Collagen-Targeting Self-Assembled Nanoprobes for Multimodal Molecular Imaging and Quantification of Myocardial Fibrosis in a Rat Model of Myocardial Infarction.

Currently, inadequate early diagnostic methods hinder the prompt treatment of patients with heart failure and myocardial fibrosis. Magnetic resonance imaging is the gold standard noninvasive diagnostic method; however, its effectiveness is constrained by low resolution and challenges posed by certain patients who cannot undergo the procedure. Although enhanced computed tomography (CT) offers high resolution, challenges arise owing to the unclear differentiation between fibrotic and normal myocardial tissue. Furthermore, although echocardiography is real-time and convenient, it lacks the necessary resolution for detecting fibrotic myocardium, thus limiting its value in fibrosis detection. Inspired by the postinfarction accumulation of collagen types I and III, we developed a collagen-targeted multimodal imaging nanoplatform, CNA35-GP@NPs, comprising lipid nanoparticles (NPs), encapsulating gold nanorods (GNRs) and perfluoropentane (PFP). This platform facilitated ultrasound/photoacoustic/CT imaging of postinfarction cardiac fibrosis in a rat model of myocardial infarction (MI). The surface-modified peptide CNA35 exhibited excellent collagen fiber targeting. The strong near-infrared light absorption and substantial X-ray attenuation of the nanoplatform rendered it suitable for photoacoustic and CT imaging. In the rat model of MI, our study demonstrated that CNA35-GNR/PFP@NPs (CNA35-GP@NPs) achieved photoacoustic, ultrasound, and enhanced CT imaging of the fibrotic myocardium. Notably, the photoacoustic signal intensity positively correlated with the severity of myocardial fibrosis. Thus, this study presents a promising approach for accurately detecting and treating the fibrotic myocardium.

Utilization of commercial collagens for preparing well-differentiated human beta cells for confocal microscopy.

Introduction: With technical advances, confocal and super-resolution microscopy have become powerful tools to dissect cellular pathophysiology. Cell attachment to glass surfaces compatible with advanced imaging is critical prerequisite but remains a considerable challenge for human beta cells. Recently, Phelps et al. reported that human beta cells plated on type IV collagen (Col IV) and cultured in neuronal medium preserve beta cell characteristics. Methods: We examined human islet cells plated on two commercial sources of Col IV (C6745 and C5533) and type V collagen (Col V) for differences in cell morphology by confocal microscopy and secretory function by glucose-stimulated insulin secretion (GSIS). Collagens were authenticated by mass spectrometry and fluorescent collagen-binding adhesion protein CNA35. Results: All three preparations allowed attachment of beta cells with high nuclear localization of NKX6.1, indicating a well-differentiated status. All collagen preparations supported robust GSIS. However, the morphology of islet cells differed between the 3 preparations. C5533 showed preferable features as an imaging platform with the greatest cell spread and limited stacking of cells followed by Col V and C6745. A significant difference in attachment behavior of C6745 was attributed to the low collagen contents of this preparation indicating importance of authentication of coating material. Human islet cells plated on C5533 showed dynamic changes in mitochondria and lipid droplets (LDs) in response to an uncoupling agent 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) or high glucose + oleic acid. Discussion: An authenticated preparation of Col IV provides a simple platform to apply advanced imaging for studies of human islet cell function and morphology.

Construction of CNA35 Collagen-Targeted Phase-Changeable Nanoagents for Low-Intensity Focused Ultrasound-Triggered Ultrasound Molecular Imaging of Myocardial Fibrosis in Rabbits.

Myocardial fibrosis plays an important role in the development of heart failure and malignant arrhythmia, which potentially increases the incidence of sudden cardiac death. Therefore, early detection of myocardial fibrosis is of great significance for evaluating the prognosis of patients and formulating appropriate treatment strategies. Late gadolinium-enhanced magnetic resonance imaging is considered as the currently effective strategy for noninvasive detection of myocardial fibrosis, but it still suffers from some critical issues. In this work, multifunctional CNA35-labeled perfluoropentane nanoparticles (CNA35-PFP NPs) have been elaborately designed and constructed for molecular imaging of fibrotic myocardium based on ultrasound imaging. These as-constructed CNA35-PFP NPs are intravenously infused into rabbit circulation with an animal model of myocardial infarction. Especially, these targeted CNA35-PFP NPs with nanoscale size could efficiently pass through the endothelial cell gap and adhere to the surface of fibroblasts in the fibrotic myocardium. Importantly, followed by low-intensity focused ultrasound irradiation on the myocardium, these intriguing CNA35-PFP NPs could transform from liquid into gaseous microbubbles, which further significantly enhanced the ultrasound contrast in the fibrotic area, facilitating the detection by diagnostic ultrasound imaging. Therefore, this work provides a desirable noninvasive, economical, and real-time imaging technique for the assessment of cardiac fibrosis with diagnostic ultrasound based on the rational design of liquid-to-gas phase-changeable nanoplatforms.

CT imaging of myocardial scar burden with CNA35-conjugated gold nanoparticles.

Management of patients suffering from myocardial infarction (MI) is based on the extent of coronary artery disease and myocardial scar burden. We have developed a potentially clinically-useful X-ray molecular imaging contrast agent based on gold nanoparticle (AuNPs) functionalized with collagen-binding adhesion protein 35 (CNA35) with the capabilities of achieving prolonged blood pool enhancement for vascular imaging of the coronary arteries and specific targeting of collagen within myocardial scar. At a concentration of ~ 45 mg Au/ml, AuNPs maintained a stable blood pool enhancement at 142-160 HU within an hour of intravenous administration. At 6 hours, specific signal enhancement was detected in the myocardium scar in rats injected with CNA35-AuNPs, but not with control AuNPs or in control animals. In conclusion, CNA35-AuNPs may be considered as a CT contrast agent for both vascular imaging of coronary artery disease and molecular imaging of myocardial scar in the heart.

Collagen fibril organization in chicken and porcine skeletal muscle perimysium under applied tension and compression.

The tension/compression asymmetry observed in the stress-stretch response of skeletal muscle is not well understood. The collagen network in the extracellular matrix (ECM) almost certainly plays a major role, but the details are unknown. This paper reports qualitatively and quantitatively on skeletal muscle ECM reorganization during applied deformation using confocal imaging of collagen through use of a fluorescently-tagged specific collagen binding protein (CNA35-EGFP) of porcine and chicken muscle samples under tensile and compressive deformation in both the fibre and cross-fibre directions. This reveals the overall three-dimensional structure of collagen in perimysium in planes perpendicular and parallel to the muscle fibres in both species. Furthermore, there is clear evidence of the reorganization of these structures under compression and tension applied in both the muscle fibre and cross-fibre directions. These observations improve our understanding of perimysium structure and response to three-dimensional deformations and are an important basis for constitutive models of passive skeletal muscle. Although overall behaviour was similar, some differences in perimysium structure were observed between chicken and porcine muscle tissue. Further work is required to better understand which structures are responsible for the tension and compression stress-strain asymmetry previously observed in the mechanical response of passive skeletal muscle.

Visualisation of Collagen in fixed skeletal muscle tissue using fluorescently tagged Collagen binding protein CNA35.

Detection and visualisation of Collagen structure are important to understand the relationship between mechanical behaviour and microstructure in skeletal muscle since Collagen is the main structural protein in animal connective tissues, and is primarily responsible for their passive load-bearing properties. In the current study, the direct detection and visualization of Collagen using fluorescently tagged CNA35 binding protein (fused to EGFP or tdTomato) is reported for the first time on fixed skeletal muscle tissue. This Technical Note also establishes a working protocol by examining tissue preparation, dilution factor, exposure time etc. for sensitivity and specificity. Penetration of the binding protein into intact mature skeletal muscle was found to be very limited, but detection works well on tissue sections with higher sensitivity on wax embedded sections compared to frozen sections. CNA35 fused to tdTomato has a higher sensitivity than CNA35 fused to EGFP but both show specific detection. Best results were obtained with 15µm wax embedded sections, with blocking of non-specific binding in 1% BSA and antigen retrieval in Sodium Citrate. There was a play-off between dilution of the binding protein and time of incubation but both CNA35-tdTomato and CNA35-EGFP worked well with approximately 100µg/ml of purified protein with overnight incubation, while CNA35-tdTomato could be utilized at 5 fold less concentration. This approach can be applied to study the relationship between skeletal muscle micro-structure and to observe mechanical response to applied deformation. It can be used more broadly to detect Collagen in a variety of fixed tissues, useful for structure-functions studies, constitutive modelling, tissue engineering and assessment of muscle tissue pathologies.