Circulation Time-Optimized Albumin Nanoplatform for Quantitative Visualization of Lung Metastasis via Targeting of Macrophages

Abstract

The development of molecular imaging probes to identify key cellular changes within lung metastases may lead to noninvasive detection of metastatic lesions in the lung. In this study, we constructed a macrophage-targeted clickable albumin nanoplatform (CAN) decorated with mannose as the targeting ligand using a click reaction to maintain the intrinsic properties of albumin in vivo. We also modified the number of mannose molecules on the CAN and found that mannosylated serum albumin (MSA) harboring six molecules of mannose displayed favorable pharmacokinetics that allowed high-contrast imaging of the lung, rendering it suitable for in vivo visualization of lung metastases. Due to the optimized control of functionalization and surface modification, MSA enhanced blood circulation time and active/passive targeting abilities and was specifically incorporated by mannose receptor (CD206)-expressing macrophages in the metastatic lung. Moreover, extensive in vivo imaging studies using single-photon emission computed tomography (SPECT)/CT and positron emission tomography (PET) revealed that blood circulation of time-optimized MSA can be used to discern metastatic lesions, with a strong correlation between its signal and metastatic burden in the lung.

Keywords: albumin nanoplatform; blood circulation; lung metastasis; macrophage; noninvasive imaging.

Figure 1

Construction and characterization of MSA. (A) Schematic representation of MSAs using click reaction in AD-Alb with other functional molecules. (B) MALDI-TOF data of AD-Alb according to the reaction ratio. (C) Comparison of DOF on each albumin sample using UV-based analysis and MALDI-TOF-based calculation. (D) UV spectrum of the same albumin sample used in MALDI-TOF measurement of AD-Albs. (E) UV-based calculated data of each type of AD-Albs. (F) UV-spectrum of Man-Alb and AD-Alb according to reaction ratio. (G) Size data of all AD-Alb and Man-Alb using DLS after averaging five measurements. (H) TEM image analysis using selected samples to be used for in vitro and in vivo experiments. Scale bar = 500 nm; 250 nm for the magnified images.

Figure 2

Pharmacokinetics and biodistribution of MSA. (A) Image-based evaluation strategy according to the number of ADIBO groups and mannose for optimized image agent. (B) Alteration of biodistribution according to DOF. The biodistribution of AD-Alb(6) and AD-Alb(11) was compared using serial in vivo PET imaging as a control for selecting the optimal number of Man-Albs. (C) Time–activity curve of AD-Albs and Man-Albs in the blood, liver, and lung. The graph shows the in vivo pharmacokinetics of the imaging agent. n = 5 mice/group. (D) Representative confocal immunofluorescent images of Man(6)-Alb-FL (red) and DAPI (nucleus, blue) in GM-BMM, M-BMM, and 4T1 cells. Scale bar = 50 μm. (E) Quantification of in vitro uptake of AD-Alb(11)-FL or Man(6)-Alb-FL as measured using flow cytometry after incubation for 1 h. n = 4–6/group. Data are normalized to each cell (GM-BMM, M-BMM, and 4T1) treated with AD-Alb(11)-FL. Data show means ± SEM. ****P < 0.0001 using Student’s t test. N.S.: nonsignificant.

Figure 3

Increase in [¹¹¹In]In-Man(6)-Alb-FL signal directly correlates with the metastatic burden in the lung. (A) Schematic showing SPECT/CT imaging of lung metastases in mice after intravenous injection of different numbers of 4T1 cancer cells (low met vs high met). (B,C) Different metastatic burdens induced by intravenous injection of low (5 × 10⁴) or high (1 × 10⁵) number of 4T1 cells were confirmed using H&E staining (B) and determining the lesion area of lung metastatic foci and number of lung metastases (C) 14 days after tumor injection. Scale bar = 100 μm. (D) Representative SPECT/CT images (coronal, sagittal, and transverse views) of mice with lower metastases and higher metastases 24 h after [¹¹¹In]In-Man(6)-Alb-FL injection. [¹¹¹In]In-Man(6)-Alb-FL was able to detect lung metastases (LM) with significantly higher signal in lungs from the high met group than in those from the low met group. (E) Ex vivo biodistribution of [¹¹¹In]In-Man(6)-Alb-FL in various organs of mice with lung metastases, expressed as % ID/g. (F) Correlation between [¹¹¹In]In-Man(6)-Alb-FL signal and metastatic burden, as determined by the number of metastatic foci in lungs from 4T1-bearing mice after intravenous tumor injection. n = 3–4 mice/group. Data represent the mean ± SEM. *P < 0.05, **P < 0.01 using Student’s t test.

Figure 4

In vivo imaging of lung metastases in orthotopic mouse breast tumors. (A) Illustration of orthotopic injection of 4T1-luciferase cells, followed by the intravenous injection of [¹¹¹In]In-Man(6)-Alb-FL and SPECT/CT imaging. (B) Tumor-free mice (TF, control) and 4T1-bearing mice (TB, day 28) were injected with [¹¹¹In]In-Man(6)-Alb-FL, and SPECT/CT images were acquired 24 h postinjection. Representative SPECT/CT images (coronal and sagittal views) imaged with [¹¹¹In]In-Man(6)-Alb-FL revealed strong signals in the lung metastases (day 28). (C,D) Representative luminescence (C) and fluorescence (D) images of multiple dissected organs (heart, lung, liver, stomach, spleen, kidney intestine, and tumor) from tumor-free (TF) and 4T1-bearing mice (TB) after SPECT/CT imaging on day 28. (E) SPECT/CT images of 4T1-bearing mice (TB) injected with [¹¹¹In]In-Alb(11)-FL or [¹¹¹In]In-Man(6)-Alb-FL on day 28. While [¹¹¹In]In-Man(6)-Alb-FL signals were detected in the lungs (LM), [¹¹¹In]In-Alb(11)-FL signals were only detected in the heart (He), but not in the lungs. (F) Ex vivo biodistribution of [¹¹¹In]In-Alb(11)-FL and [¹¹¹In]In-Man(6)-Alb-FL in various organs of mice with lung metastases, expressed as % ID/g. (G) Correlation between [¹¹¹In]In-Man(6)-Alb-FL signal and metastatic burden, as determined from the number of metastatic foci in lungs from 4T1-bearing mice after orthotopic tumor injection. n = 4 mice/group. Data represent the mean ± SD **P < 0.01 using Student’s t test.

Figure 5

Confirmation of Man(6)-Alb-FL as a macrophage-targeted probe. (A) Representative H&E stained images of lungs from 4T1-bearing mice injected with Man(6)-Alb-FL 28 days after tumor injection (left). Representative confocal immunofluorescent images showing in vivo colocalization of injected Man(6)-Alb-FL (red) with the CD206⁺ macrophages (green) within the lungs from 4T1-bearing mice, as confirmed in the overlay image (yellow). The white dotted lines indicate metastatic nodules in the lung. Scale bar = 75 μm. Higher magnification view (B) further demonstrates CD206⁺ macrophage-specific uptake (yellow arrowheads) in vivo. DAPI, blue. Scale bar = 10 μm. (C,D) Quantification of CD206⁺ macrophage-specific uptake in vivo using flow cytometry. (C) Man(6)-Alb-FL uptake by each gated CD206^– macrophage (CD45⁺CD11b⁺F4/80⁺CD206^–) and CD206⁺ macrophage (CD45⁺CD11b⁺F4/80⁺CD206⁺) subset was determined from the fold change in the mean fluorescence intensity (MFI) of colabeled fluorescent dye (FNR-648). (D) Fold change in MFI of colabeled FNR-648 on CD206⁺ macrophages from 4T1-bearing mice following injection with Alb(11)-FL or Man(6)-Alb-FL. n = 4–7 mice/group. Data represent the mean ± SEM. *P < 0.05, ****P < 0.0001 using Student’s t test.

Figure 6

Multimodal imaging of Man(6)-Alb-FL in lung metastasis models. (A) Representative SPECT/CT images (MIP) with [¹¹¹In]In-Man(6)-Alb-FL in tumor-free (TF) and 4T1-bearing mice (TB) on days 21 and 28. The images revealed that [¹¹¹In]In-Man(6)-Alb-FL was able to detect lung metastases (LM, yellow arrowheads) at an earlier time (day 21) when metastatic burden was relatively low. A strong signal was also observed in the lymph node (white arrowhead). (B) Quantification of [¹¹¹In]In-Man(6)-Alb-FL signal in the resected lung, expressed as % ID/g. (C) Ex vivo biodistribution of [¹¹¹In]In-Man(6)-Alb-FL in various organs of 4T1-bearing mice 21 days after tumor injection. (D–H) Simultaneous PET/MRI imaging of [⁶⁴Cu]Cu-Man(6)-Alb-FL and CT imaging were performed in tumor-free (TF) and 4T1-bearing mice (TB) on days 21 and 28. (D) Representative PET/MRI images of [⁶⁴Cu]Cu-Man(6)-Alb-FL. (E) Quantification of [⁶⁴Cu]Cu-Man(6)-Alb-FL signal in the resected lung, expressed as % ID/g. (F) Representative H&E stained images and quantification of metastatic lesion area in lungs from 4T1-bearing mice on days 21 and 28. Scale bar = 50 μm. (G,H) MRI (G) and CT (H) images (coronal, transverse, and sagittal views) are also shown. Representative MRI and CT images show strong signals from lung metastases on day 28, while no significant change was detected at an earlier stage (day 21). n = 5–10 mice/group. Data represent mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 using Student’s t test.

Figure 7

Fluorescence imaging of human breast cancer tissues with Man(6)-Alb-FL. (A) Illustration of fluorescence imaging of Man(6)-Alb-FL in resected tumor tissues from breast cancer patients. (B,C) Representative confocal immunofluorescent images of CD206 (green), Man(6)-Alb-FL (red), and DAPI (nucleus, blue) in ER⁺/PR⁺ (n = 12) (B) and triple-negative (TN) (n = 10) (B) human breast tumor sections 2 h after Man(6)-Alb-FL administration. Yellow arrowheads indicate Man(6)-Alb-FL-loaded CD206⁺ cells (B). Higher magnification images of the yellow-outlined area also show Man(6)-Alb-FL uptake in CD206⁺ cells (yellow arrowheads) in a triple-negative breast tumor section (C). Scale bar = 50 μm for images in (B) and (C), and 10 μm for the yellow magnified images in (C). (D) Pie chart indicating the percentage of Man(6)-Alb-FL uptake by CD206⁺ cells in human breast tumor sections. (E) Correlation analysis of CD206⁺ cells and Man(6)-Alb-FL-positive cells (Man(6)⁺ cells) in human breast tumor sections. Black and blue dots indicate ER⁺/PR⁺ (n = 12) and triple-negative (TN) (n = 10) breast tumor sections, respectively. (F) The number of Man(6)-Alb-FL-positive (Man(6)⁺) CD206⁺ cells in ER⁺/PR⁺ (n = 12, black) and triple-negative (TN) (n = 10, blue) human breast tumor sections. (G) The average number of Man(6)⁺CD206⁺ cells in ER⁺/PR⁺ and TN human breast tumor sections. For quantification, 3–6 nonoverlapping images per section were counted. Data show means ± SEM. ****P < 0.0001 using Student’s t-test.