ВЫСОКОМОЛЕКУЛЯРНЫЕ СОЕДИНЕНИЯ, Серия А, 2012, том 54, № 5, с. 716-721
_ БИОПОЛИМЕРЫ
УДК 541.64:539.199:547.96
SELF-ASSEMBLY OF COUMARIN-CONJUGATED ACIDIC PROTEINOIDS1 © 2012 г. Hee Jin Seo and Jin-Chul Kim
Division of Biotechnology & Bioengineering and Institute of Bioscience and Biotechnology, Kangwon National University, 192-1, Hyoja 2dong, Chuncheon-si, Kangwon-do 200-701, Korea
e-mail: jinkim@kangwon.ac.kr Received July 4, 2011 Revised Manuscript Received December 20, 2011
Abstract—Photo- and pH-responsive self assembly was prepared by dispersing coumarin-conjugated acidic proteinoid in an aqueous phase. Proteinoid composed of Asp, Ser and Leu (PASL) was prepared as an acidic one by a melt-condensation method. Epoxypropoxy coumarin (EPC) was conjugated to PASL through its reaction with Ser. Assuming all Ser residues reacted with EPC, the calculated molar ratio of Asp/Ser-EPC/Leu in EPC-PASL conjugate was about 202/18/1 on 1H NMR spectrum. EPC-PASL conjugates exhibited the dimerization and the de-dimerization of their EPC residues under cyclic irradiation of 365 nm and 254 nm. They were self-assembled into microparticle (about 500 nm), possibly due to the hydrophobic interaction among Leu residues and EPC residues. The EPC-PASL assemblies was linked each other by the photo-dimerization under the UV irradiation of 365 nm. EPCs are the hydrophobic pendants of the PASL conjugates and they could be easy to be hydrophobically assemblied. This may account for why EPC-PASL assemblies were much greater than PASL assemblies. On the contrary, EPC of PASL conjugates was cleav-aged by the photo-dedimerization under the UV irradiation of 254 nm. The size of the assemblies increased and decreased in a cyclic manner under the cyclic irradiation, and the size of the photo cross-linked assembly was larger at a higher pH, possibly due to stronger intermolecular repulsion.
INTRODUCTION
Coumarin and its derivative have been used in the preparation of photo-responsive vehicles, due to their photochemical properties. Upon the irradiation of X > 365 nm, cyclobutane bridge is formed between two molecules of coumarins, leading to the formation of dimers. The bridge is cleaved and monomers are formed under the irradiation of X > 365 nm [1—3]. Several kinds of polymeric vehicles which respond to UV irradiation were prepared by taking advantage of coumarin. An amphiphilic having coumarin in its hy-drophobic block were dispersed in a polar solvent and the resulting polymeric micelles was subjected to the irradiation of X > 310 nm for the photo cross-linking of cores [4]. The enhance release of a hydrophobic compound was observed upon the irradiation of a light of X < 260 nm. The photo cleavage of the cross-linked core was reported to promote release [5]. Also, photo-responsive vesicle and nanogel were prepared using block copolymers having hydrophilic blocks and ther-mo-sensitive blocks containing coumarins [4, 6].
On the other hand, proteinoids have attracted much attention in the field of drug delivery systems because of easy preparation and their versatility as a drug carrier material. Especially, an acidic proteinoid protected drug in oral delivery from gastric juice [7, 8]. It may give low toxicity to human body because the pro-
1 Статья печатается в представленном авторами виде.
teinoid is composed of amino acids. Proteinoids are prepared simply by heating the mixture of amino acids (so called, a melt-condensation method) [9—11]. They can form microspheres in aqueous phase with aid of inter/intra-molecular hydrogen bonding and hydrophobic interactions. At low pH values (pH 1—4), an acidic proteinoid could form self-assembled microspheres. On the other hand, at high pH values (pH > 7), the acidic proteinoid microsphere is disassembled [8]. Depending on the compositions, the microspheres exhibited a different pH-dependent release profiles. Microsphere of acidic proteinoids can suppress the release of their contents under an acidic condition but they promote the release under a neutral and an alkali condition due to the ionization of acid proteinoids [11, 12]. Recently, proteinoids were included in liposomal preparations [13] and monoolein cubic phase to achieve a pH-responsive release profile [14].
In this study, a coumarin derivative was conjugated to an acid proteinoid, and photo- and pH-responsive microspheres were prepared by dispersing the conjugate in an aqueous phase. Epoxypropoxy coumarin (EPC) was derivatized, as a coumarin derivative for the conjugation, from hydroxyl coumarin, and proteinoid composed of Asp, Ser and Leu was prepared as an acidic proteinoid by a melt-condensation method. Asp was included for the acidity, Ser was included for the EPC conjugation site, and Leu was for the hydro-phobicity. In order to investigate the photo-dimeriza-
tion/de-dimerization property of EPC residue in EPC-proteinoid conjugate, the dimerization degree was observed under a cyclic irradiation of 365 nm and 254 nm. In order to investigate the photo-response of EPC-proteinoid conjugate assemblies, the size change was investigated under the cyclic irradiation. To inves-
tigate the pH-responsive property of the photo cross-linked assemblies, the size changes of the assemblies being subjected to 30 min-irradiation of 365 nm, and 30 min-irradiation of 365 nm/10 min-irradiation of 254 nm were investigated with varying the pH of medium (Scheme 1).
Scheme 1. Photo-responsive and pH responsive structural change of EPC-PASL self-assembly. EPC-PASL is self-assembled in an aqueous phase due to the hydrophobic interaction among leucine residues (a). The self-assembly is photo-crosslinked by the dimerization of EPC residues under the irradiation of X = 365 nm ((a) ^ (b)). The crosslinking density of the crosslinked self-assembly decreases by the dedimerization of EPC residues under the irradiation of X = 254 nm ((b) ^ (a)). The crosslinked self assembly de-swells at a low pH because Asp residues is unionized and no intermolecular electrostatic repulsion is developed ((b) ^ (c)). The crosslinked self-assembly swells at a high pH because Asp residues is ionized and intermolecular electrostatic repulsion is developed ((b) ^ (d)).
EXPERIMENTAL
Materials
Epichlorohydrin, 7-hydroxycoumarin, L-Aspartic acid, DL-Leucine and L-Serine were purchased from Sigma Chemical Co. (St. Louis, Mo). Sodium bicarbonate was provided by BioShop Canada Inc. (Burlington, Vermont, USA). Dialysis membrane (MWCO: 1,000) was purchased from Spectrum (CA, USA). All other reagents were in analytical grade.
Preparations of Coumarin-Conjugated Acidic Proteinoids
Peptide composed of Asp, Ser and Leu (PASL) was prepared as an acidic proteinoid [9—11]. Asp (10 g), Ser (2.52 g) and Leu (0.15 g) were mixed with glycerol (3 ml) in a 3 neck- flask of 250 ml. After the flask was
purged with nitrogen gas, the mixture was melted and reacted for 12 h by holding the flask in a silicone bath kept around 160°C. In order to dissolve the mixture, sodium bicarbonate solution (10% in distilled water), 40 ml, was poured into the flask pre-cooled to a room temperature. For the purification, the mixture solution was put in a dialysis membrane (MWCO 1,000, Spectrum (CA, USA)) and it was dialyzed against 1 l of distilled water for 48 h with 8 time exchanges. The purified proteinoid was freeze-dried for further use.
Epoxypropoxy coumarin (EPC) was prepared by a method reported elsewhere [15]. 100 ml of 7-hydrox-ycoumarin solution (3.24%, w/v) in ethanol was combined with 5 ml of KOH solution (25 %, w/v) in distilled water in a 3-neck round bottom flask of 250 ml, and the mixture solution was stirred at room temperature for 30 min. Epichlorohydrin, 20 ml, was put to the
solution and the mixture was reacted at 95—100°C for 3 h with a reflux. The product was obtained by a series of operations (solvent evaporation, dissolution of dry residue in the two phase of water/chloroform for partition of EPC into oil phase, separation of oil phase, extraction of impurity from oil phase using water phase, separation of oil phase, oil phase evaporation, recrystalization of EPC in ethanol, and filtration).
EPC was conjugated to PASL as follows 0.2 g of PASL was dissolved in 10 ml of distilled water at a room temperature. PASL solution was mixed with 5 ml of NaOH solution (20%, w/v) in distilled water. And then, 5 ml of EPC solution (16.24%, w/v) in dim-ethylsulfoxide was dropped to 15 ml of PASL solution. The mixture was reacted for 48 h at room temperature. For the purification, the reaction mixture was dialyzed against distilled water using a dialysis membrane (MWCO 1,000, Spectrum (CA, USA)) and the purified solution was freeze-dried for further use.
Spectroscopy
The FTIR spectrum of PASL was taken in KBr pellet on a Perkin Elmer Fourier Transformed Infrared spectrophotometer instrument (EXCALIBER Series, U.S.A.) in the Central Laboratory of Kangwon National University. The 1H NMR spectrum EPC was taken in CDCH3 and the 1H NMR spectrum of EPC/PASL conjugate was taken in D2O on a Bruker Avance 600 spectrometer (Karlsruhe, Germany) in the Central Laboratory of Kangwon National University.
Dimerization of EPC Residue of EPC-PASL Conjugates Under Cyclic UVIrradiation
EPC-PASL was dissolved in distilled water so that the concentration was 0.01 mg/ml. The solution was exposed to the irradiation of X = 254 nm (6 W) for 10 min and the irradiation of X = 365 nm (400 W) for 30 min in a cyclic manner. The degree of dimerization was determined as follows [16].
Dimerization (%) = (1 - At/A0) x 100, where, A0 is the absorbance of EPC residue of EPC-PASL conjugate at 327 nm before irradiating a UV light, and At is the absorbance after irradiating a UV light for a certain period.
Size Change of PASL Assembly and EPC-PASL
Conjugate Assembly under Cyclic UV Irradiation
Each ofPASL and EPC-PASL was dissolved in distilled water so that the concentration was 2% (w/v). Each solution was subjected to the cy
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.