Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis

Abstract

Sepsis, a condition caused by severe infections, affects more than 30 million people worldwide every year and remains the leading cause of death in hospitals^1,2. Moreover, antimicrobial resistance has become an additional challenge in the treatment of sepsis³, and thus, alternative therapeutic approaches are urgently needed^2,3. Here, we show that adoptive transfer of macrophages containing antimicrobial peptides linked to cathepsin B in the lysosomes (MACs) can be applied for the treatment of multidrug-resistant bacteria-induced sepsis in mice with immunosuppression. The MACs are constructed by transfection of vitamin C lipid nanoparticles that deliver antimicrobial peptide and cathepsin B (AMP-CatB) mRNA. The vitamin C lipid nanoparticles allow the specific accumulation of AMP-CatB in macrophage lysosomes, which is the key location for bactericidal activities. Our results demonstrate that adoptive MAC transfer leads to the elimination of multidrug-resistant bacteria, including Staphylococcus aureus and Escherichia coli, leading to the complete recovery of immunocompromised septic mice. Our work provides an alternative strategy for overcoming multidrug-resistant bacteria-induced sepsis and opens up possibilities for the development of nanoparticle-enabled cell therapy for infectious diseases.

Fig. 1 |. Schematic illustration of adoptive macrophage transfer and chemical structures of the vitamin-derived lipids.

a, Construction of MACs for sepsis therapy. MACs stands for macrophages containing antimicrobial peptides linked to cathepsin B in the lysosomes (MACs). The AMP-CatB mRNA is encapsulated in the vitamin C lipid nanoparticle (V_CLNP) and delivered to the macrophage where the mRNA is translated in the cytoplasm and translocated into the lysosomes. Within the lysosomes, the cleavable linker is cleaved by the lysosomal CatB, releasing the AMP-IB367. After phagosomes carrying MDR bacteria fuse with the lysosomes, the ingested MDR bacteria are eradicated by the pre-stored AMP-IB367. b, Chemical structures of vitamin-derived lipids including V_B3-Lipid, V_C-Lipid, V_D-Lipid, V_E-Lipid, and V_H-Lipid.

Fig. 2 |. Screening, optimization, and characterization of vitamin lipid nanoparticles (VLNPs).

a, mRNA delivery efficiency of VLNPs in RAW264.7 cells. b, Expression kinetics of mRNA delivered by V_CLNPs in RAW264.7 cells. c, The first round of optimization: four levels and impact trend of each V_CLNP’s component. d, Formulation table for the validation of the predicted formulation, and the second round of optimization: the mass ratio of V_C-Lipid:mRNA. e, Fold changes of luminescence intensity in the two rounds of optimization. f, Characterization of the optimal V_CLNP formulation, including size, polydispersity index (PDI), encapsulation efficiency, zeta potential, and Cryo-TEM image (Scale bar = 50 nm). g, Confocal imagines of the RAW264.7 cells incubated with V_CLNPs encapsulating eGFP-CatB mRNA (Scale bar = 10 μm). Data in g are representative images from n = 3 independent experiments. h, Intracellular survival of MDR Staphylococcus aureus (MDRSA) in the RAW264.7 cells. Data in a, b, c, e, and h are from n = 3 biologically independent samples. All data are presented as mean ± s.d. Statistical significance was analyzed by the two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3 |. Therapeutic effects of MAC-RAWs in MDRSA-induced sepsis mice with immunosuppression.

a, Bacterial burden in blood at 24 h after cell transfer. n = 6, 8, 7, and 11 live mice for PBS, PBS-RAWs (i.p. + i.v.), MAC-RAWs (i.p.), and MAC-RAWs (i.p. + i.v.) groups, respectively. b, Percentage survival of mice with sepsis induced by i.p. bacterial inoculation. c-e, The body weights (BWs), white blood cells (WBCs), and lymphocytes (LYMs) of mice. c, BWs; d, WBCs; e, LYMs. Data in b, c, d, and e (except time point 792 h), n = 8, 10, 10, and 12 for PBS, PBS-RAWs (i.p. + i.v.), MAC-RAWs (i.p.), and MAC-RAWs (i.p. + i.v.) groups, respectively. f, g, Bacterial burden in blood of each survived mouse treated by MAC-RAWs (i.p. + i.v.). Data in a, c, d, and e are the mean ± s.d. Statistical significance was analyzed by the two-tailed Student’s t-test. Data in b were analyzed by the log-rank (Mantel–Cox) test. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; ND, not detectable.

Fig. 4 |. Therapeutic effects of MAC-BMDMs in mixed MDR bacteria (Staphylococcus aureus and Escherichia coli)-induced sepsis mice with immunosuppression.

a, Bacterial burden in blood at 24 h after cell transfer. n = 6, 10, and 12 live mice for PBS, PBS-BMDMs (i.p. + i.v.), and MAC-BMDMs (i.p. + i.v.) groups, respectively. b, Percentage survival of mice with sepsis induced by i.p. bacterial inoculation. c-e, The BWs, WBCs, and LYMs of mice. c, BWs; d, WBCs; e, LYMs. Data in b, c, d, and e (except time point 792 h), n = 8, 10, and 12 for PBS, PBS-BMDMs (i.p. + i.v.), and MAC-RAWs (i.p. + i.v.) groups, respectively. f, Bacterial burden in blood of each survived mouse treated by MAC-BMDMs. Data in a, c, d, and e are the mean ± s.d. Statistical significance was analyzed by the two-tailed Student’s t-test. Data in b were analyzed by the log-rank (Mantel–Cox) test. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; ND, not detectable.