Synthesis and evaluation of cyclosporine A-loaded polysialic acid–polycaprolactone micelles for rheumatoid arthritis
Graphical abstract
Introduction
Due to the high cost and lengthy implementation timeline involved with drug discovery, drug delivery may serve as an alternative method to advance pharmaceutical sciences and human health. In addition, a numbers of therapeutics are characterized by poor bioavailability, unfavorable biodistribution, and high cytotoxicity, particularly when administered systemically. Drug carrier systems are needed to improve drug efficacy and reduce cytotoxicity in the human body. To date, many studies have been conducted based on the theory proposed by Paul Ehrlich (Strebhardt and Ullrich, 2008) to develop therapeutics which can be site-specifically delivered to the target tissue, with reduced accumulation in the healthy tissue. Most of these delivery systems are designed with the advantages of increased drug solubility, prolonged circulatory stability, and high tissue specificity (Torchilin, 2000).
Among various nanoparticle systems, polymeric micelles have drawn much attention for the encapsulation of hydrophobic drugs since first reported in 1984 (Bader et al., 1984). These micelles are formed from macromolecules composed of hydrophobic and hydrophilic segments. In an aqueous environment, the amphiphilic polymers self-assemble into a core–shell structure due to the aggregation of the hydrophobic moieties. Thus, hydrophobic drugs can be physically encapsulated into the core via hydrophobic interactions. In general, micelles provide therapeutics with improved solubility, enhanced stability, and an extended circulation time (Xiong et al., 2011).
When administered systemically, drug delivery systems without sufficient hydrophilicity to reduce the recognition and binding of plasma proteins are often eliminated rapidly by the reticuloendothelial system (RES). Thus, by increasing the surface hydrophilicity, the rate of elimination can be decreased and the circulation time can be prolonged, thereby improving the likelihood that the drugs will reach the target disease tissues. To date, poly(ethylene glycol) (PEG)-based modification has been the most common method to improve hydrophilicity and provide the drugs with so-called “stealth” properties to evade detection by the RES (Chaudhari et al., 2012, Cole et al., 2011, Parveen and Sahoo, 2011). However, PEG may not be the ideal solution due to a non-biodegradable backbone, evidence of continuous accumulation inside the body, and problems with immunogenicity (Caliceti and Veronese, 2003, Gregoriadis et al., 2005, Knop et al., 2010). Moreover, the PEG coating is known to interfere with some of the steps involved in drug delivery. After localization to the diseased tissue, PEG coatings have been reported to hinder drug release from the carrier systems and reduce requisite drug–cell interactions (Erbacher et al., 1999, Holland et al., 1996, Hong et al., 1999).
As an alternative, polysialic acid (PSA) is a relatively unexplored natural, non-toxic, and biodegradable polysaccharide that has the potential to prolong the circulation time of associated drugs and provide additional benefits. PSA is a linear homopolymer of α-2,8-linked 5-N-glycolyneuraminic acid (Neu5Ac) and is widely produced by pathogenic bacteria, as well as the cells of vertebrates and higher invertebrates. Thus, PSA is highly involved and has multifarious roles in a wide variety of biological, immunological, and pathological processes (Chen and Varki, 2010, Varki, 1992, Varki and Gagneux, 2012, Varki and Schauer, 2009). Some pathogenic bacteria can escape the host immune system and evade the host tissues by producing a thick PSA coating on the cell wall (Gregoriadis et al., 2005). In mammals, the major function of PSA is believed to be the anti-adhesive properties that can change the cell–cell and cell–extracellular matrix interaction and promote neural plasticity. PSA acts as a post-translational modification of neural cell adhesion molecules (NCAM), and the fifth Ig domain of NCAM is able to carry PSA at a high loading capacity (Johnson et al., 2005, Rutishauser, 2008). The significant negative charge and large hydrated volume of PSA can reduce NCAM-mediated adhesion and enable neuron cell migration. Typically, PSA expression is down-regulated in most tissues of the adults (Johnson et al., 2005, Rutishauser, 2008, Rutishauser and Landmesser, 1996). However, during the neural injuries (Ghosh et al., 2012, Mikkonen et al., 1998) and tumorigenesis (Hanahan and Weinberg, 2000, Hanahan and Weinberg, 2011), PSA is expressed on the cell surfaces, which serves to alter cellular interactions to abrogate cell adhesion and facilitate cell migration. The anti-adhesive properties are further supported by immune studies that demonstrate that the removal of PSA generates an “eat me” signal to macrophages to recognize and clear the uncoated bacteria, excess proteins, or dead cells (Elward and Gasque, 2003).
Due to the natural anti-adhesive properties highlighted above, PSA has drawn attention in the field of drug delivery. Based on a series of studies on sialyated or polysialylated proteins, Gregoriadis et al. proposed that PSA was a potential material to increase the stability and circulation time of therapeutics inside the bodies (Gregoriadis et al., 1993). PSA-drug conjugation has been used to increase the half-life of insulin (Jain et al., 2003), asparaginase (Fernandes and Gregoriadis, 1997, Fernandes and Gregoriadis, 2001), and catalase (Fernandes, 1994, Fernandes and Gregoriadis, 1996). As a result, immunogenicity and antigenicity were reduced, and the efficacy of the proteins was improved. To date, several PSA–protein conjugates and Neu5Ac derivatives have been developed as vaccines and therapeutic agents (Morris et al., 2008, von Itzstein et al., 1993).
Compared to other drug delivery systems, nanoparticle-based targeted drug delivery systems have been shown to accumulate passively within tumor tissue and inflamed tissue due to the enhanced permeability of the leaky vasculature (Duncan, 2003). Previously, our lab developed micelles from PSA modified with a long chain hydrocarbon, decylamine (Bader et al., 2011). Despite possessing the necessary physical properties in regards to size and surface charge, the micelles were cytotoxic towards a synovial fibroblast cell line. Here in, we develop and characterize non-cytotoxic, PSA-based micelles via grafting of the PSA with amine-terminated polycaprolactone (PCL), a hydrophobic, non-toxic polymer that can be degraded in the human body by ester hydrolysis (Fig. 1) (Kumari et al., 2010). The protective role the PSA is thought to play in vivo was preliminarily evaluated by measurement of the fixed aqueous layer thickness (FALT). FALT has previously been correlated with the ability of carrier systems to prevent opsonization and, consequently, uptake by the RES (Fang et al., 2006, Sadzuka et al., 2003, Sadzuka et al., 2006). Cyclosporine A (CyA), a therapeutic used in the treatment of rheumatoid arthritis, was used as a model compound to demonstrate the capacity of the micelles to encapsulate hydrophobic molecules. Fluorescence microscopy experiments were used to demonstrate cellular uptake of the drug-loaded micelles and release of the CyA from the micelles.
Section snippets
Materials
Colominic acid sodium salt (PSA, isolated from E. coli, Mw 30 kDa) was obtained from Nacalai USA (San Diego, CA). N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI), N,N′-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), ethylenediamine, m-chloro-peroxybenzonic acid, benzyl alcohol, 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), ε-caprolactone (ε-CL), and Boc-gly-OH were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Trifluoroacetic acid (TFA) (peptide synthesis
Synthesis and characterization of PSA–PCL micelles
PSA–PCL was successfully synthesized via amide bond formation between PSA and amine-terminated PCL (Fig. 1). Low molecular weight PCL (Mw ∼ 8000) was synthesized via dual activation of the benzyl alcohol initiator and ε-caprolactone monomer with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) catalyst (Lohmeijer et al., 2006). The terminal hydroxyl group of the PCL polymer was subsequently conjugated to Boc-protected glycine. Acid-catalyzed deprotection yielded the desired amine-terminated PCL (Lu et
Conclusion
In this study, PSA–PCL micelles were developed as carrier systems for CyA, a disease-modifying anti-rheumatic drug that is associated with significant, adverse side-effects. Physical characterization, including size and zeta potential, demonstrated that the micelles possess favorable properties for drug delivery. The PSA gave rise to an aqueous layer, similar to that found with PEGylated liposomes, that is expected to confer “stealth”-like properties in vivo. In vitro studies verified that
Acknowledgements
The authors thank R.P. Smith at SUNY College of Environmental Science and Forestry for assistance with TEM, and M. Maye and B. Hudson for providing use of the ZetaSizer Nano and the Fluorimeter respectively. Liposomes were prepared by I. McCabe and fixed aqueous layer thickness was determined by J. Liu. This work was supported by NSF grant CBET-1032506.
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