Virus Spike and Membrane-Lytic Mimicking Nanoparticles for High

Jun 22, 2018 - Virus Spike and Membrane-Lytic Mimicking Nanoparticles for High Cell Binding and Superior Endosomal Escape. Shuai Liu† , Jixiang Yang...
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Virus Spike and Membrane-Lytic Mimicking Nanoparticles for High Cell Binding and Superior Endosomal Escape Shuai Liu,† Jixiang Yang,† Huiting Jia,† Hao Zhou,‡ Jiatong Chen,‡ and Tianying Guo*,† †

Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, College of Chemistry and Department of Biochemistry and Molecular Biology, College of Life Science, Nankai University, Tianjin 300071, China

‡

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ABSTRACT: Virus-inspired mimics for gene therapy have attracted increasing attention because viral vectors show robust efficacy owing to the highly infectious nature and efficient endosomal escape. Nonetheless, until now, synthetic materials have failed to achieve high “infectivity,” and especially, the mimicking of virus spikes for “infection” is underappreciated. Herein, a virus spike mimic by a zinc (Zn) coordinative ligand that shows high affinity toward phosphate-rich cell membranes is reported. Surprisingly, this ligand also demonstrates superior functionality of destabilizing endosomes. Therefore, the Zn coordination is more likely to imitate the virus nature with high cell binding and endosomal membrane disruption. Following this, the Zn coordinative ligand is functionalized on a bioreducible cross-linked peptide with alkylation that imitates the viral lipoprotein shell. The ultimate virus-mimicking nanoparticle closely imitates the structures and functions of viruses, leading to robust transfection efficiency both in vitro and in vivo. More importantly, apart from targeting ligand- and cell-penetrating peptide, the metal coordinative ligand may provide another option to functionalize diverse biomaterials for enhanced efficacy, demonstrating its broad referential significance to pursue nonviral vectors with high performance. KEYWORDS: virus spike mimic, membrane-lytic, cell binding, endosomal escape, zinc coordinative ligand

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INTRODUCTION Gene therapy holds great potential in contemporary medicine; however, to date, its clinical translation has not been ideally realized with regard to insufficient gene delivery.1−4 Natureinspired pursuit has elicited viruses that demonstrate high efficiency for cell transduction both in vitro and in vivo.5−7 Nonetheless, viruses are associated with immunogenicity and sophisticated manufacture.8−10 Alternatively, nonviral vectors of low cost and safety profiles have raised up11−14 but are perplexed with poor efficacy especially in vivo. The defect is largely attributed to their lack of the viral, infectious nature and endosomal escape functionality. Chemistry and polymer advances have made it possible to integrate the advantages of viruses and nonviruses through designing artificial virus-imitating biomaterials. Encouragingly, some mimics with viral morphologies and functions, such as capsid, surface topography, and nucleic acid release, have been constructed.15−18 However, the major obstacle is that these mimics are essentially different from viruses. It is difficult to achieve virus infectivity by synthetic polymers, and the mechanism in viral endosomal escape is not dependent on © XXXX American Chemical Society

the pH-responsive proton sponge effect of cationic polymers.16,19 Both virus functionalities of infectivity and endosomal escape are underappreciated until now. Therefore, rational design strategies of far more “infectious” virusmimicking viral structures and functionalities are of great significance. Viruses, such as lentiviruses, consist of nucleic acid, an envelope-like shell, and patched glycoprotein spikes.20 Their great promise lies in their initial cell recognition by glycoprotein spikes, cellular uptake facilitation by the lipoprotein envelope, subsequent endosomal escape by exposed membrane-lytic protein or peptide, and ultimate nucleic acid release from the protein shell into the cytoplasm.20,21 Understanding the structure−function relationship of viruses can benefit the design of virus-like polymers with both high efficacy and safety profile. Virus spike is regarded as the first touch toward cell membranes and is identified to be significant Received: April 29, 2018 Accepted: June 22, 2018 Published: June 22, 2018 A

DOI: 10.1021/acsami.8b06934 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

in methanol, and the mixture was stirred at 40 °C for 48 h. DDAC purification was conducted using the silica gel column (54% yield). For PC1, PC2, and PC3 polymer synthesis, calculated PLL and CBA and excess triethylamine were mixed in methanol, and the reaction continued at 60 °C for 48 h (Table S1). To synthesize PCE, ET was added to the mixture that had prepared PC2, and the reaction continued at 60 °C for another 48 h. Equimolar DDAC and zinc nitrate hexahydrate (Zn(NO3)2·6H2O) were dissolved in methanol and stirred at 40 °C for 3 h to obtain Zn-DDAC. To prepare ZnPCED, Zn-DDAC was added to the mixture that had prepared PCE, and the reaction continued at 60 °C for 48 h. All the products were precipitated in ethyl ether three times and dried under vacuum. Hemolysis Assay. The membrane-disruptive activity of ZnDDAC and Zn-PCED was evaluated using a hemolysis model. The sheep red blood cells (RBC) were washed with phosphate-buffered saline three times and resuspended at a pH of 7.4 and 5.7, respectively. Materials at various concentrations were added to RBC suspensions in a 96-well plate. After 1 h incubation at 37 °C, solutions were centrifuged and the released hemoglobin within the supernatant was measured by a microplate reader at a wavelength of 541 nm. Two controls were prepared by resuspending RBC either in buffer alone (negative control) or in deionized water (positive control). In Vitro Gene Transfection. To assay gene transfection, cells (293T, HCT116, CT26, and primary SC) were seeded in 96-well plates at a density of 1 × 104 cells per well and cultured for 24 h. Afterward, polyplexes were prepared, and 0.25 μg DNA was utilized for each well. Fresh cell culture media were mixed with formulated polyplexes and used to replace the previous media in 96-well plates. For commercial transfection reagents, polyethylenimine (PEI) was utilized at N/P = 23:1 (w/w = 3:1), and Lipo2000 was used as per the protocol. After 48 h incubation, green fluorescent protein expression was evaluated and imaged with an inverted fluorescence microscope. Gluciferase activity was detected according to the protocol and plotted as relative light units (RLU) per 10 000 cells. Mechanistic Studies. HCT116 cells were seeded in a 96-well plate at a density of 1 × 104 cells per well and cultured for 24 h. Prior to transfection, cells were preincubated with 50 μM CPZ, 2.5 mM methyl-β-cyclodextrin (MβCD), and 75 μM amiloride hydrochloride (AM) for 1 h. Then, PC2/Cy3-DNA, PCE/Cy3-DNA, and ZnPCED/Cy3-DNA polyplexes at a weight ratio of 10:1 were prepared and added to each well. DNA (0.1 μg) was utilized for each well. After 2 h incubation, Cy3-positive cells were compared using flow cytometry to determine whether inhibitors influenced the polyplex cellular uptake. In addition, to decide whether the cellular uptake of polyplexes was energy-dependent, polyplexes were added to cells and incubated at 4 °C for 2 h. Prior to the polyplex addition, both polyplexes and cells were incubated at 4 °C for 15 min. For inhibitor influence on gene transfection, HCT116 cells were preincubated with respective inhibitors for 1 h, and polyplexes were added to each well. After 2 h incubation, previous cell culture media were replaced and cells were incubated for another 46 h. In Vivo Gene Transfection. All experimental procedures were approved and in accordance with the Animal Experimentation Ethics Committee of Nankai University. Female BALB/c mice (4−5 weeks old, ∼20 g) were chosen for the experiment. For CT26 tumor inoculation, 200 μL of CT26 cell suspensions (1.0 × 106 cells/mL) was subcutaneously injected into the right back of the mice. When tumor sizes reached 80−100 mm3, 100 μL polyplexes (PC2/DNA, PCE/DNA, Zn-PCED/DNA, PEI/DNA, and Lipo2000/DNA) (15 μg luciferase plasmid was used for each sample) was injected into the tumor. The synthetic polymer/DNA weight ratio was 10:1. PEI was utilized at N/P = 23:1 (w/w = 3:1), and Lipo2000 was used as per the protocol. After 48 h, the tumor tissues were harvested from mice, collected in lysis buffer, and mechanically homogenized. Lysate was cleared by spinning at 12 000g for 15 min at 4 °C and assayed for luminescence. Luminescence was measured and normalized by protein content, determined using a BCA Protein Assay Kit, and reported as RLU per milligram of protein.

to the infectious nature. However, to date, the actual functionality of virus spike mimicking high cell binding is underrated, and the synthetic materials mimicking the viral infectivity performance are still far from satisfactory. Niu et al.20 mimicked the virus topological structure and constructed rough particle surfaces imitating the patched spikes. Nonetheless, it is difficult to induce high infectivity only by the spike topography; therefore, developing a new type of spike mimics from functionality seems more promising. Previously, a zincdipicolylamine (Zn-DPA) analog was demonstrated to have high affinity to phosphate moieties.22−24 Considering that there are numerous phosphate components in the lipid bilayer, we speculate that Zn-DPA analogs may perfectly mimic the virus spikes to recognize cell membranes, particularly beneficial to the “infectivity” behavior. To the best of our knowledge, metal coordination imitating these infective spikes for gene delivery has never been reported. Moreover, this continual perturbation of the membrane microenvironment attributed to a strong interaction between the Zn coordinative ligand and the biological membrane may destabilize the endosomes. To date, no metal coordination facilitating endosomal escape has ever been reported. However, for the purpose of enhanced membrane fusion and permeability, long alkyl chains have been exploited to mimic lipids.25−27 After all, the alkyl chains and Zn coordinative ligand are aimed to be attached on envelope-like shells, which are usually tailored to release the nucleic acid timely by biological responses.28−31 As a proof of concept, natural ε-polylysine (PLL, Mw < 5000) was first cross-linked by disulfide bonds to obtain PC polymers. Afterward, PC was modified with 1,2-epoxytetradecane (ET) and a disulfide-containing Zn coordinative DPAbased ligand (Zn-DDAC) to give a virus-mimicking vector (Zn-PCED). The Zn-DDAC coordinative ligand was aimed to imitate the virus spikes for cell recognition, and the strong interactions between the ligand and cell membranes might largely benefit the “infectivity.” As a surprising benefit, this ligand might also destabilize the endosomes by continuous perturbation of endosomal membranes. Next, the cross-linked peptide with alkyl chain functionalization was expected to mimic the virus lipoprotein shell composition and functionality of packaging DNA from enzymatic degradation and facilitating the membrane fusion activity. Ultimately, DNA release after internalization could be achieved through disulfide cleavage between PLL and PLL as well as PLL and Zn-DDAC. The viral-mimicking structure of Zn-PCED is anticipated to overcome the in vitro/in vivo disconnects shown by many other cationic polymer vectors.

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EXPERIMENTAL SECTION

Synthesis of Virus-Mimicking Zn-PCED. To synthesize ZnPCED (Figures S1−S4), ligand DDAC was first prepared. Alpha, alph′-dichloro-p-xylene (Dx, 16 mmol), and 2,2′-dipicolylamine (DPA, 8 mmol) were dissolved in dichloromethane, followed by excess anhydrous potassium carbonate (K2CO3) addition. After stirring at room temperature for 48 h under N2 atmosphere, the product (DxDA) was purified by a silica gel column (75% yield). Afterward, 5-amino-1-pentanol (AP, 10 mmol) and excess K2CO3 were mixed in ethanol, and DxDA (2.4 mmol) was added dropwise. The reaction occurred at 60 °C for 12 h. The product was purified by suspending it in water and extracting with dichloromethane (CH2Cl2). Then, the organic phase was collected, dried over anhydrous sodium sulfate (Na2SO4), and concentrated under vacuum to give DxDA-AP (82% yield). To prepare DDAC, DxDA-AP (0.75 mmol) and cystamine bisacrylamide (CBA; 1.5 mmol) were dissolved B

DOI: 10.1021/acsami.8b06934 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION Synthesis of Virus-Mimicking Polymers. The compositions and functions of the virus-mimicking Zn-PCED polymer are shown in Figure 1. PLL shows superior biocompatibility

protection. PC1 and PC2 were prepared with different crosslinking densities and similar molecular weights (Figure S5); however, more cross-linking degree (PC3) would induce gel formation (Tables S1 and S2). The obtained PC2 polymer was sequentially functionalized with alkyl chains (termed PCE polymer) and the Zn-DDAC ligand (termed Zn-PCED polymer) for enhanced membrane fusion and “infectivity” functionalities, respectively (Figures S1−S3). The structure and composition were determined by 1 H NMR characterization (Figure S4). Characteristic peaks of methyl groups in alkyl chains appeared at 0.86 ppm, and peaks at 7.0−8.5 ppm were assigned to phenyl and pyridyl groups of DDAC residues. Efficient Zn coordination of DDAC was confirmed by thin-layer chromatography (Figure S6). Next, PC, PCE, and Zn-PCED polymers were utilized to condense DNA for further polyplex characterization and gene expression. Zn Coordinative Functionalization Improves DNA Package, Cellular Uptake, and Endosomal Escape. For efficient delivery systems, cargos should be first protected by vectors and released in an intracellular environment.34−37 As depicted in Figure S7, PLL, PC, and Zn-PCED condensed DNA tightly at w/w of 5:1 and 10:1. Afterward, disulfide bond linkage provided a convenient route for material biodegradation, as reductive reagent GSH largely exists in intracellular compartments (1−10 mM).38−40 Consequently, GSH-triggered degradation facilitated DNA release from both the PC/ DNA and Zn-PCED/DNA polyplexes, demonstrating the potential of DNA unpacking in an intracellular environment (Figure 2a). Zn-PCED condensed the DNA to formulate polyplexes with low particle sizes and moderate zeta potentials (Figure 2b,c), both appropriate for cellular uptake.38,41 It is worth noting that Zn-PCED/DNA polyplexes exhibited smaller sizes than the PC and PCE counterparts, owing to their high binding of the metal coordinative ligand toward phosphate moieties of DNA. Characterized by transmission

Figure 1. Design of the virus-mimicking gene delivery system. (a) Chemical compositions and functions of the Zn-PCED polymer. (b) Zn-PCED condenses DNA to formulate a polyplex with viral structures and functions. Both the high affinity between Zn coordinative residue and cell membrane and the lipopeptide shell that facilitates the polyplex cellular uptake contribute to high “infectivity.” DNA release is achieved by GSH-triggered disulfide cleavage once in the cytoplasm.

and biodegradability; furthermore, it enables further functionalization.32,33 Natural PLL with low molecular weight was cross-linked by glutathione (GSH)-reducible disulfide bonds (termed PC polymers), acting as the major shell for DNA

Figure 2. Biophysical characterization of the virus-mimicking Zn-PCED vector. (a) DNA release assay by GSH. “+” represents the presence of 10 mM GSH, “−” represents the absence of GSH, and the polymer/DNA weight ratio is 10:1. (b) Particle sizes of polyplexes. Zn-PCED/DNA polyplexes show the smallest sizes. (c) Zeta potentials of the polyplexes. (d) Adsorption behavior of QCM electrodes, DDAC, and Zn-DDAC− immobilized electrodes exposed to 293T cells. *p < .05 indicates superior cell affinity compared to DDAC. C

DOI: 10.1021/acsami.8b06934 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 3. Virus-mimicking Zn-PCED mediates robust endosomal escape. (a) Hemolysis activity of DDAC and Zn-DDAC at different concentrations (pH = 7.4). *p < .05 indicates superior hemolysis compared to DDAC. (b) Hemolysis activity of polymers at different concentrations (pH = 7.4 or 5.7). Statistical significances are indicated at *p < 0.05. (c) Endosomal escape of PCE/Cy3-DNA and Zn-PCED/Cy3DNA polyplexes in Hela cells. The polymer/DNA weight ratio is 10:1, and 0.1 μg Cy3-DNA per well is used. The scale bar is 20 μm.

shown in Figure 3a, the hemolysis activity of DDAC was drastically enhanced after Zn coordination; meanwhile, ZnDDAC preserved much higher cell viability than DDAC (Figure S9). The reason is that relatively toxic pyridyl groups are occupied by Zn coordination, avoiding their direct cell touching. These results indicate that Zn-DDAC can accelerate the endosomal escape without inducing high cytotoxicity. As expected, Zn-PCED with Zn-DDAC residues showed higher hemolysis activity than PCE; however, the membranedisruptive functionality at pH 5.7 was similar to that of pH 7.4 (Figure 3b). We deduce that hemolysis promotion is not dependent on the acidity-triggered proton sponge effect but is caused by the perturbation of interactions between Zn-DDAC and membranes. As a consequence, on the one hand, alkylation and Zn coordination both facilitated the polyplex cellular uptake, and strong red fluorescence was observed for ZnPCED/DNA polyplexes (Figure S10). On the other hand, ZnPCED/DNA polyplexes escaped more at 2 h than 0.75 h incubation (Figure S11), and they showed superior endosomal escape functionality than their PCE counterparts (Figure 3c), indicating facilitated endosomal escape by Zn coordination. These results demonstrate the availability of virus-mimicking Zn-PCED as an efficient gene vector. Moreover, the enhanced endosomal escape (Figure 3c) together with the smaller size (Figure 2b) after Zn coordination reveals that some Zn-DDAC ligands are incorporated inside the nanoparticles, but some are

electron microscopy, Zn-PCED/DNA polyplexes showed spherical morphology (Figure S8). Furthermore, virus spikemimicking Zn-DDAC residues showed high affinity toward cells. As shown in Figure 2d, the interaction strength between 293T cells and Zn-DDAC or DDAC was quantified by quartz crystal microbalance (QCM). Zn-DDAC or DDAC is modified on the gold electrode of QCM via Au−S bond formation, and the frequency shift is proportional to the mass change on the QCM electrode.42,43 As strong interaction exists between gold and cells,44,45 the naked gold electrode exposed to cells resulted in a frequency shift of 29.9 Hz. However, the frequency shift decreased to 23.5 Hz after DDAC modification, which might be attributed to the spatial repulsive effect against cell adsorption. It is worth noting that the Zn-DDAC− immobilized electrode led to a dramatically larger frequency shift of 34.3 Hz. These results indicate that DDAC exhibits significantly enhanced cell affinity after Zn coordination, even superior to the gold−cell interaction. Therefore, Zn-DDAC functionalization may endow polymers with high “infectivity”, just as viruses do. Zn-DDAC residues bind tightly with cell membranes, and the dynamically continuous perturbation of the membrane microenvironment may promote the polyplex cellular uptake. Furthermore, endosomal membrane disruption may also be achieved by the perturbation. A hemolysis assay was conducted to evaluate the endosomal destabilization of Zn-DDAC. As D

DOI: 10.1021/acsami.8b06934 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. Zn-PCED shows high transfection efficiency. (a) Gluciferase activity post transfection in 293T, HCT116, and Hela cells. Zn-PCED mediates higher transfection efficiency than PC2, PCE, and commercial PEI and Lipo2000. Statistical significances are indicated at *p < 0.05. (b) GFP expression in 293T cells with a polymer/DNA weight ratio at 10:1. The scale bar is 200 μm.

Figure 5. Mechanism of the internalization pathway. (a) Temperature effect on polyplex internalization. *p < .05 as compared with 37 °C. (b) Temperature effect on gluciferase activity after transfection. *p < .05 as compared with 37 °C. (c) Polyplex internalization in the presence of different inhibitors. *p < .05 as compared with respective polyplexes without inhibitors. (d) Gluciferase activity after transfection in the presence of different inhibitors. *p < .05 as compared with respective polyplexes without inhibitors. HCT116 cells are used here, and the polymer/DNA weight ratio is 10:1. Per well, 0.1 μg Cy3-DNA is used.

Moreover, unlike toxic commercial transfection reagents, ZnPCED preserved high cell viability (Figure S12). The GFP expression also demonstrated that Zn-PCED led to high transfection efficacy (Figure 4b). These results prove that virus-mimicking Zn-PCED can ultimately act as a highly efficient and safe gene vector, contributing to its high “infectivity” and endosomal escape functionality. Therefore, Zn-PCED may have the potential of transducing those hard-totransfect primary cells and even in vivo, just like the efficient but immunogenic viral vectors. Mechanism of Cellular Uptake. The biological process remains elusive, and to illuminate the mechanism of gene delivery, a specific internalization pathway is considered to benefit the design of next-generation gene vectors. The hydrophobic chain and Zn-DDAC functionalization have been proven to accelerate the cellular uptake and increase the fluorescence intensity (Figure S13); however, the specific internalization pathway needs further verification. Low incubation temperature is first used to determine whether the polyplex cellular uptake is by energy-dependent endocy-

exposed on the polyplex surface. The respective proportion needs further exploration. Virus Mimicking Zn-PCED Mediates High Transfection Efficacy in Vitro. The embryonic kidney 293T and colon carcinoma HCT116 cells were first used to evaluate the transfection performance of Zn-PCED. To bolster the superiority, two commercial gene transfection reagents, polyethylenimine (25 kDa) and Lipofectamine2000 (Lipo2000), were used as positive controls. Figure 4a shows that cross-linked PC1 and PC2 mediated hundreds of higher gluciferase activity than PLL, and PC2 with better transfection performance was chosen for further functionalization. As hydrophobic alkyl chains have been testified to improve the membrane fusion and cellular uptake, PCE showed fivefold higher transfection efficiency compared to PC2 at w/w of 5:1 in 293T cells. Afterward, the Zn-DDAC ligand mimicking virus spikes and membrane-lytic components was modified on PCE, and the transfection efficacy was further improved. Even compared to commercial PEI and Lipo2000, up to 20-fold higher gluciferase activity was achieved using Zn-PCED. E

DOI: 10.1021/acsami.8b06934 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 6. Zn-PCED shows high transfection efficiency in primary cells and in vivo. (a) Gluciferase activity after transfection in primary SC cells. Statistical significances are indicated at *p < .05. (b) In vivo transfection efficiency of mice bearing CT26 tumor tissues. *p < 0.05 as compared to Lipo2000. For PC2, PCE, and Zn-PCED, the polymer/DNA weight ratio is fixed at 10:1, while PEI is used at w/w = 3:1, and Lipo2000 is utilized as per the protocol.

for in vivo applications, gene transfection was carried out in BALB/c mice with the colon carcinoma (CT26) xenograft model. The small polymer and Zn-DDAC residue dosages avoided erythrocyte destruction, and no mice weight loss or tumor damage was observed after transfection. As expected, the virus-like structure and “infectivity” functionality imparted Zn-PCED robust transfection efficacy in vivo, 19.1- and 2.6fold higher than PEI and Lipo2000 treated tumors, respectively (Figure 6b). These results are in accordance with the in vitro tests (Figure S16). We can conclude that Zn-PCED is a highly efficient gene vector both in vitro and in vivo, which can benefit from its internal virus-like structure and functions for enhanced “infectious” feature and endosomal escape.

tosis. As shown in Figure 5a, PC2/DNA, PCE/DNA, and ZnPCED/DNA polyplexes were all efficiently taken into the cells at 37 °C, with efficacies nearly 100%. Nevertheless, the active transport process was inhibited at 4 °C, and cellular uptake efficiencies were all dramatically decreased. Furthermore, the transfection efficiency was also largely inhibited at 4 °C (Figure 5b). These results demonstrate that endocytosis is involved in the polyplex cellular uptake. Endocytosis can usually be classified as clathrin-dependent endocytosis (CDE), clathrinindependent endocytosis (CIE), and micropinocytosis.46 The assay of each pathway contribution to polyplex internalization can help to understand the structure−function relationship and, more importantly, provide references for the design of other gene delivery systems. Herein, chlorpromazine (CPZ), MβCD, and AM were utilized to inhibit CDE, CIE, and macropinocytosis, respectively. Figure 5c shows that CDE, CIE, and macropinocytosis all contributed to the cellular uptake of PC2/DNA and PCE/DNA polyplexes. Regarding the Zn-PCED counterpart, the cellular uptake efficiency decreased from 97 to 88 and 93% in the presence of CPZ and AM, respectively. Nevertheless, MβCD largely inhibited the Zn-PCED/DNA cellular uptake and the efficacy reduced to 63%. Figure 5d further confirmed that CIE accompanied by the CDE pathway conducted the Zn-PCED/DNA transfection, with no obvious cytotoxicity observed (Figure S14). This can be explained as follows: The decreased particle size and other endocytosis pathway enhancement may weaken the effect of macropinocytosis, and CIE and CDE still yield high cellular uptake efficiency when macropinocytosis is inhibited. More importantly, Zn coordinative residues show the function of specifically benefiting the CIE pathway. Gene Transfection in Primary Cells and In Vivo. Numerous attempts have been made to explore gene vectors; most, however, still face difficulty for in vivo applications.16,41,47,48 Previous in vitro/in vivo disconnects showed that many cationic polymers (e.g., poly(2-(dimethylamino)ethyl methacrylate, cationic polyester, and PEI) could achieve efficient gene delivery in vitro, whereas sharply decreased efficacy was observed when turning to in vivo.16,49 The transfection efficiency of virus-mimicking Zn-PCED is further tested in primary cells and in vivo. As depicted in Figure 6a, Zn-PCED greatly outperformed PC2, PCE, and commercial PEI and Lipo2000 for gene delivery in primary schwann cells (SC). In detail, at w/w of 10:1, Zn-PCED mediated 4.9-, 2.9-, 34.8-, and 2.6-fold higher gluciferase activity compared to PC2, PCE, PEI, and Lipo2000 with high cell viability preserved (Figure S15). To further confirm the feasibility of Zn-PCED

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CONCLUSIONS We report a highly “infectious” and endosome-disruptive virus mimic for gene delivery. Bioreducible cross-linked PLL with alkylation is aimed to mimic the virus lipoprotein envelop. Subsequent Zn-DDAC modification is designed to mimic the virus spikes and membrane-lytic protein or peptide. After the Zn-DDAC functionalization, four benefits are observed: (i) tight DNA condensation: Zn-PCED/DNA polyplexes show smaller sizes compared with their PCE counterparts. (ii) High cellular uptake: Zn-DDAC exhibits high binding to cells, and this functionality drives it to imitate virus spike for “infection” very well. (iii) High endosomal escape: Zn-DDAC efficiently induces endosomal destabilization, and this property makes it suitable for virus membrane-lytic component mimicking. (iv) Efficient DNA release: disulfide bonds in Zn-DDAC can facilitate its dissociation from the polymer bone, accelerating DNA release into the cytoplasm. Thus, this metal coordinative functionalization strategy, on the one hand, achieves the virus spike and membrane-lytic mimicking for high “infectivity” and superior endosomal escape; on the other hand, it can lead inspiration to further modify broad of biomaterials in pursuit of excellent vectors. Ultimately, a well-tailored structure makes Zn-PCED a highly efficient and safe nonviral vector both in vitro and in vivo, demonstrating a great potential to develop virus mimics for clinical gene therapy.

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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/acsami.8b06934. Experimental procedures, 1H NMR spectra, polyplex characterization, and transfection efficiency and cytotoxicity (PDF) F

DOI: 10.1021/acsami.8b06934 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Tianying Guo: 0000-0001-6587-6466 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the doctoral fund from the Ministry of Education of China (RFDP, 20130031110012), National Natural Science Foundation of China (NSFC, 20874052), PCSIRT (IRT1257), NFFTBS (J1103306), and the Fundamental Research Funds for the Central Universities for financial support.

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REFERENCES

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DOI: 10.1021/acsami.8b06934 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX