Tumor-Penetrable Nitric Oxide-Releasing Nanoparticles Potentiate Local Antimelanoma Therapy.

Although nitric oxide (NO) has been emerging as a novel local anticancer agent because of its potent cytotoxic effects and lack of off-target side effects, its clinical applications remain a challenge because of the short effective diffusion distance of NO that limits its anticancer activity. In this study, we synthesized albumin-coated poly(lactic-co-glycolic acid) (PLGA)-conjugated linear polyethylenimine diazeniumdiolate (LP/NO) nanoparticles (Alb-PLP/NO NPs) that possess tumor-penetrating and NO-releasing properties for an effective local treatment of melanoma. Sufficient NO-loading and prolonged NO-releasing characteristics of Alb-PLP/NO NPs were acquired through PLGA-conjugated LP/NO copolymer (PLP/NO) synthesis, followed by nanoparticle fabrication. In addition, tumor penetration ability was rendered by the electrostatic adsorption of the albumin on the surface of the nanoparticles. The Alb-PLP/NO NPs showed enhanced intracellular NO delivery efficiency and cytotoxicity to B16F10 murine melanoma cells. In B16F10-tumor-bearing mice, the Alb-PLP/NO NPs showed improved extracellular matrix penetration and spatial distribution in the tumor tissue after intratumoral injection, resulting in enhanced antitumor activity. Taken together, the results suggest that Alb-PLP/NO NPs represent a promising new modality for the local treatment of melanoma.

Nuclear Factor Kappa-B/Homeobox A9-Mediated Modulation of Leucine-Rich Repeat Flightless-Interacting Protein 1 Is Involved in Advanced Glycation End Product-Induced Endothelial Dysfunction.

BACKGROUND: Pathogenesis of cardiovascular diseases begins with endothelial dysfunction. Our previous study has shown that advanced glycation end products (AGE) could inhibit the expression of homeobox A9 (Hoxa9), thereby inducing endothelial dysfunction. Leucine-rich repeat flightless-interacting protein 1 (LRRFIP1) has been found to participate in a variety of pathological processes, but reports of its role in endothelial dysfunction are rare. OBJECTIVES: This study aims to investigate whether LRRFIP1 is involved in AGE-induced endothelial dysfunction through Hoxa9-mediated transcriptional activation. METHODS: Chromatin immunoprecipitation was used to detect the transcriptional regulation of Hoxa9 on LRRFIP1 promoters. Human umbilical vein endothelial cells were treated with AGE or pyrrolidinedithiocarbamate (nuclear factor kappa-B [NF-κB] inhibitor). Moreover, changes in apoptosis, proliferation, migration, release of nitric oxide, and angiogenesis were detected. RESULTS: Hoxa9 promotes LRRFIP1 expression by binding to the -LRRFIP1 promoter. Meanwhile, overexpression of LRRFIP1 inhibited phosphorylation of P65 and elevated expression of Hoxa9. Overexpression of LRRFIP1 or/and Hoxa9 reversed the effects of AGE on HUVEC. AGE-induced inhibition on the expression of LRRFIP1 and Hoxa9 could be reversed by the NF-κB inhibitor. CONCLUSION: LRRFIP1 is involved in AGE-induced endothelial dysfunction via being regulated by the NF-κB/Hoxa9 axis.

Advanced glycation end-products disrupt brain microvascular endothelial cell barrier: The role of mitochondria and oxidative stress.

During diabetes mellitus, advanced glycation end-products (AGEs) are major contributors to the development of alterations in cerebral capillaries, leading to the disruption of the blood-brain barrier (BBB). Consequently, this is often associated with an amplified oxidative stress response in microvascular endothelial cells. As a model to mimic brain microvasculature, the bEnd.3 endothelial cell line was used to investigate cell barrier function. Cells were exposed to native bovine serum albumin (BSA) or modified BSA (BSA-AGEs). In the presence or absence of the antioxidant compound, N-acetyl-cysteine, cell permeability was assessed by FITC-dextran exclusion, intracellular free radical formation was monitored with H2DCF-DA probe, and mitochondrial respiratory and redox parameters were analyzed. We report that, in the absence of alterations in cell viability, BSA-AGEs contribute to an increase in endothelial cell barrier permeability and a marked and prolonged oxidative stress response. Decreased mitochondrial oxygen consumption was associated with these alterations and may contribute to reactive oxygen species production. These results suggest the need for further research to explore therapeutic interventions to restore mitochondrial functionality in microvascular endothelial cells to improve brain homeostasis in pathological complications associated with glycation.

In Situ One-Pot Synthesis of Fe2O3@BSA Core-Shell Nanoparticles as Enhanced T1-Weighted Magnetic Resonance Imagine Contrast Agents.

Ultra-small-sized iron oxide nanoparticles with good biocompatibility are regarded as promising alternatives for the gadolinium-based contrast agents, which are widely used as a positive contrast agent in magnetic resonance imaging (MRI). However, the current preparation of the iron oxide magnetic nanoparticles with small sizes usually involves organic solvents, increasing the complexity of hydrophilic ligand replacement and reducing the synthesis efficiency. It remains a great challenge to explore new iron oxide nanoparticles with good biocompatibility and a high T1 contrast effect. Here, we reported a cage-like protein architecture self-assembled by approximately 6-7 BSA (bovine serum albumin) subunits. The BSA nanocage was then used as a biotemplate to synthesize uniformed and monodispersed Fe2O3@BSA nanoparticles with ultra-small sizes (∼3.5 nm). The Fe2O3@BSA nanoparticle showed a high r1 value of 6.8 mM-1 s-1 and a low r2/r1 ratio of 10.6 at a 3 T magnetic field. Compared to Gd-DTPA, the brighter signal and prolonged angiographic effect of Fe2O3@BSA nanoparticles could greatly benefit steady-state and high-resolution imaging. The further in vivo and in vitro assessments of stability, toxicity, and renal clearance indicated a substantial potential as a T1 contrast agent in preclinical MRI.

Integration of Fe3O4 with Bi2S3 for Multi-Modality Tumor Theranostics.

The combination of reactive oxygen species (ROS)-induced chemodynamic therapy (CDT) and photothermal therapy (PTT) holds a promising application prospect for their superb anticancer efficiency. Herein, we created a novel Fe3O4@polydopamine (PDA)@bovine serum albumin (BSA)-Bi2S3 composite as a theranostic agent, by chemically linking the Fe3O4@PDA with BSA-Bi2S3 via the amidation between the carboxyl groups of BSA and the amino groups of PDA. In this formulation, the Fe3O4 NPs could not only work as a mimetic peroxidase to trigger Fenton reactions of the innate H2O2 in the tumor and generate highly cytotoxic hydroxyl radicals (•OH) to induce tumor apoptosis but also serve as the magnetic resonance imaging (MRI) contrast agent to afford the precise cancer diagnosis. Meanwhile, the PDA could prevent the oxidization of Fe3O4, thus supporting the long-term Fenton reactions and the tumor apoptosis in the tumor. The Bi2S3 component exhibits excellent photothermal transducing performance and computed tomography (CT) imaging capacity. In addition, the PDA and Bi2S3 endow the Fe3O4@PDA@BSA-Bi2S3 composite with an excellent photothermal transforming ability which could lead to tumor hyperthermia. All of these merits play the synergism with the tumor microenvironment and qualify the Fe3O4@PDA@BSA-Bi2S3 NPs for a competent agent in the MRI/CT-monitored enhanced PTT/CDT synergistic therapy. Findings in this research will evoke new interests in future cancer therapeutic strategies based on biocompatible nanomaterials.

IR780 loaded sulfobetaine methacrylate-functionalized albumin nanoparticles aimed for enhanced breast cancer phototherapy.

New insights about nanomaterials' biodistribution revealed their ability to achieve tumor accumulation by taking advantage from the dynamic vents occurring in tumor's vasculature. This paradigm-shift emphasizes the importance of extending nanomaterials' blood circulation time to enhance their tumor uptake. The classic strategy to improve nanomaterials' stability during circulation relies on their functionalization with poly(ethylene glycol). However, recent reports have been showing that PEGylated nanomaterials can suffer from the accelerated blood clearance phenomenon, emphasizing the importance of developing novel coatings for functionalizing the nanomaterials. To address this limitation, the modification of natural carriers' surface to enhance their stability appears to be a promising strategy. Herein, sulfobetaine methacrylate (SBMA)-functionalized bovine serum albumin (BSA) was synthesized for the first time to investigate the capacity of this modification to improve the resulting nanoparticles' physicochemical properties, colloidal stability and in vitro performance. This novel polymer was then employed in the formulation of nanoparticles loaded with IR780 for application in breast cancer phototherapy (IR/SBMA-BSA NPs). When compared to their non-functionalized equivalents, the IR/SBMA-BSA NPs presented a neutral surface charge and a higher stability in biologically relevant media. Due to these features, the IR/SBMA-BSA NPs could achieve a 1.9-fold greater uptake by breast cancer cells than IR/BSA NPs. Furthermore, the IR/SBMA-BSA NPs were cytocompatible towards normal cells and reduced breast cancer cells' viability up to 42%. The phototherapy mediated by IR/SBMA-BSA NPs could further decrease cancer cells' viability to about 12%. Overall, the IR/SBMA-BSA NPs have enhanced features that propel their application in breast cancer phototherapy.

Involvement of prohibitin 1 and prohibitin 2 upregulation in cBSA-induced podocyte cytotoxicity.

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, when not effectively treated. The aim of this study was to discover new targets for the diagnosis and treatment of MN. A reliable mouse model of MN was used by the administration of cationic bovine serum albumin (cBSA). Mice with MN exhibited proteinuria, histopathological changes, and accumulation of immune complexes in the glomerular basement membrane. Label-free proteomics analysis was performed to identify changes in protein expression, and the overexpressed proteins were evaluated. There were 273 proteins that showed significantly different expression in mice with MN, as compared to the controls. String analysis showed that functions related to cellular catabolic processes were downregulated in MN. Among the differentially expressed proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2) were upregulated in the kidneys of mice with MN, as demonstrated by immunohistochemistry (IHC), and this upregulation was observed in both the tubular cells and glomeruli. Both shRNA-mediated knockdown of PHB1 or PHB2 inhibited tumor suppressor p53 expression and significantly promoted podocyte proliferation. In addition, both PHB1 and PHB2 were responsible for cBSA-induced cytotoxicity. Microarray analysis further revealed that the upregulation of PHB1 and PHB2 may be due to a blockage of proteasome activity. These data demonstrate that the upregulation of PHB2 is involved in cBSA-mediated podocyte cytotoxicity, which may lead to MN development.

Oligochitosan modified albumin as plasmid DNA delivery vector: Endocytic trafficking, polyplex fate, in vivo compatibility.

Cationic macromolecules condense DNA into small nanoparticles and form polyplex. The composition of the polyplex determines the endocytic process, the intracellular routing and the fate of the polyplex. Previously, oligochitosan-modified vectors with different protein moieties are used as gene delivery vector and the types of protein moiety can influence the endosome escape ability and transfection efficiency. Among the modified vectors, oligochitosan-modified bovine serum albumin (BSA) showed 90% transfection efficeincy compared to the modified zein and ovalbumin. These data encouraged us to investigate the mechanism of internalization involved in the superior transfection efficiency of modified BSA/ plasmid polyplex. The effect of specific endocytic inhibitors was studied in two adherent cell lines. The caveolae-mediated and lipid-mediated pathways play a significant role in the polyplex internalization. Next, a colocation of polyplex with lysosome was investigated in the presence of LysoTracker using confocal microscopy. Up to 70% of polyplex successfully escaped the lysosome without degradation. Four non-adherent cell lines showed above than 60% transfection efficiency at an optimized vector/plasmid ratio. Moreover, no significant hemolytic effect was observed up to 500 µg/mL of cationic BSA, indicating no detectable cell membrane disruption. Overall, the hybrid biomacromolecule showed good intracellular delivery and safety in a mice model.

Neurotoxicity of silver nanoparticles stabilized with different coating agents: In vitro response of neuronal precursor cells.

Silver nanoparticles (AgNPs) represent one of the most abundant biocidal nanomaterials contained in more than 30% of nano-enabled consumer products and 75% of nanomedical products. The cumulative exposure of the general population may therefore reach critical and potentially hazardous levels. Due to data gaps on AgNP effects in humans, it is urgent to further evaluate their possible toxicity, particularly in vulnerable systems like the nervous one. As AgNPs may cross the blood brain and placental barriers, this study evaluated the in vitro effect of different AgNPs on neuronal precursor cells. For this purpose, 10 nm-sized AgNPs were stabilized with five different coating agents rendering a neutral, positive and negative surface charge. Murine neural stem cells (mNSCs) were used as cellular model to test AgNP neurotoxicity by evaluating the range of toxicity endpoints including cellular viability, apoptosis induction, oxidative stress response, cellular and mitochondrial membrane damages, DNA damage, inflammation response, and neural stem cell regulation. Our results clearly showed that the neurotoxic potential of AgNPs was not dependent on their surface charge or coating agents used for their surface stabilization. All AgNP types exhibited significant toxicity in neuronal precursor cells at an in vitro dose of 5 mg Ag/L or lower.

FOXO1 Mediates Advanced Glycation End Products Induced Mouse Osteocyte-Like MLO-Y4 Cell Apoptosis and Dysfunctions.

Osteocyte plays an essential role in bone metabolism by regulating osteoblast and osteoclast activities. Dysfunction or apoptosis of osteocyte will severely endanger the bone homeostasis and result in bone diseases such as osteoporosis. Osteoporosis has been considered as one of the diabetes complications; however, the mechanism is still to be discovered. Advanced glycation end products (AGEs), as the main pathogenic factor of diabetes mellitus, have the capacity to induce osteocyte apoptosis thus sabotaging bone homeostasis. Here, we examined the role of AGE during osteocyte apoptosis and how this effect would affect osteocyte's regulation of osteoblast and osteoclast. Mouse osteocyte-like MLO-Y4 cells were used to study the properties of osteocyte and to examine its biological and pathological function. MTT assay and Annexin V assay showed that AGE significantly induce MLO-Y4 cell apoptosis. qPCR and Western blot results have shown that AGE upregulates proapoptotic gene p53 and its downstream target gene Bax, which leads to enhanced activation of caspase-3, thus inducing apoptosis in MLO-Y4 cells. Increased expression of sclerostin and RANKL in osteocytes has shown that AGE induces osteocyte dysfunction thus severely damaging the bone homeostasis by decreasing osteoblast and increasing osteoclast activities. Furthermore, the role of the transcription factor FOXO1, which is intensely associated with apoptosis, has been determined. Western blot has shown that AGE significantly decreases Akt activities. Immunofluorescence has shown that AGE promotes FOXO1 nuclei localization and enhances FOXO1 expression. Silencing of FOXO1 suppressed AGE-enhanced apoptosis; mRNA and protein expressions of cleaved caspase-3, sclerostin, and RANKL were downregulated as well. Moreover, exogenous FOXO1 increased caspase-3 mRNA levels and caspase-3 transcriptional activity. Lastly, ChIP assay has established the capacity of FOXO1 binding directly on the caspase-3, sclerostin, and RANKL promoter region in AGE environment, providing the mechanism of the AGE-induced osteocyte apoptosis and dysfunction. Our results have shown that FOXO1 plays a crucial role in AGE-induced osteocyte dysfunction and apoptosis through its regulation of caspase-3, sclerostin, and RANKL. This study provides new insight into diabetes-enhanced risk of osteoporosis given the critical role of AGE in the pathogenesis of diabetes and the essential part of osteocyte in bone metabolism.

Quantitative and specific detection of cancer-related microRNAs in living cells using surface-enhanced Raman scattering imaging based on hairpin DNA-functionalized gold nanocages.

Variations in the intracellular expression level of cancer-related microRNAs (miRNAs) are connected with worsening tumor progression. A simple, accurate, and sensitive analytical method for the imaging and detection of intracellular miRNA is still a great challenge due to the low abundance of miRNAs and the complexity of intracellular environments. In this work, target miRNA (miRNA)-mediated catalytic hairpin assembly (CHA)-induced gold nanocage (GNC)-hairpin DNA1 (hpDNA1)-hpDNA2-GNC nanostructures were designed for surface-enhanced Raman scattering (SERS) detection and imaging of the specific miR-125a-5p in the normal lung epithelial cell line (BEAS-2B cells) and lung cancer cell line (A549 cells). The finite difference time domain (FDTD) simulations showed that the polymer of GNCs possessed a much stronger electromagnetic field in nanogaps than that of single GNC, theoretically confirming the rational design of the CHA assembly strategy. Using this method, miR-125a-5p can be detected in a wide linear range with a detection limit of 43.96 aM and high selectivity over other miRNAs in vitro. Moreover, SERS imaging successfully detected and distinguished the expression levels of intracellular miR-125a-5p in BEAS-2B cells and A549 cells. The results obtained by the SERS assay were consistent with those obtained by the real-time quantitative polymerase chain reaction (qRT-PCR). This method can offer a powerful strategy for the imaging and quantitative detection of various types of biomolecules in vitro as well as in living cells.

Development of an interactive tumor vascular suppression strategy to inhibit multidrug resistance and metastasis with pH/H2O2 responsive and oxygen-producing nanohybrids.

An ideal cancer therapeutic strategy should not only reverse multidrug resistance (MDR), but also prevent cancer metastasis. In this study, bovine serum albumin (BSA) was hybridized with Mn2+via biomineralization to develop a hybrid protein oxygen nanocarrier, which contained doxorubicin (DOX) and small interfering RNA (siRNA). The nanohybrid has the function of producing oxygen and chemotherapy synergistic gene therapy. FA-BSA-MnO2/DOX/siRNA was favorable for increasing the sensitivity of MCF-7/ADR cells to DOX. Moreover, FA-BSA-MnO2/DOX/siRNA NPs were also able to generate oxygen (O2) by reaction with endogenous hydrogen peroxide (H2O2) in tumor, thereby down-regulating the expression of hypoxia inducible factor-1α (HIF-1α), and then the expression of the vascular endothelial growth factor (VEGF) was down-regulated. At the same time, siRNA can directly or indirectly suppress the expression of the VEGF and HIF-1α. Therefore, the combination of two pathways and the chemo-gene therapy strategy can interactively overcome tumor hypoxia-associated MDR and metastasis, which will enhance therapeutic efficacy in the future.

Effect of structure in ionised albumin based nanoparticle: Characterisation, Emodin interaction, and in vitro cytotoxicity.

A γ-irradiated bovine albumin serum-based nanoparticle was characterised structurally, and functionally. The nanoparticle was characterised by A.F.M., D.L.S, zeta potential, T.E.M., gel-electrophoresis, and spectroscopy. We studied the stability of the nanoparticle at different pH values and against time, by fluorescence spectroscopy following the changes in the tryptophan environment in the nanoparticle. The nanoparticle was also functionalized with Folic Acid, its function as a nanovehicle was evaluated through its interaction with the hydrophobic drug Emodin. The binding and kinetic properties of the obtained complex were evaluated by biophysical methods as well as its toxicity in tumor cells. According to its biophysics, the nanoparticle is a spherical nanosized vehicle with a hydrodynamic diameter of 70 nm. Data obtained describe the nanoparticle as nontoxic for cancer cell lines. When combined with Emodin, the nanoparticle proved to be more active on MCF-7 cancer cell lines than the nanoparticle without Emodin. Significantly, the albumin aggregate preserves the main activity-function of albumin and improved characteristics as an excellent carrier of molecules. More than carrier properties, the nanoparticle alone induced an immune response in macrophages which may be advantageous in vaccine and cancer therapy formulation.

Dipeptidyl peptidase-4 plays a pathogenic role in BSA-induced kidney injury in diabetic mice.

Diabetic kidney disease (DKD) is appeared to be higher risk of declining kidney function compared to non-diabetic kidney disease with same magnitude of albuminuria. Epithelial-mesenchymal transition (EMT) program of tubular epithelial cells (TECs) could be important for the production of the extracellular matrix in the kidney. Caveolin-1 (CAV1), dipeptidyl peptidase-4 (DPP-4) and integrin ß1 have shown to be involved in EMT program. Here, we found diabetic kidney is prone for albuminuria-induced TECs damage and DPP-4 plays a vital role in such parenchymal damages in diabetic mice. The bovine serum albumin (BSA) injection induced severe TECs damage and altered expression levels of DPP-4, integrin ß1, CAV1, and EMT programs including relevant microRNAs in type 1 diabetic CD-1 mice when compared to non-diabetic mice; teneligliptin (TENE) ameliorated these alterations. TENE suppressed the close proximity among DPP-4, integrin ß1 and CAV1 in a culture of HK-2 cells. These findings suggest that DPP-4 inhibition can be relevant for combating proteinuric DKD by targeting the EMT program induced by the crosstalk among DPP-4, integrin ß1 and CAV1.

Design of ruthenium-albumin hydrogel for cancer therapeutics and luminescent imaging.

Improving cell uptake of metal compounds has became an important goal in the field of metal-based anticancer agents. This may combat platinum resistance and side effects seen commonly in current anticancer chemotherapy regimes. Here, we explore a novel degradable ruthenium-albumin hydrogel, which shows strong luminescence for cell imaging and high selectivity for cancer cells versus non-cancer cells. This is an early indication of the possibility of reducing unwanted side effects of metals by using bovine serum albumin hydrogel as a delivery strategy. This work provides a strong basis for development of a new class of metal-based cancer therapeutic agents.

Self-Assembled Reduced Albumin and Glycol Chitosan Nanoparticles for Paclitaxel Delivery.

Cancer continues to pose health problems for people all over the world. Nanoparticles (NPs) have emerged as a promising platform for effective cancer chemotherapy. NPs formed by the assembly of proteins and chitosan (CH) through noncovalent interactions are attracting a great deal of interest. However, the poor water solubility of CH and low stability of this kind of NP limit its practical application. Herein, the formation of reduced bovine serum albumin (rBSA) and glycol chitosan (GC) nanoparticles (rBG-NPs) stabilized by hydrophobic interactions and disulfide bonds was demonstrated for paclitaxel (PTX) delivery. The effects of the rBSA:GC mass ratio and pH on the particle size, polydispersity index (PDI), number of particles, and surface charge were evaluated. The formation mechanism and stability of the NPs were determined by compositional analysis and dynamic light scattering. Hydrophobic and electrostatic interactions were the driving forces for the formation of the rBG-NPs, and the NPs were stable under physiological conditions. PTX was successfully encapsulated into rBG-NPs with a high encapsulation efficiency (∼90%). PTX-loaded rBG-NPs had a particle size of ∼400 nm with a low PDI (0.2) and positive charge. rBG-NPs could be internalized by HeLa cells, possibly via endocytosis. An in vitro cytotoxicity study revealed that PTX-loaded rBG-NPs had anticancer activity that was lower than that of a Taxol-like formulation at 24 h but had similar activity at 48 h, possibly because of the slow release of PTX into the cells. Our study suggests that rBG-NPs could be used as a potential nanocarrier for hydrophobic drugs.

A Cell-Surface-Specific Ratiometric Fluorescent Probe for Extracellular pH Sensing with Solid-State Fluorophore.

Extracellular acidity is correlated with the development of various pathological states and bulk pH measurements could not report surface acidity. In this study, we have developed a ratiometric fluorescent probe that aggregates upon interaction with cells, allowing persistent labeling of cells and in situ measurement of cell surface pH. The ternary nanoplatform is constructed by a convenient noncovalent combination of bovine serum albumin protected gold nanoclusters (BSA-AuNCs), fluorescein isothiocyanate (FITC) labeled cationic peptides (CPs), and FITC-free CPs. The red fluorescent AuNCs serve as reference fluorophore, while FITC labeled peptides act as specific recognition element for H+ and FITC unlabeled peptides are used for delivery. The probe displays a sensitive fluorescence ratiometric response for pH in the range of 5.0-9.5 with calculated p Ka of 7.2. Further studies have demonstrated that this nanosensor also has properties of high selectivity, reversibility to pH fluctuations, as well as low cytotoxicity. The new surface pH-measurement tool was validated in mapping extracellular pH and monitoring acidification regarding cell metabolism, demonstrating its potential for bioimaging and biosensing.

Bovine serum albumin nanoparticles induce histopathological changes and inflammatory cell recruitment in the skin of treated mice.

Albumin is a natural, biocompatible, biodegradable and nontoxic polymer and due to these features, nanoparticles made of albumin are a good system for drug or antigen delivery. Polymeric nanoparticles are being widely explored as new vaccines platforms due to the capacity of those nanoparticles to prime the immune system by providing sustained release of the antigen after injection. Biodegradable nanoparticles associated with proteins represent a promising method for in vivo delivery of vaccines. In our previous studies, bovine serum albumin nanoparticles (BSA-NPs) were identified as a promising system for in vivo delivery of microbial antigens. The aim of this work was to show the effect of BSA-NPs on skin after nanoparticles administration. The pro-inflammatory activity of BSA-NPs was evaluated using in vivo models. BSA-NPs are easily uptake by macrophagic RAW 264.7 and BHK-21 cells without any significant cytotoxicity. Histological examination of skin sections from BSA-NPs-treated mice revealed intense cellular infiltration, increased skin thickness, follicular hypertrophy, vascular congestion and marked collagenesis. Mice immunized with recombinant non-structural protein 1 (rNS1) from Dengue virus 1 and BSA-NPs showed a high seroconversion rate if compared to animals immunized only with rNS1. Therefore, the effect of BSA-NPs on skin after BSA-NPs administration has a biotechnological relevance to the rational design of vaccine formulations based on albumin nanocarriers. However in the next years future studies should be carried out to best characterize the effect of BSA-NPs on dendritic cells and establish the role of these nanoparticles as a new vaccine platform for infectious diseases or cancer.

Chrysin Ameliorates Malfunction of Retinoid Visual Cycle through Blocking Activation of AGE-RAGE-ER Stress in Glucose-Stimulated Retinal Pigment Epithelial Cells and Diabetic Eyes.

Diabetes-associated visual cycle impairment has been implicated in diabetic retinopathy, and chronic hyperglycemia causes detrimental effects on visual function. Chrysin, a naturally occurring flavonoid found in various herbs, has anti-inflammatory, antioxidant, and neuroprotective properties. The goal of the current study was to identify the retinoprotective role of chrysin in maintaining robust retinoid visual cycle-related components. The in vitro study employed human retinal pigment epithelial (RPE) cells exposed to 33 mM of glucose or advanced glycation end products (AGEs) in the presence of 1⁻20 µM chrysin for three days. In the in vivo study, 10 mg/kg of chrysin was orally administrated to db/db mice. Treating chrysin reversed the glucose-induced production of vascular endothelial growth factor, insulin-like growth factor-1, and pigment epithelium-derived factor (PEDF) in RPE cells. The outer nuclear layer thickness of chrysin-exposed retina was enhanced. The oral gavage of chrysin augmented the levels of the visual cycle enzymes of RPE65, lecithin retinol acyltransferase (LRAT), retinol dehydrogenase 5 (RDH5), and rhodopsin diminished in db/db mouse retina. The diabetic tissue levels of the retinoid binding proteins and the receptor of the cellular retinol-binding protein, cellular retinaldehyde-binding protein-1, interphotoreceptor retinoid-binding protein and stimulated by retinoic acid 6 were restored to those of normal mouse retina. The presence of chrysin demoted AGE secretion and AGE receptor (RAGE) induction in glucose-exposed RPE cells and diabetic eyes. Chrysin inhibited the reduction of PEDF, RPE 65, LRAT, and RDH5 in 100 µg/mL of AGE-bovine serum albumin-exposed RPE cells. The treatment of RPE cells with chrysin reduced the activation of endoplasmic reticulum (ER) stress. Chrysin inhibited the impairment of the retinoid visual cycle through blocking ER stress via the AGE-RAGE activation in glucose-stimulated RPE cells and diabetic eyes. This is the first study demonstrating the protective effects of chrysin on the diabetes-associated malfunctioned visual cycle.

Advanced glycation end products evoke inflammatory reactions in proximal tubular cells via autocrine production of dipeptidyl peptidase-4.

We have previously shown that albuminuria and renal levels of advanced glycation end products (AGEs), receptor for AGEs (RAGE), and oxidative stress are suppressed in dipeptidyl peptidase-4 (DPP-4)-deficient diabetic rats, thus suggesting the crosstalk between AGE-RAGE axis and DPP-4 in experimental diabetic nephropathy. Therefore, we examined here the role of DPP-4 in AGE-evoked inflammatory reactions in human proximal tubular cells. Proteins were extracted from proximal tubular cells, and conditioned medium was collected, both of which were subjected to western blot analysis using anti-DPP-4 antibody. RAGE-aptamer was prepared using a systemic evolution of ligands by exponential enrichment. NF-κB p65 and monocyte chemoattractant protein-1 (MCP-1) gene expression was analyzed by reverse transcription-polymerase chain reaction. AGEs significantly increased DPP-4 expression and soluble DPP-4 production by tubular cells, the latter of which was attenuated by RAGE-aptamer or an anti-oxidant, N-acetylcysteine. AGEs or DPP-4 up-regulated NF-κB p65 or MCP-1 mRNA levels in tubular cells, which were suppressed by linagliptin, an inhibitor of DPP-4. AGEs stimulated NF-κB p65 gene expression in tubular cells isolated from control rats, but not from DPP-4-deficient rats. Our present results suggest that the AGE-RAGE-mediated oxidative stress could evoke inflammatory reactions in proximal tubular cells via autocrine production of DPP-4.