Synthesis, Loading, and Application of Individual Nanocapsules for

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Synthesis, Loading, and Application of Individual Nanocapsules for Probing Single-Cell Signaling Bingyun Sun and Daniel T. Chiu* Department of Chemistry, University of Washington, Seattle, Washington 98195-1700 Received December 22, 2003. In Final Form: March 1, 2004 This paper describes the synthesis and loading of silica and polystyrene-acrylic based nanocapsules with small molecules. The nanocapsules are used for delivering defined packages of stimuli to single cells with both high spatial and temporal resolutions. To introduce molecules into the capsules, we characterized two approaches. The first approach is based on a base-swell process in which the shell of the capsule is swelled so small molecules can diffuse into the interior of the capsule and be trapped inside once the capsules are de-swelled. The second approach is based on a dry-swell-dry process in which the solution containing the molecules of interest and the nanocapsules is physically dried to promote more molecules to enter into the interior of the capsule. We characterized both methods by monitoring the content of and the release from individual capsules with confocal microscopy and wide-field imaging. To illustrate the biological applications of such nanocapsules, we used optical trapping to position a single carbachol-loaded capsule adjacent to a single CHO cell that has been transfected with muscarinic acetylcholine (M1) receptors, released the carbachol from the capsule with a single 3-ns N2 laser pulse, and then monitored the subsequent intracellular signaling triggered by the binding of carbachol to the M1 receptors.

Introduction The transduction of signals inside cells and among cells is a central and basic process in biological systems. An understanding of this process will offer new routes to drugs and new therapies for diseases.1,2 Because of the complexity of biological systems and variations among individual cells, the ability to study signaling pathways at the level of single cells may offer a better understanding of cell signaling than that of bulk studies in which important signaling events may be masked by the actions of the population.1,3 The small sizes of single mammalian cells and the presence of highly heterogeneous functional domains of the cell1,4,5 necessitate techniques that can initiate controllably a signaling event at a defined location on the cell and methods capable of detecting all the responses of the cell with high sensitivity and resolution. Here we describe our work that uses individual nanocapsules for delivering stimuli to single cells and to activate the cell with both high spatial (sub-micrometers) and temporal (sub-microseconds) resolutions.6 A wide range of strategies exist for delivering bioactive molecules to cells, such as perfusion,7,8 microinjection,9,10 iontophoresis,11,12 and light-induced uncaging of caged molecules.13,14 To evoke, with high spatial resolution, a ligand-induced response from a single cell, laser photolysis of a caged compound using two-photon excitation is * To whom correspondence should be addressed. (1) Hunter, T. Cell 2000, 100, 113-127. (2) Berridge, M. J.; Lipp, P.; Bootman, M. D. Nat. Rev. Mol. Cell Biol. 2000, 1, 11-21. (3) Weng, G.; Bhalla, U. S.; Iyengar, R. Science 1999, 284, 92-96. (4) Edidin, M. Trends Cell Biol. 2001, 11, 492-496. (5) Pierce, S. K. Nat. Rev. Immunol. 2002, 2, 96-104. (6) Sun, B.; Chiu, D. T. J. Am. Chem. Soc. 2003, 125, 3702-3703. (7) Pelc, R.; Ashley, C. C. Eur. J. Physiol. 1997, 435, 174-177. (8) Spitzer, K. W.; Bridge, J. H. B. Am. J. Physiol. 1986, 256, C441C447. (9) Angelantonio, S. D.; Nistri, A. J. Neurosci. Methods 2001, 110, 155-161. (10) Akaoka, H.; Saunier, C. F.; Chergui, K.; Charlety, P.; Buda, M.; Chouvet, G. J Neurosci. Methods 1992, 42, 119-128. (11) Junginger, H. E. Adv. Drug Delivery Rev. 2002, 54, S57-S75. (12) Awenowicz, P. W.; Porter, L. L. J. Neurophysiol. 2002, 88, 34393451.

perhaps the current most promising approach, as demonstrated in recent works to map the distribution of receptors and ion channels on the membrane15,16 and calcium fluxes in cells.17 Despite the power of two-photon excitation and microscopy, the use of a caged compound suffers from a number of chemical drawbacks. Chief among them lies in the design and synthesis of caged compounds, which can be complex and time-consuming.6,18 To complement the use of chemical cages, we explored the use of nanocapsules to physically confine the molecules of interest, in which we used lipid vesiclesswhose size can be varied from tens of nanometers to micrometerssto encapsulate bioactive compounds. Exposure to a single focused UV laser pulse releases these enclosed molecules.6,19 This strategy is especially useful for the highly localized delivery of stimuli to a single cell, since individual nanocapsules can be optically trapped and positioned precisely at the cell surface and then photolyzed selectively to release the contained molecules.6 Although lipid vesicles provide a flexible platform to explore this strategystheir sizes can be tuned, they can encapsulate small membraneimpermeable molecules as well as large proteins and DNAs, their surface can be functionalized with a wide range of recognition molecules, and the lipid bilayer can be doped with high concentrations of dyes or other sensitizerssthey suffer one important drawback: namely, they cannot be stored for a long period of time.20 This (13) Albota, M.; Beljonne, D.; Bre´das, J.-L.; Ehrlich, J. E.; Fu, J.-Y.; Heikal, A. A.; Hess, S. E.; Kogej, T.; Levin, M. D.; Marder, S. R.; McCordMaughon, D.; Perry, J. W.; Ro¨ckel, H.; Rumi, M.; Subramaniam, G.; Webb, W. W.; Wu, X.-L.; Xu, C. Science 1998, 281, 1653-1656. (14) Brown, E. B.; Shear, J. B.; Adams, S. R.; Tsien, R. Y.; Webb, W. W. Biophys. J. 1999, 76, 486-499. (15) Pettit, D. L.; Wang, S. S.; Gee, K. R.; Augustine, G. J. Neuron 1997, 19, 465-471. (16) Denk, W. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 6629-6633. (17) Li, W. H.; Llopis, J.; Whitney, M.; Zlokarnik, G.; Tsien, R. Y. Nature 1998, 392, 936-941. (18) Morgan, C. G.; Bisby, R. H.; Johnson, S. A.; Mitchell, A. C. FEBS Lett. 1995, 375, 113-116. (19) Srinivasan, R. Science 1986, 234, 559-565. (20) Kates, M. In Laboratory techniques in biochemistry and molecular biology; Work, T. S., Work, E., Eds.; Elsevier: New York, 1972; Vol. 3.

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Figure 1. Schematic drawing that shows the layout of our experimental setup: M, mirror; BS, beamspliter; DC, dichroic; SF, spatial filter; OJ, objective; CS, coverslip; NF, neutral-density filter; TS, telescope; D, diffuser; PH, pinhole; BF, band-pass filter; L, lens; SPCM, single-photon counting module.

paper investigates alternatives to lipid vesicles that are more robust and have longer shelf lives. Capsules of both inorganic (metal, metal oxide, and ceramic) and organic (polymeric) materials have been extensively studied and reviewed in the literature21-23 because of the wide range of potential applications, including drug delivery, gene therapy, and heterogeneous catalysis. The synthesis of hollow particles has employed a number of strategies, such as self-assembly, templateassisted approaches, emulsion/suspension polymerization, and dendrimers. In contrast to the design and synthesis of hollow particles, comparatively less work has focused on the loading into and release from such capsules,24,25 especially at the level of individual capsules that is important for our applications. Here we explore the synthesis and loading of silica and polystyrene-acrylic based capsules and characterized the release from single capsules upon laser photolysis. Using such capsules, we also demonstrate the spatially defined delivery of carbachol to initiate calcium signaling in a Chinese hamster ovary (CHO) cell transfected with muscarinic acetylcholine receptors. Experimental Section Optical Setup. Figure 1 is a schematic of our homebuilt optical setup. We used a Nd:YAG laser (wavelength at 1064 nm) for the optical trapping and manipulation of micro- and nanocapsules, a pulsed N2 laser (wavelength at 337 nm) for photolysis of single capsules, and an Ar+ ion laser (wavelength at 488 nm) for both single-molecule fluorescence confocal microscopy and wide-field fluorescence imaging. To achieve good beam quality, the output of the N2 laser was first passed through a spatial filter, before being aligned collinearly with the YAG trapping laser and sent into the microscope through the front port. (21) Meier, W. Chem. Soc. Rev. 2000, 29, 295-303. (22) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967-973. (23) McDonald, C. J.; Devon, M. J. Adv. Colloid Interface Sci. 2002, 99, 181-213. (24) Ding, J.; Liu, G. J. Phys. Chem. B 1998, 102, 6107-6113. (25) Marinakos, S. M.; Novak, J. P.; Brousseau, L. C., III; House, A. B.; Edeki, E. M.; Feldhaus, J. C.; Feldheim, D. L. J. Am. Chem. Soc. 1999, 121, 8518-8522.

Single-molecule confocal detection was achieved by sending the output of the Ar+ ion laser into the microscope through a side port; the emitted photons were collected by a 100× oil immersion objective (N.A. 1.3) and was passed through a 1:1 relay lens that projected the primary image plane onto a pinhole; the light through the pinhole was sent through a band-pass filter and was detected by a single-photon counting module (SPCM; Perkin-Elmer, Wellesley, MA). A scanning mirror was positioned at the Fourier plane of the relay lens between the lens and the pinhole. The placement of the scanning mirror permitted the facile and accurate displacement of the confocal probe volume from the foci of the YAG and N2 laser in the object plane. Fluorescence imaging was accomplished by scattering the output of the Ar+ laser with a diffuser prior to sending it through the front port and into the objective; fluorescence was collected by the objective and projected onto a CCD camera after passing through dichroic and band-pass filters. Preparation of Nanocapsules. A polyol process26-28 was used to synthesize silver nanoparticles with different sizes. Silver nitrate, the precursor, was reduced by ethylene glycol (EG), which also served as the solvent. Poly(vinylpyrrolidone) (PVP, MW ≈ 10 000) was used as the protecting agent to prevent the aggregation of silver particles. In a typical experiment, 2 g of PVP was added slowly into 15 mL of an EG solution of 80 mg of AgNO3 under continuous magnetic stirring. This reaction mixture was heated to 120 °C at a rate of ∼1 °C/min, after which the reaction was allowed to proceed for 1 h at 120 °C. The reaction mixture was allowed to cool to room temperature and acetone (>200 mL) was then added to dilute this reaction mixture. The silver colloidal particles formed were separated from this solution by centrifugation and were then re-dispersed in ethanol. To form silica nanocapsules, the Sto¨ber method29-31 was used to coat the silver colloids with amorphous silica. (26) Silvert, P.-Y.; Herrera-Urbina, R.; Duvauchelle, N.; Vijayakrishnan, V.; Elhsissen, K. T. J. Mater. Chem. 1996, 6, 573-577. (27) Silvert, P.-Y., Herrera-Urbina, R., Elhsissen, K. T. J. Mater. Chem. 1997, 7, 293-299. (28) Ducamp-Sanguesa, C.; Herrera-Urbina, R.; Figlarz, M. J. Solid State Chem. 1992, 100, 272-280.

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Approximately 2.4 mg of the silver colloids was dispersed into a mixture of 20 mL of 2-propanol and 4 mL of deionized (DI) water, after which 1 mL of 30% ammonia solution and 0.01 mL of tetraethyl orthosilicate (TEOS) were added consecutively to this solution. After the reaction had proceeded for 30 min, the final mixture was centrifuged at 8000 rpm to isolate the silica-coated silver nanoparticles, which were then re-dispersed in DI water. Silica nanocapsules were formed from the silica-coated silver nanoparticles by dissolving the silver core overnight in a solution of ammonia at pH ∼ 10. Loading of Molecules into Nanocapsules. We used two methods to introduce molecules into the interior of the nanocapsules. The first method is based on a baseswell process (wet method),32 in which a concentrated stock solution (5.3% solids in water) of capsules was added directly into a 1-mL solution containing 1.5 mg/mL fluorescein in 2-propanol/chloroform (95%/5% (v/v)), and 0.02 mL of 30% ammonia solution. The mixture was sonicated for 15 min, and the loading process was allowed to proceed at 45 °C for 2 h. We used this procedure to encapsulate both fluorescein and carbachol into the nanocapsules, in which we first dissolved carbachol in a small amount (50 nm) achieved by decreasing the PVP to AgNO3 ratio will broaden this size range. A number of methods exist for the formation of other metal colloidal particles with good homogeneity in size, such as gold nanoparticles that have a less than 10% variation in diameter,35 but it is in general difficult with these methods to form large particles (>100 nm) while maintaining this narrow distribution in sizes. Once silver nanoparticles of a desired size were formed, we coated the surface of the particle with a layer of silica using the Sto¨ber method,29-31 which is based on the basecatalyzed hydrolysis of TEOS and the subsequent condensation of silica onto the surface of the silver colloids. Parts D-F of Figure 2 show the results of this process, which are TEM images showing silica-coated silver nanoparticles of different sizes. The thickness of the silica shell can be tuned by adjusting the amount of NH4OH and the concentration of TEOS present in the reaction mixture, as well as by controlling the reaction time.29,30 More NH4OH or TEOS or longer reaction time leads to a thicker silica coating on the silver particles (heterogeneous nucleation process) as well as the formation of pure silica particles (homogeneous process) (Figure 2D-F). By increasing the concentration of silver colloids or by decreas(34) Fievet, F.; Lagier, J. P.; Blin, B.; Beaudoin, B.; Figlarz, M. Solid State Ionics 1989, 198, 32-33. (35) Giersig, M.; Ung, T.; Liz-Marza´n, L. M.; Mulvaney, P. Adv. Mater. 1997, 9, 570-575.

Figure 3. (A) TEM image of polystyrene-based capsules. Nomarski (B) and fluorescence (C) images of capsules loaded with fluorescein using the wet method. The scale bar represents 1 µm. Confocal scans in the xy (D) and xz (E) dimensions of the fluorescein-loaded capsules.

ing the concentration of TEOS, the homogeneous process can be essentially eliminated (Figure 2E). Formation of Nanocapsules. To form nanocapsules, the silver core was etched away in an ammonia solution, thereby leaving behind a silica shell. The insets in Figure 2D-F show examples of such nanocapsules. Because the silica shell was formed in a basic environment in which silica can also experience slow hydrolysis, a thin silica coating cannot withstand the ammonia solution and will also be etched away. To overcome this problem, we performed the etching in the silica-coating solution so that silica condensation onto the silver nanoparticle can compensate for the dissolution of silica. In addition to silica nanocapsules, we have also explored the use of polymeric capsules. The polystyrene-acrylic based hollow beads in particular have the advantage that they can be purchased commercially, although their sizes (which are from hundreds of nanometers to 1 µm) are larger than the silica nanocapsules. For applications in which small sizes and good size tunability are important, silica nanocapsules may prove to be more versatile. Loading of Nanocapsules. We have explored several strategies for loading the nanocapsules. To encapsulate small molecules into the polystyrene-acrylic containers, we characterized two loading methods by monitoring the content of and the release from individual capsules. The first method is based on a base-swell process, which we called the “wet” method, in which the shell of the capsule is swelled in an organic solution with volatile base (ammonia) so small molecules can diffuse into the interior of the capsule23 and be trapped inside once the capsules are reimmersed back into a nonbasic solution. The second method is based on a dry-swell-dry process, which we called the “dry” method, in which the solution contains the molecules of interest and the nanocapsules are physically dried to promote more molecules to enter into the interior of the capsule. Figure 3 shows the results of loading of small molecules into the capsules. Figure 3A is a TEM image of the capsule, while parts B and C of Figure 3 show the Nomarski and

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Figure 4. (A) TEM image of polystyrene-based capsules after loading with fluorescein using the dry method. The scale bar represents 1 µm. (B) A sequence of CCD images showing the release of fluorescein upon UV photolysis of the capsule that was loaded using the dry method. The solution outside the capsule contained 1 mM NaOH. The scale bar represents 2 µm. (C) Schematic illustration of the experimental procedure we used to obtain the result in D. Here the optically trapped capsule and the confocal probe volume were displaced laterally from each other by 4 µm (left illustration). The capsule was then photolyzed with a single 3-ns pulse at 0.25 µJ from the N2 laser (middle). The diffusion of fluorescein molecules (expanding green sphere) was monitored with confocal detection (right), which was carried out using the 488-nm line of an Ar+ laser at 2 mW; the data integration time was set at 1 ms. (D) Photon trace showing the arrival and passage of fluorescein through the confocal probe volume upon photolysis of the capsule and release of fluorescein. The arrow points to the firing of the N2 laser pulse. The solid line is the simulated result based on the three-dimensional diffusion equation and our experimental conditions.

fluorescence images after the capsules had been loaded with fluorescein using the wet method. To ensure fluorescein was loaded inside the capsule and not just attached to the surface of the capsule, we spatially profiled the fluorescence emission from the capsule with confocal microscopy. Parts D and E of Figure 3 are the xy and xz confocal scans, which clearly show fluorescein is localized inside the capsule. The xy and xz scans appear different because the resolution in the xy, which is defined by the diffraction-limited focus (∼0.3 µm) of the laser probe volume, is greater than the resolution in the xz, which is determined by the size of the confocal pinhole and the spherical aberration present in the objective (∼1 µm). We observed similar spatial fluorescence intensity profiles from capsules loaded with fluorescein using the dry method. The amount of molecules loaded into the capsules can be controlled either by varying the initial concentration of the loading molecules or by varying the size of the capsules. At room temperature, the loaded capsules are stable for days in DI water. Over longer periods (>1 week), we have observed a slow leakage of the capsules’ contents into solution. These molecules that leaked into solution can be easily removed by passing the solution containing the capsules through a prepacked size-exclusion column. To prevent this leakage and to improve long-term storability, we are exploring a number of strategies, including the introduction of coatings or a thin shell of polymer around the capsule. Release from Nanocapsules. To demonstrate that the encapsulated small molecules can be physically released from the nanocapsule, we monitored the release and diffusion of fluorescein from individual capsules upon

laser photolysis. Figure 4A shows a TEM image of the capsule after being loaded with fluorescein using the dry method. Dimples on the surface of the capsule are clearly visible. The exact mechanism of the dimple formation is not clear but was likely caused by the drying process that resulted in the partial collapse of the shell of the capsule. The presence of the dimple does not, however, seem to negatively affect molecules from being encapsulated into or released from the capsules. In fact, the amount of encapsulated fluorescein was found to be greater using the dry method than with the wet method where no such dimple or deformation was observed. We monitored the release upon laser photolysis from individual optically trapped nanocapsules using both widefield fluorescence imaging and confocal point detection. Figure 4B is a sequence of fluorescence images showing the release of fluorescein from the capsule. Here a single capsule loaded with fluorescein was first optically trapped, followed by photolysis of the capsule with a single 3-ns pulse from a N2 laser, the output of which was aligned collinearly with the trapping laser beam. The observed fluorescence intensity increased as fluorescein diffused away from the volume defined by the capsule because the high concentration of fluorescein present inside the capsule caused it to self-quench,36,37 and also because the medium contained sodium hydroxide that increased the quantum yield of fluorescein, once it was released from the capsule. Wide-field fluorescence imaging is a simple and convenient method to visualize the release of fluorescein, but to ensure the observed fluorescence intensity was not (36) Walter, B. Ann. Phys. (Berlin) 1888, 34, 316-326. (37) Chen, R. F.; Knutson, J. R. Anal. Biochem. 1988, 172, 61-77.

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Figure 5. Response of a fluo-3-loaded CHO-M1 cell to the local release of carbachol from a polystyrene-based capsule. (A) Schematic illustration of the experimental design. Bright-field (B-I) and fluorescence (B-II) images of the capsule and cell before the photolysis of the capsule, which was loaded with fluorescein (in addition to carbachol) for ease of visualization; (B-III) increase in intracellular concentration of calcium as visualized by fluo-3 after photolysis of the capsule and release of carbachol.

caused by excitation from the UV laser pulse, we also used confocal point detection to monitor the release and diffusion of fluorescein after photodestruction of the capsule. Figure 4C schematically illustrates our experiment, in which the diffraction-limited probe volume of the confocal microscope was displaced a calibrated distance (4 µm) away from the collinearly aligned trapping (YAG) and photolysis (N2) laser foci in which the capsule is located. Upon photolysis of the capsule, fluorescein began to diffuse away (expanding green sphere in the inset) from the volume defined by the capsule toward the confocal probe volume. Figure 4D is the detected photon trace that shows this arrival and passage of fluorescein through the confocal detection volume. The simulated curve (solid line) using a three-dimensional diffusion equation fits well with our experimental observation (Figure 4). To monitor release from nanocapsules, the advantage of using wide-field fluorescence imaging is its ease of use, while confocal detection provides a more sensitive and quantitative measurement. We observed similar signals after photolysis of capsules loaded with the wet method, albeit the amount of fluorescein present was less than that from capsules loaded with the dry method. The drying process increases the loading efficiency, but it also promotes attachment of the molecules to the surface of the capsule, which need to be removed with multiple steps of washing and purification. The dry method is useful for applications in which high concentrations of stimuli are required, but, for most applications, the wet method may be more appropriate because of the relatively mild conditions under which loading occurs. Delivery of Stimulus to Single Cells. To illustrate the use of nanocapsules for studying single cells, we loaded

the capsule with carbachol, an agonist to the muscarinic acetylcholine receptor type 1 (M1), and studied the calcium signaling in Chinese hamster ovary cells (CHO) transfected with M1 receptors. Figure 5A shows our experimental procedure, in which a single capsule loaded with carbachol was optically trapped and positioned adjacent to a single cell (CHO M1) (left panel) followed by application of a single N2 laser pulse to release the encapsulated carbachol (right panel). The binding of carbachol by the receptor initiates a calcium-signaling cascade, which ultimately results in the increase of intracellular calcium, a process that we monitored with the calcium indicator, fluo-3.6 Parts B-I and B-II of Figure 5 show the bright-field and fluorescent images of the capsule and the cell prior to release of carbachol, respectively, and Figure 5B-III shows the resultant increase of intracellular concentrations of calcium upon activation by carbachol. We have consistently observed that the amount of carbachol present within the capsule is more than sufficient to trigger a cellular response. Conclusions In comparison to caged compounds, nanocapsule-based chemical delivery is versatile and easy to implement from a design and synthetic perspective. Capsules that are hundreds of nanometers to micrometers in diameter are most suitable for in vitro studies in which individual capsules can be optically manipulated and photolyzed to release the chemicals for studying specific biological responses. For in vivo experiments or brain slices, capsules that are tens of nanometers in diameter are likely more useful. To attain high spatial resolution in such applications, it would be necessary to also develop chemistry for

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sensitizing the shell of the nanocapsule with the desired molecules, such as dyes having good two-photon absorption cross sections so the nanocapsules can be photolyzed selectively using multiphoton excitation. In comparison with lipid vesicles, the main advantage to the use of silicaand polystyrene-based capsules is their inherent robustness that lends themselves to long-term storability, which is pertinent in making this technique easy to use and widely adopted. The drawback with our current methods of loading is the inability to encapsulate large compounds, such as protein and DNA molecules. This drawback may be overcome by loading such large molecules during the synthesis of hollow particles, provided the conditions are

Sun and Chiu

sufficiently mild to retain biological functionality. Besides the study of cell signaling, polymeric capsules of microand nanometer dimensions are broadly useful in applications such as drug delivery, gene therapy, and catalysis. Acknowledgment. B.S. acknowledges support from the Center for Nanotechnology at the University of Washington for a UIF fellowship. We thank Prof. Yongnan Xia and Dr. Yugang Sun for the helpful suggestions on synthesis of silica nanocapsules. We gratefully acknowledge the support of this work by NIH. LA0364340