Thermoresponsive polymers as gene delivery vectors: Cell viability, DNA transport and transfection studies

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Journal of Controlled Release 108 (2005) 472 – 483 www.elsevier.com/locate/jconrel

Thermoresponsive polymers as gene delivery vectors: Cell viability, DNA transport and transfection studies Beverley R. Twaites a,b, Carolina de las Heras Alarco´n a,b, Matthieu Lavigne a,b, Annabelle Saulnier a,b, Sivanand S. Pennadam c, David Cunliffe a,b, Dariusz C. Go´recki a,b, Cameron Alexander c,* a

School of Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth PO1 2DT (UK) b Institute of Biomedical and Biomolecular Science, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth PO1 2DT (UK) c School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK Received 12 May 2005; accepted 9 August 2005 Available online 7 October 2005

Abstract A range of gene delivery vectors containing the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAm) was evaluated for effects on cell viability, intracellular trafficking and transgene expression in C2C12 mouse muscle cells. Polymers were complexed with plasmid DNA at pH 7.4 and the ability of the resulting particles to transfect cells was assessed via confocal microscopy and protein expression studies in tissue culture. Cell viability assays indicated that these polymers were toxic at high concentrations when not complexed to DNA or at certain polymer : DNA ratios. Poly(ethyleneimine) co-polymers with side-chain grafted PNIPAm were shown to be less toxic than poly(ethyleneimine) alone or PNIPAm-co-(N,NV-dimethylaminoethylmethacrylate) linear co-polymers and the effects were concentration dependent. Confocal micrographs of labeled polymers and DNA indicated rapid cellular entry for all the complexes but expression of Green Fluorescent Protein was achieved only when the branched PEI–PNIPAm co-polymers were used as vectors. The results indicate that design of appropriate co-polymer components and overall polymer architecture can be used to mediate, and perhaps ultimately control, DNA transport and transgene expression. D 2005 Elsevier B.V. All rights reserved. Keywords: Thermoresponsive polymers; Gene delivery; Confocal microscopy; MTT assay; Cell viability; Transgene expression

1. Introduction * Corresponding author. Tel.: +44 115 951 5100; fax: +44 115 951 5102. E-mail address: [email protected] (C. Alexander). 0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2005.08.009

Synthetic polymers are under intense development as non-pathogenic and non-immunogenic alternatives to viruses as vectors for gene delivery [1–10]. These

materials also have advantages over viral vectors in terms of their low cost and ease of production. As a consequence, a great variety of these polymers have been prepared and their ability to transfect many different cell types studied [11–15]. However, none to date have shown transfection efficiency comparable to viruses and the most effective synthetic vector in widespread use for in vitro gene delivery, poly(ethyleneimine) (PEI), is relatively toxic or requires modification to transfect cells in vivo[16–22]. There is accordingly a need to prepare synthetic vectors that can rapidly and efficiently transfect cells without a toxic effect, but a complete understanding of the design criteria needed for active but non-toxic vectors is still elusive. There have now been a number of studies [23–30] focusing on enhancing transfection efficiencies through the use of polymers that can complex strongly with DNA during transport to target sites but which can potentially bind with lower affinity to DNA once inside a cell. These materials contain temperature responsive components arranged in various ways within, or attached to, a polymer chain. Phase transitions at the Lower Critical Solution Temperature (LCST) of the polymer result in coil-to-globule chain collapse of the responsive components, resulting in changes of binding affinity for plasmid DNA and in the structures of the resulting complexes. In theory, the polymer collapse to a compressed hydrophobic globule might protect DNA from nucleases prior to and during intracellular trafficking, while the expanse to a more open hydrophilic state should enable efficient disassembly of the polymer–DNA complexes leading to enhanced transfection. Polymers of this type prepared to date include derivatives of branched PEI containing end-grafted poly (N-isopropylacrylamide) (PNIPAm) homopolymers, or linear polycations synthesized from N-isopropylacrylamide, N,NV-dimethylaminoethylmethacrylate (DMAEMA) and hydrophobic side-chain monomers. In a previous paper [31] we reported the physico-chemical and DNA complexation properties of some of these polymers and here we describe experiments to assess intracellular trafficking of DNA by these and related polymers in conjunction with their effects on the viability of mammalian cells. For these experiments we utilised C2C12 mouse muscle cells to enable comparison of the effects of polymer vectors with direct gene transfer of plasmid DNA as previously performed on this cell line [32].

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2. Materials and methods High purity reagents, monomers and solvents for chemical synthesis were purchased from Aldrich, Acros or Fisher Scientific (UK) and used as received. Branched poly(ethyleneimine) (PEI, f.w. ~ 25 kDa, Aldrich), was dialysed (3 kDa cutoff) against deionised water (5  1000 ml) and lyophilized prior to use. PEI–octanamide, PEI–PNIPAM co-polymer (PEI– PNIPAm1) and polymers based on poly(N-isopropylacrylamide), (N,NV-dimethylaminoethyl)methacrylate (DMAEMA) and hexylacrylate (HA) (polymers PNDHA1–3) were prepared as described previously [31]. We also synthesised a further linear co-polymer (coded PNDD) containing N-isopropylacrylamide and DMAEMA together with the fluorescent marker monomer N-[2-[[[5-(N,N-dimethylamino)-1-naphthalenyl]sulfonyl]amino]propyl]-2-propenamide (DANSAPP) to monitor intracellular trafficking. 2.1. Synthesis of DANSAPP This was synthesised by adapting the procedure of Ren et al. [33]. Dansyl chloride (1 g, 3.70 mmol) in THF (100 ml) was added dropwise to a solution of diaminopropane (2.74 g, 37.03 mmol) in THF (150 ml) at 0 8C. The reaction was stirred at 0 8C for 4 h, then KOH (1 M, 10 ml) was added. The solvent was evaporated and the aqueous layer was extracted with CH2Cl2 (4  50 ml). The organic layer was dried with Mg2SO4 and evaporated to leave a pale yellow green oil as an intermediate. The intermediate was dissolved in THF and to this solution (1.136 g, 3.70 mmol) in THF (30 ml) at room temperature, acryloyl chloride (0.33 g, 3.70 mmol) and triethylamine (0.373 g, 3.70 mmol) were added. The reaction was stirred at room temperature overnight. The salts were filtered and washed with THF. The combined filtrates were evaporated, and the residue was chromatographed on silica using ethyl acetate/petroleum ether (80 / 20). The structure was confirmed by 1H and 13C-NMR. 2.2. Synthesis of polymer PNDD In a Schlenk vessel, monomer NIPAm (466 mg, 4.11 mmol), DMAEMA (409 mg, 2.40 mmol), fluorescent DANSAPP monomer (124 mg, 0.34 mmol)

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and 2,2-azo-bis(isobutyronitrile), initiator (50 mg) and mercaptoethanol (23 mg, 0.30 mmol) as chain transfer reagent were dissolved in THF (10 ml). This mixture was degassed by three freeze–thaw cycles under vacuum. The vessel was then placed in a thermostatted oil bath at 65 8C for 24 h. Once cooled to room temperature, the solution was concentrated under reduced pressure and the residue added to petroleum ether (500 ml) to precipitate the polymer. The precipitated materiel was dissolved in THF and precipitated again into light petroleum ether (b.p. 40–60 8C) three times to obtain the purified co-polymer as a colourless precipitate, which was then dried in vacuo overnight. DNA stains ethidium bromide (Sigma) and YOYO-1 (Molecular Probes) were used as received. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma. Solutions for biological assays (phosphate buffered saline, PBS, containing 150 mM NaCl) were sterilized prior to use. High purity endotoxin-free pX61 plasmid containing the human a-galactosidase A cDNA controlled by the human CMV immediate early promoter region and the rat myosin light chain 1 / 3 enhancer was prepared using a Qiagen kit as described previously [32]. 2.3. Preparation of labelled DNA The intracellular trafficking of DNA–polymer complexes was followed using pDNA labelled with the intercalating dye YOYO-1, at a concentration such that one molecule of dye was bound for every 100 base pairs of plasmid. 2.4. Preparation of polymer: DNA complexes A stock solution of pDNA was diluted into OptiMEM-1R to give a working solution of 0.4 Ag / 20 Al (4.9 nM). Polymer stock solutions were diluted into a total volume of 20 Al in Opti-MEM-1R, so that the final N : P ratios of polymer : DNA were between 0.1– 8 : 1. Complexes were formed by adding the polymer solution (20 Al) rapidly to the DNA solution (20 Al) in sterile microcentrifuge tubes (nuclease free, Fischer Scientific) with gentle shaking of the contents. The resultant suspensions were allowed to stand for 30 min at room temperature prior to addi-

tion of further Opti-MEM-1R (160 Al) with gentle mixing. 2.5. Cell culture C2C12 cells (mouse C3H muscle myoblasts ECAXX 91031101) were maintained in Dulbecco’s Modified Eagles Medium (DMEM, Sigma) supplemented with l-glutamine (200 mM, Sigma), penicillin (10,000 units), streptomycin (10 mg, Sigma) and 10% Fetal Calf Serum (FCS, Sigma). Cells were incubated at 37 8C in a humidified atmosphere containing 5% CO2 and used at appropriate degrees of confluence. Only cells that had been passaged b10 times were used in experiments, in order to maintain cell characteristics. 2.6. Cell viability (MTT) assay C2C12 cells were seeded into 96 well plates, and were allowed to grow to around 90% confluence. The wells were washed with PBS (200 Al per well) followed by Opti-MEM 1R (200 Al). Polymer–DNA complex, or polymer (200 Al) was then added to each well. Opti-MEM 1R (with and without DNA) was used as the control. Plates were incubated at 33 or 37 8C for 20 h. After this time, the media was removed, and the wells were rinsed with PBS and Opti-MEM 1R. MTT was dissolved in PBS to a concentration of 5 mg/ml. A working solution was prepared by dilution of the MTT solution in OptiMEMR (GibcoBRL) (1 : 4). Opti-MEM 1R (150 Al) and MTT (50 Al) were then added to each well, and plates were left to incubate at 37 8C for a further 3.5 h. Following incubation with MTT all media was removed, and dimethylsulfoxide (DMSO) was added to dissolve formasan crystals, with gentle mixing on a shaker-incubator (Jencons PLS) for 5 min at 750 rpm. Plates were then transferred to a plate reader (Anthos Labtec) and read at 550 nm, with reference at 620 nm. Plates were returned to the shaker-incubator for a further 5 min and re-measured. Absorbances of free polymer and polymer–DNA complexes were compared with Opti-MEM 1R and Opti-MEM 1RDNA controls. Experiments were carried out in triplicate sets of 96 well plates and repeated on consecutive days; results quoted are averages of at least 6 measurements.

2.7. Polymer–DNA complex transport and transfection in C2C12 cell line Autoclaved small microscope slides were transferred into the wells of a 12 well sterile plate, and were coated with collagen solution (0.1 mg/ml) for 1 h at room temperature. Wells were then rinsed  3 with PBS. Newly passaged C2C12 cells (~50,000 cells) were added to each well, and the plate was returned to incubate at 37 8C to allow cell growth and attachment onto the coverslips. Transport studies commenced when cells were ~45–50% confluent. The required polymer solution (50 Al N : P 2, 4) was added to YOYO-1 DNA (1 Ag in 50 Al PBS) and the resultant complexes were gently shaken before being allowed to stand at room temperature for 30 min. The incubation media of C2C12 cells was discarded, and plates were rinsed with PBS and OptiMEMR. Opti-MEMR (400 Al) was added to the complexes, and samples were incubated with cells for the appropriate time at 33 or 37 8C. Control experiments were performed with Opti-MEMR without DNA while LipofectAMINER (GibcoBRL)–DNA complexes were used as positive controls. Cells were incubated with the polymer–DNA complexes for either 0, 2, 4, 6, 8 or 24 h and transfection was stopped by the addition of ice cold PBS (2  1 ml). Paraformaldehyde (1 ml, 4% w/v in PBS) was added to each well for 30 min to fix the samples. Coverslips were carefully dried and mounted with Vectorshield mounting medium (Vector Labs), onto glass microscope slides, and sealed. Samples were imaged using a Zeiss LSM510 confocal microscope with He, HeNe and Ar lasers. For transfection experiments C2C12 cells (2  105 per well) were seeded in a 6 well plate 24 h before the transfection. Different amounts of polymer were diluted in 100 Al OPTIMEM before being complexed to a fixed amount (2 Ag/well) of pCS2*mt-SGP diluted in 100 Al OPTIMEM (final concentration = 6 nM) in order to span N / P ratios from 1 / 1 to 4 / 1. Complexes were formed by vortexing for 10 s and incubating for 30 min at the appropriate temperature before being diluted in OPTIMEM (final volume = 1 ml) and added onto the pre-washed sterile PBS culture. The cells were incubated with the transfection mixture at 37 8C/5% CO2 for 24 h (1 ml of DMEM containing 10% FCS was added after 5 h to allow the

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cells to restart growth). The culture medium was changed after 24 h and replaced with fresh DMEM + 10% FCS and cells were incubated another 24 or 48 h to allow GFP expression. Positive controls were carried out using jetPEIk (Poly-Plus transfection) at N / P= 5, 7.5 or 9. To evaluate the efficiency of gene expression, cells were observed using a Zeiss LSM 510 Meta confocal laser scanning microscope (GFP was excited via Argon laser at 488 nm/emission at 520 nm ) or by flow cytometry. For flow cytometry analyses, cells were trypsinised (300 ul Trypsin/well) 24 h post transfection, washed in 3 ml sterile PBS, pelleted at 1400 rpm, and resuspended in 1 ml PBS. Cells were analysed on a FACScalibur instrument, (BD Biosciences) using a detection method for GFP and propidium iodide (note propidium iodide was added to the cell suspension prior to running the sample on the flow cytometer). Data were analysed to calculate % GFP positive cells and dead cells among a particular cell population.

3. Results and discussion 3.1. Characterization of polymers Thermoresponsive polymers based on poly(N-isopropylacrylamide) (PNIPAm) were prepared via conventional radical co-polymerisation procedures and carboxyl-tipped PNIPAm homopolymer was grafted to branched PEI by carbodiimide coupling. The key properties of these polymers are given in Table 1. The degree of modification (and hence reduction in amine content) of PEI was 1 mol% for both PEI– octanamide and PEI–PNIPAm1 as determined by 1H NMR through peak area ratios of 2–5–3.2 ppm (PEI methylene protons) and d = 1.18 and 3.98 ppm (isopropyl sidechains of PNIPAm). Potentiometric pH titrations confirmed the reduction in titratable primary amine groups of PEI on modification with carboxyltipped PNIPAm. For polymers PNDHA1–3 (poly(Nisopropylacrylamide co-N,NV-(dimethylaminoethyl)methacrylate) co-hexylacrylate) amine content was quantified directly from 1H NMR peak integrals at d = 1.18 and 3.98 ppm (PNIPAm), 2.29 ppm (DMAEMA), and 0.89 ppm (HA). All the LCST transitions took place over a greater temperature range compared to PNIPAm homopolymer, as ex-

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pected owing to the presence of both strongly hydrophilic (DMAEMA) and hydrophobic (HA) components in the co-polymers. Variations in the onset and range of LCST in the PNDHA1 and PNDHA3 polymers were also in accord with predictions based on monomer content, with PNDHA1 exhibiting a reduced LCST owing to the increased hexylacrylate component compared to PNDHA3. The difference in LCST between PNDHA2 and PNDHA3 was not expected considering their very similar functional group content. It is most likely that this variation was due to differences in dblockinessT and hence changes in local pKa of amine groups in DMAEMA segments in the two polymers arising from the use of non-living free radical polymerization techniques. 3.2. Cell viability (MTT) assay with polymers and DNA–polymer complexes Polyethyleneimine has been reported to be cytotoxic as a result of its membrane disruptive properties, and through the induction of apoptotic pathways [20,34,35] but the exact mechanisms are not yet fully understood. We therefore used the MTT assay to evaluate the effects of polymer vectors on metabolic activity and because of its high sensitivity, reliability of quantitation and ease of use. Viability was evaluated following incubation of cells with linear PNIPAm-co–DMAEMA co-polymers and PNIPAm grafted to branched PEI as free polymers and also in complexes with plasmid DNA. Unmodified PEI and PEI octanamide were used as positive controls. These assays were carried out at either 33 or 37 8C in order to minimize the effects of temperature alone on cell viability and because the range of phase transition temperatures included polymers with LCSTs, above, below, or spanning the 33–37 8C interval. As expected for polycations, PEI and PEI-derivatives when not complexed to DNA proved toxic to C2C12 cells at 33 and 37 8C, with a loss in viable cell numbers of up to 40% within 4 h. The linear polymers PNDHA1–3 also reduced the viable cell count when uncomplexed, though to a lesser extent (20–30%) than the PEI-based materials. There were no significant differences in cell viability over the 33–37 8C range in the presence of these polymers and thus subsequent MTT assays with the polymer–DNA complexes were carried out at 37 8C only.

Polymer–pX 61 plasmid complexes were markedly less damaging to cells than the free polymers, with only PNDHA1-based complexes causing similar reductions in numbers of viable cells. The toxicity profiles of polymer–DNA complexes (Fig. 1) did not show any significant correlations with vector amine nitrogen to DNA phosphate backbone (N : P) ratios. At higher N : P ratios, the complexes were less prone to aggregation as indicated by dynamic light scattering suggesting a stabilising effect of increased overall positive charge, but, despite the expectation that this might lead to increased damage to cell membranes, there was no trend to decreased cell viability at N : P= 2–4. These results were partially in accord with those of Turk et al. [24] and Hinrichs et al. [36], who observed reduced toxicity with cationic PNIPAm copolymers compared to cationic homo-polymers (PEI and pDMAEMA); all the co-polymers in our study were less toxic to C2C12 cells than branched PEI homopolymer at N : P= 1. However, changes in cell viability through incorporation of PNIPAm chains in the PEI co-polymers were not significant compared to PEI alone over all the N : P ratios, reflecting the low degree of grafting in our PEI conjugate PEI–PNIPAm1. Additional experiments with polymer–DNA complexes at N : P 8 : 1 did show the expected decreases in cell viability (up to 30% for both the linear and branched sets of polymers), and therefore DNA transport and transfection experiments were carried out at N : P 1–4. 3.3. Intracellular transport We previously showed that polymer DNA complexes were compacted above LCST, and reasoned therefore that in the absence of specific uptake mechanisms, there might be differences in cell entry and intra-cellular trafficking between complexes prepared above and below LCST. Initial studies were carried out only at 37 8C in order to minimize the effects of temperature alone on cell viability. However, polymer complexes with labeled pDNA at N : P ratios of 2 : 1 and 4 : 1 entered cells rapidly irrespective of which polymer had been used to prepare complexes or whether the complexes were initially prepared below or above LCST. At 37 8C PEI–PNIPAm1 complexes as formed at N : P 4 : 1 exhibited particle sizes

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Table 1 Properties of polymers used in cell viability and transport assays [31] Polymer

Composition

PEI

x:y=1:2

H2N

H N

N x

¯ w (kDa) M

¯w M ¯n M

LCST/8C (pH 7.4)

25

2.0

–

x : y : z = 33 : 6 6 : 1

25

2.1

–

x : y : z = 33 : 66 : 1

257

3.1

30–33

n : m : p = 58 : 28 : 14

234

2.1

22–30

n : m : p = 56: 33 : 11

51

2.2

37–50

NH2 y

H2N PEI–octanamide

O HN H2N

C7H15

N N x–z z

N H y

NH2

H2N

PEI–PNIPAm1

O O

NH

HN H2N

N N x–z z

n N H y

NH2

H2N

PNDHA1

O n O

NH O

m O O

OH

p

O (CH2)5CH3

NMe2 PNDHA2

O n O

NH O

m O O

p

OH

O (CH2)5CH3

NMe2 (continued on next page)

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Table 1 (continued) PNDHA3

n : m : p= 54 : 36 : 10

54

3.5

31–34

n : m : p = 58 : 38 : 4

19

2.8

38–40

O n O

m

NH O

OH

p

O O

O (CH2)5CH3

NMe2

PNDD

O n O

NH O

m O O NMe2

OH

p NH O S O

NMe2

of 50–60 nm, while corresponding PNDHA1–3– pDNA complexes were in the 50–70 nm range and therefore no difference in cell entry in the absence of specific uptake mechanisms would have been predicted once the polymer–DNA complexes had reached 37 8C. YOYO-1 DNA was detectable within the cytosol within 3 h of initial incubation with polymer–DNA complexes and discrete compartments

within the cells became fluorescent within 6 h (Fig. 2). Representative micrographs of PNDHA1–pDNAYOYO-1 complexes at N : P 4 are shown in Fig. 2a and b, after 3 and 6 h, respectively, while Fig. 2c and d depict PNDHA3–pDNA-YOYO-1 complexes at N : P 4 at the same incubation times; qualitatively similar images were obtained for PNDHA2 complexes and at N : P ratios of 2 : 1. After 24 h many

700 N:P = 1

Number of viable cells /103

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600

N:P = 2 N:P = 4

500 400 300 200 100 0 PEI

PEI-octanamide PEI-PNIPAm1

PNDHA1

PNDHA2

PNDHA3

Polymer Fig. 1. Viability of C2C12 cells following incubation with polymer-DNA complexes for 20 h. PEI=poly(ethyleneimine), PNIPAm=poly(Nisopropylacrylamide), PNDHA=poly(N-isopropylacrylamide-co-N,NV-(dimethylamino)ethylmethacrylate-co-hexylacrylate).

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Fig. 2. Confocal microscopy of C2C12 myoblasts following incubation with polymer–DNA complexes at N : P= 4 : 1. Images recorded 400: (a, b) PNDHA1 + pX61 labelled with YOYO-1 after 3 and 6 h respectively; (c, d) PNDHA3 after 3 and 6 h. Magnification 400 (a–c),  100 (d).

cells incubated with the complexes exhibited fluorescent cell nuclei whereas cells treated with YOYO-1 DNA alone or mixtures of polymers with YOYO-1 did not exhibit strong nuclear fluorescence after this same time period. These results demonstrated that the observed emissions were due to pDNA-bound YOYO-1 introduced into the cells via complexation with the cationic polymers and not due to free dye that had entered the nucleus, although YOYO-1 migration following polymer–DNA complex breakdown in the cytosol and/or nuclear membrane damage could not be ruled out after longer incubation times. Double-labelling experiments with dansyl-functionalised polymer PNDD and YOYO-DNA showed colocalisation of labels in the cells, and retention of label even after 24 h. Estimates of uptake efficiency based on confocal micrographs suggested that complexes of polymers PNDHA1–3 with DNA entered over 80% of cells, while PEI–PNIPAm1–DNA complexes were taken up by between 60% and 80% of the C2C12 cells (Fig. 3). There were no discernable differences in intracellular transport apparent over the 33–37 8C range and so transfection experiments were carried out over a greater temperature range (25–37 8C) as we reasoned that polymer LCST transitions would be broadened when complexed to DNA. Transfection studies utilis-

Fig. 3. Confocal micrographs of C2C12 cells following incubation with polymer PNDD and YOYO-labelled DNA. Images (a, c) visualization of dansyl fluorescence after 24 h transfection following complex formation below (a) and above (c) LCST. Images (b, d) visualization of YOYO-1 fluorescence after 24 h transfection following complex formation below (b) and above (d) LCST. Magnification 20.

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ing a plasmid encoding GFP indicated that although entry of DNA into the cells was efficient with linear polymers PNDHA1–3 and PNDD, there was no transgene expression irrespective of the temperatures at which polymer–DNA complexes were formed or incubated with cells. By contrast, branched polymer PEI– PNIPAm1 complexed with pDNA at both 25 and 37 8C was able to transfect C2C12 cells, with expression of GFP within a proportion of cells after 3 h. Extension of incubation time to 72 h did not result in significant differences in the numbers of cells transfected, however, expression of GFP was still detectable after this time as evidenced by confocal micrographs (Fig. 4). Preliminary analysis by flow cytometry confirmed that up to 3-fold increases in GFP expression could be obtained by complexation of DNA above the LCST transition of PEI–PNIPAm1 polymer (37–45 8C) followed by a period during the transfection assay below LCST (up to 1 h at 25 8C). However, the overall transgene expression efficiency was no greater than 6% of total cells even with the varied temperature regime.

Fig. 4. Representative images showing expression of GFP following transfection with branched polymer PEI–PNIPAm1 at N : P= 4. Image (a) complexation of polymer with DNA at 25 8C followed by transfection for 4 h; (b) complexation at 37 8C followed by transfection for 4 h; (c) complexation at 25 8C followed by transfection for 72 h; (d) complexation at 37 8C followed by transfection for 72 h. Magnification 60.

The overall level of transgene expression was low compared to viral vectors, but of a similar magnitude to that observed by Turk et al. [24] who observed GFP expression in up to 10% of HeLa cells with branched PEI–PNIPAm polymers at 37 8C. No data on temperature dependence of transgene expression across the LCST range was reported by Turk and co-workers, although the importance of polymer architecture and size of the resultant complexes with DNA on cell uptake were noted. The highest uptake, obtained with branched PEI–PNIPAm derivatives, was attributed by Turk et al. to the ability of the responsive polymers to compact complexes above their LCST, whereas their lower transfection efficiency compared to linear PEI was considered to arise from the tighter binding as a consequence of high charge density in the branched PEI derivatives. In our case the high uptake of all the polymer–DNA complexes also indicated tight complexation but independent of polymer architecture. Our previous data had shown similar particle sizes for the PEI–PNIPAm1 polymer and PNDHA1–3 polymers complexed with pDNA at N : P= 4 below LCST (R H = 60–70 nm at 20 8C) and some compaction above LCST (R H ~ 50 nm at 45 8C): thus the similar rates and levels of polymer uptake for PEI– PNIPAm1 and PNDHA1–3 were expected assuming passive endocytosis. Differences in transfection efficiency of the linear PNDHA1–3 polymers and the branched PEI–PNIPAm1 polymers and the low transgene expression were thus most likely due to poor protection of DNA from cytosolic nucleases, difficulties in unpackaging of DNA from complexes within the cell or through barriers to nuclear entry. The ability of these polymers to interact with DNA was manifest in our prior study in ethidium bromide displacement and gel retardation assays: at N : P= 4 (i.e. the ratio used in transfection experiments) the highest degree of dye displacement was observed for PEI– PNIPAm1 suggesting this polymer may have offered the best protection of DNA during intracellular trafficking. The higher pKa range of PEI amine groups (~5.5–6.9) compared to DMAEMA (~7.3) and corresponding differences in polymer–DNA complex stability in the cellular environment may also have accounted for the variations in transgene expression observed. PNDHA1–DNA complexes were stable over several 20–45 8C cycles in DLS but aggregation of complexes occurred after more prolonged times at

37 8C. By contrast, PEI–PNIPAm1 complexes were less prone to this aggregation, and this too may have accounted for the ability of these complexes to effect GFP expression. It should also be noted that aggregation of complexes may have been much more pronounced within cells owing to adsorption of cytosolic proteins. The enhancement of GFP expression following incubation of cells with the PEI–PNIPAm1–pDNA complexes above LCST followed by a transfection period of 1 h below LCST was expected based on the hypothesis that chain expansion of PNIPAm grafts would increase the hydration of the complexes and reduce their stability once in the cell. Gel retardation assays [31] had indicated that at intermediate N : P ratios PEI–PNIPAm1 complexes were least retarded when formed and assayed below LCST and most strongly retarded when formed or when gels were run above LCST. This indicated that the pDNA was less strongly bound below LCST at these N : P ratios, but the possibility of complex aggregation above LCST and consequent inability to migrate in the gels, and the fact that at N : P= 4 all the polymer– DNA complexes were completely retarded irrespective of temperature, rendered further consideration of polymer–DNA binding affinities problematic through gel retardation data alone. Experiments designed to enhance DNA release by destabilisation of the complexes through more prolonged reductions in temperature during transfection assays were carried out. However, these did not lead to higher levels of GFP expression. In the case of the linear polymers PNDHA1–3, no transgene expression was observed with or without a short-term dcold shockT at 25 8C. These data contrasted with the enhancements in protein expression observed by Kurisawa et al. [37,38] using linear PNIPAm-co–DMAEMA based vectors in COS-1 cell lines. Again, the differences in cell type and cationic co-monomer content of the polymers in our study and those of Kurisawa may have accounted for the lack of protein expression with PNDHA1–3–DNA complexes. The results considered together indicated that the branched PEI–PNIPAm polymers were the most effective gene delivery vectors in the study, affording the best transgene expression while exhibiting reduced toxicity in the C2C12 cell line compared to PEI. While it is likely that PEI–PNIPAm co-polymers

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are not sufficiently bio-compatible for in vivo use, at least as far as gene function restoration or replacement strategies are concerned, nevertheless the design features incorporated in these model responsive vectors may be adapted in the future to generate responsive architecture polymers that are both safe and efficient vectors.

4. Conclusions These experiments showed that thermoresponsive cationic polymers were able to transport DNA into mouse myoblasts and that GFP was expressed when branched PEI-polymers with pendant PNIPAm chains were used as DNA delivery vectors. No transgene expression irrespective of temperature was observed with the analogous linear thermoresponsive cationic polymers. Cell viability was reduced by all the polymers when administered without DNA, but thermoresponsive polymer–DNA complexes were of lower toxicity compared to PEI homopolymer. Overall, this study indicates that appropriate molecular design of responsive polymers can be used to improve both the toxicological properties and the efficiency of gene delivery vectors.

Acknowledgements We thank the Engineering and Physical Sciences Research Council (EPSRC), the Wellcome Trust, (Grants 067484, GR/N AF/001572) the Royal Society and the Institute of Biomedical and Biomolecular Sciences (University of Portsmouth) for financial support. We also acknowledge the help of Sajida Jaffer and Theodore Sakopoulos in preliminary experiments, Jeannette Beveridge for assistance with confocal microscopy, Jill Rice for cell culture, Nigel Armstrong for recording NMR spectra and Dr. Steve Holding (RAPRA) for gel permeation chromatography.

References [1] A.K. Pannier, L.D. Shea, Controlled release systems for DNA delivery, Mol. Ther. 10 (2004) 19 – 26.

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