Colloids and Surfaces B: Biointerfaces 126 (2015) 580–584
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short Communication
Self-illuminative cascade-reaction-driven anticancer therapeutic cassettes made of cooperatively interactive nanocomplexes Woo Chul Song a , Seung Won Shin a , Kyung Soo Park a , Min Su Jang a , Jin-Ha Choi c , Byung-Keun Oh c , Soong Ho Um a,b,∗ a
School of Chemical Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea c Department of Chemical & Biomolecular Engineering and Interdisciplinary Program of Integrated Biotechnology, Sogang University, 35 Baekbeom-Ro, Mapo-gu, Seoul 121-742, South Korea b
a r t i c l e
i n f o
Article history: Received 19 October 2014 Received in revised form 2 December 2014 Accepted 5 December 2014 Available online 15 December 2014 Keywords: Cancer therapy Self-illuminative thermal cascade reaction Nanocomplex
a b s t r a c t Therapeutic options based on near-infrared (NIR) wavelengths have attracted attention owing to in vivo lowest-background interventions and the development of several nano-architectures with localized surface plasmon resonance. Because of their limited tissue penetration, the clinical use of NIR light-driven treatments is not widespread; this technology is inapplicable to infection sites in the deeper areas of internal tissues. In this study, we demonstrate a self-illuminative therapeutic cassette able to exert anticancer effects via a series of enzymatic, chemical, and optical cooperative cascade reactions. It consists of (1) NIR-illuminative nanocomplexes and (2) NIR-sensitive therapeutic cassettes, which demonstrate a 60% chemically-induced killing effect in a prostate cancer model without external NIR irradiation. This technology can also be actively exploited as an imaging agent due to adaptation of a self-illuminating nanocomplex. Consequently, these novel therapeutic cassettes, which work not only as a powerful internal NIR stimulant, but also as a biological imaging platform, provide a new rational design concept for biomedical use. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer has been preserved with a variety of therapeutic methods. With the advent of nanotechnology, new anticancer nano-kits have been developed. Light-sensitive therapies working at nearinfrared (NIR) wavelengths have been emphasized due to both their synergetic effects and minimal in vivo physiological interference, as well as various functional modules [1,2]. These therapeutics include gold nanorods [3], gold nanoshells [4], carbon nanotubes [5,6], and supramolecular nanostructures [7,8] possessing specific properties of NIR-specialized optical resonance. NIR-induced combinational nanoarchitectures have enabled spatiotemporal control of theragnostic effects with sustainable drug release [9], localized thermal ablation [10,11] and minimized off-target effects [12,13], and have been found to be effective for treating a variety of solid cancers, including those of the skin [14], breast [15] and liver [16]. Despite
∗ Corresponding author at: School of Chemical Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea. Tel.: +82 312907348; fax: +82 312907272. E-mail address: [email protected] (S.H. Um). http://dx.doi.org/10.1016/j.colsurfb.2014.12.011 0927-7765/© 2014 Elsevier B.V. All rights reserved.
the outstanding characteristics of light-driven cancer therapeutics, their use is limited due to the difficulty of penetrating deeply into tissues; generally NIR can penetrate up to 50 mm in physiological environments [17], indicating that the effectiveness of NIR for solid tumors is limited by depth. Here we demonstrate a novel self-illuminative and reactioncascade-driven therapeutic cassette that addresses the inherent drawbacks of traditional light-driven cancer treatments, particularly the restricted penetration of NIR. This system is comprised of two different compartments: (1) a self-illuminative NIR nanocomplex and (2) an NIR-responsive anticancer therapeutic cassette (Fig. 1). The first compartment contains a complex of both mutant Renilla luciferase (mRluc8) and quantum dots (QD800). Without direct exposure to NIR light, mRluc8 QD800 immediately converts the blue light of mRluc8 into a strong NIR emission of QD800 via bioluminescence resonance energy transfer (BRET) [18]. The second compartment contains a complex of doxorubicin (DOX), doublestranded DNA with 40 bases (DNA40) and gold nanorods (AuNRs). The therapeutic cassette, which is abbreviated DOX DNA40 AuNR, acts via conformational changes in the structure of DOX and DNA40 caused by the localized surface plasmon resonance (LSPR) effect of AuNRs under NIR irradiation, resulting in a sudden release of
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Louis, MO, USA) into DNA40 (DOX DNA40), DOX in 20-fold molar excess was incubated with DNA40 for 16 h at room temperature. Tris(2-carboxyethyl)phosphine (TCEP, Cat. no. 68957, Sigma–Aldrich, St. Louis, MO, USA) in 10-fold molar excess was reacted with DOX DNA40 for the reduction of the sulfhydryl group. Cetyltrimethylammonium bromide (CTAB)-stabilized AuNRs (Cat. no. A12-25-808, Nanopartz, Loveland, CO, USA) was centrifuged (5000 × g, 5 min) to remove excess CTAB. AuNR pellets were resuspended with distilled water containing 1 M DOX DNA40. After 3-h incubation at room temperature, the reaction solution was readjusted to 10 mM phosphate buffer composition to give ionic strengths in reacting environments. After a further 16 h of incubation at room temperature, the DOX DNA40 AuNR were centrifuged (5000 × g and 5 min) and resuspended with 10 mM phosphate buffer three times. 2.3. In vitro cell viability tests
Fig. 1. Schematic diagram of a self-illuminative cascade-reaction-driven therapeutic cassette. A series of cooperative enzymatic, chemical and optical cascade reactions create harmonic and synergetic therapeutic effects. Step 1: the oxidative enzymatic reaction of mRluc8 emits a strong blue light of 487 nm. Step 2: as a BRET acceptor, QD800 is excited by this blue light and emits NIR light at 800 nm. Step 3: NIR light stimulates AuNRs via the localized surface plasmon resonance (LSPR) effect. Step 4: the locally stimulated surface of AuNRs induces DOX release from DNA40. After a drop-down of cassettes and successive coelenterazine-h, the prostate cancer model is apoptized by self-illuminative-cascade-driven DOX release.
DOX molecules [19]. In principle, the self-illuminative therapeutic cassette induces the release of an anticancer drug via a series of enzymatic (mRluc8), chemical (DOX DNA40 AuNR) and optical (BRET and LSPR) cascade reactions and, in this study, the therapeutic effect of this complex is evaluated using an in vitro prostate cancer model. 2. Materials and methods 2.1. Preparation of mRluc8 QD800 mRluc8 is a mutant Renilla reniformis luciferase and was prepared as described in a previous study [20]. mRluc8 QD800 was prepared by conjugating mRluc8 to QD800 (Cat. no. Q21371MP, Invitrogen, Carlsbad, CA, USA). Next, 20 pmole QD800, 10,000 pmole mRluc8 and 2 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Cat. no. 22980, Thermo Fischer Scientific, Hudson, NH, USA) were reacted in 10 mM phosphate buffer for 1 h at room temperature. Unreacted EDC and mRluc8 were removed using a 100-kDa cutoff spin filter column (Cat no. UFC510096, Merck KGaA, Darmstadt, Germany) under 2000 g for 1 min, and this washing step was repeated 20 times. Final products were quantified using a UV/VIS spectrophotometer (Cat. no. 6132000008, Eppendorf, Hamburg, Germany). 2.2. Preparation of DOX DNA40 AuNR Two complementary, single-stranded DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) were annealed at 65 ◦ C for 10 min to form DNA40. One was 5 -/Thiol/ GTCGCAGGCACATCAGTACAGCCAGATCCGTACCTACTCG-3 and the other was 5 -CGAGTAGGTACGGATCTGGCTGTACTGATGTGCCTGCGAC-3 . To intercalate DOX (Cat. no. 44583, Sigma–Aldrich, St.
WST-1 assay was conducted using a Quick Cell Proliferation Assay Kit II (Cat. no. K-302-500, BioVision, Milpitas, CA, USA). PC3 cells were seeded on a 96-well culture plate and cultured until the confluence reached 80% in complete growth media (RPMI 1640 supplemented with 10% fetal bovine serum, Cat. no. 11875-093 and 16000-044, Gibco-Invitrogen, Carlsbad, CA, USA). After each cell was washed with 1× phosphate buffered saline solution (PBS), the cells were treated with free DOX (10 M), mRluc8 QD800 (5 pmole or 10 pmole) and DOX DNA40 AuNR (0.322 pmole, corresponding to 10 M total DOX) in 100 L complete growth media, and incubated for 2 h. Then, 5 g coelenterazine-h (Co-h, Cat. no. S2011, Promega, Madison, WI, USA) was added for mRluc8 QD800 and cells were irradiated with an NIR laser (either 2.5 W, 4 W or 5.5 W) for 5 min. The cells were then incubated for 24 h in the dark at 37 ◦ C. After incubation, each sample was treated with 10 L WST reagent and further incubated for 30 min. Finally, the absorbance intensities at 440 nm were measured using a SpectraMax M5 Microplate Reader (Cat. no. 89212-398, Molecular Devices, Sunnyvale, CA, USA). 3. Results and discussion 3.1. Characterization of the self-illuminative nanocomplex (mRluc8 QD800) NIR-illuminative nanocomplexes were constructed via simple conjugation of mRluc8 and QD800. mRluc8 is an protein with light output 5.56-fold stronger than that of the native Renilla luciferase. mRluc8 also shows substantial enzymatic functions in various acidic (cancerous) conditions (Fig. S1) and enhanced long-term stability in physiological environments [20]. Indeed, mRluc8 may be an ideal light resource for the conversion of chemical signals into photon emission. When engaged with QD800, it exhibits a wavelength transformation from 487 nm (mRluc8) to 800 nm (QD800) via BRET (Fig. 2a). To form the mRluc8 and QD800 complexes (mRluc8 QD800), mRluc8 was conjugated with QD800 through carbodiimide chemistry; mRluc8, with an average of 21 primary amines, can bind with the corresponding carboxyl ligands of QD800 [18]. To form mRluc8 QD800 with 800-nm photon emission, several parameters, including varying the molar ratios of mRluc8 and QD800 (Fig. 2a and d), were tested. For example, mRluc8 in molar ratios of 80–600 was mixed with 10 pmole of QD800 with 2 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a crosslinker. The incubation time for the conjugation reaction was limited to 1 h because of the fatal deactivation of used mRluc8s after 1 h of EDC reaction (Fig. S2) [21]. It was concluded that increased mRluc8 molar ratios resulted in the conjugate form producing
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Fig. 2. Characterization of NIR-illuminative and NIR-sensitive therapeutic nanocomplexes. (A) BRET spectra of mRluc8 QD800 with various conjugation ratios of mRluc8 to QD800 (the red line represents 500-fold molar excess). (B) Normalized absorbance spectra of AuNRs (black line), DNA40 AuNR (blue line) and DOX DNA40 AuNR (red line). (C) TEM image of DOX DNA40 AuNR (inset shows the aggregation of AuNRs during the surface modification and substitution process). The scale bar is 200 nm. (D) Change in mRluc8 QD800 electrophoretic mobility with stepwise increase in the conjugation ratio of mRluc8 to QD800 from 0 to 600. (E) Change in the electrophoretic mobility of NIR-sensitive therapeutic nanocomplexes; AuNR (lane 1), DNA40 AuNR (lane 2) and DOX DNA40 AuNR (lane 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
stronger blue light (487 nm) and NIR light (800 nm) output (Fig. 2a). However, the diminished efficiency of the BRET effect between mRluc8 and QD800 resulted in a decreased BRET ratio, which is defined as the spectral area from 650 to 850 nm divided by the spectral area from 350 to 650 nm (Fig. S3). This may be attributed to the increased distances between QD800 and the outermost mRluc8. Eventually, NIR light at 800 nm ceases to increase, despite the continuous increase in blue light at 487 nm. To prepare mRluc QD800 with the maximum degree of NIR light emission, QD800 must be adequately coated with mRluc8 in a 500-fold molar excess ratio of mRluc8, at which the emission of the QD800 is maximized (Fig. 2a). In addition, a used buffer condition was tested for efficient conjugation. Both 10 mM phosphate buffer and 10 mM Tris buffer were tested as reaction buffer candidates (Fig. S4); Tris buffer was not suitable for mediation of peptide bond formation in carbodiimide reactions because Tris ions have a primary amine for self-ligation of byproducts. 3.2. Characterization of the therapeutic cassette (DOX DNA40 AuNR) NIR-responsive anticancer therapeutic cassettes were designed and manufactured via functionalization of AuNRs with DNA40, and were rationally designed to efficiently embed DOX in a previous study [19]. Because AuNRs have an aspect ratio of 4 (20-nm width and 80-nm length) and respond to NIR wavelengths between 750 and 850 nm, the therapeutic cassettes can act as an NIR signal transducer of LSPR thermal vibrations [10,11]. This permits atomic vibration, generates heat and induces sustained DOX release, all within a restricted area [19]. To maximally intercalate DOX to DNA40, we titrated the DOX intercalating trends with the increasing molar ratios of DOX to DNA40 (Fig. S5). This resulted in a sudden increase of DOX fluorescence intensity to around 10 molar ratios. Intercalated DOX typically loses its inherent fluorescence intensity
[22]. This means that one DNA40 possesses a maximum of 10 DOX molecules. In the case of DOX DNA40 AuNR, pristine AuNRs stabilized by CTAB were replaced successively with DOX DNA40. DOX DNA40 possesses a sulfhydryl group at one end. The 1500-fold molar excess of DOX DNA40 was treated with AuNRs via goldsulfhydryl chemistry [23]. During the AuNR surface substitution process, total ionic strength may be a significant factor in efficient AuNR surface functionalization. If the ionic strength is too high, AuNRs can easily aggregate before stabilization by DOX DNA40. In contrast, if the ionic strength is too low, AuNRs will not be covered by enough DOX DNA40 for effective stabilization. To investigate these and assess the higher package of DNA40s on the gold surface, the substitution process was optimized in different media with different ion concentrations. The reaction was conducted sequentially in two steps. DOX DNA40 was bound to the AuNR surface within DI water for 3 h followed by an additional reaction in 10 mM phosphate buffer for 16 h. This enabled approximately 311 DOX DNA40 molecules to be bound to one AuNR (Fig. S6) while maintaining the non-aggregated and intact form of the DOX DNA40 AuNR at higher yields (>70%) (Fig. 2b and c). The physicochemical features of NIR-sensitive therapeutic cassettes were further investigated by gel electrophoretic mobility shift assay (GEMSA) [24] (Fig. 2e) and measured by zeta-potential values (Fig. S7). At a physiological pH of 7.4, DOX exists in protonated form (pKa ≈ 9), and its electric properties exhibit a positive charge. Even when DOX is intercalated into DNA40s or is physically adhered to them via a charged interaction, the net DNA40 negativities are affected, which could be the main driving factor under an applied electrophoretic field. In Fig. S8, DOX intercalation was observed to diminish the electrophoretic mobility of DNA40. Prior to GEMSA, the pH of the running buffer (0.5× Tris–acetate-EDTA buffer) was adjusted to pH 7.5 to protonate the DOX. The resulting electrophoretic mobility and surface negativity of DOX DNA40 AuNR were less than that of DNA40 AuNR. 3.3. DOX release via cascade reaction of the self-illuminative therapeutic cassette To identify whether the NIR-illuminative nanocomplex of mRluc8 QD800 works efficiently with the NIR-sensitive therapeutic cassette of DOX DNA40 AuNR, the cooperation of these entities was tested. For the test model, 0.75 pmole of DOX DNA40 AuNR were stimulated by NIR photons generated from either mRluc8 QD800 or only NIR (808 nm) laser irradiation. As a substrate of mRluc8, 10 ng/g coelenterazine-h was added dropwise to achieve the strongest NIR light from mRluc8 QD800. It was also expected that once DOX DNA40 AuNR was stimulated, the conformational change of DOX DNA40 would induce DOX release from DNA40 AuNR. By measuring in situ DOX fluorescence intensities, the free DOX quantities could be profiled (Fig. 3a and b). Interestingly, when 10 pmole of mRluc8 QD800 was used, the DOX release efficiency reached 36% compared with that of 5.5 W (Watt) laser irradiation. Also, Fig. 3a shows that the free DOX increased suddenly on the addition of coelenterazine-h and went on during 60 s. After 60 s, the DOX release profiles remained constant at the equilibrium state. Furthermore, DOX releases were patterned similarly to the illuminative kinetics of mRluc8 QD800 (Fig. S9). This indicates that sustainable DOX releases are precisely triggered by NIR photons excreted by mRluc8 QD800. However, there were no significant temperature variations within the solution when mRluc8 QD800 was used, whereas NIR laser irradiation led to outstanding temperature escalations (Fig. S10). This may be due to the different stimulation mechanisms of DOX DNA40 AuNR with mRluc8 QD800 and NIR laser irradiation. Using a three-dimensional perspective, we speculated that the NIR light generated from a laser device stimulates only one side of DOX DNA40 AuNR. However, with the cooperative system
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system is very expressive and achieves therapeutic effects without any external stimulation, addressing several intrinsic drawbacks of traditional light-driven therapy. 4. Conclusions
Fig. 3. DOX release profile and in vitro synergetic cancer-killing effects of selfilluminative therapeutic cassettes. (A) and (B) represent DOX release profiles from 0.1 pmole of DOX DNA40 AuNR. (A) mRluc8 QD800 was used as a light resource; 5 pmole: closed diamonds, 10 pmole: closed circles. (B) In comparison, NIR laser (808 nm) alone was used as a light source; 2 W: open squares, 4 W: closed diamonds, 5.5 W: closed circles). (C) WST-1 (water soluble tetrazolium salt) cell viability assay results of the synergetic therapeutic cassettes for the PC3 prostate cancer cell line. Cells were treated with free DOX (10 M), mRluc8 QD800 (10 pmole) and DOX DNA40 AuNR (0.322 pmole, corresponding to a total of 10 M DOX) in 100 L of complete growth media, and incubated for 2 h. Then, 5 g Co-h was added to mRluc8 QD800. The cells were subsequently incubated for 24 h in the dark at 37 ◦ C. Error bars represent standard deviation (n = 3, *P < 0.05). DOX: doxorubicin, RQ: mRluc8 QD800, DDA: DOX DNA40 AuNR, Co-h: coelenterazine-h.
of mRluc8 QD800, the DOX DNA40 AuNR can be stimulated temporally in all directions because mRluc8 QD800 is well dispersed around DOX DNA40 AuNR. Obviously, the NIR photons excreted by mRluc8 QD800 may not be sufficient to induce a sudden temperature increase, which is caused by conductive heat transfer from oscillating molecules on the surface of AuNRs by LSPR. However, it is possible that such a stimulation mechanism enables mRluc8 QD800 to induce nanoscale conformational change of DOX DNA40, followed by efficient DOX release from DOX DNA40 AuNR. As a result, it is likely that there are augmentative interactions between mRluc8 QD800 and DOX DNA40 AuNR. 3.4. Anticancer effects of the self-illuminative therapeutic cassette To validate the therapeutic effects of self-illuminative therapeutic cassettes in a prostate cancer model, a cell viability test was conducted via WST-1 assay (Fig. 3c and Fig. S11). The PC3 prostate cell line which is one of the most fatal male cancer species over the world [25] was used as a model. PC3 cells were prepared within a 96-well culture plate and each cell was treated with free DOX (10 M), mRluc8 QD800 (5 pmole or 10 pmole) and DOX DNA40 AuNR (0.322 pmole, which corresponds to a total of 10 M DOX) within 100 L of complete growth media. After 2 h of incubation, 5 g of coelenterazine-h (Co-h) was added to mRluc8 QD800 and irradiated with a 2.5-, 4- or 5.5-W NIR laser for 5 min. As a result, a significant drop in target cell viability (from 80.6 to 54.7%) was observed from a control set including mRluc8 QD800 and DOX DNA40 AuNR compared with that of a complete set including mRluc8 QD800, DOX DNA40 AuNR, and Coh (Fig. 3c; mRluc8 QD800 and DOX DNA40 AuNR were termed RQ and DDA, respectively). Because Co-h alone did not cause any significant cytotoxicity (Fig. S11), the toxicity of the complete set was attributed to the augmentative interaction of mRluc8 QD800 and DOX DNA40 AuNR. Although the synergetic therapeutic effects of this cassette were not equal to those of the NIR laser (Fig. S11), this
In summary, we have proposed a novel self-illuminative and cascade-reaction-driven cooperative therapeutic cassette platform in which there are two different nanocomplexes: an NIR-illuminative nanocomplex and an NIR-sensitive therapeutic module. The NIR-sensitive therapeutic cassettes were demonstrated to have anticancer properties. In principle, the NIR-sensitive therapeutic module was proposed to play a prominent role as a transducer for light-converted thermal vibrations. It is emphasized that the need for external stimulation such as NIR irradiation for conventional thermo-induced cancer therapies can be circumvented. Instead, NIR-illuminative nanocomplexes emit strong NIR light independently. The induced cascade reaction is based on a serial working mechanism of chemo-enzymatic mRluc8, optical BRET, LSPR effects, and DOX intercalation. This cascade finally results in strong anticancer therapeutic effects in a prostate cancer model. In addition, this self-illuminative therapeutic platform has the potential to be altered for target cell selectivity (Fig. S12) [19]. If these nanocomplexes can be successfully accumulated within a target tumorigenic area, all of the therapeutic mechanisms could be induced successfully without any external stimulus. This may be the first attempt to overcome the intrinsic limitations of conventional light-driven cancer therapeutics using a singular perspective. Furthermore, this technology may be applicable to both in vitro and in vivo theragnostic research areas, including molecular imaging and optogenetics, because of the adoption of strongly illuminative modules. Acknowledgement This work was supported by the Basic Science Research Program of the National Research Foundation (NRF) funded by the Ministry of Science ICT and Future Planning (Grant Nos. 2013R1A1A1058670 and 2013R1A1A2016781). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.12.011. References [1] R.A. Weissleder, Nat. Biotechnol. 19 (2001) 316–317. [2] V. Ntziachristos, J. Ripoll, L.V. Wang, R. Weissleder, Nat. Biotechnol. 23 (2005) 313–320. [3] T.R. Kuo, V.A. Hovhannisyan, Y.C. Chao, S.L. Chao, S.J. Chiang, S.J. Lin, C.Y. Dong, C.C. Chen, J. Am. Chem. Soc. 132 (2010) 14163–14171. [4] M. Bikram, A.M. Gobin, R.E. Whitmire, J.L. West, J. Control. Release 123 (2007) 219–227. [5] N.M. Iverson, P.W. Barone, M. Shandell, L.J. Trudel, S. Sen, F. Sen, V. Ivanov, E. Atolia, E. Farias, T.P. McNicholas, N. Reuel, N.M.A. Parry, G.N. Wogan, M.S. Strano, Nat. Nanotech. 8 (2013) 873–880. [6] N.W. Shi Kam, M. O’Connell, J.A. Wisdom, H. Dai, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 11600–11605. [7] G.H. Wu, A. Mikhailovsky, H.A. Khant, C. Fu, W. Chiu, J.A. Zasadzinski, J. Am. Chem. Soc. 130 (2008) 8175–8177. [8] M.L. Viger, W. Sheng, K. Dore, A.H. Alhasan, C.-J. Carling, J. Lux, C.D.G. Lux, M. Grossman, R. Malinow, A. Almutairi, ACS Nano 8 (2014) 4815–4826. [9] Y.-X.J. Wang, X.-M. Zhu, Q. Liang, C.H.K. Cheng, W. Wang, K.C.-F. Leung, Angew. Chem. Int. Ed. 53 (2014) 4812–4815. [10] H.K. Moon, S.H. Lee, H.C. Choi, ACS Nano 3 (2009) 3707–3713. [11] X. Huang, P.K. Jain, I.H. El-Sayed, M.A. El-sayed, Lasers Med. Sci. 23 (2008) 217–228. [12] J.-O. You, D.T. Auguste, Biomaterials 29 (2008) 1950–1957. [13] J.-O. You, P. Guo, D.T. Auguste, Angew. Chem. Int. Ed. 52 (2013) 4141–4146.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short Communication
Self-illuminative cascade-reaction-driven anticancer therapeutic cassettes made of cooperatively interactive nanocomplexes Woo Chul Song a , Seung Won Shin a , Kyung Soo Park a , Min Su Jang a , Jin-Ha Choi c , Byung-Keun Oh c , Soong Ho Um a,b,∗ a
School of Chemical Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea c Department of Chemical & Biomolecular Engineering and Interdisciplinary Program of Integrated Biotechnology, Sogang University, 35 Baekbeom-Ro, Mapo-gu, Seoul 121-742, South Korea b
a r t i c l e
i n f o
Article history: Received 19 October 2014 Received in revised form 2 December 2014 Accepted 5 December 2014 Available online 15 December 2014 Keywords: Cancer therapy Self-illuminative thermal cascade reaction Nanocomplex
a b s t r a c t Therapeutic options based on near-infrared (NIR) wavelengths have attracted attention owing to in vivo lowest-background interventions and the development of several nano-architectures with localized surface plasmon resonance. Because of their limited tissue penetration, the clinical use of NIR light-driven treatments is not widespread; this technology is inapplicable to infection sites in the deeper areas of internal tissues. In this study, we demonstrate a self-illuminative therapeutic cassette able to exert anticancer effects via a series of enzymatic, chemical, and optical cooperative cascade reactions. It consists of (1) NIR-illuminative nanocomplexes and (2) NIR-sensitive therapeutic cassettes, which demonstrate a 60% chemically-induced killing effect in a prostate cancer model without external NIR irradiation. This technology can also be actively exploited as an imaging agent due to adaptation of a self-illuminating nanocomplex. Consequently, these novel therapeutic cassettes, which work not only as a powerful internal NIR stimulant, but also as a biological imaging platform, provide a new rational design concept for biomedical use. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer has been preserved with a variety of therapeutic methods. With the advent of nanotechnology, new anticancer nano-kits have been developed. Light-sensitive therapies working at nearinfrared (NIR) wavelengths have been emphasized due to both their synergetic effects and minimal in vivo physiological interference, as well as various functional modules [1,2]. These therapeutics include gold nanorods [3], gold nanoshells [4], carbon nanotubes [5,6], and supramolecular nanostructures [7,8] possessing specific properties of NIR-specialized optical resonance. NIR-induced combinational nanoarchitectures have enabled spatiotemporal control of theragnostic effects with sustainable drug release [9], localized thermal ablation [10,11] and minimized off-target effects [12,13], and have been found to be effective for treating a variety of solid cancers, including those of the skin [14], breast [15] and liver [16]. Despite
∗ Corresponding author at: School of Chemical Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea. Tel.: +82 312907348; fax: +82 312907272. E-mail address: [email protected] (S.H. Um). http://dx.doi.org/10.1016/j.colsurfb.2014.12.011 0927-7765/© 2014 Elsevier B.V. All rights reserved.
the outstanding characteristics of light-driven cancer therapeutics, their use is limited due to the difficulty of penetrating deeply into tissues; generally NIR can penetrate up to 50 mm in physiological environments [17], indicating that the effectiveness of NIR for solid tumors is limited by depth. Here we demonstrate a novel self-illuminative and reactioncascade-driven therapeutic cassette that addresses the inherent drawbacks of traditional light-driven cancer treatments, particularly the restricted penetration of NIR. This system is comprised of two different compartments: (1) a self-illuminative NIR nanocomplex and (2) an NIR-responsive anticancer therapeutic cassette (Fig. 1). The first compartment contains a complex of both mutant Renilla luciferase (mRluc8) and quantum dots (QD800). Without direct exposure to NIR light, mRluc8 QD800 immediately converts the blue light of mRluc8 into a strong NIR emission of QD800 via bioluminescence resonance energy transfer (BRET) [18]. The second compartment contains a complex of doxorubicin (DOX), doublestranded DNA with 40 bases (DNA40) and gold nanorods (AuNRs). The therapeutic cassette, which is abbreviated DOX DNA40 AuNR, acts via conformational changes in the structure of DOX and DNA40 caused by the localized surface plasmon resonance (LSPR) effect of AuNRs under NIR irradiation, resulting in a sudden release of
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Louis, MO, USA) into DNA40 (DOX DNA40), DOX in 20-fold molar excess was incubated with DNA40 for 16 h at room temperature. Tris(2-carboxyethyl)phosphine (TCEP, Cat. no. 68957, Sigma–Aldrich, St. Louis, MO, USA) in 10-fold molar excess was reacted with DOX DNA40 for the reduction of the sulfhydryl group. Cetyltrimethylammonium bromide (CTAB)-stabilized AuNRs (Cat. no. A12-25-808, Nanopartz, Loveland, CO, USA) was centrifuged (5000 × g, 5 min) to remove excess CTAB. AuNR pellets were resuspended with distilled water containing 1 M DOX DNA40. After 3-h incubation at room temperature, the reaction solution was readjusted to 10 mM phosphate buffer composition to give ionic strengths in reacting environments. After a further 16 h of incubation at room temperature, the DOX DNA40 AuNR were centrifuged (5000 × g and 5 min) and resuspended with 10 mM phosphate buffer three times. 2.3. In vitro cell viability tests
Fig. 1. Schematic diagram of a self-illuminative cascade-reaction-driven therapeutic cassette. A series of cooperative enzymatic, chemical and optical cascade reactions create harmonic and synergetic therapeutic effects. Step 1: the oxidative enzymatic reaction of mRluc8 emits a strong blue light of 487 nm. Step 2: as a BRET acceptor, QD800 is excited by this blue light and emits NIR light at 800 nm. Step 3: NIR light stimulates AuNRs via the localized surface plasmon resonance (LSPR) effect. Step 4: the locally stimulated surface of AuNRs induces DOX release from DNA40. After a drop-down of cassettes and successive coelenterazine-h, the prostate cancer model is apoptized by self-illuminative-cascade-driven DOX release.
DOX molecules [19]. In principle, the self-illuminative therapeutic cassette induces the release of an anticancer drug via a series of enzymatic (mRluc8), chemical (DOX DNA40 AuNR) and optical (BRET and LSPR) cascade reactions and, in this study, the therapeutic effect of this complex is evaluated using an in vitro prostate cancer model. 2. Materials and methods 2.1. Preparation of mRluc8 QD800 mRluc8 is a mutant Renilla reniformis luciferase and was prepared as described in a previous study [20]. mRluc8 QD800 was prepared by conjugating mRluc8 to QD800 (Cat. no. Q21371MP, Invitrogen, Carlsbad, CA, USA). Next, 20 pmole QD800, 10,000 pmole mRluc8 and 2 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Cat. no. 22980, Thermo Fischer Scientific, Hudson, NH, USA) were reacted in 10 mM phosphate buffer for 1 h at room temperature. Unreacted EDC and mRluc8 were removed using a 100-kDa cutoff spin filter column (Cat no. UFC510096, Merck KGaA, Darmstadt, Germany) under 2000 g for 1 min, and this washing step was repeated 20 times. Final products were quantified using a UV/VIS spectrophotometer (Cat. no. 6132000008, Eppendorf, Hamburg, Germany). 2.2. Preparation of DOX DNA40 AuNR Two complementary, single-stranded DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) were annealed at 65 ◦ C for 10 min to form DNA40. One was 5 -/Thiol/ GTCGCAGGCACATCAGTACAGCCAGATCCGTACCTACTCG-3 and the other was 5 -CGAGTAGGTACGGATCTGGCTGTACTGATGTGCCTGCGAC-3 . To intercalate DOX (Cat. no. 44583, Sigma–Aldrich, St.
WST-1 assay was conducted using a Quick Cell Proliferation Assay Kit II (Cat. no. K-302-500, BioVision, Milpitas, CA, USA). PC3 cells were seeded on a 96-well culture plate and cultured until the confluence reached 80% in complete growth media (RPMI 1640 supplemented with 10% fetal bovine serum, Cat. no. 11875-093 and 16000-044, Gibco-Invitrogen, Carlsbad, CA, USA). After each cell was washed with 1× phosphate buffered saline solution (PBS), the cells were treated with free DOX (10 M), mRluc8 QD800 (5 pmole or 10 pmole) and DOX DNA40 AuNR (0.322 pmole, corresponding to 10 M total DOX) in 100 L complete growth media, and incubated for 2 h. Then, 5 g coelenterazine-h (Co-h, Cat. no. S2011, Promega, Madison, WI, USA) was added for mRluc8 QD800 and cells were irradiated with an NIR laser (either 2.5 W, 4 W or 5.5 W) for 5 min. The cells were then incubated for 24 h in the dark at 37 ◦ C. After incubation, each sample was treated with 10 L WST reagent and further incubated for 30 min. Finally, the absorbance intensities at 440 nm were measured using a SpectraMax M5 Microplate Reader (Cat. no. 89212-398, Molecular Devices, Sunnyvale, CA, USA). 3. Results and discussion 3.1. Characterization of the self-illuminative nanocomplex (mRluc8 QD800) NIR-illuminative nanocomplexes were constructed via simple conjugation of mRluc8 and QD800. mRluc8 is an protein with light output 5.56-fold stronger than that of the native Renilla luciferase. mRluc8 also shows substantial enzymatic functions in various acidic (cancerous) conditions (Fig. S1) and enhanced long-term stability in physiological environments [20]. Indeed, mRluc8 may be an ideal light resource for the conversion of chemical signals into photon emission. When engaged with QD800, it exhibits a wavelength transformation from 487 nm (mRluc8) to 800 nm (QD800) via BRET (Fig. 2a). To form the mRluc8 and QD800 complexes (mRluc8 QD800), mRluc8 was conjugated with QD800 through carbodiimide chemistry; mRluc8, with an average of 21 primary amines, can bind with the corresponding carboxyl ligands of QD800 [18]. To form mRluc8 QD800 with 800-nm photon emission, several parameters, including varying the molar ratios of mRluc8 and QD800 (Fig. 2a and d), were tested. For example, mRluc8 in molar ratios of 80–600 was mixed with 10 pmole of QD800 with 2 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a crosslinker. The incubation time for the conjugation reaction was limited to 1 h because of the fatal deactivation of used mRluc8s after 1 h of EDC reaction (Fig. S2) [21]. It was concluded that increased mRluc8 molar ratios resulted in the conjugate form producing
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Fig. 2. Characterization of NIR-illuminative and NIR-sensitive therapeutic nanocomplexes. (A) BRET spectra of mRluc8 QD800 with various conjugation ratios of mRluc8 to QD800 (the red line represents 500-fold molar excess). (B) Normalized absorbance spectra of AuNRs (black line), DNA40 AuNR (blue line) and DOX DNA40 AuNR (red line). (C) TEM image of DOX DNA40 AuNR (inset shows the aggregation of AuNRs during the surface modification and substitution process). The scale bar is 200 nm. (D) Change in mRluc8 QD800 electrophoretic mobility with stepwise increase in the conjugation ratio of mRluc8 to QD800 from 0 to 600. (E) Change in the electrophoretic mobility of NIR-sensitive therapeutic nanocomplexes; AuNR (lane 1), DNA40 AuNR (lane 2) and DOX DNA40 AuNR (lane 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
stronger blue light (487 nm) and NIR light (800 nm) output (Fig. 2a). However, the diminished efficiency of the BRET effect between mRluc8 and QD800 resulted in a decreased BRET ratio, which is defined as the spectral area from 650 to 850 nm divided by the spectral area from 350 to 650 nm (Fig. S3). This may be attributed to the increased distances between QD800 and the outermost mRluc8. Eventually, NIR light at 800 nm ceases to increase, despite the continuous increase in blue light at 487 nm. To prepare mRluc QD800 with the maximum degree of NIR light emission, QD800 must be adequately coated with mRluc8 in a 500-fold molar excess ratio of mRluc8, at which the emission of the QD800 is maximized (Fig. 2a). In addition, a used buffer condition was tested for efficient conjugation. Both 10 mM phosphate buffer and 10 mM Tris buffer were tested as reaction buffer candidates (Fig. S4); Tris buffer was not suitable for mediation of peptide bond formation in carbodiimide reactions because Tris ions have a primary amine for self-ligation of byproducts. 3.2. Characterization of the therapeutic cassette (DOX DNA40 AuNR) NIR-responsive anticancer therapeutic cassettes were designed and manufactured via functionalization of AuNRs with DNA40, and were rationally designed to efficiently embed DOX in a previous study [19]. Because AuNRs have an aspect ratio of 4 (20-nm width and 80-nm length) and respond to NIR wavelengths between 750 and 850 nm, the therapeutic cassettes can act as an NIR signal transducer of LSPR thermal vibrations [10,11]. This permits atomic vibration, generates heat and induces sustained DOX release, all within a restricted area [19]. To maximally intercalate DOX to DNA40, we titrated the DOX intercalating trends with the increasing molar ratios of DOX to DNA40 (Fig. S5). This resulted in a sudden increase of DOX fluorescence intensity to around 10 molar ratios. Intercalated DOX typically loses its inherent fluorescence intensity
[22]. This means that one DNA40 possesses a maximum of 10 DOX molecules. In the case of DOX DNA40 AuNR, pristine AuNRs stabilized by CTAB were replaced successively with DOX DNA40. DOX DNA40 possesses a sulfhydryl group at one end. The 1500-fold molar excess of DOX DNA40 was treated with AuNRs via goldsulfhydryl chemistry [23]. During the AuNR surface substitution process, total ionic strength may be a significant factor in efficient AuNR surface functionalization. If the ionic strength is too high, AuNRs can easily aggregate before stabilization by DOX DNA40. In contrast, if the ionic strength is too low, AuNRs will not be covered by enough DOX DNA40 for effective stabilization. To investigate these and assess the higher package of DNA40s on the gold surface, the substitution process was optimized in different media with different ion concentrations. The reaction was conducted sequentially in two steps. DOX DNA40 was bound to the AuNR surface within DI water for 3 h followed by an additional reaction in 10 mM phosphate buffer for 16 h. This enabled approximately 311 DOX DNA40 molecules to be bound to one AuNR (Fig. S6) while maintaining the non-aggregated and intact form of the DOX DNA40 AuNR at higher yields (>70%) (Fig. 2b and c). The physicochemical features of NIR-sensitive therapeutic cassettes were further investigated by gel electrophoretic mobility shift assay (GEMSA) [24] (Fig. 2e) and measured by zeta-potential values (Fig. S7). At a physiological pH of 7.4, DOX exists in protonated form (pKa ≈ 9), and its electric properties exhibit a positive charge. Even when DOX is intercalated into DNA40s or is physically adhered to them via a charged interaction, the net DNA40 negativities are affected, which could be the main driving factor under an applied electrophoretic field. In Fig. S8, DOX intercalation was observed to diminish the electrophoretic mobility of DNA40. Prior to GEMSA, the pH of the running buffer (0.5× Tris–acetate-EDTA buffer) was adjusted to pH 7.5 to protonate the DOX. The resulting electrophoretic mobility and surface negativity of DOX DNA40 AuNR were less than that of DNA40 AuNR. 3.3. DOX release via cascade reaction of the self-illuminative therapeutic cassette To identify whether the NIR-illuminative nanocomplex of mRluc8 QD800 works efficiently with the NIR-sensitive therapeutic cassette of DOX DNA40 AuNR, the cooperation of these entities was tested. For the test model, 0.75 pmole of DOX DNA40 AuNR were stimulated by NIR photons generated from either mRluc8 QD800 or only NIR (808 nm) laser irradiation. As a substrate of mRluc8, 10 ng/g coelenterazine-h was added dropwise to achieve the strongest NIR light from mRluc8 QD800. It was also expected that once DOX DNA40 AuNR was stimulated, the conformational change of DOX DNA40 would induce DOX release from DNA40 AuNR. By measuring in situ DOX fluorescence intensities, the free DOX quantities could be profiled (Fig. 3a and b). Interestingly, when 10 pmole of mRluc8 QD800 was used, the DOX release efficiency reached 36% compared with that of 5.5 W (Watt) laser irradiation. Also, Fig. 3a shows that the free DOX increased suddenly on the addition of coelenterazine-h and went on during 60 s. After 60 s, the DOX release profiles remained constant at the equilibrium state. Furthermore, DOX releases were patterned similarly to the illuminative kinetics of mRluc8 QD800 (Fig. S9). This indicates that sustainable DOX releases are precisely triggered by NIR photons excreted by mRluc8 QD800. However, there were no significant temperature variations within the solution when mRluc8 QD800 was used, whereas NIR laser irradiation led to outstanding temperature escalations (Fig. S10). This may be due to the different stimulation mechanisms of DOX DNA40 AuNR with mRluc8 QD800 and NIR laser irradiation. Using a three-dimensional perspective, we speculated that the NIR light generated from a laser device stimulates only one side of DOX DNA40 AuNR. However, with the cooperative system
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system is very expressive and achieves therapeutic effects without any external stimulation, addressing several intrinsic drawbacks of traditional light-driven therapy. 4. Conclusions
Fig. 3. DOX release profile and in vitro synergetic cancer-killing effects of selfilluminative therapeutic cassettes. (A) and (B) represent DOX release profiles from 0.1 pmole of DOX DNA40 AuNR. (A) mRluc8 QD800 was used as a light resource; 5 pmole: closed diamonds, 10 pmole: closed circles. (B) In comparison, NIR laser (808 nm) alone was used as a light source; 2 W: open squares, 4 W: closed diamonds, 5.5 W: closed circles). (C) WST-1 (water soluble tetrazolium salt) cell viability assay results of the synergetic therapeutic cassettes for the PC3 prostate cancer cell line. Cells were treated with free DOX (10 M), mRluc8 QD800 (10 pmole) and DOX DNA40 AuNR (0.322 pmole, corresponding to a total of 10 M DOX) in 100 L of complete growth media, and incubated for 2 h. Then, 5 g Co-h was added to mRluc8 QD800. The cells were subsequently incubated for 24 h in the dark at 37 ◦ C. Error bars represent standard deviation (n = 3, *P < 0.05). DOX: doxorubicin, RQ: mRluc8 QD800, DDA: DOX DNA40 AuNR, Co-h: coelenterazine-h.
of mRluc8 QD800, the DOX DNA40 AuNR can be stimulated temporally in all directions because mRluc8 QD800 is well dispersed around DOX DNA40 AuNR. Obviously, the NIR photons excreted by mRluc8 QD800 may not be sufficient to induce a sudden temperature increase, which is caused by conductive heat transfer from oscillating molecules on the surface of AuNRs by LSPR. However, it is possible that such a stimulation mechanism enables mRluc8 QD800 to induce nanoscale conformational change of DOX DNA40, followed by efficient DOX release from DOX DNA40 AuNR. As a result, it is likely that there are augmentative interactions between mRluc8 QD800 and DOX DNA40 AuNR. 3.4. Anticancer effects of the self-illuminative therapeutic cassette To validate the therapeutic effects of self-illuminative therapeutic cassettes in a prostate cancer model, a cell viability test was conducted via WST-1 assay (Fig. 3c and Fig. S11). The PC3 prostate cell line which is one of the most fatal male cancer species over the world [25] was used as a model. PC3 cells were prepared within a 96-well culture plate and each cell was treated with free DOX (10 M), mRluc8 QD800 (5 pmole or 10 pmole) and DOX DNA40 AuNR (0.322 pmole, which corresponds to a total of 10 M DOX) within 100 L of complete growth media. After 2 h of incubation, 5 g of coelenterazine-h (Co-h) was added to mRluc8 QD800 and irradiated with a 2.5-, 4- or 5.5-W NIR laser for 5 min. As a result, a significant drop in target cell viability (from 80.6 to 54.7%) was observed from a control set including mRluc8 QD800 and DOX DNA40 AuNR compared with that of a complete set including mRluc8 QD800, DOX DNA40 AuNR, and Coh (Fig. 3c; mRluc8 QD800 and DOX DNA40 AuNR were termed RQ and DDA, respectively). Because Co-h alone did not cause any significant cytotoxicity (Fig. S11), the toxicity of the complete set was attributed to the augmentative interaction of mRluc8 QD800 and DOX DNA40 AuNR. Although the synergetic therapeutic effects of this cassette were not equal to those of the NIR laser (Fig. S11), this
In summary, we have proposed a novel self-illuminative and cascade-reaction-driven cooperative therapeutic cassette platform in which there are two different nanocomplexes: an NIR-illuminative nanocomplex and an NIR-sensitive therapeutic module. The NIR-sensitive therapeutic cassettes were demonstrated to have anticancer properties. In principle, the NIR-sensitive therapeutic module was proposed to play a prominent role as a transducer for light-converted thermal vibrations. It is emphasized that the need for external stimulation such as NIR irradiation for conventional thermo-induced cancer therapies can be circumvented. Instead, NIR-illuminative nanocomplexes emit strong NIR light independently. The induced cascade reaction is based on a serial working mechanism of chemo-enzymatic mRluc8, optical BRET, LSPR effects, and DOX intercalation. This cascade finally results in strong anticancer therapeutic effects in a prostate cancer model. In addition, this self-illuminative therapeutic platform has the potential to be altered for target cell selectivity (Fig. S12) [19]. If these nanocomplexes can be successfully accumulated within a target tumorigenic area, all of the therapeutic mechanisms could be induced successfully without any external stimulus. This may be the first attempt to overcome the intrinsic limitations of conventional light-driven cancer therapeutics using a singular perspective. Furthermore, this technology may be applicable to both in vitro and in vivo theragnostic research areas, including molecular imaging and optogenetics, because of the adoption of strongly illuminative modules. Acknowledgement This work was supported by the Basic Science Research Program of the National Research Foundation (NRF) funded by the Ministry of Science ICT and Future Planning (Grant Nos. 2013R1A1A1058670 and 2013R1A1A2016781). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.12.011. References [1] R.A. Weissleder, Nat. Biotechnol. 19 (2001) 316–317. [2] V. Ntziachristos, J. Ripoll, L.V. Wang, R. Weissleder, Nat. Biotechnol. 23 (2005) 313–320. [3] T.R. Kuo, V.A. Hovhannisyan, Y.C. Chao, S.L. Chao, S.J. Chiang, S.J. Lin, C.Y. Dong, C.C. Chen, J. Am. Chem. Soc. 132 (2010) 14163–14171. [4] M. Bikram, A.M. Gobin, R.E. Whitmire, J.L. West, J. Control. Release 123 (2007) 219–227. [5] N.M. Iverson, P.W. Barone, M. Shandell, L.J. Trudel, S. Sen, F. Sen, V. Ivanov, E. Atolia, E. Farias, T.P. McNicholas, N. Reuel, N.M.A. Parry, G.N. Wogan, M.S. Strano, Nat. Nanotech. 8 (2013) 873–880. [6] N.W. Shi Kam, M. O’Connell, J.A. Wisdom, H. Dai, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 11600–11605. [7] G.H. Wu, A. Mikhailovsky, H.A. Khant, C. Fu, W. Chiu, J.A. Zasadzinski, J. Am. Chem. Soc. 130 (2008) 8175–8177. [8] M.L. Viger, W. Sheng, K. Dore, A.H. Alhasan, C.-J. Carling, J. Lux, C.D.G. Lux, M. Grossman, R. Malinow, A. Almutairi, ACS Nano 8 (2014) 4815–4826. [9] Y.-X.J. Wang, X.-M. Zhu, Q. Liang, C.H.K. Cheng, W. Wang, K.C.-F. Leung, Angew. Chem. Int. Ed. 53 (2014) 4812–4815. [10] H.K. Moon, S.H. Lee, H.C. Choi, ACS Nano 3 (2009) 3707–3713. [11] X. Huang, P.K. Jain, I.H. El-Sayed, M.A. El-sayed, Lasers Med. Sci. 23 (2008) 217–228. [12] J.-O. You, D.T. Auguste, Biomaterials 29 (2008) 1950–1957. [13] J.-O. You, P. Guo, D.T. Auguste, Angew. Chem. Int. Ed. 52 (2013) 4141–4146.
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