Application of fluorocarbon nanoparticles of 131I-fulvestrant as a targeted radiation drug for endocrine therapy on human breast cancer

Abstract

Background: Breast cancer is the most prevalent malignant tumor among women, with hormone receptor-positive cases constituting 70%. Fulvestrant, an antagonist for these receptors, is utilized for advanced metastatic hormone receptor-positive breast cancer. Yet, its inhibitory effect on tumor cells is not strong, and it lacks direct cytotoxicity. Consequently, there's a significant challenge in preventing recurrence and metastasis once cancer cells develop resistance to fulvestrant.

Method: To address these challenges, we engineered tumor-targeting nanoparticles termed ¹³¹I-fulvestrant-ALA-PFP-FA-NPs. This involved labeling fulvestrant with ¹³¹I to create ¹³¹I-fulvestrant. Subsequently, we incorporated the ¹³¹I-fulvestrant and 5-aminolevulinic acid (ALA) into fluorocarbon nanoparticles with folate as the targeting agent. This design facilitates a tri-modal therapeutic approach-endocrine therapy, radiotherapy, and PDT for estrogen receptor-positive breast cancer.

Results: Our in vivo and in vitro tests showed that the drug-laden nanoparticles effectively zeroed in on tumors. This targeting efficiency was corroborated using SPECT-CT imaging, confocal microscopy, and small animal fluorescence imaging. The ¹³¹I-fulvestrant-ALA-PFP-FA-NPs maintained stability and showcased potent antitumor capabilities due to the synergism of endocrine therapy, radiotherapy, and CR-PDT. Throughout the treatment duration, we detected no notable irregularities in hematological, biochemical, or histological evaluations.

Conclusion: We've pioneered a nanoparticle system loaded with radioactive isotope ¹³¹I, endocrine therapeutic agents, and a photosensitizer precursor. This system offers a combined modality of radiotherapy, endocrine treatment, and PDT for breast cancer.

Keywords: Breast cancer; Cerenkov radiation; Fulvestrant; Nanomedicine; Nuclear medicine; Photodynamic therapy.

Fig. 1

Synthesis and identification of ¹³¹I-fulvestrant: A Results of paper chromatography for ¹³¹I-fulvestrant. B Results of paper chromatography for Na¹³¹I control group. C Chemical structure of ¹³¹I-fulvestrant. D Mass spectrometry identification results for ¹³¹I-fulvestrant. E Molecular sieve column chromatography purification results for ¹³¹I-fulvestrant. F Identification of the stability of ¹³¹I-fulvestrant at different time points using paper chromatography. (Values are means ± s.d., n = 3)

Fig. 2

Characteristics of ¹³¹I-fulvestrant-ALA-PFP-FA-NPs: A structural identification of ¹³¹I-fulvestrant-ALA-PFP-NPs using transmission electron microscopy and light microscopy. B Determination of nanoparticle size using nanoparticle size analyzer. C Measurement of nanoparticle zeta potential using malvern instrument. D Determination of nanoparticle stability (changes in particle size at different time points). E Changes in nanoparticle zeta potential at different time points. F Determination of nanoparticle encapsulation efficiency (assessment of nanoparticle radioactivity). (Values are means ± s.d., n = 3)

Fig. 3

In vitro characteristics of ¹³¹I-fulvestrant-ALA-PFP-FA-NPs: A Ultrasound imaging of nanoparticle morphology before and after LIFU irradiation. B DFY-type ultrasound image quantitative analysis diagnostic device for quantitative analysis of nanoparticle B-mode ultrasound and contrast-enhanced mode grayscale values. C Drug release kinetics of nanoparticles at different time points following LIFU irradiation. D Using confocal microscopy to observe in vitro targeting and transmembrane properties of DiI-labeled nanoparticles (Scale: 10 μm)(Values are means ± s.d., n = 3)

Fig. 4

Cytotoxicity study of ¹³¹I-fulvestrant-ALA-PFP-FA-NPs: A Flow cytometry analysis of apoptosis in different cell groups. B Semi-quantitative analysis of apoptosis in different cell groups. C Cell viability assessment of different cell groups using CCK-8 assay. a: PBS, b: Fulvestrant, c: 131I-fulvestrant, d: 131I-fulvestrant-PFP-NPs,e: 131I-fulvestrant-PFP-FA-NPs, f: 131I-fulvestrant-ALA-PFP-FA-NPs(Values are means ± s.d., n = 3)

Fig. 5

Cerenkov radiation: A Cherenkov radiation signal detection: Cherenkov radiation imaging and semi-quantitative analysis of different drug solutions. B Cherenkov radiation imaging and semi-quantitative analysis of different drug solutions added to MCF-7 cell culture dishes. C Fluorescence imaging of PpIX signal in excised tissues 24 h after nanoparticle injection. D Semi-quantitative analysis of PpIX signal in excised tissues

Fig. 6

Detection of ALA to PpIX conversion and ROS levels: A Observation of red fluorescence of PpIX using confocal microscopy after 2 h of incubation with nanoparticles and MCF-7 cells. Scale = 50 μm. B flow cytometry analysis of ROS Levels in MCF-7 tumor cells after different treatment methods. C Quantitative analysis of ROS levels in MCF-7 tumor cells after different treatment methods using flow cytometry. (Values are means ± s.d., n = 5)

Fig. 7

In vivo and in vitro imaging of ¹³¹I-fulvestrant-ALA-PFP-FA-NPs: A In vivo fluorescence imaging of tumors in mice bearing tumors at different time points following intravenous injection of ¹³¹I-fulvestrant-ALA-PFP-NPs. B Changes in fluorescence signal intensity at corresponding time points within the tumor region. C Fluorescence imaging of excised major organs 24 h after intravenous injection of.¹³¹I-fulvestrant-ALA-PFP-NPs in mice bearing tumors. D Semi-quantitative analysis of average fluorescence intensity in various organs and tumors (Values are Mean ± S.D., n = 5)

Fig. 8

In vivo tumor-targeting capability and antitumor efficacy of ¹³¹I-fulvestrant-ALA-PFP-FA-NPs: A Detection and biodistribution of nanoparticle targeting to tumors at different time points after intravenous injection of ¹²⁵I-fulvestrant-ALA-PFP-NPs Using SPECT/CT. B PET/CT analysis of FDG uptake in tumor tissues before and after various treatment groups. (Values are Mean ± S.D., n = 5)

Fig. 9

In vivo antitumor efects of ¹³¹I-fulvestrant-ALA-PFP-FA-NPs: A Evaluation of in vivo antitumor effects in different drug treatment groups: changes in tumor size following different treatments. B Growth curves of tumors in different drug treatment groups following various treatments. C Survival curves of tumor-bearing mice in different drug treatment groups following various treatments. D Histological evaluation of tumor tissues in different drug treatment groups: he staining, Ki67 staining, and TUNEL staining. a: PBS, b: Fulvestrant, c: 131I-fulvestrant, d: 131I-fulvestrant-PFP-NPs,e: 131I-fulvestrant-PFP-FA-NPs, f: 131I-fulvestrant-ALA-PFP-FA-NPs(Scale bar represents 50 μm for all panels, Values are Mean ± S.D., n = 5)

Fig. 10

In vivo toxicity test: A Safety assessment of drug: HE staining of major organs. B Changes in body weight of experimental mice in each treatment group during administration. C Impact of different drug treatments on liver function biomarkers in mice: alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). D Impact of different drug treatments on renal function biomarkers in mice: blood urea nitrogen (BUN) and creatinine (CRE). E Impact of different drug treatments on hematological parameters in mice: white blood cells (WBC), red blood cells (RBC), platelets (PLT).a:PBS, b:Fulvestrant, c:131I-Fulvestrant, d:131I-fulvestrant-PFP-NPs,e:131I-fulvestrant-PFP-FA-NPs, f:131I-fulvestrant-ALA-PFP-FA-NPs(Scale bar represents 50 μm for all panels, Values are Mean ± S.D., n = 5)