Synthesis and purification of LEL triblock copolymers

The synthesis of the tri-block copolymers denoted LEL-20 and LEL-37 was carried out via conventional ring-opening polymerization (ROP) of 0.102 mol D, L-lactide (Polysciences Europe GmbH, Germany) with 0.0043 mol poly(ethylene glycol) (PEG, Mn = 1000 g·mol− 1 and 1500 g·mol− 1, Merck KGaA, Germany) in the presence of 0.001 mol stannous octoate (Merck KGaA, Germany) as a catalyst. The reaction proceeded on the Schlenk line without a solvent, i.e., in the melt, under an inert nitrogen atmosphere according to the modified procedure described by Michlovská et al. 2010. Subsequently, the copolymer melt was purified from unreacted monomers and catalyst. The single purification step required the dissolution of the copolymer in water and subsequent heating of the solution to 80 °C, which caused the copolymer to precipitate from the solution, leaving unreacted monomers and the catalyst in the solution. The residual water with impurities was discarded, and the purification procedure was repeated 3 times in total. The final purified product was freeze-dried (Martin Christ EPSILON 2-10D, Germany) until constant weight.

Gel permeation chromatography (GPC)

The number-average molecular weight (Mn), mass-average molecular weight (Mw), and polydispersity index (PDI = Mw/Mn) of the copolymers were measured using the gel permeation chromatography (GPC) method on a Waters Alliance e2695 GPC system (Waters Corporation, Milford, MA, USA) equipped with an Agilent PLgel 5 μm MIXED-D column at 65 °C and a refractive index detector at 40 °C, with dimethylformamide (DMF, 99.8%, Biosolve BV, The Netherlands) with 10 mM LiCl (≥ 99%, Sigma‒Aldrich, The Netherlands) as the eluent at a flow rate of 1 ml/min against linear poly(ethylene glycol) standards. The data were evaluated using Empower 3 Software 2010. Approximately 3 mg of copolymers were dissolved in 1 ml of DMF + 10 mM LiCl eluent, filtered prior to measurement, and analyzed.

Preparation of LEL tri-block copolymer solutions

The copolymers were weighed into vials and dispersed in Dulbecco’s modified Eagle medium (DMEM, Sigma‒Aldrich, USA) at the desired concentration to buffer the acidic reaction and ensure cellular survival in a hydrogel matrix. The dissolution of copolymers was carried out on a magnetic stirrer at 300–500 rpm for 3 days at a decreased temperature in the range of 4 to 12 °C until a completely homogeneous solution was obtained. For in-cell NMR experiments, LEL-20 and LEL-37 concentrations of 25 and 15 w/w %, respectively, were used.

Test tube inversion method (TTIM) and pH measurement

The sol-gel phase transitions of tri-block copolymer solutions were investigated visually using the test tube inversion method on the thermal block (DITABIS AG, Germany). The range for the experiments was set from 10 to 40 °C with a 2 °C temperature increment and an equilibration time of 5 min. The sol-gel transition was evaluated based on the criterion of flow. When the solution exhibited flowing characteristics, it was determined to be in the solution state. When the solution stayed in the bottom of the test tube after inversion, it was concluded that the solution was in a gel state. The pH value was obtained by reading the laboratory pH meter (Verkon, Czech Republic) after 1 min of equilibration.

Rheological analysis

Rheological properties of copolymer dispersions were determined using a controlled stress rheometer (Discovery HR-2, TA Instruments, USA) equipped with steel plate-plate geometry (40 mm) with a solvent trap and a Peltier plate temperature system. The temperature sweep measurements were carried out within the linear viscoelastic region of the material with a geometry gap set to 200 μm applying a constant 1 Hz frequency (6.28 rad·s− 1), oscillation strain of 1%, temperature ramp of 1 °C per minute and a temperature range from 10 to 40 °C.

DNA oligonucleotide preparation

The DNA oligonucleotides 5’-FAM-labeled and unlabeled 5’-GCT TCT AGT CAA TCC CCC CTC CCC CCT TCC CCC CTC CCC CC-3’ and unlabeled 5’-TTG ACT AGA AGC-3’ were purchased from Generi Biotech (Czech Republic). The nonlabelled oligonucleotides were first dissolved in H2O and subjected to standard n-butanol precipitation to remove contaminants from the solid-state synthesis as described by Viskova et al. 2019. Briefly, 30 ml of n-butanol (≥ 99%, Sigma‒Aldrich, USA) was added to ~ 1 ml of an aqueous solution of the DNA oligonucleotide. The resulting mixture was then vigorously shaken for 10 minutes and centrifuged at 30 000×g at 4°C for 1 hour. After centrifugation, the supernatant was removed, and upon drying, the pellet was redissolved in 1 ml of H2O. The DNA concentration was determined spectrophotometrically, and the strands were mixed at a ratio of 1:1.1 and annealed by incubation at 95°C for 5 minutes, followed by gradual cooling to room temperature. To form the 5’-FAM-labeled double-stranded DNA, the reverse strand was mixed with the FAM-labeled forward strand in the same ratio as mentioned above. The samples were then annealed for 10 min at 37 °C.

Cell culture

HeLa cells (Sigma‒Aldrich, USA) and HEK293T cells (ATCC, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma‒Aldrich, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, GE Life Sciences, USA) and penicillin‒streptomycin solution (P/S) (100 units penicillin and 0.10 mg streptomycin/ml) (Sigma‒Aldrich, USA) under a 5% CO2 atmosphere at 37 °C. At 80–90% confluency, the cells were passaged by washing with Dulbecco’s phosphate-buffered saline (DPBS) (Sigma‒Aldrich, USA) and by harvesting with 0.05% trypsin and 0.02% EDTA (Sigma‒Aldrich, USA) in 1 × DPBS.

Sample preparation for DNA in-cell NMR spectra acquisition

A total of 8 × 107 pelleted HeLa cells were resuspended in 2 ml of electroporation buffer (140 mM sodium phosphate, 5 mM KCl, 10 mM MgCl2, pH = 7.0) containing 400 µM DNA and 10 µM FAM-labeled DNA. The cell suspension was divided into five 4-mm electroporation cuvettes (Cell Projects, UK). After a 5-minute incubation on ice, electroporation was conducted with a BTX-ECM 830 system (Harvard Apparatus, USA) using two square-wave pulses (100 µs/1000 V; 30 ms/350 V) separated by a 5-second interval. After electroporation, the cells were incubated for 2 min at room temperature. The suspension from electroporation cuvettes was transferred to a centrifugation tube containing 10 ml of DMEM. The cells were pelleted by centrifugation at 200×g for 5 min and washed with 10 ml of DMEM. An aliquot of cells (~ 1 × 105) was used for flow cytometry analysis to determine cell survival and transfection efficiency. For 1 H NMR DNA spectra acquisition, the cell pellet was mixed either with 2 w/w % SeaPrep® agarose in DMEM at 37 °C or with 25 w/w % LEL-20 in DMEM at 4 °C, both in a v:v ratio of 1:1 to obtain a final volume of 500 µl.

Sample preparation for protein in-cell NMR spectra acquisition

HEK293T cells were transfected with plasmid pHL-sec, bearing the coding sequence for human ubiquitin (Tanaka et al. 2019), using poly(ethylenimine) (PEI) as described in Banci et al. 2013. Briefly, 25 µg of plasmid DNA was mixed with 50 µg of PEI in 5 ml of 15 N-labeled BIOEXPRESS-6000 media (Cambridge Isotope Laboratories, UK). The culture medium in a subconfluent 75 cm2 culture flask was replaced with the transfection mixture, followed by the addition of 15 ml BIOEXPRESS-6000 medium with 2% FBS. The cells were incubated for 48 h without a medium change to express the protein. For the acquisition of 15 N-labeled protein spectra, the cells were harvested with 0.05% trypsin and 0.02% EDTA, washed with DMEM, and centrifuged for 5 min at 200×g. To obtain a total volume of 500 µl, the cell pellet was mixed with 15 w/w % LEL-37 in DMEM or 10 w/w % Mebiol® gel in DMEM at a v:v ratio of 1:2 at 4 °C.

Sample preparation for intracellular ATP monitoring

For monitoring of cell metabolic activity via ATP level determination from 31P NMR spectra, HeLa cells were harvested with 0.05% trypsin and 0.02% EDTA (Merck KGaA, Germany), washed with DMEM, and centrifuged for 5 min at 200×g. To obtain a total volume of 500 µl, the cell pellet was mixed with 4 w/w % SeaPrep® agarose (Lonza, Switzerland) in DMEM in a v:v ratio of 3:1 at 37 °C, 10 w/w % Mebiol® gel (Cosmo Bio, USA) in DMEM 1:1 at 4 °C, 15 w/w % LEL-37 in DMEM 2:1 at 4 °C and 25 w/w % LEL-20 DMEM 2:1 at 4 °C.

Bioreactor setup

A PEEK capillary with a 0.75 mm inner diameter was filled with the mixture of polymer with cells. To solidify, the samples in SeaPrep® agarose were incubated on ice for 10 min, the samples in Mebiol® gel and LEL-37 at 37 °C for 20 min, and LEL-20 at room temperature for 20 min. A thread of the gel was pushed with a syringe from the capillary into a 5 mm screw-cap NMR tube filled with bioreactor media: DMEM without NaHCO3 (Sigma‒Aldrich, USA), 10% D2O (Eurisotop, France), 70 mM HEPES (Sigma‒Aldrich, USA), ZellShield (Minerva Biolabs, Germany). The NMR cuvette with the sample was connected to a tubing system ensuring media flow. The flow of medium was driven by HPLC pump (ECP2010, ECOM, Czech Republic) from a reservoir incubated in a water bath at 37 °C or at room temperature, through vacuum degassing system (DG 4014, ECOM, Czech Republic), to the bottom of the NMR cuvette via a glass capillary connected to the inlet tubing. The fresh medium was flowing up through the sample, thereby displacing the nutrient-depleted medium, which was drained via the outlet tubing connected to an orifice in the cuvette lid. The flow rate was set to 50 µl/min.

Release of cells from thermosensitive matrices

Gel threads with cells were transferred into a Falcon tube, containing 25 ml of DPBS at 4 °C. To dissolve the matrix, the tube was incubated for 10 min on a rotator at 4 °C. Subsequently, the suspension was filtered through 70 μm cell strainer (Corning, USA) to remove undissolved pieces of gel.

1D 1 H and 31P NMR experiments

1D 1 H NMR spectra for copolymer characterization and 1D 31P NMR spectra for ATP level measurement were recorded on a Bruker Avance NEO 500 MHz NMR (Bruker Corporation, Billerica, MA, USA) equipped with a 5 mm nitrogen-cooled dual (BB-1 H) cryoprobe (Prodigy). For the copolymer (LEL-20 and LEL-37) measurement, approximately 10 mg of sample was weighed on analytical balances and consecutively dissolved in 500 µl of deuterated chloroform (CDCl3). The sample was transferred into a 5 mm NMR cuvette and measured by a 1D 1 H zg pulse sequence with 128 scans at 20 °C. The number-average molecular weight was established from the ratio of PEG signal intensity (-CH2-O-CH2-) from δ = 3.34–3.84 ppm to the PLA (-CH-) signal from δ = 5.29–5.05 ppm. For ATP level measurements, 500 µl of a sample (see the NMR sample preparation) was transferred into a 5 mm NMR cuvette and measured by a 1D 31P zgdc pulse sequence with 4 × 512 scans at 20 °C (LEL-20 and SeaPrep® agarose) and 37 °C (LEL-37 and Mebiol® gel) with a bioreactor system. The obtained spectra were processed with the QSIN (sine squared) function with the SSB parameter set to 2.2. The NMR spectra were processed, integrated, and analyzed using MNova v14.2.1 software (Mestrelab Research, Spain).

In-cell 1D 1 H and 2D 1 H – 15 N NMR experiments

In-cell 1D 1 H NMR spectra for DNA and 2D 1 H – 15 N NMR spectra for protein investigation were recorded on a Bruker Avance NEO 950 MHz NMR (Bruker Corporation, Billerica, MA, USA) equipped with a 5 mm triple-resonance (1 H/19F-13 C-15 N) inverse cryoprobe with cooled 1 H and 13 C preamplifiers and a bioreactor system. A 1D 1 H JR-echo (1–1 echo) pulse sequence with zero excitation set to the resonance of water and the excitation maximum set to 13 ppm was used for the in-cell DNA measurement. Spectra were measured at 20 °C (LEL-20 and SeaPrep® agarose) with 5 × 1024 scans. Spectra were processed by the exponential apodization function with the line-broadening parameter (LB) set to 30 and baseline corrected. A 2D 1 H – 15 N SOFAST-HMQC pulse sequence was used for the in-cell protein measurement. Spectra were recorded at 37 °C (LEL-37 and Mebiol® gel) with five independent measurements consisting of 100 scans and 128 increments. The obtained spectra were processed by an exponential apodization function with the line-broadening parameter (LB) set to 10. The NMR spectra were processed and analyzed using Bruker Topspin 4.0 (Bruker Corporation, Billerica, MA, USA) and MNova v14.2.1 software (Mestrelab Research, Spain).

Cell viability analysis using trypan blue staining

HeLa cells were embedded in the tested polymers and incubated in 1 ml of the bioreactor media (DMEM without NaHCO3 + 10% D2O + 70 mM HEPES + ZellShield) at room temperature. At the specified time points, aliquots of the samples were homogenized in 100 µl of DPBS, and 20 µl of Trypan blue (Sigma‒Aldrich, USA) was added. After 2 min of incubation, 20 µl of the suspension was transferred to a microscope slide, and pictures of three random areas of each sample were taken. From each image, an area containing at least 100 cells was selected, where blue (dead) and white (living) cells were counted. The percent viability was calculated as the number of living cells/total number of cells×100, and the results from the three areas were averaged.

Flow cytometry analysis

Approximately 1 × 105 of HeLa or HEK293T cells were resuspended in 200 µl of DPBS, and propidium iodide (Sigma‒Aldrich, USA) was added to a final concentration of 2.5 µg/µl. Samples were analyzed using a BD FACS Verse Cell Analyzer (BD Biosciences, USA). For identification of propidium iodide-positive cells, the excitation wavelength was set to 488 nm, and emission at 700 ± 54 nm was detected. To determine the DNA transfection efficiency, the FAM label was detected as emission at 572 ± 32 nm upon excitation at 488 nm. A total of 1 × 104 cells were analyzed in each experiment. The results were processed with BD FACSsuite software (BD Biosciences, USA).

The membrane integrity of HeLa cells immobilized in thermosensitive copolymer gels was further tested under bioreactor conditions equipped with constant media flow. Threads of the cells embedded in Mebiol® gel, LEL-37, and LEL-20 were placed into an NMR bioreactor and incubated at 37 °C (Mebiol® gel, LEL-37) or 20 °C (LEL-20) at a media flow of 50 µl/min. As a control representing conditions without a polymer matrix, cells were incubated on dishes with nonadhesive surfaces in excess of the bioreactor media (instead of continuous flow) without a CO2-enriched atmosphere. After 4 h of incubation, the cells were isolated from the gel matrices and stained with propidium iodide (PI) to determine their membrane integrity using fluorescent flow cytometry as described previously.

Confocal microscopy

After NMR measurement, HeLa cells were released from the immobilization matrices and washed with cold DPBS. Approximately 5 × 105 cells were transferred to a 35 mm glass-bottom iBidi dish (Ibidi GmbH, Germany) precoated with 0.2% gelatin (Sigma‒Aldrich, USA) supplemented with 2 ml of DMEM containing 25 ng/ml SiR-DNA far-red dye (Tebubio, France) to visualize the cell nuclei. All microscopy images were obtained using a Zeiss Axio Observer. Z1 with an LSM 800 confocal unit, CO2 incubation chamber, and Plan-Neofluar 10×/0.30 AIR objective. Images were taken every 10 min in eight different positions during a 16-hour period in transmission mode with 640 nm excitation and emission detection at 650–750 nm. Confocal images were processed with ZEN Blue software (Zeiss, Germany).