Nonviolent Self-Catabolic DNAzyme Nanosponges for Smart

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Nonviolent Self-Catabolic DNAzyme Nanosponges for Smart Anticancer Drug Delivery Jing Wang,† Huimin Wang,† Hong Wang,† Shizhen He,† Ruomeng Li,† Zhao Deng,‡ Xiaoqing Liu,† and Fuan Wang*,† Downloaded via UNIV OF SOUTHERN INDIANA on July 18, 2019 at 07:53:57 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, People’s Republic of China ‡ State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430072, People’s Republic of China S Supporting Information *

ABSTRACT: The development of self-assembled DNA nanomedicine requires a facile and accurate DNA degradation strategy for precisely programmable drug release. Conventional DNA catabolic strategies are restrained with the fragile and unclear enzymatic reactions that might lead to inefficient and uncontrollable digestion of DNA scaffolds and thus might bring undesirable side effects to the sophisticated biosystems. Herein we reported a versatile selfsufficient DNAzyme-driven drug delivery system consisting of the rolling circle polymerized DNAzyme−substrate scaffolds and the encapsulated pH-responsive ZnO nanoparticles (NPs). The full DNAzyme nanosponges (NSs) were also encoded with multivalent tandem aptamer sequences to facilitate their efficient delivery into cancer cells, where the acidic endo/lysosomal microenvironment stimulates the dissolution of ZnO into Zn2+ ions as DNAzyme cofactors and therapeutic reactive oxygen species generators. The supplement Zn2+ cofactors mediated the nonviolent DNAzyme-catalyzed cleavage of DNA scaffolds for precise and efficient drug administrations with synergistically enhanced therapeutic performance. The facile design of DNAzyme, together with their cost-effective and intrinsic robust features, is anticipated to provide extensive insights for the development of DNA-based therapeutic platforms by activating the specific intracellular biocatalytic reactions. As an intelligent and nonviolent self-driven drug delivery platform, the present DNAzyme NS system could be engineered with more therapeutic sequences and agents and was anticipated to show exceptional promise and versatility for applications in biomedicine and bioengineering. KEYWORDS: rolling circle amplification, DNAzyme, ZnO NPs, DNA hydrogel, controlled drug release

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ing of diverse DNA assemblies, especially fully addressable DNA nanostructures, to encapsulate, transport, and release molecular cargos into cells.20,21 It is thus highly desirable to develop facile and efficient approaches for synthesizing largescale DNA nanoassemblies with desired built-in functionalities. Recently, isothermal rolling circle amplification (RCA) has emerged as a promising tool for assembling functional DNA nanostructures.22−28 Driven by liquid crystallization and dense packaging of building blocks, these RCA strands are likely to noncanonically self-assemble into monodispersed and sophisticated yarn-like DNA nanosponges (NSs). These NSs can be endowed with diverse therapeutic properties (i.e., antisense

anotechnology is changing the therapeutic landscape in terms of diagnosis, treatment, and prognosis.1−3 Although exogenous organic or inorganic nanomaterials are extensively studied, they are challenged by nontargeted cytotoxicity and accumulation, as well as inflexible preparations and postmodifications.4−9 Recently, endogenously generated biomaterials, whose catabolite can be assimilated or eliminated by the hosting body, have received extensive attention for biomedical research.10−14 Among them, DNA biomaterials are particularly attractive due to their intrinsic biocompatibility and low-immunogenicity features. The specific and predictable Watson−Crick base-pairing of DNA contribute to the self-assembly of various sophisticated DNA architectures15−17 for precisely positioning other biologically important molecular components at the nanometer scale.18,19 These interesting properties facilitate the engineer© 2019 American Chemical Society

Received: February 26, 2019 Accepted: May 1, 2019 Published: May 1, 2019 5852

DOI: 10.1021/acsnano.9b01589 ACS Nano 2019, 13, 5852−5863

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Cite This: ACS Nano 2019, 13, 5852−5863

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Figure 1. (a) Schematic illustration of RCA-based assembly and acid-triggered disassembly of DOX@RCA-ZnO-NSs. (b) Intracellular ZnO dissociation induced ROS generation and activated DNAzyme cleavage accompanied by stimulated drug release after specific cellular uptake.

DNAzyme provides an ideal candidate for realizing the effective and programmable self-powered disassembly of DNA NSs.35 The encoded DNAzyme and substrate sequences could realize the accurate and programmable digestion of NS nanocarriers with auxiliary Zn2+ ion cofactors. The all-DNAbased DNAzyme is more simple and robust under complicated intracellular environments, making it more appealing in biomedical applications. This makes the built-in self-hydrolyzing DNAzyme a promisingly facile and nonviolent stimulus in regulating necessary biomedical situations through selfsustained drug delivery. Especially, the moderate and accurate self-sacrificing DNAzyme NSs could minimize or even prevent undesired toxicity and severe side effects upon their systemic administration. Herein, we fabricated compact and hierarchical ZnOencapsulated DNAzyme NSs with multiple built-in functionalities for intelligent and self-sufficient cancer therapy (Figure 1). The RCA-generated multivalent sgc-8c aptamers can specifically recognize protein tyrosine kinase 7 (PTK7) of cancer cells, thus enabling the precise delivery of various therapeutic agents through NS nanocarriers. The RCAassembled DNA/ZnO NSs stay in a stabilized state at normal physiological conditions through potent electrostatic and coordination interactions, yet drive the efficient dissolution of ZnO in an acidic endo/lysosomal microenvironment.39,40 This leads to the supplement of a sufficiently high concentration of Zn2+-ion DNAzyme cofactors for immediately catalyzing the hydrolytic cleavage of RCA products and for generating intracellular destructive reactive oxygen species (ROS). As a versatile delivery vehicle, the collapsed NSs spark the effective and programmable administration of encapsulated chemotherapeutic drugs into the cytosol, originating from the

DNA and immunostimulatory CpG) for tumor-specific gene silencing and immunotherapy.29,30 As robust accommodation materials, DNA NSs could retain the intact activity and integrity of loading small molecule drugs and biomacromolecule proteins without leakage and show great potential in nanomedicine and bioimaging applications.23,26,31−33 Despite the increasing interest in NS-mediated therapeutic strategies, the efficiency and programmability of these drug administrations remain critical hurdles for practical applications. For example, the stimulated drug release from these NSs is rather limited by the intracellular nuclease-mediated digestion process, which is constrained by the varied spatiotemporal distribution of native nucleases. It is highly desirable to develop more compact and self-sufficient strategies through the built-in stimuli-responsiveness and disassembly functionalities for ondemand NS-shed drug delivery. The built-in DNA-digesting enzyme is initially introduced to address these self-responsive drug release issues, yet these exogenous and fragile enzymatic stimuli have the limitations of elaborate design and tedious preparation.26 The encapsulated enzyme also tends to execute violent and stochastic digestion of DNA, which might result in an ineffective delivery and undesired side effects. Thus, the nonviolent built-in stimuli with self-sustainable, programmable, and accurate digestion of exogenous DNA carriers are more appealing for biological studies. DNAzymes are catalytic nucleic acids that can mimic the functions of protein−enzymes, including endonucleases,34−38 which inspired us to simultaneously encode the DNAzyme and substrate sequences into functional RCA NSs. Traditional RNA-cleaving DNAzymes are incompatible since their chimeric substrate could not be incorporated into the RCA NSs. Luckily, a recently discovered DNA-hydrolyzing 5853


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The physically homogeneous RCA NSs were achieved with a ∼220 nm diameter and narrow size distribution, which was consistent with the dynamic light scattering (DLS) results (Table S2). The successfully DNA-incorporated NSs were further demonstrated by their negatively charged surface (a zeta potential of −25.8 mV, Table S2) and their characteristic DNA absorbance of 260 nm (Figure S1). Then the ZnO-embedded RCA NSs (termed RCA-ZnONSs hereafter) were constructed by simply introducing ZnO nanoparticles (NPs, ∼5 nm in diameter, Figure S1 inset) into the RCA reaction mixture, followed by thoroughly washing with nuclease-free water. The homogeneously encapsulated ZnO could facilitate the sufficient supply of DNAzyme cofactors (Zn2+ ions) and stimulate the robust generation of ROS with therapeutic functions. The fast and efficient encapsulation of ZnO NPs into NSs is attributed to the electrostatic interaction between negatively charged DNA and positively charged ZnO NPs and a possible coordination interaction between DNA base and ZnO.39 The high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) investigation revealed that the RCA NSs maintained their sponge-like structure after ZnO incorporation (Figure 2c). Furthermore, the STEM-based EDS mapping and spectra confirmed an efficient ZnO encapsulation into the RCA NSs (Figures 2c, S2). As indicated by DLS, the ZnO NPs did not dramatically change the morphological features of NSs (Table S2). The increased zeta potential of RCA-ZnO-NSs (−12.6 mV), as compared with that of bare RCA NSs (−25.8 mV), also implies the successful incorporation of ZnO NPs into the RCA NSs (Table S2). The tightly packed inorganic−organic hybrid was anticipated to be more stable than conventional DNA nanostructures since the condensed structure was expected to protect the nucleic acids from enzymatic attack. Indeed, the as-prepared RCA-ZnO-NSs showed long-term colloidal stability under simulated physiological conditions (Figure S3). Moreover, the RCA-ZnO-NSs were treated with 10% human serum to study its serum resistance at 37 °C. The serum-treated NSs showed no obvious degradation, as revealed by gel electrophoresis (Figure S4), and retained their nanosponge structure (Figure S5). The extremely high stability of RCA-ZnO-NSs could maintain the integrity of DNA sequences, thus avoiding undesired drug leakage during circulation. The high surface area of our robust RCA-ZnO-NSs makes the nanohybrids an ideal drug (e.g., doxorubicin, DOX) carrier for therapeutic applications (Figure S6). The DOX loading capacity of RCAZnO-NSs was calculated to be 5 mmol·g−1, which is higher (1.3-fold) than the original RCA NSs (3.9 mmol·g−1). It is attributed to the ZnO-condensed RCA NS packing and the Zn2+-DOX chelate complex assembly.41,42 Note that the RCAassembled NSs are too small to be counted and also cannot be completely dried for quantification in case of adhesion and agglomeration; thus the amount of RCA NSs was determined through quantifying the representative DNA content by UV− vis spectroscopy. The facile construction of DOX@RCA-ZnO-NSs ensures the stimuli-responsive DNAzyme-driven drug release. Here the exogenous Zn2+ ions were introduced as prerequisite cofactors to initiate the DNAzyme-mediated fragmentation of NSs through the catalyzed cleavage of the DNA substrate. As shown in Figure 3a, the aptamer-encoded NSs stayed intact without Zn2+ ions, yet showed an efficient DNA cleavage in the presence of Zn2+-ion cofactors, which is consistent with the gel

fully accessible and uniform distribution of self-catabolic DNAzyme and cofactors. As a facile stimuli-responsive drug delivery system, the present intelligent DNAzyme-sufficient synergistic chemotherapy benefits efficient tumor-specific ablation without obvious recurrence and is anticipated to play an important role in tumor diagnosis and treatment.

RESULTS AND DISCUSSION Our functional DNA nanosponges were synthesized by polymerase-mediated rolling circle polymerization on a circular DNA template. Here the DNA template was integrated with the complementary sequence of the sgc-8c aptamer for cellspecific drug delivery and was also encoded with the complementary sequences of Zn2+-dependent DNAzyme and its all-DNA substrates for self-sufficient drug administration (Table S1). The phi29 DNA polymerase efficiently moves around the circular DNA template to continuously generate long concatemeric ssDNA with each repeated sequence of the sgc-8c aptamer recognition unit and tandem DNAzyme/ substrate trigger unit. The preparations of the circular ssDNA template and RCA product were verified by denaturing gel electrophoresis (Figure 2a). Meanwhile, the morphological feature of the as-obtained RCA product was investigated by scanning electron microscopy (SEM) (Figure 2b) and transmission electron microscopy (TEM) (Figure 2b inset).

Figure 2. (a) Denaturing gel electrophoresis characterization of the RCA process: (i) primer, (ii) template, (iii) primer and template, (iv) ligation of templates, (v) RCA products, (vi) DNAzymemediated hydrolysis of RCA NSs. (b) SEM characterization of the RCA NSs. Scale bar = 500 nm. Inset shows the corresponding TEM image. Scale bar = 100 nm. (c) STEM and EDS mapping of the as-generated RCA-ZnO-NSs. Scale bar = 100 nm. 5854


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Figure 3. (a) Denaturing PAGE analysis of the controlled RCA NS degradation. (b) DOX release profiles of DOX@RCA-ZnO-NSs pretreated with different pHs. (c) Confocal microscopic images of HeLa cells and Raji cells incubated with Cy5-labeled RCA-ZnO-NSs. Scale bar = 20 μm. (d) Determining the endocytic uptake of Cy5-labeled RCA-ZnO-NSs via CLSM. Lysosome was stained with Lysotracker Green; the nucleus was stained with Hoechst. Scale bar = 10 μm.

and ZnO NPs was acquired to be 4.3 μM:1 mM, which is sufficient to guarantee an efficient DNA-hydrolyzing DNAzyme reaction. Accordingly, the dissolved Zn2+ ions could specifically bind to DNAzyme to activate the DNA cleavage and the consequent NSs fragmentation for programmable drug release. As anticipated, the intercalated DOX shows an increased releasing rate with decreasing pH of the surrounding conditions (Figure 3b). For example, 7.4% of DOX was released at pH 7.4, while more than 95% of DOX was released at pH 5.0 within 4 h. Clearly, the insufficient DNAzyme cofactors could not promote the efficient DOX release at the neutral condition. In addition, the non-DNAzyme-simulated cumulative DOX release was also investigated by using DOX@ RCA (MT)-ZnO-NSs, where the active DNAzyme was substituted with a nonactive DNAzyme (Figure S10). Similarly, scarcely any DOX release was observed in neutral conditions, while a slightly higher DOX release was revealed at pH 5.0, which was mainly attributed to the DOX protonationmediated high aqueous solubility in an acidic environment.43,44 However, the present DOX release is much slower than that from DOX@RCA-ZnO-NSs, demonstrating the intrinsic DNAzyme-burst DOX release. From this perspective, the endogenously self-driven DOX@RCA-ZnO-NS therapeutic platform can achieve an enhanced intracellular drug release, but can also avoid premature drug release at neutral pH during circulation, which is definitely beneficial for their effective therapeutic applications. Note that our self-powered DNAzyme-driven system revealed a more robust feature, while the DNase-driven strategy showed a substantially decreased activity for disassembling NSs with exposure to a moderately high temperature (Figures S11 and S12). The smart self-catabolic DNAzyme-mediated disassembly of DOX@RCA-ZnO-NSs could be exploited as an efficient intracellular chemotherapeutic vehicle for delivering drugs into tumor cells. To enhance the tumor-specific delivery of NSs, the multivalent tandem chain of sgc-8c aptamers was

electrophoresis experiment (Figure 2a). The partial digestion of NSs generates a variety of ssDNA with defined lengths (n × template), while the full digestion of NSs leads to the production of ssDNA with only a unit length (1 × template). This DNAzyme-mediated DNA cleavage was further demonstrated by introducing the mutant RCA NSs (RCA (MT) NSs) encoded with a nonactive DNAzyme sequence that displayed no obvious cleavage with Zn2+-ion cofactors. The DNAzyme cleavage was also observed in nonaptamer-integrated NSs (RCA (NA) NSs), demonstrating the facile applicability of our DNAzyme strategy. The nonviolent DNAzyme degradation progressed smoothly with the majority of DNA digestion within 15 min and full DNA cleavage after 1 h of DNAzyme reaction (0.5 mM Zn2+ ions, Figure S7). This fast DNAzymestimulated degradation of RCA NSs could promote their efficient drug release after encapsulating therapeutic agents. The Zn2+-dependent DNAzyme cleavage was then extensively investigated by gel electrophoresis (Figure S8). Slight digestion of RCA NSs was observed for 0.25 mM Zn2+ ions, while most DNA sequences were cleaved by Zn2+ ions ranging from 0.5 to 1 mM. These results indicated that the DNAzyme reaction could be realized with high efficiency in a moderate concentration of Zn2+ ions, which was especially beneficial for their practical applications. The efficient DNAzyme-driven NSs fragmentation encouraged us to further explore the DNAzyme-simulated drug release from DOX@RCA-ZnO-NS therapeutic agents. Here the stimulated acidic microenvironment was introduced to dissolve the incorporated ZnO NPs, generating the supplementary Zn2+-ion cofactors to promote the DNAzymemediated DOX payload release. An inductively coupled plasma atomic emission spectrometric (ICP-AES) analysis demonstrated that ZnO NPs were completely dissolved in pH 5.0 (late endo/lysosomal pH), partially dissolved in pH ranging from 5.5 to 7.0, and nearly undissolved in physiological pH 7.4 (Figure S9). Note that the molar ratio between the RCA NSs 5855


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Figure 4. (a) Quantification of time-dependent cellular uptake for DOX@RCA (NA) NSs and DOX@RCA NSs as determined by flow cytometry; **p < 0.01, ***p < 0.001, and ****p < 0.0001. (b) In vitro cell viability of HeLa cells and (c) in vitro cell viability of Raji cells treated with DOX@RCA NSs and DOX@RCA (NA) NSs. (d) Confocal microscopic images of HeLa cells incubated with Cy5-labeled DOX@RCA-ZnO-NSs for different time intervals. Scale bar = 10 μm.

integrated to specifically recognize PTK7,45 which is overexpressed in many tumor cells (including CEM and HeLa cells). The multivalent binding affinity of tandem aptamer chains could substantially enhance the interactions between NSs and target cells.46 First, the selective internalization of NSs was investigated by confocal microscopy imaging of the Cy5incorporated RCA-ZnO-NSs (Figure 3c). Obviously, the Cy5labeled RCA-ZnO-NSs were effectively internalized by HeLa cells, yet were unable to be taken up by Raji cells (acute lymphoblastic leukemia B-cells) that showed negligible PTK7 expression. The specific interaction between sgc-8c and PTK7 was further demonstrated by using a competitive inhibition assay in Hela cells, where PTK7 receptors were initially blocked by an excess amount of free sgc-8c aptamers prior to their incubation with RCA-ZnO-NSs. An obviously lower cellular uptake of functional NSs was observed in HeLa cells (Figure S13), confirming the specific interaction between sgc8c aptamers and PTK7 receptors. In addition, the internalization pathway of NSs into HeLa cells was then investigated by selectively staining lysosomes with LysoTracker Green

(Figure 3d). It showed that most of the NSs were colocalized with lysosomes, indicating that NSs were specifically internalized by endocytosis.47 The efficient internalization of our DOX-incorporated NSs was further demonstrated by flow cytometry where the DOX internalization was quantitatively analyzed (Figures 4a and S14). The RCA NSs showed an enhanced DOX uptake in HeLa cells as compared with the RCA (NA) NSs, thus verifying the specifically sgc-8c-promoted receptor-mediated endocytosis into target cells. As expected, the aptamerintegrated NSs showed a substantially enhanced therapeutic performance as compared to the RCA (NA) NSs (Figure 4b). There was no therapeutic difference between DOX@RCA NSs and DOX@RCA (NA) NSs for Raji cells (Figure 4c), further demonstrating aptamer-mediated DOX delivery and cell treatment. Our multifunctional NS hybrids facilitated not only efficient drug delivery but also subsequent intracellular drug release, as revealed by the intracellular distribution of DOX in HeLa cells (Figure 4d). The internalized Cy5-labeled NSs showed a good colocalization with DOX after a 15 min 5856


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Figure 5. (a) Confocal microscopic images of intracellular ROS in the presence of RCA NSs or RCA-ZnO-NSs. Scale bar = 25 μm. (b) In vitro anticancer effects of RCA NSs, RCA-ZnO-NSs, DOX@RCA NSs, DOX@RCA (MT)-ZnO-NSs, and DOX@RCA-ZnO-NSs; **p < 0.01 and ****p < 0.0001. (c) Combination index (CI) values of DOX@RCA (MT)-ZnO-NSs and DOX@RCA-ZnO-NSs at different concentrations.

was estimated to be 19 μM, while that of DOX@RCA-ZnONS-treated cells was acquired to be 683 μM, which is adequately high to activate the DNAzyme-mediated disassembly of NSs (Figure S15). The concomitantly delivered ZnO-generated Zn2+ ions were examined by a Zn2+-specific fluorogenic probe, Zinquin ethyl ester. A faint blue fluorescence of a Zn2+-specific probe was revealed in intact nontreated HeLa cells, while a significantly enhanced fluorescence was shown in DOX@RCA-ZnO-NSs as well as ZnCl2-treated HeLa cells (2 h of incubation) (Figure S16). Thus, the encapsulated ZnO is indeed dissolved to Zn2+ ions in an acidic intracellular environment. In addition, the resulting Zn2+ ions could disrupt endosomes and induce apoptosis by ROS via a p53 pathway.51−53 The Zn2+-mediated generation of ROS was examined by dichlorofluorescin diacetate (DCFHDA), a nonfluorescent fluorescein analogue. The DCFH-DA probe could easily penetrate cellular membranes and then can be successively hydrolyzed by esterase and oxidized by ROS to produce highly green fluorescent dichlorofluorescein (DCF). As expected, no fluorescence of DCF was revealed in the RCA NS-treated HeLa cells, while a bright green fluorescence of DCF was predominantly observed in RCA-ZnO-NS-treated HeLa cells, implying an efficient production of intracellular ROS by Zn2+ ions (Figure 5a). The RCA-ZnO-NSs showed dose-dependent cytotoxicity to HeLa cells, while the bare RCA

incubation with HeLa cells, and the Pearson correlation coefficient was acquired to be 0.92. After a prolonged incubation of 30 min, the fluorescence of DOX became enhanced and separated from that of Cy5 with a reduced colocalization coefficient of 0.14. Thus, DOX was already released into the cytosol and partially internalized into the nucleus. Ultimately, a large proportion of DOX was accumulated in the nucleus with few colocalized DOX with NSs after 1 h (about 80% colocalization of DOX with the nucleus). These results indicate that the self-driven DOX@ RCA-ZnO-NS disassembly could facilitate effective payload release, resulting in an enhanced therapeutic efficacy of the system. The present NS carriers not only facilitated the efficient delivery of DOX but also simultaneously enabled the transferring of ZnO NP payloads into cells. The DNAzymesustained tumor-specific drug administration requires a sufficient supply of DNAzyme cofactors, which are especially important for the indispensable DNAzyme-mediated digestion of NSs. Although zinc is the second most abundant transition metal element in the human body,48 the accessible Zn2+ content is rather limited in picomolar amounts, as most Zn2+ ions are conjugated with other ligands,49,50 which is insufficient for activating our DNAzyme NS platform. ICP-AES measurements showed that the concentration of intracellular Zn2+ ions 5857


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Figure 6. (a) Dynamic biodistributions of free Cy5, Cy5-RCA (NA)-ZnO-NSs, and Cy5-RCA-ZnO-NSs. (b) Ex vivo fluorescent images of major tissues and tumors after free Cy5, Cy5-RCA (NA)-ZnO-NS, or Cy5-RCA-ZnO-NS administration. (c) Semiquantitative biodistribution of free Cy5, Cy5-RCA (NA)-ZnO-NSs, or Cy5-RCA-ZnO-NSs in nude mice determined by the average fluorescent intensity of major organs and tumors.

synergism in most cases, while the concomitantly DOX/ ZnO-encapsulated RCA NSs (DOX@RCA-ZnO-NSs) revealed a more obvious synergism, implying that DNAzyme plays an indispensable role in the synergistically enhanced combined chemotherapy. In particular, a strong synergism (CI = 0.1−0.3) could be identified for a higher dosage of DOX (>4 μM), which was possibly attributed to a more thorough destruction of RCA NSs under these conditions. This synergistically enhanced therapeutic effect of our compact NS nanohybrids was attributed to the dissolved Zn2+-mediated ROS chemotherapeutic operation and the concomitantly Zn2+DNAzyme-accelerated drug release. The detailed therapeutic performance of these different systems was further examined by flow cytometry, by which the apoptotic/necrosis cells were quantified after double-staining with an annexin V-FITC/PI apoptosis detection kit. As shown in Figures S17 and S18, the RCA NSs caused no damage to cells, which was consistent with the MTT assay (Figure 5b). Nevertheless, the apoptosis/necrosis ratios were acquired to be 10.9% and 21.0% for RCA-ZnO-NSs and DOX@RCA NSs, respectively. An additive 27.6% apoptosis was achieved for ZnO/DOX-loaded mutant DNAzymes NSs (DOX@RCA (MT)-ZnO-NSs). As expected, the DOX@RCA-ZnO-NSs displayed the highest apoptosis of 37.4%, which is attributed to the cooperatively ZnO-participating DNAzyme fragmentation of NSs. Meanwhile, the cell viability of each system was investigated by the live/dead staining method, where live and dead cells were stained with calcein AM and propidium iodide (PI), respectively (Figure S19). Similarly, the intuitive identification of the DOX@RCA-ZnO-NS therapeutic system was revealed by the highest percentage of dead cells. Inspired by the excellent therapeutic performance in vitro, we further investigated the multifunctional DOX@RCA-ZnONSs nanosystem in vivo. A HeLa-tumor-bearing xenograft was established to investigate the biodistribution of NSs nanohybrids. Here the Cy5-labeled RCA-ZnO-NSs were injected intravenously at a single dosage for bioimaging, and the wholebody distribution of Cy5 was obtained via an IVIS imaging system, which enabled us to monitor its real-time biodis-

NSs showed no cytotoxicity (Figure 5b), indicating a biocompatible feature of our NS carriers and a moderate therapeutic function of RCA-ZnO-NSs. After confirming the respective therapeutic functions of the DOX and ZnO constructs, we then extensively explored the overall in vitro antiproliferation efficacy of our NS-based therapeutic platform. As shown in Figure 5b, all of these DOX@RCA NS, RCA-ZnO-NS, DOX@RCA (MT)-ZnO-NS, and DOX@RCA-ZnO-NS systems showed an increased therapeutic performance with an elevated dosage of each NS agent. As compared with the control RCA NSs, the RCA-ZnONSs showed a slightly enhanced therapeutic performance (23.4% at 32 μg/mL of RCA NSs), which is attributed to the Zn2+-induced ROS generation. The therapeutic performance of DOX@RCA NSs is higher (45.0%) than that of RCA-ZnONSs, implying that the DOX therapeutic agent is more efficient than the ROS agent. Moreover, the DOX@RCA (MT)-ZnONSs, encoded with tandem nonactive DNAzyme sequences, showed a substantially promoted therapeutic performance (60.2%), yet it was still much lower than our compact DOX@ RCA-ZnO-NS system (84.6%). This is mainly attributed to an enhanced disassociation of ZnO NPs that immediately stimulates a high localized concentration of Zn2+ ions for stimulating the DNAzyme-burst efficient administration of chemotherapeutic agents. Since ZnO concomitantly facilitated the ROS chemotherapeutic operation and the DNAzyme-burst DOX release, we anticipated that there existed a correlation between DOX and ZnO agents in this compact NS-based platform. This inherent relevancy was assessed by combination index (CI) parameters that were acquired through the Chou−Talalay approach by using CalcuSyn software.54 The CI value indicates the intrinsic drug interactions: CI = 1 indicates an addictive effect, CI > 1 indicates an antagonistic effect, and CI < 1 indicates a synergistic effect. The lower the CI value, the stronger the synergistic effect. Based on the hypothesis that RCA NSs and RCA NSs (MT) induced no cytotoxicity, the CI values were plotted against the dosages of drugs (Figure 5c). The DOX@RCA (MT)-ZnO-NSs showed insignificant 5858


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Figure 7. (a) Administration schedule of DOX@RCA-ZnO-NSs in the subcutaneous HeLa tumor model. (b) Tumor growth curve, (c) representative tumor images, (d) body weight profile, and (e) TUNEL analysis of tumors after treatment with (i) PBS, (ii) free DOX, (iii) DOX@RCA NSs, (iv) DOX@RCA (MT)-ZnO-NSs, (v) DOX@RCA (NA)-ZnO-NSs, and (vi) DOX@RCA-ZnO-NSs. Scale bar = 100 μm. *p < 0.05 and **p < 0.01.

tribution and to provide guidance for tumor-specific chemotherapy. As shown in Figure 6a, the intravenously injected free Cy5 was cleared out quickly with no distinct fluorescence in the tumor site at 24 h postinjection. However, the fluorescent Cy5-RCA-ZnO-NSs were initially distributed among the whole body and then mainly transferred into the liver region. Subsequently, the Cy5-RCA-ZnO-NSs accumulated in the tumor region at 2 h postinjection and reached a maximum value at 12 h postinjection. Meanwhile, a much lower accumulation of Cy5-RCA (NA)-ZnO-NSs was shown in the tumor site. Thus, the significantly enhanced retention of our compact Cy5-RCA-ZnO-NSs was attributed to the aptamerfavored cellular uptake as well as the EPR effect. In addition, the ex vivo biodistributions of free Cy5, Cy5-RCA (NA)-ZnONSs, and Cy5-RCA-ZnO-NSs were quantitatively evaluated in tumors and major organs (heart, liver, spleen, lung, kidney), which were finally extracted and imaged. As shown in Figure 6b and c, the fluorescence signal of the Cy5-RCA-ZnO-NS-

treated tumor group was 3.8-fold and 2.1-fold higher than that of the free Cy5-treated and Cy5-RCA (NA)-ZnO-NS-treated tumor groups, respectively. This further demonstrated the robust and aptamer-mediated tumor-addressable characters of the compact Cy5-RCA-ZnO-NS system. Also, the fluorescence of the Cy5-RCA-ZnO-NS-treated tumor group was 17.9-fold, 5.6-fold, 6.4-fold, 14.4-fold, and 5.3-fold higher than that of the heart, liver, spleen, lung, and kidney, respectively. These results convincingly proved the superior tumor accumulation of our in vivo DOX@RCA-ZnO-NS administration approach. After confirming the good biocompatibility and tumorspecific accumulation of DOX@RCA-ZnO-NSs in vivo, we attempted to evaluate their antitumor effect in the HeLa mouse model (with a tumor volume of ∼50 mm3). The tumorbearing mice were randomly divided into six groups (n = 4) and respectively treated with phosphate-buffered saline (PBS), free DOX, DOX@RCA NSs, DOX@RCA-ZnO-NSs, DOX@ RCA (MT)-ZnO-NSs, and DOX@RCA (NA)-ZnO-NSs via 5859


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distinct inflammation or disorganization was observed in these main organs in DOX@RCA-ZnO-NS-treated mice. The histological images of these organs were the same as those of healthy mice, which again confirmed the long-term histocompatibility of our DOX@RCA-ZnO-NS therapeutic platform.

tail-vein injection according to the administration procedure, as schematically illustrated in Figure 7a. The changes of the relative tumor volume were plotted to investigate the in vivo therapeutic effects (Figure 7b). In contrast to free DOX, the tumor growth was suppressed after successive administration of DOX@RCA NSs, indicating a passive accumulation of these therapeutic reagents endowed by the intrinsic EPR effect of NSs and an additional recognition effect of multivalent sgc-8c aptamers. Yet the most significant antitumor effect was revealed in DOX@RCA-ZnO-NS-treated mice, of which the tumor volume was only 1.8-fold larger than the initial tumor after 21 days of treatment (Figure 7b and c). Meanwhile, the tumor increased to 2.7-fold larger in volume for the DOX@ RCA (NA)-ZnO-NS-treated mice, where the tumor-targeted drug delivery is only based on the EPR effect. This indicates that the tandem multivalent aptamer recognition unit affects the therapeutic performance. Moreover, the tumor increased to 4.4-fold larger in volume for the DOX@RCA (MT)-ZnO-NStreated mice, where the mutant DNAzyme failed to trigger an efficient drug release. It should be noted that the antitumor performance of our DOX@RCA (MT)-ZnO-NS system (4.4fold) is promoted as compared with that of the DOX@RCA NS system (6.3-fold), which is attributed to the extra encapsulated ZnO-generated ROS. The significantly improved therapeutic effect of our DOX@RCA-ZnO-NSs was attributed to the synergistic effect of Zn2+-mediated ROS generation and Zn2+-DNAzyme-facilitated drug release. As a key indicator of systemic cytotoxicity, the body weight variation was monitored during these therapeutic procedures. Neither body weight loss nor apparent abnormality was noticed in all groups (Figure 7d), indicating the negligible systemic toxicity of DOX@RCAZnO-NSs in vivo. Histological examination was performed to further evaluate the in vivo antitumor activity of our NS agents. Hematoxylin and eosin (H&E) staining was employed in tumor tissue at the end of each in vivo therapy. As shown in Figure S20, the tumor tissue exhibited severe destruction after DOX@RCA-ZnO-NS treatment, but only limited necrosis or apoptosis was observed in DOX@RCA (NA)-ZnO-NS- and DOX@RCA (MT)-ZnONS-treated tumors. A further terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was also applied to study the apoptosis level of tumor cells as treated above (Figure 7e), which agreed well with the above discoveries. In addition, hematology and blood biochemical analyses were carried out to demonstrate our biocompatible NSs. As anticipated, hematology analysis indicated that the measured parameters (red blood cell count (RBC), white blood cell count (WBC), platelet count (PLT), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemoglobin (MCH)) were within normal ranges (Figure S21) and showed no obvious trend associated with various treatments. As compared with the healthy normal group, the biochemical indexes of the DOX@RCA-ZnO-NS-treated group showed no distinct increase in aspartate transaminase (AST), alanine transaminase (ALT), creatinine (CREA), blood urea nitrogen (BUN), albumin (ALB), total protein (TP), total bilirubin (TBIL), and globulin (GLOB), confirming a minimal toxicity of our DOX@RCA-ZnO-NSs agents (Figure S21). Moreover, the major organs, such as the heart, liver, spleen, lung, and kidney, were collected after three weeks of therapy and stained with H&E for histological examination (Figure S22). No

CONCLUSIONS In summary, we have developed a nonviolent self-sustained drug delivery system consisting of built-in DNAzyme NSs and pH-responsive ZnO NPs. The robust DNAzyme NSs were encoded with a multivalent tandem aptamer sgc-8c sequence to facilitate the efficient and tumor-specific drug administration into cells, where the intracellular acid-stimulated Zn2+ ions could readily stimulate the efficient and accurate fragmentation of the as-constructed DNAzyme NSs for programmable drug release. As a general and versatile drug delivery platform, the present nonviolent DNAzyme NS system shows a synergistically enhanced therapeutic effect, originating from the coassembled ZnO NPs and DOX constructs. The exquisite self-catabolic DNA nanostructures integrated with therapeutic ZnO NPs and self-hydrolyzing DNAzymes provide insights for the development of DNA-based therapeutic platforms, thus showing extensive prospects for the construction of other multifunctional theranostic platforms by encoding with other additional therapeutic sequences. EXPERIMENTAL SECTION Materials. Zinc acetate, magnesium acetate, sodium hydroxide, 3aminopropyltriethoxysilane (APTES), DCFH-DA, calcein-AM solution, and propidium iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA). All oligonucleotides were synthesized and HPLCpurified by Sangon Biotechnology Co., Ltd. (Shanghai, China). The sequences of oligonucleotides are listed in Table S1. Quick ligation kit (M2200S) and phi29 DNA polymerase were obtained from New England Biolabs (Ipswich, MA, USA). dNTPs were purchased from Takara Bio (Kusatsu, Japan), and Cy5-dCTP was obtained from GE Healthcare (Pittsburgh, PA, USA). Doxorubicin hydrochloride (98%) was obtained from Aladdin Reagents (Shanghai, China). Hoechst 33342 and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Dulbecco’s modified Eagle’s medium, RPMI 1640 medium, penicillin−streptomycin, and trypsin were obtained from Gibco (NY, USA). Zinquin ethyl ester was obtained from Abcam (Cambridge, MA, USA). Annexin V-FITC/PI double staining kit was purchased from BestBio Biotechnology Co., Ltd. (Shanghai, China). The one-step TUNEL apoptosis assay kit was obtained from KeyGEN (Nanjing, China). Instrumental Methods. Scanning electron microscopy (Zeiss Merlin Compact, Germany) and transmission electron microscopy (Hitachi HT-7700 microscope, Japan) were used to investigate the morphology of nanoparticles. Elemental mapping of obtained RCAZnO-NSs was performed on a Talos F200S microscope. The hydrodynamic size and zeta potential of the nanoparticles were measured by a Zetasizer Nano ZS (Malvern, UK). ICP-AES (IRIS Intrepid II XSP, USA) was used for quantifying Zn2+ ions. A Cary 100 UV−vis spectrophotometer (Varian Inc.) and a Cary Eclipse spectrometer (Varian Inc.) were utilized for UV−vis spectra and fluorescence spectra, respectively. The gels were imaged with FluorChem FC3 (ProteinSimple, USA). Cytofluorimetric analyses were obtained via a flow cytometer (BD FACSVerse, CA, USA). Cell and mice imaging was performed with a Leica SP8 confocal microscope and an IVIS Imaging System, respectively. Cell Culture and Animal Models. HeLa cells were cultured in T25 cm2 flasks using Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) containing 10% fetal bovine serum (FBS, PAN) and 100 U· 5860


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ACS Nano mL−1 penicillin−streptomycin in a humidified atmosphere at 37 °C with 5% CO2. Raji cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 100 U·mL −1 penicillin− streptomycin. Animal experiments were approved by the Animal Care and Use Committee of Wuhan University. Balb/c mice (5 weeks old) bearing HeLa tumors were subcutaneously inoculated with 5 × 106 cells on the right side of the back. Tumor volume was calculated according to the following equation: V = (L × W2)π/6 (L and W are the longest and shortest diameters of the tumor, respectively). Synthesis of RCA-ZnO-NSs and DOX Loading. ZnO NPs were synthesized as follows. Zinc acetate (1.0 mmol) and magnesium acetate (0.1 mmol) were mixed with 30 mL of anhydrous ethanol; then the mixture was heated to 70 °C and stirred vigorously until dissolution. Subsequently, 50 mg of NaOH was dissolved in 8 mL of refluxing ethanol in a separate flask. These solutions were then cooled in an ice bath and injected into the solution containing zinc acetate and magnesium acetate. The resultant mixture was stirred thoroughly for 8 h, and ZnO NPs were then precipitated. Afterward, the obtained ZnO NPs were redispersed in anhydrous dimethylformamide (DMF) (15 mL) under sonication, and 50 μL of APTES was then added and stirred at 120 °C for 15 min. The amine-functionalized ZnO NPs were washed with DMF three times and dried in a vacuum at room temperature. For RCA NS synthesis, linear templates (2 μM) were ligated with 400 U·μL−1 quick ligase at 25 °C for 30 min in the presence of 4 μM primer. To construct RCA NSs, circularized template−primer complex (containing 1 μM template) was added to the reaction mixture containing 33 mM Tris-acetate pH 7.9, 10 mM Mg-acetate, 66 mM K-acetate, 0.1% Tween 20, 1 mM dithiothreitol, 1 mM of each dNTP, and 1 U·μL−1 phi29 polymerase. The proposed RCA reaction was performed at 30 °C for 3 h and stopped by inactivating polymerase at 95 °C for 10 min. RCA-ZnO-NSs were synthesized by adding extra 10 mM ZnO NPs in the RCA mixture. The resultant products were washed with ultrapure water and centrifuged at 8000 rpm for 15 min three times and then were sonicated and pipetted several times to redisperse the NSs. For DOX intercalation, DOX was mixed with RCA-ZnO-NSs (RCA NSs: 1 mg·mL−1) overnight and the mixture was purified by dialysis against deionized water in an 8 kDa dialysis unit. The loading capability of DOX was determined via UV−vis spectroscopy, and the loaded DOX was calculated by the following equation: cDOX = (nfeed − cdialysate × Vdialysate)/VRCA, where nfeed is the total amount of feed DOX, cdialysate is the DOX concentration of the dialysate, and Vdialysate and VRCA are the volume of the exterior dialysate and the original RCA reaction, respectively. Zn2+-Responsive Self-Sustained NS Fragmentation for Programmable Drug Release. RCA-ZnO-NSs, RCA (NA)-ZnONSs, and RCA (MT)-ZnO-NSs were preincubated in acetate buffer (pH 5.0) for 30 min; then the buffer was adjusted to 10 mM HEPES (pH 7.4, 100 mM NaCl) and incubated for 2 h. For kinetic analysis, these NS reactions were stopped by adding 95% formamide (20 mM EDTA). The self-cleavage reaction of RCA NSs was initiated by different concentrations of Zn2+ (0.25, 0.5, 0.75, and 1 mM) at 37 °C. The DOX release kinetics were evaluated through a dialysis method. The purified DOX@RCA-ZnO-NSs or DOX@RCA (MT)ZnO-NSs was preincubated for 30 min at pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4, respectively; then they were separately placed into dialysis bags (8 kDa) and incubated in 5 mL of HEPES (10 mM, pH 7.4, 100 mM NaCl). The medium outside the dialysis bag was collected and analyzed with UV−vis spectroscopy at different time intervals (0, 0.5, 1, 2, 3, and 4 h). Stability Study of the Compact NSs. For the stability assay, 1 mg·mL−1 RCA-ZnO-NSs were added into 10% human serum and incubated at 37 °C for 0, 2, 4, 6, 8, and 10 h, respectively. Then these samples were heat-inactivated and analyzed by agarose gel electrophoresis. For the thermal stability assay, RCA-ZnO-NSs were preincubated at 20, 40, or 60 °C for 1 h; then the responsive DOX release was monitored as well. For comparison, DNase I and RCA NSs were preincubated as well; then DNase I and DOX were coloaded onto

RCA NSs, and DOX release from the resultant mixture was monitored. Specific Cellular Uptake of DOX@RCA-ZnO-NSs. For the celltargeting study, Cy5-labeled RCA-ZnO-NSs were incubated with HeLa cells or Raji cells at 4 °C for 2 h, followed by washing with RPMI 1640 medium twice. Cells were finally suspended in cold PBS prior to flow cytometric analysis or confocal imaging. For the inhibition assay, HeLa cells were pretreated either with or without excess free aptamer sgc-8c (500 nM) at 4 °C for 30 min, with intermediate washing three times. These cells were further incubated with Cy5-labeled RCA-ZnO-NSs (5 μg·mL−1) at 4 °C for 2 h. For lysosome staining, HeLa cells were incubated with Cy5-labeled RCAZnO-NSs for 10 min; then 5 μM Lysotracker Green was added and incubated for another 5 min. Then the cells were observed immediately with confocal laser scanning microscopy (CLSM) after washing with cold PBS twice. Promoted Drug Release from NSs. To monitor the intracellular DOX release, Cy5-labeled DOX@RCA-ZnO-NSs were added to the seeded HeLa cells on glass-bottomed microscope dishes for 15 min, 30 min, and 1 h, respectively. At the predetermined time intervals, the cells were washed with cold PBS twice and stained with 1× Hoechst 33342 for 5 min. Then the cells were washed again and visualized with CLSM immediately. Intracellular Degradation of ZnO NPs. For intracellular Zn2+ quantification, Cells were seeded in 125 mm2 culture dishes and treated with DOX@RCA-ZnO-NSs or PBS after being grown to 80% confluency. Then the cells were washed three times and collected for ICP-AES quantification. For intracellular Zn2+ imaging, Zinquin ethyl ester was applied to image the intracellular Zn2+ ions. DOX@RCA-ZnO-NSs or ZnCl2 (RPMI 1640 medium with 10% FBS) was added to the HeLa cells and incubated for 2 h at 37 °C. Then the cells were washed with incubation buffer three times and incubated with 25 μM Zinquin ethyl ester for another 30 min. Prior to CLSM, the cells were washed with bovine serum albumin (BSA)-free incubation buffer three times. Intracellular ROS Imaging. HeLa cells were incubated with RCA NSs or RCA-ZnO-NSs for 4 h; then the cells were washed and incubated with DCFH-DA instead. After 4 h, the cells were washed again and incubated for another 12 h before confocal imaging. Fluorescent imaging of DCF was done with 488 nm excitation and 510−540 nm emission. Cell Viability Test. HeLa cells were seeded on 96-well plates at a density of 5 × 103 cells per well and grown to 80% confluency before treatment. The cells were washed with PBS and respectively treated with RCA NSs, RCA (NA) NSs, DOX@RCA NSs, RCA-ZnO-NSs, DOX@RCA (MT)-ZnO-NSs, or DOX@RCA-ZnO-NSs for 24 h. The cells were then washed with PBS, and 100 μL of 0.5 mg·mL−1 MTT was added to each well and further incubated in the dark for 4 h. Finally, the medium was removed and the generated purple formazan crystals were dissolved by DMSO to acquire the absorbance of 570 nm using a microplate reader. The cell viability was calculated as follows: cell viability = (ODTreated − ODBlank/ODControl − ODBlank) × 100%. Apoptosis Assays by Annexin V/PI Staining and Fluorescent Staining of Live/Dead Cells. HeLa cells were seeded in sixwell plates (5 × 105 cells per well) and then treated with PBS, RCA NSs, RCA-ZnO-NSs, DOX@RCA NSs, DOX@ RCA (MT)-ZnONSs, or DOX@RCA-ZnO-NSs for 24 h. All cells were harvested and stained with an FITC-annexin V/PI double-staining kit. Finally, these samples were analyzed immediately by flow cytometry within 1 h. For fluorescent staining, these cells were washed with PBS gently and stained with a calcein-AM/PI double-staining kit. Live (490 nm of excitation, emission range: 505−530 nm) and dead (545 nm of excitation, emission range: 560−590 nm) cells were imaged under a confocal microscope. Fluorescence Imaging in Vivo/ex Vivo. For the biodistribution investigation, HeLa tumor-bearing mice were injected with free Cy5, Cy5-labeled RCA (NA)-ZnO-NSs, or Cy5-labeled RCA-ZnO-NSs through a tail vein. At 2, 6, 12, and 24 h postadministration, the in vivo fluorescent images were obtained. At 24 h postinjection, the mice 5861


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ACS Nano were euthanized, and the internal organs (heart, liver, spleen, lung, kidney) and tumor tissues were harvested and subjected to ex vivo imaging as well as semiquantitative biodistribution analysis. In Vivo Tumor Growth Inhibition. When the tumor size reached 50 mm3, the balb/c nude mice were divided into six groups (n = 4 per group) randomly and treated as follows: (i) PBS, (ii) free DOX, (iii) DOX@RCA NSs, (iv) DOX@RCA (MT)-ZnO-NSs, (v) DOX@ RCA (NA)-ZnO-NSs, and (vi) DOX@RCA-ZnO-NSs. The mice in each group were intravenously injected with different formulations at a DOX concentration of 2 mg·kg−1 (100 μL) except for the control group. All mice groups were administrated twice a week for 3 weeks. The weight of the mice and the volume of the tumors were measured every 3 days until the completion of treatment. Finally, the mice were euthanized, and the tumors were dissected and fixed in 10% neutral buffered formalin. After 6 h, dissected tumors were embedded in paraffin blocks and processed into sections with a thickness of 4 μm. After deparaffinization, tumor sections were stained with H&E or TUNEL according to the manufacturer’s protocol, and the slices were visualized with the microscope. Assessment of Biocompatibility. To evaluate the biocompatibility of DOX@RCA-ZnO-NSs, the blood samples were collected after euthanization using a standard ocular vein blood collection technique, followed by hematology analysis and blood biochemical assay. The major organs, including heart, lung, liver, kidney, and spleen, and tissue sections (4 μm) were collected and stained with H&E, and the tissue damage was examined with a light microscope.

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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/acsnano.9b01589. DNA sequences, characterization of NSs and DOX intercalation, demonstration of DNAzyme-mediated RCA NS cleavage and DOX release, acid-responsive ZnO dissociation, therapeutic effects of DOX@RCAZnO-NSs in vitro and in vivo (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Xiaoqing Liu: 0000-0002-1309-5454 Fuan Wang: 0000-0002-3063-2485 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is supported by National Natural Science Foundation of China (21503151, 81602610), National Basic Research Program of China (973 Program, 2015CB932601), and Jiangsu Provincial Natural Science Foundation of China (BK20161248, BK20160381). REFERENCES (1) Brannon-Peppas, L.; Blanchette, J. O. Nanoparticle and Targeted Systems for Cancer Therapy. Adv. Drug Delivery Rev. 2012, 64, 206− 212. (2) Sun, T.; Zhang, Y. S.; Pang, B.; Hyun, D. C.; Yang, M.; Xia, Y. Engineered Nanoparticles for Drug Delivery in Cancer Therapy. Angew. Chem., Int. Ed. 2014, 53, 12320−12364. (3) 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. 5862


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Nonviolent Self-Catabolic DNAzyme Nanosponges for Smart

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