Lipophilic Polycation Vehicles Display High Plasmid DNA Delivery to

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Lipophilic Polycation Vehicles Display High Plasmid DNA Delivery to Multiple Cell Types Yaoying Wu,†,‡ Adam E. Smith,†,∥ and Theresa M. Reineke*,† †

Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States Department of Chemical Engineering, University of Mississippi, 134 Anderson, University, Mississippi 38677, United States

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S Supporting Information *

ABSTRACT: A class of cationic poly(alkylamidoamine)s (PAAAs) containing lipophilic methylene linkers were designed and examined as in vitro plasmid DNA (pDNA) delivery agents. The PAAAs were synthesized via step-growth polymerization between a diamine monomer and each of four different diacid chloride monomers with varying methylene linker lengths, including glutaryl chloride, adipoyl chloride, pimeloyl chloride, and suberoyl chloride, which served to systematically increase the lipophilicity of the polymers. The synthesized polymers successfully complexed with pDNA in reduced serum medium at N/P ratios of 5 and greater, resulting in polyplexes with hydrodynamic diameters of approximately 1 μm. These polyplexes were tested for in vitro transgene expression and cytotoxicity using HDFa (human dermal fibroblast), HeLa (human cervical carcinoma), HMEC (human mammary epithelial), and HUVEC (human umbilical vein endothelial) cells. Interestingly, select PAAA polyplex formulations were found to be more effective than Lipofectamine 2000 at promoting transgene expression (GFP) while maintaining comparable or higher cell viability. Transgene expression was highest in HeLa cells (∼90% for most formulations) and lowest in HDFa cells (up to ∼20%) as measured by GFP fluorescence. In addition, the cytotoxicity of PAAA polyplex formulations was significantly increased as the molecular weight, N/ P ratio, and methylene linker length were increased. The PAAA vehicles developed herein provide a new delivery vehicle design strategy of displaying attributes of both polycations and lipids, which show promise as a tunable scaffold for refining the structure−activity−toxicity profiles for future genome editing studies. he field of genome editing is of high interest to the biomedical community due to the promise of providing selective therapeutics for a variety of untreatable genetic diseases.1−3 Significant clinical advances have been made in the field of in vivo and ex vivo gene editing therapies; however, optimization of delivery vehicles to provide safe and highly efficient delivery to multiple cell types is still desired to make further clinical progress with these therapeutic methods.4−6 Nonviral vectors have shown promise in circumventing several drawbacks associated with viral carriers, including immunogenicity and limited scale-up.5−10 Among the various classes of synthetic vehicles, cationic polymers and lipids have been extensively studied for nucleic acid complexation and delivery. 11−14 For example, one of the more popular commercial agents, Lipofectamine 2000,15 comprises a cationic lipid, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,Ndimethyl-1-propanaminium trifluoroacetate (DOSPA), and a neutral lipid, dioleoylphosphatidylethanolamine (DOPE).16 This vehicle forms liposomal vesicles; the electrostatic interaction between cations on the hydrophilic head groups and negatively charged nucleic acid drives lipoplex assembly upon mixing, where the nucleic acid can be subsequently associated with either the surface, interior, or both of the liposomal vesicles.17−19 The resulting lipoplexes bind to cell

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membranes mainly by a nonspecific electrostatic interaction and are then internalized by cells via endocytosis.17 It is hypothesized that the cationic lipids then destabilize the endosomal membrane, resulting in a flip-flop reorganization of phospholipids and the subsequent release of the cargo into the cytoplasm.8 Although Lipofectamine 2000 remains one of the most efficient transfection agents, significant research effort has been devoted to developing more efficient alternative systems to promote nucleic acid delivery due to the structural heterogeneity and toxicity of lipoplexes.12,20−22 Several research groups have taken advantage of lipophilicity to improve the efficiency of various classes of transfection agents, such as polyethylenimine (PEI) and poly(glycoamidoamines).16,23−26 For example, Bansal et al. have shown that the modification of PEI with lipophilic groups such as (4-bromobutyl)triphenylphosphonium bromide (BTP) grafted on linear PEI improved the cellular uptake and transfection efficiency (up to 3.6-fold higher in A549 cells and 7.1-fold higher in MCF-7 cells) of pDNA, as compared to linear PEI.23 Valencia-Serna et al. found that 1.2 kDa PEIs Received: June 1, 2017 Revised: June 27, 2017

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DOI: 10.1021/acs.bioconjchem.7b00306 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Scheme 1. Synthetic Scheme of Poly(alkylamidoamine)s (PAAAs) via Step-Growth Polymerization of a Series of Acid Chlorides with Tetra-tert-Butyloxycarbonyl Pentaethylhexamine

was incorporated to provide cationic charge (after deprotection) to enable binding the negatively charged phosphodiester backbone of the nucleic acid cargo. The PAAAs were synthesized via step growth polymerization between the oligoamine and one of four alkyl diacid chloride monomers (Scheme 1) according to our previously published procedures.31 A typical synthesis consisted of adding a dichloromethane (DCM) solution of one diacid chloride monomer (0.22 mmol/mL) into a DCM solution containing the Bocprotected oligoethyleneamine monomer (0.22 mmol/mL). The mixture was stirred for 3 h at room temperature. The Boc group was deprotected by adding 4 M HCl in dioxane into the solid residue after the DCM was evaporated. The polymers were purified via dialysis against ultrapure water to remove residual monomers and short oligos and then lyophilized to dryness yielding white solids. Eight total PAAA copolymers consisting of four structure types were synthesized to study the impact of lipophilicity (3, 4, 5, and 6 methylenes in the repeat unit) on pDNA delivery: poly(glutaramidopentaethylenetetramine) (Glut4), poly(adipamidopentaethylenetetramine) (A4), poly(pimelamidopentaethylenetetramine) (Pim4), and poly(suberamidopentaethylenetetramine) (Sub4). In addition, for two of the structure types, Glut4 and Sub4, three similar degrees of polymerization were created to examine the role of polymer molecular weight on in vitro transgene delivery performance. It should be noted that two other more lipophilic polycations were also synthesized using the diacid chloride monomers sebacoyl chloride (C8) and dodecanedioyl chloride (C10). However, after Boc deprotection, the subsequent polycations exhibited poor water solubility and were thus excluded from further study. The molecular weight (Mw) and dispersity index (Đ) of the eight polymer variants were characterized by size exclusion chromatography (SEC) using a TSK-GEL GMPWXL column and a Wyatt HELEOS II static light scattering (SLS) detector. The weight-average molecular weight (Mw), dispersity index (Đ), and degree of polymerization (DP) data are summarized in Table 1 (see Supporting Information, Figure S3 for SEC traces). It is worth noting that the relatively lower Đ values obtained for these step growth polymers is likely due to low reaction conversion and the dialysis purification of these polymers. Polyplexes were formed by mixing equal volumes of OptiMEM solutions containing pDNA and each of the eight PAAA polycation variants at various N/P ratios (amine group numbers/phosphate group number in pDNA backbone). The reduced serum medium was selected (instead of water) to formulate the polyplexes in order to optimize the cell viability

substituted with palmitic acid, a 16 carbon fatty acid, effectively delivered siRNA to K562 chronic myeloid leukemia cells and exhibited higher gene silencing efficiency than 25 kDa PEI.24 In addition, Kuchelmeister et al. conjugated lipophilic C18 tails onto two cationic peptide arms to form cationic molecular tweezers that shuttled DNA into HEK293T cells. The presence of the C18 tail significantly elevated the transfection efficiency of the molecular tweezers in HEK293T cells to a level comparable to Lipofectamine 2000.16 Previously, we developed poly(glycoamidoamine)s (PGAA)s and examined their transfection efficiency both in vitro and in vivo.27−29 PGAAs feature various types of carbohydrate groups that are copolymerized with oligoethyleneamines and have exhibited transfection efficiency comparable to that of PEI yet with significantly lower cytotoxicity.27 Recently, Anderson and co-workers have further modified PGAAs with various lengths of lipophilic alkyl side chains.26 The obtained PGAA brushes were combined with siRNA and mRNA to form nanoparticles using microfluidic techniques. These vehicles exhibited significant transfection efficiency in vivo, which demonstrated that the alkyl tails could improve the transfection performance of PGAA.26 In this current work, a family of linear poly(alkylamidoamine)s (PAAA)s have been created by copolymerizing diacid chloride monomers with varying methylene linker lengths, including glutaryl chloride (Glut), adipoyl chloride (A), pimeloyl chloride (Pim), and suberoyl chloride (Sub) with a protected pentaethylenehexamine monomer. Upon deprotection, as shown in Scheme 1, a family of lipophilic polycations (Glut4, A4, Pim4, and Sub4) were prepared and explored for their ability to deliver pDNA into a variety of cell types. This work aims to examine the hypothesis that adding lipophilicity to polycation-based nucleic acid delivery vehicles in a controlled fashion facilitates interaction between polyplexes and the cellular membrane and subsequently improves the delivery of plasmids to cultured cells (similar to lipid vehicles). This design principal also provides a tunable scaffold for refining the structure−activity−toxicity profile for future genome editing studies with this class of lipopolymers. To systematically investigate the impact of lipophilicity on pDNA delivery, a series of alkyl diacid chloride monomers with a number of methylene linkers systematically varying from 3 to 6 (e.g., glutaryl chloride, adipoyl chloride, pimeloyl chloride, suberoyl chloride, respectively) were purified via distillation prior to polymerization. The tert-butyloxycarbonyl (Boc) protected oligoethyleneamine monomer was synthesized according to our previously published procedure,30 and characterized via NMR and mass spectrometry (Supporting Information, Scheme S1, Figures S1 and S2). This monomer B

DOI: 10.1021/acs.bioconjchem.7b00306 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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various N/P ratios ranging from 10 to 80 in four cell types: HDFa (human dermal fibroblasts, adult), HeLa (human cervical carcinoma), HMEC (human mammary epithelial cells), and HUVEC (human umbilical vein endothelial cells). The four cell lines were also transfected with Lipofectamine 2000, which was used as the positive control for these assays. Polyplexes were prepared at N/P ratios between 10 and 80 to assess a large range of formulations and aid identification of maximal transfection conditions with all cell types and polymer variants. The transfections were carried out in Opti-MEM for 24 h, after which the media was replaced with complete growth media containing 10% serum. It should be noted that all polymers and controls were examined at the same concentrations and formulations in the cell lines to assess and compare the biological properties of this new polymer series. The percentage of GFP positive cells for each polymer at various N/ P ratios and control was determined via flow cytometry (Figure 1, columns) 48 h after transfection. Although the transfection efficiency level varied between cell types, all four analogues exhibited higher transfection ability than Lipofectamine 2000 for the cell lines tested. As shown in Figure 1, with all vehicles and controls, the delivery efficiency was highly cell-type dependent. With HDFa cells (Figure 1a), the delivery efficiency was low for the Lipofectamine 2000 control (90% cells positive for GFP expression with minimal toxic effects. The PAAAs with shorter linker lengths (Glut4−37 and A4−27) offered slightly lower transfection at low N/P ratios. However, at higher N/P ratios, transfection efficiency received a significant boost. PAAAs with longer linker lengths yielded ∼90% cells positive for GFP at most N/P ratios. Cell viability did tail off with increasing N/P ratios, which was particularly evident with the PAAAs containing the longer methylene linkers (e.g., Sub4−28). In HeLa cells, Lipofectamine was also similarly effective with low toxicity. Overall, the PAAAs and control were highly effective at promoting GFP transgene expression in HeLa cells with little toxicity. As shown in Figure 1c, with HMEC cells, the results were dramatically different. Most of the formulations appeared to yield effective transgene expression (over 50% of the cells exhibiting GFP expression). In general, transgene expression appeared to slightly increase with methylene linker length and N/P ratio. However, at higher N/P ratios of 60 and 80, expression tailed off in most formulations likely due to toxicity, which was also found to increase with increasing methylene linker length. Interestingly, polyplexes formulated with the Sub4−28 N/P = 60 formulation yielded GFP expression in almost 100% of cells, yet viability was significantly impacted at this condition (∼20% cells viable). One particularly promising formulation appeared to be Pim4−45 at an N/P ratio of 20; close to 80% of cells were positive for GFP expression with about 60% cell viability. In comparison to Lipofectamine 2000, only about 50% cells were positive for GFP and about 60% cell viability was found with this lipid control formulation. Thus, with HMEC cells, the PAAA formulations also exhibited promising delivery efficiency and transgene expression and the Pim4−45 formulation at N/P ratios 10−40 offered overall the highest transfection and cell viability. The transfection results with HUVEC cells also varied from those of the other cell types. Lipofectamine 2000 formulated with pDNA using the manufacturer’s recommended conditions was ineffective at promoting expression in this cell type, similar to the results found with this control in HDFa cells. Polyplexes formulated with the PAAAs were found to promote transgene expression with HUVEC cells and GFP expression increased with N/P ratio. Unfortunately, a significant increase in toxicity was also noticed with increasing N/P ratios. While some PAAAs appeared to be very efficient at promoting transgene expression, for example, Pim4−45 at N/P = 80 offered ∼100% GFP positive cells, viability was dramatically impacted (less than 20% cell survival) for these formulations. Moreover, HUVEC cell viability also decreased with the increase in methylene linker length. In general, polyplexes formed with A4−27 at N/P = 10 appeared to yield about 25% cells positive for GFP and full cell viability, which appeared to be the most promising formulation. To further understand the role of polymer length on transfection efficiency and cytotoxicity, three variants each of Glut4 (3 methylene groups per repeating unit) and Sub4 (6 methylene groups per repeating unit) were created at three similar degrees of polymerization of approximately 30, 40, and 60. Transgene expression and toxicity of the Glut4 and Sub4 variants were examined as a function of N/P ratio with the HeLa cell model. The study of delivery efficiency was further

Figure 2. Transfection efficiency of Glut4 and Sub4 series of polymers at various N/P ratios in HeLa cells. Error bars represent the standard deviation of analyzed data from three replicates. All raw data is available in Table S1 in the Supporting Information.

very high delivery efficiency where ∼80% cells positive for GFP were found. However, when toxicity of the vehicles were collectively compared, several trends emerged from this study (Figure 3 and Table S2 in the Supporting Information). First, it

Figure 3. Cytotoxicity profile of Glut4 and Sub4 series of polymers at various N/P ratios in HeLa cells. Error bars represent the standard deviation of analyzed data from three replicates. All raw data is available in Table S2 in the Supporting Information.

was apparent that as the molecular weight/length of the PAAA increased, the toxicity of the polyplex formulation also increased in HeLa cells. For instance, at an N/P ratio of 10, the percentage of cell survival dropped from 92% to 58% as the degree of polymerization of Glut4 increased from 30 to 57. This trend was consistently found for each N/P ratio tested with the Glut4 and Sub4 polymer series. Second, as the N/P ratio of polyplex formulation was increased for each length within the Glut4 and Sub4 polymer series, the toxicity clearly increased. Low N/P ratios yielded the highest cell survival D

DOI: 10.1021/acs.bioconjchem.7b00306 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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where ∼60−80% cell survival was found at an N/P ratio of 5. However, at an N/P ratio of 40, viability dropped off significantly (∼5−40%). Third, when comparing the Glut4 and Sub4 series at similar degrees of polymerization and N/P ratios, it was clear that the Sub4 polymers exhibited an overall higher cytotoxicity profile than Glut4 polymers. While this trend was not evident at the lowest N/P ratio of 5, when polyplexes were formed at higher N/P ratios such as 10, the longer length variants of the Sub4 vehicles (e.g., 43 and 65) exhibited lower cell survival (63% and 37%, respectively) than with Glut4 at similar lengths (82% for Glu4−40 and 58% for Glut4−57). Toxicity further increased dramatically with increasing N/P ratios when comparing the Glut4 and Sub4 polymer series. These results indicate that with HeLa cells, the shorter polymers at lower N/P ratios containing the lower methylene linkers result in high transgene expression while maintaining lower cytotoxicity with HeLa cells. Collectively, these results demonstrate that lipopolycations have the ability to promote effective expression of transgenes with multiple cultured cell types in a more effective manner than Lipofectamine 2000. While most PAAA formulations offered excellent transgene expression, toxicity was significantly impacted by increasing the molecular weight, N/P ratio, and methylene linker length. These results are consistent with previous work showing that lipophilic modification of PEI and other polymers can enhance membrane-disruptive properties and transgene expression of the delivery agents.33 However, a fine balance exists between enhanced membrane disruption and improving delivery efficiency with the unfortunate effect of increased cytotoxicity of these reagents.34,35 Overall, this work demonstrates that PAAAs show promise for future exploration of genome editing in a number of in vitro and ex vivo cell models, which is our current focus.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00306. All synthetic and cell culture procedures, NMR spectra, MS spectra, SEC traces, gel images, and LS data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Adam E. Smith: 0000-0003-1733-443X Theresa M. Reineke: 0000-0001-7020-3450 Present Address ‡

Department of Biomedical Engineering, Duke University, 101 Science Dr., Durham, North Carolina 27705, United States Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Young Joon Kim and Luis Ugozzoli at Bio-Rad, Inc. for their support of the flow cytometry experiments and the National Institutes of Health (NIH) Director’s New Innovator Award Program (DP2OD006669-01) and Bio-Rad, Inc. for financial support of this project. E

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Bioconjugate Chemistry (23) Bansal, R., Tripathi, S. K., Gupta, K. C., and Kumar, P. (2012) Lipophilic and cationic triphenylphosphonium grafted linear polyethylenimine polymers for efficient gene delivery to mammalian cells. J. Mater. Chem. 22, 25427−25436. (24) Valencia-Serna, J., Gul-Uludağ, H., Mahdipoor, P., Jiang, X., and Uludağ, H. (2013) Investigating siRNA delivery to chronic myeloid leukemia K562 cells with lipophilic polymers for therapeutic BCR-ABL down-regulation. J. Controlled Release 172, 495−503. (25) Rheiner, S., Rychahou, P., and Bae, Y. (2015) Effects of the lipophilic core of polymer nanoassemblies on intracellular delivery and transfection of siRNA. AIMS Biophys. 2, 284−302. (26) Dong, Y., Dorkin, J. R., Wang, W., Chang, P. H., Webber, M. J., Tang, B. C., Yang, J., Abutbul-Ionita, I., Danino, D., DeRosa, F., Heartlein, M., Langer, R., and Anderson, D. G. (2016) Poly(glycoamidoamine) brushes formulated nanomaterials for systemic siRNA and mRNA delivery in vivo. Nano Lett. 16, 842−848. (27) Liu, Y., and Reineke, T. M. (2005) Hydroxyl stereochemistry and amine number within poly(glycoamidoamine)s affect intracellular DNA delivery. J. Am. Chem. Soc. 127, 3004−3015. (28) Reineke, T. M. (2006) Poly(glycoamidoamine)s: Cationic glycopolymers for DNA delivery. J. Polym. Sci., Part A: Polym. Chem. 44, 6895−6908. (29) Ingle, N. P., Malone, B., and Reineke, T. M. (2011) Poly(glycoamidoamine)s: A broad class of carbohydrate-containing polycations for nucleic acid delivery. Trends Biotechnol. 29, 443−453. (30) Srinivasachari, S., and Reineke, T. M. (2009) Versatile supramolecular pDNA vehicles via “click polymerization” of βcyclodextrin with oligoethyleneamines. Biomaterials 30, 928−938. (31) Liu, Y., and Reineke, T. M. (2010) Degradation of poly(glycoamidoamine) DNA delivery vehicles: polyamide hydrolysis at physiological conditions promotes DNA release. Biomacromolecules 11, 316−325. (32) Won, Y.-Y., Sharma, R., and Konieczny, S. F. (2009) Missing pieces in understanding the intracellular trafficking of polycation/DNA complexes. J. Controlled Release 139, 88−93. (33) Piest, M., and Engbersen, J. F. J. (2010) Effects of charge density and hydrophobicity of poly(amido amine)s for non-viral gene delivery. J. Controlled Release 148, 83−90. (34) Grandinetti, G., Smith, A. E., and Reineke, T. M. (2012) Membrane and nuclear permeabilization by polymeric pDNA vehicles: Efficient method for gene delivery or mechanism of cytotoxicity? Mol. Pharmaceutics 9, 523−538. (35) Grandinetti, G., and Reineke, T. M. (2012) Exploring the mechanism of plasmid DNA nuclear internalization with polymerbased vehicles. Mol. Pharmaceutics 9, 2256−2267.

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DOI: 10.1021/acs.bioconjchem.7b00306 Bioconjugate Chem. XXXX, XXX, XXX−XXX