HBpep peptide, resins and Fmoc protected amino acids used in solid phase peptide synthesis were purchased from GL Biochem, China. N-Hydroxysuccinimide, tetrahydrofuran, triphosgene, sodium azide, triphosgene and benzoic acid were purchased from Tokyo Chemical Industry (TCI), Japan. N,N’-Diisopropylcarbodiimide, acetic acid, 2-hydroxyethyl disulfide, N,N-diisopropylethylamine, piperidine, trifluoroacetic acid, triisopropylsilane, 2,4,6-trinitrobenzenesulfonic acid, glutathione, bovine serum albumin, lysozyme, saporin, β-galactosidase, R-phycoerythrin, methylthiazolyldiphenyl-tetrazolium bromide, Hoechst 33342, methyl-β-cyclodextrin, chlorpromazine hydrochloride, amiloride chloride were obtained from Sigma-Aldrich, USA. Dichloromethane, N,N-dimethylformamide, LysoTracker Red DND-99, Opti-MEM, Ni-NTA His bind resin and 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside were purchased from Thermo Fisher Scientific, USA. Organic solvents including ethyl acetate, hexane and diethyl ether were purchased from Aik Moh Paints & Chemicals Pte Ltd, Singapore. Dulbecco's modified Eagle medium, fetal bovine serum, phosphate buffered saline and Antibiotic-Antimycotic (100X) liquid were purchased from Gibco, USA. Nano-Glo® Dual-Luciferase® kit used for luciferase detection was purchased from Promega, USA. Enhanced green fluorescent protein (EGFP) was expressed by E. Coil BL21 strain and purified with Ni-NTA His bind resin. Luciferase-encoding mRNA encoded and EGFP-encoding mRNA used for mRNA transfection experiments were obtained from Trilink.
Self-immolative moiety synthesis
The self-immolative moieties conjugated to HBpep-K peptide were designed based on the literature28, the synthesis routes of the amine-reactive species are shown in Extended Data Fig. 10. First, for the synthesis of the side blocked intermediate product, HO-SS-R, 2-hydroxyethyl disulfide (1 equiv, 10 mmol) was dissolved in 15 mL tetrahydrofuran (THF), and another 15 mL THF containing a carboxylic acid reactant such as acetic acid or benzoic acid (0.9 equiv, 9 mmol) was added. Then, under an ice bath, 15 mmol of N,N’-Diisopropylcarbodiimide (DIC) was slowly added into the reaction mixture. The reaction was kept at 0 °C for another 0.5 hours and then increased to room temperature. After the overnight reaction, the mixture as filtered, and the supernatant was evaporated under reduced pressure. The raw products were then purified using silica gel chromatography with ethyl acetate/hexane (1/4) as elute. The purified products were isolated by rotary evaporation (R-215 Rotavapor, BUCHI, Switzerland).
Then, the intermediate products HO-SS-R and N-hydroxysuccinimide (NHS) were coupled by using triphosgene. Specifically, HO-SS-R (1 equiv, 5 mmol), and 4-dimethylaminopyridine (DMAP, 0.1 equiv, 0.5 mmol) was dissolved in 10 mL of THF. Then, triphosgene (0.37 equiv, 1.85 mmol) in 10 mL THF was added into the prior solution dropwise under an ice bath. After another 0.5 hours on the ice bath, the reactions were continued at 40 °C for 4 hours, followed by evaporation under reduced pressure to remove excess phosgene. NHS (1.5 equiv, 7.5 mmol) in 20 mL THF, and N,N-Diisopropylethylamine (DIEPA, 1.5 equiv, 7.5 mmol) was then pipetted in the prior mixtures. The reactions were kept at 40 °C for 24 hours before evaporation. The raw products were purified using silica gel chromatography with ethyl acetate/hexane (1/3) as elute. The purified products were isolated by rotary evaporation. The amine-reactive products NHS-SS-Ac and NHS-SS-Ph were synthesized from acetic acid and benzoic acid. The chemical structures of the HO-SS-R and NHS-SS-R were verified by 1H nuclear magnetic resonance (NMR) as shown in Extended Data Fig. 11. The NMR spectra were collected on a Bruker Advance 400 spectrometer (USA).
Peptide synthesis and purification
The peptides used in this study were synthesized by the classical Merrifield solid phase peptide synthesis (SPPS) technique44. Wang resin (1.0 g, 0.56 mmol) was first swollen in 15 mL of dichloromethane (DCM) for 0.5 hours with nitrogen flow bubbling. Then, the DCM was drained with increased pressure, and the resin was washed three times with DMF.
For N-terminal protected amino acid (Fmoc-AA-OH) coupling, Fmoc-AA-OH (2 equiv, 1.12 mmol) was dissolved in 5 mL of N,N-dimethylformamide (DMF), then 5 mL of DMF with 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosph-ate (HATU, 1.9 equiv, 1.064 mmol) and DIPEA (5 equiv, 2.80 mmol) was added into the prior solution. The mixture was reacted for 2 min at room temperature before being added onto the resin for 1 hour of coupling reaction with nitrogen flow bubbling. The resin was washed with DCM and then DMF three times each after the coupling reaction. The coupling efficiency was evaluated by using 2,4,6-trinitrobenzenesulfonic acid (TNBS).
For deprotection of N-terminal amine, 15 mL of 20% piperidine in DMF (volume ratio) was added onto the resin. The deprotection continued for 0.5 hour at room temperature with nitrogen flow bubbling. After that, the resin was washed with DCM then DMF three times and the deprotection efficiency was also evaluated using 2,4,6-trinitrobenzenesulfonic acid (TNBS).
After all amino acids in the peptide sequence were coupled onto the resin by performing coupling/deprotection cycles from the C- to the N termini direction, peptides were cleaved from the resins by using a cocktail containing 95% of trifluoroacetic acid (TFA), 2.5% of H2O and 2.5% of triisopropylsilane (TIPS). After 2 hours of cleavage, the reaction mixtures were filtered. The supernatants were concentrated by using nitrogen flow and precipitated into 50 mL of cold diethyl ether. After centrifugation, the pellets were dried under vacuum and re-dissolved by using 90% of 10 mM acetic acid and 10% acetonitrile for purification by High Performance Liquid Chromatography (HPLC, 1260 Infinity, Agilent Technologies, USA) equipped with a C8 column (Zorbax 300SB-C8, Agilent Technologies, USA). The purified peptides were isolated by lyophilization (FreeZone 4.5 Plus, Labconco, USA) from HPLC elutes.
The redox responsive peptides were synthesized by reacting the ε-amine of the single Lys residue of the N-terminal protected peptide (Fmoc-HBpep-K, Fmoc-GHGVY-GHGVY-GHGPY-K-GHGPY-GHGLYW) with the amine-reactive species NHS-SS-R, followed by deprotection. First, the Fmoc-HBpep-K peptide (1 euqiv, 15 μmol) was dissolved in 5 mL of DMF containing DIPEA (15 equiv, 225 μmol). After 30 minutes of deprotonation, NHS-SS-R (1.5 equiv, 22.5 μmol) in 0.5 mL of DMF was added into the solution. The mixture solutions were allowed to react at room temperature for 24 hours before precipitation by adding 50 mL of cold diethyl ether. The raw products were collected from the pellets by centrifugation, and dried under reduced pressure. The purification of modified peptides was conducted on an HPLC system equipped with a C8 column. The purified Fmoc protected peptides were isolated by lyophilization from the HPLC fractions.
Then, the purified Fmoc protected peptides were dissolved in 5 mL of DMF containing 20% piperidine. The mixture was stirred at room temperature for 2 hours of N-terminal deprotection. The raw products were collected from the precipitates after adding 50 mL of cold diethyl ether into the reaction mixtures and purified by HPLC. The final products were isolated by lyophilization as white solids. Two modified peptides were synthesized, namely HBpep-SA from NHS-SS-Ac and HBpep-SP from NHS-SS-Ph. The MWs of Fmoc-HBpep-K and modified peptides were verified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix (Extended Data Fig. 12). Both MWs weree consistent with the expected MWs of the peptides. The MALDI-TOF spectra were collected on an AXIMA Performance spectrometer (Shimadzu Corporation, Japan). The modified peptides HBpep-SA and HBpep-SP were dissolved in 10 mM acetic acid solution at 10 mg/mL as stock solution.
Coacervation of peptide and therapeutic recruitment
The phase separation behavior of HBpep-K and HBpep-SR peptides at various pH was monitored turbidity measurements using a UV-Vis spectrometer (UV-2501PC, Shimadzu, Japan). The absorbance at 600 nm (A600) was used to calculate the relative turbidity as below22:
The recruitment of the macromolecules within the peptide coacervates was conducted during the coacervation process at the optimal pH (7.5 for HBpep, 6.5 for HBpep-SR). The therapeutics were dissolved or diluted in 10 mM phosphate buffers (pH = 7.5 or 6.5, ionic strength = 100mM) to achieve the target concentrations. Then, the peptides stock solutions were mixed with the therapeutics containing the buffer at a 1:9 volume ratio to induce coacervation and recruitment of the therapeutics. As shown in Extended Data Fig. 7, the recruitment efficiency of HBpep-SP coacervates was calculated by comparing the supernatant fluorescence in the buffer solution before and after coacervation and centrifugation using microplate reader (Infinite M200 Pro, Tecan, Switzerland). The fluorescence of EGFP (or FITC) and R-PE were detected using 488 nm/519 nm and 532 nm/584 nm for the excitation/emission wavelengths, respectively.
Confocal microscopy of EGFP delivery mediated by HBpep coacervates
For 3D view of coacervate-treated cells, cells treated overnight with EGFP-loaded coacervates (0.01 mg/mL of EGFP, 0.2 mg/mL of HBpep) were first stained with a plasma membrane stain and fixed prior to image acquisition. Briefly, cells were rinsed with HBSS buffer and stained with either 1x CellTrackerTM CM-DiI (C7000) (ThermoFisher) for 5 min at 37 ºC followed by 15 min at 4 ºC or 1x CellMaskTM Deep Red Plasma membrane stain (ThermoFisher) for 10 min at 37 ºC. Cells stained with membrane dyes were rinse once with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After fixing, cells were washed three times with PBS and finally resuspended in PBS. Confocal Z-stack images were collected on Olympus FV1000 inverted scanning confocal microscope using 40× oil immersion objective (NA 1.3). The Z-stacks were reconstructed into 3D images or animations with the aid of Imaris software 3D view and Animation modes.
For live cell imaging, T22 cells treated with EGFP-loaded DgHBP-2 coacervates were split and seeded on day 3 and day 7 to achieve 50-60% confluency and images acquired after cells adhered about 4hrs after seeding. Z-stack Images (Differential interference contrast (DIC) or fluorescence) were acquired on Nikon Eclipse Ti inverted microscope, using a 40× oil immersion objective (NA 1.3) and sum slices projection was applied to all the stack images using ImageJ Software.
Characterization of redox-responsive peptide coacervates
Optical and fluorescence microcopy images of HBpep-SP coacervates and fluorescence image of macromolecules-loaded HBpep-SP coacervates were taken using an invert fluorescence microscope (AxioObserver.Z1, Zeiss, Germany). Dynamic light scattering (DLS, ZetaPALS, Brookhaven, USA) system was employed to measure the size of pristine HBpep-SR coacervates and macromolecules-loaded HBpep-SR coacervates. The fresh prepared pristine or macromolecules-loaded coacervates (with or without 0.1 mg/mL of macromolecules, 1 mg/mL of modified peptides) was diluted into PBS with a volume ratio of 1:9 before the DLS test. The redox-responsivity of HBpep-SA and HBpep-SP was evaluated by measuring the decrease in concentration in the presence of glutathione (GSH). The fresh prepared HBpep-SA or HBpep-SP coacervates (50 μL, 1 mg/mL of peptide) were diluted in 450 μL of PBS containing 1 mM of GSH. The mixtures were incubated at 37 °C before adding 25 μL of acetic acid to dissolve all the unreacted peptides, and their concentration was measured by HPLC.
Delivery of proteins and peptides
For protein delivery into cells, 105 of cells were suspended in 1 mL of Dulbecco's modified Eagle medium (DMEM) supplemented with 10% of fetal bovine serum, 100 units/mL of penicillin and 100 μg/mL of streptomycin, and then transferred into 35 cm2 culture dishes. After 24 hours of incubation at 37 °C with 5% of CO2, the media was replaced with 900 μL of Opti-MEM. Then, 100 μL of freshly prepared peptide- or protein-loaded HBpep-SA or HBpep-SP coacervate suspensions (0.05 mg/mL of peptide or 0.1 mg/mL of protein, 1 mg/mL of HBpep-SR) were added into the media. After 4 hours of incubation, the media was removed and the cells were washed with PBS twice before adding 1 mL of fresh media (DMEM, 10% FBS, antibiotics). The cells were incubated for another 20 hours and then washed twice at pH 5.0 in phosphate buffer to remove any coacervates that had not entered the cells, before being imaged under the fluorescence microscope (AxioObserver.Z1, Zeiss, Germany).
Delivery and transfection of mRNA
Two reporter genes including luciferase and enhanced green fluorescent protein (EGFP), were used to evaluate the mRNA transfection efficiency of the HBpep-SR coacervates. Before transfection, HepG2 or HEK293 cells were incubated in 96-wells plates with a density of 104 cells per well for 24 hours. Then, the media were replaced with 90 μL of Opti-MEM, followed by the addition of 10 μL of freshly prepared mRNA-loaded coacervate suspensions (1 or 2 mg/mL of modified peptides). The final concentration of luciferase-encoding mRNA used in transfection was 3.3 μg/mL. After 4 hours of incubation, the media were removed and the cells were washed by PBS twice before adding 100 μL of media (DMEM, 10% FBS, antibiotics). Then transfection was continued for another 20 hours before testing the luminescence using the Nano-Glo® Dual-Luciferase® kit and a microplate reader. For EGFP-encoding mRNA (Cy5 labelled) transfection, the cultures were conducted in 35 cm2 dish in which 100 μL of mRNA loaded HBpep-SP coacervates (1 mg/mL of HBpep-SP) was added to achieve the final mRNA concentration of 1 μg/mL. The transfection was conducted for 4 hours of uptake and 20 hours of expression before imaging the cells under a fluorescence microscope and testing the transfection efficiency by FACS (LSR Fortessa X20, BD Biosciences, USA).
The cytotoxicity of the therapeutics-loaded or pristine peptide coacervates was evaluated by using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Following literature protocols45,46, 104 of HepG2 or HEK293 cells in 100 μL of media (DMEM, 10% FBS, antibiotics) were transferred into 96-wells plates and incubated for 24 hours. Then, the media were replaced with 100 μL of Opti-MEM containing therapeutics-loaded coacervates (various concentration of therapeutics, 0.1 mg/mL HBpep-SP) or various concentrations of pristine coacervate suspensions. After 4 hours of uptake, the media were removed and the cells were washed by PBS twice before adding 100 μL of media (DMEM, 10% FBS, antibiotics). The cells were incubated for another 20 hours before 10 μL of 5mg/mL MTT dissolved in PBS was added. The media were removed after 4 hours of incubation with MTT, and the cells were washed by PBS twice. After that, 100 μL of DMSO per well was added for absorbance measurements at 570 nm using a microplate reader (Infinite M200 Pro, Tecan, Switzerland). The relative cell viability was calculated as below.
where At, Ab, and Ac represent the absorbance of tested cells, untreated cells and no cell, respectively.
Internalization mechanism study
The LysoTracker staining was conducted by following the manual from the manufacturer. Similar to protein delivery, 105 of HepG2 cells were incubated in 35 cm2 dish with DMEM for 24 hours. Then the media were replaced with 900 μL of Opti-MEM and 100 μL of EGFP-loaded HBpep-SP coacervates (0.1 mg/mL of EGFP, 1 mg/mL of HBpep-SP). The cells were cultured for another 2 hours before being washed twice with a pH 5.0 phosphate buffer to remove any coacervates that had not entered the cells. After that, 1 mL of Opti-MEM containing 50 nM of LysoTracker was added for 30 minutes of staining at cell culture condition. The treated HepG2 cells were washed by PBS twice and fixed with 4% formaldehyde solution. Before being imaged by confocal microscopy (LSM 780, Zeiss, Germany), the cells were treated with 1 μg/mL of Hoechst 33342 for 10 minutes to stain the nucleus.
Based on the literature13,41,42, various inhibitors were used to study the pathway of the coacervates internalization. HepG2 cells were treated with chlorpromazine (CPM, 30 μM), amiloride chloride (AM, 20 μM), sodium azide (NaN3, 100 mM) or methyl-β-cyclodextrin (MβCD, 2.5 mM) separately for 1 hour. Then 100 μL of EGFP loaded HBpep-SP coacervates (0.1 mg/mL of EGFP, 1 mg/mL of HBpep-SP) was added. After another 4 hours of incubation, the cells were washed twice with a pH 5.0 phosphate buffer followed by PBS twice. Then the treated cells were imaged by fluorescence microscopy or dissociated by trypsin for FACS. For the 4 °C treated group, the HepG2 cells were pre-incubated for 1 hour and kept at low temperature during the 4 hours of uptake process. Two control groups including totally untreated cells (negative control, NC) and cells treated by EGFP-loaded coacervates without any inhibitors (blank) were also conducted.
All experiments were repeated three times. The data are presented as means ± standard deviation (SD). Statistical significance (p < 0.01) was evaluated by using two-sided Student's t-test when only two groups were compared.