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

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.


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The reaction was assessed through Thin Layer Chromatography (TLC-acetic acid/chloroform/methanol 1/89/10). The product was characterized through HPLC. Analyses were performed with a YL9100 HPLC system equipped with a YL9110 quaternary pump, a YL9101 vacuum degasser and a YL9160 PDA detector, linked to YL-Clarity software for data analysis (Young Lin, Hogyedong, Anyang, Korea). Column was a Teknocroma C18 mediterranea Sea 25 × 0.46 cm, PDA wavelengths were set at 220-290 nm, eluent flow was 1 mL/min, gradient was 0 min: 100% water, 20 min: 100% acetonitrile, 30 min: 100% acetonitrile, 35 min: 100% water. 1 H and 13 C NMR spectra were recorded on a Jeol ECZ-R 600, at 600 and 150 MHz, respectively, using SiMe4 as internal standard. The following abbreviations are used to designate peak multiplicity: s = singlet, d = doublet, t = triplet, m = multiplet, bs = broad singlet. ESI-MS spectra were recorded on a Micromass Quattro API micro (Waters Corporation, Milford, MA, USA) mass spectrometer. Data were processed using a MassLynx System (Waters

PEGylated Linker Stearyl-PEG-Maleimide (ST-PEG-MBS) Synthesis and Characterization
In the first step, 10 mg stearic acid (140 mM), 1.25 mg N-hydroxysuccinimide (NHS-43 mM), 1 mg 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC-26 mM) were dissolved in 0.25 mL dimethylformammide (DMF) anhydrous and kept reacting overnight at 45 °C. The reaction mixture was diluted with 1 mL chloroform and washed with 4 mL 0.1 M HCl to eliminate water soluble compounds. The organic phase was treated with MgSO4 and dried under nitrogen steam and checked through TLC (0.5% triethylamine in chloroform): being in excess of stearic acid, it was a mixture of the reagent and of activated stearic acid (36%). In a second step, 6.1 mg of first step reaction product (corresponding to 2.2 mg activated stearic acid-12 mM), 2 mg MBS (12 mM), 12 mg diamino-PEG (12 mM), 12 μL triethylamine (237 mM) were dissolved in 0.5 mL chloroform and kept reacting overnight at 45 °C. The reaction mixture was dried under nitrogen steam; the residue was dissolved in 400 μL ethanol, and 1.6 mL 1 M HCl was added to precipitate un-reacted stearic acid from the first step reaction. The obtained solution underwent size exclusion through a Sephadex G10 column to eliminate compounds with Mw < 500. ST-PEG-MBS was purified from un-reacted diamino-PEG through Dowex 50WX8-200 resin. The purified solution was freeze dried. The obtained conjugate (6.4 mg) was checked through TLC (acetic acid/chloroform/methanol 1/84/15) and characterized by 1 H-NMR (300 MHz 1 H were recorded on a Bruker 300 Avance instrument at 25 °C). The product was characterized through HPLC. Analyses were performed with a YL9100 HPLC system equipped with a YL9110 quaternary pump, a YL9101 vacuum degasser and a YL9160 PDA detector, linked to YL-Clarity software for data analysis (Young Lin, Hogye-dong, Anyang, Korea), and, alternatively, with a LC10 HPLC UV system (Shimadzu, Tokyo, Japan) equipped with a ELSD detector, linked to a Class LC10 software for data analysis. Column was a Teknocroma C18 mediterranea Sea 25 × 0.46 cm, PDA wavelengths were set at 220-290 nm, eluent flow was 1 mL/min, gradient was 0 min: 100% water, 20 min: 100% acetonitrile, 30 min: 100% acetonitrile, 35 min: 100% water.
TLC ( 1 H NMR. The maleimide protons were detected at 6.5 ppm, and the stearic moiety could be easily evidenced at high field (1.2 ppm multiplet for the alkylic chain and 0.9 ppm for the methyl moiety). Both maleimide and stearic moiety were linked to diamino-PEG in 1:1 molar ratio. Figure S1. 1 H-NMR spectrum of ST-PEG-MBS.

7-Hydroxymethotrexate (7OH-MTX) Synthesis and Characterization
5 mg MTX were dissolved in 33 mL 0.133 M Sorensen buffer brought at pH = 7.8 with 5 M sodium hydroxide. 10 mL enzyme solution (corresponding to 112 mg enzyme) were slowly unfrozen in ice bath and then added to the MTX solution. The mixture was kept at 37 °C for 2 h. Afterwards the pH was brought to 8.4 with 0.5 M sodium hydroxide and kept at 40 °C overnight. The mixture was centrifuged at 5000 rpm (Rotofix 32A centrifuge, Hettich, Tuttlingen, Germany) and the supernatant was isolated, heated until 70 °C and centrifuged. Complete deproteinization was obtained with Amicon Centriflo (Millipore, Burlington, MS, USA) equipped with 50.000 Da dialysis membrane. The dialysed solution was brought to pH = 4.0 with acetic acid and kept at 4 °C overnight in order to obtain the complete precipitation of 7OH-MTX. The obtained suspension was centrifuged at 25,000 rpm (Allegra ® 64R centrifuge, Beckmann Coulter, Paolo Alto, CA, USA), supernatant was discarded and the precipitate was dried under vacuum. Nearly 2 mg 7OH-MTX were obtained. Reaction product was assessed through PDA-HPLC described below: eluted fractions were collected and analyzed with mass spectroscopy (

UPLC-MS Analysis of MTX and its Metabolites
MTX, 7OH-MTX and urine samples underwent also UPLC-MS analysis owing to the following conditions. UPLC-MS analysis was performed with an Acquity UPLC (Waters Corporation, Milford MA, USA) equipped with BSM, SM, CM and PDA detectors. The analytical column was a Zorbax Eclipse XDB-C18 150 × 4.6 mm. The mobile phase consisted of methanol and ammonium acetate buffer pH = 6.0 (30/70). UPLC retention time (Rt) was obtained at flow of 0.4 mL/min, and the column effluent was monitored using Micromass Quattro microTM API Esci multi-mode ionization enabled as detector. The MS conditions were: purging gas (nitrogen) heated at 350 °C at a flow rate of 800 L/h; nebulizer gas (nitrogen) at 80 L/h; capillary voltage in negative mode at 3000 V; fragmentor voltage 20 V. UPLC

Stability and Drug Release Experiments in Cell Culture Medium and Rat Plasma
ddMTX loaded behenic acid SLN, purified through size exclusion (Sepharose CL-4B), were diluted 50 folds in cell culture medium or in rat plasma and kept under magnetic stirring. At scheduled times, 50 μL of the release medium were centrifuged at 25,000 rpm (Allegra® 64R centrifuge, Beckmann Coulter, Paolo Alto, CA, USA) and the supernatant was diluted with 200 μL water, before being derivatized for fluorescence HPLC detection. Particle size was measured before and at the end of the experiment in cell culture medium or in rat plasma.