Pharmaceutical nanotechnologySynthesis and characterization of silk fibroin microparticles for intra-articular drug delivery
Graphical abstract
Introduction
Osteoarthritis (OA) is a degenerative disease of articular joints that impacts nearly 27 million Americans or 12.1% of US adults (Bitton, 2009). The disease is characterized by progressive deterioration of the cartilage lining, subchondral bone destruction and thickening of the joint capsule (Gerwin et al., 2006, Hough Jr., 2005, Poole and Howell, 2001). These tissue changes lead to symptomatic joint pain and joint dysfunction, leading to restrictions on daily life activities. The first and most widely prescribed pharmacological treatment for osteoarthritis is the oral administration of non-steroidal anti-inflammatory drugs (NSAIDs) to reduce inflammation and treat symptomatic pain. While NSAIDs have been shown to have an effect on pain management, the long term use of systemic NSAIDs does not modify OA progression and has been associated with side-effects. When NSAID therapy provides no benefit, intra-articular injection of corticosteroids or hyaluronan may be prescribed (Zhang et al., 2008). While corticosteriods and other disease-modifying drugs may have benefits when administered to the affected joint, their benefits are limited by the rapid clearance of small-molecule drugs from the knee joint (Evans et al., 2014, Gerwin et al., 2006, Wallis et al., 1985). The benefits of glucocorticoid injections are often short-lived (1-4 weeks), necessitating frequent injections to maintain efficacy (Ayral, 2001, Jones and Doherty, 2003). Small-molecule drugs, such as methotrexate and diclofenac may also have therapeutic potential for treatment of arthritis, but have also been shown to have intra-articular half-lives as short as one to two hours following intra-articular injection (Owen et al., 1994, Wigginton et al., 1980).
In order to increase the residence time of small-molecule drugs that may have efficacy in treating arthritis, a broad set of drug delivery strategies have been developed that can allow for the slow and sustained release of drug into the articular cavity (Kang and Im, 2014). These strategies include encapsulation of drug in natural materials, such as chitosan, alginate, albumin protein, and elastin-like polypeptides, as well as synthetic materials such as polymers and lipids. Polymeric, drug-loaded particles, such as those constructed from materials such as poly-(lactide-co-glycolide) acid (PLGA) have been most widely studied for the delivery of small molecules to the joint space, and are now the subject of a clinical trial for the delivery of triamcinolone (Butoescu et al., 2009a, Butoescu et al., 2009b, Horisawa et al., 2002a, Horisawa et al., 2002b, Kumar et al., 2013, Liang et al., 2004). Similarly, liposomes have been developed to release dexamethasone following delivery to the joint space (Butoescu et al., 2009a, Butoescu et al., 2009b, Lopez-Garcia et al., 1993, Turker et al., 2005). These clinical studies demonstrate that synthetic microparticles have utility in releasing drug following intra-articular delivery, yet to date no polymeric drug delivery system has been approved for intra-articular administration in humans (Butoescu et al., 2009a, Butoescu et al., 2009b, Gerwin et al., 2006). While polymeric and lipid carriers have been most widely proposed, naturally derived delivery vehicles may provide the benefit of biocompatibility and low cost. One of the earliest demonstrations of a biologically-based drug carrier systems for intra-articular injection were albumin microparticles, formed by emulsion and cross-linking, for the delivery of rose bengal (Ratcliffe et al., 1987). In vitro release studies showed that drug was poorly retained within the microparticles during incubation in serum, which was further confirmed by in vivo studies that revealed little difference in joint concentration between free drug injection and drug-loaded particles after 24 h. Elastin-like polypeptides (ELPs), recombinant proteins based on a native elastin sequence that can transition into drug depots in situ, provide another example of a pseudo-naturally derived delivery vehicle that has utility in increasing protein drug residence times in the joint space (Adams et al., 2009, Allen and Cott, 2010, Betre et al., 2006).
Silk fibers from the mulberry silkworm, Bombyx mori, have a long history of clinical use as a biomaterial, particularly as sutures and more recently as new FDA-approved medical devices. Silk fibroin, the de-gummed fibers of native silk, are a natural protein material that has proven to be non-immunogenic and robust against hydrolytic degradation (Meinel and Kaplan, 2012). The protein is very hydrophobic, readily forming β-sheet secondary structures resulting in a high density of crystalline regions. These regions are nearly impenetrable to water and yield desirable degradation properties in vivo (Cao and Wang, 2009, Numata and Kaplan, 2010, Vepari and Kaplan, 2007). Silk fibroin can be used to fabricate microparticles to entrap and release drug by emulsification (Wang et al., 2010). Using this method, silk microparticles (∼1–20 μm) can be easily and reproducibly formed with high loading efficiencies for small-molecule drugs and slow release rates (5–50% in first 168 h). In this study, we explored the utility of silk fibroin microparticles developed for intra-articular drug delivery of small-molecule drugs by adapting the method for silk microparticle fabrication to incorporate conjugation of a fluorescent dye. In vitro studies of fluorophore release and measures of the intra-articular clearance of fluorophore from rat knee joints using live-animal, fluorescence in vivo imaging (Bowles et al., 2013, Whitmire et al., 2012), were performed to assess the potential for the silk microparticles to contribute to sustained release in this application.
Section snippets
Study design
In order to assess the utility of silk fibroin (SF) microparticles for intra-articular drug delivery of small molecules, particles were characterized for physical properties, in vitro release kinetics, and in vivo intra-articular retention in rat knee joints. The physical properties of particle size, dispersity and surface morphology were assessed by scanning electron microscopy (SEM) imaging. The in vitro release of a model tracer, Cy7 near-infrared dye, was used to assess the effects of
Particle characterization
Cy7 dye was conjugated to SF at a molar ratio of 0.35:1 (Cy7:SF), forming Cy7-bound (SF-Cy7) solution for particle fabrication alone or in combination with unmodified SF solution. Cy7-labeling of silk protein impacts the formation of SF microparticles by emulsification, such that different formulations of SF-Cy7 and SF solution were investigated to identify a formulation with maximal Cy7 retention. Resulting SF-Cy7 microparticles of all formulations were found to be mainly spherical in shape
Particle size characterization
Drug carrier size is an important consideration for intra-articular drug delivery, that to date has not been fully resolved. Early work by Horisawa et al. examined the effect of particle size through the use of poly(lactic-co-glycolic acid) (PLGA) microparticles and nanoparticles. They determined that fluorescein-loaded nanoparticles (∼265 nm in diameter) were readily phagocytosed and thus caused a very minimal immunogenic response from local tissue. However, microparticles (∼26 μm average
Conclusion
SF microparticle drug carriers can be used to overcome the shortcomings of the intra-articular delivery of small-molecule drugs for the treatment of OA. The formed SF particles are of micron dimensions sufficient to increase joint residence time and limit rapid clearance of loaded drug. Live-animal fluorescence imaging is a useful tool for measuring the radial biodistribution and joint clearance properties of a drug delivery system when ROI image analysis is employed. Utilizing this tool for
Conflict of interest
There are no conflicts of interest to report.
Author contributions
TKM made significant contributions to the design, execution, and analysis of this work. TKM led the experiments and was in charge of analyzing the data. TKM drafted the manuscript and contributed to the revision of the manuscript to ready it for submission. RD Bowles provided significant contribution in the design and implementation of the experiment. RD Bowles helped to acquire the necessary imaging data for the work and contributed to the revision of the manuscript. DMT provided substantial
Acknowledgement
This work was supported by the NIH (R01AR047442, P41EB002520, T32GM008555, F31AR063610-01 and F32AR063012).
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