Programmed pH-Responsive Microcapsules for the Controlled

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Programmed pH-Responsive Microcapsules for the Controlled Release of CdSe/ZnS Quantum Dots Wei-Ching Liao,† Marianna Riutin,† Wolfgang J. Parak,‡ and Itamar Willner*,† †

Institute of Chemistry, Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Fachbereich Physik, Philipps-Universität Marburg, Renthof 7, 35037 Marburg, Germany

‡

S Supporting Information *

ABSTRACT: Two methods for the preparation of pHresponsive all-DNA microcapsules loaded with CdSe/ZnS quantum dots (QDs) are discussed. One approach involves the construction of DNA microcapsules composed of nucleic acid layers that include, at pH 7.2, “dormant” C− G·C+ triplex sequences. The formation of the C−G·C+ triplex structures at pH 5.0 leads to the cleavage of the microcapsules and to the release of the QDs. A second approach involves the synthesis of CdSe/ZnS QD-loaded DNA microcapsules, stabilized at pH 7.2 by T−A·T interlayer triplex bridges. The dissociation of the bridges at pH 9.0 separates the bridging triplex units, resulting in the degradation of the microcapsules and to the release of the QDs. The programmed pH-stimulated release of luminescent QDs, emitting at 620 and 560 nm, from the C−G·C+ or T−A·T triplexresponsive microcapsules is demonstrated by subjecting the QD-loaded microcapsule mixtures to pH 5.0 or pH 9.0, respectively. KEYWORDS: DNA, triplex, switch, nanotechnology, nanoparticle complexes was reported,32 and the cytotoxicity of the microcapsules toward cancer cells was examined. The pH-stimulated switchable formation of triplex DNA structures is well-established.33−35 The C−G·C+ triplex DNA structure is stabilized at pH 5.0, and the structure reconfigures into the C−G duplex configuration at neutral pH values. In turn, the triplex T−A·T is stabilized at neutral pH values, and it dissociates under basic conditions, pH ≥9. The pH-stimulated reconfiguration of triplex DNA structures has been implemented to design switchable DNA devices,36−38 to control the reversible aggregation/deaggregation of metal nanoparticles,39 to stimulate reversible hydrogel/solution transitions,40 and to design shape-memory hydrogels.41 In the present study, we present the assembly of quantum dot (QD)-loaded pHresponsive microcapsules that are stabilized or unlocked by the predesigned dissociation or formation of T−A·T or C−G· C+ triplex structures, respectively. Specifically, we describe that upon mixing T−A·T and C−G·C+ microcapsules loaded with two different sized CdSe/ZnS QDs, the pH-programmed release of specific QDs proceeds.

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he information encoded in the base sequence of DNA provides a rich arsenal to construct stimuli-responsive materials.1,2 Indeed, substantial research efforts have been directed to the development of stimuli-responsive DNAbased materials and carriers.3,4 For example, stimuli-responsive DNA-based hydrogels,5 stimuli-responsive DNA origami nanostructures6−9 or nanoparticle aggregates,10 and stimuliresponsive DNA-functionalized nanocarriers, such as SiO2 nanoparticles11−13 or micellar aggregates,14 were reported. Different triggers have been implemented to switch the functions of the different DNA-based materials or nanostructures, and these included pH,15−17 strands/antistrands,18 K+stabilized G-quadruplexes/crown ether,19 metal ion/ligands,20 enzymes or DNAzymes,21 light,22 or thermal stimuli.23 Different applications of stimuli-responsive DNA-based materials and nanostructures were suggested, including their use for controlled drug delivery,24 sensors,25 actuators,26 nanoreactors for programmed synthesis,27 and the fabrication of shapememory materials.28,29 Recently, the design of stimuliresponsive DNA microcapsules has attracted growing interest. Besides the enzymatic cleavage of the shells of DNA-based capsules,30 the organization of DNA microcapsules being unlocked by ligand−aptamer complexes was reported.31 Specifically, the unlocking of anticancer drug-loaded microcapsules by cancer cell biomarkers (ATP, VEGF)−aptamer © 2016 American Chemical Society

Received: June 19, 2016 Accepted: August 15, 2016 Published: August 15, 2016 8683

DOI: 10.1021/acsnano.6b04056 ACS Nano 2016, 10, 8683−8689

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Figure 1. (A) Schematic preparation of QD-loaded pH-responsive microcapsules that include “dormant” C−G·C+ triplex DNA units. (B) Detailed composition of three-layer constituents that build up the C−G·C+ triplex pH-responsive shell of the microcapsules (red sequences represent the C−G·C+ triplex domains).

Figure 2A depicts the fluorescence and bright-field confocal microscopy images of the DNA-coated QD620-loaded CaCO3 particles, panel I, and of the DNA shell-protected microcapsules, panel II. After dissolution of the particles, the microcapsules are still fluorescent due to the entrapped CdSe/ZnS QDs. Figure 2A, panel II, shows that the brightfield image reveals a phase contrast of the shells, since the inner

RESULTS AND DISCUSSION Figure 1 depicts the synthesis of the C−G·C+ triplex-responsive DNA microcapsules loaded with CdSe/ZnS QDs, emitting at λem = 620 nm (QD620). CaCO3 microparticles loaded with the CdSe/ZnS QDs were coated with positively charged poly(allylamine hydrochloride), PAH. Subsequently, a hybrid DNA structure consisting of the sequences (1), (2), (3), (4), (5), and (6) was deposited on the primary positively charged PAH layer. Note, the nucleic acids (1), (2), (3), and (4) include the sequences capable of forming the C−G·C+ triplex structure at pH 5.0, yet, since the deposition of the hybrid onto the microparticles is driven at pH 7.2, these sequences exist in a “dormant” nontriplex structure. Also note that strand (5) acts as an axle to connect the different components, and strand (6) acts as an interlayer linker. Subsequent to the deposition of the first nucleic acid layer, the second nucleic acid hybrid, composed of (1), (2), (3), (4), (6), and (7), was deposited on the first layer to yield the second layer coating. By the stepwise interaction of the particles with the first and second hybrid constituents, a shell composed of six layers was constructed. After the deposition of each of the layers, the microcapsules were precipitated by centrifugation, followed by a washing step, and then the microcapsules were subjected to the deposition of the subsequent layer. (For a detailed description of the conditions applied to construct the layers, see the Experimental Section). Afterward, the CaCO3 core was dissolved with ethylenediaminetetraacetic acid (EDTA) to yield the CdSe/ZnS QD-loaded microcapsules.

Figure 2. (A) Confocal fluorescence and bright-field microscopy images corresponding to (I) QD-impregnated CaCO3 microparticles coated with the pH-responsive shells shown in Figure 1; (II) QD-loaded microcapsules after the dissolution of the CaCO3 cores. Scale bar is 10 μm. (B) SEM images corresponding to (a) CaCO3 microparticles prior to their functionalization; (b) CaCO3 microcapsules coated with a six-layer shell composed of the pHresponsive sequences shown in Figure 1; (c) microcapsules after dissolution of the CaCO3 cores. Scale bar is 2 μm. 8684

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Figure 3. (A) Schematic mechanism for the pH-stimulated degradation of the microcapsules through the separation of C−G·C+ triplexstabilized fragments of the shell (at pH 5.0). (B) Fluorescence spectra corresponding to the released QDs620 from the microcapsules shown in (A) upon (a) subjecting the microcapsules to pH 5.0; (b) subjecting the particles to pH 7.2; (c) subjecting the microcapsules to pH 9.0. In all experiments, the fluorescence spectra of the released QDs were recorded after a fixed time interval of 30 min. (C) Time-dependent fluorescence changes upon the release of the QDs620 from the microcapsules at (a) pH 5.0, (b) pH 7.2, and (c) pH 9.0.

discs or highly distorted structures). We find almost identical anisotropic fluorescence intensities for the CaCO3-loaded microcapsules and the hollow microcapsules (after dissolution of the core), implying that the hollow microcapsules are essentially spherical. Further support for the formation of the QD-loaded microcapsules was obtained by the concomitant loading of the microcapsule shells with a fluorophore (Figure S1). In these experiments, the microcapsules were formed by AlexaFluor488-labeled nucleic acids (λem = 520 nm) in the presence or absence of the QDs, and the results were compared to those depicted in Figure 2A, panel II. The QDs-loaded AlexaFluor488-labeled DNA microcapsules (Figure S1, panel I) reveal the formation of hollow microcapsules upon following the fluorescence of the fluorophore at λ = 520 nm (panel I, a) and the luminescence of the QDs at λ = 620 nm (panel I, b). In Figure S1, panel II, we provide the fluorescence features of the shell-unlabeled QDs-loaded microcapsules. Evidently, only the luminescence of the QDs at λ = 620 nm is observed. In Figure S1, panel III, confocal microscopy images of the AlexaFluor488labeled microcapsules that are not loaded with the QDs are displayed. Evidently, the formation of the fluorophore-labeled hollow microcapsules is observed at λ = 520 nm (panel III, a), and no luminescence corresponding to the QDs is observed (panel III, b). Finally, Figure S1, panel IV, depicts the merged confocal microscopy image of a mixture of the AlexaFluor488modified microcapsules that are nonloaded and the shellunlabeled QDs-loaded microcapsules. Evidently, both types of microcapsules can be identified. The nucleic acid-coated CaCO3 particles and the resulting microcapsules, generated after the dissolution of the cores, revealed indistinguishable sizes that corresponded to 3.5 ± 0.8 μm.

microcapsule region is transparent. Note that the CdSe/ZnS QDs tend to concentrate at the shell boundary. Interestingly, we find that the CdSe/ZnS QDs coat the CaCO3 microparticles prior to the deposition of the DNA shell (Figure 2A, panel I), and after the EDTA dissolution of the CaCO3 particles, the QDs are preferentially localized at the inner shell boundary rather than being diffusionally distributed in the microcapsule volume. The adsorption of the CdSe/ZnS QDs at the inner shell boundary after dissolution of the core CaCO3 can be attributed to the electrostatic adsorption of the mercaptopropionic acid (MPA)-functionalized QDs to the primary, positively charged PAH layer associated with the inner part of the shell. In turn, the localization of the QDs on the surface of the CaCO3 is, at present, not understood. Nonetheless, we note that the molar ratio of CdSe/ZnS QDs to Ca2+/CO32− is 1:200. Thus, presumably, the high concentration of Ca2+/ CO32− results in the rapid crystallization of the CaCO3 microparticles, favoring the adsorption of the dilute constituent of the MPA-modified QDs onto the surface of the microcrystals (eventually by H-bonds). The scanning electron microscopy (SEM) images of the CaCO3 microparticles prior to the deposition of the DNA layers and after the deposition of the DNA layer are presented in Figure 2B, panels a and b, respectively. The uncoated microparticles appear as smooth spheres, whereas the DNA-coated microparticles reveal a porous “hairy” surface, consistent with the formation of a modifying coating shell on the particles. Figure 2B, panel c, depicts the SEM image of the capsules after dissolution of the core. Complementary fluorescence anisotropy measurements suggest that the microcapsules after the dissolution of the core retain their spherical structure (rather than being squeezed into 8685

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Figure 4. (A) Schematic preparation of QDs560-loaded pH-responsive microcapsules stabilized by T−A·T triplex units. (B) Schematic mechanism for the pH-stimulated degradation of the microcapsules through the separation of T−A·T triplex-stabilized fragments from the shell (at pH 9.0). (C) Time-dependent fluorescence changes upon the release of the QD560 from the microcapsules at (a) pH 9.0, (b) pH 7.2, and (c) pH 5.0.

demonstrate that the DNA microcapsules are selectively unlocked at pH 5.0, due to the formation of the C−G.C+ complex that dissociates the microcapsules. The variations in the fluorescence intensities of the CdSe/ZnS QDs in the pH region of 5−9 are small. Figure S3A,B shows the effect of different pH values on the QDs. One may realize that the fluorescence of the 620 nm emitting QDs decreases by ca. 25% upon acidification to pH 5.0, whereas the luminescence of the 560 nm emitting QDs decreases only by ca. 18% upon switching the pH from 7.2 to 9.0. From the fluorescence changes observed upon unlocking the microcapsules, Figure 3B, curve a, and using an appropriate calibration curve (taking into account the pH effect on the fluorescence intensities of the respective QDs), and evaluating the number of microcapsules in the reaction volume (by flow cytometry), we estimate the amount of released QD620 to be 53 amole·capsule−1 (or 3.2 × 107 QDs·capsule−1). For further discussion on the effect of pH on the unlocking of the microcapsules and the release of the QDs, see Figure S4 and accompanying discussion. Confocal microscopy images of the QDs-loaded microcapsules before and after treatment at pH 5.0 support the suggested release mechanism (Figure S4). We find that the fluorescence intensities associated with the microcapsules decreased by ca. 70%, consistent with the release of the QDs. Also, the resulting fluorescent microcapsules reveal a non-homogenous distribution of the QDs on the shell interface, suggesting the pHinduced formation of pores (or holes) in the shell, through which the QDs were released. Figure 4A depicts the synthesis of the T−A·T pH-responsive microcapsules loaded with CdSe/ZnS QDs of a second size, emitting at λem = 560 nm (QD560). The PAH-coated QD560loaded CaCO3 microparticles were functionalized by a first DNA hybrid consisting of the sequences (8), (9), (10), (11),

Figure 3A shows the schematic pH-stimulated unlocking of microcapsules at pH 5.0 and the release of the QD620. Subjecting the microcapsules to pH 5.0 results in the reconfiguration of the nucleic acids (1), (2), (3), and (4) into the C−G·C+-stabilized triplex structure. This leads to the dissociation of the microcapsules and to the release of the QD620. The time-dependent release of the QDs from the microcapsules subjected to pH 5.0 was followed by precipitation of the microcapsules (via centrifugation) from aliquots of microcapsules at different time intervals, and fluorescence analysis of the supernatant phase that contains only the released QDs (for further details, see the Experimental Section). Figure 3B, curve a, depicts the fluorescence spectrum of the QD620 released by the pH trigger (pH 5.0), that correspond to the fluorescence from the supernatant above the opened capsules. For comparison, the spectra depicted in curves b and c show the fluorescence of the QDs released from the microcapsules subjected to pH 7.2 (b) and pH 9.0 (c) for a similar time interval (30 min). Evidently, effective release of the QD loads is observed only under acidic conditions that lead to the dissociation of the microcapsules via the formation of the C−G·C+ triplex structures. Figure 3C shows the timedependent fluorescence changes in the solution (λem = 620 nm) upon the release of the QDs (curve a) at pH = 5.0. After a time interval of ca. 30 min, the fluorescence intensities level off to a saturation level. For comparison, the time-dependent fluorescence changes in the solution at pH 7.2 or 9.0, as a result of leakage of the QDs, curves b and c, are minute. Furthermore, treatment of QD-loaded microcapsules stabilized by DNA shells composed of layers bridged by nucleic acids that cannot generate the triplex C−G·C+ structure at pH 5.0 did not yield to the unlocking of the microcapsules and to the release of the QDs (see Supporting Information Figure S2). These results 8686

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ACS Nano (12), and (13). The strand (12) acts as an axle to connect the different components by forming the triplex T−A·T structure with nucleic acids (8), (9), (10), and (11) at pH 7.2, and strand (13) acts as an interlayer linker. Subsequently, the second nucleic acid hybrid composed of (8), (9), (10), (11), (13), and (14) was deposited on the first layer to yield the second layer coating. After the sequential deposition of six DNA cross-linking layers, the CaCO3 core was dissolved with EDTA and the QD560-loaded microcapsules were generated. (Detailed sequence information is shown in Figure S5 in the Supporting Information. For the microscopic characterization of the resulting microcapsules, see Figures S6 and S7.) Figure 4B presents the schematic pH-induced separation of the microcapsules and the release of the QD560. Subjecting the microcapsules to pH 9.0 dissociates the T−A·T bridging units, resulting in the release of the QD560. Figure 4C shows the timedependent fluorescence changes upon the release of the QD560 at pH = 9.0, curve a. Evidently, after a time interval of ca. 30 min, the release of the QDs is completed. Using an appropriate calibration curve, and knowing the concentration of the microcapsules, we evaluated the amount of released QD560 to be 140 amole·capsule−1 (or 8.5 × 107 QDs·capsule−1). Control experiments revealed that minute leakage of the QD560 from the microcapsules proceeds at pH 7.2 and 5.0, curves b and c, respectively, implying that the release of the QD560 is stimulated only upon the dissociation of the T−A·T shell bridges under basic conditions (for the selective pH-induced release of the QDs, see Figure S2). The QD620- and QD560-loaded microcapsules that are responsive to pH 5.0 and 9.0, respectively, were further implemented as a functional mixture for the programmed release of the QDs (Figure 5). Treatment of the microcapsule mixture at pH 5.0 results in the release of the QD620, whereas at pH 9.0, the selective release of the QD560 is observed.

Figure 5. (A) Fluorescence spectra corresponding to the programmed pH-stimulated release of QDs from a mixture of QD620-loaded C−G·C+-responsive microcapsules and QD560-loaded T−A·T-responsive microcapsules upon subjecting the mixture to (a) pH 5.0, (b) pH 7.2, and (c) pH 9.0. (B) Fluorescence intensities of the release of QD620 and QD560 from the mixture of the two kinds of the microcapsules subjected to different pH values for a fixed time interval of 30 min. obtain CdSe/ZnS QD-loaded CaCO3 particles, 0.33 M Na2CO3 solution (300 μL) was mixed with a similar volume of 0.33 M CaCl2 solution (300 μL) while being stirred at room temperature and in the presence of 0.5 μM QD620 or 1.5 μM QD560, which had been transferred from toluene to water. A final volume of 1000 μL was adjusted with water. After being stirred for 110 s followed by 70 s of rest, the resulting colloidal solution was washed three times with water by repeated centrifugation (900 rpm, 20 s), and the supernatant was removed. Synthesis of Layer-by-Layer DNA-Assembled Capsules. The CdSe/ZnS QD-loaded CaCO3 microparticles serving as a core were modified with six layers of DNA (as found to be optimal, Figure S4, Supporting Information) as follows: The microparticles, 6 mg, were suspended in 300 μl of 1 mg·mL−1 PAH solution (10 mM HEPES, pH 7.2, including 500 mM NaCl and 50 mM MgCl2) and kept in continuous shaking for 30 min, after which the particles were washed twice with buffer (10 mM HEPES, pH 7.2, including 500 mM NaCl) through centrifugation (900 rpm, 20 s) and removal of the supernatant. The PAH-modified particles were alternately incubated with 300 μl of DNA sequence mixtures for a time interval of 30 min each, in the following order A1 [(1), (3), (5), (6)]/A2 [(2), (4), (5)] and A3 [(1), (3), (6), (7)]/A4 [(2), (4), (7)] for the C−G·C+ triplex and B1 [(8), (10), (12), (13)]/B2 [(9), (11), (12)] and B3 [(8), (10), (13), (14)]/B4 [(9), (11), (14)] for the T−A·T triplex. Each DNA mixture consists of 10 μM of the mentioned DNA sequence in 10 mM HEPES, pH 7.2, containing 500 mM NaCl and 50 mM MgCl2. For the first deposited layer, sequences (6) and (13) were added to mixtures A2 and B2, respectively. After each adsorption cycle, one washing step was applied to remove the unbound nucleic acids. The formed DNA-coated microparticles were introduced to 0.1 M EDTA (pH 7.2) for 1 h to dissolve the CaCO3 core. Subsequently, when the suspension became clear, the resulting capsules were washed with buffer (10 mM HEPES, pH 7.2, including 500 mM NaCl) using slow

CONCLUSIONS In conclusion, the present study has introduced the preparation of pH-responsive triplex DNA-based microcapsules. Specifically, we have implemented the C−G·C+ and T−A·T triplex DNA motifs to induce the unlocking of the microcapsules and selectively release QD loads incorporated in the microcarriers. Such mixtures of microcapsules may be applied as pH sensors and imaging agents, as pH-controlled drug release systems, or as microreactors for controlled synthesis. EXPERIMENTAL SECTION Reagents and Instruments. Magnesium chloride, sodium chloride, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES base), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES acid), poly(allylamine hydrochloride) (PAH, 58 kDa MW), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), glacial acetic acid, and ammonium hydroxide solution were purchased from Sigma−Aldrich. Red-Orange CdSe/ZnS quantum dots and Hops-Yellow CdSe/ZnS quantum dots in toluene were purchased from Evident Technologies. All oligonucleotides were synthesized, standard desalting purified, and freeze-dried by Integrated DNA Technologies, Inc. (Table S1, Supporting Information). Ultrapure water from a NANOpure Diamond (Barnstead Int., Dubuque, IA) source was used in all experiments. Magellan XHR 400L scanning electron microscopy and a FV-1000 confocal microscope (Olympus, Japan) were implemented to characterize microparticles. Preparation of CdSe/ZnS QD-Loaded CaCO3 Microparticles. Spherical CaCO3 microparticles were fabricated with a colloidal crystallization method as described previously by Volodkin.42 To 8687

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ACS Nano centrifugation (500 rpm, 20 min) while discarding the supernatant. (We note that this centrifugation speed, yielding a g value corresponding to 30 rcf is sufficient to precipitate the microcapsules without structural destruction, cf. Figure S10.) It should be noted that, during all of the steps of preparation of the microcapsule shells and the subsequent dissolution of the microcapsule shells, we applied centrifugation steps to purify the structures. We did not observe any aggregation of the microcapsules during these steps. Presumably, electrostatic repulsive interactions between microcapsules and the lack of complementarity between the exterior nucleic acid components constituting the shells prohibit such aggregation phenomena. pH-Induced Unlocking of the Microcapsules and the Release of the Loaded CdSe/ZnS QDs. To perform the pHinduced dissociation of the microcapsules, 40 μL of the triplex sequence-coated microcapsule solution, including 2000 capsules μL−1, was treated with 1 μL of 10% acetic acid or 15% ammonium hydroxide to adjust the pH. The microcapsules were incubated under different pH conditions (pH 5.0, 7.2, and 9.0) for various time intervals. Then, a centrifugation at 500 rpm for 20 min proceeded on the microcapsule solution to precipitate the residual capsules. The fluorescence of released CdSe/ZnS QDs in the supernatant was measured by a Cary Eclipse fluorescence spectrophotometer (Varian, Inc.). Programmed Release of the pH-Responsive Microcapsules. The C−G·C+ and T−A·T triplex-bridged microcapsule mixture containing 2000 capsules μL−1 of each was implemented for pHinduced programmed release of loads. Forty microliters of microcapsule solution was incubated under different pH values, which were adjusted by adding 1 μL of 10% acetic acid or 15% ammonium hydroxide, for 30 min. The released QD620 and QD560 were separated from residual capsules by being centrifuged at 500 rpm for 20 min. Subsequently, the fluorescence spectra of the released QD620 and QD560 in the supernatant were measured.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b04056. Nucleic acids sequences; preparation of MPA-capped CdSe/ZnS QDs; control experiments of non-pHresponsive DNA microcapsules; effect of pH on the fluorescence of the QDs; detailed composition of T−A·T triplex shells; SEM images, confocal images of microcapsules; optimization of DNA layer number of capsules; calibration curves of QD620 and QD560 (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research is supported by the Israel Science Foundation. Support from the Council for Higher Education in Israel is acknowledged by W.-C.L. W.J.P. acknowledges funding from the German Research Foundation (DFG, project PA794/21-1). REFERENCES (1) Wang, F.; Liu, X.; Willner, I. DNA Switches: From Principles to Applications. Angew. Chem., Int. Ed. 2015, 54, 1098−1129. (2) Lu, C.-H.; Willner, I. Stimuli-Responsive DNA-Functionalized Nano-/Microcontainers for Switchable and Controlled Release. Angew. Chem., Int. Ed. 2015, 54, 12212−12235. (3) Roh, Y. H.; Ruiz, R. C. H.; Peng, S.; Lee, J. B.; Luo, D. Engineering DNA-Based Functional Materials. Chem. Soc. Rev. 2011, 40, 5730−5744. 8688

DOI: 10.1021/acsnano.6b04056 ACS Nano 2016, 10, 8683−8689

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DOI: 10.1021/acsnano.6b04056 ACS Nano 2016, 10, 8683−8689