Nonviolent Self-Catabolic DNAzyme Nanosponges for Smart

Subscriber access provided by Bethel University

Article

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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b01589 • Publication Date (Web): 01 May 2019 Downloaded from http://pubs.acs.org on May 1, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Nonviolent Self-Catabolic DNAzyme Nanosponges for Smart Anticancer Drug Delivery Jing Wang,1 Huimin Wang,1 Hong Wang,1 Shizhen He,1 Ruomeng Li,1 Zhao Deng,2 Xiaoqing Liu,1 Fuan Wang*1 1

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China 2 State

Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan

University of Technology, Wuhan 430072, P. R. China * To whom correspondence should be addressed. E-mail: [email protected]

ACS Paragon Plus Environment

1

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

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 self-sufficient DNAzyme-driven drug delivery system consisting of the rolling circle polymerized DNAzymesubstrate 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 ROS generators. The supplement Zn2+ cofactors mediated the nonviolent DNAzymecatalyzed 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 NSs 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


2

Page 3 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Nanotechnology is changing the therapeutic landscape in terms of diagnosis, treatment, and prognosis.1-3 Albeit the exogenous organic or inorganic nanomaterials are extensively studied, they are challenged by non-targeted cytotoxicity and accumulation, as well as inflexible preparations and post-modifications.4-9 Recently, the endogenously generated biomaterials, whose catabolite can be assimilated or eliminated by the hosting body, have received extensive attentions for biomedical researches.10-14 Among them, DNA biomaterials are particularly attractive due to their intrinsic biocompatibility and low-immunogenicity features. The specific and predictable WatsonCrick base-pairing of DNA contributes 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 engineering of diverse DNA assemblies, especially the 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 large-scale DNA nanoassemblies with desired built-in functionalities. Recently, the 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 DNA and immunostimulatory CpG) for tumorspecific 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 NSs-mediated therapeutic strategies, the efficiency and programmability of these drug administration remain the critical hurdles for


3

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 37

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-responsibility and disassembly functionalities for on-demand NSs-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 the 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 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 NSs nanocarriers with auxiliary Zn2+ ions cofactors. The all-DNA-based DNAzyme is more simple and robust under complicated intracellular environment, 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 the self-sustained drug delivery. Especially,


4

Page 5 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

the moderate and accurate self-sacrificing DNAzyme NSs could minimize or even prevent the undesired toxicity and severe side effects upon their systemic administrations. Herein, we fabricated compact and hierarchical ZnO-encapsulated DNAzyme NSs with multiple built-in functionalities for intelligent and self-sufficient cancer therapy (Figure 1). The RCAgenerated multivalent sgc-8c aptamers can specifically recognize protein tyrosine kinase 7 (PTK7) of cancer cells, thus enables the precise delivery of various therapeutic agents through NSs nanocarriers. The RCA-assembled DNA/ZnO NSs stay in a stabilized state at normal physiological condition through potent electrostatic and coordination interactions, yet drive the efficient dissolution of ZnO in acidic endo/lysosomal microenvironment.39,40 This leads to the supplement of 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 sparkle the effective and programmable administration of encapsulated chemotherapeutic drugs into cytosol, originating from the 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 the 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 (NSs) were synthesized by polymerase-mediated rolling circle polymerization on a circular DNA template. Here the DNA template was integrated with the complementary sequence of sgc-8c aptamer for cell-specific 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


5

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 37

around the circular DNA template for continuously generating long concatemeric ssDNA with each repeated sequence of sgc-8c aptamer recognition unit, and tandem DNAzyme/substrate trigger unit. The preparations of circular ssDNA template and RCA product were verified by denaturing gel electrophoresis (Figure 2a). Meanwhile, the morphological feature of the asobtained RCA product was investigated by SEM (Figure 2b) and TEM (Figure 2b inset). The physically homogeneous RCA NSs were achieved with ~220 nm in diameter and narrow size distribution, which was consistent with the DLS results (Table S2). The successfully DNAincorporated 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-ZnO-NSs 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 reactive oxygen species (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 its sponge-like structure after ZnO incorporation (Figure 2c). Furthermore, the STEMbased EDS mapping and spectra confirmed an efficient ZnO encapsulation into the RCA NSs (Figure 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


6

Page 7 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 for studying its serum resistance at 37 °C. The serum-treated NSs showed no obvious degradation as revealed by gel electrophoresis (Figure S4), and retained its 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 circulations. The high surface area of our robust RCA-ZnO-NSs makes the nanohybrids an ideal drug (e.g., DOX) carrier for therapeutic applications (Figure S6). The DOX loading capacity of RCA-ZnO-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 NSs package and the Zn2+-DOX chelate complex assembly.41,42 Noted that the RCA-assembled 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 DNAzymedriven 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 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


7

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 37

the gel 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 furtherly demonstrated by introducing the mutant RCA NSs (RCA (MT) NSs) encoded with nonactive DNAzyme sequence that displayed no obvious cleavage with Zn2+-ion cofactors. The DNAzyme cleavage was also observed in non-aptamer-integrated NSs (RCA (NA) NSs), demonstrating the facile applicability of our DNAzyme strategy. The nonviolent DNAzyme degradation was progressed smoothly with majority DNA digestion within 15 min, and full DNA cleavage after one-hour 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 furtherly explore the DNAzyme-simulated drug release from DOX@RCA-ZnO-NSs therapeutic agents. Here the stimulated acidic microenvironment was introduced to dissolve the incorporated ZnO NPs, generating the supplementary Zn2+-ions cofactors to promote the DNAzyme-mediated 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). Noted that the molar ration between the RCA NSs and ZnO NPs was acquired to


8

Page 9 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 for pH 7.4 while more than 95% of DOX was released for 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 non-active DNAzyme (Figure S10). Similarly, scarcely any DOX release was observed in neutral condition, while a slightly higher DOX release was revealed at pH 5.0, which was mainly attributed to the DOX protonationmediated high aqueous solubility at 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-NSs 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. Noted 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 (Figure 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 integrated to specifically recognize protein tyrosine kinase 7 (PTK7),45 which was


9

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

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 Firstly, the selective internalization of NSs was investigated by confocal microscopy imaging of the Cy5-incorporated RCA-ZnO-NSs (Figure 3c). Obviously, the Cy5-labeled RCAZnO-NSs were effectively internalized by HeLa cells, yet were unable to be uptake 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 sgc8c 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 sgc-8c 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 furtherly demonstrated by flow cytometry where the DOX internalization was quantitatively analyzed (Figure 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 aptamer-integrated NSs showed a substantially enhanced therapeutic performance as compared to the RCA (NA) NSs (Figure 4b). There showed no therapeutic difference between DOX@RCA NSs and DOX@RCA (NA) NSs for Raji cells (Figure 4c), furtherly demonstrating aptamer-mediated DOX delivery and cells treatment. Our multifunctional NSs hybrids facilitated not only the efficient drug delivery but also the subsequent intracellular drug release, as revealed


10

Page 11 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 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 is getting 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 nucleus with few colocalized DOX with NSs after 1 h (about 80% colocalization of DOX with nucleus). These results indicate that the self-driven DOX@RCA-ZnO-NSs disassembly could facilitate effective payload release, resulting in an enhanced therapeutic efficacy of the system. The present NSs carriers not only facilitated the efficient delivery of DOX, but also simultaneously enabled the transferring of ZnO NPs payloads into cells. The DNAzyme-sustained 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 amount for most of Zn2+ ions are conjugated with other ligands,49,50 which is insufficient for activating our DNAzyme NSs platform. ICP-AES measurements showed that the concentration of intracellular Zn2+ ions was estimated to be 19 μM while that of DOX@RCA-ZnO-NSs-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-generate Zn2+ ions were examined by a Zn2+-specific fluorogenic probe, zinquin ethyl ester. A faint blue fluorescence of Zn2+-specific probe was revealed in intact non-treated 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


11

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

indeed dissolved to Zn2+ ions in acidic intracellular environment. In addition, the resulting Zn2+ ions could disrupt endosome and induce apoptosis by reactive oxygen species (ROS) via a p53 pathway.51-53 The Zn2+-mediated generation of ROS was examined by dichlorofluorescin diacetate (DCFH-DA), a nonfluorescent fluorescein analog. 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 NSs-treated HeLa cells while a bright green fluorescence of DCF was predominantly observed in RCA-ZnO-NSs-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 NSs showed no cytotoxicity (Figure 5b), indicating a biocompatible feature of our NSs carriers and a moderate therapeutic function of RCA-ZnONSs. After confirming the respective therapeutic functions of the DOX and ZnO constructs, we then extensively explored the overall in vitro antiproliferation efficacy of our NSs-based therapeutic platform. As shown in Figure 5b, all of these DOX@RCA NSs, RCA-ZnO-NSs, DOX@RCA (MT)-ZnO-NSs, and DOX@RCA-ZnO-NSs systems showed an increased therapeutic performance with an elevated dosage of each NSs agent. As compared with the control RCA NSs, the RCA-ZnO-NSs 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-ZnO-NSs, implying that the DOX therapeutic agent is more efficient than the ROS agent. Moreover, the DOX@RCA (MT)-ZnONSs, encoding with tandem non-active DNAzyme sequences, showed a substantially promoted therapeutic performance (60.2%), yet it was still much lower than our compact DOX@RCA-ZnO-


12

Page 13 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

NSs 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 DNAzymeburst efficient administration of chemotherapeutic agent. Since the ZnO concomitantly facilitated the ROS chemotherapeutic operation and the DNAzyme-burst DOX release, we anticipated that there existed a correlationship between DOX and ZnO agents in this compact NSs-based platform. This inherent relevancy was assessed by Combination Index (CI) parameters that were acquired through the Chou-Talalay approach by using a CalcuSyn software.54 The CI value indicates the intrinsic drug interactions: CI=1 indicates an addictive effect, CI>1 indicates an antagonistic effect, and CI4 μM), which was possibly attributed to a more thorough destruction of RCA NSs under these conditions. This synergistically enhanced therapeutic effect of our compact NSs 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 furtherly examined by flow cytometry, by which the apoptotic/necrosis cells were quantified after double-staining with Annexin V-FITC/PI apoptosis detection kit. As shown in Figure S17 and S18, the RCA NSs exposed no damage to cells, which was consistent with the MTT assay (Figure 5b). Nevertheless,


13

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 37

the apoptosis/necrosis ratios were acquired to be 10.9% and 21.0% for RCA-ZnO-NSs and DOX@RCA NSs, respectively. An addictive 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-participated DNAzyme-fragmentation of NSs. Meanwhile, the cell viability of each system was investigated by 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-NSs 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-ZnO-NSs nanosystem in vivo. A HeLa-tumor-bearing xenograft was established to investigate the biodistribution of NSs nanohybrids. Here the Cy5-labeled RCAZnO-NSs were injected intravenously at a single dosage for bioimaging, and the whole-body distribution of Cy5 was obtained via an IVIS imaging system which enabled us to monitor its realtime biodistribution 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 tumor site after 24 h of postinjection. However, the fluorescent Cy5-RCA-ZnONSs were initially distributed among the whole body and then mainly transferred into the liver region. Subsequently, the Cy5-RCA-ZnO-NSs accumulated in tumor region after 2 h postinjection and reached a maximum value after 12 h postinjection. Meanwhile, a much lower accumulation of Cy5-RCA (NA)-ZnO-NSs was shown in tumor site. Thus the significantly enhanced retention of our compact Cy5-RCA-ZnO-NSs was attributed to the aptamer-favored cellular uptake as well as the EPR effect. In addition, the ex vivo biodistributions of free Cy5, Cy5-RCA (NA)-ZnO-NSs and Cy5-RCA-ZnO-NSs were quantitively evaluated in tumors and major organs (heart, liver, spleen,


14

Page 15 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

lung, kidney), which were finally extracted and imaged. As shown in Figure 6b and 6c, the fluorescence signal of Cy5-RCA-ZnO-NSs-treated tumor group was 3.8-fold and 2.1-fold higher than that of free Cy5-treated and Cy5-RCA (NA)-ZnO-NSs-treated tumor groups, respectively. This furtherly demonstrated the robust and aptamer-mediated tumor-addressable characters of the compact Cy5-RCA-ZnO-NSs system. Also, the fluorescence of Cy5-RCA-ZnO-NSs-treated tumor group was 17.9-fold, 5.6-fold, 6.4-fold, 14.4-fold and 5.3-fold higher than that of heart, liver, spleen, lung, and kidney, respectively. These results convincingly proved the superior tumor accumulation of our in vivo DOX@RCA-ZnO-NSs administration approach. After confirming the good biocompatibility and tumor-specific accumulation of DOX@RCAZnO-NSs in vivo, we attempted to evaluate their antitumor effect in HeLa mouse model (with a tumor volume of ~50 mm3). The tumor-bearing mice were randomly divided into six groups (n=4) and respectively treated with PBS, free DOX, DOX@RCA NSs, DOX@RCA-ZnO-NSs, DOX@RCA (MT)-ZnO-NSs and DOX@RCA (NA)-ZnO-NSs via tail-vein injection according to the administration procedure as schematically illustrated in Figure 7a. The changes of the relative tumor volume were plotted for investigating 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-NSs-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 7c). Meanwhile, the tumor increased to 2.7-fold larger in volume for the DOX@RCA (NA)ZnO-NSs-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


15

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

performance. Moreover, the tumor increased to 4.4-fold larger in volume for the DOX@RCA (MT)-ZnO-NSs-treated 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-NSs system (4.4-fold) is promoted as compared with that of DOX@RCA NSs 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 neglectable systemic toxicity of DOX@RCA-ZnO-NSs in vivo. Histological examination was performed to furtherly evaluate the in vivo antitumor activity of our NSs agents. Hematoxylin and eosin (H&E) staining was employed to 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-NSs treatment, but only limited necrosis or apoptosis was observed in DOX@RCA (NA)-ZnO-NSs- and DOX@RCA (MT)-ZnO-NSs-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 corroborated 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


16

Page 17 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

associated with various treatments. As compared with the healthy normal group, the biochemical indexes of DOX@RCA-ZnO-NSs-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 heart, liver, spleen, lung, and kidney were collected after three-week therapy and stained with H&E for histological examination (Figure S22). No distinct inflammation or disorganization was observed in these main organs of DOX@RCA-ZnO-NSs-treated mice. The histological images of these organs were the same as those of healthy mice, which again confirmed the longterm histocompatibility of our DOX@RCA-ZnO-NSs therapeutic platform. CONCLUSIONS In summary, we have developed a nonviolent self-sustained drug delivery system consisting of the built-in DNAzyme NSs and the pH-responsive ZnO NPs. The robust DNAzyme NSs were encoded with a multivalent tandem aptamer sgc-8c sequence to facilitate the efficient and tumorspecific 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 NSs system shows a synergistically enhanced therapeutic effect, originating from the co-assembled 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 showed extensive prospects for the construction of other multifunctional theranostic platforms by encoding with other additional therapeutic sequences.


17

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 37

EXPERIMENTAL SECTION Materials. Zinc acetate, magnesium acetate, sodium hydroxide, 3-Aminopropyltriethoxysilane (APTES), DCFH-DA, calcein-AM solution and Propidium iodide (PI) were purchased from Sigma-Aldrich (St Louis, MO, USA). All oligonucleotides were synthesized and HPLC-purified 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 were obtained from GE healthcare (Pittsburgh, PA, USA). Doxorubicin hydrochloride (98%) was obtained from Aladdin Reagent (Shanghai, China). Hoechst 33342 and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zoliumbromide (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). 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 RCA-ZnO-NSs was performed on a Talos F200S microscope. The hydrodynamic size and zeta potential of nanoparticles were measured by a Zetasizer Nano ZS (Malvern, UK). ICP-AES (IRIS Intrepid II XSP, USA) was used for quantifying Zn2+ ions. Cary 100 UV-Vis Spectrophotometer (Varian Inc.) and 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


18

Page 19 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

were obtained via flow cytometer (BD FACSVerse, CA). Cell and mice imaging were performed with Leica SP8 confocal microscope and IVIS Imaging System, respectively. Cell culture and animal models. HeLa cells were cultured in T-25 cm2 flasks using Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) containing 10% fetal bovine serum (FBS, PAN), 100 U·mL−1 penicillin-streptomycin in a humidified atmosphere at 37 ℃ with 5% CO2. Raji cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 100 U·mL−1 penicillinstreptomycin. Animal experiments were approved by Animal Care and Use Committee of Wuhan University, balb/c mice (5 weeks old) bearing HeLa tumor were subcutaneously inoculated with 5 × 106 cells at the right back of mice. Tumor volume was calculated according to the following equation: V = (L × W2)π/6 (L and W are the longest and shortest diameter of 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 anhydrous ethanol, then the mixture was heated to 70 ℃ and stirred vigorously until dissolution. Subsequently, 50 mg of NaOH was dissolved in 8 mL 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 DMF (15 mL) under sonication, 50 μL of APTES was then added and stirred at 120 ℃ for 15 min. The amine-functionalized ZnO NPs were washed with DMF three times and dried in vacuum at room temperature. For RCA NSs synthesis, linear templates (2 μM) were ligated with 400 U·μL-1 quick ligase at 25 ℃ for 30 minutes in the presence of 4 μM primer. To construct RCA NSs, circularized templateprimer 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 DTT, 1 mM of


19

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 37

each dNTP, and 1 U·μL-1 phi29 polymerase. The proposed RCA reaction was performed at 30 ℃ for three hours and stopped by inactivating polymerase at 95 ℃ 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 8 kDa dialysis unit. The loading capability of DOX was determined via UV-Vis spectroscopy and the loading 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 dialysate, Vdialysate and VRCA are the volume of exterior dialysate and original RCA reaction, respectively. Zn2+-responsive self-sustained NSs fragmentation for programmable drug release. RCAZnO-NSs, RCA (NA)-ZnO-NSs and RCA (MT)-ZnO-NSs were preincubated at 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 hours. For kinetic analysis, these NSs 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 mM, 0.5 mM, 0.75 mM, and 1 mM) at 37 ℃. The DOX release kinetics were evaluated through a dialysis method. The purified DOX@RCAZnO-NSs or DOX@RCA (MT)-ZnO-NSs was pre-incubated 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 HEPES (10 mM, pH 7.4, 100 mM NaCl). The medium outside dialysis bag was collected and analyzed with UV-Vis spectroscopy at different time-intervals (0, 0.5 h, 1 h, 2 h, 3 h, 4 h).


20

Page 21 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Stability study of the compact NSs. For stability assay, 1 mg·mL-1 RCA-ZnO-NSs were added into 10% human serum and incubated at 37 ℃ for 0 h, 2 h, 4 h, 6 h, 8 h and 10 h, respectively. Then these samples were heat-inactivated and analysed by agarose gel electrophoresis. For thermal stability assay, RCA-ZnO-NSs were preincubated at 20 ℃, 40 ℃ or 60 ℃ for 1 hour, 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 co-loaded onto RCA NSs, and DOX release from the resultant mixture was monitored. The specific cellular uptake of DOX@RCA-ZnO-NSs. For the cell-targeting study, Cy5labeled RCA-ZnO-NSs were incubated with HeLa cells or Raji cells at 4 ℃ 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 inhibition assay, HeLa cells were pretreated either with or without excess free aptamer sgc-8c (500 nM) at 4 ℃ for 30 min, with intermediate three times of washing, these cells were furtherly incubated with Cy5-labelled RCA-ZnO-NSs (5 μg·mL−1) at 4 ℃ for two hours. For lysosome staining, HeLa cells were incubated with Cy5labeled RCA-ZnO-NSs for 10 min, then 5 μM Lysotracker Green was added and incubated for another 5 min, and then the cells were observed immediately with 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.


21

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 37

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 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 for imaging the intracellular Zn2+ ions. DOX@RCA-ZnO-NSs or ZnCl2 (RPMI 1640 medium with 10% FBS) were added to HeLa cells for incubating 2 h at 37 ℃, 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 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 imaged 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 grew to 80% confluence 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 0.5 mg·mL-1 MTT was added to each well and further incubated in 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 6-well plates (5×105 cells per well) and then treated with PBS, RCA NSs, RCA-ZnO-NSs, DOX@RCA NSs, DOX@ RCA (MT)-ZnO-NSs, or DOX@RCA-ZnO-NSs


22

Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

for 24 h. All cells were harvested and stained with 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 calceinAM/PI double staining kit. Live (490nm of excitation, emission range: 505-530 nm) and dead (545 nm of excitation, emission range: 560-590 nm) cells were imaged under confocal microscope. Fluorescence imaging in vivo/ex vivo. For the biodistribution investigation, HeLa tumorbearing mice were injected with free Cy5, Cy5-labeled RCA (NA)-ZnO-NSs or Cy5-labeled RCAZnO-NSs through a tail vein. At 2 h, 6 h, 12 h, and 24 h post administration, the in vivo fluorescent images were obtained. At 24 h post-injection, the mice were euthanized, the internal organs (heart, liver, spleen, lung, kidney) and tumor tissues were harvested and subjected to the ex vivo imaging as well as semi-quantitative 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 three weeks. The weight of mice and the volume of tumor were measured every three 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 hematoxylin and eosin (H&E) or terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) according to the manufacturer’s protocol, and the slices were visualized with the microscope.


23

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

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, tissue sections (4 μm) were collected and stained with H&E and the tissue damages were examined with a light microscope. ASSOCIATED CONTENT Supporting Information Available: DNA sequences, characterization of NSs and DOX intercalation, demonstration of DNAzyme-mediated RCA NSs cleavage and DOX release, acidresponsive ZnO dissociation, therapeutic effects of DOX@RCA-ZnO-NSs in vitro and in vivo. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author To whom correspondence should be addressed. E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT 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).


24

Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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. 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. (4) Dykman, L.; Khlebtsov, N. Gold Nanoparticles in Biomedical Applications: Recent Advances and Perspectives. Chem. Soc. Rev. 2012, 41, 2256-2282. (5) Li, Z.; Barnes, J. C.; Bosoy, A.; Stoddart, J. F.; Zink, J. I. Mesoporous Silica Nanoparticles in Biomedical Applications. Chem. Soc. Rev. 2012, 41, 2590-2605. (6) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Quantum Dots for Live Cells, In Vivo Imaging, and Diagnostics. Science 2005, 307, 538-544. (7) Elsabahy, M.; Wooley, K. L. Design of Polymeric Nanoparticles for Biomedical Delivery Applications. Chem. Soc. Rev. 2012, 41, 2545-2561. (8) Zhao, X.; Yuan, Z.; Yildirimer, L.; Zhao, J.; Lin, Z. Y.; Cao, Z.; Pan, G.; Cui, W. TumorTriggered Controlled Drug Release from Electrospun Fibers Using Inorganic Caps for Inhibiting Cancer Relapse. Small 2015, 11, 4284-4291. (9) Tang, Z.; Kotov, N. A. One-Dimensional Assemblies of Nanoparticles: Preparation, Properties, and Promise. Adv. Mater. 2005, 17, 951-962. (10) Seeman, N. C. DNA in a Material World. Nature 2003, 421, 427-431. (11) Kahn, J. S.; Hu, Y.; Willner, I. Stimuli-Responsive DNA-Based Hydrogels: From Basic Principles to Applications. Accounts Chem. Res. 2017, 50, 680-690. (12) Hou, W.; Zhao, X.; Qian, X.; Pan, F.; Zhang, C.; Yang, Y.; de La Fuente, J. M.; Cui, D. PHSensitive Self-Assembling Nanoparticles for Tumor Near-Infrared Fluorescence Imaging and Chemo-Photodynamic Combination Therapy. Nanoscale 2016, 8, 104-116. (13) Chen, Y.-J.; Groves, B.; Muscat, R. A.; Seelig, G. DNA Nanotechnology from the Test Tube to the Cell. Nat. Nanotechnol. 2015, 10, 748-760. (14) He, Y.; Ye, T.; Su, M.; Zhang, C.; Ribbe, A. E.; Jiang, W.; Mao, C. Hierarchical SelfAssembly of DNA into Symmetric Supramolecular Polyhedra. Nature 2008, 452, 198-201.


25

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 37

(15) Hong, F.; Zhang, F.; Liu, Y.; Yan, H. DNA Origami: Scaffolds for Creating Higher Order Structures. Chem. Rev. 2017, 117, 12584-12640. (16) Halo, T. L.; McMahon, K. M.; Angeloni, N. L.; Xu, Y.; Wang, W.; Chinen, A. B.; Malin, D.; Strekalova, E.; Cryns, V. L.; Cheng, C. Nanoflares for the Detection, Isolation, and Culture of Live Tumor Cells from Human Blood. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17104-17109. (17) Tan, S. J.; Campolongo, M. J.; Luo, D.; Cheng, W. Building Plasmonic Nanostructures with DNA. Nat. Nanotechnol. 2011, 6, 268-276. (18) Li, S.; Jiang, Q.; Liu, S.; Zhang, Y.; Tian, Y.; Song, C.; Wang, J.; Zou, Y.; Anderson, G. J.; Han, J.-Y. A DNA Nanorobot Functions as a Cancer Therapeutic in Response to a Molecular Trigger In Vivo. Nat. Biotechnol. 2018, 36, 258-264. (19) Douglas, S. M.; Bachelet, I.; Church, G. M. A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads. Science 2012, 335, 831-834. (20) Zhang, Q.; Jiang, Q.; Li, N.; Dai, L.; Liu, Q.; Song, L.; Wang, J.; Li, Y.; Tian, J.; Ding, B.; Du, Y. DNA Origami as an In Vivo Drug Delivery Vehicle for Cancer Therapy. ACS Nano 2014, 8, 6633-6643. (21) Chen, C.; Geng, J.; Pu, F.; Yang, X.; Ren, J.; Qu, X. Polyvalent Nucleic Acid/Mesoporous Silica Nanoparticle Conjugates: Dual Stimuli-Responsive Vehicles for Intracellular Drug Delivery. Angew. Chem. 2011, 50, 882-886. (22) Fire, A.; Xu, S. Q. Rolling Replication of Short DNA Circles. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 4641-4645. (23) Kim, E.; Zwi-Dantsis, L.; Reznikov, N.; Hansel, C. S.; Agarwal, S.; Stevens, M. M. One-Pot Synthesis of Multiple Protein-Encapsulated DNA Flowers and Their Application in Intracellular Protein Delivery. Adv. Mater. 2017, 29, 1701086. (24) Lee, J. B.; Hong, J.; Bonner, D. K.; Poon, Z.; Hammond, P. T. Self-Assembled RNA Interference Microsponges for Efficient siRNA Delivery. Nat. Mater. 2012, 11, 316-322. (25) Jang, M.; Kim, J. H.; Nam, H. Y.; Kwon, I. C.; Ahn, H. J. Design of a Platform Technology for Systemic Delivery of siRNA to Tumours Using Rolling Circle Transcription. Nat. Commun. 2015, 6, 7930. (26) Sun, W.; Jiang, T.; Lu, Y.; Reiff, M.; Mo, R.; Gu, Z. Cocoon-Like Self-Degradable DNA Nanoclew for Anticancer Drug Delivery. J. Am. Chem. Soc. 2014, 136, 14722-14725.


26

Page 27 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(27) Lv, Y.; Hu, R.; Zhu, G.; Zhang, X.; Mei, L.; Liu, Q.; Qiu, L.; Wu, C.; Tan, W. Preparation and Biomedical Applications of Programmable and Multifunctional DNA Nanoflowers. Nat. Protoc. 2015, 10, 1508-1524. (28) Lee, J. B.; Peng, S.; Yang, D.; Roh, Y. H.; Funabashi, H.; Park, N.; Rice, E. J.; Chen, L.; Long, R.; Wu, M. A Mechanical Metamaterial Made from a DNA Hydrogel. Nat. Nanotechnol. 2012, 7, 816-820. (29) Roh, Y. H.; Lee, J. B.; Shopsowitz, K. E.; Dreaden, E. C.; Morton, S. W.; Poon, Z.; Hong, J.; Yamin, I.; Bonner, D. K.; Hammond, P. T. Layer-by-Layer Assembled Antisense DNA Microsponge Particles for Efficient Delivery of Cancer Therapeutics. ACS Nano 2014, 8, 97679780. (30) Wang, C.; Sun, W.; Wright, G.; Wang, A. Z.; Gu, Z. Inflammation-Triggered Cancer Immunotherapy by Programmed Delivery of CpG and Anti-PD1 Antibody. Adv. Mater. 2016, 28, 8912-8920. (31) Sun, W.; Ji, W.; Hall, J. M.; Hu, Q.; Wang, C.; Beisel, C. L.; Gu, Z. Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-Cas9 for Genome Editing. Angew. Chem. 2015, 54, 12029-12033. (32) Hu, R.; Zhang, X.; Zhao, Z.; Zhu, G.; Chen, T.; Fu, T.; Tan, W. DNA Nanoflowers for Multiplexed Cellular Imaging and Traceable Targeted Drug Delivery. Angew. Chem. 2014, 126, 5931-5936. (33) Sun, W.; Ji, W.; Hu, Q.; Yu, J.; Wang, C.; Qian, C.; Hochu, G.; Gu, Z. Transformable DNA Nanocarriers for Plasma Membrane Targeted Delivery of Cytokine. Biomaterials 2016, 96, 1-10. (34) Breaker, R. R.; Joyce, G. F. A DNA Enzyme That Cleaves RNA. Chem. Biol. 1994, 1, 223229. (35) Gu, H.; Furukawa, K.; Weinberg, Z.; Berenson, D. F.; Breaker, R. R. Small, Highly Active DNAs That Hydrolyze DNA. J. Am. Chem. Soc. 2013, 135, 9121-9129. (36) Feng, J.; Xu, Z.; Liu, F.; Zhao, Y.; Yu, W.; Pan, M.; Wang, F.; Liu, X. Versatile Catalytic Deoxyribozyme Vehicles for Multimodal Imaging-Guided Efficient Gene Regulation and Photothermal Therapy. ACS Nano 2018, 12, 12888-12901. (37) Wang, F.; Lu, C.; Willner, I. From Cascaded Catalytic Nucleic Acids to Enzyme-DNA Nanostructures: Controlling Reactivity, Sensing, Logic Operations, and Assembly of Complex Structures. Chem. Rev. 2014, 114, 2881-2941.


27

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 37

(38) Lu, Y.; Liu, J. Functional DNA Nanotechnology: Emerging Applications of DNAzymes and Aptamers. Curr. Opin. Biotech. 2006, 17, 580-588. (39) Ma, L.; Liu, B.; Huang, P.-J. J.; Zhang, X.; Liu, J. DNA Adsorption by ZnO Nanoparticles Near Its Solubility Limit: Implications for DNA Fluorescence Quenching and DNAzyme Activity Assays. Langmuir 2016, 32, 5672-5680. (40) He, Z.-M.; Zhang, P.-H.; Li, X.; Zhang, J.-R.; Zhu, J.-J. A Targeted DNAzymeNanocomposite Probe Equipped with Built-in Zn2+ Arsenal for Combined Treatment of Gene Regulation and Drug Delivery. Sci. Rep. 2016, 6, 22737. (41) Muhammad, F.; Guo, M.; Guo, Y.; Qi, W.; Qu, F.; Sun, F.; Zhao, H.; Zhu, G. Acid Degradable ZnO Quantum Dots as a Platform for Targeted Delivery of an Anticancer Drug. J. Mater. Chem. 2011, 21, 13406-13412. (42) Ye, D.-X.; Ma, Y.-Y.; Zhao, W.; Cao, H.-M.; Kong, J.-L.; Xiong, H.-M.; Möhwald, H. ZnOBased Nanoplatforms for Labeling and Treatment of Mouse Tumors without Detectable Toxic Side Effects. ACS Nano 2016, 10, 4294-4300. (43) Wang, C.; Cheng, L.; Liu, Z. Drug Delivery with Upconversion Nanoparticles for MultiFunctional Targeted Cancer Cell Imaging and Therapy. Biomaterials 2011, 32, 1110-1120. (44) Guo, X.; Wei, X.; Jing, Y.; Zhou, S. Size Changeable Nanocarriers with Nuclear Targeting for Effectively Overcoming Multidrug Resistance in Cancer Therapy. Adv. Mater. 2015, 27, 64506456. (45) Shangguan, D.; Tang, Z.; Mallikaratchy, P.; Xiao, Z.; Tan, W. Optimization and Modifications of Aptamers Selected from Live Cancer Cell Lines. Chembiochem 2010, 8, 603606. (46) Chen, T.; Shukoor, M. I.; Wang, R.; Zhao, Z.; Yuan, Q.; Bamrungsap, S.; Xiong, X.; Tan, W. Smart Multifunctional Nanostructure for Targeted Cancer Chemotherapy and Magnetic Resonance Imaging. ACS Nano 2011, 5, 7866-7873. (47) Shangguan, D.; Cao, Z.; Meng, L.; Mallikaratchy, P.; Sefah, K. H.; Li, Y.; Tan, W. CellSpecific Aptamer Probes for Membrane Protein Elucidation in Cancer Cells. J. Proteome Res. 2008, 7, 2133-2139. (48) Wolfgang, M.; Yuan, L. Coordination Dynamics of Zinc in Proteins. Chem. Rev. 2009, 109, 4682-4707.


28

Page 29 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(49) Claudia, A.; Lucia, B.; Ivano, B.; Antonio, R. Counting the Zinc-Proteins Encoded in the Human Genome. J. Proteome Res. 2006, 5, 196-201. (50) Anzellotti, A. I.; Farrell, N. P. Zinc Metalloproteins as Medicinal Targets. Chem. Soc. Rev. 2008, 37, 1629-1651. (51) Mihai, C.; Chrisler, W. B.; Xie, Y.; Hu, D.; Szymanski, C. J.; Tolic, A.; Klein, J. A.; Smith, J. N.; Tarasevich, B. J.; Orr, G. Intracellular Accumulation Dynamics and Fate of Zinc Ions in Alveolar Epithelial Cells Exposed to Airborne ZnO Nanoparticles at the Air-Liquid Interface. Nanotoxicology 2015, 9, 9-22. (52) Cho, W.-S.; Duffin, R.; Howie, S. E. M.; Scotton, C. J.; Wallace, W. A. H.; MacNee, W.; Bradley, M.; Megson, I. L.; Donaldson, K. Progressive Severe Lung Injury by Zinc Oxide Nanoparticles; the Role of Zn2+ Dissolution inside Lysosomes. Part. Fibre. Toxicol. 2011, 8, 2727. (53) Provinciali, M.; Donnini, A.; Argentati, K.; Stasio, G. D.; Bartozzi, B.; Bernardini, G. Reactive Oxygen Species Modulate Zn-Induced Apoptosis in Cancer Cells. Free Radic. Biol. Med. 2002, 32, 431-445. (54) Chou, T.-C. Drug Combination Studies and Their Synergy Quantification Using the ChouTalalay Method. Cancer Res. 2010, 70, 440-446.


29

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

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 with stimulated drug release after specific cellular uptake.


30

Page 31 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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.


31

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 37

Figure 3. (a) Denaturing PAGE analysis of the controlled RCA NSs 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.


32

Page 33 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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.


33

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 37

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.


34

Page 35 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 6. (a) Dynamic biodistributions of free Cy5, Cy5-RCA (NA)-ZnO-NSs, Cy5-RCA-ZnONSs. (b) Ex vivo fluorescent images of major tissues and tumors after free Cy5, Cy5-RCA (NA)ZnO-NSs or Cy5-RCA-ZnO-NSs 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.


35

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 37

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, (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.


36

Page 37 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Table of Contents Graphic


37

Nonviolent Self-Catabolic DNAzyme Nanosponges for Smart

Recommend Documents