Terminal Deoxynucleotidyl Transferase-Catalyzed Preparation of pH

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Biological and Medical Applications of Materials and Interfaces

Terminal Deoxynucleotidyl Transferase-Catalyzed Preparation of pHResponsive DNA Nanocarrier for Tumor-Targeted Drug Delivery and Therapy Guo-Ying Sun, Yi-Chen Du, Yunxi Cui, Jing Wang, Xiao-Yu Li, An-Na Tang, and De-Ming Kong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05358 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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ACS Applied Materials & Interfaces

Terminal Deoxynucleotidyl Transferase-Catalyzed Preparation of pH-Responsive DNA Nanocarrier for Tumor-Targeted Drug Delivery and Therapy Guo-Ying Sun, Yi-Chen Du, Yun-Xi Cui, Jing Wang, Xiao-Yu Li, An-Na Tang, De-Ming Kong* State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, 300071, P R China

This work is dedicated to 100th anniversary of Nankai University

KEYWORDS:

DNA

nanocarrier,

anticancer

therapy,

deoxynucleotidyl transferase (TdT), pH-responsive, AS1411

ACS Paragon Plus Environment

doxorubicin

(Dox),

terminal

ACS Applied Materials & Interfaces 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

ABSTRACT: Developing a highly efficient carrier for tumor-targeted delivery and site-specific release of anticancer drugs is a good way to overcome the side effects of traditional cancer chemotherapy. Benefiting from the nontoxic and biocompatible characteristics, DNA-based drug carriers have attracted increasing attention. Herein, we reported a novel and readily manipulated strategy to construct spherical DNA nanocarrier. In this strategy, terminal deoxynucleotidyl transferase (TdT)-catalyzed DNA extension reaction is used to prepare a thick DNA layer on a gold nanoparticle (AuNP) surface by extending long poly(C) sequences from DNA primers immobilized on AuNP. The poly(C) extension products can then hybridize with G-rich oligonucleotides to give CG-rich DNA duplexes (for loading anticancer drug doxorubicin, Dox) and multiple AS1411 aptamers. Via synergic recognition of multiple aptamer units to nucleolin proteins, biomarker of malignant tumors, Dox-loaded DNA carrier can be efficiently internalized in cancer cells and achieve burst release of drug in acidic organelles due to i-motif formationinduced DNA duplex destruction. As-prepared pH-responsive drug carrier was demonstrated to be promising for highly efficient delivery of Dox and selective killing of cancer cells in both in vitro and in vivo experiments, thus showing a huge potential in anticancer therapy.

Introduction Cancer is one of the main causes of death and brings enormous challenges to the therapeutic process. As a result, many treatments such as radiotherapy1-4, chemotherapy5-7 and other synergistically combined therapy appeared8-10. Nevertheless, these strategies usually suffer from low therapeutic efficacy and inevitable side effects due to inefficient drug delivery to target sites and inability to discriminate cancer from healthy cells. To address these issues, various drug delivery vehicles, such as organic or inorganic nanoparticles11-14, nanosheets15-17, polymers18-20 and hydrogels21,22, have been developed. Although greatly improved cancer cell-targeted drug


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delivery is achieved, additional biotoxicity caused by some of these vehicles severely limit their applications. Compared with the exogenous materials, DNA possesses biologically benignancy with highly biocompatibility, which make it a predominant material for biotherapy and biomedical applications23-27. With the advance of DNA nanotechnology, many DNA nano-assemblies such as DNA origami28-30, DNA tetrahedron31,32 and DNA cages33,34 have been successfully constructed and applied to the delivery of anticancer drugs. But these nano-assemblies require for a lot of synthetic DNAs that need to be designed carefully35-38. Meanwhile, the low DNA density largely limits their drug-loading capacity. To address these issues, Tan group first put forward a tightly packed DNA nanostructure—DNA nanoflower using rolling circle amplification (RCA) technique and applied it to tumor-targeted delivery of anticancer drug doxorubicin (Dox)39. One problem however, is that the tightly packed structure of DNA nanoflower (due to the presence of large amounts inorganic salts) always drag the speed of drug release in cells40. In addition, to obtain highly dispersed DNA nanoflowers, the RCA template sequence should be elaborately designed to ensure to form highly ordered secondary structures. Therefore, it is undoubtedly ideal to establish a simple method to gain DNA-based nanocarriers, which can achieve high drug payload, targeting delivery and burst intracellular release simultaneously. As we know, DNA sequences always suffer from enzymolysis when exposed in complex physiological environments. This may help in the drug release process in cells partly but could also cause the premature release and loss of drugs41. Recent researches have revealed that tight packing of nucleic acids on nanoparticle surface can resist enzymolysis and facilitate cellular internalization in the meanwhile41-45. Therefore, easily prepared DNA-modified gold nanoparticles (AuNPs) have been applied as drug delivery carriers46-48. The large surface areas of AuNPs could



immobilize hundreds of DNA sequences and protect DNAs from the complicated environments through steric effect.49-51 Nevertheless, due to the short chain DNA modification, the DNA layer on the surface of AuNP is relatively thin and cannot achieve efficient drug payload. When it comes to the long chain DNA modification, the greatly increase of DNA synthesis cost and the big damage for DNA assembly efficiency on AuNP also bother us. In this work, we proposed a simple way to increase the length of DNA oligonucleotides immobilized on AuNP surface, and thus prepared a promising DNA-based drug carrier, which shows attractive characteristics of high drug payload, cancer cell-targeted delivery and pHcontrolled burst release. As-prepared nanocarrier was demonstrated to work well for highly efficient drug delivery and cancer-targeted therapy both in vitro and in vivo.

Experimental Section Materials. DNA oligonucleotides (Table S1) were synthesized and purified by Sangon Biotech. Co. Ltd (Shanghai, China). Hydrogen tetrachloroaurate(III) (HAuCl4·4H2O, 99.99%) was bought from China National Pharmaceutical Group Corporation (Shanghai, China). Sodium chloride (NaCl), sodium citrate (C6H5Na3O7·2H2O), disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were obtained from Heowns Biochem Technologies. LLC. (Tianjin, China). Terminal deoxynucleotidyl transferase (TdT), dCTP and cobalt chloride (CoCl2) were purchased from New England Biolabs (Beijing, China). Dulbecco’s modifed Eagle’s medium (DMEM, Hyclone), penicillin-streptomycin, fetal bovine serum (FBS, Hyclone), 0.25% TrypsinEDTA (1), phenol red were purchased from Gibco (Tianjin, China). 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) was bought from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) and 4′,6-diamidino-2-phenylindole (DAPI) were bought from Beyotime Institute of Biotechnology (Shanghai, China).


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Preparation of AuNPs. Citrate reduction method was used to synthesis AuNPs. All the glasswares used were completely immersing in aqua regia (HNO3/HCl, 1:3, v/v) and rewashed by ultrapure water extensively. 100 mL of 0.01% HAuCl4 was firstly heated to boil with rapid stir. Then 4.5 mL of 1% sodium citrate was quickly mixed in and further stirred for 15 min until the solution color turned to deep red. The products were collected after natural cooling and stored at 4 oC. Preparation of the AuNP@Primers. AuNP@Primers were prepared through salt aging process. 20 μL of 100 μM thiol-modified Primer (HS-Primer, Table S1) was mixed with 1 mL of 4 nM AuNP and gently shaken for 3 h under room temperature for preliminary link. Then, sodium citrate-hydrochloric acid buffer solution (100 mM, pH = 3) was added to adjust the solution’s pH value. After shaken for another one hour, 2 M NaCl was slowly added in the mixture stepwise to a final concentration of 0.4 M and then vibrated at room temperature overnight. The products were centrifuged at 4 oC for 30 min (12000 rpm) and washed three times with 10 mM PB buffer (pH 7.4). At last, the obtained AuNP@Primers were redispersed in 150 μL PB buffer and stored at 4 oC

for further use. The concentration of AuNP@Primers was measured by the absorption at 524

nm (ε = 3.0  108 L·mol-1·cm-1). Synthesis of Dox-loaded AuNP@Primers/(CG)n/AS1411s. A mixture of 5 nM AuNP@Primers, 1 mM dCTP, 0.25 mM CoCl2 and 20 U TdT was prepared in 1  TdT reaction buffer consisting of 10 mM magnesium acetate, 20 mM Tris-acetate and 50 mM potassium acetate, pH 7.9. The reaction was under 37 oC for 4 h. Later, 5 μM of AS1411-containing rich-G oligonucleotide (rG-AS1411, Table S1) was mixed in and reacted at 25 oC for one hour. After that, 100 μM of Dox was added in and gently vibrated at room temperature overnight. At last, products



were centrifuged at 4 oC for 30 min (12000 rpm) and washed with ultrapure water to remove the uncombined DNAs and Dox. Gel electrophoresis assay. The products of amplification reaction were analyzed by 3% agarose gel in 1 × TAE buffer at a 85 V constant voltage for 40 min at room temperature to confirm the successful assembly of AuNPs with HS-Primer, TdT-catalyzed primer extension on AuNP surface, hybridization of poly(C) extension product with rG-AS1411 and thus to ensure the feasibility of the proposed system. For comparison, we did Primer extesion experiment on/without AuNPs at the same time. As for Primer extension without AuNPs, a mixture of 0.5 μM Primer without thiol modification (Primer, Table S1), 1 mM dCTP, 0.25 mM CoCl2 and 20 U TdT was prepared in 1  TdT buffer. It was reacted at 37 oC for 4 h and then added in 1  loading buffer. After stained with GelRed for 20 min, the gel was imaged by Gel Documentation system (Huifuxingye, Beijing, China). The Primer extension on AuNP surface was performed as above excepted that Primers were replaced by AuNP@Primers, and the gel was directly photographed under white light. CD spectroscopy assay. A mixture of 1 μM Primer (3 mL), 1 mM dCTP, 0.25 mM CoCl2 and 20 U TdT was prepared in 1  TdT buffer. Then the reaction was under 37 oC for 4 h. After that, adjusted the pH value to 5.0, 5.5, 6.0, 6.5, 7.0 and 7.4, respectively, with the help of HCl and NaOH. The CD spectra (scanning speed = 100 nm/min, bandwidth = 0.5 nm, response time = 1 s) were recorded in the range of 210-340 nm. Final results were the average of three times-scanning. Cell culture. There were two kinds of experimental cells in this work. HeLa (Human epithelial carcinoma) and NIH-3T3 (Human embryo fibroblast) cells were cultured in DMEM supplemented with 10% FBS and 100 U/mL 1% penicillin/streptomycin at 37 oC in a 100% humidified atmosphere containing 5% CO.


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Laser confocal imaging. The experimental cells were seeded with DMEM medium in 24-well plate (4  104 cells per well) and cultured in a humidified atmosphere containing 5% CO2 at 37 oC overnight. With the final concentration of 1 nM, Dox-loaded nanocarriers prepared before were incubated with two kinds of cells respectively for 1 h, 2 h or 3 h. Then, the plate was washed with PBS buffer. Next, DAPI (0.5 mg mL-1) was used to stain cell nucleus. After being washed with PBS buffer, confocal laser scanning microscopy (CLSM) was used for cells imaging with 425 nm excitation. Cell viability test. MTT assay was used to determine the in vitro cell viability. Brieﬂy, the experimental cells were respectively seeded with DMEM medium (100 mL) at 96-well plates (5000 cells per well) and incubated in a humidified atmosphere containing 5% CO2 overnight. Next, the original medium was abandoned and fresh medium containing free Dox, AuNP@Primers/(C)n,

AuNP@Primers/(CG)n/AS1411s

or

Dox-loaded

AuNP@Primrs/(CG)n/AS1411s was added. The cells incubated with medium alone set as the control. After incubation for 36 h or 48 h, the plate was washed with PBS buffer, and then 100 µL culture medium with MTT (0.5 mg/mL) contained was added and incubated for another 4 h. Next, the medium was abandoned to wash away remaining MTT with addition of 150 µL DMSO followed. To dissolve the formazan crystals completely, the plate was vibrated for 5 min. The cell survival rate was calculated from the absorbance at 490 nm determined by a microplate reader (Bio-Rad iMark, America). Six parallel experiments were conducted for each group. In vivo experiment. All animal experiments were approved by the POSTECH Biotech Center Ethics Committee. Experimental mice were our-week-old Balb/c nu/nu female mice. 107 of Hela cells were collected and subcutaneously inoculated at the right ﬂank of mice. The experimental mice were divided into 3 groups randomly once the tumor volume reached about 200 mm3. One



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group was set as control that was injected with 0.9% saline. Other two groups were treated with AuNP@Primers/(CG)n/AS1411s or Dox-loaded AuNP@Primers/(CG)n/AS1411s, respectively, via tail intravenous injection every day for totally two weeks. The quantity of loaded Dox was controlled to 0.5 mg kg-1. The body weight and tumor volume were monitored and recorded during three weeks. Histological analysis of visceral organs and tumor tissues were achieved through hematoxylin and eosin (H&E) staining. A biological microscope (Leica DM3000) were applied for the final images.

Results and Discussion Design and preparation of DNA nanocarrier. The proposed pH-responsive DNA nanocarrier was prepared from DNA-modified AuNPs (AuNP@Primers) (Scheme 1). Via the formation of Au-S bonds, short DNA strands (HS-Primer, Table S1) can be easily immobilized on the AuNP surface with high intensity (about 100 HS-Primers/AuNP). Then, terminal deoxynucleotidyl transferase (TdT)-catalyzed DNA extension reaction is conducted on the AuNP surface to increase the thickness of DNA layer. TdT is a special enzyme that can extend single-stranded or doublestranded DNA primers in a template-independent manner and its extension product is depended on the identity and ratio of added deoxynucleotide(s)52,53. Only if dCTP added, a long poly(C) sequence with several hundreds of nucleotides could be easily linked at the 3′-end of DNA primers in a few of hours54,55. Then, via a simple DNA hybridization reaction, a G-rich oligonucleotide containing an AS1411 aptamer (rG-AS1411, Table S1) can be further assembled on aboveprepared

AuNP@Primers/(C)n

to

give

the

final

DNA

nanocarrier

AuNP@Primers/(CG)n/AS1411s. Herein, AuNPs play the following roles: (1) they are the cores of the nanocarriers. One AuNP can connect hundreds of TdT-catalyzed DNA extension products and thus assemble them into one nanosystem to form a nanocarrier; (2) they can protect DNAs


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from enzymolysis in complicated biological environments,49-51 thus endowing the nanocarrier with improved biostability; (3) due to the presence of AuNPs, the prepared nanocarrier can be collected by centrifugation, thus making the preparation of highly purified nanocarrier possible. AS1411 is the aptamer of nucleolin, a cancer biomarker shows overexpression on most malignant cells’ plasma membrane56. The assembly of AS1411 can promote the targeted aggregation of the DNA nanocarrier towards cancer cells and enhance its cancer cell uptake efficiency through the synergy of endocytosis and AS1411-induced micropinocytosis pathways56,57. Poly(C) extension product was selected for two reasons: one is to achieve high drug loading capacity since it is reported that CG-rich DNA duplexes are better carriers of Dox than AT-rich ones58-61. The other is to realize pH-controlled burst release of drug in cancer cells62-64, thus overcoming the contradiction between carrier biostability and drug release rate in nucleases-controlled release pathway. That is, when Dox is delivered into cancer cells by the DNA nanocarrier, the poly(C) sequence might fold into “i-motif” structure in acidic organelles (e.g. lysosome (pH 4.5–5.5) and endosome (pH 5.4–6.2)), leading to the destruction of CG-rich DNA duplexes and burst release of loaded Dox.



Scheme 1. Illustration of the preparation of Dox-loaded AuNP@Primers/(CG)n/AS1411s nanocarrier and its cancer cell-targeted delivery, internalization and pH-responsive drug release. Characterization of the DNA nanocarrier. Several ways was applied to verify the successful preparation of pH-responsive DNA nanocarrier. “Step-by-step” assembly of DNA on AuNP surface was determined by UV-vis absorption spectroscopy (Figure 1a). Bare AuNPs had a characteristic absorption peak at 519 nm. After modificated with short-stranded TdT primers, the peak red-shifted to 524 nm, and an obvious absorbance peak, corresponding to the characteristic


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absorption of DNA, emerged at 260 nm meanwhile. The absorption peak at 260 nm became more and more obvious after successive treatment of AuNP@Primers with TdT enzyme and rGAS1411, thus strongly verified the successful performance of TdT-catalyzed primer extension on AuNP surface and subsequent highly efficient assembly of rG-AS1411. Transmission electron microscopy (TEM) results (Figure 1b) show that AuNPs kept in dispersive and highly stable state during above treatments. The negatively stained results of AuNP@Primers/(CG)n/AS1411s clearly showed the DNA strands extended from AuNP surface (Figure 1c and Figure S1). Dynamic light scattering (DLS) revealed the average diameter of bare AuNPs was about 15 nm (Figure 1d). It was increased to 36 nm with primer modification, and further increased to 104 nm then to 144 nm after TdT-catalyzed primer extension and subsequent hybridization reaction. These results indicated that the DNA layer on AuNP surface became thicker and thicker as expected. Agarose gel electrophoresis (Figure 1e and 1f) demonstrated that TdT-catalyzed primer extension could be performed both in solution and on AuNP surface. Bare AuNPs tended to self-aggregate in TAE buffer and could not migrate from the origin point. AuNP@Primers showed a good dispersity, and a clear band was observed in gel. The successful performance of TdT-catalyzed primer extension on AuNP surface was demonstrated by the observation that AuNP@Primers/(C)n showed a smeared band with slower migration rate than AuNP@Primers. After further incubation with rGAS1411, the migration rate of smeared band further decreased, thus representing the successful assembly of AuNP@Primers/(C)n with aptamer-containing complimentary strand. Collectively, all

of

above

characterization

experiments

demonstrated

AuNP@Primers/(CG)n/AS1411s nanocarrier was successfully prepared.


that

the

proposed


1.6

AuNPn AuNP@Primersn AuNP@Primers/(C)n

1.2


(d)

0.8 0.4 0.0 200

300

400

500

600

700

Wavelength (nm)

AuNPn AuNP@Primersn AuNP@Primers/(C)n AuNP@Primers/(CG)n/AS1411s

25

Number (%)

(a) Absorbance

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

800

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20 15 10 5 0

10

100

Size (d.nm)

1000

Figure 1. (a) UV-vis absorption spectra, (b) TEM images and (d) DLS characterization for AuNP, AuNP@Primers, AuNP@Primers/(C)n and AuNP@Primers/(CG)n/AS1411s, respectively. (c) Negatively stained TEM image of AuNP@Primers/(CG)n/AS1411s. An enlarged figure can be found in Figure S1. (e,f) Electrophoresis analysis of TdT-catalyzed reaction in solution (d) and on AuNP surface (e). M: DNA Marker; Line 1: Primer; Line 2: Primers/(C)n; Line 3: AuNP; Line 4: AuNP@Primers; Line 5: AuNP@Primers/(C)n; Line 6: AuNP@Primers/(CG)n/AS1411s. Biostability and pH-responsive characteristic of DNA nanocarrier. Our aim is to construct a pH-responsive drug carrier that is stable in blood circulation systems but can perform pHcontrolled drug release in cancer cells. As-designed nanocarrier was expected to display improved biostability owing to the protection of DNA layer by AuNPs. To verify this, 10% fetal bovine serum was used to mimic the physiological fluid to incubate with the constructed nanocarrier AuNP@Primers/(CG)n/AS1411s for a series of time at 37 oC. Gel electrophoresis analysis showed that the nanocarrier bands barely changed from o to 6 h (Figure S2), indicating that the prepared nanocarrier was stable in biological environments and showed great potential in drug delivery application.


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The pH-responsive characteristic of the proposed nanocarrier mainly comes from the formation of i-motif structure by poly(C) extension products under acidic conditions, which was firstly demonstrated by recording the circular dichroism (CD) spectral changes of Primers/(C)n under different pH conditions. As shown in Figure 2a, the CD spectrum of Primers/(C)n showed a positive peak at 277 nm and a negative one at 216 nm at pH of 7.4, which implies the ordinary single-stranded DNA structure. When the pH value was decreased to 7.0, however, significant CD spectral changes were observed. That is, a positive peak and a negative peak appeared at 286 nm and 265 nm, respectively, which means the successful formation of i-motif structure65-67. Decreasing pH could further promote the construction of i-motif structure, which was reflected on the CD signal intensities’ increase of both positive and negative peaks. i-motif structure formation by AuNP@Primers/(C)n under acidic conditions was further verified by DLS analysis (Figure 2b). With the decrease of pH from 7.4 to 5.0, a sharp decrease in nanoparticle diameter was observed, which could be interpreted by the shrinkage of poly(C) strands due to i-motif formation. And the sudden change in the pH range of 7.4~7.0 was perfectly consistent with CD results. After further assembly with rG-AS1411, the obtained AuNP@Primers/(CG)n/AS1411s particles retained the pH-responsive property, but the pH value required for i-motif formation was delayed. This might be reasonably explained by the competition between CG-rich duplex and i-motif. As shown in Figure 2b, AuNP@Primers/(CG)n/AS1411s showed a sharp size change in the pH range of 7.0~6.5. Such a delay of pH can ensure that the drug carrier keeps stable in transmission path but performs rapid drug release in acidic organelles of cancer cells.



6 4 2

(b) 160

pH 5.0 pH 5.5 pH 6.0 pH 6.5 pH 7.0 pH 7.5


140

Size (d.nm)

(a) 8 CD/medg

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

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0

120 100

AuNP@Primers/(C)n

80

-2 220 240 260 280 300 320 340

Wavelength (nm)

60

5.0

5.5

6.0

6.5

pH

7.0

7.5

Figure 2. (a) CD spectra of Primers/(C)n under different pH values; (b) DLS characterization of the size distribution of AuNP@Primers/(C)n and AuNP@Primers/(CG)n/AS1411s under different pH conditions. Above experiments were performed immediately after sample preparation; TEM images of (c) AuNP@Primers/(C)n and (d) AuNP@Primers/(CG)n/AS1411s at different time points under the pH condition of 5.0. pH-Responsive behavior of AuNP@Primers/(C)n could also be observed in TEM images. In acidic conditions, both intramolecular and intermolecular i-motif structures might be formed. Intermolecular i-motif was formed by two or more than two poly(C) strands, which might be provided either by same AuNP@Primers/(C)n or by different ones. Therefore, it is reasonable to predict that aggregation of AuNP@primer/(C)n particles might be observed due to the i-motif


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formation. TEM images confirm this speculation. That is, different from the long-term stability and dispersity displayed by AuNP@Primers/(C)n solution at pH of 7.4 (Figure S3), time-dependent aggregation behavior was clearly observed under acidic conditions (e.g., pH 5.0), accompanied by the color change of solution from red to purple (Figure S4). What needs to be highlighted is that this kind of aggregation is quite different from commonly observed stability reduction-induced AuNP coagulation that is irreversible, here formed aggregates could be easily broken and return to dispersible state when the solution pH was adjusted from 5.0 to 7.4. Correspondingly, the solution changed back to homogeneous and transparent red color (Figure S4). Such a reversible aggregation-disaggregation behavior indicated that it was really caused by pH-induced i-motif formation and destruction. And again, due to the competition between CG-rich duplex and i-motif, a delay of time was also observed for i-motif formation-induced aggregation of AuNP@Primers/(CG)n/AS1411s (Figure 2d). Drug payload and pH-controlled release. pH-induced conversion from CG-rich duplex to imotif makes as-prepared AuNP@Primers/(CG)n/AS1411s a promising carrier to realize burst release of drug in cancer cells. Then, its anticancer drug loading capacity and pH-induced drug release behavior were investigated. Dox was used as the model drug since it is a well-known anticancer drug widely used in clinical therapy. As expected, Dox could be successfully loaded in the nanocarrier with high efficiency (about 70% loading efficiency), which was confirmed by the greatly reduced Dox fluorescence after mixing with the nanocarrier (Figure 3a). The loading capability was calculated to be ~14000 Dox molecules per AuNP@Primers/(CG)n/AS1411s nanoparitcle. Before assembling with rG-AS1411, the precursor AuNP@Primers/(C)n could only load ~ 2400 Dox molecules. These results are accordingly with the previous reports that Dox preferentially intercalates into repeat CG base pairs58-61. Then, Dox release from the nanocarrier



was investigated (Figure 3b). Under neutral pH conditions (e.g. pH 7.4), CG-rich duplex in nanocarrier kept intact and only a low level of drug was release, which was reflected by the very slight Dox fluorescence recovery. These results suggested that the drug carrier would keep stable before entering cancer cells, and premature drug leakage into transmission paths can be efficiently overcome68,69. On the contrary, a sharply increase of Dox fluorescence was observed under acidic conditions with pH of 5.0, which is consistent with the proposed i-motif formation-induced drug release mechanism under acidic conditions. Such a pH-controlled way could release about 75% Dox in 1 h. Other Dox molecules, which might be wrapped in the DNA strands immobilized on AuNP surface, could be gradually released subsequently via DNA strand digestion by various nucleases in cancer cells. All the results indicated that the prepared nanocarrier might achieve burst drug release inside cancer cells, thus efficiently overcoming the challenge faced by DNA nanoflowers on drug release24. The drug-loading capability of the proposed DNA nanocarrier was further investigated by employing methylene blue (MB) as another modal drug. MB is a clinical drug for methemoglobinemia treatment, it is also a good candidate of photosensitizer for photodynamic therapy of tumors. Via the interactions between MB and DNA duplexes, this drug could also be efficiently loaded in the DNA nanocarrier with a loading capabilityof ~8000 MB molecules per AuNP@Primers/(CG)n/AS1411s nanoparticle (Figure S5). Under acidic condition, the loaded MB was rapidly released and the release efficiency could reach about 70% MB in 1 h at pH of 5.0. These results suggested the prepared DNA nanocarrier might be employed as a universal carrier for the delivery and pH-controlled release of drugs that can interact with DNA.


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(b)100

(a) 700 600

Dox release (%)

Dox fluorescence (a.u.)

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


500 400 300 200 0

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pH 5.0

60 40 pH 7.4

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Time (min)

240

Figure 3. (a) Rapid loading of Dox on AuNP@Primers/(CG)n/AS1411s and (b) different release behaviors under different pH conditions. Cancer cell-targeted delivery and release of Dox. To endow the nanocarrier with cancer celltargeted recognition and accumulation abilities, thus to enhance the drug delivery efficiency and weaken side effects, nucleolin aptamer—AS1411 was conveniently assembled on the nanocarrier via a simple hybridization between poly(C) products and rG-AS1411 oligonucleotides. After loading Dox, the prepared nanocarrier was incubated with HeLa cells and NIH-3T3 cells separately for 1, 2 and 3 h (Figure 4). We found that the Dox fluorescence given by nucleolinoverexpressed HeLa cells was obviously brighter than that given by nucleolin-negative NIH-3T3 cells, thus confirming the important roles played by AS1411. It was calculated that one AuNP@Primers/(CG)n/AS1411s carried about 676 AS1411 units. Since these AS1411 units might synergically interact with nucleolin proteins on the HeLa cell surface, the nanocarrier could accumulate towards HeLa cells and be efficiently internalized in them. Once the drug carrier reached the acidic organelles such as lysosome and endosome, the decreased pH environments promoted the conversion of poly(C) sequences in the nanocarrier from duplex to i-motif structure, thus resulting in the rapid release of loaded Dox. Time-dependent Dox fluorescence increase



suggested that the loaded Dox molecules were successfully released, and the released Dox molecules spread in both cytoplasm and nucleus, mainly in nucleus. Such a sustained accumulation in the nucleus makes the released Dox a good killer of cancer cells. These results demonstrated that the prepared drug carrier could efficiently distinguish cancer cells from healthy ones, and thus might efficiently overcome the side effect problem faced by traditional Dox-based chemotherapy.

Figure 4. Fluorescence images of HeLa and NIH-3T3 cells incubated with 1 nM Dox-loaded AuNP@Primers/(CG)n/AS1411s for 1 h, 2 h and 3 h at 37 oC. Selective killing of cancer cells. To investigate whether the utility of the nanocarrier can endow Dox with the selective killing cancer cells ability or not, HeLa and NIH-3T3 cells were cultured with same concentrations of free Dox or Dox-loaded AuNP@Primers/(CG)n/AS1411s, respectively, and the cell viabilities were analysed by the MTT assay (Figure 5). Different from free Dox that had nearly identical cytotoxicity towards HeLa and NIH-3T3 cells, Dox-loaded AuNP@Primers/(CG)n/AS1411s nanocarrier showed much more obvious cytotoxicity towards HeLa than towards NIH-3T3 cells, which is consistent with above-observed different drug delivery efficiencies of the prepared nanocarrier towards nucleolin-positive cancer cells and nucleolinnegative healthy cells, thus confirming that the nanocarrier has the potential to reduce the side


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effects of loaded drugs. Their IC50 values also supported the conclusion (Figure S6). Free Dox showed comparable IC50 values for HeLa and NIH-3T3 cells (0.47 vs 0.44 μM). On the contrary, Dox-loaded AuNP@Primers/(CG)n/AS1411s gave obviously lower IC50 value towards HeLa than towards

NIH-3T3

cells

(0.62

vs

1.01

μM).

Interestingly,

without

loading

Dox,

AuNP@Primers/(CG)n/AS1411s was also able to kill HeLa cells in some degree but showed little effects on NIH-3T3 cells. We speculated that such a cancer cell-specific cytotoxicity might be attributed to AS1411-induced proliferation inhibition considering that AS1411 was the first anticancer aptamer found in phase II clinical trials70-72. Such a speculation was confirmed by negligible cytotoxicity of AuNP@Primers/(C)n to the two kinds of experimental cells. That is, the assembly of AS1411 aptamers not only confers the nanocarrier with cancer cell recognition specificity,57,73 but also might be helpful to improve the therapeutic effect in some degree. In addition, this DNA nanocarrier preparation strategy might also provide a prospective way for the delivery of aptamer drugs and antisense drugs considering that these nucleic acid-based drugs are difficultly internalized in cells by themselves.

36 h

100

Cell Viability (%)

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


48 h NIH-3T3

Hela

80 60 40 20 0

1

2

3

4

1

2

3

4

Figure 5. Cell viability of Hela and NIH-3T3 cells cultured with (1) Free Dox, (2) AuNP@Primers/(C)n,

(3)



and

(4)

Dox-loaded


AuNP@Primers/(CG)n/AS1411s for 36 h or 48 h. Error bars are standard deviations of 6 samples (n = 6). The concentration of each material is equivalent of 1 μM Dox. In vivo cancer therapy application. Having demonstrated that the prepared nanocarrier is promising for cancer cell-targeted drug delivery in in vitro experiments, we next to investigate its feasibility of in vivo cancer therapy using HeLa tumor-bearing mice as the model. When the average tumor volume reached about 200 mm3 (the 6th day in Figure 6), 9 mice were divided into 3 groups randomly and respectively treated with 0.9% saline, AuNP@Primers/(CG)n/AS1411s or Dox-loaded AuNP@Primers/(CG)n/AS1411s via tail intravenous injection. Before treatment, all the mice showed a similar tumor development tendency. After treatment, however, significant difference was observed for the three groups. Saline-treated group showed the most rapid tumor growth. AuNP@Primers/(CG)n/AS1411s could delay the tumor development in some degree, but continuous increase in tumor size could still be observed. On the contrary, Dox-loaded AuNP@Primers/(CG)n/AS1411s showed distinct tumor cure potential. After treatment for 7 days (the 13th day in Figure 6), tumor growth was efficiently controlled and the tumor size turned from increase to decrease. After treatment for 13 days, the tumor was even smaller than that before treatment, indicating that such a treatment could not only inhibit the proliferation of cancer cells, but also lead to ablation of tumor. During the whole treatment, all the mice were in a good shape. their weight went up steadily and didn’t show any exception (Figure 6c), confirming the significantly reduced side effects due to the loading of Dox in prepared DNA nanocarrier.


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Relative tumor volume (%)

(a)

0.9% Salinen AuNP@Primers/(CG)n/AS1411s

1000

Dox-loaded AuNP@Primers/(CG)n/AS1411s

800 600

Therapy

400 200 0

2

4

6

8 10 12 14 16 18 20

Day

(c) 30 Weight (g)

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


28 26 24 22 20 18 16 14 12

0.9% Saline AuNP@Primers/(CG)n/AS1411s

Dox-loaded AuNP@Primers/(CG)n/AS1411s

0

5

10

15

Day

20

25

30

Figure 6. (a) Growth curves of tumor volume with different treatments. (b) Photos of tumorbearing mouse with different treatments and corresponding histological analysis results of dissected tumor tissues. (c) Mouse body weight changes after different treatments. Histological analysis. Histological analysis indicated that the main visceral organs (intestine, lung, liver, spleen, kidney, stomach, heart) of all tested mice are in healthy stage (Figure S7), which means our drug delivery system possesses favorable biocompatibility and negligible side effects. However, significantly different results were given by tumors of different groups (Figure 6b). The tumor tissue of saline-treated group was tightly connected and stayed in a healthy stage. On the contrary, Dox-loaded AuNP@Primers/(CG)n/AS1411s-treated group showed incompact tissue structure and obvious apoptotic cells in tumor tissue. Correspondingly, the cell apoptosis rate was increased from 8.30% to 62.29% when Dox-loaded AuNP@Primers/(CG)n/AS1411s, instead of 0.9% saline, was used to treat tumor-bearing mice (Figure S8), thus further demonstrating that our nanocarrier could specifically delivery Dox to tumor sites and effectively kill cancer cells.

Conclusion



In summary, a novel strategy was reported for the construction of DNA-based drug carrier via TdT enzyme-catalyzed DNA extension on AuNP surface and simple DNA hybridization. TdTcatalyzed DNA extension reaction not only provides a simple way to increase the thickness of DNA layer on AuNP surface, but also endows the nanocarrier with pH-responsive characteristic by producing i-motif structure-forming poly(C) extension products. Via subsequent DNA hybridization between poly(C) products and aptamer-containing G-rich oligonucleotides, CG-rich DNA duplexes (good carriers of anticancer drug Dox) and multiple AS1411 aptamer units, which confer the nanocarrier with cancer cell-targeted recognition and accumulation capabilities, are successfully assembled. Experiments both in vitro and in vivo demonstrated that as-prepared DNA nanocarrier is promising for highly efficient delivery of Dox and selective cancer cells killing, thus showing a huge potential in anticancer therapy. ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.” Sequence of the oligonucleotides used in this work, supporting Figures S1–S8. AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21728801, 21874075), the National Natural Science Foundation of Tianjin (No. 16JCYBJC19900) and the Fundamental Research Funds for Central Universities (Nankai University, China). REFERENCES (1) Baumann, M.; Krause, M.; Overgaard, J.; Debus, J.; Bentzen, S. M.; Daartz, J.; Richter, C.; Zips, D.; Bortfeld, T. Radiation Oncology in the Era of Precision Medicine. Nat. Rev. Cancer 2016, 16, 234–249. (2) Schaue, D.; McBride, W. H. Opportunities and Challenges of Radiotherapy for Treating Cancer. Nat. Rev. Clin. Oncol. 2015, 12, 527–540. (3) Pottier, A.; Borghi, E.; Levy, L. Metals as Radio-Enhancers in Oncology: The Industry Perspective. Biochem. Biophys. Res. Commun. 2015, 468, 471–475. (4) Nelson, D. F. Radiotherapy in the Treatment of Primary Central Nervous System Lymphoma (PCNSL). J. Neuro-Oncol. 1999, 43, 241–247. (5) Iwao, O. Guided Molecular Missiles for Tumor-Targeting Chemotherapy—Case Studies Using the Second-Generation Taxoids as Warheads. Acc. Chem. Res. 2008, 41, 108–119.



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Terminal Deoxynucleotidyl Transferase-Catalyzed Preparation of pH

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