Journal: Advanced Science

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia–Reperfusion Injury

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Schematic illustration of PL720 cascade targeted therapy for MI/R in mice. When PL720 is administered at ischemia‐ reperfusion phase, it effectively targets the site of injury. Upon reaching the lesion site, the encapsulated L ‐arginine is released to facilitate the production of NO for timely vasodilation. Simultaneously, the slow‐released FTY720 within the injured myocardium activates the AKT pathway in cardiomyocytes to alleviate cardiomyocyte apoptosis. When administered at the late perfusion inflammation phase, the PL720 captured monocyte (monocyte‐PL720 aggregates) is recruited to the heart lesion. Recruited monocytes in the heart differentiate into macrophages, and subsequently, the macrophages phagocytize PL720. With the sustained release of FTY720, the STAT3 signaling pathway of macrophages is activated, thereby promoting macrophage polarization. This process reduces sprouting vessel degeneration caused by long M1 subtype macrophages while increasing M2 subtype macrophages to promote the maturation and quiescence of sprouting vessels.

Article Snippet: The hydrodynamic size distribution and surface zeta potential (ξ) of PLTs, PNVs, and PL720 were determined using a Malvern NanoSizer (Zeta–Sizer, Malvern Instruments, UK).

Techniques:

Journal: Advanced Science

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia–Reperfusion Injury

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Preparation and characterization of PL720. a) Schematic diagram of PL720 fabrication. b) TEM image of PNVs and PL720. c) Mean diameter of PLTs, PNVs, and PL720 ( n = 5). d) Zeta potential (ξ) analysis of PLTs, PNVs, and PL720 ( n = 5). e) Polymer dispersity index (PDI) of PLTs, PMVs, and PL720 ( n = 3). f. Release profiles of L‐arginine and FTY720 from PL720 within 24 h. g) Quantification of proteins in PLTs, purified platelet membrane, and PL720 identified by LC‐MS/MS. The Venn diagram illustrates the protein overlap among PLTs, purified platelet membrane, and PL720. h) Volcano plot displaying differential proteins between purified platelet membrane and PL720. The red points indicate significantly up‐regulated proteins. The blue points represent significantly down‐regulated proteins ( n = 3; fold change > 2 and adj. Pval < 0.05); and the gray points represent proteins without significant differential changes. i) Expression abundance of proteins associated with platelet adhesion and immune escape properties in purified platelet membrane and PL720 (n = 3). j) Classification of PL720 proteins by molecular function. k) Classification of PL720 Proteins by biological process. Results are presented as mean ± SD.

Article Snippet: The hydrodynamic size distribution and surface zeta potential (ξ) of PLTs, PNVs, and PL720 were determined using a Malvern NanoSizer (Zeta–Sizer, Malvern Instruments, UK).

Techniques: Zeta Potential Analyzer, Polymer, Purification, Membrane, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: Advanced Science

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia–Reperfusion Injury

doi: 10.1002/advs.202308727

Figure Lengend Snippet: In vitro assessment of the anti‐apoptotic and NO‐generating capacity of PL720. a) CLSM images of HUVECs stained with DAF‐FM DA after interacting with PL720 at predetermined time points. b) Quantitative analysis of the mean fluorescence intensity of HUVECs in (a) ( n = 3). c) Relative nitrite levels in the culture supernatants of HUVECs using the Griess reaction ( n = 3). d) Evaluation of the anti‐apoptotic effects of PBS, PNVs, FTY720, L‐arginine, and PL720 on H/R‐treated H9C2 cardiomyocytes. e) Quantitative analysis of apoptosis percentage of H9C2 cells in (d) ( n = 3). f) WB analysis of p‐AKT, AKT, BAX, and BCL‐2 protein levels. g) Quantitative analysis of p‐AKT/AKT levels in (f) ( n = 3). h) Quantitative analysis of BAX/BCL‐2 levels ( n = 3). Results are reported as mean ± SD. Data were analyzed using one‐way ANOVA followed by a two‐tailed Student's t ‐test. ns indicates non‐significant ( p > 0.05). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The hydrodynamic size distribution and surface zeta potential (ξ) of PLTs, PNVs, and PL720 were determined using a Malvern NanoSizer (Zeta–Sizer, Malvern Instruments, UK).

Techniques: In Vitro, Staining, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia–Reperfusion Injury

doi: 10.1002/advs.202308727

Figure Lengend Snippet: In vitro monocyte‐binding ability and macrophage uptake ability of PL720. a) Representative CLSM images of THP‐1 monocytes bound with DiO‐labeled PL720 and anti‐CD62P antibody blocked PL720 after pre‐incubation with or without LPS+INF‐γ stimulation (red: cell membrane, green: PL720, blue: nuclei). b) Semi‐quantification of DiO fluorescence intensity in (a) ( n = 4). c) Flow cytometry analysis of THP‐1 monocytes bound with DiO‐labeled PL720 and PL720 62Pblock after pre‐incubation with or without inflammatory activation. d) Quantification of DiO normalized fluorescence intensity in (c) ( n = 3). e) Representative CLSM images of monocytes (THP‐1) and macrophages (THP‐1 (MΦ)) after incubation with DiO‐labeled PL720 for 0.5 and 3 h. (Green: PL720, Red: cell membrane, blue: nuclei). f) The Pearson's correlation coefficient of membrane and PL720 was measured at different time points ( n = 8). g) Schematic diagram of Transwell assay for endothelial penetration capacity of monocytes. h) Representative images of monocytes in the lower chamber after migration through the endothelial layer (green: PL720). i) Quantitative analysis of monocytes in the lower chamber after migration across endothelial layer ( n = 3). Results are reported as mean ± SD. Data were analyzed using one‐way ANOVA followed by a two‐tailed Student's t ‐test. ns indicates non‐significant ( p > 0.05). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The hydrodynamic size distribution and surface zeta potential (ξ) of PLTs, PNVs, and PL720 were determined using a Malvern NanoSizer (Zeta–Sizer, Malvern Instruments, UK).

Techniques: In Vitro, Binding Assay, Labeling, Incubation, Membrane, Fluorescence, Flow Cytometry, Activation Assay, Transwell Assay, Migration, Two Tailed Test

Journal: Advanced Science

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia–Reperfusion Injury

doi: 10.1002/advs.202308727

Figure Lengend Snippet: PL720 activates STAT3 pathway to induce immunomodulation in vitro. a) CLSM images indicate phenotypes of inflammatory BMDMs after being treated by PBS, FTY720, and PL720. b) Quantitative analysis of fluorescence intensity of iNOS (M1) and CD206 (M2) in (a) ( n = 3). c) Flow cytometry assay of inflammatory BMDMs after treated by PBS, FTY720, and PL720. d) Statistical analysis of flow cytometry results in (c) ( n = 3). e) WB of p‐STAT3, STAT3, INOS, and CD206 protein. f–h). Quantification of p‐STAT3/STAT3, INOS/GAPDH and CD206/GAPDH levels ( n = 3). i–k) Concentration of cytokine M1 markers (IL‐1β, TNF‐α) and M2 markers (IL‐10, TGF‐β) in supernatants ( n = 3). Results are reported as mean ± SD. Data were analyzed using one‐way ANOVA followed by a two‐tailed Student's t ‐test. ns indicates non‐significant ( p > 0.05). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The hydrodynamic size distribution and surface zeta potential (ξ) of PLTs, PNVs, and PL720 were determined using a Malvern NanoSizer (Zeta–Sizer, Malvern Instruments, UK).

Techniques: In Vitro, Fluorescence, Flow Cytometry, Concentration Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia–Reperfusion Injury

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Targeting specificity, cardiomyocyte anti‐apoptotic effects, and the ability to rescue damaged myocardium after treatment with a single dose of PL720 during the ischemic phase. a) The typical ex vivo NIR fluorescence images of hearts from sham, sham+PL720, and MI/R + PL720 groups post‐injection for 6 h. b) The mean fluorescence intensity was obtained from the heart regions of mice described in (a) ( n = 3). c) The CLSM image of the heart lesion 12 h after injection of DiI‐labeled PL720 for healthy (sham) and MI/R mice. d) Quantitative analysis of DiI fluorescence signal intensities in hearts in (c) ( n = 3). e) Representative images of TUNEL staining 2d after surgery to detect apoptotic nucleus (white arrows point to apoptotic cardiomyocytes). f) The number of TUNEL positive staining nucleus in (e) ( n = 3). g) WB of p‐AKT, AKT, BAX, and BCL‐2 protein. h) Quantification of p‐AKT/AKT levels ( n = 3). i) Quantification of BAX/BCL‐2 levels ( n = 3). j) Heart rates of mice in each group at different times (0, 1, 2, 3, 4, 5, 6, and 24 h) after the first injection of PBS, PNVs, L720, and PL720 ( n = 6). k) TTC staining images of mouse heart in each group to evaluate myocardial ischemia injury. l) TTC staining was quantified to determine infarct size ( n = 4). Results are reported as mean ± SD. Data were analyzed using one‐way ANOVA followed by a two‐tailed Student's t ‐test. ns indicates non‐significant ( p > 0.05). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The hydrodynamic size distribution and surface zeta potential (ξ) of PLTs, PNVs, and PL720 were determined using a Malvern NanoSizer (Zeta–Sizer, Malvern Instruments, UK).

Techniques: Ex Vivo, Fluorescence, Injection, Labeling, TUNEL Assay, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia–Reperfusion Injury

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Targeting specificity, and macrophage polarization reprogramming effect of a single dose of PL720 during the late reperfusion inflammation phase. a) The typical ex vivo NIR images of the heart for sham group or MI/R mice injected with DiR‐labeled PL720 or PL720 62Pblock. b) Quantitative analysis of DiR fluorescence signal in hearts for different groups of mice ( n = 3). c) The CLSM image of the injury site in the heart after injection of DiI‐labeled PL720 and PL720 62Pblock. d) Quantitative analysis of DiI fluorescence signal in hearts for different groups of mice ( n = 3). e) CLSM images of MI/R injured heart sections showing the total (F4/80) and M2 subtype (CD206) macrophages after being treated by PBS, PNVs, L720, and PL720, respectively. f) Fluorescence intensity quantification of F4/80 in (e) ( n = 4). g) Fluorescence intensity quantification of CD206 in (e) ( n = 4). h) WB of p‐STAT3, STAT3, INOS, and CD206 protein levels. i–k) Quantification of p‐STAT3/STAT3, INOS/GAPDH and CD206/GAPDH levels ( n = 3). l–o) Quantification of L‐1β, TNF‐α, IL‐10, and TGF‐β related mRNA expression ( n = 3). Results are reported as mean ± SD. Data were analyzed using one‐way ANOVA followed by a two‐tailed Student's t ‐test. ns indicates non‐significant ( p > 0.05). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The hydrodynamic size distribution and surface zeta potential (ξ) of PLTs, PNVs, and PL720 were determined using a Malvern NanoSizer (Zeta–Sizer, Malvern Instruments, UK).

Techniques: Ex Vivo, Injection, Labeling, Fluorescence, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia–Reperfusion Injury

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Cardioprotection effect after two doses of PL720 treatment. a) Echocardiograms obtained by M‐mode ultrasound at different times post‐operation (pre, day 3, day 7, and day 28). b–g) Cardiac function of the mice was evaluated according to the EF, FS, LVID; d, LV Vol; d, LVID; s and LV Vol; s measured by the echocardiography of the mice in each group ( n = 5). h) Masson staining of MI/R heart paraffin sections. i)Fibrosis remodeling percentage of MI/R mice after treatment with PBS, PNVs, L720, and PL720 based on Masson staining images. j) Sections of the injury sites in each group of mice were subjected to CD31 and WGA staining. CD31 positivity was utilized to visualize microvessel at the boundary areas of the injury sites, while WGA staining was utilized to visualize myocyte in the border regions of the injury area. k) Quantification of the number of CD31 positive and myocyte within the equivalent area of the injury border region, and computation of their ratio ( n = 3). Results are reported as mean ± SD. Data were analyzed using one‐way ANOVA followed by a two‐tailed Student's t ‐test. ns indicates non‐significant ( p > 0.05). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The hydrodynamic size distribution and surface zeta potential (ξ) of PLTs, PNVs, and PL720 were determined using a Malvern NanoSizer (Zeta–Sizer, Malvern Instruments, UK).

Techniques: Staining, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 1. Schematic illustration of PL720 cascade targeted therapy for MI/R in mice. When PL720 is administered at ischemia- reperfusion phase, it effectively targets the site of injury. Upon reaching the lesion site, the encapsulated L-arginine is released to facilitate the production of NO for timely vasodilation. Simultaneously, the slow-released FTY720 within the injured myocardium activates the AKT pathway in cardiomyocytes to alleviate cardiomyocyte apoptosis. When administered at the late perfusion inflammation phase, the PL720 captured monocyte (monocyte-PL720 aggregates) is recruited to the heart lesion. Recruited monocytes in the heart differentiate into macrophages, and subsequently, the macrophages phagocytize PL720. With the sustained release of FTY720, the STAT3 signaling pathway of macrophages is activated, thereby promoting macrophage polarization. This process reduces sprouting vessel degeneration caused by long M1 subtype macrophages while increasing M2 subtype macrophages to promote the maturation and quiescence of sprouting vessels.

Article Snippet: Characterization of PL720 Nanocarriers: The microscopic morphology of PL720 was examined via TEM (JEM-2100, JEOL, Japan) following negative staining with 1% phosphotungstic acid.

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 2. Preparation and characterization of PL720. a) Schematic diagram of PL720 fabrication. b) TEM image of PNVs and PL720. c) Mean diameter of PLTs, PNVs, and PL720 (n = 5). d) Zeta potential (𝜉) analysis of PLTs, PNVs, and PL720 (n = 5). e) Polymer dispersity index (PDI) of PLTs, PMVs, and PL720 (n = 3). f. Release profiles of L-arginine and FTY720 from PL720 within 24 h. g) Quantification of proteins in PLTs, purified platelet membrane, and PL720 identified by LC-MS/MS. The Venn diagram illustrates the protein overlap among PLTs, purified platelet membrane, and PL720. h) Volcano plot displaying differential proteins between purified platelet membrane and PL720. The red points indicate significantly up-regulated proteins. The blue points represent significantly down-regulated proteins (n = 3; fold change > 2 and adj. Pval < 0.05); and the gray points represent proteins without significant differential changes. i) Expression abundance of proteins associated with platelet adhesion and immune escape properties in purified platelet membrane and PL720 (n = 3). j) Classification of PL720 proteins by molecular function. k) Classification of PL720 Proteins by biological process. Results are presented as mean ± SD.

Article Snippet: Characterization of PL720 Nanocarriers: The microscopic morphology of PL720 was examined via TEM (JEM-2100, JEOL, Japan) following negative staining with 1% phosphotungstic acid.

Techniques: Zeta Potential Analyzer, Polymer, Membrane, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 3. In vitro assessment of the anti-apoptotic and NO-generating capacity of PL720. a) CLSM images of HUVECs stained with DAF-FM DA after interacting with PL720 at predetermined time points. b) Quantitative analysis of the mean fluorescence intensity of HUVECs in (a) (n = 3). c) Relative nitrite levels in the culture supernatants of HUVECs using the Griess reaction (n = 3). d) Evaluation of the anti-apoptotic effects of PBS, PNVs, FTY720, L-arginine, and PL720 on H/R-treated H9C2 cardiomyocytes. e) Quantitative analysis of apoptosis percentage of H9C2 cells in (d) (n = 3). f) WB analysis of p-AKT, AKT, BAX, and BCL-2 protein levels. g) Quantitative analysis of p-AKT/AKT levels in (f) (n = 3). h) Quantitative analysis of BAX/BCL-2 levels (n = 3). Results are reported as mean ± SD. Data were analyzed using one-way ANOVA followed by a two-tailed Student’s t-test. ns indicates non-significant (p > 0.05). * p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Characterization of PL720 Nanocarriers: The microscopic morphology of PL720 was examined via TEM (JEM-2100, JEOL, Japan) following negative staining with 1% phosphotungstic acid.

Techniques: In Vitro, Staining, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 4. In vitro monocyte-binding ability and macrophage uptake ability of PL720. a) Representative CLSM images of THP-1 monocytes bound with DiO-labeled PL720 and anti-CD62P antibody blocked PL720 after pre-incubation with or without LPS+INF-𝛾stimulation (red: cell membrane, green: PL720, blue: nuclei). b) Semi-quantification of DiO fluorescence intensity in (a) (n = 4). c) Flow cytometry analysis of THP-1 monocytes bound with DiO-labeled PL720 and PL720 62Pblock after pre-incubation with or without inflammatory activation. d) Quantification of DiO normalized fluorescence intensity in (c) (n = 3). e) Representative CLSM images of monocytes (THP-1) and macrophages (THP-1 (MΦ)) after incubation with DiO-labeled PL720

Article Snippet: Characterization of PL720 Nanocarriers: The microscopic morphology of PL720 was examined via TEM (JEM-2100, JEOL, Japan) following negative staining with 1% phosphotungstic acid.

Techniques: In Vitro, Binding Assay, Labeling, Incubation, Membrane, Flow Cytometry, Activation Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 5. PL720 activates STAT3 pathway to induce immunomodulation in vitro. a) CLSM images indicate phenotypes of inflammatory BMDMs after being treated by PBS, FTY720, and PL720. b) Quantitative analysis of fluorescence intensity of iNOS (M1) and CD206 (M2) in (a) (n = 3). c) Flow cytometry assay of inflammatory BMDMs after treated by PBS, FTY720, and PL720. d) Statistical analysis of flow cytometry results in (c) (n = 3). e) WB of p-STAT3, STAT3, INOS, and CD206 protein. f–h). Quantification of p-STAT3/STAT3, INOS/GAPDH and CD206/GAPDH levels (n = 3). i–k) Concentration of cytokine M1 markers (IL-1𝛽, TNF-𝛼) and M2 markers (IL-10, TGF-𝛽) in supernatants (n = 3). Results are reported as mean ± SD. Data were analyzed using one-way ANOVA followed by a two-tailed Student’s t-test. ns indicates non-significant (p > 0.05). * p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Characterization of PL720 Nanocarriers: The microscopic morphology of PL720 was examined via TEM (JEM-2100, JEOL, Japan) following negative staining with 1% phosphotungstic acid.

Techniques: In Vitro, Flow Cytometry, Cytometry, Concentration Assay, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 7. Targeting specificity, and macrophage polarization reprogramming effect of a single dose of PL720 during the late reperfusion inflammation phase. a) The typical ex vivo NIR images of the heart for sham group or MI/R mice injected with DiR-labeled PL720 or PL720 62Pblock. b) Quantitative analysis of DiR fluorescence signal in hearts for different groups of mice (n = 3). c) The CLSM image of the injury site in the heart after injection of DiI-labeled PL720 and PL720 62Pblock. d) Quantitative analysis of DiI fluorescence signal in hearts for different groups of mice (n = 3). e) CLSM images of MI/R injured heart sections showing the total (F4/80) and M2 subtype (CD206) macrophages after being treated by PBS, PNVs, L720, and PL720, respectively. f) Fluorescence intensity quantification of F4/80 in (e) (n = 4). g) Fluorescence intensity quantification of CD206 in (e) (n = 4). h) WB of p-STAT3, STAT3, INOS, and CD206 protein levels. i–k) Quantification of p-STAT3/STAT3, INOS/GAPDH and CD206/GAPDH levels (n = 3). l–o) Quantification of L-1𝛽, TNF-𝛼, IL-10, and TGF-𝛽related mRNA expression (n = 3). Results are reported as mean ± SD. Data were analyzed using one-way ANOVA followed by a two-tailed Student’s t-test. ns indicates non-significant (p > 0.05). * p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Characterization of PL720 Nanocarriers: The microscopic morphology of PL720 was examined via TEM (JEM-2100, JEOL, Japan) following negative staining with 1% phosphotungstic acid.

Techniques: Ex Vivo, Injection, Labeling, Fluorescence, Expressing, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 8. Cardioprotection effect after two doses of PL720 treatment. a) Echocardiograms obtained by M-mode ultrasound at different times post- operation (pre, day 3, day 7, and day 28). b–g) Cardiac function of the mice was evaluated according to the EF, FS, LVID; d, LV Vol; d, LVID; s and LV

Article Snippet: Characterization of PL720 Nanocarriers: The microscopic morphology of PL720 was examined via TEM (JEM-2100, JEOL, Japan) following negative staining with 1% phosphotungstic acid.

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 1. Schematic illustration of PL720 cascade targeted therapy for MI/R in mice. When PL720 is administered at ischemia- reperfusion phase, it effectively targets the site of injury. Upon reaching the lesion site, the encapsulated L-arginine is released to facilitate the production of NO for timely vasodilation. Simultaneously, the slow-released FTY720 within the injured myocardium activates the AKT pathway in cardiomyocytes to alleviate cardiomyocyte apoptosis. When administered at the late perfusion inflammation phase, the PL720 captured monocyte (monocyte-PL720 aggregates) is recruited to the heart lesion. Recruited monocytes in the heart differentiate into macrophages, and subsequently, the macrophages phagocytize PL720. With the sustained release of FTY720, the STAT3 signaling pathway of macrophages is activated, thereby promoting macrophage polarization. This process reduces sprouting vessel degeneration caused by long M1 subtype macrophages while increasing M2 subtype macrophages to promote the maturation and quiescence of sprouting vessels.

Article Snippet: Both inflammatory and non-inflammatory activated THP-1 cells were incubated with green DiO-labeled PL720 or CD-62P-inhibited PL720 for 60 min. THP-1 cells were stained using a DiI (red) cell membrane staining kit (Beyotime, China) and observed using CLSM.

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 2. Preparation and characterization of PL720. a) Schematic diagram of PL720 fabrication. b) TEM image of PNVs and PL720. c) Mean diameter of PLTs, PNVs, and PL720 (n = 5). d) Zeta potential (𝜉) analysis of PLTs, PNVs, and PL720 (n = 5). e) Polymer dispersity index (PDI) of PLTs, PMVs, and PL720 (n = 3). f. Release profiles of L-arginine and FTY720 from PL720 within 24 h. g) Quantification of proteins in PLTs, purified platelet membrane, and PL720 identified by LC-MS/MS. The Venn diagram illustrates the protein overlap among PLTs, purified platelet membrane, and PL720. h) Volcano plot displaying differential proteins between purified platelet membrane and PL720. The red points indicate significantly up-regulated proteins. The blue points represent significantly down-regulated proteins (n = 3; fold change > 2 and adj. Pval < 0.05); and the gray points represent proteins without significant differential changes. i) Expression abundance of proteins associated with platelet adhesion and immune escape properties in purified platelet membrane and PL720 (n = 3). j) Classification of PL720 proteins by molecular function. k) Classification of PL720 Proteins by biological process. Results are presented as mean ± SD.

Article Snippet: Both inflammatory and non-inflammatory activated THP-1 cells were incubated with green DiO-labeled PL720 or CD-62P-inhibited PL720 for 60 min. THP-1 cells were stained using a DiI (red) cell membrane staining kit (Beyotime, China) and observed using CLSM.

Techniques: Zeta Potential Analyzer, Polymer, Membrane, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 3. In vitro assessment of the anti-apoptotic and NO-generating capacity of PL720. a) CLSM images of HUVECs stained with DAF-FM DA after interacting with PL720 at predetermined time points. b) Quantitative analysis of the mean fluorescence intensity of HUVECs in (a) (n = 3). c) Relative nitrite levels in the culture supernatants of HUVECs using the Griess reaction (n = 3). d) Evaluation of the anti-apoptotic effects of PBS, PNVs, FTY720, L-arginine, and PL720 on H/R-treated H9C2 cardiomyocytes. e) Quantitative analysis of apoptosis percentage of H9C2 cells in (d) (n = 3). f) WB analysis of p-AKT, AKT, BAX, and BCL-2 protein levels. g) Quantitative analysis of p-AKT/AKT levels in (f) (n = 3). h) Quantitative analysis of BAX/BCL-2 levels (n = 3). Results are reported as mean ± SD. Data were analyzed using one-way ANOVA followed by a two-tailed Student’s t-test. ns indicates non-significant (p > 0.05). * p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Both inflammatory and non-inflammatory activated THP-1 cells were incubated with green DiO-labeled PL720 or CD-62P-inhibited PL720 for 60 min. THP-1 cells were stained using a DiI (red) cell membrane staining kit (Beyotime, China) and observed using CLSM.

Techniques: In Vitro, Staining, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 4. In vitro monocyte-binding ability and macrophage uptake ability of PL720. a) Representative CLSM images of THP-1 monocytes bound with DiO-labeled PL720 and anti-CD62P antibody blocked PL720 after pre-incubation with or without LPS+INF-𝛾stimulation (red: cell membrane, green: PL720, blue: nuclei). b) Semi-quantification of DiO fluorescence intensity in (a) (n = 4). c) Flow cytometry analysis of THP-1 monocytes bound with DiO-labeled PL720 and PL720 62Pblock after pre-incubation with or without inflammatory activation. d) Quantification of DiO normalized fluorescence intensity in (c) (n = 3). e) Representative CLSM images of monocytes (THP-1) and macrophages (THP-1 (MΦ)) after incubation with DiO-labeled PL720

Article Snippet: Both inflammatory and non-inflammatory activated THP-1 cells were incubated with green DiO-labeled PL720 or CD-62P-inhibited PL720 for 60 min. THP-1 cells were stained using a DiI (red) cell membrane staining kit (Beyotime, China) and observed using CLSM.

Techniques: In Vitro, Binding Assay, Labeling, Incubation, Membrane, Flow Cytometry, Activation Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 5. PL720 activates STAT3 pathway to induce immunomodulation in vitro. a) CLSM images indicate phenotypes of inflammatory BMDMs after being treated by PBS, FTY720, and PL720. b) Quantitative analysis of fluorescence intensity of iNOS (M1) and CD206 (M2) in (a) (n = 3). c) Flow cytometry assay of inflammatory BMDMs after treated by PBS, FTY720, and PL720. d) Statistical analysis of flow cytometry results in (c) (n = 3). e) WB of p-STAT3, STAT3, INOS, and CD206 protein. f–h). Quantification of p-STAT3/STAT3, INOS/GAPDH and CD206/GAPDH levels (n = 3). i–k) Concentration of cytokine M1 markers (IL-1𝛽, TNF-𝛼) and M2 markers (IL-10, TGF-𝛽) in supernatants (n = 3). Results are reported as mean ± SD. Data were analyzed using one-way ANOVA followed by a two-tailed Student’s t-test. ns indicates non-significant (p > 0.05). * p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Both inflammatory and non-inflammatory activated THP-1 cells were incubated with green DiO-labeled PL720 or CD-62P-inhibited PL720 for 60 min. THP-1 cells were stained using a DiI (red) cell membrane staining kit (Beyotime, China) and observed using CLSM.

Techniques: In Vitro, Flow Cytometry, Cytometry, Concentration Assay, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 7. Targeting specificity, and macrophage polarization reprogramming effect of a single dose of PL720 during the late reperfusion inflammation phase. a) The typical ex vivo NIR images of the heart for sham group or MI/R mice injected with DiR-labeled PL720 or PL720 62Pblock. b) Quantitative analysis of DiR fluorescence signal in hearts for different groups of mice (n = 3). c) The CLSM image of the injury site in the heart after injection of DiI-labeled PL720 and PL720 62Pblock. d) Quantitative analysis of DiI fluorescence signal in hearts for different groups of mice (n = 3). e) CLSM images of MI/R injured heart sections showing the total (F4/80) and M2 subtype (CD206) macrophages after being treated by PBS, PNVs, L720, and PL720, respectively. f) Fluorescence intensity quantification of F4/80 in (e) (n = 4). g) Fluorescence intensity quantification of CD206 in (e) (n = 4). h) WB of p-STAT3, STAT3, INOS, and CD206 protein levels. i–k) Quantification of p-STAT3/STAT3, INOS/GAPDH and CD206/GAPDH levels (n = 3). l–o) Quantification of L-1𝛽, TNF-𝛼, IL-10, and TGF-𝛽related mRNA expression (n = 3). Results are reported as mean ± SD. Data were analyzed using one-way ANOVA followed by a two-tailed Student’s t-test. ns indicates non-significant (p > 0.05). * p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Both inflammatory and non-inflammatory activated THP-1 cells were incubated with green DiO-labeled PL720 or CD-62P-inhibited PL720 for 60 min. THP-1 cells were stained using a DiI (red) cell membrane staining kit (Beyotime, China) and observed using CLSM.

Techniques: Ex Vivo, Injection, Labeling, Fluorescence, Expressing, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Platelet Membrane Nanocarriers Cascade Targeting Delivery System to Improve Myocardial Remodeling Post Myocardial Ischemia-Reperfusion Injury.

doi: 10.1002/advs.202308727

Figure Lengend Snippet: Figure 8. Cardioprotection effect after two doses of PL720 treatment. a) Echocardiograms obtained by M-mode ultrasound at different times post- operation (pre, day 3, day 7, and day 28). b–g) Cardiac function of the mice was evaluated according to the EF, FS, LVID; d, LV Vol; d, LVID; s and LV

Article Snippet: Both inflammatory and non-inflammatory activated THP-1 cells were incubated with green DiO-labeled PL720 or CD-62P-inhibited PL720 for 60 min. THP-1 cells were stained using a DiI (red) cell membrane staining kit (Beyotime, China) and observed using CLSM.

Techniques: