Early Detection of Brain Metastases from Triple-Negative Breast Cancer with a Tumor-Targeting Dual-Modal MR/NIRF Imaging Probe
Brain metastases (BMs) present a substantial treatment challenge in breast cancer patients, particularly in those with triple-negative breast cancer (TNBC). TNBC, characterized by the absence of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor (HER2) in histochemical examination, represents the most aggressive breast cancer subtype and accounts for approximately half of all breast cancer brain metastases cases. , The blood–brain barrier (BBB) complicates early imaging and effective drug delivery, leading to a poor prognosis of brain metastasis. Patients with TNBC brain metastases have a median survival of only 4.9 months post-diagnosis, significantly shorter than the 10 to 18 months observed in other subtypes. , Therefore, early detection of TNBC brain metastases is crucial in clinical practice. Crossing the BBB is essential for the efficacy of targeted nanoprobes in the molecular imaging of early-stage brain metastasis. Angiopep-2, a 19-amino acid peptide, serves as a ligand for the low-density lipoprotein receptor-related protein 1 (LRP1) on the BBB. This interaction facilitates the crossing of the barrier, enabling the delivery of therapeutic agents to the brain. Angiopep-2 has been explored in preclinical studies for delivering therapeutics across the BBB in the treatment of various neurological disorders, including cancer, Alzheimer’s disease, and Parkinson’s disease. Clinical trials have demonstrated that Angiopep-2 effectively enhances the brain penetration of paclitaxel when conjugated with drugs. However, data remain insufficient regarding the capacity of Angiopep-2 to facilitate the transport of imaging probes across the BBB for early detection of brain metastases. Identifying molecular targets remains a secondary consideration in the molecular imaging of early brain metastasis due to the limited number of recognized targets. The epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein member of the ErbB family. Studies have indicated that EGFR is frequently overexpressed in TNBC, more so than in other subtypes. Previous research has also demonstrated that EGFR plays a significant role in the proliferation and brain metastasis of TNBC. , Moreover, EGFR has been used as a molecular target in molecular imaging and therapeutic applications. For instance, antibodies against EGFR have been conjugated with gold or fluorescent nanoparticles for computed tomography (CT) or optical imaging of lung cancers exhibiting EGFR overexpression. , The GE11 peptide, characterized by the amino acid sequence YHWYGYTPQNVI, has been shown to have high affinity for the breast cancer cell line MDA-MB-231 and the lung cancer cell lines H1299, both known for their EGFR overexpression. Importantly, the small molecular weight of GE11 enhances its ability to cross BBB, offering significant advantages over antibodies for targeting the EGFR in the imaging of early brain metastases. Recently, nanotechnology has demonstrated several advantages in preclinical tumor imaging, such as easy assembly, controllable particle size, tunable circulation lifetime, and enhanced permeability and retention at tumor sites. In this study, we utilized the fifth generation (G5) polyamidoamine (PAMAM) dendrimer to develop an EGFR-targeting nanoprobe for brain metastasis imaging. Nanoparticles based on G5 PAMAM are characterized by their smaller size, high drug loading capacity, and biodegradability. The nanoprobe was designed to facilitate both near-infrared (NIR) and magnetic resonance imaging (MRI) by conjugating NIR783 and Gd 3+ -DTPA to the PAMAM dendrimer, respectively. Brain metastasis was induced in mice using MDA-MB-231 brain-seeking cells through intracardiac injection, with lesion progression monitored via T2-weighted MRI. Imaging of brain metastases with the EGFR-targeted, dual-modality nanoprobe was performed before and after the lesions were detected in T2-weighted MR images. 

Materials All chemical reagents were obtained from Aladdin Reagent (Shanghai, China) unless otherwise specified. The fifth-generation polyamidoamine dendrimer (G5 dendrimer) (MW: 28826Da) and Rhodamine-NHS were purchased from Dendritech Inc. (Midland, MI, USA). The Maleimide-PEG 2k -NHS and PEG-NHS were purchased from JenKem Technology Co. Ltd. (Beijing, China). GE11 peptide and Angiopep-2 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Recombinant human EGFR protein (ab155639) was from Abcam (Cambridge, MA, USA). The human breast cancer brain-seeking cell line MDA-MB-231-BR was kindly provided by Dr. Patricia S. Steeg (National Cancer Institutes of Health, Bethesda, MD, USA). U-87 MG, BT-474 and mouse brain microvascular endothelial cell bEnd.3 were obtained from the American Type Culture Collection (ATCC). GL261 cell lines were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co. Ltd. (Shanghai, China). Fetal bovine serum (FBS), trypsin, penicillin and streptomycin were purchased from ThermoFisher Scientific (New York, USA). Synthesis of the Nanoprobes The nanoprobes were synthesized as described previously. First, the fifth generation (G5) dendrimer was reacted with NHS-PEG 2k -OPSS (in PBS, pH=7.4) (got intermediate 1), then continued to be treated with N-hydroxysuccinimidyl (NHS) esters of rhodamine and IR783 in PBS (pH=7.4, got intermediate 2). Subsequently, intermediate 2 was reacted with DTPA-dianhydride in NaHCO 3 (pH=9.5, got intermediate 4). Then, intermediate 4 was linked with Angiopep-2 or/and GE11 peptide (in PBS, pH=7.4, got intermediate 5). After that, the intermediate 5 was chelated with Gd 2 (CO 3 ) 3 in HEPES (pH=8.3) to get Den-Angio or Den-Angio-GE11. Den-PEG contains G5 dendrimer, PEG, rhodamine, IR783, Gd-DTPA but not Angiopep-2 and GE11 when compared to Den-Angio or Den-Angio-GE11. Characterization of Nanoprobes The average molar ratios among G5 dendrimer (PAMAM), DTPA, Gd 3+ , PEG, Angiopep-2 and GE11 in nanoprobes were determined using the 1 H NMR (Mercury 400 spectrometer, Varian, Germany). The particle size and zeta potentials were measured using a dynamic light scattering detector (Zetasizer, Malvern, USA). The binding constants between the GE11 peptides or Den-Angio-GE11 and EGFR proteins were measured using Surface Plasmon Resonance (SPR). The molecular weight of nanoprobes was determined by Gel Permeation Chromatography (GPC). The nanoparticle shape was determined by transmission electron microscopy (JEOL F200, Japan). Cell Culture All cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (complete medium). Cells were maintained at 37°C in a humidified atmosphere and 5% CO 2 . Brain Metastasis and Glioma in Mice All animals were received care in accordance with Guide for the Care and Use of Laboratory Animals (8th edition) and approved by the Institutional Animal Use and Care Committee of the Medical School of Southeast University. Brain metastasis model was induced by intracardiac injection of MDA-MB-231-BR cells in BALB/c Nude mice (Charles River). Specifically, mice 6-week-old were housed in an SPF environment for one week with a 12-hour day/night rotation before experiments. The mice were then anesthetized by 1% isoflurane in oxygen and injected into the left ventricle (Blood pulsations were visible) with 1 × 10 5 MDA-MB-231BR cells in 100 µL of phosphate-buffered saline (PBS). Brain metastasis progression was monitored by T2-weighted MRI every week after injection. The orthotopic glioma model was induced by GL261 cells in C57BL/6 mice (Charles River). Mice 6-week-old were housed for one week before experiments. The mice were then anesthetized by 1% isoflurane in oxygen and stereotaxic injected 2 × 10 5 GL261 cells into the brains (volume is 20 µL). The lesions of glioma were detected by T2-weighted MRI. Cell Viability Assay The proliferation ability of cells was assayed by CCK-8 agents. Specifically, MDA-MB-231-BR and bEnd.3 cells were collected and plated in 96-well plates at a density of 5,000 cells per well. The cells were incubated at 37°C in a 5% CO 2 atmosphere for 24 hours. After removing the supernatant, nanoprobes were diluted to designated concentrations in complete cell culture medium and added to each well. The cells were then incubated for additional 48 hours. CCK-8 (Cell Counting Kit-8, KeyGEN BioTECH, CN) was then added to each well and incubated for an additional 3 hours. The optical density of each well was subsequently measured at a wavelength of 450 nm (OD450 nm) using a microplate spectrophotometer (ThermoFisher Scientific, USA). Cell viability of normal control group was set 100%, and each group was compared with the normal control. Invasion Assay In vitro Invasion assays of MDA-MB-231-BR cells were conducted using invasion chambers in a 24-well plate (BD, USA). MDA-MB-231-BR cells were collected upon reaching confluency and plated into the upper chamber at a density of 50,000 cells in medium without fetal bovine serum (FBS). The lower chambers were filled with complete medium containing 20% FBS. Once cells are attached to the bottom of the upper chamber, nanoprobes were diluted to designated concentrations and added to the upper chamber. The cells were then incubated at 37°C in a 5% CO 2 atmosphere for an additional 24 hours. After removing the supernatant, cells still attached to the bottom of the upper chamber were scratched with a cotton swab, and the migrated cells on the other side of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. The membrane was then cut down, attached to a slide, and finally examined under a microscope. Targeting of MDA-MB-231-BR Cells The binding rate of nanoprobes to EGFR was measured by flow cytometry analysis. MDA-MB-231-BR, U87, and BT-474 cells were collected, and the cell density was adjusted to 1 × 10 6 cells/mL. Triplicates of no particles, Den-Angio-GE11, Den-Angio, and Den-PEG, diluted to designated concentrations, were added to the cell suspension and incubated for 1 hour at 4°C. Following incubation, the cells were washed twice with PBS, and the binding rate was assessed through rhodamine fluorescence on the cells using the FACSCalibur analysis system (Becton Dickinson, CA, USA). Western Blot Studies Cells growing into confluency were lysed with RIPA buffer (ThermoFisher Scientific, USA); then, protein concentrations were quantified by BCA Protein Assay Kits (ThermoFisher Scientific, USA). The protein was subjected to SDS-PAGE and then transferred to polyvinylidenefluoride membranes (Millipore, USA). Blots were blocked with 5% skim milk (BD Bioscience, USA) and then incubated with primary antibodies to EGFR and beta-actin (Cambridge, MA, USA) overnight at 4°C. Horseradish peroxidase conjugated secondary antibodies (ThermoFisher Scientific, USA) were incubated with blots for 1 hour at room temperature. Binding was detected by chemiluminescence image analyzer (Tano, CN). Immunofluorescence Analysis MDA-MB-231-BR cells were seeded at approximately 2 × 10 4 cells per well in 12-well plates and cultured overnight to reach around 80% confluency. The nanoprobes were diluted in complete cell culture medium to designated concentrations and incubated at 37°C for 30 minutes. After incubation, the cells were washed three times with PBS and fixed with 4% formaldehyde/PBS for 10–15 minutes. The cell nuclei were stained with DAPI Fluoromount-G, and the cells were imaged using a fluorescence microscope. Near-Infrared Fluorescence (NIRF) Imaging For in vitro imaging, MDA-MB-231-BR cells were seeded at approximately 4 × 10 4 cells per well in 96-well plates and cultured overnight. The cells were then incubated with 5 μM of nanoprobes for 4 hours at 37°C. Following incubation, the cells were washed twice with PBS and then fixed with 1% Biowest Agarose for imaging. For in vivo imaging, mice were anesthetized with a continuous supply of 1% isoflurane, subsequently injected with 100 μL of targeted or control nanoprobes through tail vessels (Each mouse was given by 0.1 μmol of probes or controls). NIRF imaging was performed at various time point (0.5, 1, 2, 4, 8, 12, 24, 36 h) using the IVIS Spectrum In vivo Imaging System (Perkin Elmer, USA). The NIRF images were collected at an excitation wavelength of 745 nm and an emission wavelength of 810 nm. To investigate the distribution of nanoprobes in mice, mice were sacrificed by dislocation of cervical vertebrae after 36 h from probes administration, and organs were taken out for imaging by NIRF. MR Imaging MRI was performed by a 7.0-Tesla small animal MR scanner (Bruker PharmaScan, Germany). For cell phantom imaging, 2 × 10 6 cells were suspended in 1 mL of 1% agarose (Oxford, UK) with 5 μmol/L nanoprobes or controls and placed in 10-mm-diameter Eppendorf tubes. A Rapid Acquisition Relaxation with Enhancement sequence (RARE) was used for T1-weighted imaging, with a repetition time of 725 ms and an echo time of 9 ms. The imaging parameters included a matrix size of 256 × 256, a field of view of 2 × 2 cm, and a slice thickness of 1 mm. For in vivo imaging, mice were anesthetized with a continuous supply of 1% isoflurane, and their respiratory rate and body temperature were monitored using a physiology monitor. A spin echo sequence (500/9 ms of repetition time/echo time, 4 averages) was used for T1-weighted imaging, while a fast spin echo sequence (3,000/45 ms of repetition time/echo time, 3 averages) was used for T2-weighted imaging. Twelve axial slices with a slice thickness of 1 mm, a matrix of 256 × 256, and a field of view of 2 × 2 cm was positioned over the brain. Brain Section and Staining Mice were ordinally perfused with normal saline and 4% paraformaldehyde after anesthetization with isoflurane. Brain tissues were obtained after perfusion and fixed in formaldehyde again. Then, brain tissues were dehydrated for 48 hours in 30% sucrose solution. After that, brains were embedded by OTC and quickly frozen in liquid nitrogen. Brain tissues were cut into serial slices with 10 µm in thickness. The sections were then performed with hematoxylin and eosin staining. For fluorescence investigation, sections were washed by PBS and then stained with DAPI. Finally, sections were taken images by fluorescence microscope. Permeability of BBB in BMS Mice were administrated 100 µL of 2% Evans’ blue via tail vein (2 mg/kg). After 12 hours, mice were anesthetized with isoflurane and perfused with normal saline. Brain tissues were dissected, and 100 mg of tissue was minced and then incubated in dimethylformamide at 60°C for 24 hours. The supernatants were subsequently obtained by centrifugation at 1000 revolutions per minute, and the absorbance was measured at 620 nm. Statistical Analysis The statistical analysis was performed using SPSS Statistics 20 software. Numerical data are expressed as means ± standard deviations (SD). For statistical comparisons, an independent-sample t -test was employed. A P value less than 0.05 was considered statistically significant. 

All chemical reagents were obtained from Aladdin Reagent (Shanghai, China) unless otherwise specified. The fifth-generation polyamidoamine dendrimer (G5 dendrimer) (MW: 28826Da) and Rhodamine-NHS were purchased from Dendritech Inc. (Midland, MI, USA). The Maleimide-PEG 2k -NHS and PEG-NHS were purchased from JenKem Technology Co. Ltd. (Beijing, China). GE11 peptide and Angiopep-2 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Recombinant human EGFR protein (ab155639) was from Abcam (Cambridge, MA, USA). The human breast cancer brain-seeking cell line MDA-MB-231-BR was kindly provided by Dr. Patricia S. Steeg (National Cancer Institutes of Health, Bethesda, MD, USA). U-87 MG, BT-474 and mouse brain microvascular endothelial cell bEnd.3 were obtained from the American Type Culture Collection (ATCC). GL261 cell lines were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co. Ltd. (Shanghai, China). Fetal bovine serum (FBS), trypsin, penicillin and streptomycin were purchased from ThermoFisher Scientific (New York, USA). 

The nanoprobes were synthesized as described previously. First, the fifth generation (G5) dendrimer was reacted with NHS-PEG 2k -OPSS (in PBS, pH=7.4) (got intermediate 1), then continued to be treated with N-hydroxysuccinimidyl (NHS) esters of rhodamine and IR783 in PBS (pH=7.4, got intermediate 2). Subsequently, intermediate 2 was reacted with DTPA-dianhydride in NaHCO 3 (pH=9.5, got intermediate 4). Then, intermediate 4 was linked with Angiopep-2 or/and GE11 peptide (in PBS, pH=7.4, got intermediate 5). After that, the intermediate 5 was chelated with Gd 2 (CO 3 ) 3 in HEPES (pH=8.3) to get Den-Angio or Den-Angio-GE11. Den-PEG contains G5 dendrimer, PEG, rhodamine, IR783, Gd-DTPA but not Angiopep-2 and GE11 when compared to Den-Angio or Den-Angio-GE11. 

The average molar ratios among G5 dendrimer (PAMAM), DTPA, Gd 3+ , PEG, Angiopep-2 and GE11 in nanoprobes were determined using the 1 H NMR (Mercury 400 spectrometer, Varian, Germany). The particle size and zeta potentials were measured using a dynamic light scattering detector (Zetasizer, Malvern, USA). The binding constants between the GE11 peptides or Den-Angio-GE11 and EGFR proteins were measured using Surface Plasmon Resonance (SPR). The molecular weight of nanoprobes was determined by Gel Permeation Chromatography (GPC). The nanoparticle shape was determined by transmission electron microscopy (JEOL F200, Japan). 

All cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (complete medium). Cells were maintained at 37°C in a humidified atmosphere and 5% CO 2 . 

All animals were received care in accordance with Guide for the Care and Use of Laboratory Animals (8th edition) and approved by the Institutional Animal Use and Care Committee of the Medical School of Southeast University. Brain metastasis model was induced by intracardiac injection of MDA-MB-231-BR cells in BALB/c Nude mice (Charles River). Specifically, mice 6-week-old were housed in an SPF environment for one week with a 12-hour day/night rotation before experiments. The mice were then anesthetized by 1% isoflurane in oxygen and injected into the left ventricle (Blood pulsations were visible) with 1 × 10 5 MDA-MB-231BR cells in 100 µL of phosphate-buffered saline (PBS). Brain metastasis progression was monitored by T2-weighted MRI every week after injection. The orthotopic glioma model was induced by GL261 cells in C57BL/6 mice (Charles River). Mice 6-week-old were housed for one week before experiments. The mice were then anesthetized by 1% isoflurane in oxygen and stereotaxic injected 2 × 10 5 GL261 cells into the brains (volume is 20 µL). The lesions of glioma were detected by T2-weighted MRI. 

The proliferation ability of cells was assayed by CCK-8 agents. Specifically, MDA-MB-231-BR and bEnd.3 cells were collected and plated in 96-well plates at a density of 5,000 cells per well. The cells were incubated at 37°C in a 5% CO 2 atmosphere for 24 hours. After removing the supernatant, nanoprobes were diluted to designated concentrations in complete cell culture medium and added to each well. The cells were then incubated for additional 48 hours. CCK-8 (Cell Counting Kit-8, KeyGEN BioTECH, CN) was then added to each well and incubated for an additional 3 hours. The optical density of each well was subsequently measured at a wavelength of 450 nm (OD450 nm) using a microplate spectrophotometer (ThermoFisher Scientific, USA). Cell viability of normal control group was set 100%, and each group was compared with the normal control. 

Invasion assays of MDA-MB-231-BR cells were conducted using invasion chambers in a 24-well plate (BD, USA). MDA-MB-231-BR cells were collected upon reaching confluency and plated into the upper chamber at a density of 50,000 cells in medium without fetal bovine serum (FBS). The lower chambers were filled with complete medium containing 20% FBS. Once cells are attached to the bottom of the upper chamber, nanoprobes were diluted to designated concentrations and added to the upper chamber. The cells were then incubated at 37°C in a 5% CO 2 atmosphere for an additional 24 hours. After removing the supernatant, cells still attached to the bottom of the upper chamber were scratched with a cotton swab, and the migrated cells on the other side of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. The membrane was then cut down, attached to a slide, and finally examined under a microscope. 

The binding rate of nanoprobes to EGFR was measured by flow cytometry analysis. MDA-MB-231-BR, U87, and BT-474 cells were collected, and the cell density was adjusted to 1 × 10 6 cells/mL. Triplicates of no particles, Den-Angio-GE11, Den-Angio, and Den-PEG, diluted to designated concentrations, were added to the cell suspension and incubated for 1 hour at 4°C. Following incubation, the cells were washed twice with PBS, and the binding rate was assessed through rhodamine fluorescence on the cells using the FACSCalibur analysis system (Becton Dickinson, CA, USA). 

Cells growing into confluency were lysed with RIPA buffer (ThermoFisher Scientific, USA); then, protein concentrations were quantified by BCA Protein Assay Kits (ThermoFisher Scientific, USA). The protein was subjected to SDS-PAGE and then transferred to polyvinylidenefluoride membranes (Millipore, USA). Blots were blocked with 5% skim milk (BD Bioscience, USA) and then incubated with primary antibodies to EGFR and beta-actin (Cambridge, MA, USA) overnight at 4°C. Horseradish peroxidase conjugated secondary antibodies (ThermoFisher Scientific, USA) were incubated with blots for 1 hour at room temperature. Binding was detected by chemiluminescence image analyzer (Tano, CN). 

MDA-MB-231-BR cells were seeded at approximately 2 × 10 4 cells per well in 12-well plates and cultured overnight to reach around 80% confluency. The nanoprobes were diluted in complete cell culture medium to designated concentrations and incubated at 37°C for 30 minutes. After incubation, the cells were washed three times with PBS and fixed with 4% formaldehyde/PBS for 10–15 minutes. The cell nuclei were stained with DAPI Fluoromount-G, and the cells were imaged using a fluorescence microscope. 

For in vitro imaging, MDA-MB-231-BR cells were seeded at approximately 4 × 10 4 cells per well in 96-well plates and cultured overnight. The cells were then incubated with 5 μM of nanoprobes for 4 hours at 37°C. Following incubation, the cells were washed twice with PBS and then fixed with 1% Biowest Agarose for imaging. For in vivo imaging, mice were anesthetized with a continuous supply of 1% isoflurane, subsequently injected with 100 μL of targeted or control nanoprobes through tail vessels (Each mouse was given by 0.1 μmol of probes or controls). NIRF imaging was performed at various time point (0.5, 1, 2, 4, 8, 12, 24, 36 h) using the IVIS Spectrum In vivo Imaging System (Perkin Elmer, USA). The NIRF images were collected at an excitation wavelength of 745 nm and an emission wavelength of 810 nm. To investigate the distribution of nanoprobes in mice, mice were sacrificed by dislocation of cervical vertebrae after 36 h from probes administration, and organs were taken out for imaging by NIRF. 

MRI was performed by a 7.0-Tesla small animal MR scanner (Bruker PharmaScan, Germany). For cell phantom imaging, 2 × 10 6 cells were suspended in 1 mL of 1% agarose (Oxford, UK) with 5 μmol/L nanoprobes or controls and placed in 10-mm-diameter Eppendorf tubes. A Rapid Acquisition Relaxation with Enhancement sequence (RARE) was used for T1-weighted imaging, with a repetition time of 725 ms and an echo time of 9 ms. The imaging parameters included a matrix size of 256 × 256, a field of view of 2 × 2 cm, and a slice thickness of 1 mm. For in vivo imaging, mice were anesthetized with a continuous supply of 1% isoflurane, and their respiratory rate and body temperature were monitored using a physiology monitor. A spin echo sequence (500/9 ms of repetition time/echo time, 4 averages) was used for T1-weighted imaging, while a fast spin echo sequence (3,000/45 ms of repetition time/echo time, 3 averages) was used for T2-weighted imaging. Twelve axial slices with a slice thickness of 1 mm, a matrix of 256 × 256, and a field of view of 2 × 2 cm was positioned over the brain. 

Mice were ordinally perfused with normal saline and 4% paraformaldehyde after anesthetization with isoflurane. Brain tissues were obtained after perfusion and fixed in formaldehyde again. Then, brain tissues were dehydrated for 48 hours in 30% sucrose solution. After that, brains were embedded by OTC and quickly frozen in liquid nitrogen. Brain tissues were cut into serial slices with 10 µm in thickness. The sections were then performed with hematoxylin and eosin staining. For fluorescence investigation, sections were washed by PBS and then stained with DAPI. Finally, sections were taken images by fluorescence microscope. 

Mice were administrated 100 µL of 2% Evans’ blue via tail vein (2 mg/kg). After 12 hours, mice were anesthetized with isoflurane and perfused with normal saline. Brain tissues were dissected, and 100 mg of tissue was minced and then incubated in dimethylformamide at 60°C for 24 hours. The supernatants were subsequently obtained by centrifugation at 1000 revolutions per minute, and the absorbance was measured at 620 nm. 

The statistical analysis was performed using SPSS Statistics 20 software. Numerical data are expressed as means ± standard deviations (SD). For statistical comparisons, an independent-sample t -test was employed. A P value less than 0.05 was considered statistically significant. 

Synthesis and Characterization of the Nanoprobes The chemical structures and synthetic pathways of nanoprobes are presented in . Briefly, the PAMAM G5 dendrimer was selected as a platform and modified with polyethylene glycol to enhance biocompatibility and prolong the half-life in circulation. The rhodamine and NIR fluorophore NIR783 were conjugated to enable visualization of brain sections in vitro and NIR imaging of the brain in vivo. Then, the EGFR targeting peptide GE11 and BBB-permeable angiopep-2 were functionalized into the PAMAM dendrimer. Additionally, Gd 3+ -DTPA was synthesized to the dendrimer for MR imaging due to its high thermodynamic stability and kinetic inertness. The key physical and chemical properties of the nanoprobes were listed in . It is shown that the average hydrodynamic diameter of the nanoprobe Den-Angio-GE11 was about 8.8nm, which was slightly larger than that of the control groups Den-Angio (7.3 ± 0.16 nm) and Den-PEG (5.7 ± 0.18 nm) ( Figure S1 ). The polydispersity index (PDI) of all nanoprobes was below 0.3, and the surface charges ranged from +7.3 to +7.8 mV ( Figure S1 ). The shape of Den-PEG, Den-Angio and Den-Angio-GE11 was close to circular as shown in Figure S2 . The molecular weight of Den-Angio-GE11 was approximately 115 kDa. The molar ratios of PAMAM/DTPA/Gd/PEG/Angiopep-2/GE11 in Den-Angio-GE11 were 1/84/41/12.6/4.5/6.7 calculated according to the data of 1 H NMR Den-PEG, Den-Angio and Den-Angio-GE11 ( Figure S3 ). The T1-weighted relaxation times of nanoprobes ranged from 82.9 to 90.3 ms. Cytotoxicity of Nanoprobes The cytotoxicity of the nanoprobes was evaluated by measuring the viability of MDA-MB-231-BR and mouse brain endothelium cells bEnd.3. As shown in and , cell viability was not significantly declined, as the nanoprobe concentration gradually increased. At a nanoprobe concentration of 10 μM, the cell viability of bEnd.3 cells was approximately 94% compared to the normal control, indicating negligible cytotoxicity of the nanoprobes in vitro. Furthermore, the effect of nanoprobes on the invasion of MDA-MB-231-BR cells was assessed. As shown in and , Den-Angio-GE11 and its control nanoprobes, Den-Angio and Den-PEG, did not significantly affect metastatic cell invasion. Targeting of Nanoprobes to EGFR The physical affinity of nanoprobes for MDA-MB-231-BR cells was tested in vitro using Surface Plasmon Resonance (SPR). The dissociation constant of the nanoprobe Den-Angio-GE11 was 8.344 × 10 −10 mol/L (M), significantly lower than GE11 peptide (7.946 × 10 −7 M) ( ). Immunofluorescence staining results indicated a dose-dependent increase in red fluorescence signals in the Den-Angio-GE11 group, while no significant increase was observed in the control group (Den-Angio and Den-PEG) ( ). Flow cytometry quantification showed that the binding of Den-Angio-GE11 to MDA-MB-231-BR cells reached 64.2% at 5 μmol/L, which was significantly higher than that of the control groups ( and , P <0.05). Further, to verify the specificity of this binding, the binding ratio of Den-Angio-GE11 was also assayed in EGFR-low expressed BT-474 cells and moderately expressing U87 cells ( ). As shown in , the binding ratio of Den-Angio-GE11 at 5 μM was 0.7% with BT-474 and 47.5% with U87 cells, respectively. The statistics result indicated that the binding rate of Den-Angio-GE11 with MDA-MB-231-BR cells was significantly higher than that with BT-474 cells ( ), which supported the specific recognition of MDA-MB-231-BR cells by Den-Angio-GE11. Imaging Capability in Vitro of Nanoprobes The imaging capability of nanoprobes was first investigated in MDA-MB-231 BR cells. When incubated with Den-Angio-GE11 and control vectors for 5 minutes (the time at which the binding ratio reached its maximum), MDA-MB-231 BR cells exhibited significantly higher signal intensity on T1-weighted MRI compared to the control nanoprobe groups or the PBS group ( and , P <0.05). Similarly, NIR imaging results ( and , P <0.01) indicated that the Den-Angio-GE11 group had significantly higher fluorescence intensity than the control nanoprobe groups and the PBS control group. These assays demonstrated that Den-Angio-GE11 could image metastatic MDA-MB-231-BR cells. BBB Permeability in Mice With Brain Metastasis The brain metastases in mice were monitored by T2-weighted MRI on days 7, 14, 21, 24, 28 and 35 after brain metastases induced. When lesions were detected in T2-weighted images, BBB permeability was evaluated using Gd-DTPA-enhanced T1-weighted MRI. As shown, six lesions were detected in all mice by T2-weighted images on day 21 after brain metastases preparation. On day 24, seventeen lesions (about one-half of total lesions) appeared on T2-weighted images. And on day 35, total 30 lesions appeared on T2-weighted images in our study (10 mice in BMS group). The BBB permeability was evaluated weekly through T1-enhancement by Gd-DTPA. In addition to two lesions that were enhanced by Gd-DTPA on day 21, most lesions will be enhanced after day 35. Some individual lesions remained non-enhancement even on day 50 ( and ). This result indicates that brain metastasis induced by MDA-MB-231-BR cells progressed slowly in the early stage and BBB in this model remained functions for one or two weeks after detection by T2-weighted MRI. In contrast, glioma lesions were enhanced by Gd-DTPA on day 7 after grafting. Results of Evans’ blue leakage assay showed that, in most cases of brain metastases, BBB was functional before day 35 ( ) in this study. Histological examination revealed that the metastatic cells clustered in the hyperintense area on T2-weighted images, separated by numerous voids ( ). Imaging of Early Brain Metastases by Den-Angio-GE11 As shown, early brain metastases could not be enhanced by Gd-DTPA when they were initially detected by T2-weighted MRI. However, significant enhancement was observed following administration of Den-Angio-GE11 on T1-weighted MRI. Similar enhancement was not observed in the control nanoprobe groups ( ). In NIRF imaging, the fluorescence signals in the heads were significantly more intense in the Den-Angio-GE11 group than that in the control groups. The signal peaked 2 hours post-administration of the nanoprobes or control vectors and gradually decreased thereafter ( and ). The nanoprobes were primarily distributed in the liver and kidneys, with smaller amounts found in the spleen ( ). The results of hematoxylin and eosin staining of brain sections confirmed the presence of brain metastases. Examination under a fluorescence microscope showed that Den-Angio-GE11 was enriched in the lesions compared to the Den-Angio control group ( ). Imaging of Extreme-Early Brain Metastases by Den-Angio-GE11 Additionally, we imaged extreme-early brain metastases that were undetectable by T2-weighted MRI. As shown in , T2-weighted images did not reveal any significant lesions. After administration of Den-Angio-GE11, although no positive signals were observed on T1-weighted images, NIRF imaging showed significantly higher fluorescence intensity in the BMS group compared to the sham group, regardless of Den-Angio-GE11 presence (the average radiation efficiency value is 46.27 × 10 6 in BMS group with Den-Angio-GE11, 10.28 × 10 6 in sham group with Den-Angio-GE11 and 1.31 × 10 6 without nanoprobe, respectively. P = 0.005) ( and ). Histological examination indicated that metastatic cells were dispersed in the brain parenchyma at this stage, with no clusters in the lesions. Most nanoprobes bound to metastatic cells in the BMS group, demonstrated by the overlap of red fluorescence from Den-Angio-GE11 with green fluorescence in metastatic cells. This result demonstrates that Den-Angio-GE11 can cross the BBB and target brain metastatic MDA-MB-231 BR cells in vivo ( ). 

The chemical structures and synthetic pathways of nanoprobes are presented in . Briefly, the PAMAM G5 dendrimer was selected as a platform and modified with polyethylene glycol to enhance biocompatibility and prolong the half-life in circulation. The rhodamine and NIR fluorophore NIR783 were conjugated to enable visualization of brain sections in vitro and NIR imaging of the brain in vivo. Then, the EGFR targeting peptide GE11 and BBB-permeable angiopep-2 were functionalized into the PAMAM dendrimer. Additionally, Gd 3+ -DTPA was synthesized to the dendrimer for MR imaging due to its high thermodynamic stability and kinetic inertness. The key physical and chemical properties of the nanoprobes were listed in . It is shown that the average hydrodynamic diameter of the nanoprobe Den-Angio-GE11 was about 8.8nm, which was slightly larger than that of the control groups Den-Angio (7.3 ± 0.16 nm) and Den-PEG (5.7 ± 0.18 nm) ( Figure S1 ). The polydispersity index (PDI) of all nanoprobes was below 0.3, and the surface charges ranged from +7.3 to +7.8 mV ( Figure S1 ). The shape of Den-PEG, Den-Angio and Den-Angio-GE11 was close to circular as shown in Figure S2 . The molecular weight of Den-Angio-GE11 was approximately 115 kDa. The molar ratios of PAMAM/DTPA/Gd/PEG/Angiopep-2/GE11 in Den-Angio-GE11 were 1/84/41/12.6/4.5/6.7 calculated according to the data of 1 H NMR Den-PEG, Den-Angio and Den-Angio-GE11 ( Figure S3 ). The T1-weighted relaxation times of nanoprobes ranged from 82.9 to 90.3 ms. 

The cytotoxicity of the nanoprobes was evaluated by measuring the viability of MDA-MB-231-BR and mouse brain endothelium cells bEnd.3. As shown in and , cell viability was not significantly declined, as the nanoprobe concentration gradually increased. At a nanoprobe concentration of 10 μM, the cell viability of bEnd.3 cells was approximately 94% compared to the normal control, indicating negligible cytotoxicity of the nanoprobes in vitro. Furthermore, the effect of nanoprobes on the invasion of MDA-MB-231-BR cells was assessed. As shown in and , Den-Angio-GE11 and its control nanoprobes, Den-Angio and Den-PEG, did not significantly affect metastatic cell invasion. 

The physical affinity of nanoprobes for MDA-MB-231-BR cells was tested in vitro using Surface Plasmon Resonance (SPR). The dissociation constant of the nanoprobe Den-Angio-GE11 was 8.344 × 10 −10 mol/L (M), significantly lower than GE11 peptide (7.946 × 10 −7 M) ( ). Immunofluorescence staining results indicated a dose-dependent increase in red fluorescence signals in the Den-Angio-GE11 group, while no significant increase was observed in the control group (Den-Angio and Den-PEG) ( ). Flow cytometry quantification showed that the binding of Den-Angio-GE11 to MDA-MB-231-BR cells reached 64.2% at 5 μmol/L, which was significantly higher than that of the control groups ( and , P <0.05). Further, to verify the specificity of this binding, the binding ratio of Den-Angio-GE11 was also assayed in EGFR-low expressed BT-474 cells and moderately expressing U87 cells ( ). As shown in , the binding ratio of Den-Angio-GE11 at 5 μM was 0.7% with BT-474 and 47.5% with U87 cells, respectively. The statistics result indicated that the binding rate of Den-Angio-GE11 with MDA-MB-231-BR cells was significantly higher than that with BT-474 cells ( ), which supported the specific recognition of MDA-MB-231-BR cells by Den-Angio-GE11. 

The imaging capability of nanoprobes was first investigated in MDA-MB-231 BR cells. When incubated with Den-Angio-GE11 and control vectors for 5 minutes (the time at which the binding ratio reached its maximum), MDA-MB-231 BR cells exhibited significantly higher signal intensity on T1-weighted MRI compared to the control nanoprobe groups or the PBS group ( and , P <0.05). Similarly, NIR imaging results ( and , P <0.01) indicated that the Den-Angio-GE11 group had significantly higher fluorescence intensity than the control nanoprobe groups and the PBS control group. These assays demonstrated that Den-Angio-GE11 could image metastatic MDA-MB-231-BR cells. 

The brain metastases in mice were monitored by T2-weighted MRI on days 7, 14, 21, 24, 28 and 35 after brain metastases induced. When lesions were detected in T2-weighted images, BBB permeability was evaluated using Gd-DTPA-enhanced T1-weighted MRI. As shown, six lesions were detected in all mice by T2-weighted images on day 21 after brain metastases preparation. On day 24, seventeen lesions (about one-half of total lesions) appeared on T2-weighted images. And on day 35, total 30 lesions appeared on T2-weighted images in our study (10 mice in BMS group). The BBB permeability was evaluated weekly through T1-enhancement by Gd-DTPA. In addition to two lesions that were enhanced by Gd-DTPA on day 21, most lesions will be enhanced after day 35. Some individual lesions remained non-enhancement even on day 50 ( and ). This result indicates that brain metastasis induced by MDA-MB-231-BR cells progressed slowly in the early stage and BBB in this model remained functions for one or two weeks after detection by T2-weighted MRI. In contrast, glioma lesions were enhanced by Gd-DTPA on day 7 after grafting. Results of Evans’ blue leakage assay showed that, in most cases of brain metastases, BBB was functional before day 35 ( ) in this study. Histological examination revealed that the metastatic cells clustered in the hyperintense area on T2-weighted images, separated by numerous voids ( ). 

As shown, early brain metastases could not be enhanced by Gd-DTPA when they were initially detected by T2-weighted MRI. However, significant enhancement was observed following administration of Den-Angio-GE11 on T1-weighted MRI. Similar enhancement was not observed in the control nanoprobe groups ( ). In NIRF imaging, the fluorescence signals in the heads were significantly more intense in the Den-Angio-GE11 group than that in the control groups. The signal peaked 2 hours post-administration of the nanoprobes or control vectors and gradually decreased thereafter ( and ). The nanoprobes were primarily distributed in the liver and kidneys, with smaller amounts found in the spleen ( ). The results of hematoxylin and eosin staining of brain sections confirmed the presence of brain metastases. Examination under a fluorescence microscope showed that Den-Angio-GE11 was enriched in the lesions compared to the Den-Angio control group ( ). 

Additionally, we imaged extreme-early brain metastases that were undetectable by T2-weighted MRI. As shown in , T2-weighted images did not reveal any significant lesions. After administration of Den-Angio-GE11, although no positive signals were observed on T1-weighted images, NIRF imaging showed significantly higher fluorescence intensity in the BMS group compared to the sham group, regardless of Den-Angio-GE11 presence (the average radiation efficiency value is 46.27 × 10 6 in BMS group with Den-Angio-GE11, 10.28 × 10 6 in sham group with Den-Angio-GE11 and 1.31 × 10 6 without nanoprobe, respectively. P = 0.005) ( and ). Histological examination indicated that metastatic cells were dispersed in the brain parenchyma at this stage, with no clusters in the lesions. Most nanoprobes bound to metastatic cells in the BMS group, demonstrated by the overlap of red fluorescence from Den-Angio-GE11 with green fluorescence in metastatic cells. This result demonstrates that Den-Angio-GE11 can cross the BBB and target brain metastatic MDA-MB-231 BR cells in vivo ( ). 

Brain metastasis is devastating, and early detection is crucial for improving patient prognosis. In this study, we developed a nanoprobe, Den-Angio-GE11, capable of crossing the BBB and targeting metastatic cells by conjugating Angiopep-2 and GE11 peptides. The early detection capability of this nanoprobe was evaluated in mouse model of brain metastasis induced by MDA-MB-231-BR cells. Our data indicated that Den-Angio-GE11 significantly enhanced the signals of brain metastases in the early stage through T1-weighted MRI and NIRF imaging, while Gd-DTPA could not. In the extreme-early stage, when brain metastases were not apparent on T2-weighted MRI, Den-Angio-GE11 enhanced signals of brain metastases on NIRF imaging but not on T1-weighted MRI. Taken together, early TNBC brain metastasis can be detected using Den-Angio-GE11. The “early stage” in brain metastases lacks a clear definition and definitive quantitative criteria. For instance, human micrometastases are generally defined as lesions less than 2 mm in diameter or comprising fewer than 200 cells, whereas mouse micrometastases consist of no more than 10 cells and lack angiogenesis. , However, there is no similar definition for the early brain metastasis. In clinic, the gold standard for diagnosis of brain tumors is T1 enhancement in MRI which is based on Gd-DTPA leaking from compromised BBB. However, the average survival of TNBC brain metastases after diagnosis is very short (only 4.9 months), , implying that they are probably in advanced stage when diagnosed by conventional T1 enhancement in MRI. Therefore, detection of early brain metastases was supposed to occur before BBB compromise in patients. It is reported brain metastasis induced by MDA-MB-231-BR cells develops slowly. This makes it suitable for imaging or drug delivery study of brain tumors because it provides a long time window for BBB investigation. Indeed, the time of BBB functioning in brain metastasis by MDA-MB-231-BR was obviously longer compared to orthotopic gliomas in this study. Almost all brain metastasis lesions required about three weeks to be detected on 7.0T MR by T2-weighted images, then the BBB still could remain functional for an additional one or two weeks. Even in some individuals, the BBB remained functional for 50 days post-brain metastasis preparation. On the contrary, the orthotopic gliomas by GL261 just kept BBB functional for several days from cells planted in our study. Detecting early brain metastases requires a good signal-to-noise ratio in imaging. Targeting strategies can enhance probe concentration in lesions, subsequently improving the signal-to-noise ratio of images. However, due to the heterogeneity of triple-negative breast cancer brain metastases, well-known molecular targets for imaging are lacking. EGFR is known to be overexpressed in various tumor types, including triple-negative breast cancer. , , Our previous study showed that EGFR plays a significant role in brain metastases of MDA-MB-231-BR cells in mice. In this study, EGFR was significantly overexpressed in MDA-MB-231-BR cells, moderately expressed in U87 cells, and not expressed in BT-474 cells. The probe Den-Angio-GE11 is selectively bound to MDA-MB-231-BR and U87 cells but not BT-474 cells. This specific binding demonstrates the targeting capability of Den-Angio-GE11 for metastatic MDA-MB-231-BR cells in vivo. Optimizing the efficacy of nanoprobes to traverse the BBB is crucial for enhancing the signal-to-noise ratio. Various factors of probes, such as size, charge properties, and shape, affect their ability to cross the BBB. Among these factors, nanoparticle size significantly affects the ability to cross the BBB. Oshra et al reported that smaller nanoprobes (20–70 nm) exhibit superior BBB penetration capabilities, with Ohta et al identifying that the optimal size for brain entry is 15 nm. , Additionally, the brain extracellular space (ECS) and interstitial fluid (ISF) drainage within the ECS determine whether a probe can reach its target cells after crossing BBB. , Given that the physiological ECS in brain tissue is approximately 20 nm, smaller nanoprobes diffuse more efficiently, making enhanced enrichment in metastatic sites and increased signal-to-noise ratios of images. , Den-Angio-GE11 was 8.8 nm in diameter and significantly outperformed Gd-DTPA in detecting brain metastases on T1-weighted MRI. However, the small size of Den-Angio-GE11 also led to rapid clearance by kidneys and reduced circulation time. Peak fluorescence in brain metastases was observed at 1–2 hours post-administration, followed by a gradual decline. This highlights the challenge of optimizing nanoprobe size for effective BBB penetration while maintaining sufficient circulation time to maximize diagnostic potential. In clinical settings, MR plain scans are commonly used for screening brain metastases following primary tumor resection or treatment with radiotherapy and chemotherapy. The fact that these extreme-early brain metastases were undetectable by T2-weighted imaging in 7.0T MR suggests that the conventional plain MR scans will also miss those tiny metastases. Therefore, utilizing other highly sensitive imaging techniques is promising for the detection of these extreme-early foci. For example, PET-MR leveraged both high sensitivity and high spatial resolution, so it is probably useful for the detection of extreme-early brain metastases in future. Although near-infrared fluorescence (NIRF) imaging by Den-Angio-GE11 indicated metastatic lesions and histological examination of brain metastasis specimens confirmed this result, however, the T1-enhancement performance of Den-Angio-GE11 on extreme-early lesions was not ideal. This limitation results from the low cell density in extreme-early foci. Therefore, in addition to the more sensitive imaging modality as mentioned above, more effective techniques to help imaging agents cross uncompromised BBB are also necessary. In addition, save and effective nanoparticles are promising for clinical transformation. Liposome nanoparticles have been widely used for drug delivery and have been transformed in clinical practice. Recently, Chen et al fused platelet cell membranes with liposomes and targeted tumors that highly expressed CD44 by P-selectin on the surface of platelet membrane. This is an inspiration for us to enhance abilities of crossing BBB and targeting TNBC brain metastasis in future studies. In this study, the BBB in brain metastasis induced by MDA-MB-231-BR cells in mice exhibited prolonged functions. Den-Angio-GE11, a two-order nanoprobe created by conjugating Angiopep-2 and GE11 peptides to G5 PAMAM dendrimers, successfully crossed the BBB and targeted metastatic MDA-MB-231BR cells in the brain, enhancing the T1 and NIRF signals of brain metastases that just detectable by T2-weighted imaging. However, for extreme-early brain metastases undetectable by T2-weighted imaging, its T1-enhancement effect was limited, highlighting the need for a more sensitive imaging technique or further improve the gadolinium load in the probes to detect extreme-early TNBC brain metastases. 

