Highly Branched Poly(Î²-Amino Esters): Synthesis and Application in

Article pubs.acs.org/Biomac

Highly Branched Poly(β-Amino Esters): Synthesis and Application in Gene Delivery Lara Cutlar,†,§ Dezhong Zhou,*,†,§ Yongsheng Gao,† Tianyu Zhao,† Udo Greiser,† Wei Wang,†,‡ and Wenxin Wang*,†,‡ †

Charles Institute of Dermatology, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland School of Materials Science and Engineering, Tianjin University, Tianjin 300072, China

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Downloaded by NEW YORK MEDICAL COLLEGE on September 6, 2015 | http://pubs.acs.org Publication Date (Web): August 25, 2015 | doi: 10.1021/acs.biomac.5b00966

S Supporting Information *

ABSTRACT: Highly branched poly(β-amino esters) (HPAEs) are developed via a facile and controllable “A2+B3/B2” strategy successfully. As nonviral gene delivery vectors, the performance of HPAEs is superior to the wellstudied linear counterpart as well as the leading commercial reagent Superfect. When combined with minicircle DNA construct, HPAEs can achieve ultrahigh gene transfection efficiency, especially in keratinocytes.

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features.15,16 Vast efforts have clearly shown that finely tuning the structures can lead branched/dendritic polymers to be more efficient gene vectors compared to their linear counterparts;15 branched poly(ester amines)17 and polyesters18 have achieved higher gene transfection efficiency. Although several branched poly(amino esters) were developed via the “A2+BB′B″” and “A3 + 2BB′B” strategies19−21 previously, nevertheless, the synthesis was restricted to specialized monomers in which the functional groups are of unequal reactivity such as [1-(2-aminoethyl) piperazine, AEPZ], and some of the reactions were carried out under reduced pressure and at high temperature (140 °C) in the presence of a special catalyst [Al(OiPr)3]. The limited choice of starting monomers and complicated synthesis steps resulted in limited diversity of branched structure and difficulties in controlling the polymerization. Most importantly, the transfection efficiency of these polymers did not exceed the recombinant protein expression achieved by the standard controls (Lipofectamine, PEI, etc.). Therefore, to develop a novel type of highly branched poly(β-amino esters) (HPAEs) with high transfection efficiency and low cytotoxicity from various commercially available monomers via a facile synthesis process is of high importance to advance nonviral gene therapy to the clinic. Beyond the vector, the DNA cassette itself is also an important tool for enhancing recombinant protein expression. The multiple accessory sequences that allow for plasmid propagation have adverse effects in the human body.22 The bacterial or viral sequences hold the possibility of generating an immune response. In addition, any antibiotic resistance sequences used in production must be removed as the possibility of a transfer to the host is clinically unacceptable. To achieve this, multiple methods to generate minicircle (MC) DNA constructs were developed by removing the prokaryotic sequences with intra-

INTRODUCTION The identification of the causative factors of monogenic diseases has resulted in ebullient research efforts aimed at correction of these erroneous sequences. This need for an efficient, safe gene delivery vector to transport corrective nucleic acids has fueled multiple fronts of research. Despite progress in engineering viruses to increasingly benign vectors, they still have many safety and production concerns.1,2 In 2000, Langer and his colleagues first designed and synthesized linear poly(β-amino esters) (LPAEs) as gene vectors by the conjugate addition of amines to diacrylates in a one-step process.3 Following this pioneering work, Anderson et al. developed a library of over 2350 LPAEs and identified several high performance candidates relying on advanced synthesis and screening technology. Among these, the LPAEs based on a C32 backbone have specifically demonstrated highly favorable properties for gene transfection.4−7 The cationic nature allows them to form compact polyplexes with DNA,7 the terminal amine groups can enhance the interaction of polyplexes with cells effectively,8,9 and the polyester bond backbone can be hydrolytically degraded to smaller units under physiological conditions within hours to reduce the cytotoxicity.10 Most notably, the C32 backbone has generated promising results in vivo utilizing a murine model of prostate cancer.11 Members of the C32 backbone family that are end-capped with 1,3diaminopropane (designated C32-103) are found to be especially efficient in gene transfections of a wide range of mammalian cell types.8,12 However, the linear structure allows for only two end groups and thus prevents LPAEs from further performance enhancements. Branched/dendritic polymers compose one of the most important categories of gene delivery vectors; the commercially available branched polyethylene imine (PEI, Mw = 25 kDa)13 and dendritic Superfect14 have been widely used as positive controls in various studies. Previous studies have shown that branched/ dendritic polymers, due to their higher number of end groups and relatively high molecular flexibility, have many superior © XXXX American Chemical Society

Received: February 10, 2015

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DOI: 10.1021/acs.biomac.5b00966 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Synthesis of HPAEs via a “A2+B3/B2” strategy by conjugate addition of 4-amino-1-butanol (S4) to trimethylolpropane triacrylate (TMPTA) and bisphenol A ethoxylate diacrylate (BE), and then end-capped with 3-morpholinopropylamine (MPA).

molecular recombination resulting in a supercoiled minimally sized transgene cassette.23−25 The FDA recommends a supercoiled DNA fraction of over 80% for clinical applications (U.S. Food and Drug Administration, Guidance for Industry: Considerations for Plasmid DNA Vaccines for Infectious Disease Indications 2007). These MC DNA cassettes have shown a 10− 100-fold higher and more stable nonintegrative transgene expression while increasing the safety profile of gene therapy.23,26 However, to the best of our knowledge, MC for epidermal gene therapy has never been reported despite that epidermal cells, for example, the keratinocytes (NHK), play important biological roles. Some devastating skin diseases such as recessive dystrophic epidermolysis bullosa (RDEB) are due to the genetic defect of epidermal cells’ inability to produce a single protein.27 Previous studies have indicated that, after finely adjusting the polymer composition and structure to a three-dimensional architecture with multiple terminal groups, branched polymers can exhibit better transfection ability than their linear counterparts. We hypothesize that HPAEs would have similar molecular structure advantages in gene transfection compared to LPAE. Herein, we report the design and synthesis of multifunctional HPAEs as novel gene delivery vectors via a facile as well as controllable “A2+B3/B2” strategy using commercially available monomers. Triacrylate was employed in tandem with diacrylate to copolymerize with amines via Michael addition to produce two HPAEs whose structure and composition can be well controlled while in tandem effectively circumventing gelation.

The well-studied LPAE C32-103 was also synthesized as a positive control for comparison along with the standard commercial reagent Superfect to systematically illustrate the effectiveness of HPAEs in gene transfection. Meanwhile, a “large” plasmid construct (15.8 kbp, pRFP) and a “small” MC construct (3 kbp, MCGFP) were compared by transfecting keratinocytes, as well as cancer cells, to prove the potential application of HPAEs/MC DNA combinations in an epidermal gene therapy.

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EXPERIMENTAL SECTION

Materials. All chemicals used were purchased from Sigma-Aldrich unless otherwise stated. The minicircle production system used and the parental plasmids MN511A1 and MN512A1 were purchased from Systems Biosciences. Superfect and Alamar Blue were purchased from Invitrogen and Qiagen. Gaussia Luciferase Assay Kit was purchased from New England BioLabs and used according to the protocol. HPAE Synthesis and Characterization. As illustrated in Figure 1, to synthesize the linear PAE (LPAE, C32-103), 1,4-butanediol diacrylate (B4, 0.455 g, 2.3 mmol) and 5-amino-1-pentanol (S5, 0.206 g, 2 mmol) were dissolved in DMSO (1 mL) and reacted at 90 °C. Then the mixture was cooled to RT and diluted to 100 mg mL−1 with DMSO. 1,3-Diaminopropane (DA, 288 μg, 2 mmol) was added to end-cap the acrylate terminated base polymer at RT for 24 h. After that, the product was precipitated into diethyl ether, dried under vacuum for 24 h, and then stored at −20 °C. To synthesize HPAEs, bisphenol A ethoxylate (BE) and 4-amino-1-butanol (S4) were chosen as functional monomers which were expected to bring transfection efficiency, trimethylolpropane triacrylate (TMPTA) was used as branching monomer which was B


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LPAE were dissolved in DMSO to 100 mg mL−1 stock solution, and then according to polymer/DNA weight (w/w) ratio, the solution was further diluted with commercially available sodium acetate buffer (pH 5.2, 0.025 M), under which conditions the PAEs can be well dissolved as reported in previous studies.8−12 The amount of 0.25 μg of DNA per well was employed. Weight/weight ratios of 10:1, 20:1, 30:1 were chosen for all the transfections. HPAE solution was added into DNA, vortexed, allowed to complex for 10−15 min, and then diluted with media containing 10% FBS to 100 μL. The media in the wells of cell culture plates were removed quickly, and the diluted polyplexes solution was added. The commercial transfection agent Superfect was used as per protocol. After 4 h, the media were replaced with 100 μL of fresh complete media and cells were cultured for another 44 h. Reporter Protein Expression. Green fluorescent protein of the MCGFP and red fluorescent protein of the pRFP were used for the transfection as mentioned above. Transfected cells were monitored and imaged on an Olympus IX81 inverted fluorescent microscope 48 h post transfection. Gaussia Luciferase Expression. Plasmid GLuc was used and transfection was carried out as above. The amount of Gaussia luciferase protein expressed in each well was measured by luminescence intensity 48 h post transfection. All the measurements were performed exactly as outlined by guidelines of the manufacture. The luciferase activity in the cell supernatant was directly plotting it in terms of relative light units (RLU). Results were obtained as the mean ± SD from quadruplicate values. Alamar Blue Assay. The metabolic activity of transfected cells was measured with Alamar Blue assay. The cells were washed with HBSS after the supernatant was removed, followed by addition of 100 μL of 10% Alamar Blue in HBSS. Two hours later, the Alamar Blue solution from each well was transferred to a fresh flat bottomed 96-well plate for fluorescence measurements at 590 nm. The fluorescence values of untreated cells were plotted as 100% viable. Results were obtained as the mean ± SD from quadruplicate values. Polyplex−Cell Interaction. Gluciferase plasmid was labeled with Cy3 fluorescent dye according to protocols. HeLa and RDEBK cells were seeded in 96-well plates at a density of 5000 cells per well. Polyplexes preparation and transfection were carried out as mentioned above. Four hours post transfection, cells were washed with HBSS buffer, fixed with paraformaldehyde, and then stained with DAPI and viewed under microscope; images were taken at 40× magnification. Flow Cytometry Measurement. RDEBK cells were seeded in T25 flasks at a density of 3500 cells/cm2. HPAE-2 and MCGFP were used to prepare the polyplexes. Transfection was carried out as mentioned above. An amount of 10 μg or 30 μg of MCGFP was used for each flask, respectively. After 48 h, cells were washed with HBSS and collected. Draq7 was used to gate out dead cells, and GFP positive singlet events were counted. At least 10 000 cells were counted. Cells without any treatment were used as control. Polymer Degradation and Corresponding Cytotoxity Tests. HPAE-1, HPAE-2, and LPAE were dissolved in sodium acetate buffer (pH = 5.2, 0.025 M) at a concentration of 10 mg mL−1. The solutions were kept stirring at 37 °C. At the time points of 6, 21, 30, and 48 h, 1 mL of solution was taken out and freeze-dried immediately and then dissolved in DMF and filtered with a 200 nm filter. GPC tests were carried out as above. The percentage of degradation was defined as the molecular weight of the degraded polymers divided by the molecular weight of the original polymer. To test the cytotoxicity, degraded products (from 48 h degradation) were diluted with sodium acetate to 1 mg mL−1, and then required amount of solution was directly added into RDEBK cell culture media in the plates. After 24 h, Alamar Blue assays were carried out as mentioned above.

expected to give rise to highly branched structures. In order to compare the effect of different branched structures on gene transfection efficiency and safety, two HPAEs named HPAE-1 and HPAE-2 were synthesized by utilizing different monomer feed ratios of TMPTA to BE. With the higher feed ratio of TMPTA to BE, HPAE-1 was expected to exhibit more branched structure than HPAE-2. For synthesis of the highly branched HPAE-1, TMPTA (0.346 g, 1.17 mmol), BE (0.181 g, 0.39 mmol), and S4 (0.214 g, 2.4 mmol) were dissolved in 7.15 mL of DMSO, and the reaction occurred at 90 °C for 48 h. MPA (288 μg, 2 mmol) was dissolved in 2.88 mL of DMSO and then added to end-cap the acrylate terminated base polymer at RT for 24 h. The following day the polymer product was precipitated into diethyl ether, as above dried under vacuum for 24 h and then stored at −20 °C. The synthesis of the highly branched HPAE-2 followed the same protocol. Briefly, TMPTA (0.284 g, 0.95 mmol), BE (0.29 g, 0.62 mmol) and S4 (0.178 g, 2 mmol) were dissolved in 7.52 mL of DMSO, reacted at 90 °C; end-capping and precipitation were performed as above. Gel Permeation Chromatography (GPC). Molecular weight (Mw and Mn) and polydispersity index (PDI) of HPAEs were measured by GPC. A volume of 50 μL of PAE reaction solution was collected, diluted with 1 mL of DMF, filtered with a 0.2 μm filter, and then measured on a PL-GPC 50 Integrated GPC system equipped with a refractive index detector (RI), a viscometer detector (VS DP), and a dual angle light scattering detector (LS 15° and LS 90°) at 50 °C with DMF (plus 0.1% LiBr) as elution solution at a flow rate of 1 mL min−1. Proton Nuclear Magnetic Resonance (1H NMR) Measurements. Chemical structure and composition of the HPAEs were confirmed by 1H NMR. The HPAEs were dissolved in deuterated chloroform (CDCl3), and measurements were carried out on a Varian Inova 500 MHz spectrometer and reported in parts per million (ppm) relative to the response of the solvent (7.24 ppm) or to tetramethylsilane (0.00 ppm). Gel Electrophoresis. For DNA condensation study, 1 μg of DNA was used for each sample preparation. Polyplexes were first prepared by adding the required amount of HPAE solution into the DNA solution, mixing and incubating for 10 min. Then the loading dye was added, and polyplexes were loaded into the wells in agarose gel (1%) containing SYBR Safe DNA stain. Electrophoresis was carried out at 120 mV for 30 min. For DNA protection study, polyplexes were prepared as above, and then DNase was added according to protocols. Polyplexes were incubated at 37 °C for 15 min. After that, EDTA solution was used to stop the activity of the DNase and then polyplexes were subjected to gel electrophoresis as mentioned above. Polyplex Sizes. An amount of 2 μg DNA was used for each sample preparation. Polyplexes were first prepared by adding the required amount of HPAE solution into the DNA solution, mixing, and incubating for 10 min. Then the polyplexes were diluted with 1 mL of DMEM with 10% FBS. Measurements were carried out on a Malvern Instruments Zetasizer (Nano-2590) instrument with a scattering angle of 90°. Four hours later, sizes of the polyplexes were measured again to evaluate the polyplex stability. Measured sizes are presented in the Results and Discussion as the average values of 4 runs ± standard deviations (SD). Plasmid DNA Production and Minicircle Induction. E. coli XL10 ultracompetent Gold cells (Stratagene) were transformed with the large plasmid and grown in LB media supplemented with 100 μg/mL ampicillin for positive selection. The large plasmid (15.8 kb) was purified by using an endotoxin-free Giga-plasmid preparation kit (Qiagen, West Sussex, UK). The parental plasmid MN511A1 (Systems Biosciences) was induced to produce the GFP expressing MC DNA by standard protocol. Cell Culture. All cells were cultured at 37 °C, 5% CO2 in a humid incubator. The type VII collagen null-RDEB keratinocytes-RDEB-TA4 (RDEBK) were kindly provided by Dr F. Larcher (Madrid). The NHK and RDEBK cells were cultured in Keratinocyte Growth Medium 2 (C20011 Promocell). The HeLa cells were cultured in DMEM with 10% serum. Transfection Experiments. Cells were seeded in 96-well plates at a density of 1 × 104 cells/well in 100 μL of media and cultured until 70− 80% confluence. For polyplex preparation, HPAE-1, HPAE-2, and

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RESULTS AND DISCUSSION HPAEs were synthesized in a one-pot process. Acrylate terminated base polymers were first prepared by copolymerization of trimethylolpropane triacrylate (TMPTA), diacrylate bisphenol A ethoxylate (BE), and 4-amino-1-butanol (S4). Then functional 3-morpholinopropylamine (MPA) was used to endC


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cap the base polymers to result in the HPAE-1 and HPAE-2 (Figure 1) with backbone structures of esters and tertiary amine groups. The choice of the monomers, particularly the diacrylate with bisphenol A, was based on two considerations: first, previous reports on LPAEs have shown that the combination of BE and MPA, especially diacrylate with “hydrophobic” bisphenol A groups, can bring ultrahigh gene transfection efficiency;28 second, preliminary synthesis studies have shown that the monomer combination of BE, TMPTA, and S4 can lead to wellcontrolled (in terms of both molecular weight and composition) and highly branched poly(β-amino esters) without gelation. To assess the effect of functional monomers BE, S4, and branching monomer TMPTA on the gene transfection performance of HPAEs, two monomer feed ratios (BE/TMPTA = 1:2.4 and 1:1.2) were used to produce HPAE-1 and HPAE-2 with different compositions and highly branched structures. The LPAE, linear structured C32-103 was also synthesized according to previous work8,29 (Figure S1) and treated as benchmark to evaluate the gene transfection performance of the HPAEs.11 Structures of HPAE-1 and HPAE-2 were first confirmed by gel permeation chromatography (GPC). GPC traces of HPAEs are shown in Figure 2a. HPAE-1 and HPAE-2 had weight molecular weights

H NMR analysis further verified that, by simply varying the feed ratios of triacrylate (TMPTA) to diacrylate (BE), besides the branched structures, the compositions of HPAEs can also be easily adjusted (Figure 3 and Table 1). All these results indicate

Figure 3. 1H NMR spectra analysis of HPAE-1 and HPAE-2. Functional groups are assigned to the corresponding signal peaks on the spectra, respectively: 0.8 (s, CH3CH2−), 1.3−1.7 (m, CH3CH2−, −C(CH3)2−, −NHCH2CH2CH2N−, and −NCH2(CH2)2CH2OH), 2.3−2.6 (m, −NCH 2 CH 2 COO−, −COCH 2 CH 2 N−, −NHCH 2 CH 2 CH 2 N−, −COCH2CH2NH−, and −NHCH2CH2CH2N(CH2)2), 2.7−2.9 (m, −NCH2(CH2)2CH2OH and −NHCH2CH2COO−), 3.2−3.9 (m, −NCH2COO−, −NCH2CH2O−, and −NCH2(CH2)2CH2OH), 4.0− 4.5 (m, −O(CH2)2O−, −COOCH2C−, and −NCH2CH2COO−), 6.8 (d, −O−C(CH)2CH−), 7.1 (d, −O−C(CH)2CH−).

Table 1. Monomer Feed Ratios and Compositions of HPAE-1 and HPAE-2 feed ratio

HPAE-1 HPAE-2

composition

[BE]/ [TMPTA]a

Mw (Da)b

Mn (Da)b

PDIb

[BE]/ [TMPTA]c

αd

1:2.4 1:1.2

8553 8918

2621 3426

3.25 2.60

1:2.9 1:1.5

0.31 0.34

a

Reaction condition: DMSO as solvent; monomer concentration, 100 mg mL−1; temperature, 90 °C; end-capping condition, MPA, 0.2 mM, RT, 24 h. bDetermined by RI detector, PDI = Mw/Mn. cCalculated from 1H NMR spectrum dThe Mark−Houwink exponent, determined by VS DP detector

that the HPAEs of potentially various structures and compositions can be synthesized in a controlled manner via the facile “A2+B3/B2” strategy providing great flexibility to develop vectors of diverse properties, functionalities and thus performance in gene delivery. The LPAE has Mw of 7548 Da with PDI of 2.06, and the composition was also verified by 1H NMR (Figure S2). The different structures of HPAE-1 and HPAE-2 form the basis of a systematic comparison for their different performances in gene delivery in terms of safety and transfection efficiency, parameters including DNA condensation, polyplex size and stability, polyplex−cell interaction, vector post-transfection degradation, and cytotoxicity of the degraded products were further investigated to the enhance the mechanistic understanding.

Figure 2. (a) GPC traces of HPAE-1 and HPAE-2; (b) Mark−Houwink (MH) plots of HPAE-1 and HPAE-2.

(Mw) of 8553 and 8918 Da with PDIs of 3.25 and 2.60, respectively. The Mark−Houwink (MH) exponent alpha (α) values of HPAE-1 and HPAE-2 were 0.31 and 0.34 (Figure 2b), respectively, indicating typical highly branched structures, which were derived from the combination of branching segments formed by S4/TMPTA and linear segments formed by S4/BE. D


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DNA condensation and protection ability of HPAEs were evaluated first with gel electrophoresis. A range of polymer/DNA weight ratios (w/w) from 10:1 to 60:1 was investigated. It can be seen that, over the w/w ratio range, except at the lowest w/w ratio of 10:1 for HPAE-1, the HPAEs can condense DNA completely without any DNA migration (Figure 4a). The strong

Figure 4. (a) DNA condensation capacity of HPAE-1 and HPAE-2 at w/w ratio ranging from 10:1 to 60:1 (from left to right); (b) DNA protection properties of HPAE-1 and HPAE-2.

DNA binding ability of HPAE-1 and HPAE-2 is due to the protonation of the multiple tertiary amines on their backbones. At the same time, HPAE-1 (the α value of the Mark−Houwink plot was 0.31) exhibited more branched structure than HPAE-2 (the α value of the Mark−Houwink plot was 0.34), which resulted in more compact DNA/HPAE polyplexes (which can also be evidenced by the relatively smaller DNA/HPAE-1 polyplex sizes shown in Figure 5). Therefore, it was relatively more difficult for the SYBR Safe DNA stain dye to gain access to the DNA condensed by HPAE-1 compared to that condensed by HPAE-2. Thus, HPAE-1 exhibited better DNA shielding ability compared to HPAE-2. The ability of HPAEs to protect DNA from degradation by enzyme (DNase) was further investigated (Figure 4b). It can be seen that, at the w/w ratio of 10:1, all the three polymers cannot protect the DNA effectively featured by the obvious DNA segments from the degradation. With the w/w ratio increased to 20:1, partial DNA in the LPAE/DNA polyplexes still degraded by DNase. In contrast, under the same w/w ratio, HPAE-1 and HPAE-2 showed complete DNA protection and all the DNA in the polyplexes is retained. Previous studies by Singh and Nah et al. indicated that the increase in hydrophobicity by introducing hydrophobic molecules such as hexamoic acid and deoxycholic acid would increase the DNA protection ability of polycations.30−33 Here, the backbone of LPAE was composed of B4 units and S5 units, and the terminals were 103 units (a water-soluble molecule). Whereas the backbones of HPAEs were composed of TMPTA/BE units and S4 units, and the terminals were MPA units (a water insoluble molecule). HPAEs were relatively more hydrophobic than LPAE. Therefore, the slight difference in DNase protection is possibly derived from the relatively different hydrophobicity between the HPAEs and LPAE.34 As size is one of the leading issues for cell membrane uptake, hydrodynamic diameters of polylexes formed from HPAE-1 and HPAE-2 with DNA were measured. As shown in Figure 5a, in DMEM cell culture media with 10% FBS, the HPAE-1/DNA and HPAE-2/DNA polyplexes were seen to be very small with diameters generally below 100 nm, and the PDI of all the polyplexes was below 0.18 as shown in Figure S3a. These results

Figure 5. (a) Sizes of HPAE/DNA and LPAE/DNA polyplexes in DMEM with 10% FBS; (b) sizes of HPAE/DNA and LPAE/DNA polyplexes after incubation in DMEM with 10% FBS for 4 h.

indicate that both HPAEs can assemble with DNA to form small polyplexes without obvious aggregation even in the presence of serum. Stability of the polyplexes was further tested. After 4 h of incubation in the presence of serum, sizes of the HPAE/DNA polyplexes were still below 110 nm without obvious increase (Figure 5b). PDI of the polyplexes increased slightly but was still below 0.23 as shown in Figure S3b. This means that HPAE/DNA polyplexes were very stable. Notably, since HPAE-2 had slightly weaker DNA condensation ability, the formed HPAE-2/DNA polyplexes were greater in size and so not as compact as the HPAE-1/DNA polyplexes (which can also be evidenced by the slightly different DNA shielding ability in Figure 4). As time progressed, the DNA in the polyplexes were further condensed by HPAE-2, and the relatively looser HPAE-2/DNA polyplexes became more compact, which resulted in their slight decrease in size after 4 h incubation. The features of small diameter and good stability would be beneficial for the HPAE/DNA polyplex uptake and thus improve their gene transfection efficiency. To illustrate the biological compatibility and transfection efficiency of HPAEs, transfections were carried out with HeLa, keratinocytes (NHK) and recessive dystrophic epidermolysis bullosa keratinocytes (RDEBK) cells. Figure 6a shows the difference influences of HPAEs on cell metabolic activity. Across all the three cell types, LPAE preserved very high cell viability even at high w/w ratio of 30:1. At the w/w ratios of 10:1 and 20:1, both HPAE-1 and HPAE-2 preserved very high cell viability. Comparatively, at the highest w/w ratio of 30:1, HPAEs preserved slightly lower cell viability but still with metabolic health levels above 75%. These results in combination with the sizes indicate that the HPAE-1 and HPAE-2 can effectively E


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Figure 6. (a) Proliferative capacity of HeLa, NHK, and RDEBK cells after transfection with HPAE-1, HPAE-2, LPAE, and commercially available Superfect determined by Alamar Blue assays; (b) gluciferase activity of Hela, NHK, and RDEBK cells after transfection at various w/w ratios. *Significant difference from the SuperFect (p < 0.05 Student’s two-tailed t test).

condense DNA to nanoscaled polyplexes while not induce severe cytotoxicity and results in safe gene delivery vectors. After transfection with HPAE-1 and HPAE-2, the gluciferase activity in HeLa cells was very high, especially at the high w/w ratio of 30:1 (Figure 6b), and overall HPAE-2 exhibits higher transfection efficiency than HPAE-1 across the w/w ratio range. In contrast, although showing similar properties to HPAE-1/ DNA and HPAE-2/DNA including DNA condensation and protection, sizes, and stability, LPAE/DNA polyplexes showed relatively much lower transfection efficiency. The gluciferase activity of HeLa cells after transfection by LPAE was 2−3 orders of magnitude lower than that of HPAE-1 and HPAE-2. Moreover, HPAE-1 and HPAE-2 also exhibited ultrahigh transfection capability specifically to the keratinocyte cell lines NHK and RDEBK; the gluciferase activity from both after transfection was as much as 10 00-fold higher than that of the LPAE. This superiority in keratinocytes points to HPAE-1 and HPAE-2 as ideal candidates, in terms of both safety and efficiency, for the treatment of epithelial conditions. To further investigate the different transfection performances of HPAE-1, HPAE-2, and LPAE in different cell types, Cy3 labeled DNA was used for transfection. In HeLa cells, it clearly shows that the polyplexes formed by HPAE-1 and HPAE-2 with DNA have strong interaction with cells, evident by many polyplexes around the nucleus (Figure 7a).13 This correlated very well with the fact that HPAE-1 and HPAE-2 have very high transfection capability to HeLa cells. For RDEBK cells, HPAE-1/ DNA and HPAE-2/DNA polyplexes also had strong interactions with cells, featured by the great intensity of the Cy3 fluorescence from the polyplexes inside the cells (Figure 7b). In contrast, the interaction of LPAE/DNA polyplexes with both HeLa and RDEBK cells was relatively weaker. This indicates that the different transfection capability of HPAEs and LPAE was possibly due to the different polyplex/cell interactions.

Figure 7. Interaction of HPAE-1/DNA, HPAE-2/DNA, and LPAE/ DNA polyplexes with HeLa cells (a) and RDEBK cells (b).

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Figure 8. Fluorescence images of green and red reporter proteins from a small MC construct MCGFP and a large construct pRFP.

Figure 9. Flow cytometry histogram profiles and data of MCGFP expression from RDEBK 4 days post transfection with HPAE-2.

The transfection capability of HPAE-1 and HPAE-2 with a “large” plasmid DNA construct (pRFP) and a “small” construct (MCGFP) was further determined and compared. The size difference between these two DNA constructs is in the range of 12−13 kb (Figure S4). The fluorescence images showed a

significant difference between MC and plasmid DNA reporter gene expression over NHK and RDEBK cells (Figure 8). The MCGFP was expressed by many more individual cells regardless of cell type and both GFP and RFP expression were driven by the same promoter. This was in agreement with previous studies as G


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the uptake of the smaller minicircle DNA was easier for the cells, in both accelerating DNA diffusion through the cell cytoplasm and increasing the potential transgene copy number within the nucleus.26 Based on these results, it is conceivable that combination of high performance vector and MC DNA construct can achieve ultrahigh transfection efficacy in a clinical setting. Therefore, HPAE-2 was further combined with MCGFP to transfect RDEBK cells; transfection efficiency was quantitatively determined by flow cytometry. Unsurprisingly, 4 days after transfection, an average of 74.5% of cells were expressing GFP with 10 μg DNA treatment, and 93.1% after 30 μg (Figure 9) while 91.3 ± 4.1% and 85.6% ± 2.3% cell viability were preserved, respectively. Meanwhile, the fluorescence intensity of transfected cells was significantly enhanced by the increase of MCGFP dosage. HPAE-1 and HPAE-2 have demonstrated a much higher transfection capability compared to LPAE in the keratinocytes cell lines, this was illustrated with a Gluciferase plasmid and a “large” RFP plasmid; notably when combined with the MC DNA construct previously unattained heights of recombinant expression were achieved. Finally, HPAEs post-transfection degradation and cytotoxicity of the degraded products were investigated. A key feature of LPAE is their relatively rapid biodegradability into small, noncytotoxic products, which improves the long-term safety of the vectors.6−9,11,12 Within 48 h, both HPAE-1 and HPAE-2 can degrade into oligomers (Figure S5). Moreover, the degraded components of HPAE-1 and HPAE-2 were noncytotoxic. As shown in Figure S6, even at a concentration of 300 μg per mL, the cell viability remains above 95%. The good degradability and nontoxic degraded polymer fragments imply HPAEs have applications as safe gene delivery vectors, especially for repeat or multiple administrations in gene therapy.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by Science Foundation Ireland (SFI), Grant Number 10/IN.1/B2981(T) (SFI Principal Investigator Program), Grant Number 14/TIDA/2367 (Technology Innovation Development Award), Health Research Board of Ireland, University College Dublin, and DEBRA Ireland. This work could not have been performed without the generous donations of RDEB cells from Dr. F. Larcher. We would also like to acknowledge the very kind technical support of Dr. O. Carroll at the Network of Excellence for Functional Biomaterials (NFB), National University of Ireland, Galway and Dr. Alfonso Blanco of the Conway Flow Cytometry core, University College Dublin.

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CONCLUSION To summarize, multifunctional HPAEs were developed via a facile and controllable “A2+B3/B2” strategy as nonviral gene delivery vectors. The highly branched structure is critical to achieve high safety and efficiency in gene transfection. HPAEs preserve high cell viability and lead to the expression of significant levels of recombinant protein expression over a range of cell types regardless of the sizes of the DNA construct. In particular, HPAEs can reach ultrahigh transfection efficiency for keratinocytes when combined with the MC construct. Moreover, HPAEs are degradable and the degraded products are nontoxic. The low cytotoxicity and high efficiency infer HPAEs hold great potential for application in multiple fields, particularly in epithelial gene therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00966. Synthesis of the LPAE, DNA gel, NMR data, polymer degradation and cytotoxicity of degraded products (PDF)

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REFERENCES

(1) Wirth, T.; Parker, N.; Ylä-Herttuala, S. Gene 2013, 525, 162−9. (2) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Nat. Rev. Genet. 2014, 15, 541−555. (3) Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122, 10761−10768. (4) Kang, H. C.; Lee, M.; Bae, Y. H. Crit. Rev. Eukaryotic Gene Expression 2005, 15, 317−342. (5) Green, J. J.; Langer, R.; Anderson, D. G. Acc. Chem. Res. 2008, 41, 749−759. (6) Lächelt, U.; Wagner, E. Front. Chem. Sci. Eng. 2011, 5, 275−286. (7) Luten, J.; van Nostrum, C. F.; De Smedt, S. C.; Hennink, W. E. J. Controlled Release 2008, 126, 97−110. (8) Green, J. J.; Zugates, G. T.; Tedford, N. C.; Huang, Y. H.; Griffith, L. G.; Lauffenburger, D. A.; Sawicki, J. A.; Langer, R.; Anderson, D. G. Adv. Mater. 2007, 19, 2836−2842. (9) Akinc, A.; Thomas, M.; Klibanov, A. M.; Langer, R. J. Gene Med. 2005, 7, 657−63. (10) Anderson, D. G.; Akinc, A.; Hossain, N.; Langer, R. Mol. Ther. 2005, 11, 426−434. (11) Anderson, D. G.; Peng, W.; Akinc, A.; Hossain, N.; Kohn, A.; Padera, R.; Langer, R.; Sawicki, J. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 16028−16033. (12) Anderson, D. G.; Lynn, D. M.; Langer, R. Angew. Chem., Int. Ed. 2003, 42, 3153−3158. (13) Zhou, D.; Li, C.; Hu, Y.; Zhou, H.; Chen, J.; Zhang, Z.; Guo, T. Chem. Commun. 2012, 48, 4594−4596. (14) Newland, B.; Zheng, Y.; Jin, Y.; Abu-Rub, M.; Cao, H.; Wang, W.; Pandit, A. J. Am. Chem. Soc. 2012, 134, 4782−4789. (15) Newland, B.; Aied, A.; Pinoncely, A. V.; Zheng, Y.; Zhao, T.; Zhang, H.; Niemeier, R.; Dowd, E.; Pandit, A.; Wang, W. Nanoscale 2014, 6, 7526−7533. (16) Zhou, Y.; Huang, W.; Liu, J.; Zhu, X.; Yan, D. Adv. Mater. 2010, 22, 4567−4590. (17) Arote, R. B.; Lee, E. S.; Jiang, H. L.; Kim, Y. K.; Choi, Y. J.; Cho, M. H.; Cho, C. S. Bioconjugate Chem. 2009, 20, 2231−41. (18) Reul, R.; Nguyen, J.; Kissel, T. Biomaterials 2009, 30, 5815−24. (19) Wu; Liu, Y.; Jiang, X.; He; Goh, S. H.; Leong, K. W. Biomacromolecules 2006, 7, 1879−1883. (20) Lim, Y.-b.; Kim, S.-M.; Lee, Y.; Lee, W.-k.; Yang, T.-g.; Lee, M.-j.; Suh, H.; Park, J.-s. J. Am. Chem. Soc. 2001, 123, 2460−2461. (21) Liu, Y.; Wu, D.; Ma, Y.; Tang, G.; Wang, S.; He, C.; Chung, T.; Goh, S. Chem. Commun. 2003, 2630−2631. (22) Chen, Z. Y.; He, C. Y.; Meuse, L.; Kay, M. A. Gene Ther. 2004, 11, 856−864. (23) Darquet, A. M.; Cameron, B.; Wils, P.; Scherman, D.; Crouzet, J. Gene Ther. 1997, 4, 1341−1349. (24) Chen, Z.-Y.; He, C.-Y.; Ehrhardt, A.; Kay, M. A. Mol. Ther. 2003, 8, 495−500. (25) Bigger, B. W. J. Biol. Chem. 2001, 276, 23018−23027. (26) Keeney, M.; Ong, S. G.; Padilla, A.; Yao, Z.; Goodman, S.; Wu, J. C.; Yang, F. ACS Nano 2013, 7, 7241−50.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

§ L.C. and D.Z. contributed equally. The manuscript was written through contributions of all authors

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Article


Biomacromolecules (27) Chamorro, C.; Almarza, D.; Duarte, B.; Llames, S. G.; Murillas, R.; García, M.; Cigudosa, J. C.; Espinosa-Hevia, L.; Escámez, M. J.; Mencía, A.; Meana, A.; García-Escudero, R.; Moro, R.; Conti, C. J.; Del Río, M.; Larcher, F. Exp. Dermatol. 2013, 22, 601−3. (28) Eltoukhy, A. A.; Chen, D.; Alabi, C. A.; Langer, R.; Anderson, D. G. Adv. Mater. 2013, 25, 1487−1493. (29) Eltoukhy, A. A.; Siegwart, D. J.; Alabi, C. A.; Rajan, J. S.; Langer, R.; Anderson, D. G. Biomaterials 2012, 33, 3594−3603. (30) Layek, B.; Haldar, M. K.; Sharma, G.; Lipp, L.; Mallik, S.; Singh, J. Mol. Pharmaceutics 2014, 11, 982−994. (31) Zhu, H.; Dong, C.; Dong, H.; Ren, T.; Wen, X.; Su, J.; Li, Y. ACS Appl. Mater. Interfaces 2014, 6, 10393−10407. (32) Liu, W. G.; Zhang, X.; Sun, S. J.; Sun, G. J.; Yao, K. D. Bioconjugate Chem. 2003, 14, 782−789. (33) Chae, S. Y.; Son, S.; Lee, M.; Jang, M.-K.; Nah, J.-W. J. Controlled Release 2005, 109, 330−344. (34) Teo, P. Y.; Yang, C.; Hedrick, J. L.; Engler, A. C.; Coady, D. J.; Ghaem-Maghami, S.; George, A. J.; Yang, Y. Y. Biomaterials 2013, 34, 7971−9.

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Highly Branched Poly(Î²-Amino Esters): Synthesis and Application in

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