Engineering clustered ligand binding into nonviral vectors: alphavbeta3 targeting as an example

Abstract

The development of techniques to efficiently deliver genes using nonviral approaches can broaden the application of gene delivery in medical applications without the safety concerns associated with viral vectors. Here, we designed a clustered integrin-binding platform to enhance the efficiency and targetability of nonviral gene transfer to HeLa cells with low and high densities of alpha(v)beta(3) integrin receptors. Arg-Gly-Asp (RGD) nanoclusters were formed using gold nanoparticles functionalized with RGD peptides and used to modify the surface of DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes with attached RGD nanoclusters resulted in either 5.4- or 35-fold increase in gene transfer efficiency over unmodified polyplexes for HeLa cells with low- or high-integrin surface density, respectively. The transfection efficiency obtained with the commercially available vector jetPEI-RGD was used for comparison as a vector without clustered binding. JetPEI-RGD exhibited a 1.2-fold enhancement compared to unmodified jetPEI in cells with high densities of alpha(v)beta(3) integrin receptors. The data presented here emphasize the importance of the RGD conformational arrangement on the surface of the polyplex to achieve efficient targeting and gene transfer, and provide an approach to introduce clustering to a wide variety of nanoparticles for gene delivery.

Figure 1

Schematic comparing Adenovirus type 2 and DNA/PEI– Au-Cap-RGD-ASA polyplex. (a) Type 2 Adenovirus with penton-base proteins (reproduced from ref. 9). (b) DNA/PEI-Au-Cap-RGD-ASA polyplex with RGD nanoclusters. (c) Au-Cap-RGD-ASA nanoparticles are conjugated to preformed DNA/PEI polyplexes when exposed to UV light.

Figure 2

Salt stability of modified and unmodified nanoparticles measured using UV-Vis absorbance spectroscopy. Salt concentration was increased using 2 µl for unmodified nanoparticles and 15 µl of 4.11 mol/l NaCl every 15 minutes. Wavelength scans of (a) 5 nm unmodified Au nanoparticles and (b) Au-Cap nanoparticles in solution with increasing NaCl concentration. Shaded area under the curves in a and b were used to calculate aggregation parameter. (c) Aggregation parameter plotted with mean ± SD for n = 3 for increasing NaCl concentration for 5 nm unmodified Au and Au-Cap. Aggregation parameter of unmodified Au show a large increase with small increases in NaCl concentration.

Figure 3

Characterization of DNA/PEI and DNA/PEI-Au-Cap-RGD-ASA polyplexes. (a) Mean size ± SD (n = 3) of 5 nm unmodified Au and Au-Cap measured using dynamic light scattering (DLS). (b) Ethidium bromide (EtBr) competition assay for DNA/PEI polyplex formation. Plasmid DNA (26.6 µg/ml in Tris–EDTA buffer) and EtBr (1.1 µg/ml) were added to a fluorimeter vial to a final volume of 150 µl and read before and after the addition of each aliquot of PEI (0.5 µl of 0.1 mg/ml). (c) Size of DNA/PEI polyplexes modified with 5 nm Au-Cap-RGD nanoparticles were measured using DLS for low (13.3 µg DNA/ml) and high concentration (40 µg DNA/ml). (d) Electron micrographs of negatively stained DNA/PEI-Au-Cap-RGD-ASA. All the polyplexes were observed to contain Au particles. Bar = 100 nm. (e) Number distribution of the amount of Au nanoparticles per surface area of polyplex (n = 17).

Figure 4

Cell toxicity (proliferation) assay for Au-Cap nanoparticles and DNA/PEI-Au polyplexes. HeLa cells were plated in 96-well plates (7,000 cells/well) for 12 hours before exposing the cells to Au-Cap nanoparticles or DNA/PEI-Au-Cap-RGD-ASA polyplexes at varying concentrations of Au nanoparticles (1.7 µl/well) and testing for cell proliferation. The data were normalized to the absorbance of untreated samples to calculate the percent viability for each sample (n = 3).

Figure 5

Effects of multivalent nanoparticles on HeLa cells. (a) Flow cytometry of α_vβ₃ integrin stained of HeLa cells passaged by scrapping or trypsinization measured 12 hours after plating. A gate that included 5% of cells in the unstained control was used in determining the percent of cells that were α_vβ₃ integrin positive. The amount of trypsin and scrapped α_vβ₃ integrin-positive cells are plotted with ***P < 0.0001 using a two-tailed t-test (n = 3). Mean fluorescence of all trypsin or scrapped cells were compared. **P < 0.001 using a two-tailed t-test (n = 3). (b) Antiadhesion assay of RGD peptide, Au-Cap, and Au-Cap-RGD nanoparticles with HeLa cells (n = 3). The multivalent Au-Cap-RGD nanoparticles required less of the equivalent amount of RGD than the monovalent free RGD peptide.

Figure 6

Transfection efficiency in high- and low-α_vβ₃ integrin-expressing HeLa cells measured using a luciferase reporter gene. (a) Luciferase expression in low-α_vβ₃-integrin-expressing HeLa cells transfected with DNA/PEI-Au-Cap-RGD-ASA (with covalent linker) and DNA/PEI-Au-Cap-RGD (without covalent linker) (n = 3). (b) Luciferase expression in high-α_vβ₃-integrin-expressing HeLa cells transfected with DNA/PEI-Au-Cap-RGD-ASA (with covalent linker), DNA/PEI-Au-Cap-RGD (without covalent linker), and DNA/PEI-Au-Cap (n = 3). The symbol *** represents statistical significance to the level of P < 0.001 for DNA/PEI-Au-Cap-RGD-ASA compared to DNA/PEI-Au-Cap-RGD and DNA/PEI-Au-Cap using the Tukey test. (c) Luciferase expression in high-α_vβ₃-integrin-expressing HeLa cells transfected with DNA/PEI-Au-Cap-RGD-ASA (with covalent linker), DNA/PEI-Au-Cap-ASA (with covalent linker but no RGD), DNA/PEI-Au-Cap-RGD (without covalent linker), and DNA/PEI-Au-Cap (without covalent linker) (n = 3). The symbol *** represents statistical significance to the level of P < 0.001 for DNA/PEI-Au-Cap-RGD-ASA compared to DNA/PEI-Au-Cap-RGD, DNA/PEI-Au-Cap, and DNA/PEI-Au-Cap-ASA using the Tukey test. (d) Competitive binding of free RGD peptide for transfection of high-α_vβ₃-integrin-expressing HeLa cells with DNA/PEI-Au-Cap-RGD-ASA (n = 3). The symbol * represents statistical significance to the level of P < 0.05.

Figure 7

Transfection efficiency using vectors with clustered and homogeneous ligands. Luciferase transgene expression of DNA/PEI and DNA/PEI-Au-Cap-RGD-ASA compared to DNA/jetPEI and DNA/jetPEI-RGD for (a) high- and (b) low-α_vβ₃-integrin-expressing HeLa cells (n = 3). Sensitivity of the vector to integrin density can be compared using the transfection efficiency of high- and low-integrin-expressing cells by dividing the RLU/mg of the high-expressing cells to the RLU/mg of the low-expressing cells (integrin sensitivity factor). (c) integrin sensitivity factor comparison of HeLa cells for DNA/PEI, DNA/PEI-Au-Cap-RGD-ASA, DNA/jetPEI, and DNA/jetPEI-RGD (n = 3).