A covalently stabilized lipid-polycation-DNA (sLPD) vector for antisense oligonucleotide delivery

Abstract

Antisense oligonucleotide G3139 is designed for Bcl-2 downregulation and is known to induce toll-like receptor activation. Novel stabilized lipid-polycation-DNA (sLPD) nanoparticles were constructed and evaluated for the delivery of G3139 to human carcinoma KB cells and for bioactivity in vivo. Polyethylenimine (PEI) was incorporated as a DNA condensing agent. The lipid composition used was DOTAP/DDAB/Chol/TPGS/linoleic acid/hexadecenal at molar ratios of 30/30/34/1/5/0.2. The nanoparticles were stabilized by the formation of a reversible covalent bond between the aldehyde group on the cis-11-hexadecenal and amines on the PEI. When sLPDs were used to transfect KB cells, 90.4% Bcl-2 downregulation was observed, compared to no significant downregulation by free G3139 and 54.6% downregulation by nonstabilized LPD-G3139. The sLPDs were then evaluated for therapeutic efficacy in mice bearing KB subcutaneous tumors and were found to trigger a strong antitumor response, inhibiting tumor growth and prolonging survival with 72% increase in lifespan (ILS). Consistent with previous reports on other G3139 nanoparticles, the increased antitumor activities of sLPDs in vivo were found to be associated with increased cytokine induction rather than Bcl-2 downregulation, suggesting an immunological mechanism.

Figure 1

Design of sLPD Panel A. Formation of a Schiff’s base between PEI-2k and hexadecenal. Panel B. A model for the structure of sLPD

Figure 2

Colloidal and serum stability of sLPD Panel A. Colloidal stability of sLPDs. Panel B. Stability of sLPD-G3139 in serum. The sLPD-G3139 was mixed with serum at 1:4 volume ratio and incubated at 37 °C for different times. The samples were then analyzed by electrophoresis in 3% low melting point agarose gel and stained with ethidium bromide.

Figure 3

Internalization of sLPD-G3139 in KB cells The cells were incubated with 500 nM sLPD-G3139 (containing 10% fluorescent FAM-G3139) at 37 °C. At different time points, unbound sLPDs were removed by washing with PBS and cellular nuclei were stained by DAPI. The cell were then mounted to the slide and observed under a fluorescence microscope. Blue color indicated nuclei stained by DAPI and green color indicated FAM-labeled G3139. Panel A. Cells were treated with sLPD-G3139 spiked with 10% FAM-G3139 (green) at 37°C for 30, 120 and 240 min., respectively, stained by DAPI (blue) and visualized on a confocal microscope. Panel B. Optical sections (14 total) of cells were collected after 4 hr by incremental scanning along the z-axis at a spacing of 0.45 μm. Panel C. Cells were treated with sLPD-G3139 and visualized on a fluorescence microscope.

Figure 4

Bcl-2 protein expression in KB cells treated with sLPD-G3139 Panel A. Bcl-2 down-regulation by Western blot. Cells were treated with PBS, free G3139 or sLPD-G3139 at 1 μM. Bcl-2 protein levels were analyzed at 48 hrs by Western blot. Upper panel represents the results of Western blot and lower represents its corresponding densitometry data. Panel B. Concentration-dependent Bcl-2 down-regulation by sLPD-G3139. Cells were treated with sLPD-G3139 at different concentrations. Bcl-2 protein levels were analyzed at 48 hrs by Western blot. Upper panel shows the results of Western blot and lower its corresponding densitometry data.

Figure 5

Induction of caspases in KB cells treated with sLPD-G3139. Cells were incubated 1 μM sLPD-G3139 or control formulations. Cellular apoptosis was evaluated via caspase-9, -3 and -8 activities, as described in the Materials and Methods section. The values in the plot represent the means of 4 separate experiments. Error bars were standard deviations, n=4. *, p < 0.05, when compared to the free G3139 group. **, p < 0.05, when compared to the LPD-G3139 group.

Figure 6

Therapeutic efficacy of sLPD-G3139s Nude mice were inoculated s.c. with human KB cells 5 days prior to treatment. The mice received i.v. injections of 5mg/kg sLPD-G3139 or control formulations on every 3^rd day. Five mice were used in each group. Panel A. Effect of treatment on tumor size. Panel B. Effect of treatment on animal survival.

Figure 7

G3139 accumulation and Bcl-2 expression in tumors Panel A. Tumor accumulation profile of FAM-G3139. Following tail i.v. bolus administration of 5 mg/kg of FAM-G3139 loaded sLPD, LPD, or free FAM-G3139 in tumor-bearing mouse (n=3). Each point represents Mean ± SD of three mice. Panel B. Immunohistochemical staining of Bcl-2 in tumors. Frozen sections were prepared from tumors after 4 times of 5 mg/kg treatment with free G3139 or sLPD-G3139 and stained with anti-human Bcl-2 antibodies.

Figure 8

Serum cytokine production in mice. After the treatment by sLPD-G3139 or controls (free G3139, LPD-G3139, empty sLPD or empty LPD), serum from the mice was collected and cytokine IL-6 and IFN-γ were detection by ELISA. The values in the plot represent the means of 3 separate experiments. Error bars were standard deviations, n=3. Panel A. IL-6 measurement Panel B. IFN-γ measurement.