Cell penetrating peptide conjugated bioreducible polymer for siRNA delivery

doi:10.1016/j.biomaterials.2011.03.058

Biomaterials

Volume 32, Issue 22, August 2011, Pages 5213-5222

https://doi.org/10.1016/j.biomaterials.2011.03.058 Get rights and content

Abstract

The primary cardiomyocyte–specific peptide (PCM) and the cell-penetrating peptide (CPP), HIV-Tat (49–57), were incorporated into the polymer, cystamine bisacrylamide-diaminohexane (CBA-DAH), to increase the delivery of RNAi to target cells, specifically cardiomyocytes. Interestingly, the impact of PCM and Tat conjugation on cellular uptake and transfection efficiency was greater in H9C2 rat cardiomyocytes than in NIH 3T3 cells. We examined the potential for siRNA targeting SHP-1 or Fas to inhibit the apoptosis of cardiomyocytes under hypoxic conditions using PCM and Tat-modified poly(CBA-DAH), (PCM-CD-Tat). To evaluate for efficacy in inhibiting apoptosis, either Fas siRNA/polymer or SHP-1 siRNA/polymer were transfected into cardiomyocytes treated under hypoxic and serum-deprived conditions. After incubation under hypoxic conditions, treatment with either the SHP-1 siRNA complex or the Fas siRNA complex resulted in an increase in cell viability and a reduction in LDH-cytotoxicity. The cells transfected with either of the siRNA polyplexes had a lower incidence of apoptosis as demonstrated by Annexin V–FITC/PI staining. Both the SHP-1 siRNA/PCM-CD-Tat complex and the Fas siRNA/PCM-CD-Tat complex warrant further investigation as therapeutic agents to inhibit the apoptosis of cardiomyocytes.

Introduction

Post-transcriptional gene silencing (PTGS) through RNA interference (RNAi) has been proposed as a potent biological strategy for the treatment of gene-related disease [1], [2], [3]. RNA interference is mediated by double-stranded RNA (dsRNA), 21–23 nucleotides in length, which binds to and triggers the degradation of target messenger RNA (mRNA) through RNA-induced silencing complex (RISC) in a sequence-specific manner [4], [5]. Although RNA interference is a very promising technology, there are significant barriers to its development as a clinically viable therapy including: (i) the need for high levels of siRNA to achieve efficacy; (ii) the generation of an innate immune response; (iii) the inherent instability of the siRNA; and (iv) limited efficacy with systemic delivery. Delivery of siRNA with polymeric carriers such as polyethylenimine (PEI) and bioreducible disulfide polymers has been proposed as a means to overcome the inherent instability and poor and/or selective cellular uptake of siRNA. These polymeric carriers, however, still face major barriers with regard to transfection efficiency and cell-specific targeting for siRNA delivery.

In our previous studies, we have reported the development of novel cardiomyocyte-targeting bioreducible polymers for siRNA delivery to the myocardium [1], [6]. In these studies, our primary cardiomyocyte-targeting peptide (PCM) conjugated bioreducible polymer targeted siRNA delivery to cardiomyocytes with higher transfection efficiency. To further enhance the transfection efficiency of siRNA targeted to cardiomyocytes, we designed and synthesized a new bioreducible polymer conjugated with both a PCM peptide and a Tat peptide. The Tat sequence (C-⁴⁷YGRKKRRQRRR⁵⁷), a cell-penetrating peptide (CPP) which facilitates the cellular uptake of HIV-1 [7], [8], [9], [10], was introduced to the PCM-poly(CBA-DAH) (PCM-CD) to enhance the cellular uptake of siRNA. CPPs have been successfully employed as a non-viral approach for the delivery of genetic material [11], [12], [13]. In the present study, our newly modified bioreducible polymer was evaluated for specific binding to cardiomyocytes and enhanced cellular penetration into cardiomyocytes by using the cellular uptake profile and intracellular trafficking in H9C2 cells, comparing with NIH 3T3 cells.

To investigate whether our cardiomyocyte-targeted siRNA carrier improved siRNA delivery to cardiomyocytes, we used siRNA targeting SHP-1 or Fas. We have previously shown that our Fas siRNA delivery system inhibits cardiomyocyte apoptosis [1], [6]. Along with TNF-α and Fas, the Src homology domain 2 (SH2) containing tyrosine phosphatase-1 (SHP-1) plays an important role in apoptosis and a negative regulatory role in the phosphorylation of Akt [14], [15], [16], [17], [18]. Akt activation is a pro-survival signal which inhibits cardiomyocyte apoptosis through extracellular signal–regulated kinase (ERK)-1/2 [19]. Therefore, silencing of SHP-1 or Fas expression with siRNA delivery should reduce apoptosis under in vitro or in vivo conditions of ischemia. In the present study, we evaluated the potential of siRNA targeting SHP-1 or Fas to inhibit the apoptosis of cardiomyocytes under hypoxic and serum-deprived conditions in vitro, using our newly synthesized PCM-CD-Tat for siRNA delivery to the myocardium. Gene silencing and inhibition of apoptosis under hypoxic conditions were examined by FACS analysis and real-time RT-PCR. The innate immune response to the siRNA was measured by the interferon-α response in peripheral blood mononuclear cells (PBMCs).

Section snippets

Materials

N,N′-Cystaminebisacrylamide (CBA) was purchased from PolySciences, Inc. (Warrington, PA). tert-Butyl-N-(6-aminohexyl) carbamate (N-Boc-1,6-diaminohexane, N-Boc-DAH), trifluoroacetic acid (TFA), triisobutylsilane (TIS), N,N-diisopropylethylamine (DIPEA) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO). Fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (DPBS), Roswell Park Memorial Institute 1640 medium (RPMI

Synthesis and characterization of PCM-poly(CBA-DAH)-Tat

Cell-penetrating peptides (CPP)s, such as the Tat peptide, have been used extensively in vitro and in vivo to facilitate the uptake of a range of biological chemicals. For enhanced penetration with targeting, the Tat peptide was introduced to the cardiomyocyte-targeting PCM-modified polymer. Cysteine-terminated PCM and Tat peptide were added to the SM(PEG)₂ activated poly(CBA-DAH) (Fig. 1A). The conjugation was confirmed by the NMR peaks of tryptophan (specifically, the peaks around 7.6–7.0 ppm

Conclusion

In this study, we described the development of a cardiomyocyte-targeting non-viral gene carrier and anti-apoptotic candidate siRNAs to inhibit cardiomyocyte apoptosis. Our newly synthesized PCM-poly(CBA-DAH)-Tat was evaluated to study the efficacy of combining a cardiomyocyte-targeting PCM peptide and a cell-penetrating Tat peptide. Incorporation of the PCM and Tat peptides into a bioreducible poly(CBA-DAH) polymer enhanced the transfection efficiency of the cardiomyocyte-targeting delivery

Acknowledgments

This work was financially supported by NIH grants HL HL071541 (DAB) and 065447 (SWK).

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Published by Elsevier Ltd.

Cell penetrating peptide conjugated bioreducible polymer for siRNA delivery

Abstract

Introduction

Section snippets

Materials

Synthesis and characterization of PCM-poly(CBA-DAH)-Tat

Conclusion

Acknowledgments

Biomaterials

J Biotechnol

Biomaterials

Biomed Pharmacother

Adv Drug Deliv Rev

Adv Drug Deliv Rev

J Biol Chem

J Mol Cell Cardiol

Biomaterials

Post-transcriptional gene silencing by double-stranded RNA

Nat Rev Genet

Gene silencing in mammals by small interfering RNAs

Nat Rev Genet

An RNA-directed nuclease mediates post-transcriptional gene silencing in drosophila cells

Nature

Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide

Gene Ther

Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome-DNA complexes

Proc Natl Acad Sci U S A

Modulation of muscle contraction by a cell-permeable peptide

J Mol Med

A brief introduction to cell-penetrating peptides

J Mol Recognit