General methods

Ultrasonication with Sonics VCX 130 (diameter of probe: 3 mm, Sonics & Materials, Inc., USA) was carried out during preparation of a miniemulsion (before photo-polymerization) and redispersion of nanogel powder at amplitudes of 60% (5 min) and 40% (30 s), respectively. Each purification process was followed by dialysis and lyophilization in a freeze dryer (ALPHA 1–2 LDplus, CHRIST). NMR spectra were recorded in deuterated solvents (Deutero GmbH) with internal standards using an NMR spectrometer operating at 600 MHz (Varian). Fluorescence study was conducted in SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices, USA). Both the amount of conjugated trehalose and trehalose release profile were determined enzymatically using a Trehalose Assay Kit (Megazyme International, Ireland). Phosphate buffered saline (PBS, pH 7.0 and 7.4), potassium chloride (KCl), and normal saline (NS) solutions. Deionized water (DI water) was produced using a reverse osmosis system (conductivity < 2 µS/cm).

Materials and reagents for synthesis of nanogels

Acrylamide (AM, Acros Organics), N,N’-methylenebisacrylamide (MBA, Acros Organics), 2-hydroxyethyl acrylate (HEA, Acros Organics), lithium phenyl-(2,4,6-trimethylbenzoyl) phosphinate (LAP, Carbosynth), sorbitane monooleate (Span 80, Sigma Aldrich), cyclohexane (Chempur), acetone (Chempur), sulfo-Cy5-amine (Lumiprobe), dialysis membrane (Spectrum™ Spectra/Por™ 2 RC Dialysis Membrane, MWCO: 12–14 kDa). 6-O-acryloyl-α,α’-trehalose (TreA) was synthesized following our previously described method [36]. 4-acrylamidobutanoic acid (4-AMBA) was synthesized according to Yaşayan et al. (2012) [46]. 4-acrylamidobutanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (4-AMBA-Sulfo-NHS) was synthesized based on the method reported by Tsuji et al. (2019) for the synthesis of homologous N-sulfosuccinyl-6-hexyloylacrylamide sodium salt [47] and the detailed synthesis procedure is provided in Supplementary Information.

Synthesis of trehalose-releasing nanogels (TNG) and HEA-containing nanogels (HEA1NG and HEA2NG)

TNG were synthesized via an inverse miniemulsion free-radical polymerization (FRP) according to our previously described method [33]. Briefly, a water-in-oil (w/o) miniemulsion (1:10, v/v) was created from monomers and photoinitiator-containing aqueous phase (PBS pH 6.0, 1.0 mL) and Span 80-containing organic phase (cyclohexane, 10.0 mL). MBA (20.0 mg), TreA (152.7 mg), AM (35.3 mg), and 4-AMBA (13.4 mg) were placed in a 4-mL dark vial and dissolved in PBS (pH 6.0). The solution of LAP initiator (2.3 mg, 51 µL) was added, and the aqueous phase was then transferred into a 20-mL transparent vial containing the cold organic phase (4 ºC). The miniemulsion was prepared by ultrasonication of the mixture in ice bath (60% amplitude, 5 min). After that, the vial was covered in aluminum foil and exposed for 0.5 h to high power light-emitting diodes (LEDs, 3 W, 395–405 nm) photoirradiation from the bottom of the vial. Following a precipitation step in 40 mL of acetone, the product was centrifuged at 14,610 ×g for 10 min, twice rinsed with 40 mL of acetone and left to air dry overnight. In order to purify the product, the crude nanogels were suspended in DI water and dialyzed against H3PO4 solution (pH 5.0, MWCO 12–14 kDa) for 24 h with multiple media changes and DI water as the last change. The nanogel dispersion was finally freeze-dried, producing a white fluffy powder that was kept at 4 ºC until usage.

To investigate the role of trehalose in nanogel stability, HEA-based nanogels were synthesized using a similar procedure as described earlier. However, in this case, the TreA monomer was replaced with the HEA monomer in both equimolar and equimass of TreA feed (Table S1) for obtaining HEA1NG and HEA2NG, respectively.

Synthesis of trehalose-releasing nanogels bearing active ester (NHS-TNG)

To synthesize NHS-TNG, a similar procedure as described for the synthesis of TNG was applied with the difference that 4-AMBA-sulfo-NHS (4.0 mg, 0.011 mmol) was added and the aqueous phase was prepared in PBS neutralized with NaOH to pH 6.0. The crude NHS-TNG were kept at -20 ºC without further purification for preparation of fluorescently labeled nanogels.

Synthesis of Cy5-labeled trehalose-releasing nanogels (Cy5-TNG)

NHS-TNG (10.0 mg) was redispersed in DMSO (250 µL) containing sulfo-Cy5-amine (0.25 mg, 0.00033 mmol) and triethylamine (0.068 mg, 0.00067 mmol). Then the nanogel dispersion was shaken overnight in an orbital shaker (1000 rpm, 25 ºC). On the next day, the volume was adjusted to 1.0 mL with DMSO, and then the product was precipitated with 3.6 mL of acetone. The suspension was then centrifuged at 14,610 ×g (4 ºC, 2 min) and washed six times with acetone. Nanogel precipitate was then redispersed in 800 µL of DI water, ultrasonicated at 40% amplitude (30 s), and then dialyzed against H3PO4 solution (pH 5.0, MWCO 12–14 kDa) for 24 h with multiple media changes and DI water as the last change. The pure Cy5-TNG dispersion was finally freeze-dried to obtain fine powder and kept at 4 ºC prior to use.

Dynamic light scattering (DLS) and electrophoretic light scattering (ELS)

Z-average mean hydrodynamic diameter (dH) and polydispersity index (PdI) were measured by Dynamic Light Scattering (Malvern, Zetasizer Nano 90 S) (4 mV He-Ne ion laser, λ = 633 nm, scattering angle: 90°). The sample was prepared from a TNG stock solution in water (10 mg/mL, prepared with sonication at 40% amplitude for 30 s) by dilution with 1 mM KCl (1.0 mg/mL). In addition, the ζ potential of TNG was measured by Electrophoretic Light Scattering (Malvern, Zetasizer Nano ZC).

Cryogenic transmission electron microscopy (cryo-TEM)

Cryo-TEM analysis was carried out using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, Oregon, USA). Images were recorded with a Gatan Rio 16 CMOS 4k camera (Gatan Inc., Pleasanton, California, USA) and processed with Gatan Microscopy Suite (GMS) software (Gatan Inc., Pleasanton, California, USA). Specimens were prepared from TNG dispersion in DI water (500 µg/mL) via the vitrification of aqueous solutions on oxygen plasma-activated grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, Germany).

Nuclear magnetic spectroscopy (NMR)

1H NMR spectra were recorded in deuterated solvents by using Varian NMR instrument operating at 600 MHz. Chemical shifts are reported in ppm (δ) relative to tetramethylsilane (DMSOd6) or 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (D2O) as an internal reference.

Determination of conjugated trehalose (CTre)

The content of trehalose in nanogels was determined after the alkaline hydrolysis of ester bonds in trehalose acrylate units. Briefly, 40 µL of 1 M NaOH was added to 400 µL of nanogel dispersion (100 µg/mL) in PBS (pH 7.4) and the mixture was incubated at 70 ºC for 1 h. After neutralization with 40 µL of 1 M HCl, the sample was subjected to enzymatic determination of trehalose by using Trehalose Assay Kit in a microplate assay procedure. CTre (% w/w) was calculated as the percentage of weight of trehalose in nanogel vs. weight of nanogel.

Trehalose release study by enzymatic determination

An initial stock dispersion of nanogels (10 mg/mL) was prepared by redispersing nanogel powder in DI water followed by ultrasonication (40% amplitude, 30 s). The stock was diluted to a final concentration of 100 µg/mL (30 mL) in PBS (pH 7.4) containing 1% v/v antibiotic antimycotic solution in a 50-mL glass vial. Afterward, the nanogel dispersion was placed in an incubator at 37 °C with constant shaking (332 ×g). Aliquots (800 µL) were taken every 24 h over 30 days and frozen at -20 ºC. After all samples were collected, they were thawed and the amount of trehalose was determined enzymatically.

Colloidal stability of nanogels in various media

Colloidal stability of nanogels (1.0 mg/mL, containing 1% v/v antibiotic antimycotic solution) was determined for 7 days at 37 °C in various biological media, including DI water, PBS (pH 7.4), NS, DMEM, and DMEM + 10% FBS, by measuring the optical density (OD) at 650 nm on a microplate reader. The OD value was converted to % transmittance using the following equation:

Hemolytic rate of trehalose-releasing nanogels

Fresh blood was collected from the auricular vein of healthy New Zealand white rabbits. The blood was then diluted with saline at a ratio of 4:5. In this experiment, three types of samples were prepared: a negative control (normal saline), a positive control (ultrapure water), and test samples (TNG at different concentrations). Each sample (1 mL) was immersed in a water bath at 37 °C for 30 min. After that, 20 µL of the diluted rabbit blood was added to each tube.

The tubes were further incubated in a water bath at 37 °C for 1 h. Following the incubation, the samples were centrifuged at 500 ×g for 5 min. The supernatant was carefully removed, and photographs were taken. The collected supernatant was then used to measure OD at a wavelength of 545 nm. The OD value was converted to % hemolytic rate using the following equation:

$$\% } = \frac_-_}_-_}\times 100\text$$

Where ODt is the OD test value obtained in the presence of trehalose or TNG, ODnc is the negative control and the ODpc is the positive control.

In vitro cytotoxicity of trehalose-releasing nanogels

The cell harmlessness of TNG is a prerequisite for biological research. The MTS Cell Proliferation and Cytotoxicity Detection Kit was used to measure cytotoxicity of TNG according to the manufacturer’s instructions. Briefly, HUVECs were inoculated at a density of 1 × 104 per well in 96-well plates. After 24 h, various concentrations of TNG (25, 100, 150, 200 µg/mL) were added, and the cells were co-cultured for 24 h. Then, 10 µL of MTS was added to each well and incubated for additional 2–4 h, and finally OD value at 450 nm was measured. The OD value was converted to % cell viability using the following equation:

$$\% } =\frac_-_}_-_}\times 100\text$$

Where ODt is the OD test value obtained in the presence of TNG, ODc is the OD test value obtained in the absence of TNG, and the ODb is the OD test value of blank plate.

In vitro cellular uptake of trehalose-releasing nanogels

RAW264.7 cells were cultured on 20-mm round coverslip until cell adherence to ~ 50%. Then the medium was removed and replaced with fresh serum-free culture medium containing free trehalose and TNG labeled with Cy5 which have the equal trehalose concentration. After 1 or 3 h of co-incubation, the medium was removed, washed with PBS for 3 times, then fixed with 4% paraformaldehyde for 15 min, washed with PBS for 3 times again, the nuclei were finally labeled with DAPI and observed under confocal microscope.

In vitro autophagy stimulation

Raw 264.7 cells were cultured on 20-mm round coverslip at a density of 1 × 105 and in 6-well plates until cell adherence to ~ 50%. Then the medium was removed and replaced with fresh serum-free culture medium containing 1 µg/mL of lipopolysaccharide (LPS) and 40 µg/mL of oxidized low-density lipoprotein (ox-LDL) for 12 h to induce the formation of foam cells.

Trehalose (100 µM) or TNG (at a concentration equivalent to 100 µM of free trehalose) was added to the above foam cells, incubated for 12 h, then washed 3 times with PBS at 4 °C. Cells in 96-well plates were lysed with RIPA lysis buffer and protease inhibitor (phenylmethylsulfonyl fluoride, PMSF) were added and placed at 4 °C. After manual scrapping, the lysates were collected and centrifuged at 15,000 ×g for 20 min. Then, the loading buffer was added to the supernatants at a 1:4 ratio, mixed, and then boiled for 5 min before being stored at -80 °C. The sequestosome 1 (p62) and microtubule-associated protein 1 A/1B-light chain 3 (LC3) protein expressions were quantified by Western blot.

Additionally, cells on the round coverslip were also washed three times with PBS at 4 °C, fixed with 4% paraformaldehyde for clarity, and then incubated overnight at 4 °C with LC3 rabbit polyclonal antibody or p62/SQSTM1 rabbit polyclonal antibody. After rinsing the round coverslips five times with PBST (0.1% Tween-20 PBS), they were incubated for 1 h with fluorescently-labeled rabbit secondary antibodies under darkness. Subsequently, the coverslips were washed five times before the nuclei were finally labeled with DAPI. Fluorescence signals from DAPI, p62, and LC3 were detected by SP8 confocal microscopy.

Intracellular ROS clearance in macrophages

RAW264.7 cells were cultured in 12-well plates for 12 h. After cells were pretreated with trehalose (100 µM) or TNG (at a concentration equivalent to 100 µM of free trehalose) for 4 h, they were stimulated with 0.5 µM H2O2 for 0.5 h. The negative control group was treated with fresh medium, and the positive control group was only stimulated with 0.5 µM H2O2 for 0.5 h. Subsequently, cells were rinsed and treated with DCFH-DA (10 µM) in serum-free DMEM for 30 min. After washing with PBS and the intracellular ROS clearance was observed in cell culture dishes by fluorescence microscope. Through similar procedures, the cells were harvested in PBS, intracellular fluorescent signals were measured via flow cytometry (CytoFLEX, Beckman Coulter) and analyzed using FlowJo software.

In vitro lipid efflux

Raw 264.7 cells were inoculated in 6-well plates at a cell density of 2 × 105 per well. Cells were then stimulated with 1 µg/mL of LPS and 40 µg/mL of ox-LDL to form foam cells. The cells were treated with free trehalose (100 µM) or TNG (at a concentration equivalent to 100 µM of free trehalose) for 24 h. Following this, the medium was removed, and the cells were washed twice with PBS before being fixed using a 4.0% paraformaldehyde (PFA) solution. Subsequently, the cells were incubated with Oil Red O (ORO) isopropanol working solution for 15 min. Then cells were observed by optical microscopy. In addition, the intracellular ORO was extracted by isopropanol and the ORO concentration was determined by measuring its absorbance at 524 nm via an UV-Vis spectroscopy.

Animal models

Male apolipoprotein E-deficient (ApoE−/−) mice, aged eight weeks, were obtained from the Hunan SJA Bioscience Co., Ltd. (Hunan, China). All animal care and experimental protocols comply with the relevant laws, regulations and standards concerning animal welfare ethics. This project has been supervised and approved by Laboratory Animal Welfare and Ethics Committee of Chongqing University (IACUC issue number: COU-IACUC-RE-202109-002).

Atherosclerosis treatment with nanogels

ApoE−/− mice were randomized into 3 groups (5 mice/group), and high fat diet (HFD) was given for 12 weeks. Then, the mice were subjected to different treatments for one month. The mice were injected with 0.9% saline as the untreated control group, while the other two groups were treated with either free trehalose at a dose of 2.5 g/kg of trehalose, or TNG at a concentration of 16 mg/kg every three days via tail vein injection. The body weight of mice was monitored during the treatment.

In vivo pharmacokinetics evaluation of nanogels

To evaluate the in vivo pharmacokinetics of TNG, the Cy5-labeled TNG (Cy5-TNG) was intravenously administered to C57BL/6 mice at dose of 16 mg/kg, while free Cy5 was intravenously injected at the equal concentration of Cy5 in Cy5-TNG. Then, 20 µL of blood was collected at 0.5, 1, 2, 4, 8, 12, and 24 h after injection. The blood samples were diluted with 40 µL PBS contained EDTA2K in 96-well black plates, and the fluorescence intensity was measured by fluorescence microplate reader (Hitachi, Japan).

In vivo accumulation of nanogels in atherosclerotic lesion and biodistribution

After atherosclerosis modeling, mice were injected with 150 µL Cy5 (control group) or Cy5-TNG via tail vein. After 24 h, the mice were euthanized, and the aortas were isolated after perfusion with 0.9% saline containing heparin sodium. Additionally, heart, liver, spleen, lung, and kidney were harvested to analyze the biodistribution of nanogels in the main organs. Imaging and fluorescence quantification were performed using the Xenogen IVIS 200 system.

Efficacy study and histological study of atherosclerotic plaques after treatment

Quantitative analysis of atherosclerotic plaques after treatments: After the 10th round of trehalose and TNG treatments via intravenous injections (IV), the aortas from ApoE−/− mice were harvested, spanning from the heart to the iliac bifurcation. Aortas were fixed by perfusion with 4% paraformaldehyde, dissected longitudinally, and then stained with Oil Red O (ORO) to quantify the plaque area. The extent of atherosclerotic plaque at the aortic root was also determined by the same way. Quantitative analysis of atherosclerotic plaque areas was performed using Photoshop 2020 software.

The aortic roots were fixed with 4% paraformaldehyde in PBS for 1 h. Frozen sections were prepared from the fixed samples, and ORO staining was performed to quantify the plaque area. For immunofluorescence analysis, the sections were washed with PBS and then permeabilized/blocked using a solution containing 0.5% Triton-X100 in 5% BSA. Antibodies specific to p62, LC3 and CD68 were separately incubated with the sections overnight at 4 °C. After washing the sections five times with PBST (PBS with 0.1% Tween-20), they were incubated with secondary antibodies for 1 h. Nuclei were stained with DAPI in the dark. The fluorescence signals from DAPI, p62, LC3 and CD68 were detected using SP8 confocal microscopy (Leica, Germany). Sections of the main organs including heart, liver, spleen, lung, and kidney were analyzed by hematoxylin-eosin (HE) staining.

After one month of treatments, a complete blood routine analysis and serum biochemistry analysis were conducted. Blood samples were collected and analyzed using an automated hematology analyzer (Sysmex KX-21, Sysmex Co., Japan) to obtain the complete blood count (CBC) data. The concentrations of various biochemical markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CR), uric acid (UA), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TG), and total cholesterol (TC) in the serum, were quantified using an automated analyzer platform (Roche Cobas C501, Roche Co., Switzerland).

Statistical analysis

The collected data were presented as mean ± SD (n ≥ 3). The statistical analysis was performed using GraphPad Prism Version 8.4.3 software (GraphPad, USA). Tukey’s test and one-way analysis of variance (ANOVA) were employed to identify group differences. To determine whether there is a significant difference between two specific groups, an unpaired t-test (two tails) was used. The significance thresholds for differences were set at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns, no significance.