Vingerhoeds M H, Haisma H J, Belliot S O, Smit R H, Crommelin D J, Storm G
Department of Pharmaceutics, Faculty of Pharmacy, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands.
Pharm Res. 1996 Apr;13(4):604-10. doi: 10.1023/a:1016010524510.
Immuno-enzymosomes are tumor-specific immunoliposomes bearing enzymes on their surface. These enzymes are capable of converting relatively nontoxic prodrugs into active cytostatic agents. The enzyme beta-glucuronidase (GUS)4 was coupled to the external surface of immunoliposomes directed against ovarian carcinoma cells. This study aimed at optimization of the prodrug-activating capacity of these immuno-enzymosomes by increasing the enzyme density on the immunoliposomal surface.
To achieve coupling of GUS to the liposomes, introduction of extra thiol groups was required. Two thiolating agents were examined: iminothiolane and SATA.
When iminothiolane was used, aggregation of enzymosomes was observed above enzyme densities of 10 micrograms GUS/mumol lipid (TL). An increased electrostatic repulsion of the enzymosomes, created by inclusion of additional negatively charged lipids and by lowering the ionic strength of the external aqueous medium resulted in enzyme densities > or = 20 micrograms GUS/mumol TL without aggregation. Utilizing SATA, > or = 30 micrograms GUS/mumol TL could be coupled without aggregation, even at physiological ionic strength. It was shown that the enzyme density on immuno-enzymosomes, and thus on the tumor cell surface, strongly influences the antitumor effect of the prodrug daunorubicin-glucuronide against in vitro cultured ovarian cancer cells. The antitumor effect of immuno-enzymosomes with enzyme densities of about 20 micrograms GUS/mumol TL was similar to that of the parent drug daunorubicin.
SATA-mediated thiolation of GUS-molecules enabled the preparation of immuno-enzymosomes with high enzyme densities while avoiding spontaneous aggregation. In vitro antitumor activity experiments showed that the improved immuno-enzymosome system is able to completely convert the prodrug daunorubicin-glucuronide into its parent compound.
免疫酶体是一种肿瘤特异性免疫脂质体，其表面带有酶。这些酶能够将相对无毒的前药转化为活性细胞生长抑制剂。β-葡萄糖醛酸酶（GUS）4与针对卵巢癌细胞的免疫脂质体的外表面偶联。本研究旨在通过增加免疫脂质体表面的酶密度来优化这些免疫酶体的前药激活能力。
为了实现GUS与脂质体的偶联，需要引入额外的巯基。研究了两种巯基化试剂：亚氨基硫醇和SATA。
当使用亚氨基硫醇时，在酶密度高于10微克GUS/微摩尔脂质（TL）时观察到酶体聚集。通过加入额外的带负电荷的脂质以及降低外部水介质的离子强度所产生的酶体静电排斥增加，使得酶密度≥20微克GUS/微摩尔TL时不会发生聚集。使用SATA，即使在生理离子强度下，≥30微克GUS/微摩尔TL也可以偶联而不发生聚集。结果表明，免疫酶体上的酶密度，进而肿瘤细胞表面的酶密度，强烈影响前药柔红霉素-葡萄糖醛酸对体外培养的卵巢癌细胞的抗肿瘤作用。酶密度约为20微克GUS/微摩尔TL的免疫酶体的抗肿瘤作用与母体药物柔红霉素相似。
SATA介导的GUS分子巯基化能够制备具有高酶密度的免疫酶体，同时避免自发聚集。体外抗肿瘤活性实验表明，改进后的免疫酶体系统能够将前药柔红霉素-葡萄糖醛酸完全转化为其母体化合物。
