Chaperone Copolymer-Assisted Aptamer-Patterned DNA Hydrogels

Subscriber access provided by University of South Dakota

Article

Chaperone Copolymer Assisted Aptamer-patterned DNA Hydrogels for Triggering Spatiotemporal Release of Protein Zheng Zhang, Jialun Han, Yuxuan Pei, Renzhen Fan, and Jie Du ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00450 • Publication Date (Web): 19 Sep 2018 Downloaded from http://pubs.acs.org on September 19, 2018

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

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

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

ACS Applied Bio Materials

Chaperone Copolymer Assisted Aptamer-patterned DNA Hydrogels for Triggering Spatiotemporal Release of Protein Zheng Zhang, Jialun Han, Yuxuan Pei, Renzhen Fan and Jie Du* State Key Laboratory of Marine Resource Utilization in South China Sea, College of Materials and Chemical Engineering, Hainan University, Haikou 570228, PR China. *Corresponding author: [email protected]

ABSTRACT: In this paper, a DNA hydrogel with low DNA concentration, short sticky end and good mechanical strength was simply prepared via one-pot self-assembly from two kinds of DNA building block (X- and L-shaped DNA units) chaperoned by a cationic comb-type copolymer (CCC). The gelling process was completed under physiological conditions within one minute, and the reversible sol-gel phase transition was achieved at room temperature through the continuous addition of CCC and an anionic polymer poly(sodium vinylsulfonate). Moreover, aptamer was successfully patterned into the hydrogel system via click chemistry. Upon the addition of complementary sequences (CSs) of aptamer, the aptamer was hybridized with CSs, leading to the fast dissociation of protein from aptamer with an adjustable release rate in specific regions at prospective times. The hydrogel with excellent cytocompatibility was successfully applied to human serum, a complex matrix. The aptamer-patterned DNA hydrogel is a potential candidate for controlled protein delivery. KEYWORDS: controlled release; DNA Hydrogel; patterning; self-assembly

INTRODUCTION Hydrogels are widely used as stimuli-responsive materials in drug release systems because of their responsiveness to outside stimulus such as light, temperature, pH, metal ion, and other molecules.1-4 However, the disadvantage of hydrogel as drug release carriers is the fast release of drugs due to the high hydrogel permeability, which can give rise to severe side-effects in vivo.5 Therefore, hydrogels have been extensively studied to improve their sustained-release capability. Ligands with different affinities, including metal-ion-chelating ligands,6 heparin or heparan sulfate,7 and streptavidin or biotin,8 have been widely incorporated into hydrogels to improve the affinity of the hydrogel with protein drugs and overcome burst release. Although those molecules can combine many biologics, they face many problems such as low specificity, low affinity and high toxicity. In recent decades, oligonucleotide aptamers selected from DNA/RNA libraries by SELEX have been shown to be able to combine any target molecule with high specificity and affinity.9 Moreover, unlike other affinity ligands, aptamers can hybridize with their complementary sequences (CSs) and induce the rapid dissociation of protein-aptamer complexes.10-12 Hence, aptamers have been incorporated into hydrogels as affinity ligands to expand the application of hydrogels in the field of controlled drug delivery.13-17 In addition, most studies have shown that aptamers are evenly incorporated into hydrogels,16-17 allowing all areas of the hydrogel to release proteins. However, according to practical need, the treatment of disease requires protein in specific areas. Therefore, it is important to incorporate aptamers in specific areas of hydrogels. Tedious synthetic steps and the need for toxic reagents greatly limit the application of hydrogel in the biomedical fields. DNA hydrogels have been widely studied due to their easy synthetic processes and excellent biocompatibility.18-19 However, the formation of DNA hydrogels requires long “sticky ends” or high concentrations of DNA building blocks. To solve these problems, a new strategy for building DNA hydrogels must be proposed. Atsushi Maruyama’s group confirmed that a cationic comb-type copolymer

ACS Paragon Plus Environment

ACS Applied Bio Materials 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

(CCC), poly(L-lysine)-graft-dextran (PLL-g-Dex), can stabilize the multilevel DNA structure and facilitate DNA hybridization.20 These functions of the CCC have been used for gene-delivery, MNAzyme catalysis, and single-base mismatches detection.21-23 To date, the self-assembly of DNA hydrogels adjusted using PLL-g-Dex has not been studied. Therefore, in this work, we designed an aptamer-patterned DNA hydrogel to solve the above problems. The DNA hydrogels were cross-linked via the one-pot self-assembly of two kinds of DNA building block (X- and L-shaped DNA units) with respective “sticky ends” in the presence of CCC. CCC obviously accelerated the gelation of DNA hydrogels in the physiological environment at low concentration and short “sticky ends” of DNA building block. Moreover, CCC significantly improved the mechanical strength of the DNA hydrogel, and the DNA hydrogel achieved a reversible sol-gel phase transition at room temperature through the cyclic addition of CCC and poly(sodium vinylsulfonate) (PVS). In addition, aptamer was successfully patterned into the hydrogel through click chemistry. Upon the addition of CSs of aptamer, the aptamer was hybridized with CSs, which induced the fast dissociation of protein from aptamer with an adjustable release rate in specific regions and at a predictable time. Thus, this aptamerpatterned DNA hydrogel chaperoned by CCC could be used as a controlled-release protein carrier.

EXPERIMENTAL SECTION Materials. The CCC PLL-g-Dex was prepared via the reductive amination reaction of PLL·HBr (Mn=20000, SigmaAldrich) with dextran (Mn=5900, Sigma-Aldrich) as previously described.24 The grafted amount of dextran in CCC (Mn = 68000) was 92 wt%, as detected by 1H NMR (Fig. S1). All DNA was purchased from FASMAC Co., Ltd and is listed in Table S1 and Table S2. Platelet-derived growth factor BB (PDGF-BB) was supplied by Sigma-Aldrich. Murine macrophage cells (RAW 264.7 cells), Live/Dead Assay Kit and Cell Counting Kit-8 were obtained from Sangon Biotech Co., Ltd. DNA buffer which was used to dissolve DNA contained TrisHCl (10 mM, pH 7.2), 0.5 mM ethylenediaminetetraacetic acid (EDTA) and 50 mM NaCl. The absorption medium and release medium contained Tris-HCl (50 mM, pH 7.2) and 50 mM KCl. Preparation and characterization of X- and L-shaped DNA building units. Equimolar amount of X-shaped (X1, X2, X3 and X4) and L-shaped (L1 and L2) DNA strands were separately dissolved into DNA buffer to obtain final concentrations of 50 μM for all DNA strands. The final mixture was incubated at 95 °C for 10 min and then placed at room temperature for 3 h, resulting in Xand L-shaped DNA structures. Gel electrophoresis was utilized to detect formation of DNA building units. DNA sample (7 μL) was mixed with 6×loading buffer (2 μL) and analyzed with 3% agarose gel at 90 V for approximately 40 min in 0.35×TAE buffer [14 mM tris (hydroxymethyl) aminomethane, 0.45 mM EDTA and 7 mM acetic acid, pH 8.0]. The bands were stained with GoldView I nucleic acid dye and then imaged with a GelDoc XR+ system (Bio-Rad, Hercules, USA). One-pot self-assembly of DNA hydrogel. The X-shaped and L-shaped building units were used to prepare DNA hydrogel. The molar ratio of XDNA to L-DNA was kept at 1:2. X-DNA (20 μL) at different concentrations (31.25, 62.5, 125, 250, and 500 μM) and L-DNA (20 μL) at different concentrations (62.5, 125, 250, 500, and 1000 μM) were mixed at room temperature in the absence or presence of CCC (0.2 μL, 300 μM) to form DNA hydrogels. The tubes containing the mixtures were then inverted for 30 min. Acrydite-labelled aptamer was patterned into SH-labelled DNA hydrogel. The SH-labelled DNA hydrogels were immersed into 50 µL DNA buffer with different concentrations (4, 8, 12, 16 and 20 μM) of acrydite/FAM-labelled PDGF-BB aptamer (5’-acryditeACAGGCTACGGCACGTAGAGCATCACCATGATCCTG-FAM-3’) and 1.8 mM initiator 2959 were then incubated on a shaker at ambient temperature for 4 h. Subsequently, the DNA hydrogel was irradiated with paralleled UV light (365 nm, 10 mW/cm2) by covering the hydrogel with a photomask (200 μm lines with 300 μm intervals) for 5 min. The aptamer-patterned DNA hydrogel was placed into 500 µL DNA buffer, and incubated on a shaker to wash out the unreacted aptamer. Subsequently, the hydrogel was imaged using fluorescence microscopy (Eclipsc Ti-U, Japan). The patterned hydrogel was incubated in a


Page 2 of 16

Page 3 of 16 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


solution of DABCYL-labeled CSs of the PDGF-BB aptamer at 37 °C for the prospective time and then imaged by fluorescence microscopy. Determination of the rheological properties of the DNA hydrogel. Storage modulus and loss modulus were using a dynamic mechanical analyzer (DMA Q800, USA). The frequency was changed while keeping the amplitude at 25 mm. The hydrogel sample was cut into a cylinder with a diameter of 10 mm and height of 5 mm. Aptamer-patterned DNA hydrogels for protein capture. Hydrogels were incubated in 1 mL absorption medium (with protein) to study the ability of the hydrogel to capture protein. The approach for detecting the concentrations of protein captured and released was based on a graphene oxide-based fluorescence biosensor.25 The detection theory is shown in Fig. S2 and S3. This method allowed the accurate characterization of the capture and release properties of the aptamer-patterned DNA hydrogels. Aptamer-patterned DNA hydrogels for protein release. The hydrogels that had captured PDGF-BB were incubated in 1 mL release medium, and CSs were then added. The concentrations of PDGF-BB in solution were detected using a graphene oxide-based fluorescent aptasensor. Cell culture. Cell culture test was performed with murine macrophage cells (RAW 264.7 cells). The aptamerpatterned DNA hydrogel was placed in a 24-well plate and then sterilized with 75% (v/v) ethanol for 150 min followed by three rinses with sterilized phosphate-buffered saline (PBS). Subsequently, the hydrogel was prewetted with culture medium for 120 min. After removing the culture mediums, 500 μL of a suspension of RAW 264.7 cells (1 × 105 cells well−1) was seeded on the hydrogel or culture plates and then cultured under standard conditions. After incubation for 72 h, the cells on hydrogels or culture plates were stained with the Live/Dead Assay Kit and then observed by fluorescence microscopy. The Cell Counting Kit-8 was used to evaluate the cell proliferation on the hydrogel or culture plates after culturing for 1, 3, 5, and 7 days. Briefly, at each time point, the culture medium was removed, and Cell Counting Kit8 working solutions were added at 37 °C for 120 min. Finally, the supernatant medium was extracted to detect cell quantity using a Thermo Scientific microplate reader (Thermo, USA). Statistical analysis. Data are expressed as the mean ± standard deviation of five samples. Statistical significance was determined by analysis of variance (single factor) with p < 0.05.

RESULTS AND DISCUSSION Fabrication and characterization of DNA hydrogels. As shown in Fig. 1, X-shaped DNA was obtained from four single-stranded DNAs (ssDNAs), and each strand had a “sticky end” portion (marked in black). The L-shaped DNA was a linear duplex assembled by two SH-labelled ssDNAs with “sticky ends” complementary to those of X-DNA. CCC can decrease the entropically unfavourable counterion condensation eﬀect and reduce the energy barrier associated with breakage and reassociation of the nucleic acid base pairs.21 Thus, the addition of CCC can promote the self-assembly of X-DNA and L-DNA and stabilize the structure of the DNA hydrogel. After the gel was formed, PVS polyanion was added, and PVS combined with CCC to remove CCC from DNA. This resulted in the dissociation of the three-dimensional network structure of the SH-labelled DNA hydrogels.



++++++ ++++++

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

Page 4 of 16

Fig. 1 Diagram showing the concept SH-labelled DNA hydrogel formation and dissociation. The X-shaped DNA (X-DNA) and L-shaped DNA (L-DNA) were crosslinked by hybridizing their “sticky ends” (marked in black) to form the SH-labelled hydrogel in the presence of CCC. The “sticky ends” of X-DNA were complementary to those of L-DNA. PVS then combined with CCC, resulting in the dissociation of the threedimensional network structure of the DNA hydrogel. Agarose gel electrophoresis was used to verify the formation of the X-DNA and L-DNA structures. As shown in Fig. S4, X-DNA (lane 5) did not move as far as X1, X2, X3 and X4 (lanes 1, 2, 3 and 4, respectively). Similarly, L-DNA (lane 8) did not move as far as than L1 and L2 (lanes 6 and 7, respectively). The results indicate that X-DNA and L-DNA were indeed formed as designed. The ideal molar ratio of L-DNA to X-DNA is 2:1; as this ratio, the initial DNAs are entirely crosslinked and form the most indurative network.26 Next, 20 μL of X-DNA (500 μM) and 20 μL of L-DNA (1000μM) were mixed. As shown in Fig. S5, the solution gradually lost its fluidity and appeared to be in a gel state, indicating that a sol-to-gel transition had occurred. In addition to visual observation, a frequency scanning test was carried out on the DNA hydrogels to further confirm hydrogel formation. As shown in Fig. 2A, the loss modulus (G”) was significantly smaller than the storage modulus (G’) throughout the process, which provided the obvious signal of the gel-like state.27 In addition, the G’ showed a slight frequencydependent increase, suggesting a physical gel.27 Combined with previous visual observations, this result indicates that a pure DNA hydrogel was indeed formed via self-assembly of the DNA building blocks. The effect of DNA concentrations on the rheological properties of the DNA hydrogels was studied. XDNA and L-DNA were mixed at different concentrations (X-DNA 500 μM and L-DNA 1000 μM; X-DNA 250 μM and L-DNA 500 μM; X-DNA 125 μM and L-DNA 250 μM; X-DNA 62.5 μM and L-DNA 125 μM; X-DNA 31.25 μM and L-DNA 62.5 μM) with or without CCC. The mechanical strength of the DNA hydrogel can be evaluated based on the loss factor G’’/G’. A smaller loss factor indicates a better mechanical strength.28 As shown in Fig. 2B and Fig. S5, when the concentration of X-DNA and L-DNA were 500 and 1000 μM, respectively, G” was smaller than G’, suggesting that a hydrogel was formed. After the addition of CCC, the Loss factor was reduced from 0.051 ± 0.008 to 0.031 ± 0.006, indicating that the mechanical strength of the DNA hydrogel was greatly improved. When the X-DNA and L-DNA concentrations were respectively reduced to 250 and 500 μM, the result was similar. These results indicate that CCC can significantly increase the mechanical strength of the DNA hydrogel. When the concentrations of X-DNA and L-DNA were further decreased to 125 and 250 μM or to 62.5 and 125 μM, respectively, G’ was smaller than G’’ in the absence of CCC, and hydrogel formation did not occur. This is because the crosslinking sites between X-DNA and L-DNA were not plentiful enough to form a gel. However, when CCC was added into the solution, it rapidly changed into a gel state and showed good mechanical strength. Furthermore, PLL alone, dextran alone or a mixture of PLL and dextran could not make this DNA solution (125 μM X-DNA and 250 μM L-DNA) form a hydrogel (Fig. 2C and Fig. S6). Thus, the gelation of DNA was attributed to the CCC, not to the homopolymer of PLL or dextran. These results demonstrate that CCC increased the crosslinking sites at the equal DNA concentrations and reduced the critical gelation concentration of the DNA hydrogels. However, when the concentration of X-DNA was further reduced to 31.25 μM, no hydrogel was formed due to the insufficient crosslinking sites, regardless of whether CCC was present. After 125 μM X-DNA, 250 μM L-DNA and 300 μM CCC were incubated to form a DNA hydrogel, the polyanion, PVS, was added to dissociate CCC from DNA. Fig. 2D and Fig. S7 showed that the DNA


Page 5 of 16 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


hydrogels with CCC can switch from the gel to sol state after adding PVS, and this process can be cycled many times. This indicates that the DNA hydrogel respond to CCC and PVS in a reversible way at room temperature. The effects of CCC and PVS concentrations on the DNA hydrogel (125 μM X-DNA, 250 μM L-DNA) were also studied. As shown in Fig. S8, the loss factor of the solution gradually decreased with increasing CCC concentration, and 300 μM CCC induced the solution to transform into a stable DNA hydrogel. PVS was then added into the hydrogel (with 300 μM CCC). As the PVS concentration increased, the loss factor gradually increased, and the DNA hydrogel gradually transformed from a gel into a sol. Since the self-assembly occurs via the hybridization of the “sticky ends”, we attempted to demonstrate how these “sticky ends” affect the mechanical properties of the DNA hydrogel. For this purpose, four groups of X-DNA and L-DNA containing “sticky ends” with different lengths were designed, bp12 (12-base-pair-long “sticky ends”), bp9 (9-base-pair-long “sticky ends”), bp6 (6-base-pair-long “sticky ends”) and bp3 (3-base-pair-long “sticky ends”). As shown in Fig. 2E and Fig. S9, when the “sticky ends” were bp12, 500 μM X-DNA and 1000 μM L-DNA formed a gel in the absence of CCC. After the addition of CCC, the loss factor of the hydrogel was reduced from 0.035 ± 0.007 to 0.023 ± 0.004. When the length of the “sticky ends” was reduced to nine base pairs, the result was similar. However, when the length of the “sticky ends” was further reduced to six base pairs, the hydrogel was not formed without CCC because the linkage between X-DNA and L-DNA was only six base pairs long and not strong enough to form a gel. However, after adding CCC, G” was obviously smaller than G’, and a stable gel was formed. These results suggest that CCC can shorten the critical length of the “sticky ends” for gelation in DNA hydrogels. However, when the length of the “sticky ends” was further reduced to three base pairs, a hydrogel was not formed, regardless of whether CCC was present. This is because the applied force of the three base pairs with or without CCC is too small. Next, the effect of base mismatch was studied. Four groups of X-DNA and L-DNA with different amounts of mismatched sites of “sticky ends” were designed as follows: bp9A (one base mismatch within bp9), bp9B (two base mismatches within bp9), bp9C (three base mismatches within bp9) and bp9D (four base mismatches within bp9). As shown in Fig. 2F and Fig. S10, when the number of base mismatches was one or two, G” was obviously smaller than G’, and stable gels were formed. In contrast, when the number of base mismatches increased to three, G’ was smaller than G” in the absence of CCC, and a hydrogel was not formed. After the addition of CCC, the G’ was significantly higher than the G”, and a stable hydrogel was formed. Nevertheless, when the amount of base mismatches increased further to four, hydrogel formation did not occur, regardless of whether CCC was present or absent. The above results show that CCC can increase the critical number of base mismatches for the gelation of DNA hydrogel.



Fig. 2 (A) Storage and loss modulus of DNA hydrogels formed from 500 μM X-DNA and 1000 μM L-DNA with 9-base-pair-long “sticky ends”. (B) Mixture of diﬀerent concentrations of X-DNA and L-DNA in the absence or presence of CCC at room temperature. The CCC concentration was fixed at 300 μM. (C) Rheological properties of the mixture of X-DNA (125 μM) and L-DNA (250 μM) in the absence or presence of PLL (300 μM), dextran (300 μM), a mixture of PLL and dextran (300 μM) or CCC (300 μM). Rheological tests were performed on the hydrogels at a fixed frequency (1 Hz). (D) DNA hydrogels (125 μM X-DNA and 250 μM L-DNA with 9-base-pair-long “sticky ends”) reversibly respond to CCC and PVS. (E) Effect of the length of the “sticky ends” on the rheological properties of the hydrogel (X-DNA 500 μM, L-DNA 1000 μM and CCC 300 μM). (F) Effect of the amount of mismatched sites of the “sticky ends” on rheological properties of the hydrogel (X-DNA 500 μM, L-DNA 1000 μM and CCC 300 μM). Fabrication of aptamer-patterned DNA hydrogel. Acrydite-labelled aptamers were patterned into the DNA hydrogels via thiol-ene UV initiation reaction between sulfydryl and the double bond of acrydite. The reactions were completed by UV light and I2959 as photoinitiator. DNA hydrogels were immersed in acrydite/FAM-labelled aptamer and initiator for 4 h. Finally, the gel was irradiated with UV light for 5 min by covering the hydrogel with a photomask (200 μm lines with 300 μm intervals). The gels were then washed using DNA buffer to remove unreacted aptamer and observed via fluorescence microscopy. The results demonstrate that an aptamerpatterned DNA hydrogel was indeed formed (Fig. 3).


Page 6 of 16

Page 7 of 16 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


The rheological properties of DNA hydrogel with or without aptamer are shown in Fig. S11. The results demonstrate that the rheological properties of the hydrogels with and without aptamer were not significantly different. In this work, the grafting amount of PDGF-BB aptamer in the hydrogel was calculated (Fig. S12 and Table S3).

Fig. 3 Schematic diagram of aptamer-patterned DNA hydrogel formation (photomask: 200 μm lines, 300 μm intervals; scale bars =300 μm). Examination of PDGF-BB capture from aptamer-patterned DNA hydrogels. To capture PDGF-BB, the aptamer-patterned DNA hydrogel was immersed in 1 mL absorption medium with 0.22 μM PDGF-BB. As incubation time increased, the PDGF-BB molecules gradually penetrated into the hydrogel network, leading to a decrease in the PDGF-BB concentration in the absorption medium. The protein capture ability of the hydrogels was assessed by detecting the change in PDGF-BB concentration in the absorption medium. As shown in Fig. S13, after incubation for 4.5 h, the DNA hydrogels with aptamer showed an obviously higher PDGF-BB capture capability (approximately 47%) than the DNA hydrogel without aptamer (approximately 9%) due to specific binding between PDGFBB and aptamer. These results demonstrated that the hydrogels without aptamer had a very low protein capture capacity, and protein capture was attributed only to physical diffusion. In contrast, the protein capture capacity in the hydrogel with aptamer was mainly determined by the specific affinity between the aptamer and proteins. This suggests that aptamers play a crucial role in the protein capture ability of DNA hydrogels. To further study the ability of the DNA hydrogels to capture PDGF-BB, aptamer-patterned DNA hydrogels with different characteristics were evaluated. Firstly, different concentrations of DNA building blocks were investigated (X-DNA 62.5 μM and L-DNA 125 μM, X-DNA 125 μM and L-DNA 250 μM, X-DNA 250 μM and L-DNA 500 μM, and X-DNA 500 μM and L-DNA 1000 μM). As shown in Fig. 4A, higher concentrations of DNA building units (X-DNA 500 μM and L-DNA 1000 μM) led to lower protein capture capacities. This could be because the higher concentrations of DNA units resulted in more close-knit crosslinked networks, which limited the diffusion of PDGF-BB into the hydrogel. As the concentration of DNA building units decreased, the amount of PDGF-BB captured gradually increased. In addition to the concentration of DNA building units, the concentration of PDGF-BB in the absorption medium also affected the capture capability of the hydrogel. Hydrogel samples (125 μM XDNA, 250 μM L-DNA and 300 μM CCC) were immersed into 1 mL absorption medium with different concentrations of PDGF-BB (0.0275, 0.0550, 0.0825, 0.11, 0.22 and 0.44 μM); the corresponding molar ratios of PDGF-BB to aptamers were calculated to be 0.25:1, 0.5:1, 0.75:1, 1:1, 2:1 and 4:1, respectively. The results are shown in Fig. 4B. As the molar ratios of PDGF-BB to aptamer increased from 0.25:1 to 1:1, the normalized protein capture of the hydrogel increased from 1 ± 0.07 to 3.98 ± 0.21. However, as the molar ratios of PDGF-BB to aptamers was increased from 1:1 to 4:1, no significant change in the hydrogel normalized capture was observed. This could be explained as follows. When the amount of PDGF-BB exceeded the amount of aptamer, the aptamers in the hydrogel were completely consumed. These results show that when the molar ratio of PDGF-BB to aptamer is 1:1, the hydrogels reached saturation capture. Another important factor affecting the hydrogel capture capacity was the amount of aptamer patterned into the hydrogels. As shown in Fig. 4C, as the amount of aptamer patterned into the hydrogel increased, the protein capture capability of the hydrogel increased obviously. As the amount of aptamer



increased from 0.11 nmol to 0.43 nmol, normalized capture increased from 5.21 ± 0.41 to 17.12 ± 0.81. Since the amount of PDGF-BB in the absorption medium was 0.43 nmol, when the amount of aptamer increased from 0.43 nmol to 0.52 nmol, there was no significant change in normalized capture, because the PDGF-BB in the absorption medium had been completely captured. These results indicate that specific binding between the aptamer and PDGF-BB was the main driving force for PDGF-BB capture. Therefore, by adjusting the above three parameters, the capture capability of PDGF-BB can be accurately controlled, providing an advantage over most conventional protein carriers with low and uncontrolled protein capture efficiency.29-30

Fig. 4 (A) Capture capabilities of hydrogel formed from different concentrations of building blocks. For normalization, the capture capability of the hydrogel (with 500 μM X-DNA, 1000 μM L-DNA and 300 μM CCC) is regarded as 1. The molar ratio of PDGF-BB to aptamer was 1:1. (B) Effect of PDGF-BB amount in the absorption medium on the capture capability of the hydrogel (X-DNA 125 μM, L-DNA 250 μM and CCC 300 μM). For normalization, the capture capability for the molar ratio of PDGF-BB to aptamer of 0.25:1 is regarded as 1. (C) Relationship between the amount of aptamers patterned into the hydrogel and the capture capability (X-DNA 125 μM, L-DNA 250 μM and CCC 300 μM). For normalization, the hydrogel capture capability without aptamer (0 nmol) is regarded as 1. The concentration of PDGF-BB was fixed at 0.43 μM. Sample with significantly higher normalized capture compared to the control (normalized capture = 1) are noted with * (p < 0.05). Examination of PDGF-BB release from aptamer-patterned DNA hydrogels. The hydrogels were subjected to PDGF-BB release tests in the absence of CSs to examine the binding ability between aptamers and PDGF-BB in the hydrogels. Here, controlled aptamer with improper sequences could not bind PDGF-BB. As shown in Fig. 5A, more than 90% of PDGF-BB was released from the hydrogels without aptamers within two hours. Similarly, the hydrogel with controlled aptamer also exhibited burst release. In contrast, only 10% of PDGF-BB was released from the aptamer-patterned hydrogels. These results suggest that aptamers dramatically reduce burst release and result in the effective sequestration of PDGF-BB in the hydrogel. To determine the ability of the aptamer-patterned DNA hydrogel to hybridize with CSs, the hydrogel was treated with DABCYL-labelled CS36 for different times. When the CSs diffused into the hydrogel network and hybridized with FAM-labelled aptamer, the emission of FAM was quenched by DABCYL due to Förster resonance energy transfer (FRET).31-32 Therefore, as the incubation time increased, the intensity of fluorescence decreased, as shown in Fig. 5B(a-d). At the same time, the dissociated PDGF-BB from aptamers increasingly diffused out of the hydrogels, resulting in an increase in PDGF-BB concentrations in the release medium [Fig. 5B(e)]. These results demonstrate that CSs can penetrate rapidly into the hydrogel to hybridize with aptamers and induce PDGF-BB release.


Page 8 of 16

Page 9 of 16 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


Fig. 5 (A) PDGF-BB release from hydrogel without aptamer, with aptamer and with control aptamer (XDNA 125 μM, L-DNA 250 μM and CCC 300 μM) in the absence of CSs. (B) Fluorescent images of hydrogel (with FAM-labelled aptamer) in the presence of DABCYL-labelled CS36 at different incubation times: (a) 5 min, (b) 2h, (c) 4h and (d) 6 h (scalebars =300 μm). (e) Cumulative release of PDGF-BB at different times. The molar ratio of CS36 to aptamer was fixed at 1:1. The dissociation rate of PDGF-BB triggered by CS36 could be fitted to a theoretical kinetic model according to the relation between triggering time and PDGF-BB concentration. The fitting formula is given as follows:

ln

C ∞ − Ct = kt C ∞ − C0

(1)

where, C0 is the initial concentration of PDGF-BB in the absence of CSs, Ct is that at time t, C∞ is that when the reaction reaches equilibrium in the presence of CSs, and k is a rate constant that reflects the dissociation rate of PDGF-BB triggered by CSs. The influence of CSs concentration on k for PDGF-BB dissociation was studied. Based on the fitting formula, the dissociation rate corresponded to the slope of the linear curve. Different concentrations of CSs resulted in different dissociation rate constants k. As shown in Fig. 6A, when then molar ratio of CSs to aptamer was 1:5, k was - 0.1931. When the molar ratio of CSs to aptamers was increased to 1:1, k was reduced to - 0.7947 (Fig. 6E). By comparing the slopes of the curves (Fig. 6F), we can conclude that higher CSs concentrations lead to lower rate constants (i.e., higher release capability).



Fig. 6 Dissociation dynamics of PDGF-BB triggered by CS36 for different CSs to aptamer ratios: (A) CSs: Aptamer = 1:5, (B) CSs: Aptamer = 2:5, (C) CSs: Aptamer = 3:5, (D) CSs: Aptamer = 4:5 and (E) CSs: Aptamer = 1:1. (F) The concentrations of PDGF-BB were calculated using the fitting formula In(C∞ - Ct)/(C∞ - C0) = kt to obtain the rate constant (slope of the linear curve) of PDGF-BB dissociation. To further understand the protein release triggered by CSs, the molar ratio of CSs to aptamer, the chain length of CSs and the time of CSs addition were carefully studied in this work. The percentage of CS36 hybridized with aptamer in the hydrogel is shown in Fig. S14 and Table S4. As shown in Fig. 7A, as the CSs concentration increased, the cumulative release of PDGF-BB increased from 25% to 83%. Because of specific binding between PDGF-BB and aptamer, PDGF-BB bound to aptamer did not dissociate without CSs (CSs = 0). Therefore, without CSs, PDGF-BB was released only by physical diffusion, and the cumulative release was approximately 10%. These results are similar to those shown in Fig. 6. The amount of base pairs obviously influenced the strength of DNA hybridization.33 Longer CSs resulted in stronger competition ability. As shown in Fig. 7B, when the length of CSs was nine or 15 nucleotides, there was no obvious difference in the amount of PDGF-BB released compared to the control group (without CSs). This suggests that short CSs (nine and 15 nucleotides) cannot trigger the release of PDGF-BB from aptamer. When the length of CSs changed from 15 to 26 nucleotides, the release amount was increased by four times compared to the control. When the CSs were completely complementary with the PDGF-BB aptamer (36 nucleotides), the release amount increased by about 5.5 times compared to the control. These results indicated that increasing the length of CSs can facilitate PDGF-BB separation from PDGF-BB-aptamer complexes. In addition to the reasonable design of CSs length, triggered release can be adjusted by controlling the triggering time. As shown in Fig. 7C, we used CS36 to treat the DNA hydrogel at hours 0 and 15. The release amount of PDGF-BB over 15 and 28 h were approximately 47% and 84% (the molar ratio of added CSs to aptamer was fixed at 1:2 each time). The hydrogels released approximately 10% of PDEF-BB in the absence of CS36. The cumulative release of PDGF-BB was approximately 47% for the first triggering (0 h), and about 37% for the second triggering (15 h). This might be explained as follows. The first release included two processes: the physical diffusion of PDGF-BB (10%) and triggered release by CSs (37%). However, for the second release, only included CSs-triggered release, because the physical diffusion process reached equilibrium within 4h (Fig. 7C). These results indicate that release amount of PDGF-BB can be precisely adjusted by changing the amount and length of CSs. In addition, PDGF-BB can be released at prospective times by adding CSs.


Page 10 of 16

Page 11 of 16 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


Fig. 7 (A) Effect of molar ratios of CS36 to aptamer on PDGF-BB release. The amount of grafted aptamers was 0.11 nmol. The total release time was 12h. Here, CSs: aptamer = 0 served as the control group. Experimental group with obviously higher cumulative release compared to the control group was noted with * (p < 0.05). (B) Effect of CSs length on the release amount of PDGF-BB. The normalized release of PDGF-BB in the absence of CSs (bp 0) is regarded as 1. Groups with significantly higher normalized release compared to bp are noted with * (p < 0.05). (C) Profiles of PDGF-BB cumulative release in the release medium with or without CS36 at different triggering times (0 and 15 h). Application in complex matrixes. A complex matrix, human serum, was studied to demonstrate the practical application of hydrogels in the triggered release of PDGF-BB. Hydrogels (X-DNA 125 μM, L-DNA 250 μM, CCC 300 μM) with captured PDGF-BB were immersed into human serum solution. The serum solution had been diluted 50 times before each experiment. We used CS36 to treat the DNA hydrogel at hours 0, 48, 96 and 144 (the molar ratio of CSs to aptamer was fixed at 1:4 each time). As shown in Fig. 8, the cumulative release amounts of PDGF-BB over 48, 96, 144 and 168 h were approximately 29%, 47%, 65% and 83%, respectively. For the first triggering (0 h), the cumulative release of PDGF-BB was approximately 29% (including 11% from “physical diffusion” and 18% from “CSs triggered release”). For each of the remaining three trigger times (48, 96 and 144 h), the release amount of PDGF-BB was approximately 18%. These results indicate that this hydrogel shows promise for practical applications in complex matrixes.

Fig. 8 Profiles of PDGF-BB cumulative release in human serum with or without CS36 at different triggering times (0, 48, 96 and 144 h). Cytocompatibility of the aptamer-patterned DNA hydrogels. The viability and proliferation of RAW 264.7 cells cultured on the hydrogels were studied in vitro. With increasing culture time (Fig. 9A), the RAW 264.7 cells increasingly proliferated on the DNA hydrogels and culture plate without hydrogels (control group). The proliferation rates of RAW 264.7 cells on the surface of the DNA hydrogels were higher than that of the control group. Cell viability was also



investigated using a Live/Dead Assay Kit. As shown in Fig. 9B, high cell viability (green) was achieved on both the DNA hydrogel and culture plate, but more cells were visible on the DNA hydrogel, suggesting increased cell proliferation, consistent with the cell proliferation results. Compared to other synthetic polymer hydrogel,34-35 the DNA hydrogel showed less cytotoxicity and higher cell proliferation rate. Therefore, the DNA hydrogels constructed by X-DNA, L-DNA, aptamer and CCC showed good cell compatibility and have potential application in cell research.

Fig. 9 (A) Proliferation of RAW 264.7 cells cultured on the surfaces of DNA hydrogels (X-DNA 125 μM, LDNA 250 μM and CCC 300 μM) and culture plate (without hydrogels). * p < 0.05. (B) Morphology and viability of RAW 264.7 cells cultured on surfaces of a culture plate (a) and DNA hydrogel (b) for 72 h (green spots are live cells, and red spots are dead cells; scale bars = 200 μm).

CONCLUSIONS Under the action of CCC, the critical concentration for gelation in DNA hydrogels was dramatically decreased. The critical length of the “sticky ends” for gelation in DNA hydrogels was also significantly shortened. Moreover, CCC obviously improved the mechanical strength of the DNA hydrogels. We also demonstrated that the DNA hydrogel reversibly respond to CCC and PVS by switching between the gel and sol states at room temperature. In addition, aptamer was successfully patterned into the hydrogel via click chemistry. This hydrogel possesses high protein capture capacity along with a high retention rate in the absence of triggering CSs. The severe drawback (i.e., burst release) of traditional hydrogels as protein drug carriers was resolved by using this aptamer-patterned DNA hydrogel. Furthermore, CSs can be used to control the release of PDGF-BB at prospective times and in specific areas with adjustable release rates. The hydrogel can also be applied in complex matrixes such as human serum. Moreover, in vitro experiments showed that RAW 264.7 cells cultured on the DNA hydrogels exhibited good viability and proliferation. This hydrogel with excellent biocompatibility will become a potential platform for the controlled release of protein drugs and facilitate the treatment of complex human diseases. ASSOCIATED CONTENT Supporting Information The characterization of the cationic comb-type copolymer PLL-g-Dex, detection method for the concentration of PDGF-BB, agarose gel electrophoresis analysis, the grafting amount of PDGF-BB aptamer, and the percentage of CS36 hybridized with PDGF-BB aptamer in hydrogels. AUTHOR INFORMATION Corresponding Author


Page 12 of 16

Page 13 of 16 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


* Email: [email protected] Author Contributions Z. Z. and J. L. H. contributed equally to this paper. The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant No. 21763009, 21404028), Graduate Students Innovation Research Project of Hainan Province (Hys2016-04), and Scientific Research Start-up Foundation of Hainan University (Grant No. kyqd1568).

REFERENCES 1. Anseth, K. S.; Metters, A. T.; Bryant, S. J.; Martens, P. J.; Elisseeff, J. H.; Bowman, C. N., In situ Forming Degradable Networks and Their Application in Tissue Engineering and Drug Delivery. J. Control. Release 2002, 78 (1), 199-209. DOI: 10.1016/S0168-3659(01)00500-4. 2. Xu, K.; Lee, F.; Gao, S. J.; Chung, J. E.; Yano, H.; Kurisawa, M., Injectable Hyaluronic Acid-Tyramine Hydrogels Incorporating Interferon-α2a for Liver Cancer Therapy. J. Control. Release 2013, 166 (3), 203210. DOI: 10.1016/j.jconrel.2013.01.008. 3. Lee, H. M.; Larson, D. R.; Lawrence, D. S., Illuminating the Chemistry of Life: Design, Synthesis, and Applications of “Caged” and Related Photoresponsive Compounds. ACS Chem. Biol. 2009, 4 (6), 409-427. DOI: 10.1021/cb900036s. 4. Kirschner, C. M.; Anseth, K. S., Hydrogels in Healthcare: From Static to Dynamic Material Microenvironments. Acta Mater. 2013, 61 (3), 931-944. DOI: 10.1016/j.actamat.2012.10.037. 5. Hoare, T. R.; Kohane, D. S., Hydrogels in Drug Delivery: Progress and Challenges. Polymer 2008, 49 (8), 1993-2007. DOI: 10.1016/j.polymer.2008.01.027. 6. Lin, C. C.; Metters, A. T., Metal-Chelating Affinity Hydrogels for Sustained Protein Release. J. Biomed. Mater. Res. A 2007, 83 (4), 954-964. DOI: 10.1002/jbm.a.31282. 7. Jha, A. K.; Yang, W.; Kirn-Safran, C. B.; Farach-Carson, M. C.; Jia, X., Perlecan Domain I-Conjugated, Hyaluronic Acid-Based Hydrogel Particles for Enhanced Chondrogenic Differentiation via BMP-2 Release. Biomaterials 2009, 30 (36), 6964-6975. DOI: 10.1016/j.biomaterials.2009.09.009. 8. Tian, B.; Liu, J.; Dvir, T.; Jin, L.; Tsui, J. H.; Qing, Q.; Suo, Z.; Langer, R.; Kohane, D. S.; Lieber, C. M., Macroporous Nanowire Nanoelectronic Scaffolds for Synthetic Tissues. Nat. Mater. 2012, 11 (11), 986994. DOI: 10.1038/nmat3404. 9. Jayasena, S. D., Aptamers: an Emerging Class of Molecules that Rival Antibodies in Diagnostics. Clin. Chem. 1999, 45 (9), 1628-1650. 10. Soontornworajit, B.; Zhou, J.; Snipes, M. P.; Battig, M. R.; Wang, Y., Affinity Hydrogels for Controlled Protein Release Using Nucleic Acid Aptamers and Complementary Oligonucleotides. Biomaterials 2011, 32



(28), 6839-6849. DOI: 10.1016/j.biomaterials.2011.05.074. 11. Battig, M. R.; Soontornworajit, B.; Wang, Y., Programmable Release of Multiple Protein Drugs from Aptamer-Functionalized Hydrogels via Nucleic Acid Hybridization. J. Am. Chem. Soc. 2012, 134 (30), 1241012413. DOI: 10.1021/ja305238a. 12. Soontornworajit, B.; Zhou, J.; Wang, Y., A Hybrid Particle–Hydrogel Composite for OligonucleotideMediated Pulsatile Protein Release. Soft Matter 2010, 6 (17), 4255-4261. DOI: 10.1039/c0sm00206b. 13. Wang, Z.; Xia, J.; Cai, F.; Zhang, F.; Yang, M.; Bi, S.; Gui, R.; Li, Y.; Xia, Y., Aptamer-Functionalized Hydrogel as Effective Anti-Cancer Drugs Delivery Agents. Colloids Surf. B. Biointerfaces 2015, 134, 40-46. DOI: 10.1016/j.colsurfb.2015.06.031. 14. Nishida, Y.; Ohtsuki, S.; Araie, Y.; Umeki, Y.; Endo, M.; Emura, T.; Hidaka, K.; Sugiyama, H.; Takahashi, Y.; Takakura, Y.; Nishikawa, M., Self-Assembling DNA Hydrogel-Based Delivery of Immunoinhibitory Nucleic Acids to Immune cells. Nanomed. Nanotechnol. Biol. Med. 2016, 12 (1), 123-130. DOI: 10.1016/j.nano.2015.08.004. 15. Ren, J.; Hu, Y.; Lu, C.-H.; Guo, W.; Aleman-Garcia, M. A.; Ricci, F.; Willner, I., pH-Responsive and Switchable Triplex-Based DNA Hydrogels. Chem. Sci. 2015, 6 (7), 4190-4195. DOI: 10.1039/c5sc00594a. 16. Battig, M. R.; Huang, Y.; Chen, N.; Wang, Y., Aptamer-Functionalized Superporous Hydrogels for Sequestration and Release of Growth Factors Regulated via Molecular Recognition. Biomaterials 2014, 35 (27), 8040-8048. DOI: 10.1016/j.biomaterials.2014.06.001. 17. Soontornworajit, B.; Zhou, J.; Shaw, M. T.; Fan, T. H.; Wang, Y., Hydrogel Functionalization with DNA Aptamers for Sustained PDGF-BB Release. Chem. Commun. 2010, 46 (11), 1857-1859. DOI: 10.1039/b924909e. 18. Li, J.; Zheng, C.; Cansiz, S.; Wu, C.; Xu, J.; Cui, C.; Liu, Y.; Hou, W.; Wang, Y.; Zhang, L.; Teng, I. T.; Yang, H. H.; Tan, W., Self-Assembly of DNA Nanohydrogels with Controllable size and Stimuli-Responsive Property for Targeted gene Regulation Therapy. J. Am. Chem. Soc. 2015, 137 (4), 1412-1415. DOI: 10.1021/ja512293f. 19. Zhang, L.; Lei, J.; Liu, L.; Li, C.; Ju, H., Self-Assembled DNA Hydrogel as Switchable Material for AptamerBased Fluorescent Detection of Protein. Anal. Chem. 2013, 85 (22), 11077-11082. DOI: 10.1021/ac4027725. 20. Wu, L.; Shimada, N.; Kano, A.; Maruyama, A., Poly(l-lysine)-graft-Dextran Copolymer Accelerates DNA Hybridization by Two Orders. Soft Matter 2008, 4 (4), 744-747. DOI: 10.1039/b717478k. 21. Kim, W. J.; Sato, Y.; Akaike, T.; Maruyama, A., Cationic Comb-type Copolymers for DNA Analysis. Nat. Mater. 2003, 2 (12), 815-820. DOI: 10.1038/nmat1021. 22. Kano, A.; Moriyama, K.; Yamano, T.; Nakamura, I.; Shimada, N.; Maruyama, A., Grafting of Poly(ethylene glycol) to Poly-lysine Augments its Lifetime in Blood Circulation and Accumulation in Tumors without Loss of the Ability to Associate with siRNA. J. Control. Release 2011, 149 (1), 2-7. DOI: 10.1016/j.jconrel.2009.12.007.


Page 14 of 16

Page 15 of 16 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


23. Gao, J.; Shimada, N.; Maruyama, A., MNAzyme-Catalyzed Nucleic Acid Detection Enhanced by a Cationic Copolymer. Biomater. Sci. 2015, 3 (5), 716-720. DOI: 10.1039/c4bm00449c. 24. Maruyama, A.; Katoh, M.; Tsutomu Ishihara, A.; Akaike, T., Comb-Type Polycations Effectively Stabilize DNA Triplex. Bioconjug. Chem. 1997, 8 (1), 3-6. DOI: 10.1021/bc960071g. 25. Zhang, Y.; Bai, Y.; Feng, F.; Shuang, S., A Graphene Oxide-Based Fluorescent Aptasensor for AlphaFetoprotein Detection. Anal. Methods 2016, 8 (32), 6131-6134. DOI: 10.1039/c6ay01949h. 26. Cheng, E.; Xing, Y.; Chen, P.; Yang, Y.; Sun, Y.; Zhou, D.; Xu, L.; Fan, Q.; Liu, D., A pH-Triggered, FastResponding DNA Hydrogel. Angew. Chem. Int. Ed. 2009, 48 (41), 7660-7663. DOI: 10.1002/anie.200902538 27. Borzacchiello, A.; Ambrosio, L., Structure-Property Relationships in Hydrogels. Springer Milan. 2009, 920. 28. Xing, Y.; Cheng, E.; Yang, Y.; Chen, P.; Zhang, T.; Sun, Y.; Yang, Z.; Liu, D., Self-Assembled DNA Hydrogels with Designable Thermal and Enzymatic Responsiveness. Adv. Mater. 2011, 23 (9), 1117-1121. DOI: 10.1002/adma.201003343. 29. Chung, H.-J.; Kim, J.-T.; Kim, H.-J.; Kyung, H.-W.; Katila, P.; Lee, J.-H.; Yang, T.-H.; Yang, Y.-I.; Lee, S.-J., Epicardial Delivery of VEGF and Cardiac Stem Cells Guided by 3-Dimensional PLLA Mat Enhancing Cardiac Regeneration and Angiogenesis in Acute Myocardial Infarction. J. Control. Release 2015, 205, 218-230. DOI: 10.1016/j.jconrel.2015.02.013. 30. Farhadi, M. R.; Capelle, H. H.; Erber, R.; Ullrich, A.; Vajkoczy, P., Combined Inhibition of Vascular Endothelial Growth Factor and Platelet-Derived Growth Factor Signaling: Effects on the Angiogenesis, Microcirculation, and Growth of Orthotopic Malignant Gliomas. J. Neurosurg. 2005, 102 (2), 363-370. DOI: 10.3171/jns.2005.102.2.0363 31. He, X.; Wei, B.; Mi, Y., Aptamer Based Reversible DNA Induced Hydrogel System for Molecular Recognition and Separation. Chem. Commun. 2010, 46 (34), 6308-6310. DOI: 10.1039/c0cc01392g. 32. Strehlitz, B.; Nikolaus, N.; Stoltenburg, R., Protein Detection with Aptamer Biosensors. Sensors 2008, 8 (7), 4296-4307. DOI: 10.3390/s8074296. 33. Delport, F.; Pollet, J.; Janssen, K.; Verbruggen, B.; Knez, K.; Spasic, D.; Lammertyn, J., Real-Time Monitoring of DNA Hybridization and Melting Processes Using a Fiber Optic Sensor. Nanotechnology 2012, 23 (6), 065503. DOI: 10.1088/0957-4484/23/6/065503. 34. Fu, Y.; Xu, K.; Zheng, X.; Giacomin, A. J.; Mix, A. W.; Kao, W. J., 3D Cell Entrapment in Crosslinked Thiolated Gelatin-poly(ethylene glycol) Diacrylate Hydrogels. Biomaterials 2012, 33 (1), 48-58. DOI: 10.1016/j.biomaterials.2011.09.031. 35. Nguyen, E. H.; Zanotelli, M. R.; Schwartz, M. P.; Murphy, W. L., Differential Effects of Cell Adhesion, Modulus and VEGFR-2 Inhibition on Capillary Network Formation in Synthetic Hydrogel Arrays. Biomaterials 2014, 35 (7), 2149-2161. DOI: 10.1016/j.biomaterials.2013.11.054.



Graphical Abstracts

Chaperoned by cationic copolymer PLL-g-Dex, DNA hydrogels with low DNA concentration, short sticky end and good mechanical strength were simply prepared via one-pot self-assembly from two kinds of DNA building block (X- and L-shaped DNA units). This aptamer-patterned DNA hydrogels could be used as a smart protein carrier for controlled protein release at prospective times and in specific areas.


Page 16 of 16

Chaperone Copolymer-Assisted Aptamer-Patterned DNA Hydrogels

Recommend Documents