Design and inflammation-targeting efficiency assessment of an engineered liposome-based nanomedicine delivery system targeting E-selectin

doi:10.12122/j.issn.1673-4254.2025.05.14

Abstract

Abstract:

Objective To develop an E-selectin-targeting nanomedicine delivery system that competitively inhibits E-selectin-neutrophil ligand binding to block neutrophil adhesion to vessels and suppress their recruitment to the lesion sites. Methods Doxorubicin hydrochloride (DOX)-loaded liposomes (IEL-Lip/DOX) conjugated with E-selectin-affinity peptide IELLQARC were developed using a post-insertion method. Two formulations [2-1P: Mol(PC): Mol(DPI)=100:1; 2-3P: 100:3] were prepared and their modification density and in vitro release characteristics were determined. Their targeting efficacy was assessed in a cell model of LPS-induced inflammation, a mouse model of acute lung injury (ALI), a rat femoral artery model of physical injury-induced inflammation, and a zebrafish model of local inflammation. Results The prepared IEL-Lip/DOX 2-1P and 2-3P had peptide modification densities of 4.76 and 7.57 pmoL/cm², respectively. Compared with unmodified liposomes, IEL-Lip/DOX exhibited significantly reduced 48-h cumulative release rates at pH 5.5. In the inflammation cell model, IEL-Lip/DOX showed increased uptake by activated inflammatory endothelial cells, and 2-1P exhibited a higher trans-endothelial ability. In ALI mice, the fluorescence intensity of IEL-Lip/Cy5.5 increased significantly in lung tissues by 53.71% [Z-(2-1P)] and 93.41% [Z-(2-3P)], and 2-1P had an increased distribution by 24.19% in the inflammatory lung tissue compared to normal mouse lung tissue. In rat femoral artery models, 2-1P had greater injured/normal vessel fluorescence intensity contrast. In the zebrafish models, both 2-1P and 2-3P showed increased aggregation at the site of inflammation. Conclusion This E-selectin-targeting nanomedicine delivery system efficiently targets activated inflammatory endothelial cells to increase drug concentration at the inflammatory site, which sheds light on new strategies for treating neutrophil-mediated inflammatory diseases and practicing the concept of "one drug for multiple diseases".

Key words: E-selectin, IEL short peptides, doxorubicin hydrochloride-loaded liposomes, targeted delivery

Yumeng YE, Bo YU, Shasha LU, Yu ZHOU, Meihong DING, Guilin CHENG. Design and inflammation-targeting efficiency assessment of an engineered liposome-based nanomedicine delivery system targeting E-selectin[J]. Journal of Southern Medical University, 2025, 45(5): 1013-1022.

Figures/Tables 6

Tab.1 Particle size, polydispersity index (PDI), and potential of modified liposomes for each formulation (n=3)

Groups	Size (nm)	PDI	Zeta (mV)
Control	93.84±1.96	0.11±0.01	-1.66±0.41
2-1P	81.36±0.28	0.08±0.05	-25.30±0.40
2-3P	83.21±0.52	0.04±0.03	-23.87±0.12

Tab.2 Peptide modification density on liposome surface

Groups	Moles of DPI (μmol)	Liposome's area (cm²)	DPI modification density (pmol/ cm²)
2-1P	0.04	8966.09	4.76
2-3P	0.13	17718.10	7.57

Fig.1 Release profiles of each formulation at pH 5.5, pH 6.5, and pH 7.4. A: Cumulative release rate of PEGylated DOX liposomes (P) within 48 h. B: Cumulative release rate of 2-1P formulation of IEL-Lip/DOX within 48 h. C: Cumulative release rate of 2-3P formulation of IEL-Lip/DOX within 48 h. D: Release of the different formulations at different pH values after 48 h. *P<0.05, ****P<0.0001 vs P group.

Fig.2 In vitro targeting evaluation of IEL-Lip/DOX. A: Representative fluorogram images (×400) of E-selectin expression and DOX uptake in bEnd.3 inflammatory cells with different formulations of IEL-Lip/DOX. Green represents E-selectin labeling on the cell membrane, blue represents the DAPI-stained cell nucleus, and red represents DOX. B: Fluorogram (×400) of DOX uptake by HUVEC inflammatory cells. Blue represents the DAPI-stained cell nucleus, and red represents DOX. C: Flow cytometry analysis of DOX uptake by activated bEnd.3 and HUVEC cells in response to different formulations of liposomes (****P<0.0001 vs P group). D: Percentage bar chart of variables for drug penetration into the lower chamber in the Transwell simulating an invitro endothelial barrier model (**P<0.01 vs 2-3P group). P: Unmodified PEGylated DOX liposomes; 2-1P: IEL-Lip/DOX prepared in the ratio of Mol(PC): Mol(DPI)=100:1; 2-3P: IEL-Lip/DOX prepared in the ratio of Mol(PC): Mol(DPI)=100:3.

Fig.3 In vivo targeting efficiency of the prepared liposomes in mouse models of acute lung injury (ALI). A: Representative immunofluorescence images (×200) of E-selectin expression in lung tissue sections at different time points after LPS challenge. CK represents lung tissue sections from normal mice, green represents E-selectin labeling, and blue represents nuclear staining with DAPI. B: Quantitative plots of E-selectin immunofluorescence expression and positive area in lung tissue sections at different time points (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs CK group). C: Representative tissue fluorescence images of ALI mice. D: Quantitative histogram of lung tissue fluorescence imaging in ALI mice. The upper panel shows DOX fluorescence intensity and the bottom panel shows Cy5.5 fluorescence intensity. (*P<0.05, **P<0.01 vs Z-Cy5.5 group, n=4). E: Comparison of DOX distribution in tissues and plasma after 2-1P injection in normal and ALI mice (*P<0.05, **P<0.01, ***P<0.001 vs Normal group, n=4). F: Quantification of tissue distribution of differently formulated liposomes in ALI mice (**P<0.01 vs P group, n=4).

Fig.4 In vivo targeting efficiency of the prepared liposomes in rat femoral artery physical injury inflammation model. A: Representative in vivo imaging fluorescence maps of femoral artery vasculature after injection of each formulation. B: DOX fluorescence intensity ratio of the injured side vessel to the normal side vessel for each formulation. *P<0.05 vs P group (n=4).

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