Novel formulation of a methotrexate derivative with a lipid nanoemulsion

Abstract

Background: Lipid nanoemulsions that bind to low-density lipoprotein receptors can concentrate chemotherapeutic agents in tissues with low-density lipoprotein receptor overexpression and decrease the toxicity of the treatment. The aim of this study was to develop a new formulation using a lipophilic derivative of methotrexate, ie, didodecyl methotrexate (ddMTX), associated with a lipid nanoemulsion (ddMTX-LDE).

Methods: ddMTX was synthesized by an esterification reaction between methotrexate and dodecyl bromide. The lipid nanoemulsion was prepared by four hours of ultrasonication of a mixture of phosphatidylcholine, triolein, and cholesteryloleate. Association of ddMTX with the lipid nanoemulsion was performed by additional cosonication of ddMTX with the previously prepared lipid nanoemulsion. Formulation stability was evaluated, and cell uptake, cytotoxicity, and acute animal toxicity studies were performed.

Results: The yield of ddMTX incorporation was 98% and the particle size of LDE-ddMTX was 60 nm. After 48 hours of incubation with plasma, approximately 28% ddMTX was released from the lipid nanoemulsion. The formulation remained stable for at least 45 days at 4°C. Cytotoxicity of LDE-ddMTX against K562 and HL60 neoplastic cells was higher than for methotrexate (50% inhibitory concentration [IC(50)] 1.6 versus 18.2 mM and 0.2 versus 26 mM, respectively), and cellular uptake of LDE-ddMTX was 90-fold higher than that of methotrexate in K562 cells and 75-fold in HL60 cells. Toxicity of LDE-ddMTX, administered at escalating doses, was higher than for methotrexate (LD(50) 115 mg/kg versus 470 mg/kg; maximum tolerated dose 47 mg/kg versus 94 mg/kg) in mice. However, the hematological toxicity of LDE-ddMTX was lower than for methotrexate.

Conclusion: LDE-ddMTX was stable, and uptake of the formulation by neoplastic cells was remarkably greater than of methotrexate, which resulted in markedly greater cytotoxicity. LDE-ddMTX is thus a promising formulation to be tested in future animal models of cancer or rheumatic disease, wherein methotrexate is widely used.

Keywords: cholesterol; didodecyl methotrexate; drug delivery; methotrexate; nanoparticles.

Figures

Figure 1

Esterification reaction of methotrexate in the presence of cesium carbonate.

Figure 2

Transmission electron microscopy of (A) lipid nanoemulsion and (B) LDE-ddMTX. Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion.

Figure 3

In vitro release profile of ddMTX from LDE in dialysis against human plasma at 37°C (mean ± standard deviation, n = 5). Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion.

Figure 4

Cytotoxicity of (A) LDE (o), LDE-ddMTX (●), and methotrexate (■) in (B) 562 (I) and HL60 (II) cell lines. The cells were incubated with the formulations (final concentration range 0.1–2.0 mM) for 4 hours at 37°C, and survival was quantified by enzyme-linked immunosorbent assay. Results are presented the mean ± standard error of the mean of three experiments. Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion.

Figure 5

Uptake of LDE-ddMTX (●) and methotrexate (○) 4 hours by (A) K562 and (B) HL60 cell lines. Cells were incubated with the formulations (final concentration range 0.15–2.0 mM) for 4 hours at 37°C, lysed, and the drugs were then quantified by high-performance liquid chromatography. Results are presented the mean ± standard error of the mean of three experiments. Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion.

Figure 6

(A) Red cell count, (B) platelet count, (C) leukocyte count, and (D) leukocyte differential count in mice after 24 hours of intravenous LDE-ddMTX or methotrexate (both at 180 mg/kg dose) administration and after intravenous LDE without methotrexate. Notes: ns indicates p > 0.05; *** indicates p < 0.001. Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion; LYMPH, lymphocytes; NEUTRO, neutrophyls; EOSINO, eosinophyls; MONO, monocytes; LYMPHOBL, lymphoblast.