Clinical trials exploring bolus intravenous delivery of interleukin-12 (IL-12) for treatment of solid tumors ultimately failed due to lack of clinical response and severe dose-limiting toxicities. The present study was conducted to evaluate whether recombinant murine IL-12 (rmIL-12) could be successfully encapsulated within Poly (D, l-lactide-co-glycolide) (PLGA) nanospheres (rmIL-12ns) for safe and effective systemic delivery at pharmacologic scale. Optimal fabrication of rmIL-12ns occurs with dichloromethane as the organic solvent and emulsion formation via ultrasonication at 50% power (250 W sonicator) for 10 s (50W10s). We then determined whether utilization of synthesis modifiers including fetal bovine serum (FBS), magnesium hydroxide [Mg(OH)₂], trehalose, or the surfactants polysorbate 80 and Span 60 alone or in combination could increase the encapsulation efficiency (EE) and/or modify the burst elution profile characteristic of the 50W10s rmIL-12ns formulation. The greatest EEs compared to the unmodified formulation were measured with modifications containing the surfactants polysorbate 80 and Span 60 (surfactant:​ 28.3 ± 6.10%, p = 0.29 and Surf/FBS:​ 85.4 ± 2.19%, p = 0.039). The Surf/FBS formulation was further modified for in vivo murine injection by substituting FBS with mouse serum albumin (MSA). The resulting Surf/MSA rmIL-12ns were then characterized before delivery at three doses (0.1, 1, and 10 mg rmIL-12ns) in our established murine model of metastatic osteosarcoma to assess efficacy. Preliminary results suggested no evidence of disease with delivery of the 0.1 mg dose in 75% of mice (3 of 4) versus a nontreated historical control (2 of 34).