Neutrophil/monocyte-targeted dual-ligands modified liposomes delivering puerarin for ischemia stroke treatment

Abstract

Extremely low actual biological effect of insoluble small molecule drugs in ischemia region is a pain point and aporia in Ischemia Stroke (IS) therapy, Although there are studies on single, double-ligands modified liposomes or biomimetic exogenous carriers for directly targeting IS so far, but they often have off-target effects due to they were swallowed, degraded directly (instability) and sabotaged without the help of endogenous cell in the systemic circulation. DSPE-PEG₃₄₀₀-PGP (PGP) and DSPE-PEG₃₄₀₀-cRGD (cRGD) were synthesized via michael addition reaction of maleimide (-Mal) with sulfhydryl (-SH), succinimidyl ester (-NHS) with active primary amine group (-NH₂) respectively. The cRGD and PGP were modified on liposomes by thin film hydration method. Optimal modified ratio of cRGD and PGP were achieved by cellular uptake of HL-60 cells and THP-1 cells in vitro. The precise targeting effects of cRGD/PGP-Lips were examined in a nude MCAO model by an in a vivo imaging system. Puerarin (Pue) was cleverly encapsulated using a calcium acetate gradient to construct cRGD/PGP-Pue-Lips, and its therapeutic efficiency were assessed by rat MCAO model of IS. Optimal modification ratio for both cRGD and PGP were 3 %. The cRGD/PGP-Lips had significant synergetic targeting efficiency in vitro and in vivo, and the encapsulation efficiency of Pue were greater than 80 % through calcium acetate gradient. The cRGD/PGP-Pue-Lips could effectively penetrate BBB and enhance Pue retention on the brain ischemia region in vivo, resulting in a nearly two-fold reduction significantly in cerebral infarction area and edema in rats. In addition, cRGD/PGP-Pue-Lips didn't cause systemic toxicity in major organ tissues. Precise dual-ligands modified nanocarrier targeting endogenous cells is highly competitive as a novel anti-stroke and perspective for treatment of IS.

Keywords: Ischemia stroke; Monocytes; Neutrophils; PGP; cRGD.

Graphical abstract

Fig. 1

Screening for different modification ratios of cRGD and PGP on THP-1 cells and HL-60 cells. (A) Cellular uptake of Blank-Lips, 1 % PGP-Lips, 3 % PGP-Lips, and 5 % PGP-Lips on HL-60 cells. (B) Cellular uptake of Blank-Lips, 1 % cRGD-Lips, 3 % cRGD-Lips, and 5 % cRGD-Lips on THP-1 cells. Data were presented as Mean ± SD, n = 3, ∗∗p < 0.01.1 % PGP-Lips: 1 % DSPE-PEG₃₄₀₀-PGP modified liposomes with loading 5-FAM, 3 % PGP-Lips: 3 % DSPE-PEG₃₄₀₀-PGP modified liposomes with loading 5-FAM, 5 %PGP-Lips: 5 % DSPE-PEG₃₄₀₀-PGP modified liposomes with loading 5-FAM. Similarly, 1 % cRGD-Lips: 1 % DSPE-PEG₃₄₀₀-cRGD modified liposomes with loading 5-FAM, 3 % cRGD-Lips: 3 % DSPE-PEG₃₄₀₀-cRGD modified liposomes with loading 5-FAM, 5 % cRGD-Lips: 5 % DSPE-PEG₃₄₀₀-cRGD modified liposomes with loading 5-FAM.

Fig. 2

(A) Cellular uptake of Blank-Lips, PGP-Lips, cRGD-Lips, and cRGD/PGP-Lips on HL-60 cells. (B) Cellular uptake of Blank-Lips, PGP-Lips, cRGD-Lips, and cRGD/PGP-Lips on THP-1 cells. (C) Quantitative data of cellular uptake (Mean ± SD, n = 3, ∗∗p < 0.01). cRGD/PGP-Lips: both 3 % DSPE-PEG₃₄₀₀-PGP and 3 % DSPE-PEG₃₄₀₀-cRGD modification liposomes.

Fig. 3

Evaluation of the ability of cRGD/PGP-Lips to across BBB in vitro. (A) The BBB model where neutrophils containing cRGD/PGP-Lips spanned monolayer of HUVECs, and transfer of HL-60 cells from upper chamber to basal chamber by confocal microscopy. (B) The fluorescence signal of cRGD/PGP-Lips was green, PC12 cells were stained with wathet CFSE and HL-60 cells were labeled with Hoechst (navy blue). (C, D) Quantitative efficiency of neutrophils crossing HUVECs and neutrophils transferred cRGD/PGP-Lips into neurons by flow cytometric, Q1 represents the number of HL-60 cells in upper chamber, Q2 represents the number of HL-60 cells in basal chamber, Q3 represents the number of neurons contained liposome in basal chamber, Q4 represents the number of total neurons in basal chamber. (E, F) The transmigration percentage of neutrophils with the stimulation of IL-8 in basal chamber by flow cytometric (Mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01).

Fig. 4

The process of cRGD/PGP-Lips delivery during fusion of inflammatory cells with neuronal cells in vitro. (A, B) Confocal microscopy images of HL-60 cells, cRGD/PGP-Lips, Merge, PC12 cells, and HL-60 cells + PC12 cells. Green: HL-60 cells were labeled by Hoechst. Red: cRGD/PGP-Lips. Blue: PC12 cells. Bar represents 10 μm.

Fig. 5

In vivo distribution and precise ischemia region targeting of cRGD/PGP-Lips in a nude model of cerebral ischemia/reperfusion. (A) In vivo fluorescence imaging of nude mice brain area with DIR-labeled under MCAO reperfusion 2 h after BL, cRGD-Lips, PGP-Lips, and cRGD/PGP-Lips were injected. (B) Ex vivo imaging of excised organs and brains 24 h postinjection. (C) Semi-quantitative analysis of the fluorescent intensity of brain (Mean ± SD, n = 3, NS = nonsignificant, ∗∗P < 0.01). (D) The fluorescent image of BL, cRGD-Lips, PGP-Lips, and cRGD/PGP-Lips in the ischemia area at 24 h after administration in vivo. (E) Semi-quantitative analysis of the total fluorescent intensity of brain in 24 h (Mean ± SD, n = 3, ∗∗P < 0.01). BL: Blank-Lips (DIR-labeled unmodified liposomes); cRGD-Lips: DIR-labeled cRGD-Lips; PGP-Lips: DIR-labeled PGP-Lips; cRGD/PGP-Lips: DIR-labeled cRGD/PGP-Lips.

Fig. 6

Preparation, characterization, and release of cRGD/PGP-Pue-Lips. (A) The schematic diagram of the planar structure of cRGD/PGP-Pue-Lips. (B) Morphology of cRGD/PGP-Pue-Lips were obtained by TEM after negative staining with 2 % sodium phosphotungstate solution. Bar: 100 nm. (C) Particle size distribution of cRGD/PGP-Pue-Lips were determined by DLS synchronically. (D) In vitro Pue accumulative release curves from free Pue, Pue Lip, cRGD-Pue Lip, PGP-Pue Lip, cRGD/PGP-Pue-Lips in PBS (pH 7.4) at 37 °C (n = 3, mean ± SD).

Fig. 7

Treatment efficacies of cRGD/PGP-Pue-Lips in MCAO rats. (A) Representative TTC staining of brain sections in sham group, MCAO (control) group, Pue-Lips group, cRGD-Pue-Lips group, PGP-Pue-Lips group, and cRGD/PGP-Pue-Lips group. The non-ischemia region is red, and the infarct region appears in white. (B) (C) Quantification statistics of neurological scores and cerebral infarct area of rats after IS. (D) In vivo T2W-MRI images of cerebral edema area in rats at 24 h post-injection of control, Pue-Lips, cRGD-Pue-Lips, PGP-Pue-Lips, and cRGD/PGP-Pue-Lips. (E) Quantification statistics of cerebral edema area after IS by Imaje J. Data are expressed with mean ± SD, n = 6, ∗P < 0.05, ∗∗P < 0.01.

Fig. 8

H&E microscopic images of heart, liver, spleen, lung, and kidney sections 24 h after Saline, Blank-Lips, cRGD-Lips, PGP-Lips, cRGD/PGP-Lips treatments. Original magnification:40 × .