Cyclodextrin-Based Microcapsules as Bioreactors for ATP

Aug 20, 2013 - A biomimetic energy converter was fabricated via the assembly of CF0F1-ATPase on lipid-coated hollow nanocapsules composed of α-cyclod...
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Cyclodextrin-Based Microcapsules as Bioreactors for ATP Biosynthesis Jian-Hu Li,† Yi-Fu Wang,† Wei Ha,‡ Yan Liu,† Li-Sheng Ding,‡ Bang-Jing Li,*,‡ and Sheng Zhang*,† †

State Key Laboratory of Polymer Materials Engineering, Polymer Research Institute of Sichuan University, Chengdu 610065, China Chengdu Institute of Biology, Chinese Academy of Science, Chengdu, China

‡

S Supporting Information *

ABSTRACT: A biomimetic energy converter was fabricated via the assembly of CF0F1-ATPase on lipid-coated hollow nanocapsules composed of α-cyclodextrins/ chitosan-graf t-poly(ethylene glycol) methacrylate. Upon entrapped GOD into these capsules, the addition of glucose could trigger proton-motive force and then drive the rotation of ATPase to synthesize ATP.

L

by the layer-by-layer (LBL) assembly technique into the biomimetic system design and fabricated a series of biomimetic energy converters via the assembly of ATPase on lipid-coated hollow microcapsules. However, this strategy requires a procedure to remove the core in order to create a hollow interior and needs multiple steps to produce the wall of the microcapsules. In our previous study, we reported that polymeric hollow spheres with semipermeability could be obtained directly by self-assembly of rod−coil copolymers/cyclodextrin (CD) complexes in analogy to the cell membrane.13−18 In the present work, we utilized this self-assembly approach to design a new type of biomimetic capsules for ATP synthesis. This capsule comprised a self-assembled hollow sphere as the inner support and its absorbed lipid as the out layer. Furthermore, glucose oxidases (GODs) were entrapped in the cavity of polymeric hollow spheres during the procedure for formation of hollow spheres. It is well-known that GOD can catalyze the hydrolysis of glucose coupled with the release of proton. Therefore, when glucose was added into the suspension of biomimetic capsules, proton gradients could be formed between the exterior and interior of the capsules and subsequently drove the rotation of ATPase to systhsize ATP. This self-assembly approach is much simple and straightforward. In addition, in comparison to bacteriorhodopsin (BR), a light-driven proton pump occurring in the archaebacteria Halobacterium salinarum, GOD is much cheaper and easy to get.

iving organisms make a series of metabolisms every moment. An organism that wants to maintain activities needs a continuous supply of energy: adenosine triphosphate (ATP). As the energy currency, ATP induces large conformational changes in molecules such as myosin, kinesin, and chaperonin. With inspiration from biology, biological and biomimetic devices that supply ATP as energy, such as molecular motors and artificial cell bioreactors, have been developed.1−4 However, in motility and functionality assays reported on these devices, the issue of ATP regulation is usually sidestepped by supplying ATP externally into the buffer solution. Complications can occur as high concentrations of ATP, and the devices stop operation when all the ATP runs out. For the long-term operation of more complex molecular devices, continual supply of the right amount of ATP is required. The enzyme primarily responsible for the production of ATP is the F1F0-type ATPase, which synthesizes ATP from adenosine diphosphate (ADP) and inorganic phosphate (Pi) by utilizing proton gradients.5 Several groups have been working on the reconstitution of ATPase in liposomes as biomimetic systems to carry out ATP biosynthesis.6−8 Although they have excellent assay characteristics, the future applicability of liposomes is intrinsically limited due to chemical and mechanical weaknesses. In order to get more stable and robust biomimetic membranes, polymersome and lipid bilayercoated microcapsules have been developed replacing traditional liposomes very recently.9−12 For instance, Choi et al. demonstrated that synthesized amphiphilic polymersomes can replace liposomes for ATP synthesis. However, the synthesis needs strict control, and the assembled vesicles were not sufficiently stable. Li et al. introduced microcapsules fabricated © 2013 American Chemical Society

Received: April 24, 2013 Revised: August 15, 2013 Published: August 20, 2013 2984

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Hollow spheres could be prepared by different approaches, such as LBL self-assembly and noncovalently connected micelles.19,20 However, these methods need physical or chemical procedures to remove the core or the template. Jenekhe et al. found that rod−coil copolymers could selfassemble to form hollow nanospheres directly in their selective solvent (a solvent only dissolve one block of the copolymers).21 Over the past 10 years, this convenient method has aroused many researcher’s interest.22−24 Recently, we developed a novel approach to prepare rod−coil complexes using the selective inclusion complexation between CD and copolymers. These complexes could effectively self-assemble into hollow spheres and showed good encapsulation behavior of enzyme molecules.13−18 In this study, we constructed hollow spheres using the biocompatible materials CS-g-PEGM and α-CD. Chitosan (CS) is a biocompatible and weak cationic polysaccharide with glucosamine as the basic unit. Bonding poly(ethylene glycol) methacrylate (PEGM) oligomers on the CS backbone (synthesis and characterization are provided in the Supporting Information), the resulting CS-g-PEGMs were water-soluble copolymers, which formed transparent solution in water. However, when an aqueous solution of α-CD was added to the solution of CS-g-PEGM at room temperature, the mixed solution became turbid quickly, indicating the formation of aggregates. The morphology of the aggregates was studied by transmission electron microscopy (TEM). As shown in Figure 1a, the particles had round outline shape with diameter of about

Table 1. Particle Size and Zeta Potentials of the Capsules sample non-cross-linked capsules without GOD non-cross-linked capsules with entrapped GOD cross-linked capsules without GOD cross-linked capsules with entrapped GOD lipid-coated cross-linked capsules with entrapped GOD

hydrodynamic diameter (Dh) (nm)

polydispersity index

zeta potentials (mV)

489

0.395

33.1

666

0.305

33.0

567

0.011

33.2

696

0.014

31.4

1074

0.189

−19.4

CDs are well-known as host compounds and are capable of including a range of guest molecules in their cavities with high selectivity.26,27 It has been reported that PEG chains can penetrate the inner cavity of α-CD to form inclusion complexes in water, and the resulting rod-like crystallites were waterinsoluble because the adjacent α-CD molecules packed closely through hydrogen bonding.28 CS-g-PEGM was expected to form inclusion complexes with α-CDs because of the existence of PEGM grafts, although the CS backbone was too large to penetrate the cavity of α-CD. X-ray diffraction (XRD; Figure S4, Supporting Information) results showed that CS-g-PEGM/ α-CD had typical PEGM-α-CD rod-like crystallites (2θ = 19.9°).28 When the α-CDs preferentially stacked along the PEGM side chains to form insoluble segments, water became a selective solvent for the CS-g-PEGM/α-CD. As a result, micelle-like aggregates were formed. The requirement of efficient space-filling packing of the rod-like blocks induced that the PEGM-α-CD preferred to pack radially into a sphere to form a hollow structure. The expected structure was an inner rod-like PEGM-α-CD inclusion segments surrounded by a protonated coil-like CS shell. The zeta potentials of CS-gPEGM/α-CD particles with and without GOD were all about 33 mV (Table 1), confirming that the surface of particles was covered by protonated CS indeed. Our previous studies have demonstrated that the size of this kind of assembled hollow spheres can be controlled by varying the rigid block fraction.13 There is a inclusion-dissociation equilibrium between CDs and polymers in the inclusion complex formation process. Our previous paper had demonstrated that the assemblies of rod− coil complexes composed of CD and copolymers showed a dependence on concentration and temperature.3,17 At low concentration or high temperature, the particles were unstable and disrupted due to the dissociation of CDs from the polymer chains. To produce hollow particles that were more stable and robust, we cross-linked the double bonds at the end of PEGM with UV energy. We measured the transmittance of CS-gPEGM/α-CD solution before and after cross-linking as a function of temperature. It can be seen that the transmittance of CS-g-PEGM/α-CD before cross-linking increased with the increase of temperature. When the temperature was up to 50 °C, the solution became almost transparent, indicating that most CS-g-PEGM/α-CD particles had disrupted because α-CD molecules dissociated from the PEGM chain. On the contrary, after cross-linking, CS-g-PEGM/α-CD solution kept turbid even at 60 °C, which was caused by the fact that the α-CD molecules could not slide off the PEG chain due to the crosslinking (Figure 2). DLS and TEM showed that the cross-linking did not affect the size of particles much (Figure 1 and Table 1).

Figure 1. (a) TEM images of cross-linked α-CD/CS-g-PEGM nanocapsules. (b) TEM images of cross-linked α-CD/CS-g-PEGM/ GOD nanocapsules.

500 nm and the cores of particles were a little brighter than the outlines of the particles, which was a typical TEM image of hollow spheres as reported for different kinds of hollow particles.13−18,22 After addition of GOD to the solution during the process of self-assembly of CS-g-PEGM and α-CD, the enzymes were entrapped in the CS-g-PEGM/α-CD hollow spheres. The isoelectric point of GOD and CS was around 4.2 and 6.3, respectively,25 therefore the pH of mixture was adjusted to 6.8 to make sure that the GODs could not absorb on CS-g-PEGM through electrostatic interaction. After encapsulating GOD, the zeta potential of particles almost remained constant (Table 1), indicating that the GOD molecules were entrapped in the cavity of particles but not absorbed on the surface. In Figure 1b, no bright domains were found within the spheres with entrapped GOD, which was due to the fact that the hollow spheres were filled with enzyme molecules. Dynamic light scattering (DLS) measurement showed that the size of microcapsules increased because of GOD encapsulation (Table 1). The encapsulation efficiency (EE) of GOD was 88.4%, and the leakage efficiency (LE) of GOD was 3.0%. 2985

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Figure 2. (a) Collapse experiment of non-cross-linked GOD-encapsulated capsules (1) and cross-linked GOD-encapsulated capsules (2). (b) Transmittance of non-cross-linked GOD-encapsulated capsules (■) and cross-linked GOD-encapsulated capsules (★) as a function of temperature.

interaction of phosphatidic acid with the cationic CS. Lipidmodified microcapsules with incorporated ATPase were thus obtained. To detect the adsorption of liposomes to the microcapsule surface, we observed the morphology of lipid-coated microcapsules by confocal laser scanning microscopy (CLSM) using fluorescent phospholipids of 1-palmitoyl-2-[6-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]hexa-noyl]-sn-glycero-3-phosphocholine (NBD-PC) as the reporter molecule. Figure 3 showed that continuous fluorescence and an almost constant intensity covered the entire surface of the microcapsules, which proved the successful deposition of lipids on the outer shell of the

Furthermore, because the cross-linked particles were not easy to dissociate, the particles showed much narrower size distribution after cross-linking. The zeta potentials of crosslinked CS-g-PEGM/α-CD particles remained over 30 mV. These results indicated that stable positive CS-g-PEGM/α-CD particles could be obtained by UV cross-linking. It is well-known that GOD can catalyze the oxidation of β-Dglucose and release H+.10,11 A proton gradient can be created between the interior and exterior of the GOD-encapsulated capsules by adding glucose to the solution. The quantity and catalytic activity of encapsulated GOD in CS-g-PEGM/α-CD microcapsules reflect their ability of releasing proton, which is very important for ATP synthesis since the rate of ATP synthesis is dependent on the magnitude of the proton gradient.29 The activity of entrapped GOD in cross-linked CSg-PEGM/α-CD microcapsules was 74.8% of the free enzyme activity. The small inactivation of GOD was probably caused by the UV irradiation. These results indicated that, through the self-assembly of CS-g-PEGM and α-CD, the GOD could be entrapped in semipermeable microcapsules effectively, which prevented the enzyme from diffusion into the surrounding solution while allowing substrate (glucose) and products (including H+) to pass through. ATPase-liposomes were incubated with the CS-g-PEGM/αCD microcapsule solution above the phase transition temperature of the phospholipids. The ATPase-liposomes were fabricated by reconstituting ATPase into lioposomes according to the literature.9 The proteoliposomes could adsorb favorably on the outer shell of the microcapsules through the electrostatic

Figure 3. (a) CLSM image of the stable α-CD/CS-g-PEGM/GOD microcapsules coated by lipids. (b) CLSM image of the stable α-CD/ CS-g-PEGM/GOD microcapsules coated by lipids with incorporated CF0F1-ATPase. 2986

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through ATPase from the interior solution to the exterior solution. Scheme 1 illustrates the construction of microcapsule developed in this study and the ATP synthesis process in this

microcapsules. Table 1 shows that the size of the capsules increased from 696 to 1074 nm after being coated by lipids, and the zeta potential changed from 31.4 mV to −19.4 mV, which further proved the adsorption of lipid. To detect pH change inside the lipid-coated capsules induced by the oxidation of glucose, a pH sensitive fluorescent probe, 8hydroxyprene-1,3,6-trisulphonic acid (pyranine) was introduced by mixing the pyranine with α-CD/CS-g-PEGM/GOD microcapsules, and then ATPase-liposomes coated the shell of microcapsules. After adding 10% glucose solution, the fluorescence excitation spectra of pyranine was monitored at an emission wavelength of 513 nm. The results showed that the fluorescence intensity at 406 nm increased with time, and the fluorescence intensity at 460 nm decreased (Figure 4),

Scheme 1. Schematic Illustration of the Formation of CDBased Microcapsule Containing ATPase and the ATP Synthesis Process in This Microcapsule

microcapsule. Adding glucose solution into the capsules suspension, a proton gradient between the external and internal capsules will be generated via the hydrolysis of glucose catalyzed by GOD, which will drive ATPase running and result in ATP synthesis. The amount of synthesized ATP was quantitatively determined by using high-performance liquid chromatography (HPLC). Since the ATP peak area of the HPLC was proportional to the amount of ATP concentration in the sample, we first derived a standard ATP concentration cure by plotting the ATP peak area as a function of the ATP concentration (Figure S5). Thus, the concentration of synthesized ATP could conveniently be obtained by reading the standard curve. ATP production in capsules’ exterior and interior was measured at different reaction times. The ATP in the interior of the capsules was released by destroying the lipids coating the capsules by adding 0.1% Triton X-100 to the capsule suspension. As shown in Figure 5, the ATP production continuously increaseed with time. It was interesting that the ATP yield in the capsules’ interior was almost same as that in the capsules’ exterior, which indicated that the ATPases randomly oriented into this biomimietic microcapsule system. Although the ATP synthesis rate of this capsules system is still much less than that in vivo conditions, it is higher than that of artificial systems using BR as proton pump. For instance, a polymersome system that used BR as proton pump produced ATP at a rate of about 18.3 nmol/mg/h at the first hour, and the ATP production almost had no apparent change then,12 while the ATP synthesis rate of system in this study was about 26.6 nmol/mg/h at the first hour and the ATP production kept increasing over the first hour. This result implied that proton gradient could be produced more efficiently and maintained for a longer period of time via the hydrolysis of glucose catalyzed by entrapped GOD. Furthermore, the structure of lipid-coated

Figure 4. Changes in pH with time inside the CF0F1/lipidmicrocapsules as a result of the reaction of glucose and GOD. The inset shows the fluorescence spectra of pyranine encapsulated in the CF0F1/lipid-microcapsules suspended in the mixed solution after different time intervals.

indicating that the internal solution of the microcapsules became increasingly acidic with time. It demonstrated that the glucose could pass through the membrane of lipid-coated capsules to realize the hydrolysis reaction. Considering that glucose can not be transported without special transport proteins through a lipid membrane in the real living system, it was believed that the lipid bilayer coating on the capsule surface was not highly condensed. Thus, the possible pores or defects on the surface would allow the penetration of glucose. A similar phenomenon also was observed in other lipid-coated microcapsules.12 The pH within the capsules can be calculated by determining the characteristic peak intensities at 460 and 406 nm, because the ratio of the fluororescence intensities of pyranine at 460 and 406 nm is dependent on the hydrogen ion concentration. The internal pH within the capsules can be obtained from a thirdorder equation that has been reported.30 pH = a + bx + c /x + dx 2 + e/x 2 + fx 3 + g /x 3

x is I460/I406. Calculated values for the constants are a, 6.1; b, 2.69; c, −0.108; d, −1.65; e, 0.013; f, 0.39; and g, −0.0082. Figure 4 shows the pH values as a function of time. The high concentration of proton within the capsules caused the proton transportation to occur across the membrane of capsules 2987

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microcapsules brings an advantage of stability. In comparison with liposome, such microcapsule was very stable under physiological conditions, especially in the presence of surfactants. In future studies, we will further investigate the effects of variable glucose concentration, density of crosslinking and other factors on the synthesis of ATP. In conclusion, we have successfully fabricated a new stable biomimetic system for ATP synthesis, in which ATPaseliposome coated on microcapsules encapsulated GOD. The microcapsules were prepared by self-assembly of α-CD and CSg-PEGM, which serves to make the system more mechanically robust, and prevent liposomal fusion and aggregations. The encapsulated GOD can catalyze the hydrolysis of glucose and then provide proton flow to drive the ATP synthesis. ATP will be an important fuel source for powering future nanodevices based on biomimetic technology. Therefore, this ATP generation system has great potential to play a crucial role in the operation of biologically inspired nanodevices.

ASSOCIATED CONTENT

S Supporting Information *

The methods, characterization of polymers and XRD results are in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 5. ATP synthesis catalyzed is shown as a function of reaction time: exterior solutions (□); interior and exterior solutions (■).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *Fax: (+86) 028-85403421; E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by National Natural Science Foundation of China (NSFC Grant Nos. 51373174, 51073107 and 21074138), the CAS Knowledge Innovation Program (Grant No. KSCX2-EW-J-22), West Light Foundation of CAS and Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT1026). 2988

dx.doi.org/10.1021/bm400584h | Biomacromolecules 2013, 14, 2984−2988