Bio-Orthogonal Nanogels for Multiresponsive Release

Responsive nanogel systems are interesting for the drug delivery of bioactive molecules due to their high stability in aqueous media. The development of nanogels that are able to respond to biochemical cues and compatible with the encapsulation and the release of large and sensitive payloads remains challenging. Here, multistimuli-responsive nanogels were synthesized using a bio-orthogonal and reversible reaction and were designed for the selective release of encapsulated cargos in a spatiotemporally controlled manner. The nanogels were composed of a functionalized polysaccharide cross-linked with pH-responsive hydrazone linkages. The effect of the pH value of the environment on the nanogels was fully reversible, leading to a reversible control of the release of the payloads and a “stop-and-go” release profile. In addition to the pH-sensitive nature of the hydrazone network, the dextran backbone can be degraded through enzymatic cleavage. Furthermore, the cross-linkers were designed to be responsive to oxidoreductive cues. Disulfide groups, responsive to reducing environments, and thioketal groups, responsive to oxidative environments, were integrated into the nanogel network. The release of model payloads was investigated in response to changes in the pH value of the environment or to the presence of reducing or oxidizing agents.

. (A) Synthesis of the modified dextran. (B) 1 H-NMR spectrum of native dextran (black) and oxidized dextran (red) and final modified dextran (green). (C) FTIR spectrum of native dextran (black), oxidized dextran (red) and the final levulinic acid-functionalized oxidized dextran (green). Figure S2. Molecular weight distribution of the dextran derivative measured by GPC in water, for native dextran (black), oxidized dextran (red) and the final levulinic acid-functionalized oxidized dextran (green).
The degree of oxidation of the dextran was measured before the functionalization with the levulinic acid by titration. 1 A sample of 0.1g of dextran was dissolved in 25 mL of a 0.25 N hydroxylamine hydrochloride solution in water. Each sample was reacted for 2 h at 50°C. The solution was then titrated with 0.1 M sodium hydroxide and methyl red. The change of the pH value with the addition of sodium hydroxide was recorded to determine the equivalent volume and compared to a blank sample prepared with unreacted dextran. The degree of functionalization with levulinic acid was calculated from the ratio of the NMR peak of the ketone protons of the levulinic acid and the proton on the C1 of the dextran. On average, there were 0.75 aldehydes/glucose and 0.6 ketones/glucose in the modified dextran.

Synthesis of thioketal dipropionic acid dihydrazide (TKNN)
In a 50 mL flask, butyl 3-mercaptopropionate (8.00 g) and anhydrous acetone (6.00 g) were mixed with a solution of hydrochloric acid in dioxane (HCl 4M, 4 mL). The resulting solution was stirred under nitrogen for 15 h at room temperature. The solvent was removed under reduced pressure. The residue was then diluted with diethyl ether (60 mL), transferred to an extraction funnel, washed sequentially with saturated NaHCO3 solution (30 mL) and water (30 mL). The organic phase was dried with MgSO4 and evaporated under reduced pressure to afford crude dibutyl thioketal dipropionate as a transparent, pale amber oil. The resulting dibutyl thioketal dipropionate (5.00 g) was dissolved in methanol (100 mL) at room temperature. Hydrazine monohydrate (6.30 g) was then added and the reaction mixture stirred overnight (ca. 18 h) at room temperature. The resulting reaction mixture was evaporated under reduced pressure. The residue was then diluted with water (60 mL), transferred to an extraction funnel, washed twice with diethyl ether (30 mL). The aqueous phase was evaporated under reduced pressure to obtain the desired product as a white solid. Figure Figure S5B).

Viability and uptake experiments
The HeLa cell medium was removed and the cells were detached with 7 mL of 0.25 % Trypsin-EDTA (Gibco/Thermo Fisher, Germany) for 5 min at 37 °C, 5 % CO2, and 95 % relative humidity. 7 mL of FBS supplemented medium was added to the cell suspension and the cells were centrifuged at 300 g for 5 min (5810R, Eppendorf, Germany). The supernatant was discarded and the cell pellet was resuspended in FBS supplemented medium. The cell viability and cell count were determined by mixing 20 μL of cell suspension with 20 µL of trypan blue followed by measurement with an automated cell counter (TC10, Bio-Rad, Germany).

Cell viability assay
After the cell harvesting, HeLa cells were seeded in a 96-well plate (white, item No.: 655083, GreinerBio-One, Austria) with a cell number of 5,000 cells per well. Nanogels dilutions in FBS supplemented DMEM (10 % FBS, 1 % Glutamax, 100 U mL -1 penicillin, and 100 µg mL -1 streptomycin) were prepared and added in a volume of 100 μL to the cells. Following overnight incubation at 37 °C and 5 % CO2, the medium was removed. Samples were processed in triplicates. The cellTiter-Glo® Luminescent Cell Viability Assay (Promega, Germany) was performed according to the instructions of the manufacturer. The luminescence was measured with an Infinite M1000 plate reader (Tecan, Switzerland).

Cell uptake experiments
For the cell uptake experiments, HeLa cells were seeded in a 24-well plate after harvesting with a cell number of 100000 cells per well. Cells were incubated at 37 °C and 5 % CO2 overnight to achieve the attachment. On the next day, the medium was removed and the cells