Research Article www.acsami.org
Near-Infrared Light Activation of Proteins Inside Living Cells Enabled by Carbon Nanotube-Mediated Intracellular Delivery He Li, Xinqi Fan, and Xing Chen* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China S Supporting Information *
ABSTRACT: Light-responsive proteins have been delivered into the cells for controlling intracellular events with high spatial and temporal resolution. However, the choice of wavelength is limited to the UV and visible range; activation of proteins inside the cells using near-infrared (NIR) light, which has better tissue penetration and biocompatibility, remains elusive. Here, we report the development of a single-walled carbon nanotube (SWCNT)-based bifunctional system that enables protein intracellular delivery, followed by NIR activation of the delivered proteins inside the cells. Proteins of interest are conjugated onto SWCNTs via a streptavidindesthiobiotin (SA-DTB) linkage, where the protein activity is blocked. SWCNTs serve as both a nanocarrier for carrying proteins into the cells and subsequently a NIR sensitizer to photothermally cleave the linkage and release the proteins. The released proteins become active and exert their functions inside the cells. We demonstrated this strategy by intracellular delivery and NIR-triggered nuclear translocation of enhanced green fluorescent protein, and by intracellular delivery and NIR-activation of a therapeutic protein, saporin, in living cells. Furthermore, we showed that proteins conjugated onto SWCNTs via the SA-DTB linkage could be delivered to the tumors, and optically released and activated by using NIR light in living mice. KEYWORDS: bionanotechnology, spatiotemporal control, near-infrared light, protein activation, protein delivery
1. INTRODUCTION Protein-based probes and drugs are highly valuable for both basic research and therapeutic applications.1 Various proteins including enzymes, cytokines, antibodies, and transcription factors have been engineered to probe and manipulate cellular functions, mostly by targeting cell-surface or extracellular biomolecules. In contrast, targeting intracellular molecules remains challenging, because most proteins cannot spontaneously cross the barrier imposed by the plasma membrane. Intracellular delivery of functional proteins is currently an active research area. A variety of methods such as microinjection,2 electroporation,3,4 nanoinjection,5 enhancing endocytosis and endosome escape,6−10 and nanocarrier-aided delivery11−18 have been developed and proven useful for introducing exogenous proteins into cells. Alternatively, transfection of the encoding genes can also be employed to expressusually requiring a time lag for transcription and translationthe target proteins in the cells.19 Notably, intracellular protein delivery holds advantages for applications that seek direct and immediate interception of the cellular processes without involving genetic manipulations. Upon being delivered or expressed into the cells, the proteins are meant to exert their functions at the desired locations and times, which imposes another challenge in protein engineering. © XXXX American Chemical Society
Controlling the protein activity with light has recently emerged as a powerful means to address this challenge.20 Owing to the rapid advances in the fields of optogenetics and photopharmacology, proteins can be engineered to be lightresponsive by genetic fusion with light-sensing domains21,22 or by chemical modification with photocleavable moieties.23,24 Coupling the photoreactive proteins with intracellular delivery techniques offers an appealing approach for optical modulation of cellular events inside the cells with high spatiotemporal precision. For example, photoactivatable proteins were introduced into the cells for optical control of protein translocation and enzymatic activity by using microinjection25,26 or permeabilization.27 However, the light-responsive proteins delivered into cells so far have mostly been activated using UV or visible light. Controlling protein activity inside the cells using near-infrared (NIR) light remains elusive. Comparing to UV and visible light, NIR light processes much higher tissue penetration power and hence has great promise for in vivo applications. In addition, NIR light is highly biocompatible and causes minimal photodamage to the cells. It is therefore of Received: January 10, 2016 Accepted: January 21, 2016
A
DOI: 10.1021/acsami.6b00323 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
because of the well-established protein desthiobiotinylation chemistry.
great interest to apply NIR light to control the protein activity inside the cells. On the basis of these considerations, we sought to develop a platform with dual functions, intracellular delivery and NIR activation of proteins inside the cells. Our design was inspired by two characteristic properties of single-walled carbon nanotubes (SWCNTs). Due to their nanoscale needle-like geometry, SWCNTs can effectively enter the interior of a cell with minimal damage to the cell membrane, serving as a nanocarrier for protein delivery.28−32 Furthermore, SWCNTs absorb NIR light33,34 and generate local heating through the photothermal effect, which has been utilized for killing cancer cells and modulating protein activity.35−39 Combining these two features, SWCNTs could potentially serve as a platform simultaneously enabling intracellular delivery and NIR lightcontrolled protein activation inside the cells, which has not been explored so far. In the present work, we construct the dual-functional system for intracellular delivery and NIR activation of proteins (Figure 1). Proteins of interest are
2. EXPERIMENTAL SECTION 2.1. Preparation of SWCNT-SA. Pristine HiPco SWCNTs (Unidym, Lot. P2150, 1 mg/mL) were sonicated in an aqueous solution of 3-(N-succinimidyloxyglutaryl) aminopropyl polyethylene glycol-carbamyl distearoylphosphatidyl-ethanolamine (DSPE-PEG2000NHS) (NOF Corporation, cat. no. SUNBRIGHT DSPE-020GS, 2 mg/mL) using a both sonicator for 1 h with the bath temperature kept below 20 °C. After centrifugation at 16,000 g for 30 min and filtration through a 100 kDa MWCO ultrafilter (Millipore), the resulting SWCNT-DSPE-PEG2000-NHS (10 μg, calculated based on SWCNTs) was resuspended in 0.2 mL MES buffer (pH 5.0) containing 1-ethyl-3(3-(dimethylamino)propyl) carbodiimide (EDC, 20 μg) and Nhydroxysuccinimide (NHS, 20 μg) and incubated at room temperature for 1 h. After removing excess EDC and NHS by filtration through a 100 kDa ultrafilter and resuspending in 0.2 mL of PBS buffer (pH 8.5), streptavidin (IBA, cat. no. 2−0203, 26.5 μg) was immediately added and the reaction was kept at 4 °C overnight, followed by quenching the reaction by exchanging the buffer to Tris-buffer saline (pH 8.0). 2.2. Preparation of DTB-EGFP, DTB-nEGFP, DTB-SAP, biotinEGFP, biotin-nEGFP, and biotin-SAP. Desthiobiotin (Sigma, cat. no. D1411, 50 mg), N-Hydroxysuccinimide (29.8 mg), and N,N′dicyclohexylcarbodiimide (DCC, 48.5 mg) were dissolved in 3 mL of DMF and the mixture was stirred at room temperature for 24 h, followed by removing the precipitate by filtration. The filtrate was dried with a rotary evaporator, washed with cold 2-isopropanol, and lyophilized to give desthiobiotin N-hydroxysuccinimide ester (DTBNHS). EGFP and nEGFP (containing the PKKKRKWV sequence before the N-terminal lysine of EGFP) were recombinantly expressed and purified in E. coli BL21. For preparing DTB-EGFP, 16.7 μg of DTB-NHS (10 mg/mL in DMSO) was added into PBS solution (pH 8.5) with 100 μg of EGFP. The mixture was placed on a rotating mixer and reacted for 12 h at 4 °C. Excess DTB-NHS was removed by dialysis against PBS buffer. DTB-nEGFP and DTB-SAP were prepared using a similar procedure. Biotinylated proteins were prepared by dissolving biotin-NHS (Biomatrik, cat. no. 246201) and EGFP, nEGFP, or SAP at a molar ratio of 10:1 in PBS buffer (pH 8.5) and the solution was gently mixed for 12 h at 4 °C. Excess biotin-NHS was then removed by dialysis against PBS buffer. 2.3. Preparation of SWCNT-SA-DTB-EGFP, SWCNT-SA-DTBnEGFP, SWCNT-SA-DTB-SAP, SWCNT-SA-biotin-EGFP, SWCNTSA-biotin-nEGFP, and SWCNT-SA-biotin-SAP. Twenty μg DTBEGFP, DTB-nEGFP, DTB-SAP, biotin-EGFP, biotin-nEGFP or biotin-SAP was incubated with 10 μg SWCNT-SA for 12 h at 4 °C. The excess protein was the removed by size exclusion chromatography (Sepharose CL-4B, GE Healthcare, cat. no.17−0150−01). The loading of EGFP, nEGFP, or SAP is estimated to be approximately 1.5 mg of protein per mg of SWCNTs. SDS-PAGE assay. The SWCNT-protein conjugates were mixed with SDS loading buffer, boiled for 10 min, loaded onto the 12% PAGE gel, and assayed according to standard procedures. 2.4. NIR-Triggered Release of EGFP in Vitro. A solution of 0.2 μM SWCNT-SA-DTB-EGFP was irradiated by an 808 nm laser (LOSBLD-0808-003w-C, Hi-Tech Optoelectronics Co., Ltd.) with power density of 1.33 W/cm2 for 10 min. The irradiated samples were resolved by native PAGE and the gel was directly scanned in a fluorescence imager. As a control experiment, biotin-PEG6-OH (Biomatrik, cat. no. 245406, 200 μM) was added to release DTBEGFP. The solution was then centrifuged at 11,800 g for 1 min using a 0.1 μm ultrafilter (Millipore, cat. no. UFC30VV). The filtrate was resolved by native PAGE and the gel was directly scanned using a Typhoon fluorescence scanner (GE Healthcare, FLA9500). 2.5. NIR-Triggered Release of nEGFP inside Living Cells. In this experiment, SA was fluorescently labeled by incubating with Cy5NHS (OKEANOS, cat no. OK-F-13104) at molar ratio of 1:10 in PBS buffer (pH 8.5) at 4 °C for 12 h, followed by filtration to remove the excess Cy5-NHS. The resulting Cy5-SA was then used to prepare the
Figure 1. An SWCNT-assisted strategy for NIR-triggered activation of proteins inside the cells. Proteins of interest are chemically conjugated onto the surface of SWCNTs via a desthiobiotin−streptavidin linkage. The activity of the proteins on SWCNTs are masked by the steric hindrance. SWCNTs are internalized and deliver the proteins into the interior of the cells. Once inside the cells, NIR irradiation on SWCNTs produces local heat, which cleaves the linkage between proteins and SWCNTs. The released proteins become active and perform their cellular functions.
loaded onto SWCNTs via a cleavable linkage. We reasoned that the translocation or enzymatic activities of certain proteins would be blocked once conjugated onto the surface of SWCNTs by sterically hindering protein binding. At the same time, SWCNTs serve as the intracellular delivery carrier. The other essential component of our technique is to optically release the proteins from SWCNTs and hence activate the proteins inside the cells. To realize this, we chose the streptavidin-desthiobiotin (SA-DTB) linker40 to conjugate proteins of interest onto SWCNTs. We reasoned that the SA-DTB linkage would be cleaved upon NIR irradiation by thermally denaturing the streptavidin protein. Because the heating effect is locally confined near the SWCNT surface, the released proteins and other cellular components would not be damaged. Furthermore, the choice of SA-DTB linker makes our method generally applicable to various kinds of proteins, B
DOI: 10.1021/acsami.6b00323 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces SWCNT-protein conjugates using the same procedure described above. HeLa cells cultured in 8-well chamber slides (Thermo Scientific, cat no.155409) were incubated with 0.2 μM SWCNT-SADTB-nEGFP, or SWCNT-SA-biotin-nEGFP in FBS-free medium for 6 h. After removing the medium, washing with PBS, and adding complete DMEM, we irradiated the cells with the 808 nm laser (power density, 1.33 W/cm2) for 5 min. After 30 min, the cells were then imaged by laser-scanning confocal fluorescent microscopy (LSM700, Zeiss). Quantification of nEGFP colocalized with Hoechst 33342 or Cy5 were calculated using Manders’ overlap coefficient by image J.41 2.6. NIR-Triggered Release of SAP for Inducing Cell Apoptosis. Hela cells were incubated with 0.1 μM SWCNT-SADTB-SAP, SWCNT-SA-DTB-dEGFP (denatured EGFP), or SWCNT-SA-biotin-SAP in FBS-free DMEM medium for 6 h, followed by removing the excess SWCNT-protein conjugates and replacing the complete DMEM. The cells were then irradiated with the 808 nm laser ((power density, 1.33 W/cm2) for 5 min. After incubating for another 4 h, the cells were assayed using the Alexa Fluor 488 annexin V/dead cell apoptosis kit (Invitrogen, cat. no.13241) following the manufacturer’s instructions and analyzed using a BD Accuri C6 flow cytometer. For immunoblot analysis of the cleaved caspase-3, 4 h after NIR irradiation, the cells were collected, washed twice with cold PBS, and placed in lysis buffer containing 1% Triton X-100. The cell lysates were assayed by Western blot using antibodies against β-tubulin (Easybio, cat. no. BE3312−100) and caspase-3 (Beyotime, cat. no. AC030). 2.7. Mice. Wild type Balb/c nude mice (8wk, 20−25g) were purchased from Vital River Laboratory Animal Center (Beijing, China), and kept under SPF condition with free access to standard food and water. All animal experiments were performed in accordance with guidelines approved by the ethics committee of Peking University. 2.8. In Vivo Delivery and Release of nEGFP in Tumor. Male Balb/c nude mice were inoculated with 1 × 107 HeLa cells in both flanks for 7 days. The tumor-bearing mice were intravenously injected with SWCNT-SA-DTB-nEGFP (0.75 mg kg−1, calculated based on nEGFP). Four hours after the injection, the tumor on one side was irradiated with the 808 nm laser (1.5 W/cm2 for 5 min), whereas the other tumor was not irradiated. The mice were sacrificed 60 min later and tumors were dissected for frozen sectioning. The tumor section slides were stained with Hoechst 33342 and visualized under confocal fluorescent microscopy (LSM700, Zeiss).
Figure 2. Synthesis of SWCNT-SA-DTB-EGFP conjugates and NIRtriggered release in vitro. (a) SDS-PAGE analysis on the SWCNTprotein conjugates. Lane 1: DTB-EGFP; Lane 2: SWCNT-SA-DTBEGFP; Lane 3: SWCNT-SA; Lane 4: SA. The samples were heated in an SDS solution, followed by gel electrophoresis analysis. (b) In gel fluorescence analysis of NIR-triggered release of EGFP from SWCNTSA-DTB-EGFP. The solution of SWCNT-SA-DTB-EGFP (0.2 μM, calculated based on the concentration of DTB-EGFP) was irradiated with an 808 nm NIR laser (power density, 1.33 W/cm2) for 10 min. The irradiated samples were resolved by native PAGE and the gel was directly scanned in a fluorescence imager. Lane 1: SWCNT-SA-DTBEGFP without NIR irradiation; Lane 2: SWCNT-SA-DTB-EGFP with NIR irradiation at 1.33 W/cm2 for 10 min; Lane 3: SWCNT-SA-DTBEGFP incubated with 200 μM biotin for 2 h; Lane 4: DTB-EGFP. The loading of SWCNT-SA-DTB-EGFP for lane 1−3 is equal. The loading of DTB-EGFP in lane 4 is not comparable to lane 1−3. The relative densities (RD) of bands in lane 1−3 were quantified by ImageJ.
monomer. According to the molecular weight, multiple PEGylated phospholipid molecules could be attached to one streptavidin subunit. The SWCNT-SA conjugate could then be readily used for loading with protein of interest that is desthiobiotinylated. As a model system, we prepared desthiobiotinylated enhanced green fluorescent protein (DTB-EGFP) by conjugating carboxylated DTB onto the lysine of EGFP (Figure S3). Simply incubating DTB-EGFP with SWCNT-streptavidin, followed by size exclusion chromatography to remove the excess DTB-EGFP, gave the SWCNT-SA-DTB-EGFP conjugate, as assayed by SDS-PAGE (Figure 2a, lane 2) and Western blot analysis (Figure S4). To evaluate NIR lighttriggered cleavage of SA-DTB linkage and release of EGFP in vitro, a solution of SWCNT-SA-DTB-EGFP was irradiated with an 808 nm NIR laser at the power density of 1.33 W/cm2 for 10 min. The released DTB-EGFP was purified by filtration and resolved by native PAGE. Since native PAGE does not involve the denaturing SDS detergent and maintains proteins in their native state, the release of EGFP could be visualized and quantified by in-gel fluorescence analysis. DTB-EGFP remained conjugated on the nanotube surface with no optical activation (Figure 2b, lane 1). Upon NIR irradiation, the released DTBEGFP (Figure 2b, lane 2) was observed to migrate the same way as the free DTB-EGFP (Figure 2b, lane 4). DTB-EGFP exhibited as smeared bands, probably because of the heterogeneity of the number and conjugation position of DTB on EGFP. Efficient release was observed upon competition using free biotin (Figure 2b, lane 3), confirming that the optical release was through the cleavage of the SA-DTB linkage. The fact that more DTB-EGFP was released upon biotin treatment indicates that the NIR irradiation (1.33 W/ cm2 for 10 min) resulted in partial release of DTB-EGFP. Assuming that biotin treatment resulted in complete release (Figure 2b, lane 3), the efficiency of NIR-triggered release was
3. RESULTS AND DISCUSSION 3.1. Design and Synthesis of NIR Activatable SWCNTProtein Conjugates. To functionalize SWCNTs with streptavidin, we adapted a previously reported chemical method (Figure S1).42 SWCNTs were first coated with a PEGylated phospholipid containing a carboxyl group, DSPE-PEG2000COOH, which solubilizes SWCNTs in aqueous solution and provides carboxyl functional groups for chemical conjugation. Transmission electron microscopy (TEM) revealed that the resulting SWCNT-DSPE-PEG2000-COOH was approximately 200 nm in length (Figure S2). Streptavidin (SA) was then conjugated onto the PEGylated SWCNTs by the carbodiimide condensation reaction. The ratio between SA and PEGylated SWCNTs was controlled so that no significant cross-linking of SWCNTs by SA occurred on the resulting SWCNT-SA, as confirmed by TEM (Figure S2c). The SWCNT-SA conjugate was further assayed using the SDS-PAGE analysis. Heating SWNT-SA in the SDS aqueous solution released the PEGylated phospholipid-modified streptavidin because SDS competed the binding to the nanotube surface via hydrophobic interactions. The tetrameric SA also dissociated into monomers. Gel electrophoresis on the released SA showed shifted bands in addition to the band of unmodified SA monomer (Figure 2a, lane 3). The shifted bands were attributed to the modified SA C
DOI: 10.1021/acsami.6b00323 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 3. NIR-triggered release and translocation of nEGFP into the nucleus. (a) HeLa cells were incubated with 0.2 μM SWCNT-SA-DTB-nEGFP (SA was labeled with Cy5) for 6 h, followed by confocal fluorescence microscopy. (b) After incubation with 0.2 μM SWCNT-SA-DTB-nEGFP, the cells were irradiated with the 808 nm laser (power density, 1.33 W/cm2) for 5 min, followed by confocal fluorescence microscopy. (c) The cells were incubated with SWCNT-SA-biotin-nEGFP, followed by NIR irradiation and confocal fluorescence microscopy. The nuclei were visualized by staining with Hoechst 33342. Scale bars: 20 μm. (d) Quantification of nEGFP colocalized with nucleus marker Hoechst 33342 (black) or Cy5 (gray). Colocalization coefficients were calculated using Manders’ overlap coefficient (>50 cells). The error bars indicate standard deviation.
demonstrate optically triggered protein translocation inside the cells. A nucleus-targeted EGFP (nEGFP) was generated by fusing a nuclear localization signal, the SV40 T-antigen Lys-LysLys-Arg-Lys-Val,43 to the N-terminal of EGFP. nEGFP was desthiobiotinylated and conjugated to SWCNT-SA to give the SWCNT-SA-DTB-nEGFP conjugate. To better visualize this process, SWCNT-SA was fluorescently labeled with a red fluorescent dye Cy5 before conjugating with DTB-nEGFP, which enabled two-color fluorescence imaging to evaluate the colocalization of nEGFP and SWCNTs in the cells. Incubation of HeLa cells with SWCNT-SA-DTB-nEGFP resulted in the intracellular accumulation of SWCNT-SA-DTB-nEGFP, indicating effective intracellular delivery using SWCNTs as the nanocarrier (Figure 3a). With no NIR irradiation, SWCNT-SADTB-nEGFP was excluded from the nucleus, suggesting that SWCNTs blocks the nuclear translocation of nEGFP (Figure 3d). Upon NIR irradiation at the power density of 1.33 W/cm2 for 5 min, DTB-nEGFP was released and translocated into the nucleus, as indicated by the colocalization of EGFP fluorescence and nucleus staining (Figure 3b, d). The Cy5 fluorescence indicated that SWCNT-SA remained in the cytoplasm after NIR release. To further confirm that the release was due to optical cleavage of the SA-DTB linkage, we synthesized biotinylated nEGFP and accordingly SWCNT-SA-
estimated to be approximately 70% (Figure 2b, lane 2). We then evaluated the dependence of release on laser power density and irradiation time. At the laser power density of 0.67 W/cm2, we observed an irradiation time-dependent increase in EGFP fluorescence intensity (Figure S5). The release was further supported by Western blot analysis, which also confirmed that EGFP was released in the desthiobiotinylated form (Figure S5). By increasing the laser power density to 1.33 W/cm2, DTB-EGFP was released much faster, with an observable release at the irradiation time as short as 2 min (Figure S6). The fluorescence intensity of the released DTBEGFP reached maximum at 5 min. Further increase of the irradiation time to 10 min did not significantly increase the release. When an irradiation time of 20 min was applied, both the Western blot band intensity and fluorescence intensity were decreased, indicating that prolonged irradiation at a high power density may cause not only photobleach but also degradation of EGFP. We therefore chose irradiation conditions no harsher than irradiation for 5 min at 1.33 W/cm2 for the following experiments. 3.2. NIR Light-Triggered Protein Translocation Inside the Cells. The successful photorelease of EGFP from SWCNT-SA-DTB-EGFP prompted us to evaluate the applicability of this strategy in live cells. We first sought to D
DOI: 10.1021/acsami.6b00323 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 4. NIR-activated apoptosis in HeLa cells delivered with SWCNT-SA-DTB-SAP. (a) Flow cytometry analysis of the apoptotic induction upon NIR irradiation. HeLa cells were incubated with 0.1 μM SWCNT-SA-DTB-SAP for 6 h, followed by NIR activation using the 808 nm laser (1.33 W/ cm2, 5 min). The treated cells with no NIR irradiation were used as a negative control. As two additional negative controls, cells were treated with SWCNT-SA-biotin-SAP or SWCNT-SA-DTB-dEGFP, followed by NIR irradiation. The cells undergoing apoptosis were stained with Annexin V and PI and analyzed by flow cytometry. dEGFP, denatured EGFP. (b) Immunoblot of the cleaved caspase-3 induced by NIR irradiation in cells incubated with SWCNT-SA-DTB-SAP. Anti-β-tubulin blot was used as the loading control. Relative densities of cleaved caspase 3 and β-tubulin in the blots were measured using ImageJ. Cleaved caspase 3 is normalized to β-tubulin and fold change in cleaved caspase 3 over the negative control was shown as mean ± SD from three replicate experiments.
enzymatic activity of proteins delivered into the cells. Lightactivated therapy has attracted great interest.44−48 Intracellular delivery and NIR activation of a therapeutic protein would be highly valuable for improving therapeutic efficiency and specificity owing to the spatiotemporal precision and deep tissue penetration of NIR light. Toward this goal, we constructed the SWCNT-mediated delivery and NIR activation system for saporin (SAP). Saporin is a ribosome inactivating protein (RIP) isolated from the seeds of Saponaria off icinalis.49 Categorized as a type I RIP, SAP consists only of a single catalytic polypeptide chain and lacks a cell-binding chain to insert itself into the cells.50 Once delivered into the cytosol, SAP inhibits protein synthesis by inactivating ribosomes with high potency and induces cell apoptosis.51,52 Immunotoxins prepared using SAP have been widely evaluated for targeted cancer therapy.53−55 We obtained the SWCNT-SA-DTB-SAP conjugate by preparing desthiobiotinylated SAP and conjugating it with SWCNT-SA. HeLa cells were treated with SWCNTSA-DTB-SAP for 6 h, followed by irradiation with the 808 nm laser at the power density of 1.33 W/cm2 for 5 min. Four h after the NIR irradiation, the cells were evaluated for apoptosis
biotin-nEGFP. Because biotin has approximately 5 orders of magnitude higher affinity to SA than DTB, the linkage of biotin-SA would not be cleaved as effectively as DTB-SA using the same irradiation condition. As a result, NIR irradiation on SWCNT-SA-biotin-nEGFP in the cytoplasm did not resulted in release and translocation of biotin-nEGFP, supporting that the translocation observed for SWCNT-SA-DTB-nEGFP was through the NIR light-triggered cleavage of the SA-DTB linkage (Figure 3c, d). Previously, SWCNTs have been used for photothermal killing of cancer cells.35,36 In those studies, SWCNTs were functionalized with ligands for cell-surface receptors, which resulted in efficient internalization of SWCNTs into the cells via receptor-mediated endocytosis. The amount of SWCNTs present inside the cells in our work should be much less and do not induce cytotoxicity. To confirm this, we performed MTS assay on the treated cells, which indicates that SWCNT-SADTB-EGFP and NIR irradiation did not cause apparent cytotoxicity (Figure S7). 3.3. NIR Activation of the Saporin-Induced Apoptosis. Next we sought to apply this strategy to optically control E
DOI: 10.1021/acsami.6b00323 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
whereas the right-side tumor was not irradiated. The tumor tissues were then isolated and high-resolution fluorescence imaging was performed on the tissue sections. Significant release and nuclear translocation of nEGFP was observed in tumors with NIR irradiation (Figure 5a). In contrast, when there was no NIR irradiation, nEGFP remained conjugated and localized in the cytosol (Figure 5b). These results demonstrate that proteins conjugated onto SWCNTs via the SA-DTB linkage can be delivered to the tumors in vivo and optically released and activated by using NIR light.
by the Annexin V and PI staining (Figure 4a, S8). Cells undergoing apoptosis are stained by Annexin V. As shown by the flow cytometry analysis, NIR irradiation induced 33% cell apoptosis more than cells without treatment. When the cells treated with SWCNT-SA-DTB-dEGFP or with SWCNT-SAbiotin-SAP were irradiated NIR light, no significant apoptosis induction was observed. To further investigate the apoptotic pathway triggered by SAP release, we assayed activation of caspase-3 by immunoblotting, which showed that the caspase cascade was activated upon NIR irradiation in cells treated with SWCNT-SA-DTB-SAP, but not in the control cells (Figure 4b). These results demonstrate that SWCNT-SA-DTB-SAP is able to deliver SAP into the cells and induce cell death upon NIR irradiation. 3.4. NIR Light-Triggered Release and Nuclear Translocation of nEGFP in Living Mice. Finally, we demonstrated that our strategy is applicable in living animals. As a proof-ofconcept experiment, we sought to optically trigger the intracellular release and activation of proteins delivered to HeLa tumor xenografts in living mice. We used nEGFP as a model protein because the release and nuclear translocation triggered by NIR irradiation can be readily imaged. HeLa cells were bilaterally implanted into both flanks of Balb/c mice to form tumors with a diameter of approximately 1 cm (Figure 5).
4. CONCLUSIONS In summary, we have developed an SWCNT-based approach for intracellular delivery and optical activation of proteins inside the cells. By conjugating proteins onto the surface of SWCNTs, SWCNTs serve as both a delivery vehicle and an NIR light sensitizer. The SA-DTB linkage is chosen so that proteins are attached and inactive on the nanotube surface until the NIR light triggers the cleavage of the SA-DTB linkage. We have demonstrated the generic applicability of this methodology by delivering a fluorescent protein and a therapeutic protein. It should be noted that the activity of some proteins probably cannot be blocked by steric hindrance of SWCNT conjugation. Other linkages may be explored to overcome this limitation. For example, those proteins may be conjugated onto SWCNTs using a blocking antibody. As is well-documented in the literature, NIR light processes good tissue penetration. Our preliminary studies show that our strategy can be applied for intracellular delivery and NIR-trigger activation of proteins in living animals. We envision that this approach will find interesting applications in in vivo studies using more physiologically and pathologically relevant systems.
■
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00323. Nine additional figures and experimental details (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
Figure 5. NIR-triggered release and nuclear translocation of nEGFP in living mice. The mice bearing HeLa tumor xenografts on both sides of the flank were intravenously injected with SWCNT-SA-DTB-nEGFP (0.75 mg kg−1, calculated based on nEGFP). Four hours after the injection, (a) the tumor on the left side was irradiated with the 808 nm laser (1.5 W/cm2 for 5 min), whereas (b) the right-side tumor was not irradiated. The tumor tissues were then isolated and high-resolution fluorescence imaging was performed on the tissue sections. The nuclei were visualized by staining with Hoechst 33342. Scale bars: 30 μm. The colocalization of nEGFP with Hoechst 33342 was quantified by Manders’ overlap coefficient (>100 cells). The values are shown at the left-bottom corners of the figures as means ± sem.
■
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21425204) and a DuPont Young Professor Award.
■
REFERENCES
(1) Leader, B.; Baca, Q. J.; Golan, D. E. Protein Therapeutics: A Summary and Pharmacological Classification. Nat. Rev. Drug Discovery 2008, 7, 21−39. (2) Graessmann, A.; Graessmann, M.; Hoffmann, H.; Niebel, J.; Brandner, G.; Mueller, N. Inhibition by Interferon of SV40 Tumor Antigen Formation in Cells Injected with SV40 cRNA Transcribed inVitro. FEBS Lett. 1974, 39, 249−251. (3) Knight, D. E.; Scrutton, M. C. Gaining Access to the Cytosol: The Technique and Some Applications of Electropermeabilization. Biochem. J. 1986, 234, 497−506. (4) Stephens, D. J.; Pepperkok, R. The Many Ways to Cross the Plasma Membrane. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 4295−4298.
The tumor bearing mice were administrated with SWCNT-SADTB-nEGFP (0.75 mg kg−1, calculated based on nEGFP) by intravenous injection. Owing to the enhanced permeability and retention (EPR) effect,11,56 SWCNT-SA-DTB-nEGFP can accumulate in the tumor sites (Figure S9). Four hours after the injection, the tumor on the left flank was irradiated with the 808 nm laser at the power density of 1.5 W/cm2 for 5 min, F
DOI: 10.1021/acsami.6b00323 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces (5) Chen, X.; Kis, A.; Zettl, A.; Bertozzi, C. R. A Cell Nanoinjector Based on Carbon Nanotubes. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 8218−8222. (6) Erazo-Oliveras, A.; Najjar, K.; Dayani, L.; Wang, T.-Y.; Johnson, G. A.; Pellois, J.-P. Protein Delivery into Live Cells by Incubation with an Endosomolytic Agent. Nat. Methods 2014, 11, 861−867. (7) Zuris, J. A.; Thompson, D. B.; Shu, Y.; Guilinger, J. P.; Bessen, J. L.; Hu, J. H.; Maeder, M. L.; Joung, J. K.; Chen, Z.-Y.; Liu, D. R. Efficient Delivery of Genome-Editing Proteins in Vitro and in Vivo. Nat. Biotechnol. 2015, 33, 73−80. (8) Li, M.; Tao, Y.; Shu, Y.; LaRochelle, J. R.; Steinauer, A.; Thompson, D.; Schepartz, A.; Chen, Z. Y.; Liu, D. R. Discovery and Characterization of a Peptide That Enhances Endosomal Escape of Delivered Proteins in Vitro and in Vivo. J. Am. Chem. Soc. 2015, 137, 14084−14093. (9) Liu, J.; Gaj, T.; Yang, Y.; Wang, N.; Shui, S.; Kim, S.; Kanchiswamy, C. N.; Kim, J. S.; Barbas, C. F., 3rd Efficient Delivery of Nuclease Proteins for Genome Editing in Human Stem Cells and Primary Cells. Nat. Protoc. 2015, 10, 1842−1859. (10) Postupalenko, V.; Desplancq, D.; Orlov, I.; Arntz, Y.; Spehner, D.; Mely, Y.; Klaholz, B. P.; Schultz, P.; Weiss, E.; Zuber, G. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer. Angew. Chem., Int. Ed. 2015, 54, 10583−10586. (11) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751−760. (12) Brahmachari, S.; Das, D.; Shome, A.; Das, P. K. Single-Walled Nanotube/Amphiphile Hybrids for Efficacious Protein Delivery: Rational Modification of Dispersing Agents. Angew. Chem., Int. Ed. 2011, 50, 11243−11247. (13) Yan, M.; Du, J.; Gu, Z.; Liang, M.; Hu, Y.; Zhang, W.; Priceman, S.; Wu, L.; Zhou, Z. H.; Liu, Z.; Segura, T.; Tang, Y.; Lu, Y. A Novel Intracellular Protein Delivery Platform Based on Single-Protein Nanocapsules. Nat. Nanotechnol. 2010, 5, 48−53. (14) Han, D. H.; Na, H. K.; Choi, W. H.; Lee, J. H.; Kim, Y. K.; Won, C.; Lee, S. H.; Kim, K. P.; Kuret, J.; Min, D. H.; Lee, M. J. Direct Cellular Delivery of Human Proteasomes to Delay Tau Aggregation. Nat. Commun. 2014, 5, 5633. (15) Chorny, M.; Hood, E.; Levy, R. J.; Muzykantov, V. R. Endothelial Delivery of Antioxidant Enzymes Loaded into NonPolymeric Magnetic Nanoparticles. J. Controlled Release 2010, 146, 144−151. (16) Giannotti, M. I.; Esteban, O.; Oliva, M.; Garcia-Parajo, M. F.; Sanz, F. Ph-Responsive Polysaccharide-Based Polyelectrolyte Complexes as Nanocarriers for Lysosomal Delivery of Therapeutic Proteins. Biomacromolecules 2011, 12, 2524−2533. (17) Schroeder, A.; Goldberg, M. S.; Kastrup, C.; Wang, Y.; Jiang, S.; Joseph, B. J.; Levins, C. G.; Kannan, S. T.; Langer, R.; Anderson, D. G. Remotely Activated Protein-Producing Nanoparticles. Nano Lett. 2012, 12, 2685−2689. (18) Morales, D. P.; Braun, G. B.; Pallaoro, A.; Chen, R.; Huang, X.; Zasadzinski, J. A.; Reich, N. O. Targeted Intracellular Delivery of Proteins with Spatial and Temporal Control. Mol. Pharmaceutics 2015, 12, 600−609. (19) Kim, T. K.; Eberwine, J. H. Mammalian Cell Transfection: The Present and the Future. Anal. Bioanal. Chem. 2010, 397, 3173−3178. (20) Gautier, A.; Gauron, C.; Volovitch, M.; Bensimon, D.; Jullien, L.; Vriz, S. How to Control Proteins with Light in Living Systems. Nat. Chem. Biol. 2014, 10, 533−541. (21) Airan, R. D.; Thompson, K. R.; Fenno, L. E.; Bernstein, H.; Deisseroth, K. Temporally Precise in Vivo Control of Intracellular Signalling. Nature 2009, 458, 1025−1029. (22) Levskaya, A.; Weiner, O. D.; Lim, W. A.; Voigt, C. A. Spatiotemporal Control of Cell Signalling Using a Light-Switchable Protein Interaction. Nature 2009, 461, 997−1001. (23) Ellis-Davies, G. C. Caged Compounds: Photorelease Technology for Control of Cellular Chemistry and Physiology. Nat. Methods 2007, 4, 619−628.
(24) Fortin, D. L.; Banghart, M. R.; Dunn, T. W.; Borges, K.; Wagenaar, D. A.; Gaudry, Q.; Karakossian, M. H.; Otis, T. S.; Kristan, W. B.; Trauner, D.; Kramer, R. H. Photochemical Control of Endogenous Ion Channels and Cellular Excitability. Nat. Methods 2008, 5, 331−338. (25) Pellois, J. P.; Muir, T. W. A Ligation and Photorelease Strategy for the Temporal and Spatial Control of Protein Function in Living Cells. Angew. Chem., Int. Ed. 2005, 44, 5713−5717. (26) Lee, H. M.; Xu, W.; Lawrence, D. S. Construction of a Photoactivatable Profluorescent Enzyme Via Propinquity Labeling. J. Am. Chem. Soc. 2011, 133, 2331−2333. (27) Hahn, M. E.; Muir, T. W. Photocontrol of Smad2, a Multiphosphorylated Cell-Signaling Protein, through Caging of Activating Phosphoserines. Angew. Chem., Int. Ed. 2004, 43, 5800− 5803. (28) Klumpp, C.; Kostarelos, K.; Prato, M.; Bianco, A. Functionalized Carbon Nanotubes as Emerging Nanovectors for the Delivery of Therapeutics. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 404−412. (29) Liu, Z.; Tabakman, S.; Welsher, K.; Dai, H. Carbon Nanotubes in Biology and Medicine: In Vitro and in Vivo Detection, Imaging and Drug Delivery. Nano Res. 2009, 2, 85−120. (30) Shi Kam, N. W.; Jessop, T. C.; Wender, P. A.; Dai, H. Nanotube Molecular Transporters: Internalization of Carbon Nanotube-Protein Conjugates into Mammalian Cells. J. Am. Chem. Soc. 2004, 126, 6850− 6851. (31) Kam, N. W.; Dai, H. Carbon Nanotubes as Intracellular Protein Transporters: Generality and Biological Functionality. J. Am. Chem. Soc. 2005, 127, 6021−6026. (32) Kam, N. W.; Liu, Z.; Dai, H. Carbon Nanotubes as Intracellular Transporters for Proteins and DNA: An Investigation of the Uptake Mechanism and Pathway. Angew. Chem., Int. Ed. 2006, 45, 577−581. (33) O’Connell, M. J.; Bachilo, S. M.; Huffman, C. B.; Moore, V. C.; Strano, M. S.; Haroz, E. H.; Rialon, K. L.; Boul, P. J.; Noon, W. H.; Kittrell, C.; Ma, J.; Hauge, R. H.; Weisman, R. B.; Smalley, R. E. Band Gap Fluorescence from Individual Single-Walled Carbon Nanotubes. Science 2002, 297, 593−596. (34) Iverson, N. M.; Barone, P. W.; Shandell, M.; Trudel, L. J.; Sen, S.; Sen, F.; Ivanov, V.; Atolia, E.; Farias, E.; McNicholas, T. P.; Reuel, N.; Parry, N. M.; Wogan, G. N.; Strano, M. S. In Vivo Biosensing Via Tissue-Localizable Near-Infrared-Fluorescent Single-Walled Carbon Nanotubes. Nat. Nanotechnol. 2013, 8, 873−880. (35) Shi Kam, N. W.; O’Connell, M.; Wisdom, J. A.; Dai, H. Carbon Nanotubes as Multifunctional Biological Transporters and nearInfrared Agents for Selective Cancer Cell Destruction. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 11600−11605. (36) Chakravarty, P.; Marches, R.; Zimmerman, N. S.; Swafford, A. D.; Bajaj, P.; Musselman, I. H.; Pantano, P.; Draper, R. K.; Vitetta, E. S. Thermal Ablation of Tumor Cells with Antibody-Functionalized Single-Walled Carbon Nanotubes. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 8697−8702. (37) Joshi, A.; Punyani, S.; Bale, S. S.; Yang, H.; Borca-Tasciuc, T.; Kane, R. S. Nanotube-Assisted Protein Deactivation. Nat. Nanotechnol. 2008, 3, 41−45. (38) Murakami, T.; Nakatsuji, H.; Inada, M.; Matoba, Y.; Umeyama, T.; Tsujimoto, M.; Isoda, S.; Hashida, M.; Imahori, H. Photodynamic and Photothermal Effects of Semiconducting and Metallic-Enriched Single-Walled Carbon Nanotubes. J. Am. Chem. Soc. 2012, 134, 17862−17865. (39) Lin, L.; Liu, L.; Zhao, B.; Xie, R.; Lin, W.; Li, H.; Li, Y.; Shi, M.; Chen, Y. G.; Springer, T. A.; Chen, X. Carbon Nanotube-Assisted Optical Activation of Tgf-Beta Signalling by near-Infrared Light. Nat. Nanotechnol. 2015, 10, 465−471. (40) Hirsch, J. D.; Eslamizar, L.; Filanoski, B. J.; Malekzadeh, N.; Haugland, R. P.; Beechem, J. M.; Haugland, R. P. Easily Reversible Desthiobiotin Binding to Streptavidin, Avidin, and Other BiotinBinding Proteins: Uses for Protein Labeling, Detection, and Isolation. Anal. Biochem. 2002, 308, 343−357. G
DOI: 10.1021/acsami.6b00323 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces (41) Bolte, S.; Cordelieres, F. P. A Guided Tour into Subcellular Colocalization Analysis in Light Microscopy. J. Microsc. (Oxford, U. K.) 2006, 224, 213−232. (42) Liu, Z.; Tabakman, S. M.; Chen, Z.; Dai, H. Preparation of Carbon Nanotube Bioconjugates for Biomedical Applications. Nat. Protoc. 2009, 4, 1372−1382. (43) Kalderon, D.; Roberts, B. L.; Richardson, W. D.; Smith, A. E. A Short Amino Acid Sequence Able to Specify Nuclear Location. Cell 1984, 39, 499−509. (44) Konan, Y. N.; Gurny, R.; Allemann, E. State of the Art in the Delivery of Photosensitizers for Photodynamic Therapy. J. Photochem. Photobiol., B 2002, 66, 89−106. (45) Idris, N. M.; Gnanasammandhan, M. K.; Zhang, J.; Ho, P. C.; Mahendran, R.; Zhang, Y. In Vivo Photodynamic Therapy Using Upconversion Nanoparticles as Remote-Controlled Nanotransducers. Nat. Med. (N. Y., NY, U. S.) 2012, 18, 1580−1585. (46) Choi, M.; Choi, J. W.; Kim, S.; Nizamoglu, S.; Hahn, S. K.; Yun, S. H. Light-Guiding Hydrogels for Cell-Based Sensing and Optogenetic Synthesis. Nat. Photonics 2013, 7, 987−994. (47) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. (Washington, DC, U. S.) 2014, 114, 10869−10939. (48) Chen, Q.; Ke, H.; Dai, Z.; Liu, Z. Nanoscale Theranostics for Physical Stimulus-Responsive Cancer Therapies. Biomaterials 2015, 73, 214−230. (49) Stirpe, F.; Gasperi-Campani, A.; Barbieri, L.; Falasca, A.; Abbondanza, A.; Stevens, W. A. Ribosome-Inactivating Proteins from the Seeds of Saponaria Officinalis L. (Soapwort), of Agrostemma Githago L. (Corn Cockle) and of Asparagus Officinalis L. (Asparagus), and from the Latex of Hura Crepitans L. (Sandbox Tree). Biochem. J. 1983, 216, 617−625. (50) Stirpe, F.; Barbieri, L.; Battelli, M. G.; Soria, M.; Lappi, D. A. Ribosome-Inactivating Proteins from Plants: Present Status and Future Prospects. Bio/Technology 1992, 10, 405−412. (51) Bergamaschi, G.; Perfetti, V.; Tonon, L.; Novella, A.; Lucotti, C.; Danova, M.; Glennie, M. J.; Merlini, G.; Cazzola, M. Saporin, a Ribosome-Inactivating Protein Used to Prepare Immunotoxins, Induces Cell Death Via Apoptosis. Br. J. Haematol. 1996, 93, 789−794. (52) Polito, L.; Bortolotti, M.; Farini, V.; Battelli, M. G.; Barbieri, L.; Bolognesi, A. Saporin Induces Multiple Death Pathways in Lymphoma Cells with Different Intensity and Timing as Compared to Ricin. Int. J. Biochem. Cell Biol. 2009, 41, 1055−1061. (53) Bonardi, M. A.; French, R. R.; Amlot, P.; Gromo, G.; Modena, D.; Glennie, M. J. Delivery of Saporin to Human B-Cell Lymphoma Using Bispecific Antibody: Targeting Via Cd22 but Not Cd19, Cd37, or Immunoglobulin Results in Efficient Killing. Cancer Res. 1993, 53, 3015−3021. (54) Yip, W. L.; Weyergang, A.; Berg, K.; Tonnesen, H. H.; Selbo, P. K. Targeted Delivery and Enhanced Cytotoxicity of CetuximabSaporin by Photochemical Internalization in Egfr-Positive Cancer Cells. Mol. Pharmaceutics 2007, 4, 241−251. (55) Bostad, M.; Kausberg, M.; Weyergang, A.; Olsen, C. E.; Berg, K.; Hogset, A.; Selbo, P. K. Light-Triggered, Efficient Cytosolic Release of Im7-Saporin Targeting the Putative Cancer Stem Cell Marker Cd44 by Photochemical Internalization. Mol. Pharmaceutics 2014, 11, 2764−2776. (56) Liu, Z.; Chen, K.; Davis, C.; Sherlock, S.; Cao, Q.; Chen, X.; Dai, H. Drug Delivery with Carbon Nanotubes for in Vivo Cancer Treatment. Cancer Res. 2008, 68, 6652−6660.
H
DOI: 10.1021/acsami.6b00323 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
