Integrin receptor-targeting polypeptide derivatives and molecular probes and uses

Abstract

The application relates to an integrin receptor targeting polypeptide derivative and molecular probe and application. The integrin receptor targeting polypeptide derivative has a structure shown in formula I. The novel polypeptide derivative constructed by the application has the function of simultaneously coupling multiple imaging molecules, and can be simultaneously used for preoperative diagnosis and intraoperative guidance of multiple imaging methods. The research result can provide a new idea for further development of molecular imaging, and increase the range of clinical application of molecular imaging.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an integrin receptor-targeting polypeptide derivative and molecular probe and its applications. Background Technology

[0002] Integrins are a family of heterodimeric transmembrane glycoproteins that mediate cell-cell and cell-ECM connections. The integrin family consists of 8 β and 18 α subunits, which assemble as heterodimers to form 24 distinct integrins. As important signaling molecules, they bidirectionally transmit transmembrane signals through unique transduction pathways, regulating various cellular functions such as growth and development, tissue repair, wound healing, and thrombosis. Abnormal integrin expression and function are closely related to various pathophysiological changes, such as cardiovascular and cerebrovascular diseases, hematological diseases, inflammatory diseases, and malignant tumors. Integrins also play a crucial role in tumor metastasis, participating in tumor localization in microvessels and mediating interactions between metastatic tumor cells and host cells. They are involved in organ-specific regulation of metastasis and are highly expressed in breast cancer, osteosarcoma, glioma, neuroblastoma, lung cancer, prostate cancer, bladder cancer, melanoma, and gastric cancer.

[0003] Integrin α v β3 is a complex composed of two transmembrane glycoproteins that participates in intracellular signal transduction and supports endothelial cell migration and survival. Under homeostatic conditions, integrin α... v β3 is not widely expressed. It is upregulated in cytokine-activated endothelial and vascular cells. Blocking α-β3 expression with monoclonal antibodies or low-molecular-weight antagonists can help. v β3-integrin inhibits angiogenesis in various in vivo models. Single α... v β3 and α v Small molecule inhibitors of β5 integrin can all inhibit tumor angiogenesis in animal models. These phenomena suggest that α v β3 plays a key role in angiogenesis and highlights its potential as a target for anti-angiogenic therapy.

[0004] α v β3 expression and its interaction with specific matrix ligands, such as arginine-glycine-aspartic acid (RGD) tripeptides and their derivatives, play a crucial role in tumor angiogenesis and metastasis. During angiogenesis, integrins expressed by endothelial cells regulate cell migration and survival. Integrins expressed by tumor cells promote cell invasion and passage through the blood vessel wall, facilitating tumor metastasis. α vβ3 integrin receptors are highly expressed in activated endothelial cells and tumor cells, but not in normal endothelial cells and most normal tissues, providing a potential target for anti-angiogenesis strategies. The sustained growth, invasion, and metastasis of malignant tumors are closely related to tumor angiogenesis. Studies have shown that integrin α is expressed in both malignant tumors and newly formed vascular endothelial cells. v β3 is highly expressed. α v β3 primarily functions by binding to ligands containing RGD tripeptide sequences in the extracellular matrix (ECM).

[0005] Since tumorigenesis in all solid tumor cells is inevitably accompanied by tumor angiogenesis, integrin α... v β3 receptors are important participants in tumor development and progression, and possess a certain degree of specificity; therefore, integrin α... v β3 receptors are not only potential molecular targets for tumor therapy, but also specific targets for molecular imaging. Molecular imaging enables non-invasive, visual, and quantitative analysis of integrin α. v β3 receptors, by presenting integrin levels in tumor cells and newly formed tumor blood vessels, offer new opportunities for more appropriate patient selection for anti-angiogenic therapy and for monitoring treatment efficacy. Based on integrin α... v β3 receptor imaging agents are used in various imaging techniques. Ellgela et al. used microvesicles targeting integrin αvβ3 receptors for contrast-enhanced ultrasound to evaluate tumor angiogenesis. They observed that targeted microvesicles accumulated more within tumor microvessels and almost none outside the vessels, while non-targeted microvesicles did not accumulate within microvessels. Sipkin et al. demonstrated in animal experiments that MRI and antibody-modified paramagnetic liposomes can conveniently target integrin αvβ3 receptors. v Imaging of β3 receptors. Near-infrared fluorescent dye-conjugated cyclic RGD peptides can visualize integrin-positive tumors subcutaneously inoculated in model mice. These imaging methods can visualize integrin receptor expression in tumor cells to some extent, but in practical applications, these methods all suffer from low sensitivity.

[0006] Currently, radionuclide-labeled targeting integrin α v β3-based RGD peptide radionuclide imaging (SPECT or PET) probes have been a hot topic in molecular imaging research. Haubner et al. conducted... 125 I-labeling. In experiments detecting M21 melanoma, MaCaF breast cancer, and osteosarcoma in nude mice, it was found that although the probe could accumulate in tumor tissue 10 minutes after intravenous injection, it was rapidly cleared from the tumor and remained in liver tissue for too long, making it unsuitable for clinical use. Compared with SPECT, PET has higher sensitivity and resolution. 68 Ga and 18F has a near 100% positron efficiency, a low positron energy (0.64 MeV), and a relatively short physical half-life (T). 1 / 2 With characteristics such as 67 min and 109.7 min, it is an ideal peptide labeling and PET imaging radionuclide. Beer et al. performed a study on 16 breast cancer patients (12 primary lesions and 4 metastatic lesions). 18 F-Galacto-RGDPET imaging showed that the tracer could identify all invasive lesions; this was performed on 11 patients with head and neck tumors. 18 F-Galacto-RGD PET imaging showed radioactive concentration in 10 out of 12 lesions, with 2 showing no significant uptake. Pathological analysis confirmed that the suspected tumor lesions without radioactive uptake were superficial lesions. In a preoperative PET scan of 19 patients with solid tumors (10 musculoskeletal tumors, 4 melanomas, 2 head and neck tumors, 2 gliomas, and 1 breast cancer), 17 lesions with significant uptake were malignant, while the other 2 benign lesions showed no significant radioactive uptake. To obtain probes with better pharmacokinetics and targeting function, the prodrug can be modified. GE Healthcare structurally modified an RGD-like peptide (ACDCRGDCFCG) derived from a phage peptide library, introducing multiple disulfide bonds and cyclizing it to obtain the product... 18 F-Fluciclatide ( 18 F-AHlll585) is stable in vivo. Imaging in healthy volunteers showed that... 18 F-Fluciclatide has a good safety profile and is suitable for clinical use. 18 F-Fluciclatide is rapidly cleared from the blood and normal tissues and is primarily excreted through the kidneys. 18 The effective radiation dose of F-Fluciclatide is 26 uSv / MBq, which is similar to commonly used... 18 F-FDG is comparable.

[0007] To meet the diverse medical needs of recent years, multimodal contrast agents, centered on a specific imaging technology while incorporating other imaging techniques, have emerged. Because different imaging methods utilize different imaging bases and principles, it is difficult to simultaneously perform detection using multiple imaging methods. Furthermore, a single imaging examination method cannot obtain the comprehensive diagnostic information required for certain diseases, let alone adequately meet the needs for early diagnosis and treatment of malignant diseases. To obtain more complete, sufficient, and accurate imaging information and improve the early detection rate of malignant diseases, the simultaneous diagnostic analysis using multiple imaging modalities has become a research hotspot. Therefore, in the rapid development of molecular imaging, multimodal imaging has also become an important part of research.

[0008] Compared to fluorescence imaging in the visible light region (400–700 nm) and the near-infrared I region (700–1000 nm), biological tissues exhibit weaker scattering and lower autofluorescence in the near-infrared II region (1000–1700 nm). Therefore, near-infrared II fluorescence imaging has highly competitive advantages such as deeper tissue penetration and a higher imaging signal-to-noise ratio. On the other hand, with the optimization and advancement of imaging equipment, near-infrared II equipment has already achieved clinical application functions, and its role in intraoperative guidance is gradually gaining recognition.

[0009] In recent years, researchers have designed and developed many types of near-infrared II fluorescent probes and explored their application potential in fields such as tumor imaging diagnosis, angiography, brain imaging, and immunoassay, including quantum dots, single-walled carbon nanotubes, organic dyes, and conjugated polymers. However, these fluorescent probes still face some bottlenecks in terms of stability and biosafety, limiting their further applications. In recent years, rare-earth near-infrared II fluorescent probes have attracted widespread attention due to their advantages such as good photostability and chemical stability, and narrow emission half-maximum width. Furthermore, because autofluorescence has a short lifetime (<10 ns), while rare-earth near-infrared II fluorescent probes have the unique advantages of long excited-state lifetimes and tunability, their application can be extended from spectral domain imaging to temporal domain imaging (time-resolved imaging), fluorescence lifetime multi-channel imaging, and time-gated imaging, further reducing background fluorescence interference and significantly improving the signal-to-noise ratio of the imaging.

[0010] The luminescence properties of rare-earth ions are modulated by many factors, and designing rare-earth-based luminescent materials emitting in the near-infrared II region requires meticulous design. Firstly, the candidate rare-earth elements must possess a large absorption cross-section within a suitable excitation window, such as Nd... 3+ Yb 3+ Er 3+ and Tm 3+ Under irradiation with 730nm, 808nm, or 860nm laser light, Nd... 3+ After being stimulated, it is in 4 I 9 / 2 The electrons in the ground state were excited to 4 F 7 / 2 , 4 F 5 / 2 or 4 F 3 / 2 Energy level, and then in 4 F 7 / 2 and 4 F 5 / 2 Electron nonradiative relaxation to excited state 4 F 3 / 2 Energy level, finally at 4 F 3 / 2 Excited electrons can undergo radiative transitions to...4 I 11 / 2 and 4 I 13 / 2 It emits near-infrared II fluorescence at energy levels of 1064nm and 1330nm.

[0011] Highly specific molecular imaging probes are an important component of precision medicine, with broad development prospects and market potential, and have been widely favored in the pharmaceutical translation market in recent years. Summary of the Invention

[0012] The purpose of this invention is to provide an integrin receptor-targeting peptide derivative, molecular probe, and its applications. The novel peptide derivative constructed in this invention has the ability to simultaneously couple multiple imaging molecules, and can be used for preoperative diagnosis and intraoperative guidance of various imaging methods. This research may provide new ideas for the further development of molecular imaging and expand the scope of molecular imaging in clinical applications.

[0013] To achieve the above objectives, a first aspect of the present invention provides an integrin receptor-targeting polypeptide derivative RGD-2DTPA, wherein the integrin receptor-targeting polypeptide derivative RGD-2DTPA has the structure shown in Formula I:

[0014]

[0015] A second aspect of the present invention provides a molecular imaging probe, said probe being a polypeptide derivative coupled with at least one metal ion and / or at least one radionuclide.

[0016] This invention designs and constructs a novel integrin receptor-targeting peptide derivative, RGD-2DTPA, which specifically targets integrin receptors on the surface of tumor cell membranes. Simultaneously, the probe possesses strong metal chelating ability and a high loading rate, enabling efficient coupling with various metal nuclides and imaging metal ions. This allows for the construction of a multimodal imaging probe suitable for PET, MRI, visible and near-infrared fluorescence imaging, for tumor-targeted diagnosis and intraoperative imaging guidance.

[0017] According to a preferred embodiment of the present invention, the metal ion is a lanthanide metal ion, specifically Gd. 3+ 、Nd 3+ and Eu 3+ At least one of them.

[0018] The preferred radionuclides for coupling are 68 Ga 3+ , 64 Cu 2+ , 86 Y 3+ , 90 Y 3+ ,89 Zr 4+ , 111 In 3+ and 99m Tc 3+ At least one of them.

[0019] According to the present invention, the probe may contain two or more receptors or be used in combination to achieve multimodal detection, specifically,

[0020] The probe can be coupled with two or more metal ions or used in combination. For example, the probe can be simultaneously coupled with Gd. 3+ and Nd 3+ The polypeptide derivatives of that.

[0021] The probe can also be coupled to both a metal ion and a radioactive nuclide simultaneously. For example, the probe can be coupled to both... 68 Ga 3+ and Nd 3+ The polypeptide derivatives of that.

[0022] For probes coupled with metal ions, they can be prepared by mixing metal salts with the polypeptide RGD-2DTPA and then purifying the mixture.

[0023] Probes coupled with radionuclides can be prepared using conventional radionuclide labeling methods.

[0024] A third aspect of the present invention provides the above-described molecular imaging probe for the preparation of integrin receptor α. v Applications of β3-specific imaging agents.

[0025] Specifically, for simultaneous coupling with Gd 3+ and Nd 3+ The aforementioned polypeptide derivatives can be used to prepare multifunctional imaging agents for MRI and infrared two-zone imaging. For simultaneous conjugation with... 68 Ga ions and Nd 3+ The aforementioned polypeptide derivatives can be used in the preparation of multifunctional imaging agents for PET and infrared two-zone imaging.

[0026] This invention provides a novel multifunctional molecular imaging probe that uses the novel molecule RGD-2DTPA as a precursor and flexibly couples with different metal ions to become an integrin receptor α. v β3-specific multifunctional imaging agent. This is achieved through individual or co-coupling with metal nuclides. 68 Ga 3+ Lanthanide metals Nd 3+ Gd 3+This enables the probe to possess multimodal imaging capabilities, making it suitable for PET, MRI, fluorescence imaging, and near-infrared II imaging. By combining different imaging metals, a multifunctional probe, Gd / Nd-RGD-2DTPA (MRI + near-infrared II fluorescence), can also be constructed, simultaneously providing preoperative diagnosis and intraoperative guidance. 68 This novel probe, including Ga / Nd-RGD-2DTPA (PET + near-infrared II fluorescence), is a newly designed instrument capable of detection in various functional combinations. This novel multifunctional probe offers new insights for the further development of molecular imaging, better meeting clinical needs and expectations for imaging technology. It also boasts high safety and has significant potential and value for clinical translation.

[0027] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0028] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0029] Figure 1 The results show the high-resolution mass spectrometry quality control results. (a) RGD-2DTPA; (b) Nd-RGD-2DTPA.

[0030] Figure 2 As shown 68 Results of experiments on the uptake and inhibition of Ga-RGD-2DTPA in U87MG and A549 cells (n=5).

[0031] Figure 3 As shown 68 Ga-RGD-2DTPA tail vein injection of U87MG tumor-bearing mice PET / CT imaging.

[0032] Figure 4 The image shows an in vitro MRI T1-weighted image of Gd-RGD-2DTPA, compared with the commonly used magnetic resonance contrast agent Gd-DTPA.

[0033] Figure 5 The image shows an MRI contrast-enhanced image of Gd-RGD-2DTPA applied to the U87MG tumor model.

[0034] Figure 6 The image shows Nd-RGD-2DTPA applied to the U87MG tumor model for tumor resection guided by near-infrared II fluorescence imaging. Detailed Implementation

[0035] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0036] Synthesis of RGD-2DTPA

[0037] RGD-2DTPA was successfully prepared by a probe synthesis company using solid-phase synthesis. All reagents and amino acid precursors used in the synthesis process were high-purity chemicals. After the reaction was completed, the product was lyophilized to obtain a white powder RGD-2DTPA product. High-resolution mass spectrometry was performed to ensure that the purity of the precursor was >95%.

[0038]

[0039] Chemical structural formula of RGD-2DTPA

[0040] The markings of Nd-RGD-2DTPA and Gd-RGD-2DTPA

[0041] Relying on the strong metal coupling function of RGD-2DTPA, it can directly bind to a variety of lanthanide metal ions.

[0042] (1) Synthesis of Nd-RGD-2DTPA: Excess NdCl3·6H2O was mixed with a certain amount of RGD-2DTPA (2mg) and dissolved in ultrapure water. The mixture was reacted at 50℃ for 1h. The product was purified using an activated Sep-pak column and Nd-RGD-2DTPA was collected.

[0043] (2) Synthesis of Gd-RGD-2DTPA: The method is basically the same as before. Excess GdCl3·6H2O is mixed with a certain amount of RGD-2DTPA (2mg) and dissolved in ultrapure water. The reaction is carried out at 70℃ for 30min. The product is purified using an activated Sep-pak column and Gd-RGD-2DTPA is collected.

[0044] Characterization of RGD-2DTPA and Nd-RGD-2DTPA

[0045] A small amount of RGD-2DTPA sample was thoroughly dissolved in anhydrous acetonitrile and analyzed by high-resolution ESI-MS. The ESI-MS results showed that the synthesized RGD-2DTPA exhibited a distinct product peak at 685.8 ([M+H]+). Figure 1(a) This demonstrates the successful synthesis of RGD-2DTPA. After successfully constructing the Nd-RGD-2DTPA infrared imaging probe, high-resolution ESI-MS detection was performed again, revealing that the product peak of Nd-RGD-2DTPA appeared at 826.7 ([M+H]+), proving that Nd was successfully coupled to RGD-2DTPA. Figure 1 (b) and has high purity.

[0046] 68 Marking and Characterization of Ga-RGD-2DTPA

[0047] (1) Turn on the incubator and set the temperature to 95°C.

[0048] (2) Take 2.5 mL of 0.05 M HCl solution and rinse. 68 Ga.

[0049] (3) Mix a certain amount of RGD-2DTPA with 68 The Ga solution was mixed and placed in an incubator at 95°C, and allowed to stand for 20 minutes.

[0050] (4) Take out and measure the system with a 2mL syringe, then add it to the activated Sep-pak column, rinse the impurities with 3.0mL of pure water and discard them.

[0051] (5) Add a 0.2μm microporous filter membrane, collect the product into a sterile vacuum bottle with 1.0mL 80% ethanol solution, and add a certain amount of physiological saline to the system for dilution.

[0052] Products were detected using Radio-TLC. 68 The chemical purity of Ga-RGD-2DTPA is as high as 56%, with a labeling rate of up to 56%, and its radiochemical purity is >95%.

[0053] 68 Ga-RGD-2DTPA for PET / CT imaging in tumor-bearing mice

[0054] After completing the probe cell uptake experiment and detecting integrin receptor expression in the tumor model, high-resolution micro-PET / CT imaging was first used to detect... 68 Ga-RGD-2DTPA enhances the targeted imaging function of U87MG tumor-bearing mouse models with high integrin receptor expression, while also... 68 Preliminary detection of the in vivo radioactivity distribution of Ga-RGD-2DTPA was conducted to verify the tumor-specific targeting effect of the probe and the radioactivity distribution in imaging.

[0055] (1) Mark the 68The Ga-RGD-2DTPA probe was diluted with physiological saline (0.9%) and passed through a membrane. Three U87MG tumor-bearing mice (male, 7 weeks old, 20–22g, tumor diameter approximately 1.2cm) were injected with a certain amount via the tail vein. 68 Ga-RGD-2DTPA probe (11.1 Mbq, 200 μL).

[0056] (2) High-resolution micro-PET / CT scans were performed at 30 min, 60 min and 120 min after drug injection, with a scan time of 15 min. Animals were anesthetized using an isoflurane gas anesthesia system during the acquisition process.

[0057] Micro-PET / CT imaging is shown in Figure 3 Thirty minutes after probe injection, its radioactivity was mainly concentrated in the heart, bladder, and tumor site. As time progressed, the uptake at the tumor site gradually increased in PET / CT images. Experiments have shown... 68 Ga-RGD-2DTPA exhibits tumor-specific targeting with high expression of integrin receptors and can be used for tumor-specific PET / CT.

[0058] Gd-RGD-2DTPA for PET / CT imaging in tumor-bearing mice

[0059] Using a 3.0T MRI imaging system allows for more precise visualization of the probe's magnetic resonance enhancement. This system was used to perform in vitro T1-weighted magnetic resonance imaging (MRI) of Gd-RGD-2DTPA and the commonly used contrast agent Gd-DTPA.

[0060] (1) Before probe injection, U87MG tumor-bearing mice underwent a single MRI scan. Imaging was performed using a 3.0T PET / MR imaging device with a mouse imaging coil. The scan sequence information is as follows: TR: 455ms, TE: 17.5ms; flip angle 75°; FOV: 70×60mm 2 Matrix: 256×256; Slice thickness: 1.5mm; Number of slices: 12. T1-weighted axial and coronal scans were performed on mice.

[0061] (2) After the probe was injected, each experimental group underwent MRI imaging at 5 min, 10 min, 15 min and 60 min respectively. The MR scanning steps and sequences were the same as in step (1).

[0062] Depend on Figure 4 As shown, at the same metal concentration, the T1-weighted signal intensity of Gd-RGD-2DTPA is slightly higher than that of Gd-DTPA.

[0063] The magnetic resonance imaging (MRI) enhancement capability of Gd-RGD-2DTPA was fully verified through in vitro experiments. In this invention, a certain amount of Gd-RGD-2DTPA probe was injected into U87MG tumor-bearing mice, followed by T1-weighted MRI scans.

[0064] MR imaging results as follows Figure 5 Compared with pre-injection MR imaging, 5 minutes after probe injection, the whole-body MR signal of tumor-bearing mice showed enhancement, with the most significant increase in MR signal in the heart, liver, and gallbladder. The MR signal at the tumor site was significantly enhanced. After 10 and 15 minutes, the MRI signal at the tumor site gradually increased and diffused into the tumor. By the first hour of imaging, a uniform MRI enhancement signal was visible at the tumor site. The experimental results demonstrate that Gd-RGD-2DTPA can be used for MRI enhancement imaging of tumors with high integrin receptor expression.

[0065] Nd-RGD-2DTPA for tumor removal in tumor-bearing mice guided by near-infrared 2-zone fluorescence imaging.

[0066] Near-infrared II (NIIR) imaging has become a research hotspot in the field of molecular imaging in recent years. Compared with visible light imaging and NIIR I imaging, NIIR II imaging exhibits higher contrast and imaging depth. With the advancement of portable devices, this imaging method is gradually being applied to intraoperative guidance in surgery. The Nd-RGD-2DTPA constructed in this invention possesses NIIR II fluorescence imaging capabilities. This invention utilizes intratumoral injection for intraoperative guidance experiments. After the probe is uniformly distributed within the tumor (approximately 25 minutes), NIIR II fluorescence-guided tumor resection is performed. Specifically,

[0067] (1) A certain amount of Nd-RGD-2DTPA was injected into the tumor of U87MG tumor-bearing mice. A near-infrared II camera was used to take one frame of image every 1 minute. After the probe was evenly distributed throughout the tumor, the next experiment was carried out.

[0068] (2) Under the guidance of the near-infrared II fluorescence surgical navigation system, the tumors on the tumor-bearing mice were removed.

[0069] Surgical video screenshots as follows Figure 6 As shown, the tumor site after the probe was injected is clearly visible. After the tumor was removed and separated, no imaging was observed in the surrounding organs of the mouse, proving that the probe remained only in the tumor.

[0070] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An integrin receptor-targeting polypeptide derivative RGD-2DTPA, characterized in that, The integrin receptor-targeting polypeptide derivative RGD-2DTPA has the structure shown in Formula I: Formula I.

2. A molecular imaging probe, wherein the probe is a polypeptide derivative of claim 1 coupled with at least one metal ion and / or at least one radionuclide; The metal ion is a lanthanide metal ion; the lanthanide metal ion is Gd. 3+ 、Nd 3+ and Eu 3+ At least one of them; The radioactive nuclide is 68 Ga 3+ , 64 Cu 2+ , 86 Y 3+ , 90 Y 3+ , 89 Zr 4+ , 111 In 3+ and 99m Tc 3+ At least one of them.

3. The molecular imaging probe according to claim 2, wherein, The probe is simultaneously coupled with Gd 3+ and Nd 3+ The polypeptide derivatives of that.

4. The molecular imaging probe according to claim 2, wherein, The probe is simultaneously coupled with 68 Ga ions and Nd 3+ The polypeptide derivatives of that.

5. The molecular imaging probe according to any one of claims 2-4 in the preparation of integrin receptor α v Applications of β3-specific imaging agents.

6. The application of the molecular imaging probe according to claim 3 in the preparation of a multifunctional imaging agent for MRI and infrared two-zone imaging.

7. The application of the molecular imaging probe according to claim 4 in the preparation of a multifunctional imaging agent for PET and infrared II imaging.

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