Peptide-Engineered Seliciclib Nanomedicine for Brain-Targeted Delivery and Neuroprotection

Abstract

Seliciclib, a cyclin-dependent kinase 5 (CDK5) inhibitor, has demonstrated neuroprotective potential. However, its therapeutic application is limited by poor permeability across the blood-brain barrier (BBB). In this study, polymeric nanoparticles (NPs) modified with a BBB-targeting peptide ligand (His-Ala-Ile-Tyr-Pro-Arg-His) were employed to encapsulate seliciclib. In vitro transport studies showed that the peptide-modified NPs exhibited significantly greater translocation across a bEnd.3 cell monolayer compared to unmodified NPs. Furthermore, in vivo biodistribution analysis revealed that the brain accumulation of peptide-modified NPs was 3.38-fold higher than that of unmodified NPs. Notably, the peptide-conjugated, seliciclib-loaded NPs demonstrated a significant neuroprotective effect against the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺) in differentiated SH-SY5Y cells.

Keywords: nanoparticles; neuroprotection; peptide; seliciclib.

Figure 1

(A) Proton nuclear magnetic resonance (¹H-NMR) spectra and (B) size exclusion chromatogram of PLGA-PEG-maleimide.

Figure 1

(A) Proton nuclear magnetic resonance (¹H-NMR) spectra and (B) size exclusion chromatogram of PLGA-PEG-maleimide.

Figure 2

(A) Dynamic light scattering (DLS) histograms and (B) TEM images of seliciclib@PPM NPs and seliciclib@PPM NPs-Cys-T7.

Figure 3

Cumulative release of seliciclib from seliciclib@NPs in (A) pH 7.4 phosphate-buffered release medium and (B) pH 5.5 acetate-buffered release medium at 37 °C. (n = 3, mean ± SD).

Figure 4

Transport of free seliciclib, seliciclib@PPM NPs, and seliciclib@PPM NPs-Cys-T7 across bEnd.3 monolayer via an in vitro BBB cell model for 24 h. (n = 3, mean ± SD, *** p < 0.001).

Figure 5

Cellular uptake of peptide-free PP NPs and peptide-conjugated PPM NPs-Cys-T7 in (A) L929 fibroblast cells (as control), (B) bEnd.3 endothelial cells, (C) proliferative SH-SY5Y cells, as well as (D) differentiated SH-SY5Y cells under 5% CO₂ at 37 °C for 2 h analyzed by flow cytometry (n = 3, mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, compared to PP NPs), and (E) the increase in cellular uptake of peptide-conjugated NPs relative to peptide-free NPs (calculated by Equation (4)) at 1.5 mg/mL in four cell lines. (F) Flow cytometric analysis of MFI on proliferative and differentiated SH-SY5Y cells stained with anti-human CD71 antibody (green line) and isotype IgG control (orange line), and (G) their CD71 expression levels calculated by Equation (3).

Figure 5

Cellular uptake of peptide-free PP NPs and peptide-conjugated PPM NPs-Cys-T7 in (A) L929 fibroblast cells (as control), (B) bEnd.3 endothelial cells, (C) proliferative SH-SY5Y cells, as well as (D) differentiated SH-SY5Y cells under 5% CO₂ at 37 °C for 2 h analyzed by flow cytometry (n = 3, mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, compared to PP NPs), and (E) the increase in cellular uptake of peptide-conjugated NPs relative to peptide-free NPs (calculated by Equation (4)) at 1.5 mg/mL in four cell lines. (F) Flow cytometric analysis of MFI on proliferative and differentiated SH-SY5Y cells stained with anti-human CD71 antibody (green line) and isotype IgG control (orange line), and (G) their CD71 expression levels calculated by Equation (3).

Figure 6

(A) IVIS images of Cy7.5@PPM NPs and Cy7.5@PPM NPs-Cys-T7 in mice brain over 5-day period following intravenous injection via the tail vein. (B) The fluorescence intensity versus time profiles of Cy7.5@NPs accumulated in the brain, and (C) the corresponding area under the curve AUC_{0–120 h}. (D) Ex vivo IVIS images of brains collected 72 h post-injection (n = 3, mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

(A) IVIS images of Cy7.5@PPM NPs and Cy7.5@PPM NPs-Cys-T7 in mice brain over 5-day period following intravenous injection via the tail vein. (B) The fluorescence intensity versus time profiles of Cy7.5@NPs accumulated in the brain, and (C) the corresponding area under the curve AUC_{0–120 h}. (D) Ex vivo IVIS images of brains collected 72 h post-injection (n = 3, mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7

(A) Cell viability of differentiated SH-SY5Y pretreated with 10 μg/mL of seliciclib, seliciclib@PPM NPs or seliciclib@PPM NPs-Cys-T7 for 2 h, followed by incubation with various concentrations of MPP⁺ (0.1–2.0 mM) for 48 h, and (B) the corresponding IC₅₀ values were 1.11 ± 0.19 mM, 1.78 ± 0.11 mM and 3.15 ± 0.69 mM, respectively. (n = 3, mean ± SD, ** p < 0.01, *** p < 0.001).