Enzyme-Responsive Polymeric Supra-Amphiphiles Formed by the

Article pubs.acs.org/Langmuir

Enzyme-Responsive Polymeric Supra-Amphiphiles Formed by the Complexation of Chitosan and ATP Yuetong Kang, Chao Wang, Kai Liu, Zhiqiang Wang,* and Xi Zhang* Key Lab of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China ABSTRACT: Chitosan and adenosine-5′-triphosphate (ATP) are employed as building blocks to fabricate polymeric supraamphiphiles based on electrostatic interactions, which can selfassemble to form spherical aggregates. The spherical aggregates inherit the phosphotase responsiveness of ATP. Compared to our previous work, this enzyme-responsive system can be more biocompatible and block polymers are not needed in preparation, which makes it possible to fabricate the chitosan-based enzyme-responsive assemblies in a large-scale, cheap way. Therefore, the application of the assemblies for nanocontainers and drug delivery is greatly anticipated.

■

INTRODUCTION Among a variety of stimuli-responsive polymers, enzymeresponsive polymers show intriguing properties because of the precise selectivity and high efficiency of the enzymatic catalysis.1−3 It is known that diseases are always associated with the abnormal activities of some specific enzymes, such as abnormally high enzymatic activity or concentration, in diseased tissues or cells. Therefore, the enzyme-responsive polymers may have special advantages in the utilization as targeting drug delivery systems.4,5 So far, there have been a number of reports on enzyme-responsive polymer systems.6−10 The enzyme responsiveness of these systems is generally achieved using a covalent approach, which is accomplished by covalently linking the enzyme-responsive moiety to the polymers.11−14 Recently, the noncovalent approach to fabricating enzymeresponsive systems has captured more and more attention.15−23 For example, we have constructed a phosphotase-responsive supra-amphiphile based on the electrostatic complexation of a double hydrophilic block copolymer and adenosine 5′triphosphate (ATP).24 It is found that the supra-amphiphiles can self-assemble into spherical aggregates and that spherical aggregates can be disassembled upon the treatment of phosphotase. A similar concept can be extended to the fabrication of cholinesterase-repsonsive polymeric supraamphiphiles.25,26 Huang and his co-workers have used host− guest interactions to construct an enzyme-triggering assembly system on the basis of surfactants and cyclodextrin complexes and α-amylase, which represents another noncovalent approach to fabricating enzyme-responsive systems.27 Compared to the covalent approach, no complicated synthesis is involved in this noncovalent approach, and the composition and components can be easily adjusted. In this article, we have attempted to employ chitosan and ATP as the building blocks for fabricating the polymeric supraamphiphile, as shown in Scheme 1. Chitosan, a massively available modified natural polymer, is produced via the © 2012 American Chemical Society

deacetylation of chitin, which is the most abundant natural polymeraccharide second only to cellulose. Because of its nontoxicity and biocompatibility, chitosan is widely used in pharmaceutical and biomaterial research.28−31 Additionally, it is among the few natural-based cationic polyelectrolytes because of the protonation of its free amine groups. ATP contains a hydrophobic adenine group along with four negative charges arising from the attached phosphate groups. It is a crucial molecule that strongly participates in the biological activities of living creatures, usually transferring chemical energy via the formation and cleavage of the phosphoanhydride bonds catalyzed by specific enzymes. Moreover, ATP has been introduced into artificial self-assemblies.32 In the present article, ATP is designed to be associated with chitosan to form polymeric supra-amphiphiles driven by electrostatic attractions. As the ratio between ATP and chitosan varies, part of the chitosan is associated with ATP and part of the chitosan remains cationic and hydrophilic. In this way, a polymeric supra-amphiphile is fabricated and is responsible for the formation of chitosan−ATP aggregates by self-assembly. In the presence of the active phosphatase, ATP will be hydrolyzed to single-negative-charge phosphates and a neutral adenosine, which is followed by the disassembly of the polymeric supraamphiphiles and the spherical aggregates. Considering that chitosan and ATP are natural building blocks, this study may provide a new kind of biocompatible enzyme-responsive supraamphiphile and assembly.

■

EXPERIMENTAL SECTION

Materials. Chitosan was analytical grade with a deactylation degree greater than 90%, obtained from Sinopharm Reagent Company. The molecular weight measured by a viscometric method to be 690 000 g/ mol. HCl, NaOH, and phosphotungstic acid were all analytical agents Received: August 12, 2012 Revised: September 14, 2012 Published: October 1, 2012 14562

dx.doi.org/10.1021/la303271f | Langmuir 2012, 28, 14562−14566

Langmuir

Article

Scheme 1. Schematic Illustration of the Formation and the Phosphatase-Induced Degradation of the Polymeric SupraAmphiphiles and the Spherical Aggregates

Figure 1. (a, c, d) TEM images of chitosan−ATP with r = 3, 1, and 6, respectively. (b) DLS number mean diameter of the chitosan−ATP complex with r = 3. (e) DLS count rates of the chitosan−ATP complex with different r values. In all of the samples, the concentration of the chitosan monomer unit remained at 8 mM.

■

and were purchased from the Beijing Chemical Reagents Company. Calf intestine alkaline phosphatase (CIAP) was purchased from Takara (Dalian, China). Instruments. Dynamic light scattering (DLS) data were obtained on a Malvern ZS 90 Zetasizer instrument. Transmission electron microscopy (TEM) images were performed with a Jemo 2010 electron microscope. UV−vis data were measured with a Hitachi U-3010 spectrometer with a slit width of 2.0 nm and at a scanning rate of 600 nm/min. 31P NMR spectra were performed on JNM-ECA600 equipment. To enrich ATP and its derivative compounds for 31P NMR characterization, the complex solutions (8 or 20 mL) were lyophilized first, and then 0.5 mL of purified water was added. Preparation of the Chitosan/ATP Aggregates. Chitosan was first dissolved in 1% (v/v) acetic acid. ATP solution was dropwise mingled with a stirred chitosan solution, and a certain amount of concentrated sodium hydroxide solution was added until the pH of the solution reached 5.0, with concentrations of the chitosan monomer unit from 2 to 10 mM and with a charge ratio of 1 to 10. Meanwhile, the transparency of the solution decreased, accompanying by an apparent Tyndall effect, meaning that the aggregates were obtained. The charge ratio, r, was defined as the molar amount of the chitosan monomer unit divided by 4 times the molar amount of ATP.

RESULTS AND DISCUSSION

To understand if the proportion between positive and negative charges will influence the stability of the aggregates, we varied the ratio between chitosan and ATP while keeping the concentration of chitosan constant. The samples were monitored by DLS and TEM. As shown in Figure 1a, when r is 3, spherical aggregates are formed with a size of around 50 nm. In addition, the average size of the aggregates is 68 nm, as indicated by DLS in Figure 1b. DLS data agree with the TEM observation considering the different measurement conditions. Higher or lower values of r will not ensure that the aggregates are spherical and narrowly dispersed. As presented in Figure 1c, when r reaches 1, the number of spherical aggregates is quite large, but they adhere to each other, which may explain the sharp decline in the count rate of the sample (Figure 1e). When r is greater than 6, as shown in Figure 1d, most of the structure is fiberlike and few spherical aggregates exist. As displayed in Figure 1e, with the decrease in r from 10 to 2, the count rate of the samples generally increases, indicating the enhancing tendency to form complexes, especially in samples with r = 2 14563

dx.doi.org/10.1021/la303271f | Langmuir 2012, 28, 14562−14566

Langmuir

Article

Figure 2. (a, c, d) TEM images of chitosan−ATP with c = 8, 2, and 10 mM, respectively; r is fixed at 3. (b) DLS number mean diameter of chitosan− ATP spherical aggregates with different concentrations.

Figure 3. (a) Count rate and (b) zeta potential of chitosan−ATP spherical aggregates at pH of 3, 4, 5, 6, and 7.

and 3. Therefore, r = 3 is chosen for the fabrication of the polymeric supra-amphiphiles in later studies. We wondered if the concentration of the two components could influence the self-assembly. In answer to this question, we varied the concentration of the two building blocks with the value of r fixed at 3, and the complexes were investigated using TEM and DLS. With the concentration of the chitosan monomer unit ranging from 2 to 8 mM, the number of complexes increases accordingly (Figure 2a,c), and the structure remains as spherical aggregates with a slight increase in size (Figure 2b). When the concentration of the chitosan monomer unit is 10 mM, the aggregates turn to bundlelike structures (Figure 2d). The phenomenon is in agreement with the DLS data (Figure 2b) in which the hydrodynamic diameter of 10 mM aggregates is much larger than the rest, probably resulting from the morphological transformation between 8 and 10 mM. Hence, the concentration of chitosan monomer units is fixed at 8 mM as the optimized concentration for the investigation below. The pH of the system is another crucial factor affecting the assembly because the charges on both components rely directly on the degree of protonation. As shown by the DLS results in Figure 3, with the rise in pH, the count rate of aggregates peaks at pH 5.0 and the zeta potential decreases all the way down but

remains positive under certain circumstances. The positive zeta potentials support the fact that part of the chitosan is associated with ATP and part of the chitosan remains cationic and hydrophilic. In this way, polymeric supra-amphiphiles are fabricated as designed. In the samples in which the pH is 7.0 and above, the zeta potential is below 20 mV and the aggregates cannot stably exist and therefore precipitate shortly after preparation. This may be a consequence of the shortage of charges on the spherical aggregates and the poor solubility of the deprotonated chitosan. Interestingly, ATP can be inherited by the chitosan−ATP spherical aggregates. The phosphatase responsiveness was monitored by DLS. When CIAP is added to the solution of the spherical aggregates, the transparency rises in the monitored range of wavelength and finally changes from turbid to fully transparent. In the meantime, the count rate of the aggregates decreases simultaneously (Figure 4), indicating the disassembly of the spherical aggregates. The disassembly triggered by the enzyme is further confirmed by 31P NMR. As shown in Figure 5, the signal of γ-ATP (δ −20.30) decreases after the enzyme treatment, accompanied by an increase in the signal of the hydrolyzed products, especially that of phosphate (δ +1.78), suggesting the simultaneous disassembly of the aggregates and the degradation of ATP.33,34 It should be 14564

dx.doi.org/10.1021/la303271f | Langmuir 2012, 28, 14562−14566

Langmuir

Article

(20974059), the Foundation for Innovative Research Groups of the NSFC(21121004), and an NSFC-DFG joint grant (TRR 61). We thank Dr. Fu Tang and Associate Professor Ning Ma of the Beijing University of Science and Technology for DLS characterization. We also thank Senior Engineer Ying Ma of the Chemical Experimental Center, Department of Chemistry, Tsinghua University for viscometric measurements.

■

(1) Ulijn, R. V. Enzyme-Responsive Materials: A New Class of Smart Biomaterials. J. Mater. Chem. 2006, 16, 2217−2225. (2) Börner, H. G.; Kühnle, H.; Hentschel, J. Making “Smart Polymer” Smarter: Modern Concepts to Regulate Functions in Polymer Science. J. Polym. Sci., Part A: Polym. Chem. 2009, 48, 1−14. (3) Roy, D.; Cambre, J. N.; Sumerlin, B. S. Future Perspectives and Recent Advances in Stimuli-Responsive Materials. Prog. Polym. Sci. 2010, 35, 278−301. (4) De la Rica, R.; Aili, D.; Stevens, M. M. Enzyme-Responsive Nanoparticles for Drug Release and Diagnostics. Adv. Drug Delivery Rev. 2012, 64, 967−978. (5) Lee, S. W.; Reimer, C. L.; Fang, L.; Iruela-Arispe, M. L.; Aaronson, S. A. Overexpression of Kinase-Associated Phosphatase (KAP) in Breast and Prostate Cancer and Inhibition of the Transformed Phenotype by Antisense KAP Expression. Mol. Cell. Biol. 2000, 20, 1723−1732. (6) Lam, E. W. N.; Zwacka, R.; Seftor, E. A.; Nieva, D. R. C.; Davidson, B. L.; Engelhardt, J. F.; Hendrix, M. J. C.; Oberley, L. W. Effects of Antioxidant Enzyme Overexpression on the Invasive Phenotype of Hamster Cheek Pouch Carcinoma Cells. Free Radical Biol. Med. 1999, 27, 572−579. (7) Hu, J.; Zhang, G.; Liu, S., Enzyme-Responsive Polymeric Assemblies, Nanoparticles and Hydrogels. Chem. Soc. Rev. 2012, 41, 5933−5949. (8) Price, A. D.; Zelikin, A. N.; Wang, Y.; Caruso, F. Triggered Enzymatic Degradation of DNA within Selectively Permeable Polymer Capsule Microreactors. Angew. Chem., Int. Ed. 2009, 48, 329−332. (9) Habraken, G. J. M.; Peeters, M.; Thornton, P. D.; Koning, C. E.; Heise, A. Selective Enzymatic Degradation of Self-Assembled Particles from Amphiphilic Block Copolymers Obtained by the Combination of N-Carboxyanhydride and Nitroxide-Mediated Polymerization. Biomacromolecules 2011, 12, 3761−3769. (10) Itoh, Y.; Matsusaki, M.; Kida, T.; Akashi, M. EnzymeResponsive Release of Encapsulated Proteins from Biodegragable Hollow Capsules. Biomacromolecules 2006, 7, 2715−2718. (11) Kühnle, H.; Börner, H. G. Biotransformation on Polymer− Peptide Conjugates:A Versatile Tool to Trigger Microstructure Formation. Angew. Chem., Int. Ed. 2009, 48, 6431−6434. (12) Raghupathi, K. R.; Azagarsamy, M. A.; Thayumanavan, S. GuestRelease Control in Enzyme-Sensitive, Amphiphilic-Dendrimer-Based Nanoparticles through Photochemical Crosslinking. Chem.Eur. J. 2011, 17, 11752−11760. (13) Thornton, P. D.; Mart, R. J.; Ulijn, R. V. Enzyme-Responsive Polymer Hydrogel Particles for Controlled Release. Adv. Mater. 2007, 19, 1252−1256. (14) Amir, R. J.; Zhong, S.; Pochan, D. J.; Hawker, C. J. Enzymatically Triggered Self-Assembly of Block Copolymers. J. Am. Chem. Soc. 2009, 131, 13949−13951. (15) Zhang, X.; Wang, C. Supramolecular Amphiphiles. Chem. Soc. Rev. 2011, 40, 94−101. (16) Wang, C.; Wang, Z.; Zhang, X. Amphiphilic Building Blocks for Self-Assembly: From Amphiphiles to Supra-Amphiphiles. Acc. Chem. Res. 2011, 45, 608−618. (17) Wang, C.; Wang, Z.; Zhang, X. Superamphiphiles as Building Blocks for Supramolecular Engineering: Towards Functional Materials and Surfaces. Small 2011, 7, 1379−1383. (18) Wang, G.; Wang, C.; Wang, Z.; Zhang, X. Bolaform Superamphiphile Based on a Dynamic Covalent Bond and Its SelfAssembly in Water. Langmuir 2011, 27, 12375−12380.

Figure 4. Count rate of the chitosan−ATP complex solution with the addition of CIAP (12.5 U/mL).

Figure 5. 31P NMR spectra of a chitosan−ATP aggregate solution (a) before and (b) 12 h after the addition of CIAP (6 U/mL). The phosphorus signals are assigned as follows: (A) α-AMP, (B) phosphate, (C) γ-ATP, (D) α-ATP, and (E) β-ATP.

pointed out that ATP is hydrolyzed to be adenosine and phosphate finally in this system, which can be indicated clearly by 31P NMR because there is no signal from AMP, ADP, or ATP in the spectrum after 12 h of phosphatase treatment. In addition, the free ATP can be hydrolyzed in minutes, and the different hydrolysis rate may suggest that the enzyme cannot attack the ATP inside the aggregates. Although some of the ATP remains, the interaction between ATP and chitosan is not strong enough to stabilize the aggregates and aggregates start to disassemble. In summary, we have employed chitosan, a natural polymer, and ATP as building blocks to fabricate enzyme-responsive polymeric supra-amphiphiles on the basis of electrostatic interactions. The polymeric supra-amphiphiles can selfassemble in water to form enzyme-responsive aggregates. Chitosan-based supra-amphiphiles allow for the inexpensive large-scale fabrication of assemblies. Considering the biocompatibility of the enzyme-responsive aggregates, the application of the assemblies in nanocontainers and drug delivery is greatly anticipated.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; xi@mail. tsinghua.edu.cn. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program of China (2013CB834502), the NSFC 14565

dx.doi.org/10.1021/la303271f | Langmuir 2012, 28, 14562−14566

Langmuir

Article

(19) Liu, K.; Yao, Y.; Liu, Y.; Wang, C.; Li, Z.; Zhang, X. SelfAssembly of Supra-Amphiphiles Based on Dual Charge-Transfer Interactions: From Nanosheets to Nanofibers. Langmuir 2012, 28, 10697−10702. (20) Han, P.; Ma, N.; Ren, H.; Xu, H.; Li, Z.; Wang, Z.; Zhang, X. Oxidation-Responsive Micelles Based on a Selenium-Containing Polymeric Superamphiphile. Langmuir 2010, 26, 14414−14418. (21) Wang, C.; Guo, Y.; Wang, Z.; Zhang, X. Superamphiphiles Based on Charge Transfer Complex: Controllable Hierarchical SelfAssembly of Nanoribbons. Langmuir 2010, 26, 14509−14511. (22) Liu, Y.; Liu, K.; Wang, Z.; Zhang, X. Host-Enhanced π-π Interaction for Water-Soluble Supramolecular Polymerization. Chem.Eur. J. 2011, 17, 9930−9935. (23) Han, P.; Li, S.; Wang, C.; Xu, H.; Wang, Z.; Zhang, X.; Thomas, J.; Smet, M. UV-Responsive Polymeric Superamphiphile Based on a Complex of Malachite Green Derivative and a Double Hydrophilic Block Copolymer. Langmuir 2011, 27, 14108−14111. (24) Wang, C.; Chen, Q.; Wang, Z.; Zhang, X. An EnzymeResponsive Polymeric Superamphiphile. Angew. Chem., Int. Ed. 2010, 49, 8612−8615. (25) Xing, Y.; Wang, C.; Han, P.; Wang, Z.; Zhang, X. Acetylcholinesterase Responsive Polymeric Supra-Amphiphiles for Controlled Self-Assembly and Disassembly. Langmuir 2012, 28, 6032−6036. (26) Guo, D.-S.; Wang, K.; Wang, Y.-X.; Liu, Y. CholinesteraseResponsive Supramolecular Vesicle. J. Am. Chem. Soc. 2012, 134, 10244−10250. (27) Jiang, L.; Yan, Y.; Drechsler, M.; Huang, J. Enzyme-Triggered Model Self-Assembly in Surfactant−Cyclodextrin Systems. Chem. Commun. 2012, 48, 7347−7349. (28) Lin, Y.-H.; Chang, C.-H.; Wu, Y.-S.; Hsu, Y.-M.; Chiou, S.-F.; Chen, Y.-J. Development of pH-Responsive Chitosan/Heparin Nanoparticles for Stomach-Specific Anti-Helicobacter Pylori Therapy. Biomaterials 2009, 30, 3332−3342. (29) Schütz, C. A.; Juillerat-Jeanneret, L.; Kaüper, P.; Wandrey, C. Cell Response to the Exposure to Chitosan-TPP//Alginate Nanogels. Biomacromolecules 2011, 12, 4153−4161. (30) Nasti, A.; Zaki, N. M.; Leonardis, P.; Ungphaiboon, S.; Sansongsak, P.; Rimoli, M. G.; Tirelli, N. Chitosan/TPP and Chitosan/TPP-Hyaluronic Acid Nanoparticles: Systematic Optimisation of the Preparative Process and Preliminary Biological Evaluation. Pharm. Res. 2009, 26, 1918−1930. (31) Du, Y.-Z.; Ying, X.-Y.; Wang, L.; Zhai, Y.; Yuan, H.; Yu, R.-S.; Hu, F.-Q. Sustained Release of ATP Encapsulated in Chitosan Oligosaccharide Nanoparticles. Int. J. Pharm. 2010, 392, 164−169. (32) Morikawa, M.; Yoshihara, M.; Endo, T.; Kimizuka, N. ATP as Building Blocks for the Self-Assembly of Excitonic Nanowires. J. Am. Chem. Soc. 2005, 127, 1358−1359. (33) Song, Z.; Parker, K. J.; Enoh, I.; Zhao, H.; Olubajo, O. MyosinCatalyzed ATP Hydrolysis Elucidated by 31P-NMR Kinetic Studies and 1H PFG-Diffusion Measurements. Anal. Bioanal. Chem. 2009, 395, 1453−1459. (34) Hancock, C. R.; Brault, J. J.; Wiseman, R. W.; Terjung, R. L.; Meyer, R. A. 31P-NMR Observation of Free ADP during Fatiguing, Repetitive Contractions of Murine Skeletal Muscle Lacking AK1. Am. J. Physiol. Cell Physiol. 2004, 288, 1298−1304.

14566

dx.doi.org/10.1021/la303271f | Langmuir 2012, 28, 14562−14566