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Diseases caused by viruses, such as monkeypox virus (MPXV) and human papillomavirus (HPV), pose serious threats to human health and safety. Although numerous strategies have been constructed for detecting MPXV and HPV DNA, most methods require either laborious procedures or complicated instruments involving skilled professionals. In this research, a CRISPR-Cas12a-mediated colorimetric detection platform for MPXV and HPV sensing was constructed for the first time by applying probe DNA to reprogram the catalytic properties of molybdenum disulfide quantum dots (MoS 2 QDs). In the presence of MPXV or HPV targets, the CRISPR-Cas12a trans-cleavage activity is effectively motivated to decompose the probe DNA, leading to the suppression of enzymatic activity DNA enhancer adsorbed on MoS 2 QDs, resulting in greatly decreased catalytic behaviors. The MoS 2 QDs-DNA nanohybrids displayed prominent specificity and sensitivity, with detection limits at subpicomolar levels, as well as excellent stability and accuracy for determining MPXV and HPV DNA in human sera biosamples. Furthermore, the proposed colorimetric biosensing approach can be ensembled with a smartphone platform, allowing visible analysis of DNA targets. Taken together, this colorimetric strategy offers a novel diagnosis method for MPXV and HPV DNA detection, particularly favorable for highly endemic developing countries with restricted medical and instrumental support., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Acute liver failure (ALF) is a highly fatal disease, necessitating the advancement and optimization of alternative therapeutic strategies to benefit patients awaiting liver transplantation. In this study, we innovatively established the antioxidant nanozyme-hepatocyte-like cells (HLCs) microtissue sheets (HS/N-Au@composite) for ALF therapy. We first prepared a 3D-printed hyaluronic acid/gelatin/sodium alginate scaffold with N-acetylcysteine (NAC)-capped gold nanoclusters (NAC-Au NCs), forming the N-Au@hydrogel. For the encapsulation of HLC spheroids, we used a biocompatible hybrid hydrogel composed of decellularized extracellular matrix (dECM), thrombin, and fibrinogen, resulting in the HS@dECM hydrogel. Utilizing 3D printing technology, we integrated the N-Au@hydrogel with the HS@dECM hydrogel to create the HS/N-Au@composite for in situ transplantation to treat ALF. Our results demonstrated that NAC-Au NCs effectively mitigated reactive oxygen species (ROS)-induced liver necrosis in ALF. Additionally, the N-Au@hydrogel provided mechanical support, ensuring the proper landing and effective functioning of the transplanted HLC spheroids. The HS/N-Au@composite synergistically decreased serum transaminase levels, reduced the accumulation of pro-inflammatory cytokines, accelerated liver function recovery, and promoted liver regeneration in ALF treatment. This combination of HLC spheroids and NAC-Au NCs nanozymes via 3D-printed composite scaffolds represents a promising strategy for enhancing hepatocyte transplantation and advancing stem cell regenerative medicine in ALF therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Immunotherapy through the activation of the stimulator of interferon genes (STING) signaling pathway is increasingly recognized for its robust anti-tumor efficacy. However, the effectiveness of STING activation is often compromised by inadequate anti-tumor immunity and a scarcity of primed immune cells in the tumor microenvironment. Herein, we design and fabricate a co-axial 3D-printed scaffold integrating a non-nucleotide STING agonist, SR-717, and an AKT inhibitor, MK-2206, in its respective shell and core layers, to synergistically enhance STING activation, thereby suppressing tumor recurrence and growth. SR-717 initiates the STING activation to enhance the phosphorylation of the factors along the STING pathway, while MK-2206 concurrently inhibits the AKT phosphorylation to facilitate the TBK1 phosphorylation of the STING pathway. The sequential and sustained release of SR-717 and MK-2206 from the scaffold results in a synergistic STING activation, demonstrating substantial anti-tumor efficacy across multiple tumor models. Furthermore, the scaffold promotes the recruitment and enrichment of activated dendritic cells and M1 macrophages, subsequently stimulating anti-tumor T cell activity, thereby amplifying the immunotherapeutic effect. This precise and synergistic activation of STING by the scaffold offers promising potential in tumor immunotherapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Liver fibrosis is a chronic pathological condition lacking specific clinical treatments. Stem cells, with notable potential in regenerative medicine, offer promise in treating liver fibrosis. However, stem cell therapy is hindered by potential immunological rejection, carcinogenesis risk, efficacy variation, and high cost. Stem cell secretome-based cell-free therapy offers potential solutions to address these challenges, but it is limited by low delivery efficiency and rapid clearance. Herein, an innovative approach for in situ implantation of smart microneedle (MN) arrays enabling precisely controlled delivery of multiple therapeutic agents directly into fibrotic liver tissues is developed. By integrating cell-free and platinum-based nanocatalytic combination therapy, the MN arrays can deactivate hepatic stellate cells. Moreover, they promote excessive extracellular matrix degradation by more than 75%, approaching normal levels. Additionally, the smart MN arrays can provide hepatocyte protection while reducing inflammation levels by ≈70-90%. They can also exhibit remarkable capability in scavenging almost 100% of reactive oxygen species and alleviating hypoxia. Ultimately, this treatment strategy can effectively restrain fibrosis progression. The comprehensive in vitro and in vivo experiments, supplemented by proteome and transcriptome analyses, substantiate the effectiveness of the approach in treating liver fibrosis, holding immense promise for clinical applications., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The clustered regularly interspaced short palindromic repeat (CRISPR) system, an emerging tool for genome editing, has garnered significant public interest for its potential in treating genetic diseases. Despite the rapid advancements in CRISPR technology, the progress in developing effective delivery strategies lags, impeding its clinical application. Extracellular nanovesicles (EVs), either in their endogenous forms or with engineered modifications, have emerged as a promising solution for CRISPR delivery. These EVs offer several advantages, including high biocompatibility, biological permeability, negligible immunogenicity, and straightforward production. Herein, we first summarize various types of functional EVs for CRISPR delivery, such as unmodified, modified, engineered virus-like particles (VLPs), and exosome-liposome hybrid vesicles, and examine their distinct intracellular pathways. Then, we outline the cutting-edge techniques for functionalizing extracellular vesicles, involving producer cell engineering, vesicle engineering, and virus-like particle engineering, emphasizing the diverse CRISPR delivery capabilities of these nanovesicles. Lastly, we address the current challenges and propose rational design strategies for their clinical translation, offering future perspectives on the development of functionalized EVs.

Lipid nanoparticles (LNPs) have recently emerged as successful gene delivery platforms for a diverse array of disease treatments. Efforts to optimize their design for common administration methods such as intravenous injection, intramuscular injection, or inhalation, revolve primarily around the addition of targeting ligands or the choice of ionizable lipid. Here, we employed a multi-step screening method to optimize the type of helper lipid and component ratios in a plasmid DNA (pDNA) LNP library to efficiently deliver pDNA through intraduodenal delivery as an indicative route for oral administration. By addressing different physiological barriers in a stepwise manner, we down-selected effective LNP candidates from a library of over 1000 formulations. Beyond reporter protein expression, we assessed the efficiency in non-viral gene editing in mouse liver mediated by LNPs to knockdown PCSK9 and ANGPTL3 expression, thereby lowering low-density lipoprotein (LDL) cholesterol levels. Utilizing an all-in-one pDNA construct with Strep. pyogenes Cas9 and gRNAs, our results showcased that intraduodenal administration of selected LNPs facilitated targeted gene knockdown in the liver, resulting in a 27% reduction in the serum LDL cholesterol level. This LNP-based all-in-one pDNA-mediated gene editing strategy highlights its potential as an oral therapeutic approach for hypercholesterolemia, opening up new possibilities for DNA-based gene medicine applications., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Hai-Quan Mao reports financial support was provided by National Institute of Allergy and Infectious Diseases. Kam W. Leong reports financial support was provided by National Institutes of Health. Shuting Sarah Cai reports financial support was provided by National Science Foundation. Hai-Quan Mao, Yining Zhu, Jingyao Ma, Ruochen Shen, and Leonardo Cheng has patent pending to Johns Hopkins Technology Ventures. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The membrane-fusion-based internalization without lysosomal entrapment is advantageous for intracellular delivery over endocytosis. However, protein corona formed on the membrane-fusogenic liposome surface converts its membrane-fusion performance to lysosome-dependent endocytosis, causing poorer delivery efficiency in biological conditions. Herein, we develop an antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo. Leveraging specific lipid composition at an optimized ratio, such antifouling membrane-fusogenic liposome facilitates fusion capacity even in protein-rich conditions, attributed to the copious zwitterionic phosphorylcholine groups for protein-adsorption resistance. Consequently, the antifouling membrane-fusogenic liposome demonstrates robust membrane-fusion-mediated delivery in the medium with up to 38% fetal bovine serum, outclassing two traditional membrane-fusogenic liposomes effective at 4% and 6% concentrations. When injected into mice, antifouling membrane-fusogenic liposomes can keep their membrane-fusion-transportation behaviors, thereby achieving efficient luciferase transfection and enhancing gene-editing-mediated viral inhibition. This study provides a promising tool for effective intracellular delivery under complex physiological environments, enlightening future nanomedicine design., (© 2024. The Author(s).)

Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), which were therapeutic DNA with high immunostimulatory activity, have been applied in widespread applications from basic research to clinics as therapeutic agents for cancer immunotherapy, viral infection, allergic diseases and asthma since their discovery in 1995. The major factors to consider for clinical translation using CpG motifs are the protection of CpG ODNs from DNase degradation and the delivery of CpG ODNs to the Toll-like receptor-9 expressed human B-cells and plasmacytoid dendritic cells. Therefore, great efforts have been devoted to the advances of efficient delivery systems for CpG ODNs. In this review, we outline new horizons and recent developments in this field, providing a comprehensive summary of the nanoparticle-based CpG delivery systems developed to improve the efficacy of CpG-mediated immune responses, including DNA nanostructures, inorganic nanoparticles, polymer nanoparticles, metal-organic-frameworks, lipid-based nanosystems, proteins and peptides, as well as exosomes and cell membrane nanoparticles. Moreover, future challenges in the establishment of CpG delivery systems for immunotherapeutic applications are discussed. We expect that the continuously growing interest in the development of CpG-based immunotherapy will certainly fuel the excitement and stimulation in medicine research.

Spatially resolved gene expression profiling provides insight into tissue organization and cell-cell crosstalk; however, sequencing-based spatial transcriptomics (ST) lacks single-cell resolution. Current ST analysis methods require single-cell RNA sequencing data as a reference for rigorous interpretation of cell states, mostly do not use associated histology images and are not capable of inferring shared neighborhoods across multiple tissues. Here we present Starfysh, a computational toolbox using a deep generative model that incorporates archetypal analysis and any known cell type markers to characterize known or new tissue-specific cell states without a single-cell reference. Starfysh improves the characterization of spatial dynamics in complex tissues using histology images and enables the comparison of niches as spatial hubs across tissues. Integrative analysis of primary estrogen receptor (ER)-positive breast cancer, triple-negative breast cancer (TNBC) and metaplastic breast cancer (MBC) tissues led to the identification of spatial hubs with patient- and disease-specific cell type compositions and revealed metabolic reprogramming shaping immunosuppressive hubs in aggressive MBC., (© 2024. The Author(s).)

Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) gene-editing technology shows promise for manipulating single or multiple tumor-associated genes and engineering immune cells to treat cancers. Currently, most gene-editing strategies rely on viral delivery; yet, while being efficient, many limitations, mainly from safety and packaging capacity considerations, hinder the use of viral CRISPR vectors in cancer therapy. In contrast, recent advances in non-viral CRISPR/Cas9 nanoformulations have paved the way for better cancer gene editing, as these nanoformulations can be engineered to improve safety, efficiency, and specificity through optimizing the packaging capacity, pharmacokinetics, and targetability. In this review, the advance in non-viral CRISPR delivery is highlighted, and there is a discussion on how these approaches can be potentially used to treat cancers in addressing the aforementioned limitations, followed by the perspectives in designing a proper CRISPR/Cas9-based cancer nanomedicine system with translational potential., (© 2023 Wiley‐VCH GmbH.)

The emerging field of liquid biopsy has garnered significant interest in precision diagnostics, offering a non-invasive and repetitive method for analyzing bodily fluids to procure real-time diagnostic data. The precision and accuracy offered by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) technology have advanced and broadened the applications of liquid biopsy. Significantly, when combined with swiftly advancing nanotechnology, CRISPR/Cas-mediated nanodevices show vast potential in precise liquid biopsy applications. However, persistent challenges are still associated with off-target effects, and the current platforms also constrain the performance of the assays. In this review, we highlight the merits of CRISPR/Cas systems in liquid biopsy, tracing the development of CRISPR/Cas systems and their current applications in disease diagnosis particularly in liquid biopsies. We also outline ongoing efforts to design nanoscale devices with improved sensing and readout capabilities, aiming to enhance the performance of CRISPR/Cas detectors in liquid biopsy. Finally, we identify the critical obstacles hindering the widespread adoption of CRISPR/Cas liquid biopsy and explore potential solutions. This feature article presents a comprehensive overview of CRISPR/Cas-mediated liquid biopsies, emphasizing the progress in integrating nanodevices to improve specificity and sensitivity. It also sheds light on future research directions in employing nanodevices for CRISPR/Cas-based liquid biopsies in the realm of precision medicine.

The effects of immunotherapeutics on interactions between immune and cancer cells are modulated by multiple components in the tumour microenvironment (TME), including endothelium and tumour stroma, which provide both a physical barrier and immunosuppressive stimuli. Herein, we report a recirculating chip to enable continuous immune cell recirculation through a microfluidic cell array to include these crucial players. This system consists of a three-layered cell array (μFCA) spatially emulating the TME, with tailored fluidic circuits establishing T cell recirculation. This platform enables the study of dynamics among the TME, immune cells in a circulatory system and cancer cell responses thereof. Through this system, we found that tumour endothelium hindered T cell infiltration into the reconstructed breast cancer tumour compartment. This negative effect was alleviated when treated with anti-human PD-L1 (programmed cell death ligand 1) antibody. Another key stromal component - cancer associated fibroblasts - attenuated T cell infiltration, compared against normal fibroblasts, and led to reduced apoptotic activity in cancer cells. These results confirm the capability of our tumour-on-a-chip system in identifying some key axes to target in overcoming barriers to immunotherapy by recapitulating immune cell interactions with the reconstructed TME. Our results also attest to the feasibility of scaling up this system for high-throughput cancer immunotherapeutic screening.

Targeting the activated epidermal growth factor receptor (EGFR) via clustered regularly interspaced short palindromic repeat (CRISPR) technology is appealing to overcome the drug resistance of hepatocellular carcinoma (HCC) towards tyrosine kinase inhibitor (TKI) therapy. However, combining these two distinct drugs using traditional liposomes results in a suboptimal synergistic anti-HCC effect due to the limited CRISPR/Cas9 delivery efficiency caused by lysosomal entrapment after endocytosis. Herein, we developed a liver-targeting gene-hybridizing-TKI fusogenic liposome (LIGHTFUL) that can achieve high CRISPR/Cas9 expression to reverse the EGFR-mediated drug resistance for enhanced TKI-based HCC therapy efficiently. Coated with a galactose-modified membrane-fusogenic lipid layer, LIGHTFUL reached the targeting liver site to fuse with HCC tumor cells, directly and efficiently transporting interior CDK5- and PLK1-targeting CRISPR/Cas9 plasmids (pXG333-CPs) into the HCC cell cytoplasm and then the cell nucleus for efficient expression. Such membrane-fusion-mediated pXG333-CP delivery resulted in effective downregulation of both CDK5 and PLK1, sufficiently inactivating EGFR to improve the anti-HCC effects of the co-delivered TKI, lenvatinib. This membrane-fusion-participant codelivery strategy optimized the synergetic effect of CRISPR/Cas9 and TKI combinational therapy as indicated by the 0.35 combination index in vitro and the dramatic reduction of subcutaneous and orthotopic TKI-insensitive HCC tumor growth in mice. Therefore, the established LIGHTFUL provides a unique co-delivery platform to combine gene editing and TKI therapies for enhanced synergetic therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

Rationale: Bilateral sonication with focused ultrasound (FUS) in conjunction with microbubbles has been shown to separately reduce amyloid plaques and hyperphosphorylated tau protein in the hippocampal formation and the entorhinal cortex in different mouse models of Alzheimer's disease (AD) without any therapeutic agents. However, the two pathologies are expressed concurrently in human disease. Therefore, the objective of this study is to investigate the effects of repeated bilateral sonications in the presence of both pathologies. Methods: Herein, we investigate its functional and morphological outcomes on brains bearing both pathologies simultaneously. Eleven transgenic mice of the 3xTg-AD line (14 months old) expressing human amyloid beta and human tau and eleven age-matched wild-type littermates received four weekly bilateral sonications covering the hippocampus followed by working memory testing. Afterwards, immunohistochemistry and immunoassays (western blot and ELISA) were employed to assess any changes in amyloid beta and human tau. Furthermore, we present preliminary data from our clinical trial using a neuronavigation-guided FUS system for sonications in AD patients (NCT04118764). Results: Interestingly, both wild-type and transgenic animals that received FUS experienced improved working memory and spent significantly more time in the escape platform-quadrant, with wild-type animals spending 43.2% (sham: 37.7%) and transgenic animals spending 35.3% (sham: 31.0%) of the trial in the target quadrant. Furthermore, this behavioral amelioration in the transgenic animals correlated with a 58.3% decrease in the neuronal length affected by tau and a 27.2% reduction in total tau levels. Amyloid plaque population, volume and overall load were also reduced overall. Consistently, preliminary data from a clinical trial involving AD patients showed a 1.8% decrease of amyloid PET signal 3-weeks after treatment in the treated hemisphere compared to baseline. Conclusion: For the first time, it is shown that bilateral FUS-induced BBB opening significantly and simultaneously ameliorates both coexistent pathologies, which translated to improvements in spatial memory of transgenic animals with complex AD, the human mimicking phenotype. The level of cognitive improvement was significantly correlated with the volume of BBB opening. Non-transgenic animals were also shown to exhibit similar memory amelioration for the first time, indicating that BBB opening results into benefits in the neuronal function regardless of the existence of AD pathology. A potential mechanism of action for the reduction of the both pathologies investigated was the cholesterol metabolism, specifically the LRP1b receptor, which exhibited increased expression levels in transgenic mice following FUS-induced BBB opening. Initial clinical evidence supported that the beta amyloid reduction shown in rodents could be translatable to humans with significant amyloid reduction shown in the treated hemisphere., Competing Interests: Competing Interests: Some of the work presented herein is supported by patents licensed to Delsona Therapeutics, Inc where E.E.K. serves as co-founder and scientific adviser. The rest of the authors declare that they have no competing interests., (© The author(s).)

Reactive oxygen species (ROS)-associated oxidative stress, inflammation storm, and massive hepatocyte necrosis are the typical manifestations of acute liver failure (ALF), therefore specific therapeutic interventions are essential for the devastating disease. Here, we developed a platform consisting of versatile biomimetic copper oxide nanozymes (Cu NZs)-loaded PLGA nanofibers (Cu NZs@PLGA nanofibers) and decellularized extracellular matrix (dECM) hydrogels for delivery of human adipose-derived mesenchymal stem/stromal cells-derived hepatocyte-like cells (hADMSCs-derived HLCs) (HLCs/Cu NZs@fiber/dECM). Cu NZs@PLGA nanofibers could conspicuously scavenge excessive ROS at the early stage of ALF, and reduce the massive accumulation of pro-inflammatory cytokines, herein efficiently preventing the deterioration of hepatocytes necrosis. Moreover, Cu NZs@PLGA nanofibers also exhibited a cytoprotection effect on the transplanted HLCs. Meanwhile, HLCs with hepatic-specific biofunctions and anti-inflammatory activity acted as a promising alternative cell source for ALF therapy. The dECM hydrogels further provided the desirable 3D environment and favorably improved the hepatic functions of HLCs. In addition, the pro-angiogenesis activity of Cu NZs@PLGA nanofibers also facilitated the integration of the whole implant with the host liver. Hence, HLCs/Cu NZs@fiber/dECM performed excellent synergistic therapeutic efficacy on ALF mice. This strategy using Cu NZs@PLGA nanofiber-reinforced dECM hydrogels for HLCs in situ delivery is a promising approach for ALF therapy and shows great potential for clinical translation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Effective delivery of the CRISPR-Cas9 components is crucial to realizing the therapeutic potential. Although many delivery approaches have been developed for this application, oral delivery has not been explored due to the degradative nature of the gastrointestinal tract. For this issue, we developed a series of novel phenylboronic acid (PBA)-functionalized chitosan-polyethylenimine (CS-PEI) polymers for oral CRISPR delivery. PBA functionalization equipped the polyplex with higher stability, smooth transport across the mucus, and efficient endosomal escape and cytosolic unpackaging in the cells. From a library of 12 PBA-functionalized CS-PEI polyplexes, we identified a formulation that showed the most effective penetration in the intestinal mucosa after oral gavage to mice. The optimized formulation performed feasible CRISPR-mediated downregulation of the target protein and reduction in the downstream cholesterol. As the first oral CRISPR carrier, this study suggests the potential of addressing the needs of both local and systemic editing in a patient-compliant manner.

Tumor-positive resection margins after surgery can result in tumor recurrence and metastasis. Although adjuvant postoperative radiotherapy and chemotherapy have been adopted in clinical practice, they lack efficacy and result in unavoidable side effects. Herein, a self-intensified in-situ therapy approach using electrospun fibers loaded with a biomimetic nanozyme and doxorubicin (DOX) is developed. The fabricated PEG-coated zeolite imidazole framework-67 (PZIF67) is demonstrated as a versatile nanozyme triggering reactions in cancer cells based on endogenous H 2 O 2 and •O 2 - . The PZIF67-generated •OH induces reactive oxygen species (ROS) overload, implementing chemodynamic therapy (CDT). The O 2 produced by PZIF67 inhibits the expression of hypoxia-up-regulated proteins, thereby suppressing tumor progression. PZIF67 also catalyzes the degradation of glutathione, further disturbing the intracellular redox homeostasis and enhancing CDT. Furthermore, the introduced DOX not only kills cancer cells individually, but also replenishes the continuously consumed substrates for PZIF67-catalyzed reactions. The PZIF67-weakened drug resistance strengthens the cytotoxicity of DOX. The combined application of PZIF67 and DOX also suppresses metastasis-associated genes. Both in vitro and in vivo results demonstrate that the self-intensified synergy of PZIF67 and DOX on electrospun fibers efficiently prevents postsurgical tumor recurrence and metastasis, offering a feasible therapeutic regimen for operable malignant tumors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Photothermal therapy (PTT) is an effective treatment modality that is highly selective for tumor suppression and is a hopeful alternative to traditional cancer therapy. However, PTT-induced inflammatory responses may result in undesirable side effects including increased risks of tumor recurrence and metastasis. Here we developed multifunctional MnO nanoparticles as scavengers of proinflammatory molecules to alleviate the PTT-induced inflammatory response. The MnO nanoparticles improve the PTT therapy by (1) binding and scavenging proinflammatory molecules to inhibit the proinflammatory molecule-induced Toll-like receptors (TLR) activation and nuclear factor kappa B (NF-κB) signaling; (2) inhibiting activated macrophage-induced macrophage recruitment; and (3) inhibiting tumor cell migration and invasion. In vivo experimental results showed that further treatment with MnO nanoparticles after laser therapy not only inhibited the PTT-induced inflammatory response and primary tumor recurrence but also significantly reduced tumor metastasis due to the scavenging activity. These findings suggest that MnO nanoparticles hold the potential for mitigating the therapy-induced severe inflammatory response and inhibiting tumor recurrence and metastasis.

Periodontitis is a common type of inflammatory bone loss and a risk factor for systemic diseases. The pathogenesis of periodontitis involves inflammatory dysregulation, which represents a target for new therapeutic strategies to treat periodontitis. After establishing the correlation of cell-free DNA (cfDNA) level with periodontitis in patient samples, we test the hypothesis that the cfDNA-scavenging approach will benefit periodontitis treatment. We create a nanoparticulate cfDNA scavenger specific for periodontitis by coating selenium-doped hydroxyapatite nanoparticles (SeHANs) with cationic polyamidoamine dendrimers (PAMAM-G3), namely G3@SeHANs, and compare the activities of G3@SeHANs with those of soluble PAMAM-G3 polymer. Both G3@SeHANs and PAMAM-G3 inhibit periodontitis-related proinflammation in vitro by scavenging cfDNA and alleviate inflammatory bone loss in a mouse model of ligature-induced periodontitis. G3@SeHANs also regulate the mononuclear phagocyte system in a periodontitis environment, promoting the M2 over the M1 macrophage phenotype. G3@SeHANs show greater therapeutic effects than PAMAM-G3 in reducing proinflammation and alveolar bone loss in vivo. Our findings demonstrate the importance of cfDNA in periodontitis and the potential for using hydroxyapatite-based nanoparticulate cfDNA scavengers to ameliorate periodontitis., (© 2022. The Author(s).)

Membrane fusion, a fundamental biological process of the fusion of the membrane composition between cells, is vital for cell-cell communication and cargo transport between living cells. This fusion interaction achieves the transportation of the inner content to the cellular cytosol as well as the simultaneous blending of foreign substances with the cell membrane. Inspired by this biological process, emerging membrane-fusogenic particles have been developed, opening a new area for bioengineering and biomedical applications. Especially, membrane-fusion-mediated transfer of inner cargoes can bypass endosomal entrapment to maximize the transportation efficiency, emerging as a unique cytoplasmic delivery platform distinct from those depending on conventional endocytosis-based pathways. In addition, the membrane fusion enables cell surface modification through lipid diffusion and mixing, providing a tool for direct cell membrane engineering. In this review, we focus on the development of membrane-fusogenic particles and their up-to-date progress. We briefly introduce the concept of membrane fusion, elaborate inspiring strategies of membrane-fusogenic particles, and highlight the recent advances and the promising applications of membrane-fusogenic particles as a next-generation bioengineering tool. In the end, we conclude with the present challenges and opportunities, providing insights in the future research of membrane-fusogenic particles.

Helicobacter pylori ( H. pylori ) infection is considered to be the main cause of most digestive diseases,such as chronic active gastritis, gastroduodenal ulcers, or even gastric cancer. Oral medication is a transformative approach to treat H. pylori -induced infections. However, unlike intravenous administration, orally administrated drugs have to overcome various barriers before reaching the infected sites, which significantly limits the therapeutic efficacy. These challenges may be addressed by emerging nanomedicine that is equipped with nanotechnology approaches to enable efficient and effective targeted delivery of drugs. Herein, in this review, we first discuss the conventional therapy for the eradication of H. pylori . Through the introduction of the critical barriers of oral administration, the benefits of nanomedicine are highlighted. Recently-published examples of nanocarriers for combating H. pylori in terms of design, preparation, and antimicrobial mechanisms are then presented, followed by our perspective on potential future research directions of oral nanomedicines.

Liver disease, particularly viral hepatitis and hepatocellular carcinoma (HCC), is a global healthcare burden and leads to more than 2 million deaths per year worldwide. Despite some success in diagnosis and vaccine development, there are still unmet needs to improve diagnostics and therapeutics for viral hepatitis and HCC. The emerging clustered regularly interspaced short palindromic repeat/associated proteins (CRISPR/Cas) technology may open up a unique avenue to tackle these two diseases at the genetic level in a precise manner. Especially, liver is a more accessible organ over others from the delivery point of view, and many advanced strategies applied for nanotheranostics can be adapted in CRISPR-mediated diagnostics or liver gene editing. In this review, the focus is on these two aspects of viral hepatitis and HCC applications. An overview on CRISPR editor development and current progress in clinical trials is first given, followed by highlighting the recent advances integrating the merits of gene editing and nanotheranostics. The promising systems that are used in other applications but may hold potentials in liver gene editing are also discussed. This review concludes with the perspectives on rationally designing the next-generation CRISPR approaches and improving the editing performance., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Hepatitis B virus (HBV) infection is one of the global healthcare burdens, and its early diagnosis is crucial for the prevention of HBV-induced chronic hepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although different detection approaches have been reported, most of these methods either rely on sophisticated machines or procedures, which limit their use particularly in the high endemic, developing countries. In this work, we report a dual-sensing nanoplatform built on noble metal-molybdenum disulfide (MoS 2 ) nanohybrids, and this platform can detect the HBV DNA target through either fluorometric or colorimetric readouts. The design with the silver nanocluster (AgNC)-MoS 2 nanohybrid enables multiplex fluorescent detection, while the HBV DNA-regulated growth of platinum nanoparticles (PtNPs) on the MoS 2 nanosheets offers signal-on colorimetric detection. Both AgNC-MoS 2 and PtNP-MoS 2 nanohybrids show high sensitivity with pico-molar detection limit and single nucleotide specificity, even with the spiked human serum. Collectively, the proposed nanohybrids possess their potential in the use of early HBV diagnosis, particularly suitable for the high endemic areas with limited medical and instrumental supports., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bleomycin sclerotherapy is used in the treatment of cystic lesions; however, the histopathological changes are undefined. Present animal models of cystic diseases are not adequate for the study of sclerotherapy of hepatic cysts, primarily because the established cysts in these models are too small in size. The aim of the present study was to establish a new animal model of simple hepatic cysts, and assess the histopathological changes after bleomycin sclerotherapy. Rabbit gallbladder, with ligaturing of the cholecystic duct whilst preserving cholecystic vessels, was used as a model for simple hepatic cysts. Bleomycin (2 mg dissolved in 1 ml saline) was injected into the aspirated gallbladder, gallbladder tissue was harvested (after 1, 7, 14, 28, 42, 56 and 84 days) and histopathological changes were evaluated (n=4 per group). Additionally, control rabbit gallbladders were injected with 1 ml saline and sampled after 14 days (n=4). Histopathological changes were evaluated using hematoxylin-eosin and Masson's trichrome staining, and immunohistochemistry for CD20-, CD43- and CD68-positive cells was performed. The integrated optical density (IOD) of immunohistochemical staining and average positive stained area percentage (APSAP) of collagen were quantitatively analyzed. The results revealed gallbladders in the control group had regular epithelial cells with no visible inflammation or fibrosis. In the experimental group, epithelial cells were swollen and necrotic on the first day, and were replaced gradually by single-layer flat cells from day 56. Inflammatory infiltration was found in the submucosa, and the IOD of T cells, B cells and macrophages were highest on day 1, and these parameters declined gradually, eventually disappearing. The APSAP of collagen was highest on day 7, and gradually declined thereafter. The results suggest that histopathological changes after bleomycin sclerotherapy of a simple hepatic cyst model were characterized by sequential epithelial destruction, inflammatory cell infiltration, collagen proliferation and epithelial partial regeneration., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Li et al.)

Biomimetic strategies are useful for designing potent vaccines. Decorating a nanoparticulate adjuvant with cell membrane fragments as the antigen-presenting source exemplifies, such as a promising strategy. For translation, a standardizable, consistent, and scalable approach for coating nanoadjuvant with the cell membrane is important. Here a turbulent mixing and self-assembly method called flash nanocomplexation (FNC) for producing cell membrane-coated nanovaccines in a scalable manner is demonstrated. The broad applicability of this FNC technique compared with bulk-sonication by using ten different core materials and multiple cell membrane types is shown. FNC-produced biomimetic nanoparticles have promising colloidal stability and narrow particle polydispersity, indicating an equal or more homogeneous coating compared to the bulk-sonication method. The potency of a nanovaccine comprised of B16-F10 cancer cell membrane decorating mesoporous silica nanoparticles loaded with the adjuvant CpG is then demonstrated. The FNC-fabricated nanovaccines when combined with anti-CTLA-4 show potency in lymph node targeting, DC antigen presentation, and T cell immune activation, leading to prophylactic and therapeutic efficacy in a melanoma mouse model. This study advances the design of a biomimetic nanovaccine enabled by a robust and versatile nanomanufacturing technique., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Drug screening in oncology, especially for triple-negative breast cancer (TNBC), has high demand but remains unsatisfactory. Currently available models are either nonrepresentative of the complex tumor microenvironment or only suitable for low throughput screening, resulting in a low-yield success for drug development. To tackle these issues, the L-TumorChip system is developed in this study. It is a three-layered microfluidic tumor-on-a-chip platform integrating tumor microvasculature and tumor-stromal microenvironment with high throughput screening capability. Its layered and modular design is readily scalable through simple integration of multiple units. Here, L-TumorChip is validated with a TNBC model. The L-TumorChip system emulates certain tumor-stroma complexities and tumor-endothelium interactions, including TNBC invasion through the leaky microvasculature and angiogenesis. Additionally, with this L-TumorChip, the influence of different stromal cells, including normal fibroblasts, mesenchymal stem cells, and cancer-associated fibroblasts (CAF), on cancer cell growth as well as the stromal effects on drug responses to doxorubicin treatment is investigated. The presence of CAF delays drug pharmacokinetics, while apoptotic responses indicated by caspase-3 activities are higher in coculture with normal fibroblasts. Collectively, the L-TumorChip system represents a translational high-throughput screening toolkit that enables drug screening with a scenario closer to the in vivo conditions. This potential use may therefore facilitate development of new cancer drugs., (© 2020 Wiley-VCH GmbH.)

Recent advances in CRISPR present attractive genome-editing toolsets for therapeutic strategies at the genetic level. Here, a liposome-coated mesoporous silica nanoparticle (lipoMSN) is reported as an effective CRISPR delivery system for multiplex gene-editing in the liver. The MSN provides efficient loading of Cas9 plasmid as well as Cas9 protein/guide RNA ribonucleoprotein complex (RNP), while liposome-coating offers improved serum stability and enhanced cell uptake. Hypothesizing that loss-of-function mutation in the lipid-metabolism-related genes pcsk9, apoc3, and angptl3 would improve cardiovascular health by lowering blood cholesterol and triglycerides, the lipoMSN is used to deliver a combination of RNPs targeting these genes. When targeting a single gene, the lipoMSN achieved a 54% gene-editing efficiency, besting the state-of-art Lipofectamine CRISPRMax. For multiplexing, lipoMSN maintained significant gene-editing at each gene target despite reduced dosage of target-specific RNP. By delivering combinations of targeting RNPs in the same nanoparticle, synergistic effects on lipid metabolism are observed in vitro and vivo. These effects, such as a 50% decrease in serum cholesterol after 4 weeks of post-treatment with lipoMSN carrying both pcsk9 and angptl3-targeted RNPs, could not be reached with a single gene-editing approach. Taken together, this lipoMSN represents a versatile platform for the development of efficient, combinatorial gene-editing therapeutics., (© 2020 Wiley-VCH GmbH.)

Severe sepsis represents a common, expensive, and deadly health care issue with limited therapeutic options. Gaining insights into the inflammatory dysregulation that causes sepsis would help develop new therapeutic strategies against severe sepsis. In this study, we identified the crucial role of cell-free DNA (cfDNA) in the regulation of the Toll-like receptor 9-mediated proinflammatory pathway in severe sepsis progression. Hypothesizing that removing cfDNA would be beneficial for sepsis treatment, we used polyethylenimine (PEI) and synthesized PEI-functionalized, biodegradable mesoporous silica nanoparticles with different charge densities as cfDNA scavengers. These nucleic acid-binding nanoparticles (NABNs) showed superior performance compared with their nucleic acid-binding polymer counterparts on inhibition of cfDNA-induced inflammation and subsequent multiple organ injury caused by severe sepsis. Furthermore, NABNs exhibited enhanced accumulation and retention in the inflamed cecum, along with a more desirable in vivo safety profile. Together, our results revealed a key contribution of cfDNA in severe sepsis and shed a light on the development of NABN-based therapeutics for sepsis therapy, which currently remains intractable., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

For patients carrying BRCA1 mutations, at least one-third develop triple negative breast cancer (TNBC). Not only is TNBC difficult to treat due to the lack of molecular target receptors, but BRCA1 mutations (BRCA1m) also result in chemotherapeutic resistance, making disease recurrence more likely. Although BRCA1m are highly heterogeneous and therefore difficult to target, BRCA1 gene's synthetic lethal pair, PARP1 , is conserved in BRCA1m cancer cells. Therefore, we hypothesize that targeting PARP1 might be a fruitful direction to sensitize BRCA1m cancer cells to chemotherapy. We used CRISPR/Cas9 technology to generate PARP1 deficiency in two TNBC cell lines, MDA-MB-231 ( BRCA1 wild-type) and MDA-MB-436 (BRCA1m). We explored whether this PARP1 disruption (PARP1m) could significantly lower the chemotherapeutic dose necessary to achieve therapeutic efficacy in both a 2D and 3D tumor-on-a-chip model. With both BRCA1m and PARP1m, the TNBC cells were more sensitive to three representative chemotherapeutic breast cancer drugs, doxorubicin, gemcitabine and docetaxel, compared with the PARP1 wild-type counterpart in the 2D culture environment. However, PARP1m did not result in this synergy in the 3D tumor-on-a-chip model, suggesting that drug dosing in the tumor microenvironment may influence the synergy. Taken together, our results highlight a discrepancy in the efficacy of the combination of PARP1 inhibition and chemotherapy for TNBC treatment, which should be clarified to justify further clinical testing., (© 2020 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals, Inc. on behalf of The American Institute of Chemical Engineers.)

Background/aim: Long noncoding RNAs (lncRNAs) are noncoding transcripts that are >200 nucleotides in length. However, the biological functions and regulation mechanisms of lncRNAs in gastric carcinogenesis remain unknown., Materials and Methods: The expression levels of Linc00472 were analyzed by real-time PCR. The DNA methylation status was assessed using Combined Bisulfite Restriction Analysis (COBRA). The biological role of Linc00472 was assessed in AGS cells with Linc00472 overexpression., Results: Using the next-generation sequencing approach, we identified DNA methylation-associated lncRNAs in gastric cancer cells. Among them, the expression level of Linc00472 significantly decreased in gastric cancer tissues compared to adjacent normal tissues. Furthermore, we observed a more frequent hypermethylation of CpG islands upstream of Linc00472 in gastric cancer tissues. Ectopic Linc00472 expression could significantly inhibit gastric cancer cell growth and migration., Conclusion: Epigenetically regulated Linc00472 expression plays a crucial role in modulating gastric cancer cell growth and motility., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Inflammation is ubiquitous in the body, triggering desirable immune response to defend against dangerous signals or instigating undesirable damage to cells and tissues to cause disease. Nanomedicine holds exciting potential in modulating inflammation. In particular, cell membranes derived from cells involved in the inflammatory process may be used to coat nanotherapeutics for effective targeted delivery to inflammatory tissues. Herein, the recent progress of rationally engineering cell membrane-based nanotherapeutics for inflammation therapy is highlighted, and the challenges and opportunities presented in realizing the full potential of cell-membrane coating in targeting and manipulating the inflammatory microenvironment are discussed., Competing Interests: The authors declare no conflict of interest.

Mesenchymal stem cell (MSC) has been increasingly applied to cancer therapy because of its tumor-tropic capability. However, short retention at target tissue and limited payload option hinder the progress of MSC-based cancer therapy. Herein, we proposed a hybrid spheroid/nanomedicine system, comprising MSC spheroid entrapping drug-loaded nanocomposite, to address these limitations. Spheroid formulation enhanced MSC's tumor tropism and facilitated loading of different types of therapeutic payloads. This system acted as an active drug delivery platform seeking and specifically targeting glioblastoma cells. It enabled effective delivery of combinational protein and chemotherapeutic drugs by engineered MSC and nanocomposite, respectively. In an in vivo migration model, the hybrid spheroid showed higher nanocomposite retention in the tumor tissue compared with the single MSC approach, leading to enhanced tumor inhibition in a heterotopic glioblastoma murine model. Taken together, this system integrates the merits of cell- and nanoparticle- mediated drug delivery with the tumor-homing characteristics of MSC to advance targeted combinational cancer therapy.

Breast cancer is the most common malignancy and also the second leading cause of cancer mortality in women globally. Strategies for early and precise detection of breast cancer cells are highly desired in breast cancer diagnosis and treatment. Here, we report on the efficient detection of HER2-positive (HER2+) breast cancer cells using an amplified signal scheme enabled by gold nanoclusters entrapped in mesoporous silica nanoparticles (MSNs). The utilization of MSNs as an excellent enzyme immobilization support and gold nanoclusters as an effective peroxidase mimic imparts high sensitivity to this detection platform. In addition, the inclusion of target-specific HER2 antibodies adds excellent selectivity. Determination of HER2+ cancer cells in breast cancer tissue demonstrates the potential application of this biosensor design in clinical diagnosis in particular, and bioanalysis in general.

Molecularly, breast cancer represents a highly heterogenous family of neoplastic disorders, with substantial interpatient variations regarding genetic mutations, cell composition, transcriptional profiles, and treatment response. Consequently, there is an increasing demand for alternative diagnostic approaches aimed at the molecular annotation of the disease on a patient-by-patient basis and the design of more personalized treatments. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technology enables the development of such novel approaches. For instance, in diagnostics, the use of the RNA-specific C2c2 system allows ultrasensitive nucleic acid detection and could be used to characterize the mutational repertoire and transcriptional breast cancer signatures. In disease modeling, CRISPR/Cas9 technology can be applied to selectively engineer oncogenes and tumor-suppressor genes involved in disease pathogenesis. In treatment, CRISPR/Cas9 can be used to develop gene-therapy, while its catalytically-dead variant (dCas9) can be applied to reprogram the epigenetic landscape of malignant cells. As immunotherapy becomes increasingly prominent in cancer treatment, CRISPR/Cas9 can engineer the immune cells to redirect them against cancer cells and potentiate antitumor immune responses. In this review, CRISPR strategies for the advancement of breast cancer diagnostics, modeling, and treatment are highlighted, culminating in a perspective on developing a precision medicine-based approach against breast cancer., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

Implants decorated with antimicrobial peptides (AMPs) can prevent infection and reduce the risk of creating antibiotic resistance. Yet the restricted mobility of surficial AMP often compromises its activity. Here, we report a simple but effective strategy to allow a more flexible display of AMP on the biomaterial surface and demonstrate its efficacy for wound healing. The AMP, tachyplesin I (Tac), is tagged with the polyhydroxyalkanoate-granule-associated protein (PhaP) and immobilized on haloarchaea-produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) via hydrophobic interaction. The PhaP-Tac coating effectively inhibits the growth of both Gram-negative and Gram-positive bacteria. It also increases the surface hydrophilicity to improve fibroblast proliferation in vitro, and accelerates wound healing by decreasing bacterial counts to below 10 5  CFU per gram of tissue in a deep-wound mouse model in vivo. Taken together, these findings demonstrate an effective strategy to realize the full potential of AMPs in imparting implants with an anti-microbial activity that is localized and potent., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Controlled delivery of protein therapeutics remains a challenge. Here, the inclusion of diselenide-bond-containing organosilica moieties into the framework of silica to fabricate biodegradable mesoporous silica nanoparticles (MSNs) with oxidative and redox dual-responsiveness is reported. These diselenide-bridged MSNs can encapsulate cytotoxic RNase A into the 8-10 nm internal pores via electrostatic interaction and release the payload via a matrix-degradation controlled mechanism upon exposure to oxidative or redox conditions. After surface cloaking with cancer-cell-derived membrane fragments, these bioinspired RNase A-loaded MSNs exhibit homologous targeting and immune-invasion characteristics inherited from the source cancer cells. The efficient in vitro and in vivo anti-cancer performance, which includes increased blood circulation time and enhanced tumor accumulation along with low toxicity, suggests that these cell-membrane-coated, dual-responsive degradable MSNs represent a promising platform for the delivery of bio-macromolecules such as protein and nucleic acid therapeutics., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid-based CRISPR/Cas9 system. To tackle this, a self-assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid-guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9-mediated E7 knockout leads to significant inhibition of HPV-induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine-based therapeutics.

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

A unique interaction between the cyanine dye and negatively charged quantum dot is used to construct a signal-on biaptameric quantum dot (QD) Förster resonance energy transfer (FRET) beacon for protein detection and distinct aptamer characterization. The beacon comprises a pair of aptamers, one intercalated with the cyanine dye (YOYO-3) and the other conjugated to a negatively charged, carboxyl-QD. When the target protein is present, structural folding and sandwich association of the two aptamers take place. As a consequence, YOYO-3 is displaced from the folded aptamer and transferred to the unblocked QD surface to yield a target concentration-dependent FRET signal. As a proof-of-principle, we demonstrate the detection of thrombin ranging from nanomolar to submicromolar concentrations and confirm the dye translocation using cylindrical illumination confocal spectroscopy (CICS). The proposed beacon provides a simple, rapid, signal-on FRET detection for protein as well as a potential platform for distinct aptamer screening.

Researchers have applied mesenchymal stem cells (MSC) to a variety of therapeutic scenarios by harnessing their multipotent, regenerative, and immunosuppressive properties with tropisms toward inflamed, hypoxic, and cancerous sites. Although MSC-based therapies have been shown to be safe and effective to a certain degree, the efficacy remains low in most cases when MSC are applied alone. To enhance their therapeutic efficacy, researchers have equipped MSC with targeted delivery functions using genetic engineering, therapeutic agent incorporation, and cell surface modification. MSC can be genetically modified virally or non-virally to overexpress therapeutic proteins that complement their innate properties. MSC can also be primed with non-peptidic drugs or magnetic nanoparticles for enhanced efficacy and externally regulated targeting, respectively. Furthermore, MSC can be functionalized with targeting moieties to augment their homing toward therapeutic sites using enzymatic modification, chemical conjugation, or non-covalent interactions. These engineering techniques are still works in progress, requiring optimization to improve the therapeutic efficacy and targeting effectiveness while minimizing any loss of MSC function. In this review, we will highlight the advanced techniques of engineering MSC, describe their promise and the challenges of translation into clinical settings, and suggest future perspectives on realizing their full potential for MSC-based therapy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aptamer nanomedicine, including therapeutic aptamers and aptamer nanocomplexes, is beginning to fulfill its potential in both clinical trials and preclinical studies. Especially in oncology, aptamer nanomedicine may perform better than conventional or antibody-based chemotherapeutics due to specificity compared to the former and stability compared to the latter. Many proof-of-concept studies on applying aptamers to drug delivery, gene therapy, and cancer imaging have shown promising efficacy and impressive safety in vivo toward translation. Yet, there remains ample room for improvement and critical barriers to be addressed. In this review, we will first introduce the recent progress in clinical trials of aptamer nanomedicine, followed by a discussion of the barriers at the design and in vivo application stages. We will then highlight recent advances and engineering strategies proposed to tackle these barriers. Aptamer cancer nanomedicine has the potential to address one of the most important healthcare issues of the society.

A new SELEX scheme is proposed for the selection of aptamers targeting a specific epitope of a native protein. Anti-sialic acid receptor (SAR) aptamers that inhibit H1 hemagglutination at a low picomole dose are selected accordingly.

A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg(2+), coupling time, and aptamer type on the beacon's performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This work aims for ultrasensitive detection of target proteins in complex biological matrixes based on aptamer microarrays. Two extensively studied aptamers (HTQ and HTDQ) that bind distinct epitopes of thrombin are chosen for the microarray study. Although HTQ and HTDQ have nanomolar to subnanomolar affinities, it is found that either aptamer when applied directly has difficulty in detecting a few nanomoles per liter thrombin in the presence of a 10- or 100-fold (w/w) excess of serum total protein (STP). By investigating dodecyl (12-carbon) and oligodeoxythymidine (oligo(dT)) spacers, we observe both spacers enhance the microarray signal response, but oligo(dT) is strikingly better than dodecyl. Moreover, we discover that a microarray spot coprinted with the two distinct aptamers (HTQ and HTDQ) functions like a bivalent molecular construct and exhibits an avidity effect. With the synergy of oligo(dT) spacers and the avidity effect, detection of picomolar-range thrombin in the presence of either 10% unlabeled serum or a 10,000-fold excess of labeled serum total protein is achieved. It corresponds to a 100-1000-fold sensitivity enhancement as compared to using an individual aptamer without a spacer.

Fifty cases of primary liver cancer were treated by electrochemical therapy (ECT). Tumour size ranged from 3.5 to 21.0 cm. ECT was performed by administration of 6-10 V, 50-100 mA and an electric quantity of 300-1000 C. Duration of treatment was one and a half to four hours. There was an inverse relationship between tumour size and curative effect. There was no death within three months of treatment. Six- and 12-month survival was 88% and 69%, respectively. In conclusion, ECT is an effective treatment which prolongs the patients' life. We have found the method to be of practical value in the management of liver cancers in the intermediate or advanced stage.