HF@SLN alters macrophage polarization markers and increases TFEB expression

Following successful nanoparticle fabrication, we next investigated the biological interactions of HF@SLN with macrophages in vitro. Fluorescein isothiocyanate (FITC)-labeled HF@SLN nanoparticles exhibited time-dependent association with M2 BMDMs, with substantially stronger cellular fluorescence observed after 6 h compared to 2 h incubation (Fig. 2a), indicating progressive nanoparticle internalization.

Fig. 2: The interaction between HF@SLN and M2 BMDMs in vitro. (A) Confocal images of internalized HF@SLN by the M2 BMDMs, cell nucleus was stained by 4′,6-diamidino-2-phenylindole (DAPI, blue), and HF@SLN was connected with FITC (green); scale bar 20 μm. (B) Confocal images of lysosome in M2 BMDMs treated with HW, HF@SLN or without any treatments (denoted as NC), cell nucleus was stained by DAPI (blue), and lysosome was stained with Lysotracker (red); scale bar 20 μm. (C) Quantitative analysis of lysosome. Quantitative analysis of (D) CD80+ and (E) CD206+ macrophages. (F) Confocal images of TFEB expression on the M2 BMDMs treated with HW, HF@SLN or without any treatments (denoted as NC), cell nucleus was stained by DAPI (blue) and TFEB was stained with immunofluorescence staining (red); scale bar 20 μm. (G) Western blotting images of TFEB expression on the M2 BMDMs treated with HF@SLN or without any treatment (denoted as NC). (H) The quantitative results of Western blotting images. Data are presented as means ± SD. The error bars are based on standard errors of the individual samples (n = 3), *P < 0.05 and ***P < 0.001, ANOVA with Tukey's post-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Given that PIKfyve inhibition disrupts lysosomal maturation and induces cytoplasmic vacuolization, we next evaluated lysosomal alterations using LysoTracker staining. Untreated M2 BMDMs displayed relatively weak lysosomal fluorescence, whereas both free HW and HF@SLN treatments induced markedly enhanced lysosomal accumulation after 6 h incubation (Fig. 2b). Quantitative flow cytometric analysis further confirmed a more elevated lysosomal signals in both treatment groups, with HF@SLN and free HW inducing 1.67 ± 0.52 fold and 1.74 ± 0.49 fold increases, respectively, relative to untreated controls (Fig. 2c). Notably, no statistically significant difference was observed between free HW and HF@SLN, indicating that nanoparticle assembly preserved the intrinsic PIKfyve inhibitory activity of HW.

Accumulating evidence suggests that lysosomal stress and autophagy disruption regulate multiple aspects of macrophage function, including changes in polarization-associated phenotypes [24,25]. Given the established role of PIKfyve inhibition in regulating macrophage function, we next evaluated the effects of HF@SLN treatment on macrophage polarization markers in M2-polarized BMDMs. Flow cytometric analysis revealed marked changes in macrophage polarization marker expression following treatment with either free HW or HF@SLN (Fig. 2d and e). Specifically, the proportion of CD80-positive macrophages increased from 27.9 ± 3.1% in untreated controls to 53.2 ± 5.3% and 49.6 ± 0.7% after HW and HF@SLN treatment, respectively, whereas the proportion of CD206-positive macrophages was concomitantly reduced. These findings indicate that HF@SLN treatment is associated with changes in macrophage polarization marker expression consistent with a pro-inflammatory phenotype.

Previous studies have linked PIKfyve inhibition to activation of TFEB, a master regulator of lysosomal biogenesis and macrophage functional reprogramming [[26], [27], [28]]. Consistent with these reports, immunofluorescence staining revealed markedly elevated TFEB expression following HW and HF@SLN treatment (Fig. 2f). Western blot analysis further demonstrated that HF@SLN increased TFEB expression by approximately 1.8 ± 0.5-fold compared to untreated controls (Fig. 2g and h). The increased TFEB expression was accompanied by macrophage repolarization toward a pro-inflammatory phenotype, suggesting a potential involvement of TFEB in the immunomodulatory effects of HF@SLN.