Molecular Mechanisms of Intracellular Delivery of Nanoparticles Monitored by an Enzyme-Induced Proximity Labeling

doi:10.1007/s40820-023-01313-0

Abstract

Abstract:

Achieving increasingly finely targeted drug delivery to organs, tissues, cells, and even to intracellular biomacromolecules is one of the core goals of nanomedicines. As the delivery destination is refined to cellular and subcellular targets, it is essential to explore the delivery of nanomedicines at the molecular level. However, due to the lack of technical methods, the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date. Here, we develop an enzyme-induced proximity labeling technology in nanoparticles (nano-EPL) for the real-time monitoring of proteins that interact with intracellular nanomedicines. Poly(lactic-co-glycolic acid) nanoparticles coupled with horseradish peroxidase (HRP) were fabricated as a model (HRP(+)-PNPs) to evaluate the molecular mechanism of nano delivery in macrophages. By adding the labeling probe biotin-phenol and the catalytic substrate H₂O₂ at different time points in cellular delivery, nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles. Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP (+)-PNPs in macrophages over time. Based on dynamic clustering analysis of these proteins, we further discovered that different organelles, including endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus, are involved in delivery with distinct participation timelines. More importantly, the engagement of these organelles differentially affects the drug delivery efficiency, reflecting the spatial-temporal heterogeneity of nano delivery in cells. In summary, these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.

Key words: Enzyme-induced proximity labeling, Intracellular delivery, Nano-protein interaction, Dynamic molecule profiling, Macrophages

Junji Ren, Zibin Zhang, Shuo Geng, Yuxi Cheng, Huize Han, Zhipu Fan, Wenbing Dai, Hua Zhang, Xueqing Wang, Qiang Zhang, Bing He. Molecular Mechanisms of Intracellular Delivery of Nanoparticles Monitored by an Enzyme-Induced Proximity Labeling[J]. Nano-Micro Letters, 2024, 16(1): 103-.

Junji Ren, Zibin Zhang, Shuo Geng, Yuxi Cheng, Huize Han, Zhipu Fan, Wenbing Dai, Hua Zhang, Xueqing Wang, Qiang Zhang, Bing He. [J]. Nano-Micro Letters, 2024, 16(1): 103-.

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URL: https://www.qk.sjtu.edu.cn/nml/EN/10.1007/s40820-023-01313-0

https://www.qk.sjtu.edu.cn/nml/EN/Y2024/V16/I1/103

Figures/Tables 8

Fig. 1 Schematic diagram of the construction of HRP (+)-PNPs and schematic representation of dynamic molecular profiling of the intracellular delivery of nanoparticles in macrophages by enzyme-induced proximity labeling technology (nano-EPL)

Fig. 2 Preparation and characterization of PNPs and HRP (±)-PNPs. a Schematic illustration of HRP (±)-PNPs preparation. b Particle size and zeta potential value of PNPs and HRP (±)-PNPs (n = 3). c TEM images of PNPs and HRP (±)-PNPs (scale bar, 0.1 μm). d Relative enzyme activity of HRP (−), HRP (±)-PNPs. e Schematic illustration of the labeling activity of HRP (+)-PNPs. g Coomassie staining and f western blot of BSA labeled by HRP. h Western blot of BSA labeled by HRP ( ±) incubated in PBS at various pH values (5.0-7.4). i Western blot of BSA labeled by HRP ( ±)-PNPs. BP-omitted, H₂O₂-omitted, and BSA-omitted groups were used as controls. j Silver staining analysis of BSA labeled by HRP (±)-PNPs incubated in PBS at various pH values (5.0-7.4). k Silver staining analysis of BSA labeled by HRP (+)-PNPs incubated with CTSB for 1 h. l Schematic illustration of the labeling activity of HRP (+)-PNPs within/outside of liposomes. m Western blot of BSA labeled by HRP (+)-PNPs within/outside liposomes

Fig. 3 In situ labeling activity of interacting proteins during the intracellular delivery of HRP (+)-PNPs. a Schematic illustration of the two incubation methods. b Fluorescence images and c western blot and d Coomassie staining of intracellular proteins labeled by HRP (+)-PNPs through pulse-chase treatment. BP-omitted, H₂O₂-omitted, and BSA-omitted groups were used as controls (scale bar, 10 μm). e TEM analysis of proteins (as arrows show) labeled by HRP (+)-PNPs in phagosomes (scale bar, 0.5 μm). f Fluorescence images of intracellular proteins labeled by HRP (+)-PNPs through continuous incubation. BP-omitted, H₂O₂-omitted, and BSA-omitted groups were used as controls (scale bar, 10 μm)

Fig. 4 Phagosome-centered process during intracellular delivery of HRP(+)-PNPs. a Schematic illustration of the pulse-chase approach. b Fluorescence images and d quantitative image analysis of nanoparticles incubated with J774A.1 cells through pulse-chase treatment (scale bar, 10 μm). Nanoparticles (red), nuclei (blue), cytomembrane (white). c Fluorescence images and e quantitative image analysis of intracellular vesicles containing HRP(+)-PNPs at 5 time points (scale bar, 2 μm). Nanoparticles (red). f Florescence images of 70 kD dextran, h ER, i Golgi apparatus, j lysosome and HRP (+)-PNPs (scale bar, 2 μm). g Trend of colocalization assessed by regions between 70 kD dextran, k ER, l Golgi apparatus, m lysosome, and HRP (+)-PNPs over time (n = 50). n Schematic diagram of the fusion of phagosomes with different vesicles

Fig. 5 Dynamic molecule profiling of intracellular delivery of HRP (+)-PNPs in macrophages. a Schematic illustration of the proteomic experiment. b Fluorescence image and c western blot and d Coomassie staining of intracellular proteins labeled by HRP (+)-PNPs through the pulse-chase approach over time. (scale bar, 10 μm). e Manhattan plot for the true positive proteins at 6 time points. f LFQ intensity and g count of the true positive proteins of 6 time points. h Dynamic molecular mapping of intracellular delivery of nanoparticles

Fig. 6 Proteomics analysis of the participating organelles. a Trend of proteins in each cluster after normalization. b The percentage of early endosomes (EEs), late endosomes (LEs), ER, Golgi apparatus, lysosome proteins, and enzymes in 10 clusters. c Trend of EEs, LEs, ER, Golgi apparatus, lysosome, and extracellular protein counts over time. Numbers in parentheses represent number of proteins. d Relative abundance trend of EEs, LEs, ER, Golgi apparatus, lysosome, and extracellular proteins over time

Fig. 7 Detailed organellar participation timeline during the intracellular delivery of HRP(+)-PNPs. a Fluorescence images of GOLIM4, b ECM29, c STXBP2, d LAMP1 and biotinylated proteins (scale bar, 2 μm). Trend of colocalization assessed by regions and relative abundance between e GOLIM4, f ECM29, g STXBP2, h LAMP1 and biotinylated proteins over time (n = 50). i Schematic diagram of the dynamic subcellular organelles/phagosome fusion process during intracellular delivery of nanoparticles

Fig. 8 Intracellular delivery efficiency of gene drug-loaded nanoparticles. a Schematic illustration of Ce6@PPNs@siRNA and Ce6@PPNs@pEGFP preparation and functional steps. b Particle size and zeta potential value of PPNs, Ce6@PPNs, Ce6@PPNs@siRNA, and Ce6@PPNs@pEGFP (n = 3). c Schematic diagram of phagosome disruption caused by the photodynamic properties of Ce6@PPNs (660 nm, 200 mW cm⁻², 30 s). d Fluorescence image analysis and e mean fluorescence intensity of intracytoplasmic 70 kD dextran after illumination. f Fluorescence images of cytoplasmic Fam-siRNA and g eGFP after photodynamic treatment at 5 time points (scale bar, 10 μm). Fam-siRNA and EGFP (Green). h Trend of mean fluorescence intensity of cytoplasmic Fam-siRNA and i eGFP after photodynamic treatment at 5 time points. j Schematic illustration of the optimum escape period

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