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Inflammatory bowel disease (IBD) manifests as intestinal barrier destruction, mucosal immunity dysregulation, and disrupted gut microbiome homeostasis. Conventional anti-inflammatory medications for IBD therapy partially alleviate symptoms but are unable to restore normal barrier and immune function. Here, we report a nanomedicine comprising bilirubin (BR)-attached low-molecular-weight, water-soluble chitosan nanoparticles (LMWC-BRNPs), that promotes restoration of the intestinal barrier, mucosal immunity, and the gut microbiome, thereby exerting robust therapeutic efficacy. In a mouse model of dextran sulfate sodium salt (DSS)-induced colitis, orally administered LMWC-BRNPs were retained in the GI tract much longer than other nonmucoadhesive BRNPs owing to the mucoadhesiveness of LMWC via electrostatic interaction. Treatment with LMWC-BRNPs led to considerable recovery of the damaged intestinal barrier compared with the current IBD medication, 5-aminosalicylic acid (5-ASA). Orally administered LMWC-BRNPs were taken up by pro-inflammatory macrophages and inhibited their activity. They also concurrently increased the population of regulatory T cells, thereby leading to the recovery of dysregulated mucosal immunity. An analysis of the gut microbiome revealed that LMWC-BRNPs treatment significantly attenuated the increase Turicibacter , an inflammation-related microorganism, resulting in protection of gut microbiome homeostasis. Taken together, our findings indicate that LMWC-BRNPs restored normal functions of the intestine and have high potential for use as a nanomedicine for IBD therapy.

Cancer stem-like cells (CSCs) are considered promising targets for anti-cancer therapy owing to their role in tumor progression. Extensive research is, therefore, being carried out on CSCs to identify potential targets for anti-cancer therapy. However, this requires the availability of patient-derived CSCs ex vivo, which remains restricted due to the low availability and diversity of CSCs. To address this limitation, a functional polymer thin-film (PTF) platform was invented to induce the transformation of cancer cells into tumorigenic spheroids. In this study, we demonstrated the functionality of a new PTF, polymer X, using a streamlined production process. Polymer X induced the formation of tumor spheroids with properties of CSCs, as revealed through the upregulated expression of CSC-related genes. Signal transducer and activator of transcription 3 (STAT3) phosphorylation in the cancer cells cultured on polymer X was upregulated by the fibronectin-integrin α5-Janus kinase 2 (JAK2) axis and maintained by the cytosolic LMO2/LBD1 complex. In addition, STAT3 signaling was critical in spheroid formation on polymer X. Our PTF platform allows the efficient generation of tumor spheroids from cancer cells, thereby overcoming the existing limitations of cancer research.

Patients with BRAF V600E -mutant melanoma are effectively treated with the BRAF-inhibiting drug, vemurafenib, but soon develop drug resistance, limiting vemurafenib's therapeutic efficacy. Constitutive activation of STAT3 in cancer cells and immune cells in the tumor microenvironment (TME) is a crucial contributor to the development of drug resistance and immune evasion in most cancers. Here, we investigated the antitumor efficacy and TME remodeling by APT STAT3 -9R, a cell-permeable STAT3 inhibitory peptide, as a strategy to treat vemurafenib-resistant melanoma. We found that vemurafenib-resistant melanoma remodels into immunosuppressive TME by increasing the expression of specific chemokines to facilitate the infiltration of immunosuppressive immune cells, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Intratumoral treatment of APT STAT3 -9R led to a reduction in the population of MDSCs and TAMs, while increasing infiltration of cytotoxic T lymphocytes in the TME. Moreover, combination therapy with APT STAT3 -9R and an anti-PD-1 antibody enhanced significant suppression of tumor growth by decreasing infiltration of these immunosuppressive immune cells while increasing the infiltration and cytotoxicity of CD8 + T cells. These findings suggest that combined blockade of STAT3 and PD-1 signaling pathways may be an effective treatment option for overcoming poor therapeutic outcomes associated with drug-resistant BRAF-mutant melanoma., Competing Interests: S.J. is a co-founder of BiliX and a non-executive director of Ubiquoss Holdings. None of these activities is related to the paper., (© 2022 The Author(s).)

Allogeneic stem cell transplantation is a curative immunotherapy where patients receive myeloablative chemotherapy and/or radiotherapy, followed by donor stem cell transplantation. Graft versus host disease (GVHD) is a major complication caused by dysregulated donor immune system, thus a novel strategy to modulate donor immunity is needed to mitigate GVHD. Tissue damage by conditioning regimen is thought to initiate the inflammatory milieu that recruits various donor immune cells for cross-priming of donor T cells against alloantigen and eventually promote strong Th1 cytokine storm escalating further tissue damage. Bilirubin nanoparticles (BRNP) are water-soluble conjugated of bilirubin and polyethylene glycol (PEG) with potent anti-inflammatory properties through its ability to scavenge reactive oxygen species generated at the site of inflammation. Here, we evaluated whether BRNP treatment post-transplantation can reduce initial inflammation and subsequently prevent GVHD in a major histocompatibility (MHC) mismatched murine GVHD model. After myeloablative irradiation, BALB/c mice received bone marrow and splenocytes isolated from C57BL/6 mice, with or without BRNP (10 mg/kg) daily on days 0 through 4 post-transplantation, and clinical GVHD and survival was monitored for 90 days. First, BRNP treatment significantly improved clinical GVHD score compared to untreated mice (3.4 vs 0.3, p=0.0003), and this translated into better overall survival (HR 0.0638, p=0.0003). Further, BRNPs showed a preferential accumulation in GVHD target organs leading to a reduced systemic and local inflammation evidenced by lower pathologic GVHD severity as well as circulating inflammatory cytokines such as IFN-γ. Lastly, BRNP treatment post-transplantation facilitated the reconstitution of CD4 + iNK T cells and reduced expansion of proinflammatory CD8α + iNK T cells and neutrophils especially in GVHD organs. Lastly, BRNP treatment decreased ICOS + or CTLA-4 + T cells but not PD-1 + T cells suggesting a decreased level of T cell activation but maintaining T cell tolerance. In conclusion, we demonstrated that BRNP treatment post-transplantation ameliorates murine GVHD via diminishing the initial tissue damage and subsequent inflammatory responses from immune subsets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pareek, Flegle, Boagni, Kim, Yoo, Trujillo-Ocampo, Lee, Zhang, Jon and Im.)

Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding ∼20-fold less DNA and ∼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.

The risk of fomite-mediated transmission in the clinic is substantially increasing amid the recent COVID-19 pandemic as personal protective equipment (PPE) of hospital workers is easily contaminated by direct contact with infected patients. In this context, it is crucial to devise a means to reduce such transmission. Herein, we report an antimicrobial, antiviral, and antibiofouling trifunctional polymer that can be easily coated onto the surface of medical protective clothing to effectively prevent pathogen contamination on the PPE. The coating layer is formed on the surfaces of PPE by the simple spray coating of an aqueous solution of the trifunctional polymer, poly(dodecyl methacrylate (DMA)-poly(ethylene glycol) methacrylate (PEGMA)-quaternary ammonium (QA)). To establish an optimal ratio of antifouling and antimicrobial functional groups, we performed antifouling, antibacterial, and antiviral tests using four different ratios of the polymers. Antifouling and bactericidal results were assessed using Staphylococcus aureus , a typical pathogenic bacterium that induces an upper respiratory infection. Regardless of the molar ratio, polymer-coated PPE surfaces showed considerable antiadhesion (∼65-75%) and antibacterial (∼75-87%) efficacies soon after being in contact with pathogens and maintained their capability for at least 24 h, which is sufficient for disposable PPEs. Further antiviral tests using coronaviruses showed favorable results with PPE coated at two specific ratios (3.5:6:0.5 and 3.5:5.5:1) of poly(DMA-PEGMA-QA). Moreover, biocompatibility assessments using the two most effective polymer ratios showed no recognizable local or systemic inflammatory responses in mice, suggesting the potential of this polymer for immediate use in the field., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Background Ischemia/reperfusion (I/R) injury causes overproduction of reactive oxygen species, which are the major culprits of oxidative stress that leads to inflammation, apoptosis, myocardial damage, and dysfunction. Bilirubin acts as a potent endogenous antioxidant that is capable of scavenging various reactive oxygen species. We have previously generated bilirubin nanoparticles (BRNPs) consisting of polyethylene glycol-conjugated bilirubin. In this study, we examined the therapeutic effects of BRNPs on myocardial I/R injury in mice. Methods and Results In vivo imaging using fluorophore encapsulated BRNPs showed BRNPs preferentially targeted to the site of I/R injury in the heart. Cardiac I/R surgery was performed by first ligating the left anterior descending coronary artery. After 45 minutes, reperfusion was achieved by releasing the ligation. BRNPs were administered intraperitoneally at 5 minutes before and 24 hours after reperfusion. Mice that received BRNPs showed significant improvements in their cardiac output, assessed by echocardiogram and pressure volume loop measurements, compared with the ones that received vehicle treatment. BRNPs treatment also significantly reduced the myocardial infarct size in mice that underwent cardiac I/R, compared with the vehicle-treatment group. In addition, BRNPs effectively suppressed reactive oxygen species and proinflammatory factor levels, as well as the amount of cardiac apoptosis. Conclusions Taken together, BRNPs could exert their therapeutic effects on cardiac I/R injury through attenuation of oxidative stress, apoptosis, and inflammation, providing a novel therapeutic modality for myocardial I/R injury.

Most therapeutic peptides available on the market today are naturally occurring hormones or protein fragments that were serendipitously discovered to possess therapeutic effects. However, the limited repertoire of available natural resources presents difficulties for the development of new peptide drug candidates. Traditional peptides possess several shortcomings that must be addressed for biomedical applications, including relatively low affinity or specificity toward biological targets compared to antibody- and protein scaffold-based affinity molecules, poor in vivo stability owing to rapid enzymatic degradation, and rapid clearance from circulation owing to their small size. Going forward, it will be increasingly important for scientists to develop novel classes of high-affinity and -specificity peptides against desired targets that mitigate these limitations while remaining compatible with pharmaceutical manufacturing processes. Recently, several highly constrained, artificial cyclic peptides have emerged as platforms capable of generating high-affinity peptide binders against various disease-associated protein targets by combining with phage or mRNA display method, some of which have entered clinical trials. In contrast, although linear peptides are relatively easy to synthesize cost-effectively and modify site-specifically at either N- or C-termini compared to cyclic peptides, there have been few linear peptide-based platforms that can provide high-affinity and -specificity peptide binders.In this Account, we describe the creation and development of a novel class of high-affinity peptides, termed "aptide"-from the Latin word " apt us " meaning "to fit" and "pep tide "-and summarize their biomedical applications. In the first part, we consider the design and creation of aptides, with a focus on their unique structural features and binding mode, and address screening and identification of target protein-specific aptides. We also discuss advantages of the aptide platform over ordinary linear peptides lacking preorganized structures in terms of the affinity and specificity of identified peptide binders against target molecules. In the second part, we describe the potential biomedical applications of various target-specific aptides, ranging from imaging and therapy to theranostics, according to the types of aptides and diseases. We show that certain aptides can not only bind to a target protein but also inhibit its biological function, thereby showing potential as therapeutics per se . Further, aptides specific for cancer-associated protein antigens can be used as escort molecules or targeting ligands for delivery of chemotherapeutics, cytokine proteins, and nanomedicines, such as liposomes and magnetic particles, to tumors, thereby substantially improving therapeutic effects. Finally, we present a strategy capable of overcoming the critical issue of short blood circulation time associated with most peptides by constructing a hybrid system between an aptide and a hapten cotinine-specific antibody.

Although naturally occurring, self-assembled protein nanoarchitectures have been utilized as antigen-delivery carriers, and the inability of such carriers to elicit immunogenicity requires additional use of strong adjuvants. Here, we report an immunogenic Brucella outer membrane protein BP26-derived nanoarchitecture displaying the influenza extracellular domain of matrix protein-2 (M2e) as a vaccine platform against influenza virus. Genetic engineering of a monomeric BP26 containing four or eight tandem repeats of M2e resulted in a hollow barrel-shaped nanoarchitecture (BP26-M2e nanobarrel). Immunization with BP26-M2e nanobarrels induced a strong M2e-specific humoral immune response in vivo that was much greater than that of a physical mixture of soluble M2e and BP26, with or without the use of an alum adjuvant. An anti-M2e antibody generated by BP26-M2e nanobarrel-immunized mice specifically bound to influenza virus-infected cells. Furthermore, in viral challenge tests, BP26-M2e nanobarrels effectively protected mice from influenza virus infection-associated death, even without the use of a conventional adjuvant. A mechanism study revealed that both M2e-specific antibody-dependent cellular cytotoxicity and T cell responses are involved in the vaccine efficacy of BP26-M2e nanobarrels. These findings suggest that the BP26-based nanobarrel developed here represents a versatile vaccine platform that can be used against various viral infections.

Glutathione (GSH) is produced at high levels in the normal liver, but its production is considerably reduced under certain pathological conditions. Accordingly, an imaging probe capable of visualizing the altered GSH level in the liver would be a useful tool for monitoring hepatic functions or diseases. Here, we report a gold nanoparticle (AuNP)-based computed tomography (CT) contrast agent that undergoes a change in colloidal stability in response to GSH levels, resulting in differential CT signal intensity between normal (higher intensity) and pathological (lower intensity) livers, enabling imaging of hepatic function. This GSH-responsive CT contrast agent, prepared by coating AuNPs with PEGylated bilirubin (PEG-BR), shows serum stability and high sensitivity to GSH. The resulting poly(ethylene glycol) (PEG)-BR@AuNPs preferentially accumulate in the normal liver, as evidenced by strongly enhanced CT intensity, but fail to do so in a GSH-depleted mouse model, where the CT signal in the liver was substantially decreased. In addition, injection of PEG-BR@AuNPs caused a greater reduction in CT signals in the liver in a drug-induced acute liver failure model than in healthy normal mice. These findings suggest that GSH-responsive PEG-BR@AuNPs have the potential to be used as a CT contrast agent to detect various hepatic function-related diseases and liver-metastasized tumors.

Extra-domain B of fibronectin (EDB-FN) is an alternatively spliced form of fibronectin with high expression in the extracellular matrix of neovascularized tissues and malignant cancer cells. In this study, we evaluated the practicality of using EDB-FN as a biomarker and therapeutic target for malignant gliomas (MGs), representative intractable diseases involving brain tumors. Methods: The microarray- and sequence-based patient transcriptomic database 'Oncopression' and tissue microarray of MG patient tissue samples were analyzed. EDB-FN data were extracted and evaluated from 23,344 patient samples of 17 types of cancer to assess its effectiveness and selectivity as a molecular target. To strengthen the results of the patient data analysis, the utility of EDB-FN as a molecular marker and target for MG was verified using active EDB-FN-targeting ultrasmall lipidic micellar nanoparticles (~12 nm), which had a high drug-loading capacity and were efficiently internalized by MG cells in vitro and in vivo . Results: Brain tumors had a 1.42-fold cancer-to-normal ratio ( p < 0.0001), the second highest among 17 cancers after head and neck cancer. Patient tissue microarray analysis showed that the EDB-FN high-expression group had a 5.5-fold higher risk of progression than the EDB-FN low-expression group ( p < 0.03). By labeling docetaxel-containing ultrasmall micelles with a bipodal aptide targeting EDB-FN (termed APT EDB -DSPE-DTX), we generated micelles that could specifically bind to MG cells, leading to superior antitumor efficacy of EDB-FN-targeting nanoparticles compared to nontargeting controls. Conclusions: Taken together, these results show that EDB-FN can be an effective drug delivery target and biomarker for MG., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Tubulin-based nanotubes (TNTs) to deliver microtubule-targeting agents (MTAs) for clinical oncology are reported. Three MTAs, docetaxel (DTX), laulimalide (LMD), and monomethyl auristatin E (MMAE), which attach to different binding sites in a tubulin, are loaded onto TNTs and cause structural changes in them, including shape anisotropy and tubulin layering. This drug-driven carrier transformation leads to changes in the drug-loading efficiency and stability characteristics of the carrier. TNTs coloaded with DTX and LMD efficiently deliver dual drug cargoes to cellular tubulins by the endolysosomal pathway, and results in synergistic anticancer and antiangiogenic action of the drugs in vitro. In in vivo tests, TNTs loaded with a microtubule-destabilizing agent MMAE suppress the growth of tumors with much higher efficacy than free MMAE did. This work suggests a new concept of using a drug's target protein as a carrier. The findings demonstrate that the TNTs developed here can be used universally as a delivery platform for many MTAs., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rosmarinic acid (RA), one of the most important polyphenol-based antioxidants, has received growing interest because of its bioactive properties, including anti-inflammatory, anticancer, and antibacterial activities. Despite the high therapeutic potential of RA, its intrinsic properties of poor water solubility and low bioavailability have limited its translation into the clinic. Here, we report on the synthesis and preparation of PEGylated RA-derived nanoparticles (RANPs) and their use as a therapeutic nanomedicine for treatment of inflammatory bowel disease (IBD) in a dextran sulfate sodium (DSS)-induced acute colitis mouse model. PEGylated RA, synthesized via a one-step process from RA and a PEG-containing amine, self-assembled in buffer to form nanoparticles (RANPs) with a diameter of 63.5 ± 4.0 nm. The resulting RANPs showed high colloidal stability in physiological medium up to 2 weeks. RANPs were capable of efficiently scavenging H 2 O 2 , thereby protecting cells from H 2 O 2 -induced damage. Furthermore, the corticosteroid drug, dexamethasone (DEX), could be loaded into RANPs and released in response to a reactive oxygen species stimulus. Intravenously administered RANPs exhibited significantly improved pharmacokinetic parameters compared with those of the parent RA and were preferentially localized to the inflamed colon. Intravenous administration of RANPs in DSS-induced colitis mice substantially mitigated colonic inflammation in a dose-dependent manner compared with the parent RA, as evidenced by significantly reduced disease activity index scores, body weight loss, and colonic inflammatory damage. In addition, RANPs suppressed expression and production of typical pro-inflammatory cytokines in the inflamed colon. Furthermore, DEX-loaded RANPs showed enhanced therapeutic efficacy in the colitis model compared with bare RANPs at the equivalent dose, indicating synergy with a conventional medication. These findings suggest that RANPs deserve further consideration as a potential therapeutic nanomedicine for the treatment of various inflammatory diseases, including IBD.

Rationale: Central nervous system has low vascular permeability by organizing tight junction (TJ) and limiting endothelial transcytosis. While TJ has long been considered to be responsible for vascular barrier in central nervous system, suppressed transcytosis in endothelial cells is now emerging as a complementary mechanism. Whether transcytosis regulation is independent of TJ and its dysregulation dominantly causes diseases associated with edema remain elusive. Dll4 signaling is important for various vascular contexts, but its role in the maintenance of vascular barrier in central nervous system remains unknown., Objective: To find a TJ-independent regulatory mechanism selective for transcytosis and identify its dysregulation as a cause of pathological leakage., Methods and Results: We studied transcytosis in the adult mouse retina with low vascular permeability and employed a hypertension-induced retinal edema model for its pathological implication. Both antibody-based and genetic inactivation of Dll4 or Notch1 induce hyperpermeability by increasing transcytosis without junctional destabilization in arterial endothelial cells, leading to nonhemorrhagic leakage predominantly in the superficial retinal layer. Endothelial Sox17 deletion represses Dll4 in retinal arteries, phenocopying Dll4 blocking-driven vascular leakage. Ang II (angiotensin II)-induced hypertension represses arterial Sox17 and Dll4, followed by transcytosis-driven retinal edema, which is rescued by a gain of Notch activity. Transcriptomic profiling of retinal endothelial cells suggests that Dll4 blocking activates SREBP1 (sterol regulatory element-binding protein 1)-mediated lipogenic transcription and enriches gene sets favorable for caveolae formation. Profiling also predicts the activation of VEGF (vascular endothelial growth factor) signaling by Dll4 blockade. Inhibition of SREBP1 or VEGF-VEGFR2 (VEGF receptor 2) signaling attenuates both Dll4 blockade-driven and hypertension-induced retinal leakage., Conclusions: In the retina, Sox17-Dll4-SREBP1 signaling axis controls transcytosis independently of TJ in superficial arteries among heterogeneous regulations for the whole vessels. Uncontrolled transcytosis via dysregulated Dll4 underlies pathological leakage in hypertensive retina and could be a therapeutic target for treating hypertension-associated retinal edema.

Catalytic precipitation and subsequent electrochemical oxidation or reduction of a redox-active precipitate has been widely used in electrochemical biosensors. However, such biosensors often do not allow for low detection limits due to a low rate of precipitation, nonspecific precipitation, loose binding of the precipitate to the electrode surface, and insulating behavior of the precipitate within a normal potential window. Here, we report an ultrasensitive electrochemical immunosensor for parathyroid hormone (PTH) detection based on DT-diaphorase (DT-D)-catalyzed formation of an organic precipitate and electrochemical oxidation of the precipitate. In the present study we found that DT-D can be used as a catalytic label in precipitation-based affinity biosensors because DT-D catalyzes fast reduction of 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan precipitate; the MTT reduction does not occur in the absence of DT-D; and a high electrochemical signal is obtained at low potentials during electrodissolution of MTT-formazan precipitate. The immunosensor is fabricated using a silane copolymer-modified ITO electrode surface that is suitable for both efficient and strong adsorption of MTT-formazan precipitate. When the enzymatic MTT-formazan precipitation and subsequent MTT-formazan electrodissolution is applied to a sandwich-type immunosensor, PTH can be detected over a wide range of concentrations with a very low detection limit (∼1 pg/mL) in artificial serum. The measured concentrations of PTH in clinical serum samples showed high similarity with those obtained using a commercial instrument.

Rationale : Magnetic relaxation switching (MRSw) induced by target-triggered aggregation or dissociation of superparamagnetic iron oxide nanoparticles (SPIONs) have been utilized for detection of diverse biomarkers. However, an MRSw-based biosensor for reactive oxygen species (ROS) has never been documented. Methods : To this end, we constructed a biosensor for ROS detection based on PEGylated bilirubin (PEG-BR)-coated SPIONs (PEG-BR@SPIONs) that were prepared by simple sonication via ligand exchange. In addition, near infra-red (NIR) fluorescent dye was loaded onto PEG-BR@SPIONs as a secondary option for fluorescence-based ROS detection. Resul ts : PEG-BR@SPIONs showed high colloidal stability under physiological conditions, but upon exposure to the model ROS, NaOCl, in vitro , they aggregated, causing a decrease in signal intensity in T2-weighted MR images. Furthermore, ROS-responsive PEG-BR@SPIONs were taken up by lipopolysaccharide (LPS)-activated macrophages to a much greater extent than ROS-unresponsive control nanoparticles (PEG-DSPE@SPIONs). In a sepsis-mimetic clinical setting, PEG-BR@SPIONs were able to directly detect the concentrations of ROS in whole blood samples through a clear change in T2 MR signals and a 'turn-on' signal of fluorescence. Conclusions : These findings suggest that PEG-BR@SPIONs have the potential as a new type of dual mode (MRSw-based and fluorescence-based) biosensors for ROS detection and could be used to diagnose many diseases associated with ROS overproduction., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

While conventional approaches for inflammatory bowel diseases mainly focus on suppressing hyperactive immune responses, it remains unclear how to address disrupted intestinal barriers, dysbiosis of the gut commensal microbiota and dysregulated mucosal immune responses in inflammatory bowel diseases. Moreover, immunosuppressive agents can cause off-target systemic side effects and complications. Here, we report the development of hyaluronic acid-bilirubin nanomedicine (HABN) that accumulates in inflamed colonic epithelium and restores the epithelium barriers in a murine model of acute colitis. Surprisingly, HABN also modulates the gut microbiota, increasing the overall richness and diversity and markedly augmenting the abundance of Akkermansia muciniphila and Clostridium XIVα, which are microorganisms with crucial roles in gut homeostasis. Importantly, HABN associated with pro-inflammatory macrophages, regulated innate immune responses and exerted potent therapeutic efficacy against colitis. Our work sheds light on the impact of nanotherapeutics on gut homeostasis, microbiome and innate immune responses for the treatment of inflammatory diseases.

: Although cancer stem cells (CSC) are thought to be responsible for tumor recurrence and resistance to chemotherapy, CSC-related research and drug development have been hampered by the limited supply of diverse, patient-derived CSC. Here, we present a functional polymer thin film (PTF) platform that promotes conversion of cancer cells to highly tumorigenic three-dimensional (3D) spheroids without the use of biochemical or genetic manipulations. Culturing various human cancer cells on the specific PTF, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4), gave rise to numerous multicellular tumor spheroids within 24 hours with high efficiency and reproducibility. Cancer cells in the resulting spheroids showed a significant increase in the expression of CSC-associated genes and acquired increased drug resistance compared with two-dimensional monolayer-cultured controls. These spheroids also exhibited enhanced xenograft tumor-forming ability and metastatic capacity in nude mice. By enabling the generation of tumorigenic spheroids from diverse cancer cells, the surface platform described here harbors the potential to contribute to CSC-related basic research and drug development. SIGNIFICANCE: A new cell culture technology enables highly tumorigenic 3D spheroids to be easily generated from various cancer cell sources in the common laboratory., (©2018 American Association for Cancer Research.)

A major problem with cancer chemotherapy begins when cells acquire resistance. Drug-resistant cancer cells typically upregulate multi-drug resistance proteins such as P-glycoprotein (P-gp). However, the lack of overexpressed surface biomarkers has limited the targeted therapy of drug-resistant cancers. Here we report a drug-delivery carrier decorated with a targeting ligand for a surface marker protein extra-domain B(EDB) specific to drug-resistant breast cancer cells as a new therapeutic option for the aggressive cancers. We constructed EDB-specific aptide (APT EDB )-conjugated liposome to simultaneously deliver siRNA(siMDR1) and Dox to drug-resistant breast cancer cells. APT EDB -LS(Dox,siMDR1) led to enhanced delivery of payloads into MCF7/ADR cells and showed significantly higher accumulation and retention in the tumors. While either APT EDB -LS(Dox) or APT EDB -LS(siMDR1) did not lead to appreciable tumor retardation in MCF7/ADR orthotropic model, APT EDB -LS(Dox,siMDR1) treatment resulted in significant reduction of the drug-resistant breast tumor. Taken together, this study provides a new strategy of drug delivery for drug-resistant cancer therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Despite the appreciable success of synthetic nanomaterials for targeted cancer therapy in preclinical studies, technical challenges involving their large-scale, cost-effective production and intrinsic toxicity associated with the materials, as well as their inability to penetrate tumor tissues deeply, limit their clinical translation. Here, we describe biologically derived nanocarriers developed from a bioengineered yeast strain that may overcome such impediments. The budding yeast Saccharomyces cerevisiae was genetically engineered to produce nanosized vacuoles displaying human epidermal growth factor receptor 2 (HER2)-specific affibody for active targeting. These nanosized vacuoles efficiently loaded the anticancer drug doxorubicin (Dox) and were effectively endocytosed by cultured cancer cells. Their cancer-targeting ability, along with their unique endomembrane compositions, significantly enhanced drug penetration in multicellular cultures and improved drug distribution in a tumor xenograft. Furthermore, Dox-loaded vacuoles successfully prevented tumor growth without eliciting any prolonged immune responses. The current study provides a platform technology for generating cancer-specific, tissue-penetrating, safe, and scalable biological nanoparticles for targeted cancer therapy.

In pulmonary drug delivery, the ability of an affinity molecule to bind to lung epithelium may prolong retention of therapeutic molecules within the lung and consequently yield higher overall bioavailability. To this end, we screened a library of structurally constrained peptides ('aptides') using phage-display technology and identified a high-affinity aptide for the mouse nicotinic acetylcholine receptor-α1 (nAChR-α1). The isolated aptide (APTnAChR-α1) bound to its target protein with high affinity (Kd=47nM). Alexa 488-labeled APTnAChR-α1 showed preferential binding to nAChR-α1-positive mouse lung epithelial cells and mouse muscle cells. Furthermore, the aptide exhibited substantial binding in nAChR-α1-positive tissue sections of muscle, trachea and lung, but not in liver, kidney or spleen tissues, which are nAChR-α1-negative. In an in vivo experiment, a high-intensity fluorescence signal was observed in the entire lung up to 50h after tracheal injection of Cy5.5-APTnAChR-α1, whereas most of the fluorescence signal from a Cy5.5-labeled scrambled peptide washed out within 20h after injection. Taken together, these results indicate that the high-affinity peptide for nAChR-α1 identified here bound tightly to lung epithelium and thus exhibited a long residence time in this tissue, suggesting that the peptide could be used for pulmonary delivery of active pharmaceuticals., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Computed tomography (CT) is one of the most useful diagnostic tools among commonly used biomedical imaging techniques, which also include magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasound. However, currently available CT contrast agents, which are based on small iodinated molecules, possess a number of limitations, including a lack of targeted molecular imaging, short imaging time, and renal toxicity. Here, we report a multifunctional nanoparticle for targeted molecular CT imaging and therapy of prostate cancer. By functionalizing the surface of gold nanoparticles (GNPs) with a prostate-specific membrane antigen (PSMA) RNA aptamer that binds to PSMA, we established a targeted molecular CT imaging system capable of specific imaging of prostate cancer cells that express the PSMA protein. The resulting PSMA aptamer-conjugated GNP showed more than 4-fold greater CT intensity for a targeted LNCaP cell than that of a nontargeted PC3 cell. Furthermore, the PSMA aptamer-conjugated GNPs after loading of doxorubicin were significantly more potent against targeted LNCaP cells than against nontargeted PC3 cells.

We report a combined chemoimmunotherapy vehicle consisting of plasmid loaded with doxorubicin and evaluate its efficacy in two different tumor models. A stable complex was formed with a 1300:1 ratio of doxorubicin bound to native plasmid via intercalation. Pharmacokinetics of the complex showed much slower clearance from plasma up to 3 h compared to 10 min for free doxorubicin. In mice bearing NCI-H358 xenografts, lower doses of complex (doxorubicin 0.5 mg/kg, plasmid 4 mg/kg) effectively reduced tumor growth compared to high doses (5 mg/kg) of free doxorubicin (68% versus 77%). Similar results were observed in mice bearing 4T1 murine allografts; the complex (doxorubicin 2 mg/kg, plasmid 8 mg/kg) was effective and caused similar reduction of tumor compared to free doxorubicin (4 mg/kg) (47% versus 46%). The complex showed no signs of severe systemic toxicity or cardiotoxicity compared to the free doxorubicin in mice as indicated by body weights and heart tissue histology. Elevated levels of cytokines (IL-12, IL-6, and IFN-gamma) were observed in serum as well as in tumor tissue after intravenous injection of complex when compared to plasmid or doxorubicin alone. This approach simultaneously delivers both chemotherapeutic and immunotherapeutic agents without time delay, improves pharmacokinetics of the free drug, lowers drug toxicity, upregulates a variety of cytokines, and is effective against different tumors.

The nonspecific binding of DNA-conjugated gold nanoparticles (AuNPs) to solid surfaces is more difficult to control than that of DNA molecules due to the more attractive interactions from the large number of DNA molecules per AuNP. This paper reports that the polymeric self-assembled monolayers (SAMs) formed on indium-tin oxide (ITO) electrodes significantly inhibit the nonspecific binding of DNA-conjugated AuNPs. The random copolymers used to prepare the polymeric SAMs consist of three functional parts: an ITO-reactive silane group, a DNA-blocking poly(ethylene glycol) (PEG) group, and an amine-reactive N-acryloxysuccinimide group. In order to compare the polymeric SAMs with various monomeric SAMs, the relative nonspecific binding of the DNA-conjugated AuNPs to the ITO electrodes modified with (3-aminopropyl)triethoxysilane (APTES), 3-aminopropylphosphonic acid, 3-phosphonopropionic acid, or 11-phosphonoundecanoic acid is examined by measuring the electrocatalytic anodic current of hydrazine caused by the nonspecifically absorbed AuNPs and by counting the AuNPs adsorbed onto modified ITO electrodes. Carboxylic-acid-terminated and amine-terminated monomeric SAMs cause high levels of nonspecific binding of DNA-conjugated AuNPs. The monomeric SAM modified with the carboxylic-acid-terminated poly(amidoamine) dendrimer shows low levels of nonspecific binding (2.0% nonspecific binding relative to APTES) due to the high surface density of the negative charge. The simply prepared polymeric SAM produces the lowest level of nonspecific binding (0.8% nonspecific binding relative to APTES), resulting from the combined effect of (i) DNA-blocking PEG and carboxylic acid groups and (ii) dense polymeric SAMs. Therefore, thin and dense polymeric SAMs may be effective in electrochemical detection and easy DNA immobilization along with low levels of nonspecific binding.

A novel, water-soluble trans-platinum complex was synthesized by inclusion complexation with beta-cyclodextrin. The complexation was confirmed by (1)H NMR, FT-IR, TGA, and XRD as well as by SEM and EDX. As the precursor complex is not water-soluble, it is difficult to employ it for biological applications. Here, we report that the encapsulation with cyclodextrin allowed to solubilize the complex to a solubility value of 1.6 mg/mL. Moreover, the cytotoxicity in vitro of the novel inclusion complex indicated a much higher activity after encapsulation.

We report a novel quantum dot (QD)-aptamer(Apt)-doxorubicin (Dox) conjugate [QD-Apt(Dox)] as a targeted cancer imaging, therapy, and sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed a targeted QD imaging system (QD-Apt) that is capable of differential uptake and imaging of prostate cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results in a targeted QD-Apt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET mechanism. A donor-acceptor model fluorescence resonance energy transfer (FRET) between QD and Dox and a donor-quencher model FRET between Dox and aptamer result when Dox intercalated within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD, which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.

Targeted uptake of therapeutic nanoparticles in a cell-, tissue-, or disease-specific manner represents a potentially powerful technology. Using prostate cancer as a model, we report docetaxel (Dtxl)-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2'-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the prostate-specific membrane antigen (PSMA), a well characterized antigen expressed on the surface of prostate cancer cells. These Dtxl-encapsulated nanoparticle-aptamer bioconjugates (Dtxl-NP-Apt) bind to the PSMA protein expressed on the surface of LNCaP prostate epithelial cells and get taken up by these cells resulting in significantly enhanced in vitro cellular toxicity as compared with nontargeted nanoparticles that lack the PSMA aptamer (Dtxl-NP) (P < 0.0004). The Dtxl-NP-Apt bioconjugates also exhibit remarkable efficacy and reduced toxicity as measured by mean body weight loss (BWL) in vivo [body weight loss of 7.7 +/- 4% vs. 18 +/- 5% for Dtxl-NP-Apt vs. Dtxl-NP at nadir, respectively (mean +/- SD); n = 7]. After a single intratumoral injection of Dtxl-NP-Apt bioconjugates, complete tumor reduction was observed in five of seven LNCaP xenograft nude mice (initial tumor volume of approximately 300 mm3), and 100% of these animals survived our 109-day study. In contrast, two of seven mice in the Dtxl-NP group had complete tumor reduction with 109-day survivability of only 57%. Dtxl alone had a survivability of only 14%. Saline and nanoparticles without drug were similarly nonefficacious. This report demonstrates the potential utility of nanoparticle-aptamer bioconjugates for a therapeutic application.

Purpose: Protein drugs cannot be delivered efficiently through oral routes. To address this challenge, we evaluated the effect of prolonged gastrointestinal transit on the bioavailability of insulin carried by magnetically responsive microparticles in the presence of an external magnetic field., Methods: Magnetite nanocrystals and insulin were coencapsulated into poly(lactide-co-glycolide) (PLGA) microparticles and their effects on hypoglycemia were evaluated in mice in the presence of a circumferentially applied external magnetic field., Results: A single administration of 100 U/kg of insulin-magnetite-PLGA microparticles to fasted mice resulted in a reduction of blood glucose levels of up to 43.8% in the presence of an external magnetic field for 20 h (bioavailability = 2.77 +/- 0.46 and 0.87 +/- 0.29% based on glucose and ELISA assay, respectively), significantly higher than similarly dosed mice without a magnetic field (bioavailability = 0.66 +/- 0.56 and 0.30 +/- 0.06%, based on glucose and ELISA assay, respectively)., Conclusions: A substantially improved hypoglycemic effect was observed in mice that were orally administered with insulin-magnetite-PLGA microparticles in the presence of an external magnetic field, suggesting that magnetic force can be used to improve the efficiency of orally delivered protein therapeutics.

New wedge-shaped thermotropic liquid crystalline materials containing a guanidinium moiety at the apex organize into various supramolecular structures such as hexagonal columnar, rectangular columnar and Pm3n cubic mesophases depending on anions illustrating guest-directed self-organization in mesophases.

Nanoparticles and microparticles have many potential biomedical applications ranging from imaging to drug delivery. Therefore, in vitro systems that can analyze and optimize the interaction of such particles with cells may be beneficial. Here, we report a microfluidic system that can be used to study these interactions. As a model system, we evaluated the interaction of polymeric nanoparticles and microparticles and similar particles conjugated to aptamers that recognize the transmembrane prostate specific membrane antigen (PSMA), with cells seeded in microchannels. The binding of particles to cells that expressed or did not express the PSMA (LNCaP or PC3, respectively) were evaluated with respect to changes in fluid shear stress, PSMA expression on target cells, and particle size. Nanoparticle-aptamer bioconjugates selectively adhered to LNCaP but not PC3 cells at static and low shear (<1 dyn/cm2) but not higher shear (approximately 4.5 dyn/cm2) conditions. Control nanoparticles and microparticles lacking aptamers and microparticle-aptamer bioconjugates did not adhere to LNCaP cells, even under very low shear conditions (approximately 0.28 dyn/cm2). These results demonstrate that the interaction of particles with cells can be studied under controlled conditions, which may aid in the engineering of desired particle characteristics. The scalability, low cost, reproducibility, and high-throughput capability of this technology is potentially beneficial to examining and optimizing a wide array of cell-particle systems prior to in vivo experiments.

We demonstrate that wettability of poly(ethylene glycol) (PEG) surfaces can be controlled using nanostructures with various geometrical features. Capillary lithography was used to fabricate PEG nanostructures using a new ultraviolet (UV) curable mold consisting of functionalized polyurethane with acrylate group (MINS101m, Minuta Tech.). Two distinct wetting states were observed depending of the height of nanostructures. At relatively lower heights (< 300 nm for 150 nm pillars with 500 nm spacing), the initial contact angle of water was less than 80 degrees and the water droplet easily invaded into the surface grooves, leading to a reduced contact angle at equilibrium (Wenzel state). At relatively higher heights (> 400 nm for 150 nm pillars with 500 nm spacing), on the other hand, the nanostructured PEG surface showed hydrophobic nature and no significant change in contact angle was observed with time (Cassie state). The presence of two wetting states was also confirmed by dynamic wetting properties and contact-angle hysteresis. The wetting transition from hydrophilic (bare PEG surface) to hydrophobic (PEG nanostructures) was described by the Cassie-Baxter equation assuming that enhanced hydrophobicity is due to the heterogeneous wetting mediated by an air pocket on the surface. The measured contact angles in the Cassie state were increased with increasing air fraction, in agreement with the theoretical prediction.

Novel artificial ion channels (1 and 2) based on CB[n] (n = 6 and 5, respectively) synthetic receptors with carbonyl-fringed portals (diameter 3.9 and 2.4 A, respectively) can transport proton and alkali metal ions across a lipid membrane with ion selectivity. Fluorometric experiments using large unilamellar vesicles showed that 1 mediates proton transport across the membranes, which can be blocked by a neurotransmitter, acetylcholine, reminiscent of the blocking of the K+ channels by polyamines. The alkali metal ion transport activity of 1 follows the order of Li+ > Cs+ approximately Rb+ > K+ > Na+, which is opposite to the binding affinity of CB[6] toward alkali metal ions. On the other hand, the transport activity of 2 follows the order of Li+ > Na+, which is also opposite to the binding affinity of 2 toward these metal ions, but virtually no transport was observed for K+, Rb+, and Cs+. It is presumably because the carbonyl-fringed portal size of 2 (diameter 2.4 A) is smaller than the diameters of these alkali metal ions. To determine the transport mechanism, voltage-clamp experiments on planar bilayer lipid membranes were carried out. The experiments showed that a single-channel current of 1 for Cs+ transport is approximately 5 pA, which corresponds to an ion flux of approximately 3 x 107 ions/s. These results are consistent with an ion channel mechanism. Not only the structural resemblance to the selectivity filter of K+ channels but also the remarkable ion selectivity makes this model system unique.

We report a protein attachment and patterning method based on a hydroquinone-caged biotin surface that generates bioactive biotin by mild electrochemical perturbation. The electrochemical activation proceeds under the buffered aqueous environment at neutral pH. It also allows site-selective generation of bioactive biotin for the immobilization of target protein by using prepatterned electrode arrays.

Nucleic acid ligands (aptamers) are potentially well suited for the therapeutic targeting of drug encapsulated controlled release polymer particles in a cell- or tissue-specific manner. We synthesized a bioconjugate composed of controlled release polymer nanoparticles and aptamers and examined its efficacy for targeted delivery to prostate cancer cells. Specifically, we synthesized poly(lactic acid)-block-polyethylene glycol (PEG) copolymer with a terminal carboxylic acid functional group (PLA-PEG-COOH), and encapsulated rhodamine-labeled dextran (as a model drug) within PLA-PEG-COOH nanoparticles. These nanoparticles have the following desirable characteristics: (a) negative surface charge (-50 +/- 3 mV, mean +/- SD, n = 3), which may minimize nonspecific interaction with the negatively charged nucleic acid aptamers; (b) carboxylic acid groups on the particle surface for potential modification and covalent conjugation to amine-modified aptamers; and (c) presence of PEG on particle surface, which enhances circulating half-life while contributing to decreased uptake in nontargeted cells. Next, we generated nanoparticle-aptamer bioconjugates with RNA aptamers that bind to the prostate-specific membrane antigen, a well-known prostate cancer tumor marker that is overexpressed on prostate acinar epithelial cells. We demonstrated that these bioconjugates can efficiently target and get taken up by the prostate LNCaP epithelial cells, which express the prostate-specific membrane antigen protein (77-fold increase in binding versus control, n = 150 cells per group). In contrast to LNCaP cells, the uptake of these particles is not enhanced in cells that do not express the prostate-specific membrane antigen protein. To our knowledge, this represents the first report of targeted drug delivery with nanoparticle-aptamer bioconjugates.

The ability to control the deposition and location of adherent and non-adherent cells within microfluidic devices is beneficial for the development of micro-scale bioanalytical tools and high-throughput screening systems. Here, we introduce a simple technique to fabricate poly(ethylene glycol)(PEG) microstructures within microfluidic channels that can be used to dock cells within pre-defined locations. Microstructures of various shapes were used to capture and shear-protect cells despite medium flow in the channel. Using this approach, PEG microwells were fabricated either with exposed or non-exposed substrates. Proteins and cells adhered within microwells with exposed substrates, while non-exposed substrates prevented protein and cell adhesion (although the cells were captured inside the features). Furthermore, immobilized cells remained viable and were stained for cell surface receptors by sequential flow of antibodies and secondary fluorescent probes. With its unique strengths in utility and control, this approach is potentially beneficial for the development of cell-based analytical devices and microreactors that enable the capture and real-time analysis of cells within microchannels, irrespective of cell anchorage properties.

The control of surface properties and spatial presentation of functional molecules within a microfluidic channel is important for the development of diagnostic assays and microreactors and for performing fundamental studies of cell biology and fluid mechanics. Here, we present a simple technique, applicable to many soft lithographic methods, to fabricate robust microchannels with precise control over the spatial properties of the substrate. In this approach, the patterned regions were protected from oxygen plasma by controlling the dimensions of the poly(dimethylsiloxane) (PDMS) stamp and by leaving the stamp in place during the plasma treatment process. The PDMS stamp was then removed, and the microfluidic mold was irreversibly bonded to the substrate. The approach was used to pattern a nonbiofouling poly(ethylene glycol)-based copolymer or the polysaccharide hyaluronic acid within microfluidic channels. These nonbiofouling patterns were then used to fabricate arrays of fibronectin and bovine serum albumin as well as mammalian cells. In addition, further control over the deposition of multiple proteins onto multiple or individual patterns was achieved using laminar flow. Also, cells that were patterned within channels remained viable and capable of performing intracellular reactions and could be potentially lysed for analysis.

The synthesis of a new degradable polymer system, poly(amino alcohol esters) and the resulting polymers' potential for use in gene transfection vectors are reported. The polymerization proceeded in a one step reaction from commercially available bis(secondary amines) monomers (N,N'-dimethyl-1,3-propanediamine and N,N'-dimethyl-1,6-hexanediamine, respectively) through nucleophilic addition to the diglycidyl ester of dicarboxylic acid (diglycidyl adipate). Poly(amino alcohol ester) 1 and 2 were synthesized with a yield of 89% and 91% with Mn = 24,800 and Mn = 36,400, respectively. Poly(amino alcohol ester) 1 degraded hydrolytically in phosphate buffer at pH 7.4 with a half-life of approximately 5 days. Both polymers readily self-assembled with plasmid DNA into nanometer-sized DNA/polymer complexes less than 180 nm diameter and are significantly less cytotoxic than the commonly used DNA delivery polymer, poly(ethylene imine) (PEI).