Khanh Ha, PhD
Mass General Hospital
A fluorescent probe for molecular imaging of plasmin activity in wound healing
Backgrounds: Once a wound is contained by fibrin, activation of plasmin promotes the removal of fibrin and stimulates angiogenesis, tissue remodeling, and tissue regeneration. Insufficient fibrin deposition or excessive plasmin-mediated fibrinolysis in early convalescence prevents injury containment, causing bleeding. Alternatively, excess fibrin deposition and/or inefficient plasmin activity later in convalescence impairs musculoskeletal repair, resulting in tissue fibrosis and osteoporosis. The ability to map plasminogen expression noninvasively and serially as a biomarker would thus have significant potential in improving novel therapies. Here, we describe the development of a selective plasmin-activatable near-infrared (NIR) fluorescent imaging probe
Methods: The probe was incubated with purified murine plasmin (0.01 mg/mL), thrombin (0.01 mg/mL), trypsin (0.01 mg/mL), or bovine serum albumin ([BSA] 0.1% in water), and the fluorescence increase was recorded over time. At 1 day post injury, 1 day post-injection, and 3 weeks post-injection, the WT and the Plg KO mice were used to study plasmin activation in vivo.
Results: The probe should show a 10-fold increase in fluorescence relative to the initial sample when treated with purified murine plasmin. At 5ug/mouse, there was no signal in the Plg KO or scramble probe mice. A scrambled sequence was also synthesized as a control in which the amino acid sequence was rearranged. The plasmin probe showed a signal at the fracture site 3 weeks after injury, while the signal of the scramble probe was weak. At 1d post-injury and 1d post injection, the WT and the Plg KO mice have significant signals.
Activatable fluorogenic sensor for molecular imaging of thrombin activity in osteosarcoma
The development of a thrombin imaging probe is significant in human health and disease because it can provide a platform for future therapeutics such as targeted drug delivery and diagnostic imaging. Thrombin is an enzyme that plays a vital role in bone cancer and the blood clotting process, and its activation occurs at the start of tissue injury. Once activated, it initiates platelet activation and fibrin formation. Thrombin activation deficiency can lead to ineffective wound healing, whereas overactivation of thrombin can lead to the formation of obstructive blood clots or cancer cell growth. Despite thrombin’s central role in bone repair and disease, there is still no suitable molecular imaging agent.
The study was designed to develop a fluorescent molecular imaging probe that detects thrombin activity in vivo with minimal invasion. Upon addition of the recombinant thrombin to the probe, significant fluorescence amplification was observed. Osteosarcoma, the most prevalent primary bone tumor in children and adolescents, has seen limited progress in prognosis over the past four decades, particularly for patients with metastatic or recurrent disease. Elevated plasma fibrinogen and thrombin levels correlate with poor cancer prognosis; however, their mechanistic role in osteosarcoma remains unclear.
Our study investigates the impact of fibrinogen and thrombin on osteosarcoma growth and metastasis using a murine model.
Fluorochrome-Functionalized of Feraheme for Macrophage Imaging in Biological Systems
Macrophages are a significant cell group of the innate immune system that play a critical role in pathogen clearance, wound healing, and homeostasis in various coordinated pathways. These pathways are extensively studied and have been linked to contributing factors to major diseases such as vascular inflammation, autoimmunity, and several cancers. Studying these pathways requires identifying and discerning macrophages from other cells with macrophage-specific fluorophore-labeled contrast reagents. Fluorophores widely used in research are cyanine’s with a wavelength between 450 and 800 nanometers; however, the recent push for lower-wavelength fluorophores has led to the development of dyes in the 500-nanometer wavelengths, allowing for deeper tissue penetration. Current chemical methods for developing macrophage-sensing nanoparticles are complex, low-yielding, and often yield unstable nanoparticles that decompose. Our group developed a new technique using a ferumoxytol nanoparticle (FMX) and a unique lysine-fluorophore, which was conjugated to the nanoparticle surface via EDCI chemistry. Our novel FMX nanoparticle formed microaggregates that were characterized by fluorescence and light scattering. Fluorophores developed and used in our study include cyanine Cy-AL5 and Cy-AM7, and a newly developed Alexa Fluor 555 (AF555) fluorophore. We confirmed FMX-fluorophore labeling with UV-VIS spectroscopy, designated by the respectable absorbances of several fluorophores. We also took nanoparticle diameter measurements to verify that this new nanoparticle is an acceptable candidate for in-vivo macrophage imaging.
Backgrounds: Once a wound is contained by fibrin, activation of plasmin promotes the removal of fibrin and stimulates angiogenesis, tissue remodeling, and tissue regeneration. Insufficient fibrin deposition or excessive plasmin-mediated fibrinolysis in early convalescence prevents injury containment, causing bleeding. Alternatively, excess fibrin deposition and/or inefficient plasmin activity later in convalescence impairs musculoskeletal repair, resulting in tissue fibrosis and osteoporosis. The ability to map plasminogen expression noninvasively and serially as a biomarker would thus have significant potential in improving novel therapies. Here, we describe the development of a selective plasmin-activatable near-infrared (NIR) fluorescent imaging probe
Methods: The probe was incubated with purified murine plasmin (0.01 mg/mL), thrombin (0.01 mg/mL), trypsin (0.01 mg/mL), or bovine serum albumin ([BSA] 0.1% in water), and the fluorescence increase was recorded over time. At 1 day post injury, 1 day post-injection, and 3 weeks post-injection, the WT and the Plg KO mice were used to study plasmin activation in vivo.
Results: The probe should show a 10-fold increase in fluorescence relative to the initial sample when treated with purified murine plasmin. At 5ug/mouse, there was no signal in the Plg KO or scramble probe mice. A scrambled sequence was also synthesized as a control in which the amino acid sequence was rearranged. The plasmin probe showed a signal at the fracture site 3 weeks after injury, while the signal of the scramble probe was weak. At 1d post-injury and 1d post injection, the WT and the Plg KO mice have significant signals.
Activatable fluorogenic sensor for molecular imaging of thrombin activity in osteosarcoma
The development of a thrombin imaging probe is significant in human health and disease because it can provide a platform for future therapeutics such as targeted drug delivery and diagnostic imaging. Thrombin is an enzyme that plays a vital role in bone cancer and the blood clotting process, and its activation occurs at the start of tissue injury. Once activated, it initiates platelet activation and fibrin formation. Thrombin activation deficiency can lead to ineffective wound healing, whereas overactivation of thrombin can lead to the formation of obstructive blood clots or cancer cell growth. Despite thrombin’s central role in bone repair and disease, there is still no suitable molecular imaging agent.
The study was designed to develop a fluorescent molecular imaging probe that detects thrombin activity in vivo with minimal invasion. Upon addition of the recombinant thrombin to the probe, significant fluorescence amplification was observed. Osteosarcoma, the most prevalent primary bone tumor in children and adolescents, has seen limited progress in prognosis over the past four decades, particularly for patients with metastatic or recurrent disease. Elevated plasma fibrinogen and thrombin levels correlate with poor cancer prognosis; however, their mechanistic role in osteosarcoma remains unclear.
Our study investigates the impact of fibrinogen and thrombin on osteosarcoma growth and metastasis using a murine model.
Fluorochrome-Functionalized of Feraheme for Macrophage Imaging in Biological Systems
Macrophages are a significant cell group of the innate immune system that play a critical role in pathogen clearance, wound healing, and homeostasis in various coordinated pathways. These pathways are extensively studied and have been linked to contributing factors to major diseases such as vascular inflammation, autoimmunity, and several cancers. Studying these pathways requires identifying and discerning macrophages from other cells with macrophage-specific fluorophore-labeled contrast reagents. Fluorophores widely used in research are cyanine’s with a wavelength between 450 and 800 nanometers; however, the recent push for lower-wavelength fluorophores has led to the development of dyes in the 500-nanometer wavelengths, allowing for deeper tissue penetration. Current chemical methods for developing macrophage-sensing nanoparticles are complex, low-yielding, and often yield unstable nanoparticles that decompose. Our group developed a new technique using a ferumoxytol nanoparticle (FMX) and a unique lysine-fluorophore, which was conjugated to the nanoparticle surface via EDCI chemistry. Our novel FMX nanoparticle formed microaggregates that were characterized by fluorescence and light scattering. Fluorophores developed and used in our study include cyanine Cy-AL5 and Cy-AM7, and a newly developed Alexa Fluor 555 (AF555) fluorophore. We confirmed FMX-fluorophore labeling with UV-VIS spectroscopy, designated by the respectable absorbances of several fluorophores. We also took nanoparticle diameter measurements to verify that this new nanoparticle is an acceptable candidate for in-vivo macrophage imaging.
