Conformational manipulation of scale-up prepared single-chain polymeric nanogels for multiscale regulation of cells

Folded single chain polymeric nano-objects are the molecular level soft material with ultra-small size. Here, we report an easy and scalable method for preparing single-chain nanogels (SCNGs) with improved efficiency. We further investigate the impact of the dynamic molecular conformational change of SCNGs on cellular interactions from molecular to bulk scale. First, the supramolecular unfoldable SCNGs efficiently deliver siRNAs into stem cells as a molecular drug carrier in a conformation-dependent manner. Furthermore, the conformation changes of SCNGs enable dynamic and precise manipulation of ligand tether structure on 2D biomaterial interfaces to regulate the ligand–receptor ligation and mechanosensing of cells. Lastly, the dynamic SCNGs as the building blocks provide effective energy dissipation to bulk biomaterials such as hydrogels, thereby protecting the encapsulated stem cells from deleterious mechanical shocks in 3D matrix. Such a bottom-up molecular tailoring strategy will inspire further applications of single-chain nano-objects in the biomedical area.

the solution was washed with 1 mM HCl and water and then dried over sodium sulfate. DCM was removed via rotary evaporation to afford 0.82 g of crude product, which was purified by column chromatography using petroleum ether and ethyl acetate (5:1) as an eluent to afford the product as a yellow oil. 1 H NMR (400 MHz, CDCl3): δ (ppm), 4.67 (d, 4H), 2.46 (t, 2H), 1.67 (s, 12H).
Synthesis of Vinyl-NCS (Ac-NCS): Amine (1) (0.1 mol) and base (triethylamine, 0.02 mol) were dissolved in 100 mL of THF. Then, carbon disulfide (0.3 mol) was added into the above mixture in an ice bath and stirred at low temperature for 30 min. After the reaction was finished, 5 equivalents of hydrogen peroxide (30%) were added dropwise into the reaction mixture and then neutralized with HCl (1 M). The reaction mixture was then evaporated under reduced pressure and extracted with ethyl ethanoate 3 times. The extraction solvent was subsequently evaporated under reduced pressure. Finally, a yellowish oily residue was obtained as crude product 2, which was purified by liquid chromatography on a silica gel column. 1 H NMR (400 MHz, CDCl3): δ (ppm), 3.74 (t, 2H), 3.68 (m, 4H), 3.62 (t, 2H), 2.27 (s, 1H). (Supplementary Fig. 4) In the next step, 0.01 mol of 2 and 0.02 mol of triethylamine were dissolved in 15 mL of DCM. The solution was cooled to 0 °C under a N2 atmosphere. Then, a solution of acryl chloride (0.015 mol) in DCM (5 mL) was added into above mixture dropwise. The resulting mixture was stirred at room temperature for 4 hours. After the reaction was finished, the reaction mixture was poured into 30 mL of cold water and extracted with DCM 3 times. The organic layer of the extraction was washed with HCl (0.1 M) and saline 3 times. After drying over Na2SO4, the organic solvent was removed under reduced pressure. The final product of 3 was purified by liquid chromatography on a silica gel column. 1 H NMR (400 MHz, CDCl3): δ (ppm), 6.40 (d, 1H), 6.15 (m,1H), 5.83 (d, 1H), 4.32 (m, 2H), 3.76 (t, 2H), 3.68 (m, 4H).
Synthesis of guest monomer: acrylate-type vinyl-ADA adamantane (V-ADA): First, 0.05 mol of 1-adamantylamine hydrochloride and 0.05 mol of triethylamine were dissolved in 10 mL of CHCl3. Next, 0.10 mol of Vinyl-NCS (3) S4 was dissolved in 5 mL of CHCl3 and added dropwise into the above mixture cooled in an ice bath. The reaction was monitored by thin layer chromatography. After the reaction was finished, the reaction mixture was poured into 20 mL of CHCl3 and washed with HCl (0.1 M) and saline 3 times. The organic layer was removed under reduced pressure. The final product of V-ADA was purified by liquid chromatography on a silica gel column. 1 H NMR (400 MHz, CDCl3): δ (ppm), 6.41 (d, 1H), 6.14 (m, 3H), 5.87 (d, 1H), 4.30 (t, 2H), 3.80-3.66 (m, 6H), 2.11 (s, 3H), 1.99 (m, 6H), 1.65 (m, 6H). (Supplementary Fig. 5) Synthesis of the host monomer: acrylate-type vinyl-β-cyclodextrin (V-CD) Mono-Ac-βCD: βCD grafted with a single acrylate group (mono-Ac-βCD) was synthesized through reaction between the amino group of βCD-NH2 and the isothiocyanate group of Vinyl-NCS. In this reaction, 0.05 mol of Vinyl-NCS (3) was mixed with the same amount (0.05 mol) of βCD-NH2 in 10 mL of dimethyl sulfoxide (DMSO) for 24 hours at room temperature. The crude product was precipitated in acetone and purified by precipitation in acetone three times. The final product was dried and stored in a vacuum oven at 25 °C. (Supplementary Fig. 6) Synthesis of guest monomer: acrylamide-type vinyl-ADA adamantane (V-ADA): First, 0.05 mol of 1adamantylamine hydrochloride and 0.05 mol of triethylamine were dissolved in 10 mL of CHCl3. The obtained mixture was slowly added into 0.10 mol of 1 in 20 mL of CHCl3 in an ice bath for 30 min. The reaction was monitored by thin layer chromatography. After the reaction was finished, the reaction mixture was poured into 50 mL of CHCl3 and washed with HCl (0.1 M) and saline 3 times. The organic layer was removed under reduced pressure. The product of 2 was purified by liquid chromatography on a silica gel column. Then, 0.01 mol of 2 and 0.02 mol of triethylamine were dissolved in 15 mL of DCM. The solution was cooled to 0 °C under a N2 atmosphere.
Then, a solution of acryl chloride (0.015 mol) in DCM (5 mL) was added into above mixture dropwise. The resulting mixture was stirred at room temperature for 4 hours. After the reaction was finished, the reaction mixture was poured into 30 mL of cold water and extracted with DCM 3 times. The organic layer of the extraction was washed with HCl (0.1 M) and saline 3 times. After drying over Na2SO4, the organic solvent was removed under reduced pressure. The final product of 3 was purified by liquid chromatography on a silica gel column.
( Supplementary Fig. 7a, Supplementary Fig. 8) Synthesis of host monomer: βCD grafted with a single acrylamide group was synthesized through reaction between the amino group of βCD-NH2 and the acryl chloride. In this reaction, 0.05 mol of acryl chloride was mixed with the same amount (0.05 mol) of βCD-NH2 and 0.05 mol of triethylamine in 10 mL of chloroform for 48 hours at room temperature. The crude product precipitated in acetone and purified by dialysing against water for 3 days and then precipitating in acetone for three times. The final product was dried and stored in a vacuum oven at 25 °C.
( Supplementary Fig. 7b, Supplementary Fig. 9) S5 V-ADA and V-CD self-assembly to prepare supramolecular crosslinkers (V-ADA@CD-V): First, 0.1 mol of V-CD was dissolved in 50 mL of DI water. Then, 0.9 equivalents (0.09 mol) of V-CD was dissolved in 4 mL of THF.
The V-ADA THF solution was then added into the V-CD water solution dropwise to obtain the supramolecular crosslinkers (V-ADA@CD-V). The combined molar ratio of V-ADA and V-CD was 0.9, where V-CD was added in a slight excess to ensure the full complexation of V-ADA and the formation of a water-soluble supramolecular divinyl crosslinker (V-ADA@CD-V).

Supplementary Note 1
Mechanical Test Data Analysis: All of the original experimental data recorded by the MACH-1 machine were imported into Microsoft Excel. The original data stands for displacement and force at each time point were converted to strain and stress values using the formulas listed below: (1) Strain ε = ∆ ε is the strain, ∆ is the compressed height of the hydrogel at each time point (displacement in the original data), and H is the total height of the hydrogel.
(2) Stress σ = σ is the stress, FN is the normal force, and A refers to the actual cross-sectional area of the hydrogels at each time point, which was calculated using the following equation: By assuming the volume of the hydrogel as constant the actual cross-sectional area of the hydrogels can be obtained by dividing the hydrogel volume by the actual height at each time point. The loading-unloading curve was sketched using the calculated stress and strain values.

Supplementary Note 2
Statistical Analyses: Analysis data are presented as mean ± standard error. All statistical analyses were Using Graphpad Prism 5, pvalues less than 0.05 were regarded as statistical significances. Two-tailed Student's t-test was employed to compare two groups at the same time point. One-way analysis of variance (ANOVA) including Tukey-Kramer post-hoc test S6 was used to compare multiple groups at the same time point. Energy dissipation: The area of the force vs. displacement curve relates to the loading energy. Thus, energy dissipation was determined by calculating the area in between the loading and unloading curves in the force vs. displacement plots recorded during the loading experiments. The area was calculated using Origin Pro 8.0's Integration function. (Supplementary Fig. 27)

Supplementary Note 3
Characterization of SCNGs 1 H NMR spectra was taken on a 400 MHz Bruker instrument, and the acquired NMR data were analyzed with mestrenova software. Chemical shift values were referenced using Tetramethylsilane (TMS).
Gel permeation chromatography (GPC) was carried out on a system comprising a Waters 1515 HPLC pump, Waters 2414 refractive index detector. Waters Ultrahydrogel 250 columns at 30°C for Aqueous (100 mM, PBS) phase online tests. Normally, 5 mg polymer samples were dissolved in 4 mL PBS. Then the solutions were filtered by nylon filter (0.2µm). 100 µL filtered sample solutions were injected into the Waters equipment to measure the molecular weight. For the unfolding of SCNGs, 2 mL of the filtered solution was separated after the first injection.
60µl of 1mM free ADA-NH2 solution was added directly into the solution. After 2 hours, these samples were filtered again and characterized by GPC.
Dynamic light scattering (DLS) analyses were carried on Delsa Max Pro of Beckman Coulter. The data were analysis on DelsaMax 1.0.1.6. to characterize the distribution and hydrodynamic size of the polymers. Normally, 200mg polymer was dissolved in 10 mL DI water. Half of the solution was treated with 1mM free ADA-NH2 to get the corresponding unfolded sample. All the samples were filtered by nylon filter (0.2µm) to reduce dust particles and atmospheric contaminants before measurement. 1mL sample solution was injected into the measurement cell to obtain the hydrodynamic radius and distribution of the nanoparticles.
Atomic Force Microscope (AFM) was used to observe the morphologies of the nanoparticles. The tests were operated in air on a Bruker Multimode VIII SPM equipped with a J scanner. Experiments were performed in Bruker Peak Force Tapping mode with NSC11 tip (spring constant 48 N/m, Bruker). Dilute solution (10 -4 mg/mL) of the SCNGs in water will be deposited onto a fresh silica wafer surface. The samples will be dried at room temperature for measurement. The AFM characterization will be carried out on Bruker's tapping mode with different square areas.
Scanning electron microscopy (SEM) was used to investigate the morphology of the SCNGs on the substrates.
Substrates modified with the SCNGs were dried for 24 hours before the samples were mounted onto copper studs and sputter-coated with gold/palladium for 60 s. Then, SEM images were acquired by using an ultra-high resolution S7 scanning electron microscope (SU8010, Hitachi, Tokyo, Japan). Standard scan settings were applied during scanning. The acquired images were analysed with open source software ImageJ.

Supplementary Note 4
Cytotoxicity analysis of SCNGs 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The viability of human mesenchymal stem cells (hMSCs) incubated with the SCNGs was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The hMSCs were first seeded in the wells of a 96-well plate and adhered for 24 hours before they were incubated with either 1 mg/mL of SCNG-RGD in ɑ-MEM for another 12 hours. At each time point, the supernatant was removed before 100 µL of MTT solution (0.5 mg/mL) was added to each well and incubated at 37 °C for 4 hours in a cell culture incubator. Afterwards, we discarded the MTT solution and dissolved the precipitated formazan in each well in 200 µL of. The optical density of the formazan solution at a wavelength of 570 nm was determined with a microplate spectrophotometer (Multiskan FC Microplate Photometer, Thermo Fisher). The MTT cell viability assay was performed 24, 48 and 72 hours after SCNG-RGD incubation, and at least 4 replicated wells were examined for every measurement.

Supplementary Note 5
Cytometry analysis of the cell uptake efficiency

Supplementary Note 6
Cell culture on 2D substrates All cell culture experiments throughout this study were conducted at 37 °C and 5% CO2. hMSCs (passage 4) were seeded at a constant density of 5,000 cells/cm 2 onto RGD-conjugated SCNGs glass substrates or control surfaces (without RGD) under basal medium, i.e., alpha-minimum essential medium supplemented with 10% FBS, 1% streptomycin /penicillin, and 1% L-glutamine.

S8
Cell fixation and immunofluorescence staining for analysis Cells were washed with PBS to remove non-adherent cells and fixed with 4% w/v paraformaldehyde in PBS at pH 7.2-7.4 for 15 min at room temperature. Cells were permeabilized with 0.25% v/v Triton X-100 in PBS for 10 min.
For the vinculin staining assay, cells were fixed and permeabilized with 4% w/v paraformaldehyde and 0.1% v/v Triton X-100 for 5 min without prior PBS washing. Then, the cells were fixed with 4% w/v paraformaldehyde for an additional 15 min. The non-specific binding epitopes were blocked with 1% w/v BSA in PBS for 1 hour at 37 °C.
The primary antibodies were prepared in PBS/BSA with rhodamine-phalloidin (1:500; Molecular Probes) and observations were conducted in confocal dishes after the cells were washed with PBS buffer. 3D cell-laden hydrogels were put on glass slides and washed with PBS buffer before observation.

Supplementary Note 9
Characterization of the SCNG-hydrogels.
The prepared hydrogel was put in a 30 °C oven to dehydrate it and determine the solid weight. Then, the dehydrated hydrogel was put into water at room temperature and weighed at certain time intervals until the weight become constant to obtain the swelling curve.
Compression tests of the hydrogels were carried out on a Mach-1 micromechanical system with a 17N nano detector in a Thermo Series II water jacket incubator. Prepared disc-shaped cell-laden hydrogel samples were swelled in water for 24 hours. Then, these samples were transferred to the compression site, immersed in culture medium, and compressed with defined strains and compression frequencies by using the MACH-1 machine.

S9
Rheology characterization was conducted on a Malvern Kinexus rheometer. Experiments were conducted with the predefined function sequences. The gelation behaviour was characterized by single frequency strain-controlled time sweep measurements (strain 1%). Briefly, 300 µL of pre-gel solution (20 wt%) was added into the 1 mm (plate-to-plate) gap of the 20-mm-diameter test plate. The hydrogels were homogeneously distributed between the top and bottom plates of the rheometer. The time sweep was recorded at a strain of 1% and a frequency of 10 Hz.
After stabilization for 2 min, a bottom UV lamp was turned on. The irradiation and test periods lasted for 35 min in the shear stress rheological experiment.

Supplementary Note 10
Cell culture in 3D hydrogels. To examine the quantitative gene expression level, all samples were homogenized in 1 mL TRIzol reagent (Invitrogen, Waltham, Massachusetts, USA), and whole RNA was extracted according to the manufacturer's instructions. The RNA concentration was measured by a Nanodrop One spectrophotometer (Nanodrop Technologies, Waltham, Massachusetts, USA). One microgram of whole RNA was reverse transcripted into cDNA by using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher scientific). Quantitative PCR was conducted on an Applied Biosystems 7300 Real Time PCR system (Thermo Fisher scientific) using Taqman primers and probes specific to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the osteogenic marker genes runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and type I collagen (Col 1). Gene expression levels were normalized to those of GAPDH, and the relative gene expression levels were expressed as 2 -∆∆Ct ..

Supplementary Tables
Supplementary  Fig. 1 Characterization of the chain tranfer agent. 1 H NMR spectrum of the alkynyl CTA (BDPT).