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. 2016 Jun 17;2(6):e1600102.
doi: 10.1126/sciadv.1600102. eCollection 2016 Jun.

The transition from linear to highly branched poly(β-amino ester)s: Branching matters for gene delivery

Dezhong Zhou  1 , Lara Cutlar  2 , Yongsheng Gao  2 , Wei Wang  2 , Jonathan O'Keeffe-Ahern  2 , Sean McMahon  2 , Blanca Duarte  3 , Fernando Larcher  3 , Brian J Rodriguez  4 , Udo Greiser  2 , Wenxin Wang  1
Affiliations

The transition from linear to highly branched poly(β-amino ester)s: Branching matters for gene delivery

Dezhong Zhou et al. Sci Adv. .

Abstract

Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(β-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(β-amino ester)s (HPAEs) via a one-pot "A2 + B3 + C2"-type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(β-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(β-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000. Moreover, we further demonstrate that HPAEs can correct genetic defects in vivo using a recessive dystrophic epidermolysis bullosa graft mouse model. Our findings prove that the A2 + B3 + C2 approach is highly generalizable and flexible for the design and synthesis of HPAEs, which cannot be achieved by the conventional polymerization approach; HPAEs are more efficient vectors in gene transfection than the corresponding LPAEs. This provides valuable insight into the development and applications of nonviral gene delivery and demonstrates great prospect for their translation to a clinical environment.

Keywords: Gene delivery; RDEB; highly branched polymer; non-viral vector; poly(β-amino ester).

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Figures

Fig. 1
Fig. 1. Branched poly(β-amino ester)s (HPAEs) are developed via an A2 + B3 + C2 Michael addition approach from various commercially available monomers.
(A) Scheme illustration of the development of HPAEs by branching LPAEs with triacrylate via the A2 + B3 + C2 Michael addition approach. (B) Scheme of the synthesis of conventional LPAEs. RT, room temperature. (C) Scheme of the synthesis of HPAEs. An A2-type monomer is copolymerized with a B3-type monomer and a C2-type monomer via the A2 + B3 + C2 Michael addition to first generate the acrylate-terminated base polymer and then endcapped with a second amine. (D) Structures of the monomers used for the synthesis of HPAEs and the corresponding LPAEs in this work. Detailed sets of monomers are listed in tables S1 and S3.
Fig. 2
Fig. 2. HPAEs show great superiority in gene transfection compared with the corresponding LPAEs.
(A to C) HeLa, rADSC, and SHSY-5Y cells show higher Gluciferase activity after treatment with HPAEs compared with the corresponding LPAEs. RLU, relative light units. (D) HPAEs demonstrate stronger DNA binding affinity compared with the corresponding LPAEs. (E) HPAEs condense DNA to allow for the formulation of smaller polyplexes with low PDI, compared to the corresponding LPAEs. (F) Correspondingly, HPAE/DNA polyplexes have a relatively higher ζ potential. (G) Fluorescence images show that the highly branched S4-TMPTA-BE-MPA mediates much higher cellular uptake of polyplexes compared with the linear counterpart S4-BE-MPA. Scale bars, 20 μm. (H) GFP fluorescence images show that the highly branched S4-TMPTA-BE-MPA mediates much higher gene transfection efficiency compared with the linear counterpart S4-BE-MPA. Scale bars, 200 μm. Data points marked with asterisks are statistically significant relative to the corresponding LPAEs (*P < 0.05; t test, single-tailed).
Fig. 3
Fig. 3. Characterization of the highly branched S4-TMPTA-BE-MPA with different branched structures.
(A) 1H NMR spectra of the 10 versions of HPAEs and LPAE. As the feed ratio of TMPTA/BE increased, the TMPTA units in HPAEs increased, whereas the BE units decreased correspondingly. (B) GPC traces show that the HPAEs and LPAE have an Mw of around 12,000. (C) MH plots of the HPAEs and the corresponding LPAE. Increases in the feed ratio of TMPTA/BE resulted in a sequential decrease of α values of the LPAE and HPAEs. LPAE had an α value of 0.65, confirming its typical linear structure. In contrast, the α value of HPAEs decreased from 0.48 (HPAE-1) to 0.31 (HPAE-10), proving their highly branched structures. IV, intrinsic viscosity.
Fig. 4
Fig. 4. HPAE-2 and HPAE-4 have broad utilities in gene transfection over diverse cell types.
(A) Gluciferase activity of cells after treatment with HPAE-2 and HPAE-4 shows an up to 8521-fold enhancement compared to that mediated by the LPAE, PEI, SuperFect, and Lipofectamine 2000. One-way analysis of variance (ANOVA) was used; data are shown as average ± SD; n = 4. Data points marked with asterisks (*) are statistically significant relative to the SuperFect group, and data points marked with the pound sign (#) are statistically significant relative to the Lipofectamine 2000 group. *P < 0.05, superior Gluciferase activity compared with SuperFect; #P < 0.05, superior Gluciferase activity compared with Lipofectamine 2000. (B) Representative fluorescence images of cells after transfection with HPAE-4/DNA polyplexes at 15:1 (w/w). Scale bars, 200 μm. (C) Western blotting results from the supernatant of RDEBK cells. A clearly visible band for C7 was seen after transfection with HPAE-4/COL7A1.
Fig. 5
Fig. 5. HPAE-4 mediates a long-term expression of C7 in a human RDEB graft mouse model.
(A) Human RDEB grafts 0 day (left), 21 days (middle), and 60 days (right) after surgery. (B) Immunofluorescence image clearly shows the band of C7 along the BMZ (indicated by arrows) in the “healthy” skin graft. DAPI, 4′,6-diamidino-2-phenylindole. (C) Five days after the last injection of the HPAE-4/COL7A1 polyplexes, a significant level of C7 was produced and localized along the BMZ (indicated by the arrows). (D) After 30 days, the level of C7 along the BMZ remained at a significant level (indicated by the arrows).
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References

    1. Pack D. W., Hoffman A. S., Pun S., Stayton P. S., Design and development of polymers for gene delivery. Nat. Rev. Drug Discov. 4, 581–593 (2005). - PubMed
    1. Dahlman J. E., Barnes C., Khan O. F., Thiriot A., Jhunjunwala S., Shaw T. E., Xing Y., Sager H. B., Sahay G., Speciner L., Bader A., Bogorad R. L., Yin H., Racie T., Dong Y., Jiang S., Seedorf D., Dave A., Singh Sandhu K., Webber M. J., Novobrantseva T., Ruda V. M., Lytton-Jean A. K. R., Levins C. G., Kalish B., Mudge D. K., Perez M., Abezgauz L., Dutta P., Smith L., Charisse K., Kieran M. W., Fitzgerald K., Nahrendorf M., Danino D., Tuder R. M., von Adrian U. H., Akinc A., Panigrahy D., Schroeder A., Koteliansky V., Langer R., Anderson D. G., In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight. Nat. Nanotechnol. 9, 648–655 (2014). - PMC - PubMed
    1. Minter M. A., Simanek E. E., Nonviral vectors for gene delivery. Chem. Rev. 109, 259–302 (2009). - PubMed
    1. Zhao T., Zhang H., Newland B., Aied A., Zhou D., Wang W., Significance of branching for transfection: Synthesis of highly branched degradable functional poly(dimethylaminoethyl methacrylate) by vinyl oligomer combination. Angew. Chem. Int. Ed. Engl. 53, 6095–6100 (2014). - PubMed
    1. Luo D., Saltzman M., Synthetic DNA delivery systems. Nat. Biotechnol. 18, 33–37 (2000). - PubMed

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