Cancer theranostic effect of hyaluronic acid-coated graphene oxide targeting oncogenic miR-21

Abstract

Purpose:

With less availability of antibody-based cancer therapeutic approach, oncogenic nucleic acid-targeting therapy has been spotlighted as new paradigm for cancer therapeutics. However, in vivo delivery issues and uncertainty of therapeutic antisense drug reaction remain as critical hurdles for successful targeted cancer therapy. Therefore, development of in vivo delivery system and technique to evaluate real antagomir reaction to oncogenic microRNA (miRNA) is necessary to advance the shortcomings of conventional anti-miRNA therapeutics. In this study, we developed in vivo graphene oxide-mediated antagomir delivery nanoplatform to inhibit endogenous miR-21 and monitored the inhibition efficacy of antagomir against miR-21 in cancer-bearing mice.

Method: Cy3 fluorescent dye-labeled peptide nucleic acid (PNA) containing complementary sequence for microRNA21 (Cy3-PNA21) and scramble sequence (Cy3-PNAscr) were first prepared, and simply mixed with hyaluronic acid-coated graphene oxide (HA-GO) for final formation of HA-GO-PNA21 (HGP21) and HA-GO-PNAscr (HGPscr) complex with quenched fluorescence. For observation of sequence-specific fluorescence recovery, miR-21 mimics and scramble microRNA mimics were each treated to HGP21 and HGPscr. Furthermore, HGP21 and HGPscr were treated to MDA-MB-231, a malignant human breast cancer cell known as over-expressing hyaluronic acid specific CD44 receptor on its membrane, and also high expression of miR-21. Simultaneously, HGP21 and HGPscr were treated to HeLa cell, a miR-21 low-expressing cell line. Therapeutic effect of HGP21 was evaluated by various experiments including proliferation analysis, migration assay and measurement of apoptotic activity. For in vivo application, MDA-MB-231 tumor xenograft model was established followed by injection of HGP21, HGPscr, and PNA21 (P21) for in vivo miR-21 imaging. Fluorescence image was taken at 1, 3, 6, 24 hr post-injection.

Results: Fluorescence intensity in Cy3-anti-PNA21 was decreased as HA-GO concentration was increased, showing maximum quenching dose at 0.3 µg. Different dose of miR-21 oligomer treatment gradually recovered the quenched fluorescence signals in HGP21, compared to scramble oligomer treatment or HGPscr with miR-21. When HGP21 was treated into MDA-MB-231 (miR-21 positive) cells, intense cy3 signal was found in the cytoplasmic area, compared to HGPscr-treated group. 2 hr pre-treatment of free HA to block CD44 resulted in hardly observable Cy3 signal. No fluorescence signal was also observed in HGP21-treated HeLa cells known to show low expression of miR-21. All these in vitro experiments were parallel with flow cytometry results. HGP21-transferred MDA-MB-231 cells revealed therapeutic effect of suppressing cancer proliferation and migration by lowering amount of miR-21. In addition, HGP21 treatment induced high apoptotic activity by recovering PTEN expression. In vivo fluorescence images displayed that intravenously administered HGP21 efficiently targeted the cancer site with intense fluorescence signal in nude mice, compared to HGPscr or PNA21 injected mice. Ex vivo fluorescence image and confocal microscopic data in tumor tissue section supported that HGP21 specifically delivered to tumor and anti-PNA21 was successfully blocked oncogenic miR-21 in vivo.

Conclusions: In this study, we developed in vivo delivery nanoplatform of anti-miR21 using graphene oxide, and visualized the targeting pattern of therapeutic anti-miR21 to oncogenic miR-21 in vivo. We expect that this system will be readily applicable for in vivo delivery of other therapeutic anti-microRNAs in wide variety of cancers and could provide promising in vivo nanodevice for antisense oligonucleotide-based cancer theranostics.