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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.