Protein Nanocapsule Weaved with Enzymatically Degradable

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NANO LETTERS

Protein Nanocapsule Weaved with Enzymatically Degradable Polymeric Network

2009 Vol. 9, No. 12 4533-4538

Zhen Gu,†,‡,§ Ming Yan,†,§ Biliang Hu,⊥ Kye-Il Joo,⊥ Anuradha Biswas,†,§ Yu Huang,§,| Yunfeng Lu,*,†,§ Pin Wang,*,⊥ and Yi Tang*,†,§ Department of Chemical and Biomolecular Engineering, Department of Mechanical and Aerospace Engineering, California NanoSystems Institute, Department of Material Science and Engineering, UniVersity of California at Los Angeles, Los Angeles, California 90095, and Mork Family Department of Chemical Engineering and Materials Science, UniVersity of Southern California, Los Angeles, California 90089 Received September 5, 2009; Revised Manuscript Received October 5, 2009

ABSTRACT Target proteins can be functionally encapsulated using a cocoon-like polymeric nanocapsule formed by interfacial polymerization. The nanocapsule is cross-linked by peptides that can be proteolyzed by proteases upon which the protein cargo is released. The proteasemediated degradation process can be controlled in a spatiotemporal fashion through modification of the peptide cross-linker with photolabile moieties. We demonstrate the utility of this approach through the cytoplasmic delivery of the apoptosis inducing caspase-3 to cancer cells.

Protein therapeutics, including antibodies,1 cytokines,2 transcription factors3 and enzymes,4 are indispensible in healthcare and biomedical applications.5,6 Most protein-based drugs and drug candidates suffer from poor thermal stability, serum proteolysis, and inability to penetrate cell membranes.7 Therefore, increasing the robustness and efficacy of proteins through hybrid carrier-protein materials is an important goal.8-10 To date, various approaches have been intensely explored, including liposomes,11 polymers,8,12-15 mesoporous silicates,10 or carbon nanotubes.16 Despite these, covalent approaches based on protein surface modification suffer from the irreversible modification of proteins and loss of function, while noncovalent strategies such as macromolecular selfassembly are prone to colloidal instability.8,17,18 An ideal method would therefore be (1) noncovalent encapsulation that can protect the protein from aggregation, proteolysis and denaturation; (2) reversible disassembly of the protective layer and release of the target protein upon reaching the cellular target or on demand; and (3) increased efficiency in

transport across cell membrane for proteins drugs that have cytosolic targets.

* To whom correspondence should be addressed. E-mail: (Y.L.) luucla@ ucla.edu; (P.W.) [email protected]; (Y.T.) [email protected]. Fax: (+1) 310206-4107. † Department of Chemical and Biomolecular Engineering, University of California at Los Angeles. ‡ Department of Mechanical and Aerospace Engineering, University of California at Los Angeles. § California NanoSystems Institute, University of California at Los Angeles. | Department of Material Science and Engineering, University of California at Los Angeles. ⊥ University of Southern California.

To demonstrate the utility of the degradable NC, we selected caspase-3 (CP3) as a target protein. CP3 is a peptidase in the apoptotic signaling pathway and is a potent executioner when introduced into otherwise antiapoptotic cancer cells.25,26 CP3 specifically cleaves the peptide sequence Asp-Glu-Val-Asp (DEVD) after the second aspartic acid.27,28 Successful cytoplasmic delivery of functional CP3 can therefore lead to cell apoptosis as a “read-out”.11,29 To encapsulate CP3 within a nanocapsule, we designed a

10.1021/nl902935b CCC: $40.75 Published on Web 11/04/2009

 2009 American Chemical Society

In this report, we designed a strategy to reversibly encapsulate proteins using a cocoon-like, enzymatically degradable polymeric nanocapsule (NC) by a one-pot procedure (Figure 1a). In the first step, monomers and crosslinkers are deposited to the protein surface via physical adsorption means, such as electrostatic force or van der Waals force. This process seeds the protein surface with sites for polymerization. Next, in situ free-radical polymerization is performed to form the protective shell in aqueous solution, facilitated by bisacrolylated short peptide cross-linkers19-21 that can be specifically cleaved by proteases. Upon proteolytic cleavage of the cross-linker, the polymeric shell disintegrates and the protein is released in functional form. The degradability of the polymeric shell can thus be tuned by the sequence of the cross-linker to respond to different proteases and can be spatiotemporally controlled by using photolabile22-24 caged peptide sequences.

Figure 1. Schematic diagram and characterization of protein nanocapsules. (a) One-pot preparation of protein nanocapsules by in situ free-radical polymerization. (b) Typical monomers and cross-linker used in the CP3-based nanocapsule study. (c) TEM images of fresh CP3 nanocapsules (CP3-NC13) and (d) the sample experienced self-degradation at 37 °C for 12 h in pH 7.4 PBS buffer. (e) Degradation kinetics of DLS intensity ratio It/I0 of caspase-3 nanocapsules, where the subscripts “0” and “t” represent time t ) 0 and t ) t; data were normalized to facilitate direct comparison. (f) Activity assay of free CP3 and CP3 NCs using the colorimetric substrate Ac-DEVD-pNA (p-nitroanilide) by spectrometrically monitoring the release of pNA at 409 nm. The error bars in c indicate standard deviation (SD).

DEVD-cross-linked polymeric shell that can be degraded by CP3 from within. In situ radical polymerization at 4 °C was performed in the presence of (1) mature CP3; (2) two monomers, acrylamide (AAm) and N-(3-aminopropyl) methacrylamide (APMAAm), which create a positively charged surface that can improve cell-penetrating efficiency; and a bisacryloylated VDEVDTK peptide as cross-linker (CLVDEVDTK) (Figure 1b). The cationic monomers can also bind to the CP3 surface and facilitate the desired interfacial polymerization process (pI of CP3 is ∼6.111). The lowtemperature process was selected to ensure the level of proteolysis is minimized during the polymerization process. With a fixed ratio of AAm/APMAAm/CL-VDEVDTK (8/6/1), the sizes of NCs can be fine-tuned between 8 and 20 nm by varying the ratio of protein to monomers (Supporting Information Figure S3). Specifically, when the molar ratio of protein to AAm was 1:1200, the average 4534

hydrodynamic radius of CP3 NCs was 13 nm (designated CP3-NC13). In contrast, the size of native CP3 was ∼5 nm. TEM shows that fresh CP3-NC13 in aqueous solution is compact with a spherical shape (Figure 1c). In contrast, the size of the nanoparticles decreased considerably after incubation in PBS at 37 °C for 12 h, indicative of degradation of the polymeric shell by the encapsulated CP3. The profile of nanoparticles is also less robust compared to the fresh CPNC13 and resembled that of “naked” CP3 protein (Figure 1d). The continuous self-degradation process was dynamically monitored by measuring the average count rate by DLS (Figure 1e). Within 200 min, the scattering intensity of CP3NC13 shrank to nearly the same level as that of native CP3 at 37 °C, while decreasing only slightly at 4 °C. The scattering intensity of CP3 NCs synthesized under identical conditions, except with the use of a nondegradable crosslinker N,N′-methylene bisacrylamide (designated CP3-NCN), Nano Lett., Vol. 9, No. 12, 2009

Figure 2. CP3 nanocapsules as apoptotic agents. (a) Bright-field-microscopy images of HeLa cells treated for 24 h with (i) saline; (ii) 200 nM CP3-NC13; (iii) nondegradable CP3 NCs; and (iv) native CP3. The scale bar is 50 µm. (b) CP3-induced HeLa cell apoptosis as measured by APO-BrdUTM TUNEL assay after the treatment with 200 nM CP3-NC13 for 24 h. Red fluorescence represents the PI-stained nuclei, and green fluorescence represents the Alexa Fluor 488-stained nick end label, an indicator of apoptotic DNA fragmentation. The merged pictures represent the merging of PI-stained nuclei with the Alexa Fluor 488-stained nick end label. The scale bar is 200 µm. (c) Viability of HeLa cells treated for 48 h with (i) native CP3; (ii) CP3-NC13; (iii) CP3-NCs cross-linked by CL-VDVADTK (CP3-NCDVAD); and (iv) CP3-NCN by varying concentration. (d) Growth profiles of HeLa (red), MCF-7 (blue), and M249 (green) cells cultured with 200 nM of CP3-NC13. Cytotoxicity was assessed at 24, 48, and 72 h.

remained essentially constant at 37 °C. To test the activity of CP3 NCs toward external peptidyl substrates, we used a spectrometric assay in the presence of a colorimetric substrate Ac-DEVD-pNA (p-nitroanilide), which can be rapidly cleaved by native CP3 (Figure 1f). The nondegradable CP3-NCN did not show appreciable protease activity, confirming CP3 is well shielded from outside environment and cannot access the peptidyl substrate. Interestingly, CP3-NC13 displayed a distinct activity profile indicative of a two step process (Figure 1f). An initial lag phase (30 min) in which digestion of the external substrate was slow, was followed by a linear phase parallel to that of the native CP3 reaction. Presence of the distinctive lag phase is suggestive of an initial period in which CP3 digests the DEVD cross-linkers and becomes liberated from the cocoon. The activity of CP3-NC13 was Nano Lett., Vol. 9, No. 12, 2009

dramatically attenuated at 4 °C, indicating limited selfdegradation of the polymer shell and is consistent with the DLS data (Figure 1e). Taken together, these results demonstrate the importance of the peptide cross-linker in modulating NC degradability. Armed with the CP3-NC, we next investigated its antitumor properties. As shown in Figure 2a, apoptotic hallmarks such as membrane blebbing and cell shrinkage were clearly observed in HeLa cells treated with 200 nM CP3-NC13 for 24 h. In contrast, no morphological changes were observed in cells treated with either native CP3 or CP3-NCN, or in untreated cells. Also during apoptosis, CP3 cleaves the caspase-activated-deoxyribonuclease inhibitor (ICAD) and leads to fragmentation of the nucleosome, a signature apoptotic feature.26,30,31 This was observed upon treating cells 4535

Figure 3. Preparation of peptide cross-linker containing photolabile amino acid. (a) Schematic diagram depicting the structure and function of the peptide cross-linker CL-VDEVDmTK. (b) The changes of absorption integral area of VDEVDmTK at 348 nm and the mass signal integral area weight of 50 µg (1 mg/mL) VDEVDTK upon irradiation with UV light (λ ) 365 nm, 100 W). (c) Left: a bulk hydrogel was prepared by free-radical polymerization using AAm (50 mg/mL) and APMAAm (30 mg/mL) as monomers, and CL-VDEVDmTK (2 mg/ mL) as the cross-linker. Treated with CP3 (2.5 mg/mL) for 2 h at pH 7.4 and room temperature, the gel remained the original appearance. Right: the gel was dissolved into viscous solution after irradiation by UV light for 60 s and a subsequent digestion by CP3 for 2 h. The schematic diagram below demonstrates the mechanism of the liquefaction of the hydrogel requires synergistic actions of both UV and CP3.

with 200 nM CP3-NC13 followed by visualization with the TUNEL assay (Figure 2b). HeLa cells treated with CP3NC13 showed extensive apoptotic DNA fragmentation as stained by Alexa Fluor 488, while control cells treated with native CP3 and CP3-NCN did not show these apoptosis characteristics. Cytotoxicity (IC50) of CP3-NC13 was determined to be ∼100 nM by the MTS assay as presented in Figure 2c. In contrast, native CP3 did not exhibit notable cytotoxicity up to 2 µM, demonstrating the poor cellular uptake efficiency of the naked protein. CP3-NC13 can also dramatically inhibit the proliferation of different cancer cells, including MCF-7 and melanoma cell M249 (Figure 2d). The cytotoxicity of the NCs can be tuned by varying the sequence of the peptide cross-linker. CP3-NCs cross-linked by CLVDVADTK displayed lower cytotoxicity (IC50: 400 nM) than those cross-linked by CL-VDEVDTK (Figure 2c). This can 4536

be rationalized by the higher catalytic efficiency displayed by CP3 toward DEVD than toward DVAD (kcat/Km(DEVD) ) 790.4 mM-1 min-1; kcat/Km(DVAD) ) 243.8 mM-1 min-1).32 Next, the internalization steps of the NCs were visualized using enhanced green fluorescent protein (eGFP) NC also cross-linked by CL-VDEVDTK. As expected, eGFP-NC uptake into cells was readily visualized and no cytoxicity was observed (Supporting Information Figure S5). Comparing eGFP-NC internalization efficiency in the presence of various uptake inhibitors suggests a clathrin/caveolae-mediated endocytosis pathway (Supporting Information Figure S6), which is consistent with the behavior of most cationic carriers.33,34 Collectively, these results demonstrate the potential utility of the protease-modulated reversible NCs for intracellular administration of proteins. Nano Lett., Vol. 9, No. 12, 2009

Figure 4. Phototriggering of degradation of CP3 nanocapsules. (a) Proteolytic activity of CP3-NCPD (0.02 mg/mL) to Ac-DEVD-pNA upon UV treatment with different exposure time (adsorption signal was monitored at 409 nm). (b) Cytotoxicity toward HeLa cells treated with protein nanocapsules (CP3 and BSA) cross-linked by CL-VDEVDmTK. Cells were first incubated with 800 nM nanocapsules for 1 h in 96-well plates and then treated by UV for 40 s on ice. Cell viability was quantified by the MTS assay after further incubation in medium for 48 h. The error bars indicate SD. (c) Schematic diagram of “on demand” localized UV treatment to trigger the activity of CP3-NCPD. (d) Bright-field-microscopy image and fluorescence image of live (green) and dead (red-orange) HeLa cells (by LIVE/DEAD Cytotoxicity Kit) after (i) saline treatment; (ii) 1 h co-culture with 4.0 µM CP3-NCPD at 37 °C and further 12 h culture with fresh medium after a thorough wash by PBS; (iii) 40 s UV exposure through an aluminum mask (shielded the part on the right side of the blue dash line), then another 12 h culture; (iv) 1 h coculture with 4.0 µM CP3-NCPD at 37 °C and a thorough wash by PBS followed by UV exposure for 40 s with an aluminum mask, then further 12 h culture with fresh medium. The scale bar is 200 µm.

To achieve spatiotemporal control of the CP3-NC degradation process, we conjugated the P1 aspartic acid of the CLVDEVDTK with a photolabile o-nitrobenzyl ester moiety (Dm). The resulting CL-VDEVDmTK cross-linker can be cleaved by CP3 upon decaging of the aspartic acid (Figure 3a). After brief UV exposure (λ ) 365 nm, 100 W), the absorption integral area of VDEVDmTK (50 µg, 1 mg/mL) at 348 nm steadily decreased, while the mass signal integral area of VDEVDTK correspondingly increased over time (Figure 3b). Additionally, bulk free-radical polymerization using AAm and APMAAm as monomers, and CL-VDEVDmTK as the crosslinker was performed. Figure 3c demonstrates that only upon the synergistic action of UV irradiation and CP3 hydrolysis Nano Lett., Vol. 9, No. 12, 2009

the polymeric matrix can be dissociated from a hydrogel into a free-flowing solution. We characterized the light-responsiveness of CP3 NCs cross-linked with CL-VDEVDmTK (designated CP3-NCPD, 300 nM) by monitoring the release of pNA from Ac-DEVDpNA after different durations of UV irradiation. In the absence of UV treatment, no obvious proteolysis of the peptidyl substrate was observed (Figure 4a). With increasing UV irradiation, the proteolytic activity of CP3-NCPD steadily increased and reached a maximum after 40 s of exposure, which is sufficiently brief to minimize UV-induced damage to cells. To demonstrate the temporal control of NC degradation, HeLa cells were first incubated with CP3-NCPD 4537

for 1 h and then treated with UV for 40 s. After further incubation in medium for two days, nearly all cells underwent apoptosis (Figure 4b). In contrast, cells treated with CP3NCPD without UV exposure, or cells exposed to UV without CP3-NCPD treatment did not show significant cell death. Similarly, cells treated with BSA-NCPD and irradiated with UV also maintained comparable viability to control samples, demonstrating that the combined actions of light and protease are required for degradation of the encapsulating layer. Next, to demonstrate spatial control of NC degradation, we treated cells with 4 µM CP3-NCPD for one hour to facilitate internalization (Supporting Information Figure S8). After thorough washing with PBS to remove NCs that are not endocytosed, a mask was applied to shield half of the culture area while exposing the other half to UV irradiation for 40 s (Figure 4c). After an additional 12 h of incubation, a prominent live/dead cell pattern that corresponds to the shielded/exposed pattern can be visualized by optical microscope, and further imaged by the LIVE/DEAD cytotoxicity assay (Figure 4d). As expected, the spatial control of cell viability was only detected with the synergistic treatment of protease CP3 and UV light. Taking advantages of multiphoton caging groups (i.e., intrinsic three-dimensional resolution and reduced photodamage),24,35-37 this strategy could be further extended to develop a system for real protein-therapeutics, where the irradiation process needs to be performed through the target tissue. In conclusion, we have developed a method to encapsulate and release enzymes in functional forms. While we demonstrated the encapsulation of CP3 and the resulting self-degrading nanocapsule, the protease-modulated design can be extended to applications in which external proteases can trigger the disassembly of the encapsulating shell. Furthermore, spatiotemporal control of the degradation process can be installed by shielding the protease recognition sequences. This method can be useful in the preparation and administration of protein drugs, vaccines, and other macromolecular therapeutics. Acknowledgment. This work was supported by a David and Lucile Packard Foundation to Y.T. and a DTRA Grant BRBAA07-E-2-0042 to Y.T. and Y. L. We thank Professor A. Clay Clark for pHC332, Dr. W. Zhang for cloning the eGFP expression plasmid, Professor Y. Chen, Professor T. Segura, Ms. J. Du, Mr. Q. Ng, and Mr. Y. Lei for helpful discussions, Mr. Y. Li for assistance with peptide synthesizer usage, and Mr. D. Britton for providing materials for TOC design. Supporting Information Available: Experimental details for peptide cross linker synthesis, preparation and characterization of protein nanocapsules, cell culture, apoptosis/ cytotoxicity analysis, and cellular images. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Brekke, O. H.; Sandlie, I. Nat. ReV. Drug DiscoV. 2003, 2, 52–62. (2) Homey, B.; Muller, A.; Zlotnik, A. Nat. ReV. Immunol. 2002, 2, 175– 184.

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NL902935B

Nano Lett., Vol. 9, No. 12, 2009