Engineering and Delivery of cGAS-STING Immunomodulators for the Immunotherapy of Cancer and Autoimmune Diseases.

The cyclic GMP-AMP synthase-stimulator interferon gene (cGAS-STING) pathway is an emerging therapeutic target for the prophylaxis and therapy of a variety of diseases, ranging from cancer, infectious diseases, to autoimmune disorders. As a cytosolic double stranded DNA (dsDNA) sensor, cGAS can bind with relatively long dsDNA, resulting in conformational change and activation of cGAS. Activated cGAS catalyzes the conversion of adenosine triphosphate (ATP) and guanosine triphosphate (GTP) into cGAMP, a cyclic dinucleotide (CDN). CDNs, including 2'3'-cGAMP, stimulate adapter protein STING on the endoplasmic membrane, triggering interferon regulatory factor 3 (IRF3) phosphorylation and nuclear factor kappa B (NF-κB) activation. This results in antitumor and antiviral type I interferon (IFN-I) responses. Moreover, cGAS-STING overactivation and the resulting IFN-I responses have been associated with a number of inflammatory and autoimmune diseases. This makes cGAS-STING appealing immunomodulatory targets for the prophylaxis and therapy of various related diseases. However, drug development of CDNs and CDN derivatives is challenged by their limited biostability, difficult formulation, poor pharmacokinetics, and inefficient tissue accumulation and cytosolic delivery. Though recent synthetic small molecular CDN- or non-CDN-based STING agonists have been reported with promising preclinical therapeutic efficacy, their therapeutic efficacy and safety remain to be fully evaluated preclinically and clinically. Therefore, it is highly desirable and clinically significant to advance drug development for cGAS-STING activation by innovative approaches, such as drug delivery systems and drug development for pharmacological immunomodulation of cGAS. In this Account, we summarize our recent research in the engineering and delivery of immunostimulatory or immunoregulatory modulators for cGAS and STING for the immunotherapy of cancer and autoimmune diseases. To improve the delivery efficiency of CDNs, we developed ionizable and pH-responsive polymeric nanocarriers to load STING agonists, aiming to improve the cellular uptake and facilitate the endosomal escape to induce efficient STING activation. We also codelivered STING agonists with complementary immunostimulatants in nanoparticle-in-hydrogel composites to synergetically elicit potent innate and adaptive antitumor responses that eradicate local and distant large tumors. Further, taking advantage of the simplicity of manufacturing and the established nucleic acid delivery system, we developed oligonucleotide-based cGAS agonists as immunostimulant immunotherapeutics as well as adjuvants for peptide antigens for cancer immunotherapy. To suppress the overly strong proinflammatory responses associated with cGAS-STING overactivation in some of the autoimmune disorders, we devised nanomedicine-in-hydrogel (NiH) that codelivers a cGAS inhibitor and cell-free DNA (cfDNA)-scavenging cationic nanoparticles (cNPs) for systemic immunosuppression in rheumatoid arthritis (RA) therapy. Lastly, we discussed current drug development by targeting cGAS-STING for cancer, infectious diseases, and autoimmune diseases, as well as the potential opportunities for utilizing cGAS-STING pathway for versatile applications in disease treatment.

Polymeric micelles amplify tumor oxidative stresses through combining PDT and glutathione depletion for synergistic cancer chemotherapy.

Cancer has been one of the major healthcare burdens, which demands innovative therapeutic strategies to improve the treatment outcomes. Combination therapy hold great potential to leverage multiple synergistic pathways to improve cancer treatment. Cancer cells often exhibit an increased generation of reactive oxygen species (ROS) and antioxidant species compared with normal cells, and the levels of these species can be further elevated by common therapeutic modalities such as photodynamic therapy (PDT) or chemotherapy. Taking advantage that cancer cells are vulnerable to further oxidative stress, we aim to design a drug delivery system by simultaneously increasing the cellular ROS level, reducing antioxidative capacity, and inducing anticancer chemotherapy in cancer cells. Here, we designed a star-shape polymer, PEG(-b-PCL-Ce6)-b-PBEMA, based on the Passerini three-component reaction, which can both enhance ROS generation during PDT and decrease the GSH level in cancer cells. The polycaprolactone conjugated with photosensitizer Ce6 served as hydrophobic segments to promote micelle formation, and Ce6 was used for PDT. The H2O2-labile group of arylboronic esters pendent on the third segment was designed for H2O2-induced quinone methide (QM) release for GSH depletion. We thoroughly investigated the spectral properties of blank micelle during its assembling process, ROS generation, and H2O2-induced QM release in vitro. Moreover, this polymeric micelle could successfully load hydrophobic anticancer drug Doxorubicin (DOX) and efficiently deliver DOX into cancer cells. The triple combination of ROS generation, GSH elimination, and chemotherapy dramatically improved antitumor efficiency relative to each of them alone in vitro and in vivo.

Hybrid Nanomedicine Fabricated from Photosensitizer-Terminated Metal-Organic Framework Nanoparticles for Photodynamic Therapy and Hypoxia-Activated Cascade Chemotherapy.

During photodynamic therapy (PDT), severe hypoxia often occurs as an undesirable limitation of PDT owing to the O2 -consuming photodynamic process, compromising the effectiveness of PDT. To overcome this problem, several strategies aiming to improve tumor oxygenation are developed. Unlike these traditional approaches, an opposite method combining hypoxia-activated prodrug and PDT may provide a promising strategy for cancer synergistic therapy. In light of this, azido-/photosensitizer-terminated UiO-66 nanoscale metal-organic frameworks (UiO-66-H/N3 NMOFs) which serve as nanocarriers for the bioreductive prodrug banoxantrone (AQ4N) are engineered. Owing to the effective shielding of the nanoparticles, the stability of AQ4N is well preserved, highlighting the vital function of the nanocarriers. By virtue of strain-promoted azide-alkyne cycloaddition, the nanocarriers are further decorated with a dense PEG layer to enhance their dispersion in the physiological environment and improve their therapeutic performance. Both in vitro and in vivo studies reveal that the O2 -depleting PDT process indeed aggravates intracellular/tumor hypoxia that activates the cytotoxicity of AQ4N through a cascade process, consequently achieving PDT-induced and hypoxia-activated synergistic therapy. Benefiting from the localized therapeutic effect of PDT and hypoxia-activated cytotoxicity of AQ4N, this hybrid nanomedicine exhibits enhanced therapeutic efficacy with negligible systemic toxicity, making it a promising candidate for cancer therapy.

An Albumin Sandwich Enhances in Vivo Circulation and Stability of Metabolically Labile Peptides.

The effectiveness of numerous molecular drugs is hampered by their poor pharmacokinetics. Different from previous approaches with limited effectiveness, most recently, emerging high-affinity albumin binding moieties (ABMs) for in vivo hitchhiking of endogenous albumin opens up an avenue to chaperone small molecules for long-acting therapeutics. Although several FDA-approved fatty acids have shown prolonged residence and therapeutic effect, an easily synthesized, water-soluble, and high-efficiency ABM with versatile drug loading ability is urgently needed to improve the therapeutic efficacy of short-lived constructs. We herein identified an ideal bivalent Evans blue derivative, denoted as N(tEB)2, as a smart ABM-delivery platform to chaperone short-lived molecules, through both computational modeling screening and efficient synthetic schemes. The optimal N(tEB)2 could reversibly link two molecules of albumin through its two binding heads with a preferable spacer, resulting in significantly extended circulation half-life of a preloaded cargo and water-soluble. Notably, this in situ dimerization of albumin was able to sandwich peptide therapeutics to protect them from proteolysis. As an application, we conjugated N(tEB)2 with exendin-4 for long-acting glucose control in a diabetic mouse model, and it was superior to both previously tested NtEB-exendin-4 (Abextide) and the newly FDA-approved semaglutide, which has been arguably the best commercial weekly formula so far. Hence, this novel albumin binder has excellent clinical potential for next-generation biomimetic drug delivery systems.

PET imaging of EGFR expression using an 18F-labeled RNA aptamer.

OBJECTIVE: Epidermal growth factor receptor (EGFR) is a theranostic biomarker for a variety of cancer types. The aim of the present study was to develop an 18F radiolabeled EGFR targeting RNA aptamer, and to investigate its ability to visualize and quantify EGFR in xenograft models. METHODS: Biolayer interferometry binding assay was used to detect the binding affinity of the alkyne-modified EGFR aptamer MinE07 (denoted as ME07) with recombinant human wild-type EGFR protein and the mutant EGFRvIII protein. Cy5-conjugated ME07 was used for flow cytometry and immunofluorescence staining, and an Alexa Fluor 488-labeled EGFR antibody (ab193244) was used as a control. 18F-Fluorobenzoyl (FB) azide was employed as a synthon to produce 18F-FB-ME07 via click chemistry, and the cellular uptake and internalization characteristics of 18F-FB-ME07 were investigated. Static PET scans, 60-min dynamic scans, and biodistribution study of 18F-FB-ME07 were performed in three types of tumor models. RESULTS: The Kd values of ME07 to wtEGFR and EGFRvIII proteins were 0.3 nM and 271 nM respectively. The A431, U87MG, and HCT-116 cells showed strong, weak, and negative binding with Cy5-ME07, which is consistent with EGFR expression level in these cells. Peak cell uptake values of 18F-FB-ME07 in A431, U87MG and HCT-116 cells were 2.86%, 2.19% and 0.88% of the added dose respectively. The mean internalization of 18F-FB-ME07 in these cells were 60.02%, 53.1%, and 52.8% of the total accumulated radioactivity. In static PET imaging, despite high uptake in the liver and kidneys, 18F-FB-ME07 showed reasonable accumulation in A431 tumors (1.02 ± 0.13 %ID/g at 30 min after injection). Of note, the uptake of 18F-FB-ME07 in A431 xenografts was significantly higher than that in U87MG and HCT-116 xenografts. In A431 xenografted mice, the tumor/blood ratio was 3.89 and the tumor/muscle ratio reached 8.65. CONCLUSIONS: We for the first time generated an aptamer-derived EGFR targeting PET tracer 18F-FB-ME07, which showed highly selective targeting ability in mouse tumor models expressing different levels of EGFR. Our results suggest that 18F-FB-ME07 is a potential EGFR targeting molecular imaging probe for future clinical translation.

Positron Emission Tomography Imaging of Prostate Cancer with Ga-68-Labeled Gastrin-Releasing Peptide Receptor Agonist BBN7-14 and Antagonist RM26.

Radiolabeled bombesin (BBN) analogs have long been used for developing gastrin-releasing peptide receptor (GRPR) targeted imaging probes, and tracers with excellent in vivo performance including high tumor uptake, high contrast, and favorable pharmacokinetics are highly desired. In this study, we compared the 68Ga-labeled GRPR agonist (Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2, BBN7-14) and antagonist (d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, RM26) for the positron emission tomography (PET) imaging of prostate cancer. The in vitro stabilities, receptor binding, cell uptake, internalization, and efflux properties of the probes 68Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-Aca-BBN7-14 and 68Ga-NOTA-poly(ethylene glycol)3 (PEG3)-RM26 were studied in PC-3 cells, and the in vivo GRPR targeting abilities and kinetics were investigated using PC-3 tumor xenografted mice. BBN7-14, PEG3-RM26, NOTA-Aca-BBN7-14, and NOTA-PEG3-RM26 showed similar binding affinity to GRPR. In PC-3 tumor-bearing mice, the tumor uptake of 68Ga-NOTA-PEG3-RM26 remained at around 3.00 percentage of injected dose per gram of tissue within 1 h after injection, in contrast with 68Ga-NOTA-Aca-BBN7-14, which demonstrated rapid elimination and high background signal. Additionally, the majority of the 68Ga-NOTA-PEG3-RM26 remained intact in mouse serum at 5 min after injection, while almost all of the 68Ga-NOTA-Aca-BBN7-14 was degraded under the same conditions, demonstrating more-favorable in vivo pharmacokinetic properties and metabolic stabilities of the antagonist probe relative to its agonist counterpart. Overall, the antagonistic GRPR targeted probe 68Ga-NOTA-PEG3-RM26 is a more-promising candidate than the agonist 68Ga-NOTA-Aca-BBN7-14 for the PET imaging of prostate cancer patients.

Correction to: PET imaging of EGFR expression using an 18F-labeled RNA aptamer.

The original version of this article contained a mistake in the first paragraph of "Cell uptake and internalization of 18F-FB-ME07" section. The text "The specific activity was 7.4-14.8 kBq/nmol" should have been "The specific activity was 7.4-14.8 Mbq/nmol".

Polymeric Nanoparticles with a Glutathione-Sensitive Heterodimeric Multifunctional Prodrug for In Vivo Drug Monitoring and Synergistic Cancer Therapy.

Polymeric micelle-based drug delivery systems have dramatically improved the delivery of small molecular drugs, yet multiple challenges remain to be overcome. A polymeric nanomedicine has now been engineered that possesses an ultrahigh loading (59 %) of a glutathione (GSH)-sensitive heterodimeric multifunctional prodrug (HDMP) to effectively co-deliver two synergistic drugs to tumors. An HDMP comprising of chemotherapeutic camptothecin (CPT) and photosensitizer 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-α (HPPH) was conjugated via a GSH-cleavable linkage. The intrinsic fluorogenicity and label-free radio-chelation (64 Cu) of HPPH enabled direct drug monitoring by fluorescence imaging and positron emission tomography (PET). Through quantitative PET imaging, HDMP significantly improves drug delivery to tumors. The high synergistic therapeutic efficacy of HDMP-loaded NPs highlights the rational design of HDMP, and presents exciting opportunities for polymer NP-based drug delivery.

Combinatorial Screening of DNA Aptamers for Molecular Imaging of HER2 in Cancer.

HER2, a cell membrane protein overexpressed in many types of cancers, is correlated with poor diagnosis, suboptimal treatment outcome, and low survival rate. Multiple HER2-targeted drugs have been developed for the treatment of HER2-overexpressing tumor, which can in turn down-regulate HER2 expression. It is thus significant to profile HER2 expression for cancer prognosis, patient stratification, and monitoring therapy response. Aptamers, a class of single-stranded DNA/RNA (ssDNA/ssRNA) ligands, are promising for molecular biomarker imaging. Aptamers typically have strong binding affinity, high selectivity, batch-to-batch reproducibility, and low toxicity, and systemically injected aptamers often have high tumor-to-background ratio within a short time. However, current aptamers have been mostly screened in vitro, and these aptamers may lose binding ability in vivo due to conformational change under physiological environments. Here, a DNA library was combinatorially screened in vitro and in vivo, to select HER2-targeting DNA aptamers, termed Heraptamers, and labeled with 18F for positron emission tomography (PET) imaging of HER2 in ovarian cancer. Specifically, using systematic evolution of ligands by exponential enrichment (SELEX), Heraptamer candidates were first selected and validated in vitro using HER2 extracellular domain (ECD) and HER2-positive SKOV3 cancer cells; then, aptamer candidates were modified with alkyne, radiolabeled with 18F using azide-functionalized precursors by click chemistry, and screened in SKOV3-tumor-bearing mice using PET. Two aptamers, Heraptamer1 and Heraptamer2, reached high tumor uptake ratios within as short as 1 h. At 1.5 h post injection, the tumor uptake ratio of these two aptamers was up to 0.5%ID/g (injection dose/gram tissue), with tumor-to-muscle ratio of 4.55 ± 1.63 in SKOV3 tumor. In contrast, these aptamers have low uptake ratios in control MDA-MB-231 tumors. These preclinical studies showed that Heraptamers are promising for specific HER2 imaging.

In vitro selection with artificial expanded genetic information systems.

Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson-Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS-SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS-SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS-SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS-SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.

Double-Layered Plasmonic-Magnetic Vesicles by Self-Assembly of Janus Amphiphilic Gold-Iron(II,III) Oxide Nanoparticles.

Janus nanoparticles (JNPs) offer unique features, including the precisely controlled distribution of compositions, surface charges, dipole moments, modular and combined functionalities, which enable excellent applications that are unavailable to their symmetrical counterparts. Assemblies of NPs exhibit coupled optical, electronic and magnetic properties that are different from single NPs. Herein, we report a new class of double-layered plasmonic-magnetic vesicle assembled from Janus amphiphilic Au-Fe3 O4 NPs grafted with polymer brushes of different hydrophilicity on Au and Fe3 O4 surfaces separately. Like liposomes, the vesicle shell is composed of two layers of Au-Fe3 O4 NPs in opposite direction, and the orientation of Au or Fe3 O4 in the shell can be well controlled by exploiting the amphiphilic property of the two types of polymers.

Assembling a bi-coordinated Cr complex for ferromagnetic nanorings: insight from first-principles calculations.

Motivated by the recent synthesis of bi-coordinated transition metal-organic complexes [Samuel, et al., Chem. Sci., 2015, 6, 3148], we have studied the structure and magnetic properties of a series of bi-coordinated transition metal based nanorings by folding quasi-1D chains. Among the cyclic alkyl(amino)carbine (CAAC) based quasi-1D chains (TM-CAAC, TM = Cr, Mn, Fe, Co, Ni), only Cr-CAAC is found to be ferromagnetic. First-principles calculations combined with Heisenberg-Dirac-van Vleck models were performed to understand the onset of robust ferromagnetism in Cr-based systems. With increasing size, the infrared intensity increases and the exchange energy oscillates. In particular, when the number, n, of TM-CAAC units is even and larger than 3, the magnetic coupling in nanorings is stronger than that in quasi-1D chains. The band gap changes very slowly with size. More importantly, compared with the highly coordinated Cr single molecular magnets, the low coordination of Cr ions enhances magnetic moment and stabilizes ferromagnetic coupling.

Self-assembled, aptamer-tethered DNA nanotrains for targeted transport of molecular drugs in cancer theranostics.

Nanotechnology has allowed the construction of various nanostructures for applications, including biomedicine. However, a simple target-specific, economical, and biocompatible drug delivery platform with high maximum tolerated doses is still in demand. Here, we report aptamer-tethered DNA nanotrains (aptNTrs) as carriers for targeted drug transport in cancer therapy. Long aptNTrs were self-assembled from only two short DNA upon initiation by modified aptamers, which worked like locomotives guiding nanotrains toward target cancer cells. Meanwhile, tandem "boxcars" served as carriers with high payload capacity of drugs that were transported to target cells and induced selective cytotoxicity. aptNTrs enhanced maximum tolerated dose in nontarget cells. Potent antitumor efficacy and reduced side effects of drugs delivered by biocompatible aptNTrs were demonstrated in a mouse xenograft tumor model. Moreover, fluorophores on nanotrains and drug fluorescence dequenching upon release allowed intracellular signaling of nanotrains and drugs. These results make aptNTrs a promising targeted drug transport platform for cancer theranostics.

A cell-targeted, size-photocontrollable, nuclear-uptake nanodrug delivery system for drug-resistant cancer therapy.

The development of multidrug resistance (MDR) has become an increasingly serious problem in cancer therapy. The cell-membrane overexpression of P-glycoprotein (P-gp), which can actively efflux various anticancer drugs from the cell, is a major mechanism of MDR. Nuclear-uptake nanodrug delivery systems, which enable intranuclear release of anticancer drugs, are expected to address this challenge by bypassing P-gp. However, before entering the nucleus, the nanocarrier must pass through the cell membrane, necessitating coordination between intracellular and intranuclear delivery. To accommodate this requirement, we have used DNA self-assembly to develop a nuclear-uptake nanodrug system carried by a cell-targeted near-infrared (NIR)-responsive nanotruck for drug-resistant cancer therapy. Via DNA hybridization, small drug-loaded gold nanoparticles (termed nanodrugs) can self-assemble onto the side face of a silver-gold nanorod (NR, termed nanotruck) whose end faces were modified with a cell type-specific internalizing aptamer. By using this size-photocontrollable nanodrug delivery system, anticancer drugs can be efficiently accumulated in the nuclei to effectively kill the cancer cells.

Programmable and multiparameter DNA-based logic platform for cancer recognition and targeted therapy.

The specific inventory of molecules on diseased cell surfaces (e.g., cancer cells) provides clinicians an opportunity for accurate diagnosis and intervention. With the discovery of panels of cancer markers, carrying out analyses of multiple cell-surface markers is conceivable. As a trial to accomplish this, we have recently designed a DNA-based device that is capable of performing autonomous logic-based analysis of two or three cancer cell-surface markers. Combining the specific target-recognition properties of DNA aptamers with toehold-mediated strand displacement reactions, multicellular marker-based cancer analysis can be realized based on modular AND, OR, and NOT Boolean logic gates. Specifically, we report here a general approach for assembling these modular logic gates to execute programmable and higher-order profiling of multiple coexisting cell-surface markers, including several found on cancer cells, with the capacity to report a diagnostic signal and/or deliver targeted photodynamic therapy. The success of this strategy demonstrates the potential of DNA nanotechnology in facilitating targeted disease diagnosis and effective therapy.

Evolution of functional six-nucleotide DNA.

Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add functional groups not found in natural DNA, but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-ß-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-ß-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These were added to a library of oligonucleotides used in a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.

DLISA: A DNAzyme-Based ELISA for Protein Enzyme-Free Immunoassay of Multiple Analytes.

A DNAzyme-based ELISA, termed DLISA, was developed as a novel protein enzyme-free, triply amplified platform, combining a catalytic and molecular beacon (CAMB) system with a cation exchange reaction for ultrasensitive multiplex fluorescent immunosorbent assay. Classical ELISA, which employs protein enzymes as biocatalysts to afford amplified signals, suffers from poor stability caused by the irreversible denaturation of these enzymes under harsh conditions, such as heat and acidity. Compared with proteins, nucleic acids are more stable and adaptable, and they can be easily produced using a commercial DNA synthesizer. Moreover, the catalytic and cleavage activities of DNAzyme can be achieved in solution; thus, no enzyme immobilization is needed for detection. Taken together, these attributes suggest that a DNAzyme-based ELISA detection approach will be more robust than current ELISA assays. Importantly, the proposed triply amplified DLISA immunoassay method shows ultrasensitive detection of such targets as human IgG with a detection limit of 2 fg/mL (3 × 10(-17) M), which is well within the range of many important disease biomarkers. DLISA can also be used to construct a sensing array for simultaneous multiplexed detection. With these merits, this high-throughput, stable, simple, sensitive, and low-cost multiplex fluorescence immunoassay shows promise for applications in clinical diagnosis.

Aptamer-Drug Conjugates.

Western medicine often aims to specifically treat diseased tissues or organs. However, the majority of current therapeutics failed to do so owing to their limited selectivity and the consequent "off-target" side effects. Targeted therapy aims to enhance the selectivity of therapeutic effects and reduce adverse side effects. One approach toward this goal is to utilize disease-specific ligands to guide the delivery of less-specific therapeutics, such that the therapeutic effects can be guided specifically to diseased tissues or organs. Among these ligands, aptamers, also known as chemical antibodies, have emerged over the past decades as a novel class of targeting ligands that are capable of specific binding to disease biomarkers. Compared with other types of targeting ligands, aptamers have an array of unique advantageous features, which make them promising for developing aptamer-drug conjugates (ApDCs) for targeted therapy. In this Review, we will discuss ApDCs for targeted drug delivery in chemotherapy, gene therapy, immunotherapy, photodynamic therapy, and photothermal therapy, primarily of cancer.

Automated modular synthesis of aptamer-drug conjugates for targeted drug delivery.

Aptamer-drug conjugates (ApDCs) are promising targeted drug delivery systems for reducing toxicity while increasing the efficacy of chemotherapy. However, current ApDC technologies suffer from problems caused by the complicated preparation and low controllability of drug-aptamer conjugation. To solve such problems, we have designed and synthesized a therapeutic module for solid phase synthesis, which is a phosphoramdite containing an anticancer drug moiety and a photocleavable linker. Using this module, we have realized automated and modular synthesis of ApDCs, and multiple drugs were efficiently incorporated into ApDCs at predesigned positions. The ApDCs not only recognize target cancer cells specifically, but also release drugs in a photocontrollable manner. We demonstrated the potential of automated and modular ApDC technology for applications in targeted cancer therapy.

Cell membrane-anchored biosensors for real-time monitoring of the cellular microenvironment.

Cell membrane-anchored biochemical sensors that allow real-time monitoring of the interactions of cells with their microenvironment would be powerful tools for studying the mechanisms underlying various biological processes, such as cell metabolism and signaling. Despite the significance of these techniques, unfortunately, their development has lagged far behind due to the lack of a desirable membrane engineering method. Here, we propose a simple, efficient, biocompatible, and universal strategy for one-step self-construction of cell-surface sensors using diacyllipid-DNA conjugates as the building and sensing elements. The sensors exploit the high membrane-insertion capacity of a diacyllipid tail and good sensing performance of the DNA probes. Based on this strategy, we have engineered specific DNAzymes on the cell membrane for metal ion assay in the extracellular microspace. The immobilized DNAzyme showed excellent performance for reporting and semiquantifying both exogenous and cell-extruded target metal ions in real time. This membrane-anchored sensor could also be used for multiple target detection by having different DNA probes inserted, providing potentially useful tools for versatile applications in cell biology, biomedical research, drug discovery, and tissue engineering.