In Vivo Tracking of Adipose-Derived Mesenchymal Stem Cell Therapy for Subacute Liver Injury Using Rare-Earth Down-Conversion Nanoparticles

doi:10.12300/j.issn.1674-5817.2025.199

Abstract

Abstract:

Objective To evaluate the feasibility of using near-infrared-Ⅱ (NIR-Ⅱ) rare-earth down-conversion nanoparticles (DCNPs) as a novel contrast agent for labeling adipose-derived mesenchymal stem cells (ADSCs), and to explore their potential application for in vivo tracking in a mouse model of subacute liver injury. Methods Core-shell DCNPs (NaYF₄:Yb³⁺,Er³⁺@NaYF₄) were synthesized via a high-temperature thermal decomposition method. Water-soluble DCNPs were prepared through surface modification with DSPE-PEG2000, and further conjugated with fluorescein isothiocyanate (FITC) to observe cellular uptake. The CCK-8 assay was used to detect cell viability after co-incubation of ADSCs with DCNPs at 24 h and 48 h. Confocal laser scanning microscopy was employed to evaluate the labeling efficiency of DCNPs in ADSCs across various probe concentrations and incubation periods. Flow cytometry and differentiation assays were employed to evaluate the effects of DCNPs labeling on ADSCs surface antigen phenotype and multidirectional differentiation potential. NIR-Ⅱ fluorescence imaging was adopted to evaluate the dynamic changes of NIR‑Ⅱ fluorescence signals of labeled ADSCs during continuous in vitro culture. The in vivo toxicity of DCNP-labeled ADSCs transplanted via a single tail vein injection into SPF-grade male Sprague-Dawley (SD) rats was evaluated one month after transplantation through complete blood count, serum biochemical analysis, and multi-organ histopathological examination. A subacute liver injury model was induced in SPF-grade male BALB/c mice using carbon tetrachloride (CCl₄) administration. DCNP-labeled ADSCs were administered via tail vein injection, and the homing of ADSCs in vivo was monitored using NIR-Ⅱ fluorescence imaging. The effects of tracking on ADSC therapy were assessed using biochemical analysis of liver function indicators and hematoxylin and eosin (HE) staining. Results The NaYF₄:Yb³⁺/Er³⁺ nanoparticles synthesized in this study exhibited a spherical crystal structure with particle sizes ranging from approximately 20 to 30 nm, and demonstrated good dispersion stability in water. CCK-8 results showed that DCNPs labeling exerted no cytotoxic effect on ADSCs viability after 24 and 48 h of labeling within the concentration range of 0 to 500 μg/mL used in this study. The cell labeling efficiency of DCNPs increased with dosage, while the cellular uptake remained essentially stable within 16 h. The NIR-Ⅱ fluorescence signal of DCNPs remained stable during the early stage of in vitro ADSCs culture. Although the fluorescence intensity decreased on day 3 of culture (P=0.003), detectable fluorescence signals were still observed in the cells on day 8. Flow cytometry and differentiation assays confirmed that DCNPs labeling had no significant adverse effects on the phenotype and differentiation capacity of ADSCs. One month after the transplantation of DCNP-labeled ADSCs into normal SD rats, blood routine tests, serum biochemical analysis, and multi-organ histopathological examination all revealed no obvious toxic reactions in the body. After the DCNP-labeled ADSCs were injected via the tail vein into mice with subacute liver injury, the serum levels of the liver function indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly reduced (both P<0.05), and the infiltration of inflammatory cells in the liver tissue was markedly decreased. Moreover, in vivo tracing results showed that the labeled ADSCs could home and migrate to the liver within 1 h, with the fluorescence signal gradually weakening over time. By 24 h, the fluorescence signal enriched in the liver had significantly decreased. Conclusion DCNPs possess good biocompatibility for ADSC labeling without affecting stem cell therapy. They may enable real-time, high signal-to-noise ratio in vivo tracking of stem cell homing and migration in ADSC therapy for subacute liver injury.

Key words: Mesenchymal stem cells, Rare-earth down-conversion nanoparticles, In vivo tracking, Subacute liver injury, SD rats, BALB/c mice

CLC Number:

LIAN Ronggui,LIAO Naishun. In Vivo Tracking of Adipose-Derived Mesenchymal Stem Cell Therapy for Subacute Liver Injury Using Rare-Earth Down-Conversion Nanoparticles[J]. Laboratory Animal and Comparative Medicine, 2026, 46(5): 716-726. DOI: 10.12300/j.issn.1674-5817.2025.199.

Figures/Tables 5

Figure 1 Characterization， hydrodynamic diameter and zeta potential changes as well as post-modification stability of DCNPs

Note： A， Transmission electron microscopy image； B， X-ray diffraction pattern［PDF 27-0689， standard hexagonal-phase sodium erbium fluoride （β-NaErF₄）as the positive control］； C， Fluorescence emission spectrum； D， Changes in hydrodynamic diameter of down-conversion nanoparticles （DCNPs） and DCNPs@PEG （DCNPs after modification with DSPE-PEG2000-COOH）； E， Changes in zeta potential of DCNPs and DCNPs@PEG； F， Changes in hydrodynamic diameter of DCNPs@PEG during static storage at room temperature in ultrapure water over 5 days.

Figure 2 Verification of the cytotoxicity， uptake， and stability of DCNP-labeled ADSCs

Note： A-B， Effects of different concentrations of down-conversion nanoparticles （DCNPs） on adipose-derived mesenchymal stem cells （ADSCs） viability （A， Co-incubation of DCNPs with ADSCs for 24 h； B， Co-incubation for 48 h）； C， Confocal laser scanning microscope images（CLSM） showing uptake following co-incubation of DCNPs with ADSCs at different concentrations and time points （scale bar=20 μm）； D， Time-dependent changes in the fluorescence signal of DCNP-labeled ADSCs under 980 nm excitation（one-way analysis of variance， ^** P<0.01， ^*** P<0.001， ^**** P<0.000 1）

Figure 3 Effects of DCNPs labeling on the phenotype and multilineage differentiation potential of ADSCs

Note： A， Flow cytometric characterization of surface markers in adipose-derived mesenchymal stem cells （ADSCs）（PE-A，phycoerythrin fluorescence area； FITC-A， fluorescein isothiocyanate fluorescence area. CD29， CD90， and CD105 are positive markers of ADSCs， while CD31 and CD34 are negative markers）. B， Verification of the multilineage differentiation potential of ADSCs （Osteogenesis， osteogenic differentiation confirmed by alizarin red S staining； Chondrogenesis， chondrogenic differentiation confirmed by alcian blue staining； Adipogenesis， adipogenic differentiation confirmed by oil red O staining； the scale bar=100 μm）. ADSCs group， ADSCs not incubated with down-conversion nanoparticles （DCNPs）； ADSCs+DCNPs group， ADSCs co-incubated with DCNPs for 24 h； Negative control group， the negative control sample.

Figure 4 Biocompatibility evaluation of DCNP-labeled ADSCs in vivo

Note： A， Analysis of serum biochemical parameters one month after tail vein injection of adipose-derived mesenchymal stem cells （ADSCs） into normal SD rats. B， Histopathological analysis of major organs from normal rats after ADSCs transplantation （HE staining， scale bar=200 μm）.Normal， normal rats injected with an equivalent volume of normal saline； ADSCs， normal rats injected with ADSCs； ADSCs+DCNPs， normal rats injected with down-conversion nanoparticle （DCNP）-labeled ADSCs. Crea， creatinine； TBil， total bilirubin； PCT， plateletcrit； HCT， hematocrit； LYMPH， lymphocyte percentage； Hgb， hemoglobin； RBC， red blood cell count； GRAN， granulocyte count； WBC， white blood cell count； PLT， platelet count； ALT， alanine aminotransferase； AST， aspartate aminotransferase； ALP， alkaline phosphatase； TG， triglyceride； Glu， glucose.

Figure 4 Biocompatibility evaluation of DCNP-labeled ADSCs in vivo
Note： A， Analysis of serum biochemical parameters one month after tail vein injection of adipose-derived mesenchymal stem cells （ADSCs） into normal SD rats. B， Histopathological analysis of major organs from normal rats after ADSCs transplantation （HE staining， scale bar=200 μm）.Normal， normal rats injected with an equivalent volume of normal saline； ADSCs， normal rats injected with ADSCs； ADSCs+DCNPs， normal rats injected with down-conversion nanoparticle （DCNP）-labeled ADSCs. Crea， creatinine； TBil， total bilirubin； PCT， plateletcrit； HCT， hematocrit； LYMPH， lymphocyte percentage； Hgb， hemoglobin； RBC， red blood cell count； GRAN， granulocyte count； WBC， white blood cell count； PLT， platelet count； ALT， alanine aminotransferase； AST， aspartate aminotransferase； ALP， alkaline phosphatase； TG， triglyceride； Glu， glucose.

Figure 5 Therapeutic efficacy and in vivo NIR-Ⅱ fluorescence imaging of DCNP-labeled ADSCs in mice with subacute liver injury
Note： A， Schematic diagram of adipose-derived mesenchymal stem cells （ADSCs） administration for the treatment of subacute liver injury in BALB/c mice （i.p.， intraperitoneal injection； i.v.， intravenous injection）； B-C， Serum ALT and AST levels （one-way analysis of variance， ^* P<0.05， ^** P<0.01， ^*** P<0.001）； D， Hematoxylin and eosin （HE） staining of liver tissues to evaluate the degree of liver injury and therapeutic efficacy （scale bar=200 μm）. Normal， healthy control mice； Model， mice with induced subacute liver injury； ADSCs， mice with subacute liver injury treated with ADSCs； ADSCs+DCNPs： mice with subacute liver injury treated with down-conversion nanoparticle （DCNP）-labeled ADSCs. E， In vivo tracking of DCNP-labeled ADSCs in two mice with subacute liver injury （Bright， bright-field images acquired without laser excitation； fluorescence signal intensity and in vivo distribution in mice during live imaging at different time points from 0 to 72 h）.

Figure 5 Therapeutic efficacy and in vivo NIR-Ⅱ fluorescence imaging of DCNP-labeled ADSCs in mice with subacute liver injury
Note： A， Schematic diagram of adipose-derived mesenchymal stem cells （ADSCs） administration for the treatment of subacute liver injury in BALB/c mice （i.p.， intraperitoneal injection； i.v.， intravenous injection）； B-C， Serum ALT and AST levels （one-way analysis of variance， ^* P<0.05， ^** P<0.01， ^*** P<0.001）； D， Hematoxylin and eosin （HE） staining of liver tissues to evaluate the degree of liver injury and therapeutic efficacy （scale bar=200 μm）. Normal， healthy control mice； Model， mice with induced subacute liver injury； ADSCs， mice with subacute liver injury treated with ADSCs； ADSCs+DCNPs： mice with subacute liver injury treated with down-conversion nanoparticle （DCNP）-labeled ADSCs. E， In vivo tracking of DCNP-labeled ADSCs in two mice with subacute liver injury （Bright， bright-field images acquired without laser excitation； fluorescence signal intensity and in vivo distribution in mice during live imaging at different time points from 0 to 72 h）.

References 22

[1]
MAZINI L, ROCHETTE L, AMINE M, et al. Regenerative capacity of adipose derived stem cells (ADSCs), comparison with mesenchymal stem cells (MSCs)[J]. Int J Mol Sci, 2019, 20(10): 2523. DOI: 10.3390/ijms20102523.

[2]
MAZINI L, ROCHETTE L, ADMOU B, et al. Hopes and limits of adipose-derived stem cells (ADSCs) and mesenchymal stem cells (MSCs) in wound healing[J]. Int J Mol Sci, 2020, 21(4): 1306. DOI: 10.3390/ijms21041306.

[3]
THAWLEY V. Acute liver injury and failure[J]. Vet Clin N Am Small Anim Pract, 2017, 47(3): 617-630. DOI: 10.1016/j.cvsm. 2016.11.010.

[4]
STERODIMAS A, DE FARIA J, NICARETTA B, et al. Tissue engineering with adipose-derived stem cells (ADSCs): Current and future applications[J]. J Plast Reconstr Aesthet Surg, 2010, 63(11): 1886-1892. DOI: 10.1016/j.bjps. 2009.10.028.

[5]
NADERI N, COMBELLACK E J, GRIFFIN M, et al. The regenerative role of adipose-derived stem cells (ADSC) in plastic and reconstructive surgery[J]. Int Wound J, 2017, 14(1): 112-124. DOI: 10.1111/iwj.12569.

[6]
LEE S, CHAE D S, SONG B W, et al. ADSC-based cell therapies for musculoskeletal disorders: a review of recent clinical trials[J]. Int J Mol Sci, 2021, 22(19): 10586. DOI: 10.3390/ijms221910586.

[7]
蒋励, 陈子晗, 凡勇. 近红外二区荧光探针在活体多重成像中的研究进展[J]. 化学学报, 2024, 82(10): 1069-1085. DOI: 10.6023/A24070218.

JIANG L, CHEN Z H, FAN Y. Research progress on fluorescent probes in the second near-infrared window for in vivo multiplexed imaging[J]. Acta Chim Sin, 2024, 82(10): 1069-1085. DOI: 10.6023/A24070218.

[8]
XU J M, FU M, JI C Y, et al. Plasmonic-enhanced NIR-Ⅱ downconversion fluorescence beyond 1500 nm from core–shell–shell lanthanide nanoparticles[J]. Adv Opt Mater, 2023, 11(19): 2300477. DOI: 10.1002/adom.202300477.

[9]
YANG Y L, WANG S F, LU L F, et al. NIR-Ⅱ chemiluminescence molecular sensor for in vivo high-contrast inflammation imaging[J]. Angew Chem, 2020, 132(42): 18538-18543. DOI: 10.1002/ange.202007649.

[10]
WANG P Y, FAN Y, LU L F, et al. NIR-Ⅱ nanoprobes in-vivo assembly to improve image-guided surgery for metastatic ovarian cancer[J]. Nat Commun, 2018, 9: 2898. DOI: 10.1038/s41467-018-05113-8.

[11]
CHEN H J, WANG L, ZHU H, et al. NIR-Ⅱ fluorescence imaging for in vivo quantitative analysis[J]. ACS Appl Mater Interfaces, 2024, 16(22): 28011-28028. DOI: 10.1021/acsami.4c04913.

[12]
CHEN Y, PEI P, YANG Y W, et al. Noninvasive early diagnosis of allograft rejection by a granzyme B protease responsive NIR-Ⅱ bioimaging nanosensor[J]. Angew Chem Int Ed, 2023, 62(23): e202301696. DOI: 10.1002/anie.202301696.

[13]
WANG C L, LIN H X, GE X G, et al. Dye-sensitized downconversion nanoprobes with emission beyond 1500 nm for ratiometric visualization of cancer redox state[J]. Adv Funct Mater, 2021, 31(16): 2009942. DOI: 10.1002/adfm. 202009942.

[14]
DUO Y Y, ZHAO L, WANG Z G, et al. NIR-Ⅱ fluorophores: from synthesis to biological applications[J]. J Anal Test, 2023, 7(3): 245-259. DOI: 10.1007/s41664-023-00254-2.

[15]
ZHAO M Y, CHEN X Y. Recent advances in NIR-Ⅱ materials for biomedical applications[J]. Acc Mater Res, 2024, 5(5): 600-613.DOI: 10.1021/accountsmr.4c00025.

[16]
麦丽开·阿不力米提, 艾山·艾比布勒, 史深, 等. 复方卡森糖浆缓解D-氨基半乳糖引起的小鼠急性化学性肝损伤[J]. 实验动物与比较医学, 2022, 42(3): 194-200. DOI: 10.12300/j.issn.1674-5817.2021.140.

Merika·Ablimiti, Aishan·Abibule, SHI S, et al. Compound casson syrup alleviates acute chemical liver injury in mice caused by D-galactosamine[J]. Lab Anim Comp Med, 2022, 42(3): 194-200. DOI: 10.12300/j.issn.1674-5817.2021.140.

[17]
SI Y L, ZHAO Y L, HAO H J, et al. MSCs: Biological characteristics, clinical applications and their outstanding concerns[J]. Ageing Res Rev, 2011, 10(1): 93-103. DOI: 10.1016/j.arr.2010.08.005.

[18]
张丽丽, 万鑫, 冀菁荃, 等. 四氯化碳诱导急性肝损伤大鼠模型对心脏的影响[J]. 实验动物与比较医学, 2021, 41(6): 486-492. DOI: 10.12300/j.issn.1674-5817.2021.033.

ZHANG L L, WAN X, JI J Q, et al. Effects of carbon tetrachloride-induced acute liver injury on cardiac pathological changes in a rat model[J]. Lab Anim Comp Med, 2021, 41(6): 486-492. DOI: 10.12300/j.issn.1674-5817.2021.033.

[19]
ROCHETTE L, MAZINI L, MALKA G, et al. The crosstalk of adipose-derived stem cells (ADSC), oxidative stress, and inflammation in protective and adaptive responses[J]. Int J Mol Sci, 2020, 21(23): 9262. DOI: 10.3390/ijms21239262.

[20]
孟雨, 梁冬丽, 郑琳琳, 等. 人肝肿瘤细胞的裸小鼠原位癌建模条件优化及评价[J]. 实验动物与比较医学, 2024, 44(5): 511-522. DOI: 10.12300/j.issn.1674-5817.2024.048.

MENG Y, LIANG D L, ZHENG L L, et al. Optimization and evaluation of conditions for orthotopic nude mouse models of human liver tumor cells[J]. Lab Anim Comp Med, 2024, 44(5): 511-522. DOI: 10.12300/j.issn.1674-5817.2024.048.

[21]
PAPADOPOULOS K S, PIPERI C, KORKOLOPOULOU P. Clinical applications of adipose-derived stem cell (ADSC) exosomes in tissue regeneration[J]. Int J Mol Sci, 2024, 25(11): 5916. DOI: 10.3390/ijms25115916.

[22]
ZHONG Y, WANG J, LU K Q, et al. NIR-Ⅱ luminescent nanoparticles based-Theranostic platform for drug-induced liver injury diagnoses and ameliorates[J]. Chem Eng J, 2024, 488: 151058. DOI: 10.1016/j.cej.2024.151058.

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