Methotrexate-Loaded Biodegradable Polymeric Micelles for Lymphoma Therapy in Mouse Model

Purpose: Methotrexate (MTX), a folate antimetabolite, competitively inhibits dihydrofolate reductase, the intracellular enzyme responsible for converting folic acid to folate cofactor, thus blocking the synthesis of thymine and purine, causing impairment of tumor growth and induction of cell death. High dose MTX (HD-MTX) is infused in doses of 3-5g/m² and over a period of 6-24h to maintain the serum MTX at high levels, which shows benefit in the treatment of childhood leukemia and certain adult NHL, particularly the Burkitt type. However, the clinical implication of MTX is hampered for some reasons. On the one hand, the adverse effects of HD- MTX are severe, including bone marrow suppression, mucositis, hepatotoxicity, nephrotoxicity and neurological symptoms. On the other hand, leucovorin rescue, adequate hydration, urinary alkalization and monitoring of MTX concentration is required to avoid life-threating side effects of HD-MTX, which makes the administration rather complicated. In addition, the poor water solubility also impedes the application of MTX. To overcome these obstacles, a new drug delivery system is required to reduce the side effects of HD-MTX as well as to increase the solubility of the drug. So we designed a self-assembled micellar system to load MTX to overcome water solubility and to enhance therapeutic efficacy while reduce toxicity.

Materials and Methods: Monomethyl poly (ethylene glycol)-poly (ε-caprolactone) (MPEG-PCL) our group previously synthesized was employed to load MTX through a one-step solid dispersion method. The preparation was done without any surfactants, organic solvents, or vigorous stirring.

Results: MTX loaded micelles had a small particle size of 25.64±0.99nm and polydisperse index (PDI) of 0.176±0.05. Drug loading and encapsulation efficiency of MTX loaded micelle were 5.57±0.14% and 92.46±2.38%, respectively. Compared with free MTX, MTX loaded micelles demonstrated a much slower and sustained release behavior in vitro. MTT assay and cell apoptosis study suggested that MTX loaded micelles were more effective in inhibiting proliferation (IC50 value 137.8ug/ml vs 86.5ug/ml, p<0.05) and inducing apoptosis (p<0.01) on Raji lymphoma cells than MTX treatment, which was especially distinct in high dose groups (38.78% vs 22.17%). Cellular uptake study indicated that MPEG-PCL micelle had a 1.5 times higher uptake rate in Raji cells. As for in vivo studies, MTX loaded micelles were more competent to suppress tumor growth (p<0.01) and prolong survival time than MTX injection (p<0.05) in the subcutaneous Raji lymphoma mouse model. Notably, the high dose group of micelle formulation exhibited the strongest anti-tumor effect without additional toxicity. As for body weight, which represented systematic toxicity, no difference between control group and two kinds of MTX formulation group was observed (p >0.05). Furthermore, immunofluorescent and immunohistochemical studies showed that tumors of MPEG-PCL-MTX treated mice had more apoptotic cells (p<0.05) and fewer proliferative cells (p<0.01).

Conclusions: In this work, MPEG-PCL-MTX micelle was fabricated with a thin-film hydration method to improve the water solubility of MTX and to meet the requirements of intravenous administration, which was stable, safe, effective, easy to produce and scale up, and showed potential clinical application. The enhanced therapeutic efficiency of MPEG-PCL-MTX micelle might be attributed to enhanced permeability and retention (EPR) effect, which increased drug accumulation in the tumor site. Besides, compared with MTX injection with highest concentration in our experiment, MPEG-PCL-MTX micelle with lowest concentration achieved similar or better therapeutic outcome, which indicated the amount of drug could be reduced to achieve the same therapeutic efficiency and alleviate adverse effects towards normal tissues and organs. Moreover, MPEG-PCL-MTX micelle of high dose (7mg/ml) achieved great therapeutic advantage without obvious toxicity, which makes micelle an effective and safe choice when HD-MTX is clinically employed.