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About the JournalISSN: 2331-205X Editor-in-chief: Udo Schumacher • Editorial Board medicine@cogentoa.com

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PHARMACEUTICAL SCIENCE | RESEARCH ARTICLE

5-Flurouracil microencapsulation and impregnation in hyaluronic acid hydrogel as composite drug delivery system for ocular fibrosisMeghali Bora, Raghavendra C. Mundargi, YongDe Chee, Tina T.L. Wong and Subbu S. Venkatraman

Cogent Medicine (2016), 3: 1182108

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Bora et al., Cogent Medicine (2016), 3: 1182108http://dx.doi.org/10.1080/2331205X.2016.1182108

PHARMACEUTICAL SCIENCE | RESEARCH ARTICLE

5-Flurouracil microencapsulation and impregnation in hyaluronic acid hydrogel as composite drug delivery system for ocular fibrosisMeghali Bora1*†, Raghavendra C. Mundargi1†, YongDe Chee1, Tina T.L. Wong1,2 and Subbu S. Venkatraman1

Abstract: Effective and sustained release formulations of antimetabolite 5-Flurouracil (5Fu) for ocular fibrosis are highly desirable for success of glaucoma filtration surgery (GFS). However, burst release and rapid clearance of carriers and/or drug still exist as major limitations. Here, we report the feasibility of encapsulat-ing 5Fu in sustained release carrier of poly (d,l-lactide-co-glycolide) (PLGA) micro-spheres (MPs) and impregnating in a hyaluronic acid (HA) hydrogel as unit dose formulation. In order to optimize the encapsulation process by solid-in-oil-in-water emulsion technique, the effects of PLGA end groups, PLGA concentration, and 5Fu loading were studied systematically. The MPs were extensively characterized for surface morphology, particle size distribution, thermal properties, crystallinity, re-sidual solvent, syringeability, and in vitro release. The optimized formulation of MPs was ~94 μm in size and released 5Fu in a sustained manner for up to two weeks. In our concept, MPs are dispersed first in HA solution and then cross-linked in situ after injection into the conjunctival space. Thus, syringeability experiments were

*Corresponding author: Meghali Bora, School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore E-mail: megh0005@e.ntu.edu.sg

Reviewing editor:Udo Schumacher, University Medical Center Hamburg-Eppendorf, Germany

Additional information is available at the end of the article

ABOUT THE AUTHORSThe research activities of our group are focused on polymeric delivery systems for biomedical applications. More specifically, it involves modification, design optimization, and characterization of micro/nanoparticulate carriers. Some of the major research areas include localized drug/gene delivery via stents, ocular nanomedicine, shape memory and hemocompatible polymers, cardiac regenerative medicine, injectable implants, and biomedical devices. Our team is closely working with hospitals as well as research centers toward developing therapeutic solutions for various disease targets.

The research work in this paper addresses an important issue of drug delivery. Prolonged sustained drug delivery using biocompatible and biodegradable carriers is highly desirable, as it greatly improves patient compliance. Design and characterization of the formulation reported here make a significant contribution toward the ongoing research activities of ocular drug delivery. Moreover, this work forms a platform for microparticulate ocular delivery systems for further investigation and product development suitable for a wider research audience.

PUBLIC INTEREST STATEMENTGlaucoma is a common eye disease that leads to blindness and affects large number of population worldwide. The surgical treatment often fails due to several limitations of the drug, 5-Fluorouracil (5Fu), that is injected into the eye either during or after glaucoma surgery. In our work, we have addressed these issues by developing a potential delivery system for 5Fu via combination of two different carriers. Micron-sized particles are first loaded with 5Fu and then entrapped inside a gel network. This combined system is nontoxic and gets safely disintegrated inside body after treatment. It was investigated in detail for its material properties and our study shows that 5Fu is released slowly from particle–gel combined system within the desired therapeutic range for almost two weeks. This would ensure that patients do not need multiple injections. Moreover, our system, upon injection inside eye, will provide localized treatment avoiding any side effects.

Received: 28 February 2016Accepted: 20 April 2016First Published: 09 May 2016

© 2016 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license.

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Bora et al., Cogent Medicine (2016), 3: 1182108http://dx.doi.org/10.1080/2331205X.2016.1182108

3.2. Synthesis of HA hydrogelsHA was first modified into its methacrylated derivative, HA-MA, using MA as described previously (Nettles, Vail, Morgan, Grinstaff, & Setton, 2004; Smeds et al., 2000). HA powder was dissolved in DI water to prepare 1.5% (w/v) solution and pH was adjusted to 8 using 5 M NaOH while slowly adding 20 M excess of MA to it. The reaction was allowed to continue for 2 h at RT while maintaining the pH at 8 and then stored at 4°C for 24 h. HA-MA solution was dialyzed against 0.1 M NaCl solution using a dialysis membrane (Spectra/Por 6 dialysis tubing, 10 kDa molecular weight cut-off (MWCO), 29 mm diameter, flat width 45 mm) for 48 h followed by 2 cycles of dialysis against alternating solutions of 1:4 C2H5OH– H2O (v/v) and ultrapure H2O for 24 h before lyophilization for 72 h (Christ Alpha 1–4 LSC, Germany). After freeze drying, 4% (w/v) solution of HA-MA was prepared in PBS, pH 7.4 and 0.4% (w/w) of photoinitiator I2959 was added into it. This macromer solution with initiator was then ex-posed to UV lamp (VL-8.LC, 1 × 8 W, Vilber Lourmat, France) at 365 nm for 10 min (light intensity 720 μW/cm2 at 15 cm) for cross-linking to develop HA-MA hydrogels.

3.3. Preparation of composite hydrogelsModified HA powder, HA-MA, was dissolved in PBS buffer to prepare 4% (w/v) solution and allowed to stir overnight at RT. Next day, 25 mg of 5Fu-loaded PLGA MPs (containing 1 mg/ml 5Fu) were weighed and mixed with HA-MA solution (polymer to particles mass ratio of 1.6:1). Upon mixing, HA-MA  +  5Fu-PLGA MPs solution was cross-linked using UV light as described above. Schematic in Figure  2 shows the fabrication of 5Fu-loaded PLGA MPs, incorporation in HA-MA solution, and sub-sequent cross-linking by UV light to obtain composite hydrogel system.

3.4. Characterization of 5Fu-loaded PLGA MPs

3.4.1. YieldAfter freeze drying, 5Fu-loaded MPs were sieved using 150-μm and 106-μm sieves to remove poly-meric debris and small fibers. The dry mass of MPs was recorded before and after sieving to estimate the % yield. The % yield of 5Fu-loaded MPs was calculated from the freeze-dried and sieved MPs using the following equation:

% Yield =

(

Dry mass of MPs after sieve

Initial mass of PLGA used

)

× 100

Figure 1. Schematic showing the development and ocular delivery of composite HA formulation.

Notes: (A) 5Fu-loaded PLGA MPs infused in HA solution can be administered using 25G needle and (B) 5Fu-loaded PLGA MPs infused in HA-MA solution can be administered followed by UV cross-linking to form MPs-impregnated hydrogel matrix system.

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Bora et al., Cogent Medicine (2016), 3: 1182108http://dx.doi.org/10.1080/2331205X.2016.1182108

The results show that hydrogel composite formulations can be potentially used as drug delivery car-riers for therapeutic applications not only for ocular diseases, but also for other diseases as local drug delivery systems.

Supplementary materialSupplementary material for this article can be accessed here http://dx.doi.org/10.1080/2331205X.2016.1182108

AcknowledgementsAuthors are grateful to Mr. Diels Ding from Fritsch Asia Pacific Pte Ltd, Singapore, for providing the Laser Particle Analyzer facility for size measurement of PLGA microparticles. Authors would also like to acknowledge the generous help and support from Mr. Vishal Mogal and Dr. Zviad Tsakadze, School of Materials Science and Engineering, Nanyang Technological University, Singapore, for the NMR and XRD experiments, respectively.

FundingThis work was supported by Nanyang Technological University [Tier 1 MOE AcRF Tier 1 RG72/12].

Competing interestsThe authors declare no competing interest.Author detailsMeghali Bora1

E-mail: megh0005@e.ntu.edu.sgRaghavendra C. Mundargi1

E-mail: RCMundargi@ntu.edu.sgYongDe Chee1

E-mail: YCHEE1@e.ntu.edu.sgTina T.L. Wong1,2

E-mail: ttlwong@ntu.edu.sgSubbu S. Venkatraman1

E-mail: assubbu@ntu.edu.sg1 School of Materials Science and Engineering, Nanyang

Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore.

2 Ocular Therapeutics and Drug Delivery, Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751, Singapore.

† These authors contributed equally to this work.

Citation informationCite this article as: 5-Flurouracil microencapsulation and impregnation in hyaluronic acid hydrogel as composite drug delivery system for ocular fibrosis, Meghali Bora, Raghavendra C. Mundargi, YongDe Chee, Tina T.L. Wong & Subbu S. Venkatraman, Cogent Medicine (2016), 3: 1182108.

Cover imageSource: Author.

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