Size-Dependent Drug Loading, Gene Complexation, Cell Uptake, and Transfection of a Novel Dendron-Lipid Nanoparticle for Drug/Gene Co-delivery

Abstract

Dendron micelles have shown promising results as a multifunctional delivery system, owing to their unique molecular architecture. Herein, we have prepared a novel poly(amidoamine) (PAMAM) dendron-lipid hybrid nanoparticle (DLNP) as a nanocarrier for drug/gene co-delivery and examined how the dendron generation of DLNPs impacts their cargo-carrying capabilities. DLNPs, formed by a thin-layer hydration method, were internally loaded with chemo-drugs and externally complexed with plasmids. Compared to generation 2 dendron DLNP (D2LNPs), D3LNPs demonstrated a higher drug encapsulation efficiency (31% vs 87%) and better gene complexation (minimal N/P ratio of 20:1 vs 5:1 for complexation) due to their smaller micellar aggregation number and higher charge density, respectively. Furthermore, D3LNPs were able to avoid endocytosis and subsequent lysosomal degradation and demonstrated a higher cellular uptake than D2LNPs. As a result, D3LNPs exhibited significantly enhanced antitumor and gene transfection efficacy in comparison to D2LNPs. These findings provide design cues for engineering multifunctional dendron-based nanotherapeutic systems for effective combination cancer treatment.

Figure 1.

Synthesis of multifunctional DLNPs for drug/gene co-delivery: (a) The conjugation of alkyne-functionalized PAMAM dendrons to azide-modified DOPE via click chemistry; (b) Plots of Hoechst 33342 fluorescence intensities as a function of DLNP concentration. The inflection points of the plots indicate the critical micelle concentrations (CMCs) of D2LNP (blue) and D3LNP (red); (c) Schematic illustration of the self-assembly process for the formation of dendron-lipid nanoparticles (DLNPs) with the loading of Nile Red and complexation of pGFP.

Figure 2.

Size measurements and Nile Red loading of DLNPs: (a) The size of D2LNPs and D3LNPs was monitored at cholesterol/DL-conjugate ratio of 0, 0.2, 0.5, 1.0, and 2.0 using NTA; (b-d) Particle size distributions and representative TEM images of D2LNP and D3LNP at cholesterol/DL-conjugate ratio of 0.5; (e, f) Nile Red loading and encapsulation efficiency of DLNPs. Blue and red bars represent D2LNP and D3LNP, respectively. Significance levels are indicated as ^#p <0.10, *p <0.05, **p <0.01, and ***p <0.001.

Figure 3.

Gel retardation assays of (a) D2LNP, (b) D2 dendron, (c) D3LNP, (d) D3 dendron, (e) Nile Red-loaded D2LNP, and (f) Nile Red-loaded D3LNPs at N/P ratios of 1, 5, 10, 20, 30, and 50.

Figure 4.

Cellular uptake of DLNPs by U87 cells: (a) In vitro Nile Red and pGFP co-delivery of DLNPs; (b) Cellular internalization mechanism of DLNPs. U87 cells were pre-incubated with inhibitors (chlorpromazine, methyl-β-cyclodextrin, or filipin) or under 4 °C for 30 min, followed by incubation with DLNPs; (c) GFP expression of cells after 48 h transfection with DLNPs in the presence or absence of chloroquine.

Figure 5.

In vitro cytotoxicity of Dox-loaded DLNPs on U87 cells: (a) Cytotoxicity of D2, D3, bare D2LNP, and bare D3LNP; (b) Cytotoxicity of Dox-loaded DLNPs, compared to free Dox. Significance levels are indicated as ^#p <0.10, *p <0.05, and **p <0.01.

Figure 6.

Gene transfection efficiency of DLNPs on U87 cells: (a) Transfection efficiency of pGFP plasmid complexed with D2LNP and D3LNP on U87 cells. The transfection was determined by the expression of green fluorescent protein in the cells; (b) Transfection efficiency of luciferase plasmid complexed with D2LNP and D3LNP on U87 cells. The transfection was measured by the luciferase activity of the cell lysates. Significance levels are indicated as ^#p <0.10, *p <0.05, and **p <0.01.