⁸⁹Zr-Labeled Multifunctional Liposomes Conjugate Chitosan for PET-Trackable Triple-Negative Breast Cancer Stem Cell Targeted Therapy

Figures & data

Figure 1 CD44 overexpression in TNBC CSCs and tissues. (A) 3D mammosphere after 7 days of culture (magnification, ×400). (B) CD44⁺ CSCs in TNBC 3D mammospheres. (C) CD44 staining in TNBC tissues. The red fluorescence of CD44 was observed on the membranes of most of the tumor cells (excitation 465–495 nm, emission 590 nm). The cell nuclei were visualized as blue fluorescence (excitation 330–380 nm, emission 420 nm).

Abbreviations: CD, cluster of differentiation; TNBC, triple-negative breast cancer; CSC, cancer stem cell; PE, p-phycoerythrin; APC, allophycocyanin.

Figure 2 Molecular docking of CD44 and CS. (A) Chemical structures of CD44 and CS. (B) Schema of CS binding to CD44 protein. (C) The amino acid sequence of CD44 that interacts with CS. (D) CD44 interaction with CS via hydrogen bonding.

Abbreviations: CS, chitosan; CD, cluster of differentiation; HIS, histidine; ASN, asparagine; CYS, cysteine; ILE, isoleucine; ARG, arginine; TYR, tyrosine; GLU, glutamic acid; THR, threonine; ASP, aspartic acid.

Table 1 Binding Free Energy Between CS and CD44

Force	Energy (kJ/mol)
Van der Waals force	−174.065
Electrostatic force	−485.916
Polar solvation free energy	238.583
Non-polar solvation free energy	−20.908
Total free energy	−442.306

Abbreviations: CS, chitosan; CD, cluster of differentiation.

Figure 3 Molecular dynamics simulations. (A) The RMSD of the simulation calculated over 100 ns. (B) The RMSF of the simulation calculated over 100 ns. (C) The Rg of the simulation calculated over 100 ns.

Abbreviations: RMSD, root-mean-square deviation; RMSF, root-mean-square fluctuation; Rg, radius of gyration.

Figure 4 Characterization of CS-MLPs. (A) Morphology of blank liposomes. (B) Morphology of CS-MLPs. (C) Hydrodynamic size of blank liposomes. (D) Hydrodynamic size of CS-MLPs. (E) Stability of CS-MLPs in storage at 4°C for 3 weeks.

Abbreviations: CS, chitosan; MLP, multifunctional liposome.

Figure 5 Uptake of CS-MLPs by CD44 overexpressing cells and distribution in vivo. (A) Fluorescence images of the pre-blocking and target groups. (B) Micro PET images of nude mice bearing subcutaneously implanted human xenografts of MDA-MB-231 cells at 4, 8, 24, 48, and 96 h. Images show high levels of ⁸⁹Zr-labeled CS-MLPs accumulation in the tumor (left side of axillary). Yellow arrows indicate the tumor. (C) Histological images of heart, liver, spleen, lung, and kidney.

Abbreviations: CS, chitosan; MLP, multifunctional liposome; CD, cluster of differentiation; PET, positron emission tomography; ⁸⁹Zr, zirconium-89; HA, hyaluronic acid; Rh, rhodamine B; DAPI, 4’,6-diamidino-2-phenylindole; DIC, differential interference contrast; SUV, standard uptake value

Table 2 Accumulation of Radioactivity (%ID/g) in ROI in the Tissues of Mice at Different Time Points (n = 3, Mean ± SD)

Time (h)	Tumor	Heart	Spleen	Kidney	Lung	Liver
2	3.46±0.33	10.40±0.96	8.13±0.22	7.33±1.05	4.25±1.03	17.61±1.45
6	4.13±0.24	12.27±1.01	10.69±1.22	6.43±2.14	3.83±0.83	16.12±1.24
24	6.61±0.30*	6.85±0.39	5.82±2.08	5.64±1.62	3.29±0.90	13.30±1.15
48	7.52±0.43	6.13±0.29	6.47±0.86	4.16±1.34	3.38±2.43	12.31±1.02
96	7.04±1.00	5.37±0.48	4.7±1.23	2.57±1.16	1.69±0.58	10.90±2.39

Notes: *Compared with the accumulation of radioactivity in ROI at 4 h, p<0.05.

Abbreviations: ID/g, injected dose per gram of tissue; ROI, regions of interest.

Figure 6 Anti-tumor activity of ⁸⁹Zr@CS-GA-MLPs in vivo. (A) Body weight of control group, non-target group, and target group during 28 days of treatment. (B) Tumor volume of control group, non-target group, and target group during 28 days of treatment (* p<0.05). (C) Histological images of tumors of control group, non-target group, and target group after 28 days of treatment. Black arrows indicate the tumor tissues, and red arrows indicate the necrosis tissues.

Abbreviations: CS, chitosan; MLP, multifunctional liposome; ⁸⁹Zr, zirconium-89.

Scheme 1 Scheme of formation, and accumulation of ⁸⁹Zr-labeled, GA-loaded, CS-decorated liposomes as a multifunctional targeted therapy system. PET imaging was used to trace the biodistribution of liposomes. The binding mechanism was analyzed by molecular docking.

Abbreviations: CS, chitosan; PET, positron emission tomography; CD, cluster of differentiation; TNBC, triple-negative breast cancer; GA, gambogic acid; ⁸⁹Zr, zirconium-89.