Tumor-targeted delivery of siRNA using fatty acyl-CGKRK peptide conjugates

Meenakshi Sharma,Keykavous Parang,Naglaa Salem El-Sayed,Hung Do,Rakesh Kumar Tiwari,Hamidreza Montazeri Aliabadi

doi:10.1038/s41598-017-06381-y

Meenakshi Sharma, Keykavous Parang + Show 4 more

Open Access

https://doi.org/10.1038/s41598-017-06381-y

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Abstract

Tumor-targeted carriers provide efficient delivery of chemotherapeutic agents to tumor tissue. CGKRK is one of the well-known tumor targeting peptides with significant specificity for angiogenic blood vessels and tumor cells. Here, we designed fatty acyl conjugated CGKRK peptides, based on the hypothesis that hydrophobically-modified CGKRK peptide could enhance cellular permeation and delivery of siRNA targeted to tumor cells for effective silencing of selected proteins. We synthesized six fatty acyl-peptide conjugates, using a diverse chain of saturated and unsaturated fatty acids to study the efficiency of this approach. At peptide:siRNA weight/weight ratio of 10:1 (N/P ≈ 13.6), almost all the peptides showed complete binding with siRNA, and at a w/w ratio of 20:1 (N/P ≈ 27.3), complete protection of siRNA from early enzymatic degradation was observed. Conjugated peptides and peptide/siRNA complexes did not show significant cytotoxicity in selected cell lines. The oleic acid-conjugated peptide showed the highest efficiency in siRNA uptake and silencing of kinesin spindle protein at peptide:siRNA w/w ratio of 80:1 (N/P ≈ 109). The siRNA internalization into non-tumorigenic kidney cells was negligible with all fatty acyl-peptide conjugates. These results indicate that conjugation of fatty acids to CGKRK could create an efficient delivery system for siRNA silencing specifically in tumor cells.

Highlights

Despite significant advances in cancer research and introduction of new generations of molecularly-targeted drugs, cancer is still among leading causes of morbidity worldwide
Fatty acyl conjugated peptide were cleaved from the solid support using freshly prepared cleavage cocktail reagent R followed by purification using HPLC and characterized by MALDI mass spectrometry (See Supporting Information)
We have demonstrated the development of a tumor-targeted delivery system using fatty acylation of CGKRK peptide

Summary

Introduction

Despite significant advances in cancer research and introduction of new generations of molecularly-targeted drugs, cancer is still among leading causes of morbidity worldwide. The main obstacles are the short stability of siRNA under physiological conditions, as siRNA gets readily degraded by the nucleases in the serum[9], and the negligible cellular internalization due to anionic and hydrophilic nature of the double stranded RNA Considering all these barriers, designing a safe and efficient siRNA delivery system is required. We hypothesized that the coupling of an optimized fatty acyl chain with CGKRK peptide can improve the ability of the peptide to permeate and deliver siRNA inside the cancer cell to enhance gene silencing efficiency. We further characterized and evaluated conjugated peptides by selected parameters i.e. size and zeta potential, siRNA binding, serum stability, specificity for the cancer cells, cellular uptake, and most significantly, the mRNA interference efficacy. To test the conjugated peptides, we used 3 different cancer cells lines (AU-565, a human breast cancer cell line; MDA-MB-435, a human cancer cell line considered a breast cancer cell line for many years, and recently recognized as a melanoma cell line; and MDA-MB-231, a triple negative human breast cancer cell line) and one non-tumorigenic human cell line (human embryonic kidney cells; HEK-293) to observe the specificity of the peptide for the cancer cells

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