Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Comparison of Biological Responses of Polymers Based on Imine and Disulfide Backbones for siRNA Delivery Junyi Che,† Yonger Xue,† Jia Feng, Guang Bai, and Weien Yuan* Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, No. 800, Dongchuan Road, Shanghai 200240, China ABSTRACT: To achieve a successful delivery of siRNA by carriers in vivo, the degradation of polymers in response to tiny intracellular changes should be seriously considered. In addition, the balance between degradation and stability of polymers is another key point for high performance of carriers. In this study, imine and disulfide linkages, which are sensitive to pH changes and redox environment, respectively, were constructed as the main backbone of polymers to deliver siRNA at the intracellular and animal level. Comparisons were made between performances of these two different polymers. Both of the polymers synthesized here have good ability to condense siRNA. However, polyplexes formed by the imine backbone-based polymer (TPSP) showed a larger particle size and a higher zeta potential than that of the disulfide backbone-based polymer (DTDPS). Although both TPSP and DTDPS could deliver the target siRNA into 7721 cells, polyplexes formed by TPSP showed a higher silence efficiency in vitro and accomplished more accumulation in tumors. In conclusion, we believe TPSP is superior to be used for siRNA delivery and promises a potential for widespread use. KEYWORDS: gene delivery, polycationic nucleic acid carrier, biologically degradable linkage, imine linkage, disulfide linkage
1. INTRODUCTION Gene therapy is a therapeutic way that delivers exogenous genetic substances into diseased cells to repair and compensate genetic defect or abnormality of humans that may cause serious diseases.1,2 However, the development of gene therapy remains stagnant nowadays because of the lack of efficient and safe delivery carriers.1 At present, most gene delivery carriers can be mainly divided into virus and nonvirus carriers. Despite virus carriers often having high delivery efficiency, serious immune rejection compromises its translation for clinic use.3 Nonvirus carriers (i.e., cationic polymer and liposome) usually exhibit definite chemical structures, designed functions, nonimmunogenicity, and the potential for large-scale production; thus, we believe that they would be the most promising carriers for gene delivery.4−6 Successful gene delivery requires accomplishing five tasks: (1) condensing nucleic acids into polyplexes to avoid degradation,7,8 (2) targeting to diseased cells,9 (3) rupturing endosomes to help endosomal escape of nucleic acids,10 (4) releasing nucleic acids into cytoplasm,11 and (5) metabolizing into nontoxic substances.12 To achieve these goals, we developed a core−shell delivery system, in that a polyplex was formed by cationic polymers in which the nucleic acid is the core while the selfassembled triblock copolymer is the shell. The core is dependent on the biologically responsive cationic polymers.13 The polymers should be relatively stable when in the process of delivery but degradable in response to tiny intracellular changes. However, degradation often accompanies the decrease of delivery efficiency. Therefore, it is very important to achieve the balance © XXXX American Chemical Society
between stability and degradation of cationic polymers. Meanwhile, the requirements for the delivery of siRNA and DNA are not the same because carriers need to release them in different time points and positions.14−17 That is to say, for siRNA, they can produce biological effect in cytoplasm, which requires carriers to release siRNA quickly into cytoplasm after the step of endosomal escape.16,18 Whereas for DNA, they can only accomplish transcription in nucleus;10,19 thus, it is more appropriate to release DNA near the nucleus rather than an early release. The most ideal situation is that we could use suitable biodegradable linkages that are dependent on different degradation mechanisms to realize the gene release at different release points and speed. Now, imine linkage and disulfide linkage, which can be degraded in acidic environment and redox environment, respectively, are two promising linkages applied in most nonvirus carriers.20−23 In our group, we used imine linkage-based polymers to deliver siRNA and obtained high efficiency in delivery. With regard to DNA, however, it can only be expressed when delivered into a nucleus.24 Hence, different biodegradable linkages should be considered to be directed against different genetic materials or even in a combination together to improve efficiency. Regarding this, comparison of different biodegradable bonds for delivering gene materials on cell- and animal-based levels as well as some obstacles it meets should be further explored. Received: October 24, 2017 Accepted: January 17, 2018
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DOI: 10.1021/acsami.7b16101 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
recorded in D2O with 0.03% (v/v) tetramethylsilane as the internal standard using a Varian Mercury Plus 400 MHz spectrometer, and the Fourier transform infrared (FT-IR) spectrum was recorded in a KBr pellet using a Bruker Optics FT-IR spectrometer. Meanwhile, the molecular weight and polydispersity of the TPSP and DTDPS were determined by size-exclusion chromatography (SEC), with a PEG standard using an Agilent 1260 HPLC Infinity equipped with a refractive index detector and thermostatic (40 °C) gel permeation chromatography column PL aquagel-OH. The mobile phase was ddH2O at the flow rate of 1 mL/min. 2.3. Degradation of Polymers. The degradation behavior of TPSP was determined by incubating the TPSP at 37 °C in buffers of pH 5.0, 6.0, and 7.4, followed by the measurement of molecular weight with SEC. The degradation of the DTDPS was determined by incubating this polymer at 37 °C in 10 mM GSH phosphate-buffered saline (PBS) solution and then utilizing to SEC to measure the molecular weight. 2.4. Preparation and Characterization of Polyplexes. The polyplexes were prepared by adding siRNA solution to TPSP and DTDPS aqueous solution at various mass ratios. The zeta potential and particle size of polyplexes were determined by dynamic light scattering (DLS; Brookhaven Instruments Corporation 90 Plus Particle Size Analyzer). The morphology of the particles was detected by transmission electron microscopy (TEM, FEI Tecnai G2 Spirit BioTwin). The condensing siRNA ability of the two polymers was determined by electrophoresis on a 1% (w/v) agarose gel pretreated with 0.5 mg/mL ethidium bromide in 1× tris−acetate−EDTA buffer at 110 V. The gel was visualized with a UV illuminator (Tanon 2500 Gel Image System). 2.5. In Vitro Cytotoxicity. Cytotoxicity of TPSP and DTDPS were examined by an MTT assay. The TPSP and DTDPS solutions of various concentrations were incubated with PGL3-expressed SMMC 7721 cells for 4 h. MTT solution (20 μL) was added for a further 6 h incubation. Then, the medium of each well was replaced with 150 μL of dimethyl sulfoxide and incubated for extra 10 min. Absorbance values at 570 nm and 630 nm were measured by a Microplate Reader (3M, USA). The value of untreated cells was considered as 100% cell viability. 2.6. In Vitro Silence Efficiency of PGL3 Reporter Gene. The ability of silence PGL3 reporter gene was examined by packing antiPGL3 siRNA into polyplexes with TPSP and DTDPS, followed by incubating these polyplexes in PGL3-expressed SMMC 7721 cells. The cells were seeded into 48-well plates at a density of 1 × 105 cells/well and cultured to 90% confluence in the Dulbecco’s modified Eagle’s medium (Hyclone, USA) containing 10% fetal bovine serum (HyClone, USA). Then, each well was washed by 1× PBS (Hyclone, USA) twice, after which 250 μL of serum-free medium was added. Polyplexes solutionpacked 500 ng antiPGL3 siRNA (50 μL) at various mass ratios were gently filled in each well. After incubation in a 5% CO2 at 37 °C for 4 h, the medium was replaced with a fresh medium, followed by an extra 48 h incubation under the same conditions. To determine the luciferase expressing level, the cells were lysed by lysis buffer (1×, Promega) and the cell debris was removed by centrifugation at 12 000 rpm for 3 min (Eppendorf 5810R Centrifuge, Germany). The supernatant (20 μL) was harvested and then mixed with 20 μL of the substrate solution (Luciferase Assay System, Promega). The luminescence of the expressed luciferase was measured using a Single Tube Luminometer (Berthold Detection Systems GmbH), from which the total protein concentrations in cell lysates were determined using a Micro BCA Protein Assay Kit (Thermo Scientific Pierce). Luciferase activity was determined by relative light units per protein concentrations (μg/mL). Then, the optimal polyplexes were wrapped with triblock membrane at the predetermined ratios, followed by repeating the above protocol. 2.7. In Vivo Tissue Distribution and PGL3 Silence Efficiency of Polywraplexes. Nude BALB/c mice were implanted with SMMC 7721 cancer cells in their back region to build the tumor model animal, ≈200 mm3 in size. Also, the polyplexes formed by antiPGL3 siRNA, TPSP, and DTDPS were covered by triblock copolymer (mPEG45PCL20-maltotriose) according to the reported method25 to form polywraplexes at different triblock copolymer−siRNA mass ratios (w/ w), which were then measured by DLS. Briefly, the polyplex core to form
In this study, we chose imine and disulfide linkages as the biodegradable bonds, spermine as the unit, and 1,4-phthalaldehyde and dithiodipropionic acid as the two linkers to construct two cationic carriers [imine backbone-based polymer (TPSP) and disulfide backbone-based polymer (DTDPS)].20,22 We conducted the physiochemical characterization on polymers and polyplexes formed from them. In addition, the degradability and delivery efficiency of siRNA were tested both in vitro and in vivo. Also, regarding the polyplexes we designed for this project, we added a shell we reported recently25 to form a core−shell delivery system for minimally invasive delivery of siRNA in vivo. The core is polymer-condensed siRNA polyplexes, and the shell is a triblocked copolymer. These two parts have different tasks. The shell can protect polyplexes from the attack of enzymes and other biomolecules in blood. When polywraplexes arrive the desired tissue, the shell will drop down and the polymers help siRNA get into cells. For the shell we used in this project, the anionic multicarboxyl surge block of the membrane is negatively charged, which could be absorbed around the cationic surface of the polyplex by electrostatic interaction, introducing the selfassembly of polywraplexes. The hydrophobic central block could form a 2D isolating layer and make multicarboxyl block arrange regularly. Moreover, the poly(ethylene glycol) (PEG) chain could stabilize the particle and help the nanoparticle achieve long blood circulation. The core of this system is usually designed to be biodegradable in response to specific biological changes in targeted cells, and imine and disulfide linkages were designed for the cores in this project, respectively.
2. EXPERIMENTAL SECTION 2.1. Materials. Spermine, 1,4-phthalaldehyde, glutathione (GSH), and branched polyethylenimine (PEI; 25 kDa) were purchased from Sigma-Aldrich. Bis(2-carboxyethyl) disulfide was purchased from Okyo Chemical Industry (TCI). Cellulose membranes (MWCO = 10 000 Da) were from Thermo Scientific. Anhydrous diethyl ether was produced by Aladdin. Methoxyl-poly(ethylene glycol) (mPEG) 2000 and Nile red were supplied by Alfa Aesar (Tianjin, China). NH2-PEG-COOH was purchased from JenKem Technology (Beijing, China). ε-Caprolactone (ε-CL), maltotriose, benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphatecastros reagent, and rhodamine were supplied by Aladdin Reagent Database Inc. (Shanghai). PEG standard kit (ranging from 106 to 20 100 Da in molecular weight) was acquired from PSS Polymer Standards Service GmbH. Green fluorescent protein plasmid was constructed by GenePharma (Shanghai, China). PGL3 siRNA was constructed by GenePharma and its sequences were 5′CUUACGCUGAGUACUUCGAdTdT-3′. Micro BCA protein assay kit was purchased from Thermo Scientific. 2.2. Synthesis and Characterization of Polymers. The TPSP was synthesized by condensing 1,4-phthalaldehyde and spermine. Briefly, 1 mmol 1,4-phthalaldehyde dissolved in dimethylformamide (DMF) was added dropwise into 1 mmol spermine DMF solution and heated to 120 °C under a water- and oxygen-free condition. After stirring for 24 h, the solution was evaporated and the gained residue was redissolved in deionized water to be dialyzed through a cellulose membrane (MWCO = 10 000 Da) for 48 h. The final product was harvested after lyophilization and stored in −80 °C. The DTDPS was synthesized by condensing bis-(2-carboxyethyl)disulfide and spermine. Similarly, 1 mmol bis(2-carboxyethyl)disulfide was catalyzed by adding activators and then dissolved in DMF solution under the water- and oxygen-free condition. After stirring for 12 h, this mixture solution was added into 1 mmol spermine solution and kept stirred for 24 h. Anhydrous diethyl ether was added, and the precipitation was dissolved in deionized water followed by the dialysis of the same cellulose membrane. The final product was obtained after lyophilization. Formation of two polymers was confirmed by proton nuclear magnetic resonance (1H NMR) and IR. The 1H NMR spectrum was B
DOI: 10.1021/acsami.7b16101 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 1. Reaction scheme of the polymerization for TPSP (A) and DTDPS (B).
Figure 2. Spectroscopic confirmations of TPSP by 1H NMR (A) and FT-IR (B). 2.8. Statistical Analysis. The data collected were presented as mean ± standard deviation (S.D.) from several separate experiments. Statistical analysis was tested by one-way analysis of variance and a value for *P < 0.05, **P < 0.01, or ***P < 0.001 was considered statistically significant.
polywraplexes was prepared by adding cationic polymers into a solution of siRNA at a predetermined w/w ratio. The triblock copolymer, mPEG45-PCL20-maltotriose-COO−, was added to the polyplex solution at the predetermined mass ratio, followed by mixing (for 30 s) and incubating (1 h). Six groups of PBS, naked antiPGL3 siRNA, TPSP (TPSP-anti PGL3 siRNA-copolymer) at the predetermined mass ratio, DTDPS (DTDPS-anti PGL3 siRNA-copolymer) at the predetermined mass ratio, TPSP−siRNA polyplexes, and DTDPS− siRNA polyplexes were injected to the model mice at the dose of 0.5 mg kg−1 through the tail vein. The mice were sacrificed after 24 h, and five main organs and tumors were dissected and imaged under a fluorescent microscope (ZhongKe KaiSheng Medical Technology Co., Ltd, China). At the same time, the model mice were treated with the same group dosage and sacrificed after 48 h. The tissues were dissected, homogenized in liquid nitrogen, and lysated. The luciferase expression was measured and the silencing efficiency was calculated with reference to the saline group as 100%.
3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Polymers. The biodegradable TPSP with imine linkages was synthesized through the reaction of spermine and 1,4-phthalaldehyde (Figure 1A). The structure of TPSP was verified by the disappearance of the aldehyde (−CHO) hydrogen and the newly appeared imine (−CHN−) hydrogen at δ 10.10 ppm and δ 7.55 ppm in its 1H NMR spectrum (Figure 2A). Consistently, FT-IR spectrum of TPSP, as shown in Figure 2B, confirmed the chemical change of the peak from 1693 cm−1 to 1643 cm−1 because of the C
DOI: 10.1021/acsami.7b16101 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 3. Spectroscopic confirmations of DTDPS by 1H NMR (A) and FT-IR (B).
contained no GSH, this polymer was very stable, whereas in 10 mM GSH solution, dramatic degradation could happen within merely 8 h. Compared with that of TPSP, the degradation speed of DTDPS was faster. However, one-point GSH only existed in cytoplasm, meaning that TPSP would be degraded because of the formation of endosomes and lysosomes when the TPSP-based polyplexes were phagocytosed by targeted cells, whereas DTDPS could only be degraded after the polyplexes ruptured lysosome and enter into cytoplasm. 3.2. Characterization of TPSP−siRNA and DTDPS− siRNA Polyplexes. The ability of polymers to condense siRNA into polyplexes was confirmed by gel electrophoresis assay, followed by the measurements of particle size and zeta potential. As is shown in Figure 5, the migration of siRNA was completely
disappearance of aldehyde carbonyl peak (CHO) at 1693 cm−1 and the appearance of imine carbon (CNH) at 1643 cm−1. Similarly, the DTDPS having disulfide bond was synthesized through the reaction of spemine and bis(2carboxyethyl) disulfide (Figure 1B). The DTDPS structure was confirmed by the disappearance of the active hydrogen at δ 12.5 ppm in its 1H NMR spectrum (Figure 3A). In the FT-IR spectrum, Figure 3B shows that the peak of 3423 cm−1(N−H) and 1641 cm−1 (CO) appeared at the same time, which confirmed the formation of the amido bond. The average molecular weight of TPSP and DTDPS was determined by SEC to be 21.5 kDa and 23.5 kDa, respectively, and the SEC values were 1.82 and 2.00, accordingly. Moreover, the degradation behaviors of these two polymers are shown in Figure 4. In a buffer of pH 7.4, the molecular weight of TPSP only
Figure 5. Gel electrophoresis of TPSP−siRNA polyplexes (A) and DTDPS−siRNA polyplexes (B).
stopped when the mass ratio of polymers to siRNA for both TPSP and DTDPS reached 5, which means these two polymers have almost the same strong ability to completely condense siRNA at a small concentration. The average particle size of polyplexes detected by DLS increased with the gradual increase of mass ratios (Figure 6). In detail, particle sizes of the polyplexes formed by TPSP and siRNA increased from 175 nm to 250 nm with the change of polymer to siRNA ratios from 10 to 50. The size of DTDPS−siRNA polyplexes was around 75 nm. The TPSP has a conjugated Π bond which makes it stiffer, contributing to the larger particle size when it wraps the nucleic acid. The zeta potential of the polyplexes ranged within 40−45 mV and 30−35 mV, respectively, in TPSP and DTDPS groups (Figure 6).
Figure 4. Degradation properties of TPSP and DTDPS.
had tiny changes during 24 h which means TPSP is very stable at the pH value of cytoplasm. On the contrary, with the increase of the pH value, the degradation speed accelerated, especially in the beginning 10 h. This result showed that, during the formation from endosome to lysosome, the TPSP would be degraded quickly in the gradual acidic environment."should be "This result showed that the TPSP would be degraded quickly in the gradual acidification process from endosome to lysosome, the TPSP would be degraded quickly in the gradual acidic environment. As for DTDPS, the same tendency exists. When the environment D
DOI: 10.1021/acsami.7b16101 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
cytotoxicity was always below 15 % because of its relatively high positive charge density, and it cannot be degraded in cells. 3.4. Silence Efficiency of TPSP and DTDPS. To evaluate the silence efficiency of TPSP and DTDPS, the antiPGL3 siRNA was condensed by TPSP and DTDPS at various mass ratios and then subjected to PGL3-expressed SMMC 7721 cells for future examination in vitro. Figure 9 shows that, with the increase of
Figure 6. Physiochemical properties of polyplexes formed by two polymers and siRNA.
Moreover, the TEM results were consistent with those measured by DLS (Figure 7). Figure 9. Silence efficiency of polyplexes and polywraplexes formed of siRNA and polymers under serum-free condition.
polymer−siRNA mass ratios, the efficiency of silencing PGL3 reporter gene raised accordingly. At the same time, the silence efficiency of TPSP was always higher than that of DTDPS. This was mainly due to two reasons: the higher zeta potential of polyplexes formed by TPSP and siRNA which helped better endocytosis efficiency when polyplexes surrounded cells and stronger proton sponge effect when TPSP was degraded and each unit could release two free amino groups. When the mass ratio reached 50, the silence effect of TPSP could almost compare with the positive control PEI (25 kDa). Then, polyplexes at a mass ratio of 20 were chosen to form polywraplexes on the ground that the increasing mass ratio of the polyplex from 20 to 50 did not result in significant increase of silencing efficiency. Moreover, polyplexes with higher mass ratios needed more triblock membranes to form polywraplex while the membrane solution may form precipitation at high working concentration. As is shown in Figure 9, the silencing efficiency of the formed polywraplexes was lower than the corresponding polyplexes for the neutral charge of polywraplexes caused by the decreased cellular uptake efficiency of the particles, which then resulted in poor silencing efficiency. 3.5. In Vivo Tissue Distribution and Silence Effect. To obtain the optimal polywraplexes, the zeta potential and the particle size of polywraplexes at different triblock copolymer− siRNA mass ratios were measured. The cores were prepared at the polymer−siRNA mass ratio of 20, of which the particle is 200 nm (TPSP-formed polyplexes) and 80 nm (DTDPS-formed polyplexes). As is shown in Figure 10B, the zeta potential of polywraplexes formed from TPSP−siRNA polyplexes decreased from 8.9 mV to −1.5 mV as the triblock copolymer−siRNA mass ratio increased from 40 to 80, whereas the zeta potential of DTDPS polywraplexes decreased from 12.4 mV to −4.6 mV as the triblock copolymer−siRNA mass ratio increased from 20 to 40. Therefore, the TPSP polywraplexes at a mass ratio of 80 and DTDPS polywraplexes at a mass ratio of 40, of which the corresponding particle size increased to 280 nm and 130 nm, respectively, (Figure 10A), were chosen for the further tissue
Figure 7. TEM images of polyplexes formed of polymers and siRNA (A) TPSP−siRNA polyplexes and (B) DTDPS−siRNA polyplexes.
3.3. In Vitro Cytotoxicity. The cytotoxicity of polymers was examined by MTT assay in PGL3-expressed SMMC 7721 cells under different concentrations from 50 μg/mL to 2 mg/mL and PEI (25 kDa) served as the control. Figure 8 shows that when it
Figure 8. Relative cell viability of PGL3-expressed SMMC 7721 cells treated with TPSP, DTDPS, and PEI 25 K, respectively.
was in the working concentrations (50 μg/mL to 500 μg/mL), the cell viability dropped slightly and remained above 60%, which demonstrated their good biocompatibility. Then, after we increased the test concentrations, we could see the correlated decrease of cell viability to 10 % at 2 mg/mL. However, TPSP always showed a little more cytotoxicity than DTDPS, which may be a consequence of its higher positive charge density26 and hydrophobicity. For the PEI (25 kDa) group, however, its E
DOI: 10.1021/acsami.7b16101 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 10. Particle size and zeta potential of polywraplexes (polyplexes formed of TPSP and siRNA) and polywraplexes (polyplexes formed of DTDPS and siRNA).
Figure 11. (A) In vivo imaging in SMMC 7721 tumor-bearing BALB/c nude mice after intravenous injection of naked siRNA, polywraplexes (polyplexes formed of DTDPS and siRNA) and polywraplexes (polyplexes formed of TPSP and siRNA), DTDPS polyplexes, and TPSP polyplexes after 24 h. (B) In vivo silence efficiency of polyplexes and polywraplexes (n = 6).
From chemical structures of two polymers, aromatically conjugated imine linkages were relatively stiff and contribute to larger size and zeta potential. At the same time, for imine linkage, stronger proton sponge effect, earlier degradation time, and higher zeta potential improve the activity of the polymer in delivering into cells when compared with disulfide linkage. In vitro and in vivo results confirmed the designing philosophy mentioned above. To achieve the goal of delivering siRNA in vivo, imine backbone polymers would be a better choice.
distribution measurement. The tissue distribution of polywraplexes was measured by injecting five dosage forms (naked fluorescent Cy3-siRNA, triblock copolymer−DTDPS−siRNA, triblock copolymer−TPSP−siRNA, DTDPS−siRNA polyplexes, and TPSP−siRNA polyplexes) to experimental mice implanted with SMMC-7721 tumor cells through the tail vein. The animals were sacrificed after 24 h and the organs were imaged under a fluorescent microscope. Figure 11A shows that the siRNA formed in particulate forms mainly accumulated in liver, kidney, and tumor. Further analysis showed that triblock copolymer−TPSP−siRNA polyplexes accumulated the most in the tumor, followed by the triblock copolymer-DTDPS−siRNA polyplexes, and at the same time, the groups of TPSP−siRNA polyplexes, DTDPS−siRNA polyplexes, and naked siRNA did not show any siRNA accumulation in tumor tissues. The results of Figure 11A indicated that the negative-charged siRNA was responsible for the liver accumulation, and copolymer-protected polyplexes helped tumor and cell targeting. Furthermore, Figure 11B shows similar results as we mentioned before. Triblock copolymer−TPSP−siRNA group expressed the lowest luciferase activity which may be due to the most polyplex accumulation, followed by DTDPS group. Meanwhile, naked siRNA and two kinds of polyplex cores did not show any silence efficiency.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected];
[email protected]. Phone: +86 21 34205072. Fax: +86 21 34205072. ORCID
Weien Yuan: 0000-0003-4177-7812 Author Contributions †
J.C. and Y.X. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The study was supported by the Projects of National Natural Science Foundation of China (no. 81570992, 81571261, and 81602099), Science and Technology Development Foundation of Shanghai (17401901000), SUMHS seed foundation project (no. HMSF-16-21-010), Science and Technology Development
4. CONCLUSIONS In the present study, we successfully synthesized two new cationic polymers based on imine linkage and disulfide linkage. F
DOI: 10.1021/acsami.7b16101 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
(19) Vijayanathan, V.; Thomas, T.; Thomas, T. J. DNA nanoparticles and development of DNA delivery vehicles for gene therapy. Biochemistry 2002, 41, 14085−14094. (20) Kim, Y. H.; Park, J. H.; Lee, M.; Kim, Y.-H.; Park, T. G.; Kim, S. W. Polyethylenimine with acid-labile linkages as a biodegradable gene carrier. J. Controlled Release 2005, 103, 209−219. (21) von Harpe, A.; Petersen, H.; Li, Y.; Kissel, T. Characterization of commercially available and synthesized polyethylenimines for gene delivery. J. Controlled Release 2000, 69, 309−322. (22) Cai, X.; Dong, C.; Dong, H.; Wang, G.; Pauletti, G. M.; Pan, X.; Wen, H.; Mehl, I.; Li, Y.; Shi, D. Effective gene delivery using stimulusresponsive catiomer designed with redox-sensitive disulfide and acidlabile imine linkers. Biomacromolecules 2012, 13, 1024−1034. (23) Saito, G.; Swanson, J. A.; Lee, K.-D. Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities. Adv. Drug Delivery Rev. 2003, 55, 199−215. (24) Capecchi, M. R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 1980, 22, 479−488. (25) Ge, X.; Duan, S.; Wu, F.; Feng, J.; Zhu, H.; Jin, T. Polywraplex, Functionalized Polyplexes by Post-Polyplexing Assembly of a Rationally Designed Triblock Copolymer Membrane. Adv. Funct. Mater. 2015, 25, 4352−4363. (26) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 2013, 12, 967−977.
Foundation of Pudong New District, Shanghai, China (PKJ2016Y55 and PWZxq2017-03), and National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (2017ZX09101005-008-002). The study was also partly sponsored by the Interdisciplinary Program of Shanghai Jiao Tong University (nos. YG2015MS06, YG2017MS22, YG2015QN12, YG2017QN56, and YG2016QN22). We appreciate the help from the faculties of Instrumental Analysis Centre (IAC) of Shanghai Jiao Tong University.
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REFERENCES
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DOI: 10.1021/acsami.7b16101 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
