Engineered extracellular vesicles with high collagen-binding affinity present superior in situ retention and therapeutic efficacy in tissue repair - PubMed (original) (raw)
. 2022 Aug 8;12(13):6021-6037.
doi: 10.7150/thno.70448. eCollection 2022.
Lu Lu 1, Hengyue Song 1 2, Yixin Duan 3, Jianing Chen 1, Randy Carney 4, Jian Jian Li 3, Ping Zhou 5, Jan Nolta 5, Kit S Lam 6, J Kent Leach 7, Diana L Farmer 1 2, Alyssa Panitch 1 4, Aijun Wang 1 2 4
Affiliations
- PMID: 35966577
- PMCID: PMC9373818
- DOI: 10.7150/thno.70448
Engineered extracellular vesicles with high collagen-binding affinity present superior in situ retention and therapeutic efficacy in tissue repair
Dake Hao et al. Theranostics. 2022.
Abstract
Although stem cell-derived extracellular vesicles (EVs) have remarkable therapeutic potential for various diseases, the therapeutic efficacy of EVs is limited due to their degradation and rapid diffusion after administration, hindering their translational applications. Here, we developed a new generation of collagen-binding EVs, by chemically conjugating a collagen-binding peptide SILY to EVs (SILY-EVs), which were designed to bind to collagen in the extracellular matrix (ECM) and form an EV-ECM complex to improve EVs' in situ retention and therapeutic efficacy after transplantation. Methods: SILY was conjugated to the surface of mesenchymal stem/stromal cell (MSC)-derived EVs by using click chemistry to construct SILY-EVs. Nanoparticle tracking analysis (NTA), ExoView analysis, cryogenic electron microscopy (cryo-EM) and western-blot analysis were used to characterize the SILY-EVs. Fluorescence imaging (FLI), MTS assay, ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to evaluate the collagen binding and biological functions of SILY-EVs in vitro. In a mouse hind limb ischemia model, the in vivo imaging system (IVIS), laser doppler perfusion imaging (LDPI), micro-CT, FLI and RT-qPCR were used to determine the SILY-EV retention, inflammatory response, blood perfusion, gene expression, and tissue regeneration. Results:In vitro, the SILY conjugation significantly enhanced EV adhesion to the collagen surface and did not alter the EVs' biological functions. In the mouse hind limb ischemia model, SILY-EVs presented longer in situ retention, suppressed inflammatory responses, and significantly augmented muscle regeneration and vascularization, compared to the unmodified EVs. Conclusion: With the broad distribution of collagen in various tissues and organs, SILY-EVs hold promise to improve the therapeutic efficacy of EV-mediated treatment in a wide range of diseases and disorders. Moreover, SILY-EVs possess the potential to functionalize collagen-based biomaterials and deliver therapeutic agents for regenerative medicine applications.
Keywords: Extracellular vesicle; collagen-binding; in situ retention; therapeutic efficacy; tissue repair.
© The author(s).
Conflict of interest statement
Competing Interests: The authors have declared that no competing interest exists.
Figures
Figure 1
Preparation and characterization of SILY-EVs. (A) Schematic diagram of the study design. (B) Evaluation of the SILY-EV conjugation process. Scale bar = 2 μm. (C) ExoView images of SILY-EV conjugation. Red indicated EVs stained with CD63. Green indicated SILY labeled with TAMRA. (D) Quantification of conjugation efficiency of SILY on EVs via ExoView. (E) Size distributions of EVs and SILY-EVs based on NTA measurements. (F) Cryo-EM images of EVs and SILY-EVs. Scale bar = 50 nm. (G) Western-blot analysis of EVs and SILY-EVs.
Figure 2
Binding ability of SILY-EVs to collagen and their effects on biological functions. (A) Images of attached PKH67-labeled EVs or SILY-EVs on collagen surface. Scale bar = 3 μm. (B) Quantification of the numbers of EVs or SILY-EVs attached to the collagen surface. (C) Quantification of the numbers of the EVs or SILY-EVs unattached to the collagen surface by using NTA. (D) Images and (E) quantification of HECFC viability, green for live cells and red for dead cells, cultured on the collagen surfaces decorated with EVs or SILY-EVs under the ischemic simulated environment. Scale bar = 50 μm. (F) Survival of HECFCs and (G) expression of caspase 3 and caspase 9 in HECFCs cultured on the collagen surfaces decorated with EVs or SILY-EVs under the ischemic simulated environment. (H) Images and (I) quantification of Ki67 positive HECFCs cultured on the collagen surfaces decorated with EVs or SILY-EVs. Scale bar = 10 μm. (J) Proliferation of HECFCs cultured on the collagen surfaces decorated with EVs or SILY-EVs. (K) Expression of pro-angiogenic genes (KDR and TIE2) in HECFCs cultured on the collagen surfaces decorated with EVs or SILY-EVs. (L) Expression of IFN-γ and IL10 related to inflammatory responses in the LPS-stimulated human macrophage-like THP-1 cells cultured on the collagen surfaces decorated with EVs or SILY-EVs. Data are expressed as mean ± standard deviation: *p < 0.05, **p < 0.01 (n = 6).
Figure 3
Retention of SILY-EVs in a mouse hind limb ischemia model. (A) Ex vivo evaluation for retention of EVs and SILY-EVs on the ischemic hind limb tissue section via binding to collagen. Green indicated collagen, blue indicated nucleus, red indicated DiD-labeled EVs, and gray indicated TAMRA-labeled SILY. The white arrows indicated EVs or SILY-EVs attached on the collagen of the ischemic hind limb tissue section. Scale bar = 10 μm. (B) Quantification of the EVs and SILY-EVs attached on the collagen of the ischemic hind limb tissue section. (C) Representative IVIS images of the retention of EVs or SILY-EVs at different time points after transplantation in the mouse hind limb ischemia model. (D) Quantification of the fluorescence intensity in EV group and SILY-EV group after transplantation in the mouse hind limb ischemia model. Data are expressed as mean ± standard deviation: **p < 0.01 (n = 6).
Figure 4
Effect of SILY-EVs on blood perfusion and vascular remodeling in the mouse hind limb ischemia model. (A) Representative LDPI of blood perfusion. (B) Quantification of the blood perfusion. (C) Micro-CT images of blood vessel. Scale bar = 1 mm. (D) Quantification of the blood vessel volume. Data are expressed as mean ± standard deviation: *p < 0.05, **p < 0.01 (n = 6).
Figure 5
Effects of SILY-EVs on inflammatory response and macrophage polarization in the mouse ischemic hind limb. (A) Representative immunofluorescence staining of CCR7+ M1 macrophages, CD163+ M2 macrophages and CD68+ macrophages. Scale bar = 50 μm. Quantification of the number of (B) CD68+ macrophages, (C) CCR7+ M1 macrophages, (D) CD163+ M2 macrophages and (E) M2/M1 ratio in the ischemic hind limb. Gene expression of (F) IFN-γ and (G) IL10 in different treatment groups. Data are expressed as mean ± standard deviation: *p < 0.05, **p < 0.01 (n = 6).
Figure 6
Effects of SILY-EVs on muscle repair in the ischemic hind limb. (A) Representative hematoxylin and eosin (H&E) and masson trichrome staining images of ischemic muscles treated with PBS, EVs, and SILY-EVs. The arrows indicated the vascular structures. Scale bar = 100 μm in H&E 5X images, 20 μm in H&E 20X images, and 100 μm in Masson images. Quantification of (B) average myofiber size, (C) percentage of centrally nucleated myofibers, and (D) area of collagen deposition. Gene expression of (E) Myoz1 and (F) Myoz3 in different treatment groups. Data are expressed as mean ± standard deviation: *p < 0.05, **p < 0.01, ***p < 0.001 (n = 6).
Figure 7
Effects of SILY-EVs on revascularization in the ischemic hind limb. (A) Representative immunofluorescence staining of CD31 and α-SMA. The arrows indicated the capillaries. Scale bar = 15 μm. Quantification of the density of (B) capillaries and (C) arterioles. Expression of angiogenic genes, (D) ANG II and (E) PECAM1, in different treatment groups. Data are expressed as mean ± standard deviation: *p < 0.05 (n = 6).
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