Methotrexate-Loaded Solid Lipid Nanoparticles: Protein Functionalization to Improve Brain Biodistribution

Abstract

Glioblastoma is the most common and invasive primary tumor of the central nervous system and normally has a negative prognosis. Biodistribution in healthy animal models is an important preliminary study aimed at investigating the efficacy of chemotherapy, as it is mainly addressed towards residual cells after surgery in a region with an intact blood⁻brain barrier. Nanoparticles have emerged as versatile vectors that can overcome the blood⁻brain barrier. In this experimental work, solid lipid nanoparticles, prepared using fatty acid coacervation, have been loaded with an active lipophilic ester of cytotoxic drug methotrexate, and functionalized with either transferrin or insulin, two proteins whose receptors are abundantly expressed on the blood⁻brain barrier. Functionalization has been achieved by grafting a maleimide moiety onto the nanoparticle's surface and exploiting its reactivity towards thiolated proteins. The nanoparticles have been tested in vitro on a blood⁻brain barrier cellular model and in vivo for biodistribution in Wistar rats. Drug metabolites, in particular 7-hydroxymethotrexate, have also been investigated in the animal model. The data obtained indicate that the functionalization of the nanoparticles improved their ability to overcome the blood⁻brain barrier when a PEG spacer between the proteins and the nanoparticle's surface was used. This is probably because this method provided improved ligand⁻receptor interactions and selectivity for the target tissue.

Keywords: blood–brain barrier; insulin; methotrexate; solid lipid nanoparticles; transferrin.

Figure 1

Biodistribution of the unfunctionalized ddMTX-loaded SLNs and free MTX (sacrifice 30 min after administration). Statistical analysis: ddMTX-loaded SLNs vs. free MTX; stearic acid SLNs < MTX: p < 0.005 **; behenic acid SLNs < MTX: p < 0.05 @; behenic acid SLNs > MTX: p < 0.05 #, p < 0.001 ###; behenic acid SLNs PVA120000 > MTX: p < 0.1 §, p < 0.01 §§, p < 0.05 §§§.

Figure 2

Electrophoresis. Red boxes: free proteins; blue boxes: dimerized proteins; yellow boxes: proteins grafted onto SLNs. (A) ST-MBS; 8:1 2-iminothiolane/TRF molar ratio; (B) ST-MBS; 4:1 2-iminothiolane/TRF molar ratio; (C) ST-MBS; 2:1 2-iminothiolane/TRF molar ratio; (D) ST-MBS; 1:1 2-iminothiolane/INS molar ratio; (E) ST-PEG-MBS; 2:1 2-iminothiolane/TRF molar ratio; (F) ST-PEG-MBS; 1:1 2-iminothiolane/INS molar ratio.

Figure 3

Permeation through the hCMEC/D3 cells monolayer of unfunctionalized and functionalized ddMTX-loaded behenic acid SLNs and free MTX. Statistical analysis: ddMTX-loaded SLNs vs. free MTX; SLNs TRF ST-PEG-MBSp < 0.05 *; SLNs INS ST-PEG-MBS<MTX: p < 0.1 $; SLNs > MTX: p < 0.1 #, p < 0.05 ##; SLNs TRF ST-PEG-MBS>MTX: p < 0.1 §, p < 0.05 §§; SLNs INS ST-PEG-MBS > MTX: 24 h p < 0.005 °; SLNs TRF ST-MBS>MTX: p < 0.05 ç; SLNs INS ST-MBS > MTX: p < 0.05 @, p < 0.005 @@.

Figure 4

Biodistribution of functionalized ddMTX-loaded SLNs and free MTX (sacrifice 3 h after administration): in (A) the whole body and (B) brain and capillary depletion (cap. dep.). Statistical analysis: ddMTX-loaded SLNs vs. free MTX; SLNs < MTX: p < 0.01 £ SLNs INS ST-PEG-MBS < MTX: p < 0.05 *; SLNs INS ST-MBS < MTX: p < 0.01 $, p < 0.05 $$; SLNs > MTX: p < 0.1 #, p < 0.05 ##; SLNs TRF ST-PEG-MBS > MTX: p < 0.1 §, p < 0.05 §§, p < 0.01 §§§; SLNs INS ST-PEG-MBS>MTX: p < 0.1 °, p < 0.05 °°; SLNs TRF ST-MBS > MTX: p < 0.1, ST-MBS > MTX: p < 0.1 @.