Nanogels

Nanogels were prepared by HDRP following a previously reported procedure [21 (link)]. Monomers (NIPAM, NPAM, A–Pr–OH, AMPS, AM, 4VI), template (if applicable), and MBA as crosslinker (CL), in different monomer/CL ratios were dissolved in anhydrous DMSO in a Wheaton™ bottle. The volume of the solvent was adjusted to give the appropriate total monomer concentration (C_M = 0.5, 1, or 2%). AIBN as initiator (1, 3, 5, or 10%, compared to total moles of double bonds) was then added to the solution (feed compositions for all nanogels were calculated using Equations S1–S4). The Wheaton™ bottle was sealed and the pre-polymerization solution purged with N₂. The solution was then heated to the appropriate temperature (60 or 70 °C) for 12, 24, or 48 h. The transparent nanogel solution was then dialyzed against deionized water for 3 days with frequent changes (MWCO 3500 Da, diameter 22 mm), before being frozen in liquid N₂ and lyophilized (LTE scientific Lyotrap) to yield a white fluffy powder. All the nanogels synthesized were stored at room temperature.

Four stock solutions of chitosan were prepared by dissolution of the CS powder at concentrations of 2.5 mg·mL⁻¹ or 1.5 mg·mL⁻¹ in a 10% (m/v) citric acid or acetic acid aqueous solution and stirred overnight.
Nanogels were obtained by an ionotropic gelation process [61 (link),63 (link)]. The polyanionic phase, that is, HA (0.8 mg·mL⁻¹) and TPP (1.2 mg·mL⁻¹) in water (4.5 mL), were added dropwise to the CS solution (9 mL) under sonication (750W, amplitude 32%) to obtain stable nanosuspensions. The gadolinium complex (GdDOTP or GdDOTA) was previously dissolved in the polyanion solution. At the end of the addition, magnetic stirring was maintained for 10 min. Unloaded nanogels were obtained in the same way, omitting the gadolinium complex. Nanosuspensions were then freeze-dried, using glucose 15% (m/v) as a cryoprotectant. Nanoparticle average hydrodynamic diameters (D_H) and polydispersity indexes (PdI) were determined by Dynamic Light Scattering (DLS) (Malvern Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, UK). Each nanosuspension was analysed in triplicate at 20 °C at a scattering angle of 173°, for each sample, after 1/20 dilution in water. Pure water was used as a reference dispersing medium. ζ-(zeta) potential data were collected through electrophoretic light scattering at 20 °C, 150 V, in triplicate for each sample, after 1/20 dilution in water. The instrument was calibrated with a Malvern—68 mV standard before each analysis cycle.
The shape and the surface morphology of the nanogels were investigated by atomic force microscopy (AFM) (Catalyst, BrukerNano) in tapping mode. Samples were prepared by placing a drop of nanoparticle suspension on a freshly-cleaved mica sheet and the experiments were performed in fluid tapping mode to keep the integrity of the NPs. AFM images were generated with a scan rate of 1 Hz and 512 lines per image. Experiments were performed at constant room temperature. During the scans, proportional and integral gains were increased to the value just below the feedback started to oscillate. Images were processed only by flattening to remove background slopes.
Gadolinium nanogel loading was determined on nanoparticle suspensions by ICP-OES. The nanoparticle suspension was incubated overnight in a 1:3 (v/v) mixture of HCl (37%) and HNO₃ (69%). Volumetric dilutions were carried out to achieve an appropriate Gd concentration within the working range of the method. Samples were analysed using Thermo Scientific iCAP 6300 series Duo ICP spectrometer. Gd emission intensity was correlated to Gd concentration by means of a calibration curve which was previously established from Gd(NO₃)₃ ICP-OES standard. Solutions used for the calibration were obtained by dilution of increasing amounts of Gd(NO₃)₃ standard with unloaded nanogels previously incubated under acidic conditions, as described above.

Mid-log phase bacteria were resuspended in PBS to a concentration of 5 × 10⁶ colony forming units (CFU)/mL. Next, 20 µL bacterial suspension was mixed with 30 µL PBS containing increasing concentrations of SAAP-148, PVA-lyophilized and redispersed SAAP-148-loaded nanogels or placebo nanogels and 50 µL of filtered, inactivated and centrifuged human plasma (Sanquin, Leiden, The Netherlands) in polypropylene V-shaped 96-well microplates. After incubation for 4 h or 24 h at 37 °C under rotation at 200 rpm using an orbital shaker, 10-fold serial dilutes were made and plated onto MH agar plates to determine the number of viable bacteria. Results are expressed as lethal concentration (LC)_99.9, i.e., the lowest concentration of SAAP-148 killing 99.9% of the inoculum.

HSEs were exposed to SAAP-148, lyophilized and redispersed SAAP-148-loaded nanogels or placebo nanogels at the desired concentrations in PBS for 4 h including 1% (v/v) Triton^TM X-100 as positive control and PBS as negative control. Afterwards, LDH release from dead cells to the basal medium was detected by the Cytotoxicity Detection Kit according to manufacturer’s instructions. Furthermore, the HSEs were cut out, transferred to 24-well flat-bottom culture plates, and exposed to WST-1 reagent in DMEM medium overnight to determine the metabolic activity of the cells in the models. Read-out of medium solutions without the HSEs was performed according to manufacturer’s protocol. Results are expressed as percentage cytotoxicity or metabolic activity relative to controls.

HSEs were changed to antibiotic-free medium at least two days before infection with GFP-producing MRSA. Bacteria were added to the HSEs at a concentration of 1 × 10⁸ CFU/model, spun down on top of the HSEs for 2 min at 300× g, and incubated for 1 h at 37 °C and 5% CO₂ to infect the HSEs. After infection, the bacterial suspension was removed, and the HSEs were treated with TAMRA-SAAP-148 or PVA-lyophilized and redispersed TAMRA-SAAP-148-loaded nanogels in PBS for 4 h after which the HSEs were fixed in 1% (v/v) PFA for 1 h at 4 °C. HSEs were washed and stored in PBS at 4 °C until staining. Cell culture inserts were blocked with PBS containing 1% (w/v) BSA and 0.3% (v/v) Triton^TM X-100 (PBT) for 15 min at room temperature. Transparent membranes were removed from plastic inserts with a scalpel and the membranes were incubated with Phalloidin Alexa Fluor 405 diluted 1:50 (v/v) and DAPI diluted 1:100 (v/v) in PBT for 2 h at 4 °C. Membranes were washed three times in PBS and three times in demineralized water, then placed on a glass slide, and treated with ProLong Diamond Antifade mountant. All samples were imaged with a Leica SP-8 upright confocal microscope and 3D images were produced using a Leica SP8 WLL-2 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany).