A Noncationic Biocatalytic Nanobiohybrid Platform for Cytosolic Protein Delivery Through Controlled Perturbation of Intracellular Redox Homeostasis

Abstract

Intracellular delivery of proteins has largely been relying on cationic nanoparticles to induce efficient endosome escape, which, however, poses serious concerns on the inflammatory and cytotoxic effects. Herein, a versatile noncationic nano biohybrid platform is introduced for efficient cytosolic protein delivery by utilizing a nano-confined biocatalytic reaction. This platform is constructed by co-immobilizing glucose oxidase (GOx) and the target protein into nanoscale hydrogen-bonded organic frameworks (HOFs). The biocatalytic reaction of nano-confined GOx is leveraged to induce controlled perturbation of intracellular redox homeostasis by sustained hydrogen peroxide (H₂O₂) production and diminishing the flux of the pentose phosphate pathway (PPP). This in turn induces the endosome escape of nanobiohybrids. Concomitantly, GOx-mediated hypoxia leads to overexpression of azo reductase that initiated the materials' self-destruction for releasing target proteins. These biological effects collectively induce highly efficient cytosolic protein delivery. The versatility of this delivery platform is further demonstrated for various types of proteins, different protein loading approaches (in situ immobilization or post-adsorption), and in multiple cell lines. Finally, the protein delivery efficiency and biosafety are demonstrated in a tumor-bearing mouse model. This nanohybrid system opens up new avenues for intracellular protein delivery and is expected to be extensively applicable for a broad range of biomolecuels.

Keywords: hydrogen‐bonded organic frameworks; intracellular protein delivery; nanobiohybrids.

Scheme 1

Construction of enzyme‐based nanocomposite and its mechanism of endosomal escape. Synthetic route and schematic illustration of G@HOF‐based Nanobiohybrid Platform for intracellular delivery of functional protein.

Figure 1

Characterization and catalytic activity of G@HOF. a,b) TEM of HOF, G@HOF and before and after Na₂S₂O₄ treatment (20 mm) with G@HOF for different times. c) BSA‐FITC release from GB‐FITC@HOF in PBS (pH=7.4) with or without Na₂S₂O₄ (20 mm). d) Glucose solutions (1 mg mL⁻¹) incubation with free GOx and G@HOF for different times (n=3). e) H₂O₂ concentration of glucose solutions (1 mg mL⁻¹) incubation with free GOx and G@HOF for different times (n=3).

Figure 2

The ability of endosomal escape and protein release of G@HOF‐based nano platform. a) Confocal images exhibiting the endosomal escape of BSA‐FITC, B@HOF, and GB@HOF (10 mg mL⁻¹) nanoparticles in 4T1 cells; the lysosomes were stained with Lyso‐Tracker Red probe, shown in red; and the nucleus was stained with Hoechst, shown in blue; nanoparticles was shown in green. Scale bar, 10 µm. b) Bio‐TEM images of HOF and G@HOF after incubation with 4T1 cells for 2 and 4 h, illustrating the endosomal escape of the G@HOF. Scale bar: 0.5 µm. c) Cytotoxicity studies of 4T1cells cultivating with HOF, R@HOF, G@HOF and GR@HOF with low concentration of glucose (0.1 mg mL⁻¹) for 24 h. d,e) FCM and quantitative apoptosis analysis of 4T1 cells after difference treatments for 6 h.

Figure 3

Disruption of redox homeostasis by double oxidation. a) Concentration of intracellular H₂O₂ after treatment with G@HOF for 0, 4, 12, 24 h. b) Confocal images of intracellular ROS level in 4T1 cells after 2 h treatment of low glucose, HOF or G@HOF. Scale bar, 100 µm. c) Mean fluorescence intensity (MFI) of DCFH‐DA in 4T1 cells after 2 h treatment of low glucose, HOF or G@HOF. d) Cytotoxicity studies of 4T1cells cultivating with GR@HOF with low concentration of glucose (0.1 mg mL⁻¹) with or without GSH (10 mm) for 24 h. e) Concentration of intracellular glucose solution after incubation with G@HOF for 4 or 24 h. f) Schematic diagram of partial Pentose phosphate pathway (PPP). g) The enzymatic activity of G6PDH after treatment with G@HOF for 0, 3, 6, 12 h., unit mU mL⁻¹. h) ratio of NADPH/NADP⁺ after treatment with G@HOF for 0, 3, 6, or 12 h. i,j) Concentration of i GSH and j) G6P after treatment with G@HOF for 0, 3, 6, or 12 h.

Figure 4

Evaluation of versatility of delivery platform. a–c) Cytotoxicity studies of a) B16F10, b) PC3 and c) A549 cells cultivating with HOF, G@HOF and GR@HOF for 24 h. d) Cytotoxicity studies of 4T1 cells cultivating with G@HOF, C@HOF and GC@HOF for 24 h.

Figure 5

In Vivo biodistribution and antitumor efficacy. a) Representative photographs and bioluminescence images of organs ex vivo after euthanasia show the targeting of GR@HOF‐HA toward tumor. b) Quantifications of fluorescence intensity of heart, liver, spleen, lung, kidney and tumor after intravenous injection for 24 h (n=3). c) Quantifications of fluorescence intensity intensity of tumor after intravenous injection for 2, 6, 24 h (n=3). d) Experimental outline showing the treatment steps and procedures for evaluating the therapeutic outcomes of different groups in 4T1 tumor‐bearing Balb/c mice. e) Average tumor growth curves of the 4T1 tumor‐bearing Balb/c mice after various treatments and f) gross tumor images and g) corresponding tumor weight after 14 days. The mice treated with PBS were set as the control group. Data are presented as mean ±  SEM. n=5 mice per group. h) Photograph of tumors peeled from mice at the end of treatment. i) Ki67, H&E and TUNEL‐stained images of the dissected tumor tissues after 14 days of treatment.