Research ArticleHEALTH AND MEDICINE

Accessing neuroinflammation sites: Monocyte/neutrophil-mediated drug delivery for cerebral ischemia

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Science Advances  10 Jul 2019:
Vol. 5, no. 7, eaau8301
DOI: 10.1126/sciadv.aau8301
  • Fig. 1 The uptake characterization and comigration across HBMEC of liposomes with M/Ns.

    (A) Binding affinity assay of cRGD/cRGD-PEG-DSPE with αvβ1 protein using SPR. RU, Response Unit. (B) The uptake of C6-PLs/C6-cRGDLs by M/Ns after 30 min of incubation assayed by flow cytometer. a.u., arbitrary unit. (C) Effects of blocking antibodies against integrins and free cRGD peptide on uptake of C6-PLs/C6-cRGDLs with M/Ns assayed by flow cytometer. **P < 0.01, compared with control. (D) The C6 fluorescence of the suspension in the lower chamber after the migration of C6-PLs/C6-cRGDLs across HBMEC with or without M/Ns assayed by microplate reader. PLs and cRGDLs meant non–fluorescence-labeled liposomes. **P < 0.01. (E) The C6 fluorescence of the suspension in lower chamber with treatment of blocking antibodies against ICAM-1 or VCAM-1 on HBMEC assayed by microplate reader. **P < 0.01 and *P < 0.05, compared with control. All untreated neutrophils and monocytes in (A) to (D) meant blank cell controls untreated with liposomes.

  • Fig. 2 The uptake of cRGDLs by M/Ns in vivo of I/R rats.

    (A) The percent of M/Ns loading with Cy5-cRGDLs in blood circulation of I/R rats assayed by flow cytometer (n = 3). (B) Confocal microscope images of cellular location of Cy5-NBD-cRGDLs in isolated M/Ns from I/R rats. Red: Cy5, hydrophilic core marker and green: NBD-PE, lipid membrane marker. (C) Confocal microscope images of comigration of Cy5-PLs/Cy5-cRGDLs with M/Ns across cerebral vessels in the ischemic hemisphere 12 hours after reperfusion. Blue: M/Ns stained with cell tracer CFSE; red: Cy5-PLs or Cy5-cRGDLs; and green: cerebral vessels labeled with vWF rabbit anti-rat antibody and secondary antibody of Alexa Fluor 594 goat anti-rabbit.

  • Fig. 3 Multitargeting delivery effect in vivo.

    (A) Fluorescence imaging of I/R nude mice after administration of DiR-cRGDLs/DiR-PLs at different times after reperfusion in I/R rat. (B) Coronal sections of TTC stain (white means ischemic area) or fluorescence imaging of I/R nude mice administrated with DiR-cRGDLs or DiR-PLs. (C) Radioactivity ratio of ischemic hemisphere to control hemisphere at different times after administration in I/R rats (n = 5). The bar represents 50 μm. (D) The plasma concentration-time curve and biodistribution (E) to (H) of I125-labeled PLs, cRGDLs, and PG.

  • Fig. 4 The liposome transport from M/Ns to neuron in vitro and in vivo.

    (A) Confocal microscope images of colocation of Cy5-NBD-PLs/Cy5-NBD-cRGDLs with neurons or microglia (B) in brains of I/R rats 12 hours after reperfusion. Green: NBD-PE marker; red: Cy5 marker; blue: neurons stained with Rabbit anti-rat MAP2 or mouse anti-rat Iba-1 antibody and secondary antibody of Alexa Fluor 405 goat anti-rabbit. (C) Confocal microscope images of the transfer of Cy5-cRGDLs from THP-1 or (D) HL-60 to PC12. Green: THP-1 or HL-60 stained with DiI; red: Cy5-cRGDLs; PC12 was not stained.

  • Fig. 5 Pharmacodynamic evaluation on liposome loading with ER in vivo.

    (A) Coronal sections stained with TTC of ischemic rats treated with liposomal ER 0, 3, 6, 12, and 24 hours after reperfusion. (B) Quantitative results of infarct volume of various formulations and control group. **P < 0.01 and *P < 0.05; N.S. means no significant difference. (C) Immunofluorescent labeling for NeuN in the cerebral cortex. The bar represents 50 μm. (D) Balance beam score of I/R rats treated with different formulations at 3 hours after reperfusion. (E) Latencies to find the hidden platform in the water maze during five consecutive training days. n = 5. (F) The percentage of time spent in the target quadrant within 60 s in the MWM test. n = 5. (G) Typical traces from a water maze experiment recorder. n = 5.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaau8301/DC1

    Supplementary Materials and Methods

    Fig. S1. The synthesis of cRGD peptide with PEG-DSPE assayed by MALDI-TOF.

    Fig. S2. TEM photos and size distribution of various liposomes.

    Fig. S3. Particle size of PLs/cRGDLs in different positon of the suspension or different medium by dynamic light scattering at different time.

    Fig. S4. Derived count rate of PLs/cRGDLs in different positon of the suspension or different medium by dynamic light scattering at different time.

    Fig. S5. The influence of liposomes and antibodies on the migration of M/Ns.

    Fig. S6. Location of liposomes in ischemic brains of I/R rats 12 hours after reperfusion.

    Fig. S7. Confocal microscope images of comigration of Cy5-PLs/Cy5-cRGDLs with M/Ns across cerebral vessels in the nonischemic hemisphere 24 hours after reperfusion.

    Fig. S8. Transfer mechanisms of the liposomes from immune cells to neuron cells.

    Fig. S9. TEERs of HBMEC in comigration and inhibition assay.

    Fig. S10. Confocal microscope images of cellular location of Cy5-NBD-cRGDLs in M/Ns.

    Table S1. Characterization of all liposomes used in the study.

    Table S2. The pharmacokinetics parameters of I125-labeled PG.

    Reference (41)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. The synthesis of cRGD peptide with PEG-DSPE assayed by MALDI-TOF.
    • Fig. S2. TEM photos and size distribution of various liposomes.
    • Fig. S3. Particle size of PLs/cRGDLs in different positon of the suspension or different medium by dynamic light scattering at different time.
    • Fig. S4. Derived count rate of PLs/cRGDLs in different positon of the suspension or different medium by dynamic light scattering at different time.
    • Fig. S5. The influence of liposomes and antibodies on the migration of M/Ns.
    • Fig. S6. Location of liposomes in ischemic brains of I/R rats 12 hours after reperfusion.
    • Fig. S7. Confocal microscope images of comigration of Cy5-PLs/Cy5-cRGDLs with M/Ns across cerebral vessels in the nonischemic hemisphere 24 hours after reperfusion.
    • Fig. S8. Transfer mechanisms of the liposomes from immune cells to neuron cells.
    • Fig. S9. TEERs of HBMEC in comigration and inhibition assay.
    • Fig. S10. Confocal microscope images of cellular location of Cy5-NBD-cRGDLs in M/Ns.
    • Table S1. Characterization of all liposomes used in the study.
    • Table S2. The pharmacokinetics parameters of I125-labeled PG.
    • Reference (41)

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