Materials and reagentsMethanol, diethyl ether, Traut’s Reagent (2-iminothiolane hydrochloride), lysozyme from chicken egg white (~70000 U mg−1), β-Galactosidase from Escherichia coli (lyophilized, powder, ~140 U mg−1), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate buffered saline (PBS), ethylenediaminetetraacetic acid (EDTA), D-cystine (DcySS), 6’-amino-D-luciferin (D-amLu), 6-amino-2-cyanobenzothiazole (NCBT), adenosine 5’-triphosphate disodium salt hydrate (ATP), magnesium chloride hexahydrate (MgCl2), recombinant luciferase from Photinus pyralis (firefly), anhydrous dimethyl sulfoxide (DMSO) and DMSO-d6 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), geneticin and fetal bovine serum (FBS) were purchased from Life Technologies Gibco, USA. Amino-dextran (10 kDa, 5.1 mole amine per mole of dextran), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), Alexa Fluor™ 647 NHS Ester (Succinimidyl Ester) were purchased from Thermo Fisher Scientific, USA. D-cysteine hydrochloride monohydrate (Dcys), L-cysteine hydrochloride monohydrate (Lcys), L-cystine dihydrochloride (LcySS), 2, 2’-dithiodipyridine, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and N-succinimidyl 3-maleimidopropionate (BMPS) were purchased from TCI Europe N.V. (Belgium).The following chemicals were obtained as indicated: Puromycin (Peprotech, USA), CellTiter-Glo® Luminescent Cell Viability Assay (Promega, USA), GSH/GSSG-Glo™ Assay (Promega, USA), Recombinant RNasin® Riboneclease Inhibitor (Promega, USA), RNase A from bovine pancreas (Roche CustomBiotech, Germany), RNA from yeast (Roche CustomBiotech, Germany), Hank’s Balanced Salt Solution (HBSS, Hyclone, USA), non-essential amino acids (NEAA, HyClone, USA), penicillin-streptomycin (HyClone, USA), L-glutamine (HyClone SpA, USA), and trypsin (HyClone SpA, USA).BioLure assay in the cell-free reaction bufferBioLure assay in the cell-free reaction buffer were adapted from the protocol reported in the literature with minor modifications33. The cell-free reaction buffer was based HBSS−HEPES buffer at pH 7.4, supplemented by recombinant firefly luciferase (10 μg mL−1), ATP (1 mM), and MgCl2 (5 mM). Then DcySS or LcySS (final concentration at 50 μM) were added to the reaction buffer, with or without TCEP (final concentration at 100 μM). NCBT (5 mM stock solution in DMSO) was added right before the luminescence detection at a final concentration at 50 μM in the reaction buffer. The luminescence was recorded at 25 °C using Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific Inc., USA). The measurement time was set at 1000 ms, and the recording lasted for 30 min at a kinetic interval of 30 s. The total luminescence was calculated based on the integral of the real-time luminescence during the 30 min.Cell cultureA375-eGFP-Fluc cells (A375-Fluc-Neo/eGFP-Puro, provided by Imanis Life Sciences, USA) were cultured in DMEM with 4.5 g L−1 glucose, supplemented with 10% of FBS, 1% of L-glutamine, 1% of NEAA, penicillin (100 IU mL−1), streptomycin (100 μg mL−1), geneticin (0.6 mg mL−1) and puromycin (1 μg mL−1). HEK293 cells (ATCC) were cultured in DMEM with 4.5 g L−1 glucose, supplemented with 10% of FBS, 1% of L-glutamine, 1% of NEAA, penicillin (100 IU mL−1) and streptomycin (100 μg mL−1). The cells were cultured in the 5% CO2-incubator at 37 °C, and 95% relative humidity. The culture media were changed every 2-3 days, and the cells were passaged at ~90% confluence using 0.25% (v/v) trypsin EDTA/PBS.Electroporation-mediated intracellular deliveryFor a typical electroporation process, 4 × 105 cells were resuspended in Lonza SF 4D-Nucleofector™ Solution (16.4 μL Nucleofector™ Solution + 3.6 μL Supplement from SF Cell Line 4D-Nucleofector™ X Kit S, Lonza Bioscience, Switzerland), followed by the addition of 2 μL molecules of interests (DcySS, LcySS, Dex-Dcys, Dex-Lcys, Dex-NC, Lyso, Dlyso, RNase, or DRNase, stock solution in MilliQ water). For negative control, only 2 μL MilliQ water was added. The mixture was transferred to one well of Nucleocuvette™ Strip carefully without air bubbles. Then, the Nucleocuvette™ Strip was electroporated with 4D-Nucleofector® X Unit using the pre-optimized program (Code: FF-120 for A375-eGFP-Fluc cells). After electroporation, the Nucleocuvette™ Strip was incubated in a cell incubator (37 °C, 5% CO2 and 95% relative humidity) for 10 min for the cell recovery. Then, 80 μL pre-warmed cell culture medium was added to resuspend the cells in the well, and the sample was transferred to an Eppendorf and centrifuged at 90 g for 10 min. The supernatant was carefully removed without disturbing the cell pellet. Then the cell pellet was resuspended in HBSS-HEPES buffer (pH 7.4) and added to 96-well microplates (white wall with transparent bottom) for BioLure assay analysis.BioLure assay of electroporated cellsBioLure assay of electroporated cells was performed after electroporation-mediated intracellular delivery. NCBT (5 mM stock solution in DMSO) was added to the cell suspension in HBSS-HEPES buffer, and electroporated cells with the same amount of DMSO but without NCBT was used as the negative control. The luminescence was recorded using Varioskan™ LUX multimode microplate reader at 25 °C. The measurement time was set at 1000 ms, and the recording lasted for 30 min at a kinetic interval of 30 s. The total luminescence was calculated based on the integral of the real-time luminescence during the 30 min.Synthesis and characterizations of Dex-Dcys, Dex-Lcys and Dex-NCSynthesis of Dex-Dcys and Dex-LcysDex-Dcys and Dex-Lcys were synthesized by a two-step reaction. First, amino-dextran (4 mg, 0.4 μmol) was conjugated with SPDP linker (0.6 mg, 1.96 μmol) in 1 mL PBS−EDTA buffer (pH 8.0) for 0.5 h at room temperature. Then, the unreacted SPDP was removed by centrifugation at 16,110 ×g for 10 min using Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa MWCO). The SPDP conjugated dextran (Dex-SPDP) was further purified by washing with PBS-EDTA buffer 3 times using Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa MWCO). Then the final products were either mixed with L-cysteine hydrochloride monohydrate (0.7 mg, 4 μmol) or D-cysteine hydrochloride monohydrate (0.7 mg, 4 μmol) in PBS−EDTA buffer. The UV-absorbance at 343 nm was measured before and at 15 min after the addition of cysteine, to estimate the number of cysteine conjugated per dextran molecule according to the method reported in literature59. After 0.5 h, the reaction mixture was again purified by centrifugation at 16,110 ×g for 10 min using Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa MWCO). The products (Dex-Dcys and Dex-Lcys) were washed 6 times by MilliQ water and freeze-dried overnight. The lyophilized dextran polymers were stored at −20 °C until use.Synthesis of Dex-NCFirst, amino-dextran (4 mg, 0.4 μmol) was conjugated with BMPS linker (0.52 mg, 1.96 μmol, in 1 mL PBS-EDTA buffer (pH 8.0) for 0.5 h at room temperature. The intermediate product (Dex-mal) was purified similarly as Dex-SPDP using Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa MWCO). Then, Dex-mal was subjected to reaction with D-cysteine hydrochloride monohydrate (0.7 mg, 4 μmol) in 1 mL PBS−EDTA buffer for 0.5 h at room temperature, and purified similarly as Dex-Dcys. The final product (Dex-NC) was freeze-dried and stored at −20 °C until use.Fluorescence labelling of Dex-Dcys, Dex-Lcys and Dex-NCThe fluorescent derivatives of Dex-Dcys, Dex-Lcys and Dex-NC were synthesized by reacting with Alexa Fluor™ 647 NHS Ester. Briefly, 2 mg Dex-Dcys, Dex-Lcys or Dex-NC was dissolved in 1 mL PBS−EDTA buffer (pH 8.0) and then Alexa Fluor™ 647 NHS Ester (0.02 μmol, 0.4 mM stock in DMSO) was added. The reaction was allowed at room temperature for 0.5 h with protection from light. The unreacted Alexa Fluor™ 647 was removed by washing with PBS-EDTA buffer 3 times using Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa MWCO), and then further washed by MilliQ water 3 times before lyophilization.Characterizations of polymersThe synthesized polymers, inducing intermediate products (Dex-SPDP and Dex-mal) were characterized by 1H-NMR on a Bruker Vertex 70 spectrometer to confirm the structure. The results were analyzed by MestreNova software and plotted in Figs. S3 and S4 in the supporting information.The polymers were also subjected to elemental analysis with an automatic elemental analyzer vario MICRO cube (HANAU Elementar Analysensysteme GmbH, Germany, Serial no. 15082023). Sulfanilamide standard (>99.9%, Elementar, Germany) was used as the standard. Analyzes were performed in CHNS mode (O2 dosing time: 70 s; Autozero delay: 10 s; Peak anticipation: N 50 s, C 120 s, H 100 s, S 70 s). Carbon was determined as CO2, hydrogen as H2O, nitrogen as N2 and sulfur as SO2. N2 is not adsorbed in the adsorption column and is the first measuring component to enter the thermal conductivity detector (TCD). CO2, H2O and SO2 are adsorbed together in the adsorption column. The adsorption column is heated stepwise to desorption temperatures of CO2 (60 °C), H2O (140 °C) and SO2 (210 °C). The measured gas enters the detector with the carrier gas (He) one by one. The percentage elemental concentration of the element in the sample is calculated using formula c % \(=\frac{a\cdot 100\cdot f}{w}\), where c is the element concentration [%], a absolute element content [mg], f the daily factor and w the sample weight [mg].Fluorescently labeled Dex-Dcys, Dex-Lcys and Dex-NC were further characterized by size exclusion chromatography (SEC). The system consists of a Waters 515 HPLC pump, Biotech DEGASi GPC Degasser, Waters 717 plus Autosampler, Shimadzu RF535 Fluorescence HPLC monitor and Waters 2410 Differential Refractometer together with Waters Ultrahydogel 120, 250, 2000 7.8 × 300 mm columns and a guard column. The column was kept at 30 °C. Fluorescence excitation was set to 650 nm and emission 675 nm. Pullulan standards by Polymer Standard Service were used for the molecular weight calibration.Confocal microscopy imagingCells electroporated with fluorescently labeled dextran derivatives were suspended in HBSS−HEPES buffer and then sediment in a 35 mm glass bottom dish before confocal imaging. Then the cells were imaged by a Leica Stellaris 8 confocal microscope (Leica Microsystems, Wetzlar, Germany). The images acquired were processed by Fiji 1.51 software.Flow cytometry analysisAll the flow cytometry analysis was performed on BD LSR-II Cell Analyzer flow cytometer, and the data was processed by FlowJoTM software. Regarding the quantitative flow cytometry analysis, Quantum™ Alexa Fluor® 647 Molecules of Soluble Fluorochrome (MESF) beads (Bangs Laboratories, Inc, USA) were used. Basically, 5 beads with pre-determined Alexa Fluor® 647 labelled molecules were run on the same day same by the same flow cytometer at the same setting with flow cytometry cell samples. The mean fluorescence intensity (MFI) of each bead (from triplicate samples) were calibrated using the QuickCal® analysis template as instructed by the manufacturer. BD FACSDiva™ logarithmic regression was selected to fit the data.Synthesis of Py-DcysPy-Dcys (S-(pyridin-2-ylthio)-D-cysteine) was synthesized by reacting 2, 2’-dithiodipyridine with D-cysteine hydrochloride following literature procedures60. Briefly, 2, 2’-dithiodipyridine (0.22 g, 1 mmol) was dissolved in 2.5 mL methanol with 0.1 mL acetic acid. Then the second reactant (Dcys HCl, 78.81 mg, 0.5 mmol), pre-dissolved in 1 mL methanol, was slowly added. The reaction was left stirring at room temperature overnight. On the following day, the reaction mixture was precipitated by cold diethyl ether (40 mL), and centrifuge at 6000 rpm for 5 mins to remove the supernatant. The precipitation was redissolved in 2 mL methanol, and precipitated in cold diethyl ether (20 mL) again. After removal of the supernatant, the precipitation was repeated for another time. Finally, the white precipitate was collected and dried overnight to remove residual diethyl ether. The synthesized Py-Dcys was characterized by 1H-NMR on a Bruker Vertex 70 spectrometer to confirm the structure.Protein labelling and characterizationsLysozyme and RNase A were labelled via two-step reactions. First, 5 mg protein was dissolved in 1 mL PBS−EDTA buffer (pH 8). Then, 0.5 mg Traut’s Reagent (2-iminothiolane hydrochloride, 3.6 μmol) in 50 μL PBS−EDTA buffer (pH 8.0) was added, and stirred at room temperature for 30 min. The proteins were purified by Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa MWCO), by washing with PBS−EDTA buffer for 4 times. During each washing step, the sample was centrifuged at 16,110 g for 10 min. The purified proteins were diluted in PBS-EDTA buffer to make the volume to 1 mL, followed by the addition of Py-Dcys (1.1 mg, 4.2 μmol). The UV-absorbance at 343 nm was measured before and at 15 min after the addition of Py-Dcys, to estimate the number of cysteine conjugated (on average 1.1 Dcys per lysozyme, and 0.7 Dcys per RNase A). The reaction was allowed at room temperature for 30 min, and the proteins were purified again by Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa MWCO) using the same centrifugation conditions. The proteins were washed 6 times by MilliQ water instead of PBS−EDTA buffer, and freeze-dried overnight. The lyophilized proteins were stored at −20 °C until use.β-Galactosidase (bGal) were labelled similarly as Dex-Dcys via two-step reactions. First, bGal (1 mg) was conjugated with SPDP linker (0.22 mg, 0.7 μmol) in 1 mL PBS−EDTA buffer (pH 8.0) for 0.5 h at room temperature. The protein was purified by washing with PBS-EDTA buffer 3 times using Amicon Ultra-0.5 Centrifugal Filter Unit (10 kDa MWCO, 16,110 g for 10 min each time). Then D-cysteine hydrochloride monohydrate (0.22 mg, 1.4 μmol) in PBS−EDTA buffer was added and the UV-absorbance at 343 nm was measured before and at 15 min after the addition of cysteine, to estimate the number of D-cysteine conjugated (11.3 Dcys on each bGal subunit, 116 kDa). After 0.5 h, the reaction mixture was again purified by centrifugation at 16,110 × g for 10 min using Amicon Ultra-0.5 Centrifugal Filter Unit (10 kDa MWCO). The purified D-cysteine conjugated bGal (DbGal) was freeze-dried overnight and stored at −20 °C until use.The proteins were characterized by gel electrophoresis on SDS-PAGE gel and Native PAGE gel. For SDS−PAGE Gel, proteins were mixed with Novex™ Tris-Glycine SDS Sample Buffer (Thermo Fisher, USA) and reduced by TCEP or β-mercaptoethanol, and heated at 95 °C for 10 min. The samples were loaded on 4–15% Mini-PROTEAN® TGX™ Precast Protein Gels along with Thermo Scientific Spectra Multicolor Broad Range Protein Ladder (10–260 kDa). Then electrophoresis was performed at 160 V for 30 min. The gel was stained by PageBlue™ protein staining solution (Thermo Fisher, USA), and imaged by GelDoc™ Imaging System (BioRad, USA).For Native-PAGE gel electrophoresis, a Tris-glycine polyacrylamide gel system consisting of a 4.5% stacking gel and a 6% separation gel was used to separate 20 µg purified protein under nondenaturing conditions in a Mini-PROTEAN electrophoresis chamber (BioRad) in running buffer (25 mM Tris, 192 mM glycine, pH ∼8.0) at 30 V overnight at 4 °C. Further gels were washed 3 times in ddH2O for 5 minutes, incubated in 10 mL of InstantBlue® Coomassie Protein Stain (ab119211) for 1 h, distained twice in 100 mL of ddH2O for 15 min. After staining, the protein bands were cut from the gel into small pieces, and the proteins were extracted in to HBSS buffer by gentle shaking at 4 °C overnight. Then, the gel pieces were removed by centrifugation, and the protein-containing supernatants were collected and washed 3 times by Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa MWCO), to remove salts and impurities diffusing from the gel during the extraction. The concentrated and purified proteins were diluted in the reaction buffers and the luminescence was recorded according to the procedures previously described in the BioLure assay in the cell-free reaction buffer section.Lysozyme activity evaluationsThe enzymatic activity of lysozymes before and after Dcys modification was evaluated by Invitrogen™ EnzChek™ Lysozyme Assay Kit (Thermo Fisher, USA), following the manufacture’s protocol. Lysozymes of different concentrations (0–200 ng per well) were diluted in 1X reaction buffer and a volume of 50 μL was added to one well in a 96-wellplate, and 50 μL lysozyme substrate working solution (50 μg mL−1 substrate in 1X reaction buffer) was added. The plate was incubated for 30 min at 37 °C protected from light, and the mean fluorescence (RLU) was measured by Varioskan™ LUX multimode microplate reader. The excitation wavelength was 494 nm and the emission was 518 nm.Regarding lysozyme quantification in the cell lysate, 100,000 electroporated cells with Dlyso were washed with cold PBS, and lyzed on ice for 10 min with 100 μL lysis buffer (containing protease inhibitor cocktail). The sample was centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was collected and analyzed by EnzChek™ Lysozyme Assay Kit following the protocol described above. A control sample consisted of electroporated cell lysate without Dlyso was also measured using the same assay, to identify the endogenous lysozyme amount. Finally, the intracellularly delivered lysozyme was calculated by subtracting negative control from the Dlyso electroporated sample.RNase activity evaluationsThe enzymatic activity of RNase was evaluated by a well-established protocol from literature61 with modifications. First, prepare crude yeast RNA stock solution at 1 mg mL−1 in 62.5 mM Tris-buffer (pH 8.0). Then 40 μL of diluted RNase or DRNase solution was added to 160 μL RNA stock; the mixture was heated at 37° for 10 min. The reaction was stopped by the addition of 200 μL of 6% HClO4 (Perchloric acid). After 5 min on ice, the samples were centrifuged at 16,110 × g for 3 min to precipitate the undegraded RNA and proteins. Then 100 μL of the supernatant was diluted with 500 μL Milli-Q water, and the UV absorbance spectra from 240 to 300 nm was measured.The RNase activity before and after Dcys labelling was further evaluated by RNA gel electrophoresis. The 16 S and 23 S rRNA from E.coli was used as the substrate for RNase. For each sample, 1 ug rRNA in 10 μL TE buffer was mixed with RNase or DRNase (0.04, 0.4, 4 and 40 ng) with 40 U Recombinant RNasin Ribonuclease Inhibitor (Promega, USA). The mixture was heated at 37° for 10 min and loaded on 2% agarose gel, run at 80 V for 2 h. Gell was imaged with ChemiDoc™ Imaging System (Bio-Rad).β-Galactosidase activity evaluationsThe enzymatic activity of β-Galactosidase before and after Dcys modification was evaluated by FACS Blue LacZ beta Galactosidase detection kit (Abcam, UK), following the manufacture’s protocol. β-Galactosidase of different concentrations (0–25 ng per well) were diluted in 1X reaction buffer and a volume of 50 μL was added to one well in a 96-wellplate, followed by the addition of 100 μL reaction buffer and 50 μL substrate reagent. The plate was incubated for 20 min at room temperature protected from light. Finally, 100 μL stop buffer was added and the plate was incubated for 10 min before reading. The mean fluorescence (RLU) was measured by Varioskan™ LUX multimode microplate reader. The excitation wavelength was 390 nm and the emission was 460 nm.Regarding β-Galactosidase quantification in electroporated cells, 25,000 electroporated cells with Dbgal were washed with cold PBS twice, and the cell suspension was diluted in 50 μL 1X reaction buffer and analyzed by FACS Blue LacZ beta Galactosidase detection kit following the protocol described above. A control sample consisted of electroporated cells without Dbgal was also measured using the same assay. Finally, the intracellularly delivered β-Galactosidase was calculated by subtracting negative control from the Dbgal electroporated sample.HEK293 transfectionHEK293 cells were transiently transfected to express luciferase using Nucleofection. The plasmid used was pLV[Exp]-EGFP/Neo-EF1A>Luciferase (ID: VB900088-2587fmv, VectorBuilder, USA). The plasmid vector was cloned in Stbl3 E.coli, and extracted by NucleoBond Xtra Midi kit (Macherey-Nage, Germany). In each transfection sample, 4.3 μg endotoxin-free plasmids were mixed with 2 × 106 cells suspended in 100 μL electroporation buffer. The mixture was transferred to a 4D-Nucleocuvette™ Vessel, and electroporated with 4D-Nucleofector® X Unit using the pre-optimized program (Code: CM-130). After electroporation, the Nucleocuvette™ Vessel was kept in a cell incubator (+37 °C, 5% CO2 and 95% relative humidity) for 10 min, and then all the cell suspension was transferred to cell culture flasks for incubation. The eGFP expression was checked at 24, and 48 h post transfection by BD LSR-II Cell Analyzer flow cytometer. The luciferase expression was checked at 48 h post transfection by incubating transfected cells with 50 μM D-amLu, and the luminescence was recorded by Varioskan™ LUX multimode microplate reader.Cell viability evaluationThe cell viability was evaluated using CellTiter-Glo® luminescent cell viability assay following the manufacturer’s protocol. The cell suspension with or without electroporation was added on a 96-well plate at density of 2 × 104 cells per well in 100 μL cell culture medium. The cell viability was detected at pre-determined time points from the luminescent intensity, in which represent the amount of ATP produced by the viable cells. The assay was carried out according to manufacturer’s protocol and the data was recorded by Varioskan™ LUX multimode microplate reader.Cellular glutathione concentration evaluationThe cellular glutathione concentration was evaluated using GSH/GSSG-Glo™ assay following the manufacturer’s protocol. 1 × 104 cells with or without electroporation were suspended in 25 μL HBSS buffer and added to wells of a 96-wellplate. Then 25 μL total glutathione lysis reagent or oxidized glutathione lysis reagent was added to the wells and the plate was gently shaked for 5 mins on a plate shaker. Afterwards, Luciferin Generation Reagent was added to the plate (50 μL per well) and the plate was incubated for 30 mins at room temperature. Finally, Luciferin Detection Reagent was added to the plate (100 μL per well), and the plate was shaked for 15 min before reading the luminescence by Varioskan™ LUX multimode microplate reader. Glutathione standard curve was prepared using serial dilution of GSH (0-20 μM) in 25 μL HBSS buffer, followed by the addition of 25 μL total glutathione lysis reagent. Then the same procedure applied as described above for cell suspensions.Statistical analysisExperiments were performed at least in triplicates and all the values were presented as mean ± standard deviation (SD). Statistical significances among different experimental groups were analyzed by using Student’s t-test (unpaired and two-tailed) and one-way ANOVA analysis. All statistical analysis were carried out using Origin 2022b software.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
