Multicomponent Encapsulation into Fully Degradable Protein Nanocarriers via Interfacial Azide-Alkyne Click Reaction Allows the Co-Delivery of Immunotherapeutics

Encapsulation of multiple adjuvants along with antigens into nanocarriers allows a co-delivery to antigen-presenting cells for the synergistic induction of robust immune responses. However, loading cargoes of different molar masses, polarities, and solubilities in high efficiencies remains a challenge. Therefore, we developed a strategy to encapsulate a triple combination of the so-called adjuvants, i.e. with Resiquimod (R848), muramyl dipeptide (MDP) and polyinosinic-polycytidylic acid (Poly(I : C)) into human serum albumin (HSA) nanocarriers. The loading is conducted in situ while the nanocarrier is formed by an orthogonal and metal-free click reaction at the interface of an inverse miniemulsion. By this unique approach, high encapsulation efficiency without harming the cargo during the nanocarrier formation process and regardless of their physical properties is achieved, thus keeping their bioactivity. Furthermore, we demonstrated high control over the encapsulation efficiency and varying the amount of each cargo did not influence the efficiency of multicomponent encapsulation. Azide-modified HSA was crosslinked with hexanediol dipropiolate (HDDP) at the interface of a water-in-oil miniemulsion. Varying the crosslinker amount allowed us to tailor the density and degradation rates of the protein shell. Additional installation of disulfide bonds into the crosslinker created redox-responsive nanocarriers, which degraded both by protease and under reducing conditions with dithiothreitol. The prepared HSA nanocarriers were efficiently taken up by dendritic cells and exhibited an additive cell activation and maturation, exceeding the nanocarriers loaded with only a single drug. This general protocol allows the orthogonal and metal-free encapsulation of various drugs or adjuvants at defined concentrations into the protein nanocarriers.

2. Synthesis of disulfide 1,6-hexanediol dipropiolate (HDDP-SS) The dialkyne crosslinker was synthesized by esterification following the literature. Briefly, bis(2-hydroxyethyl) disulfide (3.0 g, 19.5 mmol), propiolic acid (5.4 g, 78.0 mmol) and p-TsOH (360 mg, 2 mmol) were dissolved in 150 mL benzene and stirred under reflux for 3 days using a dean-stark apparatus. Afterwards, 100 mL saturated NaHCO 3 solution was added to the reaction solution and the organic phase separated. The aqueous phase was washed twice with 100 mL diethylether. The organic phases are combined and dried over Na 2 SO 4 . The solvent was removed and the product purified by column chromatography (n-hexane:EtOAc = 3:1).

Azidation of proteins with 1-imidazole-sulfonyl azide hydrochloride
The protein (1g) was dissolved in 20 mL K 2 CO 3 solution of pH 11. The azide transfer agent (276 mg) was dissolved in 2 mL water and added dropwisely to the protein solution. The pH value of the reaction solution was adjusted to pH 11 with 1 M NaOH. The reaction solution was stirred at room temperature for 48 h. The product was purified by dialysis (MWCO 1K) and lyophilized. Yield: 0.93 g. The amount of azide moieties was determined using the fluorescamine assay.

Fluorescamine Fluorescent product Amine
Scheme S1. Reaction of fluorescamine with amines to a fluorescent product at pH 8.2.
Glycine was used for the standard calibration curve and lysozyme was used as a reference. A decreased amount of amine groups was determined for the protein after azide-functionalization, indicating a successful reaction. Figure S7. Fluorescamine assay, standard calibration with glycine. Table 1. Azide-functionalization of human serum albumin at different pH value and time.
Number of azide groups (N ex (N 3 )) quantified by the theoretical (N theo (NH 2 ) and experimental (N ex (NH 2 ) number of amines, measured with the fluorescamine assay.   Figure S8. Transmission electron micrograph of human serum albumin nanocarrier.

Degradability of protein nanocarrier
The enzymatic degradation of the protein nanocapsules were performed with proteinase K and determined by release of Cy5-Oligo. A 0.1 wt% nanocarrier dispersion in PBS buffer was treated with a proteinase K solution (30 U/mL) at 37 °C. After the enzymatic degradation the dispersion is filtered by centrifugation in an Amicon centrifuge filter 3K at 500 g for 30 min and the amount of released dye in the supernatant measured by fluorescence. The degradation of the nanocarriers by proteinase K is also monitored by DLS measurements every 5 min over 13 10 h. The reduction-responsive properties of HDDP-SS-crosslinked protein nanocarriers were investigated with dithiothreitol (DTT) by release of Cy5-Oligo. A 0.1 wt% nanocarrier dispersion in PBS was treated with a DTT solution (25 mM) at 20 °C. After the reductive degradation, the dispersion is filtered by centrifugation in an Amicon centrifuge filter 3K at 500 g for 30 min and the amount of released dye in the supernatant measured by fluorescence.
The degradation of the nanocarriers by DTT is also monitored by DLS measurements every 5 min over 10 h. In both cases, enzymatic and reductive degradation, a sample treated with PBS buffer serves as a control sample and every experiment was performed in triplets.

Enzymatic activity
The enzymatic activity of HRP and HRP-HDDP nanocarriers were determined using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as substrate. Briefly, the substrate was dissolved in 100 mM potassium phosphate buffer (pH 5.0) to a concentration of 5 mg/mL. The enzyme in different stages of the nanocarrier preparation were diluted to a concentration of 0.002 mg/mL protein in a solution of 0.5% Triton X-100 in 40 mM PBS buffer (pH 6.8). The ABTS solution (190 µL) was mixed with the enzyme solution (3.3 µL) in a 96-well plate and the reaction started with addition of 0.3% (w/w) hydrogen peroxide solution (6.6 µL). The absorbance at 405 nm was monitored by a UV/Vis spectrophotometer and the enzyme activity determined by a standard calibration with native HRP. Figure S17. IR spectra of horse radish peroxidase and azide-functionalized HRP.

Quantification of adjuvants in protein nanocarriers
For the quantification of adjuvants in the protein nanocapsules (PNCs), the PNCs were degraded by proteinase K (30 U/mL) at 37 °C overnight. The PNCs remains and the enzyme were separated from the released adjuvants through a centrifuge filter (MWCO 3K, 30 min, 1500 g). The amount of Resiquimod (R848) was determined by fluorescence (λ ex = 260 nm, λ em = 360 nm) using a standard calibration curve ( Figure S20). Muramyl dipeptide (MDP) was 18 determined from the supernatant using the Morgan-Elson Reaction (Scheme S3). The supernatant (50 µL) was mixed with borate buffer (50 µL, pH 9) and incubated at 100 °C for 3 min. The mixture is cooled to room temperature and DMAB (500 µL) was added to the mixture.
The mixture was incubated again at 37 °C for 15 min and afterwards the absorbance measured at 585 nm. The MDP was quantified by a standard calibration ( Figure S21). Poly(I:C) was quantified from the full mixture after degradation as it has a too high molecular weight to be separated from the proteins. The full mixture of PNCs after degradation with proteinase K was eluted through a reverse phase HPLC column using a mixture of Acetonitrile, 0.01% formic acid and 0.02 mol/L ammoniumacetate. The Poly(I:C) signal was quantified using a standard calibration curve ( Figure S22).

In vitro experiments with BMDCs
Bone marrow-derived dendritic cells (BMDC) were differentiated from bone marrow progenitors (BM cells) of 8-to 10-week-old C57BL/6mice. Briefly, the bone marrow was FACSCanto IIflow cytometer equipped with BD FACSDiva software (BDBiosciences). Data were generated based on defined gating strategies and analyzed using FlowJo software (FlowJo,Ashland, USA). Figure S23. Cell binding/uptake into BMDC of loaded HSA-HDDP nanocarrier and free adjuvants measured by flow cytometry. Figure S24. Confocal image of bone-marrow derived dendritic cells (green) and uptaken human serum albumin nanocarriers (red).