Using fluorescent protein-coding plasmid vectors as an indicator of successful gene delivery, we sought to compare the efficacy and nonspecific cytotoxicity of PBAEs in primary GB astrocytes and BTSCs, as well as in healthy primary astrocytes and NSCs. We also explored methods of expanding the practical usage of PBAE-DNA nanoparticles via lyophilization and long-term storage. We identified several PBAE structures that could be used as efficient gene delivery agents to brain cancer cells. Although gene therapy is emerging as an attractive option with great potential to treat GB, most groups have thus far investigated viral or cellular vectors, which may carry additional safety risks. Furthermore, most synthetic gene vectors must be prepared shortly before use and are not stable for long periods of time. Here, we show that synthetic PBAEs can transfect GB astrocytes and BTSCs with high efficiency superior to their efficiency in non-cancerous astrocytes and NSCs, and we show the development of a stable form of the PBAE-DNA nanoparticles that can be easily prepared, stored, and reconstituted with little to no loss of function over three months.
3.1 DNA Nanoparticle Uptake by Glioblastoma Astrocytes
The ability of PBAEs to facilitate DNA entry into the cell was assessed by fluorescence imaging. Using peak absorbance values, one Cy3 molecule was calculated to have been conjugated approximately once per 83 nucleotides. DNA enters GB cells efficiently using a wide range of polymers with varying structures (). Within hours after transfection, most if not all cells have taken up nanoparticles. Of note, however, is that the Cy3 signal indicates only the presence of DNA in the cell and does not provide information on the plasmid’s integrity or exact location. It is known that many downstream barriers to gene delivery exist following entry into the cell, including escape from the acidic endosome, trafficking to the nucleus, and transcription and translation of the gene [29
]. Although many studies of nucleic acid delivery use uptake as a measure of successful transfection, efficient uptake is likely necessary but not sufficient for high transgene expression. In particular, although polymers 455 and 447 were among the most successful polymers to cause transgene expression, most of the other polymers tested also cause uptake in as many cells as do 455 and 447 ().
Figure 2 PBAEs facilitate efficient uptake of fluorescently-labeled DNA by GB 319 astrocytes. Different polymer structures (indicated by the numbers in each set of images) are complexed at different weight ratios (w/w) with DNA and show high Cy3 signal 4 hours (more ...)
3.2. Gene Delivery to Glioblastoma Astrocytes
PBAEs similar to some of those studied here have been found to be effective in overcoming intracellular barriers to gene delivery [13
] in addition to simply facilitating uptake. To identify the most effective of those polymers, they were used to transfect GB 319 astrocytes with EGFP plasmid DNA at polymer-to-DNA weight-to-weight (w/w) ratios from 30:1 to 120:1. Formulations of polymeric nanoparticles administered in the absence of serum are show variable transfection efficiency and viability in 319 GB astrocytes depending on polymer structure (Supplemental Figure S1
). All were used at a dose of 1.5 μg DNA per condition unless otherwise stated. Because a simple combinatorial synthesis scheme was used to design the library of PBAEs used in this study, many distinct molecules could be tested. Although some trends were observed, it was also found that changes as small as one or two atoms in the base monomers could alter the polymers’ effects on cells. For instance, all polymers tested with the E7 end cap showed transfection efficiency above 15%; however, both 447 and 457 showed significantly higher transfection at the same w/w ratios than 657 (for 30 w/w, 38.4±3% and 45.1±5% for 447 and 457, respectively, compared to 18.4±1% for 657). The same is true of 454, which was more effective than 554, and of 455, which was more effective than both 545 and 655.
To further narrow the range of PBAEs most effective in GB gene delivery, top formulations were tested again, using a wider range of dosages (0.15 to 3.0 μg DNA per condition) and in the presence of 10% FBS. In agreement with studies by other groups on lipofection [33
], the lipid-DNA complex formed by Lipofectamine 2000 was found here to be much less effective at gene transfer in the presence of serum, though its intrinsic toxicity was also lessened at lower doses ().
Figure 3 PBAEs can transfect GB 319 astrocytes with comparable or superior efficiency and safety compared to leading commercial reagents. A: Top polymers were screened at a wider range of doses (indicated on the graph as μg DNA) in 10% serum and found (more ...)
Many PBAEs, on the other hand, remained effective in the presence of serum. A subset, compared favorably to results from FuGENE HD and Lipofectamine 2000 transfection, yielding higher transfection efficiency at the optimal dose and still retaining 80% or greater viability (). For instance, one of the leading polymers found in this study was 447, used at a 30:1 w/w ratio to DNA, which transfected up to 60.6±5 % of cells in the absence of serum or, at a lower dose and in serum-containing medium, 40.0±2 % of cells while maintaining 82.5±4% viability. At doses low enough for Lipofectamine 2000 to maintain 80.1±2% viability, its transfection efficiency was decreased to 13.6±2%.
3.3. Duration of Transgene Expression
Polymeric methods of gene delivery are thought to be safer than virus-mediated delivery partly because of the reduced likelihood of inflammatory response and insertional mutagenesis. Because polymer-delivered plasmids integrate only rarely into the host cell’s genome, however, a potential concern is that expression of the transgene will be too transient. When GB 319 astrocytes were transfected with EGFP plasmids in the presence of 10% serum, expression Lipofectamine 2000 expression became visible first of all tested conditions (within 5 hr). However, it also declined the most quickly, decreasing to between 0–5 cells per microscope field (10x magnification) at the same time that FuGENE- and PBAE-mediated expression was still increasing. Of the reagents tested, PBAE 456 peaked latest in the number of cells per well (between 7–8 days), while the other PBAEs and FuGENE peaked slightly earlier, at 4–6 days (). It should be noted that this number was not normalized to the number of total cells per field; however, the consistently increasing number of EGFP+ cells beyond the first few days suggests that GFP plasmids may be retained through the cell divisions during that time and are not completely silenced even after two weeks.
Figure 4 EGFP expression peaks between 4–6 days for most PBAEs and FuGENE HD and at 1–2 days for Lipofectamine 2000 (graph, top). Few cells retain the EGFP gene for extended periods of time. All polymers tested led to persistent expression in some (more ...)
Forty days later, all groups except Lipofectamine 2000 contained some GFP+ cells, including one of the most promising polymers from initial screens, PBAE 447 (). Interestingly, in the experimental group transfected with PBAE 453, the number of GFP+ cells steadily increased up to the 40th day. Because of higher toxicity, 453 had not been included in optimization experiments; however, as a consequence of this toxicity, the increasing number of stably transfected GFP+ cells was visually clear, as shown in . In each of the other PBAE formulations, a small group of cells (0.5–1.1%) appeared to be stably GFP+ after 70 days.
This raises the possibility that delivered transgenes could be continually expressed in a small number of cells that can act as long-term reservoirs for human brain cancer therapeutics. Instead of transfection lasting only a few days or weeks, as is typical of non-viral gene delivery, long-term protein expression could be achieved. Long-term expression may be important for diseases like brain cancer, where treatment will likely need to persist to completely eradicate all tumor cells or to continually slow tumor growth. At the same time, the low frequency (<1%) of chromosomal insertion greatly reduces the probability that a tumor suppressor gene or other essential gene will be disrupted and therefore cause de novo mutations.
3.4. Optimization and Characterization of Stable Nanoparticle Formulations
To optimize nanoparticle formulation for storage in dry form while maintaining stability, we used one of our top polymers as a test case, 447 at a 30:1 weight ratio to DNA. There was very little transfection and few particles remained when particles were lyophilized without modification; by adding 30–90 mg/mL sucrose as a lyoprotectant before freezing (final concentration 15–45 mg/mL sucrose), full functionality of the particles was retained after immediate reconstitution in water, measured by transfection efficiency, viability, and sizing (Supplemental Figure S2
) in both the presence and the absence of serum. Moreover, freshly prepared particles usually begin to aggregate in suspension over time, particularly in media containing salts or serum, which can be measured by an increase in effective size and a wider size distribution (). Lyoprotectants are known to help nanoparticles retain morphology and functionality after freezing and drying processes [17
]. In this study, particles lyophilized with sucrose remained more stable in serum compared with freshly prepared particles over a period of four hours, a critical window of time for nanoparticle uptake during transfections.
Figure 5 A: DNA nanoparticle lyophilization protocol. Particles in suspension are mixed with sucrose, frozen, lyophilized, and stored for quick and easy use. B: changes in mean particle diameter over 4 hr during incubation in 10% serum. C: Nanoparticle tracking (more ...)
After 3 months, particles lyophilized with 60 mg/mL sucrose and stored at 4°C were not significantly different in transfection efficiency or toxicity compared to freshly prepared particles made with polymer stored at 4°C and fresh DNA stored at −20°C (p>0.05). Though the measured percent transfection efficiency of lyophilized particles was lower, the total fluorescence measured in each well after transfection did not show a significant decrease after 3 months compared to the starting value (). Combined with the increase in measured viability over time, this may suggest that, while the proportion of GFP+ cells decreases up to 25% after 3 months, the absolute number of GFP+ cells or the total amount of GFP produced in each well remains approximately the same. Even after 6 months, approximately 50% of the original transfection efficiency and total fluorescence per cell is retained ().
Figure 6 A–B: Transfection efficiency of particles lyophilized with 60 mg/mL sucrose and stored at 4°C is comparable to that of freshly prepared particles at 0 and 3 months. Lyophilized particles have reduced efficacy after 6 months, though approximately (more ...)
3.5. Gene Delivery to Adherent 551 GB BTSCs
Using a smaller range of polymers shown to be effective in gene delivery to GB astrocytes, the same transfection screening experiments were carried out on stably transduced GFP+ BTSCs using plasmid DsRed-Max. Dissociated BTSCs plated in monolayer on laminin could also be transfected by PBAEs with high efficacy. As with the GB astrocytes, top PBAEs showed significantly less non-specific toxicity than Lipofectamine 2000 (ANOVA, post hoc Dunnett test, p<0.05) and caused up to 43.0±7% transfection efficiency while maintaining 61.4±5% viability, or 37.6±4% transfection with 85.7±6% viability ().
Figure 7 PBAEs facilitate efficient DNA delivery to BTSCs when plated in monolayer (A, C) or as 3–D neurospheres (B, D). Some polymers were less toxic than Lipofectamine 2000 while transfecting equally well or better (*p<0.05 superior to Lipofectamine). (more ...)
When BTSCs transfected with PBAE 447 at a 30:1 w/w ratio were maintained in culture without passaging, the cells reformed adherent 3-D neurospheres over time, with DsRed-Max transgene expression still visible throughout the reformed neurosphere after 45 days (). Even after passaging the transfected BTSCs, mechanically dissociating all neurospheres, and re-plating on laminin, a subset of the cells remained stably DsRed+ at least 90 days after transfection.
Significantly, when BTSCs were plated on laminin as a mixture of single cells and neurospheres as a co-culture, PBAEs 454 and 456 were relatively effective in penetrating into the 3-D structure and transfecting cells throughout the neurosphere (up to 24.3±7% and 30.8±2% transfection, respectively), while DsRed was expressed in up to 40–45% of cells when 447 or 457 was used ().
3.6. Comparison of PBAE-mediated Gene Delivery to GB Cells and Healthy Cells
An eventual goal of this technology will be to deliver genes intracranially, where both GB cells and healthy brain cells could potentially be exposed to DNA-containing nanoparticles. Ideally, the polymer used for transfection should preferentially affect GB cells while having minimal or no effect on healthy cells. However, another possible therapeutic strategy is to deliver genes that are expressed only by GB cells using cancer-specific promoters or that have an effect only on cancer cells, such as through the induction of apoptosis. In the latter strategy, gene transfer of an exogenous paracrine factor to healthy cells would not be harmful and could even be advantageous, as these healthy cells could serve as a “factory” for secreted factors that would kill surrounding tumor cells.
In this work, we identified PBAEs that could be effective for both potential strategies. Most of the top PBAE formulations for GB astrocytes or 551 BTSCs were significantly less effective (p<0.05) on F34 fetal cortical cells, especially when the healthy cells were maintained as undifferentiated NSCs rather than cultured as astrocytes (). In particular, polymer 457 transfected nearly 20% of BTSCs but only 4.8% of F34 astrocytes, and it had no apparent effect on F34 NSCs. Polymer 456 had 13.4% efficacy on BTSCs with no quantifiable toxicity, but it had little to no effect on F34 cells (0.3% transfected with greater than 85% viability). On the other hand, 447’s efficacy in GB astrocytes and BTSCs, along with its moderate efficacy in healthy astrocytes and healthy NSCs (19.1±2% transfection), suggests that it may be particularly useful in delivery of genes whose products are secreted proteins that are specifically cytotoxic to cancer cells. Lipofectamine 2000 was almost completely ineffective on BTSCs at a standard dose (0.3 μg per well); by doubling the dose, transfection efficiency increased drastically to 41.2±6%, but viability plummeted to 42.9±4%. This effect was even more pronounced in fetal astrocytes, in which Lipofectamine 2000 treatment killed nearly all cells ().
Figure 8 In general, tumor stem cells (BTSC) and glioblastoma cells (GB) are transfected much more effectively by PBAEs than healthy fetal stem cells (NSC) and astrocytes (Ast). One exception is 447, which transfects all cell types tested, and Lipofectamine 2000, (more ...)