Investigation of Dna Spectral Conformational Changes and Polymer Buffering Capacity in Relation to Transfection Efficiency of Dna/polymer Complexes

Purpose The relation between transfection efficiency of DNA/polymer complexes and DNA conformational alterations is investigated. The buffering capacity of several synthetic polymers is also studied and related to their performance in transfection efficiency. Methods The cationic polymer/DNA interaction was evaluated by measuring the alteration of DNA secondary structures in solution before and after the addition of polymers using the ATR-FTIR technique. The degree of protonation in aqueous solutions of cationic polymers depends upon pH and the structures. A polymer capable of protonation acts like a proton sponge and reacts with H + when titrated with HCl. This characteristic was evaluated in relation to transfection efficiency because the capacity would help the release of endocytotic DNA from endosome/lysosome and subsequent gene expression. Results IR results show that the antisymmetric PO2-vibration of DNA (at 1224 cm-1) shifts toward lower frequencies when complexed with PEI or PLLys (these polymers are able to transfect DNA), but shifts toward higher frequencies or no alterations in the presence of PDAMA or dextran (these polymers are poor in DNA transfection). Interestingly, the polymers with the best performance in transfection efficiency are in this order: PEI>PDMAEMA>PLLys>PDAMA>dextran which is in the same order as their polymer buffering capacity. These facts indicate that polymers possessing better buffering capacity should result in higher transfection efficiency. Also, we have demonstrated in this paper that the alteration in the antisymmetric PO 2-stretching vibration in the IR spectra is sensitive to the binding of cationic polymers with DNA and the transfection efficiency of the formed complexes. These findings are useful for the developing polymer-based gene delivery systems with better in vitro and in vivo performance. Novelty of the Work: this work establishes a rational relation between transfection efficiency of DNA/polymer complexes and DNA conformational alterations. Also, the buffering capacity of polymers is found to be closely related to their performance in transfection efficiency.

Fourier transformed IR spectroscopy with attenuated total reflectance technique has been used to characterize the secondary structure of DNA and to assess DNA interactions with drugs, organic chemicals and cationic metal ions in aqueous solution (18)(19)(20)._________________________________________ Recently, there have been studies made of the relationship of DNA with cationic lipid/DNA complexes and their effect on the hydration state of cationic lipid/DNA complexes (21,22).However, only a few attempts of characterizing the nature of cationic polymer/DNA complexation in aqueous solutions by IR spectroscopy and correlating their transfection efficiency have been made (23,24).Moreover, in order to mimic polymer/DNA complexes within the acidic endosomal/lysosomal compartments of cells, the buffering capacity of the polymers was performed in this study in a milieu of decreasing pH and in relation to their transfection efficiency.

IR spectroscopic studies of DNA/polymer complexes
Attenuated total reflectance (ATR) IR spectroscopy was used to explore the structural characterization of cationic polymer/DNA complexes in solutions.10 mM of an acetate buffer (pH 5) and 10 mM of Tris buffer solutions of pH 7.4 and 9 were selected to avoid possible disturbance of the phosphate vibrations of DNA in the IR spectra.Stock solutions of DNA (2 mg/ml) and different polymer solutions were prepared in buffers and then adjusted to the desired pH with HCl or NaOH.The complexation of DNA with the polymer was carried out by adding 400 μl of the polymer into 400 μl of DNA solution.After mixing for 5 seconds, the resulting dispersions were incubated at room temperature for one-hour to complete complexation.Next, DNA/polymer samples were spread onto the ZnSe trough of ATR crystal so as to cover the ZnSe surface.The cationic polymer/DNA weight ratios (r) used in the experiments were selected at 4/1, 2/1, 1/1, 1/2, 1/20, and 1/40 to cover the range of forming smaller particles in the transfection studies and were no cytotoxicity observed.The IR spectra were obtained using a BioRad FTS6000 FTIR spectrometer equipped with a MID-IR DTGS detector and KBr beam splitter under a nitrogen purge.ATR-IR spectra were recorded after 1024 scans with settings of 2 cm -1 resolution, 5 kHz scan speed, and 2 cm -1 optic aperture from a 10-reflection, 45 o -incidence angle ZnSe horizontal crystal.Spectra were collected and manipulated with Bio-Rad Win-IR Pro software.A flat base line in the region of 2300-1850 cm -1 was obtained after buffer subtraction.Also peaks in the region from 3800 to 3700 cm -1 were minimized in order to have a good water vapor subtraction before spectral variations in DNA absorbance were compared.For obtaining spectral data, measurements were carried out at least in triplicate and were within ±1 cm -1 in wavenumber deviation.

Titration behavior of polymers with acid
The ability of cationic polymers to be protonated and carrying positive charges over the pH range from 12 to 2 was determined by titration with acids.A greater buffering capability of a polymer indicates less [H + ] in solution and less changes in pH values during HCl titration.Briefly, each polymer (1 mg/ml) was dissolved in 150 mM NaCl and titrated to pH11 with 1N NaOH. 10 ml of each solution was titrated with increasing volume of 0.1 N HCl and pH values were recorded with a pH meter (pH/mV/Temp meter 6171).To compare the titration behavior of polymers alone with the behavior of polymer/plasmid DNA complexes, 0.1 mg polymer (e.g.PEI) was mixed with 0.1 mg DNA to form complexes in 150 mM NaCl (10 ml) and titrated to pH 11.The polymer/DNA complexes were then titrated with 0.01N HCl.

Transfection studies and β-galactosidase assay
COS-7 cells were used to evaluate the transfection efficiency of the polymer/DNA complexes.The cells were seeded in a 96-well plate (1×10 4 cells per well) under complete DMEM and incubated for 24 hours before transfection.The polymer/DNA complexes (volume 200 μL) were then added to these cells.The DNA concentration was kept constant at 5 μg/mL and the amounts of polymer/DNA were expressed as mass ratios.After 1 hour at 37C, the transfection mixture was removed and replaced with complete DMEM for additional 48 hours.Then a histochemical staining X-Gal assay was used to evaluate the transfection efficiency of the delivered gene (expression of β-galactosidase).
Transfection results (mean ± SEM of 3 experiments) expressed as relative transfection efficiency were normalized to the number of transfected cells found after incubation of the cells with a positive control (PDMAEMA /plasmid DNA complexes; optimal ratio(r = 4/1) at 20 and 5 μg/ml, respectively).The optimal ratio of PLLys/DNA and PDAMA/DNA complexes for their transfection is also at r = 4/1.The PEI/DNA complexes show an optimum transfection at r = 2/1.The ratio of dextran and DNA in experiments is at 4/1.The zeta potential of PDMAEMA, PDAMA and PLLys in complexes with DNA at their optimal ratios in transfection is circa +16mV and the zeta potential of PEI/DNA complexes is around +20 mV.The dextran has a neutral charge and naked DNA has the zeta potential of -20mV (measured by Malvern instrument Zetasizer Nano ZS)

IR spectra of DNA in aqueous buffers at various pH
The infrared spectrum of DNA in the region of 1800-800 cm -1 contains a variety of information on the conformational arrangement.Polymers with amido or amine groups can interact with DNA via electrostatic attractions after their protonation.However, the nitrogen base region (1700-1500 cm -1 ) in the DNA spectrum overlaps with the amine signals of the polymers, requiring spectra between 1300 and 800 cm -1 to be designated for monitoring this possible interaction.Typical IR spectra of DNA (2 mg/ml) in acetate (pH 5) and Tris buffer (7.4 and 9) are shown in Figure 1A.The secondary conformation of DNA in aqueous solution clearly remains in B-form in the pH range 5 to 9 because the assignments of DNA at 1224, 1086, 1056, and 969cm -1 were observed.The absorption bands at 1224 and 1086cm -1 are known to be the antisymmetric and symmetric stretching vibration of the phosphate groups (PO 2 -), respectively.IR absorption at 1056cm -1 is typically assigned to the vibration of ribose (C-C sugar) and absorption at 969cm -1 is as an indication of the existence of DNA.The band at 1224 cm -1 is a marker of B-form DNA (25).The bands at 938, 894, and 836 cm -1 are also signals of the B-form DNA (26) but they are not prominent in the ATR spectra, making it difficult to observe possible conformational alterations after complexating with polymers.A transition from Bto C-form (all belong to B-family structures) was observed by circular dichroism as we previously reported (27)(28)(29) but the addition of polycation polymer, in many cases, could not lead to any significant changes in CD spectra and DNA remains in the B-form.(28,30,31).However, the antisymmetric vibration of PO 2 -(1224 cm -1 ) exhibits a greater change either in band position or intensity before and after DNA condensation (32).In this study, the sensitive band at 1224 cm -1 is used to evaluate the changes before and after complexation with polymers as it is chosen to monitor the changes for the interaction of DNA with liposomes (33,22).No significant changes in the DNA symmetric vibration of PO 2 -at 1086 cm -1 were observed either in the presence of polymers or liposomes.

Effect of PEI and PLLys ratios on IR spectra
The IR spectrum of PEI indicates that its absorbance bands at circa 1224cm -1 are distinguished from the DNA phosphate peak (Figure 1B).For PEI/DNA complexes, the optimal transfection efficiency was found at a PEI/DNA ratio of 2/1 to 1/1 where the smallest particles were formed.The typical IR spectra of PEI/DNA complexes at various pH (from 5 to 9) are shown in Figure 2A and show that DNA still remains in the B conformation in the presence of PEI as indicated by a band at 937 cm -1 .At low PEI/DNA ratios (r = 1/40) in pH 5, 7.4 and 9 buffers, the spectra of DNA in complexes were nearly identical to naked DNA and the antisymmetric phosphate band stands is close to 1224 cm -1 (Figure 2B).This indicates that there is fairly less interaction between DNA and PEI, which is in agreement with the evidence of found large particles (>1μm).By increasing the PEI/DNA ratio to r =2/1, a shift of the antisymmetric phosphate band from 1224 to 1220 cm -1 was observed.The shift to a lower frequency at lower pH means an occurrence of a stronger interaction between PEI and DNA and smaller particles were formed.Also, this shift to a lower frequency suggests an increase of DNA hydration state (22,34,35).These alterations are all factors which result in the higher transfection efficiency of polyplexes.
The IR spectra of PLLys/DNA complexes at various pH are shown in Figure 3A.The PLLys/DNA complexes were studied at r =4/1 where smallest particles and their optimal transfection efficiency were achieved (36).When PLLys/DNA ratios increased from r =1/20 to r =4/1, a similar trend as in PEI was observed, i.e., a shift of the antisymmetric phosphate band at 1224 cm -1 to a lower frequency (1220 cm -1 , Figure 3A and 3B).This also means an increased occurrence of hydration around DNA PO 2 -groups when DNA is complexated with PLLys.At pH 7.4 and 9 with PLLys, DNA still remains in the B conformation (Figure 3A).The IR spectrum of DNA is also affected by the presence of cations (e.g.Pb2 + , Cu2 + , H + ) (18,37), and as shown, it can also be affected by cationic polymers.With PEI or PLLys at pH 9, the DNA PO 2 -antisymmetric band was nearly identical to naked DNA and unchanged in position because the limited protonated positive charges of the polymer can barely interact with DNA (Figure 2B and Figure 3B).It has been previously reported by our group that the particle size of polymer/DNA complexes is dependent on pH and the polymer/DNA ratios (16).The particle size of complexes formed at pH 9 was larger and was responsible for the loss of transfection efficiency (15).
Although the shift of the PO2 -antisymmetric band to lower frequencies was found after complexation, the presence of cationic polymers does not alter the secondary structure of DNA from the B conformation in buffer solutions.Previous studies have reported that DNA in solution appears to be B form in the presence of following milieu: DOTAP/cholesterol liposomes or DOTAP/DOPE liposomes (22) or cation ions or polymers (26,32,34,38), in spite of the different chemical structures of the interacted polycations, are in agreement with the findings of this study (Figure 2A, 3A).

Effect of PDAMA, PDMAEMA and dextran on IR spectra
For the complexes prepared at a PDAMA/DNA ratio of 4/1, the DNA absorptive band at 1224 cm -1  was not altered after complexation as shown in Figure 4. Interestingly, the formed PDAMA/DNA complexes were found in vitro to bear a very low activity in transfection.PDMAEMA, the polymer/DNA ratio at r =4/1 could bring out an optimal transfection and a small particle size as we have reported (16,39).Since the position of the DNA antisymmetric phosphate band at 1224 cm -1 was found to overlap with the polymer absorbance peaks, the possible spectral change of DNA in the presence of PDMAEMA is difficult to analyze (vibration at 1234 cm -1 is assigned to PDMAEMA, Figure 4, line c).Nevertheless, the observation of the sugar vibration of DNA assigned at 894 cm -1 is another indication that the B form DNA is present in the complexes in a hydration state and its transfection ability is preserved.The spectrum of the dextran polymer with DNA shows an unchanged antisymmetric phosphate band at 1224 cm -1 as naked DNA (Figure 4, line a), which means that the interaction between DNA and the dextran polymer is minimal and consistent with very low transfection efficiency found in experiments.

The buffering capacity of PEI, PDMAEMA, PLLys, PDAMA, dextran and NaCl
In Figure 5, it shows that PEI possesses the best buffering capacity within the pH range.NaCl and the dextran polymer containing no amino groups in their structures demonstrate no buffering effect in the range of pH 5 to 8. The polymers with the best performance in buffering capacity are in this order: PEI > PDMAEMA > PLLys > PDAMA > dextran =NaCl.Interestingly, the same order was found for the transfection efficiency of these polymers (Figure 5 and Figure 6).This demonstrates that the higher transfection potential of a polymer shows, the better buffering capacity is to acids.

Correlation between the alterations of DNA spectra in the presence of polymers and their transfection efficiency
The relative transfection efficiency of PEI and PLLys with respect to the reference polymer PDMAEMA is about 1.3 and 0.5, respectively (Figure 6).The antisymmetric PO 2 -vibration of DNA shifts to a lower frequency from 1224 cm -1 to 1220 cm -1 in the presence of PEI or PLLys, which is attributable to the electrostatic interactions between cationic polymers and DNA, yielding a hydration effect in the region of DNA phosphate groups (21,24).Moreover, the formed small particles of polymer/DNA complexes that result from strong electrostatic interaction and hydration effect could make these complexes better for transfection into cells.Noteworthy is the fact that Hackl et al. has reported a shift of the DNA antisymmetric PO 2 -band to a higher frequency (from 1224 cm -1 ) in the presence of bivalent ions (such as Ca 2+ ) (38,25).These co-precipitates of bivalent ions have an effect on the dehydration of DNA resulting in a very low transfection activity.Since the zeta potential of studied complexes is similar (from +16 to +20mV, except the dextran) and only non-specific interaction to the studied cells is discussed, the effect of cell surface charge with the counter-charge complexes is minimal.In a series of polycationic polymers (based on the same framework in structure), the higher molecular weight of a polymer has, the better transfection efficiency would be (40).Results show that the lowest Mw (19 kDa) PDAMA bears the worst transfection but the PEI in a similar Mw (25 kDa) showed the best transfection activities.This suggests that PDAMA is likely lack of the interaction to DNA antisymmetric PO2 - group because the absorption peak at 1224 cm -1 remains unchanged.However, a possibility that a substantial increase in Mw of PDAMA resulting in a higher transfection activity can not be excluded.

Correlation between the buffering capacity of polymers and their potential in transfection efficiency
The transfection potential is found to be related to the polymers' buffering capacity although overlapping of polymer bands in the DNA region around 1224 cm -1 could occur (ex.PDMAEMA).Titration studies were performed to determine the buffering capacity of the studied polymers within the cellular endosomal/lysosomal compartments.Protonable polymers e.g., PEI with a plenty of amino groups in the structure could not only mediate the DNA binding and DNA condensation but also help endosomal release through their ability to act as a "proton sponge".Evidence shows that the retardation of pH change in lysosome vesicles by lowering the pH to around pH 5 leads to vesicle swelling, rupture and eventually DNA/polymer complexes are released from the entrapment of endosome/lysosome vesicles (41).Therefore, the buffering capacity of polymers is related to the prevention of DNA degradation in lysosomes and the ease of delivery of DNA into nucleus from lysosomes which results in better transfection activities.
Since dissociation of DNA and polymer must be occurred at nucleus for a successful transfection and negatively charged proteins would compete the binding to cationic polymers with DNA, electrophoretic studies were conducted to show DNA dissociation of PEI/DNA complexes in the presence of negatively charged albumin (at pH 5 and pH 8) (Figure 7).There is no free DNA band visible in lanes 2, 3 and 4 (Figure 7A, 7B) for PEI/DNA complexes.However, in the presence of various amounts of albumin, DNA bands and albumin bands were observed (lanes 5, 6 and 7) in comparison to only albumin (lane 8).These results suggest that DNA can be dissociated from complexes and provide good transfection potential even though a strong interaction occurs between PEI and DNA especially at pH 5.

CONCLUSION
In this paper, we have demonstrated the structure-function relationship in polymer/DNA complexes and the DNA antisymmetric PO 2 vibration is sensitive to the binding of cationic polymers.The alterations in shifting frequencies could be used to interpret the performance in transfection after polymer complexation.Also, polymers that possess better buffering capacity have higher transfection efficiency.These findings could be very useful for the development of polymer-based gene delivery systems with better performance in vitro and in vivo.