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DNA Polymer Nanoparticles Programmed via Supersandwich Hybridization for Imaging and Therapy of Cancer Cells Na Li, Mei-Hao Xiang, Jin-Wen Liu, Hao Tang, and Jian-Hui Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03253 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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Analytical Chemistry

DNA Polymer Nanoparticles Programmed via Supersandwich Hybridization for Imaging and Therapy of Cancer Cells Na Li, Mei-Hao Xiang, Jin-Wen Liu,* Hao Tang, and Jian-Hui Jiang* Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, P. R. China ABSTRACT: Spherical nucleic acid (SNA) constructs are promising new single entity materials, which possess significant advantages in biological applications. Current SNA-based drug delivery system typically employed single-layered ss- or ds-DNA as the drug carriers, resulting in limited drug payload capacity and disease treatment. To advance corresponding applications, we developed a novel DNA-programmed polymeric SNA, a long concatamer DNA polymer that is uniformly distributed on gold nanoparticles (AuNPs), by self-assembling from two short alternating DNA building blocks upon initiation of immobilized capture probes on AuNPs, through a supersandwich hybridization reaction. The long DNA concatamer of polymeric SNA enables to allow high-capacity loading of bioimaging and therapeutics agents. We demonstrated that both of the fluorescence signals and therapeutic efficacy were effectively inhibited in resultant polymeric SNA. By further modifying with the nucleolin-targeting aptamer AS1411, this polymeric SNA could be specifically internalized into the tumor cells through nucleolin-mediated endocytosis, and then interact with endogenous ATP to cause the release of therapeutics agents from long DNA concatamer via a structure switching, leading to the activation of the fluorescence and selective synergistic chemotherapy and photodynamic therapy. This nanostructure can afford a promising targeted drug transport platform for activatable cancer theranostics.

Nucleic acid-based drug delivery systems that show both efficient intracellular drug transport and precise diseases treatment have become increasingly important drug candidates in recent years thanks to the advent of nanoparticle drug carriers.1-3 By virtue of its programmable and sequence-specific interactions, DNA has been widely used as versatile and powerful ligand for modifying nanomaterials, and, with rational self-assembly, DNA has also emerged as a favorable building block for constructing one-, two-, and three-dimensional nanostructures with controllable size, architecture, and surface chemistry.4,5 Such unique and controllable properties together with inherent biocompatibility endow DNA nanostructures with promising applications in biomedicine and biotechnology. Notably, spherical nucleic acids (SNA), a three-dimensional nanostructures comprise of densely functionalized and highly oriented nucleic acids covalently attached to the surface of spherical nanoparticles, have been extensively explored.6-10 These SNA nanostructures are demonstrated to exhibit unusual biological properties that have enabled a variety of applications in the fields of biodiagnostics, gene regulation, and nanomedicine.11-13 One attractive property is that, despite being highly negatively charged, SNA can easily and quickly enter cells of diverse types via scavenger receptor-mediated endocytosis without the need for ancillary transfection agents, making them an excellent candidate for nucleic acids and other payloads delivery applications.11,14-16 However, a single-layered ss- or ds-DNA was typically employed as the drug delivery carriers in a majority of these SNA, and as a result they tend to have limited drug payload capacity

and the attendant high cost, hampering production scaleup.17-19 In addition, the lack of targeted transport or activatable designs for theranostics agents often leads to limited imaging sensitivity toward targeted cancer cells and increased side effects.20-22 The pursuit of highperformance SNA-based targeted drug delivery system for cancer activatable theranostics, therefore, remains a great challenge. Herein, we report a novel DNA-programmed polymeric SNA (PSNA), which is a long concatamer DNA nanostructure that is uniformly distributed on gold nanoparticles (AuNPs), and it is easily formed by self-assembled from two short alternating DNA building blocks (P1 and P2) upon initiation of immobilized capture probes (CP) on AuNPs, through a supersandwich hybridization reaction2325 (Scheme 1A). In our design, the P1 contains a Ce6labeled aptamer sequence (Ce6-P1-apt) that specifically bind to ATP, and P2 is a BHQ2-labeled partially complementary DNA for P1. Therefore, the resultant supersandwich DNA nanostructures, a long dsDNA concatamers containing periodically aligned repeated units of P1 and P2 partially hybridized on different regions, in which the GC pairs of the dsDNA motif provide a large number of loading sites for anticancer drug doxorubicin (Dox),26-28 leading to the quenching of the fluorescence and the decrease of the cytotoxicity of Dox. Meanwhile, the fluorescence and singlet oxygen (1O2) generation ability of Ce6 could be also efficiently quenched by BHQ2 due to the energy transfer between them. It is noted that, previous reports had revealed the intracellular ATP concentration (1–10 mM) was much greater than that in the extracellular

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environment (18.25 MΩ. Transmission electron microscope (TEM) images of AuNPs were obtained using a 2100F TEM (JEOL, Japan). Dynamic light scattering (DLS) experiments were carried out in a Malvern Nano-ZS system (Malvern Instruments, UK) equipped with a He Ne laser working at 633 nm to examine the hydrodynamic diameter of AuNPs. The UV absorption spectra were obtained with a UV-2450 spectrophotometer (Shimadzu, Japan). The fluorescence spectra were recorded at room temperature in a quartz cuvette on an F-7000 fluorescence spectrophotometer (Hitachi, Japan). All fluorescence images were acquired using an oil dipping objective (60×) on Nikon TI*E+A1 SI confocal laser scanning microscope (Japan). Preparation of functionalized PSNA. Citratestabilized gold nanoparticles (13 nm) and capture probe functionalized gold nanoparticles (AuNPs-CP) were prepared according to previous report method.33 To prepara-


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Analytical Chemistry tion of Ce6 and AS1411 functionalized PSNA (Ce6-PSNAAS1411), the mixtures of AuNPs-CP (5 nM), Ce6-P1-apt (5 μM), and P2-BHQ2 (5 μM) was left in 5 mM HEPES buffer (100 mM Na+, 5 mM Mg2+) for 24 h. Then, the mixture was centrifuged at 18000 rpm for 30 min and resuspended in HEPES buffer. Afterwards, 5 μM AS1411-BHQ2 was added and incubated for another 2 h. To preparation of Ce6P SNA-Dox-AS1411, 10 μM Dox was added to Ce6-PSNAAS1411 solution and then excess Dox were removed by centrifugation. The concentration of Ce6-PSNA-DoxAS1411 was determined by measuring their extinction at 523 nm. The Ce6, Dox and AS1411 functionalized TSNA (Ce6-TSNA-Dox-AS1411) were prepared by the same method described above, except that without adding of P2BHQ2. In vitro ATP-triggered Drug Release. We prepared two groups of sample, each group had seven parallel samples. One group contained 5 nM Ce6-PSNA-Dox-AS1411 and 5 mM ATP in 100 μL HEPES buffer (5 mM, pH 7.4, 100 mM NaCl, 5 mM MgCl2). Another group was the blank which only contained 5 nM Ce6-PSNA-Dox-AS1411 in 100 μL HEPES buffer and without addition of 5 mM ATP. After incubation of different time, the supernatant was collected by centrifugation (18000 rpm, 30 min). The fluorescence intensity was measured using F-7000 fluorescence spectrophotometer, 488 nm excitation for Dox and 405 nm excitation for Ce6. The concentration of released drug was calculated from the fluorescence intensity according to standard curve. Detection Singlet Oxygen (1O2) Generation Based on Ce6-PSNA-Dox-AS1411. Singlet Oxygen (1O2) generation of Ce6-PSNA-Dox-AS1411 was measured with DPBF, a probe molecule reacted with 1O2 to cause the fluorescence decrease at about 485 nm. Briefly, 50 μL of Ce6-PSNADox-AS1411 was incubated with or without ATP in HEPES buffer at 37 °C for 15 min. Then 50 μL of 50 μg/mL DPBF was added. The mixture was irradiated by 650 nm LED lamp. After irradiation, the fluorescence intensity at 485 nm was recorded under an excitation wavelength of 403 nm. The solution of DPBF without Ce6-PSNA-Dox-AS1411 was used as the control. Cell Culture and Intracellular ATP-responsive Drug Release. HeLa cells and Hacat cells were cultured in RPMI 1640 cell culture medium supplemented with 10% FBS, 100 IU/mL penicillin and 100 IU/mL streptomycin. The cells were maintained at 37 °C under a humidified atmosphere containing 5% CO2. For the cell uptake of Ce6-PSNA-AS1411, HeLa cells were seeded in culture dish overnight, then, the cells were washed with PBS twice and Ce6-PSNA-AS1411 in fresh medium containing 10% FBS was added. After culture for 4h, followed by washing twice with PBS to remove unbinding probes, the nuclei were stained with Hoechst (10 μg/mL) for 10 min. Finally, the cells were observed by confocal laser scanning microscope. Hoechst was excited at 405 nm, and the fluorescence emission was collected from 420 to 470 nm; Ce6 was excited at 405 nm, and fluorescence emission was collected from 660 to 750 nm. For the detection of intracellular ATP-responsive drug release of Ce6-PSNA-Dox-AS1411, HeLa cells were incubat-

ed with Ce6-PSNA-Dox-AS1411 for 4 h, then the cell nuclei were stained with Hoechst for 10 min and subjected to confocal microscopic observation. The fluorescence of Hoechst and Ce6 were collected with the same method described above. Dox was excited at 488 nm, and the fluorescence imaging was collected from 570-620 nm. To simultaneously collect the fluorescence signals of Dox and Ce6, we used series scan mode to eliminate the cross interference between Ce6 and Dox, and the color of Ce6 channel was set as green (a pseudo color) to distinguish with the red Dox fluorescence signals. For the investigation of intracellular ATP level to the influence on intracellular drug release of Ce6-PSNA-DoxAS1411, before adding Ce6-PSNA-Dox-AS1411, the HeLa cells were treated with 10 μM oligomycin or 5 mM Ca2+ for 30 min. Then the cells were stained with Hoechst for 10 min and subjected to confocal microscopic observation. Fluorescence Imaging of Intracellular 1O2 Levels. 1 O2 generation inside cells was detected using DCFH-DA. HeLa cells were seeded in culture dish for 24 h. Then the medium was removed and Ce6-PSNA-Dox-AS1411 in fresh medium containing 10% FBS was added. After 4 h, the cells were washed twice with PBS and further stained with 10 μM DCFH-DA for 30 min. Finally, the cells were washed with PBS again and irradiated at 650 nm for 15 min with the power intensity of 10 mW•cm-2. The cells treated without Ce6-PSNA-Dox-AS1411 were used as the control. The fluorescence images were acquired on a confocal laser scanning microscope with the excitation laser at 488 nm and collected emission wavelength from 500 to 520 nm. Cell Viability Assays. The toxicity of different functionalized PSNA was investigated with HeLa cells and Hacat cells by MTS assay. The cells were seeded in 96-well plates and incubated at 37 °C in 5% CO2 atmosphere for 24 h. After incubating with different concentrations of functionalized PSNA for another 12 h, the cells were washed with PBS and irradiated at 650 nm for 15 min with the power intensity of 10 mW•cm-2. The cell without laser irradiation were used as the control. The cells were further incubated for 24 h. Finally, the culture medium was replaced with 100 μL fresh medium contained 20 μL CellTiter reagent and incubated for another 2 h in cell incubator. The absorbances at 490 nm of each well were acquired on a Thermo Scientific Multiskan Microplate Reader (Thermo Fisher, U.S.A.). Cell viability was calculated as described by the manufacturer. Apoptosis Assay. Flow cytometry evaluation of cell apoptosis induced by different functionalized PSNA were detected using the Annexin V-FITC apoptosis detection kit. The HeLa cells were seeded in culture dish and incubated for 48 h. The cells were incubated with different functionalized PSNA for 12 h and then exposed to 650 nm laser irradiation for 15 min. Afterwards, the cells were digestion using trypsin and washed twice with PBS by centrifugation. The cells were collected and resuspended in 100 µL binding buffer contained 5 μL Annexin V-FITC and 10 μL PI. After incubated for 15 min in incubator, the apoptosis of cells in all the samples were respectively analyzed by flow cytometer.



RESULTS AND DISCUSSION

Drug Loading and Release of DNA Supersandwich Structures. The DNA duplex scaffold by hybridizing an ATP aptamer and its cDNA have been demonstrated to hold GC-rich motif for Dox loading, and the interaction of ATP aptamer with ATP would result in the dissociation of the DNA duplex, and then the releasing of Dox.26-28,34 To amplify the drug loading event, two alternating DNA building blocks (P1 and P2) were rationally engineered, in that case they were successively hybridized to each other, creating long concatamers, forming ATP-responsive DNA supersandwich structures. Agarose gel electrophoresis showed that a ladder of different lengths of the DNA concatamers with the maximum length being ~500 base pairs, indicating the formation of the proposed supersandwich structures (lane 4 in Figure S1). As anticipated, these bands disappeared when the DNA supersandwich structures was exposed to 5 mM ATP (lane 5 in Figure S1), which reveals that the presence of ATP result in disassembly of the supersandwich structures. These may result from the fact that the ATP aptamer change its conformation when binding ATP, which consequently disassembles the supersandwich structures. Using the ATPresponsive DNA supersandwich structures as the carriers for Dox, the fluorescence spectral signals for Dox decrease with increasing DNA supersandwich concentrations (Figure S2A). Dox as a chemotherapeutic agent easily intercalate into the double-stranded GC or CG sequences of DNA supersandwich, in which the fluorescence can be quenched and the cytotoxicity of Dox was decreased. Moreover, a DNA supersandwich concentration of 0.5 μM gave a quenching ratio up to ~5.5, which implied that a high loading of DNA supersandwich structures for Dox. When adding 5 mM ATP, a remarkable fluorescence recovery was observed (Figure S2B), suggesting that Dox/supersandwich is responsive to ATP. These results may provide a theoretical basis for constructing an ATPresponsive system for Dox drug delivery. Construction Functionalized PSNA for Drug Loading and Release. To develop a high-efficient drug loading system for intracellular transport, AuNPs-based SNA were selected as building blocks to incorporate DNA supersandwich structures for targeted delivery and therapy in living cells based on its excellent properties. To enhance the therapeutic effect, furthermore, chemotherapy and photodynamic therapy (PDT) are employed together to specifically kill cancer cells. A photosensitizer, Ce6, was modified to the P1 to target intracellular ATP. Starting from the functionalized capture probe on AuNPs, the consecutive DNA hybridization creates long concatamers containing repeated units of Ce6-P1-apt and P2-BHQ2 probe. TEM images showed that the surface functionalization both with CP and CP-P1-P2 did not affect the size and size distribution of AuNPs (Figure S3), but the functionalization led to a slightly red shift of the surface plasmon band of the AuNPs (Figure S4A), which was attributed to the change in the dielectric constant of the surrounding environment of the AuNPs due to the surface DNA functionalization.35 DLS measurements further

demonstrated the PSNA were discrete colloidal particles with a diameter of ~85 nm (Figure S4B). Interestingly, by changing the concentration of P1 and P2, PSNA with controllable size can be realized. As the concentration of P1 and P2 changed (from 0 to 5 μM), the DLS hydrodynamic size of PSNA became larger and larger (Figure S5), which was attributed to the fact that higher concentration of P1 and P2 can trigger more efficient supersandwich hybridization reaction corresponding to DNA-programmed polymerization into a long dsDNA concatamers. In addition, PSNA exhibited a more negative zeta potential as compared to that of only CP probes modified AuNPs, indicating that the negatively charged long P1-P2 concatamers were indeed functionalized on the surface of the P SNA (Figure S6).

Figure 1. (A) The fluorescence spectra of Dox in the presence P of different concentrations of SNA. The concentration of Dox was 5 μM. (B) Agarose gel electrophoresis analysis of P P SNA: lane 1, DNA marker; lane 2, AuNPs-CP; lane 3, SNA; P lane 4, SNA+ATP. (C) Fluorescence recovery ratios of Ce6P SNA-AS1411 in the presence of different concentrations of ATP (red line), GTP (blue line), CTP (black line) and UTP (purple line), respectively. (D) Fluorescence recovery ratios P of Ce6- SNA-AS1411 in the presence of 4 mM ATP, GTP, CTP and UTP. All error bars indicated standard deviations across four repetitive assays.

After demonstrating the successful assembly of DNA polymer on the AuNPs surface, the protection effect of DNA polymer on AuNPs against enzymatic cleavage was investigated. In contrast to corresponding DNA selfassembled nanostructure, the developed pSNA was found to exhibit obvious resistance to degradation by nuclease such as DNase I (Figure S7). This enhanced nuclease resistance of pSNA was understood to be a consequence of the tight packing of the DNA polymer on the AuNPs surface causes steric inhibition of nuclease degradation.11 The finding of nuclease resistance ability of pSNA may offer remarkable potential for bioanalytical and biomedical applications. When 5 μM Dox was incubated with an increasing concentration of the PSNA, a sequential decrease was observed in the fluorescence intensity of Dox, which were similar to that of P1-P2 supersandwich structures (Figure 1A). Moreover, the amount of Dox loading on the P SNA was calculated to be ~ 1138 Dox molecules per na-


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Analytical Chemistry noparticle, which much more than the traditional sandwich SNA structures (~ 278 Dox molecules per nanoparticle). Next, we investigated the response of Ce6-PSNA-AS1411 toward ATP in HEPES buffer. The fluorescence signals of Ce6 displayed a dynamic increase with increasing ATP concentrations in the range of 0 mM to 4 mM, and a saturated response achieved at 4 mM (Figure 1C). This saturated signal was ascribed to complete dissociation of Ce6P1-apt from P1-P2 dsDNA concatamers, and ATP can effectively activate Ce6-PSNA-AS1411. Gel electrophoresis analysis further revealed that supersandwich structure can be efficiently assembled on AuNPs and ATP enable specifically disassemble the supersandwich structure (Figure 1B and Figure S8). In addition, good selectivity is exhibited for ATP, and the fluorescence recovery ratios of Ce6 induced by 4 mM ATP was found to be around 35-fold (Figure 1D), however, when incubated Ce6-PSNA-AS1411 with the three analogues CTP, GTP, or UTP with same concentration for 15 min, the fluorescence intensity of Ce6 was almost the same with the blank. This result gave clear evidence that the Ce6-PSNA-AS1411 was specific for ATP, and successfully facilitated the following ATP-responsive Ce6-PSNAAS1411 in living cells.

Figure 2. (A)Time-dependent release of Ce6 and Dox from

Ce6-PSNA-Dox-AS1411 with or without ATP. (B)Time de1 P pendent O2 generation of Ce6- SNA-Dox-AS1411 with or without ATP. (C) HeLa cells confocal images of (I) DCFH-DA only, (II) DCFH-DA with laser irradiation, (III) DCFH-DA P and Ce6- SNA-Dox-AS1411 without laser irradiation, and (IV) P DCFH-DA and Ce6- SNA-Dox-AS1411 with laser irradiation.

Further investigation of time-dependent Dox and Ce6 releasing behavior of Ce6-PSNA-Dox-AS1411 induced by ATP was performed by measuring the fluorescence of Dox and Ce6 in the supernatants after centrifuging the mixtures at indicated time points. Kinetic studies showed that the Ce6-PSNA-Dox-AS1411 responded rapidly to 5 mM ATP within 60 min, and the release ratios of Dox and Ce6 reached a plateau at 120 min with a 34% and 46% release, respectively (Figure 2A). In contrast, no obvious fluorescence enhancement of Dox and Ce6 was observed in the absence of ATP. These data implied that Ce6-PSNA-DoxAS1411 was stable in HEPES buffer, while ATP could efficiently activate it, and then result in a fast Dox and Ce6 releasing. More importantly, this ATP-dependent drug-

loading and releasing properties are favorable for cancer therapy, since intracellular ATP in cancer cells are overexpressed,29 facilitating active drug release from the P SNA-based drug delivery vehicles. ATP Triggered 1O2 Generation. The 1O2 generation ability is critical in cancer cell photodynamic therapy, as it directly leads to target cell apoptosis. To evaluate ATPinduced generation of 1O2 in the PSNA-based PDT system, DPBF was employed as ROS indicator. As shown in Figure 2B, in the absence of ATP, no obvious decrease of the DPBF fluorescence was detected, which indicates that the suppression of 1O2 production by energy transfer between Ce6 and BHQ2. However, the time-dependent DPBF fluorescence decrease was observed in the Ce6-PSNA-DoxAS1411 system upon the addition of 5 mM ATP and then irradiation with 650 nm laser, verifying the 1O2 generation. These results suggested that the 1O2 generation of Ce6P SNA-Dox-AS1411 can be effectively activated by the ATP. Furthermore, the 1O2 generation within cells was also investigated by measuring the fluorescence of DCFH-DA, a cell penetrated ROS indicator, which can be specifically oxidized by 1O2 to produce enhanced fluorescence upon oxidation.36 It was observed that negligible DCFH-DA fluorescence in cell itself or in Ce6-PSNA-Dox-AS1411 incubated cells without laser irradiation (Figure 2C), while strong fluorescence appearing after laser irradiation in Ce6-PSNA-Dox-AS1411-incubated cells, confirming the 1O2 generation in cells. These intracellular 1O2 generation may provide a good PDT platform for cancer cells therapy. Cytotoxicity Investigation of Functionalized PSNA. Prior to cell imaging experiment, the cytotoxicity of PSNA was assessed with standard MTS assay in HeLa cell lines. We chose the cells only incubated with culture medium as blank sample, and the ratio of the cells incubated with the PSNA to the blank sample was used to evaluate the cell viability. After incubation of HeLa cells with PSNA (without Dox and Ce6) at concentrations of 0-10 nM for 12, 24, or 48 h, above 97% cell viability was observed even at the PSNA levels up to 10 nM over 48 h (Figure S9), suggesting the excellent biocompatibility and nontoxicity of the PSNA. The negligible cytotoxicity may ascribe to low toxicity of AuNPs itself and the presence of abundant DNA biopolymer on the surface of AuNPs, which could enhance the biocompatibility in vitro and in vivo.37 Target-Cell-Specific Delivery. To evaluate the specific cell-targeting ability of the Ce6-PSNA-AS1411, two different human cell lines, human cervical carcinoma cell (HeLa) that overexpress nucleolin receptor and human normal immortalized epidermal cells (Hacat) that deficient in nucleolin receptor, were incubated with Ce6P SNA-AS1411 individually for 4 h under the same conditions. The intracellular uptake efficiency of Ce6-PSNAAS1411 in two cells was then determined by measuring intracellular gold content using ICP-MS (Figure S10). The results demonstrated that the uptake amounts of Ce6P SNA-AS1411 in HeLa was much higher than that in Hacat, which were consistent with the fact that the nucleolin receptor was overexpressed in HeLa but not in Hacat. Interestingly, for HeLa cells incubated with the Ce6T SNA-AS1411 for 4 h, we also observed similar uptake



amounts with Ce6-PSNA-AS1411. Moreover, an additional cell imaging and flow cytometric experiment further confirmed the functionalization of AS1411 can significantly enhance cancer cells specific delivery (Figure S11). In addition, the endocytosis pathway of Ce6-PSNAAS1411 were further investigated. We compared the cellular uptake of Ce6-PSNA-AS1411 on HeLa cells at 37 °C and 4 °C (Figure S12). Confocal laser microscopy imaging revealed that, at the temperature of 4°C or the pretreatment with NaN3 at 37°C, the uptake of Ce6-PSNA-AS1411 was significantly inhibited, evidencing that the Ce6-PSNAAS1411 permeated into the cells through a typical internalization pathway. All those results manifested that Ce6P SNA-AS1411 enabled specific targeting at nucleolin receptor through AS1411, which may promote the entry of Ce6P SNA-AS1411 into cells via receptor-mediated endocytosis and then induce activatable drug release.

P

Figure 3. Intracellular delivery of Ce6- SNA-Dox-AS1411 (A) T and Ce6- SNA-Dox-AS1411 (B) on HeLa cells observed by P confocal images. The cells were incubated with Ce6- SNAT Dox-AS1411 or Ce6- SNA-Dox-AS1411 at 37 °C for 4 h respectively.

Monitoring Endogenous ATP-Triggered Drug Release within Cancer Cells. Next, we investigated the ability of the targeted drug release from the Ce6-PSNADox-AS1411 triggered by ATP in HeLa cells. Ce6-PSNADox-AS1411 were incubated with the HeLa cells to visualize the release of Dox and Ce6. Fluorescence collection gates of Dox (570−620 nm) and Ce6 (660−750 nm) were carefully controlled to eliminate cross-color interference between Dox and Ce6 channels. After a 4 h incubation at 37 °C, it was observed that the fluorescence intensity of Dox and Ce6 were both strong in HeLa cells, and Ce6 fluorescence in the cytoplasm and Dox in the nucleus (Figure 3A). The Dox in the nucleus mainly binds with chromosomes to exert an anticancer function. These results demonstrated that the Ce6-PSNA-Dox-AS1411 was an efficient drug (Dox and Ce6) loading and delivery system, and the ATP in the HeLa cells could induce an intracellular drug release. Of note, although it has similar targeted capability with Ce6-TSNA-Dox-AS1411, traditional sandwich SNA was used as carriers (Figure S10), both relatively weak fluorescence of Ce6 and Dox were observed, which were consistent with the fact that the higher drug loading of developed PSNA (Figure 3B). To confirm the extent of Dox and Ce6 release was indeed dependent on endogenously produced ATP in HeLa cells, the cells were pretreated with some stimulants of ATP and then probed the influence of Dox and Ce6 re-

lease. It was found that, after HeLa cells pretreated with 5 mM Ca2+, a commonly used ATP inducer,38 a significant fluorescence enhancement of Dox and Ce6 compared with the untreated cells. Whereas, much weaker Dox and Ce6 fluorescence signals were obtained upon treatment with 10 μM oligomycin, a well-known inhibitor of ATP39 (Figure 4). These results revealed that Ce6-PSNA-DoxAS1411 was able to give fluorescence signals dynamically correlated to the level of ATP, confirming that this strategy had great potential for controllably releasing of Dox and Ce6 in response to varying levels of ATP.

Figure 4. Images of HeLa cells treated with medium (A), 5 2+ mM Ca (B), or 10 μM oligomycin (C) followed by incubation P with 1 nM Ce6- SNA-Dox-AS1411 for 4 h at 37 °C.

Chemotherapeutic and Photodynamic Synergistic Therapy. Having demonstrated the ability of functionalized PSNA for trageted drug delivery and controlled drug release in cancer cells, we then investigated the chemotherapy and PDT synergistic effect of functionalized PSNA on HeLa cells and Hacat cells using a MTS assay. As shown in Figure 5A, In the absence of 650 nm laser irradiation, the Ce6-PSNA-AS1411 was basically noncytotoxic to HeLa cells. Under the 15 min irradiation with laser, the Ce6-PSNA-AS1411 exhibited enhanced phototoxicity, which could be ascribed to PDT led to a decrease of HeLa cells viability. Moreover, in HeLa cells, a significant decrease of cell viability without laser irra-diation was also observed after Ce6-PSNA-DoxAS1411 treatment, owing to ATP-specific release of Dox for chemotherapy in cancer cells. For HeLa cells incubated with Ce6-PSNA-Dox-AS1411, and then with laser irradiation, the viability of HeLa dropped dramatically to below 20% at a high concentration of 1 nM. Compared to the only chemotherapy or PDT group, the combination therapy of chemotherapy and PDT could remarkablly enhance antitumor effects due to the chemotherapy−PDT synergistic therapy. It should be pointed out that, the inhibition ratios of functionalized PSNA were obviously higher than TSNA, as a result of enhanced Dox and Ce6 release (Figure S13). In addition, no significant loss of viability was found in normal Hacat cells regardless of single modal therapy or synergistic therapy (Figure 5B), which was consistent with aforementioned findings that


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Analytical Chemistry lower uptake efficiency of Ce6-PSNA-Dox-AS1411 in Hacat cells. To further verify this result, the cell apoptosis efficacy induced by each PSNA formulations were also interrogated by flow cytometry. The data obtained by flow cytometry also demonstrated the specific cancer cell inhibition (Figure S14).

ACKNOWLEDGMENT This study was financially supported by the National Natural Science Foundation of China (21527810, 21705041), National Key Basic Research Program (2011CB911000) and China Postdoctoral Science Foundation funded project (2017M622566).

REFERENCES (1) (2) (3) (4) (5) Figure 5. Cytotoxicity assay for HeLa and Hacat cells using different approaches.

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CONCLUSIONS

In summary, we have developed an a novel PSNA nanoplatform for targeted drug-controlled release and high-performance cancer therapy. The nanoplatform could be easily prepared by self-assembled from two short alternating DNA building blocks upon initiation of immobilized capture probes on AuNPs, through a supersandwich hybridization reaction. Compared with traditional sandwich SNA, the developed PSNA showed advantageous performance including size controllability, high specificity, and high drug loading. More importantly, the mulifunctional PSNA enabled selectively target the tumor cells and specifically interact with endogenous ATP, resulting in efficient releasing of Dox and Ce6 for chemotherapy−PDT synergistic therapy. The high payload drugs capacity make this PSNA a promising targeted drug transport platform for cancer theranostics. We believe that this work provides a successful theranostic paradigm to develop the new drug delivery PSNA system for cancer imaging and therapy.

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ASSOCIATED CONTENT

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website. Experimental method, DNA sequence, scheme of traditional sandwich SNA, agarose gel electrophoresis, fluorescence assay, TEM image, UV-vis spectroscopy, DLS, zeta potential, stability assay, cell viability, ICP-MS, confocal fluorescence image, apoptosis assay (PDF)

AUTHOR INFORMATION

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Corresponding Author *Fax: +86-731-88821916. Email: [email protected];

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[email protected].

ORCID (25)

Jin-Wen Liu: 0000-0001-9340-8109 Jian-Hui Jiang: 0000-0003-1594-4023

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Notes The authors declare no competing financial interest.

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