A MALDI-TOF-based Method for Studying the Transport of BBB Shuttles-Enhancing Sensitivity and Versatility of Cell-Based In Vitro Transport Models

Abstract

In recent decades, peptide blood-brain barrier shuttles have emerged as a promising solution for brain drugs that are not able to enter this organ. The research and development of these compounds involve the use of in vitro cell-based models of the BBB. Nevertheless, peptide transport quantification implies the use of large amounts of peptide (upper micromolar range for RP-HPLC-PDA) or of derivatives (e.g. fluorophore or quantum-dot attachment, radiolabeling) in the donor compartment in order to enhance the detection of these molecules in the acceptor well, although their structure is highly modified. Therefore, these methodologies either hamper the use of low peptide concentrations, thus hindering mechanistic studies, or do not allow the use of the unmodified peptide. Here we successfully applied a MALDI-TOF MS methodology for transport quantification in an in vitro BBB cell-based model. A light version of the acetylated peptide was evaluated, and the transport was subsequently quantified using a heavy internal standard (isotopically acetylated). We propose that this MALDI-TOF MS approach could also be applied to study the transport across other biological barriers using the appropriate in vitro transport models (e.g. Caco-2, PAMPA).

Figure 1

The H₂N-HAIYPRH-CONH₂ peptide BBB shuttle (1 L): (a) light and (b) heavy versions containing an isotopically labeled acetyl moiety; and (c) the library of analogs of the parent (HAI or 1 L) on the left, and the retro-D-version (rD-HAI or 6D) and its analogous modifications on the right.

Figure 2

Scheme of the transport quantification method by MALDI-TOF MS. The in vitro BBB cell-based model (a) is performed in a transwell system where a membrane cultured with endothelial cells delimits two compartments (donor and acceptor). The donor compartment contains the light peptide before starting the experiment; at the end of the assay, a certain amount of peptide has been transported to the acceptor compartment (if 200 μM is assayed in the donor compartment, around 2 μM needs to be quantified in the acceptor compartment). Thus, (b) two aliquots of the heavy peptide are prepared, one at 200 μM and another at 2 μM; 10 μL of the assayed light peptide (from acceptor or donor compartments) and 10 μL of the heavy version, at similar concentrations, are mixed. Subsequently, 1 μL of this mixture and 1 μL of an appropriate MALDI matrix (e.g. ACH matrix solution) are placed on a MALDI plate. Finally, the spectra are acquired. A spectrum (c) obtained from a solution containing light and heavy versions of 9D peptide at 2 μM is shown. Light and heavy peptides are observed as the m/z of the peptides plus H⁺, Na⁺ or K⁺. In all cases, isotopic homolog peaks between light and heavy peptides display a 4-amu mass difference.

Figure 3

Peptide transport results from the in vitro bovine BBB cell-based model assay obtained through RP-HPLC-PDA (in blue) or MALDI-TOF MS (in green) quantification; Data are expressed as mean ± SD, n = 3. (a) RP-HPLC-PDA transport quantification significance between L- and D-peptides (additionally, peptide 9D showed significant differences (*) with 7 + D and 8 D); (b) MALDI-TOF MS transport quantification results and significance. Differences in group means were assessed using a linear model fitted for each measurement method. In each case, Wald tests derived from the linear models were used to perform pairwise comparisons between experimental conditions. A 5% level was chosen for significance of group. In vitro human BBB model: (c) transport of 1 L, 7 − D and 8D peptides when assayed with Ringer-HEPES buffer or supplemented ECM medium. Extremely significant differences (****) are observed when peptides are assayed in buffer or medium; within peptides assayed in buffer, significant differences (*) are only observed between 1 L and 8 D; within peptides assayed in medium, significant differences (* or **) are observed between 1 L both retro-D-analogs (7 − D and 8 D). Significance: ns ≡ not significant (p ≥ 0.05), *significant (0.01 ≤ p < 0.05), **very significant (0.001 ≤ p < 0.01), ***extremely significant (0.0001 ≤ p < 0.001), ****extremely significant (p < 0.0001). Data analysis was done using the T test (non-parametric).

Figure 4

Determination of the limit of quantification of MALDI-TOF MS and RP-HPLC-PDA using the peptides 1 L, 6D and 9D.