Multivalent cyclic RGD conjugates for targeted delivery of small interfering RNA

Abstract

We have designed, synthesized, and tested conjugates of chemically modified luciferase siRNA (Luc-siRNA) with bi-, tri-, and tetravalent cyclic(arginine-glycine-aspartic) (cRGD) peptides that selectively bind to the αvβ3 integrin. The cellular uptake, subcellular distribution, and pharmacological effects of the cRGD-conjugated Luc-siRNAs compared to those of unconjugated controls were examined using a luciferase reporter cassette stably transfected into αvβ3 positive M21(+) human melanoma cells. The M21(+) cells exhibited receptor-mediated uptake of cRGD-siRNA conjugates but not of unconjugated control siRNA. The fluorophore-tagged cRGD-siRNA conjugates were taken up by a caveolar endocytotic route and primarily accumulated in cytosolic vesicles. The bi-, tri-, and tetravalent cRGD conjugates were taken up by M21(+) cells to approximately the same degree. However, there were notable differences in their pharmacological effectiveness. The tri- and tetravalent versions produced progressive, dose-dependent reductions in the level of luciferase expression, while the bivalent version had little effect. The basis for this divergence of uptake and effect is currently unclear. Nonetheless, the high selectivity and substantial "knock down" effects of the multivalent cRGD-siRNA conjugates suggest that this targeting and delivery strategy deserves further exploration.

FIGURE 1. Chemical structure of cysteine-bearing cyclic RGD (cRGD) peptide

The cysteine is connected to a 6-aminohexanoic acid linker that joins two, three and four cyclic RGD moieties respectively for the bi-, tri and tetravalent versions. Only the trivalent peptide is shown here. Arrow indicates the site of conjugation of the peptide (thiol) to the maleimide functionality on the luciferase siRNA sense strand.

FIGURE 2. The conjugation of multivalent cyclic RGD peptides to the maleimide containing sense strand of the luciferase siRNA

The oligonucleotides (A-59841.1 and A-59842.1) were purified by HPLC after each step in the synthesis. (I) 6-Maleimidohexanoic acid N-hydroxysuccinimide ester, 1x PBS buffer, pH 7.2, 20 min, room temperature (RT); (II) Cysteine-bearing multivalent cRGD peptides, 0.4M potassium chloride, 40% aqueous acetonitrile, overnight, RT.

FIGURE 3. HPLC Analysis

AKTA RP-HPLC purification profiles for the crude reaction mixtures of (a) bivalent (b) trivalent and (c) tetravalent RGD-sense oligonucleotide conjugates. Arrows indicate the relative change of retention time of the conjugates (A-69252.1, A-69253.1, and A-69254.1) from the maleimide-RNA sense strand (A-59842.1). HPLC analysis was performed as described in the materials and methods section.

FIGURE 4. Possible degradation product

A possible mechanism for the degradation of cRGD-sense strand conjugate in 1X PBS buffer, pH 7.4 during annealing (95°C, 3min, then cooled to RT).

FIGURE 5. Uptake dose-response for control and cRGD- conjugated siRNAs

M21+GL₃ cells were treated with various concentrations of control or RGD-conjugated Alexa 488 labeled siRNA for 4 h at 37°C in OPTI-MEM without serum. Thereafter the cells were harvested and analyzed for fluorescence levels by flow cytometry. Results are the means and standard errors of triplicate determinations.

FIGURE 6. (A) Uptake of cRGD-conjugated siRNA by M21+ or M21- cells

M21+GL₃ cells (avb3 positive) or M21- cells (avb3 negative) were treated with 100 nM control or tetravalent RGD conjugated siRNA for 4 h. Uptake of Alexa 488 labeled siRNA was measured by flow cytometry. Data normalized on M21+ cells with RGD-siRNA as 100%. (B) Uptake of tetravalent RGD-siRNA conjugates plus or minus the presence of cycloRGD inhibitor. M21+GL₃ cells were pretreated with 10 mg cycloRGD peptide for 15 min followed by treatment with 50 nM control siRNA or tetravalent cRGD-siRNA for 4 h at 37°C. Thereafter the cells were harvested and analyzed for Alexa 488 fluorescence levels by flow cytometry. Data normalized on RGD-siRNA (no inhibitor) as 100%. (C) Uptake of RGD-conjugated siRNA with or without serum. M21+GL₃ cells were treated with 100 nM control or RGD conjugated siRNAs for 4 h at 37°C in OPTI-MEM with 5% FBS or without serum. Thereafter the cells were harvested and analyzed for fluorescence levels by flow cytometry. Data normalized on tetra-RGD-siRNA (no serum) as 100%. Results A–C are means and standard errors of triplicate or quadruplicate determinations.

FIGURE 7. Knockdown dose-response for cRGD conjugated siRNAs

M21+GL₃ cells were treated with various concentrations of control siRNA or RGD-conjugated siRNA for 4 h at 37°C in OPTI-MEM without serum. The control siRNA complexed with Lipofectamine 2000 was used as a positive control. After 4 h serum was added to 2% to all wells with the exception of the Lipofectamine 2000 in which the medium was replaced with DMEM-H with 2% serum. After 96 h the cells were lysed and luminescence analyzed on a Fluostar Omega microplate reader and normalized to total cellular protein. Results are the means of pentuplicate determinations and are expressed as percent of untreated control cells (ordinate). The abcissa shows siRNA concentrations (nM). The unconjugated siRNA had no effect at any concentration tested (not shown) unless it was complexed with Lipofectamine 2000, in which case an 80% reduction was attained at 100 nM for the original sequence while a 99% reduction was obtained for the ‘B’ sequence (not shown).

FIGURE 8. (A) Cellular distribution of siRNAs

M21+GL₃ cells were incubated with control siRNA-Alexa 488 (50 nM) for 4 hours or Tetravalent RGD-siRNA-Alexa 488 (50 nM) for 4 hours and 24 hours. Live cells were observed by confocal fluorescence microscopy as described in the materials and methods section. B. Co-localization with endocytosis markers. Tetravalent RGD-siRNA-Alexa 488 (50 nM) was co-incubated with Alexa-594 labeled transferrin (Tfn) (20 μg/ml) and cholera toxin B (CTB) (4 μg/ml). Live cells were observed by confocal fluorescence microscopy as above. Selected vesicles showing co-localization are marked with white arrows.

Scheme 1^a

Conjugation of cRGD peptides to siRNA ^a (i) Solid phase oligonucleotide synthesis and deprotection; (ii) PBS buffer, pH 7.2, rt, 20 min;(iii) 0.4M KCl, 40% MeCN in H₂O, rt, overnight; (iv) antisense strand, annealing at 95 °C in H₂O;(v) Alexa 488 NHS ester, PBS buffer, pH 7.2, 20 min.

Scheme 2^a

^a Intra-molecular amine assisted retro Michael addition of cRGD-Oligonucleotide conjugate at elevated temperature (>90 °C) in PBS buffer at pH 7.4.