Cancer immunotherapy by immune checkpoint blockade and its advanced application using bio-nanomaterials.

Cancer is the second leading cause of death worldwide. Traditional approaches, such as surgery, chemotherapy, and radiotherapy have been the main cancer therapeutic modalities in recent years. Cancer immunotherapy is a novel therapeutic modality that potentiates the immune responses of patients against malignancy. Immune checkpoint proteins expressed on T cells or tumor cells serve as a target for inhibiting T cell overactivation, maintaining the balance between self-reactivity and autoimmunity. Tumors essentially hijack the immune checkpoint pathway in order to survive and spread. Immune checkpoint inhibitors (ICIs) are being developed as a result to reactivate the anti-tumor immune response. Recent advances in nanotechnology have contributed to the development of successful, safe, and efficient anticancer drug systems based on nanoparticles. Nanoparticle-based cancer immunotherapy overcomes numerous challenges and offers novel strategies for improving conventional immunotherapies. The fundamental and physiochemical properties of nanoparticles depend on various cancer therapeutic strategies, such as chemotherapeutics, nucleic acid-based treatments, photothermal therapy, and photodynamic agents. The review discusses the use of nanoparticles as carriers for delivering immune checkpoint inhibitors and their efficacy in cancer combination therapy.

Structures and Applications of Nucleic Acid-Based Micelles for Cancer Therapy.

Nucleic acids have become important building blocks in nanotechnology over the last 30 years. DNA and RNA can sequentially build specific nanostructures, resulting in versatile drug delivery systems. Self-assembling amphiphilic nucleic acids, composed of hydrophilic and hydrophobic segments to form micelle structures, have the potential for cancer therapeutics due to their ability to encapsulate hydrophobic agents into their core and position functional groups on the surface. Moreover, DNA or RNA within bio-compatible micelles can function as drugs by themselves. This review introduces and discusses nucleic acid-based spherical micelles from diverse amphiphilic nucleic acids and their applications in cancer therapy.

High-Performance Near-Infrared Chlorinated Rylenecarboximide Fluorophores via Consecutive C-N and C-C Bond Formation.

A new class of near-infrared (NIR) fluorophores, PAI, is obtained by consecutive C-N/C-C bond formation between diphenylamines and 9,10-dibromoperylenecarboximide. Owing to the rigid structure, extended π-conjugation and pronounced push-pull substitution, these fluorophores show emission maxima up to 804 nm and large Stokes shifts. The extraordinarily high fluorescence quantum yields from 47 % to 70 % are attributed to chloro substitution in the bay positions of the perylene core. These characteristics, together with high photostability, qualify them as useful NIR emitters for applications as biomarkers and security inks.

Improvement of luminescence properties of NaYF4 :Yb3+ /Er3+ upconversion materials by a cross-relaxation mechanism based on co-doped Ho3+ ion concentrations.

NaYF4 :Yb3+ /Er3+ /Ho3+ nanophosphors were successfully synthesized using a solvothermal method and with various concentrations of Ho3+ ions. The crystal structure, grain size, morphology, and luminescence properties were analyzed by X-ray diffraction, field-emission scanning electron microscopy, and photoluminescence measurements. All samples were crystallized as a cubic structure; it was confirmed that all samples exhibited strong green emission and weak red emission generated at a particular level of the activated ions. The strongest upconversion fluorescence intensity was observed in the Ho3+ and Er3+ ions co-doped NaYF4 materials with a Ho3+ ion concentration of 0.005 mol. Only the green fluorescence intensity at the 542 nm centre increased strongly due to the 4 S3/2 →4 I15/2 energy transfer. This increase in upconversion fluorescence intensity at a selected wavelength was described as a cross-relaxation mechanism due to the addition of Ho3+ ions.

Enhancement of Immune Checkpoint Inhibitor-Mediated Anti-Cancer Immunity by Intranasal Treatment of Ecklonia cava Fucoidan against Metastatic Lung Cancer.

Although fucoidan, a well-studied seaweed-extracted polysaccharide, has shown immune stimulatory effects that elicit anticancer immunity, mucosal adjuvant effects via intranasal administration have not been studied. In this study, the effect of Ecklonia cava-extracted fucoidan (ECF) on the induction of anti-cancer immunity in the lung was examined by intranasal administration. In C57BL/6 and BALB/c mice, intranasal administration of ECF promoted the activation of dendritic cells (DCs), natural killer (NK) cells, and T cells in the mediastinal lymph node (mLN). The ECF-induced NK and T cell activation was mediated by DCs. In addition, intranasal injection with ECF enhanced the anti-PD-L1 antibody-mediated anti-cancer activities against B16 melanoma and CT-26 carcinoma tumor growth in the lungs, which were required cytotoxic T lymphocytes and NK cells. Thus, these data demonstrated that ECF functioned as a mucosal adjuvant that enhanced the immunotherapeutic effect of immune checkpoint inhibitors against metastatic lung cancer.

Self-assembled DNA nanoparticles loaded with travoprost for glaucoma-treatment.

Lipid DNA nanoparticles (NPs) exhibit an intrinsic affinity to the ocular surface and can be loaded by hybridization with fluorophore-DNA conjugates or with the anti-glaucoma drug travoprost by hybridizing an aptamer that binds the medication. In the travoprost-loaded NPs (Trav-NPs), the drug is bound by specific, non-covalent interactions, not requiring any chemical modification of the active pharmaceutical ingredient. Fluorescently labeled Trav-NPs show a long-lasting adherence to the eye, up to sixty minutes after eye drop instillation. Biosafety of the Trav-NPs was proved and in vivo. Ex vivo and in vivo quantification of travoprost via LC-MS revealed that Trav-NPs deliver at least twice the amount of the drug at every time-point investigated compared to the pristine drug. The data successfully show the applicability of a DNA-based drug delivery system in the field of ophthalmology for the treatment of a major retinal eye disease, i.e. glaucoma.

Activation of Human Dendritic Cells by Ascophyllan Purified from Ascophyllum nodosum.

In our previous study, we showed that ascophyllan purified from Ascophyllum nodosum treatment promotes mouse dendritic cell (DC) activation in vivo, further induces an antigen-specific immune response and has anticancer effects in mice. However, the effect of ascophyllan has not been studied in human immune cells, specifically in terms of activation of human monocyte-derived DCs (MDDCs) and human peripheral blood DCs (PBDCs). We found that the treatment with ascophyllan induced morphological changes in MDDCs and upregulated co-stimulatory molecules and major histocompatibility complex class I (MHC I) and MHC II expression. In addition, pro-inflammatory cytokine levels in culture medium was also dramatically increased following ascophyllan treatment of MDDCs. Moreover, ascophyllan promoted phosphorylation of ERK, p38 and JNK signaling pathways, and inhibition of p38 almost completely suppressed the ascophyllan-induced activation of MDDCs. Finally, treatment with ascophyllan induced activation of BDCA1 and BDCA3 PBDCs. Thus, these data suggest that ascophyllan could be used as an immune stimulator in humans.

USP14 Inhibition Regulates Tumorigenesis by Inducing Autophagy in Lung Cancer In Vitro.

The ubiquitin-proteasome system is an essential regulator of several cellular pathways involving oncogenes. Deubiquitination negatively regulates target proteins or substrates linked to both hereditary and sporadic forms of cancer. The deubiquitinating enzyme ubiquitin-specific protease 14 (USP14) is associated with proteasomes where it trims the ubiquitin chain on the substrate. Here, we found that USP14 is highly expressed in patients with lung cancer. We also demonstrated that USP14 inhibitors (IU1-47 and siRNA-USP14) significantly decreased cell proliferation, migration, and invasion in lung cancer. Remarkably, we found that USP14 negatively regulates lung tumorigenesis not only through apoptosis but also through the autophagy pathway. Our findings suggest that USP14 plays a crucial role in lung tumorigenesis and that USP14 inhibitors are potent drugs in lung cancer treatment.

Microwave-assisted sintering synthesis of greenish-yellow emitting Sr2 SiO4 :Eu2+ phosphors.

Europium ion (Eu2+ ) doped Sr2 SiO4 phosphors with greenish-yellow emission were synthesized using microwave-assisted sintering. The phase structure and photoluminescence (PL) properties of the obtained phosphor samples were investigated. The PL excitation spectra of the Sr2 SiO4 :Eu2+ phosphors exhibited a broad band in the range of 260 nm to 485 nm with a maximum at 361 nm attributed to the 5f-4d allowed transition of the Eu2+ ions. Under an excitation at 361 nm, the Sr2 SiO4 :Eu2+ phosphor exhibited a greenish-yellow emission peak at 541 nm with an International-Commission-on-Illumination (CIE) chromaticity of (0.3064, 0.4772). The results suggest that the microwave-assisted sintering method is promising for the synthesis of phosphors owing to the decreased sintering time without the use of additional reductive agents.

Sub-100 nm gold nanohole-enhanced Raman scattering on flexible PDMS sheets.

Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive vibrational spectroscopy technique enabling detection of multiple analytes at the molecular level in a nondestructive and rapid manner. In this work, we introduce a new approach to fabricate deep subwavelength-scaled (sub-100 nm) metallic nanohole arrays (quasi-3D metallic nanoholes) on flexible and highly efficient SERS substrates. Target structures have been fabricated using a two-step process consisting of (i) direct pattern transfer of spin-coated polymer films onto polydimethylsiloxane (PDMS) substrates by plasma etching with transferred anodic aluminum oxide masks, and (ii) producing SERS-active substrates by functionalization of the etched polymeric films followed by Au deposition. Such an all-dry, top-down lithographic approach enables on-demand patterning of SERS-active metallic nanoholes with high structural fidelity even onto flexible and stretchable substrates, thus making possible multiple sensing modes in a versatile fashion. For example, metallic nanoholes on flexible PDMS substrates are highly amenable to their integration with curved glass sticks, which can be used in optical fiber-integrated SERS systems. Au surfaces immobilized by probe DNA molecules show a selective enhancement of Raman scattering with Cy5-labeled complementary DNA (as compared to flat Au surfaces), demonstrating the potential of using the quasi-3D Au nanohole arrays for bio-sensing applications.

Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information-processing systems.

Purification of DNA-origami nanostructures by rate-zonal centrifugation.

Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1-1 µg purified DNA nanostructures, obtaining >100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40-80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1-100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.

Extracellular synthesis of gold bionanoparticles by Nocardiopsis sp. and evaluation of its antimicrobial, antioxidant and cytotoxic activities.

Advancement of biological process for the synthesis of bionanoparticles is evolving into a key area of research in nanotechnology. The present study deals with the biosynthesis, characterization of gold bionanoparticles by Nocardiopsis sp. MBRC-48 and evaluation of their antimicrobial, antioxidant and cytotoxic activities. The gold bionanoparticles obtained were characterized by UV-visible spectroscopy, X-ray diffraction analysis, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis and transmission electron microscopy (TEM). The synthesized gold bionanoparticles were spherical in shape with an average of 11.57 ± 1.24 nm as determined by TEM and dynamic light scattering (DLS) particle size analyzer, respectively. The biosynthesized gold nanoparticles exhibited good antimicrobial activity against pathogenic microorganisms. It showed strong antioxidant activity as well as cytotoxicity against HeLa cervical cancer cell line. The present study demonstrated the potential use of the marine actinobacterial strain of Nocardiopsis sp. MBRC-48 as an important source for gold nanoparticles with improved biomedical applications including antimicrobial, antioxidant as well as cytotoxic agent.

Functionalization of fatty acid vesicles through newly synthesized bolaamphiphile-DNA conjugates.

The surface functionalization of fatty acid vesicles will allow their use as nanoreactors for complex chemistry. In this report, the tethering of several DNA conjugates to decanoic acid vesicles for molecular recognition and synthetic purposes was explored. Due to the highly dynamic nature of these structures, only one novel bola-amphiphile DNA conjugate could interact efficiently with or spontaneously pierce into the vesicle bilayers without jeopardizing their self-assembly or stability. This molecule was synthesized via a Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (click reaction), and consists of a single hydrocarbon chain of 20 carbons having on one end a triazole group linked to the 5'-phosphate of the nucleic acid and on the other side a hydroxyl-group. Its insertion was so effective that a fluorescent label on the DNA complementary to the conjugate could be used to visualize fatty acid structures.

Immunosuppressive nanoparticles containing recombinant PD-L1 and methotrexate alleviate multi-organ inflammation.

Multi-organ inflammatory diseases are one of the most serious autoimmune diseases worldwide. The regulation of immune responses by immune checkpoint proteins influences the development and treatment of cancer and autoimmune diseases. In this study, recombinant murine PD-L1 (rmPD-L1) was used for controlling T cell immunity to treat multi-organ inflammation. To enhance the immunosuppressive effect, we incorporated methotrexate, an anti-inflammatory drug, into hybrid nanoparticles (HNPs) and decorated the surface of HNPs with rmPD-L1 to produce immunosuppressive HNPs (IsHNPs). IsHNP treatment effectively targeted PD-1-expressing CD4 and CD8 T cells in the splenocytes; additionally, it promoted the production of Foxp3-expressing regulatory T cells, which suppressed the differentiation of helper T cells. IsHNP treatment also inhibited anti-CD3 antibody-mediated activation of CD4 and CD8 T cells in mice in vivo. This treatment protected mice from multi-organ inflammation induced by the adoptive transfer of naïve T cells to recombination-activating gene 1 knockout mice. The results of this study imply the therapeutic potential of IsHNPs in the treatment of multi-organ inflammation and other inflammatory diseases.

Suppression of Lung Cancer Malignancy by Micellized siRNA through Cell Cycle Arrest.

UBA6-specific E2 conjugation enzyme 1 (USE1) is frequently overexpressed in lung cancer patients. Moreover, the critical role of USE1 in the progression of human lung cancer is also indicated. As the next step, the authors aim to develop USE1-targeted therapeutic agents based on RNA interference (RNAi). In this study, a lipid-modified DNA carrier, namely U4T, which consists of four consecutive dodec-1-ynyluracil (U) nucleobases to increase the cell permeability of siRNA targeting of USE1 is introduced. The U4Ts aggregate to form micelles, and the USE1-silencing siRNA-incorporated soft spherical nucleic acid aggregate (siSNA) can be created simply through base-pairing with siRNA. Treatment with siSNA is effective in suppressing tumor growth in vivo as well as cell proliferation, migration, and invasion of lung cancer cells. Furthermore, siSNA inhibited tumor cell growth by inducing cell cycle arrest in the G1 phase and apoptosis. Thus, the anti-tumor efficacy of siSNA in lung cancer cell lines and that siSNA possesses effective cell-penetrating ability without using cationic transfection moieties are confirmed. Collectively, these results suggest that siSNA can be applied to the clinical application of RNAi-based therapeutics for lung cancer treatment.

Pyropia yezoensis-derived porphyran attenuates acute and chronic colitis by suppressing dendritic cells.

Porphyran is known to inhibit immune cell function. Previously, porphyran was shown to prevent lipopolysaccharide-induced sepsis in mice. However, studies on the inhibitory effects of porphyran during colitis are currently lacking. In this study, we evaluated the effects of Pyropia yezoensis-derived porphyran on dextran sodium sulfate (DSS)-induced acute and chronic colitis. The oral or intraperitoneal administration of porphyran inhibited the progression of DSS-induced colitis in mice, with the former also preventing immune cell infiltration in the colon. The levels of intracellular interferon-γ and interleukin-17 in T cells decreased when porphyran was administered orally. Porphyran inhibited T cell activation by suppressing dendritic cells (DCs) and macrophages. Porphyran prevented pathogen-associated molecular pattern and damage-associated molecular pattern-dependent DC and macrophage activation. Finally, porphyran attenuated chronic colitis caused via the long-term administration of DSS. These findings indicate that the oral administration of porphyran can inhibit DSS-induced colitis by suppressing DC and macrophage activation.

Non-covalent monolayer-piercing anchoring of lipophilic nucleic acids: preparation, characterization, and sensing applications.

Functional interfaces of biomolecules and inorganic substrates like semiconductor materials are of utmost importance for the development of highly sensitive biosensors and microarray technology. However, there is still a lot of room for improving the techniques for immobilization of biomolecules, in particular nucleic acids and proteins. Conventional anchoring strategies rely on attaching biomacromolecules via complementary functional groups, appropriate bifunctional linker molecules, or non-covalent immobilization via electrostatic interactions. In this work, we demonstrate a facile, new, and general method for the reversible non-covalent attachment of amphiphilic DNA probes containing hydrophobic units attached to the nucleobases (lipid-DNA) onto SAM-modified gold electrodes, silicon semiconductor surfaces, and glass substrates. We show the anchoring of well-defined amounts of lipid-DNA onto the surface by insertion of their lipid tails into the hydrophobic monolayer structure. The surface coverage of DNA molecules can be conveniently controlled by modulating the initial concentration and incubation time. Further control over the DNA layer is afforded by the additional external stimulus of temperature. Heating the DNA-modified surfaces at temperatures >80 °C leads to the release of the lipid-DNA structures from the surface without harming the integrity of the hydrophobic SAMs. These supramolecular DNA layers can be further tuned by anchoring onto a mixed SAM containing hydrophobic molecules of different lengths, rather than a homogeneous SAM. Immobilization of lipid-DNA on such SAMs has revealed that the surface density of DNA probes is highly dependent on the composition of the surface layer and the structure of the lipid-DNA. The formation of the lipid-DNA sensing layers was monitored and characterized by numerous techniques including X-ray photoelectron spectroscopy, quartz crystal microbalance, ellipsometry, contact angle measurements, atomic force microscopy, and confocal fluorescence imaging. Finally, this new DNA modification strategy was applied for the sensing of target DNAs using silicon-nanowire field-effect transistor device arrays, showing a high degree of specificity toward the complementary DNA target, as well as single-base mismatch selectivity.

Nucleic acid amphiphiles: synthesis and self-assembled nanostructures.

This review provides an overview of a relatively new class of bio-conjugates, DNA amphiphiles, which consist of oligonucleotides covalently bonded to synthetic hydrophobic units. The reader will find the basic principles for the structural design and preparation methods of the materials. Moreover, the self-assembly into superstructures of higher order will be highlighted. Finally, some potential applications will be described.

Rylene Dye-Loaded Polymeric Nanoparticles for Photothermal Eradication of Harmful Dinoflagellates, Akashiwo sanguinea and Alexandrium pacificum.

In the era of climate changes, harmful dinoflagellate outbreaks that produce potent algal toxins, odor, and water discoloration in aquatic environments have been increasingly reported. Thus, various treatments have been attempted for the mitigation and management of harmful blooms. Here, we report engineered nanoparticles that consist of two different types of rylene derivatives encapsulated in polymeric micelles. In addition, to avoid dissociation of the aggregate, the core of micelle was stabilized via semi-interpenetrating network (sIPN) formation. On two types of the marine red-tide dinoflagellates, Akashiwo sanguinea and Alexandrium pacificum, the nanoparticle uptake followed by fluorescence labeling and photothermal effect was conducted. Firstly, fluorescence microscopy enabled imaging of the dinoflagellates with the ultraviolet chromophore, Lumogen Violet. Lastly, near-infrared (NIR) laser irradiation was exposed on the Lumogen IR788 nanoparticle-treated Ak. Sanguinea. The irradiation resulted in reduced cell survival due to the photothermal effect in microalgae. The results suggested that the nanoparticle, IR788-sIPN, can be applied for potential red-tide algal elimination.