In this work we investigated how small molecular changes to a lead poly(beta-amino ester), poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol) (referred to here as B4S5) affect gene delivery in two dimensional mammary cell culture compared to three dimensional primary organotypic cultured mouse mammary tissue. To vary gene delivery efficacy, we tune the polymer molecular weight and polymer terminal group of the polymer by varying the synthesis conditions. Polymer end groups were chosen so that they can facilitate intracellular delivery compared to the parent polymer and the 5-amino-1-pentanol side-chain. End group 2-methylpentane-1,5-diamine (E4) contains a primary amine as a terminal group and can increase polymer-DNA binding [33
]. Polymer end-groups 1-(3-aminopropyl)-4-methylpiperazine (E7) and 1-(3-aminopropyl)pyrrolidine (E8) contain tertiary amines and were recently reported to enhance intracellular nucleic acid delivery of DNA [35
] and siRNA [36
] in 2-D cultures, presumably by buffering the endosome and increasing endosomal escape. Terminal groups 4-amionphenyl disulfide (E9) and cystamine (E10) we introduce here as new end groups that could enable a “smart” triggered release of DNA to the cytoplasm. These new terminal groups can increase DNA binding while the polymers are in an oxidizing space outside the cell or in an endosome, but also decrease DNA binding when the polymers are in the reducing space of the cytoplasm, facilitating intracellular release. The combined effects of changes to base polymer molecular weight and terminal group alterations on the same base PBAE have not been previously investigated and differences between two-dimensional and three-dimensional efficacy are unknown. Here, we show how these changes to molecular structure affect nanoparticle formation and gene delivery in both two-dimensional and three-dimensional epithelial cultures. In both cell systems, the polymers synthesized here are more effective for gene delivery than FuGENE® HD, one of the leading commercially available reagents for non-viral gene delivery. These biomaterials are useful as tools to study the development of mammary epithelial cells and may also be promising as therapeutics for breast cancer.
Although all ten polymers tested were composed of the same base polymer, poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol) (B4S5), small changes to synthesis conditions or to terminal group generated biomaterials with widely different transfection efficacies. These studies highlight how changes to molecular weight (MW) within the same polymer structure (including the same side chain and end-groups such as with B4S5E4s, B4S5E10s, and B4S5s) affect gene delivery. They also show that if the same base polymer with the same molecular weight only has a minor change to its terminal groups (such as B4S5 1:1.1 compared with similar polymers B4S5E4, B4S5E8, and B4S5E10), the result on gene delivery can also be dramatic. Once synthesized, these polymers were used to construct polymeric nanoparticles and the particle sizes were characterized. Subsequently, these nanoparticles were utilized in 2-D culture to find optimal transfection conditions including formulation weight ratio of polymer to DNA and DNA loading per well. Finally, they were used in 3-D culture to transfect organotypic epithelial cells.
3.1. Polymer and Nanoparticle Synthesis and Characterization
Polymers were synthesized according to . Through varying the monomer ratios used during synthesis, B4S5-based polymers could be synthesized with molecular weight ranging from less than 10 kDa to greater than 110 kDa as shown in . As expected, monomer ratios closest to unity and higher temperatures produced the largest polymers. However, despite these dramatic changes to polymer size, the nanoparticles formed through self-assembly of these polymers with plasmid DNA were more similar in size and only varied by approximately twofold. Interestingly, the nanoparticle detection method itself was critical in evaluating size of these nanoparticles in aqueous solution. The standard approach to size polymeric gene delivery particles such as these is dynamic light scattering (DLS) [21
]. This method gives a Z-average hydrodynamic diameter that is an intensity-weighted mean diameter derived from a single-exponential fit of the intensity autocorrelation function. While this method is very accurate for monodisperse samples, in non-monodisperse samples, the intensity-averaging counts larger particles significantly more than smaller particles. A newer technique for nanoparticle sizing in aqueous conditions is Nanoparticle Tracking Analysis (NTA) [38
]. In this method, each individual particle is independently sized so that a direct number-averaged mean can be calculated. As each particle is counted, a mode, or the peak in the number distribution, can also be calculated. shows the size of each of the nanoparticles by DLS (intensity-average) and NTA (number average and mode). While some of the DLS measurements reach into the 250 nm range, it is clear from the NTA data that the majority components of almost all the particle formulations are between 100–150 nm in size. The exceptions are formulations which are smaller than 100 nm and include B4S5 1:1.1 which has a mode of 69 nm and B4S5E9 which has a mode of 77 nm. This analysis also shows that certain distributions are monodisperse (B4S5E4s and B4S5E7), while others are polydisperse to varying degrees (non-end-capped versions of B4S5 formulated near unity as well as B4S5E8 and B4S5E9). However, polymers of a certain type formulated at different monomer ratios and possessing different molecular weights (different versions of B4S5E4s, B4S5E10s, and B4S5s) have equivalent particle uniformity. Thus, the uniformity of particle distribution may be a property of the polymer terminal group itself. All of these formulations are of a size known to facilitate particle uptake by clatharin-mediated endocytosis as well as other internalization mechanism [39
]. To our knowledge, this is the first time that NTA has been used for self-assembled polymer/DNA particles, but highlights its utility, especially when combined with traditional DLS analysis.
Figure 2 (A) The molecular weight s of the ten PBAE polymers used for transfecting EPH4 cells and organoids. Molecular weight is in Dalton (Da). Ratios are molar monomer ratios (B4:S5) used during polymer synthesis. (B) The particle sizing data of the nanoparticles (more ...)
3.2. Gene delivery to 2-D EPH4 cells
The EPH4 cells were initially screened in a 96-well plate by varying polymer to DNA weight ratio (wt/wt) and luciferase DNA dose per well. Cells were plated at a cell density of 150,000 cells/mL and transfected with polymer/DNA complexes formed at two different wt/wt ratios of 60 wt/wt and 100 wt/wt. For 60 wt/wt three different DNA doses per well were tested, 0.6 μg/100 μL, 1.2 μg/100 μL and 1.8 μg/100 μL and for 100 wt/wt two different DNA doses were tested, 0.6 μg/100 μL and 1.2 μg/100 μL. The 60 wt/wt and 1.2 μg/100 μL per well condition gave the highest RLU/g protein (Figure S2
). Hence these conditions were chosen for the scale up 24-well plate transfections.
Transfections were next conducted in 24-well plates using GFP DNA so that delivery to individual cells, as well as the cell population overall, could be better ascertained. shows transfection efficacy in 2-D EPH4 cells by confocal microscopy and shows transfection efficacy by flow cytometry. The transfection efficacy is quantified in terms of the percent of live cells positive for GFP expression (% positive). The efficacy of the PBAE polymers is compared to FuGENE® HD, a leading non-viral commercially-available gene delivery vector for cell biology applications. The confocal images show that the EPH4 cells are healthy and agree qualitatively with the quantitative flow cytometry data. The % viability data of EPH4 cells shown in Figure S3
indicates that the PBAE vectors did not generally cause high cytotoxicity, except for B4S5E9 which had a significantly low % viability and low transfection efficacy.
Figure 3 (A) 20X confocal stacks (8 μM) showing transfection efficiency of the ten PBAE polymers in 2-D EPH4 cells 2 days post-transfection. Transfected cells contain EGFP expression plasmid. Cells were fixed with 4% PFA and stained with Phalliodin-Alexa (more ...)
Figure 4 (A) Flow cytometry data showing the transfection efficiency of the ten PBAE polymers in 2-D EPH4 cells. The efficiency is quantified in terms of percentage of live EPH4 cells that are expressing EGFP (mean + SEM, n=4). Fugene HD (white) was used as a (more ...)
3.3. Synthesis conditions and polymer molecular weight
We studied the effect of varying polymer synthesis conditions on the transfection efficiency of the PBAE polymers. The synthesis conditions were varied by changing synthesis temperature (40°C and 90°C), diacrylate:amine monomer ratio (1:1.05, 1:1.1 and 1.1:1,) and the type of solvent. The polymer molecular weight (MW) and particle size were determined at different synthesis conditions to analyze how synthesis conditions affect the biophysical properties of the non-viral delivery vectors.
The non-end-capped amino-alcohol terminated B4S5 polymer was synthesized at a lower temperature of 40°C (monomer ratio 1:1.05) and at a higher temperature of 90°C (monomer ratio 1:1.1). While monomer ratio closer to unity also increases molecular weight, synthesis temperature was found to more strongly increase molecular weight in this monomer ratio range (1.05–1.20). The polymer synthesized at a higher temperature showed a four-fold increase in percent transfected cells when compared to the polymer synthesized at a lower temperature. Similarly, for the end-capped cystamine-terminated B4S5E10 polymer, synthesis at a higher temperature of 90°C (at 1.1:1 monomer ratio) resulted in three-fold higher positively transfected cells as compared to synthesis at a lower temperature. Interestingly, the MW data () shows that polymers synthesized at 90°C have higher MWs than those synthesized at 40°C for both the non-end-capped and end-capped PBAE polymers. This indicates that increasing the synthesis temperature from 40°C to 90°C increases the MW of the polymer and thereby improves the polymer transfection efficiency. This partially mirrors findings with polyethylenimine (PEI), where increased polymer size (molecular weight) was shown to directly increase gene delivery efficacy over a wider range from ~1 kDa – 70 kDa [40
]. However, while for a given polymer structure, increasing molecular weight appears to increase transfection efficacy, polymer molecular weight is not the main driver of transfection efficacy overall between polymers with different terminal groups. This is evident as similarly structured polymers with the same MW of ~10 kDa have divergent transfection properties depending on the polymer terminal group. Polymers above ~12 kDa had lower, non-optimal efficacy, possibly due to reduced rates of intracellular DNA release from the polymer as has been demonstrated with higher molecular weight polylysine [41
3.4. Synthesis procedure
Another variable that can affect the transfection efficiency is the solvent and procedure used during the end-capping step of polymer synthesis. This effect was analyzed with just polymer B4S5E4. In the standard preparation (monomer ratio of 1.1:1), B4S5E4 is end-capped in DMSO (room temperature) and used directly. In the alternative procedure (monomer ratio 1.2:1), B4S5E4 is end-capped in THF (room temperature) and then washed with ethyl ether to remove the THF before subsequently dissolving the polymer in DMSO. While we hypothesized that this extra step would enhance the transfection efficiency of the PBAE polymer in part due to achievement of a higher molecular weight, the THF-synthesized B4S5E4 was found to be lower at delivery (18±5%) compared to the DMSO synthesized B4S5E4 (40±5%) in these cells.
3.5. Terminal group
Six different versions of the PBAE polymer were synthesized at 90°C and monomer ratio of 1.1:1 by varying the small molecule terminal group of the polymers during synthesis. These six PBAE polymers include the amino-alcohol terminated B4S5 version and the five amine terminated end-capped versions B4S5E4, B4S5E7, B4S5E8, B4S5E9 and B4S5E10. These polymers were synthesized at the same monomer ratio 1.1:1 (or 1:1.1 for B4S5) and generally had similar final molecular weights (~10 kDa), although there were differences due to the end-capping groups (most notably B4S5E9 which had a larger 24 kDa size). In terms of particle size, B4S5E7, B4S5E8, and B4S5E10 were very similarly sized at 100–120 nm. B4S5E4 was slightly larger (~150 nm) and B4S5 and B4S5E9 were smaller (~69–77 nm) (). Yet despite these similarities in polymer size and particle size, the small changes to terminal group show dramatic effect on the transfection efficiencies of these polymers (). The B4S5E7 end-capped version had the highest percent GFP positive cells (57±6%), followed by B4S5E8 (44±3%) and B4S5E4 (40±5%). These three best performing polymers showed about 10-fold higher transfection efficiency as compared to that of FuGENE® HD (4±2%). Interestingly, both the E7 and E8 end-capping monomers contain tertiary amine groups, indicating that the presence of tertiary amines could potentially aid in buffering of the endosomal pH and thereby enhance the transfection efficiency. The other amine-terminated versions, B4S5 1:1.1, B4S5E9 1.1:1 and B4S5E10 1.1:1 showed relatively lower transfection efficiencies of 16±2%, 0% and 16±3%, respectively. The B4S5E9 1.1:1 polymer showed high cytotoxicity (Figure S3
), which likely limited its efficacy for gene delivery.
The cytoplasmic environment inside the cell is highly reductive. The presence of disulfide-linked end-groups susceptible to degradation by the reductive intracellular environment would enable these polymers to have an enhanced DNA release capacity. The B4S5E9 and B4S5E10 polymers are end-capped with small molecules containing disulfide linkages, 4-aminophenyl disulfide (E9) and cystamine (E10), respectively. The addition of these degradable moieties could allow fine tuning the degradation of the PBAEs and possibly avoid some of the DNA release problems associated with higher molecular weight polymers [41
]. While the transfection efficiency of B4S5E10 was four-fold higher than FuGENE® HD for EPH4 cells, additional work is needed to further tune polymeric vectors with multimodal degradation capacities for enhanced intracellular nucleic acid delivery.
3.6. Gene delivery to 3-D organoids
Gene therapy requires delivery to cells within intact tissues. Accordingly we also tested the ability of PBAE based polymers to transfect mammary epithelial cells within intact epithelial fragments (“organoids”). We hypothesized that we might see differences in the magnitude of transfection efficiency and/or in the optimal transfection polymer in the 3-D organoids culture when compared to 2-D EPH4 culture.
Qualitative pre-screening trials performed to optimize the transfection conditions indicated that the most efficient transfection results were obtained for the conditions with high MW versions of polymer, 60 wt/wt formulations, and 6 μg of DNA dose per well in a 24-well plate. These conditions were then used to conduct transfection experiments for quantitative analysis using flow cytometry. shows the flow cytometry data for the transfection efficiency of the ten PBAE polymers used to transfect the organoids culture with GFP plasmid. The transfection efficiency is quantified in terms of percent of live cells positive for GFP expression (% positive). The efficacy of the PBAE polymers is compared to FuGENE® HD as a positive control. Confocal images () agree with flow cytometry data, highlight multiple transfected cells per organoid, and show good morphology.
3.7. Synthesis conditions and comparison of 3-D to 2-D
In general, the polymers synthesized at a higher temperature had a higher molecular weight (MW) than those synthesized at a lower temperature. The values of positively transfected live cells seen for the organoid cultures are low compared to the 2-D cultures (< 10%) (). This can be explained in part by the fact that the nanoparticles are primarily taken up by the cells in the periphery of the 3-D structure, while most of the cells in the interior remain untransfected. In isolating the organoids into single cells, viable cell recovery is low and many of the transfected cells (as well as the untransfected cells) are lost (data not shown). The non-end-capped B4S5 40°C polymer (MW = 9.6±0.1 kDa) transfected at 1±0.4% transfection efficiency while the B4S5 90°C version (MW = 12.5±0.2 kDa) transfected at 3±1% efficiency. Similarly, the B4S5E10 90°C version (MW = 14.0 kDa±0.4 kDa) gave a slightly higher % GFP positive cells than the 40°C version (MW = 8.63±0.01 kDa). These conditions indicate that for the same polymer, the transfection efficiency increases with increasing molecular weight in 3-D as well as 2-D.
3.8. Terminal groups and comparison of 3-D to 2-D
Changes in the small molecule used for end-capping the base (diacrylate terminated) polymer can have a significant effect on the transfection efficiency of the polymers. To investigate the effect of such small molecular changes on 3-D organoid transfection, six versions of the PBAE polymer were screened. These included the amino-alcohol terminated B4S5 version and the five amine terminated end-capped versions B4S5E4, B4S5E7, B4S5E8, B4S5E9 and B4S5E10 synthesized at 90°C and 1.1:1 monomer ratio. The flow cytometry data () of the transfected organoid cultures show that the B4S5E8 end-capped version had the highest percent GFP positive cells (6±1%), followed by B4S5E7 (4.0±0.6%) and B4S5 (3.0±0.3%). The transfection efficiency of the leading commercially-available agent, FuGENE® HD, was 3±1%. While these polymers are promising for gene delivery to organoids compared to other reagents, there is a significant need for improved design to allow for increased nanoparticle penetration into the organoids as well as and into tissues in vivo
. One possibility is to form nanoparticles dramatically smaller in size and/or employ coating strategies [42
] to improve uptake through cell specificity [43
] or tissue specificity [44
Importantly, this work shows that mosaic GFP expression is maintained in cultures transfected with these polymer even after 7 days culture, while GFP expression is absent in the FuGENE® HD condition (). Morphology and viability are both good. Continued expression of mosaically labeled cells undergoing morphogenesis is very desirable to contrast individual cell behaviors in the tissue as a whole while studying epithelial polarity and migration. While the absolute level of transfection in 3D organoids is modest, this technology can be used to deliver Cre recombinase mosaically to mammary epithelium from transgenic mice carrying targeted loxp flanked alleles.
Figure 5 Confocal stacks of B4S5E8 1.1:1 90°C (polymer) based EGFP transfection of 3D organoid mammary epithelium culture. Mosaic EGFP expression is maintained in cultures transfected with PBAE even after 7 days culture. Epithelial fragment was fixed with (more ...)
In comparing transfection efficacy of 2-D to 3-D cultures, generally data follows a positive linear relationship where transfection in three-dimensional organotypic culture is approximately 10% of transfection efficacy in two dimensional cell culture (). Exceptions to this trend include FuGENE® HD (pink downward triangle) which has low overall efficacy, but similar efficacy between 2-D and 3-D cultures. Outliers on the other end are B4S5E4 1.1:1 90°C (blue triangle) and B4S5E7 1.1:1 90°C (red triangle) that although have high 2-D efficacy, have lower 3-D efficacy than predicted. B4S5E8 1.1:1 90°C (green circle) was the most effective for 3-D transfection overall. Interestingly, this polymer was three times as effective for 3-D transfection as B4S5E4 1.1:1 90°C (blue triangle) even though their polymer structures and 2-D efficacies were almost identical.
Figure 6 Scatter-plot comparing the transfection efficacy (% positive) of polymeric nanoparticles in 2-D culture and 3-D culture. The colored data points include FuGENE® HD (pink downward triangle), B4S5E4 1.1:1 90°C (blue triangle), B4S5E7 1.1:1 (more ...)
The low transfection efficiency in 3-D organotypic cultures achieved with the PBAE vectors is consistent with the results reported by other studies employing 3-D tissue culture models to develop gene delivery strategies for in vivo
applications. In one study, Mellor et al. used PEI polyplexes to transfect plasmid DNA into multicellular tumor spheroids (MCTS), an in vitro
model of solid tumor. They found spatially limited transfection to cells at the MCTS periphery, as the complexes failed to reach the deeper quiescent regions, even with the use of electroporation [45
]. Such spatial limitation of transgene expression is not seen in 2-D monolayer cultures. In another study with the MCTS model, the use of 3-D spheroids revealed the difference in cytotoxicity of PEGylated and non-PEGylated polyplexes that was not detected with the 2-D monolayer cultures, indicating that vital cytotoxicity data can be excluded if vectors are screened solely in 2-D culture systems [46
]. Important differences in gene expression have also been noted between 2-D and 3-D systems [47
]. In some studies, hydrogels have been used as in vitro
scaffolds to support tissue formation by enhanced presentation of specific biological cues through locally controlled gene delivery of tissue inductive factors. Rieux et al. reported the use of fibrin based hydrogel, with cells encapsulated within or seeded onto the hydrogel, to investigate gene delivery efficacy using lipoplexes. They found that the transgene expression increased with time over a period of 11 days in culture and was significantly higher in 2-D as compared to 3-D cultures. Their study suggested that the hydrogel component fibrin interacts with and sequesters the lipoplexes thus limiting cellular internalization and causing a delay in expression [48
The comparison of transfection efficacy between 2-D and 3-D mammary epithelial cultures indicates that (a) transfection of the 3-D organotypic cultures is more difficult than transfection of 2-D cultures, but likely models some of the key challenges for in vivo
gene therapy and (b) many of the same variables that affect transfection efficiency in 2-D (e.g. MW, solvent, polymer structure) also effect transfection efficiency similarly in 3-D, but the magnitude of transfection is lower in 3-D. This study identified highly effective reagents for 2-D transfection and useful reagents and important parameters for 3-D transfection. As a part of future studies, developing improved 3-D assays would be useful for high throughput screening applications [24
] to identify next-generation materials. Use of these agents for partial transfection of mammary epithelial cells in vivo
would be interesting to study mosaic delivery of Cre recombinase [49
]. Partial transfection of a similar cell population may be sufficient for other in vivo
applications as well. One such application is suicide gene therapy for tumor regression. In this approach, a gene is delivered that encodes an enzyme that converts a prodrug to its active form. Thus, there is catalytic generation of a cytotoxic anti-cancer agent at the tumor site itself and this agent can diffuse to adjacent cells and deeper into the tumor. This approach has shown promise for breast cancer in early clinical trials [50
]. A related strategy is to introduce the suicide genes to cells of a preneoplastic tissue at risk, and these drug susceptible cells will then sensitize all the cells produced in the clonal population [51
]. In either case, there is an amplified anti-cancer response following initial cell transfection.