A Guanidinium-Rich Polymer for Efficient Cytosolic Delivery of Native

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A Guanidinium-rich Polymer for Efficient Cytosolic Delivery of Native Proteins chunlei yu, Echuan Tan, Yangyang Xu, Jia Lv, and Yiyun Cheng Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00753 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 2, 2018

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Bioconjugate Chemistry

A Guanidinium-rich Polymer for Efficient Cytosolic Delivery of Native Proteins Chunlei Yu1, Echuan Tan1, Yangyang Xu1, Jia Lv2,*, Yiyun Cheng1,2,* 1Shanghai

Key Laboratory of Regulatory Biology, School of Life Sciences, East China

Normal University, Shanghai 200241, P. R. China. 2South

China Advanced Institute for Soft Matter Science and Technology, South China

University of Technology, Guangzhou 510640, P. R. China. *E-mail: [email protected] (J.L.); [email protected] (Y.C.) ABSTRACT Cytosolic protein delivery is critical for the development of protein-based therapeutics. However, an efficient and robust carrier that can deliver native proteins without biological or chemical modifications into cells is highly desired. Here, we developed a guanidinium-rich polymer consisting of a cationic polymer scaffold modified with both phenyl and biguanide moieties. The polymer showed much higher protein binding affinity and endocytosis and reduced cytotoxicity compared to a control polymer by replacing the biguanide with monoguanide moieties. The guanidinium-rich polymer is capable of transporting proteins with various molecular weights and charge properties into the cytosol of living cells efficiently, while maintaining their bioactivities. This study permits the development of cationic polymers modified with phenylbiguanide moieties for efficient intracellular protein delivery. Keywords: guanidium, rational design, cytosolic protein delivery, cationic polymer, robust efficacy INTRODUCTION Intracellular protein delivery is very important in biological sciences and biotherapeutics.1-4 A carrier is needed in intracellular protein delivery to protect the proteins from degradation, and initiate uptake by target cells.3,

5-7

Binding between

proteins and carriers is the first and critical step for protein delivery but is still a key obstacle due to the innate properties of proteins, like uncertain charge property, large

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

molecular size and hydrophilicity.8 Modification with anionic tags such as anionic proteins and peptides, nucleic acids, polymers, and aconitic acids on proteins may strengthen their binding affinity with cationic species including lipids, polymers or nanoparticles. 3, 5, 7, 9, 10 Alternatively, proteins were modified with electron-deficient phenylboronic acid to improve their binding capability with the cationic carriers via a boron-nitrogen coordination.6,

11, 12

Besides, protein molecules were decorated with

protein transduction domains (PTD), such as cell penetrating peptides,13 toxins,14 and cationic polymers.15,

16

However, protein modification always involves complicated

synthesis, and bioactivity and specificity of the protein might be altered after chemical modification. Although several strategies have been reported for cytosolic delivery of native proteins without chemical or biological modifications,17-22 a convenient, robust and efficient carrier for cytosolic delivery of native proteins is still highly required in this area. In a recent study, we rationally designed a polymer composing of an easily modified cationic polymer scaffold,

23

a phenyl group, and a guanidium ligand for

efficient intracellular protein delivery.24 The guanidium moieties on the polymer surface are responsible for high binding affinity to various proteins via both hydrogenbonding and salt bridge interactions, while the phenyl groups are critical for escape from endosomes after endocytosis. Biguanide is a ligand with much stronger hydrogenbonding and salt-bridge interactions with proteins than the corresponding monoguanide by a divalent effect.25 Besides, biguanides are capable of interacting with cell membranes and initiate efficient endocytosis, which is benefical for intracellular delivery of biomacromolecules.26,

27

We could expect a more efficient polymer for

cytosolic protein delivery by grafting both phenyl and biguanide moieties on a cationic polymer. In this study, we conjugated phenylbiguanide on branched polyethylenimine (bPEI) via a facile chemistry, and investigated the potential of phenylbiguanide-grafted polymer for efficient cytosolic protein delivery. RESULTS AND DISCUSSION bPEI was conjugated with biguanidinebenzoic acid (BGBA) or guanidinebenzoic acid (GBA) (Figure 1a) and the products were characterized by 1H NMR (Figure 1b). Around 60 BGBA or GBA molecules were conjugated on each bPEI, and the products were named PBGBA60 and PGBA60, respectively. Both PBGBA60 and PGBA60 could bind with fluorescein isothiocyanate labeled bovine serum albumin (BSA-FITC)


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(Figure 1c, 1d) and form uniform nanoparticles at different polymer to protein weight ratios (Figure 1e, 1f).

Figure 1. (a) Synthesis of PBGBA60 and PGBA60. (b) 1H NMR spectra of the products. Fluorescence spectra of rhodamine B isothiocyanate (RBITC) labeled PBGBA60 (PBGBA60-RBITC)/BSA-FITC (c) and PGBA60-RBITC/BSA-FITC (d) complexes. (e) Dynamic light scattering (DLS) analysis of the complexes at different polymer to protein weight ratios. (f) Transmission electron microscope (TEM) images of the complexes at optimal protein transduction conditions. The protein delivery efficiencies of the polymers were screened on HeLa cells. As shown in Figure 2 and Figure S1, PBGBA60 showed significantly higher efficiency than PGBA60 at different polymer concentrations. The efficiencies of PBGBA60 and PGBA60 were futher evaluated at different incubation times. The cells treated with PBGBA60 exhibited much higher fluoresence intensity than those treated with PGBA60 for various times (Figure 2c, Figure S2 and Figure S3). Strong fluorescence



was distributed evenly in the cells as early as 2 h after treated with the PBGBA60/BSAFITC complexes and the internalized proteins were not co-localized with acidic compartments (Figure S4). This result proved that PBGBA60 is capable of efficiently transporting BSA-FITC across the cell membrane and escaping from endosomes efficiently. The cells incubated with the complexes for 6 h exhibited the highest efficacy, which was much superior to Pulsin, unmodified bPEI, and DGBA60 reported in the previous study (Figure 3a).23 The addition of a fluorescence quencher trypan blue could not decrease the fluorescence of cells transducted by the polymer/protein complexes, which confirmed the BSA-FITC transducted by PBGBA60 or PGBA60 were mainly internalized by the cells rather than distributed on the surface of cell membrane (Figure S5). To investigate mechanism of PBGBA60 in efficient intracellular protein delivery, we tested the cellular uptake of RBITC-labeled PBGBA60 and PGBA60 in the absence of proteins. Both RBITC-labeled polymers showed a similar fluorescence intensity at an equal polymer concentration (Figure S6). However, HeLa cells treated with PBGBA60-RBITC showed higher fluorescence intensity than those with an equal molar concentration of PGBA60-RBITC (Figure 3b). This result indicated that PBGBA60 itself could induce more efficient endocytosis than PGBA60. Besides, DLS experiments showed that PBGBA60 is capable of forming small and uniform nanoparticles with BSA at a wide range of polymer to protein weight ratios (Figure 1e). Besides, the nanoparticles consisting of PBGBA60-RBITC and BSA-FITC showed more obvious fluorescence resonance energy transfer (FRET) signals than the PGBA60 containing complexes, no matter at an equal polymer molar concentration or at optimal protein transduction conditions (Figure 1c-1d, and Figure S7). Our previous study has proved that the hydrogen-bonding and salt-bridge interactions between the proteins and the guanidyl on the polymer surface are the major force driving the protein-polymer complexation.23 The more compact structure for PBGBA60 and BSA complex can be explained by the enhanced hydrogen-bonding and salt-bridge interactions due to biguanide groups on the polymer. Surprisingly, though PBGBA60 showed higher binding affinity with proteins and more efficient cellular uptake, the polymer was less toxic than PGBA60 and unmodified bPEI both in the absence or presence of BSA (Figure 3c-3d). The low toxicity of biguanide-modified polymers was in accordance with a previous finding observed on polyMetformin.27


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Figure 2. (a) Fluorescent microscopy images of HeLa cells transducted by polymer/BSA-FITC complexes for 4 h at various polymer concentrations. (b) Flow cytometry analysis of HeLa cells in (a). (c) Flow cytometry analysis of HeLa cells transducted by the polymer/BSA-FITC complexes for various times. The concentrations of PBGBA60, PGBA60 and BSA-FITC were 20 μg/mL, 10 μg/mL and 33 μg/mL, respectively. Data are presented as the mean ± SD (n=3).



Figure 3. (a) Flow cytometry analysis of HeLa cells treated with polymer/BSA-FITC complexes for 6 h, and the concentrations of PBGBA60, PGBA60, DGBA60, bPEI and BSA-FITC were 20, 10, 40, 5 and 33 μg/mL, respectively. (b) Flow cytometry analysis of HeLa cells treated with 0.5 μM PBGBA60-RBTIC and PGBA60-RBTIC for 1-6 h (n=3). Cytotoxicity of polymer (c) or polymer/BSA complexes (d) at different polymer concentrations on HeLa cells (n=5). The weight ratios of PBGBA60, PGBA60 and bPEI to BSA were 0.6:1, 0.3:1 and 0.15:1, respectively. 20 μg/mL PBGBA60, 10 μg/mL PGBA60 and 5 μg/mL bPEI were used in protein transduction experiments. We further tested the robustness of PBGBA60 as a protein carrier. As shown in Figure 4a and 4b, PBGBA60 successfully delivered R-phycoerythrin (R-PE), Green fluorescent protein (GFP), FITC labeled cytochrome c (Cyt C-FITC), lysozyme-RBITC and FITC labeled ribonuclease A (RNase A-FITC) into HeLa cells. The fluorescence intensity of cells treated by the PBGBA60/protein complexes was stronger than those treated with the PGBA60/protein complexes. Both PBGBA60 and PGBA60 could form nanoparticles with lysozyme at a wide range of weight ratios (polymer/protein), indicating the robust protein binding capability of the polymers (Figure S8). RNase A is a cytotoxic protein which could induce cytotoxic effects to cells by cleaving intracellular RNAs.12 It is a membrane-impermeable protein, and thus non-toxic on


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HeLa cells at concentrations up to 21.6 μM. The polymer PBGBA60 and PGBA60 caused minimal toxicity to the cells at the tested concentrations (Figure 4c). However, both the PBGBA60/RNase A and PGBA60/RNase A complexes induced obvious toxicity to the cells with an IC50 around 0.7 and 4.5 μM, repectively, suggesting the efficient internalization of RNase A and preservation of enzyme activity. The higher anticancer acitivty of PBGBA60/RNase A complex in comparision with that of PGBA60 complex is attributed to the higher efficiency of PBGBA60 in the cytosolic delivery of RNase A (Figure S9). These results were futher comfirmed on MDA-MB231 cell and PC-9 cell (Figure S10).

Figure 4. (a) Fluorescent microscopy images of HeLa cells treated with PBGBA60 or PGBA60 complexes with R-PE, GFP, lysozyme-RBITC, Cyt C-FITC and RNase AFITC, respectively. The concentrations of PBGBA60, PGBA60, R-PE, GFP, lysozymeRBITC, Cyt C-FITC and RNase A-FITC were 20, 10, 10, 5, 10, 10 and 20 μg/mL, respectively. (b) Properties of the model proteins. (c) RNase A delivery efficacies of the polymers on HeLa cells were tested by MTT (n=5). The concentrations of PBGBA60 and PGBA60 were 10 and 5 μg/mL, respectively.



In summary, we developed a guanidium-rich polymer for efficient cytosolic protein delivery by grafting a ligand containing both phenyl and biguanide moieties on bPEI. The polymer was much more efficient and less toxic than a control polymer grafted with an equal number of phenylguanide moieties. It is capable of translocating proteins with different molecular weights and isoelectric points into cells with preservation of protein bioactivity. This study permits the development of phenylbiguanide-grafted polymers for efficient and non-toxic delivery of native proteins without the need of chemical modification. ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (21725402 and 20174030). SUPPORTING INFORMATION Experimental procedures and more data about protein transduction results and characterization of the polymers or the polymer/protein complexes are supplied as Supporting Information. The materials are available free of charge via the Internet at http://pubs.acs.org.


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REFERENCES (1)

(2) (3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

D'Astolfo, D. S., Pagliero, R. J., Pras, A., Karthaus, W. R., Clevers, H., Prasad, V., Lebbink, R. J., Rehmann, H., and Geijsen, N. (2015) Efficient intracellular delivery of native proteins. Cell 161, 674-690. Gu, Z., Biswas, A., Zhao, M., and Tang, Y. (2011) Tailoring nanocarriers for intracellular protein delivery. Chem. Soc. Rev. 40, 3638-3655. Zuris, J. A., Thompson, D. B., Shu, Y., Guilinger, J. P., Bessen, J. L., Hu, J. H., Maeder, M. L., Joung, J. K., Chen, Z. Y., and Liu, D. R. (2015) Cationic lipidmediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73-80. Postupalenko, V., Desplancq, D., Orlov, I., Arntz, Y., Spehner, D., Mely, Y., Klaholz, B. P., Schultz, P., Weiss, E., and Zuber, G. (2015) Protein delivery system containing a nickel-immobilized polymer for multimerization of affinity-purified His-tagged proteins enhances cytosolic transfer. Angew. Chem. Int. Ed. 54, 10583-10586. Wang, M., Zuris, J. A., Meng, F., Rees, H., Sun, S., Deng, P., Han, Y., Gao, X., Pouli, D., Wu, Q. et al. (2016) Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 113, 28682873. Wang, M., Sun, S., Neufeld, C. I., Perez-Ramirez, B., and Xu, Q. (2014) Reactive oxygen species-responsive protein modification and its intracellular delivery for targeted cancer therapy. Angew. Chem. Int. Ed. 53, 13444-13448. Brodin, J. D., Sprangers, A. J., McMillan, J. R., and Mirkin, C. A. (2015) DNAmediated cellular delivery of functional enzymes. J. Am. Chem. Soc. 137, 14838-14841. Mitragotri, S., Burke, P. A., and Langer, R. (2014) Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat. Rev. Drug. Discov. 13, 655-672. Wang, M., Alberti, K., Sun, S., Arellano, C. L., and Xu, Q. (2014) Combinatorially designed lipid-like nanoparticles for intracellular delivery of cytotoxic protein for cancer therapy. Angew. Chem. Int. Ed. 53, 2893-2898. Mout, R., Ray, M., Tay, T., Sasaki, K., Yesilbag Tonga, G., and Rotello, V. M. (2017) General strategy for direct cytosolic protein delivery via proteinnanoparticle co-engineering. ACS Nano 11, 6416-6421. Tang, R., Wang, M., Ray, M., Jiang, Y., and Jiang, Z. (2017) Active targeting of the nucleus using nonpeptidic boronate tags. J. Am. Chem. Soc. 139, 85478551. He, H., Chen, Y., Li, Y., Song, Z., Zhong, Y., Zhu, R., Cheng, J., and Yin, L. (2018) Effective and selective anti-cancer protein delivery via all-functions-inone nanocarriers coupled with visible light-responsive, reversible protein engineering. Adv. Funct. Mater. 1706710. Nischan, N., Herce, H. D., Natale, F., Bohlke, N., Budisa, N., Cardoso, M. C., and Hackenberger, C. P. (2015) Covalent attachment of cyclic TAT peptides to



(14) (15)

(16)

(17)

(18)

(19)

(20)

(21) (22)

(23)

(24)

(25)

(26)

GFP results in protein delivery into live cells with immediate bioavailability. Angew. Chem. Int. Ed. 54, 1950-1953. Rabideau, A. E., Liao, X., and Pentelute, B. L. (2015) Delivery of mirror image polypeptides into cells. Chem. Sci. 6, 648-653. Fu, J., Yu, C., Li, L., and Yao, S. Q. (2015) Intracellular delivery of functional proteins and native drugs by cell-penetrating poly(disulfide)s. J. Am. Chem. Soc. 137, 12153-12160. Ng, D. Y., Arzt, M., Wu, Y., Kuan, S. L., Lamla, M., and Weil, T. (2014) Constructing hybrid protein zymogens through protective dendritic assembly. Angew. Chem. Int. Ed. 53, 324-328. Zhang, Z., Shen, W., Ling, J., Yan, Y., Hu, J., and Cheng, Y. (2018) The fluorination effect of fluoroamphiphiles in cytosolic protein delivery. Nat. Commun. 9, 1377. Tang, R., Kim, C. S., Solfiell, D. J., Rana, S., Mout, R., Velazquez-Delgado, E. M., Chompoosor, A., Jeong, Y., Yan, B., Zhu, Z. J. et al. (2013) Direct delivery of functional proteins and enzymes to the cytosol using nanoparticle-stabilized nanocapsules. ACS Nano 7, 6667-6673. Lee, K., Rafi, M., Wang, X., Aran, K., Feng, X., Lo Sterzo, C., Tang, R., Lingampalli, N., Kim, H. J., and Murthy, N. (2015) In vivo delivery of transcription factors with multifunctional oligonucleotides. Nat. Mater. 14, 701706. Ghosh, P., Yang, X., Arvizo, R., Zhu, Z. J., Agasti, S. S., Mo, Z., and Rotello, V. M. (2010) Intracellular delivery of a membrane-impermeable enzyme in active form using functionalized gold nanoparticles. J. Am. Chem. Soc. 132, 2642-2645. Cheng, Y. (2017) Fluorinated polymers in gene delivery. Acta Polym. Sin. 8, 1234-1245. Lv, J., He, B., Yu, J., Wang, Y., Wang, C., Zhang, S., Wang, H., Hu, J., Zhang, Q., and Cheng, Y. (2018) Fluoropolymers for intracellular and in vivo protein delivery. Biomaterials 182, 167-175. Kannan, RM., Nance, E., Kannan, S., Tomalia, DA. (2014) Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications. J. Intern. Med. 276, 579-617. Chang, H., Lv, J., Gao, X., Wang, X., Wang, H., Chen, H., He, X., Li, L., and Cheng, Y. (2017) Rational design of a polymer with robust efficacy for intracellular protein and peptide delivery. Nano Lett. 17, 1678-1684. Mogaki, R., Hashim, P. K., Okuro, K., and Aida, T. (2017) Guanidinium-based "molecular glues" for modulation of biomolecular functions. Chem. Soc. Rev. 46, 6480-6491. Ikeda, T., Ledwith, A., Bamford, C. H., and Hann, R. A. (1984) Interaction of a polymeric biguanide biocide with phospholipid membranes. Biochim. Biophys. Acta. 769, 57-66.


Page 10 of 16

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(27)

Zhao, Y., Wang, W., Guo, S., and Wang, Y. (2016) PolyMetformin combines carrier and anticancer activities for in vivo siRNA delivery. Nat. Commun. 7, 11822.



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A guanidinium-rich polymer with robust protein delivery efficacy


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Figure 1. (a) Synthesis of PBGBA60 and PGBA60. (b) 1H NMR spectra of the products. Fluorescence spectra of rhodamine B isothiocyanate (RBITC) labeled PBGBA60 (PBGBA60-RBITC)/BSA-FITC (c) and PGBA60RBITC/BSA-FITC (d) complexes. (e) Dynamic light scattering (DLS) analysis of the complexes at different polymer to protein weight ratios. (f) Transmission electron microscope (TEM) images of the complexes at optimal protein transduction conditions. 152x137mm (300 x 300 DPI)



Figure 2. (a) Fluorescent microscopy images of HeLa cells transducted by polymer/BSA-FITC complexes for 4 h at various polymer concentrations. (b) Flow cytometry analysis of HeLa cells in (a). (c) Flow cytometry analysis of HeLa cells transducted by the polymer/BSA-FITC complexes for various times. The concentrations of PBGBA60, PGBA60 and BSA-FITC were 20 μg/mL, 10 μg/mL and 33 μg/mL, respectively. Data are presented as the mean ± SD (n=3). 169x95mm (300 x 300 DPI)


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Figure 3. (a) Flow cytometry analysis of HeLa cells treated with polymer/BSA-FITC complexes for 6 h, and the concentrations of PBGBA60, PGBA60, DGBA60, bPEI and BSA-FITC were 20, 10, 40, 5 and 33 μg/mL, respectively. (b) Flow cytometry analysis of HeLa cells treated with 0.5 μM PBGBA60-RBTIC and PGBA60RBTIC for 1-6 h (n=3). Cytotoxicity of polymer (c) or polymer/BSA complexes (d) at different polymer concentrations on HeLa cells (n=5). The weight ratios of PBGBA60, PGBA60 and bPEI to BSA were 0.6:1, 0.3:1 and 0.15:1, respectively. 20 μg/mL PBGBA60, 10 μg/mL PGBA60 and 5 μg/mL bPEI were used in protein transduction experiments. 121x86mm (300 x 300 DPI)



Figure 4. (a) Fluorescent microscopy images of HeLa cells treated with PBGBA60 or PGBA60 complexes with R-PE, GFP, lysozyme-RBITC, Cyt C-FITC and RNase A-FITC, respectively. The concentrations of PBGBA60, PGBA60, R-PE, GFP, lysozyme-RBITC, Cyt C-FITC and RNase A-FITC were 20, 10, 10, 5, 10, 10 and 20 μg/mL, respectively. (b) Properties of the model proteins. (c) RNase A delivery efficacies of the polymers on HeLa cells were tested by MTT (n=5). The concentrations of PBGBA60 and PGBA60 were 10 and 5 μg/mL, respectively. 169x136mm (300 x 300 DPI)


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A Guanidinium-Rich Polymer for Efficient Cytosolic Delivery of Native

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