Molecular Strings Significantly Improved the Gene Transfection

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Molecular Strings Significantly Improved the Gene Transfection Efficiency of Polycations Huapan Fang, Zhaopei Guo, Lin Lin, Jie Chen, Pingjie Sun, Jiayan Wu, Caina Xu, Huayu Tian, and Xuesi Chen J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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Journal of the American Chemical Society

Molecular Strings Significantly Improved the Gene Transfection Efficiency of Polycations Huapan Fang,†,‡ Zhaopei Guo,† Lin Lin,† Jie Chen,† Pingjie Sun,† Jiayan Wu,†,‡ Caina Xu,† Huayu Tian,*,†,‡ and Xuesi Chen*,†,‡ †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, China ‡ University of Science and Technology of China, Hefei, Anhui 230026, China ABSTRACT: High transfection efficiency and low cytotoxicity are the two key factors to be considered in the design of gene carriers. Herein, a novel and versatile gene carrier (PLL-RT) was prepared by introducing “molecular string” RT (i.e., p-toluylsulfonyl arginine) onto the polylysine backbone. The introduction of RT string contributed to the formation of multiple interactions between the polycationic gene carriers and cell membrane or DNA, as well as adopting α-helix conformation, all of which would be beneficial to enhance the gene transfection. In addition, RT string grafted onto other polycations such as hyperbranced PEI25k and dendrimer PAMAM could also acquire improved transfection efficiency and low cytotoxicity. Moreover, PLL-RT presented significant tumor inhibition effect in vivo. This work provided an effective strategy for constructing novel gene carriers with high transfection and low cytotoxicity.

1. Introduction Cancers are malignant diseases that pose a serious threat to human health. Common treatments including surgery, chemotherapy and radiotherapy cannot efficiently cure cancer. However, gene therapy is a novel therapeutic tool that has shown great potential for curing cancer thoroughly.1-4 Usually, gene carriers are essential for the success of gene therapy. Polycationic gene carriers have attracted increasing attention in virtue of non-immunogenicity, easy manufacture, flexible properties.5-7 Although some polycations such as polyethyleneimine (PEI),8-10 polylysine (PLL),11 and polyamidoamine (PAMAM),12,13 have already been used for gene transfer, such polycationic gene carriers were urgently needed to increase transfection performance and decrease cytotoxicity for further clinical application. It is generally accepted that the electrostatic interactions (EIs) between gene carriers and DNA or cell membrane will influence the transfection efficiency of polycationic gene carriers.14-16 In general, appropriate EIs contribute to DNA loading, DNA condensation and endocytosis, which are beneficial to efficient transfection. However, too large charge density could result in deadly cytotoxicity, which will hinder the transfection performance. One alternative strategy should be considered to introduce other types of interactions or characteristics by modifying gene carriers to acquire efficient transfection and low cytotoxicity. Cheng’s group introduced hydrophobic perfluorinated alkyl hydrocarbon to dendrimer PAMAM, which significantly improved transfection efficiency.17,18 Schmuck’s group utilized hydrogen bond interactions (HbIs) between oligopeptide and DNA or cell membrane to acquire efficient transfection.19 In addition, Cheng’s group synthesized a series of cationic

polypeptides containing α-helix structure with excellent transfection performance.20,21 However, there were few reports on constructing high efficient gene carrier by simultaneously endowing multiple interactions or characteristics to one polycationic molecule. Combining multiple interactions or characteristics into one polycation could hardly be accomplished unless various kinds of molecules are involved in the construction of the polycation. It usually needs multistep reactions that are very tedious and difficult to repeat. Moreover, the introduction of various types of molecules is likely to consume amino groups of polycations, which would damage the transfection efficiency of gene carriers. Herein, a comprehensive “all in one” strategy was developed to construct a highly efficient gene carrier by introducing multiple interactions or characteristics into one polycation. A “molecular string”, arginine protected by tosyl group (abbreviated as RT string, R refers to arginine, T refers to tosyl), was grafted onto polylysine (PLL) to improve the transfection performance (Figure 1). This strategy possessed the following advantages. (1) In addition to the transformation of the conformation of the polycationic gene carrier (i.e. from a random coil to a α-helix conformation) after “introducing RT string”, multiple interactions were introduced simultaneously including electrostatic interaction (EI), hydrogen bond interaction (HbI) and hydrophobic interaction (HpI). The multilple interactions could synergistically promote the transfection performance. (2) The amount of amino groups of the polycations would not be reduced. An amino group was consumed, meanwhile, another amino group would be supplemented. (3) “Introducing RT string” onto polycations not only acquired significant DNA transfection but also possessed excellent serum resistance and RNA silencing capacity. (4) “Introducing RT string” could be a universal

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transfection enhancing strategy for different kinds of polycationic gene carriers, such as PEI25k and PAMAM. (5) RT string modified PLL could accomplish excellent in vivo tumor inhibition effect by combining therapeutic genes. Molecular structure of RT modified polymers was

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characterized in detail, and the influences of RT groups on transfection behaviors were systematically investigated. “Introducing RT string” provided an enlightened strategy for designing polycationic gene carriers with high transfection efficiency and low cytotoxicity.

Figure 1. Construction of highly efficient gene carriers by introducing multiple interactions and α-helix characteristic into polycations. 2. Results and discussions 2.1 Synthesis and characterization of PLL-RT PLL has been used as polycationic gene carrier for more than ten years because of its excellent biodegradability.22-24 However, its poor transfection efficiency failed to live up to expectations. Therefore, in order to improve the transfection efficiency of PLL, a variety of strategies were designed and implemented.25-28 Herein, PLL was selected as the skeleton, and the effect of introducing RT string onto PLL on the transfection performance was studied. The synthetic route of RT modified PLL (PLL-RT) is shown in Figure 2A. First, Lys(Z)-NCA was synthesized by the method in previous studies29,30 and confirmed by 1H NMR characterization (Figure S1). Then PLL was synthesized through ring-opening polymerization of Lys(Z)-NCA with n-hexylamine as initiator, and de-protected in the presence of

33% HBr/CH3COOH. The degree of polymerization of PLL was 120 based on 1H NMR (Figure S2), and the polymer distribution index (PDI) was 1.23 according to the GPC (Figure S3). PLL-RT was obtained by amide condensation reaction of PLL and Boc-Arg(Tos) and followed by de-protection. “Molecular string” RT was successfully grafted onto PLL based on 1H NMR (Figure S4). Six different PLL-RTs were synthesized, namely, PLL-RT1, PLL-RT2, PLL-RT3, PLL-RT4, PLL-RT5 and PLL-RT6 with 17.1, 33.2, 40.1, 64.1, 91.4 and 117.1 RT groups grafting onto PLLs. The detailed information of PLL-RTs was summarized in Table S1. 2.2 In vitro transfection capacity After the successful synthesis of PLL-RTs, we evaluated the DNA transfection capacity of six different PLL-RTs in different cell lines (MCF-7, HeLa, B16F10, and CT26 cells). The transfection behaviors of PLL-RTs presented the


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following characteristics shown in Figure 2B. All the PLL-RTs possessed better transfection efficiency than PLL, namely, the transfection efficiency was increased once modified with “molecular string” RT. The grating amount of RT groups exhibited obvious effects on the transfection efficiency of PLL-RTs. As increasing number of RT groups were grafted onto PLLs, the transfection efficiency first improved and then reduced. The best transfection efficiency was achieved by PLL-RTs when the PLL-RT4/pDNA mass ratio was 2.5/1. The optimized transfection was 5,000 times higher than that of PLL and one order of magnitude higher compared with PEI25k in MCF-7 cells. In other types of cells, the transfection efficiency

of modified PLL-RT4 was 1,400 times in HeLa cells, 1,600 times in B16F10 cells and 2,000 times in CT26 cells of that of PLL (Figure S5). Moreover, the transfection efficiency of PLL-RT4 was also compared with commercial lipofectamine 2000 (abbreviated as Lipo2000) in different cell lines (MCF-7, HeLa, B16F10, and CT26 cells). As shown in Figure S6, the optimized transfection efficiency of PLL-RT4 was 3.6 times in MCF-7 cells, 4.1 times in HeLa cells, 3 times in B16F10 cells, and 10 times in CT26 cells of that of Lipo2000. Transfection results could clearly demonstrate that the RT string was a powerful transfection enhancer for the PLL, which had long been suffering from poor transfection ability.

Figure 2. (A) Synthetic route of PLL-RT. (B) Transfection efficiency of PLL-RTs in MCF-7 cells, PEI25k and PLL were included as control.



2.3 Verification of multiple interactions between PLL-RT and DNA or cell membrane According to the transfection experiment results in vitro, the transfection efficiency was dramatically improved after introducing RT string to the PLL skeleton. A clear and detailed mechanism is necessary. Phospholipid was the main component of cell membrane, which contained long hydrophobic alkyl chains and phosphate groups. Cardiolipin (CL) also contained long hydrophobic alkyl chains and phosphate groups. The molecular structure of cardiolipin had a great similarity with phospholipid. Therefore, CL could be used as model molecule of cell membrane to study the interactions between PLL-RT4 (or PLL) and cell membrane by surface enhanced infrared absorption spectroscopy (SEIRAS) (Supporting Information Section 2.13, Figure S7). SEIRA spectra were recorded upon the addition of polymers at 1, 5, 10, 30, 60 and 90 min intervals after taking CL membrane as background spectrum. As shown in Figure S8A&B, with the addition of PLL or PLL-RT4, a negative peak appeared at 1706 cm which should belong to the stretching vibration of carbonyl group of CL in hydrophilic environment. In addition, the peak intensity at 1706 cm for PLL-RT4 became increasingly stronger over time while the peak intensity for PLL remained unchanged, which demonstrated that the carbonyl content of CL in the hydrophilic environment decreased with time. Moreover, we observed a blue shift of positive peak from 1743 cm-1 to 1745 cm-1 with the addition of PLL-RT4 and the peak intensity increased with time, which should result from the stretching vibration of dehydrated carbonyl group. The above results fully demonstrated the strong hydrophobic interactions (HpIs) that existed between PLL-RT4 with CL. Similarly, as there were strong hydrophobic groups such as thymine base groups on DNA skeleton,31-33 it should contain strong HpIs between DNA and PLL-RT4 as well. Besides, compared with the addition of PLL, addition of PLL-RT4 induced a small peak at 1220 cm-1, which was obviously red shifted compared to the asymmetric stretching vibration of phosphate groups of CL (1230 cm-1, Figure S7B and Table S2), due to the hydrogen-bonding between PO4- of CL and guanidine group of PLL-RT4.19 Similarly, it can be speculated that strong hydrogen bond interactions exist between PO4- of DNA and guanidine group of PLL-RT4. It was concluded that there existed three types of interactions, namely EIs, HbIs, HpIs between PLL-RT4 and cell membrane or DNA. The significant transfection efficiency improvement was attributed to the RT string introduced onto the PLL backbone, which was beneficial to the multiple interactions between PLL-RT and cell membrane or DNA. Isothermal titration calorimetry (ITC) is another important means of determining the interactions between a molecule and protein or DNA.19,34,35 However, measuring the thermodynamic parameters of interactions between polymer and DNA or cardiolipin was difficult due to the complicated structures of the polymer. Herein, the oligomers Lys4 and Lys4-RT2 shown in Figure S9A were regarded as the model molecules of PLL and PLL-RT4 for studying the interactions of PLL-RT with cell membrane or DNA. Firstly, thermodynamic parameters of interactions between model molecules and cardiolipin were determined by ITC (Figure 3A&B). The corresponding thermodynamic parameters were presented in Table S3. Similar to the SEIRAS results, the thermodynamic parameters of Lys4-RT2 with cardiolipin were significantly different from those of Lys4 with cardiolipin. There were three binding sites

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between Lys4-RT2 with cardiolipin while two binding sites between Lys4 and cardiolipin, demonstrating that the binding mode of PLL-RT4 with cardiolipin was significantly different from that of PLL with cardiolipin. Moreover, we also determined thermodynamic parameters of Lys4 or Lys4-RT2 with DNA (Figure S9B&C). The thermodynamic parameters of interaction between Lys4-RT2 or Lys4 with DNA were presented in Table S4. Three binding sites existed for Lys4-RT2 with DNA but only two binding sites for Lys4 with DNA, demonstrating the significant difference of binding mode between PLL-RT4/DNA and PLL/DNA. According to the experimental results above, it was concluded that it contained HpIs and HbIs between PLL-RT4 and cell membrane or DNA. When the traditional strategy of “improving electrostatic interaction” had encountered bottleneck, the introduction of multiple non-electrostatic interactions would be a useful solution to overcome the difficulty. 2.4 Secondary structure of PLL-RT4 For some carriers such as poly(amino acid)s or peptides, the transfection efficiency was possibly influenced by its secondary structure.20,36 Cheng’s group found that the transfection efficiency of polycationic peptide in a α-helix conformation was considerably higher than that of the peptide in a random coil conformation.20 In general, PLL adopted random coil conformation.37 But the secondary structure deserved investigating after introducing RT string onto PLL. As shown in Figure 3C, not surprisingly, PLL adopted random coil conformation. It was regarded that ε-amino groups of PLL were close to the polymer backbone, resulting in high charge density and strong electrostatic repulsion along the molecular backbone, which drove PLL to be a random coil conformation. However, PLL-RT4 adopted α-helix conformation in aqueous solution at pH 7.4 (Figure 3C). The reason was that RT string grafting onto PLL increased the distance between amino groups and polymer chain, reducing the electrostatic repulsion along the molecular backbone of PLL-RT4. Another important reason should be that HpIs between hydrophobic RT groups would partially offset electrostatic repulsion between amino groups or guanidine groups in PLL-RT4. Due to the above two factors, a kind of water-soluble polypeptide was obtained. The polypeptide not only contained positively charged groups but also adopted α-helix conformation. In addition, interestingly, PLL-RT4 adopted random coil conformation at pH 6.0 (Figure S10). Changes of secondary structures possibly affected the interactions between polymer and cell membrane. 2.5 Cellular internalization According to the above result, PLL-RT4 presented different secondary structures at different pH values. Whether the changes of the secondary structures of PLL-RT4 would affect the interactions between PLL-RT4/DNA and cells must be clarified. The cellular internalization of carriers/DNA complexes was determined by flow cytometry in MCF-7, HeLa, B16F10, and CT26 cells. DNA was labeled with Cy5. The mass ratio of PLL-RT4/DNA and PLL/DNA was fixed at 2.5/1. The uptake efficiency of PLL-RT4/DNA at different pH values was determined in MCF-7 (Figure 4A&B) and HeLa cells (Figure S11). The uptake efficiency of PLL-RT4/DNA at pH 7.4 was almost four times higher in MCF-7 cells and three times higher in HeLa cells than that at pH 6.0. It was well known that zeta potential, particle size and cytotoxicity were


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Figure 3. ITC curves obtained by titrating (C) Lys4 or (D) Lys4-RT2 into cardiolipin in sodium cacodylate buffer (0.01 M) at pH 7.4 and 25 °C. (E) CD spectra of PLL and PLL-RT4 in aqueous solution at pH 7.4. important characteristics that were closely related to the endocytosis behaviors of the carriers/DNA complexes. PLL-RT/DNA complexes showed very similar charge, size and cytotoxicity at pH 7.4 and 6.0 (Figure S12&S13). However, PLL-RT4 adopted α-helix conformation at pH 7.4 and random coil conformation at pH 6.0 (Figure S10). These results clearly demonstrated that the difference in endocytosis of PLL-RT4/DNA at pH 7.4 and pH 6.0 should certainly be attributed to the different conformations of PLL-RT4 at pH 7.4 and 6.0, that is, α-helix at pH 7.4 was helpful for the efficient endocytosis. Furthermore, the uptake efficiency of PLL-RT4/DNA complexes was 11 times higher than that of PLL/DNA

complexes in MCF-7 cells (Figure 4C&D). The high endocytosis efficiency of PLL-RT4/DNA complexes was one key factor of its excellent transfection performance (Figure 2B). Similarly, the uptake efficiency of PLL-RT4/DNA complexes was considerably higher than that in PLL/DNA in HeLa, B16F10, and CT26 cells (Figure S14). The confocal laser scanning microscopy (CLSM) results further demonstrated that PLL-RT4/DNA possessed higher endocytosis than PLL/DNA in various cell lines (Figure S15). Yet, astonishingly enough, the magnitude of the zeta potential of PLL-RT4/DNA was lower than that of PLL/DNA (Figure S16A), which was not beneficial to the cellular uptake of PLL-RT4/DNA. In addition, the particle size of PLL-RT4/DNA was smaller than that of



PLL/DNA (Figure S16B, Figure S17). Nevertheless, to a certain extent, larger size would contribute to endocytosis.38,39 Thus, lower potential and smaller size of PLL-RT4/DNA would be detrimental for the cellular uptake. But the flow

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cytometry results of PLL-RT4/DNA were widely divergent (Figure S14), which demonstrated that the multiple interactions and α-helix structure played a vital role in improving the endocytosis efficiency of PLL-RT4/DNA.

Figure 4. (A) Flow cytometry of PLL-RT4/DNA complexes at different pH values in MCF-7 cells and (B) corresponding fluorescence intensity. (C) Flow cytometry of PLL/DNA and PLL-RT4/DNA complexes in MCF-7 cells and (D) corresponding fluorescence intensity. 2.6 Endosome escape After endocytosis, effective endosomal escape was essential for successful transfection. Herein, CLSM was applied to observe the endosome escape of PLL-RT4/DNA complexes in four cell lines (MCF-7, HeLa, B16F10, and CT26 cells). Nucleus was stained with DAPI (blue), endo/lysosome was stained with Lysotracker Green (green) and DNA was labeled with Cy5 (red). The yellow dots represented the overlap of polymer/DNA complexes (red) and endo/lysosome (green), that is, yellow dots could reflect the co-location of complexes and endo/lysosome. The CLSM images were taken at different time points (1, 3, and 6 h). The yellow dots could be obviously observed at 1 h for PLL-RT4/DNA group in MCF-7 cells, demonstrating PLL-RT4/DNA entering endo/lysosome (Figure S18A). And significantly increased yellow dots were observed in 3 h, which demonstrated more PLL-RT4/DNA had entered endo/lysosome. It was remarkable that a sharp decrease of the yellow dots for PLL-RT4/DNA group was observed from 3 h to 6 h. In the “merge” mode, only the red color was observed, this was because the fluorescence intensity of green channel for PLL-RT4/DNA group decreased at 6 h, which was due to the eventual rupture of endo/lysosome. Therefore, the changes

of the number of yellow dots from 3 h to 6 h could confirm the endosomal escape process of PLL-RT4/DNA, and PLL-RT4/DNA had the ability to escape from endo/lysosome. In contrast, for the PLL/DNA group, there were only a few yellow dots throughout the process, thus the endosomal escape ability of PLL/DNA and PLL-RT4/DNA could not be directly compared through the CLSM images. For this reason, correlation coefficient R value was used to evaluate the endosomal escape ability of polymer/DNA complexes (Figure S18B). R value obtained from CLSM images represented the co-localization degree of polymer/DNA complexes and endo/lysosome. The greater R value was, the more significant the co-localization degree was. Thus the endosomal escape ability of PLL/DNA and PLL-RT4/DNA could be compared through the changes of R value. For PLL/DNA group, the R value was almost unchanged throughout whole process, indicating that PLL/DNA possessed poor endosomal escape ability. However, for PLL-RT4/DNA group, the R value first increased and then decreased. Especially from 3 h to 6 h, the decrease of R value demonstrated that PLL-RT4/DNA had excellent endosomal escape ability. In addition, the similar results were obtained in HeLa, B16F10, and CT26 cells (Figure S19-S21).


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According to the obtained experimental results above, we concluded that RT string introduced into PLL significantly promoted the uptake efficiency and endosomal escape ability, which consequently boosted gene transfection in various cell lines. 2.7 Cell viability Except for high transfection efficiency, low cytotoxicity was another requisite characteristic for outstanding gene carriers. Herein the cell viability of PLL-RT4/DNA and PLL/DNA was determined by MTT assay in various cell lines. As shown in Figure S22A, the cell viability of PLL-RT4/DNA complexes at mass ratio of 20/1 in MCF-7 cells was nearly 100%, indicating that its cytotoxicity was negligible, which was considerably higher that of PLL/DNA complexes. The reason should be that the higher charge density for PLL/DNA complexes led to more cell death.40-42 Similarly, PLL-RT4/DNA complexes did not show obvious cytotoxicity at various mass ratios in HeLa, B16F10, and CT26 cells (Figure S22B-D). According to MTT results, although introducing RT string increased multiple interactions between PLL-RT and cell membrane, it did not result in significant cytotoxicity. 2.8 Antiserum assay The application of polycationic gene carriers in vivo was challenged, because the transfection of polycationic carriers is mainly affected by serum in the blood. This reason was that a lot of negatively charged proteins were included in serum, which preferred to form large particles with positively charged gene carriers, hindering the transfection.43 The antiserum ability was studied by evaluating the transfection efficiency in serum-containing media in four cell lines (MCF-7, HeLa, B16F10, and CT26 cells) (Figure S23A-D). With increasing fetal bovine serum (FBS) content, both of the transfection efficiency of PEI25k/DNA and PLL-RT4/DNA were decreased, but the transfection efficiency of PEI25k/DNA was decreased faster than that of PLL-RT4/DNA. In addition, PLL-RT4/DNA complexes exhibited outstanding serum stability whose particle size remained approximately 100 nm (Figure S24). This phenomenon indicated PLL-RT4/DNA exhibited excellent serum resistance in MCF-7, HeLa, B16F10, and CT26 cells (Supporting Information Section 2.25). 2.9 RNA silence RNA interference (RNAi) is an important technology to treat genetic diseases and cancers.44-48 The requirements of RNA silencing and DNA transfection on gene vectors are different. It would be very fascinating if a gene carrier could both deliver plasmid DNA (pDNA) and siRNA. We determined RNA silencing efficiency in Huh-7 Luc cells (Figure S25). Excitedly, we found that PLL-RT4 had excellent RNA silencing effect and the silencing efficiency was 88% when the mass ratio of PLL-RT4/siRNA was 2.5/1. Nevertheless, the silencing efficiency of PEI25k was only 48% and PLL exhibited negligible silencing efficiency. Generally speaking, pDNA needed to be transported nearby nucleus for DNA transfection, while the RNA sequence needed to be released in the cytoplasm during RNA delivery. Based on DNA transfection and RNA silencing results, PLL-RT4 exhibited an omnipotent carrier which could properly balance the ability of delivering pDNA and siRNA. 2.10 Different kinds of “molecular strings” grafted onto PLL

PLL-RT4 exhibited excellent transfection efficiency in four kinds of cells (MCF-7, HeLa, B16F10, and CT26 cells). A close relationship existed between the properties of a compound and its structure.49-53 The structural characteristic of “molecular string RT” was researched, and other types of “molecular strings” were introduced onto PLL (Figure 5A). The corresponding synthetic methods were shown in Figure S26-S32. Based on the 1H NMR spectra, the “molecular string” numbers of PLL-MS, PLL-Too, PLL-Tos, PLL-Orn, PLL-Arg, PLL-Orn(Tos) and PLL-Arg(NO2) were 67.0, 68.9, 62.0, 66.1, 64.2, 64.1 and 67.8, respectively (Figure S33-S39 and Table S5). The DNA transfection efficiencies of these polymers containing different types of “molecular strings” were determined in MCF-7, HeLa, B16F10, and CT26 cells. As shown in Figure 5B, the transfection results of these polymers in MCF-7 cells exhibited the following characteristics: (1) The transfection efficiency was very different after grafting different types of “molecular strings” onto PLL. (2) Merely hydrophobic “molecular strings” being introduced into PLL would lower the transfection efficiency of PLL, such as PLL-MS, PLL-Too and PLL-Tos. This was because of the reduced charge density after introducing MS string, Too string or Tos string. (3) The transfection efficiency was improved after merely introducing hydrophilic cationic “molecular strings” onto PLL, such as PLL-Arg and PLL-Orn. For PLL-Arg, HbIs between carrier and cell membrane or DNA would be increased after introducing Arg string, which accelerated the transfection. For PLL-Orn, introducing Orn string increased the surface charge of polycation/DNA, boosting the transfection. (4) If the cationic “molecular strings” containing hydrophobic groups were grafted onto PLL, such as PLL-RT4, PLL-Arg(NO2) and PLL-Orn(Tos), the optimal transfection efficiencies were considerably higher than that of PLL-Arg or PLL-Orn, which only contained hydrophilic cationic “molecular string”. This was owing to that the introduction of hydrophobic groups increased the HpIs between carriers and cell membrane or DNA, contributing the cellular uptake. (5) For the cationic “molecular strings” containing hydrophobic groups, if the only differences lay in hydrophobic groups, for example PLL-RT4 and PLL-Arg(NO2), the transfection efficiency between PLL-RT4 and PLL-Arg(NO2) was significantly different, that is, the transfection efficiency of PLL-RT4 was 1.7 times of PLL-Arg(NO2). The reason was that the hydrophobicity of p-toluene sulfonyl group was stronger than nitro group, which would contribute to the cellular uptake of PLL-RT4/DNA complexes. (6) When the hydrophobic groups for molecular string” were the same, the difference only existed in category of amino acids, such as PLL-RT4 and PLL-Orn(Tos), the transfection efficiency of PLL-RT4 was 2.1 times of PLL-Orn(Tos) in MCF-7 cells, which was due to strong HbIs between guanidine group of PLL-RT4 and cell membrane. (7) For cationic “molecular string” containing hydrophobic groups such as PLL-RT4, PLL-Arg(NO2) and PLL-Orn(Tos), their transfection efficiency were several times higher than that of the “golden standard” PEI25k. (8) The DNA transfection experiment was also carried out in HeLa, B16F10, and CT26 cells, exhibiting similar transfection results to the transfection in MCF-7 cells (Figure S40). After different types of “molecular strings” were grafted onto PLLs, these polymers exhibited significant differences in transfection efficiency. This phenomenon should be closely related to the physicochemical properties of polymers/DNA



complexes. First, the zeta potentials of polymers/DNA complexes were determined at the mass ratio of 2.5/1 (Figure S41A). When these “molecular strings” only containing the hydrophobic groups were grafted onto PLL, such as PLL-MS, PLL-Too and PLL-Tos, the reduced zeta-potentials were observed. This was because the number of amine groups of PLL was reduced when PLL was grafted with hydrophobic “molecular strings”, which led to the sharp decline of charge density. For “molecular strings” with two cationic groups such as that in PLL-Arg and PLL-Orn, the slight change of zeta potential was observed, which was because the introduction of “molecular strings” increased the molecular weight of structural units and the zeta potential would not change a lot at the same mass ratio. Similarly, PLL-Arg(NO2) and PLL-RT4, which contained “molecular strings” with hydrophobic group, resulted in a larger molecular weight of their structural units than PLL-Arg. Therefore, the surface charges of PLL-Arg(NO2)/DNA and PLL-RT4/DNA were lower than that of PLL-Arg/DNA at the same mass ratio. For PLL-Orn(Tos), Orn(Tos) string only included one positive charge, the zeta potential of PLL-Orn(Tos)/DNA was lower than that of PLL-RT4/DNA and PLL-Arg(NO2)/DNA. Based on the transfection efficiency and the corresponding zeta potential results, appropriate surface charge of polymer/DNA complexes was the fundamental guarantee of high efficient transfection. For PLL-MS, PLL-Too and PLL-Tos, the zeta potentials of polymer/DNA complexes were too low to transfect efficiently. For PLL-Orn(Tos), PLL-Arg(NO2) and PLL-RT4, the surface charge of polymer/DNA complexes was suitable for efficient transfection. At the same time, the particle sizes of these polymers/DNA complexes were determined (Figure S41B). For PLL-MS, PLL-Tos and PLL-Too group, the particle sizes of their corresponding complexes were considerably larger than other groups. It was because the charge density for PLL-MS, PLL-Tos and PLL-Too was too small to effectively condense DNA. In addition, the particle size of PLL-Orn/DNA complexes was considerably smaller than that of PLL/DNA complexes, which was due to the larger charge density of PLL-Orn than PLL. Compared with PLL, the guanidine groups in PLL-Arg preferred to form strong hydrogen bond with phosphate groups of DNA, which could effectively compress DNA into smaller size. For PLL-Orn(Tos), strong HpIs existed between phenyl group of PLL-Orn(Tos) and DNA, which contributed to the formation of nanoparticles. For PLL-Arg(NO2) and PLL-RT4, there existed strong HpIs and HbIs between these two kind of polymers and DNA, which could condense DNA efficiently. According to the transfection and the particle size results, the particle size of polymer/DNA complexes was an important factor for transfection. For PLL-MS, PLL-Too and PLL-Tos, the particle size of polymer/DNA was too large, thus, efficient transfection could not achieved. On the contrary, for PLL-Orn(Tos), PLL-Arg(NO2), and PLL-RT4, the suitable particle size of polymer/DNA complexes was the prerequisite for their high transfection efficiency. In addition, the CD spectra of these polymers with different types of “molecular strings” in aqueous solution were measured (Figure S42). Random coil conformation was observed for PLL-MS, which was attributed to the small hydrophobic groups of PLL-MS that barely promoted the

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formation of α-helix structure. PLL-Too and PLL-Tos exhibited standard α-helix structure, which owed to strong hydrophobicity of phenyl group and the weak electrostatic repulsion of the side chains of PLL-Tos or PLL-Too. For PLL-Orn and PLL-Arg, the strong electrostatic repulsion between the side chains of PLL-Orn or PLL-Arg hindered the formation of α-helix structure. PLL-Orn(Tos) and PLL-Arg(NO2) adopted α-helix conformation, whose reason was that the strong hydrophobicity of phenyl group of PLL-Orn(Tos) or nitro group of PLL-Arg(NO2) enhanced the helicity. According to the previous statements (Figure 4A&B, Figure S10), α-helix conformation of PLL-RT4 was conducive to improving transfection efficiency. Nevertheless, PLL-Too or PLL-Tos which adopted standard α-helix conformation showed poor transfection efficiency. Therefore, the helical structure could play a positive role in transfection efficiency only when combined with multiple interactions such as EIs and HbIs. We had determined the transfection efficiency, zeta potential, particle size and CD spectra of the polymers with different “molecular strings”. The relationship between transfection efficiency and the structural characteristics of “molecular strings” was summarized in Table 1. The structural characteristics of “molecular strings” included the interactions between polymers with cell membrane or DNA, such as electrostatic interaction (EI), hydrogen bonding interaction (HbI) and hydrophobic interaction (HpI), as well as the α-helix conformation of the polymers. “Solid star” represented that there existed this kind of interactions or α-helix characteristics. The larger the number of “solid star” was, the stronger the strength of interactions or α-helix characteristics was. On the contrary, the smaller number of “solid star” represented that the strength of interactions or α-helix characteristics was much low or negligible. For example, PLL possessed high charge density, therefore, there exist strong EIs between PLL and cell membrane (or DNA), the corresponding number of “solid star” was two. The HpIs between PLL and cell membrane (or DNA) were negligible, and the corresponding number of “solid star” was zero. Although PLL possessed high charge density, negligible HpIs and very weak HbIs occurred between PLL and cell membrane or DNA, and the degree of helix was very low, all of which resulted in poor transfection efficiency. For PLL-MS, PLL-Too and PLL-Tos, the transfection efficiency was very low, which was because their low charge density was detrimental to compression of DNA. The transfection efficiency for PLL-Orn/DNA or PLL-Arg/DNA was considerably higher than PLL/DNA, which should be due to higher charge density of PLL-Orn and strong HbIs between PLL-Arg and cell membrane or DNA. The transfection efficiency of PLL-Orn(Tos) or PLL-Arg(NO2) was considerably higher than that of PLL-Orn or PLL-Arg. The reason was that the introduction of hydrophobic group improved the HpIs between carriers and cell membrane or DNA and enhanced helicity of PLL-Orn(Tos) and PLL-Arg(NO2), which contributed to the transfection efficiency. The transfection efficiency of PLL-RT4 was higher than that PLL-Orn(Tos) and PLL-Arg(NO2). This finding should be attributed to the fact that the hydrophobicity of phenyl group of PLL-RT4 was stronger than that of nitro group of PLL-Arg(NO2); the HbIs and EIs between PLL-RT4 and cell membrane or DNA were stronger than that between PLL-Orn(Tos) and cell membrane or DNA.


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Figure 5. (A) Different types of “molecular strings” grafted onto PLL backbone. (B) DNA transfection of PLL grafted with different types of “molecular strings” in MCF-7 cells. Based on the above results and analysis, the introduction of RT string could significantly enhance the transfection efficiency of carriers by introducing multiple interactions (EIs,

HbIs, and HpIs) simultaneously.


and

promoting

α-helix

formation


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Table 1. Relationship between transfection efficiency and multiple interactions or secondary structure for PLL grafted with different types of “molecular strings”. EI: electrostatic interaction, HbI: hydrogen bond interaction, HpI: hydrophobic interaction, DS: degree of the spiral, TF: transfection efficiency.

2.11 Other polycations grafted with RT string Given that the transfection efficiency was enhanced significantly after introducing RT string onto PLL, whether RT string introduced to other conventional polycationic gene carriers could increase the transfection efficiency remained unclear. Herein, RT string was introduced into PEI25k and dendrimer G4 PAMAM, respectively (Figure S43-S46, Table S6&S7). The transfection assay of PEI25k-RTs or G4-RTs was carried out in various cell lines. The optimal transfection efficiency of PEI25k-RT2 was 6 times in MCF-7 cells, 5 times in HeLa cells, 5.6 times in B16F10 cells, and 5.8 times in CT26 cells of PEI25k/DNA (Supporting Information Section 2.27, Figure S47A-D). In addition, the optimal transfection efficiency of G4-RT2/DNA was 3.5 times in MCF-7 cells, 5 times in HeLa cells, 7.4 times in B16F10 cells, and 6 times in CT26 cells of G4 PAMAM/DNA (Supporting Information Section 2.27, Figure S48A-D). In addition, the cell viability of PEI25k-RT2/DNA (or G4-RT2/DNA) was considerably higher than that of PEI25k/DNA at a high mass ratio such as 20/1 in MCF-7, HeLa, B16F10, and CT26 cells (Supporting Information Section 2.27, Figure S49A-D, Figure S50A-D). Based on the above results, the RT string introduced into conventional polycationic gene carriers would be an ideal strategy of promoting the transfection efficiency and decreasing the cytotoxicity. This strategy provided a universal platform for developing more high-performance polycationic gene carriers. 2.12 In vivo antitumor treatment The in vivo antitumor activity of PLL-RT4 was evaluated in the CT26 tumor-bearing mice model. The tumor model was

established by subcutaneously inoculating CT26 cells into BABL/c mice. shVEGF (plasmid DNA expressing shVEGF, the same as below) was exploited as therapeutic gene. Vascular endothelial growth factor (VEGF) could stimulate the proliferation of endothelial cells in tumor tissue, promote the formation of tumor vessels and accelerate the growth of tumor.54-56 shVEGF could inhibit the expression of VEGF in the tumor tissue and suppress tumor growth. The tumor-bearing mice were intratumorally administered with PBS, shVEGF, PLL/shVEGF complexes and PLL-RT4/shVEGF complexes, respectively. All groups received injections every other day and were injected for six times in total. The tumor volume and body weight changes were recorded every other day. As shown in Figure 6, we observed that the antitumor effect of different treatment groups exhibited obvious differences. PBS and shVEGF groups both presented very rapid tumor growth, which demonstrated that the injection of simple therapeutic gene without carrier couldn’t effectively suppress the tumor growth. For the PLL/shVEGF group, the growth of tumor was inhibited to some extent. Moreover, the inhibition of tumor growth was the most significant for PLL-RT4/shVEGF group, which exhibited negligible tumor growth. Besides, the tumor weight of PLL-RT4/shVEGF complexes group was the smallest of all the groups (Figure S51). These results suggested that PLL-RT4/shVEGF complexes possessed an excellent anti-tumor effect. In addition, the body weight of the mice of PLL-RT4/shVEGF complexes group during the treatment remained stable (Figure S52), demonstrating no systemic toxicity.


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The hematoxylin-eosin (H&E) results demonstrated normal tissue morphology and no obvious pathological abnormalities for the main organs (heart, liver, spleen, lung, and kidney) of PLL-RT4/shVEGF group (Supporting Infomation Section 2.30, Figure S53). PLL-RT4/shVEGF group showed the most significant tumor tissue destruction, and exhibited an excellent therapeutic effect. Likewise, immunofluorescent staining assay indicated that the tumor neovascularization of PLL-RT4/shVEGF group was significantly inhibited (Supporting Information Section 2.31, Figure S54). In addition, the antitumor effect of the PLL-RT4/shVEGF in the VEGF

mRNA level was evaluated by qRT-PCR technology. The VEGF mRNA content of PLL-RT4/shVEGF group was the lowest, which indicated that PLL-RT4 could effectively mediate the silence of VEGF mRNA to achieve the most efficient antitumor effect (Supporting Information Section 2.32, Figure S55). Moreover, the ELISA assay results demonstrated that PLL-RT4/shVEGF could remarkably inhibit the expression of VEGF in tumor tissue, which ultimately significantly suppressed the tumor growth (Supporting Information Section 2.33, Figure S56).

Figure 6. (A) Changes of tumor volume of BABL/c mice administered with PBS, shVEGF, PLL/shVEGF and PLL-RT4/shVEGF. (B) Images of excised tumors at the end of treatment. 3. Conclusions In summary, an efficient strategy of introducing “molecular string” RT to traditional gene carriers was developed to significantly improve the transfection efficiency and reduce the cytotoxicity. RT string grafted onto PLL would introduce multiple interactions including EIs, HbIs, and HpIs between polymer and cell membrane or DNA. In addition, PLL grafted with RT string adopted α-helix conformation, which would also be helpful for gene transfection by improving the cellular uptake. In addition, the RT string introduced into PEI25k and PAMAM could also improve the transfection efficiency and decrease the cytotoxicity. Finally, in vivo experiment showed that PLL-RT4/shVEGF exhibited an excellent antitumor effect and negligible pathological abnormalities. This work provided an ideal strategy for constructing polycationic gene carriers with high transfection efficiency and low cytotoxicity. More polycations modified by introducing RT string will be developed for researching high-performance gene carriers in the future.

Corresponding Author *E-mail: [email protected] *E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (21474104, 51520105004, 51390484, 51403205 and 51503200), National program for support of Top-notch young professionals, Jilin province science and technology development program (20160204032GX, 20180414027GH).

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Molecular Strings Significantly Improved the Gene Transfection

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