CANADIAN RESEARCH FOCUS
May 11, 2010
Rafat, M., Cleroux, C., Tsilfidis, C. et al. et al. just published a new study
"PEG-PLA microparticles for encapsulation and delivery of Tat-EGFP to retinal cells”, Biomaterials, 31:3414-3421, 2010. doi:10.1016/j.biomaterials.2010.01.031.
Interview with co-author Dr. Mehrdad Rafat
Dr. Rafat received his Masters and Ph.D. degrees in Chemical Engineering from the University of Ottawa with specialization in Biomaterials and Tissue Engineering. After completing his Ph.D., Dr. Rafat joined Dr. Tsilfidis’s group as a post-doctoral fellow (PDF) at the Ottawa Hospital Research Institute (OHRI) and worked on the development of nanoparticles for controlled gene delivery to retinal cells for prevention of retinal blindness. While doing his PDF at OHRI, he received a fellowship grant from Health Canada and developed a hybrid microspheres system for encapsulation and target delivery of stem cells for biomedical applications (e.g. treatment of myocardial infarction). Dr. Rafat is currently a PDF at OHRI/UOttawa with Dr. Tsilfidis (Don and Joy Maclaren Chair for Vision) and Dr. Isabelle Catelas (Canada Research Chair in Bioengineering) working on the development of nanoparticles systems for controlled release and delivery of proteins/drugs for retina, and bone regeneration applications, respectively.
During the course of his academic career to-date Dr. Rafat has been the co-inventor of four patents and the senior author of several refereed publications. He co-invented the first clinically-tested bioengineered cornea developed in Dr. May Griffith’s lab at the University of Ottawa Eye Institute. As a result of this achievement he was awarded NSERC’s 2008 Innovation Challenge Award and the Ontario Centers of Excellence Industrial Fellowship Award (OCE/CMM) in 2006.
In addition to his academic endeavors, Dr. Rafat has also worked with the Medical Devices Bureau at Health Canada for evaluation and regulation of medical devices, as well as been a senior scientific consultant to biotech industries including the Hawaii-based firm, Cellular Bioengineering Inc, and the start-up company, Bioconstrux Inc. of Ottawa.
Dr. Rafat’s research interests are mainly focused on the development of bioengineered materials as implantable scaffolds and nano and microparticles systems for controlled delivery of cells, drugs, and proteins for biomedical applications. More specifically, he is interested in the application of hybrid nanomaterials in regenerative medicine – particularly that involving ocular and cardiovascular therapies. Beyond the development of bioengineered materials he is also interested in the evaluation, regulation, and commercialization of medical device and therapeutic technologies for various medical applications.
The delivery of an effective therapy to the retinal cells in order to treat various retinal diseases remains a challenge. Concerns for such delivery include restricted permeability of the corneal and conjunctival epithelia, and the presence of the blood-retina barrier. In addition, the size of any delivery vehicle must be small enough not to negatively impact the sensitive retinal cells. Microparticles and nanoparticles in particular offer the advantage of a controlled and sustained subcellular drug release. In this study, Rafat et al. evaluated polyethylene glycol-polylactic acid (PEG–PLA) microparticles for encapsulation and delivery of a transactivator of transcription-enhanced green fluorescent protein fusion (Tat-EGFP) to retinal cells. Their proposed delivery mechanism is depicted in Figure 1.
Figure 1. Tat-EGFP encapsulated nano/microparticles subretinally injected into the outer nuclear layer of the retina (courtesy of Rafat, M., University of Ottawa; and Kolb, E., University of Utah, 2010)
CC-CRS
Could you please outline the current need for controlled or sustained release technologies in the eye? What special challenges does the eye present for such delivery? What are the key target diseases?
Dr. Rafat
Drugs and therapeutic agents have been traditionally administered to the eye as topical liquid drops. One of the main problems in ocular therapeutics is the delivery of an optimal concentration of a therapeutic agent at the target site for a prolonged period of time. It is believed that less than 5% of a therapeutic agent administered topically is ocularly absorbed due to the loss in the tear film, and the corneal layers as well as the restricted permeability of the corneal and conjunctival epithelia. These limitations are more critical for the retina, as most of the retinal diseases involve cells in the back of the eye. In addition, due to the presence of the blood-retina barrier, drug delivery to the retina by conventional methods poses a challenge.
Therefore, there is a vital need for an optimized drug delivery vehicle that can provide sustained intraocular release of therapeutic agents. Because the eye is a highly sensitive organ and contains delicate and intricate structures, the delivery vehicle must be designed in a sub-cellular size (e.g. nano- or micro- scale size range) to allow both intravitreal and subretinal modes of delivery without ocular complications. The delivery vehicle must not block the passage of light into the eye and cause permanent vision loss.
The key target diseases may include age-related macular degeneration (AMD), diabetic retinopathy, glaucoma, retinal ischemia, retinal detachment, cataract, and ocular herpes.
CC-CRS
Could you please explain more on how the microparticles can be injected into the eye and where in the retina they are delivered? What other characteristics of the microparticles, aside from size and morphology, influence the Tat-EGFP delivery to the retina?
Dr. Rafat
The microparticles can be delivered into the eye by subretinal injection. It is performed by creating a sclerotomy (surgical incision of the sclera) about 2 mm posterior to the limbus. A coverslip coated with 0.3% hypromellose is placed on top of the eye to provide magnification and visualization of the back of the eye. Tat-EGFP encapsulated microparticles is dispersed in Dulbecco's Phosphate Buffered Saline (DPBS) and transferred to a 10-mL syringe (Hamilton, Reno, NV) with a 33-gauge blunt needle attached. The needle is then inserted through the scleral puncture, guided lateral to the lens, and inserted through the retina and microparticles are injected to the subretinal space of the eye (care must be taken to avoid lens contact because this could induce cataract development).
Polarity and hydrophilicity of the microparticles influence the delivery and release mechanisms. For example, more hydrophilic polymers tend to absorb more water resulting in faster degradation of microparticles and faster release of therapeutic agents.
CC-CRS
Clinically, what would you replace Tat-EGFP with to treat a retinal disease such as age-related macular degeneration (AMD)?
Dr. Rafat
Our plan is to replace Tat-EGFP with x-linked inhibitor of apoptosis protein (XIAP) for clinical applications. We have previously reported that XIAP confers structural neuroprotection of photoreceptors for at least 2 months after retinal detachment, which is also associated with AMD. (Invest Ophthalmol Vis Sci. 2009; 50: 1448–1453). XIAP is a key member of the inhibitors of apoptosis (IAP) gene family and is a promising therapeutic agent as it suppresses caspases 3, 7, and 9, whose activation has been shown to cause apoptotic cell death in retinal detachment animal models (Invest Ophthalmol Vis Sci. 2003; 44:1262–1267).
CC-CRS
In considering the cellular uptake of Tat-EGFP release from PEG-PLA (see Figure 4), it seems that at 96 hours there is much more fine dispersion of the fluorescence than that at 48hours – do you know what could have happened between those two times to cause this?
Dr. Rafat
This phenomenon may be caused by the biodegradation of PEG-PLA nano/microparticles resulting in the break down of larger particles into smaller ones. Also, please note that there might be some image to image variations as the images for various time points were not taken at exactly the same spot in the culture dish.
CC-CRS
Could you please further explain how electroretinograms (i.e. your analysis of A-waves and B-waves) helped you determine the biocompatibility of the micro-particles in the eye?
Dr. Rafat
Electroretinography (ERG) measures the electrical responses of various retinal cell types, including the photoreceptors, inner retinal cells (bipolar cells), and the ganglion cells. For example, an A-wave is generally caused by extracellular ionic currents generated by photoreceptors, and the B-wave is generated by bipolar cell activity. If microparticles had caused any cell toxicity or death, the responses that we would get on ERG would have been different than those of normal healthy cells. Because no significant differences between PEG-PLA-treated eyes and control healthy eyes were observed in our ERG study, it suggested that the particles were biocompatible toward the retinal cells.
CC-CRS
Does the presence of the microparticles in the outer nuclear layer of the retina pose any potential risks to the retina? Is the goal to have the particles release the drug before they become embedded in this layer?
Dr. Rafat
According to our findings so far, presence of PEG-PLA microparticles in the outer nuclear layer of the retina did not cause toxicity or adverse side effects. However, one potential risk factor for these particles is their non-transparent nature. This phenomenon may temporarily cause blurred vision until the particles are fully degraded in the eye, which may take up to few months. Overall, I believe that further studies need to be conducted in other animal models as well as in human to confirm these findings.
One of the goals is to have the particles release their protein once they become embedded in the retina. The microparticles and their release profile, however, need to be customized for each ocular disease. For example, diseases such as AMD, diabetic retinopathy, and glaucoma are chronic, progressive disorders for which we need a continuous moderate release of the therapeutic agents over time while others (e.g. retinal ischemia and retinal detachment) are acute insults requiring immediate intervention such that you need to have the particles release a big dose of therapeutic agent upfront (e.g. initial burst) followed by a controlled slow release.
CC-CRS
Are we correct in thinking there is no fluid turnover in the vitreous humour of the eye? If this is so, how are the polymer degradation products removed from the eye?
Dr. Rafat
This is true that the gel in the vitreous humour is stagnant and is not replenished; however, we are not delivering the particles into the vitreous humour, e.g., via intravitreal injection. These particles are directly injected into the retina (subretinal injection). We also know that there is blood circulation in the retina, e.g., it is continuously supplied with oxygenated blood via the retinal artery and drained of deoxygenated blood via the central retinal vein. Therefore, it is very likely that biodegradation products leave the eye via the retinal vein and capillaries.
CC-CRS
What major hurdles remain to be overcome in this technology before patients can benefit from its application? What research are you planning to do next in terms of ocular drug delivery?
Dr. Rafat
Despite the promising nature of this technology, we need to conduct 3-5 more years of research to refine and engineer various formulations using different proteins (e.g. XIAP) and polymers and tailoring them for various ocular diseases. We also need to test these formulations in bigger animal models for the proof of concept prior to moving into human trials. To achieve these goals we will need to get more funding from both public and private sectors.
Other obstacles include the regulatory matters involved with clinical trials to assess the safety and effectiveness of the optimum formulations in human subjects and meet the standards of regulatory bodies, the high cost of clinical trials, and development of manufacturing facilities and protocols that are in compliance with GMP (Good Manufacturing Practice) requirements and satisfy the volume required to serve the market.
CCCRS:
Thank you for the interview!