Multifunctional Peptide-PEG Intercalating Conjugates: Programmatic of Gene Delivery to the Blood-Brain Barrier

ABSTRACT

Purpose

To enhance transfection efficacy of pDNA through the application of multifunctional peptide-PEG-tris-acridine conjugates (pPAC) and the formation of biodegradable core-shell polyplexes for gene delivery to the blood-brain barrier (BBB).

Methods

pPAC-mediated transfection was compositionally optimized in mouse BBB cells (bEnd.3). Cellular uptake and trafficking, and brain accumulation of pDNA was evaluated by fluorescent imaging and histochemistry. We constructed anti-MRP4 siRNA-producing vectors and evaluated the efficacy of MRP4 down-regulation of MRP4 by Western blot and qPCR, and its effect on the uptake of 3H-AZT, an MRP4 substrate.

Results

A core-shell gene delivery system (GDS) was assembled from pDNA and pPAC, carrying multifunctional peptides with NLS, TAT, and brain-specific BH, or ApoE sequences, and biodegradable pLPEI polyamine. This GDS demonstrated better cellular and nuclear accumulation, and a 25-fold higher transfection efficacy in slow-dividing bEnd.3 cells compared to ExGen500. Inclusion of brain-targeting pPAC enhanced in vivo accumulation of functional pDNA in brain capillaries. Treatment by encapsulated anti-MRP4 siRNA-producing pDNA caused transient down-regulation of MRP4, and, after intravenous injection in Balb/c mice, enhanced AZT uptake in the brain by 230–270%.

Conclusions

The pPAC represent novel efficient components of GDS that could find various gene therapy applications, including genetic modulation of the BBB.

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Abbreviations

Acr:

acridine

ApoE:

Apolipoprotein E-derived peptide

BBB:

blood-brain barrier

BCEC:

brain capillary endothelial cells

BH:

brain-homing peptide

BSA:

bovine serum albumin

DAB:

Diaminobenzidine

DET:

drug efflux transporters

DLS:

dynamic light scattering

ECL:

chemoluminescence detection kit

GDS:

gene delivery system

GFP:

green fluorescent protein

HAART:

highly active antiretroviral therapy

HPLC:

high performance liquid chromatography

LPEI:

linear polyethylenimine

NLS:

SV40 nuclear localization signal peptide

NRTI:

Nucleoside reverse transcriptase inhibitors

PAGE:

Polyacrylamide gel electrophoresis

PEG:

Poly(ethylene glycol)

TAT:

HIV-1 trans-activator of transcription peptide

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ACKNOWLEDGMENTS

This work was supported by NIH grants NS050660 and NS063879 (S.V.V.). Authors are grateful to Mrs. Huai-Yun Han, Arin Zeman, and Galya Warren for excellent technical help in conducting some experiments. The assistance of UNMC Confocal Microscopy and Protein Analysis Core facilities is greatly appreciated.

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Correspondence to Serguei V. Vinogradov.

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Zhang, H., Gerson, T., Varney, M.L. et al. Multifunctional Peptide-PEG Intercalating Conjugates: Programmatic of Gene Delivery to the Blood-Brain Barrier. Pharm Res 27, 2528–2543 (2010). https://doi.org/10.1007/s11095-010-0256-x

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KEY WORDS

  • ApoE peptide
  • AZT
  • blood-brain barrier
  • brain capillary endothelial cells
  • brain-homing peptide
  • intercalating complex
  • MRP4
  • NLS peptide
  • peptide-PEG conjugate
  • plasmid DNA
  • TAT peptide