Delivery of Antisense Oligonucleotide LOR-2501 Using Transferrin-conjugated Polyethylenimine-based Lipid Nanoparticle

Materials and Methods

Materials. LOR-2501 (5’-CTC TAG CGT CTT AAA GCC GA-3’) and 5’-Cy3-LOR-2501 were synthesized by Biomics Biotechnologies (Jiangsu, China). Egg phosphatidylcholine (ePC) and cholesterol were purchased from Avanti Polar Lipids, Inc. (Shanghai, China). Branched PEI 25kDa, hexadecylamine, Traut's Reagent (2-iminothiolane•HCl) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). SPDP was obtained from Dojindo (Dojindo Laboratory, Kumamoto, Japan). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and 0.25% (w/v) trypsin were purchased from HyClone (Logan, UT, USA). Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and 4’,6-Diamidino-2-phenylindole (DAPI) was purchased from Thermo Fisher Scientific (Rockford, IL, USA). HepG2 cells were purchased from American Type Culture Collection (ATCC). All other analytical reagents were commercially obtained in reagent grade.

Synthesis of PEI-SS-HA. PEI-SS-HA was synthesized as described previously (20). Firstly, 0.8 μmol PEI 25 kDa and 20 μmol SPDP were dissolved into ethanol to achieve concentrations of 20 mg/ml and 100 mg/ml, respectively. The SPDP solution was added to the PEI 25 kDa solution rapidly under stirring. The mixture was incubated for 4h at room temperature (25°C) to prepare the PEI-PDP solution. Then, 30 μmol hexadecylamine (HA) was dissolved in 1 ml ethanol, and 60 μmol Traut's Reagent (2-Iminothiolane•HCl) was dissolved into 0.5 ml water. The Traut's Reagent solution was added to the HA solution under stirring and the mixture was incubated for 3 h at room temperature (25°C) in the dark, yielding HA-SH. Then, the HA-SH solution was added to the PEI-PDP solution slowly under vortex mixing. The reaction mixture was then stirred at room temperature (25°C) for 4 h. Then, the product was transferred into a dialysis tube (MWCO 10 kDa) and dialyzed against deionized water for 24 h. Finally, the solution was freeze-dried to obtain PEI-SS-HA (PSH), with the structural formula shown in Figure 1A.

Cell culture. HepG2 cells were grown and propagated in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% FBS and 1% antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin, Sigma). Cells were grown at 37°C in a humidified atmosphere containing 5% CO₂.

Buffer capacity determination of PSH. To assess the buffering capacity of the synthetic polymer, the ability of PEI-SS-HA (PSH) to buffer from pH 10-2 was determined by acid-base titration (20-21). Firstly, 0.5 mg/ml PEI 25 kDa and PSH solutions were prepared. Then the pH of the solution was raised to 10 using 1 mol/l NaOH. Then, 0.1 mol/l HCl was added in 10 μl increments into 2 ml polymer solution. pH values were measured after stirring thoroughly. The curve of hydrochloric acid volume vs pH value was plotted.

Preparation and characterization of LPs. An ethanol dilution method was used for the preparation of LPs containing LOR-2501 (L-LPs) (22-24). Firstly, PEI-SS-HA (PSH), ePC and cholesterol were dissolved in ethanol at molar ratio 40/18/35. Then, the ethanolic lipid solution was injected into the PBS buffer (pH 6.8) under stirring at a ratio of 1/3 (v/v) to obtain LPs containing PSH (PSH-LP). LOR-2501 was separately dissolved in diethyl pyrocarbonate-treated water. To obtain LPs containing LOR-2501 (L-PSH-LP), the LOR-2501 solution was added into the PEI-SS-HA/lipid mixture and vortexed for 30 sec, at varying nitrogen-to-phosphate (N/P) ratios, ranging from 1:1 to 10:1. “N” represented the molarity of nitrogen in PEI or PSH, and “P” represented the molarity of phosphorus in LOR-2501. To prepare LPs containing transferrin (Tf), post-insertion was used. Tf-Chol was incubated with the PSH-LP or L-PSH-LP for 1 h at 37 °C at Tf-Chol/total lipid ratios from 1/60 to 1/140. The resulting LPs were named TPSH-LP and L-TPSH-LP. A schematic diagram of the preparation process of LPs is shown in Figure 1B. Finally, all the LPs were concentrated by ultrafiltration with a tubular polysulfone ultrafiltration membrane (MWCO 100 kDa).

The particle sizes and zeta potentials of L-PSH-LP and L-TPSH-LP at various N/P ratios or Tf-Chol/total lipid ratios were determined on a Zetasizer Nano ZS 90 instrument (Malvern Instruments, Ltd., Malvern, UK). Each data point was calculated averaging 3 measurements. The structure of L-TPSH-LP was investigated by field emission scanning electron microscope (FE-SEM) (JSM-6700F, JEOL, Tokyo, Japan). SEM images were taken at 3.0 kV accelerating voltage.

Gel retardation assay for determining complexes of LPs with PSH and TPSH. PEI 25 kDa, PSH-LP and TPSH-LP were combined with LOR-2501 to form L-PEI, L-PSH-LP and L-TPSH-LP. Two microliter of 6×sample buffer (50% glycerol, 1% bromophenol blue, and 1% xylene cyanol FF in Tris-borate EDTA (TAE) buffer) was added to each sample. The samples were loaded onto 3% agarose gel containing 0.2% mg/ml ethidium bromide, and the electrophoresis was run in TAE running buffer at 120 V for 20 min. The resulting gels were photographed under UV-illumination.

Cytotoxicity assay. HepG2 cells were seeded at a density of 1×10⁴ cells/well in a 96-well plate and cultured for 24 h in 100 μl DMEM containing 10% FBS. Before adding the samples, cells were washed for three times with PBS. Then, 100 μl of PEI 25 kDa, PSH, PSH-LP, or TPSH-LP in medium without FBS were added at various concentrations from 0.5 μg/ml to 8 μg/ml. After incubation for 4 h, transfection media was removed and the cells were incubated in fresh DMEM for an additional 20 h. Then, each well was incubated with 20 μl MTT solution (5 mg/ml in PBS) for 4 h. The medium was removed and 100 μl/well of DMSO was added to dissolve the emergent formazan crystals. The optical density was measured at 570 nm in a plate reader. The results were converted into % viability and the mean±SD of 6 replicates for each sample were calculated.

Cellular uptake of LPs. HepG2 cells were plated on 24 well plates at a density of 1×10⁵ cells/well and cultivated overnight in 1 ml of DMEM medium containing 10% FBS. The medium was then removed, PBS was added to wash the cells three times and fresh serum-free DMEM medium was added. LOR-2501 5’-labeled with Cy3 (Cy3L) was formulated in the LPs. HepG2 cells were treated with Cy3L-PEI, Cy3L-PSH-LP or Cy3L-TPSH-LP for 4 h. The cells were washed with PBS, digested and fixed in 4% paraformaldehyde solution for 24 h. Cy3L positive HepG2 cells were determined by EPICS XL flow cytometer (Beckman Coulter Corp., Tokyo, Japan). Untreated cells were used as negative control. Each sample was tested by 3 serial measurements at a minimum.

Visualization of internalization of LPs by confocal microscopy. HepG2 cells were seeded in a glass bottom cell culture dish at a density of 1×10⁵ cells/well for 24 h. LOR-2501 was labeled with 5’-Cy3 (Cy3L) was mixed with LPs, and the cells were treated with naked Cy3L, Cy3L-PSH-LP, or Cy3L-TPSH-LP for 4 h at 37°C. Cells were washed 3 times with PBS and fixed in 4% paraformaldehyde for 10 min. Cellular nuclei were stained with DAPI (2 μg/ml) for 3 min followed by washing 3 times with PBS. The cells were observed by Zeiss 710 LSMNLO Confocal Microscope (Carl Zeiss; Jena, Germany).

Determination of R1 protein expression. The expression of R1 protein was assayed using Western blot. Briefly, HepG2 cells were plated on 6 well plates at a density of 1×10⁵ cells/well for 24 h. Cells were treated with L-PEI, L-PSH-LP, or L-TPSH-LP for 4 h. The medium was removed and the cells were cultured for an additional 44 h. The cells were homogenized in RIPA buffer (Sigma) supplemented with 2% PMSF and 1% protease inhibitor cocktail (Sigma). The homogenates were then centrifuged at 10,000 rpm for 5 min at 4°C. The protein was collected and quantified by Bradford protein assay. Next, 40 μg protein samples were separated by 10% SDS-PAGE gel for electrophoresis and transferred onto PVDF membrane. The membrane was blocked with 5% BSA for 3 h and immunoblotted using primary antibodies to GAPDH or R1 (Abcam Inc., Cambridge, MA, USA) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz, Santa Cruz, CA, USA) for 4 h at 4°C. Chemiluminescence was detected using ECL detection kits (GE Healthcare, Waukesha, WI, USA). The intensity of bands was quantified by scanning densitometry using software Quantity one-4.5.0.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

The proposed structure of PSH and schematic diagram of the preparation process of LPs. (A) The structure of PSH polymer; (B) The schematic diagram of the preparation process of L-PSH-LP and L-TPSH-LP.

Statistical analysis. All the data were expressed as mean±standard deviation (SD). The identification of significant differences between groups was carried out with t-test. p<0.05 stands for significant difference while p<0.01 indicates a highly significant difference.