Title: An intravaginal ring for sustained release of lysozyme

Authors: Christine Navarro, Michael B. Guerrero, Christine S. Miller, Manjula Gunawardana, Marc M.
Baum
Subject: Biomedical Engineering

Abstract
Intravaginal Rings (IVR)(Fig. 1) are traditionally used as a means of sustained release for contraceptives.
The IVR concept has been previously experimented with different Pre-Exposure Prophylaxis (PrEP, HIV
prevention) and has had success due to sustaining the release.If an enzyme can be utilized in an IVR, it
would increases the efficacy of fighting bacterial infections. A proteolytic enzyme, Lysozyme, holds
promise for cancer therapy and fighting bacterial infections. It specializes in breaking down bacterial cell
wall which contain peptidoglycan (2). The ability to effectively control the release of prophylactic
proteolytic enzymes such as lysozyme can be beneficial as a therapeutic mechanism. The main focus is on
utilizing the design of the IVR to deliver specific amounts of the enzyme in a controlled manner.

Introduction
Intravaginal Rings (IVR)(Fig. 3) are traditionally used as a means of sustained release for contraceptives
and sexually transmitted diseases (1). These rings are inexpensive to fabricate and only need to be
replaced at longer time intervals. Lysozyme is an enzyme that specializes in breaking down bacterial cell
wall which contain peptidoglycan (2). If an enzyme can be utilized in an IVR, it would increases the
efficacy of fighting bacterial infections which is only one example of enzymatic release in IVRs. future
developments project a possibility of putting other proteolytic enzymes with different functions in IVRs.

Fig 3. Human-sized polydimethylsiloxane (PDMS; silicone) (3). Channels with a diameter of 1.0 mm. Used in the in vitro release
study for lysozyme.

Methods
The formulation of the pods (30mg) used in the intravaginal rings (IVRs) were determined. The pods
contained lysozyme and Hydroxypropylmethyl Cellulose( HPMC) as an excipient to control release. The
concentrations used were 25%, 50% and 100% lysozyme. The formulation was tumbled on a horizontal
vortex for 30 minutes to ensure even distribution. Drug tablets were fabricated with a hand pellet press
and dip coated with 5% L-PLA. The process of dip coating was repeated the next day. The tablets were
inserted into separate pods in the IVR which contained one pre-punched release channel (1.0 mm Dia)
and were sealed with silicone. These cured drug pods -were used in dissolution studies with 10 ml of 1X
PBS (pH 6.8)(3). 150ul daily aliquots were analyzed at 280 nm with a spectrophotometer (4) and
quantified using a ten point Lysozyme protein standard. Bio Assays were performed with Micrococcus
luteus to show the efficacy of the lysozyme after sustained release.

Results

Discussion
The ability to effectively control the release of prophylactic proteolytic enzyme such as lysozyme can be
beneficial as a therapeutic mechanism. The main focus is on utilizing the design of the IVR to deliver
specific amounts of the enzyme in a controlled manner. Sustained release can be further optimized by
changing the sizes of the delivery channels and testing different excipients that prevents the rapid release
of the enzyme. Other methods such as dip coating the lysozyme tablet with other polymers can also
augment the rate of release. The IVR concept has been previously experimented with different
Pre-Exposure Prophylaxis (PrEP) and has had success due to sustaining the release. Future possibilities
such as inserting prescribed medication into an IVR can help deliver multiple medications so the user
does not need to take tablets daily ,which can increase the efficacy of the medication.

Conclusions
Analyzed data from the sustained release showed that with varying amounts of HPMC enabled to control
the release of the enzyme lysozyme. During in vitro evaluation, 75% of lysozyme showed a release rate
of 0.6 mg/day, 50% lysozyme showed 0.08 mg/day, and 100% lysozyme showed 2.0 mg/day. In effective
sustained release 50% of lysozyme is available when using 25% HPMC as an excipient. Bio Assays that
were done with the bacterial strain M.luteus showed that even after 15 days 5 days of sustained release the
enzyme lysozyme was 20-26% active in breaking down bacterial cell walls.

References
1. Baum, M.M., An intravaginal ring for the sustained delivery of tenofovir disoproxil fumarate. Int
J Pharmaceut (2015).
2. Goodsell, D. Lysozyme. PDB-101. RCSB PDB. Sept. 2000. Web. August 2016.
3. Gunawardana, M., Sustained Delivery of Commensal Bacteria from Pod-Intravaginal Rings.
Antimicrob. Agents Chemother. 2014.
4. Moss, J.A., et al., 2012b. Safety and pharmacokinetics of intravaginal rings delivering tenofovir
in pig-tailed macaques. Antimicrob. Agents Chemother. 56, 5952 5960.

Acknowledgments
This experience was made possible by Marianne Smith, the director of the Summer Research Experience
(SRE) program at Citrus Community College and Oak Crest Institute of Science. The SRE was funded by
Citrus Community College STEM program.