Self-assembled nanoparticles based on glycol chitosan bearing 5β-cholanic acid for RGD peptide delivery

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Abstract

The synthetic peptide bearing Arg–Gly–Asp (RGD) sequence is considered to specifically bind to αvβ3 integrin expressed on endothelial cells in the angiogenic blood vessels, which provides a potential to inhibit the tumor growth. As a carrier for the RGD peptide, hydrophobically modified glycol chitosan (HGC) capable of forming nano-sized self-aggregates was prepared by the chemical conjugation of 5β-cholanic acid to the main backbone of glycol chitosan. The RGD peptide labeled with fluoresein isothiocyanate (FITC-GRGDS) was loaded into self-aggregates in three different conditions: simple mixing, sonication, and solvent evaporation methods. Of different methods applied, solvent evaporation method showed the most promising results for peptide loading, as judged by the yield (>70%) and loading efficiency (>75%). It was found that the presence of FITC-labeled peptides makes the self-aggregates to be compact, possibly due to the role of both hydrophobic FITC and peptides containing carboxylic acids that allow hydrogen bonding and electrostatic interaction with the primary amino groups in the main backbone of glycol chitosan. FITC-labeled peptides were released from self-aggregates in a physiological solution (pH 7.4) for up to 1 day. From the cell adhesion and migration assays, it was demonstrated that FITC labeling of peptides does not significantly deteriorate biological activity of the parent peptide drug (GRGDS). Overall, the self-aggregates loaded with FITC-GRGDS might be useful for monitoring or destroying the angiogenic vessels surrounding the tumor tissue.

Introduction

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is a representative hallmark of cancer which cannot grow beyond a critical size (∼5 mm in diameter) and metastasize to another organ without blood supply [1], [2], [3]. Endothelial cells in the angiogenic vessels are known to express several markers, being either barely detectable or entirely absent in normal blood vessels [4], [5], [6], [7]. Of the makers expressed, αvβ3 integrin has drawn increasing attention since its level of expression in the tumor vasculature correlates with the grade of malignancy [8], [9], which may provide the opportunity to destroy tumor vessels by drug targeting without affecting the microvasculature of normal tissues. In recent years, several in vivo studies exhibited that peptide antagonists of αvβ3 integrin have a potential to inhibit the tumor growth by promoting apoptosis of the angiogenic vascular cells [3], [8], [10]. It, however, should be noted that most macromolecular drugs including peptides and proteins are susceptible to enzymatic degradation, when exposed to the biological environment. Furthermore, they cannot reach enough the target site via systemic administration because of their rapid elimination by renal filtration and the reticuloendothelial system, resulting in poor bioavailability and toxicity; therefore, the appropriate drug carriers that can surmount various biological barriers are obviously needed [11].

For past two decades, significant effort has been devoted to develop novel polymeric carriers that form compact micellar structure in an aqueous milieu, including amphiphilic block copolymers [12], [13], [14] and hydrophobically modified water-soluble polymers [15]. Among different carriers for controlled drug delivery, there has been a rising interest in nano-sized self-aggregates composed of natural polysaccharides such as pullulan [16], [17], curdlan [18], and chitosan [19]. In particular, chitosan and its derivatives have generated significant attraction due to its specific structure and physicochemical properties, leading to excellent biocompatibility, biodegradability, low immunogenicity, and biological activities [20], [21]. It is known that hydrophobically modified chitosan capable of forming nano-sized self-aggregates can imbibe hydrophobic drugs with a high efficacy and release them in a sustained manner [22]. Further, since chitosan possesses primary amino groups in the main backbone that can produce positively charged surfaces in a biological fluid, various macromolecular drugs containing negatively charged ones (e.g., peptides, proteins and genes) can be incorporated by the electrostatic interactions for their delivery into the site of action [19], [23], [24]. It, however, should be emphasized that most studies on nanoparticles for macromolecular drug delivery have focused on the control of the release rate in a sustained manner, whereas there have been no significant efforts for targeting of macromolecular drugs to specific cells, tissues, and organs.

Recently, our group developed hydrophobically modified glycol chitosans (HGCs), prepared by covalent attachment of 5β-cholanic acid to glycol chitosan through amide formation [25], as a potential drug carrier. Depending on the degree of substitution of 5β-cholanic acid, the HGCs formed self-aggregates (210–859 nm in diameter) in an aqueous phase by intra- and/or intermolecular association between hydrophobic 5β-cholanic acids. Also, they exhibited substantially lower critical aggregation concentrations (0.047–0.219 mg/ml) in phosphate-buffered saline (PBS, pH 7.4) than those of other low-molecular-weight surfactants, indicating high thermodynamic stability [26]. In an attempt to develop the effective peptide delivery system for the inhibition of angiogenesis, we herein prepared HGC-based self-aggregates loaded with peptides containing Arg–Gly–Asp (RGD) sequence, specifically binding to αvβ3 integrin expressed on endothelial cells in the angiogenic blood vessels [3], [10], [27], [28], [29].

Section snippets

Materials

Glycol chitosan (Mn=2.5×105, degree of deacetylation=88%), 5β-cholanic acid, bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). The HGC (degree of substitution=12%) was synthesized in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimid (NHS), as described previously [25]. RGD-bearing synthetic peptides such as GRGDS and its derivative labeled with fluoresein isothiocyanate (FITC-GRGDS) were the products of AnyGen

Characterization of HGC

To endow hydrophilic glycol chitosan with amphiphilic nature, hydrophobic 5β-cholanic acid was covalently conjugated by the reaction with primary amino groups, belonging to the main backbone of glycol chitosan, in the presence of EDC and NHS (see Fig. 1 for the detailed chemical structure). The degree of substitution (DS) was adjusted to 12% because this extent of DS resulted in formation of stable self-aggregates, low critical aggregation concentration (0.047 mg/ml), and high hydrophobicity of

Discussion

Self-assembled nanoparticles, formed by polymeric amphiphiles, have arisen as a promising carrier for various low-molecular-weight and macromolecular drugs, since they can solubilize hydrophobic drugs, increase bioavailability, and stay in the body long enough to allow gradual accumulation in the target region [12], [26], [34]. In an attempt to deliver peptide drug effectively, containing RGD sequence that specifically interacts with the αvβ3 integrin in endothelial cells, we herein prepared

Conclusion

The self-aggregates, formed by HGC, were assessed as a carrier for FITC-labeled peptide drugs. By the solvent evaporation method, FITC-labeled peptides were successfully loaded with a high efficiency. They exhibited comparable biological activity to parental peptides, as demonstrated by the cell adhesion and migration assays. In spite of their hydrophilic characteristics, FITC-labeled peptides were released from self-aggregates in a sustained manner for approximately one day.

Acknowledgements

This study was supported by the Ministry of Commerce, Industry and Energy, South Korea.

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