Periodic Reporting for period 1 - TRoMBONE (Therapy for regeneration of Heart Muscle based on targeted delivery of exosomes)
Période du rapport: 2017-04-01 au 2019-03-31
Worldwide, 26 million people suffer from heart failure (HF) with 3.6 million newly diagnosed every year solely in Europe. Heart transplantation remains the only treatment capable of addressing the fundamental problem, in spite of important advances in pharmacologic therapy, invasive treatments (e.g. revascularization) and device therapy.
Cell-based therapies have shown promising results on improving heart function but the poor survival and/or engraftment hinder their clinical efficacy. Additionally, identification of the cell type(s) to use remains an unresolved issue with bone marrow stem cells and different populations of cardiac stem cells being the most commonly used.
Exosomes is a good alternative to cell therapies for regeneration of injured myocardial tissue, e.g. after myocardial infarction. These vesicles, with a diameter of 50-200 nm, are secreted by cells, contain proteins, lipids, RNA, and are thought to play an important role in intercellular communication. Importantly, exosomes collected from stem cells and their progenies seem to represent a significant component of their paracrine effect after transplantation in the context of therapeutic angiogenesis and myocardial infarction.
The translation of this therapy to patients will require the (i) development of platforms for the sufficient delivery of exosomes into the heart and (ii) enhancement of the regenerative potential of exosomes.
The main objective of the current project is to develop an effective strategy to deliver exosomes by intravenous (IV) injection into the heart (targeting) and to maximize exosome regenerative potential (bioactivity). Therefore, the project aims to develop a non-cellular-based therapy to regenerate the heart after infarction based on the targeted delivery of exosomes.
Cell-based therapies have shown promising results on improving heart function but the poor survival and/or engraftment hinder their clinical efficacy. Additionally, identification of the cell type(s) to use remains an unresolved issue with bone marrow stem cells and different populations of cardiac stem cells being the most commonly used.
Exosomes is a good alternative to cell therapies for regeneration of injured myocardial tissue, e.g. after myocardial infarction. These vesicles, with a diameter of 50-200 nm, are secreted by cells, contain proteins, lipids, RNA, and are thought to play an important role in intercellular communication. Importantly, exosomes collected from stem cells and their progenies seem to represent a significant component of their paracrine effect after transplantation in the context of therapeutic angiogenesis and myocardial infarction.
The translation of this therapy to patients will require the (i) development of platforms for the sufficient delivery of exosomes into the heart and (ii) enhancement of the regenerative potential of exosomes.
The main objective of the current project is to develop an effective strategy to deliver exosomes by intravenous (IV) injection into the heart (targeting) and to maximize exosome regenerative potential (bioactivity). Therefore, the project aims to develop a non-cellular-based therapy to regenerate the heart after infarction based on the targeted delivery of exosomes.
The aim of the project was to develop a new exosome based therapy for regeneration of heart muscle. For this purpose, we isolate the exosome from the mono nuclear cells of umbilical cord blood. We characterised those exosome using TEM, DLS, Zeta potential, NTA, flow cytometry etc. Results from NTA and DLS show that MNC-exosomes were 100±5.0 nm 117±14.45 nm in size respectively (Figure 1). Exosomes have strong negative zeta potentials of -34.95±2.12 mV due to negatively charged phospholipid membrane. The morphological characterization of exosomes was performed using a transmission electronic microscope that allowed the observation of single vesicles. Exosomes had a round shape and presented bilayer membrane (Figure 1). The majority of exosomes (96.06%±3) expressed the most well documented exosome marker CD9 (Figure 1).
This exosome shows bio-activity on the survival of the HUVECs. Further we modified those exosomes with the targeted peptide. The exosomes modified with the targeted peptide had much higher accumulation in in vitro testing with HUVECs. Later exosome and the modified exosome both were tested in vivo. Results shows that both the exosome and the exosome modified with targeted peptide showed higher recovery than the control (PBS) after 28 days. The recovery of the modified exosomes was similar to the sham (positive control) after 28 days (Figure 2).
The another major outcome of this project was to develop a new technique for in vivo monitoring of exosomes which was a big challenge. Specially, detecting exosome in heart or brain was difficult. High sensitivity was needed to detect exosome in those organs. Here, we developed a new strategy after modifying the surface of the exosome with a radio-nuclide and used it for PET/MRI imaging. Our technique had very high sensitivity and combination of MRI gives us more anatomical details which helps to monitor exosome in organ line heart or brain. We also tested that our modification strategy didn’t damage the surface as well as the core of those exosomes. Due to high sensitivity and combination of the MRI helps this technique for detecting exosome in heart and brain where we had very low accumulations. (Figure 3)
This exosome shows bio-activity on the survival of the HUVECs. Further we modified those exosomes with the targeted peptide. The exosomes modified with the targeted peptide had much higher accumulation in in vitro testing with HUVECs. Later exosome and the modified exosome both were tested in vivo. Results shows that both the exosome and the exosome modified with targeted peptide showed higher recovery than the control (PBS) after 28 days. The recovery of the modified exosomes was similar to the sham (positive control) after 28 days (Figure 2).
The another major outcome of this project was to develop a new technique for in vivo monitoring of exosomes which was a big challenge. Specially, detecting exosome in heart or brain was difficult. High sensitivity was needed to detect exosome in those organs. Here, we developed a new strategy after modifying the surface of the exosome with a radio-nuclide and used it for PET/MRI imaging. Our technique had very high sensitivity and combination of MRI gives us more anatomical details which helps to monitor exosome in organ line heart or brain. We also tested that our modification strategy didn’t damage the surface as well as the core of those exosomes. Due to high sensitivity and combination of the MRI helps this technique for detecting exosome in heart and brain where we had very low accumulations. (Figure 3)
At the end of the project we will have nice platform for developing regenerative medicine for MI. The only effective therapy available for MI is heart transplantation. The main difficulties of this technique is the availability of the donor heart. Beside that it also needed a complicated surgery. The implementation of our therapeutic strategy is very simple. We can say that exosome based therapy will be helping to recover several several heart problems in future. So, we can say that our therapy will help to improve quality of life of the human beings.
Beside during the project, we develop a highly sensitive imaging platform for monitoring exosome in vivo. Sensitivity was one of the issue for the in vivo monitoring exosomes specially in the organ like brain or heart. But our new technique was sensitive enough to detect exosome in heart or brains which will help to develop exosome based therapy for different brain and heart diseases like, MI, Alzheimer, ischemic stroke etc.
Beside during the project, we develop a highly sensitive imaging platform for monitoring exosome in vivo. Sensitivity was one of the issue for the in vivo monitoring exosomes specially in the organ like brain or heart. But our new technique was sensitive enough to detect exosome in heart or brains which will help to develop exosome based therapy for different brain and heart diseases like, MI, Alzheimer, ischemic stroke etc.