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

The carboxylic ionophore monensin was formulated into long‐circulating nanoparticles with the help of polyethylene glycol/poly (dl‐lactide‐co‐glycolide) diblock copolymers, in an attempt to enhance the cytotoxicity of a ricin‐based immunotoxin, anti‐My9, and anticancer drugs like adriamycin and tamoxifen. This study looked into various aspects involving the preparation (using a homogenizer and an EmulsiFlex homogenizer‐extrusion device) and lyophilization of long‐circulating monensin nanoparticles (LMNP) of particle size < 200 nm in diameter. The particle size of LMNP was reduced from 194 nm to 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 freeze‐drying, which was dependent upon the manufacturing conditions such as use of the EmulsiFlex for size reduction before freeze‐drying, the freezing method (rapid/slow) and the concentration of lyoprotectant (mannitol or trehalose) employed for freeze‐drying. LMNP freeze‐dried with 2.4% of trehalose showed minimal size change (< 9%) after freeze‐drying. Further, the freezing method was found to have negligible effect on the particle size of LMNP 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 by atomic force microscopy. In‐vitro release of monensin from LMNP in phosphate 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 first 8 h followed by a fairly slow release (about 20%) in the next 88 h. In‐vivo 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 there was 500 times and 5 times potentiation of the cytotoxicity of anti‐My9 immunotoxin by LMNP (5 times 10−8 m of monensin) in HL‐60 sensitive and resistant human tumour cell lines, respectively. Further, LMNP (5 times 10−8 m of monensin) 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 fold in MCF 7 cell line as assessed by crystal violet dye uptake assay. Our results suggest that it is possibleto prepare LMNP possessing appropriate particlesize (< 200 nm), mo