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Complexation of Cell-Penetrating Peptide–Polymer Conjugates with Polyanions Controls Cells Uptake of HPMA Copolymers and Anti-tumor Activity

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ABSTRACT

Purpose

Cell penetrating peptides (CPPs) can mediate effective delivery of their associated drugs and drug carriers intracellularly, however their lack of cell specificity remains a major obstacle for their clinical development. We aimed at improving the cell specificity and therapeutic efficacy of HPMA copolymer-octaarginine (R8) conjugate (P-R8) in cells at the tumor micro-environment.

Methods

To avoid premature cell-penetration, the positively charged R8 moieties were masked via electrostatic complexation with various polyanionic molecules (heparin sulfate, hyaluronic acid, fucoidan and poly-glutamic acid). We followed the kinetics of the FITC-labeled P-R8 penetration into endothelial and cancer cells over-time after its complexation in vitro and further tested whether the in situ addition of a stronger polycation can trigger the release of P-R8 from the complexes to resume cell penetration activity. A murine model of B16-F10 lung metastasis was then used as an in vivo model for assessing the therapeutic efficacy of the P-R8, loaded with doxorubicin (P-R8-DOX), after its complexation with PGA.

Results

The intracellular penetration of P-R8-FITC was reversibly inhibited by forming electrostatic interactions with counter polyanions, and can be restored either gradually over time by dissociation from the polyanions, or promptly following the addition of protamine sulfate. Mice injected with B16-F10 cells and treated with P-R8-DOX/PGA complexes, exhibited a significant prolonged survival times when compared with DOX-treated mice or relative to mice treated with either P-R8-DOX or P-DOX alone.

Conclusions

The gradual release of P-R8 from P-R8-DOX/PGA may improve the therapeutic efficacy of water-soluble based nanomedicines for the treatment of solid lung tumors.

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Abbreviations

AIBN:

2,2’-azobis(isobutyronitrile)

CPPs:

Cell-penetrating peptides

DOX:

Doxorubicin

EPR:

Enhanced permeability and retention

Fi:

Fucoidan

FITC:

Fluorescein-5-isothiocyanate

HA:

Hyaluronic acid

Hep:

Heparin

HMW-Hep:

High molecular weight heparin

HPMA:

N-(2-hydroxypropyl)methacrylamide

LMW-Hep:

Low molecular weight heparin

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolinium bromide

PFA:

Paraformaldehyde

PGA:

Poly-(l)-glutamic acid

PMA:

Poly(methacrylic acid)

R8:

Octaarginine

SEC:

Size-exclusion chromatography

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ACKNOWLEDGMENTS AND DISCLOSURES

This work was supported in part by grants from the Israel Science Foundation (ISF) (Grant No. 418/10 to AD) and the Israeli National Nanotechnology Initiative for a Focal Technology Area (FTA) on Nanomedicines for Personalized Theranostics (to AD). GA acknowledges support from the E.J. Safra center.

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Authors and Affiliations

  1. Department of Pharmacology, Faculty of Health Sciences, Ilse Katz Institute for Nanoscale Science and Technology Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Yosi Shamay, Lina Shpirt & Ayelet David

  2. Department of Chemistry, Faculty of Natural Sciences, Ilse Katz Institute for Nanoscale Science and Technology Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Gonen Ashkenasy

  3. Department of Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Ayelet David

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  1. Yosi Shamay
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Correspondence to Ayelet David.

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Shamay, Y., Shpirt, L., Ashkenasy, G. et al. Complexation of Cell-Penetrating Peptide–Polymer Conjugates with Polyanions Controls Cells Uptake of HPMA Copolymers and Anti-tumor Activity. Pharm Res 31, 768–779 (2014). https://doi.org/10.1007/s11095-013-1198-x

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  • DOI: https://doi.org/10.1007/s11095-013-1198-x

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