Pentalysine-Grafted ROMP Polymers for DNA Complexation and

Jul 30, 2008 - In the case of polymer 1e (average degree of polymerization of 206), protein expression levels 48 h post-transfection were found to be ...
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Biomacromolecules 2008, 9, 2495–2500

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Pentalysine-Grafted ROMP Polymers for DNA Complexation and Delivery Rebecca B. Breitenkamp and Todd Emrick* University of Massachusetts, Polymer Science and Engineering Department, 120 Governors Drive, Conte Center for Polymer Research, Amherst, Massachusetts 01003 Received May 8, 2008; Revised Manuscript Received June 20, 2008

Amphiphilic graft polymers, containing oligolysine groups pendent to a hydrophobic polycyclooctene backbone, were used to form polyplexes with plasmid DNA pZsGreen1-N1. These poly(cyclooctene-graft-pentalysine) structures were found to be effective transfection reagents for COS-1 and HeLa cells. In the case of polymer 1e (average degree of polymerization of 206), protein expression levels 48 h post-transfection were found to be comparable to, or better than, commercial transfection reagents jetPEI and SuperFect. With HeLa cells, GFP expression levels were better than Lipofectamine 2000. Of particular interest was the excellent cell viability seen in experiments with polyplexes formed from the pentalysine-grafted polymers. In the example of the highest molecular weight graft copolymer, polymer 1e, cell viability relative to untreated cells was 99% with COS-1 cells and 92% with HeLa cells in contrast to the commercial reagents, which gave 67-80% with COS-1 cells and 17-52% with HeLa cells. The effectiveness of these polyolefin-graft-pentalysine structures as DNA delivery vehicles is attributed to their amphiphilic nature and branched architecture.

Introduction Synthetic polymer materials tailored for biology have become critically important components of modern medicine.1,2 In gene delivery, nonviral polymer vehicles for DNA complexation and transfection are of increasing interest due to safety and immunogenicity concerns associated with virus-mediated delivery.3–6 Cationic lipids and polymers hold promise as safer delivery vectors, but these materials are generally less effective in transfection than viral systems and still carry some toxicity concerns due to their cationic nature.7 An ideal delivery vehicle would combine high gene expression with cell-specific entry and low toxicity. Recent studies along these lines are promising.8–11 The design of synthetic polymers for effective and safe transfection can benefit from consideration of both macromolecular architecture and functionality. DNA binding affinity, endosomal escape, and biocompatibility are key features in transfection that can be addressed by synthetic vectors. The binding affinity of DNA to cationic polymers is important as premature dissociation reduces cellular entry or leads to nucleic acid degradation in the extracellular environment or in the endosome. Studies of DNA interactions with functional polyamides and peptides provide insight into the utility of such interactions toward development of gene therapy reagents with optimal DNA binding affinity.9,12–15 Moreover, heterocyclic and secondary amines aid in endosomal buffering, promoting release from the endosome and increasing protein expression.16 The functionality upon which DNA-polymer complex (polyplex) interactions are based contributes to polyplex stability and efficiency of cellular entry as seen in studies of lysine, ornithine, histidine, and arginine-containing transfection reagents.10,17,18 While polylysine-DNA complexes perform transfection, they do so with low efficiency relative to reagents such as linear and branched polyethyleneimine (PEI)16 and polyamidoamine * To whom correspondence should be addressed. Phone: (413) 577-1613. Fax: (413) 545-0082. E-mail: [email protected].

(PAMAM) dendrimers.19 The recent introduction of branched and dendritic polymers as transfection reagents provides insight into the role of macromolecular architecture in transfection efficiency and cytotoxicity.16,19,20 A systematic study of linear versus branched lysine and histidine oligopeptides provides an example of the importance of both architecture and functionality in plasmid DNA and short interfering RNA (siRNA) complexation and transfection.10 Branched polymers gave smaller polyplexes while higher histidine-to-lysine ratios improved protein expression. Here we report our studies of cationic amphiphilic graft copolymers as DNA delivery vectors. These graft copolymers were prepared by ring-opening metathesis polymerization (ROMP) of cyclooctene macromonomers, giving a structure composed of a hydrophobic, polycyclooctene backbone with pendent oligolysine grafts (Figure 1). Nonionic, hydrophilic poly(ethylene glycol) (PEG) grafts were incorporated into copolymer structures by copolymerization of PEG-functionalized cyclooctene macromonomers. To evaluate the role of architecture in transfection, these amphiphilic graft copolymers were compared with linear polylysine (PLL) and commercial transfection reagents with respect to protein expression and cell viability following transfection.

Experimental Section Materials. COS-1 (African green monkey kidney cells, Cercopithecus aethiops) and HeLa (human adenocarcinoma epithelial cervical cells) cell lines were purchased from American Type Cell Culture (ATCC, Manassas, VA) or donated by the Schwartz Research Group (University of Massachusetts, Department of Biology). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, sodium pyruvate, and Lipofectamine 2000 were purchased from Invitrogen Corporation (Carlsbad, CA). Agarose (molecular biology grade, low-EEO), ethidium bromide (1% solution, molecular biology grade), tris-acetate-EDTA (TAE) buffer (10× solution, electrophoresis grade), agarose gel-loading dye (6×, contains 15% Ficoll 400, molecular biology grade), Costar white-walled 96-well plates,

10.1021/bm800511p CCC: $40.75  2008 American Chemical Society Published on Web 07/30/2008

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Figure 1. Materials used for transfection experiments.

Nalgene PES syringe filters (0.2 µm, 13 mm diameter), and low retention Eppendorf tubes were purchased from Thermo Fisher Scientific (Waltham, MA). Poly-L-lysine hydrobromide was obtained from Sigma-Aldrich (Saint Louis, MO). CellGro Eagle’s Minimum Essential Medium, trypsin with EDTA, and non-natural amino acid supplements were purchased from Mediatech, Inc. (Herndon, VA). Gel loading dye was purchased from Biorad (Hercules, CA). The jetPEI was received from PolyPlus Transfection (San Marcos, CA). EndoFree Plasmid Maxi Kit and SuperFect transfection reagent were obtained from Qiagen (Valencia, CA). DH5R cells were donated by Jeff Kane and Professor Larry Schwartz (University of Massachusetts, Department of Biology). Nuclease-free water and CellTiter-Glo Luminescent Cell Viability Assay were obtained from Promega Corporation (Madison, WI). Prior to the encapsulation and cell transfection experiments, the reporter gene pZsGreen1-N1, a GFP-expressing plasmid with 4700 base pairs, was purchased from Clontech (Mountain View, CA) and then amplified and purified. DH5R cells, Escherichia coli cells which had been prepared for transformation, were used as hosts for bacterial transformation according to standard molecular biology procedures.21 The amplified DNA was isolated and purified with a EndoFree Plasmid Maxi Purification kit. UV analysis (Pharmacia Biotech GeneQuant II) was used to determine the concentration and purity (260/280 ratio) of the amplified DNA stock solution prepared by bacterial transformation. Gel Electrophoresis. Agarose (0.48 g) was added to 1× TAE buffer (60 mL) to prepare a 0.8 wt % agarose gel. The solution was heated until boiling and apparent dissolution. After cooling for five minutes, 3 µL of 1 wt % ethidium bromide solution was added to the agarose solution. This mixture was poured into a gel cassette containing a 10well comb and allowed to set for 30 min.

To prepare the polyplex solutions, 1 µg of DNA was diluted in nuclease-free water to obtain a final volume of 10 µL. Separately, polymer solutions of various concentrations were prepared. A total of 10 µL of each polymer solution was added to the DNA solution and allowed to equilibrate for 30 min. A total of 2 µL of nucleic acid loading buffer was mixed with the polyplex solution, and 18 µL of the final solution was loaded onto the gel. The gel was run using a FisherBiotech horizontal minigel (7 × 10 cm) setup and an Invitrogen PowerEase500 power supply at 60 V for 60-90 min in 1× TAE buffer. The gel was then imaged with a Spectroline Slimline UV transilluminator equipped with a Canon Powershot A620 camera. Dynamic Light Scattering. Polyplexes were prepared in filtered, nuclease-free water in a black-walled 96-well plate with a total well volume of 100 µL each. Prior to analyses, the plate was centrifuged at 1000 rotations per minute for 2 min to remove excess air bubbles. The plate was inserted into a Wyatt DynaPro Plate Reader equipped with a temperature control module (set to analyze at 37 °C) and allowed to equilibrate for 10 min. Acquisition time was set to 60 s, and three runs were performed in each well, which were averaged together. The wells were analyzed every 6 h over a 24 h period. Cell Transfection Experiments. COS-1 and HeLa cells were cultured according to standard protocol, and low passage number cells (less than 20) were used for cell transfection experiments. COS-1 and HeLa cells were plated in white-walled 96-well plates at seeding densities of 7 × 103 cells per well and incubated in 5% CO2 at 37 °C 24 h prior to transfection. After 24 h, the cells were 70-80% confluent and were transfected as described below. The polymers and macromonomer (1-2 mg) were weighed into low retention Eppendorf tubes, dissolved in nuclease-free water, and sterilized by filtration. The stock solutions were diluted to the

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Figure 2. Polyplex characterization: (a) gel electrophoresis and (b) dynamic light scattering of polymer 1e-DNA complexes (N/P ratio 3).

Figure 3. Relative fluorescence (left) and cell viability (right) of COS-1 cells treated with polymers 1a-e at their optimal N/P ratio for protein expression. Fluorescence was measured 48 h post-transfection (plate reader filters λexc ) 485 nm, λem ) 520 nm).

Figure 4. Relative fluorescence (left) and cell viability (right) of COS-1 cells treated with polymers 1e and 2, deprotected macromonomer 3, linear PLL (DP 262), and commercial reagents jetPEI, SuperFect, and Lipofectamine 2000. Fluorescence was measured 48 h post-transfection (plate reader filters λexc ) 485 nm, λem ) 520 nm).

appropriate concentrations to enable complexation with DNA at various N/P ratios (i.e., the ratio of protonatable nitrogens in the polymer (N) to DNA phosphates (P)). DNA solutions (4× solution for each polymer concentration) were prepared in a 96-deep well plate. DNA (1 µg) was added to nuclease-free water (40 µL). A total of 40 µL of the diluted polymer solutions were added to the DNA solutions and allowed to equilibrate for a minimum of 30 min. Prior to transfection, the cells were washed with serum-free media. The polyplexes were diluted with 120 µL serum-free media (DMEM for COS-1 cells, MEM for HeLa cells), and 60 µL of the diluted polyplex solution was added directly to the cells. Each condition was repeated in triplicate. Commercial reagents PLL, jetPEI, SuperFect, and Lipofectamine 2000 were also used as transfection reagents according to each manufacturer’s recommended protocol. After 4 h of incubation at 5% CO2 and 37 °C, 120 µL of serumcontaining growth media (10% fetal bovine serum, 1% penicillin/ streptamyocin, 1% sodium pyruvate, and 1% non-natural amino acids) was added to each well. The cell media was replaced with 150 µL of fresh serum-containing growth media 24 h after transfection, and the cells were analyzed after 48 h by fluorescence microscopy (Olympus IX71 fluorescence inverted microscope equipped with an Olympus DP71 digital camera), by plate reader in fluorescence mode (BMG Labtech FLUOstar OPTIMA plate reader) and by the CellTiter-Glo

Luminescent Cell Viability Assay. Transfection data shown in Figures 3, 4, and 6 are averaged values obtained over multiple experiments. In each experiment, each unique condition (i.e., different polymer, N/P ratio, etc.) was tested in triplicate. Cell Viability Assay. To determine cell viability 48 h after transfection, Promega’s CellTiter-Glo Luminescent Cell Viability Assay was performed according to the recommended commercial protocol. The CellTiter-Glo buffer and substrate were equilibrated to room temperature, and the buffer was added to the substrate to form the CellTiter-Glo reagent. After equilibrating the 96-well plate to room temperature, the cell media was replaced with 70 µL of serum-free media, and 70 µL of the mixed CellTiter-Glo reagent was added to each well. The plate was mixed on an orbital shaker for 2 min and then allowed to sit for 10-20 min. Luminescence was recorded on a plate reader (BMG Labtech FLUOstar OPTIMA plate reader). The average luminescence values for each condition were divided by the average luminescence of the control cells (cells treated with media only) to determine the percent cell viability. As with the transfection data, cell viability results shown in Figures 3, 4, and 6 are averaged values obtained over multiple experiments. In each experiment, each unique condition (i.e., different polymer, N/P ratio, etc.) was tested in triplicate.

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Figure 5. Fluorescence microscope images (λexc ) 467-497 nm, λem ) 516-556 nm) of HeLa cells transfected with complexes of pZsGreen1N1 (GFP-expressing plasmid DNA) and various transfection reagents: polymer 1e (N/P 3), jetPEI, Superfect, and Lipofectamine 2000.

Results and Discussion Amphiphilic graft, or comb-like, structures, containing hydrophobic polycyclooctene backbones decorated with pendent oligolysines, were evaluated for their ability to facilitate DNA transfection into cells. ROMP of tert-butyloxy-protected (t-Boc) lysine and PEG-substituted cyclooctenes gave polymers 1 and 2, as shown in Figure 1. In polymer 1, prepared by ROMP of the Boc-protected 5-pentalysine-1-cyclooctene macromonomer (3), the pentalysine grafts are placed on average at every eighth backbone carbon atom. Macromonomer 3 was used in transfection experiments for comparative purposes. Polymer 2 integrates PEG grafts into the structure by copolymerization with a PEG1200-substituted cyclooctene macromonomer to reduce the charge density but maintain water solubility. The solid phase peptide synthesis of macromonomer 3 and the syntheses of polymers 1 and 2 are described in detail elsewhere.22 Polymer 1 was first studied for its ability to complex plasmid DNA. Nuclease-free aqueous polymer solutions were added to DNA solutions at various N/P ratios. After mixing for 30 min, the solutions were analyzed by agarose gel electrophoresis. For polymer 1, complete DNA complexation was obtained at N/P g 2, as indicated by the absence of free DNA bands at these ratios as shown in Figure 2 (representative data for polymer 1e shown). Dynamic light scattering was used to evaluate the size of the polymer-DNA complexes. As shown in Figure 2, polyplexes formed by complexation of plasmid DNA and polymer 1e are ∼80 nm diameter particles with relatively low size distribution. These polyplexes were found to very stable over a 24 h time frame in water; no aggregation or size change was observed by light scattering. Cell Transfection. Initial transfection experiments were performed with COS-1 cells. Polymers 1 and 2 of various molecular

weights (Figure 1) were complexed with the GFP-expressing reporter vector pZsGreen1-N1 and were analyzed 48 h posttransfection using a plate reader, fluorescence microscope, and the CellTiter-Glo Luminescent cell viability assay. For comparison, commercial transfection reagents jetPEI (PolyPlus Transfection), Lipofectamine 2000 (Invitrogen), and SuperFect (Qiagen) were also used for COS-1 transfection according to the optimized protocols supplied by the manufacturers. Cells transfected with polyplexes containing polymers 1b-e at their optimal N/P ratios demonstrated substantial protein expression and typically higher cell viability than those treated with the commercial reagents (Figure 3). The degree of polymerization (DP) of 1 proved important with increasing average DPs of 63, 75, 89, and 206 (polymers 1b, c, d, and e, respectively), providing better transfection efficiency than the lower molecular weight polymer 1a. Polymer 1a (average DP 30) showed almost no transfection when analyzed by fluorescence microscopy. In contrast, the highest molecular weight derivative polymer 1e showed greater overall gene expression than commercial transfection reagents jetPEI and SuperFect, but lower expression than Lipofectamine 2000, while maintaining excellent cell viability. Increased cytotoxicity with higher molecular weight polymer derivatives was not observed as 1e at the optimal N/P for transfection still maintained 99% cell viability in COS-1 cells. However, increased toxicity was observed at higher N/P as in the case of 1b. To obtain significant protein expression with this material, an N/P ratio of 4 was required, but resulted in only 77% cell viability. Polymers 1b-d also facilitated DNA transfection, as indicated by protein expression levels, but with lower effectiveness than 1e. When evaluated at comparable N/P ratios as polymer 1, pentalysine macromonomer 3 proved unsuitable for transfection

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Figure 6. Relative fluorescence (left) and cell viability (right) of HeLa cells transfected with polymer 1e (N/P 3), jetPEI, SuperFect, and Lipofectamine 2000. Fluorescence was measured relative to positive controls 48 h post-transfection (plate reader filters λexc ) 485 nm, λem ) 520 nm). Deprotected macromonomer 5-pentalysine-1-cyclooctene 3, polymer 2, and PLL were ineffective transfection reagents in HeLa cells (data not shown).

(Figure 4). This is in agreement with reports by Szoka and coworkers on the poor transfection performance of polyplexes formed from very short oligolysine segments, which require substantially higher N/P ratios to complex DNA.17 However, when 3 was polymerized to give polymer 1, the resulting material behaved much differently, thus demonstrating the benefit of the multiple interactions achievable with tailored macromolecules and, in this case, a graft or comb-like structure. Linear PLLs, containing on average 126, 219, and 262 lysine repeat units, were also analyzed in COS-1 transfection experiments. Despite having similar overall net charge as polymers 1a-c, which had on average 120, 254, and 301 net charges per polymer chain, these linear polymers exhibited minimal transfection ability. The highest molecular weight PLL (DP 262) showed the best transfection (Figure 4) but was still inferior to polymer 1 derivatives and the commercial reagents. The differing abilities of PLL versus polymer 1 as transfection reagents can be attributed to the role of macromolecular architecture (linear versus graft structure), as well as the amphiphilic nature of 1 with its hydrophobic backbone and hydrophilic oligolysine grafts. We found the PEG-containing cationic graft copolymers of type 2 to be inferior to 1 in transfection experiments in accord with literature reports on PEGylated cationic polymer transfection reagents.23–26 This can be attributed to several factors, including molecular weight (2 had an average DP of only 35), as well as poorer complexation due to a combination of increased steric hindrance and reduced charge density. Also, the presence of the PEG grafts may shield charge on the surface of the polyplexes, reducing cellular entry and protein expression. Although adding the PEG segments reduces the transfection in vitro, the incorporation of such groups would be advantageous in vivo for systemic delivery applications because PEG chains prevent nonspecific protein adsorption. Therefore, current efforts are focused on evaluating higher molecular weight derivatives of polymer 2 and determining an optimum PEGto-pentalysine graft ratio to maximize transfection efficiency for these structures. With HeLa cells, which are more difficult to transfect and more sensitive to toxicity than COS-1 cells, polymer 1e-based polyplexes compete favorably with commercial reagents. This is seen qualitatively in the fluorescence microscope images of the transfected cells in Figure 5. When polymer 1e served as the transfection reagent, little change in the cell morphology was observed, indicating minimal alteration to cell growth and minimal cytotoxicity. This contrasts the fluorescence images obtained in experiments with jetPEI, SuperFect, and Lipofectamine 2000, in which significant morphological alterations

and substantial cell death was observed. Figure 6 provides a quantitative comparison of 1e-containing polyplexes with commercial reagents in terms of GFP expression and cell viability. Even relative to Lipofectamine 2000, which outperformed all polymer 1 derivatives in the COS-1 experiments, 1e proved to be an excellent transfection reagent (relative fluorescence units for Lipofectamine 2000 ) 12000 vs 19000 for polymer 1e) while maintaining high levels of cell viability (17% for Lipofectamine 2000 vs 92% for polymer 1e). Synthetic polymer delivery vectors are known to be less toxic than lipid-based and other systems, but they typically do not reach comparable protein expression levels. However, 1e was found to be extraordinarily effective in facilitating DNA delivery while still maintaining excellent cell viability. The appeal of amphiphilic graft copolymer 1e as a transfection reagent can be attributed to its balance of transfection efficiency and biocompatibility, a balance which becomes increasingly important in vitro for sensitive cell lines as well as in vivo for systemic delivery. The described experiments indicate that the success of polymer 1e as a DNA delivery vector can be attributed to multiple factors including molecular weight, amphiphilicity, and branching. Polymer molecular weight is known to influence transfection performance as demonstrated with polymers 1a-1e and with other systems.27–30 The high molecular weight of 1e also provides additional stabilization to the polyplexes, preventing premature release of DNA and subsequent degradation.31 Studies by Schaffer, et al. with PLL derivatives demonstrate that intermediate molecular weight polymers are advantageous as delivery vehicles because they balance the need for polyplex stability with the requirement of eventual polyplex dissociation.28 In the case of high molecular weight PLL with a DP ∼180, the DNA was unable to dissociate from the delivery vector, preventing eventual protein expression. However, in our studies with the molecular weight series for polymer 1, rather than a decrease, a substantial increase in protein expression was observed with the highest molecular weight derivative 1e (DP ∼206). This implies that despite the large DP of 1e, which provides stability for the polymer-DNA complex, other features of this graft copolymer facilitate DNA release from the polyplex once it is within the cell. Ongoing intracellular trafficking studies will be reported in the future to better elucidate structureproperty relationships. Moreover, we note that amphiphilicity is characteristic of naturally occurring fusogenic peptide segments, which aid proteins in cellular entry and endosomal escape.32 Because 1e contains both hydrophobic and hydrophilic domains, this polymer vector may be considered to enhance cellular entry and endosomal escape in a manner related to fusogenic peptide

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action. In addition, the hydrophobic backbone of 1e might also interact with the hydrophobic domains of DNA, further stabilizing the polyplex. The role of branching in these structures must also be considered as contributing to their performance as delivery vectors. Pentalysine grafts are present on average at every eighth carbon, and the close proximity of the lysines within the graft and along the polymer backbone may lead to incomplete protonation of the structure. Studies by Suh et al. with PEI, hydrophobically modified PEI, and polyallylamine show that the continued protonation in a multiamine-containing compound suppresses additional protonation events.33 They also determined that hydrophobic environments decrease the propensity for protonation. Therefore, we speculate that in 1e the close packing of the lysine groups, as well as close proximity of the grafts to the hydrophobic backbone, may make for incomplete protonation that offers a buffering mechanism that might aid in endosomal escape in vitro. In summary, we have described a novel and effective transfection reagent based upon an amphiphilic cationic polyelectrolyte with a comb-like architecture. These polyplexes effectively transfect COS-1 and HeLa cell lines, while maintaining impressively high cell viability. These reagents are clearly competitive with commercial polymer-, dendrimer-, and lipidbased transfection reagents in terms of both protein expression and cell viability, and as such open possibilities for new transfection reagent choices for the drug and gene delivery communities. Current efforts seek to optimize the oligolysine graft length for DNA transfection and further understand the structure-property relationship of these unique materials. Acknowledgment. This work was supported by the Department of Defense (MURI Award) and the National Science Foundation-supported Materials Research Science & Engineering Center (MRSEC) on Polymers at UMass Amherst. The authors acknowledge helpful discussions and assistance with early experiments from Theresa Reineke and Yemin Liu (University of Cincinnati); Jeff Kane, Larry Schwartz, Sangram Parelkar, Kim Wojeck, and Kurt Breitenkamp (University of Massachusetts).

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References and Notes (1) (2) (3) (4) (5)

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