In the current study, we have investigated several potential mechanisms of cytotoxicity for five different polymeric gene delivery vehicles, linear PEI, T46, T412, T443, and A442. These polymeric delivery vehicles vary in their chemistry (containing or lacking hydroxyls) and molecular weight. The goal of this study was to understand the role of these structural parameters on the biological behavior during pDNA delivery. These behaviors include polyplex size and charge, transfection efficiency, potential harmful interactions with the plasma membrane, ability to induce apoptosis, generation of ROS, and whether polyplexes were capable of directly permeabilizing the nuclear membrane. Herein, it was found that the length of the polymer as well as the composition of the polymer influence cytotoxicity and cell interactions to a large degree.
The first noticeable difference between the different polymers were their interactions with pDNA. Based on this data, it appears that the polymer structure plays a role in how plasmid DNA is complexed. The absence of hydroxyls on the A442
polymer seemed to result in a smaller, more positively-charged polyplex. However, linear PEI, which also lacks hydroxyl groups, forms polyplexes similar in size to T412
, however, forms particles double in size compared to all other polymers, indicating that this polymer is unable to compact pDNA into as small a nanoparticle as the other polymers tested. Prior research in our group has indicated that T4 exhibits significant hydrogen bonding interactions when complexing with plasmid DNA as compared with analogues that do not contain hydroxyl groups.54, 55
The different binding mechanism of T4 vs. A442
may account for their difference in polyplex size and charge.17, 54
In addition, when comparing polymers of similar molecular weight (T443
), we observe that the hydroxyl-containing T443
formed polyplexes of larger size and lower charge (), indicating that the hydroxyls do play a role in the complexation of pDNA. We also observed differences in transfection efficiency between these two polymers, with T443
having slightly higher expression efficiency than A442
(). Previous work in our group has shown that A4 with a degree of polymerization (DP) of 12 exhibits lower transfection efficiency when compared to T4 with a similar molecular weight (DP of 14).17
This trend is also seen here when studying the transfection efficiency of the higher molecular weight derivatives of these polymers. It is likely that the higher transfection efficiency of T443
(as compared to A442
) results from its ability to degrade, thus providing a mechanism for plasmid DNA release from the polyplex.17
also exhibits statistically higher luciferase expression than PEI (). We were also surprised to find that despite the degradability of T443
, it actually appeared to exhibit a statistically significant higher toxicity profile than A442
and PEI, which do not degrade. This led us to hypothesize that in this case, degradability does not necessarily decrease toxicity. Rather, we propose that the higher toxicity observed with T443
compared to A442
could be a result of T443
’s increased induction of nuclear envelope permeability; this would not only account for the increased toxicity of T443
, but the higher degree of observed gene expression as well. In fact, when comparing the cytotoxicity of all of the polymers, it was found that high protein expression also correlated with high cytotoxicity and cell death. The cell viability data for polymers T412
were not statistically significant from the cells only and DNA only controls (); it appears that the polymers with the highest degree of protein expression (PEI, T442
, and A443
) exhibited the highest toxicity (). To this end, we were interested in the further investigation of the apparent link between expression efficiency and cytotoxicity. Therefore, it is of interest to study the timing and mechanisms of toxicity with these polymeric vehicles, and how the polymer structure influences the cellular mechanisms related to apoptosis.
Previous studies in our group indicate that the hydroxyl groups present in T4 may contribute to pDNA release (via
hydrolysis) and therefore result in higher expression efficiency than non-hydroxyl containing polymers.17
To further investigate the role polymer structure has on biological behaviors, we tested the effect of molecular weight and structure on cytotoxicity. Because some of the structures are able to induce apoptosis within 30 minutes of transfection (), it is prudent to study the mechanisms of cellular uptake for these polymers since the cell membrane is the first obstacle to polymer-based gene delivery and could present the first negative interaction leading to toxic effects. We did find that the larger molecular weight polymers were able to induce membrane permeability () and apoptosis () to a larger extent than the smaller molecular weight polymers within 4 hours of transfection. The larger molecular weight polymers also had higher interactions with the plasma membrane within 30 minutes of transfection when compared to the shorter polymers (). It is possible that since the higher molecular weight polymers are able to condense plasmid DNA into smaller polyplexes than the lower molecular weight polymers, the mechanism of uptake for the smaller polyplexes will be altered, resulting in different cytotoxic profiles. To further investigate this, intracellular uptake of the polyplexes was studied in the presence of EGTA to determine the role of extracellular calcium. Extracellular calcium is required to maintain membrane fluidity,56
and without it cells are not able to recover as well after membrane penetration.57–59
Since cells without extracellular calcium exhibited improved polyplex uptake in the case of PEI, it appears that membrane fluidity could influence PEI transfection. Interestingly, the cells did not exhibit an increase in propidium iodide permeability in the presence of EGTA (Supporting Information, Figure S1
), thus discounting the idea that membrane permeability, in this case, helps to aid polyplex internalization in the cells. Supporting this observation, a recent study by Prevette et al.
indicates that membrane permeability does not play a role in PEI expression efficiency.33
Collectively considering the current and published results, we hypothesize that a more likely toxicity mechanism is that the increased basolateral surface of the cells as a result of EGTA treatment could play a role in PEI uptake, and that the different mechanism of cellular uptake plays a role in early cytotoxicity (< 30 minutes after transfection). EGTA had a slight inhibitory effect on T412
uptake, further illustrating that the poly(glycoamidoamine)s are entering the cell via
different endocytic mechanisms than PEI.15
It should be noted that the inhibitory effect of EGTA on the uptake of T412
is similar to results obtained for several lipid-DNA complexes.60, 61
The detailed mechanism as to how decreasing extracellular Ca+2
decreases the intracellular uptake of certain gene delivery vehicles while enhancing others remains unknown, but it is speculated that chelation of extracellular calcium could decrease rates of endocytosis.60, 62, 63
Collectively, these studies support previous findings that structure of the polymers influence their endocytic routes.15
Furthermore, it supports the conclusion that polymer structure influences plasma membrane interactions; the T4 analogues may take a different endocytic route than linear PEI or A442
that involves calcium-mediated endocytosis. The difference in intracellular uptake mechanisms could contribute to the different toxicity profiles for these polymers.
Also, direct plasma membrane permeabilization may be a mechanism of uptake and of cytotoxicity for the larger molecular weight polymers, as evidenced by the increase in plasma membrane permeability. The differences in plasma membrane permeability are apparent as early as 30 minutes after transfection (). There is also a loss of mitochondrial membrane potential at this time point (), indicating apoptosis has been initiated. An increase in phosphatidylserine exposure was observed within 30 minutes of transfection for T443 and A442 polyplex-treated cells (), another sign of early apoptosis. Different mechanisms of cellular entry may account for the different cytotoxic profiles of the polymers. However, although some apoptotic responses are evident 30 minutes after transfection, the time point where colocalization of polyplexes with the plasma membrane is observed (), we find that the cytotoxic profiles of the polymers tested become more apparent 4 hours after transfection, prompting us to investigate other mechanisms of polymer-induced cytotoxicity.
Previous research in our group has shown that the PGAAs are able to degrade in water and other biologically-relevant buffers.17
Here, we examined if the different cytotoxic profiles exhibited by the polymers tested were a result of T4’s ability to degrade into non-toxic byproducts. To this end, we have synthesized different molecular weights of T4 to observe the role that molecular weight plays on T4 toxicity. We do find that the higher molecular weight T4 shows higher cytotoxicity and expression efficiency than its lower molecular weight counterparts, indicating that if T4 does in fact possess the ability to degrade into smaller molecular weight polymers once inside the cell, it could alleviate some of the toxicity observed during polymer-based DNA delivery. We have also tested the toxicity of model T4 degradation products (the monomers) and have found that they do not exhibit cytotoxicity 24 hours after treatment at a concentration of 100 µg/mL. It is possible that, depending on the degradation mechanism, there could be toxic by-products that lead to apoptosis, as is the case with naturally-occurring polyamines found in cells.64–67
Specifically, intracellular catabolism of natural polyamines that are similar in structure to the monomer used to synthesize the T4 structures, such as spermine and spermidine, causes a rise in hydrogen peroxide, leading to oxidative damage and apoptosis initiation.65
Thus, the degradation products could lead to increased toxicity. Previous studies have revealed that polymer vehicles can illicit oxidative stress; interestingly, cells transfected with chitosan68
and a PEGylated branched PEI polymer69
show upregulated oxidative stress genes when compared to 25 kDa branched PEI-treated cells. In the case of the PEGylated branched PEI, the PEGylated polymer induced higher upregulation of more oxidative stress genes than non-PEGylated branched PEI within 6 hours of transfection, and it was suggested that this resulted from the higher molecular weight of the PEGylated PEI-based polymers compared to the branched PEI.69
In light of this previous data, it was of interest to us to study oxidative damage as a potential mechanism of cytotoxicity for the PGAAs. Cells were transfected with the polymers in the presence or absence of aminoguanidine, an inhibitor of several forms of oxidative-induced apoptosis.70, 71
Aminoguanidine was unable to prevent any cell death from the polyplexes (Figure S2
). Furthermore, no increase in reactive oxygen species in PGAA-treated cells was found compared to the cells only control (Figure S3
). Taken together, these results reveal that oxidative damage is not a major mechanism of toxicity for the gene delivery polymers tested.
Another potential mechanism of cytotoxicity investigated in the current work is nuclear membrane permeabilization. If the polyplexes are able to induce plasma membrane permeability, than we hypothesized that they may also be able to induce nuclear membrane permeability and thus provide a mechanism of nuclear entry for their plasmid DNA cargo. In addition, the ability to directly permeabilize the nuclear envelope may provide a link between the high toxicity observed with the polymers exhibiting the highest amount of protein expression (). We do see polyplex-nucleus interactions only 4 hours after transfection, the same time point where we see drastic differences in the cytotoxic profiles of the polymers. Our data shows increased propidium iodide fluorescence in nuclei isolated from PEI, T443
, and A442
-treated cells, indicating increased nuclear membrane permeability (). Not only do these three polymers exhibit the most cytotoxic responses four hours after transfection ( and ), but these polymers also have the highest expression efficiency (). It is possible that their high transfection efficiency correlates with their ability to permeabilize the plasma membrane, but it is also possible that the induction of apoptosis in cells treated with these polymers induces plasma membrane and nuclear envelope permeability. If this is the case, then these three polymers do not need to wait for nuclear envelope breakdown during mitosis to deliver their plasmid DNA into the nucleus. To determine whether increased membrane permeability (resulting from induction of apoptosis) was the cause of increased transfection efficiency with polyplexes formed with the higher molecular weight polymers, cells were transfected in the presence or absence of the apoptosis-inducing agent camptothecin. In the presence of camptothecin, we observed a slight increase in transfection efficiency for cells treated with naked plasmid DNA, but a slight reduction in transfection efficiency for all the polyplex types (Supporting Information Figure S6
). This indicates that increased nuclear envelope permeability due to apoptosis is likely not aiding polymer transfection. Furthermore, we also observed increased nuclear permeability to the dye trypan blue in the presence of polyplexes compared to nuclei treated with plasmid DNA only (), indicating that the polyplexes are able to induce nuclear envelope permeability in a cell-free system. Further study on the mechanism of nuclear uptake for polyplex-delivered plasma DNA is the focus of ongoing studies in our group.
Overall, we conclude that early cytotoxicity (within 30 minutes of transfection) is due to plasma membrane permeabilization, and that toxicity four hours after transfection is due in part to nuclear membrane permeabilization. We do notice colocalization between polyplexes and the nuclear envelope at this timepoint, and polyplexes are able to induce nuclear envelope permeability in isolated nuclei as well as in nuclei isolated from transfected cells. If these polymers are in fact able to permeabilize the nuclear membrane, then it is possible that they are able to permeabilize the membrane of different organelles. Further study on polyplex interaction with other organelles is necessary to make definite conclusions on this matter, since polyplexes are present throughout cells 4 hours after transfection () and could potentially interact with a myriad of intracellular processes. Although we predict that nuclear membrane permeabilization may be a mechanism of toxicity, a polymer’s ability to permeabilize the nuclear membrane may be desirable for efficient transfection; however, a careful balance between cytotoxicity and high expression efficiency needs to be established. It may be possible to tailor polymers that permeabilize the nuclear envelope to increase pDNA delivery to the nucleus but do not damage the nuclei to such an extent that the cells commit apoptosis.