Natural Polyphenols Augment Cytosolic Protein Delivery by a

Article Cite This: Chem. Mater. XXXX, XXX, XXX−XXX

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Natural Polyphenols Augment Cytosolic Protein Delivery by a Functional Polymer Chongyi Liu,† Wanwan Shen,† Bonan Li,§ Tianfu Li,§ Hong Chang,† and Yiyun Cheng*,†,‡ †

Shanghai Key Laboratory of Regulatory Biology, East China Normal University, Shanghai 200241, China South China Advanced Institute for Soft Matter Science and Technology, South China University of Technology, Guangzhou 510640, China § China Institute of Atomic Energy, Beijing 102413, China Downloaded via EAST CAROLINA UNIV on March 14, 2019 at 22:05:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Cytosolic protein delivery is of great importance for basic cell biology and the discovery of novel protein-based biotherapeutics. It remains a challenging task because of the limited binding sites on proteins and their relatively large size. As a result, most current approaches for cytosolic protein delivery need covalent modification on native proteins, which is usually involved with complicated synthesis, reduced protein bioactivity, and unexpected safety concerns. In this study, we proposed a novel strategy to deliver proteins of different molecular sizes and isoelectric points by specific recognitions between natural polyphenols and boronic acid-containing polymers. Protein molecules were decorated with polyphenols via noncovalent hydrogen-bond/hydrophobic interactions or reversible dynamic covalent bonds. The natural polyphenols increase the binding affinity between proteins and boronic acid-containing polymers, allow the release of bound proteins in acidic environments because of pH-sensitive property of catechol−boronate esters, and thus greatly promote the cytosolic delivery efficiency. This strategy showed robust efficiency in the delivery of various proteins such as bovine serum albumin, phycoerythrin, and ribonuclease A and maintained the protein bioactivity after intracellular release. The reported strategy permits the development of a polyphenol-involved polymer platform for cytosolic protein delivery.

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INTRODUCTION Protein-based therapeutics such as enzymes, growth factors, antibodies, cytokines, and peptides have shown great promise in biomedical applications.1−3 Examples include monoclonal antibodies against tumor necrosis factor α to treat inflammatory bowel disease, nerve growth factors to treat neurodegenerative diseases, and recombinant human cytokine interferon-α to treat chronic hepatitis C virus.4 However, all of these protein drugs bind to extracellular targets such as membrane-associated or secreted proteins to exert their functions. This limitation arises from the membrane impermeability of protein molecules because of their relatively large size, hydrophilicity, and limited positive charges.5 It is reported that nearly 70% of the genome-encoded proteins located inside a cell, which hampers their use as targets for the discovery of new protein-based therapeutics.6 Besides, direct cytosolic delivery of proteins provides a powerful and emerging © XXXX American Chemical Society

tool to investigate the functions of new proteins in molecular and cell biology.7 As a result, there is an urgent need to develop techniques for efficient intracellular delivery of protein molecules.8−10 Previous approaches for cytosolic protein delivery include (1) physical techniques such as electroporation,11 (2) incubating cells with endosomolytic chemicals,12,13 (3) decoration of proteins with cell penetrating peptides or other ligands,8,14−17 and (4) using protein transduction carriers such as liposomes, peptides, polymers, or nanoparticles.18−24 Among these approaches, the use of protein carriers was the most widely adopted method for intracellular protein transduction.2,5,25 Generally, protein has limited binding sites on Received: November 6, 2018 Revised: February 28, 2019

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DOI: 10.1021/acs.chemmater.8b04672 Chem. Mater. XXXX, XXX, XXX−XXX

Article

Chemistry of Materials

Figure 1. Polyphenol-mediated cytosolic protein delivery by boronic acid-decorated polymers. (a) Polyphenol facilitates the formation of protein transduction complexes. (b) Structures of the investigated polyphenols. (c) Structures of the boronic acid-decorated polymer P1 and the control polymer P0. (d) Intracellular release of proteins from the nanoparticles triggered by lysosomal acidity.

the surface and relatively large molecular size, which hinder the formation of stable complex with the carrier. In addition, the diversities of protein sequence, structure, and isoelectric point make it difficult to develop universal carriers that are applicable for the delivery of various proteins. To address these problems, proteins were genetically engineered or chemically conjugated with ligands to strengthen the binding affinity between proteins and the carriers.18,26,27 For example, proteins were fused with anionic green fluorescence protein tags, peptides, or nucleic acids to generate anionic charged proteins, also termed “supercharged proteins”, to ensure efficient binding with cationic lipids, polymers, or nanoparticles.18,22,28 Alternatively, the proteins were chemically modified with anionic polymers or aconitic acids to reversibly convert cationic amine groups on the protein surface to anionic species.6,29,30 These delivery systems have shown great promise for cytosolic protein delivery, but genetically engineering or covalent decoration of native proteins may involve with complicated synthesis, reduced protein bioactivity, and unexpected safety concerns. Although several approaches for cytosolic delivery of native proteins without the need of modification have been reported,5,7,31−36 efficient and facile strategies based on new concepts still pose a substantial challenge and attract increasing interest in recent years. Here, we reported a facile and efficient approach for cytosolic protein delivery based on catechol−boronate complexation (Figure 1a). It is reported that natural polyphenols have strong binding affinity with various biomolecules such as proteins (Figure 1b), nucleic acids, and cell membranes via hydrophobic/hydrogen-bond interactions.37−40 The interactions of polyphenols such as (−)-epigallocatechin-3-O-gallate (EGCG, A1), (+)-catechin hydrate (A2), procyanidin (A3), and ellagic acid (A4) with proteins decorate the biomolecules with catechol moieties and ensure efficient protein binding with a boronic acid-containing polymer (Figure 1c) via catechol−boronate interaction.41

Alternatively, proteins could be modified with natural catechols such as 3,4,5-trihydroxybenzaldehyde (B1) and 3,4-dihydroxybenzaldehyde (B2) via reversible Schiff-bases. The used six polyphenols significantly promote the cytosolic delivery of proteins such as bovine serum albumin (BSA), R-phycoerythrin (R-PE), and ribonuclease A (RNase A) by boronic aciddecorated polymers, escape of polymer−protein complexes from vesicular compartments such as endosomes, and maintain the bioactivity of proteins after intracellular release (Figure 1d). Considering the safety of natural polyphenols such as EGCG in food constituents and their beneficial properties for various diseases, the proposed approach allows efficient protein transduction without inducing cytotoxicity.

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RESULTS AND DISCUSSION The proposed concept was first tested on HeLa cells using BSA as the model protein. BSA was labeled with fluorescein isothiocyanate (FITC) to visualize and quantitatively determine the internalized proteins by the cells. BSA was incubated with the polyphenols A1−A4 to yield catechol-decorated protein nanoparticles (Figure 2a). Taken A1 for example, A1 was complexed with BSA at a molar ratio of 10:1. The addition of A1 into BSA solution leads to significant decrease of fluorescence intensity of BSA (Figure 2b), which is caused by the alternation of the fluorescent residue microenvironment inside the protein and can be considered as an indication of A1−BSA interaction. Similarly, the natural polyphenols A2− A4 interact with BSA and cause the decrease of fluorescence intensity of proteins (Figure 2c−e). The small-angle X-ray scattering (SAXS) results in Figure 2f further confirmed the formation of protein−polyphenol aggregate in aqueous solution.38 The binding of A1−A4 with BSA is similar to previous reports for BSA−procyanidin,42 collagen−catechin,43 α-synuclein−EGCG,44 siRNA−EGCG,45 herceptin−oligomerized EGCG,37 and trypsin−EGCG interactions.38 Both hydrogen bond and hydrophobic interaction drive the B


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Figure 2. (a) Polyphenol-decorated protein nanoparticles via noncovalent interactions. Polyphenol facilitates the formation of protein transduction complexes. (b−e) Quenching of BSA fluorescence in the presence of polyphenols. The molar ratios of A1 to BSA in (b) were 0:1, 0.5:1, 1:1, 2:1, 4:1, 8:1, and 10:1. The molar ratios of A2 to BSA in (c) were 0:1, 1:1, 2:1, 4:1, 6:1, 8:1, and 10:1. The molar ratios of A3 to BSA in (d) were 0:1, 1:1, 2:1, 3:1, 4:1, 6:1, and 10:1. The molar ratios of A4 to BSA in (e) were 0:1, 0.5:1, 1:1, 2:1, 4:1, 8:1 and 10:1. The excitation and emission wavelengths of λex = 280 nm and λem = 300−450 nm, respectively. (f) SAXS intensity profiles of BSA−A1 complexes prepared at A1 to BSA molar ratios of 10:1 and 20:1. (g) ITC data for the titration of A1 into BSA solution. (h) CD spectra of BSA and BSA−A1 complexes. The molar ratio of A1 to BSA was 10:1. Size (i) and zeta potential (j) of the BSA−A1, BSA−P1, and BSA−A1−P1 complexes determined by DLS. (k) TEM images of the BSA, BSA−A1, BSA−A1−P1, and BSA−P1 complexes. (l) Fluorescence intensity of BSA−FITC, BSA−FITC−A1, BSA−FITC−P1, and BSA− FITC−A1−P1 complexes. The concentrations of BSA−FITC and P1 were 6 and 8 μg/mL, respectively. The molar ratio of A1 to BSA−FITC is 10:1. The excitation and emission wavelengths were λex = 490 nm and λem = 500−700 nm, respectively. (m) Size of the BSA−A2−P1, BSA−A3− P1, and BSA−A4−P1 complexes determined by DLS. The molar ratio of polyphenols to BSA was 10:1.

complexes in the absence of polyphenols as determined by DLS (Figure 2i) and transmission electron microscopy (TEM) (Figure 2k). The role of polyphenol in BSA−FITC binding is further confirmed by more significant FITC fluorescence quenching compared with the complexes without polyphenol or polymer (Figure 2l). Similarly, the natural polyphenols A2− A4 and BSA formed positively charged nanoparticles (∼100 nm), as shown in Figure 2m. We then tested the efficiency of prepared transduction complexes on HeLa cells. As shown in Figures 3a,b and S2a,b, BSA−FITC alone is membraneimpermeable and the boronic acid-modified dendrimer P1 showed weak efficacy in the delivery of BSA−FITC; however, the presence of A1−A4 significantly promotes the cytosolic BSA delivery by P1 (polyphenol−BSA molar ratio of 10:1). The FITC fluorescence was uniformly distributed in the cytosol of transduced cells after 4 h, suggesting the successful endosomal escape (Figure 4). The internalization of the nanoparticles is mediated by micropinocytosis-, clathrin-, and

formation of polyphenol−BSA complexes. Isothermal titration calorimetry (ITC) results reveal that the binding of EGCG with BSA is an exothermic reaction (Figure 2g). This indicates that the interaction is dominated by hydrogen-bond interactions. The addition of A1 into BSA scarcely changed the secondary structure of the protein, which is essential to maintain the bioactivity of proteins after intracellular release (Figure 2h). Negatively charged nanoparticles around 100 nm consisted of A1 and BSA were formed as determined by dynamic light scattering (DLS) (Figure 2i,j). After confirming the binding of polyphenols A1−A4 with BSA, we incubated the polyphenol−BSA complex with a boronic acid-modified dendrimer (P1, Figure S1) to prepare the transduction complexes. The catechol−boronate complexation between polyphenol−BSA and P1 turned the zeta potential of polyphenol−BSA nanoparticles from negatively to positively charged (Figure 2j)46,47 and yielded more condensed nanoparticles (∼100 nm) in comparison with the P1−BSA C


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Figure 3. Confocal images (a) and mean fluorescence intensity (b) of transduced HeLa cells for 4 h. The doses of protein and polymer in each well were 6 and 8 μg, respectively. The molar ratio of A1, A2, A3, and A4 to BSA was 10:1, respectively. PULSin was measured as a positive control. (c) Relative fluorescence intensity of transduced cells before and after trypan blue quenching. The mean fluorescence intensity of cells treated without trypan blue was defined as 100%. (d) Cell viability of HeLa cells treated with BSA−polyphenol−P1 complexes at optimal transduction conditions for 24 h.

Figure 4. Confocal images of HeLa cells treated with BSA−A1−P1 complexes for 0.5, 1, 2, 3, 4, 6, 8, and 12 h. The nucleus was stained by Hoechst 33342 (blue), and the acidic organelles were stained by LysoTracker (red). The molar ratio of A1 to BSA was 10:1. The doses of BSA and P1 in each well were 6 and 8 μg, respectively.

unmodified dendrimer P0 and the yielding complex showed extremely low protein transduction efficiency (Figure 3b). These results confirmed that the polyphenols A1−A4 promote the cytosolic delivery of BSA−FITC by boronic acid-decorated polymers such as P1. To confirm the efficient cytosolic delivery of BSA−FITC by the proposed approach, we treated the transduced cells with trypan blue, a cell membrane-impermeable dye, to quench the BSA−FITC adsorbed on the cell surface.5 As shown in Figure 3c, more than 90% of fluorescence intensity of transduced cells by P1−A1 was maintained after the treatment with trypan blue, suggesting the successful internalization of BSA−FITC into the cytosols. It is worth noting that the polyphenols A1−

lipid raft-dependent pathways (Figure S3). The bound BSA− FITC in the nanoparticles could be released into cytosol in the presence of anionic polymers such as heparin sodium and free BSA (Figure S4a). More importantly, the protein release could be triggered in responsive to lysosomal acidity, which was due to the acid-labile property of catechol−boronate complexation (Figure S4b). The transduction efficiencies by the polyphenolassisted approach are much superior to a commercial protein transduction reagent PULSin. In addition, the polyphenols themselves failed to deliver BSA−FITC into the cytosol of HeLa cells, suggesting the importance of boronic acid-modified polymer P1 (Figure S5). To confirm the role of catechol− boronate recognition in BSA delivery, we replaced P1 with an D


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Figure 5. (a) Catechol-modified protein via reversible Schiff-base linkage. (b)1H NMR of BSA incubated with B1 at pH 7.0 or 5.0. The molar ratio of B1 to BSA was 9:1. (c) Size and zeta potential of the BSA−B1−P1 complex determined by DLS and TEM. The molar ratio of B1 to BSA is 50:1. The polymer to BSA weight ratio is 1.3:1. (d) Fluorescence intensity of BSA−FITC−B1−P1 complexes at pH 7.0 and pH 5.0. Free BSA−FITC at pH 5.0 was measured as a control. The concentrations of BSA−FITC and P1 were 6 and 8 μg/mL, respectively. The molar ratio of B1 to BSA− FITC is 50:1. The excitation and emission wavelengths were λex = 490 nm and λem = 500−700 nm, respectively. Confocal images (e) and mean fluorescence intensity (f) of transduced HeLa cells for 4 h. The doses of protein and polymer in each well were 6 and 8 μg, respectively. The molar ratio of B1 and B2 to BSA was 50:1.

A4 and its complex with BSA and polymer P1 showed minimal toxicity on HeLa cells and normal cell lines such as NIH3T3 cells at the optimal transduction conditions (Figures 3d and S6). The addition of polyphenols A1−A4 to the polymer or complex does not bring additional cytotoxicity. This is due to the safety of natural polyphenols such as A1 in this study. A1 is the major constituent of green tea catechins, and daily administration of A1 at a dose of 300 mg per person is proved to be safe in clinics.48 Therefore, the polyphenol−P1mediated cytosolic protein delivery system can achieve both high transduction efficiency and minimal cytotoxicity. As an alternative strategy, we can reversibly modify proteins with catechols such as B1 and B2 via amine−aldehyde condensation (Figure 5a). B1 or B2 can be conjugated to the amine residue of cationic amino acids such as lysine via the formation of reversible Schiff-bases (Figures 5b and S7). The addition of B1 to BSA scarcely changed the secondary structure of BSA (Figure S8). BSA−B1 and P1 formed positively charged nanoparticles (Figure 5c), and the nanoparticles also showed significantly reduced BSA−FITC fluorescence (Figure S9). The nanoparticles were stable during

a period of 6 h, and the complex formed by dynamic covalent bond was relatively more stable than that formed by noncovalent interactions (Figure S10). The release of proteins from B1-involved nanoparticles was also responsive to lysosomal acidity (Figures 5d and S11a) and anionic polymers such as heparin sodium and free BSA (Figure S11b). BSA delivered by P1 showed significantly higher efficiency in the presence of B1 and B2, suggesting the successful internalization of BSA−FITC into the cytosols (Figures 5e and S12− S14). The addition of polyphenols B1 and B2 to the complexes does not bring additional cytotoxicity (Figure S15). Similarly, benzaldehyde without catechol moieties and catechol without the aldehyde group failed to increase the performance of P1 in BSA delivery (Figure 5e,f). These results together suggest the critical roles of both aldehyde and catechol moieties in the efficient cytosolic protein delivery (Figure 5f). We further tested applicability of the polyphenol−P1 approach for the delivery of other membrane-impermeable proteins such as R-PE. R-PE is a large-sized protein (240 kDa) with an isoelectric point (pI) of 4.25. It is a fluorescent protein acting as a photosynthetic accessory pigment in red algae.20 E


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Figure 6. Cytosolic delivery of R-PE into HeLa cells by P1 or P0 in the presence or absence of polyphenols for 4 h. The dose of R-PE and polymer P1 or P0 in each well was 1 and 8 μg, respectively. The molar ratios of A1 and B1 to R-PE are 200:1 and 1000:1, respectively. PULSin was used as a positive control.

Figure 7. (a) Cytosolic delivery of RNase A induces cell death of MDA-MB-231 cells. (b) CD spectra of RNase A and RNase A−B1 complex. (c) Enzymatic activity of native RNase A and RNase A−B1 complex. The molar ratio of B1 to RNase A was 10:1. (d) Cytotoxicity of the RNase A− B1−P1 or RNase A−P1 complexes on MDA-MB-231 cells determined by MTT. BSA was used as a negative control, and its concentration was equal to that of RNase A in the complex. (e) AO−EB staining of the transduced MDA-MB-231 cells. The concentrations of RNase A and P1 were 5 and 6 μg/mL, respectively. The molar ratio of B1 to RNase A was 10:1.

The protein does not need to be modified with fluorescent dyes to visualize its internalization. Similar to the demonstrated polyphenol−BSA system, the polyphenols such as A1 significantly reduced the fluorescence intensity of R-PE,

suggesting the binding of A1 to the fluorescent protein (Figure S16). The release of R-PE from B1-involved nanoparticles was also responsive to acidic conditions (Figure S17). The most efficient polyphenols A1 and B1 in the delivery of BSA also F


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study) with a theoretical molecular weight of 28 826 Da was purchased from Dendritech (Midland MI). EGCG (A1) and rhodamine B isothiocyanate (RBITC) were purchased from SigmaAldrich (St. Louis, MO). (+)-Catechin hydrate (A2), 3,4,5trihydroxybenzaldehyde (B1), and RNase A were purchased from J&K Scientific Ltd. (Shanghai, China). Procyanidin (A3) was purchased from Dalian Meilun Biotechnology (Dalian, China). Ellagic acid (A4) and FITC were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). 4-(Bromomethyl)phenylboronic acid (PBA) and 3,4-dihydroxybenzaldehyde (B2) were purchased from Meryer Chemical Technology Co., Ltd. (Shanghai, China). Poly-L-lysine (PLL) trifluoroacetate salt with molecular weight of 15 000−20 000 Da was purchased from COLCOM (France). R-PE was purchased from Cayman Chem. (Michigan, USA). BSA was purchased from Aladdin Inc. (Shanghai, China). Trypan blue was purchased from Yesen (Shanghai, China). HRP and tetramethylbenzidine (TMB) were purchased from Yuanye Bio. (Shanghai, China). PULSin was purchased from Polyplus-transfection (France). Synthesis and Characterization of Boronic Acid-Conjugated Polymers. PBA was reacted with G5 PAMAM dendrimer in anhydrous methanol at 70 °C for 24 h. The feeding molar ratio of PBA to G5 PAMAM dendrimer was 96:1. The yielding product was intensively dialyzed against methanol and distilled water, followed by lyophilization to obtain PBA-decorated G5 PAMAM dendrimer (P1). RBITC-labeled P1 was synthesized by stirring the mixture of P1 and RBITC in dark at an RBITC−P1 molar ratio of 3:1 for 24 h. Then, the product was ultrafiltrated and concentrated to obtain RBITClabeled P1. PBA-modified PLL (P2) was synthesized by a similar procedure, the feeding molar ratio of PBA to PLL was 70:1, and trimethylamine was added as an acid-binding agent. The product was intensively dialyzed against dimethyl sulfoxide and distilled water and lyophilized to obtain PBA-modified PLL. The purified products were characterized by 1H NMR spectroscopy (Varian, 699.804 MHz) in D2O to determine the conjugated number of PBA on each polymer. Synthesis of FITC-Labeled BSA. Briefly, BSA was dissolved in phosphate-buffered saline (PBS) (pH 7.4) and mixed with FITC at a FITC−BSA molar ratio of 3:1. The reaction mixture was stirred for 24 h in the dark at room temperature. The yielding product was intensively dialyzed against PBS and distilled water, followed by lyophilization to obtain FITC-labeled BSA (BSA−FITC) as yellow powders. The product was dissolved in deionized water and stored at −20 °C before use. Fluorescence Quenching Assay. The fluorescence spectra of proteins were recorded by a Hitachi F-4500 fluorescence spectroscopy (Hitachi Ltd., Japan) at excitation and emission wavelengths of λex = 280 nm and λem = 300−450 nm, respectively at room temperature. The polyphenols were added into protein solutions at different molar ratios and incubated for 1 h to allow efficient complexation. The mixture solutions were diluted to 1 mL with deionized water and equilibrated for 1 min before measurement. The concentrations of BSA, R-PE, and RNase A were 0.3, 0.02, and 14.6 μM, respectively. SAXS and Analysis. The experiments were carried out at Shanghai Synchrotron Radiation Facility. The SAXS data of protein solutions and complexes were obtained by subtraction of solvent background through the average of 20 measurements. The wavelength of X-ray radiation was 0.103 nm, and a short exposure of 2 s was used to acquire the scattering data at ambient temperature. The concentration of BSA solution was 5 mg/mL in Tris-buffer (pH = 7). A1 was complexed with BSA at molar ratios of 10:1 and 20:1. Isothermal Titration Calorimetry. ITC measurement was carried out on MicroCal ITC (GE Healthcare, Milwaukee) with a cell volume of 200 μL. BSA and EGCG solutions were prepared in 10 mM PBS buffer at pH 7.0. EGCG (10 mM) was injected into a BSA solution (200 μM) with 2 μL in each injection. The interval time between each injection was 2 min to allow equilibration of the polyelectrolyte system. Control experiments were performed by titrating EGCG solution into PBS buffer without BSA to subtract the dilution heat of EGCG. ITC data showed the total heat generated per second as a function of time.

significantly promote the cytosolic delivery of R-PE into HeLa cells by P1 (Figure 6). On the contrary, the unmodified polymer P0 (Figure 6) and the complexes without polymer (Figure S18) failed to deliver R-PE in the presence of A1 or B1. The polyphenol-facilitated cytosolic delivery system also works when using a boronic acid-modified polylysine (P2) to replace P1 (Figures S19 and S20). Not limited to negatively charged proteins such as BSA and R-PE, we also evaluated the efficiency of the approach for cytosolic delivery of positively charged proteins such as RNase A (pI 9.6, Figure 7a). RNase A is widely used to kill cancer cells.49−51 Free RNase A is membrane-impermeable and thus shows low toxicity on cancer cells. RNase A also binds with the polyphenols such as A1 as determined by the fluorescence quenching assay (Figure S21). The addition of B1 or A1 into RNase A solution slightly changed its secondary structure (Figures 7b and S25a) and enzymatic activity determined by an RNase A activity assay (Figures 7c and S25b). The release of RNase A from the complex showed a pH-responsive behavior (Figure S22), and the enzyme activity of RNase A could be recovered after release (Figure S23). The P1−RNase A complex showed low toxicity on the breast cancer MDAMB-231 cells, suggesting low cytosolic delivery efficiency of P1 in the absence of polyphenols. On the contrary, both the polyphenols B1 and A1 significantly decrease the viability of cells because of the internalization of RNase A into the cells (Figures 7d and S24−S25c). In addition, the increased toxicity is not induced by the polyphenols themselves as the B1− RNase A and A1−RNase A complexes are not toxic on the cells. The acridine orange (AO)−ethidium bromide (EB) double staining assay also confirmed the high efficiencies of P1−B1 and P1−A1 systems in the delivery of toxic protein RNase A into the cytosol of MDA-MB-231 cells (Figures 7e and S25d). Besides RNase A, another enzyme horseradish peroxidase (HRP, 40 kDa, pI 7.2)52 was also successfully delivered into HeLa cells with maintained enzyme activity by the proposed strategy (Figure S26). The results confirmed the maintenance of protein bioactivity after the intracellular delivery by the P1−polyphenol approach. Taken together, the polyphenol−P1 system showed robust efficiency in the cytosolic delivery of membrane-impermeable proteins.

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CONCLUSIONS In summary, we reported a novel approach for cytosolic protein delivery by boronic acid-containing polymers based on catechol−boronate complexation. Proteins such as negatively charged proteins BSA and R-PE, as well as positively charged protein RNase A at physiological condition, were facilely decorated with natural polyphenols such as A1 and B1 via noncovalent interactions or reversible dynamic bonding. The decorated proteins form stable transduction complexes with boronic acid-containing polymers with minimal cytotoxicity. The approach allows efficient internalization of various proteins into the cytosols and maintains the bioactivity of transduced proteins as well. This study provides a new strategy to strengthen the binding affinity between protein molecules and polymer carriers and permits the development of a polyphenol-involved polymer platform for intracellular protein delivery.

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EXPERIMENTAL SECTION

Materials. Ethylenediamine-cored and amine-terminated generation 5 (G5) polyamidoamine (PAMAM) dendrimer (P0 in this G


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Chemistry of Materials Circular Dichroism Measurements. BSA or RNase A was mixed with polyphenols at room temperature for 1 h, and the mixture was diluted to 1 mL with deionized water (final protein concentration is 3 μM) before the measurement. The secondary structure of the proteins was measured by a circular dichroism (CD) spectrometer J-815 (JASCO, Japan). Free protein solutions were measured as a control. Complex Formation and Characterization. The polyphenols (A1−A4 and B1−B2) were added into protein solutions (1 mg/mL, pH 7.0) at different molar ratios. The mixed samples were incubated at room temperature for 1 h. Then, the polyphenol−protein complexes were mixed with the boronic acid-modified polymers and incubated for 30 min. The morphology of the formed nanostructures was characterized by a transmission electron microscope (TEM, HT7700, Hitachi Ltd., Japan). The size and zeta potential of the complexes were measured by DLS using a Malvern Zetasizer (Nano ZS 90, Malvern, UK) at 25 °C. Polymer−protein complexes in the absence of polyphenols as well as protein−polyphenol complexes without polymers were prepared as controls and characterized by TEM and DLS as described above. To evaluate the formation of protein complexes and protein release in vitro, RBITC-labeled P1 was used to quench the fluorescence of BSA−FITC in the complexes. The FITC fluorescence quenching and recovery were recorded at excitation and emission wavelengths of λex = 490 nm and λem = 500−700 nm, respectively. Native BSA and heparin sodium were used to trigger the release of proteins from the complexes. Proteins in Vitro Release. For investigating the in vitro release kinetics of protein under different pH values, we used the differential centrifugation to isolate the free protein without combination with the complex. Generally, the complex formation was conducted as described above and diluted with 2-[4-(2-hydroxyethyl)piperazin-1yl]ethanesulfonic acid buffer at different pH values. After being incubated for different times, the complex solutions were centrifuged at 13 000 rpm for 30 min. The fluorescence intensity of supernatants was recorded by fluorescence spectroscopy. Free proteins without polyphenols or polymer at the same concentration were measured as a control, and the fluorescence intensity was defined as 100%. Cell Culture. HeLa cells (a human cervical carcinoma cell line, ATCC) and MDA-MB-231 cells (a human mammary cancer cell line, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) and minimum essential medium (MEM, GIBCO) respectively, containing 10% fetal bovine serum (FBS, Gemini), 100 μg/mL streptomycin, and 100 μg/mL penicillin at 37 °C under 5% CO2. Cytosolic Delivery of BSA and R-PE. HeLa cells were cultured in 48-well plates overnight before cytosolic protein delivery. The protein complexes were prepared as described above, and the solution was diluted with 100 μL of serum-free DMEM and incubated for 30 min at room temperature. Then, the complex solutions were further diluted with 150 μL of serum-free DMEM and added into the wells. After 4 h incubation, the fluorescence intensity of the transduced cells was analyzed by flow cytometry (BD FACSCalibur, San Jose). The transduced cells were also observed by a laser scanning confocal microscopy (Leica SP5, Germany). The commercial protein transduction reagent PULSin was served as a positive control and used according to the manufacturer’s protocol. In a separate experiment, trypan blue (0.04%) was added to quench the fluorescence of BSA− FITC physically adsorbed on the cell membrane before measurement by flow cytometry. To determine the intracellular trafficking of P1− A1−BSA−FITC nanoparticles, the protein transduction experiments were conducted for 1−12 h as described above, the cell nucleus was stained by Hoechst 33342, and the acidic organelles were stained by LysoTracker (DND-99, Invitrogen) before confocal imaging. To investigate the internalization mechanism of P1−A1−BSA−FITC nanoparticles, the HeLa cells were pre-incubated with various endocytosis inhibitors including cytochalasin D (10 μM), chlorpromazine (20 μM), genistein (700 μM), and methyl-β-cyclodextrin (10 mM) or incubated at 4 °C for 2 h before the protein transduction experiments. The cells treated without inhibitors were tested as controls.

Cell Viability Assay. The viability of cells incubated with protein, polyphenols, and the complexes was evaluated by a standard 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HeLa cells were cultured in 96-well plates overnight. Then, the cell culture medium was replaced by 100 μL of serum-free DMEM-containing protein, polyphenols, or the complexes. After 4 h incubation, the medium was replaced by 100 μL of fresh DMEM containing 10% FBS. The cells were further cultured for another 20 h and tested by MTT assay. The concentrations of protein, polyphenols, and polymer in the MTT assay were equal to those in optimal protein transduction experiments. Cytosolic Delivery of RNase A. MDA-MB-231 cells were cultured in 96-well plates overnight before cytosolic RNase A delivery. The RNase A complexes were prepared as described above. The complex solutions were diluted with 100 μL of serum-free MEM and further incubated for 30 min. The mixture solutions were diluted with 500 μL of serum-free MEM, and 100 μL of the diluted solution was added into each well. After 6 h incubation, the medium in each well was replaced with 100 μL of fresh MEM containing 10% FBS and the cells were further incubated for 42 h. The cytotoxicity of MDA-MB231 cells treated with RNase A complexes was evaluated by MTT assay. Five repeats were conducted for each sample. Naked RNase A, polyphenols A1 and B1, and PBA-decorated dendrimer P1, as well as RNase A−polyphenol complexes at equal protein, polyphenol, or polymer concentrations were tested as controls. The viability of cells treated with the complexes was also determined by AO−EB doublestaining assay. The transduced MDA-MB-231 cells were washed with PBS and stained with AO (5 μg/mL) and EB (5 μg/mL) for 10 min. Then, the cells were washed with PBS and observed by a fluorescent microscope (Olympus, Japan). RNase A Activity Assay. RNase A activity was measured by an RNaseAlert Kit according to the manufacture’s protocol. Briefly, 2 μL of RNaseAlert substrate dissolved in assay buffer was incubated with RNase A (0.1 μg/mL) complex in RNase-free water. The molar ratios of A1−RNase A and B1−RNase A were 0.2:1 and 10:1, respectively. The fluorescence intensity was recorded at 5 min intervals for 40 min. The excitation and emission wavelengths were λex = 490 nm and λem = 520 nm, respectively. HRP Staining and Activity Assay. The cytosolic HRP delivery was determined by intracellular HRP enzymatic activity assay. TMB was used as a colorless substrate and catalyzed into a dark green product in the presence of HRP. Generally, cytosolic HRP delivery was conducted as described above, and the treated cells were washed three times. A TMB substrate (0.5 mL, 10 μg/mL) dissolved in acetate buffer (pH 5.0) with 3 mM H2O2 was added into each well. The enzyme activity was determined by measurement of the absorbance of solution at 630 nm by a microplate reader at 30 s intervals for 16 min. Statistical Analysis. The data for mean fluorescence intensity and cell viability were presented as mean ± standard deviation. Statistically significant difference was analyzed by Students’ t-test. The p-value less than 0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001.

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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/acs.chemmater.8b04672. Characterization of polymers; screening the optimal cytosolic protein delivery conditions; characterization of complex; 1H NMR spectrum; CD spectrum; fluorescence spectrum; stability of complex; in vitro release of protein; enzyme activity; cytosolic delivery of HRP; and other supporting figures (PDF) H


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Tianfu Li: 0000-0002-4222-7022 Yiyun Cheng: 0000-0002-1101-5692 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors appreciate the grants from the National Natural Science Foundation of China (21725402 and 21474030) and the Shanghai Municipal Science and Technology Commission (17XD1401600).

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Natural Polyphenols Augment Cytosolic Protein Delivery by a

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