In vitro and in vivo characterization of nanoparticles made of MeO-PEG amine/PLA block copolymer and PLA
Introduction
Recently, polymeric nanoparticles, liposomes, solid-lipid nanoparticles etc. have been extensively investigated to optimize medication with drugs including drug targeting (Yang et al., 1999, Nakanishi et al., 2001, Charrois and Allen, 2004, Brannon-Peppas and Blanchette, 2004, Shenoy et al., 2005, Abraham et al., 2005). Polymeric nanoparticles have attracted much attention because the chemical modification of their polymers is possible, leading to nanoparticles with various biological behaviors. Chemical modification of PLA or PLGA, being biocompatible and biodegradable, has been made actively in an attempt to modify the physicochemical and biological properties of the nanoparticles (Otsuka et al., 2000, Nagasaki et al., 2001, Yamamoto et al., 2001, Jule et al., 2003, Miura et al., 2004).
The particle size, electrical charge and surface structure affect the biological behaviors of micro- and nano-particulate dosage forms (Sugibayashi et al., 1979, Kanke et al., 1980, Unezaki et al., 1995, Tabata et al., 1998, Ishida et al., 1999). For nanoparticulate dosage forms, in particular, the surface structure is a very important factor determining biological fate in the body. Plain PLA or PLGA nanoparticles administered intravenously are delivered quickly and mostly into the liver and spleen via the reticuloendothelial system (Gref et al., 1994, Dunn et al., 1997, Mosqueira et al., 1999, Panagi et al., 2001). However, polyethylene glycol–polypropylene glycol–polyethylene glycol block copolymers such as poloxamer are useful for raising the systemic retention of nanoparticles with PLA as a core component (Dunn et al., 1997, Onishi et al., 2003, Machida et al., 2003). Polyethylene glycol–PLA block copolymer (PEG–PLA), which is a diblock copolymer with hydrophilic and hydrophobic blocks, has been reported to allow the formation of nanoparticules more stable in biological circumstances. In PEG–PLA nanoparticles, PLA chains form the core and PEG chains are located on the outer side. PEG–PLA nanoparticles vary in size from several dozen to a few hundred nanometers, and possess a hydrophilic and inactive surface of PEG, leading to a longer systemic circulation (Gref et al., 1994, Bazile et al., 1995, Yamamoto et al., 2001, Panagi et al., 2001, Avgoustakis et al., 2003, Miura et al., 2004). The PEG shell prevents PLA core from interacting with biomolecules, cells and tissues (Gref et al., 1994, Bazile et al., 1995, Mosqueira et al., 1999, Panagi et al., 2001), and suppresses opsonization (Nguyen et al., 2003). Recently, Kataoka et al. have reported various kinds of PEG–PLA nanoparticles with modified surface (Otsuka et al., 2000, Nagasaki et al., 2001, Yamamoto et al., 2001, Jule et al., 2003), in which sugars, amino acids, peptides etc. are attached to the PEG terminal of PEG–PLA. In these derivatives, an acetal-ended PEG-PLA was synthesized by sequential polymerization of ethylene oxide and dl-lactide, and utilized for further modification. On the other hand, we developed a novel approach to obtain PLA derivatives (Sasatsu et al., 2005). Our method involves the synthesis of acetal-ended PLA (PLA-acetal), its conversion to PLA with a formyl terminal end (PLA-aldehyde), and the modification of the formyl group using reductive amination. Namely, ring polymerization of dl-lactide using diethoxypropanol as an initiator was performed to obtain PLA-acetal. PLA-acetal was subsequently hydrolyzed to PLA with a formyl group, then PLA-aldehyde was reacted with the molecules having amino groups by reductive amination. As this approach allows the modification of PLA directly, various kinds of PLA derivatives are expected to be produced. A block copolymer of PLA and methoxypolyethylene glycol amine (MeO-PEG(N)), called PLA–(MeO-PEG), was reported previously to be prepared by reductive amination between PLA-aldehyde and MeO-PEG(N), but the degree of introduction of MeO-PEG(N) into PLA was not necessarily reproducible or extensive (Sasatsu et al., 2005). In the present study, the preparative conditions of PLA–(MeO-PEG) were refined to improve the introduction of MeO-PEG(N) into PLA. Further, as the present PLA–(MeO-PEG) has a secondary amino group, different from the conventional block copolymer made of PLA and methoxypolyethylene glycol (Gref et al., 1994, Bazile et al., 1995, Quellec et al., 1998), the nanoparticles prepared here were characterized in vitro and in vivo. A lipophilic fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) perchlorate was used as a model drug. As DiD is a very lipophilic, it is considered possible to evaluate the incorporation properties of the nanoparticles for lipophilic drugs. Further, since DiD seems to be incorporated stably in the core of PLA or PLA–PEG nanoparticle and little diffused to the outer aqueous solution (Mosqueira et al.), the pharmacokinetic features of the nanoparticles may markedly affect the biodistribution of DiD. The present study has dealt with the loading properties of the PLA/PLA–(MeO-PEG) mixture nanoparticles for DiD and the effect of the nanoparticles on the biodistribution of DiD.
Section snippets
Materials
Methoxypolyethylene glycol amine (MeO-PEG(N); MW 2000), 3,3-diethoxy-1-propanol and stannous octoate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). dl-Lactide was obtained from Tokyo Kasei Kogyo Co., Ltd. (Japan). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) perchlorate was purchased from Molecular Probes, Inc. (OR, USA). All other chemicals used were of reagent grade.
Animal
Male ddY mice (6–7 weeks old, 30 g) were purchased from Tokyo Laboratory Animals Science Co., Ltd
Preparation and characteristics of PLA–(MeO-PEG)
As to PLA-aldehyde(n) (Table 1), the MWn and formyl ratio were obtained from 1H NMR spectra (CDCl3), in which relative integrated intensities for the specific protons, appearing at 4.32 (q, J = 7 Hz), 5.05–5.30 (m) and 9.72 (s), were 1/99/0.70, 1/186/0.60, 1/154/0.55 and 1/221/0.55 for PLA-aldehyde(1), PLA-aldehyde(2), PLA-aldehyde(3) and PLA-aldehyde(4), respectively. Furthermore, the MWn and introduction degree of MeO-PEG(N) to PLA for PLA–(MeO-PEG)(n) were calculated based on 1H NMR spectra
Conclusion
The synthetic procedure for PLA–(MeO-PEG) could be refined. It was obtained more easily and reproducibly by using a new solvent system, a mixture of dichloromethane and methanol (1:1, v/v), in a reductive amination between PLA-aldehyde and MeO-PEG(N) and by collecting the whole first peak in the GPC separation. The DiD-loaded nanoparticles prepared using the mixture of PLA–(MeO-PEG) and PLA (55:45 (mol/mol)) had a size of 154 nm and a little positive zeta potential. The nanoparticles exhibited a
Acknowledgments
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors thank Mr. Ken-ichi Ueki for his technical assistance.
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