Development of polymeric nanoparticulate drug delivery systems: evaluation of nanoparticles based on biotinylated poly(ethylene glycol) with sugar moiety

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Abstract

Liver specific polymeric nanoparticles were designed and synthesized from biotinylated poly(ethylene glycol) conjugated with lactobionic acid containing a galactose moiety (abbreviated as BEL). Synthesized BEL conjugate was identified by Fourier transform-infrared (FT-IR) and 1H-nuclear magnetic resonance (NMR) spectroscopy. The fluorescence spectroscopy data showed that BEL conjugate was self-assembled in water to form core-shell structure nanoparticles, and the critical association concentration (CAC) value was estimated as 0.028 g/l. From the transmission electron microscope (TEM) observation, the BEL nanoparticles were spherically shaped and ranged in size between 30 and 60 nm. The particle size distribution was measured by photon correlation spectroscopy (PCS), and the result was 41.2±11.7 nm. Anti-cancer drug all-trans-retinoic acid (ATRA) was loaded into the BEL nanoparticles for evaluating its efficacy as a drug delivery carrier. The crystallinities of ATRA and ATRA-loaded nanoparticles were examined by X-ray diffraction (XRD) patterns. The ATRA release kinetics from the BEL nanoparticles showed a pseudo zero-order pattern during 1-month period.

Introduction

Nanoparticles are colloidal particles of a size below 1 μm, and widely employed in various fields of life sciences, such as separation technologies, histological studies, clinical diagnostic assays, and drug delivery system (DDS). In the DDS applications, nanoparticles have several merits, like ease purification and sterilization, drug targeting possibility, and a sustained release action (Allemann et al., 1993). We synthesized various core-shell structure nanoparticles in our previous studies (Kim et al., 2000, Kim and Kim, 2001, Kim and Kim, 2002). Core-shell structure nanoparticles present advantages in regard of a long circulation in the body, drug solubility, drug stability, and relatively high level of hydrophobic drug encapsulation into the hydrophobic core (Gref et al., 1994, Peracchia et al., 1997, Yokoyama et al., 1990, Yokoyama et al., 1991, Kwon et al., 1995).

Tissue-specific molecular delivery has been a valuable technique for biological medical research and for the diagnosis and cancer therapy. To achieve this goal, there needs to be an approach that can enable the chemical modification of carriers to allow the attachment of targeting ligands. The recognition of ligands on the surface of delivery systems by organ-specific or cell-specific receptors may result in the organ-targeted drug delivery (Takakura and Hashida, 1996, Daemen et al., 1988), or in improved drug uptake through ligand-induced endocytosis (Sato et al., 1996, Brown and Goldstein, 1986). In recent years, there has been growing interest in the area of liver cell-specific DDS. A great deal of effort has been made to achieve an appropriate liver targeting of chemotherapeutic agents with liposomes (Kim and Han, 1995), microspheres (Kim et al., 1993), and drug-carrier molecule conjugates (Seymour et al., 1991). Among the liver-associated surface receptors, the asialoglycoprotein receptor (ASGP-R: galactose receptor) is well known to be present on hepatocytes (Ashwell and Harford, 1982). It is also retained on several human hepatoma cell lines (Ciechanover et al., 1983). If a ligand binds to a galactose receptor, the ligand-receptor complex is rapidly internalized and the receptor recycles back to the surface (Nishikawa et al., 1993). Therefore, designing a DDS for galactose receptor-mediated endocytosis would be useful for targeting the hepatocyte/liver and hepatoma cells (Goto et al., 1994). The biotin content of cancerous tumors is higher than that of normal tissue. So, we expected that the biotin and the galactose moiety of the lactobionic acid could target together to the tumor cells in the liver. Biotin is a 244 Da, water-soluble vitamin (Vitamin H) composed of a ‘head’, represented by a complex aliphatic heterocycle, and an aliphatic ‘tail’, ending with a carboxyl group. The ‘head’ provides the most important contribution to the binding of biotin to avidin, while the carboxyl group can be rendered capable of reacting with several different functional groups (Elo and Korpela, 1984). To this carboxyl group, we conjugated the one terminal amino group of diamine-terminated poly(ethylene glycol) (PEG). And, subsequently the other amino group was coupled with the carboxyl group of lactobionic acid with galactose moiety. With this galactose moiety, it can be expected that the BEL nanoparticles are endocytosed by liver cells within several minutes, suggesting its preferential uptake mediated by asialoglycoprotein (ASGP) receptor as previous report (Hashida et al., 1995).

Retinoids have been shown to have substantial anticancer activity in a number of preclinical and clinical situations. All-trans-retinoic acid (ATRA), an active metabolite of retinol (Vitamin A) was chosen as a model anti-cancer drug. ATRA plays essential roles in the regulation of differentiation and proliferation of various cell types. It has been proved that ATRA is effective in the treatments of epithelial and hematologic malignancies such as head and neck cancer, lung cancer, breast cancer, ovarian adenocarcinoma, and acute promyelocytic leukemia (Han and Choi, 1996, Sccks et al., 1995, Trump et al., 1997).

In the present study, we prepared polymeric conjugate nanoparticles composed of biotin and diamine-terminated poly(ethylene glycol) with a galactose moiety from lactobionic acid. The polymeric conjugate (abbreviated as BEL) was physico-chemically characterized by FT-IR spectroscopy, fluorescence spectroscopy, PCS, and TEM. Anti-cancer agent ATRA loading was confirmed by XRD, and the release was studied in vitro to evaluate the possibility for DDS.

Section snippets

Materials

Biotin, lactobionic acid (LA) and N,N′-dicyclohexyl carbodiimide (DCC) were purchased from the Aldrich Chemical Company (Milwaukee, WI). Diamine-terminated poly(ethylene glycol) (ATPEG) with a number-average molecular weight of 2000 was supplied by Texaco Chem. Co. (Ballaire, TX). N-Hydroxysuccinimide (NHS) was purchased from Sigma Chemical Co. (St. Louis, MO). Dialysis membrane with a molecular weight cutoff (MWCO) of 2000 g/mol was obtained from Spectra/Por™ membranes. Dimethylsulfoxide (DMSO)

Results and discussion

The BEL conjugate was synthesized by two-step coupling with biotin, ATPEG, and LA. Synthesized BEL conjugate was confirmed by FT-IR and 1H-NMR spectroscopy as shown in Fig. 1, Fig. 2. The characteristic peaks of FT-IR were listed in Table 1. Especially, two characteristic peaks on this spectrum, i.e. amide stretch absorption at 3330 cm−1, and amide bending at 1570 cm−1, may be used to verify the BEL conjugate. In Fig. 2, the proton signal of biotin appeared at approximately 3.3 ppm, the proton

Acknowledgements

This work was supported by Korea Research Foundation Grant (KRF-2001-015-FP0127).

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