Zinc Coordination Substitute Amine: A Noncationic Platform for

Jul 2, 2018 - Amines have been extensively involved in vector design thus far, however, their clinical translation has been impeded by several obstacl...
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Letter Cite This: ACS Macro Lett. 2018, 7, 868−874

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Zinc Coordination Substitute Amine: A Noncationic Platform for Efficient and Safe Gene Delivery Shuai Liu,† Huiting Jia,† Jixiang Yang,† Jianping Pan,† Huiyun Liang,† Liheng Zeng,† Hao Zhou,‡ Jiatong Chen,‡ and Tianying Guo*,† †

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

ABSTRACT: Amines have been extensively involved in vector design thus far, however, their clinical translation has been impeded by several obstacles: cytotoxicity, polyplex serum instability and low efficacy in vivo. In pursuit of functional groups to substitute amines in vector design to address these disadvantages is of great significance. Herein, we report welltailored noncationic copolymers that contain hydrophilic, hydrophobic, and zinc coordinative moieties through reversible addition−fragmentation chain transfer (RAFT) polymerization for efficient and safe gene delivery. These polymers are capable of condensing DNA, enabling the formation of uncharged polyplexes. Especially, the zinc coordinative ligand can simultaneously benefit strong DNA binding, robust cellular uptake, efficacious endosomal destabilization, low cytotoxicity, and avoidance of serum protein adsorption. The coordinative module holds great promise to substitute amines and inspires the development of next-generation gene vectors. More importantly, the coordinative copolymers illuminate the possibility and potential of noncationic gene delivery systems for clinical applications.

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group used the polyethylene glycol to construct a brush polymer−DNA conjugate for gene regulation.12 This exquisite conjugate is indeed an innovative design, however to date, no amine substitute that can achieve all the amine properties has been discovered from the point of functional groups. The truth stands that apart from amines, to develop the substitutes that are conducive to the cellular uptake and endosomal escape indeed remains challenging. Encouragingly, zinc(II)-dipicolylamine (Zn-DPA) analogues have been discovered to show high affinity with phosphate derivatives.20,21 Since that large amounts of phosphate moieties exist in DNA and biological membranes, we speculate Zn-DPA analogs might benefit the DNA condensation and polyplex internalization acceleration. Additionally, the high affinity between Zn-DPA analogs and membranes might lead to continuous oscillation of the endosomal membrane microenvironment, facilitating polyplex trafficking to cytoplasm. Briefly, this Zn-coordinated ligand might be capable of acting as the substitute of amines to condense DNA, bind cell membranes, and induce endosomal disruption. Previously, Zn-DPA based analogs are extensively used as small molecules for phosphorylated chemsensors and cell imaging.22,23 Nonetheless, here Zn-DPA residues are aimed to

ene regulation provides an option to the treatment of extensive human incurable diseases by specific nucleic acid delivery and controlled factor expression.1,2 Considering the strong tendency of enzymatic degradation, nucleic acids require carriers to reach the target.3−5 To date, amine-based cationic vectors have been extensively explored.6−8 Nonetheless, the positively charged nanoparticles tend to adsorb the negatively charged proteins in serum, and the formed aggregates can be rapidly cleared by the reticuloendothelial system (RES).9,10 Furthermore, excess positive charges are associated with anabatic cytotoxicity, inflammation, and cell apoptosis.11,12 Facing these undesirable properties, Zhou et al.13 designed cationic polymers with low charge density for gene transfection. Mastrobattista et al.14 further highlighted the significance of low charges in vector design. Motivated by this attempt, noncationic polymers might demonstrate a more ideal candidate to address the long-standing issues of polycationbased gene vehicles. To this end, to pursue a functional group to substitute the amine roles in the field of nonviral vector design will be greatly significant. Current gene delivery favoring factors, such as efficacious DNA encapsulation, cellular uptake, and endosomal escape are mostly related to positive charges.15−18 To impede each of these processes would lead failure of gene expression, raising the difficulty of exploring amine substitutes and developing noncationic gene delivery systems. Some efforts have been made to explore uncharged vectors.12,19 For instance, Zhang © XXXX American Chemical Society

Received: May 14, 2018 Accepted: June 25, 2018

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DOI: 10.1021/acsmacrolett.8b00374 ACS Macro Lett. 2018, 7, 868−874

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Figure 1. (a) Synthesis of noncationic Zn-HDB polymers. (b) Noncationic Zn-HDB is utilized to fully condense DNA to formulate neutral polyplexes.

cytoplasm.28 The functions of Zn coordination were balanced through introduction of hydrophilic and hydrophobic moieties, and these components synergistically constructed noncationic gene vectors. Zn coordination acts as a critical role in the development of amine substitutes and the integral noncationic gene delivery systems. Additionally, the coexistence of hydrophilic and hydrophobic moieties shows profound implications in efficacious gene delivery systems, benefiting material solubility, particle stability, and cellular uptake.29,30 Therefore, DPA was first transformed to a monomer (DPASS) that had a disulfide bond and vinyl group (Figures S1 and S2). Afterward, RAFT polymerization was conducted using DPASS, hydrophilic HEMA, and hydrophobic BMA (Figure 1). Varying the feedratio, HDB1 and HDB2 polymers were first synthesized with similar molecular weights (Table S1). HDB2 exhibited higher HEMA and BMA contents and lower DPASS content compared to HDB1 (Figure S3). Furthermore, the structure and composition of noncationic polymers was flexible. Replacing BMA by NIPAM, HDN polymer was synthesized (Figures S4 and S5). Next, easily coordinative Zn2+ (Figure S6) was utilized to coordinate DPASS residues of polymers. In this noncationic system, Zn-DPASS moieties are first aimed to benefit DNA encapsulation. Figures S7 and S8 showed that HDB1 without Zn coordination failed to package DNA, while Zn-HDB polymers condensed DNA efficiently, attributed to strong interaction between Zn-DPASS ligand and phosphodiesters in nucleic acids. Previous polyplexes usually had positive zeta potentials,31,32 which was the source of cytotoxicity and destabilization in serum. Circumventing this, noncationic Zn-HDB polymers condensed DNA to form particles with extremely low zeta potentials around 0 mV (Figure 2a). While noncoordinative HDB1 polymer failed to

be functionalized on macromolecules to obtain gene carriers, which however, are associated with adverse steric effect, restricted macromolecule species, limited modification sites, and poor reaction activity. For the purpose of synthesizing polymers with well-tailored architectures and well-defined functionalities, reversible addition−fragmentation chain transfer (RAFT) polymerization stands out.24,25 Polymer compositions, structures, and molecular weights can be adjusted and controlled to screen exquisite nonviral vectors and further study structure−activity relationships.26,27 Moreover, transformation of Zn-DPA analogs to acrylates or acrylamides enables their incorporation into macromolecules via RAFT polymerization. This strategy expands the functionalization strategy of sterically hindered metal coordinated ligands and is capable of introducing large amounts of Zn-DPA moieties in a controlled polymer. In this study, a Zn-DPA derived acrylamide that contains disulfide bond is designed (termed Zn-DPASS). Coupled with hydrophilic monomer (2-hydroxyethyl methacrylate, HEMA) and hydrophobic monomer (butyl methacrylate, BMA or Nisopropylacrylamide, NIPAM), nonviral vectors (Zn-HDB or Zn-HDN) were synthesized via RAFT polymerization. The noncationic polymers showed high DNA packaging ability and the resultant polyplexes mediated high cellular uptake efficiency, both owing to high binding affinity of Zn-DPASS residues to phosphate groups. It is worth noting that, unlike traditional positively charged particles, the ultimate polyplexes remained almost uncharged, drastically benefiting the polyplex serum stability. Surprisingly, Zn-DPASS could also destabilize the endosomal membranes and release the nanoparticles into cytosol with negligible cytotoxicity observed. Afterward, disulfide bonds in polymers were cleaved by glutathione (GSH), facilitating efficient and timely DNA release into the 869

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Figure 2. (a) Zeta potentials of the polyplexes. (b) Unlike PEI/DNA polyplexes, Zn-HDB/DNA polyplexes are stable incubating for 4 h in the presence of serum. (c) Zn-HDB polymers show much lower cytotoxicity than PEI in 293T cells. *P < 0.05 as compared to PEI group. (d) Polyplex dissociation to release DNA with (or without) 10 mM GSH pretreated. Zn-HDBs is used at w/w = 60:1. PEI is utilized at w/w = 3:1.

Figure 3. (a) Hemolysis activity of DPASS and Zn-DPASS at different pHs. *P < 0.05 as compared to DPASS group. (b) Cell viability of DPASS and Zn-DPASS at different concentrations incubated in 293T cells. *P < 0.05 as compared to DPASS group. (c) Hemolysis activity of HDB1, ZnHDB1, and Zn-HDB2 at pH of 5.7. *P < 0.05 as compared to HDB1 group. (d) Hemolysis activity of Zn-HDB1/DNA polyplexes at different polymer concentrations and pH values. *P < 0.05 as compared to pH 7.4 group. (e) Cellular uptake of Zn-HDB1/Cy3-DNA polyplexes at different time intervals in HCT116 cells. Zn-HDB1/DNA is used at w/w = 60:1. Scale bar, 10 μm.

uncharged Zn-HDB/DNA polyplexes still remained stable post 4 h incubation in serum (Figure 2b), and this is indeed one of the critical advantages for noncationic gene delivery systems. Another advantage lies that noncationic Zn-HDB polymers mediated almost no cytotoxicity, largely outperforming cationic PEI (Figure 2c). In terms of particle cellular uptake, amine-based vectors interact with negatively charged components in cell membranes, facilitating polyplex internalization.33,34 Thus, non-

condense DNA, and the resultant zeta potentials reached around −30 mV, similar to that of DNA alone. Zn-HDB1 itself showed nearly neutral electrical characteristic with a zeta potential of −0.23 mV, further testifying its noncationic property. To demonstrate the superiority of this uncharged system, the typical cationic gene vector polyethylenimine (PEI, Mw ∼ 25 kDa) was utilized as control. The PEI/DNA polyplex showed high zeta potential, and was destabilized in the presence of serum (Figure 2a and 2b). On the contrary, 870

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Figure 4. (a) Gluciferase activity of polyplexes in 293T cells. *P < 0.05 as compared to Zn-HDB2 group. (b) Cell viability of polyplexes post transfection. (c) Gluciferase activity at high serum concentrations and (d) gluciferase activity at low DNA doses. *P < 0.05 as compared to Lipo2k. Zn-HDB1/DNA weight ratio is 60:1. (e) In vivo transfection efficiency in CT26 xenograft tumor model. *P < 0.05 as compared to PEI. Zn-HDB1/ DNA weight ratio is 60:1.

polyplex. Once into acidic environment, protonation of pyridine groups turned Zn-DPASS residues to hydrophilic and stretched structure, therefore Zn coordinated moieties had greater possibility to expose on the polyplex surface to further interact with phosphate bilayer for endosomal disruption. This functionality can ensure the erythrocyte viability post material injection, while endosomal escape occurs once the material enters acidic endosomes. Figure 3e showed that Zn-HDB1/ DNA polyplexes mediated high cellular uptake efficacy, attributed to the high affinity of Zn-HDB1 to cells. In addition, much more DNA colocalized with cell nucleus post 4 h incubation compared to that of 1 h culture, confirming the efficacious polyplex endosomal escape and DNA release. Briefly, noncationic Zn-HDB1 simultaneously demonstrates functionalities of DNA encapsulation, polyplex serum stability, high cellular uptake, robust endosomal escape, and superior DNA release, therefore, efficient gene transfection is expected. For the purpose of gene therapy, extensive endeavors have been tried to design nonviral vectors, including “gold standard” PEI.42 However, PEI shows low efficacy in high serum concentrations, making it failed to be translated in vivo.43,44 Unlike severely toxic PEI and Lipofectamine 2000 (Lipo2k), Zn-HDB1 showed both high transfection efficiency and low cytotoxicity in diverse cell lines (Figures 4a,b and Figures S10−S12). Varying the polymer composition, Zn-HDN showed similar Zn-DPASS content with Zn-HDB1 (Table S1). Zn-HDN also condensed DNA to formulate polyplexes with appropriate sizes and nearly neutral zeta potentials (Figure S13). However, it lagged behind in transfection efficacy compared to Zn-HDB1 (Figure S14). Considering the large amount of different monomers, structural flexibility of this strategy will lead inspiration to develop numerous efficient and safe polymeric vectors. Since the noncationic polyplexes would not bind negatively charged serum proteins, robust transfection efficacy in high serum concentrations might be achieved. As shown in Figure 4c, Zn-HDB1 still retained robust efficiency, while that of commercial PEI and Lipo2k sharply decreased in high serum

cationic systems need another strategy to bind cell membranes. Quartz crystalmicrobalance (QCM) is utilized here to evaluate the interaction strength between biomaterials and cells, and the frequency shifts are proportional to QCM electrode adsorption.35 Figure S9 showed that Zn coordination dramatically enhanced the binding affinity of DPA-based ligand toward cells, providing new moieties to substitute cytotoxic amines. Post internalization and endosomal escape, DNA should be released from the polyplexes for protein expression. For this purpose, disulfide linkage was designed between Zn coordinated ligands and Zn-HDB backbone, which could be cleaved by GSH in cytoplasm.36−38 Figure 2d showed that polyplex dissociation occurred to release DNA in the presence of GSH, while that maintained stable without GSH pretreated. One of the key obstacles noncationic systems faces is deficient endosomal escape. Cationic polymers usually enable endosomal disruption by proton sponge effect.39,40 Surprisingly, we discovered that Zn-DPA analog could destabilize the endosomes. Erythrocyte hemolysis assay was performed to evaluate the endosomal escape behavior.41 Zn-DPASS ligand exhibited much higher hemolysis activity compared to its noncoordinated counterpart (Figure 3a). This can be explained that the robust interaction between Zn-DPASS and phosphate groups of lipid layer exists all the time, and the continuous perturbation induces a gap on the membrane to facilitate endosomal escape. Unlike cationic polymer vectors, this endosome disruptive functionality was not pH responsive. Encouragingly, the perturbation would not cause cell death, and Zn-DPASS still exhibited high cell viability even incubated for 48 h (Figure 3b). Also, Zn coordination decreased the cytotoxicity of DPA analogs. In consequence, Zn-HDB polymers mediated high hemolysis activity in endosomal acidic environment (Figure 3c). Interestingly, the hemolysis of Zn-HDB1/DNA polyplexes decreased in neutral condition compared to that of acidic pH (Figure 3d). Zn-DPASS residues are hydrophobic in physiological pH, and they might mostly hide inside the 871

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Figure 5. (a) Cellular uptake efficiency and (b) gluciferase activity of Zn-HDB1/DNA polyplexes at different temperatures. *P < 0.05 as compared to 37 °C. (c) Cellular uptake efficiency and (d) gluciferase activity of Zn-HDB1/DNA polyplexes with respective inhibitors pretreated. “Polyplex” indicates Zn-HDB1/DNA polyplex without inhibitors pretreated. *P < 0.05 as compared to standard “Polyplex”. Zn-HDB1/DNA weight ratio is 60:1.

Therefore, we can conclude CIE and micropinocytosis pathway both influence the gene transfection of Zn-HDB1/ DNA polyplexes, especially CIE route contributed the most in this noncationic gene delivery system. The decreases of cellular uptake and transfection efficiency were not mediated by inhibitor cytotoxicity (Figure S15). In summary, Zn-DPASS ligand was developed to substitute amines with the functionalities of DNA binding, cell membrane binding, and endosomal destabilization. ZnDPASS containing noncationic Zn-HDB polymers were synthesized by RAFT polymerization with high transfection efficacy and safety profile. The Zn-DPASS residues demonstrated high affinity to phosphate groups. As a result, first, noncationic Zn-HDB polymers condensed DNA to formulate nearly neutral polyplexes, accelerating particle serum stability; second, the Zn-HDB1/DNA polyplexes mediated high cellular uptake efficacy and efficient endosomal escape; third, DNA release could be achieved in cytosol by GSH triggered disulfide cleavage. In consequence, the Zn coordination endows so many benefits in gene delivery processes, highlighting the promise to substitute amines and develop noncationic gene delivery systems with therapeutical potential in clinic.

concentrations. As a result, 1 order of magnitude higher efficacy was achieved by Zn-HDB1 compared to PEI and Lipo2k in 50% serum concentration. In addition, due to high binding affinity between Zn-DPASS residues and cell membranes, Zn-HDB1 could still mediate high gluciferase activity at low DNA doses, drastically outperforming PEI and Lipo2k for this functionality (Figure 4d). Next, in vivo transfection was evaluated using a CT26 xenograft tumor model. Noncationic Zn-HDB1 mediated higher efficacy than PEI in vivo, attributed to its stability in serum, high affinity to cell membranes, efficient endosomal escape and GSH triggered DNA release (Figure 4e). These results highlight the great potential of this noncationic gene delivery system for further clinical applications. Although plenty of efforts have been made in pursuit of nonviral carriers, little mechanistic studies have been conducted.45 To clear the mechanisms of gene transfection, particularly internalization route has become critical for the design of future therapeutically potential gene vectors. A low temperature (4 °C) is utilized to limit the active transports, judging whether the polyplex cellular uptake is proceeded via endocytosis. Figure 5a,b showed that the cellular uptake and transfection efficacy of Zn-HDB1/DNA polyplexes drastically decreased a lot at 4 °C, manifesting that endocytosis was included in the particle internalization. Endocytosis can be achieved by different pathways: micropinocytosis, clathrindependent endocytosis (CDE), and clathrin-independent endocytosis (CIE).45 Amiloride hydrochloride (AM), chlorpromazine (CPZ), and methyl-β-cyclodextrin (MβCD) can act as micropinocytosis, CDE and CIE inhibitors, respectively. As shown in Figure 5c, the cellular uptake was only largely inhibited in the presence of MβCD, demonstrating that CIE played the most important role in endocytosis of Zn-HDB1/ DNA polyplexes. Figure 5d exhibited that MβCD still decreased the gluciferase activity the largest, while transfection efficacy was also reduced obviously with AM pretreated.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.8b00374. Experimental section including the polymer and polyplex characterization, cell studies, and gene transfection (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 872

DOI: 10.1021/acsmacrolett.8b00374 ACS Macro Lett. 2018, 7, 868−874

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Tianying Guo: 0000-0001-6587-6466 Notes

The authors declare no competing financial interest.

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

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DOI: 10.1021/acsmacrolett.8b00374 ACS Macro Lett. 2018, 7, 868−874

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DOI: 10.1021/acsmacrolett.8b00374 ACS Macro Lett. 2018, 7, 868−874