The synthesis of methotrexate-loaded F127 microemulsions and their in vivo toxicity in a rat model

Methotrexate (MTX) has been often formulated as nano and micro-emulsions, nominally to address its poor solubility and off-target effects. Nanoformulated MTX is universally reported to be a more efficacious anti-cancer agent than direct-dissolved drug; however, these investigations generally fail to screen for in vivo toxicity. This study aims to remedy this oversight. MTX was formulated as a standard Pluronic oil-in-water microemulsion with good drug encapsulation efficiency (73.0% +/- 8.4). Preliminary in vitro free radical scavenging studies found that formulation reduces drug oxidation four-fold. The toxic effects of formulated and unformulated MTX were investigated in a Wistar rat model. Rats received 0.05 mg/kg MTX as either the microemulsion or directly dissolved in phosphate-buffered saline. A drug-free microemulsion, PBS solution, and saline solution were used as controls. After 28 days, serum levels of enzymes indicative of kidney and liver damage were quantified. Significantly higher serum liver, and serum kidney enzymes were observed in the rats that received the directly dissolved MTX drug (P < 0.05) compared to those who received the encapsulated form. Following sacrifice, the levels of catalase and superoxide dismutase (SOD) were significantly lower and the level of malondialdehyde higher, in rats who received either form of MTX relative to untreated controls. However, the SOD levels were lower in those who received the microemulsion than those who received free MTX. Histology supported the observation that the microemulsion formulation caused no gross structural toxicity to the liver, unlike the free drug. Although toxicity was reduced compared to the free drug, the microemulsion still caused damage to the kidneys. This organ-specific toxicity is consistent with the mode of clearance of the drug. This data demonstrates that the toxicity of formulated drugs must be considered when discussing the relative merits of formulations: encapsulation almost always improves efficacy but may not improve safety. (C) 2020 Elsevier B.V. All rights reserved.