Magnetic DNA Nanogels for Targeting Delivery and Multi-stimuli

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Magnetic DNA Nanogels for Targeting Delivery and Multi-stimuli Triggered Release of Anticancer Drugs Chi Yao, Ye Yuan, and Dayong Yang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00516 • Publication Date (Web): 07 Nov 2018 Downloaded from http://pubs.acs.org on November 8, 2018

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

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ACS Applied Bio Materials

Magnetic

DNA

Nanogels

for

Targeting

Delivery

and

Multi-stimuli Triggered Release of Anticancer Drugs Chi Yao†#, Ye Yuan†# and Dayong Yang†*

† School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering (Ministry of Education), Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, P. R. China. # Chi Yao and Ye Yuan contributed equally to this work. * E-mail: [email protected], [email protected]

KEYWORD: DNA nanogel; magnetic nanoparticle; enzymatic polymerization; drug delivery; multi-stimuli triggered release

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ABSTRACT DNA polymeric materials possess many unique properties such as programmable molecular sequences and excellent biocompatibility, and have been demonstrated high potential in an assortment of biomedical applications. Herein, a magnetic DNA nanogel is constructed, aiming at the applications of targeting drug delivery and triggered drug release. Centering on magnetic nanoparticles, DNA nanogel layer is synthesized via rolling circle amplification, which equips abundant sites for the loading of anticancer drugs. Guided by external magnetic field, the magnetic DNA nanogel can target tumor cells efficiently. Moreover, DNA nanogel is responsive to multi-stimuli including temperature, pH and nuclease, enabling controlled release of anticancer

drugs.

Our

system

achieves

magnet-controlled

delivery

and

stimuli-triggered drug release, and provides a new strategy for the development of precision medicine.

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1. INTRODUCTION DNA polymeric materials have been attracting extensive attention as both a genetic and a generic material. Compared with synthetic polymers, DNA possesses many unique properties, including its sequence designability, material programmability, molecular recognition capacity, nanoscale controllability as well as excellent biocompatibility and biodegradability.1-4 DNA polymeric material therefore has been utilized in an assortment of biomedical applications such as single-cell analysis,5,6 multiplexed diagnosis,7-10 drug delivery11-13 and tissue engineering.14 DNA nanogel is a nano-sized hydrogel constructed with pure DNA15-17 or hybridized DNA with other components. 18-21 Recently, DNA nanogels have been developed and employed in the area of biomedicines due to their programmable nanostructure, controllable size and shape as well as excellent biocompatibility. Luo and coworkers pioneered the synthesis of a range of multifunctional nanoarchitectures from an DNA monomer, and the obtained DNA nanogels were designed to deliver both drugs and tracers simultaneously.15 Tan and colleagues designed a self-assembled pure DNA nanogel with Y-shaped monomers and linker, which possessed of controllable size and stimuli-responsive properties for targeted cancer therapy.16 Groll and coworkers synthesized a chemically cross-linked DNA nanogel and introduced a new strategy for pollutants scavenging through DNA intercalation by the network of the water-swollen nanogel.18 Zhang and colleagues synthesized water-soluble DNA-polymer brushes, by which crosslinked nanogels were further assembled through nucleic acid hybridization to deliver siRNA. 19 Gu and coworkers 3



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developed a bioinspired yarn-like DNA nanoclew system, realizing the delivery of antitumor drug or genome editing tool for targeted cancer treatment.20,21 It has been demonstrated that DNA nanogels exhibited the resistance to nuclease degradation, enhanced cellular uptake, and more effective drugs or siRNA delivery to the tumor sites. As a result, the research of DNA nanogels is emerging and becomes one of the active research fields of DNA based materials. a) Primer

Annealing

φ29

T4 DNA ligase

ssDNA

Circ-DNA template

RCA

b) FeCl3 · 6H2O

NaOH Solution

TEOS

APTS

+ 80 oC FeCl2 · 4H2O

NH2

SiO2

AMNP RCA

Magnetic guiding

Circ-DNA template

DOX

Intracellular low pH, or nuclease

RCA Products M-DNA nanogel /DOX

M-DNA nanogel

Scheme 1. Schematic diagram of the synthesis of M-DNA nanogel. a) Synthesis of circ-DNA RCA template and process of RCA. b) Synthetic route of M-DNA nanogel for loading and intracellular triggered release of doxorubicin guided by magnetic field. (TEOS: Tetraethyl orthosilicate; APTS: (3-Aminopropyl) triethoxysilane.)

Herein, we develop magnetic DNA (M-DNA) nanogels as nanovectors for controlled drug delivery and triggered drug release. Magnetic Fe 3O4 nanoparticle (MNP) was employed as the core because of its unique superparamagnetic property under external magnetic field, nano-sized particle diameter and biocompatibility. 22-24 4


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The shell of DNA nanogel was constructed via rolling circle amplification (RCA), an enzymatic polymerization of DNA chain, in situ coating on the surface of magnetic core by physical crosslinking. Doxorubicin (DOX), an anticancer drug, was efficiently loaded in the M-DNA nanogel by absorption with the bases of DNA chain. Our M-DNA nanogel could be guided to tumor site under external magnetic field, and release drugs precisely by responding to multi-stimuli, including temperature, pH and nuclease. By virtue of the remote magnet-controlled delivery and stimuli-triggered drug release of DNA nanogel, we could achieve high therapeutic efficacy and low side effects, therefore providing a new strategy for the development of precision medicine. 2. RESULTS AND DISCUSSION The synthesis route of M-DNA nanogel included three steps: the synthesis of amino modified Fe3O4 nanoparticle (Fe3O4 @SiO2-NH2, AMNP) as the magnetic core, the synthesis of circular DNA (circ-DNA) as the RCA template, and the synthesis of DNA hydrogel layer via RCA (Scheme 1). The AMNPs were synthesized according to a reported method.25 Amino was modified on SiO2 coated Fe3O4 nanoparticle (MNP) to generate the positive charged surface (Figure S1). Upon this process, the magnetism of Fe3O4 was well maintained (Figure S2). All the DNA sequences used for RCA were listed in Table S1. The composition of circ-DNA was designed as follows: a linear single-stranded DNA (ssDNA) as template with a phosphate group at the 5’ end for the ligating of the circ-DNA by T4 DNA ligase; and a shorter ssDNA as primer for both the formation of circ-DNA template and the start site of RCA

5



(Scheme 1a). The circ-DNA was attached on AMNP by electrostatic interaction. In an RCA reaction, plenty of long ssDNA were synthesized under the catalysis of DNA polymerization enzyme φ29 and finally coated on AMNPs by physical crosslinking26,27. DOX was loaded in the M-DNA nanogel by adsorbing to the bases of DNA (Scheme 1b). The verification of the circ-DNA formation was performed by native-PAGE gel electrophoresis. As shown in Figure 1a, circ-DNA exhibited slower mobility than the linear one, because of the steric hindrance after cyclization. The RCA product remained in the well of PAGE gel, which confirmed the generation of long DNA chain via RCA using the circ-DNA as RCA template (Figure 1a). The TEM (transmission electron microscopy) and SEM (scanning electron microscopy) images showed that the particle size of the synthesized AMNPs nanoparticles was 100 nm on average (Figure 1b, d). After RCA, the surface of nanoparticles changed from smooth to rough observed by SEM (Figure 1d, e), and the DNA layer of M-DNA nanogel could be seen obviously on the surface of the AMNPs from the TEM images (Figure 1b, c, Figure S3), indicating the successful DNA layer coating and formation of a core-shell structure. The crosslinked DNA networks of DNA nanogel is constructed by the physical entanglement of long ssDNA produced by RCA around the magnetic nanoparticle. Dynamic light scattering (DLS) measurement demonstrated the average hydrodynamic diameter of MNPs and AMNPs was 313 ± 44 nm and 191 ± 8 nm, respectively (Figure 1f, Figure S4a). It was notable that the statistical diameter of nanoparticles significantly reduced after silica and amino functionalization, which 6


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could be attributed to the decreased surface energy and increased electrostatic repulsion.28,29 And the diameter of M-DNA nanogels increased to 216 ± 25 nm due to the DNA layer coating (Figure 1f, Figure S4a). The size of nanogel increases with the prolongation of reaction time (Figure S4b), indicating that the production of DNA chains can be controlled via tuning the reaction time of RCA, therefore the thickness of the DNA gel layer of the M-DNA nanogel is controllable. As expected, the corresponding zeta potential value of the nanoparticles displayed more positive after amino modification, and turned more negative due to the DNA functionalization (Figure 1g, Figure S5).

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a)

b)

bp 622

50 nm

c)

DNA layer

180 147 123 110 90 76 67

50 nm

d)

e)

200 nm

200 nm

g) 0

300

-10

Zeta potential (mV)

f) 400

Size (d.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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200

100

0

-20

-30

-40

MNPs

AMNPs

M-DNA nanogels

MNPs

AMNPs

M-DNA nanogels

Figure 1. Characterization of magnetic DNA nanogels. a) 12% Native-PAGE gel electrophoresis image of circ-DNA template and RCA product. b&c) TEM images of AMNPs (b) and M-DNA nanogels (c). d&e) SEM images of AMNPs (d) and M-DNA nanogels (e). f&g) The size distribution (f) and zeta potential (g) of MNPs, AMNPs and M-DNA nanogels. Statistical approach of DLS: intensity. Error bars represent standard deviations from three measurements.

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a)

b) o

25 C o 37 C o 50 C

50

Cumulative release (%)


60

40 30 20 10 0

40

30

20 pH 7.4 pH 6.0 pH 5.0

10

0 0

1

2

3

4

5

0

1

Release time (h)

2

3

4

5

Release time (h)

d)

c) 70

70

0 U/mL 70 U/mL 140 U/mL 280 U/mL 560 U/mL

60 50



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


40 30 20 10 0

60 50 40 30 20

0

1

2

3

4

5

0

100

Release time (h)

200

300

400

500

600

CDNase I (U/mL)

Figure 2. a-c) Release behavior of M-DNA nanogels/DOX under different stimuli: different temperatures at pH 7.4 (a); different pH at 37 oC (b); different concentrations of DNase I at 37 oC, pH 7.4 (c). d) Release amount of DOX in 5 h with different concentrations of DNase I. Error bars represent standard deviations from three measurements.

Next, we tested the FTIR (fourier transform infrared spectroscopy) spectra of MNPs,

AMNPs

and

M-DNA

nanogels

to

verify the

successful

amino

functionalization of MNPs and the DNA coating of AMNPs (Figure S6). The characteristic absorption peak of Fe-O located at 582 cm-1 confirmed the component of MNPs. The vibrational bands observed at 1100 cm-1, 952 cm-1 indicated the bonds of Si-O and Si-OH, respectively, and the intense absorption peak located at 1489 cm-1 confirmed the successful amino functionalization on the surface of SiO 2 coated MNPs. The presence of DNA on nanogels was also confirmed by the CO-NH band at 1543 cm-1. Furthermore, we used thermogravimetric analysis (TGA) to determine the 9



content of DNA on nanogels (Figure S7). The weight loss of MNPs at temperature lower than 200 oC was due to the removal of trace water in the samples. Weight loss of 14% in AMNPs at approximately 250 oC could be attributed to the removal of chemical groups on the surface, such as hydroxyl. As for M-DNA nanogel nanogels, weight loss increased by 2.6%, attributed to the presence of the DNA layers. Further, we investigated the performance of our M-DNA nanogel in response to multi-stimuli including pH, temperature and nuclease. Anticancer drug doxorubicin hydrochloride (DOX), as cationic molecules, can be adsorbed by negative charged DNA molecules. We loaded the M-DNA nanogels with DOX, whose encapsulation efficiency was 64%. The drug release behavior of DOX loaded M-DNA nanogel (M-DNA nanogel/DOX) under different stimuli conditions via measuring the absorbance spectra of DOX was studied (Figure S8). First, we tested the drug release of the M-DNA nanogel/DOX system at different temperatures. With the increase of temperature, the release rate increased, and release amount of DOX at pH 7.4 within 5 h was 13%, 25%, 59% at 25 oC, 37 oC and 50 oC, respectively (Figure 2a). Second, we analyzed the drug release of our nanogel system at different pH conditions. With the decrease of pH, the release rate increased, and the release amount of DOX at 37 o

C within 5 h was 25%, 31%, 38% at pH of 7.4, 6.0 and 5.0, respectively (Figure 2b).

Thirdly, we assessed the drug release effect of the nanogel in the presence of nuclease. The release amount of DOX at 37 oC within 5 h rapidly increased with the increase of DNase I concentration, up to 68% at 560 U/mL DNase I (Figure 2c, d). Further, we investigated the mechanism of drug release by studying the morphology of M-DNA 10


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nanogels after incubation under different conditions via TEM (Figure S9). The DNA layer remained integrity after the treatment of low pH or high temperature, indicating that the pH and temperature rarely affect the disintegration of DNA nanogels. We propose that H+ may compete with DOX to weaken the interaction between cation DOX and negative charged DNA molecules; the high temperature may trigger the motion and diffusion of DOX molecules. As a result, DOX dissociates from DNA nanogels at low pH or high temperature. While the DNA layer was disintegrated after the treatment of nuclease, leading to the drug release of DOX. Since the characteristic in tumor microenvironment included abnormal physiochemical conditions and dysregulated biosynthetic intermediates,30-32 it was demonstrated that our M-DNA nanogel/DOX system could respond to multi-stimuli and consequently realize controlled drug release. a)

b) 120

120

100

100

Cell viability (%)

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


80 60 40 20

* 80 60 40 20

0

0 Control

25

50

75

100

Control

200

CM-DNA nanogel (g/mL)

25

50

75

100

200

CM-DNA nanogel/DOX (g/mL)

Figure 3. In vitro evaluation of cytotoxicity and efficacy. a&b) Cell viability of human glioma U87MG cells incubated with (a) M-DNA nanogel and (b) M-DNA nanogel/DOX, as measured by MTT assay. ＊p < 0.05. Error bars represent standard deviations from three measurements.

In order to evaluate the in vitro cytotoxicity of the M-DNA nanogel system, the cell viability of human glioma U87MG cells incubated with M-DNA nanogel was tested 11



by MTT assay. As shown in Figure 3a, after incubation with a series of concentration (25, 50, 75, 100, 200 μg/mL) of M-DNA nanogel for 48 h, cell viabilities remained above 90 % compared to the control group, indicating the low cytotoxicity and good biocompatibility of M-DNA nanogel as drug delivery carrier. After loading with DOX, M-DNA nanogel/DOX showed significant toxicity to U87MG cells. The cell viabilities reduced with the increase of M-DNA nanogel/DOX concentration, and was down to 37 % at the concentration of 200 μg/mL, which indicated the good therapeutic efficacies of the M-DNA nanogel/DOX system (Figure 3b). Compared to free DOX with the same doses, the M-DNA nanogel as drug delivery carrier increased the therapeutic effects significantly (Figure S10). We also compared the cytotoxicity of DOX-loaded AMNPs (AMNPs/DOX) and M-DNA nanogel/DOX (Figure S10). The AMNPs/DOX showed much lower cytotoxicity than M-DNA nanogel/DOX, because AMNPs have much weaker load capacity for DOX by surface adhesion.

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a)

DAPI

FITC

DOX

Merge

1h

3h

6h

12 h

24 h

c) Control 1h 3h 6h 12 h 24 h

FITC intensity (a.u.)

Control 1h 3h 6h 12 h 24 h

Normalized counts

b) Normalized counts

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


DOX intensity (a.u.)

Figure 4. a) Fluorescence microscope images of human glioma U87MG cells after 1, 3, 6, 12 and 24 h of incubation with FITC labeled M-DNA nanogel/DOX. Scale bar represents 50 μm. b&c) Flow cytometry measurements for the cellular uptake of FITC-labeled M-DNA nanogel (b), and intracellular accumulation of DOX released from M-DNA nanogel/DOX (c).

Encouraged by the above demonstration of M-DNA nanogel as drug delivery carrier, we investigated the cellular uptake of the nanogel by U87MG cells. The 13



M-DNA nanogel/DOX was stained with fluorescein isothiocyanate (FITC) and incubated with U87MG cells, and imaged by fluorescence microscopy at different time intervals of 1, 3, 6, 12 and 24 h. As shown in Figure 4a, the blue fluorescence signal indicated the nucleus of cell, the red fluorescence signal denoted DOX, and the green fluorescence signal showed nanogels. The green signal observed in the cytoplasm of cells suggested that the nanogels began to be internalized by cells and gradually accumulated in cells during the incubation. The red signal could be observed both in cytoplasm and nucleus, which suggested DOX could be delivered into cells by the nanogels and subsequently released into nucleus. It could be obviously observed that the green and red fluorescence signal enhanced with the extension of incubation time in 24 h, indicating the efficient internalization and drug release of the nanogels in cells. The corresponding quantitative analysis was performed by flow cytometry, which supported the cellular uptake and intracellular accumulation results (Figure 4b, c).

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a)

M-DNA nanogel/DOX

Cell culture insert 24 well plate

Cells

N S b)

DAPI

FITC

Magnet DOX

Merge

2h Without magnet

2h With magnet

4h Without magnet

4h With magnet

Figure 5. a) Illustration of device to simulate the M-DNA nanogel penetrating tumor cells under external magnetic field with human glioma U87MG cells in culture medium. b) Fluorescence microscope images of human glioma U87MG cells in 24 well plate after 2 and 4 h of incubation with FITC labeled M-DNA nanogel/DOX added in the cell culture insert with or without magnetic field. Scale bar represents 50 μm. To study the behavior of penetration and accumulation of our M-DNA nanogels in the tumor site guided by external magnetic field, we employed a transwell model. As shown in Figure 5a, the transwell filter was seeded with a compact U87MG cell monolayer, and then FITC-labeled DOX-loaded M-DNA nanogels in the filter were guided to penetrate the cell monolayer by a magnet. The penetrated M-DNA nanogels were internalized by the U87MG cells on the underlayer plate, and imaged at different 15



time intervals of 2 and 4 h by fluorescence microscopy (Figure 5b). It was obviously observed that stronger green and red fluorescence signal appeared in cells with external magnetic field than that of control group without magnet at the same incubation time. In addition, with the extension of incubation time, green and red fluorescence signal enhanced which verified the efficient penetration and enhanced accumulation of the nanogels within tumor cells guided by magnetic field. 3. CONCLUSION In this study, we developed a novel magnetic DNA nanogel as anti-tumor drug nanovector for controlled delivery and triggered release. By virtue of the structure and function of DNA hydrogel, the drug release responded to multi-stimuli including temperature, pH, and nuclease. The nanogels possessed suitable particle size and good biocompatibility, and could be efficiently internalized by tumor cells. In addition, with the guidance of external magnetic field, the nanogels could be accumulated in tumor region to realize targeted delivery, and showed therapeutic efficacy with drug loaded. Therefore, our magnetic DNA nanogel provided a promising platform to realize targeted chemotherapy.

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Materials and Methods Synthesis of circ-DNA template. The same volume of 100 µM ssDNA and primer were mixed adequately and was successively heated at 95 oC for 2 min, 65 oC for 2 min and cooled from 60 oC to 20 oC at a rate of 1 oC/min. The circ-DNA template with a gap was synthesized. Then T4 DNA ligase and the buffer (300 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, and 10 mM adenosine triphosphate，pH 7.8) were added and incubated together for 3 h at 22 oC. Finally, the mixture was kept to 70 oC for 10 min to inactivate T4 DNA ligase. Synthesis of amino functionalized Fe3O4@SiO2 nanoparticle (AMNP). The prepared Fe3O4@SiO2 nanoparticles (1 g) was dispersed in ethanol (300 mL) with sonication for 1 h and then (3-Aminopropyl) triethoxysilane (6 mL) was added for amine functionalization with continue sonication for another 1 h. The precipitate was washed by dispersing in ethanol (40 mL) with sonication four times and collected by magnetic separation. The product was dried in a vacuum oven at 60 °C overnight. Synthesis of magnetic DNA nanogel (M-DNA nanogel). 40 μL of circ-DNA RCA templates (291 nM) were mixed with 50 μL of 10 × φ29 DNA polymerase buffer (500 mM Tris-HCl, 100 mM MgCl2, 100 mM (NH4)2SO4 and 40 mM DTT), dNTPs (4 μL, 25mM), AMNPs (100 μL, 5 mg/mL, dispersed in MQ) and 200 μL MQ with sonication for 30 min. Then 5 μL of bovine serum albumin (BSA, 20 mg/mL) and 10 U φ29 DNA polymerase (1 μL, 10 U/μL) was added in the mixture. The total volume of the system was 500 μL. Then the mixture was incubated at 30 °C for 24 h. After the reaction, the product was separated by magnetic separation and washed with MQ water several times. 17



Controlled drug release. In vitro release of DOX from M-DNA nanogel/DOX was carried out under different conditions. To test the response of M-DNA nanogel/DOX to pH, M-DNA nanogel/DOX were dispersed into 0.5 mL PBS with different pH (pH 7.4, pH 6.0, pH 5.0) with continuous shaking at 37 oC. To test the response to temperature, M-DNA nanogel/DOX were dispersed into 0.5 mL PBS (pH 7.4) with continually shaking at different temperature (25 oC, 37 oC, 50 oC). To test the response to enzyme, M-DNA nanogel/DOX were dispersed into 0.5 mL DNase I buffer (pH 7.4) added different concentration of DNase I (70 U/mL, 140 U/mL, 280 U/mL, 560 U/mL) with continually shaking at 37 oC. At predetermined time intervals, M-DNA nanogel/DOX was attracted by a magnet to the bottom of tube, and then the supernatant was all taken out and was replaced with the same volume of fresh buffer each time. 300 μL of supernatant was transferred to the well of 96-microwell plate to determine the absorbance of DOX by a microplate reader for calculating the released DOX amount. Before the determination, a calibration curve was recorded by measuring the absorbance values of a series concentration of DOX at 488 nm. Cytotoxicity. The standard MTT method was performed to assess the cytotoxicity of M-DNA nanogel and M-DNA nanogel/DOX for human glioma U87MG cells respectively. The cells were seeded into a 96-well plate at a density of 4000 cells per well and incubated overnight. The M-DNA nanogel or M-DNA nanogel/DOX dispersion solution with different final concentrations (0, 25, 50, 75, 100, and 200 μg/mL) were added to the plate wells (10 μL/well) and incubated for 48 h. Afterward, the culture medium was removed and each well was washed with PBS solution (pH 18


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7.4) twice. 90 μL fresh medium and 10 μL MTT solution were added to each well successively and still incubated for another 4 h. Next, the supernatant was discarded and 110 μL DMSO was added to each well. The plate was shaken slightly for 10 min to dissolve formazan crystals. And then the formazan solvent solution in each well was collected to each Eppendorf tube and was centrifuged at 6000 rpm/min for 3 minutes. 80 μL supernatant was transferred from each tube to each wells of a new 96-well plate. The absorbance of each wells was measured at 490 nm using a BioTek Synergy/H1 microplate reader. Cell uptake assay. For the investigation on cell uptake, M-DNA nanogel/DOX was stained by fluorescein isothiocyanate (FITC, 100 μg/mL) overnight. 8 × 104 human glioma U87MG cells were seeded into each cell culture dish and cultured overnight to allow the cells attach onto the glass bottom. Afterward, the strained M-DNA nanogel/DOX were added to each well with a concentration of 100 μg/mL. After incubation for predetermined intervals (1, 3, 6, 12, 24 h), the cells were fixed with 4% fixative solution (paraformaldehyde) for 20 min and stained by DAPI (1 μg/mL) for another 10 min by standard procedure. Finally, the samples were imaged using a fluorescence microscope. Cell penetration. To investigate the ability of M-DNA nanogel/DOX for cell penetration under external magnetic field, 3 × 104 human glioma U87MG cells were seeded into a 24-well plate with glass bottom and cell culture inserts with PET track-etched membrane (8.0 μm pore size, Falcon). The cell culture inserts were set on the well of 24-well plate and the cells were cultured overnight to attach onto the 19



glass bottom and PET track-etched membrane, respectively. Afterward, the FITC-labeled M-DNA nanogel/DOX were added to cell culture insert with a concentration of 100 μg/mL. After incubation for predetermined intervals (2, 4 h) with or without putting a magnet under the 24-well plate, the cells were fixed with 4% fixative solution (paraformaldehyde) for 20 min and stained by DAPI (1 μg/mL) for 15 min by standard procedure. Finally, the samples were imaged on an inverted fluorescence microscope. Characterization. Transmission electron microscopy (TEM) images were obtained using a FEI TECNAI G2 T20 ST transmission electron microscope at acceleration voltage of 200 kV. SEM images were obtained by Hitachi-S4800 FESEM. Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer Nano ZS90. Fourier transform infrared (FTIR) spectra were recorded on a Nikon Tensor 27 spectrometer in transmission mode. Thermogravimetric analysis (TGA) was performed on a TA Q50. The samples were tested at a heating rate of 10 oC/min in a nitrogen atmosphere. Fluorescent images were obtained using a Biotech Ti-E inverted fluorescence microscope. The absorbance of DOX was measured by a Biotech SYNERGY H1 microplate reader.

ASSOCIATED CONTENT

Supporting Information Supporting Information. Materials and methods, oligonucleotide sequences, TEM 20


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images, optical images, size distribution by DLS, zeta potential distribution, FTIR spectra, TGA measurement, absorbance spectra, cell viability assay.

AUTHOR INFORMATION Corresponding Author *Dayong Yang, [email protected]; [email protected]. ORCID Dayong Yang: 0000-0002-2634-9281 Author Contributions Chi Yao and Ye Yuan contributed equally to this work. Funding This work was supported in part by National Natural Science Foundation of China (grant no. 21621004, 21575101, 21622404 and 21704074), and China Postdoctoral Science Foundation (grant no. 2018M631735).

ACKNOWLEDGMENTS We thank Han Tang, Jinhui Geng, Jianpu Tang, and Jigang Lv at Tianjin University for their help on experiments and discussion. We are grateful to Prof. Lei Zhang, Prof. Jiayan Luo and Prof. Wei Feng in Tianjin University for DLS test, TGA test and FTIR test, respectively.

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Magnetic DNA Nanogels for Targeting Delivery and Multi-stimuli

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