Long-circulating monensin nanoparticles for the potentiation of immunotoxin and anticancer drugs

The carboxylic ionophore monensin was formulated into long-circulating nano particles with the help of polyethylene glycol/poly (DL-lactide-co-glycolid e) diblock copolymers, in an attempt to enhance the cytotoxicity of a ricin -based immunotoxin, anti-My9, and anticancer drugs like adriamycin and tamo xifen. This study looked into various aspects involving the preparation (us ing a homogenizer and an EmulsiFlex homogenizer-extrusion device) and lyoph ilization of long-circulating monensin nanoparticles (LMNP) of particle siz e < 200 nm in diameter. The particle size of LMNP was reduced from 194 nm t o 160 nm by passing the nanoparticles through an EmulsiFlex, before freeze- drying. There was a 4.8-83.7% increase in the particle size of LMNP after f reeze-drying, which was dependent upon the manufacturing conditions such as use of the EmulsiFlex for size reduction before freeze-drying, the freezin g method (rapid/slow) and the concentration of lyoprotectant (mannitol or t rehalose) employed for freeze-drying. LMNP freeze-dried with 2.4% of trehal ose showed minimal size change (< 9%) after freeze-drying. Further, the fre ezing method was found to have negligible effect on the particle size of LM NP freeze-dried with trehalose in comparison with mannitol. The entrapment efficiency of monensin in LMNP was found to be 14.2 +/-0.3 %. The LMNP were found to be spherical in shape and smooth in surface texture as observed b y atomic force microscopy. In-vitro release of monensin from LMNP in phosph ate buffered saline (PBS) pH 7.4 or PBS supplemented with 10 % human serum indicated that there was an initial rapid release of about 40 % in the firs t 8 h followed by a fairly slow release (about 20 %) in the next 88 h. In-v ivo studies conducted with Sprague-Dawley rats showed that 20% of monensin remained in circulation 4-8 h after the intravenous administration of LMNP. An in-vitro dye-based cytotoxicity assay (MTS/PMS method) showed that ther e was 500 times and 5 times potentiation of the cytotoxicity of anti-My9 im munotoxin by LMNP (5 x 10(-8) M of monensin) in HL-60 sensitive and resista nt human tumour cell lines, respectively. Further, LMNP (5 x 10(-8) M of mo nensin) potentiated the cytotoxicity of adriamycin in MCF 7 and SW 620 cell lines by 100 fold and 10 fold, respectively, and that of tamoxifen by 44 f old in MCF 7 cell line as assessed by crystal violet dye uptake assay. Our results suggest that it is possible to prepare LMNP possessing appropriate particle size (< 200 nm), monensin content and in-vitro and in-vive release characteristics with the help of a homogenizer and an EmulsiFlex homogeniz er-extrusion device. LMNP can be freeze-dried with minimal increase in part icle size by using a suitable concentration of a lyoprotectant like trehalo se. Furthermore, LMNP could potentiate the cytotoxicity of immunotoxin, adr iamycin and tamoxifen by 5-500 fold in-vitro, which will be further investi gated in-vivo in a suitable animal model.