Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Jul 2022Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery Zhe Zhang†, Xin Gao†, Yanwen Li†, Jia Lv, Hui Wang and Yiyun Cheng Zhe Zhang† Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241 †Z. Zhang, X. Gao, and Y. Li contributed equally to this work.Google Scholar More articles by this author , Xin Gao† Department of Orthopedic Oncology, The Second Affiliated Hospital of Naval Medical University, Shanghai 200003 †Z. Zhang, X. Gao, and Y. Li contributed equally to this work.Google Scholar More articles by this author , Yanwen Li† South China Advanced Institute for Soft Matter Science and Technology, School of Emergent Soft Matter, South China University of Technology, Guangzhou 510640 †Z. Zhang, X. Gao, and Y. Li contributed equally to this work.Google Scholar More articles by this author , Jia Lv Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241 Google Scholar More articles by this author , Hui Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] South China Advanced Institute for Soft Matter Science and Technology, School of Emergent Soft Matter, South China University of Technology, Guangzhou 510640 Google Scholar More articles by this author and Yiyun Cheng *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241 Department of Orthopedic Oncology, The Second Affiliated Hospital of Naval Medical University, Shanghai 200003 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.022.202202098 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Polymers have been widely proposed as carriers for cytosolic protein delivery despite multiple barriers such as protein binding, cell internalization, and endosome escape during cytosolic delivery. Inspired by the strong binding affinity of natural polyphenols with proteins and cell membranes, herein we propose polyphenol modification to improve the efficacy of the protein delivery of cationic polymers. Catechol-modified dendrimers with balanced hydrophobic and hydrogen-bonding interactions show the highest efficacy for various cargo proteins and peptides while the pyrogallol-grafted ones exhibit the lowest efficacy due to increased ligand hydrophilicity. The catechol-based polymers efficiently deliver various bioactive proteins into the cytosol of live cells, exerting biofunctions after intracellular release, and successfully transmitting α-chymotrypsin into tumor cells in vivo to inhibit tumor growth. This study proves that polycatechols can serve as a family of highly efficient carriers for delivery of macromolecular biopharmaceuticals. Download figure Download PowerPoint Introduction Therapeutic proteins have achieved remarkable success in the treatment of cancers, diabetes, and many other disorders.1 About half of the top-selling drugs globally in 2020 are protein biologics, all of which bind to extracellular targets to exert their functions.2 Although a huge number of drug targets locate inside cells, there is an almost total lack of protein biologics acting on intracellular targets due to the inability of most proteins to penetrate across cell membranes.1 The commonly used strategy for enabling exogenous proteins to reach intracellular targets is the delivery of DNA or mRNA encoding the proteins, which might be associated with problems such as poor delivery efficiency, risks of gene insertion and integration, and immunogenicity.3,4 For direct cytosolic delivery of proteins into living cells, numerous approaches have been proposed.5–10 One conventional protein delivery method relies on the fusion or conjugation of a protein transduction domain (PTD) to the protein of interest (POI).11–14 The introduction of PTD improves the endocytosis of cargo proteins; however, the proteins are easily degraded by endolysosomal proteases due to limited endosome escape capability.15 Nanocarriers have been proposed as another promising method for intracellular protein delivery with facilitated cellular uptake and endosome escape.16–23 For example, metal–organic frameworks (MOFs) can be loaded with cargo proteins during nanoparticle synthesis.24,25 However, the strategy is associated with insufficient protein release and probable protein toxification by the abundant metal ions during MOF preparation. For those nanocarriers loaded or coassembled with proteins, one of the major obstacles to protein delivery is the structural complexity of POIs, such as diverse molecular size, shape, and isoelectric points (pIs). The designed nanocarriers are usually applicable for certain proteins with proper charge properties and sizes. To expand the scope of applicable proteins, the biomolecules are chemically modified or genetically engineered with anionic molecules or specific functional groups to increase the binding strength between nanocarriers and cargo proteins.4,26–28 Although some modified ligands can be cleaved from the POIs upon specific external or internal triggers, the modifications on proteins may be accompanied with altered protein activity and undesirable safety concerns. Therefore, the development of efficient strategies for robust delivery of native proteins without modification into cytosols of cells is highly desirable. Recently, researchers have designed highly efficient cytosolic protein delivery polymers by introducing protein-binding ligands into polymers to strengthen their interactions with proteins.29,30 The reported ligands include guanidinium groups that can interact with glutamic acid or aspartic acid residues via a salt bridge,31,32 electron-deficient ligand such as phenylboronic acid that can interact with lysine and histidine residues via nitrogen-boronate coordination,30,33,34 and metal chelates such as zinc(II)-dipicolylamine that can interact with multiple amino acid residues via coordination and ionic interactions.34,35 The introduction of these ligands can greatly improve the protein loading and complex stability of cationic polymers and allow the delivery of unmodified proteins with distinct pIs into live cells. Though great advances in protein delivery have been achieved for these materials, the development of polymeric carriers that can address the multiple barriers—that is, protein binding, cell internalization, and endosome escape in cytosolic delivery—is still a highly challenging task. Natural polyphenols with multiple phenolic units, including phenol, catechol, and pyrogallol, in the structure have gained increasing attention in biomedical applications.36–40 These biomolecules have been reported to interact with proteins via multiple hydrogen bonds, π–π stacking, and hydrophobic interactions to form nanoparticles, nanocapsules, and coatings.36,41–43 Previous reports showed that natural polyphenols such as tannic acid could directly assemble with proteins into nanoparticles for cytosolic protein delivery.41 Alternatively, natural polyphenols were interacted with proteins to form nanoparticles, followed by coating the nanoparticles with boronated polymers via dynamic catechol-boronate linkage for protein delivery. Inspired by the strong binding of natural polyphenols with protein molecules, herein we proposed that direct grafting of phenolic moieties such as phenol, catechol, and pyrogallol onto polymers may generate efficient materials to facilitate protein binding, cell internalization, and endosome escape during cytosolic delivery (Figure 1a). We hope to develop a novel class of functionalized polymers for cytosolic delivery of POIs with robust efficacy. Figure 1 | Catechol-modified dendrimers exhibited high efficiency in cytosolic protein delivery. (a) Dendrimers grafted with phenyl (DP0), phenol (DP1), catechol (DP2), and pyrogallol (DP3) for cytosolic protein delivery. Cargo proteins with different molecular weights (Mw) and pIs were used in this study. Catechol modification improves protein binding, complex stability, cell internalization, and endosomal escape of polymers during cytosolic delivery. Confocal images (b) and mean fluorescence intensity (c) of HeLa cells treated with the complexes for 6 h. The doses of protein and polymer in each well were both 8 μg. ***p < 0.001 analyzed by student’s t test. Download figure Download PowerPoint Experimental Methods Materials Amine-terminated polyamidoamine (PAMAM) dendrimer was purchased from Dendritech, Inc. (Midland, MI). Benzaldehyde, 4-hydroxy benzaldehyde, 3,4-Dihydroxybenzaldehyde, and α-chymotrypsin (CT) were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). 3,4,5-Trihydroxybenzaldehyde, sodium cyanoborohydride, and β-galactosidase (β-Gal) were purchased from J&K Scientific, Ltd. (Shanghai, China). Fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RBITC), bovine serum album (BSA), and saporin from Saponaria officinalis seeds were obtained from Sigma-Aldrich (St. Louis, MO). Phycoerythrin (RPE) was purchased from Cayman Chemical Company (Ann Arbor, MI). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sangon Biotech (Shanghai, China). PULSin was obtained from Polyplus Transfection (Illkirch, France). A polyacrylamide gel electrophoresis (PAGE) Gel Fast Preparation Kit (15%) was purchased from Epizyme (Shanghai, China). Coomassie blue, a β-Gal staining kit, and Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit were purchased from Beyotime (Jiangsu, China). Trypan blue was purchased from Yesen (Shanghai, China). The peptides were synthesized by GL Biochem. (Shanghai, China). Synthesis and characterization of polyphenol-decorated dendrimers The chemicals benzaldehyde, 4-hydroxy benzaldehyde, 3,4-dihydroxybenzaldehyde, and 3,4,5-trihydroxybenzaldehyde were reacted with G5 PAMAM dendrimer in anhydrous methanol at 4 °C for 24 h, respectively. The feeding molar ratios of benzaldehyde, 4-hydroxy benzaldehyde, 3,4-dihydroxybenzaldehyde, and 3,4,5-trihydroxybenzaldehyde to G5 dendrimer were 128∶1, 96∶1, 96∶1, and 96∶1, respectively. Then, sodium cyanoborohydride with a molar ratio of 1.5∶1 to the chemicals was added, and the reaction was further carried out for 6 h. The synthesized products were intensively dialyzed and lyophilized to obtain polymers DP0, DP1, DP2, and DP3. 1H NMR spectroscopy (Varian, 500 MHz) was carried out to determine the conjugated number of each dendrimer. Catechol conjugated dendrimers with the feeding ratio of 3,4-dihydroxybenzaldehyde to G5 dendrimer at 32∶1, 64∶1, and 128∶1, respectively, were also synthesized. Synthesis of RBITC labeled dendrimers To phosphate-buffered saline (PBS) solution containing the synthesized dendrimers, RBITC dissolved in dimethyl sulphoxide (DMSO) was added dropwise at a dye/polymer molar ratio of 3∶1. After reacting in the dark for 24 h, the samples were intensively dialyzed, lyophilized and then stored at −20 °C before further use. The polymer solutions were measured by fluorescence spectroscopy (Excitation wavelength: 500 nm, Emission wavelength: 550–700 nm, Hitachi F-4500, Tokyo, Japan). Synthesis of FITC labeled proteins To a PBS buffer solution containing 2 μmol BSA, a DMSO solution containing 6 μmol FITC was added. After reacting overnight, the samples were intensively dialyzed, lyophilized, and then stored at −20 °C before further use. FITC-labeled CT was synthesized by the same procedure. Polymer-protein complex preparation and characterization The polymers were mixed with a certain amount of proteins at room temperature for 30 min. Dynamic light scattering (DLS, Nano ZS 90, Malvern, UK) and transmission electron microscopy (TEM, HT7700, Hitachi Ltd., Tokyo, Japan) were used to measured the size and zeta potential of the complexes and morphology of the formed nanoparticles, respectively. The amount of BSA bound by the polymers was detected by PAGE. In brief, the prepared complexes were centrifuged (12,500 rpm, 20 min), and the sediments were collected and redissolved by 1x loading buffer, which were then electrophoresed on a 15% polyacrylamide gel according to the protocol of a PAGE gel fast preparation kit. The protein bands were stained with Coomassie blue and imaged using a Gel Imaging System (Tanon 2500B, Shanghai, China). Free BSA solution was run as a control. The protein binding constant Kb of the polymers was analyzed as follows: Polymer and BSA-FITC were mixed in 50 μL deionized water or 6.4 mg/mL NaCl solution and incubated at room temperature for 30 min. 950 μL deionized water or NaCl solution were used to further dilute the polymer/protein complexes. Then the fluorescence intensity of the mixture solution was measured using a fluorescence spectrometer (Ex: 480 nm, Em: 518 nm). The concentration of BSA-FITC in the final solution was 100 nM. Fractional saturation (FS) was calculated according to eq 1. FS = ( F 0 − F X ) / ( F 0 − F S ) (1) F0 is the fluorescence intensity of free BSA-FITC, FX is the fluorescence intensity of complexes at different polymer concentrations, and FS is the fluorescence intensity at saturation. Kb was calculated by eq 2. FS = ( n K b C + x K b + 1 ) − ( n K b C + x K b + 1 ) 2 − 4 n x K b 2 C 2 n K b C (2)where x is molar concentration of polyphenol modified dendrimers (DP). C is the molar concentration of BSA-FITC. n is the binding sites number. Kb and n were obtained by a nonlinear curve module fitting with eq 2. Cell culture and protein delivery HeLa cells (a human cervical carcinoma cell line, ATCC) were cultured in 48-well plates overnight at 37 °C in a 5% CO2 atmosphere before protein delivery. The prepared polymer/protein complexes were incubated with 50 μL of serum-free media for 30 min. After dilution with 150 μL of serum-free media, the complexes were added into the cells. The culture media were removed after 6 h incubation. Trypan blue (0.04%) was added to quench the fluorescence of green fluorescent protein (GFP), BSA-FITC and CT-FITC were physically adsorbed on the cell surface. The treated cells were analyzed by flow cytometry (BD LSRFortessa, San Jose, CA), and laser scanning confocal microscopy (Leica SP5, Frankfurt, Germany). PULSin, a commercial reagent, was used according to the manufacturer’s protocol. The β-Gal enzyme activity of HeLa cells was tested by a β-Gal staining kit according to the manufacturer’s protocol.30 To test the influence of polymers on β-Gal activity, 8 μg polymers were incubated with 5 μg β-Gal in 50 μL H2O for 30 min. Then, 50 μL working solution was added, and the complex solutions were further incubated at 37 °C for 1 h. 100 μL DMSO was added, and the optical density of solution at 633 nm was measured using a microplate reader. Free β-Gal solutions in the absence of polymers were tested as a control. To confirm that the β-Gal activity in the complexes can be recovered after cytosolic delivery, the complex solutions were also incubated with 2× PBS for 1 h, and then the β-Gal activity was measured as described above. Gal8 recruitment assay To generate cells stably expressing yellow fluorescent protein-tagged galectin 8 (YFP-Gal8), HEK 293 cells were transfected with YFP-Gal8 plasmid and packaging plasmids pCMV-VSV-G using lipofectamine 2000 to generate pseudotyped lentiviral particles which were applied to HeLa cells. Gal8-YFP transduced cells were obtained by 1-week selection with puromycin, followed by single clonal expansions obtained through the limiting dilution method in puromycin-containing media; clonal populations were used to ensure consistent expression of YFP constructs. For Gal8 recruitment assay, HeLa cells stably expressing YFP-Gal8 were cultured in confocal dishes overnight. After 2, 4, or 6 h treatment of the cells with the polymer/BSA complexes, the media were aspirated and replaced with PBS. Images were acquired by confocal microscope. Cell viability assay A well-known MTT assay was carried out to investigate cell viability. Briefly, HeLa cells cultured in 96-well plates overnight were incubated with the samples for 6 h. Then, the complexes were removed, and fresh Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) was added to further incubate the cells for 18 h. Five repetitions were conducted for each sample. For LDH release assay, the cells cultured in 48-well plates were incubated with the polymer-protein complexes for 6 h. The media were replaced with fresh DMEM containing 10% FBS for 18 h at 37 °C. Then, the supernatant was treated with lysis buffer at 37 °C for 1 h. Three repetitions were conducted for each sample. In vivo protein delivery The animal experiments were approved by the ethics committee of East China Normal University and performed in compliance with National Institutes of Health guidelines. BALB/c mice (4 weeks old, about 18–19 g) were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). For in vivo therapy, the mice were subcutaneously injected with 143B cells (∼106 cells in PBS, 100 μL) at right back and randomly divided into five groups (four mice per group) when the tumors grew up to around 250 mm3. The mice were intravenously injected with 100 μL PBS, DP2, CT, and DP2/CT complexes and 143B cell membrane-coated DP2/CT complexes ([email protected]/CT) every other day, respectively. A total number of three injections were administrated to each mouse. The dosage of CT and polymer was 14 and 10 μg per mouse. The mice were sacrificed on the seventh day, and the tumors from each mouse were harvested and weighed. Statistical analysis The data were presented as the mean ± SD. The significant differences between two groups were analyzed by the Student’s one-tailed t-test. n.sp ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001. Results and Discussion Polyamidoamine dendrimers with well-defined surface functionality and excellent endosomal escape capability were used as the scaffold to synthesize nanocarriers for cytosolic protein delivery.44,45 A series of phenol-modified dendrimers were synthesized by reacting generation 5 (G5) polyamidoamine dendrimers with 4-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, and 3,4,5-trihydroxybenzaldehyde, respectively, followed by the reduction of yielding Schiff-base using sodium cyanoborohydride. Phenyl-modified dendrimer was synthesized as a control material by reacting the dendrimer with benzaldehyde according to the same procedure. The average numbers of phenyl, phenol, catechol, and pyrogallol ligands modified on each PAMAM dendrimer were measured to be 71, 73, 73, and 73, respectively according to 1H NMR. The synthesized materials were named DP0, DP1, DP2, and DP3, respectively ( Supporting Information Figure S1). D represents dendrimer, P represents phenol, and the Arabic figures represent the number of hydroxyl groups on the ligand. We initially investigated the efficacy of the obtained polymers in cytocolic delivery using FITC-labeled BSA (Mw 69.3 kDa, pI 4.7) as the model protein (BSA-FITC). The complexes prepared by gently mixing the polymers with BSA-FITC were incubatated with HeLa cells for 6 h. As shown in Figure 1b,c and Supporting Information Figure S2, DP2 exhibited the highest efficacy in the delivery of BSA-FITC, followed by DP1, while DP0, DP3, and unmodified dendrimer (D) showed very poor delivery efficacies on the cells. Moreover, DP2 exhibited much higher efficacy than PULSin™, a commercial protein delivery reagent. The mean fluorescence intensity of DP2/BSA-FITC treated cells was nearly nine times higher than those with PULSin™/BSA-FITC. Confocal images in Figure 1b also show that BSA-FITC delivered by DP2 were generally dispersed throughout the cells, suggesting sufficient release of the bound proteins by DP2 after the complexes were internalized by cells. We further investigated the reason why DP2-bearing catechol moieties showed the highest efficacy among the polymers. Since DP0 has shown significant cytotoxicity in the treated cells (Figure 2a), this polymer will not be investigated in detail in the following study. According to our previous studies, protein binding and complex stability play crucial roles in polymer-mediated cytosolic protein delivery.35 According to the DLS study, all the polymers formed 100–200 nm-ssized and positively charged nanoparticles with BSA in water ( Supporting Information Table S1). However, the complexes showed decreased stability in the presence of NaCl (6.4 mg/mL, 110 mM) due to weakened ionic interactions between the polymers and proteins ( Supporting Information Figure S3). The size of DP1 and DP3 complexes were gradually increased within 2 h in NaCl solution. In comparison, the DP2 complex was relatively stable during this period ( Supporting Information Figure S3). The stability of DP2/BSA complexes in the presence of salt was also confirmed by TEM (Figure 2b). We also quantitatively calculated the protein-binding ability of the polymers by fluorescence titration (Figure 2c–e). The binding constant for DP2 with BSA-FITC was found to be much higher than those for DP1 and DP3. The introduction of hydroxyl groups on the phenyl ring greatly increased the hydrophilicity of the ligand which weakened the protein-binding capability. On the other hand, the presence of hydrogel moieties also brought about hydrogen-bonding interactions. Moreover, the phenolic hydroxyl group is a hydrophilic group with a certain negative charge. Increase in the number of hydroxyl groups on the phenyl group will not only affect the protein binding of polymers via hydrophobic and hydrogen-bonding interactions but also weakens the electronic interactions between the cationic dendrimers and negative BSA (pI = 4.7). Therefore, a balanced hydrophobic interaction, hydrogen-bonding interaction, and electronic interaction on the polyphenol groups are beneficial for efficient protein binding. Figure 2 | Characterization of polyphenol dendrimers and polymer/protein complexes. (a) Viability of HeLa cells treated with different conjugations of D, DP0, DP1, DP2, and DP3, respectively for 24 h measured by an MTT assay. (b) TEM images of the polymer/BSA-FITC complexes prepared in NaCl solution. (c–e) FS and fitting curves of the DP1/BSA-FITC (c), DP2/BSA-FITC (d), and DP3/BSA-FITC (e) complexes at different polymer concentrations in NaCl solution at room temperature. The concentration of BSA-FITC was fixed at 100 nM. Download figure Download PowerPoint Cellular internalization is another key step in successful cytosolic delivery. For quantitative analysis, we conjugated the polymers D, DP0, DP1, DP2, and DP3 with a red fluorescent dye RBITC ( Supporting Information Figure S4). The polymers showed comparable fluorescent intensity at an equal polymer mass concentration. The cells were then treated with the RBITC-labeled polymers in the absence or presence of BSA-FITC and followed by analysis of internalized fluorescent dyes by confocal microscopy and flow cytometry (Figure 3a,b and Supporting Information Figure S5). DP2 exhibited the highest cellular uptake among the five polymers with strong red fluorescence throughout all of the cells, suggesting efficient endocytosis of DP2. On the contrary, cells treated with D, DP0, DP1, and DP3 were observed with much weaker fluorescence, and the red fluorescence appeared as multiple punctuate in the cells for these materials, suggesting poor endosomal escape. We then investigated the intracellular trafficking of polymer/BSA complexes in HeLa cells stably expressing YFP-Gal8. Gal8 is a cytosolic protein that can immediately recognizes the damaged endosome through selective binding with the glycosylation moieties on the inner membrane of endosomes (Figure 3c).46–48 As shown in Figure 3d,e and Supporting Information Figure S6, obvious and yellow punctate fluorescent spots were observed for DP2/BSA complexes while negligible Gal8 recruitment was detected for the other complexes. The successful endosome escape happened as early as 2 h post complex incubation. We then plotted the data from cellular uptake and Gal8 recruitment against protein delivery efficacy to clarify the factors affecting intracellular protein delivery. As shown in Figure 3f, it was clearly demonstrated that catechol modification on polymers is beneficial for enhanced cell internalization and endosomal escape. The possible reason for the highest cellular uptake and endosomal escape of catechol among the ligands is due to balanced hydrophobic and hydrogen-bonding interactions with cell membranes and endosomal membrane components. Moreover, after escaping from endosomes, the cargo proteins could be released from DP2 by the competitive effects of high-concentration proteins and polyanions in the cytosol, which is in accordance with previous reports on functional cationic polymers.8,29,30,35 Figure 3 | Cell internalization and endosomal escape of polyphenol dendrimers. Confocal images (a) and mean fluorescence intensity (b) of HeLa cells incubated with RBITC-labeled D, DP0, DP1, DP2, and DP3 for 6 h. The doses of polymer were 8 μg. (c) Schematic of intracellular Gal8 recruitment. Gal8-YFP is dispersed in the cytoplasm. However, when endosomal membranes are disrupted, it binds to the intraendosomal glycans and aggregates into punctuated fluorescent spots. (d) Confocal images of HeLa-Gal8-YFP cells treated with the polymer/BSA complexes for 6 h. (e) Quantification of Gal8 recruitment confocal images of HeLa-Gal8-YFP cells treated with the polymer/BSA complexes for 2, 4, and 6 h. (f) Relationship of cellular uptake and endosome disruption with protein delivery efficacy. **p < 0.01 and ***p < 0.001 analyzed by student’s t test. Download figure Download PowerPoint Since DP2 showed the highest efficacy in protein delivery among the synthesized polymers, we then synthesized a series of catechol-conjugated dendrimers with different catechol-grafting density. According to the 1H NMR spectra in Supporting Information Figure S7, the conjugated molecules on each dendrimer were calculated to be 23, 45, and 78, respectively, and the products were named DP2-23, DP2-45, and DP2-78, respectively. As shown in Supporting Information Figure S8, the efficacy of DP polymers increased with the increase of the catechol-grafting number. DP2-23 and DP2-45 showed much weaker efficacies in the cytosolic BSA-FITC delivery compared to DP2 conjugated with 73 catechol groups. Further increase in the catechol-grafting ratio (DP2-78) led to decreased delivery efficacy due to decreased positive charges on the polymer after conjugation of excessive catechols. The delivery of GFP (Mw 26.9 kDa, pI 6.2) and RPE (Mw 240 kDa, pI 4.3) were also investigated. As shown in Supporting Information Figure S9, DP2 formed 200–300 nm-sized nanoparticles with both GFP and RPE. As expected, DP2 exhibited the highest delivery efficacy of both the two fluorescent proteins in HeLa cells, followed by DP1, and was much more efficient than D, DP0, and DP3 as well as PULSin ( Supporting Information Figure S10). We then investigated whether the bioactivity of POIs can be maintained after intracellular delivery. β-Gal (Mw 430 kDa, pI 5.0), an enzyme capable of hydrolyzing colorless substrate 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) into a blue dye, was chosen as the model protein. The enzymatic activity of β-Gal was significantly inhibited in the presence of polymers ( Supporting Information Figure S11), which is due to the condensation of β-Gal in the complexes. However, the β-Gal