Study of Fabrication of Two Novel Methotrexate-Loaded Micelles and Their Anti-Lymphoma Effects

Background and Objectives: High-dose methotrexate (HD-MTX) is integrated in treatment of highly aggressive non-Hodgkin lymphoma (NHL), including Burkitt's lymphoma. However, current MTX injection is inadequate due to poor water solubility, low concentration in tissues, high toxicity and drug resistance. Copolymers self-assemble in water to form micelles with hydrophilic shell and hydrophobic core, which load hydrophobic chemotherapeutics. Particle sizes of micelles are in nanometer range, therefore they are enriched in tumor tissues due to high permeability and retention effect (EPR effect). The co-loading nanoparticles can deliver multiple agents as a whole. The efficacy of the encapsulated agents are maximized by controlling the drug ratio in synergistic range continuously, such as CPX351 (liposome with a fixed molar ratio (5:1) of cytarabine to daunorubicin). We hope to prepare MTX miclles to address the above mentioned issues and further enhance its anti-lymphoma efficacy by constructing co-loading micellar systems. Gambogic acid (GA) is a small molecule isolated from Garcinia hanburyi withbroad spectrum effects. GA exerts antitumor effects by inducing cell cycle arrest, inducing apoptosis, and inhibiting cell growth signaling pathways and so on. It has been reported that GA has synergistic anti-cancer effects with MTX.

Methods: Firstly, we synthesized a variety of polymeric nanomaterials with excellent biosafety and biocompatibility by ring-opening methods . After careful screening, we utilized monomethyl polyethylene glycol-polycaprolactone (MPEG-PCL) to encapsulate MTX by thin film method. After characterizing parameters MTX loaded micelle, we investigated its anti-lymphoma efficacy in vitro and in vivo. On this basis, we co-loaded MTX and GA and finely adjusted the feeding ratio to obtain a series of MTX/GA-MPEG-PCL micelles with different loading mass ratios. The pharmacological parameters were characterized. And the cell viability of lymphoma cells treated with MTX/GA-MPEG-PCL micelles with different mass ratios were detected by MTT experiments. Finally, a subcutaneous lymphoma model of Burkitt lymphoma in NOD/SCID mice was established and tumor burden was monitored to evaluate the anti-lymphoma effect of MTX/GA-MPEG-PCL micelles in vivo.

Results: MPEG-PCL-MTX micelles have small particle size (PS 25.96 nm), uniform dispersion (PDI 0.135±0.03), high encapsulation rate and drug loading capacity (DL 3.83%, EE 96.62%), good stability, and slow release effect in vitro. MTT experiments revealed that MPEG-PCL-MTX micelles showed significantly better inhibitory effects on human lymphoma Raji cells than free drug at high concentrations and induced more cell apoptosis. In mouse Burkitt lymphoma model, anti-lymphoma effects of MPEG-PCL-MTX were more potent than that of MTX injection at the same dosage . And the low-dose (3 mg/kg) MPEG-PCL-MTX micelles could achieve anti-lymphoma effect comparable to that of the high-dose (7 mg/kg) MTX injection. By comhensive sreening and optimization, we obtained a series of MTX/GA-MPEG-PCL micelles with different mass ratios (MTX:GA (W/W) of 1:10, 1:5, 1:1, 5:1, 10:1, 50:1, 100:1). The obtained micelles displayed small particle size (PS 20.36-30.14 nm) ,uniform distribution (PDI 0.129-0.199), and spherical morphology. Measurement of tumor burden in mouse Burkitt's lymphoma showed that the MTX/GA-MPEG-PCL micelles exhibited the strongest anti-lymphoma effect compared with the single drug-loaded micelles (MTX-MPEG-PCL and GA-MPEG-PCL) and the mixture of free drugs (MTX injection + GA solution). Compared mice treated with saline, there were no significant change in body weight and systemic toxicities in the co-loading micelle group.

Conclusions: We developed two novel MTX micellar formulations, MPEG-PCL-MTX and MTX/GA-MPEG-PCL, which are well-targeted, highly effective, safe, and well accessible. With enhanced anti-lymphoma efficacy and minimal systemic toxicity, they can be solutions to current problems of MTX in clinical treatment and have good translational prospects.

Funding:This work was supported by 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (No. ZYJC21007）.

eywords: lymphoma, methotrexate, gambogic acid, micelles, MPEG-PCL

No relevant conflicts of interest to declare.