Multiblock Reducible Copolypeptides Containing Histidine-Rich and

Bioconjugate Chem. 2006, 17, 1395−1403

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Multiblock Reducible Copolypeptides Containing Histidine-Rich and Nuclear Localization Sequences for Gene Delivery Devika Soundara Manickam and David Oupicky´* Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48202. Received April 23, 2006; Revised Manuscript Received September 3, 2006

Polyplexes sensitive to redox potential gradients represent promising gene delivery vectors. High molecular weight polypeptides containing disulfide bonds in the backbone were synthesized by an oxidative copolymerization of a histidine-rich peptide (HRP) and a nuclear localization sequence (NLS) peptide. The synthetic approach allowed an easy synthesis of reducible copolypeptides (rCPP) with different relative contents of the HRP and NLS sequences. Cytotoxicity and transfection activity of rCPP-based DNA polyplexes were evaluated in vitro. In comparison with control polyethylenimine (PEI), only minimum toxic effects of rCPPs were observed on the metabolic activity and membrane integrity of human endothelial cells. These findings are predominantly ascribed to favorable structural features like lower charge density and higher chain rigidity of the rCPPs compared to PEI and also to a reductive intracellular and plasma membrane degradation. Transfection activity in all tested cell lines increased with increasing content of the HRP sequence in the rCPPs, while no clearly measurable effect of the NLS sequence was found.

INTRODUCTION Polyelectrolyte complexes of nucleic acids with polycations (polyplexes) are investigated as promising delivery vectors for a variety of nucleic acid therapeutics. In particular, polyplexes capable of responding to environmental changes or stimuli by altering their properties and behavior promise a significant improvement of the efficacy of the delivery process. One of the several microenvironmental features, which could be exploited for improving the efficiency of nucleic acid delivery, is the redox potential gradient existing between extracellular environment and various subcellular organelles in normal as well as pathological states. The existence of such gradients has been exploited by incorporating disulfide bonds into the structure of a variety of delivery vectors to provide them with a capability to release the therapeutic cargo selectively in the subcellular reducing space (1-5). The easy intracellular reversibility of disulfide bonds in redoxsensitive polyplexes proved to be a highly advantageous feature for delivering a variety of nucleic acids, including plasmid DNA, mRNA, antisense oligonucleotides, and siRNA. Delivery of plasmid DNA has been by far the most investigated application of the redox-sensitive polyplexes. A number of studies in a variety of cell lines showed transfection activities comparable to or better than those observed for polyethylenimine (PEI) and/ or commercially available lipid formulations (6-10). The benefits of intracellular reduction of disulfides in the redoxsensitive polyplexes for efficient delivery of nucleic acids have been demonstrated also for mRNA, siRNA, and antisense oligonucleotides (4, 11). Redox-sensitive polyplexes based on cationic peptides are attractive vectors because of the possibility of combining DNA condensation capability with an inherent biological activity of the peptides (12-14). Here, we synthesized reducible copolypeptides (rCPP) composed of two cationic peptide sequences that should provide the copolymers with endosomal buffering capability and nuclear localization capability. It is hypothesized that the combination of the two features in a single * To whom correspondence should be addressed. E-mail: oupicky@ wayne.edu.

carrier will improve the overall efficiency of the gene delivery process. The copolypeptides are synthesized by an oxidative polycondensation using an engineered histidine-rich peptide (HRP) and a nuclear localization sequence (NLS) derived from the SV40 virus. The copolypeptides show minimal cytotoxicity and transfection activity comparable to or better than control PEI polyplexes.

EXPERIMENTAL PROCEDURES Materials. Peptides, NLS (CGAGPKKKRKVC, Mr 1274), and HRP (CKHHHKHHHKC, Mr 1431) were custom-synthesized by Sigma-Genosys. Branched PEI (25 000 g/mol), polyL-lysine (PLL, 240 000 g/mol), dithiothreitol (DTT), chloroquine, genistein (Gen) and bafilomycin A1 (Baf.A1) were from Sigma-Aldrich. Plasmid DNA vectors, gWiz high-expression luciferase (gWiz-Luc) containing luciferase reporter gene, and gWiz high-expression GFP (gWiz-GFP) containing enhanced green fluorescent protein reporter gene were from Aldevron (Fargo, ND). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and Roswell Park Memorial Institute 1640 (RPMI 1640) medium were from Invitrogen. All other chemicals were obtained from Fisher Scientific unless otherwise noted. Synthesis of Reducible Copolypeptides. The rCPPs were synthesized by DMSO-mediated oxidative polycondensation described previously (15). Five rCPPs were prepared using NLS and HRP peptides at different molar ratios in the reaction mixture. Relative content of the NLS and HRP peptides in the synthesized copolypeptides was determined by amino acid analysis (Michigan State University Macromolecular Core) using the average ratio of histidine residues of HRP to valine and alanine residues of NLS peptide. The concentration of the polypeptides in solution was estimated from the amino acid analysis by adding L-norleucine as an internal standard. Formulation of DNA Polyplexes. DNA solution (20 µg/mL) in 10 mM HEPES buffer (pH 7.4) was used to prepare complexes with PEI, and DNA solution in 30 mM sodium acetate buffer (pH 5) was used to prepare polyplexes with rCPPs at the desired N/P mixing ratio (c(basic amino acid residues of a peptide or amino groups of PEI)/c(DNA phosphates)) and mixed by vigorous vortexing for 10 s.

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1396 Bioconjugate Chem., Vol. 17, No. 6, 2006

Hydrodynamic Radius and ζ Potential Determination. The determination of hydrodynamic diameters and ζ potentials of polyplexes was performed using ZetaPlus Particle Size and Zeta Potential analyzer (Brookhaven Instruments Inc.) as described previously (14). Salt Dissociation Assay. Polyplexes were formulated as described above and incubated in 25 mM DTT for 1 h at 37 °C. Then, increasing concentration of NaCl (0-1 M) was added and samples were loaded onto 0.8% agarose gel containing 0.5 µg/mL ethidium bromide and run for 90 min at 120 V in 0.5× TBE buffer. The amount of DNA released from the polyplexes at a given NaCl concentration was analyzed using Kodak 1D image analysis software. Cell Lines. Murine melanoma cell line B16F10, mouse mammary tumor cell line 4T1, human cervical cancer cell line HeLa, human breast carcinoma MCF-7, and African green monkey kidney fibroblasts COS-7 were obtained from ATCC. Human endothelial cell line EA.hy926, derived by a fusion of human umbilical vein endothelial cells with a lung carcinoma A549, was a kind gift from Dr. Edgell (University of North Carolina). B16F10, HeLa, EA.hy926, and COS-7 were maintained in DMEM and MCF-7 and 4T1 in RPMI 1640. Both media were supplemented with 4 mM L-alanyl-L-glutamine and 10% FBS. Cytotoxicity Evaluation. Cytotoxicity of rCPPs and control PEI in EA.hy926 cells were determined by MTS and LDH assays using commercially available kits. The MTS assay was performed using the CellTiter 96 aqueous cell proliferation assay (Promega). Twenty-thousand cells seeded in a 96-well plate 2 days before the experiment were incubated with increasing concentration of the polycations in 150 µL of DMEM/FBS. The medium was removed after 16 h and replaced with a mixture of 100 µL of fresh DMEM and 20 µL of MTS reagent solution. The cells were incubated for 1 h at 37 °C in a CO2 incubator. The absorbance of each sample was then measured at 505 nm to determine cell viability. The results are expressed as the mean percentage cell viability relative to untreated cells ( SD. The cytotoxicity of the polycations was also determined by a CytoTox 96 nonradioactive cytotoxicity assay, an assay that measures LDH release from damaged cells. EA.hy926 cells were incubated with increasing concentrations of the polycations in DMEM/FBS lacking phenol red. After 2 h of incubation at 37 °C, the medium was removed and centrifuged at 250g at 4 °C for 5 min. An amount of 50 µL of the supernatant was diluted with 50 µL of DMEM/FBS without phenol red. The LDH substrate (50 µL) was added to each sample and incubated for 30 min in the dark. An amount of 50 µL of the stop solution was added to each sample, and the absorbance was measured at 490 nm. The maximum LDH release was quantified by freeze-thaw lysis of untreated cells followed by medium processing as described above. The results are expressed as the mean % LDH release ( SD. Transfection Efficiency in Vitro. Transfection activity was tested using polyplexes prepared as described above at the indicated N/P ratio. All transfection studies were performed in 48-well plates with cells plated 24 h before transfection at a seeding density of 30 000 cells per well using previously described protocols (10, 14). On a day of transfection, the cells were incubated with the complexes in 150 µL of FBSsupplemented media or medium lacking FBS as indicated. After 3 h of incubation, the transfection mixture was completely removed and the cells were cultured for an additional 24 h in fresh media supplemented with 10% FBS prior to analysis of reporter gene expression. Transfections in the presence of chloroquine and Baf.A1 were performed in the same way except that the drug was added to the culture medium before addition of the polyplexes. Caveolae inhibition experiments were con-

Manickam and Oupicky´

Figure 1. Schematic representation of the multiblock reducible copolypeptides (rCPP). Table 1. Composition and Molecular Weight of Multiblock Reducible Copolypeptides (rCPP) polycation

% HRP in reaction

% HRP in polymera

Mw

rCPP-A rCPP-B rCPP-C rCPP-D rCPP-E

0 25 50 75 100

0 27 52 80 100

∼100 000

a

∼60 000 ∼200 000

Determined from amino acid analysis.

ducted by preincubating the cells for 30 min with the respective inhibitor or combination of inhibitors (200 µM genistein, 100 nM bafilomycin A1) before the addition of polyplexes. The inhibitors were present in the transfection medium throughout the 3 h of incubation. The luciferase transfection results are expressed as relative light units (RLU) per milligram of cellular protein ( SD of triplicate experiments. The GFP transfection results are expressed as the percentage of GFP-positive cells determined by flow cytometry ( SD of triplicate experiments.

RESULTS Synthesis of rCPP and Characterization of rCPP/DNA Polyplexes. To combine two functional peptides into a single polymeric carrier, multiblock reducible copolypeptides were synthesized by randomly connecting HRP and NLS peptides via disulfide bonds into a linear polypeptide chain (Figure 1). Mild oxidation of the terminal Cys residues with DMSO was used as described in our earlier work (14, 15). Five different compositions of the rCPP were prepared by varying the amount of the NLS and HRP peptides in the polycondensation reaction (Table 1). Amino acid analysis was used to determine the relative content of the two peptides in the rCPPs. Molecular weights of selected rCPPs were estimated by size exclusion chromatography and were found to range from ∼60 000 to ∼200 000 (Table 1). The ability of the rCPP polycations to condense DNA was confirmed by ethidium bromide exclusion (data not shown) and gel retardation assays (Figure 2a). All rCPPs were able to fully condense DNA in buffers at pH 7.4 and 5 at N/P ratios of g2. Hydrodynamic size and ζ potential of the rCPP polyplexes were measured by dynamic light scattering using optimized polyplex compositions determined in the transfection assay (Table 2). All rCPPs formed polyplexes with sizes and ζ potentials fully comparable to those of control PEI. The sensitivity of the rCPP/DNA polyplexes to reducing environment was tested by following the polyplex dissociation with NaCl (16). No detectable dissociation was observed under nonreducing conditions up to 1 M NaCl (Figure 2b, data shown for rCPP-C/DNA only). Reduction of the disulfide bonds in rCPP-C in 25 mM DTT resulted in the polyplex dissociation already starting in ∼0.3 M NaCl. Cytotoxicity of rCPP Polycations. The cytotoxicity of the rCPP polycations was evaluated using MTS and LDH assays in human endothelial cells (Figure 3). The results show that the rCPP polycations exhibit only a negligible cytotoxicity in the MTS assay after 16 h of incubation in the concentration range 0-300 µg/mL, while control PEI shows an IC50 value less than 10 µg/mL (Figure 3a). The LDH release assay, which was used to estimate the early-stage cytotoxicity associated with

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Multiblock Reducible Copolypeptides

Figure 2. (a) DNA condensation by rCPPs. Polyplexes were prepared at N/P ratios of 0.5 and 2.0 (left and right lanes) in 30 mM sodium acetate buffer (pH 5.0) (upper panel) and 10 mM HEPES buffer (pH 7.4) (lower panel). (b) Salt dissociation of rCPP-C/DNA polyplexes under reducing conditions of 25 mM DTT. Table 2. Molecular Parameters of rCPP-Based DNA Polyplexesa polycation

hydrodynamic diameter (nm)

ζ potential (mV)

rCPP-A rCPP-B rCPP-C rCPP-D rCPP-E PEI

105 ( 3 125 ( 1 145 ( 2 135 ( 1 96 ( 1 115 ( 1

47 ( 1 49 ( 4 43 ( 1 46 ( 1 55 ( 1 47 ( 5

a The polyplexes were formulated at optimum molar mixing ratios. Results are presented as the mean ( SD (n ) 3).

plasma membrane disruption, shows results similar to the MTS data. When the LDH is measured after 2 h of incubation with the polycations, PEI exhibits a similar IC50 value as in case of the MTS assay. The rCPPs show minimal LDH release at concentrations below 100 µg/mL, but unlike in the MTS assay considerable cytotoxicity is observed at the highest concentration (300 µg/mL) tested (Figure 3b). Light transmission micrographs were obtained after a 16 h incubation of the cells with the polycations to directly document the differential effects of PEI and rCPP on the human endothelial cells (Figure 3c). The images show the cells incubated with the rCPP polycations as healthy, attached to the surface, and of similar appearance as untreated cells. The cells incubated with PEI, on the other hand, show clear signs of toxicity indicated by changed morphology and a large number of rounded and detached cells. Transfection Activity of rCPP/DNA Polyplexes. Initial transfection experiments were conducted to determine the optimum N/P ratio for each rCPP/DNA formulation to achieve maximum transgene expression levels in B16F10 cells (Figure 4). The N/P ratio that resulted in maximum luciferase expression was then used in all subsequent experiments. The N/P dependence of the transfection activity is mostly influenced by the content of the pH-buffering HRP peptide. For example, trans-

fection activity of rCPP-B with 27% of HRP gradually decreases with increasing N/P ratio, while the activity of rCPP-E (100% HRP) peaks at N/P ) 8 and then slowly declines, similar to the control PEI/DNA. The transfection activity of the optimized rCPP/DNA formulations was further tested in a panel of six cell lines (Figure 5). A general trend of gradual increase in the transfection activity with increasing content of HRP is observed in all the cell lines. The best performing rCPP-E polyplexes exhibit between 2- and 7-fold higher transfection activity than control PEI polyplexes depending on the cell line. It is worth noting that the difference between the lowest and the highest observed transfection is almost 5 orders of magnitude. Similar results were obtained across the same panel of cell lines in GFP transfection experiments (data not shown). The effect of chloroquine on the luciferase reporter expression of the rCPP/DNA polyplexes is plotted in Figure 6a as a chloroquine enhancement ratio ([RLU/mg protein with chloroquine]/[RLU/mg protein without chloroquine]). In fast-growing B16F10 cells, the presence of 100 µM chloroquine results in an almost 10000-fold increase in the transfection activity of rCPP-A, which consists of 100% NLS and thus lacks any buffering capability. As expected, increasing the HRP content in the polypeptides leads to a decrease in the potentiation of the transfection activity by chloroquine. The two other cell lines tested show a slightly different pattern of chloroquine effect, with maximum enhancement seen for rCPP-C/DNA polyplexes. Complementary results are obtained for the GFP reporter plasmid in B16F10 cells (Figure 6b). The dependence of transfection activity of polyplexes on pHbuffering capability of the polypeptides was examined by conducting transfections in the presence of Baf.A1, a potent inhibitor of vacuolar ATPases (Figure 7). It has been reported that, depending on the cell line used, the transfection activity of PEI can be reduced 7- to 74-fold in the presence of 175 nM Baf.A1 (17). Unexpectedly, rCPP-A polyplexes that lack any buffering capability exhibited almost a 1000-fold increase in the transfection activity in the presence of 100 nM Baf.A1 in B16F10 cells (Figure 7a). A decreasing effect of Baf.A1 on transfection enhancement is observed when moving from rCPP-A to rCPP-E. The transfection activity of control PEI/ DNA increased almost 2-fold under the same conditions. Baf.A1 has a smaller but similar enhancing effect on the transfection of rCPP-A polyplexes in HeLa cells (Figure 7b). A 2- to 4-fold, statistically insignificant, increase in the transfection activity of the remaining four rCPP polyplexes is observed in the presence of 10% FBS and 175 nM Baf.A1 (Figure 7b). There was a 2-fold decrease in the transfection activity of control PEI, which is in close agreement with the results reported previously (17). Unlike HeLa and B16F10 cells, Baf.A1 has no enhancing effect on the transfection activity of rCPP polyplexes in MCF-7 cells (Figure 7c). In experiments conducted in the absence of FBS (Figure 7df), Baf.A1 causes the expected decrease of transfection activity of polyplexes capable of endosomal pH buffering. Especially in the HeLa and MCF-7 cells, the Baf.A1 effect is directly correlated with the content of HRP (Figure 7e,f). For example, the decrease in the transfection activity of rCPP-E polyplexes in HeLa cells is almost 2 orders of magnitude. In addition, Baf.A1 has a more pronounced effect on the transfection activity of rCPP-E than PEI polyplexes. No significant differences were observed in the transfection activity of control PLL polyplexes in the presence and absence of Baf.A1 (data not shown). In B16F10 cells, the enhancing effect of Baf.A1 was preserved for polyplexes with no (rCPP-A) or minimum buffering capability (rCPP-B) (Figure 7d). There was almost a 10-fold increase in the transfection activity of rCPP-A. In agreement with the



Figure 3. Cytotoxicity evaluation in human endothelial EA.hy926 cells. (a) Metabolic activity was measured by MTS assay following 16 h of incubation of the cells with the polycations (mean ( SD, n ) 3). (b) Membrane toxicity was measured by LDH release assay after 2 h of incubation of the cells with the polycations (mean ( SD, n ) 3). (c) Light transmission micrographs of the cells after 16 h of incubation with the polycations (upper left, rCPP-E; upper right, rCPP-C; lower left, rCPP-D; lower right, PEI).

Figure 4. Influence of N/P ratio on transfection activity of rCPP/DNA in B16F10 cells. Results are shown as mean relative light units (RLU) of luciferase per milligram of cellular protein ( SD (n ) 3).

Figure 6. Effect of chloroquine on transfection activity of rCPP/ DNA: (a) chloroquine-induced enhancement of luciferase expression; (b) effect of chloroquine on eGFP expression in B16F10 cells (n ) 3).

Figure 5. Transfection activity of optimized rCPP/DNA in a panel of cell lines. Luciferase activity was measured after 24 h. Results are shown as mean RLU per milligram of cellular protein ( SD (n ) 3).

original expectations, polycations with significantly higher buffering capabilities (rCPP-D and rCPP-E) demonstrated a decrease in the transfection activity of the polyplexes in the presence of Baf.A1, 3- and 5-fold, respectively. Since the effects of Baf.A1 in B16F10 cells showed a strong dependence on the presence or absence of serum, we conducted transfections using medium supplemented with gradually increasing concentrations of bovine serum albumin (BSA) (Figure 8a). The results show that in the presence of Baf.A1, rCPP-A with no buffering capability exhibits a gradual increase in the

transfection activity with increasing concentration of BSA in the incubation medium and rCPP-E with the maximum buffering capability among the tested compositions shows no significant increase. Additional transfection studies were conducted to further probe the unexpected effect of Baf.A1 on the activity of rCPP-A polyplexes. It has been reported that cellular uptake of polyplexes proceeds via a clathrin- or caveolae-dependent endocytic pathway (18). To determine an involvement of an alternative endocytosis pathway, caveolae inhibition studies were carried out using genistein, a specific inhibitor of tyrosine kinase involved in receptor-induced formation of caveolae (Figure 8b) (19, 20). In the presence of Baf.A1, there was a 100-fold increase in transfection activity of rCPP-A polyplexes, an 8-fold increase in the case of rCPP-C polyplexes, while no effect was observed for rCPP-E polyplexes (Figure 8b). Unlike Baf.A1, genistein had no marked effect on the transfection activity of the NLS-containing rCPP polyplexes, but it led to a decrease of transfection activity of rCPP-E polyplexes. It has been



Figure 8. (a) Effect of BSA on Baf.A1-induced enhancement of luciferase expression in B16F10 cells (Baf.A1 enhancement ) [RLU/ mg with Baf.A1]/[RLU/mg without Baf.A1]). (b) Effect of Baf.A1 and caveolae inhibitor genistein (Gen) on the transfection activity of polyplexes in B16F10 cells. Luciferase activity was measured after 24 h, and results are shown as mean RLU per milligram of cellular protein and SD from triplicate samples.

Figure 7. Effect of Baf.A1 on transfection activity of rCPP polyplexes. Transfections were performed in the presence (a) or absence (d) of 10% FBS in B16F10 cells, in the presence (b) or absence (e) of 10% FBS in HeLa cells, and in the presence (c) or absence (f) of 10% FBS in MCF-7 cells. Luciferase activity was measured after 24 h, and results are shown as mean RLU per milligram of cellular protein and SD from triplicate samples (empty bars, absence of Baf.A1; solid bars, presence of Baf.A1).

reported that transfection activity of polyplexes is mediated by both clathrin-dependent and caveolae-dependent endocytosis pathways whereas lipoplex transfection is mediated only by the clathrin endocytosis pathway and is unaffected by inhibiting the caveolae endocytosis pathway (18). DOTAP lipoplexes were therefore included as a control (not shown) to verify if the observed decrease in the transfection activity of rCPP-E polyplexes in the presence of genistein was due to inhibition of the caveolae endocytosis pathway. The transfection activity of DOTAP lipoplexes was not affected by genistein, confirming published observations (18). Treatment of cells with both genistein and Baf.A1 prevented the Baf.A1-induced increase in the transfection activity of rCPP-A polyplexes. The transfection activity of NLS-containing (rCPP-A and rCPP-C) polyplexes was almost equal to levels mediated in inhibitorfree conditions. However, there was a significant decrease in the transfection activity of rCPP-E polyplexes in the presence of both genistein and Baf.A1.

DISCUSSION Polyplexes sensitive to redox potential gradients represent promising gene delivery vectors because of a favorable safety profile and flexibility for use in delivery of a wide range of nucleic acids. Reducible polypeptides used in the preparation of such polyplexes are typically based on a single peptide

sequence and are prepared either by DNA-template polymerization or by DMSO-mediated polycondensation (7, 11, 14, 21). The only example of a reducible polypeptide composed of several peptides of different amino acid sequences was reported by Rice et al. and used the DNA-template polymerization (22). Here, we use a polycondensation approach to demonstrate the feasibility of synthesizing reducible sequential polypeptides consisting of two peptides with distinct biological functions (Figure 1). This approach represents a viable alternative of preparing high molecular weight repetitive polypeptides with a better control over the amino acid sequence and molecular weight than traditional chemical synthesis and an easier alternative to polypeptide synthesis using genetic engineering methods, which provide the ultimate control over the properties of polypeptides (23). Peptide selection for the synthesis of rCPPs was based on recognizing the importance of endosomal escape and nuclear transport for the efficacy of DNA polyplexes. To enable endosomal escape of the polyplexes, HRP peptide containing histidyl residues capable of buffering in the endo/lysosomal pH range was used. The sequence of the HRP peptide was selected on the basis of previous findings that high His content is required for efficient transfection (7, 24). Two trihistidyl blocks were separated by lysyl residues to improve DNA binding capability. To provide the polyplexes with potential nuclear localization capability, NLS peptide was used that was derived from the importin R binding SV40 T antigen sequence (13, 25). Neither of the two peptides used here requires a free terminus for their activity. In cases where the N- or C-terminus of a peptide is required for its activity, a single Cys residue can be placed at the opposite end and used in the oxidative polycondensation scheme as an end-capping functionality. The copolymerization proceeded readily for all five HRP/NLS ratios tested and resulted in the formation of high molecular weight linear polypeptides. No information could be obtained about the distribution of the HRP and NLS blocks in the polypeptides. The fact that the content of the two peptides in rCPP is close to that in the


reaction mixture (Table 1) suggests similar reactivity of both peptides, and therefore, it is likely that HRP and NLS blocks are randomly distributed in the rCPPs. As expected, the HRP-containing rCPPs exhibited better DNA condensation ability at slightly acidic pH of 5 because of higher protonation levels of the His residues. The principle of the redox sensitivity of rCPP/DNA polyplexes was demonstrated in cellfree conditions by determining the concentration of NaCl required to dissociate the polyplexes in reducing and nonreducing environment. It is assumed that the disulfide bonds in the backbone of the rCPP chains are cleaved in the reducing environment of DTT. The resultant short cationic HRP and NLS peptide residues then display lower affinity to the DNA and allow subsequent dissociation of the polyplexes at lower NaCl concentration. The concentration of NaCl required to dissociate the polyplexes is directly related to the affinity between DNA and polycations, which in turn depends, among other factors, on the molecular weight of the polycations (16, 26). Because of the cooperative nature of polycation binding to DNA, the molecular weight dependence of the dissociating NaCl concentration is especially strong in the low-to-medium molecular weight range of the polycations and is therefore highly suitable to follow the degradation of rCPPs in the polyplexes. The observed NaCl concentration required to dissociate the polyplexes in reducing conditions (∼0.3 M) was close to that of polyplexes based on control small peptides, confirming that reduction of the disulfide bonds of the rCPPs proceeds readily in the polyplexes. Cellular delivery of intact nucleic acids is not the only goal in designing a successful delivery vector. The vector should be able to accomplish this without negatively affecting normal functions of the cell. The toxicity of polycations like PEI and their DNA polyplexes has been reported by a number of investigators in both in vitro and in vivo experiments. The studies suggest two types of cytotoxic action of PEI. The first type of toxicity originates from compromised plasma membrane integrity and occurs in the early stages of incubating the cells with PEI. The first phase of the cytotoxic activity of PEI is then followed by apoptosis caused by PEI-induced channel formation in the mitochondrial membrane (27, 28). Structural features that most affect the way polycations interact with cells include charge density, molecular weight, molecular flexibility, and type of charged center. Rigid molecules have more difficulties attaching to the membranes than flexible molecules. Therefore, high cationic charge densities and highly flexible polymers, such as PEI, are expected to cause greater cytotoxic effects than those with low cationic charge density and more rigid chains. We have hypothesized that intracellular degradation of rCPP into short cationic fragments will significantly reduce the cytotoxicity because of the dependence of polycation toxicity on the molecular weight. The results obtained from the MTS assay, which measures metabolic activity of cells, seem to confirm the hypothesis because no significant toxicity was observed for any of the tested rCPPs up to 300 µg/mL, while PEI showed IC50 ≈ 10 µg/mL. To further assess the short-term acute cytotoxicity of rCPPs, studies were performed to measure the amount of LDH released in the early stages of incubation. It is expected that during the early stages of incubation, the plasma membrane disruption by extracellular polycations will be the main contributing factor to the observed LDH release. Even though the reducible polycations are not degraded in the cell culture medium, their acute toxicity measured in the LDH assay is minimal when compared to control PEI. Only at the highest rCPP concentrations (300 µg/mL) do the levels of LDH release suggest substantial membrane perturbation. Because of the strong dependence of polycation toxicity on their molecular weight, it


is important to analyze possible cellular sites of rCPP degradation before drawing any conclusions. The intracellular reduction of disulfide bonds of rCPPs is most likely mediated by small redox molecules like glutathione (GSH), either alone or with the help of redox enzymes. GSH is the most abundant intracellular sulfhydryl present in millimolar concentrations inside the cell but only in micromolar concentrations in the blood plasma (29). The intracellular glutathione concentration is an additive function of both the oxidized (GSSG) and the reduced forms (GSH) of glutathione. The glutathione concentration and redox ratio (GSH/GSSG) are different in the various subcellular compartments, with the lowest redox potential in the cell usually found in the nucleus, where it is required for DNA synthesis and DNA repair as well as to maintain a number of transcription factors in a reduced state. The concentration of GSH has been reported to be about 4 mM in the cytoplasm and about 20 mM in the nucleus (30, 31). The subcellular distribution of GSH suggests that intracellular reduction of disulfide bonds will proceed preferentially in the cytoplasm and nucleus. Even though some evidence also suggests the possibility of endosomal and lysosomal reduction of disulfide bonds (32), a recent report indicates that both the endosomal and lysosomal environments are oxidizing and that disulfide bond reduction proceeds rather inefficiently (33). Despite the oxidizing nature of the extracellular environment, the presence of redox-active sulfhydryl groups in numerous proteins on the cellular plasma membrane suggests that at least the microenvironment of the cell surface can also support redox reactions in and between certain surface proteins (34, 35). On the basis of our data and the above analysis, we assume that the reduced cytotoxicity of the rCPP polycations observed in the MTS assay is due to the intracellular reduction of the disulfide bonds resulting in short fragments with decreased binding affinity to membranes, nucleic acids, and vital proteins inside the cell. The limited ability of rCPPs to damage cell membrane and release the LDH is predominantly due to favorable (compared to PEI) structural features such as lower charge density and higher chain rigidity. It might be also possible that plasma membrane reduction of rCPPs contributes to their favorable cytotoxic properties. To enable more direct and unambiguous evaluation of the contribution of disulfide bond reduction to the cytotoxicity of reducible polycation, reducible and nonreducible polycations with similar chemical structures should be used instead of comparing peptide-based polycations with PEI. Such studies are currently underway in our lab using reducible and nonreducible forms of poly(L-lysine) (15). One of the disadvantages in employing short bioactive cationic peptides as carriers in gene delivery lies in their nonspecific interactions with nucleic acids, which can hinder their biological activity. Polyplexes formed using short cationic peptides also show less efficient transfection (7) and are less stable because of the less prominent cooperative effect (16). These major limitations in utilizing bioactive peptides can be overcome by the use of reversible polymeric forms of the peptides, such as demonstrated here. Analysis of the transfection data of the rCPP/DNA polyplexes reveals that of the two peptides used in rCPP synthesis, HRP is more important than NLS for the transfection activity. The activity increases in all cell lines with increasing content of HRP. The increase in transfection activity of HRP-rich polyplexes with increasing N/P ratio suggests an important contribution of the free polypeptides to the polyplex activity (36). It was expected that an optimum HRP/NLS composition might exist at which maximum transfection activity would be observed. The results, however, suggest no clearly measurable effect of an NLS role in the rCPP/DNA activity. The apparent lack of NLS contribution to enhanced activity of the rCPP may be explained by several factors. The



NLS activity in DNA polyplexes is limited, as suggested by numerous reports demonstrating only modest enhancing effect on the transfection (25, 37-39). The large size of the plasmid DNA and the large particle size of the polyplexes might be another factor, which limits the nuclear delivery. The notion that the size of the nucleic acid may play a role in the nuclear transport of polyplexes is supported by recent findings that rCPP containing the NLS sequence (rCPP A, B, C, D) can mediate nuclear delivery of siRNA (40). Yet another reason for the apparent lack of NLS effect on the rCPP activity may lie in the loss of NLS sequences from the surface of the polyplexes in the reducing environment of cytosol by the cleavage of the disulfide bonds. Finally, it may be possible that even if a small increase in efficiency of nuclear delivery mediated by the NLS is achieved, it may not manifest itself by a significant enhancement of reporter gene expression (41, 42). The contribution of the buffering capability of HRP to the overall transfection activity of rCPP polyplexes was tested by conducting the experiments in the presence of chloroquine and Baf.A1. The lysosomotropic activity of chloroquine is known to preferentially enhance activity of polyplexes based on polycations with no buffering potential, and as such, the chloroquine-mediated enhancement of transfection can serve as a measure of the importance of the buffering component in the polyplexes. This is clearly documented especially in the fastgrowing B16F10 cells where the chloroquine enhancement ratio decreases with increasing HRP content. In the other two cell lines tested, the same effect is observed only in the three rCPPs with the highest HRP content. To complement the chloroquine data, we conducted the transfection experiments also in the presence of the inhibitor of vacuolar type H+ ATPases, Baf.A1. Since polycations with pH buffering properties require exposure to an acidic environment for their activity (43), inhibition of the vacuolar ATPases by Baf.A1 should inhibit activity of the polyplexes based on such polycations (17). Dependent on the cell line and conditions used, our data show great qualitative and quantitative differences in the effect of Baf.A1 on the transfection activity of rCPP polyplexes. When the experiments were conducted under our standard transfection conditions (i.e., in the presence of 10% FBS), only the results in MCF-7 cells confirm the expected effect of Baf.A1 and show a significant decrease of levels of transgene expression for polyplexes with high HRP content and control PEI polyplexes (Figure 7c). Qualitatively different results are observed in B16F10 cells where a large increase of luciferase expression induced by Baf.A1 is observed, especially for rCPP with no or limited buffering capability, while no significant effect is seen for control PEI (Figure 7a). Most previous studies on the effects of Baf.A1 on transfection activity of polyplexes were conducted in serum-free conditions. Under serum-free conditions in B16F10 cells (Figure 7d), we observed that Baf.A1 exhibited the expected effect on the transfection activity of rCPP/DNA polyplexes in compositions containing comparatively higher amounts of the HRP peptide (D and E), which was compatible with a mechanism of action that depends on proton capture during the acidification process during endocytosis. Also, in HeLa and MCF-7 (Figure 7e,f), Baf.A1 demonstrated the expected effect on transfection activity of rCPP/DNA complexes compatible with the mechanism mentioned above. In all three cell lines, Baf.A1 decreases the luciferase expression of the polyplexes with the highest HRP content. The effect is best shown in HeLa and MCF-7 cells, while the data obtained in B16F10 cells are less clear and a Baf.A1-mediated increase of luciferase expression is still observed for rCPP-A even in serumfree conditions. This effect seems to be at least partially dependent on the amino acid sequence of the NLS peptide because no effect of such magnitude was observed for similar

nonbuffering reducible polypeptides based on CKKKKKKKKKKC or CGRKKRRQRRRGC peptides (data not shown). In addition, Baf.A1 caused no increase in cellular uptake of the polyplexes in B16F10 (data not shown). As shown in Figure 7, the Baf.A1 effect on the activity of rCPP-A polyplexes in B16F10 cells is strongly associated with the presence of serum. To determine if there is a specific component of the serum responsible for the observed effect, we have conducted the experiment in the presence of bovine serum albumin (BSA) (Figure 8a). Increasing the albumin concentration leads to increased transfection induced by Baf.A1 in rCPP-A polyplexes but has almost no effect on rCPP-E polyplexes. As reported previously, Baf.A1 is toxic to the B16F10 cells and causes significant levels of apoptotic cell death at 100 nM (44). To limit the adverse toxic effects of Baf.A1, control experiments were also conducted at 20 nM Baf.A1. In the presence of 10% FBS, the observed results showed similar effects of Baf.A1 on the transfection activity of rCPP-A demonstrated by ∼75-fold increase of luciferase expression (data not shown). The data obtained with the caveolae inhibitor genistein (Figure 8b) seem to suggest that Baf.A1 treatment may redirect at least some of the NLS-containing polyplexes to a caveolae-mediated uptake mechanism. Since this mechanism of cellular uptake avoids routing to the lysosomal degradative environment, it may be possible that avoidance of the lysosomal degradation of the polyplexes may be the reason for the observed Baf.A1 effect (45). It should be noted, however, that detailed understanding of the unexpected behavior of the NLS-containing polyplexes in the presence of Baf.A1 is not available at this moment and further detailed investigation of this phenomenon is beyond the scope of this manuscript.

CONCLUSION In conclusion, the present studies demonstrate that reducible multiblock copolypeptides can be readily synthesized by an oxidative polycondensation. This synthetic approach allows an effective control of the relative content of individual building blocks of the copolypeptides, which is demonstrated here on the copolypeptides of the histidine-rich and NLS peptides. As such, this versatile approach should be suitable for generation of chemical libraries of copolypeptides consisting of a variety of bioactive peptide sequences. By use of the reducible copolypeptides, polyplexes sensitive to redox potential gradients can be formulated as promising gene delivery vectors. In comparison with control polyethylenimine, only minimum toxic effects of rCPPs are observed on the metabolic activity and membrane integrity of human endothelial cells. These findings are explained by a reductive intracellular and plasma membrane degradation and lower charge density and higher chain rigidity of the rCPPs than PEI. Transfection activity in all tested cell lines increases with increasing content of the HRP sequence in the rCPPs, while no clearly measurable effect of the NLS sequence is found. Overall, we believe that reducible copolypeptides possess a number of advantageous features to make them an attractive tool for gene delivery.

ACKNOWLEDGMENT This work was supported by NIH Grant CA109711. We thank David Bassett for help with FACS analysis and Harender Bisht and Yezi You for help with the polypeptide characterization.

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