Biophysical Characterization of an Integrin-Targeted Lipopolyplex Gene Delivery Vector

Abstract

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.

Effect of inserted spacer in hepatic cell-penetrating multifunctional peptide component on the DNA intracellular delivery of quaternary complexes based on modular design

“…17 In the present study, we found that the transfection efficiency of HLPD is closely related to the inserted spacer of the multifunctional peptides, between KRPTMRFRYTWNPMK and R 16 . Thus, we investigated the effect of spacer element on the in vitro DNA transfection of HLPD and mainly explored the characteristics and internalization mechanism of HLPD containing the peptides with different spacers between KRPTMRFRYTWNPMK and R 16 . The cell uptake and gene expression of Q-complexes after incubation with different inhibitors were analyzed to validate the successful intracellular gene delivery pathway.…”

“…The HA coated at the surface of ternary complexes is in charge of shielding the negative charge of the nanoparticle surface, as well as the oriented binding to the tumor tissues and cells because HA actively targets receptors (such as CD44) distributed on the surfaces of the tumor cells. 14,15 From the similar reported formulation, 16 we could conclude that some of KRPTMRFRYTWNPMK in the multifunctional peptide protruded at the surface of LPD, which possibly induces the nanoparticles to penetrate into the tumor cells, and the lipid component destroys the endosomal membranes through membrane fusion function of DOPE. 17 In the present study, we found that the transfection efficiency of HLPD is closely related to the inserted spacer of the multifunctional peptides, between KRPTMRFRYTWNPMK and R 16 .…”

“…14,15 From the similar reported formulation, 16 we could conclude that some of KRPTMRFRYTWNPMK in the multifunctional peptide protruded at the surface of LPD, which possibly induces the nanoparticles to penetrate into the tumor cells, and the lipid component destroys the endosomal membranes through membrane fusion function of DOPE. 17 In the present study, we found that the transfection efficiency of HLPD is closely related to the inserted spacer of the multifunctional peptides, between KRPTMRFRYTWNPMK and R 16 . Thus, we investigated the effect of spacer element on the in vitro DNA transfection of HLPD and mainly explored the characteristics and internalization mechanism of HLPD containing the peptides with different spacers between KRPTMRFRYTWNPMK and R 16 .…”

“…Thus, the crystal morphologies of complexes and some conformational changes in multifunctional peptides might shield their penetrating function or weaken their penetrating ability. However, when the multifunctional peptide was formed without spacer, the strong cell-penetrating function of KRPTMRFRYTWNPMK and tightly wrapping nucleic acid capability of R 16 could be fully manifested in LP a D and HLP a D. Moreover, the LP a D and HLP a D formulations showed unified spherical morphology, and the P a in the ternary and Q-complexes maintained unchangeable configuration. The function of HA was mainly in shielding the positive charge and pushing the nanoparticles to bind to the tumor cells.…”

“…The ternary complex was associated with negatively charged HA and formulated quaternary complexes (HLPD, also named as Q-complexes). Specifically, we applied a hepatic tumorspecific cell-penetrating peptide (KRPTMRFRYTWNPMK) 13 to polyarginine 16 (R 16 ) in the absence or presence of spacer to create a multifunctional peptide, which could specifically permeate hepatic tumor cells and tightly condense nucleic acids. The HA coated at the surface of ternary complexes is in charge of shielding the negative charge of the nanoparticle surface, as well as the oriented binding to the tumor tissues and cells because HA actively targets receptors (such as CD44) distributed on the surfaces of the tumor cells.…”