Encapsulation of polyprodrugs enables an efficient and controlled release of dexamethasone

Water-soluble low molecular weight drugs, such as the synthetic glucocorticoid dexamethasone (DXM), can easily leak out of nanocarriers after encapsulation due to their hydrophilic nature and small size. This can lead to a reduced therapeutic efficacy and therefore to unwanted adverse effects on healthy tissue. Targeting DXM to inflammatory cells of the liver like Kupffer cells or macrophages is a promising approach to minimize typical side effects. Therefore, a controlled transport to the cells of interest and selective on-site release is crucial. Aim of this study was the development of a DXM-phosphate-based polyprodrug and the encapsulation in silica nanocontainers (SiO2 NCs) for the reduction of inflammatory responses in liver cells. DXM was copolymerized with a linker molecule introducing pH-cleavable hydrazone bonds in the backbone and obtaining polyprodrugs (PDXM). Encapsulation of PDXMs into SiO2 NCs provided a stable confinement avoiding uncontrolled leakage. PDXMs were degraded under acidic conditions and subsequently released out of SiO2 NCs. Biological studies showed significantly enhanced anti-inflammatory capacity of the polyprodrug nanoformulations over non-encapsulated DXM or soluble polyprodrugs. These results demonstrate the advantage of combining the polyprodrug strategy with nanocarrier-mediated delivery for enhanced control of the delivery of water-soluble low molecular weight drugs.

Release of DXM from SiO 2 NCs. Release of DXM from SiO2 NCs was performed by using dialysis membranes with a molecular weight cut-off of 14 kDa. 2 mL of dispersion of SiO2 NCs encapsulating DXM or PDXM with different molecular weights were placed in a dialysis bag that was immersed in 20 mL PBS (pH 7.4) or acetate buffer solutions (pH 5.4) at 37 °C. 2 mL of the supernatants were taken from the incubation media at given intervals and an equal volume of fresh buffer solution was added to keep the volume constant. The pH-responsive release of DXM from SiO2 NCs was expressed as the cumulative ratio of released drug to encapsulated drug. The drug release experiments were performed in triplicates for each sample. To investigate the release mechanism of DXM from nanocapsules, the release profiles were fitted with different mathematical models including zero order, first order, and Higuchi kinetics by using DDsolver, a menu-driven add-in program for Microsoft Excel written in Visual Basic. [21] Analytical Tools. 1  Gel permeation chromatography (GPC) measurements were performed to determine the apparent molecular weight of the synthesized polymer prodrugs and their molar weight distribution. The polymers were first dissolved in water to reach a concentration of 5 mgꞏmL −1 and were then filtered through a 0.45 μm Teflon filter. The measurements were performed on a Waters 515 pump with a refractive index detector (ERC RI 101). Three columns (0.8 × 30 cm, 10 µm) with different porosities (10 6 , 10 4 and 500 Å) from SDV (PSS, Germany) were used at room temperature. The elution rate of water was 1.0 mLꞏmin −1 . The resulting apparent molecular weights were calculated using poly(ethylene oxide) with narrow molecular weights as standards. The UV-Vis absorption spectra of DXM and PDXM were obtained using a UV-Vis spectrometer (Lambda 16, Perkin Elmer). The calibration curve of DXM in water is displayed in Figure S15. The average size and size distribution of SiO2 NCs were measured by dynamic light scattering (DLS) at 25 °C on a Nicomp 380 submicron particle sizer (Nicomp Particle Sizing Systems, USA) at a fixed scattering angle of 90°. For the determination of the size of PDXM, dynamic light scattering measurements were performed on an ALV spectrometer equipped with a thermostat and consisting of a goniometer and an ALV-5004 multiple-tau full-digital correlator (320 channels). A He-Ne Laser operating at a laser wavelength of 632.8 nm was used as light source. Measurements were performed at 20 °C at different angles ranging from 30° to 150°. PDXM were dissolved at a concentration of 2 mg mL -1 were used for the measurement. The aggregation behavior of the SiO2 NC-PDXM in blood plasma was studied by multi-angle DLS using a method reported previously. [13] Before the measurements, PBS or undiluted human plasma was filtered through Millex-LCR filters (Merck Millipore, Billerica, USA) with 450 nm pore size into quartz cuvettes with an inner radius of 9 mm for light scattering from Hellma (Müllheim, Germany). 10 µL of SiO2 NC-PDXM100k dispersion (solid content: 0.2 wt%) was unfiltratedly pipetted in a light scattering cuvette containing 200 µL undiluted human plasma. The mixture was then diluted with filtered PBS up to 1 mL total sample volume in the light scattering cuvette. Prior to use, the quartz cuvettes were cleaned with acetone using a Thurmond apparatus. For DLS analysis of the SiO2 NCs alone, 10 µL of the SiO2 NC-PDXM100k solution (0.2wt%) was added (without filtration) in 990 µL of filtered PBS. Plasma alone was prepared by adding 800 µL of PBS to 200 µL undiluted human plasma. After mixing, the samples were incubated for 20 min on a shaker at room temperature (20 °C) prior to the measurement. DLS measurements were performed at 20 o C. For data analysis, a robust multicomponent fit method was used as previously reported. [13] The morphology of nanocapsules was characterized with a JEOL 1400 (JEOL Ltd., Tokyo, Japan) transmission electron microscope (TEM) operating at an accelerating voltage of 120 kV.
Typically, the samples were prepared by diluting the dispersions in demineralized water to obtain a solid content of  0.01 wt%. One drop of diluted dispersion was placed on 300 mesh carbon-coated copper grids and left to dry overnight at room temperature. SEM measurements were performed with a Gemini 1530 (Carl Zeiss AG, Oberkochen, Germany) field emission scanning electron microscope at an accelerating voltage of 170 V. Samples were prepared by diluting dispersions in demineralized water to obtain a solid content of  0.01 wt%. One drop of diluted dispersion was deposited on silica wafers and left to dry. Nitrogen adsorptiondesorption measurements were carried out on a Quantachrome Autosorb-1 analyzer (Boynton Beach, FL) at 77.3 K. The capsule dispersions were freeze-dried for 48 h and degassed at 70 °C for 12 h under high vacuum before measurements. The specific surface area was calculated using the Brunauer-Emmett-Teller (BET) equation based on adsorption data points in the P/P0 range of 0 < P/P0 < 0.25. Pore size distributions were estimated from adsorption branches of the isotherms using the Barrett-Joyner-Halenda (BJH) method. Prior to HPLC analysis (Agilent Technologies 1200 Series) at 20 °C, samples were filtered gently through a 0.45 μm filter. DXM was detected with a photodiode array detector at 240 nm. An Agilent Eclipse plus C18 column was used with a flow rate of 1 mL min -1 . The gradient was composed of acetonitrile/water with 0.1% trifluoroacetic acid, starting with 20%/80% and reaching 100% acetonitrile after 10 min.
Sample (PDXM100k@pH=5.4) was prepared by incubating PDXM100k in acetate buffer solution at pH 5.4 sealed in a dialysis bag with a molecular weight cut-off of 14 kDa for 48 h.
The dialysis medium was taken for HPLC measurements. DXM-hexanohydrazide conjugate was synthesized as follows. DXM (1.55 g, 3 mmol) and hexanohydrazide (26.0 mg, 2 mmol) were first dissolved in milli-Q water (25 mL). Acetic acid (2.5 mL) was added to the solution.
The mixture was purged with argon and stirred at 50 °C for 96 h. The mixture (DXMhexanohydrazide and unreacted DXM) powder was then obtained by freeze-drying the obtained solution and the product was measured by HPLC. The institutional ethics committee approved the study (Landesärztekammer Rheinland-Pfalz, 837.439.12 (8540-F)). Written informed consent was obtained for any experimentation with materials from human subjects. A plasma pool from ten volunteers was prepared and stored at -80 °C.
Protein Corona Analysis. SiO2 NCs (1 mg) were incubated with 1 mL of human citrate plasma 1 h at 37 °C under constant agitation. Hard corona-coated nanocapsules were isolated via centrifugation and washing as previously described. [22] To detach the corona proteins from the nanocapsules´ surface, the pellet was incubated with 2% sodium dodecyl sulfate (SDS) and in Tris-HCl (62.5 mM) for 5 min at 95 °C. The dispersion was centrifuged (20,000 g, 1 h, 4 °C) and the supernatant containing hard corona proteins was recovered. Subsequently, the isolated proteins were analyzed by proteomics.

Liquid Chromatography Coupled to Mass Spectrometry (LC-MS). For proteomic analysis, SDS was removed from the protein sample via Pierce Detergent Removal Spin
Columns. Digestion of corona proteins was performed as described in our previous reports. [23] Finally, isolated peptides were diluted with 0.1% formic acid spiked with 50 fmol µL -1 Hi3 Ecoli (Waters) for absolute protein quantification by LC-MS. Measurements were performed on a nanoACQUITY UPLC system coupled to a Synapt G2-Si mass spectrometer. Data was analyzed with the MassLynx 4.1 software and Progenesis QI (2.0). A reviewed human database was downloaded from Uniprot for protein identification.   Table SS2 and S11/S12. The median fluorescence intensity of Cy5 and the frequency of Cy5 positive gated cells of each population of interest were exported as csvfile and evaluated using Excel (Microsoft) and Rstudio.  Images were acquired using the confocal laser scanning microscope LSM510 (Zeiss) using pinhole width of one airy unit. Laser Power and digital gain were adjusted on the maximum intensity of a Cy5-SiO2 NC-treated liver section using the range indicator. The images were then further processed in Fiji ImageJ.

Isolation of Non-Parenchymal Liver Cells and Stimulation with PDXM-Containing
SiO 2 NCs for Functional Experiments. For the functional experiments (Figure 7), murine non-parenchymal liver cells (NPCs) were isolated from livers as described previously. [24] Briefly, mice were anesthetized with Ketamin/Xylazin and livers were perfused with 20 mL            The CD11c negative population was gated for CD163, which is a marker expressed on Kupffer cells (KC). [16] The negative population was gated for Ly6C, a marker for monocytes (Mono).

Results
The pan-macrophage marker CD68 was used to determine a further macrophage population    Figure 5e, S11 and S12). If the population included less than 50 cells, the value was excluded. In each group 3 animals were analyzed. (d) Confocal microscopy of Cy5-NHS ester and CD68 staining of liver sections. For the acquisitions the same laser settings as for the liver sections (Figure 5d, S10) has been used.

Figure S15. Toxicity of PDXM-loaded SiO 2 NCs on non-parenchymal liver cells in vitro.
NPCs were incubated with different concentrations of PDXM-SiO2 NC formulations for 24 h and dead cells were subsequently stained with propidium iodide for 5 min followed by flow cytometric analyses to determine the frequency of dead cells.