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DNAzymes are DNA oligonucleotides that have catalytic activity without the assistance of protein enzymes. In particular, RNA-cleaving DNAzymes were considered as ideal candidates for gene therapy due to their unique characteristics. Nevertheless, efforts to use DNAzyme as a gene therapeutic agent are limited by issues such as their low physiological stability in serum and intracellular delivery efficiency. In this study, we developed a nanosized synthetic DNA hydrogel functionalized with a DNAzyme tetrahedron (TDz Dgel) to overcome these limitations. We observed remarkable improvement in the gene-silencing effect as well as intracellular uptake without the support of gene transfection reagents using TDz Dgel. The improved catalytic activity of the DNAzyme resulted from the combination of the cell-penetrating DNA tetrahedron structure and high stability of DNA hydrogel. We envision that this approach will become a convenient and efficient strategy for gene-silencing therapy using DNAzyme in the future.
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ADN Catalítico , Silenciador del Gen , ADN Catalítico/química , ADN Catalítico/genética , ADN Catalítico/metabolismo , Humanos , ADN/química , ADN/genética , Hidrogeles/química , Células HeLa , Transfección/métodosRESUMEN
Recent decades have experienced tough situations due to the lack of reliable diagnostic facilities. The most recent cases occurred during the pandemic, where researchers observed the lack of diagnostic facilities with precision. Microorganisms and viral disease's ability to escape diagnosis has been a global challenge. DNA always has been a unique moiety with a strong and precise base-paired structure. DNA in human and foreign particles makes identification possible through base pairing. Since then, researchers have focused heavily on designing diagnostic assays targeting DNA in particular. Moreover, DNA nanotechnology has contributed vastly to designing composite nanomaterials by combining DNA/nucleic acids with functional nanomaterials and inorganic nanoparticles exploiting their physicochemical properties. These nanomaterials often exhibit unique or enhanced properties due to the synergistic activity of the many components. The capabilities of DNA and additional nanomaterials have shown the combination of robust and advanced tailoring of biosensors. Preceding findings state that the conventional strategies have exhibited certain limitations such as a low range of target detection, less biodegradability, subordinate half-life, and high susceptibility to microenvironments; however, a DNA-nanomaterial-based biosensor has overcome these limitations meaningfully. Additionally, the unique properties of nucleic acids have been studied extensively due to their high signal conduction abilities. Here, we review recent studies on DNA-nanomaterial-based biosensors, their mechanism of action, and improved/updated strategies in vivo and in situ. Furthermore, this review highlights the recent methodologies on DNA utilization to exploit the interfacial properties of nanomaterials in DNA sensing. Lastly, the review concludes with the limitations/challenges and future directions.
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Nanopartículas , Nanoestructuras , Ácidos Nucleicos , Humanos , Nanoestructuras/química , Nanotecnología/métodos , ADN/químicaRESUMEN
Aptamers have been spotlighted as promising bio-recognition elements because they can be tailored to specific target molecules, bind to targets with a high affinity and specificity, and are easy to chemically synthesize and introduce functional groups to. In particular, fluorescent aptasensors are widely used in biological applications to diagnose diseases as well as prevent diseases by detecting cancer cells, viruses, and various biomarkers including nucleic acids and proteins as well as biotoxins and bacteria from food because they have the advantages of a high sensitivity, selectivity, rapidity, a simple detection process, and a low price. We introduce screening methods for isolating aptamers with q high specificity and summarize the sequences and affinities of the aptamers in a table. This review focuses on aptamer-based fluorescence detection sensors for biological applications, from fluorescent probes to mechanisms of action and signal amplification strategies.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Ácidos Nucleicos , Aptámeros de Nucleótidos/química , Colorantes Fluorescentes , Técnicas Biosensibles/métodos , BacteriasRESUMEN
miRNAs are endogenous small, non-coding RNA molecules that function in post-transcriptional regulation of gene expression. Because miRNA plays a pivotal role in maintaining the intracellular environment, and abnormal expression has been found in many cancer diseases, detection of miRNA as a biomarker is important for early diagnosis of disease and study of miRNA function. However, because miRNA is present in extremely low concentrations in cells and many types of miRNAs with similar sequences are mixed, traditional gene detection methods are not suitable for miRNA detection. Therefore, in order to overcome this limitation, a signal amplification process is essential for high sensitivity. In particular, enzyme-free signal amplification systems such as DNAzyme systems have been developed for miRNA analysis with high specificity. DNAzymes have the advantage of being more stable in the physiological environment than enzymes, easy to chemically synthesize, and biocompatible. In this review, we summarize and introduce the methods using DNAzyme-based biosensors, especially with regard to various signal amplification methods for high sensitivity and strategies for improving detection specificity. We also discuss the current challenges and trends of these DNAzyme-based biosensors.
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Nanotechnology and polymer engineering are navigating toward new developments to control and overcome complex problems. In the last few decades, polymer engineering has received researchers' attention and similarly, polymeric network-engineered structures have been vastly studied. Prior to therapeutic application, early and rapid detection analyses are critical. Therefore, developing hydrogel-based sensors to manage the acute expression of diseases and malignancies to devise therapeutic approaches demands advanced nanoengineering. However, nano-therapeutics have emerged as an alternative approach to tackling strenuous diseases. Similarly, sensing applications for multiple kinds of analytes in water-based environments and other media are gaining wide interest. It has also been observed that these functional roles can be used as alternative approaches to the detection of a wide range of biomolecules and pathogenic proteins. Moreover, hydrogels have emerged as a three-dimensional (3D) polymeric network that consists of hydrophilic natural or synthetic polymers with multidimensional dynamics. The resemblance of hydrogels to tissue structure makes them more unique to study inquisitively. Preceding studies have shown a vast spectrum of synthetic and natural polymer applications in the field of biotechnology and molecular diagnostics. This review explores recent studies on synthetic and natural polymers engineered hydrogel-based biosensors and their applications in multipurpose diagnostics and therapeutics. We review the latest studies on hydrogel-engineered biosensors, exclusively DNA-based and DNA hydrogel-fabricated biosensors.
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Cancer disease has outgrown a life-threatening disease. Having reference to preceding reports provided by the International Agency for Research on Cancer, an estimated 9.6 million deaths transpired from cancer worldwide in 2018. Similarly, about 18.1 million new cases of cancer are being reported. The rise in conventional treatments akin to surgeries, chemotherapies, and radiotherapies was enormously observed to eradicate cancer tumors. These studies have shown unfavorable side effects in clinical treatments. Drug resistivity and drug cytotoxicities are also major issues to overcome. Considering these, researchers are developing alternative methods that are robust, economical, and safe. The use of light for therapeutic purposes shows a great history in vitiligo treatment. The combination of an effective activating agent and phototherapy could result as the best alternative with a great outcome to minimize adverse effects on healthy tissues. The utilization of light in the deletion of tumors using photothermal agents, and photosensitizers, hence the phototherapies in oncology were discovered and rapidly involved in the advancement of clinical approach. Here, in this article, we tried to highlight the recent trends in phototherapy and reviewed different types of phototherapy methods in cancer treatments and their latest clinical, preclinical, and in vivo studies.
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Neoplasias , Fototerapia , Humanos , Neoplasias/terapia , Fármacos FotosensibilizantesRESUMEN
Cancer therapies based on chemotherapeutic drug delive ries have been the most facilitated studies. Recently, peptide drugs have emerged as anticancer drugs due to their less immunogenicity and lower production costs compared with other synthetics. However, still, the side effects of these chemotherapeutics on healthy tissues have been a great concern to deal with, and these side effects are usually caused by off-targeted delivery and unwanted leakage. In addition, peptides are easily degraded by enzyme attacks during delivery. To address these concerns, here, we developed a robust, cancer-specific peptide drug delivery system with negligible cytotoxicity in in vitro. A peptide drug delivery vehicle (Dgel-PD-AuNP-YNGRT) was constructed by stepwise functionalization on a nanoscale DNA hydrogel (Dgel). A cell-penetrating anticancer peptide drug, Buforin IIb, was loaded within the Dgel network via electrostatic attraction followed by AuNP assembly. The AuNPs were employed as photothermal reagents for light-triggered peptide drug release. An additional peptide, including a cancer-targeting YNGRT sequence, was also bound on the Dgel for cancer-cell-targeted delivery. According to the results obtained from the studies employing cancer cells as well as normal cells, Dgel-PD-AuNP-YNGRT nanocomplexes could be delivered specifically to cancer cells, activated by light illumination, and release anticancer peptide drugs to kill cancer cells with no cytotoxicity and negligible hazardous effect on normal cell lines. The obtained cell viability assay suggests that at a high intensity (15 W/cm2), photothermally triggered released peptide drug has shown up to 44% higher kill than only peptide drug treatments in cancer cells. Similarly, the Bradford assay demonstrated that up to 90% of peptide drugs were released with our engineered Dgel-PD-AuNP-YNGRT nanocomplex. The Dgel-PD-AuNP-YNGRT nanocomplex may serve as an ideal anticancer peptide drug delivery platform for safe, cancer-specific targeting and efficient peptide drug delivery in cancer therapy.
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Antineoplásicos , Nanopartículas del Metal , Neoplasias , Humanos , Liberación de Fármacos , Oro/química , Hidrogeles/química , Nanopartículas del Metal/química , Péptidos/química , Neoplasias/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , ADN/química , Línea Celular TumoralRESUMEN
In attempts to effectively improve RNAi function, we herein report a new RNAi approach using X-shaped RDNA and Dgel (RNA interfering DNA hydrogel, Ri-Dgel). X-shaped RDNA is a 4 branched nanostructure which was composed of three dsDNA branches and one dsRNA branch, and the structure was made by annealing partially complementary ssDNAs and chimeric RNA-DNA oligonucleotides. Ri-Dgel was synthesized through the ligation of the X-shaped RDNAs using their palindromic sticky ends. In MDCK/GFP cells transfected with 1 µM of each format of siRNA, Ri-Dgel and X-RDNA, the intensity of GFP fluorescence was significantly reduced by 65% and 56%, respectively, while dsRNA which is a conventional siRNA format showed a relatively weak reduction intensity of 7% compared with a negative control. We also observed the decreased intensity of GFP fluorescence by approximately 59% in MDA-MB-231/GFP cells transfected with 5 nM Ri-Dgel. Furthermore, the Ri-Dgel showed persistent RNAi efficiency up to 6 days from the treatment. The use of Ri-Dgel to trigger RNAi resulted in enhanced efficacy and longer duration at lower concentration compared to traditional dsRNA implying the nanostructured DNA-RNA hybrid materials have great potential as a platform technology for RNAi-based therapy.
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In the era of nanotechnology, the synthesis of nanomaterials for advanced applications has grown enormously. Effective therapeutics and functionalization of effective drugs using nano-vehicles are considered highly productive and selectively necessary. Polymeric nanomaterials have shown their impact and influential role in this process. Polymeric nanomaterials in molecular science are well facilitated due to their low cytotoxic behavior, robust functionalization, and practical approach towards in vitro and in vivo therapeutics. This review highlights a brief discussion on recent techniques used in nanohydrogel designs, biomedical applications, and the applied role of nanohydrogels in the construction of advanced therapeutics. We reviewed recent studies on nanohydrogels for their wide applications in building strategies for advantageously controlled biological applications. The classification of polymers is based on their sources of origin. Nanohydrogel studies are based on their polymeric types and their endorsed utilization for reported applications. Nanotechnology has developed significantly in the past decades. The novel and active role of nano biomaterials with amplified aspects are consistently being studied to minimize the deleterious practices and side effects. Here, we put forth challenges and discuss the outlook regarding the role of nanohydrogels, with future perspectives on delivering constructive strategies and overcoming the critical objectives in nanotherapeutic systems.
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Hidrogeles/química , Nanopartículas/química , Nanoestructuras/química , Polímeros/química , Materiales Biocompatibles/química , Sistemas de Liberación de Medicamentos/métodos , Humanos , Nanotecnología/métodosRESUMEN
Noble metals have always fascinated researchers due to their feasible and facile approach to plasmonics. Especially the extensive utilization of gold (Au) has been found in biomedical engineering, microelectronics, and catalysis. Surface plasmonic resonance (SPR) sensors are achievable by employing plasmonic nanoparticles. The past decades have seen colossal advancement in noble metal nanoparticle research. Surface plasmonic biosensors are advanced in terms of sensing accuracy and detection limit. Likewise, gold nanoparticles (AuNPs) have been widely used to develop distinct biosensors for molecular diagnosis. DNA nanotechnology facilitates advanced nanostructure having unique properties that contribute vastly to clinical therapeutics. The critical element for absolute control of materials at the nanoscale is the engineering of optical and plasmonic characteristics of the polymeric and metallic nanostructure. Correspondingly, AuNP's vivid intense color expressions are dependent on their size, shape, and compositions, which implies their strong influence on tuning the plasmonic properties. These plasmonic properties of AuNPs have vastly exerted the biosensing and molecular diagnosis applications without any hazardous effects. Here, we have designed nanoscale X-DNA-based Dgel scaffolds utilized for tuning the plasmonic properties of AuNPs. The DNA nanohydrogel (Dgel) scaffolds engineered with three different X-DNAs of distinct numbers of base pairs were applied. We have designed X-DNA base pair-controlled size-varied Dgel scaffolds and molar ratio-based nano assemblies to tune the plasmonic properties of AuNPs. The nanoscale DNA hydrogel's negatively charged scaffold facilitates quaternary ammonium ligand-modified positively charged AuNPs to flocculate around due to electrostatic charge attractions. Overall, our study demonstrates that by altering the DNA hydrogel scaffolds and the physical properties of the nanoscale hydrogel matrix, the SPR properties can be modulated. This approach could potentially benefit in monitoring diverse therapeutic biomolecules.
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Técnicas Biosensibles , Nanopartículas del Metal , Oro/química , Nanopartículas del Metal/química , Hidrogeles , ADN , Resonancia por Plasmón de SuperficieRESUMEN
A continuous-flow microchip enabling high-accuracy DNA analysis was developed. Serial consecutive analysis for multiple amplified DNA samples was demonstrated. The sample segments were continuously introduced to the microchip from the PCR device which was interfaced to the microchip through capillary tubing. Electrokinetic co-injection of the DNA samples with size marker enabled reproducible and reliable injection of the DNAs into the gel-filled separation channel providing accurate size determination of the DNA samples. Cross-contamination between serially introduced DNA samples was minimized by plugging a washing solution segment following the previous sample segment between two sample plugs. Using this microchip, continuous separation of multiple samples was performed without any inconvenient and labor-intensive sample preparation steps such as sample mixing, staining, and gel loading which are necessary for conventional gel electrophoresis. It has taken about 4 min to separate single DNA sample and taken 37 min for three serially injected samples which implies that this microchip can be a platform device for fast as well as highly accurate DNA analysis.
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ADN/análisis , Electroforesis por Microchip/métodos , Diseño de Equipo , Límite de Detección , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Stimuli-responsive nanoparticles are favorable for improving the selective delivery and rational vocation that easily avoids the undesirable barriers or side effects, leading to a further improved therapeutic efficiency. Furthermore, multifunctional nanomaterials have been extensively developed as attractive candidates for theranostic reagents for cancer treatment. In this article, we developed reversibly pH-responsive gold nanoparticles (AuNPs) with an enhanced Raman scattering signal as well as an efficient photothermal effect and demonstrated their applications as a theranostic reagent for cancer treatment. Surfaces of these AuNPs were modified with mixed layers of Cy3-modified single-stranded DNA (ssDNA-Cy3) for Raman probing and a negative charge supply and cytochrome C (Cyt C) for pH-responsive charge inversion. This combination of pH-responsive ligands and Raman probes played an important role in inducing the assembly or disassembly of AuNPs corresponding to the neighboring pH, accompanied by an additional highly distinguished Raman signal intensity. An operative reversible response of the AuNPs to pH is endowed with the characteristic behavior of AuNPs with the cancerous cell's acidic microenvironment of low pH. The responsive aggregation of AuNPs in a lower pH medium provides highly amplified signals attributed to well-formed hot spots between the particle surfaces that deliver better Raman scattering signals. The acidic pH-responsive aggregation of the particles also provided efficient photothermal treatments using a long-wavelength laser light with the benefit of deeper penetration for cancer cells. In vitro experiments employing cancer cells and control normal cells well-demonstrated the specificity of the particles to cancer cells in terms of highly enhanced Raman imaging and therapeutic efficiency.
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Nanopartículas del Metal , Neoplasias , Oro/farmacología , Concentración de Iones de Hidrógeno , Nanopartículas del Metal/uso terapéutico , Neoplasias/diagnóstico por imagen , Espectrometría Raman/métodos , Microambiente TumoralRESUMEN
Microenvironment responsive nanomaterials are attractive for therapeutic applications with regional specificity. Here we report pH responsive gold nanoparticles which are designed to aggregate in acidic condition similar to cancer environment and returned to its original disassembled states in a physiological pH. The pH responsive behavior of the particles is derived by change of electrostatic interaction among the particles where attraction and repulsion play a major role in low and high pH of the environment, respectively. Since different electrostatic interaction behavior of the particles in varied pH is induced not by irreversible chemical change but by simple protonation differences, the pH responsive process of assembly and disassembly is totally reversible. The low pH specific aggregation of gold nanoparticles resulted in red shift of plasmonic absorption peak and showed higher photothermal efficacy in acidic pH than in normal physiological pH. The low pH specific photothermal effect with long wave laser irradiation was directly applied to cancer specific photothermal therapy and resulted higher therapeutic effect for melanoma cancer cells than non-pH responsive gold nanoparticles.
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Oro/química , Concentración de Iones de Hidrógeno , Hipertermia Inducida/métodos , Nanopartículas del Metal/química , Neoplasias/terapia , Fototerapia/métodos , Humanos , Neoplasias/patología , Microambiente TumoralRESUMEN
RNA interference (RNAi) is a mechanism in which small interfering RNA (siRNA) silences a target gene. Herein, we describe a DNA hydrogel capable of producing siRNA and interfering with protein expression. This RNAi-exhibiting gel (termed I-gel for interfering gel) consists of a plasmid carrying the gene transcribing siRNA against the target mRNA as part of the gel scaffold. The RNAi efficiency of the I-gel has been confirmed by green fluorescent protein (GFP) expression assay and RNA production quantification. The plasmid stability in the I-gel results in an 8-times higher transcription efficiency than that of the free plasmid. We further applied the I-gel to live cells and confirmed its effect in interfering with the GFP expression. The I-gel shows higher RNAi effect than plasmids in free form or complexed with Lipofectamine. This nanoscale hydrogel, which is able to produce RNA in a cell, provides a platform technology for efficient RNAi system.
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ADN/química , Hidrogeles/química , Interferencia de ARN , ARN/metabolismo , Animales , Perros , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , ARN Interferente PequeñoRESUMEN
We present a new class of electrically conductive, mechanically moldable, and thermally self-healable hybrid hydrogels. The hybrid gels consist of polypyrrole and agarose as the conductive component and self-healable matrix, respectively. By using the appropriate oxidizing agent under conditions of mild temperature, the polymerization of pyrrole occurred along the three-dimensional network of the agarose hydrogel matrix. In contrast to most commercially available hydrogels, the physical crosslinking of agarose gel allows for reversible gelation in the case of our hybrid gel, which could be manipulated by temperature variation, which controls the electrical on/off behavior of the hybrid gel electrode. Exploiting this property, we fabricated a hybrid conductive hydrogel electrode which also self-heals thermally. The novel composite material we report here will be useful for many technological and biological applications, especially in reactive biomimetic functions and devices, artificial muscles, smart membranes, smart full organic batteries, and artificial chemical synapses.
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Hidrogeles/química , Hidrogeles/síntesis química , Polímeros/química , Pirroles/química , Sefarosa/química , ElectrodosRESUMEN
A parallel-processing four-station polymerase chain reaction (PCR) device has been developed, which performs continuous-flow PCR without optimization of the annealing temperature. Since the annealing temperature of each station can be controlled independently, the device covers an annealing temperature range of 50-68 °C, which is wide enough to perform PCR for any DNA fragment regardless of its optimum annealing condition. This arrangement lets us continuously obtain an amplified amount of a DNA fragment at least from one of the stations. The device consists of four identical cylindrical stations (diameter 20 mm, height 55 mm). A polytetrafluoroethylene capillary reactor (length 2 m, I.D. 100 µm, O.D. 400 µm) is wound helically up around each station. The whole assembly is designed to minimize the number of heating blocks (for providing temperatures of denaturation, annealing, and extension) to be seven and to shape a compact cube (height 55 mm, base 60 mm × 60 mm). The reproducibility for continuous-flow PCR is reasonably high (run-to-run and station-to-station relative standard deviation of their amplification is lower than 6 % and about 4 %, respectively). Performance on the optimization-free DNA amplification has been evaluated with four DNA samples with different annealing conditions and product sizes (323, 608, 828, and 1101 bp), which has demonstrated that in all cases, PCR is successful at least on one station. In addition, three DNA fragments with different lengths (323, 1101, and 2836 bp) have been successfully amplified in a segmented-flow mode without the carry-over contamination between segments. This result suggests that this device could serve as the PCR module of a continuous-flow high-throughput on-line total DNA analysis system integrating all necessary modules from cell lysis/DNA extraction to PCR product analysis.
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ADN/genética , Reacción en Cadena de la Polimerasa/instrumentación , Diseño de Equipo , Ensayos Analíticos de Alto Rendimiento/economía , Ensayos Analíticos de Alto Rendimiento/instrumentación , Técnicas Analíticas Microfluídicas/economía , Técnicas Analíticas Microfluídicas/instrumentación , Reacción en Cadena de la Polimerasa/economía , TemperaturaRESUMEN
Microfluidic devices have been extensively developed as methods for microscale materials fabrication. It has also been adopted for polymeric microsphere fabrication and in situ drug encapsulation. Here, we employed multi-inlet microfluidic channels for DNA hydrogel microsphere formation and in situ protein encapsulation. The release of encapsulated proteins from DNA hydrogels showed different profiles accordingly with the size of microspheres.
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A DNA hydrogel is reported as a delivery vehicle for gold nanorods and doxorubicin. The two photothermal and chemo cancer agents were co-loaded using electrostatic and DNA binding interactions, respectively. Light-triggered and highly synergistic combination cancer therapy was demonstrated in cellular and animal models.
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ADN/química , Portadores de Fármacos/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Oro/química , Humanos , Luz , Ratones , Nanotubos/química , Nanotubos/ultraestructura , Electricidad EstáticaRESUMEN
Assembled AuNPs in a DNA hydrogel (Dgel) showed strongly coupled plasmon modes, and the Dgel vehicle can co-load anticancer drugs such as doxorubicin (Dox) as a light-controlled releasing cargo by DNA intercalations. Upon laser excitation, local heat shock generation was accompanied by the release of Dox. A highly synergistic combination of thermo- and chemotherapy was demonstrated in cellular and animal models. Our Dgel vehicle can be fragmented after the excitation-induced heat generations, which subsequently causes the dispersion of the AuNPs. Our system may be less toxic because it uses small sizes of AuNPs, and the inherently biocompatible scaffold may reduce the long-term toxicity by rapid clearance.
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Conductive hydrogels are a class of composite materials that consist of hydrated and conducting polymers. Due to the mechanical similarity to biointerfaces such as human skin, conductive hydrogels have been primarily utilized as bioelectrodes, specifically neuroprosthetic electrodes, in an attempt to replace metallic electrodes by enhancing the mechanical properties and long-term stability of the electrodes within living organisms. Here, we report a conductive, smart hydrogel, which is thermoplastic and self-healing owing to its unique properties of reversible liquefaction and gelation in response to thermal stimuli. In addition, we demonstrated that our conductive hydrogel could be utilized to fabricate bendable, stretchable, and patternable electrodes directly on human skin. The excellent mechanical and thermal properties of our hydrogel make it potentially useful in a variety of biomedical applications such as electronic skin.