An Intracellular pH-Actuated Polymer for Robust Cytosolic Protein Delivery

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Dec 2021An Intracellular pH-Actuated Polymer for Robust Cytosolic Protein Delivery Wei Xu, Feng-Qin Luo, Qi-Song Tong, Jia-Xian Li, Wei-Min Miao, Yue Zhang, Cong-Fei Xu, Jin-Zhi Du and Jun Wang Wei Xu School of Biomedical Sciences and Engineering, Guangzhou International Campus, South China University of Technology, Guangzhou 511442 , Feng-Qin Luo School of Medicine, South China University of Technology, Guangzhou 510006 , Qi-Song Tong School of Biomedical Sciences and Engineering, Guangzhou International Campus, South China University of Technology, Guangzhou 511442 , Jia-Xian Li School of Medicine, South China University of Technology, Guangzhou 510006 , Wei-Min Miao School of Biomedical Sciences and Engineering, Guangzhou International Campus, South China University of Technology, Guangzhou 511442 , Yue Zhang School of Biomedical Sciences and Engineering, Guangzhou International Campus, South China University of Technology, Guangzhou 511442 , Cong-Fei Xu School of Biomedical Sciences and Engineering, Guangzhou International Campus, South China University of Technology, Guangzhou 511442 Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, Innovation Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006 , Jin-Zhi Du *Correspondence author: E-mail Address: [email protected] School of Medicine, South China University of Technology, Guangzhou 510006 National Engineering Research Center for Tissue Restoration and Reconstruction, Key Laboratory of Biomedical Engineering of Guangdong Province, South China University of Technology, Guangzhou 510006 Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005 and Jun Wang School of Biomedical Sciences and Engineering, Guangzhou International Campus, South China University of Technology, Guangzhou 511442 Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, Innovation Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006 National Engineering Research Center for Tissue Restoration and Reconstruction, Key Laboratory of Biomedical Engineering of Guangdong Province, South China University of Technology, Guangzhou 510006 https://doi.org/10.31635/ccschem.021.202000696 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Robust cytosolic protein delivery requires both efficient protein binding with delivery vehicles and effective protein release after cell internalization. Although a variety of stimuli-responsive carriers have been designed, simultaneously integrating these two functions in one versatile carrier is challenging. Herein, we developed a polyamidoamine (PAMAM)-based polymer with an intracellular pH-actuated hydrophobic-to-hydrophilic transition for this purpose. The polymer (designated as G5-C4) was synthesized by appending N,N-dibutylaminoethyl moieties to the peripheral amino groups of PAMAM dendrimer generation 5. Assisted by hydrophobic interactions, G5-C4 formed condensed nanoparticles with proteins and remained stable under physiological conditions. After efficient cell internalization, G5-C4 underwent a hydrophobic-to-hydrophilic transition at acidic endosomal pH, and thus promoted protein release and endosomal escape, which was verified by structure–activity relationship studies. Furthermore, both in vitro and in vivo studies indicated that G5-C4 achieved superior antitumor effect. This study provides a simple but effective design for stimuli-responsive cytosolic protein delivery. Download figure Download PowerPoint Introduction Proteins are critical in cell signal transduction and metabolism. Abnormal protein mutations or dysfunction may lead to various diseases including cancers.1 Recombinant protein therapies, including cytokines, peptide hormones, and monoclonal antibodies, have been used to treat these diseases.2 Compared with small molecule drugs, protein drugs offer advantages of higher specificity, fewer adverse effects, and faster clinical development.1 However, all current protein drugs on the market are engineered for extracellular targets, which account for 30% of genome proteins.3,4 The remaining 70% intracellular targets are not accessible due to the protein drugs' hydrophilic and macromolecular nature, which prevents their efficient transport across the cell membrane. One approach for rapid cytosolic delivery is to conjugate proteins with protein transduction domains (PTDs) or other ligands.5–7 In addition, the employment of self-assembled materials as protein and peptide delivery vehicles has attracted extensive attention,8–11 such as inorganic nanoparticles,12–17 lipid materials,18–22 nanogels,23 and polymers.24,25 A number of robust delivery systems based on polymers have been identified from screening libraries, which include guanidyl-rich polymers,26–30 fluoropolymers,31 boronate-rich polymers,32 carboxylated branched poly(β-amino ester)s,33 and polymers that can coordinate metal cation.34,35 In these examples, various methods were utilized to enhance the binding affinity of polymeric carriers with cargo proteins. Despite these advances, the mechanism of intracellular protein release from the delivery vehicles is still not well documented. It is well known that efficient cytosolic protein delivery requires carriers to (1) form condensed complex with proteins and be stable in serum, (2) enter cells effectively, (3) escape from the endosomal compartment, and (4) release intact cargos. The overwhelming binding interaction between cargo proteins and carriers may hinder their release from the complexes. To overcome the dilemma between enhanced loading and efficient release, stimuli-responsive systems for protein delivery have been developed,25,36–42 in which the carriers often undergo a rapid degradation in response to intracellular acidic or redox microenvironments, and thus facilitate the release of the bound proteins into cytosol. However, these designs conventionally incorporated a variety of functional modules into one carrier to simultaneously achieve protein loading, endosomal escape, and cargo release, usually making the delivery carriers complicated in functions and tedious to synthesize. A more simple and straightforward strategy that can fulfill all the functions for robust cytosolic protein delivery is urgently needed. In this study, we propose that a polymer with an intracellular pH-triggered hydrophobic-to-hydrophilic transition around endosomal pH may meet the requirements. As a proof-of-concept study, we modified polyamidoamine (PAMAM) dendrimer generation 5 (G5) with N,N-dialkylaminoethyl moieties and investigated the influence of the length of alkyl chain on the delivery efficacy as well as the underlying mechanisms. The structure–activity study indicated that PAMAM polymer with N-dibutylaminoethyl moieties (G5-C4) outperform its counterparts with similar structures. The incorporation of N-dibutylaminoethyl moieties may have the following advantages: First, the locally high density of hydrophobic moieties could strengthen the binding affinity of polymers with proteins (Figure 1a). Second, the tertiary amine structure undergoes hydrophobic-to-hydrophilic transition under acidic pH conditions due to protonation of the nitrogen atom, which reduces the hydrophobic interaction and facilitates protein release. Third, the protonation of the tertiary amine in endosomes improves association of the polymer with endosomal membranes due to the more negative nature of endosomal membranes compared with cell membranes.43,44 This unique "turn on" feature makes the polymer a selectively membrane destabilizing carrier that is "mild" to cell membranes but "aggressive" to endosomal membranes, which facilitates endosomal deconstruction and escape (Figure 1c). Collectively, G5-C4 polymer may fulfill all the requirements for effective protein delivery. This intracellular pH-actuated strategy provides opportunities for constructing efficient protein delivery vehicles. Figure 1 | Design of N,N-dialkylaminoethyl modified polymers for cytosolic protein delivery. (a) Coassembly of polymers with proteins. (b) Structures of tertiary amine units C1 to C5, listed in order of increasing hydrophobicity. (c) Proposed working mechanism for cytosolic protein delivery. Download figure Download PowerPoint Experimental Methods Materials Amine-terminated and ethylenediamine-cored G5-PAMAM dendrimer was purchased from Dendritech, Inc. (Midland, MI). 2-(Dimethylamino)ethanol (DMEA; 98%), 2-(diethylamino)ethanol (DEEA; 99%), 2-(dibutylamino)ethanol (DBEA; 98%), 2-(dipentylamino)ethanol (DnPEA; 98%), N,N′-carbonyldiimidazole (CDI; 99%), Rhodamine B isothiocyanate (RBITC), and bovine serum albumin (BSA) were purchased from Aladdin (Shanghai, China). 2-(Dipropylamino)ethanol (DPEA) was synthesized according to reported literature.45Cis-aconitic anhydride (cis-Aco) and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide (DMSO) and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sephadex LH-20 was purchased from Pharmacia (Uppsala, Sweden). PULSin was purchased from Polyplus (Strasbourg, France). β-Galactosidase (β-Gal) and Ribonuclease A (RNase A, from bovine pancreas) were purchased from J&K Scientific (Shanghai, China). In situ β-Gal staining kit and LysoTracker Red were purchased from Beyotime (Shanghai, China). Synthesis of CDI activated alcohol The synthesis of DBEA-CDI is described as a general procedure for the construction of CDI activated alcohols. CDI (1.945 g, 12.0 mmol) was charged into a dried round-bottom flask and dissolved with 10 mL dichloromethane. Then DBEA (1.386 g, 8.0 mmol) dissolved in 10 mL dichloromethane was added dropwise to the CDI solution under stirring. The reaction was stirred for 24 h at room temperature. The reaction mixture was washed with water three times. The organic layer was collected, dried over MgSO4, and filtered. After the removal of dichloromethane under reduced pressure, a pale-yellow oil, DBEA-CDI, was obtained (yield 84.7%). Synthesis of surface-modified dendrimers Surface-modified dendrimers were synthesized through a one-step reaction. G5-PAMAM dendrimer (20.0 mg in 1.0 mL DMSO) was reacted with DBEA-CDI (6.5 mg) at a molar ratio of 1:35. The mixture was stirred at 40 °C for 24 h. The product was collected by passing the solution through a Sephadex LH-20 column with methanol as the eluent. After the removal of solvent by reduced pressure overnight, product was obtained with a yield of 80%, denoted as G5-C4. Other polymers, including G5-C1, G5-C2, G5-C3, and G5-C5, were synthesized following a similar procedure. G5-C4 polymers of different graft numbers were obtained by changing molar ratios of PAMAM to DBEA-CDI (1∶22 for G5-C420 and 1∶58 for G5-C440). Polymer characterization: NMR and pH titration Polymer structure was characterized by 1H NMR spectroscopy in CDCl3 or in D2O (400 MHz; Bruker, Billerica, MA). The pH titrations were performed using a microelectrode pH meter (Mettler Toledo, Columbus, OH); polymer (2–5 mg) was dissolved in 0.1 N HCl (100–200 μL) and titrated from pH 2.0–11.0 using 0.1 N NaOH added stepwise; pH was recorded after each addition. Synthesis of BSA-FITC BSA (150 mg) was dissolved in 15 mL of 0.1 M carbonate buffer (pH 9.5), and FITC (20 mg/mL) dissolved in DMSO was added to the solution at a BSA/FITC molar ratio of 1∶6. The mixture was stirred overnight at 4 °C and dialyzed against phosphate-buffered saline (PBS; pH 7.4) and distilled water for 2 days. BSA-FITC was obtained as yellow powder after lyophilization. Protein modification with cis-Aco Cis-Aco-modified RNase A (RNase A-Aco) was prepared according to a reported procedure with slight modification.37 Briefly, 10 mg of RNase A was dissolved in 2 mL of 0.5 M NaHCO3 buffer (pH 9.5), two molar equivalents of cis-Aco were added stepwise to avoid rapid pH decrease. The mixture was stirred for 2 h at room temperature. After that, the solution was purified with ultrafiltration [molecular weight cut-off (MWCO) = 3000, Millipore] several times to remove excess cis-Aco and salt. The RNase A-Aco obtained was a pale yellow after lyophilization. Preparation and characterization of polymer/protein complexes The tertiary amine-modified dendrimers were mixed with native proteins or cis-Aco-modified proteins at different weight ratios (w/w) in water via vortex. Before measurements, complexes were incubated in water or PBS (20 mM) of different pH for 30 min. The size and zeta potential of prepared complexes were analyzed by dynamic laser scattering (DLS; Litesizer 500; Anton Paar, Graz, Steiermark, Austria). The morphology of complexes was observed by transmission electron microscopy (TEM; Talos F200X; Thermo Scientific, Waltham, MA). Cell culture and animals HeLa cells [American Type Culture Collection (ATCC) CCL-2] were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS Gibco) and 1% streptomycin/penicillin at 37 °C under 5% CO2. Female BALB/c nude mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). Tumors were engrafted when the mice were 6–8 weeks old. All mouse studies were carried out following a protocol (2018018) approved by the Institutional Animal Care and Use Committee at South China University of Technology and complied with all relevant ethical regulations. Intracellular protein delivery The cells were seeded on 24-well plates at a density of 8 × 104 cells per well overnight before protein delivery. Polymer/protein complexes at different weight ratios were diluted with serum-free medium and added to cells. After incubation for 4 h, the cells were washed with PBS and collected. The mean fluorescence intensity (MFI) of complex treated cells was quantitatively analyzed by flow cytometry (BD FACSCelesta, San Jose, CA); the fluorescence distributions were observed by confocal laser scanning microscope (CLSM; Nikon Ti-E A1, Tokyo, Japan). The commercial protein delivery reagent PULSin was used as positive control following the manufacturer's guidelines. The fluorescence of BSA-FITC absorbed on cell membranes was quenched with 0.2 mg/mL of trypan blue before measurement. For observation by CLSM, the cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) and the acidic organelles were stained with LysoTracker Red (Beyotime). To study the endocytosis mechanism for G5-C4/BSA-FITC complex, HeLa cells were pretreated with endocytosis inhibitors including chlorpromazine (20 μM), genistein (700 μM), and amiloride (150 μM) for 1 h. The cells were cultured with complexes for 4 h, and protein delivery at 4 °C was carried out before quantitative analysis by flow cytometry. The cells treated with complexes without inhibitor treatment were set as a control. β-Gal activity assay and intracellular staining For detection of β-Gal enzyme activity in vitro, free β-Gal or the polymer/β-Gal complexes dissolved in water were preincubated in PBS (20 mM) of different pH for 30 min and added to the working solutions of in situ staining kit, incubating at 37 °C for 1 h. After removal of the supernatant, the blue-colored hydrolysis product was dissolved in 100 μL of DMSO, and the absorbance at 633 nm was detected using a microplate reader. The relative enzyme activity of free β-Gal in water or PBS was defined as 100%. Intracellular delivery of β-Gal into HeLa cells was determined by intracellular enzymatic activity using the in situ β-Gal staining kit. Briefly, the cells were treated with polymer/β-Gal complexes for 4 h, washed twice with PBS, and fixed for 15 min at room temperature. Then the cells were incubated with the working solution containing X-Gal overnight at 37 °C according to the protocol. The blue-colored hydrolysis products distributed within cells were observed by an optical microscope (Olympus, Tokyo, Japan). Hemolytic activity assay The endosomal escape ability of the polymers and complexes were evaluated by hemolysis assay according to reported procedures.46 Briefly, whole blood was collected from the orbit of BALB/c nude mouse, washed with PBS, and centrifuged (3500 rpm) several times to separate red blood cells. The collected cells were counted and diluted to 5 × 108 cells/mL, and polymers and complexes were incubated with red blood cells in PBS of varying pH (7.4 and 5.5) mimicking the normal physiological environment and acidic endosomes for 90 min at 37 °C. PBS and Triton X-100 (1% v/v) were employed as negative and positive controls, respectively. Then, the cells were centrifuged at 3500 rpm for 10 min, and the absorbance of supernatant at 405 nm was recorded using a microplate reader. The hemolytic activity was calculated according to the following formula: H = (A − A0)/(ATX − A0), where A, ATX, and A0 denote the absorbance of the sample well, positive control, and negative control, respectively. Cell viability assay The viability of cells incubated with native proteins, cis-Aco-modified proteins, polymers, or complexes was evaluated using a well-established 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, Hela cells were seeded in 96-well plates at 1 × 104 cells per well overnight. Then cells were incubated with 100 μL of serum-free DMEM containing free proteins, polymers, or complexes for 4 h. After that, the media were replaced by fresh DMEM containing 10% FBS. The cells were tested by MTT assay after another 44 h of culture to determine the cell viability. Three repeats were conducted for each sample. In vivo RNase A delivery and tumor growth inhibition HeLa xenograft tumor model was established by subcutaneous injection of 100 μL HeLa cells suspension (8 × 106 cells in 100 μL PBS) into the right flank of BALB/c nude mouse, the tumor volume reached approximately 90 mm3 at 12 days after inoculation. Tumor-bearing mice were randomly divided into four groups (n = 5) and treated with 50 μL PBS, free RNase A-Aco, G5-C4, and G5-C4/RNase A-Aco complex solution, respectively, via intratumoral injection every other 2 days. The doses of RNase A-Aco and polymer were 500 μg/kg and 4.5 mg/kg, respectively. The tumor size and body weight of mice were measured every other day. The tumor volume was calculated using the following formula: V = L × W × W/2 (L, the longest dimension; W, the shortest dimension). The tumors were excised and weighed at the end of therapeutic experiment. Statistical analysis All the data are presented as mean ± standard deviation. Students' t test was used to analyze the significant difference. Statistical significance was set at *P < 0.05, **P < 0.01, and ***P < 0.001. Results and Discussion Synthesis and characterization of N,N-dialkylaminoethyl modified polymers We chose G5 PAMAM as a model polymer because of its well-defined structure and highly dense surface functionality. The tertiary amine-modified polymers were synthesized by a one-step reaction between primary amine groups of PAMAM and N,N-dialkylaminoethyl carbamate moieties bearing alkyl chains of varying lengths.45 Polymers were purified by the LH-20 column and characterized by 1H NMR. As shown in Supporting Information Figure S1, the presence of characteristic peaks around 4.10 ppm as well as peaks between 0.8 and 1.4 ppm indicated the introduction of tertiary amines on the dendrimer surface, and an average of 30 tertiary amine moieties was grafted on each G5 PAMAM. The dendrimer derivatives with different alkyl chains were denoted as G5-C1, G5-C2, G5-C3, G5-C4, and G5-C5, respectively (Figure 1b). Structure-dependent cytosolic protein delivery The resultant polymers were tested for efficient intracellular protein delivery using BSA-FITC as a model protein. The polymers were mixed with BSA-FITC at different polymer/BSA weight ratios (1/8, 1/4, 1/2, 1/1, and 2/1). Then, the complexes were cultured with HeLa cells with a fixed protein dose. The MFI of HeLa cells was analyzed by flow cytometry. As shown in Figure 2a, each polymer showed the maximal MFI at 2/1, indicating the essential role of polymers in intracellular protein delivery. Notably, among the five polymers, G5-C4 exhibited superior efficacy over the others, including G5-C3 and G5-C5, whose are similar to G5-C4. G5-C4 was more efficient G5 PAMAM and a commercial protein transduction reagent The MFI of HeLa cells treated with G5-C4/BSA-FITC complex was approximately higher that achieved by G5-C4 and its complex on HeLa cells in the tested Supporting Information Figure In addition, changing the graft weight and generation of PAMAM the delivery of the polymer Supporting Information and We conducted confocal to the fluorescence signal within cells. As shown in Figure fluorescence were observed in cells G5-C4 complex other groups exhibited Figure 2 | Intracellular protein delivery by various N,N-dialkylaminoethyl modified (a) MFI of HeLa cells treated with complexes for 4 h after trypan blue from to right for each complex polymer/BSA weight ratios of and respectively. PULSin was used as a positive control. (b) of HeLa cells treated with complexes weight ratio of after trypan blue (c) X-Gal staining of HeLa cells after treatment with complexes weight ratio of for 4 h. The of proteins in all was 4 per Download figure Download PowerPoint To the of the proteins after intracellular β-Gal was β-Gal is a charged enzyme with high weight of a for into and a blue enzymatic hydrolysis Supporting Information Figure HeLa cells were incubated with polymer/β-Gal complexes for 4 h, by X-Gal As shown in Figure more blue the cells treated with the of the robust enzymatic activity of In a small of blue cells of the other blue product was observed in cells treated with β-Gal were with the confocal both of which that G5-C4 protein delivery of complexes by polymer critical including stable and condensed complex high efficient endosomal escape, and protein release, determined the delivery these polymers showed intracellular delivery we investigated the tertiary amine the delivery To study the protein binding and of size and zeta potential of the complexes were measured by and The of the complexes in water from to nm with a All complexes zeta which indicated that and formed complexes with around 100 G5-C1, G5-C2, and G5-C3 to form complexes (Figure After incubation in PBS (pH 7.4) for 30 min, complexes formed by G5 and were into small the of and G5-C3 complexes to over In G5-C4 and complexes showed size that the of hydrophobic to the of more condensed and stable complexes (Figure Figure | of the polymer structure on of the complexes. (a) of polymer/BSA complexes. (b) of the of polymer/BSA complexes in water or PBS (pH 7.4) by (c) In vitro relative β-Gal activity in water or PBS (pH fluorescence intensity of HeLa cells treated with complexes for 4 h before and after trypan blue The fluorescence intensity without trypan blue was defined as 100%. The polymer/BSA weight ratio of complexes was for all Download figure Download PowerPoint Then, an in vitro β-Gal activity assay was performed to study the release of proteins from the complexes. The polymer/β-Gal complexes were incubated in water or PBS (pH respectively. As shown in Figure all complexes showed β-Gal activity in but G5-C1, G5-C2, and G5-C3 complexes exhibited β-Gal release in PBS due to their In β-Gal release from G5-C4 and complexes was that G5-C1, G5-C2, and G5-C3 with or hydrophobic alkyl chains were as protein delivery However, the in delivery efficacy between G5-C4 and complexes as both were stable and To test we compared the MFI of HeLa cells treated with the two complexes before and after trypan blue As shown in Figure HeLa cells treated with complex showed MFI after trypan blue G5-C4 of the This indicated that the complexes were on the cell which was with the confocal Supporting Information Figure a may be to the affinity of to the cell membrane due to its higher hydrophobicity. and escape, and protein release the endocytosis of the G5-C4/BSA-FITC complex was (Figure As shown in Figure the intracellular delivery of BSA-FITC by G5-C4 was at 4 °C or by inhibitors including chlorpromazine and by that the complexes were into cells via and are to the delivery efficacy because the complexes escape endosomes The of and the acidic environment are to proteins, which makes endosomal escape an essential feature for protein delivery We stained the acidic endosomal of HeLa cells with LysoTracker Red to the endosomal escape of G5-C4/BSA-FITC As shown in Figure of BSA-FITC in cell were observed with incubation the into the over the efficient endosomal escape of G5-C4/BSA-FITC Figure 4 | Intracellular of G5-C4/BSA-FITC complex in HeLa cells. (a) Proposed endocytosis for (b) fluorescence intensity of HeLa cells treated with complex for 4 h. The cells were pretreated with different endocytosis are presented as mean ± standard (n = Students' t test was used to analyze the significant difference. Statistical significance was set at *P < 0.05, **P < 0.01, and < and of HeLa cells treated with complex for different The acidic were stained with LysoTracker G5-C4 polymer was with Rhodamine B The doses of

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