Development of PLGA microsphere formulations for the sustained release of growth factors
Lead Research Organisation:
King's College London
Department Name: Craniofacial Dev and Stem Cell Biology
Abstract
Osteoarthritis (OA) is a common joint disorder that carries inflammation, degeneration of the cartilaginous surface of joints that allow smooth joint movement, and eventually, chronic pain and locomotor disability. Current OA treatments aim to alleviate the symptoms, but to date, there are no effective treatments to cure or prevent the condition. Recently, researchers have resorted to tissue engineering in an attempt to reform the degraded tissue, but no successful regeneration of native cartilage has been yet achieved, suggesting that current strategies miss a relevant step.
Most cartilage tissue engineering approaches focus on selecting a stem cell population and on developing a biomaterial that serves as a 3D scaffold for tissue regeneration, but lesser attention is paid on giving inductive cues for cells to correctly reform the tissue. This fact is particularly important in tissues with complex structures, such as cartilage, where asymmetrical gradients of growth factors (GF) are present and must be maintained for stem cell survival and correct cell differentiation.
External GF injection into joint cavities is not sufficient, as proteins quickly wash away in synovial fluid, and recurrent supplementations are impractical in clinical practice. Therefore, the encapsulation of GFs in biodegradable microspheres (MSs) and their sustained release within biomaterial scaffolds is a much more feasible approach for the generation and maintenance of GF gradients.
Pouya Rezai's lab has vast expertise on the generation of MSs by microfluidics. Microfluidics studies the behaviour of fluids at the microscale and requires specialized knowledge, expertise and expensive equipment that very few laboratories have access to. Harnessing microfluidics for the synthesis of microparticles allows full control in microparticle properties (i.e diameter size, number of layers, high efficiency of molecule encapsulation, etc.) that would be unattainable otherwise.
Eileen Gentleman's lab has developed a photocrossinkable hyaluronan-based biomaterial for the repair of damaged cartilage. We have demonstrated that this biomaterial is injectable and can sustain cartilage cells, postulating it as an ideal system for clinical in situ tissue engineering of cartilage.
Here, we propose the use of poly(lactic-co-glycolic acid) (PLGA), a FDA-approved biodegradable polymer, to synthesise different MS formulations for GF sustained release. MSs can be used in concert with biomaterials and stem cells for the regeneration of cartilage in the context of OA. Thus, we will overcome current limitations on cartilage regenerative medicine and bring about the next generation of tissue engineering approaches.
To achieve that, we will first generate MSs of different sizes (ranging from nanometric to micrometric diameters) and different layers (mono or bilayered MSs). Then, we will assess which formulations are suitable to be used along with biomaterials and with cells. Lastly, we will encapsulate model proteins in relevant MS formulations to investigate their degradation and release kinetics under physiological conditions, and subsequently, to figure what formulations are more adequate to replicate cartilage natural GF gradients.
The result of this multidisciplinary project is the fulfilment of the current limitation in tissue engineering. This opportunity will bring us closer to a successful therapy in the context of regenerative medicine. The project not only brings in concert two completely different disciplines (tissue engineering, from Gentleman lab, and microfluidics and microparticle generation, from Rezai lab) into the development of a novel, revolutionizing and promising approach, but also sets the way for a new fruitful collaboration between Canadian and UK-based laboratories.
Most cartilage tissue engineering approaches focus on selecting a stem cell population and on developing a biomaterial that serves as a 3D scaffold for tissue regeneration, but lesser attention is paid on giving inductive cues for cells to correctly reform the tissue. This fact is particularly important in tissues with complex structures, such as cartilage, where asymmetrical gradients of growth factors (GF) are present and must be maintained for stem cell survival and correct cell differentiation.
External GF injection into joint cavities is not sufficient, as proteins quickly wash away in synovial fluid, and recurrent supplementations are impractical in clinical practice. Therefore, the encapsulation of GFs in biodegradable microspheres (MSs) and their sustained release within biomaterial scaffolds is a much more feasible approach for the generation and maintenance of GF gradients.
Pouya Rezai's lab has vast expertise on the generation of MSs by microfluidics. Microfluidics studies the behaviour of fluids at the microscale and requires specialized knowledge, expertise and expensive equipment that very few laboratories have access to. Harnessing microfluidics for the synthesis of microparticles allows full control in microparticle properties (i.e diameter size, number of layers, high efficiency of molecule encapsulation, etc.) that would be unattainable otherwise.
Eileen Gentleman's lab has developed a photocrossinkable hyaluronan-based biomaterial for the repair of damaged cartilage. We have demonstrated that this biomaterial is injectable and can sustain cartilage cells, postulating it as an ideal system for clinical in situ tissue engineering of cartilage.
Here, we propose the use of poly(lactic-co-glycolic acid) (PLGA), a FDA-approved biodegradable polymer, to synthesise different MS formulations for GF sustained release. MSs can be used in concert with biomaterials and stem cells for the regeneration of cartilage in the context of OA. Thus, we will overcome current limitations on cartilage regenerative medicine and bring about the next generation of tissue engineering approaches.
To achieve that, we will first generate MSs of different sizes (ranging from nanometric to micrometric diameters) and different layers (mono or bilayered MSs). Then, we will assess which formulations are suitable to be used along with biomaterials and with cells. Lastly, we will encapsulate model proteins in relevant MS formulations to investigate their degradation and release kinetics under physiological conditions, and subsequently, to figure what formulations are more adequate to replicate cartilage natural GF gradients.
The result of this multidisciplinary project is the fulfilment of the current limitation in tissue engineering. This opportunity will bring us closer to a successful therapy in the context of regenerative medicine. The project not only brings in concert two completely different disciplines (tissue engineering, from Gentleman lab, and microfluidics and microparticle generation, from Rezai lab) into the development of a novel, revolutionizing and promising approach, but also sets the way for a new fruitful collaboration between Canadian and UK-based laboratories.
Technical Summary
In order to develop PLGA microsphere (MS) formulations that can reform cartilage growth factor (GF) gradients and aid in the regeneration of degraded cartilage, we divided the project in three aims:
Aim 1: Generation of various MS formulations
We aim to generate MSs of sizes ranging from 0.1 to 10micrometer in diameter and 1 and 2 layers. The size of MSs is a key factor on MS degradation and, ultimately, gradient concentration and duration. MS can also have 2 layers (i.e MSs containing MSs) which allows the sequential release of several molecules.
Aim 2: Assessment of MS formulations' resistance to photooxidation
Photocrosslinking (instead of chemical reaction-based crosslinking) renders the biomaterial clinically feasible for in aqueous injection as it is the case of joints full of synovial liquid, but photocrosslinking yields mild photooxidation that inactivates naked proteins. An additional advantage of employing MSs is the protection of the encapsulated growth factors by the physical barrier that MS shell provides. We plan to encapsulate phenol red, a pH indicator that degrades and loses its colour upon photooxidation, to study how MS formulations protect the cargo from biomaterial photooxidation. Changes in colour will be measured by spectroscopy before and after photooxidation.
Aim 3: Study release kinetics of encapsulated GFs
We will encapsulate bovine serum albumin (BSA) according to relevant GF concentrations (0.2-2mg/ml) inside photoresistant MS formulations to study release kinetics of BSA over time in physiological conditions. We will take MS supernatants and detect protein content at different time points over 3 weeks with BCA protein assay kit.
Following these aims, we will be able to obtain a MS formulation that releases the correct amount of GF during the correct amount of time to replicate gradients in native cartilage and regenerate the tissue back to its original condition.
Aim 1: Generation of various MS formulations
We aim to generate MSs of sizes ranging from 0.1 to 10micrometer in diameter and 1 and 2 layers. The size of MSs is a key factor on MS degradation and, ultimately, gradient concentration and duration. MS can also have 2 layers (i.e MSs containing MSs) which allows the sequential release of several molecules.
Aim 2: Assessment of MS formulations' resistance to photooxidation
Photocrosslinking (instead of chemical reaction-based crosslinking) renders the biomaterial clinically feasible for in aqueous injection as it is the case of joints full of synovial liquid, but photocrosslinking yields mild photooxidation that inactivates naked proteins. An additional advantage of employing MSs is the protection of the encapsulated growth factors by the physical barrier that MS shell provides. We plan to encapsulate phenol red, a pH indicator that degrades and loses its colour upon photooxidation, to study how MS formulations protect the cargo from biomaterial photooxidation. Changes in colour will be measured by spectroscopy before and after photooxidation.
Aim 3: Study release kinetics of encapsulated GFs
We will encapsulate bovine serum albumin (BSA) according to relevant GF concentrations (0.2-2mg/ml) inside photoresistant MS formulations to study release kinetics of BSA over time in physiological conditions. We will take MS supernatants and detect protein content at different time points over 3 weeks with BCA protein assay kit.
Following these aims, we will be able to obtain a MS formulation that releases the correct amount of GF during the correct amount of time to replicate gradients in native cartilage and regenerate the tissue back to its original condition.