Controlled Drug Delivery - American Chemical Society

C h a p t e r 35

Supramolecular-Structured Polymers for Drug Delivery Tooru Ooya and Nobuhiko Yui

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Downloaded by CORNELL UNIV on December 17, 2014 | http://pubs.acs.org Publication Date: May 15, 2000 | doi: 10.1021/bk-2000-0752.ch035

School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan

Polyrotaxanes as a supramolecular-structured polymer were characterized aiming at a drug carrier, a drug permeation enhancer, an implantable material, and a stimuli-responsive material. Biodegradable polyrotaxanes exhibit their supramolecular architectures: many α-cyclodextrins (α-CDs) are threaded onto a single poly(ethylene glycol) (PEG) chain capped with biodegradable bulky end-groups. Further, a stimuli-responsive polyrotaxane, in which many β-CDs are threaded onto a triblock-copolymer of PEG and poly(propylene glycol) (PPG) capped with fluoresce in-4 -isothiocyanate, was designed as a novel smart material.

Supramolecular sciences have been getting fascinating field whereby nanostructures in nature are constructed by self-assemblies of small molecules (1). A family of polyrotaxanes has been recognized as a molecular assembly in which many cyclic compounds are threaded onto a linear polymeric chain capped with bulky endgroups (1). The name of rotaxane was givenfromthe Latin words for wheel and axle, and thus it refers to a molecular assembly of cyclic and linear molecules. Inclusion complexation of oc-cyclodextrins (α-CDs) threaded onto poly(ethylene glycol) (PEG) has been well studied as a polypseudorotaxane by Harada et al since 1990(2). Our recent studies have focused on the design of biodegradable polyrotaxanes aiming at biomedical and/or pharmaceutical applications. This paper deals with the structural characteristics of our designed polyrotaxanes as a supramolecular-structured polymer and their feasibility for biomedical and/or pharmaceutical applications.

Biodegradable Polyrotaxanes for D r u g Delivery During last two decades, water-soluble and/or biodegradable polymers have been widely demonstrated as polymeric carriers for biologically active agents (3). 'Corresponding author.

© 2000 American Chemical Society

In Controlled Drug Delivery; Park, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

375


376 Chemical immobilization of any drug onto an water-soluble polymeric backbone via biodegradable spacer has been a general strategy to design polymeric drug carriers since Rigsdorf proposed his idea on a polymeric drug carrier in 1975. We have proposed the design of biodegradable polyrotaxanes as novel candidates for drug carriers. The most attractive character of such a polyrotaxane will involve the control of drug release utilizing the dissociation of the supramolecular structure (Figure 1). Generally, drugs in polymeric prodrugs were covalently bound to the polymeric mainchain via biodegradable moieties. Those macromolecular prodrugs are to provide prolonged release action of the drugs by the hydrolysis or biological scission of the covalent bonds (4). In our approach, drugs can be introduced to α-CDs via another biodegradable spacers in the polyrotaxane. Thus, regulating the degradability of the terminal degradable moiety and the spacers, it may be likely that the terminal moieties of the polyrotaxanes are firstly degraded by hydrolytic enzymes to release the drugimmobilized α-CDs. The covalent bonds between the drug and the α-CDs are then subjected to hydrolysis to release the active drugs.

Figure 1. α-CD release via terminal hydrolysis of the polyrotaxane.

Synthesis of Biodegradable Polyrotaxanes and Drug-Polyrotaxane Conjugates We have synthesized a series of biodegradable polyrotaxanes in which many α-CDs are threaded onto a PEG chain capped with an L-phenylalanine (L-Phe) moiety via a peptide linkage (5-8). The synthesis was carried out by i) preparing a polypseudorotaxane consisting of α-CD and amino-terminated PEG as previously reported by Harada et al. (9,10), and ii) capping both terminals of amino-groups in the polypseudorotaxane with benzyloxycarbonyl (Z-) L-Phe-succinimide in dry DMSO. The obtained polyrotaxanes were hy droxy propyl ate d using propylene oxide in a IN NaOH solution, and Z-groups were deprotected with palladium-carbon. The hydroxypropylation of the α-CDs improved the solubility of the polyrotaxanes in a phosphate-buffered saline. The purity of the polyrotaxanes was evaluated by H NMR and GPC (7). The threading of α-CDs onto a PEG chain has been confirmed by detecting the hydrophobic cavity of α-CDs when the terminal peptide linkages were cleaved by papain (5, 6). The conjugation of the polyrotaxane with a model drug (theophylline) was carried out (11). Hydroxyl groups of the hydroxypropylated (HP-)a-CDs in the polyrotaxane (ca. 20-22 α-CDs were threaded onto PEG, Mn=^4000, with the degree of HP substitution being 8-9/a-CD) were activated by 4-nitrophenyl chloroformate in DMSO/pyridine at 0°C. The coupling of N-aminoethyl-theophylline-7-acetoamide with the activated polyrotaxane was carried out using DMSO/pyridine (1:1) as a l


377


solvent. Z-groups were deprotected to obtain a drug-polyrotaxane conjugate (Figure 2). The synthesis was confirmed by Ή-NMR and GPC. The number of ΗΡ-α-CDs in the drug-polyrotaxane conjugate was determined to be ca. 13-14 by 'H-NMR spectrum. From the GPC peaks, one theophylline molecule was found to be introduced into every two α-CD molecules in the drug-polyrotaxane conjugate.

Figure 2. Schematic representation of the drug (theophylline) polyrotaxane conjug

Drug Release from Biodegradable Polyrotaxanes via Supramolecular Dissociation and its Relation to Solution Properties

Table 1. Solution Parameters Determined by Light Scattering Measurements Sample

The Number of Association A2 · lu HP-a-CDs Number (mlmolg ) 2

Drug-polyrotaxane conjugate

13-14

6

-8.03

Polyrotaxane

20-30

2

3.40

L-Phe-terminated PEG

0

46

0.25

N O T E : Rl„ of the used P E G is 4000 for all samples. The number of Η Ρ - α - C D s was determined by H - N M R . !

Solution parameters of the polyrotaxane and the drug-polyrotaxane conjugate, which were determined by static light scattering measurements, were summarized in Table. 1. Association number of the polyrotaxane and the drug-polyrotaxane conjugate was found to be much smaller than that of L-Phe-terminated PEG. This result indicates that hydroxyl groups in ΗΡ-α-CDs participate in preventing the association of such a supramolecular-structured polymer. The second virial coefficient (A ) value is a measure of the extent of polymer-polymer or polymer-solvent 2


378 interaction. Polymers are avoiding one another as a result of their van der Waals "excluded volume" when the A value is large, and they are attaching one another in preference to the solvent when it is small or negative. Taking the characteristics of the A value into account, it is suggested that the polyrotaxanes are considered to exhibit a loosely packed association, presumably due to their supramolecular structure. On the other hand, L-Phe-terminated PEG seems to exhibit a tightly packed association, because the terminal hydrophobic moieties can associate easily due to the flexibility of the PEG chains (12). In addition, the drug-polyrotaxane shows less association state with strong molecular interaction of theophylline (11). Furthemiore, the ratio of hydrodynamic radius and radius of gyration (Rh/Rg) of the polyrotaxane revealed an anisotropic shape, indicating its rod-like structure (data not shown) (12). The rod-like structure of the polyrotaxane is likely to affect the reduced association state. 2


2

Time (h) Figure 3. HP-a-CD release via terminal hydrolysis of the polyrotaxane in the presence ofpapain as a model enzyme

As for the polyrotaxane, ΗΡ-α-CD was found to be released by the terminal hydrolysis in the presence of papain as a model enzyme (Figure 3). The terminal hydrolysis in the polyrotaxanes proceeded to over 85 %, in contrast to the limited hydrolysis in L-Phe-terminated PEG (-50%) (12). Taking the A values of the polyrotaxane into account (Table. 1), the complete dissociation of the polyrotaxanes by hydrolysis maybe due to the less association state related to the rod-like structure. On the other hand, the high association state of L-Phe-terminated PEG with strong molecular interaction is considered to decrease the accessibility of papain to the terminals. In vitro degradation of the drug-polyrotaxane conjugate by papain was examined in order to clarify the terminal hydrolysis of the drug-polyrotaxane 2


379


conjugate and drug-immobilized HP-α-CDs (drug-HP-α-CDs) release. As shown in Figure 4, the release of drug-HP-α-CD from Hie drug-polyrotaxane conjugate was found to be completed around 200 h. HP-α-CD release from the polyrotaxane proceeded to reach 100% at 320 h under a similar experimental condition. Considering the solution properties of the drug-polyrotaxane conjugate as mentioned above, these results indicate that the association of the conjugate does not induce the steric hindrance but rather enhances the accessibility of enzymes to tie terminal peptide linkages. Presumably, the terminal peptide linkages in the drug-polyrotaxane conjugate are likely to be exposed to the aqueous environment as a result of the specific association nature.

Potential of the Polyrotaxanes as a D r u g Penetration E n h a n c e r Cellular response to synthetic polymers is of fundamental importance to their possible therapeutic application as pharmacologically active polymers and polymeric carriers for biologically active molecules (13). Our recent studies revealed that our designed polyrotaxanes have the ability of enhancing the fluidity of red blood cell ghosts, although the mixture of the constituent molecules (HP-α-CD + L-Pheterminated PEG) shows less effect (8, 14). Here, the potential of the polyrotaxanes as a drug penetration enhancer is described in relation to the effects of the polyrotaxane structure on cellular interaction.


380


Effect of the Polyrotaxanes on Drug Permeation through Hairless Rat Skin Our previous studies revealed that the treatment of skin stratum corneum with the polyrotaxanes induces tie preferential extraction of polar lipid components but maintains ordered lipid bilayers in the stratum corneum (15). This finding indicates that the structure of the polyrotaxane is important for the effective interaction with the skin stratum corneum as well as red blood cell ghosts (8, 14). Indomethacin (IND) permeation through hairless rat skin treated with the polyrotaxanes was evaluated using a two-chamber cell at 37°C for 18 h. The enhanced permeation of IND through the full-thickness skin was observed when tie skin surface was pre-treated with the polyrotaxane solution in PBS at 37°C for 18 h (16). Increased permeability coefficient (P, cm/sec) values were obtained, corresponding to a decrease in the bound water content. Therefore, the interaction of the polyrotaxanes with the stratum corneum may induce an enhanced IND permeation. Similar effects were observed when the dermis side of the skin was pre-treated with the polyrotaxane solution under the same condition (16). This suggests the possibility of the polyrotaxane penetration into not only the stratum corneum but also subcutaneous tissue. We have not determined yet how the polyrotaxanes can increase the permeability coefficient whether it is a partition- or diffusion-controlled mechanism. Because the polyrotaxanes were degraded into their constituent molecules by a protease (data not shown), the selection of biodegradable moieties seems to enable the appropriate metabolism of polyrotaxanes after they have penetrated into subcutaneous tissues.

Biodegradable Polyrotaxanes for Tissue E n g i n e e r i n g Biodegradable polymers have been studied as implantable materials for cell growth and tissue regeneration (17). Aliphatic polyesters such as poly(L-lactic acid) (PLA) is a representative of implantable materials for tissue engineering. However, high crystallinity of PLA reduces water intrusion into tie crystalline regions resulting in incomplete hydrolysis; crystalline oligomers remain in the tissue for a long time. Such a complex situation causes chronic inflammatory reactions at the implanted sites (18), Our next concern of biodegradable polyrotaxanes is in the design of implantable materials for tissue engineering. A novel design for biodegradable polymers has been proposed by constructing a supramolecular structure of a polyrotaxane; α-CDs threaded onto a PEG chain are capped with benzyloxycarbonyl (Z-) L-Phe via ester linkages (19). The most favorable characteristics of the polyrotaxanes as implantable materials will involve their excellent biocompatibility, mechanical properties and instantaneous dissociation properties. The polyrotaxane may have high crystallinity which is due to intermoleeular hydrogen bonds between tie hydroxyl groups of the aCDs. This high crystallinity is postulated to be disappeard due to terminal hydrolysis, although the crystallinity of aliphatic polyesters usually increases as a result of their hydrolysis. The hydrolysis of the ester linkage in a certain PEG terminal will trigger rapid dissociation of the supramolecular structure into α-CDs, PEG and Z-L-Phe, thus, this may not induce any inflammatory reactions.


381


Supramolecular Dissociation of Biodegradable Polyrotaxanes with Ester Linkages The biodegradable polyrotaxane with ester linkages as a terminal stopper has been synthesized and characterized (19). In vitro hydrolysis of the polyrotaxane was performed, and the α-CD release via terminal hydrolysis was evaluated by HPLC using a reverse phase column. As for the supramolecular dissociation of the polypseudorotaxane (without the Z-L-Phe moiety), it was found that α-CD release was completed within 10 min in the buffer atanypH (Figure 5a). On the other hand, as for the polyrotaxane, the completion of the α-CD release required several days (Figure 5b). These results indicate that the supramolecular dissociation of the polyrotaxane was prolonged in comparison with the polypseudorotaxane. Further, the α-CD release rate at pH 8.0 was found to be faster than those at pH 7.4 and 5.7 (Figure 5b). The terminal hydrolysis at each pH was well correlated with the α-CD release behavior (data not shown). Taking these results into account, it is suggested that the terminal hydrolysis of the polyrotaxane is a dominant process in the supramolecular dissociation. Recently, it was found that the induction period observed before the onset of hydolysis in the polyrotaxanes was prolonged by chemical modifications of α-CDs such as acetylation. This finding enables us to design implantable materials which are to be disappeared in a predicted period.

(a)

100

P=3

T i m e (h)

Figure 5. Cumulative α-CD release (%) from the polypseudorotaxane (a) and the polyrotaxane (b).


382


Feasibility of Polyrotaxanes for Stimuli-Responsive Materials New synthetic methods have been investigated over the past two decades in order to design "intelligent" or "smart" materials which exhibit large property changes in response to small physical or chemical stimuli. Our alternative concern regarding polyrotaxanes is how such a molecular assembly can be utilized as a material with molecular dynamic functions: threading of many CDs onto a polyrotaxane might change the location along a linear polymeric chain in response to external stimuli which would be perceived as the action of mechanical pistons (20). Because the driving force for such inclusion complexation of CDs with a polymeric chain is due to intermolecular hydrogen bonding between neighboring CDs as well as steric fittings and hydrophobic interaction between host and guest molecules, several stimuli such as temperature and dielectric change may be used to control the assembled state of the CDs onto a polyrotaxane (Figure 6).

h dispersed

®

0

assembled

Figure 6. Stimuli-responsive change in CD location in a polyrotaxane.

Synthesis and Characterization of a Polyrotaxane Consisting of β-Cyclodextrins and a Poly(ethylene glycol)-Poly(propylene glycol) Triblock-Copolymer

)H

Scheme L A polyrotaxane in which many β-CDs are threaded onto the triblockcopolymer capped withfluorescein-4-isothiocyanate(FITC)

A polyrotaxane, in which many β-CDs are threaded onto PEG- b Iock-VVG-blockPEG triblock-copolymer (Mn = 4200, PPG segment Mn = 2250, PEG segment Mn = 975 x2) named Pluronic® P-84 capped with fluorescein-4-isothiocyanate (FITC), was synthesized as a model of stimuli-responsive molecular assemblies (21). Terminal hydroxyl groups of the triblock-copolymer were modified to obtain amino-terminated triblock-copolymer. The polypseudorotaxane between the amino-terminated triblock-


383 copolymer and β-CDs was prepared in 0.1 M phosphate buffered saline at 40 °C. The terminal amino-groups in the polypseudorotaxane were allowed to react with FITC in DMF at 5 °C to obtain the polyrotaxane (Scheme 1). In this condition, a side reaction of β-CDs and FITC was prevented (21, 22). The threading of β-CDs in the polyrotaxane was confirmed by GPC, Ή-NMR and 2D NOES Y NMR spectroscopies using a 750 MHz FT-NMR spectrometer (21). The number of β-CDs in the polyrotaxane was determined to be ca. 7 from the H -NMR spectrum. 5


Change in the Location of β-CDs in Response to Temperature The interaction of β-CDs with terminal FITC moiety was analyzed by means of induced circular dichroism (ICD) measurement in 0.01 Ν NaOH at 25°C (21). A positive ellipticity [0] of the polyrotaxane around 490 nm decreased when the temperature increased from 20 to 40 °C. This result indicates that the interaction between β-CD and the terminal FITC moiety is considered to decrease with increasing temperature. The interaction of β-CD hydrophobic cavity with the PPG segment of the triblock-copolymer was analyzed by means of 750MHz H-NMR spectroscopy. With increasing temperature, the peak of the methyl protons in the PPG segment shifted to the lower field and slightly broadened for the polyrotaxane (21). The methyl proton peak shifted to the lower field is considered to be due to the interaction between the methyl protons of propylene glycol (PG) units and the cavity of the β-CDs. Assuming that the shifted methyl proton peak is the included methyl protons with β-CDs, the number of β-CDs located on the PG units can be estimated (21). As a result, it is considered that ca. 5.2 β-CD molecules were localized on the PPG segment at 50 °C, although ca 1.3 β-CD molecules existed on the PPG segment at 10 °C. It was found that the association number of the polyrotaxane was decreased although that of the model tribloek-copolymer was slightly increased from 35 °C to 50 °C (23). These results indicate that the dissociation behavior of the polyrotaxane may be related to the localization of β-CDs along the tribloek-copolymer. Thus, stimuliresponsive polyrotaxanes can be one of candidates to create novel smart materials acting in a similar manner to natural chemomechanical proteins of myofibrils where myosin headpeices slide along actin filaments to initiate the contraction process. !

Conclusions It is concluded that our designed biodegradable polyrotaxanes were demonstrated to be promising as a drug carrier as well as a drug permeation enhancer, and an implantable materials for tissue engineering (24, 25). Stimuli-responsive polyrotaxanes will provide new fields of smart materials which are constructed by mimicking natural supramolecular assemblies. Supramolecular dissociation of the polyrotaxanes by their terminal hydrolysis will be the most unique characteristic when considering biomedical and pharmaceutical applications. Further, effective interaction of the polyrotaxanes with cellular systems is consideredto be related to tie supramolecular structure. These polyrotaxanes are designed as to be degraded after meanwhile or by completing their medical functions in living body, and thus, to be excreted as soluble compounds. In near future, biodegradable and/or stimuli-


384 responsive polyrotaxanes will enable us to achieve novel biomedical pharmaceutical properties.

and

Acknowledgement The authors are grateful to Mr. Fujita, H., Mr. Kamimura, W., Mr. Watanabe, J. and Mr. Kumeno, T. (JAIST) for their help in the experiments on biodegradable polyrotaxanes. A part of this study was financially supported by a Grant-in-Aid for Scientific Research (B) (No.851/09480253) and a Grant-in-Aid for Scientific Research on Priority Areas New Polymers and Their Nano-Organized System" (No. 277/09232225), from The Ministry of Education, Science, Sports and Culture, Japan. Downloaded by CORNELL UNIV on December 17, 2014 | http://pubs.acs.org Publication Date: May 15, 2000 | doi: 10.1021/bk-2000-0752.ch035

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