Polysaccharide-Based pH-Responsive Nanocapsules Prepared with Bio-Orthogonal Chemistry and Their Use as Responsive Delivery Systems

Bio-orthogonal reactions have become an essential tool to prepare biomaterials; for example, in the synthesis of nanocarriers, bio-orthogonal chemistry allows circumventing common obstacles related to the encapsulation of delicate payloads or the occurrence of uncontrolled side reactions, which significantly limit the range of potential payloads to encapsulate. Here, we report a new approach to prepare pH-responsive nanocarriers using dynamic bio-orthogonal chemistry. The reaction between a poly(hydrazide) crosslinker and functionalized polysaccharides was used to form a pH-responsive hydrazone network. The network formation occurred at the interface of aqueous nanodroplets in miniemulsion and led to the production of nanocapsules that were able to encapsulate payloads of different molecular weights. The resulting nanocapsules displayed low cytotoxicity and were able to release the encapsulated payload, in a controlled manner, under mildly acidic conditions.

(Cy5-SE) was purchased from Tocris Bioscience. Polyglycerol polyricinoleate (PGPR) was provided by Danisco and was purified first by dissolution in hexane followed by centrifugation (2000 rpm) to precipitate solid particles, then the supernatant was recover and the purified PGPR was dried by rotary evaporation. Styrene was purified with a column of basic aluminum oxide to remove the inhibitor. 2, 2'-Azobis (2-methylbutyronitrile) was recrystallized in ethanol; the resulting crystals were recovered and dried under vacuum and stored until use at -20 o C.

Synthesis of oxidized dextran (OxD)
The oxidation of dextran was carried out by dissolving 5.0 g of dextran (Mn: 16 kDa, Ð: 2.18) and 1.75 g of KClO4 in 200 mL of water. The resulting solution was stirred for 24 h at room temperature and then dialyzed for 3 days against water and finally lyophilized ( Figure S1). The resulting oxidized dextran was characterized by NMR spectroscopy, FTIR spectroscopy and GPC.
For in vitro cell viability and cell uptake OxD was labeled with cyanine-5. Cy5-OxD was synthesized by reacting 0.5 g of OxD with 6 mg of Cy5-SE, 0.5 mg of 4-dimethylaminopyridine and 7 µL trimethylamine in 50 mL of DMSO. The solution was reacted for 24 h at 50 °C under a nitrogen atmosphere. The resulting solution was dialyzed for 3 days against water and finally lyophilized. The degree of oxidation (DO) of the OxD defined as the number of oxidized residues per 100 glucose residues was determined by titration. A sample of 0.1g of OxD 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 sodium hydroxide was recorded to determine the equivalent volume and compared to a blank sample prepared with unreacted dextran. The degree of oxidation measured by titration was 24.7%.

Synthesis of levulinate-functionalized dextran (KeD)
Levulinate dextran was prepared by the reaction of 3 g of dextran (Mn: 16 kDa, Ð: 2.18) with 4.30 g of levulinic acid, 7.50 g of DCC, 1.60 g of DMAP and 1.50 g of pyridine in 100 mL of dry DMSO ( Figure S2). The solution was reacted for 24 h at 60 °C under a nitrogen atmosphere and then precipitated with ethanol. Then, the precipitate was dissolved in water, filtered through a 200 nm pore size cellulose acetate filter, and then dialyzed over seven days, and freeze-dried. The resulting levulinate dextran (Mn: 18 kDa, Ð: 2.12) was characterized by NMR spectroscopy, FTIR spectroscopy and GPC. The degree of functionalization 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, 100% of the glucose units in KeD were functionalized with one levulinic acid.
For in vitro cell viability and cell uptake study KeD was labeled with cyanine-5. Cy5-KeD was synthesized by reacting 0.5 g KeD with 6 mg Cy5-SE, 0.5 mg 4-dimethylaminopyridine and 7 µl in 50 mL of dry DMSO. The solution was reacted for 24 h at 50 °C under a nitrogen atmosphere. The resulting solution was dialyzed for 3 days against water and finally lyophilized.
Methacrylic anhydride (51.75 g) was dissolved in chloroform (250 mL) and added dropwise to a stirred solution of hydrazine monohydrate (70 mL, 1.44 mol) at 0 °C and then stirred at room temperature overnight. The organic layer was recovered and the aqueous layer washed three times with chloroform. The chloroform aliquots were combined and the solvent removed by rotary evaporation to yield a white crystalline solid. The resulting solid was recrystallized from a mixture of 10:1 toluene-dichloromethane to yield the pure monomer in the form of fine needle-like crystals ( Figure S3). In the second step, the methacryloyl hydrazide (1 g) was copolymerized with styrene (2.4 g) by free-radical polymerization in the presence of AIBN (100 mg) in DMSO at 70 °C overnight. For the polymer purification, water was added to the reaction mixture to precipitate the PSH. The polymer was recovered and dried, redissolved in THF and reprecipitated twice, once in water and once in hexane. The resulting polymer was characterized by NMR, FTIR and GPC (Mn : 1.8 kDa, Ð : 1.87).
Titration was used to quantify the hydrazide in PSH. A sample of PSH (50 mg) was dissolved in 20 mL of glacial acetic acid, then one drop of crystal violet indicator was added, and the violetcolored solution was titrated with standard 0.01 N perchloric acid which was prepared in glacial acetic acid and compared to a blank sample prepared without PSH. The endpoint was reached when a definite green coloration was produced. The molar fraction of methacryloyl hydrazide in the polymer was 20.5 mol%. Figure S3. (A) Synthesis of poly(styrene-co-methacryloyl hydrazide). (B) 1 H-NMR spectrum of methacryloyl hydrazide (red) and poly(styrene-co-methacryloyl hydrazide) (black). (C) FTIR spectrum of methacryloyl hydrazide (red)) and poly(styrene-co-methacryloyl hydrazide) (black).

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

Cell Viability Assay
After cell harvesting, HeLa cells were seeded in a 96-well plate (Item No.: 655083, Greiner Bio-One, Austria) with a cell number of 5,000 cells per well. Following overnight incubation at 37 °C and 5% CO2, the medium was removed. Nanocarrier dilutions in FBS supplemented DMEM were prepared and added in a volume of 100 µL to the cells. Samples were processed in triplicates.

Cell Uptake Experiment by Flow Cytometry
For cell uptake experiments, HeLa cells were seeded in a 24-well plate after harvesting with a cell number of 150 000 cells per well. Cells were incubated at 37 °C and 5% CO2 overnight to achieve attachment. On the next day, the medium was removed and the cells washed once with