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Self-assembled “DNA Nanocentipede” as Multivalent Drug Carrier for Targeted Delivery Wenshan Li, Xiaohai Yang, Leiliang He, Kemin Wang, Qing Wang, Jin Huang, Jianbo Liu, Bin Wu, and Congcong Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08210 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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ACS Applied Materials & Interfaces

Self-assembled “DNA Nanocentipede” as Multivalent Drug Carrier for Targeted Delivery

Wenshan Li, Xiaohai Yang*, Leiliang He, Kemin Wang*, Qing Wang, Jin Huang, Jianbo Liu, Bin Wu and Congcong Xu

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China

* Address correspondence to these authors at: State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P.R. China. Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [email protected]; [email protected].

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Abstract An idea drug carrier, with good binding affinity, selectivity, drug payload capacity and cellular internalized capability, will greatly improve the efficiency of target delivery. Herein a self-assembled and multivalent DNA nanostructure was developed as drug carrier for efficient and targeted delivery. The DNA structure was similar to centipede and composed of “trunk” and “legs”: the trunk was self-assembled DNA scaffold via hybridization chain reaction (HCR) from two biotinylated hairpin monomers upon initiation by a trigger DNA, and the legs were biotinylated aptamers which were conjugated to the trunk via streptavidin-biotin affinity interaction. The long trunk of “DNA nanocentipede” was loaded with doxorubicin (Dox) and the legs were SMMC-7721 cell-binding aptamers (Zy1) which functioned as targeting moieties to firmly and selectively grasp target cells. The results of agarose gel electrophoresis and fluorescence anisotropy confirmed that Zy1-based DNA nanocentipedes (Zy1-Nces) were successfully constructed. Flow cytometric analyses demonstrated that Zy1-Nces were more effective than free Zy1 in binding affinity and selectivity due to multivalent effect. Confocal microscopy studies demonstrated that the internalization was highly dependent on the higher valences of DNA nanocentipedes without the loss of selectivity. Meanwhile, Zy1-Nces exhibited high drug loading capacity and selective drug transport. The results of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT) assay showed enhanced cellular cytotoxicity of the Dox-loaded Zy1-Nces (Zy1-Nces-Dox) to the target SMMC-7721 cells, but not negative control L02 cells. This approach is


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applicable to prepare drug carriers for other targets just by reconstruction the nanocentipedes with relevant nucleic acid fragments. Keywords: self-assembly; multivalent; aptamer; targeted delivery; cancer theranostics

Introduction Nanomedicine has been considered to be a precise and efficient strategy for diagnosis and therapy of severe diseases.1-2 Multivalency, an important phenomenon in biological processes,3 played an essential role in this strategy due to its higher affinity and selectivity binding for ligand-receptor complexes than the monovalent counterparts.4-6 For instance, the multivalent binding can promote cellular internalization

of

nanocarriers.7-8

Intriguingly,

multivalency

also

means

multifunctional nanomedicine platform, such as combination therapy, theranostics, etc.9-10 Several artificial nanometer-sized multivalent systems were successfully designed and synthesized for biological applications. For example, Herlem et al. reported that multivalent ligands conjugated single wall carbon nanotubes greatly enhanced apoptosis effect, up to 10-20 fold than monovalent ligands;11 Martín et al. showed that giant globular multivalent glycofullerenes exhibited strong inhibition of cell infection by Ebola-pseudotyped viral;12 Zhao and co-workers demonstrated that a Poly-Aptamer-Drug system using rolling circle amplification significantly increased therapeutic efficiency due to multivalent effects.13 In general, multivalent systems can be achieved by binding multiple ligands on a scaffold. The scaffold can be a polymer,14 nanoparticle,1, 15 dendrimer16 and DNA,17-18



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etc. The ligands, including folate,19 carbohydrate,20 antibody,21 peptide22 and aptamer,23 etc. are attached by covalent or non-covalent method to a scaffold. Among these methods, self-assembly has been demonstrated as a convenient and effective approach for developing multivalent systems in nanomedicine,24 because of the ease of synthesis/assembly and the facile organization of multiple ligands.25-28 Usually, self-assembly could be operated under mild physiological conditions in aqueous solutions. This feature enables the construction of multivalent systems which contain thermal-sensitive elements, such as antibodies and peptides.14,

29-30

Therefore,

self-assembly based multivalent systems are promising platforms for nanomedicine. Herein, we introduced a self-assembled “DNA nanocentipede” as multivalent drug carrier to address the multiple challenges of targeted delivery. The nanocentipede’s trunk was full of drug and its legs could firmly and selectively grasp target cell (As shown in Scheme 1). Thus, the drug-loaded DNA nanocentipede showed high killing activity to the target cell. In order to construct such a DNA nanostructure, a DNA scaffold was firstly obtained via hybridization chain reaction (HCR)31-32, i.e. a short trigger DNA initiates autonomous cross-opening of biotin-modified hairpin monomers H1 and H2. The DNA scaffold served as the trunk of nanocentipede and contained multiple biotins. Then, multiple biotinylated aptamers were linked to the trunk as the legs of nanocentipede through streptavidin-based linkage,33-34 which resulted in the formation of the DNA nanocentipedes. The trunk of nanocentipede can be tailor-designed to intercalate chemotherapeutic drugs (e.g. doxorubicin, Dox) and fluorogenic dyes (e.g. SYBR Green I), while these legs are expected to selectively


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bind to target cells. We hypothesized that the simultaneously binding of these legs to receptors on target cells could achieve high binding affinity and enhanced cellular internalization without the loss of selectivity due to multivalent effects. The self-assembly also implied resultant flexibility for DNA nanocentipede to integrate various functionalities.10,

24

Overall, the self-assembled and multivalent DNA

nanocentipede was promising for selective cancer cell recognition, bioimaging and targeted anticancer drug delivery. Results and discussion Construction and Characterization of DNA Nanocentipedes Construction of the DNA nanocentipedes was illustrated in Scheme 1 (detail procedures were described in Experimental Section S3 of Supporting Information; all DNA sequences were listed in Table 1). The building blocks of the nanocentipedes contain biotin-modified hairpin DNA strands (H1 and H2), DNA trigger (Trigger), streptavidin and biotinylated aptamers (Zy1) which showed specific binding to target SMMC-7721 cells (human hepatocellular carcinoma cells), but not to L02 cells (normal liver cells).35-36 The Trigger initialized mutual hybridization of H1 and H2 to form a DNA scaffold, aptamers were attached to the scaffold through streptavidin-biotin affinity interaction. Thus, a Zy1-based DNA nanocentipede (Zy1-Nce) could be constructed through self-assembly. We investigated the assembly reaction in solution by agarose gel electrophoresis (detail procedures were described in Experimental Section S4 of Supporting Information). Figure 1A showed the HCR products in the presence of various



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concentrations of Trigger. It could be observed that the average length of HCR products was inversely related to the concentration of Trigger. When we used higher concentration

of

Trigger,

more parallel polymerization

reactions occurred

spontaneously between alternating H1 and H2, resulting in shorter HCR products.37 In contrast, if we decreased the concentration of Trigger, the length of HCR products became longer, which could increase drugs payload capacity and conjugate to more aptamers. Moreover, a narrower band were observed under lower concentration, indicating the better homogeneity of HCR products (Figure 1A), so 250 nM Trigger was used in subsequent studies. Interestingly, when biotinylated Zy1 and streptavidin was introduced in the assembly system, the bright band was observed in the sample inlet without any migration (Figure 1B), indicating that biotinylated Zy1 and streptavidin were assembled on HCR products to form a much larger composite. In addition, the assembly process of DNA nanocentipedes was further verified by fluorescence anisotropy which is dependent on the size and mass of the fluorophore38-39 (detail procedures were described in Experimental Section S5 of Supporting Information). As shown in Figure 1C, with the introduction of each assembly unit into solution, the value of fluorescence anisotropy increased gradually, indicating the growth of size and mass of nanostructures. Moreover, the atomic force microscopy image and cross-section analysis of DNA nanocentipedes were shown in Figure S1 of Supporting Information (detail procedures were described in Experimental Section S6 of Supporting Information). Thus, these above results demonstrated that the DNA


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nanocentipedes were successful constructed as our design scheme. Nuclease Stability of DNA Nanocentipedes DNA molecules are widely applied in biomedical field. However, the major limitation of DNA is their short half-life due to the rapid nuclease hydrolysis, which hindered their applications.40 To assess the stability of DNA nanocentipedes under nuclease treatment, DNA nanocentipedes and their corresponding control HCR products (i.e. the trunk of nanocentipedes) were incubated with Exonuclease III (Exo III) at 37 °C for different durations (detail procedures were described in Experimental Section S7 of Supporting Information). As shown in Figure 2A, the DNA nanocentipedes structure remained intact after treatment with Exo III (0.05 units/µL) for 2 h, while HCR products were digested extensively at the same time (Figure 2B). The DNA nanocentipedes showed higher stability in comparison with HCR products. The improved stability of the DNA nanocentipedes suggested that biotinylated Zy1 and streptavidin attached on trunks probably prevent the structure from cleavage due to increased steric hindrance.41 Multivalent Binding Ability of DNA Nanocentipedes Multivalent binding events would greatly enhance binding affinity.42-44 In this study, the multivalent binding ability of DNA nanocentipedes was dependent on the length of nanocentipede’s trunk and the numbers of aptamer per nanocentipede. Flow cytometric analyses were performed to investigate the binding affinity between DNA nanocentipedes and SMMC-7721 cells (detail procedures were described in Experimental Section S8 of Supporting Information). FITC-labeled Zy1 was used to



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provide a means of fluorescent tracing. Dissociation constants (Kd, where Kd is inversely related to affinity) were used to quantify affinity of DNA nanocentipedes to SMMC-7721 cells. First, we investigated the effects of the length of nanocentipede’s trunk on affinity. As shown in Figure 3A, the decreasing concentration of Trigger led to a higher affinity to SMMC-7721, indicating the close dependence of the binding affinity and the length of nanocentipede’s trunk (more detail data were provided in Figure S2 of Supporting Information). No measurable change was observed when the concentration of Trigger was lower than 500 nM. Considered the agarose gel electrophoresis in Figure 1A, 250 nM of Trigger was used in subsequent experiments to investigated the effects of the numbers of aptamer per nanocentipede on affinity. As shown in Figure 3B, an increased concentration of Zy1 contributed to a higher affinity (more detail data were provided in Figure S3 of Supporting Information). No measurable change was observed when the concentration of Zy1 reached 12.5 µM. We chose 50 µM of Zy1 in the subsequent studies, and the molar ratio of H1, H2, Zy1, and Trigger was 100 : 100 : 200 : 1 (i.e. each H1/H2 was conjugated to a Zy1). It should be noted that if there was only one aptamer per nanocentipede, a lower affinity of nanocentipede was obtained compared to the aptamer alone, because of the large molecular weight of the aptamer plus scaffold complex compared to the aptamer alone.45 As shown in Figure 3B, when the concentration of Zy1 was 0.25 µM, i.e. the molar ratio of H1, H2, Zy1, and Trigger was 100 : 100 : 1 : 1 (i.e. mono-Zy1 tethered DNA nanocentipede), the Kd of nanocentipede was 50.52±6.77 nM which was higher than that of free Zy1 (Kd = 7.55±1.10 nM, as shown in Figure S4 of Supporting


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Information). Nevertheless, the addition of more aptamer to the DNA scaffold offset and overcome any loss in binding affinity. Selective Recognition Ability of DNA Nanocentipedes Flow cytometric analyses were also used to investigate the selectivity of DNA nanocentipedes (detail procedures were described in Experimental Section S9 of Supporting Information). As shown in Figure 4A and 4B, target SMMC-7721 cells and control L02 cells were incubated with random DNA library (Lib), free Zy1 (Zy1), random DNA library-based DNA nanocentipede (Lib-Nces) and Zy1-Nces at 4 °C for 30 min, respectively. Random DNA library and free Zy1 were all labeled with FITC. The results showed that the free Zy1 and Zy1-Nces were all capable of binding to SMMC-7721 selectively. Compared to free Zy1, the lower concentration of Zy1-Nces showed the same fluorescence intensity, which suggested that higher affinity could be achieved when multiple ligands were simultaneously bond to multiple receptors on the cell surface (Figure S5A of Supporting Information).46 It is known that aptamer is easily degradable under physiological environment, resulting in loss in selectivity. Therefore, the selective recognition ability of Zy1-Nces was also investigated in binding buffer and cell culture medium (containing 10% FBS) at 37 °C (detail procedures were described in Experimental Section S9 of Supporting Information). After treated with Zy1-Nces, SMMC-7721 cells showed significantly fluorescence signal shifts both in binding buffer (Figure 4C) and cell culture medium (containing 10% FBS) (Figure 4E), while no significant shift was observed for Zy1-Nces treated L02 cells (Figure 4D and 4F). This indicated that



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Zy1-Nces remained the selective recognition of target cell under simulated physiological environment. In addition, the fluorescence signals of SMMC-7721 cells treated with Zy1-Nces were 5.6-fold higher than that of SMMC-7721 cells treated with free Zy1 (Figure S5B and S5C of Supporting Information), which also suggested the higher affinity of multivalent DNA nanocentipedes.19 Cellular Internalization of DNA Nanocentipedes The cellular internalization is one of the key points for delivering chemotherapeutic drugs into target cells to induce cell destruction effectively.13 Therefore, we investigated the cellular internalization of Zy1-Nces using confocal microscopy (detail procedures were described in Experimental Section S10 of Supporting Information). The time-dependent internalization of Zy1-Nces in target SMMC-7721 cells was first investigated. As shown in Figure 5A, when SMMC-7721 cells treated with Zy1-Nces, intracellular fluorescence was gradually enhanced with culture time, which was much stronger than that of free Zy1 under the same conditions (Figure 5B). These results revealed that Zy1-Nces could facilitate cellular internalization due to multivalent effects. In addition, when SMMC-7721 cells treated with Zy1-Nces for more than 1 h, most of the green fluorescence from Zy1-Nces could overlap with the blue fluorescence from LysoTracker Blue (a marker for lysosome), which indicated that Zy1-Nces were localized to lysosomes.28 Therefore, we inferred that Zy1-Nces were internalized by target SMMC-7721 cells via endocytosis.47-48 To further investigated the effects on cellular internalization of increasing the valences of DNA nanocentipedes, Zy1-Nces and Mono-Zy1-Nces (i.e. mono-Zy1


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tethered DNA nanocentipedes) were employed. SYBR Green I, a fluorescent dye that binds specifically to double-stranded DNA,49 was used to stain Zy1-Nces and Mono-Zy1-Nces to provide fluorescent tracing (detail procedures were described in Experimental Section S11 of Supporting Information). Optimized concentration of DNA nanocentipedes (5 nM) and constant amount of SYBR Green I (1 ×) solutions were used in subsequent studies (Figure S6 of Supporting Information). As shown in Figure 6, when treated with Zy1-Nces, SMMC-7721 cells exhibited much stronger green fluorescence signal than those treated with Mono-Zy1-Nces. L02 cells were used as the control, and no fluorescence signal of DNA nanocentipedes was observed. These results revealed that the cellular internalization was highly dependent on the higher valences of DNA nanocentipedes without the loss of selectivity. Selective Transport of Anticancer Drug based on DNA Nanocentipedes A high drug-loaded and selective nanocarrier can efficiently increase intracellular anticancer drug concentration. The drug loading capability was defined as the number of Dox loaded per Zy1-Nce.50 Theoretically, the truck of DNA nanocentipede contained 100 pairs of H1 and H2, and each pair of H1 and H2 was able to provide 16 drug loading sites (C-G) for Dox, thus exhibit high drug capability due to its 1600 drug loading sites. To evaluate the amount of Dox loaded into the DNA nanocentipedes, fluorescence intensities of Dox (2 µM) were determined under different concentrations of Zy1-Nces (0 - 10 nM, detail procedures were described in Experimental Section S12 of Supporting Information).51-52 The spectra in Figure 7 showed that when the concentration of Zy1-Nces was 4 nM (i.e. molar ratio of



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Zy1-Nces/Dox was 1/500), Dox fluorescence was dramatically quenched. Therefore, approximately 500 Dox molecules were complexed with one Zy1-Nce. This ratio (1/500) was used in subsequent studies, and the Dox loaded Zy1-Nces were named Zy1-Nces-Dox. We then evaluated the selectivity transport of Zy1-Nces-Dox. Both target SMMC-7721 cells and control L02 cells were treated with Zy1-Nces-Dox and free Dox, respectively, followed by confocal microscopy observation of Dox fluorescence signals (detail procedures were described in Experimental Section S12 of Supporting Information). As shown in Figure 8 and Figure S7 of Supporting Information, when cells were treated with free Dox, strong Dox fluorescence signals were observed in both SMMC-7721 cells and L02 cells. In contrast, when cells were treated with Zy1-Nces-Dox, fluorescence of Dox was observed only in SMMC-7721 cells, but not in control L02 cells. This indicates the selectivity transport of Dox based on DNA nanocentipedes. Cytotoxicity of Anticancer Drug Delivered by DNA Nanocentipedes Based on the selectivity transport of Zy1-Nces-Dox, the cytotoxicity of Zy1-Nces-Dox

was

evaluated

through

3-(4,

5-Dimethylthiazol-2-yl)-2,

5-Diphenyltetrazolium Bromide (MTT) assay (detail procedures were described in Experimental Section S13 of Supporting Information). In this assay, both target SMMC-7721 cells and control L02 cells were treated with free Dox and Zy1-Nces-Dox, respectively. Free Dox showed high cytotoxicity on both SMMC-7721 cells and L02 cells (black lines in Figure 9A and 9B). In contrast,


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Zy1-Nces-Dox showed a much stronger cytotoxicity in SMMC-7721 cells compared with L02 cells (red lines in Figure 9A and 9B). In addition, Zy1-Nces without drug-loaded did not influence the cell viability of either target or control cells (Figure 9C). These results revealed that the drug delivered by DNA nanocentipedes could improve the therapeutic efficacy and reduce the side-effects of drugs. Conclusions In summary, a versatile multivalent targeted drug carrier has been developed based on the self-assembled DNA nanocentipede. The carrier present three advantages. First, compared to carriers constructed by covalent bonds, this self-assembly method for fabricating a functional carrier was easily to be performed. Second, the carrier could enhance binding affinity and facilitate cellular internalization without the loss of selectivity due to multivalent effects. Third, the carrier showed high drug loading capacity and targeted drug delivery, which result in improved therapeutic efficacy and reduced side-effects of drugs. Therefore, our DNA nanocentipede has the potential to become highly efficient nanocarrier for targeted delivery. Besides, the DNA nanocentipede might be used for other applications where multivalent binding is essential. AUTHOR INFORMATION Corresponding Author *Phone: 86-731-88821566. Fax: 86-731-88821566. E-mail: [email protected]; [email protected] Notes



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The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported in part by the Natural Science Foundation of China (21190040,

21175035,

21375034),

National

Basic

Research

Program

(2011CB911002). Supporting Information Experimental details including materials and methods, atomic force microscopy image, binding of Zy1-Nces with target SMMC-7721 cells, detailed data of the affinity, SYBR Green I payload capacity and fluorescence intensity of intracellular Dox. This material is available free of charge via the Internet at http://pubs.acs.org.

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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


A H1

H2 Aptamer 5’

Trigger 5’

3’

≡

Drug Biotin Streptavidin

B

Scheme 1. Illustration of self-assembled aptamer-based “DNA Nanocentipede” as multivalent drug carrier for targeted delivery. (A) The trunk of nanocentipede was formed via HCR from two short biotinylated DNA monomers (H1 and H2) upon initiation by a trigger DNA. The legs of nanocentipede were aptamers as a function of targeting moieties to target cells, and the multiple biotins on the trunk facilitated binding to biotinylated aptamers through streptavidin-based linkage. The trunk of nanocentipede was loaded with therapeutic drugs. (B) The DNA nanocentipede specifically bound to target receptors on target cells followed by enhanced cellular internalization due to multivalent effects. Drug efficiently unloaded from the nanocentipede and induced cytotoxicity to target cells.



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Table 1. Sequences of DNA probes. Probes H1

Sequences (5’-3’) Biotin-CGT CGT GCA GCA GCA GCA GCA GCA ACG GCT TGC TGC TGC TGC TGC TGC

FITC-labeled H1

Biotin-CGT CGT GCA GCA GCA GCA GCA GCA ACG GCT TGC TGC TGC TGC TGC TGC-FITC

H2

Biotin-TGC TGC TGC TGC TGC TGC ACG ACG GCA GCA GCA GCA GCA GCA AGC CGT

FITC-labeled H2

Biotin-TGC TGC TGC TGC TGC TGC ACG ACG GCA GCA GCA GCA GCA GCA AGC CGT-FITC

Trigger

TGC TGC TGC TGC TGC TGC ACG ACG

Zy1

ACG CGC GCG CGC ATA GCG CGC TGA GCT GAA GAT CGT ACC GTG AGC GCG T(T)10-Biotin

FITC-labeled Zy-1

FITC-ACG CGC GCG CGC ATA GCG CGC TGA GCT GAA GAT CGT ACC GTG AGC GCG T (T)10-Biotin

Lib

(N)49(T)10-Biotin

FITC-labeled Lib

FITC-(N)49(T)10-Biotin

a

FITC, if applicable, was labeled at the 5’-ends of Zy1 and Lib, 3’-end of H1 and H2.

b

The underlined of DNA probes represent the stem complementary sequence of the hairpin.


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A

B

5 000

5 000 1 000

1 000

100

100

bp

bp

Concentration of Trigger (nM)

0.08

C 0.06

0.04

H 1+ H

2+ Tr

ig g

2 1+ H H

H

1

0.00

er 1+ H +S 2+ A Tr +Z ig y1 ge r

0.02

H

Fluorescence Anisotropy

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


Figure 1. Characterization of the formation of DNA nanocentipedes. (A) Characterization of the self-assembly of trunks of nanocentipedes by agarose gel electrophoresis. Different concentrations of Trigger were incubated with mixtures of H1 and H2 (25 µM each) for 24 h, then diluted 25-fold with PBS, followed by 2% agarose gel electrophoresis and visualized with SYBR Gold staining. The average length of trunks of nanocentipedes is inversely related to the concentrations of Trigger. (B) Characterization of the self-assembly of Zy1-based DNA nanocentipedes (Zy1-Nces) by agarose gel electrophoresis. From left to right, lanes: (1) DNA Markers; (2) 50 µM H1; (3) 50 µM H2; (4) 25 µM H1 + 25 µM H2; (5) 25 µM H1 + 25 µM H2 + 250 nM Trigger; (6) 25 µM H1 + 25 µM H2 + 250 nM Trigger + 2 µM streptavidin (SA) + 2 µM biotinylated Zy1, then the samples were diluted 25-fold with PBS before



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

use. (C) Characterization of the self-assembly of DNA nanocentipedes by fluorescence anisotropy. H1 and H2 were labeled with FITC at the 3’-end. From left to right: (1) 100 nM H1; (2) 50 nM H1 + 50 nM H2; (3) 50 nM H1 + 50 nM H2 + 0.5 nM Trigger (4) 50 nM H1 + 50 nM H2 + 0.5 nM Trigger + 100 nM streptavidin (SA) + 100 nM biotinylated Zy1. Excitation: 488 nm, emission: 520 nm. The error bars indicated the standard deviations of three experiments.


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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


A 5000 1000 bp

B 5000 1000 bp

0

1

2

4 8 Time (h)

12

24 Mark

Figure 2. Nuclease stability of (A) the DNA nanocentipedes (Zy1-Nces) compared with (B) HCR products. Samples were incubated with Exo III (0.05 units/µL) at 37 °C for different times. The reaction mixtures were analyzed using 2% agarose gel electrophoresis and visualized with SYBR Gold staining.



A 1

0.1

50

00

00 25

50 12

0 50

0 25

Concentration of Trigger (nM)

100

Kd Value (nM)

10

Kd Value (nM)

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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B

10

1

0.1 0.25 1.25 2.5

5

12.5 25

50

Concentration of Zy1 (µM)

Figure 3. Characterization of the strong binding of Zy1-Nces with target SMMC-7721 cells at 4 oC. (A) Effects of the length of trunk of nanocentipede on dissociation constant (Kd). At a given concentration of H1/H2 (25 µM each), lower concentration of Trigger (i.e. longer trunk of nanocentipede) induced higher affinity of DNA nanocentipedes. (B) Effects of the numbers of aptamer per nanocentipede on Kd. At a given concentration of H1/H2 (25 µM each) and Trigger (250 nM), higher concentration of aptamer Zy1 (i.e. larger numbers of aptamer per nanocentipede) induced higher affinity of DNA nanocentipedes. The error bars indicated the standard deviations of three experiments.


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L02 in Binding Buffer

SMMC-7721 in Binding Buffer

A

Normalized Intensity

Cell only Lib 50 nM Lib-Nces 3 nM Zy1 50 nM

4 oC

Cell only Lib 50 nM Lib-Nces 3 nM Zy1 50 nM Zy1-Nces 3 nM

Zy1-Nces 3 nM

FITC

FITC SMMC-7721 in Binding Buffer

L02 in Binding Buffer

C Cell only Lib 100 nM

Lib-Nces 100 nM Zy1 100 nM Zy1-Nces 100 nM

37 o C


37 o C


B


4 oC

FITC

E Cell only Lib 100 nM Lib-Nces 100 nM Zy1 100 nM

Zy1-Nces 100 nM

FITC

Lib 100 nM Lib-Nces 100 nM Zy1 100 nM Zy1-Nces 100 nM

L02 in Cell Culture Medium 37 oC


37 o C

D Cell only

FITC

SMMC-7721 in Cell Culture Medium


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


F Cell only Lib 100 nM

Lib-Nces 100 nM Zy1 100 nM Zy1-Nces 100 nM

FITC

Figure 4. Characterization of the selective binding of Zy1-Nces with target SMMC-7721 cells at 4 oC (A and B) or 37 oC (C-F). Flow-cytometric results showed the selective recognition ability of Zy1-Nces to SMMC-7721 cells (A, C and E), but not to L02 cells (B, D and F), either in binding buffer or in cell culture medium (containing 10% FBS). Black peak, cell only; pink peak, Lib (50 nM or 100 nM FITC-labeled Lib); orange peak, Lib-Nces (3 nM or 100 nM FITC-labeled Lib equivalents); blue peak, free Zy1 (50 nM or 100 nM FITC-labeled Zy1); red peak, Zy1-Nces (3 nM or 100 nM FITC-labeled Zy1 equivalents).



B

2h

2h

1h

1h

30 min

30 min

15 min

15 min

A

3h

3h

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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LysoT racker Blue

FIT C

BrightField

Merged

LysoT racker Blue

FIT C

BrightField

Merged

Figure 5. The time-dependent internalization of Zy1-Nces (A) and free Zy1 (B) in SMMC-7721 cells revealed by confocal microscopy. SMMC-7721 cells were incubated with Zy1-Nces or free Zy1 (200 nM FITC-labeled Zy1 equivalents) at 37 o

C for 15 min, 30 min, 1 h, 2 h and 3 h, respectively. (Scale bar: 20 µm).


L02+ Mono-Zy1-Nces L02+ Zy1-Nces

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


SMMC-7721+ SMMC-7721+ Mono-Zy1-Nces Zy1-Nces

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LysoT racker Blue

Sybr Green

BrightField

Merged

Figure 6. Comparison of the internalization of Zy1-Nces and Mono-Zy1-Nces (i.e. only one aptamer per nanocentipede in average) in different cells. Cells were incubated with probes at 37 oC for 2 h. The confocal microscopy images revealed that Zy1-Nces can internalize into target cells more specifically and rapidly. (Scale bar: 20 µm)



105

Fluorescence Intensity (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

Concentration of Zy1-Nces 0 nM 0.2 nM 0.4 nM 1 nM 2 nM 4 nM 10 nM

7.5x104

5.0x104

2.5x104

0 520

560

600

640

680

Wavelength (nm)

Figure 7. Characterization of the high drug payload capacity of Zy1-Nces. Fluorescence spectra of Dox (2 µM) with increasing concentration of Zy1-Nces (shown by values from top to bottom). The fluorescence quenching indicates drug loading into Zy1-Nces.


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SMMC-7721+ Free Dox L02+ Zy1-Nces-Dox L02+ Free Dox

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


SMMC-7721+ Zy1-Nces-Dox

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LysoT racker Blue

Dox

BrightField

Merged

Figure 8. Characterization of the selective drug transport of Dox-loaded DNA nanocentipedes (Zy1-Nces-Dox) against targeted cancer cells. Cells were incubated with Zy1-Nces-Dox (2 µM Dox equivalents) or free Dox (2 µM) at 37 oC for 2 h. For Zy1-Nces-Dox, the confocal microscopy images showed stronger intracellular Dox fluorescence in SMMC-7721 cells than L02 cells; in contrast, for free Dox, the intracellular Dox fluorescence was comparable in SMMC-7721 and L02 cells. (Scale bar: 20 µm).


A

SMMC-7721

Cell Viabiltiy (%)

1.0 0.8 0.6 0.4 0.2

Free Dox Zy1-Nces-Dox

0.0

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B

1.0

L02

0.8 0.6 0.4

Free Dox Zy1-Nces-Dox

0.2 0.0

0.05 0.1 0.25 0.5

1

2.5

5

0.05 0.1

Concentration of Dox (µM) Cell Viabiltiy (%)

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

Cell Viabiltiy (%)


1.0

0.25 0.5

1

2.5

5

Concentration of Dox (µM)

C

0.8 0.6 0.4

SMMC-7721 L02

0.2 0.0 0.1

0.2

0.5

1

2

5

10

Concentration of Zy1-Nces (nM)

Figure 9. Characterization of the selective cytotoxicity of molecular drugs (Dox) delivered by Zy1-Nces. MTT assay results showing selective and potent cytotoxicity of Dox delivered by Zy1-Nces (red lines, A and B), in contrast to nonselective cytotoxicity of free Dox (black lines, A and B). Zy1-Nces without drug-loaded exhibited good biocompatibility (C). The selective cytotoxicity of Zy1-Nces-Dox implied the potential capability of DNA nanocentipedes for targeted delivery. The error bars indicated the standard deviations of three experiments.


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Table of Contents

H1

H2 Aptamer 5’

Trigger 5’

3’

≡

Drug Biotin Streptavidin


Self-Assembled DNA Nanocentipede as ... - ACS Publications

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