Peptide-functionalized poly(ethylene glycol) star polymers: DNA delivery vehicles with multivalent molecular architecture

Abstract

Exploring the development of nonviral nucleic acid delivery vectors with progressive, specific, and novel designs in molecular architecture is a fundamental way to investigate how aspects of chemical and physical structure impact the transfection process. In this study, macromolecules comprised of a four-arm star poly(ethylene glycol) and termini modified with one of five different heparin binding peptides have been investigated for their ability to bind, compact, and deliver DNA to mammalian cells in vitro. These new delivery vectors combine a PEG-derived stabilizing moiety with peptides that exhibit unique cell-surface binding ability in a molecular architecture that permits multivalent presentation of the cationic peptides. Five peptide sequences of varying heparin binding affinity were studied; each was found to sufficiently bind heparin for biological application. Additionally, the macromolecules were able to bind and compact DNA into particles of proper size for endocytosis. In biological studies, the PEG-star peptides displayed a range of toxicity and transfection efficiency dependent on the peptide identity. The vectors equipped with peptides of highest heparin binding affinity were found to bind DNA tightly, increase levels of cellular internalization, and display the most promising transfection qualities. Our results suggest heparin binding peptides with specific sequences hold more potential than nonspecific cationic polymers to optimize transfection efficiency while maintaining cell viability. Furthermore, the built-in multivalency of these macromolecules may allow simultaneous binding of both DNA at the core of the polyplex and heparan sulfate on the surface of the cell. This scheme may facilitate a bridging transport mechanism, tethering DNA to the surface of the cell and subsequently ushering therapeutic nucleic acids into the cell. This multivalent star shape is therefore a promising architectural feature that may be exploited in the design of future polycationic gene delivery vectors.

Figure 1

(a) Chemical structure of PEG-star peptides depicting their molecular architecture. “PEPTIDE” refers to one of 5 different peptides listed in Table 1. n = 60. (b) Proposed scheme for the transfection of mammalian cells with pDNA using PEG-star HBP vectors. The star polymers are multivalent and peptides functionalized on the termini of the PEG-star should be competent to bind both DNA in the core of the polyplex and heparan sulfate on the surface of cells simultaneously.

Figure 2

Results of the gel shift assays. (a) PEG-PF4_ZIP. (b) PEG-RRP. (c) PEG-HIP. (d) PEG-sHIP. (e) PEG-ATIII. Polyplexes were created at the indicated charge ratios in water. Samples were electrophoresed in an agarose gel under a 60 V electric field.

Figure 3

Particle sizes of PEG-HBP/pDNA polyplexes in various dispersants. Polyplexes were formed by adding an equal volume of PEG-HBP solution to pDNA at a charge ratio of 5 in water. Dynamic light scattering was used to measure the hydrodynamic diameter of the polyplexes at time 0 (immediately after addition of dispersant) and thereafter consecutively at 20, 40, 60, and 120 min. (a) Sizes of the polyplexes dispersed in water. (b) Sizes of the polyplexes dispersed in serum-free Opti-MEM. (c) Sizes of the polyplexes dispersed in DMEM containing 10% FBS. Overlaid bars are indicative of bimodal data. * denotes ~2 µm, ** denotes ~3 µm, † denotes ~4 µm, ‡ denotes 4.5–5 µm.

Figure 4

Viability of BHK-21 cells after transfection with polyplexes created with PEG-HBPs in both (a) nonserum and (b) 10% serum conditions at the indicated charge ratios for 48 h. Cells were lysed after 48 h, and protein content was assayed and normalized against a non-transfected control to obtain the fraction of cell survival. Data are reported as a mean ± standard of deviation of three replicates.

Figure 5

The transfection efficiency of PEG-HBP/pDNA polyplexes in (a) non-serum- and (b) serum-containing conditions. BHK-21 cells were transfected with polyplexes containing the reporter gene for luciferase and incubated for 48 h. A luciferase assay was performed on the cell lysates and data is displayed as RLU/mg protein. Data are reported as a mean ± standard of deviation of three replicates.

Figure 6

The cellular uptake of Cy5-labeled DNA via polyplexes in non-serum-containing media. BHK-21 cells were transfected with polyplexes formulated with Cy5-labled DNA. Four hours after transfection, the cells were washed well with PBS and allowed to traffic for 2 more hours. The cells were then trypsinzed and analyzed via flow cytometry. Each sample represents the mean fluorescence in 50 000 cells.