Aptamer-incorporated hydrogels for visual detection, controlled drug release, and targeted cancer therapy

Abstract

Hydrogels are water-retainable materials, made from cross-linked polymers, that can be tailored to applications in bioanalysis and biomedicine. As technology advances, an increasing number of molecules have been used as the components of hydrogel systems. However, the shortcomings of these systems have prompted researchers to find new materials that can be incorporated into them. Among all of these emerging materials, aptamers have recently attracted substantial attention because of their unique properties, for example biocompatibility, selective binding, and molecular recognition, all of which make them promising candidates for target-responsive hydrogel engineering. In this work, we will review how aptamers have been incorporated into hydrogel systems to enable colorimetric detection, controlled drug release, and targeted cancer therapy.

Introduction

Hydrogels are water-retainable materials generated from cross-linked polymers. Because of their biocompatibility and low toxicity, hydrogel systems have been increasingly recognized as important for bioanalytical detection, drug delivery, cancer therapy, and tissue engineering. In particular, stimuli-responsive hydrogels have captured the attention of researchers because of their sensitive response to a variety of physical and chemical changes, such as pH [1–4], ionic strength [5, 6] ultrasound irradiation [7], and electrical [8, 9], thermal [10], and magnetic stimuli [11, 12]. This leads to changes in gel structure, to fulfill the desired bioanalytical and biomedical functions.

Hydrogel systems can be divided into two types: chemically or physically crosslinked. In chemically cross-linked hydrogels, conversion from the semi-solid gel form to the liquid sol state is controlled by covalent bonds between different polymer chains. In physically crosslinked hydrogels, the gel–sol conversion is controlled by physical interactions, for example ionic or antigen–antibody interactions. Because most of these designs do not have an appropriate screening method incorporated into the gel structure, the universality of these crosslinking approaches is limited. In addition, human physiological properties do not vary widely; therefore, pH-triggered and temperature-triggered hydrogels have limited biomedical applications. Finally, because there is a limit to the types of materials that can be used to engineer hydrogels, the length of a given crosslinker is always restricted by the size of the cross-linking molecule [13]. These limitations have prompted scientists to develop more flexible and biocompatible hydrogel designs [14].

Because of its inherent biocompatibility, DNA is very suitable for in-vivo applications. DNA can fold into unique secondary or tertiary structures, which result in high binding affinities for specific targets, leading to structural changes. Moreover, DNA is easy to synthesize, suitable for in-vitro design, and predictable in its molecular behavior, which is based upon the base-pairing principle of cDNA. For these reasons, DNA has recently been regarded as an excellent material for engineering hydrogel systems [13, 15].

Aptamers are special types of single-stranded DNA able to specifically recognize targets ranging from ions to cells. Aptamers are generated by a process termed “SELEX” (systematic evolution of ligands by exponential enrichment). Since their introduction 20 years ago, aptamers have gradually become a focus for bioanalytical and cancer-related research. Their high avidity, high affinity, easy fabrication, low cost, and easy modification have made aptamers excellent candidates for hydrogel engineering. An aptamer can specifically recognize its target, even at low concentrations, and undergo a structural change upon binding. This structural change can be utilized as a trigger for specific physical and chemical interactions within hydrogel systems, for example hydrogel structure dissociation or breakdown, resulting in chemical or drug release. The availability of a wide range of aptamers will make hydrogel systems more versatile.

Another important application of aptamer-based hydrogel systems is cancer cell-targeted delivery. Aptamer-based targeted delivery has been validated upon conjugation with drugs [16], nanoparticles [17, 18], and micelles [19]. Although these conjugates can improve the effectiveness and safety of some drugs, not all compounds can be delivered in this way because of their chemical composition. Hydrogels, especially nanogels, have long being recognized as valuable carriers for drug transport. However, lack of specificity has compromised their application. Using an aptamer specific to a certain type of cells as a recognition unit, drug loaded hydrogel complexes could be oriented toward specific cancer cells.

Motivated by the special features of aptamers, several groups have developed aptamer-functionalized hydrogel systems for visual detection, controlled drug release, and targeted drug delivery. In this paper we review approaches by which aptamers have been incorporated into hydrogels to achieve these objectives.

Aptamer cross-linked hydrogels for visual detection

Visual detection has particular appeal in applications that involve disease diagnosis, drug monitoring, and environmental surveillance, especially in situations in which sophisticated machine operators and trained practitioners are not available. Because of their high target specificity, easy synthesis, low cost, and chemical stability, aptamers have been regarded as promising recognition tools for such applications. Many strategies have been used to design aptamer-based biosensors in combination with fluorescence dyes [20, 21], electrochemical methods [22], and quantum dots [23, 24]. Aptamer-based colorimetric assays, with the aid of colorimetric reagents such as gold nanoparticles, visible dyes, enzymes, and polymers, have enabled the detection of molecular targets using the naked eye [25–28]. Using a DNA aptamer specific for adenosine, the Tan group has engineered a target-responsive hydrogel based on the structural change of an aptamer upon adenosine addition [29]. As shown in Fig. 1, two acrydite-modified oligonucleotides, strands A and B, are first separately incorporated into polyacrylamide chains. When these two oligonucleotide-conjugated polyacrylamide strands are mixed, the solution is in the fluid (sol) state. Next, a linker, termed Linker-Adap, is added to facilitate the process of sol-to-gel transition. The first 12 nucleotides (nt) (in blue) of Linker-Adap are complementary to strand A, whereas 5 nt (in red) of Linker-Adap and the first 6 nt from the adenosine aptamer are complementary to strand B. Upon addition of adenosine, the aptamer binds to its target, such that the five remaining nucleotides cannot maintain structural integrity, leading to the dissolution of the gel network. Because of their unique optical properties and chemical stability, gold nanoparticles (NPs) are used as a visual indicator. To accomplish this, NPs are mixed with strands A and B for gel aggregation before Linker-Adaps are added. After the addition of adenosine, the upper buffer solution turns from colorless to intense red, indicating that the NPs have been released into the solution, and absorption at 620 nm further verifies the result. The universality of the method was further confirmed by using cocaine–aptamer interaction as the trigger for NP release [30].

Fig. 1.

(a) Schematic diagram illustrating the formation and dissolution of hydrogel; (b) detailed structural design of Linker-Adap; (c, d) photographs of hydrogel before and after Linker-Adap is added; (e) photograph of hydrogel after adding adenosine. Reproduced, with permission, from Ref.[29]

Using an enzyme as a tool for signal amplification, Zhu et al. have devised a colorimetric technique based on aptamer–hydrogel interactions [30]. In this system, amylose was chosen as visual indicator because its color changes from yellow to dark blue when iodine is added. However, because amylase catalyzes the breakdown of amylose into oligosaccharide, there is no color change in iodine–amylose solution in the presence of amylase. Taking advantage of these characteristics, amylose was entrapped in the hydrogel system by mixing it with two linear polyacrylamide polymers, PS-A and PS-B, grafted with two pieces of cocaine DNA aptamer (Fig. 2). On addition of a linker-Apt, which is complementary to the two aptamers, the solution is transformed into the solid gel state. However, when cocaine is added, the solid gel slowly returns to the liquid state, because of the reduced cross-linking density caused by competitive cocaine–aptamer binding, this results in leakage of amylase into the upper solution. As can be seen from Fig. 2b, the color change of the solution is positively correlated with the amount of cocaine added, with 1 mmol L⁻¹ cocaine causing complete dissolution, whereas no such color change can be observed in control tubes. This work showed that the aptamer cross-linked hydrogel system enables easy-to-operate, sensitive colorimetric visual detection via appropriate signal amplification steps. With the wide range of target-specific aptamers available, this technique has a broad range of potential applications in disease diagnosis, serum biomarker monitoring, or environmental monitoring for toxic substances when regular antibody-based methods are not available.

Fig. 2.

(a) Schematic view of aptamer cross-linked hydrogel for signal amplification and visual detection; (b) photograph of color change on adding different concentrations of cocaine. Reproduced, with permission, from Ref. [30]

Aptamer-incorporated hydrogel for controlled drug release and targeted cancer therapy

Drug loading and release are critical steps in designing a hydrogel system for drug delivery. Because most hydrogels have high water-absorbing abilities, drugs can be easily released in a short time without modification. Therefore, to slow the drug-release process and prolong drug action, different strategies have been adopted, either by modulating the interaction between the drug and hydrogel matrix or by modifying the gel network. Taking advantage of interactions between ionic polymers and charged loading agents, drugs such as naphazoline [31], NSAIDs [32], anesthetics [33], and amphotericin B [34] have been successfully conjugated to different kinds of hydrogel system for sustained drug release. Drugs can also be covalently linked to polymers, but the release process is initiated by chemical or enzymatic cleavage of the polymer–drug linkage. Daunomycin [35], dexamethasone [36], and paclitaxel [37] have been conjugated and released in this manner. In addition, drug loading and release can be modulated through interpenetrating polymer networks [38], surface modification of the hydrogels [39], or incorporation of secondary delivery vehicles, including microparticles, microgels, liposomes, and micelles [40, 41], into the gel system. On the basis of these strategies, aptamers have been used in hydrogel systems for drug loading and controlled release.

Aptamer-incorporated hydrogel for controllable drug release

Protein drugs are emerging as therapeutic agents, but limited routes for protein administration have constrained their wide application. In a series of studies by the Wang group [42], aptamers selected against platelet-derived growth factor BB(PDGF-BB) have been incorporated into hydrogels for controlled protein release. In their design, PDGF-BB aptamers were first coupled with an acrydite functional group at the 5′-end. When mixed with ammonium persulfate (APS), acrylamide, bis-acrylamide, and N,N, N′,N′-tetramethylethylenediamine, the acrydite-modified aptamer was randomly incorporated into a hydrogel network via the unsaturated double bond of acrydite, as verified by electrophoresis. Later, PDGF-BB and its aptamer were simultaneously added to the hydrogel at a molar ratio of 1:625 before gel aggregation. The aptamers held their target, resulting in a decrease in the rate of release of PDGF-BB from approximately 70% to 10% in the initial 24 h and to 6% in the next 120 h.

Slowing the rate of drug release is just the initial step in drug-delivery design. New design strategies must also take specific disease conditions into account. For example, in normal physiological circumstances, many hormones (e.g. insulin) are secreted from their mother cells in a pulsatile way. In disease conditions, however, those cells may die or lose function, after which hormone replacement therapy is required. To achieve the best outcome, the hormones must be triggered at a given time and delivered in a pulsatile manner. In another design from the Wang group [43], biotinylated PGDF-BB aptamers were immobilized in streptavidin-coated polystyrene particles (Fig. 3a, b). PDGF-BB was then added to bind the aptamers to form a protein–aptamer-particle complex. After mixing in an agarose solution, this complex was entrapped within the agarose gel with an efficiency of 93±2%. A complementary oligonucleotide (CO) was then used to break down the protein–aptamer interaction, and protein release ensued. Further data suggested that the complex could reduce the initial PDGF-BB burst from approximately 70% to 8%, with average daily release of 0.75% from day 2 to day 25. By adding the CO at two time points, the authors observed a pulsatile PDGF-BB release process with pulse release of approximately 20% and 10%, respectively (Fig. 3c). Even though the materials used in the experiment were not biocompatible or may have been toxic, these studies served as a good reference for future aptamer–hydrogel related investigations.

Fig. 3.

(a) Schematic illustration of the formation of hybrid particle– hydrogel composite; (b) complementary oligonucleotides (COs) trigger the composite release process; (c) pulsatile release of PDGF-BB from hydrogel composite after adding CO at the time points indicated by green arrows. S-CO, scrambled CO. Reproduced, with permission, from Ref.[43]

Poloxamer is a polymer with good biocompatibility which has been used for a wide range of drug-delivery vehicles, including insulin [44], interleukin-2 [45], lidocaine [46], and growth factors [47]. PDGF-BB aptamer-functionalized particles have also been incorporated into poloxamer hydrogels for in-situ injection [48]. Compared with the poloxamer hydrogel without aptamer-coated particles, release of PDGF-BB from aptamer-functionalized poloxamer hydrogel during the first day was reduced from 80% to 10%. Although aptamers with high binding ability may impede the protein-release process, the rate of protein diffusion can be modulated by use of aptamers with different binding constants.

Aptamer-incorporated hydrogel for targeted cancer therapy

Severe side effects of chemotherapy have necessitated the development of cancer therapy with high selectivity for cancer cells. Recently, aptamers with high binding affinity and specificity for their cell-surface targets have been selected by using cell-SELEX technology [49–52]. Cell-SELEX differs from the SELEX by using the whole intact cells as a target, and, as a result, detailed information about targets on the cell membrane is not needed. A set of probes can be simultaneously selected through this process, and, more importantly, the aptamers selected from this process can discriminate molecular-level differences between given types of cell. The molecular targets may be cell-surface markers with physicochemical traits that could be exploited for cell type-specific drug delivery. For example, the sgc-8 aptamer specific for CCRF-CEM cells with K_d of 0.80± 0.09 nmol L⁻¹ was selected by cell-SELEX [53]. Biomarker discovery revealed that this aptamer could recognize the protein tyrosine kinase 7 (PTK7), a transmembrane receptor highly expressed on CCRF-CEM cells [54]. The sgc-8 aptamer could also be internalized after binding to its target [55]. All these features make aptamer sgc8 a good candidate for targeted cancer therapy. After conjugation with the antitumor agent doxorubicin, the sgc8-Dox complex could be efficiently delivered into CCRF-CEM cells to initiate the killing process in vitro [16]. However, simply conjugating this cytotoxic drug with the aptamer may not be sufficient to prevent these toxic drugs from diffusing into normal tissue during their targeted delivery process in vivo. Thus, more safely transporting such drug complexes to the targeted cell without damaging adjacent normal cells in vivo remains a substantial challenge.

In a preliminary study, the Tan group developed a photoresponsive DNA-cross-linked hydrogel by grafting azobenzene-incorporated DNA into polymer to form a hydrogel which encapsulated different loads in the sol state [56]. The load-release process was designed to have the sol-to-gel transition reversibly regulated by visible and UV light. Even though this method achieved sustained drug release, the need for UV exposure as trigger has restrained its application. To address this question, core–shell nano-gels for drug delivery were developed using aptamers as the recognition unit and near-infrared light as a triggering stimulus [57]. In this system, shown in Fig. 4, acrylamide monomers and acrydite-modified oligonucleotides (DP-A) are first linked with a methacryl group on gold–silver-based nanorods (Au–Ag NRs) to form a multiple linear poly-acrylamide polymer conjugate. Aptamer-sgc8 complex is then conjugated to another DNA polyacrylamide chain (DP-B). A linker, complementary to both DP-A and DP-B, was designed to link these two parts to form a core–shell nanogel. Flow cytometry results indicated that core–shell nanogels bind CEM cells with affinity equal to that of sgc8 alone, but they do not bind control Ramos cells. Next, to fulfill the drug-vehicle function, Dox was incorporated to form a Dox–sgc8-NG complex, followed by incubation with the target cells. Although Dox has high toxicity to both CEM and Ramos cells, only 3±2% of CEM cells and 8±3% of Ramos cells were killed after incubation with Dox–sgc8-NG complexes. However, after irradiation with laser light, the drug release process increased CEM cell death from 3±2% to 67±5%, whereas only 3±2% and 10± 3% cell death was observed after incubation with Dox-loaded nanogel conjugated with random DNA (Dox–lib-NGs). This study takes advantage of the aptamer as a recognition element and the nanogel as a drug carrier, demonstrating that this is a promising system for targeted therapy.

Fig. 4.

(a) Schematic illustration of aptamer-functionalized core–shell nanogel; (b) DNA sequence of linker DNA and its linkage with DP-A and DP-B; (c) working principle of aptamer cross-linked nanogel for targeted therapy. Reproduced, with permission, from Ref. [57]

Conclusions and future directions

Advances in biochemistry and materials science have sparked interest in the design of new detection and drug-delivery techniques. Aptamers, which have unique properties such as high target-binding ability, low immunogenicity, and easy modification, are promising components of these systems. Integrating aptamers into hydrogel systems has many obvious advantages over traditional hydrogels. First, based on the base-pairing principle of complementary DNA sequences, aptamers, as a designer material for hydrogel fabrication, can be easily engineered and tuned. When incorporated into a hydrogel, these DNAs can add additional properties that can be manipulated to alter the properties of the hydrogel system. This is extremely important, because most drugs must be released in a dosage and time-controlled manner. Second, considering the availability of a vast number of aptamers, a large variety of targets can be used as triggering or binding agents, greatly expanding their applications. Third, aptamers are single-stranded DNA or RNA, and, as such, they have good biocompatibility, enabling their use in vivo. Given all these advantages, aptamer-incorporated hydrogel systems merit further development. Future efforts should focus on how to improve these systems and make them clinically applicable. Most polymers into which aptamers have been incorporated are to some extent toxic. Because many biocompatible polymers are available and have been validated in vivo, incorporation of aptamers into those biocompatible polymers in the production of hydrogels will lead to in-vivo applications. Emphasis should also be placed on development and optimization of the drug incorporation and release processes. Different drugs have different pharmacokinetics and must be administered on the basis of specific disease conditions. The drug-release mode and trigger factor must be diversified to meet these requirements. It can be foreseen that in the near future aptamer-based hydrogel systems will lead to new possibilities in the development of novel detection and drug-delivery systems. Although many challenges remain, the work thus far in optimizing the use of aptamers in hydrogel systems will act to promote further development in biomedical applications, including rapid detection, drug delivery, and tissue engineering.