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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 l iposomes 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 d isadvantages and increase pat ient 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.