Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy.

BACKGROUND: Immunogenic cell death (ICD) is a crucial approach to turn immunosuppressive tumor microenvironment (ITM) into immune-responsive milieu and improve the response rate of immune checkpoint blockade (ICB) therapy. However, cancer cells show resistance to ICD-inducing chemotherapeutic drugs, and non-specific toxicity of those drugs against immune cells reduce the immunotherapy efficiency. METHODS: Herein, we propose cancer cell-specific and pro-apoptotic liposomes (Aposomes) encapsulating second mitochondria-derived activator of caspases mimetic peptide (SMAC-P)-doxorubicin (DOX) conjugated prodrug to potentiate combinational ICB therapy with ICD. The SMAC-P (AVPIAQ) with cathepsin B-cleavable peptide (FRRG) was directly conjugated to DOX, and the resulting SMAC-P-FRRG-DOX prodrug was encapsulated into PEGylated liposomes. RESULTS: The SMAC-P-FRRG-DOX encapsulated PEGylated liposomes (Aposomes) form a stable nanostructure with an average diameter of 109.1 ± 5.14 nm and promote the apoptotic cell death mainly in cathepsin B-overexpressed cancer cells. Therefore, Aposomes induce a potent ICD in targeted cancer cells in synergy of SMAC-P with DOX in cultured cells. In colon tumor models, Aposomes efficiently accumulate in targeted tumor tissues via enhanced permeability and retention (EPR) effect and release the encapsulated prodrug of SMAC-P-FRRG-DOX, which is subsequently cleaved to SMAC-P and DOX in cancer cells. Importantly, the synergistic activity of inhibitors of apoptosis proteins (IAPs)-inhibitory SMAC-P sensitizing the effects of DOX induces a potent ICD in the cancer cells to promote dendritic cell (DC) maturation and stimulate T cell proliferation and activation, turning ITM into immune-responsive milieu. CONCLUSIONS: Eventually, the combination of Aposomes with anti-PD-L1 antibody results in a high rate of complete tumor regression (CR: 80%) and also prevent the tumor recurrence by immunological memory established during treatments.

The protective effect of L-theanine addition to a cryoprotectant on the storage viability of lactic acid bacteria.

Lactic acid bacteria (LAB) that metabolize sugars to obtain energy and produce a large amount of lactate through the process are well known for their benefits. However, they can be used on a large scale only when good storage stability is guaranteed. The vitality and stability of several LAB strains were effectively protected in this investigation by L-theanine at 1% of the appropriate concentration (Lactiplantibacillus plantarum MG5023, Enterococcus faecium MG5232, Lactococcus lactis MG4668, Streptococcus thermophilus MG5140, and Bifidobacterium animalis subsp. lactis MG741). The inclusion of L-theanine as a protective agent significantly enhanced the viability of all strains throughout the freeze-drying process compared to that of the non-coated probiotics. The efficacy of L-theanine in improving bacterial stability and survivability was evaluated using accelerated stability tests, gastrointestinal (GI) tract survivability tests, and adhesion assays with intestinal epithelial cells. The cell surface was covered with substances including L-theanine, according to morphological findings, providing efficient defense against a variety of external stresses. Therefore, by exerting anti-freezing and anti-thawing properties, the adoption of L-theanine as a new and efficient protective agent may improve the stability and viability of a variety of probiotics.

Outcome of Tendon Transfer for Monomelic Amyotrophy (Hirayama Disease).

Hirayama disease is a motor neuron disease predominantly affecting adolescent males. The identifying feature of Hirayama disease is unilateral forearm and intrinsic muscle weakness that spares the brachioradialis, termed "oblique atrophy." Hirayama disease progresses slowly over several years, followed by an abrupt arrest. The pathognomonic finding is the anterior displacement of the cervical spinal cord with the detachment of the posterior dura. Systematic clinical evaluation and appropriate diagnostic studies are crucial to rule out a variety of compressive, immune-mediated, and genetic disorders. We present a patient with Hirayama disease whose hand function was improved dramatically by a tendon transfer after nearly 3 years without a definitive diagnosis and call attention to the hand surgeon's role in identifying this rare disease to enable timely functional restoration.

Perspectives for Improving the Tumor Targeting of Nanomedicine via the EPR Effect in Clinical Tumors.

Over the past few decades, the enhanced permeability and retention (EPR) effect of nanomedicine has been a crucial phenomenon in targeted cancer therapy. Specifically, understanding the EPR effect has been a significant aspect of delivering anticancer agents efficiently to targeted tumors. Although the therapeutic effect has been demonstrated in experimental models using mouse xenografts, the clinical translation of the EPR effect of nanomedicine faces several challenges due to dense extracellular matrix (ECM), high interstitial fluid pressure (IFP) levels, and other factors that arise from tumor heterogeneity and complexity. Therefore, understanding the mechanism of the EPR effect of nanomedicine in clinics is essential to overcome the hurdles of the clinical translation of nanomedicine. This paper introduces the basic mechanism of the EPR effect of nanomedicine, the recently discussed challenges of the EPR effect of nanomedicine, and various strategies of recent nanomedicine to overcome the limitations expected from the patients' tumor microenvironments.

Preclinical development of carrier-free prodrug nanoparticles for enhanced antitumor therapeutic potential with less toxicity.

BACKGROUND: Nanomedicine has emerged as a promising strategy for cancer treatment. The most representative nanomedicine used in clinic is PEGylated liposomal doxorubicin DOXIL®, which is first FDA-approved nanomedicine. However, several shortcomings, such as low drug loading capacity, low tumor targeting, difficulty in mass production and potential toxicity of carrier materials, have hindered the successful clinical translation of nanomedicines. In this study, we report a preclinical development process of the carrier-free prodrug nanoparticles designed as an alternative formulation to overcome limitations of conventional nanomedicines in the terms of technical- and industrial-aspects. RESULTS: The carrier-free prodrug nanoparticles (F68-FDOX) are prepared by self-assembly of cathepsin B-specific cleavable peptide (FRRG) and doxorubicin (DOX) conjugates without any additional carrier materials, and further stabilized with Pluronic F68, resulting in high drug loading (> 50%). The precise and concise structure allow mass production with easily controllable quality control (QC), and its lyophilized powder form has a great long-term storage stability at different temperatures (- 4, 37 and 60 °C). With high cathepsin B-specificity, F68-FDOX induce a potent cytotoxicity preferentially in cancer cells, whereas their cytotoxicity is greatly minimized in normal cells with innately low cathepsin B expression. In tumor models, F68-FDOX efficiently accumulates within tumor tissues owing to enhanced permeability and retention (EPR) effect and subsequently release toxic DOX molecules by cathepsin B-specific cleavage mechanism, showing a broad therapeutic spectrum with significant antitumor activity in three types of colon, breast and pancreatic cancers. Finally, the safety of F68-FDOX treatment is investigated after single-/multi-dosage into mice, showing greatly minimized DOX-related toxicity, compared to free DOX in normal mice. CONCLUSIONS: Collectively, these results provide potential preclinical development process of an alternative approach, new formulation of carrier-free prodrug nanoparticles, for clinical translation of nanomedicines.

The Dorsal Ligament Complex: A Cadaveric, Histology, and Imaging Study.

PURPOSE: The distinction between the dorsal intercarpal (DIC) and dorsal scaphotriquetral (DST) ligaments is imprecise and unclear in the literature. The purpose of our cadaveric study was to define the origins, insertions, and anatomic relationships of the dorsal wrist ligaments and relate these anatomic findings to magnetic resonance imaging (MRI) scans and histology. METHODS: The study included 17 unmatched fresh-frozen cadaveric specimens (7 male and 10 female), with a mean age of 67.1 years (range, 48-86 years). Wrists with arthritis or carpal malalignment were excluded. Ligaments were dissected and insertion sites were recorded in the radioulnar (width) and proximodistal (length) dimensions, centered at the midpoints of the insertion. Three cadaveric specimens underwent a histologic analysis to demonstrate ligament composition and insertion sites. Three additional cadavers underwent MRI, from which 3-dimensional models were built to model ligament topography. RESULTS: The conjoined triquetral insertion of the DIC, DST, and dorsal radiocarpal (DRC) measured 88.5 ± 6.4 mm2. In each specimen, there were 2 distinct deep and superficial components of intercarpal fibers. The deep component inserted on the lunate with an area of 59.0 ± 5.0 mm2. The deep and superficial components diverged as they coursed radially. The superficial component proceeded to the scaphoid ridge, trapezium, and trapezoid, whereas the deep component inserted on the proximal row. The deep fibers blended distally from their lunate insertion with the DST, forming a robust, 2.9 ± 0.8-mm wide extension over the dorsal capitate. The DRC inserted on the lunate, proximal to the DIC and DST insertions, with an area of 23.9 ± 5.4 mm2. CONCLUSIONS: The dorsal ligament complex forms a firm link across the proximal carpal row and the DST provides extension of the proximal row over the capitate. CLINICAL RELEVANCE: This information can guide surgeons while performing a dorsal approach to the wrist and repairing traumatic ligament disruption.

Intrinsic structure and dynamics of monolayer ring polymer melts.

We present the general structural and dynamical characteristics of flexible ring polymers in narrowly confined two-dimensional (2D) melt systems using atomistic molecular dynamics simulations. The results are further analyzed via direct comparison with the 2D linear analogue as well as the three-dimensional (3D) ring and linear melt systems. It is observed that dimensional restriction in 2D confined systems results in an increase in the intrinsic chain stiffness of the ring polymer. Fundamentally, this arises from an entropic penalty on polymer chains along with a reduction in the available chain configuration states in phase space and spatial choices for individual segmental walks. This feature in combination with the intermolecular interactions between neighboring ring chains leads to an overall extended interpenetrated chain configuration for the 2D ring melt. In contrast to the generally large differences in structural and dynamical properties between ring and linear polymers in 3D melt systems, relatively similar local-to-global chain structures and dynamics are observed for the 2D ring and linear melts. This is attributed to the general structural similarity (i.e., extended double-stranded chain conformations), the less effective role of the chain ends, and the absence of complex topological constraints between chains (i.e., interchain entanglement and mutual ring threading) in the 2D confined systems compared with the corresponding 3D bulk systems.

Thiol-Responsive Gold Nanodot Swarm with Glycol Chitosan for Photothermal Cancer Therapy.

Photothermal therapy (PTT) is one of the most promising cancer treatment methods because hyperthermal effects and immunogenic cell death via PTT are destructive to cancer. However, PTT requires photoabsorbers that absorb near-infrared (NIR) light with deeper penetration depth in the body and effectively convert light into heat. Gold nanoparticles have various unique properties which are suitable for photoabsorbers, e.g., controllable optical properties and easy surface modification. We developed gold nanodot swarms (AuNSw) by creating small gold nanoparticles (sGNPs) in the presence of hydrophobically-modified glycol chitosan. The sGNPs assembled with each other through their interaction with amine groups of glycol chitosan. AuNSw absorbed 808-nm laser and increased temperature to 55 °C. In contrast, AuNSw lost its particle structure upon exposure to thiolated molecules and did not convert NIR light into heat. In vitro studies demonstrated the photothermal effect and immunogenic cell death after PTT with AuNSW. After intratumoral injection of AuNSw with laser irradiation, tumor growth of xenograft mouse models was depressed. We found hyperthermal damage and immunogenic cell death in tumor tissues through histological and biochemical analyses. Thiol-responsive AuNSw showed feasibility for PTT, with advanced functionality in the tumor microenvironment.

In Situ One-Step Fluorescence Labeling Strategy of Exosomes via Bioorthogonal Click Chemistry for Real-Time Exosome Tracking In Vitro and In Vivo.

Exosomes are cellular components with promising uses in cancer diagnostics and therapeutics, and their imaging and tracking are essential to study their biological properties. Herein, we report on an in situ one-step fluorescence labeling strategy for exosomes via bioorthogonal click chemistry. First, exosome donor cancer cells were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to generate unnatural azide groups (-N3) on their surface via metabolic glycoengineering. Then, the azide groups were labeled with near-infrared fluorescent dye-conjugated dibenzylcyclooctyne (DBCO-Cy5) via bioorthogonal click chemistry. After 2 days of incubation, the DBCO-Cy5-labeled exosomes (Cy5-Exo) were successfully secreted from the donor cancer cells and were isolated via classical ultracentrifugation, providing a high-yield of fluorescent dye-labeled exosomes. This in situ one-step bioorthogonal click chemistry offers improved labeling efficiency, biocompatibility, and imaging sensitivy compared to standard exosomes (ST-Exo), purified with classical ultracentrifugation or carbocyanine lipophilic dye (DiD)-labeled exosomes (DiD-Exo) in vitro. In particular, the Cy5-Exo were successfully taken up by A549 cells in a time-dependent manner, and they could escape from lysosome confinement, showing their possible use as a delivery carrier of therapeutic drugs or imaging agents. Finally, intraveneously injected Cy5-Exo were noninvasively tracked and imaged via near-infrared fluorescence (NIRF) imaging in tumor-bearing mice. This new fluorescence labeling strategy for natural exosomes may be useful to provide better understanding of their theranostic effects in many biomedical applications.

Depth-Based Detection of Standing-Pigs in Moving Noise Environments.

In a surveillance camera environment, the detection of standing-pigs in real-time is an important issue towards the final goal of 24-h tracking of individual pigs. In this study, we focus on depth-based detection of standing-pigs with "moving noises", which appear every night in a commercial pig farm, but have not been reported yet. We first apply a spatiotemporal interpolation technique to remove the moving noises occurring in the depth images. Then, we detect the standing-pigs by utilizing the undefined depth values around them. Our experimental results show that this method is effective for detecting standing-pigs at night, in terms of both cost-effectiveness (using a low-cost Kinect depth sensor) and accuracy (i.e., 94.47%), even with severe moving noises occluding up to half of an input depth image. Furthermore, without any time-consuming technique, the proposed method can be executed in real-time.

Atomic layer deposition of diisopropylaminosilane on WO3(001) and W(110): a density functional theory study.

The decomposition reactions of the Si precursor, diisopropylaminosilane (DIPAS), on W(110) and hydroxylated WO3(001) surfaces are investigated to elucidate the initial reaction mechanism of the atomic layer deposition (ALD) process using density functional theory (DFT) calculations combined with ab initio molecular dynamics (AIMD) simulations. The decomposition reaction of DIPAS on WO3(001) consists of two steps: Si-N dissociative chemisorption and decomposition of SiH3*. It is found that the Si-N bond cleavage of DIPAS is facile on WO3(001) due to hydrogen bonding between the surface OH group and the N atom of DIPAS. The rate-determining step of DIPAS decomposition on WO3(001) is found to be the Si-H dissociation reaction of the SiH3* reaction intermediate which has an activation barrier of 1.19 eV. On the contrary, sequential Si-H dissociation reactions first occur on W(110) and then the Si-N dissociation reaction of the C5H7NSi* reaction intermediate is found to be the rate-determining step, which has an activation barrier of 1.06 eV. As a result, the final products in the DIPAS decomposition reaction on WO3(001) are Si* and SiH*, whereas Si* atoms remain with carbon impurities on W(110), which imply that the hydroxylated WO3 surface is more efficient for the ALD process.

Transformation of nanoparticles via the transition of functional DNAs responsive to pH and vascular endothelial growth factor for photothermal anti-tumor therapy.

This study presents a novel approach for the development of DNA-functionalized gold nanoparticles (AuNPs) capable of responding to disease-specific factors and microenvironmental changes, resulting in an effective anti-tumor effect via photothermal therapy. The AuNPs are decorated with two types of DNAs, an i-motif duplex and a VEGF split aptamer, enabling recognition of changes in pH and VEGF, respectively. The formation of VEGF aptamers on the AuNPs induces their aggregation, further enhanced by VEGF ligands. The resulting changes in the optical properties of the AuNPs are detected by monitoring the absorbance. Upon irradiation with a near-infrared laser, the aggregated AuNPs generate heat due to their thermoplasmonic characteristic, leading to an anti-tumor effect. This study demonstrates the enhanced anti-tumor effect of DNA-functionalized AuNPs via photothermal therapy in both in vitro and in vivo tumor models. These findings suggest the potential utilization of such functional AuNPs for precise disease diagnosis and treatment by detecting disease-related factors in the microenvironment.

Quality characteristics and antioxidant activity of meringue Jeung-pyun with different amounts of cacao bean husk.

In this study, we aimed to investigate the quality characteristics, antioxidant activity, and sensory traits of meringue Jeung-pyun with different amounts of cacao bean husk powder. Based on our analyses, high cacao bean husk content resulted in an increase in certain Jeung-pyun qualities, such as the L values, b values, hardness, gumminess, and number of pores, whereas the moisture content, pH, pore size, adhesiveness, cohesiveness, and chewiness significantly decreased. Electronic tongue analysis showed that the intensity of sourness, saltiness, and umami increased with the amount of cacao bean husk added. For the sensory characteristics, C6 demonstrated the highest ranking for all test items. Furthermore, it was found that the addition of cacao bean husks increased the antioxidant activity of the Jeung-pyun (p < 0.001). Therefore, these results suggest that Jeung-pyun produced with a mixing ratio of C6 has excellent qualities, antioxidant activities, and sensory characteristics.

Flavor characteristics of garlic fish cakes using electronic nose and tongue analyses.

This study investigated the utility of garlic powder as a functional ingredient. The aim was to develop fish cakes with improved functionality and sensory preference based on the antioxidant activity and quality characteristics. Increasing amounts of garlic powder in the prepared fish cakes were associated with increasing total polyphenol and flavonoid contents, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS+) radical scavenging activity, and reducing power. Furthermore, electronic tongue and electronic nose analyses showed an increased the intensity of umami and sourness and increased the levels of volatile compounds. The lowest trimethylamine peak corresponded to the highest amount of garlic powder. Sensory evaluation indicated that 3% garlic powder had the highest score for all criteria. Fishy odor decreased as the proportion of garlic powder increased. These findings suggest that the addition of 3% garlic powder improves quality characteristics, sensory preference, and antioxidant activity of fish cakes.

Zeolite Membrane-Based Low-Temperature Dehydrogenation of a Liquid Organic Hydrogen Carrier: A Key Step in the Development of a Hydrogen Economy.

Methylcyclohexane (MCH) dehydrogenation is an equilibrium-limited reaction that requires high temperatures (>300 °C) for complete conversion. However, high-temperature operation can degrade catalytic activity and produce unwanted side products. Thus, a hybrid zeolite membrane (Z) is prepared on the inner surface of a tubular support and used it as a wall in a membrane reactor (MR) configuration. Pt/C catalysts is packed diluted with quartz sand inside the Z-coated tube and applied the MR for MCH dehydrogenation at low temperatures (190-250 °C). Z showed a remarkable H2-permselectivity in the presence of both toluene and MCH, yielding separation factors over 350. The Z-based MR achieved higher MCH conversion (75.3% ± 0.8% at 220 °C) than the conventional packed-bed reactor (56.4% ± 0.3%) and the equilibrium state (53.2%), owing to the selective removal of H2 through Z. In summary, the hybrid zeolite MR enhances MCH dehydrogenation at low temperatures by overcoming thermodynamic limitations and improves the catalytic performance and product selectivity of the reaction.

Accurately Controlled Tumor Temperature with Silica-Coated Gold Nanorods for Optimal Immune Checkpoint Blockade Therapy.

Photothermal therapy (PTT) at mild temperatures ranging from 44 to 45 °C holds tremendous promise as a strategy for inducing potent immunogenic cell death (ICD) within tumor tissues, which can reverse the immunosuppressive tumor microenvironment (ITM) into an immune-responsive milieu. However, accurately and precisely controlling the tumor temperature remains a formidable challenge. Here, we report the precision photothermal immunotherapy by using silica-coated gold nanorods (AuNR@SiO2), and investigating the optimal administration routes and treatment protocols, which enabled to achieve the sustained and controlled mild heating within the tumor tissues. First, the highest photothermal performance of AuNR@SiO2 with 20-nm silica shell thickness than 5 or 40 nm was confirmed in vitro and in vivo. Then, the optimal conditions for precision immunotherapy were further investigated to produce mild temperature (44 to 45 °C) accurately in tumor tissues. The optimal conditions with AuNR@SiO2 result in a distinct cell death with high early/late apoptosis and low necrosis, leading to very efficient ICD compared to lower or higher temperatures. In colon tumor-bearing mice, intratumorally injected AuNR@SiO2 efficiently promotes a mild temperature within the tumor tissues by local irradiation of near-infrared (NIR) laser. This mild PTT substantially increases the population of mature dendritic cells (DCs) and cytotoxic T cells (CTLs) within tumor tissues, ultimately reversing the ITM into an immune-responsive milieu. Furthermore, we found that the combination mild PTT with AuNR@SiO2 and anti-PD-L1 therapy could lead to the 100% complete regression of primary tumors and immunological memory to prevent tumor recurrence. Collectively, this study demonstrates that AuNR@SiO2 with a robust methodology capable of continuously inducing mild temperature accurately within the ITM holds promise as an approach to achieve the precision photothermal immunotherapy.

Photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes to promote PD-L1 multivalent binding for effective immune checkpoint blockade therapy.

Immune checkpoint blockade (ICB) therapy targeting PD-L1 via monoclonal antibody (mAb) has shown extensive clinical benefits in the diverse types of advanced malignancies. However, most patients are completely refractory to ICB therapy owing to the PD-L1 recycling mechanism. Herein, we propose photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes (immune checkpoint blockade liposomes; ICB-LPs) to promote PD-L1 multivalent binding for inducing lysosomal degradation of PD-L1 in tumor cells. The ICB-LPs are prepared by formulation of DC8,9PC with photo-polymerized diacetylenic moiety, 1,2-dipalmitoylphosphatidylcholine (DPPC) and anti-PD-L1 peptide (D-form NYSKPTDRQYHF)-conjugated DSPE-PEG2k (anti-PD-L1-DSPE-PEG2k) in a molar ratio of 45:45:10, followed by cross-linking of liposomal bilayer upon UV irradiation. The 10 mol% anti-PD-L1-DSPE-PEG2k incorporated ICB-LPs have a nano-sized lipid bilayer structure with an average diameter of 137.7 ± 1.04 nm, showing a high stability in serum condition. Importantly, the ICB-LPs efficiently promote the multivalent binding with PD-L1 on the tumor cell membrane, which are endocytosed with aim to deliver PD-L1 to the lysosomes, wherein the durable PD-L1 degradation is observed for 72 h, in contrast to anti PD-L1 mAbs showing the rapid PD-L1 recycling within 9 h. The in vitro co-culture experiments with CD8+ T cells show that ICB-LPs effectively enhance the T cell-mediated antitumor immune responses against tumor cells by blocking the PD-L1/PD-1 axis. When ICB-LPs are intravenously injected into colon tumor-bearing mice, they efficiently accumulate within the targeted tumor tissues via both passive and active tumor targeting, inducing a potent T cell-mediated antitumor immune response by effective and durable PD-L1 degradation. Collectively, this study demonstrates the superior antitumor efficacy of crosslinked and anti-PD-L1 peptide incorporated liposome formulation that promotes PD-L1 multivalent binding for trafficking of PD-L1 toward the lysosomes instead of the recycling endosomes.

Effect of Abiotic Signals on the Accumulation of Saponarin in Barley Leaves in Hydroponics Under Artificial Lights.

Functional flavonoid production is a new agenda in the agricultural industry, and young barley leaves (YBL) are one of the highlighted crops due to their health-beneficial flavonoid, saponarin. For the year-round cultivation of a high saponarin content of YBL, abiotic signal effects on the biosynthesis and metabolism in YBL need to be understood clearly. In this research, the effects of reactive oxygen species (ROS)-related abiotic signals, such as light, potassium, and sodium, were investigated on the biosynthetic metabolism in YBL cultivation under artificial lights. A higher quantity of blue-rich white light (6500 K of light temperature) irradiation enhanced ROS levels and the related enzyme activities (APX and CAT), as well as photosynthesis and saponarin amount, while red-rich white light (3000 K of light temperature) increased the photosynthesis only. In addition, 1.0 g L-1 K+ treatment in water slightly reduced ROS levels and increased saponarin accumulation in YBL. These blue-rich light and K+ supplemental conditions relatively increased OGT expression and reduced 4-coumaric acid and isovitexin as saponarin precursors. Furthermore, the relative ratio of lutonarin as an oxidized product of saponarin increased in increments of light quantity. Finally, the abiotic conditions for saponarin production were optimized with the mixture solution treatment of 1.0 g L-1 Na+ and 1.0 g L-1 K+ under 500 PPFD of 6500 K light, and the saponarin amount per leaf was 219.5 µg plant-1; it was comparable amount with that under sunlight condition.

Solution-free and simplified H&E staining using a hydrogel-based stamping technology.

Hematoxylin and eosin (H&E) staining has been widely used as a fundamental and essential tool for diagnosing diseases and understanding biological phenomena by observing cellular arrangements and tissue morphological changes. However, conventional staining methods commonly involve solution-based, complex, multistep processes that are susceptible to user-handling errors. Moreover, inconsistent staining results owing to staining artifacts pose real challenges for accurate diagnosis. This study introduces a solution-free H&E staining method based on agarose hydrogel patches that is expected to represent a valuable tool to overcome the limitations of the solution-based approach. Using two agarose gel-based hydrogel patches containing hematoxylin and eosin dyes, H&E staining can be performed through serial stamping processes, minimizing color variation from handling errors. This method allows easy adjustments of the staining color by controlling the stamping time, effectively addressing variations in staining results caused by various artifacts, such as tissue processing and thickness. Moreover, the solution-free approach eliminates the need for water, making it applicable even in environmentally limited middle- and low-income countries, while still achieving a staining quality equivalent to that of the conventional method. In summary, this hydrogel-based H&E staining method can be used by researchers and medical professionals in resource-limited settings as a powerful tool to diagnose and understand biological phenomena.

Micro-syringe chip-guided intratumoral administration of lipid nanoparticles for targeted anticancer therapy.

BACKGROUND: Nano-sized drug delivery system has been widely studied as a potential technique to promote tumor-specific delivery of anticancer drugs due to its passive targeting property, but resulting in very restricted improvements in its systemic administration so far. There is a requirement for a different approach that dramatically increases the targeting efficiency of therapeutic agents at targeted tumor tissues. METHODS: To improve the tumor-specific accumulation of anticancer drugs and minimize their undesirable toxicity to normal tissues, a tumor-implantable micro-syringe chip (MSC) with a drug reservoir is fabricated. As a clinically established delivery system, six liposome nanoparticles (LNPs) with different compositions and surface chemistry are prepared and their physicochemical properties and cellular uptake are examined in vitro. Subsequently, MSC-guided intratumoral administration is studied to identify the most appropriate for the higher tumor targeting efficacy with a uniform intratumoral distribution. For efficient cancer treatment, pro-apoptotic anticancer prodrugs (SMAC-P-FRRG-DOX) are encapsulated to the optimal LNPs (SMAC-P-FRRG-DOX encapsulating LNPs; ApoLNPs), then the ApoLNPs are loaded into the 1 µL-volume drug reservoir of MSC to be delivered intratumorally for 9 h. The tumor accumulation and therapeutic effect of ApoLNPs administered via MSC guidance are evaluated and compared to those of intravenous and intratumoral administration of ApoLNP in 4T1 tumor-bearing mice. RESULTS: MSC is precisely fabricated to have a 0.5 × 4.5 mm needle and 1 µL-volume drug reservoir to achieve the uniform intratumoral distribution of LNPs in targeted tumor tissues. Six liposome nanoparticles with different compositions of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (PS), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)2000] (PEG2000-DSPE) are prepared with average sizes of 100-120 nm and loaded into the 1 µL-volume drug reservoir in MSC. Importantly negatively charged 10 mol% of PS-containing LNPs are very slowly infused into the tumor tissue through the micro-syringe of the MSC over 6 h. The intratumoral targeting efficiency of MSC guidance is 93.5%, effectively assisting the homogeneous diffusion of LNPs throughout the tumor tissue at 3.8- and 2.7-fold higher concentrations compared to the intravenous and intratumoral administrations of LNPs, respectively. Among the six LNP candidates 10 mol% of PS-containing LNPs are finally selected for preparing pro-apoptotic SMAC-P-FRRG-DOX anticancer prodrug-encapsulated LNPs (ApoLNPs) due to their moderate endocytosis rate high tumor accumulation and homogenous intratumoral distribution. The ApoLNPs show a high therapeutic effect specifically to cathepsin B-overexpressing cancer cells with 6.6 µM of IC50 value while its IC50 against normal cells is 230.7 µM. The MSC-guided administration of ApoLNPs efficiently inhibits tumor growth wherein the size of the tumor is 4.7- and 2.2-fold smaller than those treated with saline and intratumoral ApoLNP without MSC, respectively. Moreover, the ApoLNPs remarkably reduce the inhibitor of apoptosis proteins (IAPs) level in tumor tissues confirming their efficacy even in cancers with high drug resistance. CONCLUSION: The MSC-guided administration of LNPs greatly enhances the therapeutic efficiency of anticancer drugs via the slow diffusion mechanism through micro-syringe to tumor tissues for 6 h, whereas they bypass most hurdles of systemic delivery including hepatic metabolism, rapid renal clearance, and interaction with blood components or other normal tissues, resulting in the minimum toxicity to normal tissues. The negatively charged ApoLNPs with cancer cell-specific pro-apoptotic prodrug (SMAC-P-FRRG-DOX) show the highest tumor-targeting efficacy when they are treated with the MSC guidance, compared to their intravenous or intratumoral administration in 4T1 tumor-bearing mice. The MSC-guided administration of anticancer drug-encapsulated LNPs is expected to be a potent platform system that facilitates overcoming the limitations of systemic drug administration with low delivery efficiency and serious side effects.