Bioconjugate Chem. 2003, 14, 255−261
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TECHNICAL NOTES Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. 2. Charge Center Type Theresa M. Reineke† and Mark E. Davis* Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125. Received August 9, 2002
Recent polycation structure-gene delivery studies reveal that subtle changes in the molecular structure of polycations have substantial influences on DNA-binding and condensation and on in vitro toxicity and gene delivery efficiency. In Part 1 of this structure-property study using carbohydrate-containing polycations (1), it is demonstrated that as the amidine charge center is removed further from the carbohydrate unit within the polycation structure, the toxicity increases. Inclusion of larger carbohydrate species within the polycation backbone also reduces the toxicity. Here, the effect that polycation charge center type has on toxicity and gene delivery efficiency is investigated. A series of quaternary ammonium polycations containing N,N,N′,N′-tetramethyl-1,6-hexanediamine, D-trehalose, and β-cyclodextrin are synthesized in order to elucidate the effects of charge center type (by comparison to the data given in Part 1) on gene delivery. In all cases, it is found that the quaternary ammonium analogues exhibit lower gene expression values and similar toxicities to their amidine analogues. Additionally, transfection experiments conducted in the presence of chloroquine reveal increased gene expression from quaternary ammonium containing polycations and not from their amidine analogues.
INTRODUCTION
Cationic polymers are under investigation as alternatives to viral systems for the delivery of therapeutic genes (1-6). Several recent reports have indicated that structural differences in polycations have a considerable effect on nucleic acid condensation, polyplex stability, toxicity, and cellular uptake (1, 7-21). In Part 1 of our study (1), efforts aimed at elucidating structure-property relationships between carbohydrate-containing polyamidines and their ability to deliver plasmid DNA (pDNA) are described. It was found that the structure indeed played a role in the toxicity of the polycation in cultured cells, and as the charge center was moved away from the carbohydrate unit within the polycation backbone, the toxicity increased (1). In addition, by increasing the size of the carbohydrate moiety, a reduction in the toxicity was observed. The identification of subtle structure-property relationships prompted us to further investigate other changes within this family of polycations in hopes of enhancing the understanding of how charge variations affect the properties of carbohydrate-containing, polycationic, gene delivery vectors. A number of studies have recently indicated that there is a relationship between charge center type, toxicity, and delivery efficiency. For instance, van de Wetering et al. investigated a series of cationic methacrylate and methacrylamide polymers and determined that small changes in the structure and charge type did not have a large effect on the pDNA condensing * To whom correspondence should be addressed. mdavis@ cheme.caltech.edu. † Current address: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221.
properties, although substantial changes in the cytotoxicity and transfection capabilities were observed (14). Molecular modeling studies were used to help clarify their findings, and it was discovered that polycations having a low number of interactions with DNA possessed higher gene expression. Wolfert and co-workers also investigated a family of polymethacrylates for the influence of charge spacing, charge type, and molecular weight and found that as the pendant charge was further removed from the polymer backbone, pDNA condensation was less efficient (21). In addition, gene expression levels were influenced by charge type where polymethacrylates containing quaternary ammonium charges exhibited low transfection efficiency. Here, we describe the synthesis and characterization of a new family of carbohydrate-containing polyquaternary ammonium vectors. The polycations were synthesized from N,N,N′,N′-tetramethyl-1,6-hexanediamine, D-trehalose, and β-cyclodextrin in an effort to create a group of polycations related to the amidine carbohydrate polycations (1) and to compare the results of transfection experiments between the two polycation families. MATERIALS AND METHODS
Monomer and Polycation Synthesis. All reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) with the exception of the dimethylaminoethanethiol that was obtained from Research Organics, Inc. (Cleveland, OH) and the β-cyclodextrin, which was purchased from Wacker Biochem Corp (Adrian, MI). Electrospray mass spectra were obtained from the Beckman Institute Mass Spectrometry Center on a Perkin-Elmer SCIEX API 365 in positive ion mode. NMR spectra were collected on a Varian 300 MHz spectrometer (75.5 MHz for 13C)
10.1021/bc025593c CCC: $25.00 © 2003 American Chemical Society Published on Web 12/21/2002
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Scheme 1
Scheme 2
with trimethylsilylpropionic acid sodium salt as an internal standard. All amine products were purified over a Toyopearl SP-650M ion-exchange column using a gradient of ammonium bicarbonate (0-0.3 M) as the eluant. TLC on amine products were performed as previously described and spots were detected by using I2 (8). 6,6′-diiodo-6,6′dideoxy-D-trehalose 1 and 6A,6D-diiodo6A,6D-dideoxy-β-cyclodextrin 4 were synthesized as previously described in Part 1 of this study (1, 7, 8). N,N,N′,N′-Tetramethyl-6,6′-diamino-6,6′-dideoxy-D-trehalose 2 (Scheme 1). 1 (0.364 g, 0.648 mmol) was dissolved in 25 mL of 2.0 M dimethylamine in methanol. The reaction was stirred at 70 °C for 6 h, stirred at roomtemperature overnight, and then concentrated. The residue was redissolved in 25 mL of water, brought to a pH ) 1.8, and then purified on the cation exchange column. The appropriate fractions were combined and lyophilized to dryness yielding a white solid, 2 (0.134 g, 52%). ES/MS: m/z 397 [M + H]+, 419 [M + Na]+. 13C NMR D2O): δ 95.93, 74.77, 74.69, 73.21, 71.16, 62.13, 46.80.
N,N,N′,N′-Tetramethyl-6,6′-diaminoethanethio-6,6′dideoxy-D-trehalose 3 (Scheme 1). To a flask containing 60 mL of degassed dH2O was added dimethylaminoethanethiol (0.693 g, 6.6 mmol). Under constant stirring, 1 (0.606 g, 1.10 mmol) was then added in 4 equal installments over 30 min and then stirred at 75 °C for 5 h. This mixture was then concentrated to a residue, resuspended in 20 mL of dH2O, and acidified to a pH ) 1.8. After purification with the Toyopearl column, the appropriate fractions were combined and lyophilized to dryness yielding a white product, 3 (0.347 g, 61%) ES/ MS: m/z 517 [M + H]+. 13C NMR (D2O): δ 95.27, 74.97, 74.84, 73.58, 73.43, 59.96, 46.07, 35.23, 31.33. N,N,N′,N′-Tetramethyl-6A,6D-diamino-6A,6D-dideoxy-βcyclodextrin 5 (Scheme 2). 4 (2.169 g, 1.6 mmol) was dissolved in 2 M dimethylamine in methanol (77 mL) and was stirred at 70 °C. After 7 h the reaction was cooled to room temperature and stirred overnight. The methanol was evaporated, the crude product redissolved in methanol, evaporated again, and pumped on overnight to remove residual dimethylamine. The crude product was
Technical Notes
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Figure 1. Schematic structures of the quaternary ammonium polycations QP1-QP4. Scheme 3
then dissolved in water, acidified to pH ) 2.0, and purified yielding a white solid, 5 (0.528 g, 28%). ES/MS: m/z 1190 [M + H]+, 13C NMR (D2O): δ 104.16, 103.47, 86.39, 83.64, 82.84, 82.70, 75.45, 74.43, 74.30, 71.82, 62.72, 61.56, 47.26. N,N,N′,N′-Tetramethyl-6A,6D-diaminoethanethio-6A,6Ddideoxy-β-cyclodextrin 6 (Scheme 2). To a flask containing dH2O was added dimethylaminoethanethiol (0.630 g, 6.0 mmol), and the mixture was stirred at 80 °C. Next, 4 (1.350 g, 1.0 mmol) was added to this mixture in 4 aliquots over 30 min and then stirred for an additional 4.5 h at temperature. The mixture was then dried in vacuo, resuspended in water, acidified to a pH ) 1.5, and purified on the ion exchange column. Fractions containing the product were combined and lyophilized yielding a white solid, 6 (0.53 g, 53%). ES/MS: m/z 1310 [M + H]+. 13C NMR (D2O): 104.79, 104.55, 87.53, 84.34, 75.88, 74.65, 63.11, 60.95, 46.98, 35.95, 32.23 Polymerization. The polycations QP1-QP4 (see Figure 1) were synthesized via Menschutkin polymerization (Scheme 3) by combining a ditertiary amine comonomers with dibromohexane and mixing the reaction in a solvent for 3 days at temperature (22). For example, 3 (0.103 g, 0.20 mmol) was combined in a small scintillation vial with 0.6 mL of a 4:1 dimethylformamide:methanol solution. Next, 1,6-dibromohexane (0.055 g, 0.23 mmol) was added, and the mixture was stirred for 3 days at 40 °C. The solution was then suspended in a 50% solution of methanol in dH2O, added to a 1000 MWCO dialysis membrane (Spectrum Laboratories) (1000 MWCO was used for the tetramethyl-1,6-hexanediamine and trehalose polycations, 2000 MWCO membranes were used for the β-cyclodextrin analogues), dialyzed in 30% methanol
in water for 3 h to remove residual dibromohexane, and then dialyzed exhaustively with several water changes for 72 h. Polycation Characterization. Polycation molecular weights were determined by static light scattering. The polycations were analyzed on a Waters 515 HPLC system equipped with a Wyatt Technologies Corp. DAWN EOS in conjunction with an Optilab DSP interferometric refractometer or on a Hitachi D6000 HPLC system equipped with a ERC-7512 refractive index detector and a Precision Detectors PD2020/DLS. Each sample was prepared at a concentration between 50 and 70 mg/mL and injected onto a Polymer Labs Aquagel-OH 30 column with an eluant flow rate of 0.7 mL/min. The eluant was 0.8 M ammonium acetate, brought down to pH ) 2.8 with formic acid. The refractive index increment values were determined in the same eluant at 25 °C (633 nm). Gel Retardation Experiments. Each polycation was examined for its ability to bind pDNA through gel electrophoresis experiments as previously described (7). A 1 µg amount of pGL3-CV (10 µL of a 0.1 µg/µL in DNase free water) was mixed with an equal volume of polymer at charge ratios between 0 and 5.0 (. Each solution was incubated for approximately 30 min. A 2 µL volume of loading buffer was added to each sample, and then 10 µL of each sample was pipetted into the wells of a 0.6% agarose gel containing 6 µg of ethidium bromide/100 mL TAE buffer (40 mM Tris-acetate, 1 mM EDTA) and electrophoresed. Dynamic Light Scattering and Zeta-Potential. The polyplex size and zeta-potential were measured using a ZetaPals dynamic light scattering instrument (Brookhaven Instruments Corporation, Holtsville, NY) as
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Table 1. The Polycation Percent Yield, Refractive Index Increment, Molecular Weight, Polydispersity, and Degree of Polymerization Data for the Carbohydrate-Containing Quaternary Ammonium Polycations QP1-QP4 (Figure 1) As Determined by Static Light Scattering polycation
yield, %
dn/dc
Mw (kDa)
Mw/Mn
degree of polymerization
QP1 QP2 QP3 QP4
53 38 32 37
0.149 0.154 0.162 0.140
12.5 6.0 3.4 9.8
1.68 1.53 2.60 1.30
30 14 12 6
previously described (7, 8). A 2 µg amount of pGL3-CV was complexed with each polymer at a charge ratio of 10 ( and allowed to stand for 30 min before diluting to 1.2 mL of dH2O. The results for each sample are reported as an average of five measurements for the polyplex size and an average of 10 data points for the zeta-potential. Cell Culture Experiments. Plasmid pGL3-CV (Promega, Madison WI) containing the luciferase gene under the control of the SV40 promoter was amplified by Escherichia coli strain DH5R and was then purified using a Novagen Ultramobius 1000 plasmid kit. BHK-21 cells were purchased from ATCC (Rockville, MD) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 100 units/mg penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin at 37 °C and 5% CO2. Media and supplements were purchased from Gibco BRL (Gaithersburg, MD). BHK21 cells were plated at 50000 cells per well in 24 well plates and incubated for 24 h. A 60 µL volume of polycation dissolved in DNase free water (Gibco BRL) was added to 60 µL of plasmid DNA (0.05 µg/µL in DNase free water) at charge ratios of 3, 5, 10, 15, 20 (polymer +/DNA -). The mixtures were incubated for 30 min and then diluted to 600 µL with serum free media. Twenty four hours after cell plating, the cells were transfected with 1 µg of pGL3-CV complexed with each of the polymers above (QP1-QP4) at the various charge ratios (200 µL of each solution above) and with naked pDNA in triplicate in serum free media. After 4 h, 800 µL of DMEM was added to each well. Twenty four hours after transfection, the media was replaced with 1 mL of DMEM. Forty eight hours after transfection, cell lysates were analyzed for luciferase protein activity with results reported in relative light units (RLUs) as previously described (7). Toxicities were determined by a modified version of the Lowry protein assay with a BioRad (Hercules, CA) DC Protein Assay Kit also as previously described (7). Transfections were also completed with polyplexes prepared from pDNA and polycations AP3 and QP3 at charge ratios of 3, 5, and 10 ( in the presence of chloroquine. These experiments were carried out using the same procedure above with the exception that chloroquine was added at concentrations of 0, 50, 100, and 200 µM to the serum free media in which the polyplexes were diluted in. RESULTS
Monomer and Polycation Synthesis and Characterization. Each monomer was synthesized in accordance to the procedures described above (depicted in Schemes 1 and 2). Monomers were prepared via nucleophilic substitution of the halogenated analogue with the corresponding amine derivative. All of the polycations were prepared via the Menschutkin reaction by combining the tertiary amine-functionalized comonomers with dibromohexane in a solution of dimethylformamide and
Figure 2. Comparison of the hydrodynamic diameter of the polyplexes formed by complexing QP1-QP4 with pDNA as measured by dynamic light scattering (illustrated by the bars on the graph). The results shown are reported as an average of five measurements. The particle charge is denoted by ([) as determined by zeta-potential measurements. The results are reported as an average of 10 measurements. Polyplexes were prepared by combining 2 µg of DNA at a charge ratio of 10 ( and diluting to a total volume 1.2 mL.
methanol as shown in Scheme 3. Polymerizations proceeded accordingly with the exception of monomer 5, which would not undergo polymerization. The reaction of 5 with 1,6-dibromohexane was attempted in several solvent combinations without success. The polymer yield, molecular weight, degree of polymerization, and polydispersity index were determined by static light scattering, and the results are given in Table 1 for each polycation. pDNA Binding Studies and Polyplex Characterization. The polycations were complexed with pDNA at several charge ratios between 0 and 5.0 ( and the point of charge neutralization determined by agarose gel electrophoresis. All polycations bound and neutralized pDNA at and above a charge ratio of 1.0 (. To determine both the particle size and the surface charge of the polyplexes, each polycation was complexed with pDNA at a charge ratio of 10 ( and analyzed for particle size using dynamic light scattering and surface charge using zeta-potential. As shown in Figure 2, the polyplexes formed by each of the polycations were all between 90 and 110 nm. In addition, the surface charge of these the polyplexes was positive indicating an excess of polycation charge relative to the charge of the pDNA. Positive zeta-potentials provide for endocytosis with cultured cells (23). In Vitro Toxicity and Transfection of the Polyplexes. BHK-21 cells were plated in 24-well plates and transfected under serum-free conditions using 1 µg of pGL3-CV complexed with each polycation at charge ratios of 3, 5, 10, 15, and 20 (. Toxicities associated with the polyplexes at various charge ratios were determined by measuring the total protein concentration in the cell lysates after 48 h following initial transfection and normalizing the data with protein levels from untransfected cells. As shown in Figure 3, at a charge ratio of 3 (, the toxicity associated with polymer QP1 becomes evident where about 70% cell survival is obtained. At a charge ratio of 20 (, only about 30% of the cells survived. Polycation QP3 also shows elevated toxicity levels, and at a charge ratio of 20 (, approximately 40% of the cells survived. Polycations QP2 and QP4 were essentially nontoxic to BHK-21 cells. Toxicity comparisons between AP and QP type polycations are shown in Figure 4.
Technical Notes
Figure 3. Comparison of the relative toxicities of the carbohydrate quaternary ammonium polymers QP1-QP4 at charge ratios of 0, 3, 5, 10, 15, and 20 with BHK-21 cells. Cell survival was determined by assaying for total protein content and normalizing each sample with the protein concentration for untransfected cells. The data are reported as a mean ( SD of three samples.
Figure 4. Toxicity comparison of analogous AP and QP polyplexes at a charge ratio of 20 ( in BHK-21 cells. Data were normalized to untransfected cells for each study.
Transfection efficiencies of the polyplexes were determined by assaying for luciferase protein activity, and the results are reported in relative light units (RLUs) per mg of protein. Several polycation (+) to DNA (-) charge ratios were tested, and the results are shown in Figure 5. QP4 is found to have the highest transfection efficiency, but only at higher charge ratios. Both QP1 and QP2 give similar gene expression values at lower charge ratios; however, at 20 (, QP2 exhibits higher expression (likely due to the high toxicity of QP1). The lowest gene expression was obtained from QP3. Transfection efficiency comparisons between AP and QP type polycations are illustrated in Figure 6. Transfections were also conducted in the presence of 0, 50, 100, and 200 µM chloroquine in serum free media with naked pDNA and with polyplexes formed with polymers AP3 and QP3 at charge ratios of 3, 5, and 10 (. Polycations AP3 and QP3 were compared in this experiment because of the large difference in gene expression values obtained for these polycations (Figure 6). As depicted in Figure 7, for almost all cases, the addition of chloroquine to transfection experiments using polyplexes formed from AP3 actually resulted in a
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Figure 5. Comparison of transfection efficiencies of the QP1QP4 polyplexes with BHK-21 cells at charge ratios of 0, 3, 5, 10, 15, and 20 ( as determined by luciferase protein activity. Data are presented as a mean + SD of three replicates.
Figure 6. Comparison of transfection efficiencies for the analogous AP and QP polyplexes with BHK-21 cells as determined by luciferase protein activity. Data points represent the maximum transfection for each vector.
decrease in gene expression (most likely due to an increase in cell death from the associated toxicity of chloroquine). It is shown that at charge ratios of both 3 and 5 (, the addition of 200 µM of chloroquine did cause a small increase in gene expression. However, for polymer QP3, a substantial increase in gene expression is observed at both charge ratios of 3 and 5 (. At a charge ratio of 3 (, gene expression increases systematically with increasing concentration of chloroquine, and at a concentration of 200 µM, a 63-fold increase in gene expression is observed. At a charge ratio of 5 (, an overall 20-fold increase in gene expression is noted. At a charge ratio of 10 (, gene expression levels do not increase with QP3 and this is likely due to the high degree of cell death (between 20 and 40% cell survival) that is observed at this charge ratio. DISCUSSION
A series of carbohydrate-containing polycations with quaternary ammonium DNA-binding centers were synthesized to study the effect of charge center type on toxicity and gene delivery efficiency. The effect of charge center type was assessed by comparing the results obtained to those from polycations possessing amidine
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Figure 7. The effects of chloroquine on the transfection efficiency of polyplexes formed with polycations AP3 and QP3. Chloroquine concentrations of 0, 50, 100, and 200 µM, and polycation charge ratios of 3, 5, and 10 ( were tested with BHK-21 cells.
charges (investigated in Part 1). Synthesis of the tertiary amine monomers is accomplished through the substitution of the diiodo-carbohydrate molecule with the corresponding nucleophile as depicted in Schemes 1 and 2. Polymerizations of the tertiary amine comonomers with dibromohexane proceed efficiently through Menschutkin quaternization, as shown in Scheme 3, with the exception of monomer 5, which did not react. Previous synthetic studies with this type of polymerization reaction by Rembaum and co-workers indicate that solvent is an important variable in the kinetics of formation (22). Polymerization of 5 was attempted with several batches of monomer as well as in several solvent systems including dimethylformamide, methanol, water, and dimethyl sulfoxide without success. The polymerization of 5 may not be possible due to the steric hindrance around the amine from the bulky cyclodextrin unit. All other monomers were successfully polymerized (Figure 1). Polycation data such as molecular weights, degrees of polymerization, and polydispersity indices are displayed in Table 1. Clearly, the degree of polymerization is dependent on the structure of the diamine. As expected, all polycations neutralize pDNA above a charge ratio of 1.0 according to agarose gel electrophoresis experiments. Polyplexes prepared from the quaternary ammonium polymers QP1-QP4 were analyzed using dynamic light scattering and found to be less than 110 nm (QP4 forms the largest polyplexes at 110 nm). zeta-potential measurements show that all polyplexes have high positive surface charge (Figure 2). Toxicity studies (Figure 3) reveal that QP1 (lacks a carbohydrate unit) is the most toxic polycation to BHK21 cells where only 30% cell viability is observed at a charge ratio of 20 (. In addition, the trehalose polymer, QP3 is found to be relative toxic (40% cell survival at 20 (). These observations are consistent to those found with polyamidines (Part 1) where the amidine analogue of QP3 (AP3) was found to be the most toxic of the carbohydratecontaining polycations. QP2 exhibits only slight toxicity with 90% cell viability at 20 (, and QP4 is essentially nontoxic. Figure 4 shows the toxicity comparison between the amidine and the quaternary ammonium polycations. Both polycations that lack a carbohydrate, AP1 and QP1, are the most toxic to cells. Note that these polycations
have higher degrees of polymerization and this may influence the toxicity. However, since QP1 is approximately 3 times longer than AP1, and their toxicities to BHK-21 cells are similar, the toxicity of these noncarbohydrate polycations may not be influenced by chain length in the range investigated here. A similar trend exists between the both polycation families where toxicity increases as the charge center is moved further away from the carbohydrate moiety (AP2, AP3, QP2, QP3). However, the effect is not as severe with the β-cyclodextrin polycations. In general, the toxicity does not appear to significantly vary between each analogous quaternary ammonium and amidine polycation (Figure 4). Transfection efficiencies as determined by luciferase protein activity show that QP3 generally gives lower transfection efficiency than QP1, QP2, and QP4 (Figure 5). QP4 reveals increasing gene expression with increasing polycation amount. This effect is also noted with β-cyclodextrin polyamidines and may be attributed to the slight membrane disruption ability of cyclodextrins (24). Figure 6 shows a comparison of the transfection efficiencies for both families of polycations (polyamidines and the polyquaternary ammonium). The data displayed in Figure 6 are for the maximum transfection. In every case with carbohydrate-containing polycations, it is observed that the quaternary ammonium polycations give lower gene expression values than the amidine analogues, and the largest difference is noted between AP3 and QP3. Transfections with AP3 and QP3 polyplexes were performed with the lysosomotropic agent, chloroquine (Figure 7). Chloroquine is known to promote endosomal release of some types of polyplexes. In Part 1 (1), it was noticed that AP3 exhibited the highest transfection efficiency in the series of amidine polymers. However, here it is shown that the quaternary ammonium analogue of AP3 (QP3) displays the lowest transfection efficiency. If this discrepancy is due to the inability of the quaternary ammonium polycations to be released from the endosomes, then the addition of chloroquine may affect the gene expression. There is not a significant increase in gene expression in the presence of chloroquine for AP3, while for QP3 at charge ratios of 3 and 5 (, gene expression increases with chloroquine concentration (Figure 7). These results could indicate that the amidine
Technical Notes
analogues have improved endosomal escape properties relative to the quaternary ammonium analogues. An exception to this finding is QP4, where at a charge ratio of 20 (, relatively high transfection efficiency is noted. As previously mentioned, this may be due to the slight membrane disruption ability of the β-cyclodextrin moiety that may contribute to increase the endosomal escape (24). Reports by Wolfert et al. also reveal that quaternized methacrylate polymers transfected poorly as compared to methacrylate polymers that contain secondary amine DNA-binding centers (21). These studies indicate that although quat-based polyplexes produce efficient intranuclear transcription following injection into Xenopus oocytes, the lack of transfection could be attributed to the absence of pH responsiveness of the quaternized polycation. In addition, van de Wetering et al. also observed low transfection efficiency from a quaternized methacrylate polymer (14). The inability of the quaternized derivatives to escape from the endosomes was confirmed by an experiment in where they added an endosome-disruptive agent (adenovirus) during the transfection experiment that led to a substantial increase in transfection efficiency. In conclusion, a series of quaternary ammonium polymers containing N,N,N′,N′-tetramethyl-1,6-hexanediamine, D-trehalose, and β-cyclodextrin were synthesized and studied for their ability to bind pDNA and to deliver pDNA to BHK-21 cells. The results obtained indicate that the charge center type does not considerably influence the toxicity since no significant difference in toxicity was noted between the polycation families (quaternary ammonium versus amidine). However, it was found that the charge center type does have a significant influence on gene delivery efficiency since the amidine polycations consistently exhibit higher gene expression than their quaternary ammonium analogues. This result may be due in part to the inability of quat-based polyplexes to escape from endosomes since the addition of chloroquine to transfection experiments increased gene expression for QP3 but not with AP3. ACKNOWLEDGMENT
We thank Insert Therapeutics, Inc. for partial support of this project. T.M.R. would like to thank the NIH for a National Research Service Award (1-F32 GM64919-01). We are grateful to Jeremy Heidel for his help with some of the chloroquine transfection experiments and Swaroop Mishra for amplifying the pDNA. LITERATURE CITED (1) Reineke, T. M., and Davis, M. E. (2003) Structural effects of carbohydrate-containing polycations on gene delivery. Carbohydrate size and its distance from charge centers. Bioconjugate Chem. 14, 247-254. (2) Rubanyi, G. M. (2001) The future of human gene therapy. Mol. Aspects Med. 22, 113-142. (3) Li, S., and Ma, Z. (2001) Nonviral gene therapy. Curr. Gene Ther. 1, 201-226. (4) Han, S., Mahato, R. I., Sung, Y. K., and Kim, S. W. (2000) Development of biomaterials for gene therapy. Mol. Ther. 2, 302-317. (5) Ma, H., and Diamond, S. L. (2001) Nonviral gene therapy and its delivery systems. Curr. Pharm. Biotechnol. 2, 1-17. (6) De Smedt, S. C., Demeester, J., and Hennink, W. E. (2000) Cationic polymer based gene delivery systems. Pharm. Res. 17, 113-126.
Bioconjugate Chem., Vol. 14, No. 1, 2003 261 (7) Gonzalez, H., Hwang, S. J., and Davis, M. E. (1999) New class of polymers for the delivery of macromolecular therapeutics. Bioconjugate Chem. 10, 1068-1074. (8) Hwang, S. J., Bellocq, N. C., and Davis, M. E. (2001) Effects of structure of β-cyclodextrin-containing polymers on gene delivery. Bioconjugate Chem. 12, 280-290. (9) Hwang, S. J. and Davis, M. E. (2001) Cationic polymers for gene delivery: Designs for overcoming barriers to systematic administration. Curr. Opin. Mol. Ther. 3, 183-191. (10) Jones, N. A., Hill, I. R. C., Stolnik, S., Bignotti, F., Davis, S. S., and Garnett, M. C. (2000) Polymer chemical structure is a key determinant of physicochemical and colloidal properties of polymer-DNA complexes for gene delivery. Biochim. Biophys. Acta 1517, 1-18. (11) Neiman, Z., and Quinn, F. R. (1981) Quantitative structureactivity relationships of purines I: Choice of parameters and prediction of pKa values. J. Pharm. Sci. 70, 425-430. (12) Tang, M., and Szoka, F. (1997) The influence of polymer structure on the interactions of cationic polymers with DNA and the morphology of the resulting complexes. Gene Ther. 4, 823-832. (13) Wadhwa, M. S., Collard, W. T., Adami, R. C., McKenzie, D. L., and Rice, K. G. (1997) Peptide-mediated gene delivery: Influence of peptide structure on gene expression. Bioconjugate Chem. 8, 81-88. (14) van de Wetering, P., Moret, E. E., Schuurmans-Nieuwenbroek, N. M. E., van Steenbergen, M. J., and Hennink, W. E. (1999) Structure-activity relationships of water-soluable cationic methacrylate/methacrylamide polymers for nonviral gene delivery. Bioconjugate Chem. 10, 589-597. (15) Murphy, J. E., Uno, T., Hamer, J. D., Cohen, F. E., Dwarki, V., and Zuckerman, R. N. (1998) A combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery. Proc. Natl. Acad. Sci. U.S.A. 95, 1517-1522. (16) Fischer, D., Bieber, T., Li, Y., Elsasser, H.-P., and Kissel, K. (1999) A novel nonviral vector for DNA delivery based on low molecular weight, branched polyethylenimine: Effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res. 16, 1273-1279. (17) Ferruti, P., Manzoni, S., Richardson, S. C. W., Duncan, R., Pattrick, N. G., Mendichi, R., and Casolaro, M. (2000) Amphoteric linear poly(amido-amines)s as endosomolytic polymers: Correlation between physicochemical and biological properties. Macromolecules 33, 7793-7800. (18) Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Size matters: Molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. J. Biomed. Mater. Res. 45, 268-275. (19) Plank, C., Tang, M. X., Wolf, A. R., and Szoka, F. C. (1999) Branched cationic peptides for gene delivery: Role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes. Hum. Gene Ther. 10, 319-332. (20) Wagner, E., Cotten, M., Foisner, R., and Birnstiel, M. L. (1991) Transferrin-polycation-DNA complexes: The effect of polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. U.S.A. 88, 4255-4259. (21) Wolfert, M. A., Dash, P. R., Nazarova, O., Oupicky, D., Seymour, L. W., Smart, S., Strohalm, J., and Ulbrich, K. (1999) Polyelectrolyte vectors for gene delivery: Influence of cationic polymer on biophysical properties of complexes formed with DNA. Bioconjugate Chem. 10, 993-1004. (22) Rembaum, A., Rile, H., and Somoano, R. (1970) V. Kinetics of formation of high charge density ionene polymers. Polym. Lett. 8, 457-466. (23) Mislick, K. A., and Baldeschwieler, J. D. (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 93, 12349-12354. (24) Rodal, S. K., Skretting, G., Garred, O., Vilhardt, F., van Deurs, B., and Sandvig, K. (1999) Extraction of cholesterol with methyl-β-cyclodextrin perturbs formation of clathrincoated endocytic vesicles. Mol. Biol. Cell 10, 961-974.
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