Dually Gated Polymersomes for Gene Delivery

Letter Cite This: Nano Lett. 2018, 18, 5562−5568

pubs.acs.org/NanoLett

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

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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-isopropylacrylamide-stat-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 heterogeneous 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 the successful encapsulation of siRNA and plasmid DNA, efficient in vitro and in vivo gene transfection, and the expression of green 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

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solution.32 The polymersomes exhibited much better cancer stem cell killing and tumor growth inhibition capabilities. Battaglia et al. applied the electroporation approach to inducing the polymersome membrane rupture and to loading biomacromolecules into polymersome.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 heterogeneous membrane34 with finely controllable temperature and pH responsiveness, which is

iomacromolecules such as proteins and nucleic acids play fundamental yet vital roles in all organisms. In particular, major diseases arise from the disorder at proteins or nucleic acid level. To investigate 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.1−10 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 nanogels,22−24 and polymersomes (or polymer vesicles), etc.25−31 Among them, polymersomes are of emerging interest because their inner void can encapsulate cargoes and their membrane can protect nucleic acids. For example, we recently prepared a pH-sensitive polymersome that is capable of encapsulating siRNA and anticancer drug in pure water by adjusting the pH of aqueous © 2018 American Chemical Society

Received: May 15, 2018 Revised: July 15, 2018 Published: July 27, 2018 5562

DOI: 10.1021/acs.nanolett.8b01985 Nano Lett. 2018, 18, 5562−5568

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Scheme 1. Schematic Illustration of a Dually Gated Polymersome as a Biomacromolecular Shuttle for Gene Deliverya

The cross-linked polymersome with a “boarding gate” and a “debarkation gate” can load biomacromolecules by opening the “boarding gate” at room temperature, can lock them at body temperature in pure water, and can release them through the “debarkation gate” at acidic conditions, such as in endosomes 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. a

polymerization (Scheme S1). 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(NIPAM44-statCMA10-stat-DEA22) block copolymer was confirmed by 1H NMR spectra in CDCl3 and DMF GPC (Figures S1 and S2). The LCST of this copolymer in aqueous solution was 32 °C (Figure S3). With an initial concentration of 5.0 mg/mL, the copolymer can be self-assembled into neat polymersome (Figure S4). The polymersome solution was then placed under a UV spot curing system (8000 mw/cm2) at a λ of 365 nm for 1 min to immobilize the membrane structure at 40 °C, and the photodimerization degree of coumarin moieties is calculated to be 63.6% (Figures S5 and S6). Those cross-linked polymersomes were then used for a series of subsequent experiments unless otherwise stated. The morphology of the polymersomes above LCST was first characterized by transmission electron microscopy (TEM), as shown in Figure 1A,B. Following the same protocol as reported before, the TEM sample was prepared with a preheated copper grid and dried out at 37 °C.35 As shown in Figure 1A−C, TEM and the dynamic light scattering (DLS) results of the polymersomes at 37 °C revealed an evenly distributed size of polymersomes. The diameter of the polymersomes determined by TEM was ca. 500 nm (Figure S7), which is reasonably larger than the hydrodymic diatermer (Dh) of 462 nm determined by DLS (PD is 0.099) because large vesicles are more likely to collapse and flatten on the TEM grid.36 TEM

more suitable for the 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 °C in the mixture of neutral water and tetrahydrofuran (THF). Under these conditions, the thermoresponsive 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 °C) to load biomacromolecules in water and then closed at physiological temperature (e.g., 37 °C) to lock the payload. The pH-sensitive polyDEA moiety in the membrane of polymersome serves as the “debarkation gate”, allowing the release of biomacromolecules in acid environment (such as in endosomes) to 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 confirmed by the in vivo tests in which fluorescence signals in the nude mice were observed after injection of this dually gated polymersome encapsulated with GFP-encoding plasmid DNA. Results and Discussion. The PEO-b-P(NIPAM-statCMA-stat-DEA) block copolymer was synthesized through a reversible addition−fragmentation chain transfer (RAFT) 5563

DOI: 10.1021/acs.nanolett.8b01985 Nano Lett. 2018, 18, 5562−5568

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Figure 1. TEM and DLS studies of cross-linked polymersomes at 37 °C. (A, B) TEM images with different magnification. Samples are stained with neutral PTA (1.0 wt %) solution. (C) Typical size distribution at 37 °C determined by DLS. (D) Temperature-dependent changes in size and ζ potential.

LCST, we performed TEM study at 20 °C (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. The acid-triggered “debarkation” of these dually gated polymersomes 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 °C (Figure S12). The “debarkation gate” of polymersomes will be opened during the swelling process, accompanying the release of biomacromolecules from the polymersomes in response to the lower intracelluar pH. Before the polymersomes were employed for biological application, their cell viability and physiological stability were evaluated as well (Figures S13 and S14). The above results confirmed that the polymersomes are suitable for delivering payloads into the cells. In the following section, both siRNA and plasmid DNA were encapsulated and delivered as designed. These polymersomes are capable of cytoplasmic siRNA delivery.39 For example, 5-carboxyfluorescein (5-FAM) labeled siRNA was loaded into polymersomes at 20 °C and locked inside the polymersomes at 37 °C. 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

images also clearly confirmed the vesicular structure of polymersomes with a uniform size distribution and foldedmembrane, as indicated in Figure 1A,B. The actual thickness of this membrane was determined by a previously reported model based on the folded membrane37,38 to be 10.3 nm (Figure S8) and close to the theoretical single-layer membrane thickness (ca. 10.1 nm), indicating an interdigitated membrane structure of the polymersome. The cooling-triggered “boarding” mechanism of these dually gated polymersomes were tested by measuring the sizes and zeta potentials at various temperatures (Figure 1D). In a typical temperature reduction process from 40 to 10 °C, the Dh and the ζ potential were monitored at an interval of every 5 °C. As the temperature decreased, the size of the polymersomes increased gradually due to the growing hydration degree 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 ζ potential of the polymersomes 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 contrast, uncross-linked polymersomes dissociated below 20 °C during a similar process (Figure S10). To further verify the excellent structural stability of the cross-linked polymersomes below the 5564

DOI: 10.1021/acs.nanolett.8b01985 Nano Lett. 2018, 18, 5562−5568

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Figure 2. TEM and DLS studies of cross-linked polymersomes at 20 °C. (A) Typical size distribution at 20 °C determined by DLS. (B−-D) TEM images with different magnification. Samples are stained with neutral PTA (1.0 wt %) solution.

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 polymersomes. Therefore, GFP-encoding plasmid DNA was selected to prove the above hyphothesis on the plasmid delivery by the dually gated polymersomes. The loading process was carried out at both 20 and 37 °C to highlight the importance of the “boarding gate”. The experimental conditions and the calculated loading efficiencies were presented in Scheme S2 and Table S1 according to the method in the literature.40 At 37 °C, it is impossible to encapulate large cargo such as pDNA into polymersomes because the “boarding gate” kept shutting down. However, through electrostatic interaction between positively charged polymersomes and negatively charged plasmids, 12.1% of pDNA can be adsorbed on the membranes of the polymersomes. In contrast, when loading the pDNA at 20 °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 °C. Therefore, it is essential to encapsulate pDNA into polymersomes at lower temperature and then lock them inside at higher temperatures to protect the encapsulated pDNA from degradation during delivery, as confirmed by the following transfection and expression experiments.

free siRNA via a Sepharose column (Figures S15 and S16). Cellular uptake by L02 cells was then characterized by fluorescence microscopy 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 fluorescence signal). In contrast, the siRNA was successfully delivered into cells when encapsulated into polymersomes (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, at which point the green fluorescence corresponds to the siRNA labeled with FAM and blue to the nucleus stained with 4′,6diamidino-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). Over half of the polymersomes were internalized into cells in the first 3 h, and the efficiency increased with time. 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 5565

DOI: 10.1021/acs.nanolett.8b01985 Nano Lett. 2018, 18, 5562−5568

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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 easier 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 stained with DAPI (blue), and merged images.

Figure 4. Transfection and expression of GFP-encoding plasmid DNA that was both encapsulated into and adsorbed on the cross-linked polymersomes at 20 °C. (A−D) Green fluorescence transfected from plasmid DNA under different conditions; (E−H) corresponding bright-field photographs of panels A−D; (I−L) merged images. Groups I−III were incubated in the CO2 incubator at gradually extended time at 37 °C. Group IV was incubated outside the CO2 incubator at 25 °C for 30 min after the first 6 h of incubation in the CO2 incubator. Scale bar: 20 μm.

(Figure 4C,G,K). The time is relative longer because the opening of the debarkation gate is only slightly triggered by a small pH gradient in this specific situation. 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

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 °C, 5% CO 2 ), the green fluorescence intensity increased with incubation time (Figure 4, groups I−III). At 24 h, the green fluorescence can hardly be observed by fluorescence microscopy (Figure 4A,E,I). Up to 72 h, observable GFP signal was displayed inside the cells 5566

DOI: 10.1021/acs.nanolett.8b01985 Nano Lett. 2018, 18, 5562−5568

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

(25 °C) for half an hour after the first 6 h in the incubator. This process took advantage of the thermosensitive polyNIPAM segment. At the relatively lower temperature more DNA debarked from the temperature-controlled gates in the membranes of the polymersomes 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 polymersomes and the adsorbed pDNA on the surfaces of the polymersomes, we performed a control transfection experiment in which only 12.1% of the pDNA was adsorbed on the surfaces of the polymersomes at 37 °C and neutral pH rather than encapsulated inside the polymersomes. Unfortunately, this control group showed no evidence of GFP gene expression in the fluorescence microscope (Figure S18), demonstrating that the adsorbed pDNA is not capable of transfection in cells. Therefore, by combination with the successful transfection results in Figure 4 in which both encapsulated and adsorbed pDNA are involved, we 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 cannot successfully protect it on them. 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 polymersomes in PBS) were injected to the right axilla of nude mice subcutaneously, and the fluorescence images were acquired by live-animal molecular imaging technique at desired times (0, 2, 4, and 6 days; Figures 5 and S19). The control groups (PBS, naked pDNA, and polymersomes alone) did not show any fluorescence signal up to 6 days after injection. The experimental group nevertheless showed distinguishable fluorescence signals near the armpit, confirming that the polymersomes gave rise to the successful transfection and expression of pDNA in animal (Figure 5). Conclusions. In summary, we have designed and synthesized a multifunctional PEO-b-P(NIPAM-stat-CMAstat-DEA) diblock copolymer and self-assembled it into heterogeneous membrane polymersomes with “boarding” and “debarkation” gates. The photo-cross-linking of polyCMA 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 polymersomes have 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 RNA and DNA 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 encapsulated pDNA excelled at transfection, but the adsorbed pDNA did not get transfected at all due to better protection for pDNA inside the polymersomes than on the surfaces. In vivo experiments confirmed the ability to transfect and express pDNA in nude mice. Overall, we provide a new insight for designing a polymersome with a dual gating system as an efficient gene vector that may be applied in a wide range of fields such as bionanotechnology and nanomedicine.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b01985.

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Additional experimental details. Figures showing experimental schematics, NMR and UV−vis spectra, GPC traces, DLS results, photo-cross-linking and TEM analysis, membrane thicknesses, cytotoxicity studies, stability and thermally responsive behavior, fluorescence intensities, cellular uptake efficiencies, loading efficiencies, and the transfection and expression of GFPencoding DNA. A table showing loading efficiencies of plasmid DNA (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Fangyingkai Wang: 0000-0001-7756-6561 Jiangang Xiao: 0000-0001-9013-9898 Jianzhong Du: 0000-0003-1889-5669 5567

DOI: 10.1021/acs.nanolett.8b01985 Nano Lett. 2018, 18, 5562−5568

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.D. is supported by NSFC (grant no. 21674081), Shanghai International Scientific Collaboration Fund (grant no. 15230724500), Fundamental Research Funds for the Central Universities (grant nos. 22120180109 and 1500219107), and Shanghai 1000 Talents Plan (grant no. 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 assistance in the GPC analysis.

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DOI: 10.1021/acs.nanolett.8b01985 Nano Lett. 2018, 18, 5562−5568