Hydrophobe-substituted <italic>b</italic>PEI derivatives: boosting transfection on primary vascular cells

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SCIENCE CHINA Materials, Volume 60, Issue 6: 529-542(2017) https://doi.org/10.1007/s40843-017-9030-7

Hydrophobe-substituted bPEI derivatives: boosting transfection on primary vascular cells

Daniele Pezzoli1,*, Eleni K. Tsekoura2, Remant Bahadur K.C.2, Gabriele Candiani3, Diego Mantovani1, Hasan Uluda?2,*
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  • ReceivedMar 13, 2017
  • AcceptedMar 31, 2017
  • PublishedApr 20, 2017
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Abstract

Gene therapy targeted to vascular cells represents a promising approach for prevention and treatment of pathological conditions such as intimal hyperplasia, in-stent and post-angioplasty restenosis. In this context, polymeric non-viral gene delivery systems are a safe alternative to viral vectors but a further improvement in efficiency and cytocompatibility is needed to improve their clinical success. Herein, a library of 24 branched polyethylenimine (bPEI) derivatives modified with hydrophobic moieties was synthesised, characterised and tested in vitro on primary vascular cells, aiming to identify delivery agents with superior transfection efficiency and low cytotoxicity. Low molecular weight PEIs (0.6, 1.2 and 2 kDa) were grafted with long (C18) and short (C3) aliphatic chains, featuring different unsaturation degrees and degrees of substitution. 0.6 kDa bPEI-based derivatives were generally ineffective in transfection on vascular smooth muscle cells (VSMCs), while among the other derivatives some promising vectors were identified. Forcing polyplexes on the cell surface by means of centrifugation invariably boosted transfection levels but increased cytotoxicity as well. Of note, a propionyl-substituted derivative (PEI2-PrA1, C3:0) was the most effective on both VSMCs and endothelial cells (ECs), with higher and more sustained gene expression in combination with markedly lower cytotoxicity with respect to the gold standard 25 kDa bPEI. In addition, a linoleoyl-substituted derivative (PEI1.2-LA6, C18:2) owing to its high efficiency in VSMCs and relative inefficacy in ECs, combined with tolerable cytotoxicity was proposed as a vector for specific VSMCs targeting.


Funded by

The study was supported by the Natural Science and Engineering Research Council of Canada

the Canadian Institute for Health Research

and the Fonds de Recherche du Quebec sur les Natures et Technologies.


Acknowledgment

Pezzoli D and Tsekoura EK were awarded a post-doctoral and doctoral scholarship, respectively, from the NSERC CREATE Program in Regenerative Medicine, www.ncprm.ulaval.ca. The studies were financially supported by the Natural Science and Engineering Research Council of Canada, (Discovery Grant to Uluda? H and Mantovani D), the Canadian Institute for Health Research (Operating grant to Uluda? H), and the Fonds de Recherche du Quebec sur les Natures et Technologies (Bilateral Grant to Mantovani D). We thank Dr. Vishwa Somayaji for 1H-NMR analysis of the polymer samples


Interest statement

Bahadur KCR and Uluda? H hold ownership position in RJH Biosciences Inc. intended to commercialise the described polymers.


Contributions statement

Pezzoli D, Uluda? H, Mantovani D and Candiani G conceived the idea and designed the experiments; Pezzoli D, Tsekoura EK and Bahadur KC R performed the experiments; Pezzoli D and Uluda? H analysed the data and wrote the manuscript with support from Candiani G and Mantovani D. All authors contributed to the general discussion.


Supplement

Supplementary data are available in the online version of the paper.


References

[1] Mozaffarian D, Benjamin EJ, Go AS, et al. Executive summary: heart disease and stroke statistics—2015 update: a report from the American Heart Association. Circulation, 2015, 131: 434-441 CrossRef Google Scholar

[2] Shan L, Saxena A, McMahon R, et al. Coronary artery bypass graft surgery in the elderly: a review of postoperative quality of life. Circulation, 2013, 128: 2333-2343 CrossRef PubMed Google Scholar

[3] Marx SO, Totary-Jain H, Marks AR. Vascular smooth muscle cell proliferation in restenosis. Circ-Cardiovasc Interv, 2011, 4: 104-111 CrossRef PubMed Google Scholar

[4] Bauters C, Isner J. The biology of restenosis. Prog Cardiovasc Dis, 1997, 40: 107-116 CrossRef Google Scholar

[5] Arnold JD, Mountain DJH, Freeman MB, et al. Smooth muscle cell polymeric transfection is an efficient alternative to traditional methods of experimental gene therapy. J Surg Res, 2012, 177: 178-184 CrossRef PubMed Google Scholar

[6] Fang YL, Chen XG, Godbey WT. Gene delivery in tissue engineering and regenerative medicine. J Biomed Mater Res, 2015, 103: 1679-1699 CrossRef PubMed Google Scholar

[7] Dean DA. Nonviral gene transfer to skeletal, smooth, and cardiac muscle in living animals. AJP-Cell Physiol, 2005, 289: C233-C245 CrossRef PubMed Google Scholar

[8] Pezzoli D, Chiesa R, De Nardo L, et al. We still have a long way to go to effectively deliver genes!. J Appl Biomater Funct Mater, 2012, 2: 82-91 CrossRef PubMed Google Scholar

[9] Pezzoli D, Candiani G. Non-viral gene delivery strategies for gene therapy: a “ménage à trois” among nucleic acids, materials, and the biological environment. J Nanopart Res, 2013, 15: 1523 CrossRef Google Scholar

[10] Akinc A, Thomas M, Klibanov AM, et al. Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. J Gene Med, 2005, 7: 657-663 CrossRef PubMed Google Scholar

[11] Mintzer MA, Simanek EE. Nonviral vectors for gene delivery. Chem Rev, 2009, 109: 259-302 CrossRef PubMed Google Scholar

[12] Pezzoli D, Olimpieri F, Malloggi C, et al. Chitosan-graft-branched polyethylenimine copolymers: influence of degree of grafting on transfection behavior. PLoS ONE, 2012, 7: e34711 CrossRef PubMed ADS Google Scholar

[13] Alshamsan A, Haddadi A, Incani V, et al. Formulation and delivery of siRNA by oleic acid and stearic acid modified polyethylenimine. Mol Pharm, 2009, 6: 121-133 CrossRef PubMed Google Scholar

[14] Han S, Mahato RI, Kim SW. Water-soluble lipopolymer for gene delivery. Bioconjugate Chem, 2001, 12: 337-345 CrossRef Google Scholar

[15] Thomas M, Klibanov AM. Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells. Proc Natl Acad Sci USA, 2002, 99: 14640-14645 CrossRef PubMed ADS Google Scholar

[16] Zheng M, Zhong Y, Meng F, et al. Lipoic acid modified low molecular weight polyethylenimine mediates nontoxic and highly potent in vitro gene transfection. Mol Pharm, 2011, 8: 2434-2443 CrossRef PubMed Google Scholar

[17] Neamnark A, Suwantong O, Bahadur RKC, et al. Aliphatic lipid substitution on 2 kDa polyethylenimine improves plasmid delivery and transgene expression. Mol Pharm, 2009, 6: 1798-1815 CrossRef PubMed Google Scholar

[18] Bahadur KCR, Landry B, Aliabadi HM, et al. Lipid substitution on low molecular weight (0.6–2.0kDa) polyethylenimine leads to a higher zeta potential of plasmid DNA and enhances transgene expression. Acta Biomater, 2011, 7: 2209-2217 CrossRef PubMed Google Scholar

[19] Thapa B, Plianwong S, Remant Bahadur KC, et al. Small hydrophobe substitution on polyethylenimine for plasmid DNA delivery: optimal substitution is critical for effective delivery. Acta Biomater, 2016, 33: 213-224 CrossRef PubMed Google Scholar

[20] D'Andrea C, Pezzoli D, Malloggi C, et al. The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA. Photochem Photobiol Sci, 2014, 13: 1680-1689 CrossRef PubMed Google Scholar

[21] Pezzoli D, Kajaste-Rudnitski A, Chiesa R, et al. Lipid-based nanoparticles as nonviral gene delivery vectors. In: Bergese P, Hamad-Schifferli K (eds.). Nanomaterial Interfaces in Biology: Methods and Protocols, Methods in Molecular Biology. New York: Humana Press, 2013, 1025: 269–279. Google Scholar

[22] Chang KH, Park JM, Lee MY. Feasibility of simultaneous measurement of cytosolic calcium and hydrogen peroxide in vascular smooth muscle cells. BMB Rep, 2013, 46: 600-605 CrossRef Google Scholar

[23] Fan Z, Chen D, Deng CX. Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles. J Control Release, 2013, 170: 401-413 CrossRef PubMed Google Scholar

[24] Gresch O, Altrogge L. Transfection of difficult-to-transfect primary mammalian cells. In: Hartley JL (ed.). Protein Expression in Mammalian Cells: Methods and Protocols, Methods in Molecular Biology. New York: Humana Press, 2012, 801: 65–74. Google Scholar

[25] Hsu CYM, Hendzel M, Uluda? H. Improved transfection efficiency of an aliphatic lipid substituted 2 kDa polyethylenimine is attributed to enhanced nuclear association and uptake in rat bone marrow stromal cell. J Gene Med, 2011, 13: 46-59 CrossRef PubMed Google Scholar

[26] Pezzoli D, Giupponi E, Mantovani D, et al. Size matters for in vitro gene delivery: investigating the relationships among complexation protocol, transfection medium, size and sedimentation. Sci Rep, 2017, 7: 44134 CrossRef PubMed Google Scholar

[27] Suzuki Y, Yeung AC, Ikeno F. The representative porcine model for human cardiovascular disease. J Biomed Biotech, 2011, 2011: 1-10 CrossRef PubMed Google Scholar

[28] Malloggi C, Pezzoli D, Magagnin L, et al. Comparative evaluation and optimization of off-the-shelf cationic polymers for gene delivery purposes. Polym Chem, 2015, 6: 6325-6339 CrossRef Google Scholar

[29] Dube B, Rose L, Sawant K, et al. Cholic acid modified 2 kDa polyethylenimine as efficient transfection agent. Biotechnol Prog, 2013, 29: 1337-1341 CrossRef PubMed Google Scholar

[30] Zhang QF, Luan CR, Yin DX, et al. Amino acid-modified polyethylenimines with enhanced gene delivery efficiency and biocompatibility. Polymers, 2015, 7: 2316-2331 CrossRef Google Scholar

[31] Pezzoli D, Tarsini P, Melone L, et al. RGD-derivatized PEI-PEG copolymers: influence of the degree of substitution on the targeting behavior. J Drug Deliver Sci Tech, 2017, 37: 115-122 CrossRef Google Scholar

[32] Breunig M, Lungwitz U, Liebl R, et al. Breaking up the correlation between efficacy and toxicity for nonviral gene delivery. Proc Natl Acad Sci USA, 2007, 104: 14454-14459 CrossRef PubMed ADS Google Scholar

[33] Aliabadi HM, Landry B, Bahadur RK, et al. Impact of lipid substitution on assembly and delivery of siRNA by cationic polymers. Macromol Biosci, 2011, 11: 662-672 CrossRef PubMed Google Scholar

[34] Hsu CYM, Uluda? H. A simple and rapid nonviral approach to efficiently transfect primary tissue-derived cells using polyethylenimine. Nat Protoc, 2012, 7: 935-945 CrossRef PubMed Google Scholar

[35] Fishbein I, Chorny M, Adamo RF, et al. Endovascular gene delivery from a stent platform: gene- eluting stents. Angiol, 2013, 1: 1000109 CrossRef PubMed Google Scholar

[36] Saurer EM, Yamanouchi D, Liu B, et al. Delivery of plasmid DNA to vascular tissue in vivo using catheter balloons coated with polyelectrolyte multilayers. Biomaterials, 2011, 32: 610-618 CrossRef PubMed Google Scholar

[37] Riessen R, Rahimizadeh H, Blessing E, et al. Arterial gene transfer using pure DNA applied directly to a hydrogel-coated angioplasty balloon. Hum Gene Ther, 1993, 4: 749-758 CrossRef PubMed Google Scholar

[38] Sharif F, Hynes SO, Cooney R, et al. Gene-eluting stents: adenovirus-mediated delivery of eNOS to the blood vessel wall accelerates re-endothelialization and inhibits restenosis. Mol Therapy, 2008, 16: 1674-1680 CrossRef PubMed Google Scholar

[39] Patel SD, Waltham M, Wadoodi A, et al. The role of endothelial cells and their progenitors in intimal hyperplasia. Therapeutic Adv Cardiovascular Disease, 2010, 4: 129-141 CrossRef PubMed Google Scholar

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    Scheme 1

    Synthesis of hydrophobe-substituted bPEI derivatives.

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    Figure 1

    pDNA complexation ability of bPEI derivatives as a function of polymer:DNA ratio (w/w). (a) The complexation curves for PEI2-St6, PEI2-LA9, PEI2-αLA8, PEI2-PrA1 and PEI2-AcA1 are reported as representative examples and compared with 25 kDa bPEI. (b) BC50 values as a function of the degree of substitution for 0.6 (black squares), 1.2 (green triangles) and 2.0 kDa bPEI-based derivatives (red dots). A positive correlation was observed between the BC50 and degree of substitution, according to Pearson correlation (r = 0.91, p< 0.05; r = 0.67, p< 0.05; r = 0.84, p< 0.05 respectively for 0.6, 1.2 and 2.0 kDa bPEI-based derivatives).

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    Figure 2

    (a) Transfection efficiency and (b) cytotoxicity of bPEI derivatives at w/w 5 and 10 on PAoSMCs. Cells were transfected with pGL3 and transfection efficiency was evaluated 48 h post-transfection and expressed as relative luminescence units (RLU) normalized over the total protein content in every cell lysate. Cytotoxicity was evaluated by AlamarBlue assay and expressed as percent viability loss with respect to untreated control cells. Missing bars in (a) indicate that no significant luciferase activity was detected. Results are shown as mean ± standard deviation (n ≥ 4).

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    Figure 3

    (a) Transfection efficiency and (b) cytotoxicity of bPEI derivatives at w/w 5 and 10 on HUASMCs. Cells were transfected with pGL3 and transfection efficiency was measured 48 h post-transfection and expressed as RLU normalized over the total protein content in cell lysate. Cytotoxicity was evaluated by AlamarBlue assay and expressed as percent viability loss with respect to untreated control cells. Missing bars in (a) indicate that no significant luciferase activity was detected. Results are shown as mean ± standard deviation (n ≥ 4).

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    Figure 4

    Effect of centrifugation on transfection by bPEI derivatives on (a) PAoSMCs and (b) HUASMCs. The ratio between transfection efficiency obtained with (500 ×g) and without (1 ×g) centrifugation is reported (black bars). Cytotoxicity of centrifuged polyplexes (grey bars) is expressed as toxicity percent relative to untreated control cells. Results are shown as mean ± standard deviation (n ≥ 4).

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    Figure 5

    Kinetics of transfection efficiency of selected bPEI derivatives on (a) PAoSMCs and (b) HUASMCs. Cells were transfected with pGLuc (w/w 10 for bPEI derivatives, w/w 5 for 25 kDa bPEI) and transfection efficiency was measured at different time points and expressed as RLU. Results are shown as mean ± standard deviation (n ≥ 4).

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    Figure 6

    (a) Kinetics of transfection efficiency of selected bPEI derivatives on HUVECs. Cells were transfected with pGLuc (w/w 10 for bPEI derivatives, w/w 5 for 25 kDa bPEI) and transfection efficiency was measured at different time points and expressed as RLU. (b) Cytotoxicity of bPEI derivatives on HUVECs. Cytotoxicity was evaluated by AlamarBlue assay 48 h post-transfection and expressed as percent toxicity relative to untreated cells. Results are shown as mean ± standard deviation (n ≥ 4).

  • Table 1   Properties of the library of bPEI derivatives and of unmodified bPEIs investigated in this study. The table summarizes the type of substitute, the lipid:PEI feed ratio (mol/mol) used during the reaction, the degree of substitution calculated from 1H NMR analysis and the w/w ratio required for 50% pDNA binding during complexation (BC50), evaluated by SYBR Green I fluorophore-exclusion assay.

    Polymer

    Substitute

    Feed ratio (mol/mol)

    Degree of substitution (mol/mol)

    BC50

    PEI2-St6

    Stearic acid

    6.0

    2.14

    0.678

    PEI2-St12

    Stearic acid

    12.0

    4.53

    8.088

    PEI0.6-LA4

    Linoleic acid

    4.0

    1.09

    0.364

    PEI1.2-LA4

    Linoleic acid

    4.0

    1.84

    0.761

    PEI1.2-LA6

    Linoleic acid

    6.0

    2.55

    0.686

    PEI2-LA6

    Linoleic acid

    6.0

    2.31

    0.785

    PEI2-LA9

    Linoleic acid

    9.0

    3.20

    3.609

    PEI0.6-αLA2

    α-linoleic acid

    2.0

    0.80

    0.320

    PEI0.6-αLA4

    α-linoleic acid

    4.0

    2.30

    1.269

    PEI1.2-αLA2

    α-linoleic acid

    2.0

    0.94

    0.289

    PEI1.2-αLA4

    α-linoleic acid

    4.0

    2.45

    0.297

    PEI1.2-αLA6

    α-linoleic acid

    6.0

    3.17

    0.578

    PEI2-αLA2

    α-linoleic acid

    2.0

    1.37

    0.681

    PEI2-αLA4

    α-linoleic acid

    4.0

    2.72

    1.015

    PEI2-αLA8

    α-linoleic acid

    8.0

    3.68

    3.899

    PEI0.6-PrA1

    Propionic acid

    1.0

    0.62

    0.298

    PEI1.2-PrA0.5

    Propionic acid

    0.5

    0.28

    0.316

    PEI1.2-PrA1

    Propionic acid

    1.0

    0.76

    0.310

    PEI2-PrA0.5

    Propionic acid

    0.5

    0.15

    0.304

    PEI2-PrA1

    Propionic acid

    1.0

    0.53

    0.367

    PEI1.2-AcA1

    Acrylic acid

    1.0

    0.65

    0.343

    PEI1.2-AcA2

    Acrylic acid

    2.0

    1.21

    0.430

    PEI2-AcA1

    Acrylic acid

    1.0

    0.51

    0.355

    PEI2-AcA2

    Acrylic acid

    2.0

    0.86

    0.643

    0.6 kDa bPEI

    /

    /

    /

    0.278

    1.2 kDa bPEI

    /

    /

    /

    0.215

    2 kDa bPEI

    /

    /

    /

    0.213

    25 kDa bPEI

    /

    /

    /

    0.274
  • Table 2   Hydrodynamic diameter (DH), polydispersity index (PDI) and ζ-potential (ζP) of the polyplexes prepared using PEI1.2-LA6, PEI1.2-αLA2, PEI2-PrA2 and PEI1.2-AcA2 derivatives and of native bPEIs (0.6, 1.2, 2 and 25 kDa) at w/w 5 and 10 and measured by DLS and laser Doppler micro-electrophoresis

    ?Polymer

    w/w

    DH (nm)

    St. Dev. DH (nm)

    PDI

    St. Dev. PDI

    ζP (mV)

    St. Dev. ζP (mV)

    PEI1.2-LA6

    5

    101

    16

    0.30

    0.11

    -1.4

    1.8

    10

    135

    53

    0.40

    0.15

    15.3

    4.0

    PEI1.2-αLA2

    5

    97

    25

    0.33

    0.09

    21.0

    1.5

    10

    229

    10

    0.54

    0.01

    23.1

    0.8

    PEI2-PrA1

    5

    98

    3

    0.31

    0.02

    32.2

    0.6

    10

    112

    53

    0.41

    0.23

    27.5

    3.7

    PEI1.2-AcA2

    5

    104

    3

    0.36

    0.04

    26.0

    6.1

    10

    94

    7

    0.21

    0.12

    22.8

    4.5

    0.6 kDa bPEI

    5

    1381

    281

    1.00

    0.00

    14.4

    5.2

    10

    1885

    1715

    0.87

    0.13

    15.6

    0.2

    1.2 kDa bPEI

    5

    139

    7

    0.45

    0.03

    28.9

    0.6

    10

    88

    0

    0.03

    0.01

    14.9

    2.1

    2 kDa bPEI

    5

    115

    13

    0.27

    0.05

    29.1

    5.4

    10

    179

    152

    0.39

    0.23

    22.0

    8.0

    25 kDa bPEI

    5

    112

    20

    0.35

    0.05

    32.8

    0.7

    10

    104

    16

    0.29

    0.09

    28.2

    0.9

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