Transfecting the hard-to-transfect lymphoma/leukemia cells using a simple cationic polymer nanocomplex

Abstract

Although the development of gene delivery systems via non-viral-mediated methods is advancing rapidly, it remains a challenge to deliver plasmids into hard-to-transfect cells, such as lymphoma/leukemia cells. To develop an efficient transfection method, we formulated a simple nanocomplex by incorporating poly β-amino ester (PBAE) polymers with plasmid DNAs containing a GFP reporter gene. The formed PBAE-plasmid nanocomplexes are approximately 200nm in diameter and stable under physiological conditions, but become rapidly biodegradable when pH decreases <7.0. Cultured lymphoma/leukemia cells were used for transfection assays and resultant gene delivery rates were determined by quantifying GFP expression. Exposure of cells to the nanocomplexes composed of fractioned PBAE (>7kDa) resulted in GFP expression in 3% of cells, similar to that mediated by the standard Lipofectamine method. However, with polybrene pre-treatment, the nanocomplex could achieve GFP expression in up to 32% of lymphoma/leukemia cells, an 8-fold increase over that mediated by Lipofectamine. These findings demonstrated a simple, efficient method for in vitro gene delivery into hard-to-transfect cells. The nanocomplexes are biodegradable and have minimal cytotoxicity, suggesting the potential use for in vivo gene delivery.

Figure 1. Formulation of PBAE polymers and the PBAE polymer-plasmid nanocomplex

A, The PBAE polymers were synthesized by a simple chemical reaction of 5-amino-1- pentanol and 1,4-butanediol diacrylate monomers at 95° C for 24 hr[30]. B, The synthesized PBAE polymers were fractioned into < 7 kDa and > 7 kDa polymers by dialysis using a 7 kDa MW cut-off membrane. C, The fractioned PBAE polymers were incorporated with the plasmid pMax-GFP (~3.5 kbp) containing a GFP reporter gene to formulate a PBAE polymer-plasmid nanocomplex.

Figure 2. Characterization of the PBAE polymer-plasmid nanocomplex

A, The formed nanocomplexes composed of PBAE polymers > 7 kDa and plasmid DNA reporter were incubated in cell culture medium (pH 7.4) at 37° C and changes in size were kinetically monitored by DLS measurement. Under physiological pH conditions, the nanocomplex had a condensed size of ~200 nm in diameter and was stable for 12 hr. B, The PBAE polymer-plasmid nanocomplexes became unstable, rapidly dissociated, and enlarged under lower pH conditions (pH < 7). C, Transmission electron microscopy confirmed the condensed nanocomplexes at pH 7.4 with an average size of ~200 nm in diameter and dissociation of the nanocomplexes at pH 5.0 with an enlarged size ranging from 800 to 1200 nm.

Figure 3. The PBAE polymer-plasmid nanocomplex-mediated cell transfection

A, cultured lymphoma/leukemia Jurkat cells (5×10⁶) were transfected by the nanocomplexes composed of pMax-GFP reporter genes and PBAE (unfractioned or fractioned polymers < 7 or > 7 kDa). In control groups, cells were transfected with Lipofectamine-pMax-GFP complex or pMax-GFP alone (−). After culture for 48 hr, GFP-expressing cells were quantified by flow cytometry (%). B, Cells were pre-treated with polybrene (2 ug/mL) for 5 min, followed by the same sets of transfection treatments as described in (A). Resultant cell transfection rates were determined by quantifying GFP-expressing cells (%). C, Fluorescent microscopy confirmation of the resultant GFP expression in the transfected cells. D, Exposure of Jurkat cells to the polybrene-plasmid complexes had little cell transfection effect.

Figure 4. Optimization of the nanocomplex-mediated cell transfection

A, Dose effect of polybrene. Jurkat cells (5×10⁶) were pre-treated with polybrene for 5 min at different concentrations (0–8 μg/mL) and then exposed to the PBAE polymer (> 7 kDa)-plasmid nanocomplex. In the control experiment, reporter plasmid DNA alone (0.25 μg/mL) was used (−). After treatment for 48 hr, GFP-expressing cells were quantified by flow cytometry (%). B, Time-course of polybrene pre-treatment. Cells were pre-treated with 6 μg/mL polybrene for different times (5–120 min) and followed by exposure to the PBAE polymer-plasmid nanocomplex. Resultant GFP-expressing cells were quantified by flow cytometry (%). C, Optimal ratio of PBAE polymers and plasmid DNA reporters in the nanocomplex. Cells were pre-treated with polybrene (6 μg/mL) for 5 min and transfected by the nanocomplexes composed of PBAE > 7 kDa and pMax-GFP at different ratios (mass/mass) as indicated. Resultant GFP-expressing cells were quantified by flow cytometry (%). D, Optimal amount of reporter gene in cell transfection. Cells were pre-treated with polybrene (6 μg/mL) for 5 min and transfected by different doses of the nanocomplexes with the fixed 40:1 ratio of PBAE polymers (> 7 kDa) and pMax-GFP. The final amounts of reporter pMax-GFP were used from 0.25 to 2.0 μg/mL. Reporter plasmid DNA alone was used in control cells (−). Resultant GFP-expressing cells were quantified by flow cytometry (%).

Figure 5. The nanocomplex had little or no adverse-effect on lymphoma cells

A, No effect on expression levels of cell surface biomarkers. Jurkat cells were treated with polybrene (6 μg/mL) alone, the fractioned PBAE polymer > 7 kDa alone, or combination treatments of polybrene and the nanocomplex as described in Figure 4. Control cells received no treatment (−). After treatment for 24 hr, cells were stained with antibodies for CD2, CD3, CD5, and CD45, and expression of these surface biomarkers was then quantified by flow cytometry. Non-stained cells were used as a background control (non-staining). B, No effect on cell proliferation. Proliferation rates of the treated cells in (A) were also evaluated by MTT assay 48 hr post treatment. C, PBAE polymers had minimal effect on cell viability. Jurkat cells were treated with different concentrations of PBAE polymers > 7 kDa or PEI polymers as indicated. After culture for 48 hr, cell viability was examined by trypan blue staining.

Figure 6. The nanocomplex-mediated gene delivery in different lymphoma/leukemia cells

Cultured Jurkat leukemia/lymphoma cells, K562 myeloid leukemia cells, and Karpas 299 anaplastic large T-cell lymphoma cells were pre-treated with polybrene (6μg/mL) and then transfected with Lipofectamine containing the pMax-GFP reporter or the PBAE polymer (> 7 kDa)-plasmid nanocomplexes as described above. Plasmid pMax-GFP alone was used in control experiments (−). The total GFP-expressing cells were quantified by flow cytometry 48 hr post-transfection (%). The percentages of cells with different levels of GFP expression were also calculated (light green zone: <10¹; intermediate green: 10¹ to 10², and darker green: >10²). Cellular GFP expression was also confirmed by fluorescent microscopy.