Periodic Reporting for period 1 - ExoBBB (Design and development of exosomes with brush type architecture as RNA delivery vectors for active targeting of blood brain barrier (BBB))
Reporting period: 2022-05-01 to 2024-04-30
Small extracellular vesicles (sEVs) also known as exosomes are nanosized vesicles which are secreted by cells. These nanosized vesicles are naturally enriched with a pool of therapeutic molecules such as proteins, microRNAs (miRNAs) etc. Hence delivery of these sEVs to human body compartments is a promising approach to tackle several diseases such as cancer, chronic wounds, neurological disorders etc. But sEV delivery to the blood circulation faces several challenges. One of the biggest challenge is their very short lifetime in the blood circulation. These sEVs are rapidly cleared from the blood circulation by our body's natural defence mechanism which in turn will result in less accumulation of the sEVs in target organs such as heart, lungs, brain etc. Less accumulation of the sEVs in target organs will ultimately result in poor therapeutic efficacy. Considering the above said challenges, it is very important to do research in designing exosomes with properties to overcome the rapid elimination from blood circulation and also to accumulate significantly in target organs. The project ExoBBB aimed to solve the above said challenges by modulating the physicochemical properties of exosomes thereby enabling them to perform as more efficient therapeutic carriers. Physicochemical properties of exosomes were modulated by growing polymers in the form of brushes (polymer brushes) from the surface of exosomes using polymerization techniques.
Overall objectives of ExoBBB were
1) isolate, purify and characterize small extracellular vesicles or exosomes
2) Modulate the physicochemical properties of these exosomes using polymer brushes
3) Characterize the developed exosome-polymer brush hybrids
4) Assess the impact of various exosome-polymer brush hybrids on cells
5) Evaluate the behaviour of these exosome-polymer brush hybrids after administering to blood circulation of small animals
We have used a combinatorial approach to develop a series of exosome-polymer brush hybrids , characterized them and evaluated their interactions with cells and their behaviour after entering the blood circulation of mice. From the results generated, we could identify some exosome-polymer brush hybrids with significantly higher retention time in the blood circulation of mice.
Overall objectives of ExoBBB were
1) isolate, purify and characterize small extracellular vesicles or exosomes
2) Modulate the physicochemical properties of these exosomes using polymer brushes
3) Characterize the developed exosome-polymer brush hybrids
4) Assess the impact of various exosome-polymer brush hybrids on cells
5) Evaluate the behaviour of these exosome-polymer brush hybrids after administering to blood circulation of small animals
We have used a combinatorial approach to develop a series of exosome-polymer brush hybrids , characterized them and evaluated their interactions with cells and their behaviour after entering the blood circulation of mice. From the results generated, we could identify some exosome-polymer brush hybrids with significantly higher retention time in the blood circulation of mice.
1- Isolation and purification of small extracellular vesicles (sEVs) from human urine
Urine was used to isolate sEVs due to the feasibility to produce sEVs in high concentrations of (1E11 per mL). sEVs were isolated and characterized as depicted in figure 1A.
2- Pre- optimization of polymer brush growth using liposomes mimicking exosomal lipid compartment
Liposomes having similar lipid composition as that of sEVs were formulated (figure 2). Cationic polymer brushes were grown over these liposomes and characterized them for size, charge and stability (figure 3).
3- Synthesis of exosome-polymer brush hybrids and their characterization
sEVs were surface engineered with different polymer combinations using a combinatorial approach. Selected polymer combinations are shown in figure 4A. For evaluating the interaction of sEV formulations with THP-1 monocytes sEVs were first loaded with green fluorescence emitting carboxyfluoresceine succinimidyl ester (CFSE) probe. Uptake of the surface engineered sEVs was evaluated in both THP-1 monocyte cell lines and immune cells (figure 4 ).
4- Systemic retention pattern of surface engineered sEVs post intravenous injection
To evaluate the in vivo behavior of the surface engineered sEVs, we have employed (Cyanine 7) cy7 dye loaded sEVs mainly due to the ability of laser corresponding to cy7 dye to penetrate deep in to tissues and facilitate efficient quantification of fluorescence. The in vivo behavior of cy7 loaded sEV-polymer hybrids was evaluated using mice model as shown in figure 5.
5- Overview of the results and their exploitation and dissemination
One of the major objectives of ExoBBB was to design exosome-polymer hybrids with improved pharmacokinetics and we could identify at least two different exosome-polymer brush combinations with significantly improved lifetime in the blood. Another objective was to identify exosome-polymer hybrids with efficient brain targeting properties. In this aspect, all the exosome-polymer brush hybrids depicted more or less similar accumulation in brain. Hence we could not select a formulation which could significantly enhance brain retention following intravenous administration. But considering the significant retention of exosome-polymer hybrids in the blood circulation this could be a potential future research aspect to explore. As a future research, we could select the exosome-polymer brush hybrids with long lifetime in the blood and modify them using suitable formulation techniques to significantly enhance their accumulation in the brain.
We have disseminated the results obtained in this project (ExoBBB) at an international conference ( XXVII winter school, Venice , Italy) and a national conference on ageing conducted in Coimbra, Portugal. In addition, I have participated in dissemination activities to high school students via (i) the activities during European Researchers Night (ii) a scientific workshop covering the aspects of blood brain barrier, ageing and neurotherapeutics at FIC.A - International Science Festival 2022 (iii) the 'Science is Wonderful' competition conducted by European Commission. Currently I am preparing two manuscripts (one covering the liposome-polymer brush aspect and another detailing the exosome-polymer brush development) for publication in international peer reviewed journals.
Urine was used to isolate sEVs due to the feasibility to produce sEVs in high concentrations of (1E11 per mL). sEVs were isolated and characterized as depicted in figure 1A.
2- Pre- optimization of polymer brush growth using liposomes mimicking exosomal lipid compartment
Liposomes having similar lipid composition as that of sEVs were formulated (figure 2). Cationic polymer brushes were grown over these liposomes and characterized them for size, charge and stability (figure 3).
3- Synthesis of exosome-polymer brush hybrids and their characterization
sEVs were surface engineered with different polymer combinations using a combinatorial approach. Selected polymer combinations are shown in figure 4A. For evaluating the interaction of sEV formulations with THP-1 monocytes sEVs were first loaded with green fluorescence emitting carboxyfluoresceine succinimidyl ester (CFSE) probe. Uptake of the surface engineered sEVs was evaluated in both THP-1 monocyte cell lines and immune cells (figure 4 ).
4- Systemic retention pattern of surface engineered sEVs post intravenous injection
To evaluate the in vivo behavior of the surface engineered sEVs, we have employed (Cyanine 7) cy7 dye loaded sEVs mainly due to the ability of laser corresponding to cy7 dye to penetrate deep in to tissues and facilitate efficient quantification of fluorescence. The in vivo behavior of cy7 loaded sEV-polymer hybrids was evaluated using mice model as shown in figure 5.
5- Overview of the results and their exploitation and dissemination
One of the major objectives of ExoBBB was to design exosome-polymer hybrids with improved pharmacokinetics and we could identify at least two different exosome-polymer brush combinations with significantly improved lifetime in the blood. Another objective was to identify exosome-polymer hybrids with efficient brain targeting properties. In this aspect, all the exosome-polymer brush hybrids depicted more or less similar accumulation in brain. Hence we could not select a formulation which could significantly enhance brain retention following intravenous administration. But considering the significant retention of exosome-polymer hybrids in the blood circulation this could be a potential future research aspect to explore. As a future research, we could select the exosome-polymer brush hybrids with long lifetime in the blood and modify them using suitable formulation techniques to significantly enhance their accumulation in the brain.
We have disseminated the results obtained in this project (ExoBBB) at an international conference ( XXVII winter school, Venice , Italy) and a national conference on ageing conducted in Coimbra, Portugal. In addition, I have participated in dissemination activities to high school students via (i) the activities during European Researchers Night (ii) a scientific workshop covering the aspects of blood brain barrier, ageing and neurotherapeutics at FIC.A - International Science Festival 2022 (iii) the 'Science is Wonderful' competition conducted by European Commission. Currently I am preparing two manuscripts (one covering the liposome-polymer brush aspect and another detailing the exosome-polymer brush development) for publication in international peer reviewed journals.
The project had impact on the society, scientific community and development of the career of the researcher (Aji Alex Moothedathu Raynold ):
Scientific impact: First major scientific impact of the project was the successful development of a series of novel delivery platforms combining the physicochemical properties of exosomes and polymer brushes. Our in vivo results were very promising and it can be further investigated to get the approval for clinical trials. The second major impact was the development of exosome-polymer brush hybrids which could predominantly target specific cell populations of blood like immune cells.
Impact towards the society: Aji Alex Moothedathu Raynold was involved in some courses organized by his host institute (CNC) and other institutions (Indian Institute of Technology BHU) where he disseminated the idea of exosome based therapeutics among high school students and new researchers. He has given invited oral presentations at Indian Institute of Technology BHU), disseminated research ideas to students via 'science is wonderful' competition and took classes to MSc students of his host institute in the course 'regenerative medicine'.
Researcher career development: Aji Alex Moothedathu Raynold made significant progress in his research career during this time of the project. He could attract a highly competitive research grant worth 50000 Euros from FCT Portugal based on his research outputs from ExoBBB. In this FCT exploratory project, he hired a MSc student and supervised her thesis. He acted as jury in awarding the MSc degree and published a road map section (DOI 10.1088/2515-7639/acab88).
Scientific impact: First major scientific impact of the project was the successful development of a series of novel delivery platforms combining the physicochemical properties of exosomes and polymer brushes. Our in vivo results were very promising and it can be further investigated to get the approval for clinical trials. The second major impact was the development of exosome-polymer brush hybrids which could predominantly target specific cell populations of blood like immune cells.
Impact towards the society: Aji Alex Moothedathu Raynold was involved in some courses organized by his host institute (CNC) and other institutions (Indian Institute of Technology BHU) where he disseminated the idea of exosome based therapeutics among high school students and new researchers. He has given invited oral presentations at Indian Institute of Technology BHU), disseminated research ideas to students via 'science is wonderful' competition and took classes to MSc students of his host institute in the course 'regenerative medicine'.
Researcher career development: Aji Alex Moothedathu Raynold made significant progress in his research career during this time of the project. He could attract a highly competitive research grant worth 50000 Euros from FCT Portugal based on his research outputs from ExoBBB. In this FCT exploratory project, he hired a MSc student and supervised her thesis. He acted as jury in awarding the MSc degree and published a road map section (DOI 10.1088/2515-7639/acab88).