1) The document discusses developing a sustained release drug delivery system using stealth liposomes to treat ocular angiogenesis by encapsulating anti-VEGF antibodies and prolonging their release time in the eye. 2) Current anti-VEGF treatments require frequent, expensive injections which increase risks like endophthalmitis. The authors have created stable stealth liposomes that can encapsulate proteins and release them slowly over 200-240 days to decrease treatment frequency and costs. 3) Their best liposome formulations had a particle size of 100-150nm, encapsulation efficiency of 85-92%, and sustained protein release over 6-8 months based on in vitro studies. This drug delivery system has potential to overcome issues with current treatments

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Methods Discussion
SUSTAINED RELEASE DRUG DELIVERY FOR TREATING OCULAR ANGIOGENESIS
Zach T. Brazel*, Devi Kalyan Karumanchi, Elizabeth R. Gaillard
Northern Illinois University, Dept. of Chemistry and Biochemistry
Acknowledgment
• NIU Dept. of Chemistry – Dr. Timothy Hagen, Dr. Tao
Xu, Yesenia Valdivia Pantoja
• NIU Dept. of Physics - Dr. Laurence Lurio, Dr. Nuwan
Karunaratne, Preeti Vodnala
• NIU Dept. of Biological Sciences - Lori Bross
• NIU Technology Transfer Office
Antibodies are large and charged macromolecules;
hence have restricted movement across cell
membranes. The main disadvantages with using
liposomes as drug delivery systems is their
stability, low shelf life and rapid drug leakage. By
engineering the composition and method of
preparation, we have been able to produce stable
stealth liposomes and hence an ideal vector for the
delivery of biologics. The lipid bilayer of stealth
liposomes is coated with long chains of
polyethylene glycol, a hydrophilic polymer. This
polymer produces a hydrated shell around the
external surface of the lipid bilayer, thus creating a
steric barrier preventing interactions with
mononuclear phagocyte system. Consequently,
these liposomes remain in the circulation longer
making them excellent candidates for sustained
release drug delivery to treat angiogenesis.
Targeting ocular angiogenesis is a promising
approach to arrest diabetic retinopathy and wet
AMD. Therefore, developing drug delivery
platforms that bypass ocular barriers and deliver
the therapeutics to the posterior segment of the
eye while still protecting the drug until it is
released is a challenge. Our study dealt with the
aspects of increasing the stability of liposomes and
also, increasing the payload of the protein marker
in the process. We have been successful in
developing prototype formulations in vitro which
were able to prolong the time of release to about
200-240 days while preventing protein degradation
until release. Our technology has the potential to
overcome disadvantages of current drug delivery
systems and pave the way for a new and
promising delivery platform to reach the clinical
setting.
Introduction Results
Diabetic Retinopathy (DR) and Age-related Macular
Degeneration (AMD) are the most common ocular
diseases and a leading cause of blindness in
American adults. Angiogenesis observed in these
diseases is characterized by the growth of new
blood vessels into the retina, damaging its surface
in the process. The new blood vessels are fragile,
“leaky” and pool blood into the retinal space,
further damaging the retina. Laser treatments and
drugs like Lucentis and Avastin are available for
controlling the diseases. These drugs are anti-
VEGF antibodies that inhibit the growth of new
blood vessels.
Disadvantages of current treatment
Risk of
endophthalmitis
0.9 -1.6 %
The intravitreal injections administered every
month are inconvenient and very expensive. It has
also been shown that the risk of infectious
endophthalmitis increases due to frequent injection
of anti-angiogenic agents. In order to overcome
the disadvantages and increase patient
compliance, we are trying to encapsulate the
protein drug in liposomal nanostructures, prolong
the time of drug release into the eye, thereby,
decreasing the frequency as well as the cost factor
for these treatments.
Figure 1: Treatment of ocular angiogenesis
(http://www.dianasaville.com/192111/4245884/science/diabetic-macular-edema-program)
Figures 2-4 : Route of administration of anti-VEGF antibodies, Vitreous half life of
anti-VEGF drugs, Cost of intravitreal injections
(http://www.scienceofamd.org/treat)
Method of preparation of liposomes
Stealth liposomes were prepared using different
compositions of phospholipids and cholesterol.
Lipid hydration and extrusion method was used to
prepare the liposomes and to obtain a uniform
particle size between 100-200 nm. 9-10 freeze
thaw cycles were performed to increase the
encapsulation efficiency and the final sample was
lyophilized after removal of excess drug.
Evaluation of liposomes
Fluorescent tagging the antibody
Immunoglobulin G was used as a protein model for
this study. Succinimide amine conjugation was
used to fluorescent tag the antibody to maximize
detection sensitivity. Fluorescence intensity was
measured at 494 nm/520 nm excitation-emission.
Figure 5: Bioconjugation of fluorescent tag to the protein using amine
reactive crosslinker chemistry
(http://www.b2b.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products.html)
Figure 6 – 8: Particle size determination, Encapsulation efficiency and
Transmission electron microscopy images from formulations with
different compositions of phospholipids and cholesterol
Figure 9: Drug release profiles of stealth liposomes using fluorescein
tagged IgG as a marker from formulations with different compositions
of phospholipids and cholesterol
Patent disclosure
• ‘Timed release of substances to treat ocular
disorders’- Prov. patent 61/640557, Apr 30, 2012
• ‘Timed release of substances to treat ocular
disorders’- US 61/866810, Aug 16, 2013
• ‘Timed release of substances to treat ocular
disorders’- WO 2014051134, Aug 14, 2014
The best formulations were determined based on
the particle size, encapsulation efficiency and the
time of drug release. An average particle size of
100-200 nm was desirable. Dynamic light
scattering (DLS) of the liposomes showed that the
particle size was uniform and around 100-150 nm.
This was confirmed by imaging using Transmission
electron microscopy (TEM). The encapsulation
efficiency of the model protein in the liposomes was
about 85-92%. In our protein release studies using
SOTAX USP4 dissolution apparatus, we observed a
sustained release of the protein over a period of
6-8 months.