DNA/脂质复合体(●)或用纯化过的DNA/脂质/PLL复合体(■)处理，复合体是通过将1μgDNA与50nmol DC-Chol/DOPE脂质体(DNA/脂质复合体)或与10nmol DC-Chol/DOPE脂质体和1μg PLL(DNA/脂质/PLL复合体)混合，接着通过如方法部分中所述的蔗糖密度梯度离心而形成的。处理后36小时，将细胞溶于100μl溶胞缓冲液，并取4μl溶胞产物利用100μl荧光素酶底物溶液分析荧光素酶活性。然后在20秒内计算荧光素酶活性示于图3的结果显示：转染CHO细胞的最优选的脂质体组成为40％DC-Chol和60％DOPE。此外，在额外的1μg聚L-赖氨酸(PLL，MW＝3,000)存在下，在多数情况下均可看出转染活性增高2-7倍。特别有趣的是，当将相等量的DNA加至细胞时，则纯化后DNA/脂质复合体的活性类似于DNA/脂质体掺合复合体的活性。但是，纯化过的DNA/脂质/PLL复合体的转染活性比由掺合法制备的DNA/脂质体/PLL复合体的转染活性低约30％～50％。
为证实在CHO细胞中所得的结果不是细胞专一的，于是在两种其它细胞即BHK和小鼠肺细胞(MLC)中，将纯化过的DNA/脂质和DNA/脂质/PLL复合体的转染活性，与由掺合法形成的DNA/脂质体复合体的转染活性作了比较。
简言之，用与10nmol DC-Chol脂质体复合的1μg pRSVL(掺合复合体)转染在48孔板上生长至60％铺满度的细胞(BHK或MLC)，用在1μg/50nmol的DNA/脂质体比率下与脂质体混合的相同量的DNA转染细胞，所述二物质的混合是为了产生纯化过的DNA/脂质复合体；或用1μg/10nmol/2μg的DNA/脂质体/PLL比率下制备的、纯化过的DNA/脂质/PLL复合体转染细胞。再在转染后36小时收集细胞，按方法部分中所述测定转染过的细胞溶胞产物的荧光素酶活性。这些实验结果如表2和3中所示。
表2.用pRSVL转染的BHK细胞中荧光素酶基因的表达
荧光素酶活性(相对光单位×10-3)
脂质体组成物(DC-Chol/DOPE) DNA/脂质体掺合复合体 纯化过的DNA/脂质复合体 纯化过的DNA/脂质/PLL复合体
2∶83∶74∶65∶5 91.8±9.561.2±19.9438.2±14.4837.8±8 110.1±5.21886.8±266.71638.8±63.91015.0±41.2 214.6±41.1151.7±62.9446.3±16.9234.2±46.4
表3.用pRSVL转染的小鼠肺细胞中荧光素酶基因的表达
荧光素酶活性(相对光单位×10-3)
脂质体组成物(DC-Chol/DOPE) DNA/脂质体掺合复合体 纯化过的DNA/脂质复合体 纯化过的DNA/脂质/PLL复合体
2∶83∶74∶65∶5 1.1±0.71.5±1.03.1±0.20.1±0.0 0.4±0.20.3±0.02.0±0.31.5±1.2 0.3±0.14.1±1.31 4.6±3.110.1±2.3
有趣的是，在BHK细胞系中，纯化过的DNA/脂质复合体的转染活性显著高于经掺合而形成的DNA/脂质体复合体的转染活性。至于如MLC的细胞，因难于转染，所以由DNA/脂质体/PLL混合物制得的纯化过的复合体明显优于掺合复合体和不含PLL的、纯化过的DNA/脂质复合体。
为了确定脂质/PLL/DNA复合体是否可用不同比率的脂质/核酸和不同分子量的PLL(与先前的实施例中所用的相比)来制备，于是进行下述实验。按表4中所示的脂质与DNA的比率，由20μgpRSVL质粒DNA、10μg聚L-赖氨酸(MW25,600)和DC-Chol/DOPE脂质体(4.5/5.5摩尔比)制得脂质/聚L-赖氨酸/DNA复合体。然后将所得复合体按方法部分中所述用蔗糖梯度超离心纯化。纯化后含0.5μg DNA的复合体的等分样被用于转染CHO细胞，然后测定荧光素酶活性。该实验的结果如表4中所示。
表4.脂质/DNA比率对纯化过的含聚L-赖氨酸(MW25,600)的复合体的影响
比率(nmol脂质/μgDNA)3.36.612.5 纯化后的复合体组成物(nmol脂质/μgDNA)1.12.54.3 纯化过的复合体的大小(nm)8998101 转染活性b(计数(SD)X10-3)108(5)6,065(604)5,846(668)
20.0 9.6 35 7,633(977)
结果表明，若增大聚阳离子的存在量，则可用较低比率的脂质/DNA生产具有相当大转染活性的DNA/脂质/聚阳离子复合体。
实施例5
贮存后的复合体的转染活性
用下述各种复合体处理培养于48孔板中的CHO细胞达4小时：1μg pRSVL只和不同DC-Chol/DOPE组成的10nmol DC-Chol/DOPE脂质体的掺合物(○)；或前述掺和物中再掺有1μgPLL(MW3,000)的掺和物(□)：或者纯化过的DNA/脂质复合体(●)；或DNA/脂质/PLL(●)复合体，所述各种复合体被贮存于4℃下的10％蔗糖中达130天。纯化过的复合体是这样形成的：将1μg pRSVL只与50nmol不同DC-Chol/DOPE组成的DC-Chol/DOPE脂质体混合(DNA/脂质)，或者与10nmol DC-Chol/DOPE脂质体和1μg PLL混合(DNA/脂质/PLL复合体)，接着按方法部分中所述通过蔗糖密度梯度进行离心。结果表明，从用贮存过的DNA/脂质和DNA/脂质/PLL复合体转染的细胞制得的溶胞产物，其荧光素酶活性比得上用由掺合法配制的相应复合体转染过的细胞的溶胞产物的荧光素酶活性。
实施例6
由掺合法配制的DNA/脂质体复合体与纯化过的DNA/脂质复合体二者细胞毒性的比较
不同复合体对CHO细胞的细胞毒性研究如下。将CHO细胞分别用下列复合体处理：DNA/脂质体掺合复合体(○)，DNA/脂质体/PLL掺合复合体(□)；纯化过的DNA/脂质复合体(●)；或纯化过的DNA/脂质/PLL复合体(●)。掺合复合体是通过将1μg pRSVL DNA只与不同DC-Chol/DOPE组成的10nmol DC-Chol/DOPE脂质体混合而形成的，或还混有1μgPLL(MW＝3,000)。纯化过的复合体则是这样形成的：将1gμpRSVLDNA只与50nmol DC-Chol/DOPE脂质体混合(DNA/脂质复合体)，或者与10nmol DC-Chol/DOPE脂质体和1μg PLL混合(DNA/脂质/PLL复合体)，接着按方法部分中所述通过蔗糖密度梯度进行离心。处理后36小时，将细胞溶解，提取出蛋白质，并用考马斯蓝染料法定量。
图5显示本实验的结果，其中在实验结束之际回收的可提取的蛋白质总量，被用作所述处理之后存活细胞部分的指示剂。给出的数据显示，虽然纯化过的复合体对细胞的毒性似乎比DNA/脂质体掺合复合体稍大，但就形态学而言，转染作用在用掺合复合体或纯化过的复合体处理后的细胞中，都不会导致任何严重的细胞毒性作用。不过，用含有高mol％的DC-Chol的、纯化过的复合体处理后的细胞，在实验结束之际铺满度较小。
实施例7
用纯化过的DNA/脂质复合体在活体内转染肿瘤
在第0天，将3×106个人卵巢癌细胞皮下注入SCID小鼠。14天后，将100μl含有pUCCMVCAT DNA(30μg)、与前者复合的DC-Chol(3∶2 DC-Chol∶DOPE)脂质体(30nmol)、呈掺合物形式(1和2列)的溶液，或者相同量的呈纯化过的DNA/脂质复合体(由DNA和DC-Chol脂质体在1μg DNA/25nmol脂质比率下配制的)形式的DNA，直接注入肿瘤中。2天后杀死动物，取含有100μg蛋白质的肿瘤提取物在37℃下按Ausubel，et al.((1991)CurrentProtocols in Molecular Biology(Wiley，Boston)，Vol.1，pp.9.6.2-9.6.5)测定CAT活性。结果表明，纯化过的复合体，尽管在非最佳条件下制得，仍表现活体内转染活性。
实施例8
纯化过和未纯化的DNA/脂质/PLL复合体与DNA/脂质掺合复合体的转染活性的比较
在三种细胞系(293、BL6和C3)中，测定纯化过和未纯化的DNA/脂质/PLL复合体以及DNA/脂质掺合复合体的转染活性，方法如下：
纯化过的DNA/脂质/PLL复合体是这样形成的：将1μg PRSVLDNA与10nmol DC-Chol/DOPE脂质体(2∶3mol/mol)和1μgPLL.(MW＝25,600)混合，接着按方法部分中所述通过蔗糖密度梯度进行离心。
未纯化的DNA/脂质/PLL复合体则是这样形成的：将100μgPRSVL DNA 与 80μg PLL(MW＝25,600)和1702nmol DC-Chol/DOPE(2∶3mol/mol)脂质体(即1μg DNA/17nmol脂质/10.8μgPLL的DNA/脂质/PLK比率)混合于终体积为500μl的水中。然后取20μl未纯化的DNA/脂质/PLL复合体(即4μg DNA、3.2μg PLL和68.1nmol脂质)加入780μl适于待转染的细胞系的无血清培养基中。
将1μg PRSVL DNA与10nmol DC-Chol/DOPE(3∶2 mol/mol)脂质体混合而形成DNA/脂质掺合复合体。
用1μg DNA于37℃下转染在24孔板中生长至80％铺满度的293、C3和BL6细胞达4小时，其中的DNA呈下列形式：纯化过的DNA/脂质/PLL复合体、DNA/脂质掺合复合体或未纯化的DNA/脂质/PLL复合体。转染后，将细胞于含有10％胎牛血清的合适的培养基中又培养36-48小时。然后按方法部分中所述测定荧光素酶活性。
结果示于图7A(293细胞)、图7B(C3细胞)和图7C(BL6细胞)，结果表明，未纯化的DNA/脂质/PLL复合体在受试的三种细胞系中均表现最高的转染活性。
虽然在上文中已描述了本发明的若干实施方案，显然，可改变基本结构以提供可利用本发明的方法和设备的其它实施方案。因此，应明白本发明的范围由附上的权利要求书限定，而不是上文中通过实施例说明的特定的实施方案所限定。

Claims (30)

1.一种生产脂质的正电荷多于药物的药物/脂质复合体的方法，该方法包括：将所述药物与阳离子脂质体混合，其中药物与脂质的比率使所述药物/脂质复合体得以形成。
2.权利要求1的方法，其中混合而形成所述复合体的药物与脂质的比率为约1μg/0.1nmol～约1μg/200nmol。
3.权利要求2的方法，其中该方法进一步包括纯化所述复合体的步骤。
4.权利要求3的方法，其中所述纯化步骤是通过蔗糖密度梯度进行离心。
5.权利要求1的方法，其中的药物是核酸，而脂质体则是DC-Chol/DOPE脂质体。
6.一种生产脂质和聚阳离子的正电荷多于药物的药物/脂质/聚阳离子复合体的方法，该方法包括：将所述药物与阳离子脂质体和至少一种聚阳离子混合，其中药物与脂质与聚阳离子的比率使所述复合体得以形成。
7.权利要求6的方法，其中所述药物与脂质的比率为约1μg/0.1nmol～约1μg/200nmol。
8.权利要求6的方法，其中所述聚阳离子含量为约0.01μg～约100μg。
9.权利要求8的方法，其中的聚阳离子是分子量为约300～约200,000道尔顿的聚L-赖氨酸。
10.权利要求6的方法，其中的阳离子脂质体包括阳离子脂质和中性磷酯。
11.权利要求10的方法，其中的阳离子脂质是DC-Chol。
12.权利要求11的方法，其中的中性磷脂是二油酰磷脂酰乙醇胺。
13.权利要求12的方法，其中的药物是核酸。
14.权利要求1或6的方法，其中所述复合体的平均直径小于300nm。
15.权利要求14的方法，其中所述调和物在贮存长达一年后，其平均直径保持实质上未变化。
16.一种药物/脂质复合体，它包括：至少一种脂质和药物，这种脂质与该药物的比率使得所述复合体中脂质的正电荷多于药物。
17.权利要求16的复合体，其中所述的药物是核酸。
18.权利要求16的复合体，其中所述药物与脂质的比率为约1μg/0.1nmol～约1μg/200mmol。
19.权利要求16的复合体，其中所述的脂质是阳离子脂质。
20.权利要求19的复合体，其中该复合体进一步包括中性磷脂。
21.权利要求16的复合体，其中该复合体从过量的游离药物和游离脂质中纯化。
22.一种药物/脂质/聚阳离子复合体，它包括至少一种脂质、至少一种聚阳离子和一种药物；其中该药物与该脂质与该聚阳离子的比率使得所述复合体中脂质和聚阳离子的正电荷多于药物。
23.权利要求22的复合体，其中脂质与DNA的所述比率为约1μg/0.1nmol～约1μg/200nmol。
24.权利要求22的复合体，其中所述聚阳离子含量为约0.01μg～约100μg。
25.权利要求24的复合体，其中所述聚阳离子是分子量为约300～约200,000的聚-L-赖氨酸。
26.权利要求16和22的复合体，其中所述复合体的净正电荷在pH6.0-8.0下存在。
27.权利要求26的复合体，其中的脂质是DC-Chol和DOPE。
28.一种把药物传递给个体的方法，它包括：将权利要求16或22中任一项的复合体以治疗上有效量施药给需治疗的个体。
29.权利要求28的方法，其中的复合体是药物/脂质复合体。
30.权利要求28的方法，其中的复合体是药物/脂质/聚阳离子复合体。
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US7655468B2 (en) 2010-02-02
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