Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines

Abstract

Current nonviral genetic vaccine systems are less effective than viral vaccines, particularly in cancer systems where epitopes can be weakly immunogenic and antigen-presenting cell processing and presentation to T cells is down-regulated. A promising nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect plasmid payloads and target them to phagocytic antigen-presenting cells. However, conventional microparticle formulations composed of poly lactic-co-glycolic acid take too long to release encapsulated payload and fail to induce high levels of target gene expression. Here, we describe a microparticle-based DNA delivery system composed of a degradable, pH-sensitive poly-beta amino ester and poly lactic-co-glycolic acid. These formulations generate an increase of 3-5 orders of magnitude in transfection efficiency and are potent activators of dendritic cells in vitro. When used as vaccines in vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells.

Fig. 1.

Encapsulation of plasmid DNA using PLGA and PBAE. Molecular formulas of PBAE (Top) and PLGA (Middle). (Bottom) One percent agarose gel demonstrating DNA extracted from microparticles prepared by double emulsion. Lane 1, ladder; lanes 2 and 4, empty; lane 3, unprocessed control (88% supercoiled); lanes 5–7, aqueous extract from PLGA (lane 5), 15% PBAE (lane 6), and 25% PBAE (lane 7) microparticles after lyophilization. Percent super-coiled content for lanes 5–7 is reported in Table 1.

Fig. 2.

DCs phagocytose microparticle formulations of PLGA and PBAE in vitro. Peripheral blood mononuclear cell-derived DCs were incubated with rhodamine-conjugated dextran-encapsulated microparticles (red) for 5 h, fixed, and then stained with Hoechst dye for nucleus (blue) and phalloidin–Alexa Fluor 488 for actin (green). 3D fluorescent microscopy images indicate uptake of both PLGA microsphere formulations (Left) and 25% PBAE/75% PLGA microsphere formulations (Right). Intracellular rhodamine signals were seen as bright, localized spheres in 100% PLGA-treated DCs (Left). In 25% PBAE microsphere-treated cells, rhodamine distributions were sometimes seen as dim and dispersed, as though in the cell cytoplasm (Right).

Fig. 3.

Transfection of P388D1 macrophages with PBAE/PLGA microspheres. Results are displayed as femptograms of luciferase (luminescence assay) per mg of total protein (bicinchoninic acid assay) vs. time and formulation. Microparticle concentrations incubated with the cells were 10 μg/ml (Left)30 μg/ml (Center), and 100 μg/ml (Right). An optimal formulation of Lipofectamine 2000 (0.8:1 Lipofectamine:DNA) is displayed as a positive control in each plot (black). Twenty-five percent PBAE formulations (dark gray) consistently performed at par to 1 log unit lower than positive controls despite using 25 times less plasmid DNA, whereas 15% PBAE formulations (light gray), although lower than the 25% formulations, consistently transfected at a higher level than PLGA microparticles (white).

Fig. 4.

Activation of primary APCs by incubation with PBAE microsphere formulations. Histograms show expression levels of the indicated costimulatory molecules after 18 h of incubation with 100 ng/ml lipopolysaccharide, PLGA, 15% PBAE, and 25% PBAE microparticles encapsulating plasmid DNA (50 μg/ml), 25% PBAE microparticles without encapsulated plasmid, and an amount of free plasmid equivalent to 100% theoretical loading of microparticle treatments and instantaneous release into the supernatant. Untreated controls are shown as the open background trace in each histogram. Cells incubated with lipopolysaccharide demonstrated higher expression of costimulatory molecules but unchanged F4/80 expression. PLGA microsphere-treated cells appear to have a slightly activated phenotype, whereas cells incubated with 15% PBAE and also 25% PBAE microsphere formulations with and without plasmid DNA are activated as indicated by both down-regulation of F4/80 and up-regulation of costimulatory molecules. Results are representative of three independent experiments.

Fig. 5.

In vivo tumor rejection in B6 mice after treatment with genetic vaccine formulations. Mice were vaccinated and challenged by using the schedule shown in Left, and mean tumor size was measured by using a caliper in 2D 7 (blue), 9 (red), 11 (orange), 13 (green), and 15 (pink) days after s.c. injection of 3 × 10⁶ normal EL4 thymoma cells (Center) or EL4 cells that are transfected with SIY and express it on their surface (Right). Standard error bars are shown for comparison. ^* indicates one mouse in which the tumor expressing SIY had completely regressed by this time point.