Letter pubs.acs.org/journal/abseba
Biodegradable Highly Branched Poly(β-Amino Ester)s for Targeted Cancer Cell Gene Transfection Shuai Liu,†,‡ Yongsheng Gao,† Sigen A,† Dezhong Zhou,*,† Udo Greiser,† Tianying Guo,*,‡ Rui Guo,§ and Wenxin Wang*,† †
Charles Institute of Dermatology, School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 300071, Tianjin, China § Department of Biomedical Engineering, Jinan University, 510632, Guangzhou, China ‡
S Supporting Information *
ABSTRACT: To enhance the gene transfection efficiency to targeted cells while reducing the side effects to untargeted cells is of great significance for clinical gene therapy. Here, biodegradable highly branched poly(β-amino ester)s (HPAESS) are synthesized and functionalized with folate (HPAESSFA) and lactobionic acid (HPAESS-Lac) for targeted cancer cell gene transfection. Results show that because of the triggered degradability of the vector and enhanced receptormediated cellular uptake of polyplexes, the HPAESS-FA and HPAESS-Lac exhibit superior gene transfection capability in specific cancer cells with negligible cytotoxicity, pointing to their promise as targeted vectors for efficient cancer gene therapy. KEYWORDS: cancer cells, gene transfection, biodegradable, highly branched poly(β-amino ester)s, targeting
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end groups, limiting further functionalization through the peripheries. In contrast to linear polymers, branched polymers have exhibited superior gene transfection efficacy because of their intriguing three-dimensional (3D) architecture and their multiple peripheries.11,12 To achieve higher performance of gene transfection by integrating the advantages of LPAEs (e.g., degradability) and branched polymers (e.g., multivalence), recently our group has developed highly branched poly(βamino ester)s (HPAEs) via a one-pot “A2+B3+C2” Michael addition strategy.13−16 Results showed that HPAEs demonstrated up to 8521-fold enhancement in transfection efficacy compared to LPAEs, polyethylenimine (PEI) and SuperFect, with much lower cytotoxicity. Despite these encouraging results, the high potential of HPAEs is far away from full exploitation because the multiple terminals and backbone of HPAEs have not yet been utilized for further functionalization. We hypothesize that grafting of specific ligands onto HPAEs will provide the ability of tumor targeting and will further greatly promote their gene transfection capability in cancer cells. Moreover, the degradation of HPAEs via the hydrolysis of ester groups on the backbone is usually random and uncontrollable, which would potentially lead to off-target DNA release and thus compromise
ene therapy has tremendous potential for the treatment of tumors via induction of apoptosis of cancer cells after delivery of anticancer genes into tumors.1−3 However, safe and efficient anticancer gene delivery is the major hurdle for successful translation into the clinic.4 Although viral-based gene delivery vectors show high efficiency, safety concerns and production problems continue to limit their use as therapeutic treatment options for patients.5 Alternatively, nonviral gene vectors have attracted more attention because of their higher safety profile and ease of the manufacturing process.6,7 Nonetheless, the polyplexes formulated with vector and DNA can nonspecifically interact with various cells. Enhancement of the functional gene expression in specific cancer cells can substantially improve the therapeutic efficiency of cancers.8 It is widely reported that specific receptors are overexpressed by cancer cells. Therefore, it is of great significance to develop targeted gene delivery vectors for overexpressed markers on the surface of cancer cells, because ligand−receptor interaction can not only enhance anticancer gene expression in cancer cells but also reduce the side effects to normal cells.9 Various cationic polymers have been developed as nonviral gene vectors during the past two decades. Among them, hydrolyzable linear poly(β-amino ester)s (LPAEs) have attracted particular attention.10 LPAEs can condense DNA to form nanosized and stable polyplexes, and until now, several optimized LPAEs out of a library of more than 2350 LPAEs have proven to be highly efficient for gene transfection in various cell types. However, the linear structure imparts the LPAEs with only two © XXXX American Chemical Society
Special Issue: Biomimetic Bioactive Biomaterials: The Next Generation of Implantable Devices Received: August 30, 2016 Accepted: October 18, 2016
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DOI: 10.1021/acsbiomaterials.6b00503 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
cleavage of the disulfide bonds to release DNA in the cytoplasm. Importantly, cytotoxicity of the HPAEs post transfection can also be reduced because the low molecular weight oligomers exert lower cytotoxicity compared to the high molecular weight polymers. HPAESS were further functionalized with folate (HPAESS-FA) and lactobionic acid (HPAESS-Lac) to target folate receptor (FR) and lactose receptors (LR), which are usually overexpressed in cancer cells.18,19 We expect that the multifunctional HPAESS-FA and HAPESS-Lac would mediate enhanced gene transfection in specific cancer cells due to the combined abilities of the HPAEs for triggered degradability and cancer targeting. HPAESS was synthesized from the condensation polymerization of bisphenol A ethoxylate diacrylate (BE), disulfanediylbis(ethane-2,1-diyl) diacrylate (DSDA), trimethylolpropane triacrylate (TMPTA) and 4-amino-1-butanol (S4) via the versatile “A2+B3+C2” type Michael addition chemistry using 1,3-diaminopropane (DAP) as the end-capping agent. The copolymerization of TMPTA was chosen to give rise to a highly branched architecture, whereas the introduction of DSDA was utilized to incorporate disulfide bonds to the polymer backbone. The multiple terminal amines derived from DAP were functionalized with FA or Lac via amidation chemistry (Figure 1). LPAE, LPAE-FA (with folate ligands), LPAE-Lac (with lactose ligands), HPAE, HPAESS (with disulfide bonds), HPAESS-FA (with disulfide bonds and folate ligands) and HPAESS-Lac (with disulfide bonds and lactose ligands) of different structures, degradability and targetability were synthesized (Table S1). Gel permeation chromatography (GPC) results showed that LPAE, HPAE and HPAESS had similar weight-average molecular weights (Mw) of approximately 10 000 Da (Figure S1). According to the Mark−Houwink (MH) plot, an α value between 0.5 and 1.0 represents a linear structure, whereas a value of less than 0.5 suggests a highly branched architecture. HPAE and HPAESS exhibited α values of 0.45 and 0.47, respectively, demonstrating their typical highly branched structures. Chemical composition of the polymers were further verified by 1H NMR spectra (Figure S2). The appearance of characteristic FA and Lac signal peaks provided evidence for the successful terminal functionalization of HPAESS. LPAE-FA and
the transfection efficiency. Therefore, the development of HPAEs with triggered degradability is also highly crucial for the improvement of gene delivery systems. Here, we aim to develop HPAEs with simultaneous abilities for triggered degradation and cancer cell targeting by introducing disulfide bonds as well as specific ligands to the backbone and terminals of HPAEs, respectively (Scheme 1). Disulfide bonds Scheme 1. Ligand-Modified HPAESS Condenses DNA To Form Polyplexes, Which Are Then Taken up by Specific Cancer Cells via Ligand-Receptor Mediated Endocytosis; in the Cytoplasm, GSH Can Cleave the Disulfide Bonds of HPAESS-FA or HPAESS-Lac To Release DNA To Complete the Gene Transfection Process
can be cleaved by glutathione (GSH), a reducing tripeptide that is located mainly inside cells at a concentration of 1−10 mM, 1000-fold higher than outside cells (1−10 μM).17 It can be envisaged that polyplexes formulated by disulfide containing HPAEs (HPAESS) and DNA would be stable in the extracellular environment but that dissociation can be triggered by GSH via
Figure 1. Synthesis of HPAESS-FA or HPAESS-Lac via “A2+B3+C2” type Michael addition, followed by terminal functionalization with FA or Lac. B
DOI: 10.1021/acsbiomaterials.6b00503 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 2. (a) GPC traces of HPAESS and HPAE before and after incubation with GSH (10 mM, 1 h). (b) Gluciferase activity of Hela cells after transfection. (c) Gluciferase activity of HepG2 cells after transfection. (d) GFP expression of Hela cells after transfection with LPAE, HPAE, HPAESS, and HPAESS-FA1 at the w/w ratio of 10. Data represent mean ± SD (*p < 0.05, n = 4). Scale bars represent 200 μm.
Figure 3. Cellular uptake of various polyplexes in (a) HeLa and (b) HepG2 cells at the w/w ratio of 10:1. Scale bars represent 20 μm.
LPAE-Lac (with approximate degree of 50% terminals functionalization), HPAESS-FA1 and HPAESS-Lac1 (with approximate degree of 10% terminals functionalization), and HPAESS-FA2 and HPAESS-Lac2 (with approximate degree of 50% terminals functionalization) were prepared, respectively. DNA binding of various polymers was investigated by Picogreen assays (Figure S3). In general, the polymers exhibited high DNA binding affinity when polymer/DNA weight (w/w) ratio was equal to or greater than 10:1. Dynamic light scattering (DLS) further showed that the higher DNA affinity resulted in a smaller size of the formulated polyplexes: at the w/w ratios of 10:1 and 20:1, the polyplexes have sizes around 200 and 150 nm, respectively (Figure S4). Correspondingly, these polyplexes exhibited moderate zeta potentials (+7−15 mV). The small size and moderate zeta potential would facilitate cellular uptake of polyplexes and promote the gene transfection. Because of the low solubility in aqueous solution, the DNA binding affinity of
HPAESS-FA2 was much lower, leading to substantially bigger polyplex size and lower zeta potential. Gene transfection capability and cytotoxicity of nontargeted polymers (LPAE, HPAE and HPAESS) were first assessed in HeLa cells (FR-positive and LR-negative) and HepG2 cells (LRpositive and FR-negative). Commercial transfection reagents PEI and SuperFect were used as positive controls. In both cell types, HPAE showed 1−3 orders of magnitude higher Gluciferase activity than LPAE, indicating the superiority of the branched structure in gene transfection compared to the linear structure (Figure 2b and 2c). In comparison to HPAE, HPAESS showed up to 4-fold higher gene transfection efficiency and lower cytotoxicity (Figure 2b, c and Figure S5) supporting our hypothesis that the introduction of disulfide bonds can improve gene transfection efficiency and reduce cytotoxicity by triggered degradation of vector in the presence of GSH (Figure 2a). The ability of LPAE-FA, LPAE-Lac, HPAESS-FA, and HPAESS-Lac to target HeLa and HepG2 cells, respectively was C
DOI: 10.1021/acsbiomaterials.6b00503 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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further evaluated. In HeLa cells, HPAESS-FA1 exhibited 3-fold higher gene transfection efficiency than HPAESS at w/w ratio of 10:1, a counterpart without periphery targeting moieties (Figure 2b). To confirm the enhancement of gene transfection efficiency is derived from FA targetability, we conducted gene transfection and cellular uptake of HPAESS-FA1/DNA polyplexes in the presence of free folate. Results showed that the gene transfection efficiency and cellular uptake of HPAESS-FA1/DNA polyplexes were substantially reduced (Figure 3a and Figure S6). However, excess hydrophobic FA functionalization altered the DNA binding affinity of HPAESS-FA2, resulting in large polyplex sizes and low transfection efficiency. In contrast to the results obtained in HeLa cells, the HPAESS and HPAESS-FA1 exhibited similar gene transfection efficiency in HepG2 cells (Figure S7). These results demonstrate that the enhancement of gene transfection efficiency is indeed caused by the ability of folate moieties in HPAESS-FA1 to target overexpressed FR in HeLa cells. Similarly, the targetability of HPAESS-Lac to LR overexpressed HepG2 cells was also confirmed (Figures 2c and 3b and Figures S6 and S7). Enhanced gene transfection efficiency of targeted polyplexes was further supported by the higher GFP expression (Figure 2d). LPAE-FA and LPAE-Lac also showed to some extend the ability to target HeLa cells and HepG2 cells, respectively. However, the enhancement of gene transfection efficiency was not as strong as the HPAESS-FA and HPAESS-Lac counterparts because of the limited terminal units. In summary, highly degradable HPAEs with FR or LR targetability were developed as gene delivery vectors for cancer cell gene transfection. The branched structure, GSH triggered degradability and ligand−receptor mediated enhanced polyplex cellular uptake synergistically improved the gene transfection efficiency and safety of HPAEs. The superior gene transfection efficiency and negligible cytotoxicity highlight the great potential of HPAESS-FA and HPAESS-Lac in targeted cancer gene therapy.
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REFERENCES
(1) Zhou, J.; Liu, J.; Cheng, C. J.; Patel, T. R.; Weller, C. E.; Piepmeier, J. M.; Jiang, Z.; Saltzman, W. M. Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery. Nat. Mater. 2012, 11 (1), 82−90. (2) Samal, S. K.; Dash, M.; Van Vlierberghe, S.; Kaplan, D. L.; Chiellini, E.; van Blitterswijk, C.; Moroni, L.; Dubruel, P. Cationic polymers and their therapeutic potential. Chem. Soc. Rev. 2012, 41 (21), 7147−7194. (3) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 2013, 12 (11), 967−977. (4) Miyata, K.; Nishiyama, N.; Kataoka, K. Rational design of smart supramolecular assemblies for gene delivery: chemical challenges in the creation of artificial viruses. Chem. Soc. Rev. 2012, 41 (7), 2562−2574. (5) Kay, M. A. State-of-the-art gene-based therapies: the road ahead. Nat. Rev. Genet. 2011, 12 (5), 316−328. (6) Mintzer, M. A.; Simanek, E. E. Nonviral vectors for gene delivery. Chem. Rev. 2009, 109 (2), 259−302. (7) Liu, S.; Yang, J.; Ren, H.; O’Keeffe-Ahern, J.; Zhou, D.; Zhou, H.; Chen, J.; Guo, T. Multifunctional oligomer incorporation: a potent strategy to enhance the transfection activity of poly(L-lysine). Biomater. Sci. 2016, 4 (3), 522−532. (8) Sun, Y. X.; Zeng, X.; Meng, Q. F.; Zhang, X. Z.; Cheng, S. X.; Zhuo, R. X. The influence of RGD addition on the gene transfer characteristics of disulfide-containing polyethyleneimine/DNA complexes. Biomaterials 2008, 29 (32), 4356−4365. (9) Zhang, Q.; Liu, F.; Nguyen, K. T.; Ma, X.; Wang, X.; Xing, B.; Zhao, Y. Multifunctional mesoporous silica nanoparticles for cancer-targeted and controlled drug delivery. Adv. Funct. Mater. 2012, 22 (24), 5144− 5156. (10) Lynn, D. M.; Langer, R. Degradable poly(beta-amino esters): Synthesis, characterization, and self-assembly with plasmid DNA. J. Am. Chem. Soc. 2000, 122 (44), 10761−10768. (11) Zhao, T. Y.; Zhang, H.; Newland, B.; Aied, A.; Zhou, D. Z.; Wang, W. X. Significance of branching for transfection: synthesis of highly branched degradable functional poly(dimethylaminoethyl methacrylate) by vinyl oligomer combination. Angew. Chem., Int. Ed. 2014, 53 (24), 6095−6100. (12) Mou, Q.; Ma, Y.; Jin, X.; Zhu, X. Designing hyperbranched polymers for gene delivery. Mol. Syst. Des. Eng. 2016, 1 (1), 25−39. (13) Zhou, D.; Cutlar, L.; Gao, Y.; Wang, W.; O’Keeffe-Ahern, J.; McMahon, S.; Duarte, B.; Larcher, F.; Rodriguez, B. J.; Greiser, U.; Wang, W. The transition from linear to highly branched poly(β-amino ester)s: Branching matters for gene delivery. Sci. Adv. 2016, 2 (6), e1600102. (14) Cutlar, L.; Zhou, D.; Gao, Y.; Zhao, T.; Greiser, U.; Wang, W.; Wang, W. Highly Branched Poly(beta-Amino Esters): Synthesis and Application in Gene Delivery. Biomacromolecules 2015, 16 (9), 2609− 2617. (15) Huang, J. Y.; Gao, Y.; Cutlar, L.; O’Keeffe-Ahern, J.; Zhao, T.; Lin, F. H.; Zhou, D.; McMahon, S.; Greiser, U.; Wang, W.; Wang, W. Tailoring highly branched poly(beta-amino ester)s: a synthetic platform for epidermal gene therapy. Chem. Commun. 2015, 51 (40), 8473−8476. (16) Zhou, D.; Gao, Y.; Aied, A.; Cutlar, L.; Igoucheva, O.; Newland, B.; Alexeeve, V.; Greiser, U.; Uitto, J.; Wang, W. Highly branched poly(β-amino ester)s for skin gene therapy. J. Controlled Release 2016, in press; DOI:10.1016/j.jconrel.2016.06.014. (17) Kozielski, K. L.; Tzeng, S. Y.; Green, J. J. A bioreducible linear poly(beta-amino ester) for siRNA delivery. Chem. Commun. 2013, 49 (46), 5319−21. (18) Quan, G.; Pan, X.; Wang, Z.; Wu, Q.; Li, G.; Dian, L.; Chen, B.; Wu, C. Lactosaminated mesoporous silica nanoparticles for asialoglycoprotein receptor targeted anticancer drug delivery. J. Nanobiotechnol. 2015, 13, 89−100. (19) Zhang, C.; Gao, S.; Jiang, W.; Lin, S.; Du, F.; Li, Z.; Huang, W. Targeted minicircle DNA delivery using folate-poly(ethylene glycol)polyethylenimine as non-viral carrier. Biomaterials 2010, 31 (23), 6075− 6086.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00503 GPC traces, 1H NMR spectra, DNA binding, size, zeta potential, cell viability, and transfection efficiency in the absence or presence of FA (or Lac) (PDF)
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Letter
AUTHOR INFORMATION
Corresponding Authors
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[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by Science Foundation Ireland (SFI) (Grants 13/IA/1962, 14/TIDA/2367, 15/IFA/3037, 15/ TIDA/2969), Health Research Board of Ireland (Grant HRAPOR-2013-412), Doctoral Fund of Ministry of Education of China (Grant RFDP-20130031110012), the National Natural Science Foundation of China (Grant 20874052), and University College Dublin. D
DOI: 10.1021/acsbiomaterials.6b00503 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
