Cell penetrating peptide conjugated bioreducible polymer for siRNA delivery
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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