Enhanced Cancer Theranostics with Self-Assembled, Multilabeled siRNAs

Abstract

The integration of therapy and diagnostics, termed "theranostics", has recently gained widespread utility in the development of new and improved therapeutics that effectively diagnose and treat diseases, such as cancer. In this study, the covalent attachment of multiple fluorescent labels (i.e., fluorescein isothiocyanate (FITC)) to a wide range of siRNAs, including those adopting linear, V- and Y-shape nanostructures, was successfully accomplished by solid-phase bioconjugation for monitoring cell uptake, co-localization, and biological activity in cell culture. The FITC-labeled higher-order V- and Y-shape siRNAs maintained the requisite hybrid stabilities and A-type helical structures for invoking RNAi activity. The FITC-siRNA hybrids with sense-strand modifiers enabled efficient mRNA knockdown (∼50-90%), which also translated to increased cell death (∼20-95%) in a bone metastatic prostate cancer cell line, over a 72 h incubation period. Significantly, the Y-shaped siRNA containing three FITC probes enhanced fluorescent signaling relative to the siRNA constructs containing single and double fluorophores while retaining potent knockdown and cell death effects post-transfection. Taken together, this data highlights the theranostic utility of the multilabeled FITC-siRNA constructs for potential cancer gene therapy applications.

Scheme 1. Solid-Phase FITC Bioconjugation of Linear, V-, and Y-Shape RNA Templates

Scheme 2. Self-Assembly of (i) Linear, (ii) V-, and (iii) Y-Shape Multi-FITC-Labeled siRNA Constructs^,

The green label represents fluorescent (FL) FITC-labeled sequences for linear (L), V-shape (V), and Y-shape (Y) siRNAs containing a single (1F), double (2F), and triple (3F) fluorophores. Antisense (A) and sense (S) strands were hybridized to target a first (1) or second (2) mRNA site belonging to GRP 75, 78, and 94.

Figure 1

Native PAGE analysis of FITC-labeled siRNAs under short (A, C) and long (B, D) UV radiations. Sequences in green represent fluorescent (FL) FITC-labeled sequences for linear (L), V-shape (V), and Y-shape (Y) siRNAs. Antisense (A) and sense (S) strands hybridized to target a first (1) or second (2) mRNA sites belonging to GRP 75, 78, and 94. siRNA sequences for A and B: lane 1: dye; lane 2 A1:S1; lane 3 FL-78A1:78S1; lane 4 V-78A194A1:78S194S1; lane 5 FL-V-78A194A1:78S194S1; lane 6 V-78A194A1:78S1FL-94S1; lane 7 V-78A178A2:78S178S2; lane 8 FL-V-78A178A2:78S178S2; lane 9 V-78A178A2:FL-78S178S2; lane 10: Y-78A194A175A1:78S194S175S1; lane 11 Y-78A194A175A1:FL-78S194S175S1; lane 12 Y-78A194A175A1:78S1FL-94S175S1; lane 13 Y-78A194A175A1:FL-78S1FL-94S175S1; lane 15 dye. siRNA sequences for (C) and (D). Lane 1 dye; lane 2 empty; lane 3 A1:S1; lane 4 78A1:FL-78S1; lane 5 empty; lane 6 V-78A194A1:78S194S1; lane 7 V-78A194A1:FL-78S194S1; lane 8 V-78A194A1:FL-78S1FL-94S1; lane 9 empty; lane 10 Y-78A194A175A1:78S194S175S1; lane 11 Y-78A194A175A1:78S194S1FL-75S1; lane 12 Y-78A194A175A1:78S1FL-94S1FL-75S1; lane 13 Y-78A194A175A1:FL-78S1FL-94S1FL-75S1; lane 14 empty; lane 15 dye.

Figure 2

Characterization data of FITC-labeled siRNA hybrids. Circular dichroism spectroscopy of (A) FITC-labeled siRNAs and (B) multi-FITC-labeled siRNAs. Thermal denaturation, T_m, of FITC-labeled siRNAs (C) and multi-FITC-labeled siRNAs (D). Fluorescence emission spectra of (E) FITC-labeled siRNAs and (F) multi-FITC-labeled siRNAs. All siRNA hybrids were prepared in annealing buffer (1.25 μM, 10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH 7.5–8.0).

Figure 3

siRNAs’ uptake efficiency in PC-3 cells monitored by flow cytometry. (A) Cell internalization of linear, V- and Y-shaped FITC-labeled siRNA. (B) Time-dependent cell internalization of linear, V- and Y-shaped siRNA containing single, double or triple FITCs, respectively.

Figure 4

Time-dependent (4 and 24 h post-transfection) fluorescent and bright-field images of PC-3 cells transfected with (A) linear FITC-labeled siRNA and (B) Y-shape FL–siRNA containing multiple (3×) FITC probes.

Figure 5

qRT-PCR and Western blot analyses of GRP 75, 78, and 94. (A) Total mRNA levels were analyzed 72 h post-transfection of the antisense-labeled FL–siRNA constructs. (B) Total mRNA levels were analyzed 48 h post-transfection of the sense-strand-labeled FL–siRNA constructs containing 1, 2, and 3 FITC probes. Protein and mRNA knockdown levels of GRP 75, 78, and 94 determined by Western blot (C) and RT-PCR (D) with the Y-shape FL–siRNA containing three FITC probes. *P < 0.05 and *P < 0.01 in PC-3 cells.