In vivo Distribution and Metabolism of Calcium Phosphate Nanomaterials Based on Fluorescent Labeling with Rare Earth Europium Ions

doi:10.15541/jim20240504

Abstract

Abstract:

Nano-calcium phosphate (nCaP) has potential applications in nanomedicine fields such as drug delivery, bioimaging, antibacterial treatment, and bone formation promotion. However, its distribution and metabolic patterns within the body are not yet fully understood and require further in-depth research. This study employs a rare earth europium ion fluorescence labeling method and uses tumor-bearing mice as a model to investigate the distribution and metabolism of two sizes of nCaP (nanodots NDs: (2.53±0.63) nm; nanoparticles NPs: (107.76±25.37) nm×(17.66±1.63) nm) in the liver, spleen, lung, kidney, and tumor tissue. The results showed that after tail vein injection of 200 μL with a mass concentration of 1.5 mg/mL nCaP into tumor-bearing nude rats for 4 h, CaP NPs were primarily distributed in the liver and spleen, accounting for 65.70% and 29.32%, respectively, with 3.83% in the lung, while only 0.84% and 0.32% in the kidney and tumor. This suggests that larger CaP NPs are more easily captured by phagocytes within the reticuloendothelial system (RES). In contrast, compared to CaP NPs, accumulation of CaP NDs in the liver, spleen, and lung decreased significantly by 89.40%, 87.00%, and 88.89%, respectively, while their accumulation in the kidney and tumor increased by 3.67 and 3.06 times. This indicates that smaller particle size facilitates CaP NDs in glomerular filtration for urinary excretion and enhances their tumor-targeting capability. The clearance rates (CLz) of CaP NDs in the liver, spleen, and lung were 6.60, 4.14, and 2.40 times higher than that of CaP NPs, respectively, and 42.29% in the kidney. This indicates that reduced size of CaP NDs facilitates rapid metabolism by phagocytes in the liver, spleen, and lung but also results in reabsorption in the renal tubules. In tumor, the CLz of CaP NDs decreased by 91.9%, much smaller than that of CaP NPs, suggesting that the smaller CaP NDs exhibit significantly enhanced tumor targeting and retention capability. In the meantime, a physiologically based pharmacokinetic (PBPK) model incorporating particle size factors was preliminarily established for tumor-bearing mice to simulate the distribution of nano-calcium phosphate. The model's predictive fit (R²) for CaP NDs and CaP NPs in tumor sites reached 0.925 and 0.827, respectively. This study provides promising support for understanding in vivo distribution and metabolic patterns of nCaP and applying potential in medicine.

Key words: nano-calcium phosphate, size effect, fluorescent labeling, tissue distribution, physiologically based pharmacokinetic model

CLC Number:

TQ174

TANG Xinli, DING Ziyou, CHEN Junrui, ZHAO Gang, HAN Yingchao. In vivo Distribution and Metabolism of Calcium Phosphate Nanomaterials Based on Fluorescent Labeling with Rare Earth Europium Ions[J]. Journal of Inorganic Materials, 2025, 40(7): 754-764.

Figures/Tables 8

Fig. 1 PBPK model of tumor-bearing mice PCs indicate phagocytic cells; TCs indicate tumor cells

Fig. 2 Characterizations of CaP NDs and CaP NPs (a-d) TEM images of CaP NDs (a), Eu:CaP NDs (b), CaP NPs (c), and Eu:CaP NPs (d); (e, f) Particle size statistical distributions of CaP NDs (e) and Eu:CaP NDs (f); (g-j) Length (g, i) and width (h, j) of CaP NPs (g, h) and Eu:CaP NPs (i, j); (k) DLS particle size distribution; (l) XRD patterns; (m) FT-IR spectra; (n) Europium ion release curves of Eu:CaP NDs and Eu:CaP NPs

Table 1 Elemental content and molar ratio of Eu:CaP NDs and Eu:CaP NPs

Sample	Value	Ca/(mg·L^-1)	P/(mg·L^-1)	Eu/(mg·L^-1)	(Eu/(Ca+Eu))/%	(Ca+Eu)/P
Eu:CaP NDs	Actual	8.72	4.53	2.82	7.86	1.61
Eu:CaP NDs	Theoretical	—	—	—	8.00	1.67
Eu:CaP NPs	Actual	22.50	11.85	7.23	7.82	1.59
Eu:CaP NPs	Theoretical	—	—	—	8.00	1.67

Fig. 3 Biosafety evaluation of CaP NDs and CaP NPs (a) Cell viability; (b) Hemolysis rate; (c, d) Blood biochemistry analysis (ALT indicates alanine transaminase, AST indicates aspartate aminotransferase, CR indicates creatinine, and BUN indicates blood urea nitrogen); (e) H&E staining images of heart, liver, spleen, lung, and kidney organs in different groups with scale bar indicating 100 μm; (f) CLSM images of BMSC co-cultured with CaP NPs and CaP NDs for 24 h, respectively. Red: CaP NPs or CaP NDs fluorescence image excited at 458 nm. Blue: Cell nuclei stained with DAPI. Scale bar in (f) is 50 μm. Colorful figures are available on website

Fig. 4 Biodistributions of CaP NDs and CaP NPs in different tissues after injection for different durations Colorful figures are available on website

Fig. 5 CaP NDs and CaP NPs metabolic patterns in different tissues (a-e) Biodistributions of CaP NDs and CaP NPs in liver (a), spleen (b), lung (c), kidney (d), and tumor (e); (f) Clearance rate of CaP NDs and CaP NPs in different tissues

Table 2 Pharmacokinetic parameters of CaP NDs and CaP NPs in organ and tumor site of tumor-bearing mice

Sample	Parameter	Liver	Spleen	Lung	Kidney	Tumor
CaP NDs	AUC(0-t)/(mg·h·L^-1)	157.78	71.35	19.34	70.61	20.31
CaP NDs	CLz/(L·h^-1·kg^-1)	0.033	0.091	0.235	0.144	0.146
CaP NPs	AUC(0-t)/(mg·h·L^-1)	1347.46	578.05	66.74	19.85	6.07
CaP NPs	CLz/(L·h^-1·kg^-1)	0.005	0.022	0.098	0.341	1.796

Fig. 6 Establishment and optimization of PBPK model for tumor bearing sites (a, b) PBPK model of CaP NDs (a) and CaP NPs (b) in the tumor; (c, d) Linear regression results between logarithmically transformed measured and simulated concentrations of CaP NDs (c) and CaP NPs (d) with R2 indicating the correlation coefficient; (e) Prediction of the distribution of nCaP with different sizes in tumor

References 41

[1]	SOKOLOVA V, EPPLE M. Biological and medical applications of calcium phosphate nanoparticles. Chemistry - A European Journal, 2021, 27(27): 7471.
[2]	LI B, XU W F, LIAO X L. Research progress in calcium phosphate microspheres for bone defect repair. Journal of Inorganic Materials, 2014, 29(10): 1009. DOI
[3]	CHEN X, LI H Z, MA Y H, et al. Calcium phosphate-based nanomaterials: preparation, multifunction, and application for bone tissue engineering. Molecules, 2023, 28(12): 4790.
[4]	NIE L, HOU M J, WANG T W, et al. Nanostructured selenium- doped biphasic calcium phosphate with in situ incorporation of silver for antibacterial applications. Scientific Reports, 2020, 10: 13738.
[5]	刘静霆, 韩颖超, 李世普, 等. 羟基磷灰石纳米粒子负载阿霉素的体外抗肿瘤活性研究. 中国生物医学工程学报, 2008, 27(4): 572.
[6]	CHEN F, HUANG P, ZHU Y J, et al. Multifunctional Eu³⁺/Gd³⁺ dual-doped calcium phosphate vesicle-like nanospheres for sustained drug release and imaging. Biomaterials, 2012, 33(27): 6447.
[7]	LI C Y, DING Z Y, HAN Y C. In vitro antibacterial and osteogenic properties of manganese doped nano hydroxyapatite. Journal of Inorganic Materials, 2024, 39(3): 313.
[8]	LI C Y, DING Z Y, HAN Y C. Mn-doped nano-hydroxyapatites as theranostic agents with tumor pH-amplified MRI-signal capabilities for guiding photothermal therapy. International Journal of Nanomedicine, 2023, 18: 6101. DOI PMID
[9]	JACOBS A, RENAUDIN G, CHARBONNEL N, et al. Copper- doped biphasic calcium phosphate powders: dopant release, cytotoxicity and antibacterial properties. Materials, 2021, 14(9): 2393.
[10]	ZHAO R B, REN X Y, XIE C G, et al. Towards understanding the distribution and tumor targeting of sericin regulated spherical calcium phosphate nanoparticles. Microscopy Research and Technique, 2017, 80(3): 321. DOI PMID
[11]	KOLLENDA S A, KLOSE J, KNUSCHKE T, et al. In vivo biodistribution of calcium phosphate nanoparticles after intravascular, intramuscular, intratumoral, and soft tissue administration in mice investigated by small animal PET/CT. Acta Biomaterialia, 2020, 109: 244.
[12]	ADAMIANO A, IAFISCO M, SANDRI M, et al. On the use of superparamagnetic hydroxyapatite nanoparticles as an agent for magnetic and nuclear in vivo imaging. Acta Biomaterialia, 2018, 73: 458.
[13]	ÁLAMO P, PALLARÈS V, CÉSPEDES M V, et al. Fluorescent dye labeling changes the biodistribution of tumor-targeted nanoparticles. Pharmaceutics, 2020, 12(11): 1004.
[14]	ALTINOǦLU E I, RUSSIN T J, KAISER J M, et al. Near-infrared emitting fluorophore-doped calcium phosphate nanoparticles for in vivo imaging of human breast cancer. ACS Nano, 2008, 2(10): 2075.
[15]	LLOP J, GÓMEZ-VALLEJO V, GIBSON N. Quantitative determination of the biodistribution of nanoparticles: could radiolabeling be the answer? Nanomedicine, 2013, 8(7): 1035.
[16]	JEONG H J, LEE B C, AHN B C, et al. Development of drugs and technology for radiation theragnosis. Nuclear Engineering and Technology, 2016, 48(3): 597.
[17]	LI P Z, WANG D D, HU J, et al. The role of imaging in targeted delivery of nanomedicine for cancer therapy. Advanced Drug Delivery Reviews, 2022, 189: 114447.
[18]	WANG T T, ZHANG D, SUN D, et al. Current status of in vivo bioanalysis of nano drug delivery systems. Journal of Pharmaceutical Analysis, 2020, 10(3): 221.
[19]	XIE Y F, PERERA T S H, LI F, et al. Quantitative detection method of hydroxyapatite nanoparticles based on Eu³⁺ fluorescent labeling in vitro and in vivo. ACS Applied Materials & Interfaces, 2015, 7(43): 23819.
[20]	HAN Y C,XING Q G, WANG X Y. Quantitative detection method of rare earth doped calcium phosphate fluorescent nanometer particles in organisms: US921123B2. 2024-03-05.
[21]	DING Z Y, XING Q G, FAN Y R, et al. Polyacrylic acid complexes to mineralize ultrasmall europium-doped calcium phosphate nanodots for fluorescent bioimaging. Materials & Design, 2022, 221: 111008.
[22]	LE A D, WEARING H J, LI D S. Streamlining physiologically- based pharmacokinetic model design for intravenous delivery of nanoparticle drugs. CPT: Pharmacometrics & Systems Pharmacology, 2022, 11(4): 409.
[23]	DENG L J, LIU H, MA Y S, et al. Endocytosis mechanism in physiologically-based pharmacokinetic modeling of nanoparticles. Toxicology and Applied Pharmacology, 2019, 384: 114765.
[24]	LI M, ZOU P, TYNER K, et al. Physiologically based pharmacokinetic (PBPK) modeling of pharmaceutical nanoparticles. The AAPS Journal, 2017, 19(1): 26.
[25]	CHENG Y H, HE C L, RIVIERE J E, et al. Meta-analysis of nanoparticle delivery to tumors using a physiologically based pharmacokinetic modeling and simulation approach. ACS Nano, 2020, 14(3): 3075.
[26]	ZHANG S Q, MA X Y, SHA D Y, et al. A novel strategy for tumor therapy: targeted, PAA-functionalized nano-hydroxyapatite nanomedicine. Journal of Materials Chemistry B, 2020, 8(41): 9589.
[27]	CHENG X, XU Y R, ZHANG Y, et al. Glucose-targeted hydroxyapatite/indocyanine green hybrid nanoparticles for collaborative tumor therapy. ACS Applied Materials & Interfaces, 2021, 13(31): 37665.
[28]	BERNIER A, TOBIAS T, NGUYEN H, et al. Vascular and blood compatibility of engineered cationic cellulose nanocrystals in cell-based assays. Nanomaterials, 2021, 11(8): 2072.
[29]	SREENIVASAGAN S, SUBRAMANIAN A K, MOHANRAJ K G, et al. Assessment of toxicity of green synthesized silver nanoparticle-coated titanium mini-implants with uncoated mini- implants: comparison in an animal model study. The Journal of Contemporary Dental Practice, 2024, 24(12): 944.
[30]	高绪聪, 柴振海, 张宗鹏. 药物性肝损伤的生物标志物及其评价的研究进展. 中国药理学与毒理学杂志, 2012, 26(5): 692. DOI
[31]	KASHANI K, ROSNER M H, OSTERMANN M. Creatinine: from physiology to clinical application. European Journal of Internal Medicine, 2020, 72: 9. DOI PMID
[32]	ALMEIDA J P M, CHEN A L, FOSTER A, et al. In vivo biodistribution of nanoparticles. Nanomedicine, 2011, 6(5): 815.
[33]	祁禹鸣, 徐铭泽, 杜步婕. 纳米材料在肾脏中的应用与清除机制. 广州医药, 2023, 54(1): 1. DOI
[34]	CHOI J S, CAO J F, NAEEM M, et al. Size-controlled biodegradable nanoparticles: preparation and size-dependent cellular uptake and tumor cell growth inhibition. Colloids and Surfaces B: Biointerfaces, 2014, 122: 545.
[35]	LEDFORD B T, WYATT T G, VANG J, et al. Effects of particle size, charge, shape, animal disease state, and sex on the biodistribution of intravenously administered nanoparticles. Particle & Particle Systems Characterization, 2023, 40(7): 2300001.
[36]	MELLOR R D, UCHEGBU I F. Ultrasmall-in-nano: why size matters. Nanomaterials, 2022, 12(14): 2476.
[37]	WEI Y C, QUAN L, ZHOU C, et al. Factors relating to the biodistribution & clearance of nanoparticles & their effects on in vivo application. Nanomedicine, 2018, 13(12): 1495.
[38]	TANG L, YANG X J, YIN Q, et al. Investigating the optimal size of anticancer nanomedicine. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(43): 15344. DOI PMID
[39]	WANG J, LIU G. Imaging nano-bio interactions in the kidney: toward a better understanding of nanoparticle clearance. Angewandte Chemie International Edition, 2018, 57(12): 3008.
[40]	ZHAO Y T, WANG Y, RAN F, et al. A comparison between sphere and rod nanoparticles regarding their in vivo biological behavior and pharmacokinetics. Scientific Reports, 2017, 7: 4131.
[41]	董立乐.几种硫属及稀土纳米材料的制备与癌症诊疗探索. 合肥: 中国科学技术大学博士学位论文, 2019.