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CANADIAN RESEARCH FOCUS
Interview with Dr. Mehrdad Rafat
“PEG-PLA microparticles for encapsulation and delivery of Tat-EGFP to retinal cells”, Biomaterials (2010). 31: 3414-3421.
doi:10.1016/j.biomaterials.2010.01.031
May 7th, 2010
conducted by Patricia Comeau
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Presentation Contents
Brief background on article Slides 3 - 5 Interview with Dr.Rafat Slides 6 - 23 Dr. Rafat’s Biography Slides 24 - 27
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PEG-PLA microparticles for encapsulation and delivery of Tat-EGFP to retinal cells
• Polyethylene glycol-polylactic acid (PEG–PLA) microparticles were used for encapsulation and delivery of a Transactivator of transcription-enhanced green fluorescent protein fusion (Tat-EGFP) to retinal cells.
• Main objective was to develop a system that delivered Tat-EGFP with an initial rapid release (within 24 h) followed by a sustained release
4Figure 1: Tat-EGFP encapsulated nano/microparticles subretinally injected into the outer nuclear layer of the retina
Image courtesy of Rafat, M., University of Ottawa; and Kolb, E., University of Utah, 2010
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• Concerns for delivery of an effective therapy to
the retinal cells include restricted permeability of
the corneal and conjunctival epithelia, and the
presence of the blood-retina barrier.
• 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.
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Interview with Dr. Rafat
Vision Program, Ottawa Hospital Research Instituteand
Department of Cellular and Molecular Medicine, University of Ottawa
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What is the need for controlled release technologies in the eye?
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.
…continued on next slide →
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This low ocular absorption is 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.
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What are the key target diseases?
Key target diseases include:
• age-related macular degeneration,
• diabetic retinopathy,
• glaucoma,
• retinal ischemia,
• retinal detachment,
• cataract, and
• ocular herpes
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How and where are the microparticles injected into the retina?
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.
…continued on next slide →
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Tat-EGFP encapsulated microparticles is
dispersed in Dulbecco's Phosphate Buffered
Saline and transferred to a 10-mL syringe 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.
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What other characteristics of the microparticles, aside from size and morphology, influence
subretinal delivery?
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.
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Clinically, what would you replace Tat-EGFP with to treat a retinal disease?
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.
…continued on next slide →
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XIAP is a key member of the
inhibitors of apoptosis 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.
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Why is there a difference between the cellular uptake at 48 and 96hours?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.
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How do electroretinograms determine the biocompatibility of micro-particles?
Electroretinography (ERG) measures the
electrical responses of various retinal cell
types, including the photoreceptors, inner
retinal cells (bipolar cells), and the ganglion
cells. E.g. an A-wave is generally caused by
extracellular ionic currents generated by
photoreceptors and B-wave is generated by
bipolar cell activity.
…continued on next slide →
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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.
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Does the presence of the microparticles in the retina pose any potential risks?
According to our findings so far, the 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.
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When do you plan for the particles to release the drug?
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, chronic,
progressive disorders need a continuous
moderate release of the therapeutic agents over
time while acute insults require immediate
intervention.
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How are the polymer degradation products removed from the eye?
As the particles are directly injected into the
retina, we 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.
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What major hurdles remain to be overcome before patients can benefit from its application?
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.
…continued on next slide →
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To achieve these goals we will need more public
and private funding.
Other obstacles include the regulatory matters
involved with clinical trials, the high cost of
clinical trials, and development of manufacturing
facilities and protocols that are in compliance
with Good Manufacturing Practice requirements.
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CC-CRS Question #8
Thank you for the interview!
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Dr. Mehrdad Rafat
Biography of
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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. Dr. Rafat is currently a
PDF at OHRI/UOttawa with Dr. Tsilfidis and Dr. Isabelle
Catelas working on the development of nanoparticles
systems for controlled release and delivery of proteins/drugs
for retina, and bone regeneration applications, respectively.
…continued on next slide →
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As a result of his work towards the invention of the first
clinically-tested bioengineered cornea 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.
…continued on next slide →
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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.