pubs.acs.org/langmuir © 2009 American Chemical Society
)
Supramolecular Hydrogel of a D-Amino Acid Dipeptide for Controlled Drug Release in Vivo† )
)
Gaolin Liang,‡ Zhimou Yang,‡ Rongjun Zhang,§ Lihua Li,‡ Yijun Fan,§ Yi Kuang, Yuan Gao, Ting Wang,# W. William Lu,# and Bing Xu*,‡, ‡
)
Department of Chemistry, The Hong Kong University of Science & Technology, Clear Water Bay, Hong Kong, §Jiansu Institute of Nuclear Medicine, Wuxi, Jiangsu, China, Department of Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, and #Department of Orthopedics and Traumatology, The University of Hong Kong, Hong Kong Received December 28, 2008. Revised Manuscript Received January 21, 2009 A supramolecular hydrogel based on D-amino acids, which resists hydrolysis catalyzed by proteinase K and offers long-term biostability, exhibits controlled release in vivo, as proved by the pharmacokinetics of encapsulated 125I tracers and the SPECT imaging of the hydrogel-encapsulated 131I tracers. As the first in vivo imaging investigation of the drug release properties of the supramolecular hydrogel, isotope encapsulation serves as a valid, useful assay for characterizing the controlled release properties of supramolecular hydrogels in vivo. Our results indicate that supramolecular hydrogels promise new biomaterials for controlled drug release.
This letter reports on the development of a supramolecular hydrogel for controlled drug release in vivo. Supramolecular hydrogels,1 formed by small-molecule hydrogelators self-assembling into 3D networks of nanofibers to provide the matrices to imbibe large amounts of water, offer easily achievable biocompatibility and biodegradability and promise new biomaterials that are being actively explored for applications such as drug delivery,2 biosensing,3 tissue engineering,4 and wound healing.5 Among the molecules that gel water, peptide-based hydrogelators have attracted considerable research attention because of their biological relevance. Their applications for controlled drug release, which require that the hydrogels resist digestive enzymes and possess longterm stability, have been less explored.6 Encouraged by the † Part of the Gels and Fibrillar Networks: Molecular and Polymer Gels and Materials with Self-Assembled Fibrillar Networks special issue. *Corresponding author. E-mail:
[email protected]. Tel: 781-7365201. Fax: 781-736-2516.
(1) Estroff, L. A.; Hamilton, A. D. Chem. Rev 2004, 104, 1201–1217. Terech, P.; Weiss, R. G. Chem. Rev. 1997, 97, 3133–3159. van Esch, J. H.; Feringa, B. L. Angew Chem., Int. Ed. 2000, 39, 2263–2266. Kiyonaka, S.; Sada, K.; Yoshimura, I.; Shinkai, S.; Kato, N.; Hamachi, I. Nat. Mater. 2004, 3, 58–64. Yang, Z. M.; Gu, H. W.; Fu, D. G.; Gao, P.; Lam, K. J. K.; Xu, B. Adv. Mater. 2004, 16, 1440–1444. Yang, Z. M.; Xu, B. Chem. Commun. 2004, 2424–2425. Kostiainen, M. A.; Hardy, J. G.; Smith, D. K. Angew. Chem., Int. Ed. 2005, 44, 2556–2559. (2) Kiyonaka, S.; Sugiyasu, K.; Shinkai, S.; Hamachi, I. J. Am. Chem. Soc. 2002, 124, 10954–10955. Yang, Z. M.; Gu, H. W.; Zhang, Y.; Wang, L.; Xu, B. Chem. Commun. 2004, 208–209. (3) Hamachi, I.; Nagase, T.; Shinkai, S. J. Am. Chem. Soc. 2000, 122, 12065–12066. (4) Jayawarna, V.; Ali, M.; Jowitt, T. A.; Miller, A. E.; Saiani, A.; Gough, J. E.; Ulijn, R. V. Adv. Mater. 2006, 18, 611–614. Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D.; Kessler, J. A.; Stupp, S. I. Science 2004, 303, 1352–1355. (5) Yang, Z. M.; Xu, K. M.; Wang, L.; Gu, H. W.; Wei, H.; Zhang, M. J.; Xu, B. Chem. Commun. 2005, 441, 4–4416. Yang, Z. M.; Liang, G. L.; Ma, M. L.; Abbah, A. H.; Lu, W. W.; Xu, B. Chem. Commun. 2007, 843–845. (6) Vemula, P. K.; Li, J.; John, G. J. Am. Chem. Soc. 2006, 128, 8932–8938. Nagai, Y.; Unsworth, L. D.; Koutsopoulos, S.; Zhang, S. G. J. Controlled Release 2006, 115, 18–25. (7) Seebach, D.; Matthews, J. L. Chem. Commun. 1997, 2015–2022.
Langmuir 2009, 25(15), 8419–8422
biostability of β-peptides7 and recent progress on bioactive β-peptides,8 we have been developing supramolecular hydrogelators based on unnatural amino acids (i.e., amino acids other than the 20 natural ones) and have demonstrated that β-peptide derivatives efficiently gel water.9 This result prompted us to examine the drug release applications of hydrogels constructed from unnatural amino acids. Although the large pool of unnatural amino acids offers a range of possibilities for the exploration of supramolecular hydrogels with long-term biostability, only a few hydrogelators made from unnatural amino acids have been reported.9-11 In this letter, we report on our systematic examination of the biostability of three unnatural amino acid-based hydrogelators that share structural similarities with 1, a small-molecule hydrogelator12 that consists of naphthalene (Nap) and the phe-phe motif that is prone to self-assembly.13 In vitro, 2 and 3 showed excellent biostability against proteinase K, a powerful enzyme hydrolase for a broad spectrum of peptides. Using radioactive tracers, we found that the hydrogel formed by 2 exhibited good controlled release characteristics in vivo. After subcutaneously injecting the hydrogel into the abdomens of rats, (8) Murray, J. K.; Farooqi, B.; Sadowsky, J. D.; Scalf, M.; Freund, W. A.; Smith, L. M.; Chen, J. D.; Gellman, S. H. J. Am. Chem. Soc. 2005, 127, 13271–13280. Pomerantz, W. C.; Abbott, N. L.; Gellman, S. H. J. Am. Chem. Soc. 2006, 128, 8730–8731. Porter, E. A.; Wang, X. F.; Lee, H. S.; Weisblum, B.; Gellman, S. H. Nature 2000, 404, 565–565. Karlsson, A. J.; Pomerantz, W. C.; Weisblum, B.; Gellman, S. H.; Palecek, S. P. J. Am. Chem. Soc. 2006, 128, 12630–12631. (9) Yang, Z. M.; Liang, G. L.; Ma, M. L.; Gao, Y.; Xu, B. Small 2007, 3, 558–562. (10) Bhuniya, S.; Park, S. M.; Kim, B. H. Org. Lett. 2005, 7, 1741–1744. (11) Yang, Z. M.; Liang, G. L.; Xu, B. Chem. Commun. 2006, 738–740. (12) Yang, Z. M.; Liang, G. L.; Wang, L.; Xu, B. J. Am. Chem. Soc. 2006, 128, 3038–3043. (13) Reches, M.; Gazit, E. Science 2003, 300, 625–627.Yang, Z. M.; Liang, G. L.; Guo, Z. F.; Guo, Z. H.; Xu, B. Angew. Chem.-Int. Ed. 2007, 46, 8216– 8219. Yang, Z. M.; Xu, K. M.; Guo, Z. F.; Guo, Z. H.; Xu, B. Adv. Mater. 2007, 19, 3152–3156. Liang, G. L.; Xu, K. M.; Li, L. H.; Wang, L.; Kuang, Y.; Yang, Z. M.; Xu, B. Chem. Commun. 2007, 4096–4098.
Published on Web 03/16/2009
DOI: 10.1021/la804271d
8419
Letter Scheme 1. Chemical Structures of the Hydrogelators
gel-encapsulated 125I-NaI was released, and thereafter the blood concentration of 125I-NaI was maintained within a narrow range during the first 12 h after administration. SPECT images also showed that 4 h after subcutaneous injection only about one-third of the 131I-NaI solution remained in the injection site, whereas nearly two-thirds of the gel-encapsulated 131I-NaI remained. Using the clinically used drug epidepride to replace NaI, we also found that the gelencapsulated 125I-epidepride and 131I-epidepride showed controlled release behavior in vivo. These results suggest that supramolecular hydrogels made from unnatural amino acids may lead new types of biomaterials for controlled drug release. Because Nap-L-Phe-L-Phe12 (1) and racemic Nap-β3-HPhg3 β -HPhg11 are efficient molecular hydrogelators, their structural analogues D-phenylalanine (D-Phe), s-β3-H-phenylglycine (s-β3-HPhg), and L-4-fluorophenylalanine (L-fPhe) became the choice for the unnatural amino acids for making the hydrogelators (Scheme 1) in this study. Figure 1a-c shows transmission electron micrograph (TEM) images of cyrodried gels (gel II for 2, gel III for 3, and gel IV for 4), which provide indirect information about the aggregation of the hydrogelators in the gel phase. The nanofibers in gel II exhibit morphology similar to that of the nanofibers in gel I (Figure S1), with a length of over 10 μm, a width of about 50 nm, and an average mesh size of about 200 nm. The circular dichroism (CD) spectrum of gel II is nearly the mirror image of that of gel I (Figure S2B), agreeing with 2 being the enantiomer of 1. Gel III contains irregular fibers with width ranging from 30 nm to about 250 nm, and the fibers show a strong tendency to aggregate into bundles and leave large pores (about 500 nm) in the matrices of the gel. Its CD signal confirmed the less-ordered aggregates of 3 in the gel phase (Figure S2B). Gel IV consists of fibrils at much higher density than those in the other three gels. The fibrils (20 to 150 nm in width) tangle with each other to form a dense 3D network with 100 nm pores. The high density of the nanofibers results in the stronger elastic behavior. The CD spectrum of gel IV (Figure S2) also suggests the less ordered β-sheet arrangement in the nanofibers of 4.
Figure 1. TEM images of the nanofiber matrices in the cryo-dried (a) gel II, (b) gel III, and (c) gel IV. Scale bar 500 nm. 8420 DOI: 10.1021/la804271d
After observing the self-assembled nanofibers in the hydrogels, we used a cell viability assay to verify the biocompatibility of hydrogelators 1-4. MTT results show that the four hydrogelators all have IC50 values higher than 500 μM at 48 h on HeLa cells, indicating that they are biocompatible (Figure S4). Then, we evaluated their biostability by incubating them with proteinase K at 37 °C in HEPES buffer solutions. As shown in Figure 2a, 2 and 3 resisted enzymatic digestion, indicated by the fact that their quantities remained constant after 24 h of incubation. On the contrary, 1 and 4 hydrolyzed easily in the presence of proteinase K: only 37% of 1 and 16% of 4 remained in the solutions after being incubated for 24 h with proteinase K. To further validate that the amount of proteinase K could affect the rate of drug release, we used gel IV to encapsulate folic acid with different amounts of proteinase K and found that the release rate of folic acid depended on the amount of enzyme (Figure 2b). These results suggest that gel IV, based on L-f Phe, may find application only for short-term controlled drug release because it could not ward off the hydrolysis catalyzed by the enzyme. Because 1 is more resistant to proteinase K than 4 and 2 and 3 are almost completely resistant to proteinase K, we may extrapolate that the hydrogels of 2 or 3 could serve as biomaterials that require long-term biostability. According to the capillary model,14 the hydrogel can be considered to have fixed capillary pores perpendicular to the surface, each with the same radius. The steric hindrance at the entrance of and frictional forces within the pores in the hydrogel would impede the diffusion of solute, thus the diffusive effects of solute follows the Renkin equation.15 According to the Renkin equation (Supporting Information), because the pore sizes of gel II are smaller than those of gel III, gel II might have a better in vivo controlled drug release profile than gel III. Moreover, because hydrogelators 1 and 4 degrade quickly in the presence of proteinse K and the hydrogels of the analogs of 1 or 3 have been examined in vivo,12 we focus the investigation on the controlled release properties of gel II (based on D-amino acid dipeptides). Before starting the in vivo experiment, we investigated the biocompatibility of gel II with mouse. Each rat received a subcutaneous injection of 0.2 mL of gel II (0.8 wt% of 2) under the middorsal skin. No clinical, hematological or biochemical (including blood glucose) toxicity was observed, and there were no local or systemic bacterial, viral, or fungal infections in the mice treated over 42 days. A histological examination of the skin at the injection site also showed that there was no obvious inflammatory effect 42 days after injection (Figure S5B), compared with the control group (Figure S5A). We subcutaneously injected 125I-NaI-encapsulated gel II, which consisted of 1 wt % 2 and 100 mCi/L 125I-NaI, into the abdomens of rats at a dosage of 160 μCi/kg and detected the released 125I-NaI into the blood using a γ counter (Supporting Information). As shown in Figure 3a, subcutaneously injected 125I-NaI solution (i.e., the control) reached its Cmax (maximal drug concentration in blood) of 136.5 μCi/L at 1 h and a second peak (due to secondary absorption such as via hepatoenteral circulation16) of 92.7 μCi/L at 4 h. Unlike the control group, the 125I-NaI-encapsulated gel II reached its Cmax of 113.0 μCi/L at 3 h and a second peak C of 83.4 μCi/ (14) Krajewska, B. J. Chem. Technol. Biotechnol. 2001, 76, 636–642. (15) Renkin, E. M. J. Gen. Physiol. 1954, 38, 225–243. (16) Liang, G. L.; Bi, J. B.; Huang, H. Q.; Zhang, S.; Hu, C. Q. J. Ethnopharmacol. 2005, 101, 324–329.
Langmuir 2009, 25(15), 8419–8422
Letter
Figure 2. (a) Digestion curve of four molecular hydrogelators upon the treatment of proteinase K. (b) Controlled release of folic acid from gel IV by proteinase K.
Figure 3. (a) Profiles of the mean blood concentration of 125I-NaI vs time after s.c. administration to rats (160 μCi/kg) (9 control, 125I-NaI solution; area under the curve (AUC), 1213.3 μCi 3 h/L; b experimental, 125I-NaI in gel II; AUC, 1453.5 μCi 3 h/L). (b) Dynamic (upper two lines) and static (lower line) single photon emission computed tomography (SPECT) images of rats with 131I-NaI (500 μCi/rat; left, in solution; right, in gel II) administered s.c.. (c) Profiles of the mean blood concentration of 125I-epidepride vs time after s.c. administration to rats (160 μCi/kg) (9 control, 125I-epidepride solution; AUC, 645.5 μCi 3 h/L; b experimental, 125I-epidepride in gel II; AUC, 693.6 μCi 3 h/L). (d) Dynamic SPECT images of rats with 131I-epidepride (500 μCi/rat; left, in gel II; right, in solution) administered s.c. L at 8 h. Not only was the time of the two peaks delayed, but the concentration of the two peaks was also lower. From 1 to 12 h, the mean blood concentrations of 125I-NaI in the experimental group were maintained in a narrow range (94.7 to 61.3 μCi/L), whereas that of the control group decreased rapidly (136.5 to 38.1 μCi/L). This result confirms that gel II exhibits good behavior of controlled release in vivo. To gather more direct evidence of the controlled release property of gel II in vivo, we also subcutaneously (s.c.) injected 131 I-NaI-encapsulated gel II into the abdomens of rats and obtained dynamic (0-2 h) and static (2-4 h) SPECT images (Supporting Information). As shown in Figure 3b, the subcutaneously injected 131I-NaI solution (left rat) diffused quickly. At 4 h, most of the injected 131I-NaI was Langmuir 2009, 25(15), 8419–8422
distributed in the hypothyroid and bladder. On the contrary, the 131I-NaI in s.c. injected gel II (right rat) diffused slowly, and only a small proportion of 131I-NaI was concentrated in the hypothyroid after 4 h. The quantitative radioactivity analysis (Figure S6A) at 60 min showed that 22.3 and 53.3% radioactivity remained at the injection site of the control group and the experimental group, respectively. These results are consistent with the pharmacokinetics results shown in Figure 3a. After confirming that gel II can assist with the controlled release of encapsulated 125I-NaI and 131I-NaI in vivo, we also used epideride, a widely used dopamine D2 receptor imaging agent for clinical diagnosis, to further examine the controlled drug release properties of gel II in vivo. DOI: 10.1021/la804271d
8421
Letter
Figure 4. Cumulative percent in vivo release profiles: (9) 131I-NaI
solution, (b) 131I-epidepride solution, (2) 131I-NaI in gel II, and (1) I-epidepride in gel II
131
As indicated in Figure 3c, the curve of the mean blood concentration of the 125I-epidepride encapsulated by gel II versus time also showed controlled release behavior compared with that of the s.c. injected 125I-epidepride solution: it reached its Cmax of 53.9 μCi/L at 3 h whereas that of the injected 125I-epidepride solution (i.e., the control) reached its Cmax of 82.6 μCi/L at 0.5 h. From 0.5 to 12 h, the mean blood concentrations of 125I-epidepride in the experimental group were kept at 53.9 to 26.6 μCi/L, whereas that of the control group decreased rapidly (from 82.6 to 26.0 μCi/L). After that, we also subcutaneously injected 131I-epidepride into the abdomen of rats and obtained the dynamic (0-4 h) SPECT images. As shown in Figure 3d, the s.c. injected 131I-epidepride solution (right rat) diffused quickly. At 110 min (arrows indicated), most of the injected 131I-epidepride solution (91.5%) was absorbed and distributed in the kidney and bladder, whereas 45.6% of 131I-epidepride-encapsulated gel II was retained in the injection site and only a small proportion of 131I-epidepride was concentrated in the bladder. Even after 230 min, a considerable proportion of 131I-epidepride (about 26.5%) was retained in the injection site. The quantitative radioactivity analysis (Figure S6B) showed that at 70 and 190 min there were 57.7 and 28.4% of the radioactivity remaining at the injection sites of the experimental group and 13.3 and 6.3% remaining at the injection sites of the control group. Subtracting the remaining radioactivity at the injection site from the initial total dosage, we can get the released drug dosage. Therefore, from the analysis of radioactivity data at the injection sites (Figure S5), we can get the cumulative in vivo drug release profiles (Figure 4). As Figure 4 indicated, both the 131I-NaI and 131I-epidepride encapsulated by gel II showed controlled release properties compared with their behavior in solution. The 131I-epidepride solution showed a faster release behavior than the 131I-NaI solution subcutaneously, which is likely due to its hydrophobicity that facilitates its absorption in the subcutaneous tissue. However,
8422 DOI: 10.1021/la804271d
being encapsulated by gel II, 131I-epidepride had a slower release than did 131I-NaI. This may be ascribed to its larger molecular size than that of 131I-NaI. Moreover, the hydrophobic 131I-epidepride should have a stronger affinity for the hydrophobic domain of the nanofiber of gel II than 131I-NaI did. This affinity should slow down the release of 131I-epidepride from the gel II matrix. These results demonstrate that gel II is a good matrix for controlled drug release in vivo. In summary, besides being the first in vivo imaging of the drug release properties of supramolecular hydrogels, this letter demonstrated that a 1 wt % supramolecular hydrogelator could significantly alter the drug release profile. This work, together with previous in vitro investigation of controlled drug release of supramolecular hydrogels,17 confirms that the supramolecular hydrogel is a useful class of materials to complement the well-established drug release systems based on biodegradable polymers.18 Although gel II was effective in controlling only the release of the selected drugs on a short-term basis in this study, it is still possible to use gel II for other applications requiring long-term biostability or to prolong the duration of controlled drug release by enhancing the interactions between the hydrogelator (2) and the drug molecules. Furthermore, we demonstrated that isotope encapsulation was a valid and useful assay for characterizing the controlled release properties of supramolecular hydrogels in vivo. Moreover, unlike polymeric hydrogels having less-flexible pore sizes because their matrices are linked by covalent bonds, supramolecular hydrogels can change their pore sizes as a result of the reassembly of hydrogelators during the shrinkage/swelling processes.19 This property of self-adjusting pore sizes might render supramolecular hydrogels as “smart” matrices for controlled drug release, which is currently under investigation. Acknowledgment. This work was partially supported by RGC of Hong Kong (HKU2/05C, 604905, 600504), EHIA (HKUST), and start-up funds (Brandeis). B.X. thanks Professor Ying Chau for helpful suggestions. Supporting Information Available: Detailed experimental section, including synthesis, addition TEM data, cytotoxicity of the hydrogelators, and histology. This material is available free of charge via the Internet at http://pubs.acs.org. (17) Thornton, P. D.; Mart, R. J.; Ulijn, R. V. Adv. Mater. 2007, 19, 1252– 1256. Mahler, A.; Reches, M.; Rechter, M.; Cohen, S.; Gazit, E. Adv. Mater. 2006, 18, 1365–1370. (18) Langer, R. Acc. Chem. Res. 2000, 33, 94–101. Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Chem. Rev. 1999, 99, 3181–3198. Langer, R. Nature 1998, 392, 5–10. Bulmus, V.; Woodward, M.; Lin, L.; Murthy, N.; Stayton, P.; Hoffman, A. J. Controlled Release 2003, 93, 105–120. (19) Zhou, S. L.; Matsumoto, S.; Tian, H. D.; Yamane, H.; Ojida, A.; Kiyonaka, S.; Hamachi, I. Chem.;Eur. J. 2005, 11, 1130–1136.
Langmuir 2009, 25(15), 8419–8422
