A Substrate-Selective Enzyme-Catalysis Assembly Strategy for

Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX

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A Substrate-Selective Enzyme-Catalysis Assembly Strategy for Oligopeptide Hydrogel-Assisted Combinatorial Protein Delivery Tianyue Jiang,† Shiyang Shen,‡ Tong Wang,† Mengru Li,‡ Bingfang He,*,† and Ran Mo*,‡ †

School of Pharmaceutical Sciences and School of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China ‡ State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Advanced Pharmaceuticals and Biomaterials, China Pharmaceutical University, Nanjing 210009, China S Supporting Information *

ABSTRACT: Oligopeptide hydrogels for localized protein delivery have considerable potential to reduce systemic side effects but maximize therapeutic efficacy. Although enzyme catalysis to induce formation of oligopeptide hydrogels has the merits of unique regio- and enantioselectivity and mild reaction conditions, it may cause the impairment of function and activity of the encapsulated proteins by proteolytic degradation during gelation. Here we report a novel enzyme-catalysis strategy for self-assembly of oligopeptide hydrogels using an engineered protease nanocapsule with tunable substrate selectivity. The protease-encapsulated nanocapsule shielded the degradation activity of protease on the laden proteins due to the steric hindrance by the polymeric shell weaved around the protease, whereas the small-molecular precursors were easier to penetrate across the polymeric network and access the catalytic pocket of the protease to convert to the gelators for self-assembling hydrogel. The resulting oligopeptide hydrogels supported a favorable loading capacity without inactivation of both an antiangiogenic protein, hirudin and an apoptosis-inducing cytokine, TRAIL as model proteins. The hirudin and TRAIL coloaded oligopeptide hydrogel for combination cancer treatment showed enhanced synergistic antitumor effects both in vitro and in vivo. KEYWORDS: Protein delivery, oligopeptide hydrogel, enzyme catalysis, self-assembly, combination cancer therapy

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nonimmunogenicity,24−27 which are formed by the selfassembly of the oligopeptide-based gelators via noncovalent interactions, including hydrophobic, hydrogen bond, and π−π interactions. The assembly is generally triggered by a variety of chemical and physical stimuli,28−31 typically as change of temperature, pH, ionic strength, and addition of chemicals. Enzyme-mediated reactions as specific biological stimuli, including protease,32,33 phosphatase,34−36 and lipase,37 are considered as a preferential approach to induce hydrogel assembly, which has numerous advantages of unique regio- and enantioselectivity, and mild reaction conditions for loading proteins that are fragile to environmental change. However, the protease-catalyzed assembly strategy is a double-edged sword, which may cause the impairment of protein function or the loss of protein activity due to the digestion of proteins by proteases with low cleavage specificity.38,39 The localized delivery of therapeutic proteins using the protease-assisted self-assembled oligopeptide hydrogels still remains elusive. Here, we propose a novel substrate-selective proteasecatalyzed self-assembly strategy for an injectable oligopeptide nanofiber hydrogel without inactivation of the encapsulated proteins (Figure 1), which can be applied as an immobilized

rotein therapeutics, including cytokines, enzymes and antibodies, hold significant promise for cancer treatment on account of their superior therapeutic activities, such as apoptosis signal activation, growth signal blockage, metastasis inhibition and antiangiogenesis.1,2 Nanoparticle-based drug delivery systems (DDSs) show dramatic enhancement on the anticancer effects of systemically administered proteins, due to improved plasma stability, prolonged circulating half-life and enhanced tumor targetability.3−5 Extensive efforts have been dedicated to exploiting bioresponsive nanocarriers that can be activated by the tumor microenvironmental and/or cellular signals for efficient protein delivery,6−8 for instance, delivering tumor necrosis factor-related apoptosis inducing ligand (TRAIL) to its target death receptor on the cell membrane9−11 and caspase 3 into the cells12,13 for activation of apoptotic caspases. Despite the considerable progresses, these strategies are often limited by complicated material synthesis and formulation design. In addition, the delivery efficiency is still compromised by multiple physiological and biological barriers14−17 that are required to be overcome from the injection site to the final target. Localized drug delivery is an attractive proposition to maximize therapeutic efficacy and minimize systemic exposure-associated side effects.18−20 Peptide hydrogels offer a promising platform for local drug delivery21−23 due to favorable injectability, high biocompatibility, good biodegradability, and © XXXX American Chemical Society

Received: August 6, 2017 Revised: October 30, 2017

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DOI: 10.1021/acs.nanolett.7b03371 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 1. (a) Schematic illustration of substrate-selective protease-catalyzed self-assembly of the oligopeptide hydrogel for efficient encapsulation of hirudin and TRAIL (Hirudin/TRAIL-Gel). AAm = acrylamide, MBA = N,N-methylene bis(acrylamide), APMAAm = N-(3-aminopropyl) methacrylamide. (b) Schematic illustration of the injectable Hirudin/TRAIL-Gel as a localized delivery depot with prolonged intratumoral retention for enhanced therapeutic efficacy by the synergistic effects of hirudin-mediated antiangiogenesis and TRAIL-induced apoptosis.

Figure 2. (a) Particle size distribution and TEM image of W-NC. Scale bar is 40 nm. (b) Activity of hirudin and TRAIL after treatment with WQ9-2 or W-NC. *p < 0.05, **p < 0.01 compared with treated with WQ9-2. (c) Gelation and injectability of the obtained W-NC-catalyzed oligopeptide hydrogel. (i) The solutions containing the precursors before (left) and after (right) addition of W-NC. Inversion demonstrated the formation of the hydrogel with self-supporting capacity. (ii,iii) the resulting oligopeptide hydrogel was injectable through a conventional syringe (ii) and could recover within 5 min (iii). (d) SEM image of the obtained oligopeptide hydrogel. Scale bar is 10 μm. (e) TEM image of the obtained oligopeptide hydrogel. Scale bar is 80 nm. (f) Conversion ratio from the precursors to the gelator under the catalysis of W-NC at different concentrations over time. (g) In vitro protein release profiles of Rho-hirudin/Cy5.5-TRAIL-Gel at pH 7.4. B

DOI: 10.1021/acs.nanolett.7b03371 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 3. (a) Representative images of HUVECs after 6 h of treatment with thrombin (0.5 μg/mL) or the mixture of thrombin (0.5 μg/mL) and the released hirudin (1 μg/mL) from the hydrogel. The untreated HUVECs were taken as control. Scale bar is 100 μm. (b) Quantitative analysis on number of meshes and total tube length of HUVECs after treatment. (c) Representative flow cytometric contour plots of MDA-MB-231 cells after 12 h of treatment with the released TRAIL (10 ng/mL) or the mixture of the released TRAIL (10 ng/mL) and hirudin (350 ng/mL) from the hydrogel. The untreated HUVECs were taken as control. The cells were dually stained by Annexin V-FITC and PI. (d) Apoptosis ratio of MDA-MB231 cells after treatment. (e) Cell viability of MDA-MB-231 cells after 24 h of treatment with the released TRAIL or the mixture of the released TRAIL and hirudin from the hydrogel at different concentrations. (f) Cell viability of MDA-MB-231 and MCF-10A cells after 24 h of treatment with the blank hydrogel suspension at different concentrations.

The obtained oligopeptide hydrogel shows a commendable protein-loading capacity without protein inactivation as expected. Hirudin, an antiangiogenic protein44,45 and TRAIL, a membrane-associated cytokine as two model protein therapeutics are encapsulated into the hydrogel to acquire an injectable hirudin and TRAIL coloaded hydrogel (denoted as Hirudin/TRAIL-Gel). After local administration, Hirudin/ TRAIL-Gel as a reservoir renders prolonged retention at the tumor site for sustained release of hirudin and TRAIL, facilitating the effects of the proteins on their corresponding targets (Figure 1b). The released hirudin is able to specifically block the interaction between thrombin and its receptors on the blood vessels by strong binding to thrombin, therefore inhibiting the thrombin-induced tumor angiogenesis.46,47 On the other hand, the released TRAIL can bind to the death receptor on the cell membrane of the tumor cells for activating the caspase-mediated apoptotic signaling pathway, which combines with the antiangiogenesis effect of hirudin for enhanced synergistic antitumor activity. To verify our hypothesis, WQ9-2 with a molecular weight of 37 kDa was expressed and purified. W-NC was obtained by encapsulating WQ9-2 into a single-protein-based nanocapsule

depot for localized delivery of protein therapeutics for combination cancer therapy. A zinc metalloprotease WQ9-2 that has been screened with optimal catalysis property40 is selected as a model protease trigger to induce reverse hydrolysis reaction39,41 between precursors Fmoc-F and FF-Dopa (Figure S1) to convert to the gelator, Fmoc-FFF-Dopa, for the assembly of the oligopeptide hydrogel. However, WQ9-2 displays a broad substrate specificity, which is capable of hydrolyzing many proteins. To hinder the degradation effect of WQ9-2 on protein therapeutics but maintain the catalytic activity of WQ9-2 during the gelation process, in situ interfacial polymerization42,43 is applied to weave a polymeric shell on the surface of WQ9-2 to obtain a WQ9-2-encapsulated singleprotein-based nanocapsule (designated as W-NC) with adjustable substrate specificity (Figure 1a). The polymeric capsule shields the proteolytic activity of WQ9-2 by impeding the interaction between WQ9-2 and the laden proteins due to steric hindrance, whereas small-molecular precursors can readily penetrate through the cross-linked polymeric network and reach the catalytic pocket of WQ9-2 to transform into the gelators for the subsequent hydrogel assembly. C

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assembled hydrogel (Rho-hirudin/Cy5.5-TRAIL-Gel). The sustained release of both protein therapeutics was achieved by the hydrogel entrapment (Figure 2g). 60% of Rho-hirudin and 40% of Cy5.5-TRAIL were released from the hydrogel within 7 days. In addition, the circular dichroism spectra of the released hirudin and TRAIL, which are consistent with those of the native proteins, suggest no significant conformational variations in the secondary structure of the proteins in the encapsulation and release processes (Figure S4). The in vitro antiangiogenetic effect of hirudin released from the oligopeptide hydrogel was estimated using the tube formation assay.48,49 Human umbilical vein endothelial cells (HUVECs) were incubated with thrombin, and the mixture of thrombin with the released hirudin, respectively, followed by the microscopic observation. Thrombin, a pivotal terminal enzyme of coagulation, promotes angiogenesis, and stimulates tumor growth and metastasis.50,51 Incubation with thrombin resulted in the formation of extensive honeycomb-like structure by HUVECs (Figure 3a), which is indicative of the angiogenesis-promoting effect of thrombin. The released hirudin could significantly inhibit the thrombin-stimulated tube formation of HUVECs, as confirmed by the quantitative analysis showing both reduced number of meshes and decreased total tube length (Figure 3b), which suggests that hirudin has a powerful antiangiogenesis capability by acting on thrombin directly with a high binding affinity and selectivity. The apoptosis-inducing effect of TRAIL released from the hydrogel was evaluated using the Annexin V-FITC/propidium iodide (PI) double staining method (Figure 3c). The released TRAIL was able to greatly trigger the apoptosis of the human breast cancer (MDA-MB-231) cells with a total apoptotic ratio of 34.1% (Figure 3d), which is attributed to the efficient binding of the released TRAIL to the plasma membrane of the tumor cells (Figure S5). The presence of hirudin did not affect the apoptosis-inducing effect of TRAIL. The mixture of the released TRAIL and hirudin presented a total apoptotic ratio of 40%. These data indicate that the antitumor activity of hirudin different from that of TRAIL is not to induce the apoptosis of the cancer cells, but to produce the antiangiogenic effect for blocking the formation of blood vessels in the tumor. The in vitro cytotoxicity of the released TRAIL toward MDAMB-231 cells was further appraised using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure 3e). The half maximal inhibitory concentration of the mixture of the released hirudin and TRAIL against MDAMB-231 cells was about 22 ng/mL (TRAIL concentration), which was comparable to that of the released TRAIL alone as 26 ng/mL. By comparison, the blank oligopeptide hydrogel suspension without hirudin and TRAIL showed negligible toxicity toward MDA-MB-231 cells (Figure 3f). In addition, the exposure of the normal cells, the human normal breast epithelial (MCF-10A) cells to the blank hydrogel also had no impact on the cell proliferation within all the studied concentrations. Collectively, these results indicate that the released hirudin and TRAIL from Hirudin/TRAIL-Gel exhibit potent antiangiogenetic and proapoptotic effects, respectively, which can produce synergistic effects for enhanced antitumor efficacy. To demonstrate the enhanced retention capacity of the oligopeptide hydrogel at the tumor site, we applied the in vivo imaging technique to monitor the fluorescence variation of Cy5.5-TRAIL that was loaded in the hydrogel (Cy5.5-TRAILGel) after intratumoral administration (Figure 4a). The Cy5.5

with a cross-linked polymeric shell using the interfacial polymerization, which had an average hydrodynamic diameter of 10 nm, as evidenced by the transmission electron microscopy (TEM) examination (Figure 2a). To demonstrate the shielding effect of W-NC on the protein degradation activity of WQ9-2, a comparative evaluation of bioactivity change of hirudin and TRAIL was performed after incubation with W-NC or the native WQ9-2 (Figure 2b). The anticoagulation activity of hirudin after incubating with the native WQ9-2 was determined to markedly reduce to 72% of the untreated one using the thrombin titration assay, while treatment with W-NC did not affect the activity of hirudin. Similarly, TRAIL remained its bioactivity in the presence of W-NC, whereas the exposure to WQ9-2 caused the significant impairment in the activity of TRAIL. These results suggest that the polymeric shell as a cage enveloping WQ9-2 in the W-NC formulation restricts the unexpected degradation activity of WQ9-2 toward either hirudin or TRAIL, which provide a foothold for encapsulating them without inactivation in the hydrogel upon the WQ9-2induced self-assembling process. Next, we evaluated the catalytic activity of W-NC on the formation of peptide bonds to construct oligopeptide hydrogel. The addition of W-NC activated a quick formation of translucent hydrogel with self-supporting capability from the precursors solution containing Fmoc-F and FF-Dopa, as displayed in the inversion study (Figure 2c). The rheological evaluation was performed to investigate the W-NC-induced gelation kinetics (Figure S2). After the addition of W-NC, both of the G′ (storage modulus) and G″ (loss modulus) values significantly increased over time, and the G′ value was about 10-fold of the G″ value, which is a solid evidence of the gel formation. The obtained oligopeptide hydrogel possessed a shear-thinning and immediate recovery behavior, which was injectable and could recover within 5 min, as indicated by the comparable moduli before and after injection. The morphology of the branched network was apparently observed using the scanning electron microscope (SEM), which underpins the structure of the hydrogel (Figure 2d). The TEM image further confirmed that the basic unit of the hydrogel structure was self-assembled fibrills of about 8 nm in width and microns in length (Figure 2e). The nanofibers formed by the oligopeptides entangled with each other, resulting in the formation of 3D networks entrapping a large amount of water molecules within them. These data indicate that the physical modification of polymeric shell in W-NC endues it with substrate selectivity, which does not hamper the catalytic activity of WQ9-2, but allows the small-molecular oligopeptides rather than the macromolecular proteins to interact with the active site of enzyme for synthesis of gelators. The conversion ratio from the precursors, Fmoc-F and FFDopa to the gelator, Fmoc-FFF-Dopa under the catalysis of WNC was determined based on the change in the absorbance of Fmoc-F using high-performance liquid chromatography (Figure 2f and Figure S3). The optimal quantity of W-NC for the precursor-to-gelator conversion is about 50 U with the respective concentration of Fmoc-F and FF-Dopa as 10 mM and 20 mM. 15% of the Fmoc-FFF-Dopa gelator was generated 10 min after addition of W-NC, and the conversion ratio maintained approximately 47% after 4 h. The in vitro release profiles of proteins from the oligopeptide hydrogel were determined at pH 7.4. Rhodamine-labeled hirudin (Rho-hirudin) and Cy5.5-labeled TRAIL (Cy5.5TRAIL) were coencapsulated in the W-NC-triggered selfD

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Figure 4. (a) Representative in vivo fluorescence images of the MDA-MB-231 tumor-bearing mice over time after intratumoral administration of Cy5.5-TRAIL-Gel at a Cy5.5 dosage of 5 nmol/kg. The Cy5.5-TRAIL solution containing the precursors without the W-NC-induced gelation was taken as control. The yellow arrow symbols represent the tumor region. (b) Quantitative ROI analysis on the fluorescence signal at the tumor site of the tumor-bearing mice over time after administration. (c) Ex vivo fluorescence images of five major organs, including heart (1), liver (2), spleen (3), lung (4) and kidney (5), and the tumor (6) harvested from the tumor-bearing mice 9 days after administration. (d) CLSM images of the tumor section harvested from the tumor-bearing mice 9 days after administration. The nuclei were stained with Hoechst. Scale bar is 100 μm.

fluorescence signal of Cy5.5-TRAIL in the oligopeptide solution without W-NC-induced gelation as control reduced rapidly at the tumor region within 1 day, and almost completely disappeared 5 days after intratumoral injection. In sharp contrast, the Cy5.5 signal of Cy5.5-TRAIL-Gel remained for 9 days, which indicates that the oligopeptide hydrogel as a reservoir can efficiently hold the encapsulated protein at the tumor site for a long period of time. The quantitative result obtained from the region of interest (ROI) analysis confirmed enhancement of the oligopeptide hydrogel on the tumor retention of the protein (Figure 4b). The half-life and mean retention time of Cy5.5-TRAIL-Gel was determined to be 4.0fold and 3.5-fold that of the Cy5.5-TRAIL solution, respectively. At 9 days post administration, the tumor and five major organs were withdrawn from the mice for the ex vivo imaging (Figure 4c). A strong fluorescence signal was observed in the tumor of the mice receiving Cy5.5-TRAIL-Gel, compared with no signal detected in the Cy5.5-TRAIL solution group. Moreover, the Cy5.5 signal of Cy5.5-TRAIL-Gel was apparently and widely distributed in the tumor (Figure 4d). The in vivo antitumor efficacy of Hirudin/TRAIL-Gel was investigated on the xenograft MDA-MB-231 tumor mouse model after a single intratumoral administration. Combination treatment using Hirudin/TRAIL-Gel showed more prominent tumor-inhibiting effect than monotherapy with either HirudinGel or TRAIL-Gel (Figure 5a). The smallest size of tumor

harvested from the mice after treatment with Hirudin/TRAILGel was observed (Figure 5b). The tumor inhibition ratio is determined to be about 64% by comparison with that treated with saline as a reference (Figure 5c). This superior therapeutic efficacy resulted from the synergistic effects between antiangiogenesis of hirudin and apoptosis activation of TRAIL. The regeneration of tumor microvessels was first monitored as an intuitive index for evaluation of the antiangiogenetic effect of Hirudin/TRAIL-Gel. The image of the tumor harvested from the Hirudin/TRAIL-Gel treated mice showed a significant inhibition on the formation of the intraepidermal blood vessels in the tumor tissues (Figure 5d). The intratumoral expression level of the platelet endothelial cell adhesion molecule (PECAM-1), also known as CD31 that plays a crucial role in the adhesion of vascular endothelial cells during angiogenesis,52,53 was further evaluated using the immunefluorescence staining (Figure 5e). Treatment with Hirudin/TRAILGel resulted in a significantly reduced density of anti-CD31 staining microvessels as shown in green fluorescent lumen or linear vessel shape, which is a substantial evidence of the vascularization inhibition in the tumor tissue by Hirudin/ TRAIL-Gel. Meanwhile, the TUNEL-based immunohistochemical analysis of the tumor revealed that the highest level of apoptotic cells was found in the tumor tissue of the mice receiving Hirudin/TRAIL-Gel compared with other formulations, which confirms the apoptosis-promoting activity of E

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Figure 5. (a) Tumor growth of the MDA-MB-231 tumor-bearing mice after treatment with Hirudin-Gel (10 mg/kg hirudin), TRAIL-Gel (0.5 mg/ kg TRAIL), and Hirudin/TRAIL-Gel (10 mg/kg hirudin and 0.5 mg/kg TRAIL). Treatment with saline was taken as control. The black arrow symbol represents the time of administration. **p < 0.01. (b) Representative images of the tumors harvested from the tumor-bearing mice 15 days after treatment with saline, Hirudin-Gel, TRAIL-Gel, and Hirudin/TRAIL-Gel (from top to bottom). Scale bar is 0.5 cm. (c) Weights of the tumors harvested from the tumor-bearing mice 15 days after treatment. **p < 0.01, ***p < 0.001 compared with Hirudin/TRAIL-Gel. (d) Images of the blood vessel development in the tumors of the tumor-bearing mice 15 days after treatment with saline and Hirudin/TRAIL-Gel. The white arrow symbols represent the tumor blood vessels. (e) CLSM images of the blood vessels stained with AF488-conjugated CD31 antibody in the tumors of the tumor-bearing mice 15 days after treatment with saline and Hirudin/TRAIL-Gel. The white arrow symbols represent the tumor blood vessels. Scale bar is 100 μm. (f) TUNEL-based immunohistochemical images of the tumor harvested from the tumor-bearing mice 15 days after treatment with different formulations. The apoptotic cells were stained dark brown. Scale bar is 100 μm.

Hirudin/TRAIL-Gel (Figure 5f). The histological examination using the hematoxylin and eosin (H&E) staining presented that the Hirudin/TRAIL-Gel treatment led to the largest area of cell death (Figure S6), whereas no apparent pathological abnormalities were visualized in the major organs of the mice after applying Hirudin/TRAIL-Gel compared with the salinetreated ones (Figure S7). In addition, no remarkable variation in the body weight of the mice was determined after treatment with Hirudin/TRAIL-Gel (Figure S8). Taken together, the results suggest that the eminent antitumor effect is mainly attributed to the diminished angiogenesis and the enhanced apoptosis by Hirudin/TRAIL-Gel as a local depot with a prolonged effective period. We further performed a safety evaluation of the oligopeptide hydrogel-based formulation. To estimate the in vivo biodegradation of the oligopeptide hydrogel, the size of skin protrusion as a lump at the site of injection was monitored after subcutaneous injection of the blank oligopeptide hydrogel over time. No obvious lump can be found 3 weeks after injection (Figure S9a), which indicates that the oligopeptide hydrogel can be gradually degraded in vivo. The histological analysis displayed no noticeable inflammatory region in the treated skin 3 weeks after injection when the hydrogel had degraded completely, compared with the saline-treated group (Figure S9b). Moreover, the injected Hirudin/TRAIL-Gel did not cause any significant change in the serum concentrations of interleukin (IL)-6 and tumor necrosis factor (TNF)-α (Figure

S10). These data indicate that the oligopeptide hydrogel is safe for localized delivery of proteins within the studied time duration. In summary, we have developed a novel approach to construct an injectable oligopeptide hydrogel using an engineered enzyme nanocapsule for localized protein delivery. The protease-encapsulated single-protein-based nanocapsule with adjustable substrate selectivity was demonstrated to be able to catalyze the self-assembly of the oligopeptide hydrogel efficiently without impairing the function and activity of the encapsulated proteins. While we showed the enhanced antitumor efficacy of the hydrogel for localized codelivery of an antiangiogenetic protein and an apoptosis-inducing protein by synergistic effects, this formulation design can be generalized to delivery of other protein therapeutics,54−57 such as vaccines and hormones, for treatment of other diseases, which provides a promising delivery strategy for long-term disease management.

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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/acs.nanolett.7b03371. Experimental procedures, and additional in vitro and in vivo characterization of the hydrogel (PDF) F

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (R.M.) *E-mail: [email protected] (B.H.) ORCID

Ran Mo: 0000-0003-4010-8879 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81503012, 81673381) and the Natural Science Foundation of Jiangsu Province of China (BK20150963, BK20150029). This work was also supported by the Young Elite Scientists Sponsorship Program by CAST (2015QNRC001), the Program for Jiangsu Province Innovative Research Talents, the Program for Jiangsu Province Innovative Research Team, and the Jiangsu Specially-Appointed Professors Program to R.M.

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DOI: 10.1021/acs.nanolett.7b03371 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters (55) Luo, Z.; Wu, Q.; Yang, C.; Wang, H.; He, T.; Wang, Y.; Wang, Z.; Chen, H.; Li, X.; Gong, C.; Yang, Z. Adv. Mater. 2017, 29, 1601776. (56) Tae, G.; Kornfield, J. A.; Hubbell, J. A. Biomaterials 2005, 26, 5259−5266. (57) Mo, R.; Jiang, T.; Di, J.; Tai, W.; Gu, Z. Chem. Soc. Rev. 2014, 43, 3595−3629.

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DOI: 10.1021/acs.nanolett.7b03371 Nano Lett. XXXX, XXX, XXX−XXX