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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

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  • ReceivedMar 13, 2017
  • AcceptedMar 31, 2017
  • PublishedApr 20, 2017

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 Uludağ H and Mantovani D), the Canadian Institute for Health Research (Operating grant to Uludağ 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 Uludağ H hold ownership position in RJH Biosciences Inc. intended to commercialise the described polymers.


Contributions statement

Pezzoli D, Uludağ 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 Uludağ H analysed the data and wrote the manuscript with support from Candiani G and Mantovani D. All authors contributed to the general discussion.


Author information

Daniele Pezzoli received his PhD degree in Materials Engineering at Politecnico di Milano (Milan, Italy) in 2011, under the supervision of Prof. Gabriele Candiani. He is currently a postdoctoral fellow in the Laboratory for Biomaterials and Bioengineering of Université Laval (Quebec City, Canada), led by Prof. Diego Mantovani. His current research is focused on vascular tissue engineering using collagen gel-based scaffolds and on the development of polymeric non-viral gene delivery systems for tissue engineering applications.


Hasan Uludağ has been with the University of Alberta since 1997, designing functional biomaterials to realize the therapeutic potential of nucleic acids. He obtained dual BSc degrees in Biomedical Engineering and Biology from Brown University (USA) in 1989. He then completed his PhD degree in 1993 at the Department of Chemical Engineering at the University of Toronto. He spent four years at Genetics Institute Inc. (USA), where he contributed to development of a bone-inducing BMP device.


Supplement

Supplementary information

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


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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).

  • Scheme 1

    Synthesis of hydrophobe-substituted bPEI derivatives.

  • 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).

  • 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).

  • 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).

  • 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).

  • 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 PEI derivatives and of unmodified PEIs 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 H NMR analysis and the / ratio required for 50% pDNA binding during complexation (BC), 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 (), polydispersity index (PDI) and ζ-potential () of the polyplexes prepared using PEI1.2-LA6, PEI1.2-αLA2, PEI2-PrA2 and PEI1.2-AcA2 derivatives and of native PEIs (0.6, 1.2, 2 and 25 kDa) at / 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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