Acetylcholinesterase Responsive Polymeric Supra-Amphiphiles for

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Acetylcholinesterase Responsive Polymeric Supra-Amphiphiles for Controlled Self-Assembly and Disassembly Yibo Xing, Chao Wang, Peng Han, Zhiqiang Wang,* and Xi Zhang* Key Lab of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China S Supporting Information *

ABSTRACT: We have fabricated enzyme responsive polymeric supra-amphiphiles by mixing a block copolymer of poly(ethylene glycol)-block-poly(acrylic acid) with myristoylcholine chloride in water. The polymeric supra-amphiphiles self-assemble into spherical aggregates with sizes varying from about 40 to 150 nm. Moreover, the spherical aggregates can be disassembled triggered by acetylcholinesterase, an enzyme which can cut off the ester linkage of myristoylcholine chloride. Nile red can be loaded into the spherical aggregates and released in several hours upon the treatment of acetylcholinesterase. The releasing rate is rather fast considering that it takes more than 150 h for Nile red to diffuse out of the spherical aggregates without addition of acetylcholinesterase. It is anticipated that the new enzyme responsive polymeric supra-amphiphile may be explored as a carrier for drug delivery.

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INTRODUCTION Stimuli-responsive polymers have captured much attention recently due to their potential applications in sensors, biotechnology, drug delivery, and so forth.1−9 Among these polymers, enzyme responsive polymers are especially attractive because of their good biocompatibility, sensitivity, and therefore display of potential application in biological material and drug delivery. For example, Amir et al. have synthesized a phosphate contained water-soluble diblock copolymer, which is able to self-assemble into colloidal nanostructures when adding acid phosphatase.10 Azagarsamy et al. synthesized a series of amphiphilic biaryl dendrimers with enzyme cleavable ester moieties.11 These dendrimers are able to self-assemble into micelles and load hydrophobic guest molecules, and disassemble upon exposure to esterase, leading to the release of guest molecules. This line of research might lead to application in enzyme responsive drug delivery systems and biosensors. Compared to conventional amphiphile linked by covalent bonds, our group has recently proposed a new concept of “supra-amphiphiles” which refer to amphiphiles that are formed on the basis of noncovalent interactions, such as hydrogen bonding, electrostatic attraction, host−guest recognition, charge transfer interaction, metal coordination, and so on.12−20 Supra-amphiphiles can effectively avoid complicated synthetic procedures to some extent, and easily introduce stimuli-responsive moieties and other functional groups. Meanwhile, controlled self-assembly and disassembly for supra-amphiphiles are readily realized due to the weak © 2012 American Chemical Society

noncovalent bonds. Using this concept, we have successfully fabricated an enzyme responsive polymeric supra-amphiphile using positively charged block copolymer methoxy-poly(ethylene glycol)114-block-poly(L-lysine hydrochloride)200 and negatively charged adenosine 5′-triphosphate as building blocks, which self-assembles to spherical aggregates.21 8-Hydroxypyrene-1,3,6-trisulfonic acid, a hydrophilic model compound, can be loaded into the polymeric supra-amphiphile aggregates and released completely in about 4 h under alkaline phosphatase, which is much faster than the former reported enzyme responsive system. However, many drugs are hydrophobic; thus it is quite necessary to explore new systems for hydrophobic drugs. Herein we report a new enzyme responsive polymeric supraamphiphile formed by poly(ethylene glycol)-block-poly(acrylic acid) (PEG-b-PAA) and myristoylcholine chloride (MCC) on the basis of electrostatic interaction, as shown in Scheme 1. The polymeric supra-amphiphiles self-assemble into spherical aggregates varying from about 40 to 150 nm and disassemble by adding acetylcholinesterase (AChE), a natural enzyme in human bodies which can cut off the ester linkage of myristoylcholine chloride. Hydrophobic guest molecules such as Nile red (NR) can be loaded into the spherical aggregates and released rapidly upon the treatment of AChE. AChE is an enzyme whose function is to hydrolyze and destruct the Received: February 11, 2012 Revised: March 8, 2012 Published: March 9, 2012 6032

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Loading of NR into MCC/PEG114-b-PAA46 and MCC/PEG114-bPAA93 Aggregates. An amount of 40 μL of 5 mM NR in acetone solution was poured into 1 mL of PBS and then ultrasonicated for 15 min to remove acetone. Then 1 mL of 0.091 mM/L PEG114-b-PAA46 or 0.043 mM/L PEG114-b-PAA93 was added. After shaking for a while, 2 mL of 2 mM/L MCC was dropped into the mixed solution. After stirring for 6 h at 250 rpm and standing overnight, the mixture was centrifuged for 10 min at 10 000 rpm, and supernatant liquid, which contained MCC/PEG114-b-PAA46/NR or MCC/PEG114-b-PAA93/NR complexes, was gathered for further experiment.

Scheme 1. Schematic Illustration of Polymeric SupraAmphiphile Self-Assembly and AChE Triggered Disaggregationa

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RESULTS AND DISCUSSION The polymeric supra-amphiphiles were fabricated according to the method mentioned above. To investigate the self-assembly structure of these polymeric supra-amphiphiles, TEM was employed. As shown in Figure 1, the polymeric supraa

Firstly, MCC and PEG-b-PAA form supra-amphiphiles through electrostatic attraction, and the supra-amphiphiles self-assemble into spherical aggregates at nearly the same time, with PEG chains outside and PAA chains attached MCC inside. Upon AChE treatment, ester linkage of MCC is cut off and the aggregates disaggregate as a result.

cationic neurotransmitter acetylcholine, thus weakening or stopping the transmission of nerve impulse. AChE is widely found in the central and peripheral nervous systems, along with the acetylcholine receptor.22 It is well-known that many neural transmission barrier-related diseases, such as myasthenia gravis and Alzheimer’s disease, are closely related to the access amount of AChE in certain parts of body.23 Inhibitors of AChE are helpful in treating these diseases, while targeted delivery of such inhibitors was seldom reported.24,25 Therefore, it is anticipated that the new enzyme responsive polymeric supraamphiphile may be explored as a carrier for drug delivery.

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Figure 1. Zeta-size of MCC/PEG114-b-PAA46 decreases from ∼145 nm (R = 0.10) to ∼35 nm (R = 0.75) (a) and MCC/PEG114-b-PAA93 decreases from ∼160 nm (R = 0.10) to ∼60 nm (R = 1.0) (b). TEM images of MCC/PEG114-b-PAA46 (c) and MCC/PEG114-b-PAA93 (d) show more spherical aggregates with larger sizes are formed using PEG114-b-PAA93 than PEG114-b-PAA46.

EXPERIMENTAL SECTION

Materials. Phosphate buffered solution (PBS), H3PO4, NaOH, phosphotungstic acid, tetrahydrofuran, and acetone are all analytical grade and obtained from Beijing Chemical Reagents Company. Myristoylcholine chloride (MCC), Nile red (NR), and acetylcholinesterase (AChE, from Electrophorus electricus) were purchased from Sigma-Aldrich. Poly(ethylene glycol)114-b-poly(acrylic acid)46 (PEG114b-PAA46, Mw = 6200) and poly(ethylene glycol)114-b-poly(acrylic acid)93 (PEG114-b-PAA93, Mw = 8200) were purchased from Polymer Source. Instruments. Dynamic light scattering (DLS) data were obtained on a Malvern ZS 90 Zetasizer instrument. Fluorescence spectra were recorded on a HITACHI F-7000 apparatus at a slit of 5.0 mm and a scanning rate of 240 nm/min. The excitation wavelength was 550 nm. Transmission electron microscopy (TEM) images were taken with a JEMO 2010 electron microscope. Preparation of MCC/PEG114-b-PAA46 and MCC/PEG114-bPAA93 Polymeric Supra-Amphiphiles and Aggregates. MCC (2.0 mM/L), PEG114-b-PAA46 (0.091 mM/L) and PEG114-b-PAA93 (0.043 mM/L) were first dissolved in 10 mM/L pH 7.4 PBS, ultrasonicated for 15 min, and filtrated with a 200 nm microfiltration membrane. MCC was then dropped into PEG114-b-PAA46 or PEG114b-PAA93 slowly, standing for 6 h for further experiments. Carboxyl groups of block copolymers were almost dissociated at pH 7.4. Considering that the critical micelle concentration (CMC) of MCC is 2.5 mM/L,26 the concentration of PEG114-b-PAA46 and PEG114-bPAA93 was set at 0.023 mM/L or 0.011 mM/L, separately, and the concentration of MCC varied from 0.10 to 1.3 mM/L to fit the charge ratio of MCC and PEG114-b-PAA46 or PEG114-b-PAA93 from 0.10 to 1.3. The charge ratio of MCC and block copolymers is defined as R, which equals the value of amount of positive charges provided by myristoylcholine dividing negative charges provided by PEG-b-PAA.

amphiphiles self-assemble to spherical aggregates with controllable sizes ranging from about 40 to 150 nm. For comparative study, two kinds of PEG-b-PAA were introduced, PEG114-bPAA46 and PEG114-b-PAA93. The size of the aggregates decreases clearly when R approaches 0.75 (MCC/PEG114-bPAA46) and 1.0 (MCC/PEG114-b-PAA93). TEM images show that the aggregates are spherical in shape, and the sizes of MCC/PEG114-b-PAA46 and MCC/PEG114-b-PAA93 at R = 1.0 are 40 and 55 nm, respectively, which agree well with DLS results, as shown in Figure 1c and d. DLS results also show good dimensional homogeneity of spherical aggregates. The half-peak breadth decreases when charge ratio increases from 0.10 to 1.0, among which R values equaling to 0.50, 0.75, and 1.0 (both MCC/PEG114-b-PAA46 and MCC/PEG114-b-PAA93) show better homogeneity, with half-peak breadth as low as 15 nm. However, aggregates at low charge ratio (R < 0.35) exhibit a wide distribution of particle size and bad repeatability of zeta-size in parallel experiments, which are unfavorable but reasonable results. Supra-amphiphiles and their aggregates are dynamic in nature, which means that the aggregation and dissociation of supra-amphiphiles happen all the time. The interaction between building blocks of supra-amphiphiles determines the aggregation stability. Since electrostatic attraction of R = 0.50, 0.75, and 1.0 is stronger 6033

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than that of R = 0.10, 0.20, and 0.35, the former show better stability. Comparative study shows that the size of MCC/PEG114-bPAA93 is slightly larger than that of MCC/PEG114-b-PAA46 at the same charge ratio, and MCC/PEG114-b-PAA93 has higher count rate when R ≥ 0.50 (Supporting Information Figure S2), which means PEG114-b-PAA93 is more likely to form aggregates with MCC than PEG114-b-PAA46. Compared to PEG114-bPAA46, more carboxyl groups in PEG114-b-PAA93 make MCC/ PEG114-b-PAA93 particle shells thicker, thus leading to larger aggregate sizes. Moreover, more carboxyl groups with negative charges mean stronger electrostatic attraction and tendency to form aggregates. The disassembly of MCC/PEG114-b-PAA46 can be triggered by AChE as expected. Through analyzing the count rate variation of DLS recordings, it is found that the spherical aggregates can be disaggregated by AChE rapidly. The count rate of MCC/PEG114-b-PAA46 at R = 1.0 decreases from 135 to about 15 in less than 2 h upon 5 U/mL AChE treatment, as shown in Figure 2. At the same time, the polydispersity index of

Figure 3. Zeta-size of MCC/PEG114-b-PAA46 (a), MCC/PEG114-bPAA93 (b), MCC/PEG114-b-PAA46/NR (c), and MCC/PEG114-bPAA93/NR (d).

white (Supporting Information Figure S5), indicating the rapid release of NR. Fluorescence results show that the intensity of both MCC/PEG114-b-PAA46/NR and MCC/PEG114-b-PAA93/ NR mixtures decreases rapidly upon AChE treatment, which also confirms the release of NR from the polymeric supraamphiphiles, as shown in Figure 4A. With 2 U/mL AChE treatment, complete release of NR takes less than 8 h for MCC/PEG114-b-PAA46/NR and 2.5 h for MCC/PEG114-bPAA93/NR, as shown in Figure 4B, which is much faster than reported for synthetic polymeric enzyme-responsive systems.11 Control experiments show that, without AChE, it takes more than 150 h for NR to diffuse out of the spherical aggregates. The results in Table 1 also show that NR loaded in MCC/ PEG114-b-PAA93 is more likely to be released than in MCC/ PEG114-b-PAA46 upon AChE treatment, while much less likely to be released without AChE treatment, which means MCC/ PEG114-b-PAA93/NR owns better stability and stimuli-responsiveness. The amount of acrylic acid units in the block copolymer could be responsible for the difference. More carboxyl groups make electrostatic attraction between MCC and PEG-b-PAA stronger and thus more stable. However, since more MCC/PEG114-b-PAA93/NR complexes with larger size are formed than MCC/PEG114-b-PAA46/NR complexes with the same amount of MCC, MCC/PEG114-b-PAA93/NR complexes are looser and thus easier to be attacked by AChE. As a result, NR in MCC/PEG114-b-PAA93 is easier to be released when some MCC is decomposed by AChE.

Figure 2. Count rate change of MCC/PEG114-b-PAA46 upon 5 U/mL activated AChE treatment.

the solution increases as disaggregation goes on and reaches to almost 1 when disaggregation finished, suggesting highly dimensional heterogeneity of the solution. At the end of the enzymatic reaction, count rate gets close to about 15 and aggregate size gets to about 8 nm, meaning that hardly any spherical aggregates exist. To confirm that AChE triggered disassembly is responsible for the decrease of count rate and variation in aggregate size, deactivated AChE is used for control experiments, but almost nothing changes for 5 h (Supporting Information Figure S4). The spherical aggregates are good containers for hydrophobic guest molecules. To this extent, NR is chosen as a model. Samples of MCC/PEG-b-PAA at R = 1.0 are used to load model compound NR, for their stability and suitable sizes. About 2.5 μM/L NR (∼0.36% in MCC/PEG114-b-PAA46/NR and ∼0.38% in MCC/PEG114-b-PAA93/NR) is loaded in both MCC/PEG114-b-PAA46 and MCC/PEG114-b-PAA93 complexes. The amount is given by drawing a FL intensity−NR concentration standard curve in VTHF/VPBS = 10:3 solution. DLS results show that aggregate sizes grow clearly, from about 40 to 60 nm (MCC/PEG114-b-PAA46/NR) and 55 to 95 nm (MCC/PEG114-b-PAA93/NR), as shown in Figure 3. It is also found that more spherical aggregates are formed with PEG114-bPAA93 than with PEG114-b-PAA46 as indicated by the count rate of DLS data. We wonder whether AChE can trigger the release of NR in MCC/PEG-b-PAA polymeric supra-amphiphile. Upon AChE treatment, the color of the mixture changed from pink to milk

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CONCLUSIONS

In conclusion, we have fabricated new enzyme responsive polymeric supra-amphiphiles using negatively charged block copolymers and positively charged small molecules. Controlled self-assembly of such supra-amphiphiles is achieved through adjusting the charge ratio of building blocks. The self-assembled spherical aggregates show well responsiveness upon enzyme treatment. Hydrophobic Nile red is successfully loaded into the spherical aggregates and released rapidly upon enzyme treatment. It should be noted that the formation and selfassembly of supra-amphiphiles happen at almost the same time; however, the self-assembly takes a longer time to reach an equilibrium state. It is greatly anticipated that the new enzyme responsive polymeric supra-amphiphiles may be explored as carriers for drug delivery. 6034

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Figure 4. (A) FL spectra of MCC/PEG114-b-PAA93/NR upon 2 U/mL AChE treatment. (B)The release rate of NR under different conditions, (a) with AChE treatment and (c) without AChE treatment in MCC/PEG114-b-PAA46/NR, (b) with AChE treatment and (d) without AChE treatment in MCC/PEG114-b-PAA93/NR. (5) Yan, Q.; Yuan, J. Y.; Cai, Z. N.; Xin, Y.; Kang, Y.; Yin, Y. W. Voltage-Responsive Vesicles Based on Orthogonal Assembly of Two Homopolymers. J. Am. Chem. Soc. 2010, 132, 9268−9270. (6) Yan, Q.; Zhou, R.; Fu, C. K.; Zhang, H. J.; Yin, Y. W.; Yuan, J. Y. CO2-Responsive Polymeric Vesicles with ″Breathing″ Feature. Angew. Chem., Int. Ed. 2011, 50, 4923−4927. (7) Meng, F. H.; Zhong, Z. Y.; Feijen, J. Stimuli-Responsive Polymersomes for Programmed Drug Delivery. Biomacromolecules 2009, 10, 197−209. (8) Lowe, A. B.; McCormick, C. L. Stimuli-Responsive Water Soluble and Amphiphilic Polymers; ACS Symposium Series; American Chemical Society: Washington, DC, 2001; Chapter 1; pp 1−13. (9) Wan, P. B.; Hill, E. H.; Zhang, X. Interfacial Supramolecular Chemistry for Stimuli-Responsive Functional Surfaces. Prog. Chem. 2012, 24, 1−7. (10) 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. (11) Azagarsamy, M. A.; Sokkalingam, P.; Thayumanavan, S. Enzyme-Triggered Disassembly of Dendrimer-Based Amphiphilic Nanocontainers. J. Am. Chem. Soc. 2009, 131, 14184−14185. (12) Zhang, X.; Wang, C. Supramolecular Amphiphiles. Chem. Soc. Rev. 2011, 40, 94−101. (13) Wang, C.; Wang, Z. Q.; Zhang, X. Amphiphilic Building Blocks for Self-Assembly: From Amphiphiles to Supra-amphiphiles. Acc. Chem. Res. 2012, DOI: 10.1021/ar200226d. (14) Wang, Y. P.; Xu, H. P.; Zhang, X. Tuning the Amphiphilicity of Building Blocks: Controlled Self-Assembly and Disassembly for Functional Supramolecular Materials. Adv. Mater. 2009, 21, 2849− 2864. (15) Wang, C.; Yin, S. C.; Chen, S. L.; Xu, H. P.; Wang, Z. Q.; Zhang, X. Controlled Self-Assembly Manipulated by Charge-Transfer Interactions: From Tubes to Vesicles. Angew. Chem., Int. Ed. 2008, 47, 9049−9052. (16) Hermans, T. M.; Broeren, M. A. C.; Gomopoulos, N.; Smeijers, A. F.; Mezari, B.; Van Leeuwen, E. N. M.; Vos, M. R. J.; Magusin, P. C. M. M.; Hilbers, P. A.; Van Genderen, M. H. P.; Sommerdijk, N. A.; Fytas, J. M. G.; Meijer, E. W. Stepwise Noncovalent Synthesis Leading to Dendrimer-Based Assemblies in Water. J. Am. Chem. Soc. 2007, 129, 15631−15638. (17) Jeon, Y. J.; Bharadwaj, P. K.; Choi, S.; Lee, J. W.; Kim, K. Supramolecular Amphiphiles: Spontaneous Formation of Vesicles Triggered by Formation of a Charge-Transfer Complex in a Host. Angew. Chem., Int. Ed. 2002, 41, 4474−4476. (18) Wang, Y. P.; Han, P.; Xu, H. P.; Wang, Z. Q.; Zhang, X.; Kabanov, A. V. Photocontrolled Self-Assembly and Disassembly of Block Ionomer Complex Vesicles: A Facile Approach toward Supramolecular Polymer Nanocontainers. Langmuir 2010, 26, 709− 715. (19) Han, P.; Ma, N.; Ren, H. F.; Xu, H. P.; Li, Z. B.; Wang, Z. Q.; Zhang, X. Oxidation-responsive Micelles Based on a Seleniumcontaining Polymeric Superamphiphile. Langmuir 2010, 26, 14414− 14418.

Table 1. Time Cost (in h) with a 95% Release of NR upon Different AChE Concentration Treatment AChE concentration

10 U/mL

5 U/mL

2 U/mL

0 U/mL

MCC/PEG114-b-PAA46/NR MCC/PEG114-b-PAA93/NR

0.4 0.3

1.6 0.6

7.7 2.4

146.0 >146.0

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ASSOCIATED CONTENT

* Supporting Information S

(1) Zeta-potential, zeta-size and count rate of MCC/PEG-bPAA. (2) Zeta-size of MCC/PEG114-b-PAA46 upon 5 U/mL deactivated AChE treatment. (3) Color changing of MCC/ PEG-b-PAA/NR complexes before and after AChE treatment. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Z.W.); xi@mail. tsinghua.edu.cn (X.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by National Natural Science Foundation of China (50973051, 20974059), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (21121004). We thank Dr. Ning Ma (University of Science and Technology, Beijing) for his kindly help in DLS test, Mr. Kai Liu for his help in TEM characterization and Mr. Guangtong Wang for his helpful suggestions.

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REFERENCES

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