Arginine clustering on calix[4]arene macrocycles for improved cell penetration and DNA delivery

Abstract

Cell-penetrating peptides are widely used as molecular transporters for the internalization inside cells of various cargo, including proteins and nucleic acids. A special role is played by arginine-rich peptides and oligoarginines covalently linked or simply mixed with the cargo. Here we report cell-penetrating agents in which arginine units are clustered on a macrocyclic scaffold. Instead of using long peptides, four single arginine units were covalently attached to either the upper or lower rim of a calix[4]arene, kept in the cone conformation building a 'parallel' cyclic array. These new macrocyclic carriers show high efficiency in DNA delivery and transfection in a variety of cell lines.

Figure 1. Arginine arrays.

(a) Linear versus (b) cyclic array. Ellipse represents a macrocyclic scaffold, wavy lines represent linear hydrocarbon chains.

Figure 2. Synthesis of peptidocalix[4]arenes 3a,b and 6.

Reagents and conditions: (i) Boc-L-Arg(Pbf)-OH (N_α-Boc-N_ω-Pbf-L-arginine), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), hydroxybenzotriazole (HOBt), N,N-dimethylaminopyridine (DMAP), dry CH₂Cl₂, rt, for 2a, or Boc-L-Lys(Boc)-OH, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), dry CH₂Cl₂, rt, for 2b; (ii, 1) TFA/triisopropylsilane (TIS)/H₂O (95/2.5/2.5), rt, for 3a, or TFA/triethylsilane (TES)/CH₂Cl₂ (10/2.5/87.5) at 0 °C for 3b, (2) dil. HCl in methanol, pH 4, rt; (iii) Cbz-L-Arg-OH, N,N′-dicyclohexylcarbodiimide (DCC), HOBt, dry DMF, rt, SAX (strong anion exchange) sorbent (Cl⁻); (iv) H₂/Pd(C), HCl, ethanol, rt. Boc: t-Butoxycarbonyl, Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, Cbz: carbobenzyloxy.

Figure 3. Acyclic ligands.

Gemini-type analogues of argininocalixarenes 3a and 6. For synthetic details relative to compounds 7 and 8, and their precursors see Supplementary Information.

Figure 4. AFM images of plasmid DNA.

AFM images in tapping mode on air showing the effects induced on pEGFP-C1 plasmid DNA folding by incubation with (a) 2 μM argininocalixarene 3a; (b) 1 μM argininocalixarene 6; (c) 2 μM lysinocalixarene 3b in presence of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) 4 μM; and (d) image of pEGFP-C1 plasmid DNA 0.5 nM alone. Each image represents a 2 × 2 μm scan.

Figure 5. Cell transfection experiments.

(a) Images by fluorescence microscopy of human Rhabdomyosarcoma cells transfected (in green) upon treatment (at 48 h) with EGFP-C1 plasmid 1 nM formulated with (top) 10 μM calixarene 3a and (bottom) LTX. In histogram b, transfection efficiency (at 48 h) to Rhabdomyosarcoma cells of argininocalix[4]arenes 3a and 6 compared with the non-macrocyclic model 7 (20 μM), the lysinocalixarene 3b, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), LTX and PEI (polyethyleneimine). Error bars denote s.d. (n>3). In histogram c, transfection efficiency to other cell lines of argininocalixarene 3a (red bar) compared with argininocalixarene 3a with DOPE (pale red bar), lysinocalixarene 3b (blue bar), lysinocalixarene 3b with DOPE (light blue bar), LTX (grey bar) and PEI (light grey bar). Calixarene derivatives and DOPE were used at 10 and 20 μM, respectively. Error bars denote s.d. (n>3).