• M H Vingerhoeds, H J Haisma, S O Belliot, R H Smit, D J Crommelin, and G Storm.
• Department of Pharmaceutics, Faculty of Pharmacy, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands.
• Pharm. Res. 1996 Apr 1; 13 (4): 604-10.

PurposeImmuno-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.MethodsTo achieve coupling of GUS to the liposomes, introduction of extra thiol groups was required. Two thiolating agents were examined: iminothiolane and SATA.ResultsWhen 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.ConclusionsSATA-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.

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