Dually Gated Polymersomes for Gene Delivery - Nano Letters (ACS

Jul 27, 2018 - Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine , 301 Middle Yanchang Road, Shanghai...
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Dually Gated Polymersomes for Gene Delivery Fangyingkai Wang, Jingyi Gao, Jiangang Xiao, and Jianzhong Du Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b01985 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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Dually Gated Polymersomes for Gene Delivery Fangyingkai Wang,†‡ Jingyi Gao,‡ Jiangang Xiao‡ and Jianzhong Du*†‡ †

Department of Orthopaedics, Shanghai Tenth People’s Hospital, Tongji University School of

Medicine, 301 Middle Yanchang Road, Shanghai 200072, China ‡

Department of Polymeric Materials, School of Materials Science and Engineering, Tongji

University, 4800 Caoan Road, Shanghai, 201804, China

ABSTRACT: An ideal gene carrier requires an excellent gating system to efficiently load, protect, deliver and release environmentally sensitive nucleic acids on demand. Presented in this communication is a polymersome with a “boarding gate” and a “debarkation gate” in the membrane to complete the above important missions. This dually gated polymersome is selfassembled from a block copolymer, poly(ethylene oxide)-block-poly[N-isopropyl acrylamidestat-7-(2-methacryloyloxyethoxy)-4-methylcoumarin-stat-2-(diethylamino)ethyl

methacrylate]

[PEO-b-P(NIPAM-stat-CMA-stat-DEA)]. The hydrophilic PEO chains form the coronas of the polymersome, whereas the temperature and pH-sensitive P(NIPAM-stat-CMA-stat-DEA) block forms the dually gated heterogenous membrane. The temperature-controlled “boarding gate” can be opened at room temperature for facile encapsulation of siRNA and plasmid DNA into polymersomes directly in aqueous solution. The “debarkation gate” can be triggered by proton sponge effect for intracellular release. Biological studies confirmed successful encapsulation of siRNA and plasmid DNA, efficient in vitro and in vivo gene transfection and expression of green

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fluorescent protein (GFP) from GFP-encoding plasmid, suggesting that this kind of polymersome with a dual gating system can serve as an excellent biomacromolecular shuttle for gene delivery and other biological applications.

KEYWORDS: polymersomes, self-assembly, gene delivery, plasmid DNA, green fluorescent protein

INTRODUCTION Biomacromolecules such as proteins and nucleic acids play fundamental yet vital roles in all forms of organisms. In particular, major diseases arise from the disorder at proteins or nucleic acid level. To investigate into the sophisticated functions of biomacromolecules and thereby cure recurring

illnesses,

a

variety of

carriers

have

been

designed

to

deliver

certain

biomacromolecules, especially nucleic acids into specific sites of cells both in vitro and in vivo.110

As for gene delivery, vectors are necessary to protect nucleic acids from nuclease degradation

and to go through internalization, controlled release, and redistribution at desired subcellular sites.11,12 Complexes formed by electrostatic interaction were used for delivering negatively charged nucleic acids.2,13-16 Recently, polymeric nanoparticles have been developed as delivery vehicles,16-20 including polymer micelles,21 nanogels22-24 and polymersomes (or polymer vesicles), etc.25-31 Among them, polymersomes are of emerging interest as their inner void can encapsulate and their membrane can protect nucleic acids. For example, we recently prepared a pH-sensitive polymersome which is capable of encapsulating siRNA and anticancer drug in pure water by adjusting the pH of aqueous solution.32 The polymersomes exhibited much better cancer-stem-cells-killing and tumor-growth-inhibition capabilities. Battaglia et al. applied electroporation approach to induce the polymersome membrane rupture and to load

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biomacromolecules into polymersom.33 However, most carriers lack a smart gating system essential for the efficient transport of environment-sensitive biomacromolecules, especially for plasmid DNA and siRNA. Therefore, a gated vector with distinct responsibilities for each gate may provide more advantages for better delivery of biomacromolecules than traditional vectors without a smart gating system. Herein, we present a gene delivery polymersome with a smart dual gating system to efficiently deliver plasmid DNA and siRNA into cells. Distinct from traditional polymersomes with a homogeneous membrane, this dually gated polymersome consists of a heterogenous membrane34 with finely controllable temperature and pH responsiveness, which is more suitable for encapsulation, protection, transport and release of biomacromolecules in aqueous solution. As shown in Scheme 1, the PEO-b-P(NIPAM-stat-CMA-stat-DEA) copolymer can self-assemble into polymersomes at 40 oC in the mixture of neutral water and tetrahydrofuran (THF). Under this condition the thermo-responsive polyNIPAM moiety is dehydrated and the pH-responsive polyDEA moiety is deprotonated to form the polymersome with closed gates in the membrane. After removal of THF by dialysis, the polymersomes are photo-cross-linked in situ by the interchain covalent bond upon photodimerization of coumarin moieties to fix the membrane structure. The polyNIPAM-based “boarding gate” in the membrane will be opened below the LCST of PNIPAM (e.g., 20~30 oC) to load biomacromolecules in water, and then closed at physiological temperature (e.g., 37 oC) to lock the payload. The pH-sensitive polyDEA moiety in the membrane of polymersome serves as the “debarkation gate”, allowing release of biomacromolecules in acid environment (such as in endosome) and realize their functionalities (e.g., gene transfection). GFP-encoding plasmid DNA was successfully delivered into human L02 cells by this polymersome in vitro, transfected and expressed to proteins, and further

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confirmed by the in vivo tests where fluorescent signals in the nude mice were observed after injection of this dually gated polymersome encapsulated with GFP-encoding plasmid DNA. Scheme 1. Schematic Illustration of a Dually Gated Polymersome as a Biomacromolecular Shuttle for Gene Delivery a

a

The cross-linked polymersome with a “boarding gate” and a “debarkation gate” can load biomacromolecules by opening the “boarding gate” at room temperature, lock them at body temperature in pure water, and release them through the “debarkation gate” at acidic conditions such as in endosome at physiological temperature. The release of biomacromolecules can be further accelerated by enabling the initial “boarding gate” as a second “debarkation gate” upon slightly decreasing the temperature in cells. The colors of the bars at the top-right corner stand for the different states of the polymersome membrane, depending on the temperature and pH.

RESULTS AND DISCUSSION The PEO-b-P(NIPAM-stat-CMA-stat-DEA) block copolymer was synthesized through a reversible addition-fragmentation chain transfer (RAFT) polymerization (Scheme S1 in the

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Supporting Information). The chemical structures of the PEO-based macro chain transfer agent (macro-CTA) trithiolcarbonate and monomer 7-(2-methacryloyloxyethoxy)-4-methylcoumarin (CMA) were prepared according to our previous protocol.35 The obtained PEO43-b-P(NIPAM44stat-CMA10-stat-DEA22) block copolymer was confirmed by 1H NMR spectra in CDCl3 and DMF GPC (Figures S1-S2 in the Supporting Information). The LCST of this copolymer in aqueous solution was 32 oC (Figure S3 in the Supporting Information). With an initial concentration of 5.0 mg/mL, the copolymer can be self-assembled into neat polymersome (Figure S4 in the Supporting Information). The polymersome solution was then placed under UV spot curing system (8000 mw/cm2) at a λ of 365 nm for 1 min to immobilize the membrane structure at 40 oC and the photodimerization degree of coumarin moieties is calculated to be 63.6% (Figures S5-S6 in the Supporting Information). Those cross-linked polymersomes were then used for a series of subsequent experiments unless otherwise stated. The morphology of the polymersome above LCST was first characterized by transmission electron microscopy (TEM), as shown in Figure 1A, B. Following the same protocol as we reported before, the TEM sample was prepared with a pre-heated copper grid and dried out at 37 o

C.35 As shown in Figure 1A-C, TEM and the dynamic light scattering (DLS) results of the

polymersomes at 37 oC revealed an evenly distributed size of polymersomes. The diameter of the polymersomes determined by TEM was ca. 500 nm (Figure S7 in the Supporting Information), which is reasonably larger than the hydrodymic diatermer (Dh) of 462 nm determined by DLS (PD is 0.099) as large vesicles are more likely to collapse and flatten on the TEM grid.36 TEM images also clearly confirmed the vesicular structure of polymersomes with a uniform size distribution and folded-membrane, as indicated in Figure 1A-B. The actual thickness of this membrane was determined by a previously reported model based on the folded membrane to be

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10.3 nm (Figure S8 in the Supporting Information) and close to the theoretical single layer membrane thickness (ca. 10.1 nm), indicating an interdigitated membrane structure of the polymersome.

Figure 1. TEM and DLS studies of cross-linked polymersome at 37 oC. (A, B) TEM images with different magnification. Samples are stained with neutral PTA (1.0 wt%) solution. (C) Typical size distribution at 37 oC determined by DLS. (D) Temperature-dependent changes in size and zeta potential. The cooling-triggered “boarding” mechanism of this dually gated polymersome was tested by measuring the sizes and zeta potentials at various temperatures (Figure 1D). In a typical temperature reduction from 40 to 10 oC, the Dh and the zeta potential were monitored at an

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interval of every 5 oC. As the temperature decreased, the size of the polymersome increased gradually due to the growing degree of hydration of polyNIPAM. During this process, the polyNIPAM chains are expanded to hydrophilic coils and the positively charged polyDEA chains were exposed to water. As a result, the polymersomes swelled to 616.0 nm, and the zeta potential of the polymersome solution rose to + 35.9 mV. The size change was reversible during the heating/cooling cycles and the vesicular morphology of the polymersomes remained stable, as confirmed by DLS studies in Figure S9 in the Supporting Information. By contrast, un-crosslinked polymersomes dissociated below 20 oC during a similar process (Figure S10 in the Supporting Information). To further verify the excellent structural stability of the cross-linked polymersome below the LCST, we performed TEM study at 20 oC (Figure 2). Polymersomes expanded to around 600 nm but remained stable and neat, maintaining the required structural stability while opening their “boarding gates” in water. The membrane thickness was calculated to be 10.8 nm based on the TEM analysis in Figure S11 in the Supporting Information.

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Figure 2. TEM and DLS studies of cross-linked polymersome at 20 oC. (A) Typical size distribution at 20 oC determined by DLS. (B-D) TEM images with different magnification. Samples are stained with neutral PTA (1.0 wt%) solution. The acid-triggered “debarkation” of this dually gated polymersome is important for gene delivery. The Dh of polymersomes increases from 462.2 nm at pH 6.8 to 608.4 nm at pH 5.4 and 37 oC (Figure S12 in the Supporting Information). The “debarkation gate” of polymersomes will be opened during the swelling process, accompanying the release of biomacromolecules from the polymersome in response to the lower intracelluar pH.

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Before the polymersome was employed for biological application, its cell viability and physiological stability were evaluated as well (Figures S13-S14 in the Supporting Information). The above results confirmed that the polymersome is suitable for delivering payloads into the cells. In the following section, both siRNA and plasmid DNA were encapsulated and delivered as designed. This polymersome is capable of cytoplasmic siRNA delivery.39 For example, 5carboxyfluorescein (5-FAM) labeled siRNA was loaded into polymersome at 20 oC and locked inside the polymersome at 37 oC. The loading efficiency of FAM-siRNA is ca. 25.7% by measuring the fluorescence intensity of 5-FAM dye at 528 nm before and after removal of free siRNA via a Sepharose column (Figure S15 in the Supporting Information). Cellular uptake by L02 cells was then characterized by fluorescence microscope and flow cytometry (Figure 3). According to the control group shown in Figure 3C, the free siRNA without polymersomes was merely internalized into cells (only 0.34% of all cells showed fluorescent signal). By contrast, the siRNA was successfully delivered into cells when encapsulated into polymersome (95.0% from Figure 3D) at 24 h, confirming its excellent siRNA delivery efficiency into cells. Figure 3E-G indicated successful cell uptake at 24 h, where the green fluorescence corresponds to the siRNA labeled with FAM, blue to the nucleus stained with 4’, 6-diamidino-2-phenylindole (DAPI). Time-dependent cellular uptakes from 3 to 9 h were also studied by flow cytometry and the efficiencies are 51.8% (3 h), 60.3% (6 h) and 76.0% (9 h), respectively (Figure S17 in the Supporting Information). Over half of the polymersomes were internalized into cells in the first 3 hours and the efficiency increased with time.

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Figure 3. Cellular uptake of FAM-siRNA loaded cross-linked polymersomes at 24 h. (A-D) Flow cytometry results of free FAM-siRNA without polymersomes, and FAM-siRNA-loaded polymersomes. Uptake of FAM-siRNA without polymersomes by L02 cells is difficult, but it becomes much easy when the FAM-siRNA is loaded with polymersomes (internalized by 95.0% of cells). (E-G) Fluorescence microscopy images of green fluorescent FAM-siRNA. The nucleus is stained with DAPI (blue) and merged images. As a gene delivery vector, this dually gated polymersome also demonstrated its ability to deliver DNA into nucleus (Figure 4) as plasmid DNA is only expressed when the DNA sequence incorporated into the nucleus.30 So, in principle, after internalized by cells through endocytosis, polymersomes go through endosomal escape at acidic conditions to deliver the payloads into the cytoplasm.32 As confirmed in the previous section, the swelling of polymersomes at acidic condition in cells opens the “debarkation gate” of polymersomes and aids the release of plasmid DNA from the polymersome. Therefore, GFP-encoding plasmid DNA

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was selected to prove the above hyphothesis on the plasmid delivery by the dually gated polymersome. The loading process was carried out at both 20 and 37 oC in order to highlight the importance of the “boarding gate”. The experimental conditions and the calculated loading efficiencies were presented in Scheme S2 and Table S1 in the Supporting Information according to the literature method.40 At 37 oC, it is impossible to encapulate large cargos like pDNA into polymersomes as the “boarding gate” kept shutting down. While through electrostatic interaction between positively charged polymersomes and negetively charged plasmid, 12.1% of pDNA can be adsorbed on the membrane of the polymersome. By contrast, when loading the pDNA at 20 o

C, the overall loading efficiency of pDNA including adsorption and encapsulation increased to

34.2%, as a result of the additionally encapsulated 22.1% pDNA inside the polymersomes as the “boarding gate” is opened at 20 oC. Therefore, it is essential to encapsulate pDNA into polymersomes at lower temperature, and then lock them inside polymersomes at higher temperature to protect the encapsulated pDNA from degradation during delivery, as confirmed by the following transfection and expression experiments. To examine the transfection ability of pDNA encapsulated into and adsorbed on the polymersomes, four groups of distinct incubation conditions were carried out. Under normal cultivation conditions (37 oC, 5% CO2), the green fluorescent intensity increased with incubation time (Figure 4, groups I-III). At 24 h, the green fluorescence can hardly be observed by fluorescence microscope (Figure 4A). Up to 72 h, observable GFP signal was displayed inside the cells (Figure 4C, G, K). The time is relative longer because the opening of the debarkation gate is only slightly triggered by small pH gradient in this specific situation.

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Figure 4. Transfection and expression of GFP-encoding plasmid DNA which was both encapsulated into and adsorbed on the cross-linked polymersomes at 20 oC. (A-D) Green fluorescence transfected from plasmid DNA under different conditions; (E-H) Corresponding bright field photographs of (A-D); (I-L) Merged images. Groups I-III were incubated in the CO2 incubator at gradually extended time at 37 oC. Group IV was incubated outside the CO2 incubator at 25 oC for 30 min after the first 6 h incubation in the CO2 incubator. Scale bar: 20 µm. To improve the release efficiency of DNA and enhance the gene expression efficiency, the initial temperature-controlled “boarding gate” of polymersomes was enabled as a second “debarkation gate” of DNA. The petri culture dish was taken out of the incubator for static culture at ambient temperature (25 oC) for half an hour after the first 6 hours in the incubator. This process took advantage of the thermo-sensitive polyNIPAM segment. At the relatively

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lower temperature more DNA debarked from the temperature-controlled gate in the membrane of the polymersome without affecting the cell viability. The results in Figure 4D, H, L showed better transfection and expression efficiency than group I at the same incubation time. To compare the transfection efficiency between the encapsulated pDNA inside the polymersome and the adsorbed pDNA on the surface of the polymersome, we performed a control transfection experiment where only 12.1% of the pDNA was adsorbed on the surface of the polymersome at 37 oC and neutral pH, rather than encapsulated inside the polymersome. Unfortunately, this control group showed no evidence of GFP gene expression in the fluorescent microscope (Figure S18 in the Supporting Information), demonstrated that the adsorbed pDNA is not capable of transfection in cells. Therefore, in combination with the successful transfection results in Figure 4 where both encapsulate and adsorbed pDNA are involved, it came to the conclusion that only the encapsulated pDNA is capable of transfection, whereas the adsorbed pDNA is not able to transfect. The possible reason is that the polymersomes can effectively protect the pDNA inside them, but can’t successfully protect it on them.

Figure 5. In vivo transfection and expression of plasmid DNA encapsulated in polymersomes. Fluorescent signals were observed in pDNA in polymersome group by live animal molecular imaging technique after 6 days axillary subcutaneous injection.

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Animal tests were also carried out to confirm the in vivo performance of such gene vector and the specific operational details were provided in the experimental section. Samples (100 µL, 10.0 µg/mL pDNA with 0.5 mg/mL polymersome in PBS) were injected to the right axilla of nude mice subcutaneously and the fluorescent images were acquired by live animal molecular imaging technique at desired times (0, 2, 4, and 6 days, Figure 5 and Figure S19 in the Supporting Information). The control groups (PBS, naked pDNA, and polymersome alone) didn’t show any fluorescent signal up to 6 days after injection. The experimental group, nevertheless, showed distinguishable fluorescent signals near the armpit, confirming that the polymersome gave rise to the successful transfection and expression of pDNA in animal (Figure 5).

CONCLUSION In summary, we have designed and synthesized a multifunctional PEO-b-P(NIPAM-stat-CMAstat-DEA) diblock copolymer and self-assembled it into heterogenous membrane polymersomes with “boarding” and “debarkation” gates. The photo-cross-linking of PCMA not only improves the structural stability of the polymersome membrane but also allows better manipulation of the membrane permeability in pure water. DLS studies revealed that the polymersome has a thermo and pH dependent hydrodynamic diameter with a low polydispersity. TEM confirmed the vesicular morphology of polymersomes with a membrane thickness of ca. 10 nm. Biological experiments confirmed that the polymersomes with “boarding” and “debarkation” gates can act as gene vectors for encapsulating RNAs and DNAs directly in aqueous solution and release them under desired conditions. Furthermore, the “boarding” and “debarkation” gates can function in turn if necessary. For example, the “boarding gate” can be also enabled as a second “debarkation gate” for improving the intracellular release efficiency of DNA. We also found out that the

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encapsulated pDNA excelled at transfection but the adsorbed pDNA didn’t get transfected at all due to better protection for pDNA inside the polymersomes than on the surface. In vivo experiments confirmed the capability of transfection and expression of pDNA in nude mice. Overall, we provide a new insight for designing a polymersome with a dual gating system as an efficient gene vector, which may be applied in a wide range of fields such as bionanotechnology and nanomedicine.

ASSOCIATED CONTENT Supporting Information The Supporting Information, including Schemes S1 and S2, Figures S1-S19 and Tables S1, is available free of charge on the website. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Jianzhong Du: 0000-0003-1889-5669 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT J.D. is supported by NSFC (21674081), Shanghai International Scientific Collaboration Fund (15230724500), Fundamental Research Funds for the Central Universities (22120180109 and

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1500219107), and Shanghai 1000 Talents Plan (SH01068). Prof. Xiaoqing Zhang, Miss Beibei Fan, and Miss Yuanyuan Zhang in Tongji University School of Medicine are thanked for their assistance in the gene transfection experiments. Mr. Shaobo Xue, Ms Sujing Qiang, and Mr. Shuaikang Chang in Shanghai Tenth People’s Hospital are acknowledged for their assistance in the animal experiments. Prof. Yuanli Cai at Soochow University is appreciated for the assistance in the GPC analysis. REFERENCES (1) Guo, X.; Huang, L. Acc. Chem. Res. 2012, 45, 971-979. (2) Aoyama, Y.; Kanamori, T.; Nakai, T.; Sasaki, T.; Horiuchi, S.; Sando, S.; Niidome, T. J. Am. Chem. Soc. 2003, 125, 3455-3457. (3) Mun, J. Y.; Shin, K. K.; Kwon, O.; Lim, Y. T.; Oh, D. B. Biomaterials 2016, 101, 310-320. (4) Zhu, Y. Q.; Yang, B.; Chen, S.; Du, J. Z. Prog. Polym. Sci. 2017, 64, 1-22. (5) Hansen, M. B.; van Gaal, E.; Minten, I.; Storm, G.; van Hest, J. C.; Lowik, D. W. J. Controlled Release 2012, 164, 87-94. (6) Gaitzsch, J.; Huang, X.; Voit, B. Chem. Rev. 2016, 116, 1053-1093. (7) Kim, H. J.; Kim, A.; Miyata, K.; Kataoka, K. Adv. Drug Delivery Rev. 2016, 104, 61-77. (8) Richardson, J. J.; Choy, M. Y.; Guo, J. L.; Liang, K.; Alt, K.; Ping, Y.; Cui, J. W.; Law, L. S.; Hagemeyer, C. E.; Caruso, F. Adv. Mater. 2016, 28, 7703-7707. (9) Dong, R. J.; Zhou, Y. F.; Huang, X. H.; Zhu, X. Y.; Lu, Y. F.; Shen, J. Adv. Mater. 2015, 27, 498-526.

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(10) Cheng, R.; Meng, F. H.; Ma, S. B.; Xu, H. F.; Liu, H. Y.; Jing, X. B.; Zhong, Z. Y. J. Mater. Chem. 2011, 21, 19013-19020. (11) Xu, L.; Anchordoquy, T. J. Pharm. Sci. 2011, 100, 38-52. (12) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Nat. Rev. Drug Discovery 2005, 4, 581-593. (13) Ooya, T.; Choi, H. S.; Yamashita, A.; Yui, N.; Sugaya, Y.; Kano, A.; Maruyama, A.; Akita, H.; Ito, R.; Kogure, K.; Harashima, H. J. Am. Chem. Soc. 2006, 128, 3852-3853. (14) Zhou, G.; Xu, Y.; Chen, M.; Cheng, D.; Shuai, X. Polym. Chem. 2016, 7, 3857-3863. (15) Priegue, J. M.; Crisan, D. N.; Martínez-Costas, J.; Granja, J. R.; Fernandez-Trillo, F.; Montenegro, J. Angew. Chem., Int. Ed. 2016, 55, 7492-7495. (16) Wang, L. H.; Wu, D. C.; Xu, H. X.; You, Y. Z. Angew. Chem., Int. Ed. 2016, 55, 755-759. (17) Tu, Y.; Peng, F.; Adawy, A.; Men, Y.; Abdelmohsen, L. K. E. A.; Wilson, D. A. Chem. Rev. 2016, 116, 2023-2078. (18) Lim, E.-K.; Kim, T.; Paik, S.; Haam, S.; Huh, Y.-M.; Lee, K. Chem. Rev. 2015, 115, 327394. (19) Kowalczuk, A.; Trzcinska, R.; Trzebicka, B.; Müller, A. H. E.; Dworak, A.; Tsvetanov, C. B. Prog. Polym. Sci. 2014, 39, 43-86. (20) Tanner, P.; Baumann, P.; Enea, R.; Onaca, O.; Palivan, C.; Meier, W. Acc. Chem. Res. 2011, 44, 1039-1049.

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(21) Xu, X.; Wu, J.; Liu, Y.; Yu, M.; Zhao, L.; Zhu, X.; Bhasin, S.; Li, Q.; Ha, E.; Shi, J.; Farokhzad, O. C. Angew. Chem., Int. Ed. 2016, 55, 7091-7094. (22) Li, Y.; Maciel, D.; Rodrigues, J.; Shi, X.; Tomás, H. Chem. Rev. 2015, 115, 8564-8608. (23) McAllister, K.; Sazani, P.; Adam, M.; Cho, M. J.; Rubinstein, M.; Samulski, R. J.; DeSimone, J. M. J. Am. Chem. Soc. 2002, 124, 15198-15207. (24) Kabanov, A. V.; Vinogradov, S. V. Angew. Chem., Int. Ed. 2009, 48, 5418-5429. (25) Zhu, Y. Q.; Wang, F. Y. K.; Zhang, C.; Du, J. Z. ACS Nano 2014, 8, 6644-6654. (26) Fu, Z.; Ochsner, M. A.; de Hoog, H.-P. M.; Tomczak, N.; Nallani, M. Chem. Commun. 2011, 47, 2862-2864. (27) Kim, Y.; Tewari, M.; Pajerowski, J. D.; Cai, S.; Sen, S.; Williams, J.; Sirsi, S.; Lutz, G.; Discher, D. E. J. Controlled Release 2009, 134, 132-140. (28) Gallon, E.; Matini, T.; Sasso, L.; Mantovani, G.; Arminan de Benito, A.; Sanchis, J.; Caliceti, P.; Alexander, C.; Vicent, M. J.; Salmaso, S. Biomacromolecules 2015, 16, 1924-1937. (29) Wang, J.; Ni, Y.; Jiang, W.; Li, H.; Liu, Y.; Lin, S.; Zhou, Y.; Yan, D. Small 2015, 11, 4485-4490. (30) Lomas, H.; Canton, I.; MacNeil, S.; Du, J. Z.; Armes, S. P.; Ryan, A. J.; Lewis, A. L.; Battaglia, G. Adv. Mater. 2007, 19, 4238-4243. (31) Vriezema, D. M.; Garcia, P. M.; Sancho Oltra, N.; Hatzakis, N. S.; Kuiper, S. M.; Nolte, R. J.; Rowan, A. E.; van Hest, J. C. Angew. Chem., Int. Ed. 2007, 46, 7378-7382.

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(32) Chen, J.; Liu, Q. M.; Xiao, J. G.; Du, J. Z. Biomacromolecules 2015, 16, 1695-1705. (33) Wang, L. G.; Chierico, L.; Little, D.; Patikarnmonthon, N.; Yang, Z.; Azzouz, M.; Madsen, J.; Armes, S. P.; Battaglia, G. Angew. Chem., Int. Ed. 2012, 51, 11122-11125. (34) Xiao, Y. F.; Sun, H.; Du, J. Z. J. Am. Chem. Soc. 2017, 139, 7640-7647. (35) Wang, F. Y. K.; Du, J. Z. Chem. Commun. 2015, 51, 11198-11201. (36) Du, J. Z.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 12800-12801. (37) Zhou, C. C.; Yuan, Y.; Zhou, P. Y.; Wang, F. Y. K.; Hong, Y. X.; Wang, N. S.; Xu, S. G.; Du, J. Z. Biomacromolecules 2017, 18, 4154-4162. (38) Gao, J. Y.; Wang, M. Z.; Wang, F. Y. K.; Du, J. Z. Biomacromolecules 2016, 17, 20802086. (39) Little, S. R.; Kohane, D. S. J. Mater. Chem. 2008, 18, 832-841. (40) Bhat, S.; Curach, N.; Mostyn, T.; Bains, G. S.; Griffiths, K. R.; Emslie, K. R. Anal. Chem. 2010, 82, 7185-7192.

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