Aptamer-Patterned Hydrogel Films for Spatiotemporally

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Aptamer-patterned Hydrogel Films for Spatiotemporally Programmable Capture and Release of Multiple Proteins zheng Zhang, Chen Liu, Chunzheng Yang, Yuyang Wu, Feng Yu, Yong Chen, and Jie Du ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00191 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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ACS Applied Materials & Interfaces

Aptamer-patterned Hydrogel Films for Spatiotemporally Programmable Capture and Release of Multiple Proteins Zheng Zhang, Chen Liu, Chunzheng Yang, Yuyang Wu, Feng Yu, Yong Chen, 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.

ABSTRACT: Various hydrogels have been used as proteins delivery in the treatment of human diseases. Nevertheless, it is always difficult to control capture and release of multiple proteins in different regions and periods. This research successfully proves that multiple proteins can be captured and released from the aptamer-patterned hydrogel films with adjustable release rate at prospective time and in specific regions utilizing the complementary DNA strand (cDNA) of aptamer via photoclick chemistry and DNA hybridization. The hydrogel film is successfully applied to complex matrixes such as human serum and has excellent cytocompatibility. Thus, the aptamer-patterned hydrogel film will be a good candidate for controlled delivery of multiple proteins. KEYWORDS: chitosan; hydrogel films; patterning; multiple proteins; controlled release

INTRODUCTION A number of studies have been reported in utilizing proteins as therapeutic drugs for treatment of human diseases as proteins are a requisite biomolecule of organisms and are involved in almost all processes within the human body.1 However, it is challenging to regulate protein release rates, time points, and regions for the process of treatment. This problem can be solved by utilizing external stimuli for real-time control. These stimuli can be ultrasound, irradiation, temperature, magnetic fields or electric potentials.2 By responding to the stimuli, the polymeric system transforms its volumes, pore sizes or structural integrity and release proteins correspondingly. These principles have been widely used to accomplish release of single proteins. They are short of specificity in adjusting the release of multiple proteins, which is indispensable to cure various human diseases.3 Thus, it is important to develop an unusual polymeric delivery system to adjust the controlled release of multiple proteins at anticipative times and regions. Hydrogel, which is a three-dimensional polymeric material with high water content and various physical properties, has been widely utilized in foods, cosmetics, drug-delivery devices, and other fields.4 However, most hydrogels include complex polymer and organic cross-linkers that cannot synchronously satisfy the biocompatibility and mechanical demands of biomedical materials. In contrast, natural polymers, such as chitosan, collagen, alginate and hyaluronidase, have been regarded as the potential biomaterials.5-7 Amongst these, chitosan, a unique alkaline polysaccharide resulting from the deacetylation of chitin, is readily soluble in dilute acidic solutions, and chitosan 1

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hydrogels can be regenerated by using an alkaline coagulating bath. Thanks to their intrinsic biocompatibility, nontoxicity, biodegradability, antimicrobial activity, and low immunogenicity, chitosan hydrogels are considered to have potential applications in a wide variety of fields such as water treatment, food industry, catalysis, agriculture, and biomedicine.8-13 The applications of chitosan hydrogel for biomaterial are, nevertheless, restricted due to the difficulties in fabrication, poor solubility and mechanical properties and side-effects resulting from organic cross-linker.14-20 Therefore, a worthwhile endeavor would be to find a new strategy of constructing a high strength chitosan hydrogel without depressing its desirable properties. Aptamers have recently caused significant interest in many fields because in theory aptamers can combine all corresponding molecules with high binding specificity and affinity.21-25 They are non-toxic, and have stable structures and small sizes.26 Due to these features, aptamer have been researched in diverse biomedical and biological fields such as surface functionalization,27-30 antidote synthesis,31-33 biosensor development,34-39 and nanoparticle delivery.40-43 Furthermore, differ from other affinity ligands, the DNA aptamer can be hybridized with complementary DNA sequences (cDNA) and cause fast separation of the aptamer−proteins complex. Therefore, adding the designated cDNA can regulate and control the release of proteins from aptamers.33 Inspired by these concepts, a chitosan hydrogel film with patterned two aptamer models for the capture and release of protein was designed through combining chemical crosslinking and DNA hybridization. In this work, alkali/urea (6.5 wt % KOH/5 wt % LiOH/7 wt % urea solutions) was used in the preparation of allyl chitosan (AC). This advantage compare with past methods is that it can be achieved without organic solutions and catalysts. This soluble allyl chitosan can be cross-linked by epichlorohydrin (ECH) to prepare chitosan hydrogel films. These two aptamers have been sufficiently researched and bound to vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB). 44, 45 In recent years, Yong Wang’s group focused on many different aptamer-functionalized hydrogels to control the capture and release of cell,46 single protein47, 48 or multiple proteins49 through hybridization with complementary DNA. In addition, they achieved self-programmed protein release.50 The aptamers were incorporated into hydrogels through polymerization. Nevertheless, the distribution of the aptamers in these hydrogels is homogeneous. So far, hydrogels with spatially heterogeneous and multiple aptamers distribution have not been researched. Therefore, in our work, sulfydryl-modified these two aptamers were first grafted into the double bond-functionalized network with well-defined patterning by sulfydryl-double bond click chemistry. The diverse influence factors of the aptamer-patterned hydrogel film were researched to prove the effect of the aptamers in the capture and release of VEGF and PDGF-BB.

RESULTS AND DISCUSSION The preparation and characterization of AC hydrogel film. Scheme 1A illustrates the synthesis of AC. Allyl chloride could react immediately with chitosan in KOH/LiOH/urea/H2O solutions without other catalysts due to the basicity of the solvents. Allyl chloride were added slowly, and the all mixtures were stirred at 0 °C under away from light for protecting the double bonds from cross-linking. The reaction proceeded equably, and the transparent and clear solutions were obtained in 12 h. Sometimes, cross-linking of the chitosan hydrogel by chemical bonds is necessary to improve its acidic resistance in a harsh environment. The etherification between chitosan and ECH occurred easily in the alkaline system.51, 52 As shown in Scheme 1B, ECH was used to cross-link AC to prepare the hydrogel and then dried to get AC hydrogel film (ACF).

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Scheme 1. (A) Allylation of chitosan in KOH/LiOH/urea solution. (B) Schematic representation of ECH cross-link

AC to prepare hydrogel film.

As is well-known, the mechanical properties of film and hydrogel are very important for their applications. Figure 1A shows the ACF typical tensile stress−strain curves. The tensile strength and elongation at break values of the ACF were 5.8 MPa and 52% for ACF-1, and varied to 5.9 MPa and 71% for ACF-5. Figure 1B shows the swelling kinetics of the ACF in a PBS aqueous solution. Moreover, all of the ACF tended to swell, and reached the swelling equilibrium after 90 min. The tensile properties of ACF of swelling equilibrium were also studied. the ACF of swelling equilibrium all exhibited excellent tensile properties, and their tensile fracture stress and strain were all over 0.3 MPa and 78% (Figure 1C). As evidenced by the results of tensile and swelling properties, the ACF had the favorable properties for applications in biomaterials.

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Figure 1. (A) Typical tensile stress−strain curves of ACF. (B) Swelling kinetics of ACF in PBS aqueous solution at

37 °C. (C) Typical tensile stress−strain curves of ACF of swelling equilibrium.

The fabrication of aptamer patterned AC hydrogel film. The sulfydryl-aptamer was patterned in the hydrogel film by thiol-ene reaction between sulfydryl and double bond. Photocoupling was completed through a paralleled UV light (365 nm) and I2959 as the photoinitiator. In the first patterning process, a strip-type pattern (300 µm line with 400 µm interval) of the sulfydryl/FAM-labeled PDGF-BB aptamer was formed. The process was then repeated with sulfydryl/TAMRA-labeled VEGF aptamer. In the second patterning process, the photomask was rotated 90°. These results demonstrate that sequential patterning was formed (Figure 2).

Sequential patterning in ACF. Aqueous solution of sulfydryl/fluorophore-labeled aptamer (sulfydryl/FAM-labeled PDGF-BB aptamer and sulfydryl/TAMRA-labeled VEGF aptamer) and the photoinitiator I2959 (1.9 mM) were gradually swollen in the gel and patterned by a photomask using paralleled UV light (365 nm) to obtain a grid pattern (300 µm line, 400 µm interval; scalebars =400 µm).

Figure 2.

Examination of growth factor capture from aptamer-patterned hydrogel film. To capture PDGF-BB and VEGF, the hydrogel films were immersed into 200 µL capture buffer with 0.6 µM PDGF-BB and 0.6 µM VEGF. The detection theory of protein was shown in Scheme S1 and Figure S1. As the incubation time increased, the growth factor would penetrate into hydrogel film. As shown in Figure S2, after 3.5 h of incubation, the ACF with aptamers had the obviously higher proteins capture capability (about 54% of PDGF-BB and 41% of VEGF) than the ACF without aptamer (about 10% of PDGF-BB and 9% of VEGF) due to the specifically bound of protein to the aptamer within the ACF. Furthermore, compared with other hydrogel films (gelatin hydrogel film53 and hyaluronic acid hydrogel film54), there was no obvious difference in capture capabilities of ACF without aptamer (Figure S3), because capture capabilities of these hydrogel films were only attributed to physical diffusion. These results show that aptamer made a significant contribution to capture PDGF-BB and VEGF in the ACF. To further understand the capability of the hydrogel film to capture protein, diverse factors of the 4


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aptamer-patterned ACF were researched. Firstly, the different amounts of crosslinker (i.e., ECH) were studied. The more content of ECH gave rise to a more close-knit and higher crosslinked network, which limited diffusion of proteins. This was why the ACF-5 had a lower protein capture capability, as shown in Figure 3A. Compared with ACF-5, the normalized capture of ACF-3 was approximately 1.13±0.04 of PDGF-BB and 1.21±0.05 of VEGF, and that of the ACF-1 was about 1.25 ±0.05 of PDGF-BB and 1.35±0.051 of VEGF. The ACF-1 had the highest protein capture capacity. Therefore, ACF-1 was chosen for the next experiments. Meanwhile, the concentration of protein in capture buffer also had an influence on the capture capability of the hydrogel film. In Figure 3B, the PDGF-BB normalized capture enhanced from 1±0.05 to 3.5±0.17 when the molar ratio of PDGF-BB to aptamers increased from 1:3 to 2:1. Similarly, as VEGF to aptamer changed from 1:3 to 2:1, the VEGF normalized capture enhanced from 1±0.06 to 2.6±0.15, but when the amount of protein change continuously from 2:1 to 4:1, there was no obvious change in the normalized capture of the hydrogel film. The results showed that the hydrogel film had a saturated capture capability and reached to capture equilibrium. Another important factor that influenced the capture capability of the hydrogel film was the amount of aptamers that patterned into hydrogel film. The grafting amount of two aptamers into hydrogel film was shown in Figure S4, Table S1 and Table S2. As shown in Figure 3C, as the amount of PDGF-BB aptamer increased from 0.07 to 0.30 nmol, the PDGF-BB normalized capture was enhanced from 3.01±0.24 to 9.02±0.41. Similarly, as the amount of VEGF aptamer increased from 0.06 to 0.29 nmol, the VEGF normalized capture was enhanced from 2.58±0.25 to 7.22±0.32. Since the amount of PDGF-BB and VEGF was fixed at 0.3 and 0.29 nmol, with the amount of aptamer increasing, the normalized capture of hydrogel film was basically changeless, because the protein in the capture buffer had been entirely captured into the hydrogel film. The results suggest that the specific binding of aptamers and proteins was the primary impetus for proteins capture. Thus, through adjusting the three parameters, the amount of captured proteins can be accurately controlled as needed.

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Figure 3. The factors affecting the protein capture capability of the aptamer-patterned ACF, left: PDGF-BB, right:

VEGF. (A) The influence of the ECH content. The capture capability of ACF-5 was regarded as 1. The amounts of PDGF-BB aptamer and VEGF aptamer were fixed at 0.15 and 0.13 nmol. The molar ratio of proteins to aptamers was kept at 1:1. (B) The effect of protein content. When the molar ratio of the protein to aptamer was 1: 3, the capture capability was regarded as 1. The amounts of PDGF-BB aptamer and VEGF aptamer were fixed at 0.15 and 0.13 nmol. (C) Relationship between the concentrations of aptamer and capture capability of film. The capture capability of hydrogel film without aptamer (aptamer= 0) was represented as 1. The amounts of PDGF-BB and VEGF in the capture buffer were fixed at 0.3 and 0.29 nmol (the volume is 200 µL).The samples with obviously higher normalized capture than the control group (the normalized capture =1) were noted with (*, p

Aptamer-Patterned Hydrogel Films for Spatiotemporally

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