Near-infrared light and pH-responsive hyaluronic acid-enveloped ZIF-8 nanoparticles for the treatment of pneumonia caused by methicillin-resistant Staphylococcus aureus

2.1 Materials

Hyaluronic acid (～10 kDa), purchased from QuFu GuangLong Biochem Co., Ltd. 2-Methylimidazole, zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O), Indocyanine green (ICG) were purchased from Aladdin. Fluorescein isothiocyanate (FITC), 4′,6-Diamidino-2-phenylindole(DAPI), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), Degradation of 1,3-diphenyl-isobenzofuran (DPBF) were purchased from Sigma Co. (St. Louis, MO, USA). Analytical-grade chemicals and solvents were used without further purification.

Prof. Wang (College of Life Sciences, Northwest A&F University) generously donated Staphylococcus aureus (MRSA) and Macrophages (RAW264.7). We obtained Kunming mice from Pengyue Experimental Animal Breeding (Jinan, China).

2.2 Synthesis of IZH

Synthesis of IZH was performed using the one-pot encapsulation method with a few modifications based on previous literature descriptions (Xie et al., 2019). Specifically, 2 mL of 2-methylimidazole (2-MIM, 2.78 M) aqueous solution was added to 200 μL of ICG (10 mg/mL in DMSO) under gentle stirring at 400 rpm for 15 min at 25 °C. Then, 15 min were needed to stir the reaction mixture after 160 μL of zinc nitrate aqueous solution (0.48 M) was dropped in dropwise. Centrifugation (12,000 rpm, 25 min at 4 °C) was used to collect ICG@ZIF-8, which was then washed three times with H₂O and re-dispersed in 2 mL of H₂O. After aging for 30 min, 2 mL HA aqueous solution (2 mg /mL) was added and the mixture was aged in the dark for 30 min. Finally, the product was collected by centrifugation. According to the equation below, DLC can be defined as follows: $$\mathrm{D}\mathrm{L}\mathrm{C}\left(\%\right)=\frac{\mathrm{T}\mathrm{h}\mathrm{e}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{e}\mathrm{d}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{c}\mathrm{a}\mathrm{r}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{r}\mathrm{s}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{e}\mathrm{d}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}\times 100$$

To determine the loaded efficiency of ICG, an acidic aqueous solution was used to dissolve the IZH powder and measured at a wavelength of 780 nm according to UV–vis characteristic absorption peak.

2.3 Characterization of IZH

Scanning electron microscopy (SEM-450field emission, FEI, Hillsboro) was used to investigate the morphology of nanoparticles.

The UV–vis spectra measurement was conducted on a UV–vis spectrophotometer (Thermo Evolution 300 spectrophotometer).

The FTIR spectra were detected using an FTIR spectrometer (BRUKER TEMSOR 27, Germany).

The particle size and zeta potential were measured by a Zetasizer Nano ZS (Malvern, U.K.). In addition, the zeta potential and particle size of IZH NPs stored at 4 °C were assessed over a predetermined period (0, 0.5, 1, 2, 3, 4, 5 and 6 days) to assess their stability.

2.4 In vitro release of ICG from IZH

IZH was incubated at 37 °C with buffer solutions of pH 5.5 and 7.4 to determine in vitro ICG release. Under shaking, nanoparticles were placed in 5 mL of buffer solution to investigate ICG release from IZH. At regular time intervals, the diffusion medium was taken and supplemented with fresh buffer solution in the same amount. ICG drug release from samples was measured using a UV–vis spectrometer. There were three tests on all samples.

2.5 ROS generation detection

A reactive oxygen quencher (Wang et al., 2019), 1,3-diphenyl-isobenzofuran (DPBF), was studied with IZH. Free ICG and IZH containing the same amount of ICG were added with DPBF (20 μg/mL). Then, NIR lasers of 780 nm were used (2 W/cm²) to illuminate the mixture. At regular time intervals, a UV–vis spectrophotometer was used to measure the absorption spectra of DPBF.

2.6 Biocompatibility analysis

During this experiment, nanoparticles were examined for their toxicity to cells. Firstly, RAW 264.7 cells were cultivated at 37 °C in 96-well plates with 5 % CO₂. Then, the final IZH composite was diluted and added to the tested wells at various concentrations. A standard 3-(4, 5-dimethylthiazol-2-yl)-2, 5 di-phenyltetrazolium bromide (MTT) assay was used to assess cell viability.

As previously reported (Lu et al., 2020), the hemolysis assay was performed with IZH to check its compatibility with blood. In brief, red blood cells (RBC) solution (2 % w/w) of 0.1 mL was added to 1.9 mL of the solution of IZH (200 μg/mL). Thereafter, the specimens were continuously incubated at 37 °C for 1 h. In the following step, centrifugation at 6500 rpm for 10 min was carried out on the obtained mixtures. Finally, an automated microplate reader was used to determine the absorbance of the centrifuged supernatant at 545 nm. Among the control groups, PBS served as a negative control group and pure water served as a positive control group. $$Hemolysisratio\left(\%\right)=\left(O{D}_{\mathit{sample}}-O{D}_{\mathit{negative}}\right)/\left(O{D}_{\mathit{positive}}-O{D}_{\mathit{negative}}\right)\times 100$$

2.7 Cellular uptake of IZH

The uptake efficiency of IZH by Immune cells was evaluated by using RAW 246.7 cells. The cells were seeded in 6-well plates cell culture dishes overnight. Then, excess free HA (3 mg/mL) was added to a group of cells and incubated for 2 h. The medium was then replaced with a fresh medium containing FITC-loaded IZH (50 µg/mL) and continued cultivating for 3 h. Finally, DAPI was used to stain the cells after several rinses with PBS. Cells were observed using a fluorescence microscope (Olympus BX53).

2.8 Intracellular bacteria experiment

For the construction of infected cells, MRSA were co-incubated with RAW 264.7 cells for 1 h in a cell culture medium. Gentamicin was then added at 50 μg/mL to inhibit extracellular bacterial growth. After adding the test material and control material to the culture medium, the medium was incubated at 37 °C for further 12 h. RAW 264.7 cells were serially diluted with PBS supplemented with TritonX-100. In order to calculate colony-forming units (CFU), live bacterial numbers were plated and their numbers were determined.

2.9 In vivo activity evaluation

As a model for bacterial pneumonia in mice, the following procedures were used: briefly, after isoflurane anesthesia (1.5–2.5 %) was administered, mice were then infected with MRSA (10 μL, 10⁹ CFU/mL) through weasand. Infected mice were divided into five groups: (5 mice per group) randomly: (a) PBS; (b) ZIF-8@HA; (c) IZH; (d) ICG with laser; (e) IZH with laser. For therapeutic evaluation, PBS, ZIF-8/HA, ICG and IZH (5 mg/kg) were intravenously injected via the tail vein into mice for five days. Following administration, NIR laser (780 nm, 2 W/cm²) illumination of the mice’s chest was performed for 6 min, with 2 min between each spot of laser exposure. At the end of the experiment, further analysis was performed on the lungs collected separately. Counts of bacteria were calculated for each organ as before. Additionally, lung tissue structure showed changes by hematoxylin and eosin (H&E) staining.

2.10 Statistical analysis

Experimental data were presented as mean ± standard deviation (SD) and plotted and statistical analysis was determined using the software GraphPad Prism 7.01.

3.1 Synthesis and characterization

As shown in Fig. 1, through a simple one-pot biomineralizing method, ICG@ZIF-8 nanoparticles (NPs) were synthesized successfully. In order to improve targeting capability, HA shell was deposited on the surface of ICG@ZIF-8 via coordination bonds. By scanning electron microscope (SEM), ICG@ZIF-8 and IZH were measured in terms of size and morphology. As illustrated in Fig. 2A, the ICG@ZIF-8 NPs have a uniform diameter of about 250 nm and are monodispersed. Compared with ICG@ZIF-8 NPs, IZH NPs display rougher three-dimensional structures and obvious shell structures, which demonstrate that HA was successfully coated and IZH NPs were successfully synthesized. As indicated in Fig. 2B, DLS has determined that the average diameter of IZH NPs is approximately 375 nm. Generally, ICG@ZIF-8 is known to have a positive surface charge because of the exposed metal components (Zn²⁺) on the external surface. A significant change in zeta potential was observed after HA-modification from + 16.38 mV to −11.28 mV in inverse proportion (Fig. 2C), because HA contains many carboxyl groups which are negatively charged.

Close

Fig. 1

A schematic illustration showing ICG@ZIF-8/HA nanoplatform targeted at the infected site.

Export to PPT

Close

Fig. 2

(A) SEM images of ICG@ZIF-8 (a) and IZH (b), (B) Distribution curve of IZH particle sizes; (C) Zeta potential of ICG@ZIF-8 and IZH (D) UV–vis spectra;(E) FT-IR spectra. (F) An analysis of changes in particle size and zeta potential of IZH after 6 days of storage.

Export to PPT

As shown in Fig. 2D, compared to those of pure ZIF-8@HA, both ICG and IZH demonstrated characteristic peaks at 780 nm, indicating that ICG has been encapsulated into nanoparticles successfully. In addition, the quantitative analysis of IZH was performed, the drug loading capacity (DLC) of IZH was about 23.6 % by analysing the standard curve.

As shown in Fig. 2E, FT-IR spectra was also carried out to study the chemical structure of the products. A characteristic peak of the HA amides was also observed at 1625 cm⁻¹, 1576 cm⁻¹, and 1351 cm⁻¹, respectively, originating from the amide I and amide II and amide Ⅲ bands at the spectrum of IZH (Alkrad et al., 2003). Moreover, Zn-N stretching was demonstrated in the spectrum of ICG@ZIF-8 by a peak at 421 cm⁻¹ (Soltani et al., 2018). It is confirmed that IZH nanoparticles were synthesized successfully from these results.

The DLS method was used to monitor the change in zeta potential and size distribution of IZH over 6 days in order to investigate its storage stability. The zeta potential and average particle size of IZH did not change considerably as time progressed, as shown in Fig. 2F. It was confirmed that IZH has good stability, which is critical for the use of the drug as an antibacterial agent in clinical practice since it prevents abnormal release and inactivation.

3.2 pH-responsive drug release

As shown in Fig. 3, we conducted a controlled drug release test to determine the ICG release behavior from IZH. Incubated for 48 h in a neutral solution (pH 7.4), IZH had a lower release rate of drugs with an approximately 20 % release rate, indicating that IZH was stable in the normal physiological environment. The presence of this property can reduce the likelihood of unnecessary leakage of drugs during blood circulation. However, after incubation for 24 h at pH 5.5, 77.3 % of ICG was released from IZH, resulting from acidic environments that could break the coordination bonds between the metallic and organic active sites of ZIF-8(Zhang et al., 2022). It has been demonstrated that IZH particles are an effective pH-sensitive delivery system for releasing drugs into inflammatory sites, which has great therapeutic potential for intractable bacterial infections.

Close

Fig. 3

In vitro drug release behaviors of IZH under different pH conditions.

Export to PPT

3.3 Measurement of reactive oxygen generation

In order to determine whether IZH was capable of generating reactive oxygen precisely, 1, 3-diphenylisobenzofuran (DPBF) was used as a singlet oxygen probe under NIR irradiation at 780 nm. An analytical reagent typically employed for ROS analysis, DPBF can react with ¹O₂ quantitatively to produce an oxidation product (o-dibenzoylbenzene) that reduces DPBF's UV–vis absorption (Fig. 4A). As shown in Fig. 4B, in DMSO solutions containing DPBF, little evidence of DPBF reducing absorption was observed under laser irradiation. In comparison, DPBF's characteristic absorbance at 421 nm was clearly decreased in the presence of ICG (Fig. 4C) and IZH with an equivalent amount of ICG (Fig. 4D) after irradiation. These results suggested that IZH can be used for PDT treatment modality.

Close

Fig. 4

(A) Reaction mechanism of DPBF oxidation by ¹O₂. A spectrophotometric measurement determines ROS production. DPBF absorbance (B), and DPBF absorbance with ICG (C) or IZH (D), under irradiation with different time, respectively.

Export to PPT

3.4 Intracellular uptake of IZH

As part of our investigation, we replaced the ICG in the nanostructure with FITC to determine whether HA is involved as a mediator of cell phagocytosis. FITC payloads in nanoparticles can be used as fluorescent markers to quantify FITC@ZIF-8/HA levels within intracellular compartments. As illustrated in Fig. 5, there was a great deal of brightness in the image of cells exposed to FITC@ZIF-8/HA without free HA, compared to cells exposed to FITC@ZIF-8/HA with free HA blocking CD44 receptors. The decreased fluorescent intensity was attributed to the interaction of free-HA with CD44 receptors, causing interference with the IZH's endocytosis mediated by CD44 receptors. According to the studies above, receptor-mediated endocytosis plays a critical role in macrophage internalization of IZH.

Close

Fig. 5

Incubation of RAW 246.7 cells with FITC@ZIF-8/HA (A) and FITC@ZIF-8/HA pretreated with free-HA polymer (B) shown by fluorescence microscopy. Each image had a scale bar of 50 µm.

Export to PPT

3.5 Biocompatibility of IZH in vitro

MTT cell viability tests were used to determine the cytotoxicity of IZH in the RAW 264.7 cells. As exhibited in Fig. 6A, the survivorship of RAW 264.7 cells still remained above 80 % even at a high concentration of 60 μg/mL, which indicated that the IZH had less toxicity for normal cells. In addition, a hemolysis assay was conducted in this study to determine IZH's blood compatibility. As shown in Fig. 6B, blood compatibility was excellent with 200 μg/mL concentration in IZH group at no obvious hemolysis of RBCs was detected. It was determined that IZH was safe enough to be applied to the organism which inspired us to investigate its use as a PDT therapy for treating infections in vivo.

Close

Fig. 6

(A) RAW 246.7 cell cytotoxicity in vitro at different concentrations of IZH; (B) IZH hemolysis on red blood cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Export to PPT

3.6 Intracellular bacteria experiment

MRSA is one of the primary pathogens that is responsible for severe tissue infections, and the treatment of MRSA has become more challenging due to its ability to persist within phagocytic cells. We have already shown that IZH can eradicate intracellular bacteria in vitro in this study. MRSA-infected RAW 264.7 cells were treated with NIR radiation after being incubated with IZH. As displayed obviously in Fig. 7, Compared with the control group, ZIF-8@HA and IZH showed negligible inhibitory activity against intracellular bacteria. Additionally, the light treatment of the IZH group significantly inhibited the intracellular bacterial growth compared with the light treatment of the ICG group. It was evident that the IZH particles functionalized with HA significantly enhanced cellular uptake, allowing the antibacterial activity to be more pronounced against intracellular bacteria.

Close

Fig. 7

RAW264.7 cells infected with MRSA were treated with PBS, ZIF-8@HA, IZH, ICG + NIR irradiation, IZH + NIR irradiation, respectively. The mean (SD) values of triplicate experiments are given. *p < 0.05, **p < 0.005, ***p < 0.001.

Export to PPT

3.7 Therapeutic efficacy of IZH in vivo

Inspired by the outstanding antibacterial activity in vitro, further work was undertaken to investigate IZH's antibacterial properties in vivo. The bacterial pneumonia mouse models were divided into five groups and each group received different treatment according to the experimental design and the results were presented in the Fig. 8. Acute pneumonia mice treated with IZH + NIR, compared with those treated with control, ZIF-8@HA, IZH, or ICG + NIR, showed more effective suppression of MRSA (Fig. 8B). The better therapeutic effects might be attributed to the higher level of ICG in the lung tissues IZH + NIR treated mice than that in mice treated with ICG, which further verified the specific lung-targeting property of IZH. According to histopathological analyses, the alveoli tissue of control-treated, ZIF-8@HA-treated, and IZH-treated mice were seriously injured, with abundant inflammatory cells infiltrating compared to ICG + NIR-treated and IZH + NIR-treated group (Fig. 8C). As is notable, compared with the rest of the experiments, the lungs of the IZH under NIR irradiation-treated mice had significantly more complete alveoli. Taken together, the strategy may be useful for the development of new therapies for the treatment of pneumonia caused by MRSA.

Close

Fig. 8

(A) A schematic illustration of the construction and treatment of a pneumonia model; (B) In five animal groups, the mean number of CFU was recorded in lung tissue: PBS, ZIF-8@HA, IZH, ICG + NIR irradiation, IZH + NIR irradiation, respectively; (C) An analysis of the impact of five groups on lung tissues: PBS, ZIF-8@HA, IZH, ICG + NIR irradiation, IZH + NIR irradiation. The mean (SD) values of triplicate experiments are given. *p < 0.05, **p < 0.005, ***p < 0.001.

Export to PPT