The Proteoglycan Biglycan Modulates Platelet Adhesion and Thrombus Formation in a GPVI-Dependent Manner

Background: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions. Methods: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice. Results: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times. Conclusions: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.     

In Vitro/In Vivo Toxicity Evaluation and Quantification of Iron Oxide Nanoparticles

Increasing biomedical applications of iron oxide nanoparticles (IONPs) in academic and commercial settings have alarmed the scientific community about the safety and assessment of toxicity profiles of IONPs. The great amount of diversity found in the cytotoxic measurements of IONPs points toward the necessity of careful characterization and quantification of IONPs. The present document discusses the major developments related to in vitro and in vivo toxicity assessment of IONPs and its relationship with the physicochemical parameters of IONPs. Major discussion is included on the current spectrophotometric and imaging based techniques used for quantifying, and studying the clearance and biodistribution of IONPs. Several invasive and non-invasive quantification techniques along with the pitfalls are discussed in detail. Finally, critical guidelines are provided to optimize the design of IONPs to minimize the toxicity.     

Silver@quercetin Nanoparticles with Aggregation-Induced Emission for Bioimaging In Vitro and In Vivo

Fluorescent materials based on aggregation-induced emission luminogens (AIEgens) have unique advantages for in situ and real-time monitoring of biomolecules and biological processes because of their high luminescence intensity and resistance to photobleaching. Unfortunately, many AIEgens require time-consuming and expensive syntheses, and the presence of residual toxic reagents reduces their biocompatibility. Herein, silver@quercetin nanoparticles (Ag@QCNPs), which have a clear core–shell structure, were prepared by redox reaction of quercetin (QC), a polyphenolic compound widely obtained from plants, including those used as foods, and silver ions. Ag@QCNPs show both aggregation-induced luminescence and the distinct plasma scattering of silver nanoparticles, as well as good resistance to photobleaching and biocompatibility. The Ag@QCNPs were successfully used for cytoplasmic labeling of living cells and for computerized tomography imaging in tumor-bearing mice, demonstrating their potential for clinical applications.     

Assessing the Efficiency of Triangular Gold Nanoparticles as NIR Photothermal Agents In Vitro and Melanoma Tumor Model

Photothermal therapy (PTT) is gaining a lot of interest as a cancer treatment option with minimal side effects due to the efficient photothermal agents employed. They are based on nanomaterials that, upon laser irradiation, absorb photon energy and convert it into heat to induce hyperthermia, which destroys the cancer cells. Here, the unique light-to-heat conversion features of three different gold nanotriangular nanoparticles (AuNTs) are evaluated with respect to their absorption properties to select the most efficient nanoheater with the highest potential to operate as an efficient photothermal agent. AuNTs with LSPR response in- and out- of resonance with the 785 nm near-infrared (NIR) excitation wavelength are investigated. Upon 15 min laser exposure, the AuNTs that exhibit a plasmonic response in resonance with the 785 nm laser line show the highest photothermal conversion efficacy of 80%, which correlates with a temperature increase of 22 °C. These photothermal properties are well-preserved in agarose-based skin biological phantoms that mimic the melanoma tumoral tissue and surrounding healthy tissue. Finally, in vitro studies on B16.F10 melanoma cells prove by fluorescence staining and MTT assay that the highest phototoxic effect after NIR laser exposure is induced by AuNTs with LSPR response in resonance with the employed laser line, thus demonstrating their potential implementation as efficient photothermal agents in PTT.     

Silver Nanoparticles Biocomposite Films with Antimicrobial Activity: In Vitro and In Vivo Tests

Overuse of antimicrobials by the population has contributed to genetic modifications in bacteria and development of antimicrobial resistance, which is very difficult to combat nowadays. To solve this problem, it is necessary to develop new systems for the administration of antimicrobial active principles. Biocomposite systems containing silver nanoparticles can be a good medical alternative. In this context, the main objective of this study was to obtain a complex system in the form of a biocomposite film with antimicrobial properties based on chitosan, poly (vinyl alcohol) and silver nanoparticles. This new system was characterized from a structural and morphological point of view. The swelling degree, the mechanical properties and the efficiency of loading and release of an anti-inflammatory drug were also evaluated. The obtained biocomposite films are biocompatibles, this having been demonstrated by in vitro tests on HDFa cell lines, and have antimicrobial activity against S. aureus. The in vivo tests, carried out on rabbit subjects, highlighted the fact that signs of reduced fibrosis were specific to the C2P4.10.Ag1-IBF film sample, demonstrated by: intense expression of TNFAIP8 factors; as an anti-apoptotic marker, MHCII that favors immune cooperation among local cells; αSMA, which marks the presence of myofibroblasts involved in approaching the interepithelial spaces for epithelialization; and reduced expression of the Cox2 indicator of inflammation, Col I.     

The Impact of Multiple Functional Layers in the Structure of Magnetic Nanoparticles and Their Influence on Albumin Interaction

In nanomedicine, hybrid nanomaterials stand out for providing new insights in both the diagnosis and treatment of several diseases. Once administered, engineered nanoparticles (NPs) interact with biological molecules, and the nature of this interaction might directly interfere with the biological fate and action of the NPs. In this work, we synthesized a hybrid magnetic nanostructure, with antibacterial and antitumoral potential applications, composed of a magnetite core covered by silver NPs, and coated with a modified chitosan polymer. As magnetite NPs readily oxidize to maghemite, we investigated the structural properties of the NPs after addition of the two successive layers using Mössbauer spectroscopy. Then, the structural characteristics of the NPs were correlated to their interaction with albumin, the major blood protein, to evidence the consequences of its binding on NP properties and protein retention. Thermodynamic parameters of the NPs–albumin interaction were determined. We observed that the more stable NPs (coated with modified chitosan) present a lower affinity for albumin in comparison to pure magnetite and magnetite/silver hybrid NPs. Surface properties were key players at the NP–biological interface. To the best of our knowledge, this is the first study that demonstrates a correlation between the structural properties of complex hybrid NPs and their interaction with albumin.     

Synthesis and Characterization of Polyvinylpyrrolidone-Modified ZnO Quantum Dots and Their In Vitro Photodynamic Tumor Suppressive Action

Despite the numerous available treatments for cancer, many patients succumb to side effects and reoccurrence. Zinc oxide (ZnO) quantum dots (QDs) are inexpensive inorganic nanomaterials with potential applications in photodynamic therapy. To verify the photoluminescence of ZnO QDs and determine their inhibitory effect on tumors, we synthesized and characterized ZnO QDs modified with polyvinylpyrrolidone. The photoluminescent properties and reactive oxygen species levels of these ZnO/PVP QDs were also measured. Finally, in vitro and in vivo experiments were performed to test their photodynamic therapeutic effects in SW480 cancer cells and female nude mice. Our results indicate that the ZnO QDs had good photoluminescence and exerted an obvious inhibitory effect on SW480 tumor cells. These findings illustrate the potential applications of ZnO QDs in the fields of photoluminescence and photodynamic therapy.     

Radioiodinated Magnetic Nanoparticles Conjugated With Moxifloxacin: Synthesis and In Vitro Biological Affinities

Magnetic nanoparticles (MNPs) were synthesized and coated with tetraethyl orthosilicate and aminosilane to create functional amino groups. Moxifloxacin (MXF) was conjugated to the modified MNPs using glutaraldehyde as crosslinker. Finally, MXF conjugated magnetic nanoparticles were radiolabeled and their biological affinities such as cell incorporation ratio, cytotoxicity, and their potential as cell imaging probe were investigated using A-549 cells.     

In Vitro Structural and Functional Evaluation of Gold Nanoparticles Conjugated Antibiotics

Bactericidal efficacy of gold nanoparticles conjugated with ampicillin, streptomycin and kanamycin were evaluated. Gold nanoparticles (Gnps) were conjugated with the antibiotics during the synthesis of nanoparticles utilizing the combined reducing property of antibiotics and sodium borohydride. The conjugation of nanoparticles was confirmed by dynamic light scattering (DLS) and electron microscopic (EM) studies. Such Gnps conjugated antibiotics showed greater bactericidal activity in standard agar well diffusion assay. The minimal inhibitory concentration (MIC) values of all the three antibiotics along with their Gnps conjugated forms were determined in three bacterial strains, Escherichia coli DH5α, Micrococcus luteus and Staphylococcus aureus. Among them, streptomycin and kanamycin showed significant reduction in MIC values in their Gnps conjugated form whereas; Gnps conjugated ampicillin showed slight decrement in the MIC value compared to its free form. On the other hand, all of them showed more heat stability in their Gnps conjugated forms. Thus, our findings indicated that Gnps conjugated antibiotics are more efficient and might have significant therapeutic implications. Biswarup Saha and Jaydeep Bhattacharya authors contributed equally.     

In Vitro Hepatotoxic and Neurotoxic Effects of Titanium and Cerium Dioxide Nanoparticles,Arsenic and Mercury Co-Exposure

Considering the increasing emergence of new contaminants, such as nanomaterials, mixing with legacy contaminants, including metal(loid)s, it becomes imperative to understand the toxic profile resulting from these interactions. This work aimed at assessing and comparing the individual and combined hepatotoxic and neurotoxic potential of titanium dioxide nanoparticles (TiO₂NPs 0.75–75 mg/L), cerium oxide nanoparticles (CeO₂NPs 0.075–10 μg/L), arsenic (As 0.01–2.5 mg/L), and mercury (Hg 0.5–100 mg/L) on human hepatoma (HepG2) and neuroblastoma (SH-SY5Y) cells. Viability was assessed through WST-1 (24 h) and clonogenic (7 days) assays and it was affected in a dose-, time- and cell-dependent manner. Higher concentrations caused greater toxicity, while prolonged exposure caused inhibition of cell proliferation, even at low concentrations, for both cell lines. Cell cycle progression, explored by flow cytometry 24 h post-exposure, revealed that TiO₂NPs, As and Hg but not CeO₂NPs, changed the profiles of SH-SY5Y and HepG2 cells in a dose-dependent manner, and that the cell cycle was, overall, more affected by exposure to mixtures. Exposure to binary mixtures revealed either potentiation or antagonistic effects depending on the composition, cell type and time of exposure. These findings prove that joint toxicity of contaminants cannot be disregarded and must be further explored.     

In Vitro Cytotoxic Evaluation of MgO Nanoparticles and Their Effect on the Expression of ROS Genes

Water-dispersible MgO nanoparticles were tested to investigate their cytotoxic effects on oxidative stress gene expression. In this in vitro study, genes related to reactive oxygen species (ROS), glutathione S-transferase (GST) and catalase, were quantified using real-time polymerase chain reactions (molecular level) and molecular beacon technologies (cellular level). The monodispersed MgO nanoparticles, 20 nm in size, were used to treat human cancer cell lines (liver cancer epithelial cells) at different concentrations (25, 75 and 150 µg/mL) and incubation times (24, 48 and 72 h). Both the genetic and cellular cytotoxic screening methods produced consistent results, showing that GST and catalase ROS gene expression was maximized at 150 µg/mL nanoparticle treatment with 48 h incubation. However, the genotoxic effect of MgO nanoparticles was not significant compared with control experiments, which indicates its significant potential applications in nanomedicine as a diagnostic and therapeutic tool.     

In Vitro Comparative Study of Solid Lipid and PLGA Nanoparticles Designed to Facilitate Nose-to-Brain Delivery of Insulin

The brain insulin metabolism alteration has been addressed as a pathophysiological factor underlying Alzheimer’s disease (AD). Insulin can be beneficial in AD, but its macro-polypeptide nature negatively influences the chances of reaching the brain. The intranasal (IN) administration of therapeutics in AD suggests improved brain-targeting. Solid lipid nanoparticles (SLNs) and poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) are promising carriers to deliver the IN-administered insulin to the brain due to the enhancement of the drug permeability, which can even be improved by chitosan-coating. In the present study, uncoated and chitosan-coated insulin-loaded SLNs and PLGA NPs were formulated and characterized. The obtained NPs showed desirable physicochemical properties supporting IN applicability. The in vitro investigations revealed increased mucoadhesion, nasal diffusion, and drug release rate of both insulin-loaded nanocarriers over native insulin with the superiority of chitosan-coated SLNs. Cell-line studies on human nasal epithelial and brain endothelial cells proved the safety IN applicability of nanoparticles. Insulin-loaded nanoparticles showed improved insulin permeability through the nasal mucosa, which was promoted by chitosan-coating. However, native insulin exceeded the blood-brain barrier (BBB) permeation compared with nanoparticulate formulations. Encapsulating insulin into chitosan-coated NPs can be beneficial for ensuring structural stability, enhancing nasal absorption, followed by sustained drug release.     

Studies on Preparation of Photosensitizer Loaded Magnetic Silica Nanoparticles and Their Anti-Tumor Effects for Targeting Photodynamic Therapy

Abstract  As a fast developing alternative of traditional therapeutics, photodynamic therapy (PDT) is an effective, noninvasive, nontoxic therapeutics for cancer, senile macular degeneration, and so on. But the efficacy of PDT was compromised by insufficient selectivity and low solubility. In this study, novel multifunctional silica-based magnetic nanoparticles (SMNPs) were strategically designed and prepared as targeting drug delivery system to achieve higher specificity and better solubility. 2,7,12,18-Tetramethyl-3,8-di-(1-propoxyethyl)-13,17-bis-(3-hydroxypropyl) porphyrin, shorted as PHPP, was used as photosensitizer, which was first synthesized by our lab with good PDT effects. Magnetite nanoparticles (Fe₃O₄) and PHPP were incorporated into silica nanoparticles by microemulsion and sol–gel methods. The prepared nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and fluorescence spectroscopy. The nanoparticles were approximately spherical with 20–30 nm diameter. Intense fluorescence of PHPP was monitored in the cytoplasm of SW480 cells. The nanoparticles possessed good biocompatibility and could generate singlet oxygen to cause remarkable photodynamic anti-tumor effects. These suggested that PHPP-SMNPs had great potential as effective drug delivery system in targeting photodynamic therapy, diagnostic magnetic resonance imaging and magnetic hyperthermia therapy. Graphical Abstract   Novel multifunctional photosensitizer loaded magnetic silica nanoparticles were strategically prepared with low toxicity, good biocompatibility and remarkable photodynamic anti-tumor efficacy. The nanoparticles were believed to be of great value as drug delivery system in targeting photodynamic therapy, diagnostic magnetic resonance imaging and magnetic hyperthermia therapy.      

Biomimetic Synthesis of Gelatin Polypeptide-Assisted Noble-Metal Nanoparticles and Their Interaction Study

Herein, the generation of gold, silver, and silver–gold (Ag–Au) bimetallic nanoparticles was carried out in collagen (gelatin) solution. It first showed that the major ingredient in gelatin polypeptide, glutamic acid, acted as reducing agent to biomimetically synthesize noble metal nanoparticles at 80°C. The size of nanoparticles can be controlled not only by the mass ratio of gelatin to gold ion but also by pH of gelatin solution. Interaction between noble-metal nanoparticles and polypeptide has been investigated by TEM, UV–visible, fluorescence spectroscopy, and HNMR. This study testified that the degradation of gelatin protein could not alter the morphology of nanoparticles, but it made nanoparticles aggregated clusters array (opposing three-dimensional α-helix folding structure) into isolated nanoparticles stabilized by gelatin residues. This is a promising merit of gelatin to apply in the synthesis of nanoparticles. Therefore, gelatin protein is an excellent template for biomimetic synthesis of noble metal/bimetallic nanoparticle growth to form nanometer-sized device.     

海绵状的铂纳米粒子的合成和表征

用还原法制备了海绵状的铂纳米粒子,研究了不同的还原剂、保护剂和温度对铂纳米粒子的影响。X射线衍射和扫描电子显微镜SEM表征结果指出,铂纳米颗粒粒径小,粒径分布范围窄,平均粒径约为4.1 nm。     

Exposure of Mice to Thirdhand Smoke Modulates In Vitro and In Vivo Platelet Responses

Smoking is a risk factor for a variety of deleterious conditions, such as cancer, respiratory disease and cardiovascular disease. Thrombosis is an important and common aspect of several cardiovascular disease states, whose risk is known to be increased by both first- and secondhand smoke. More recently, the residual cigarette smoke that persists after someone has smoked (referred to as thirdhand smoke or THS) has been gaining more attention, since it has been shown that it also negatively affects health. Indeed, we have previously shown that 6-month exposure to THS increases the risk of thrombogenesis. However, neither the time-dependence of THS-induced thrombus formation, nor its sex dependence have been investigated. Thus, in the present study, we investigated these issues in the context of a shorter exposure to THS, specifically 3 months, in male and female mice. We show that the platelets from 3-month THS-exposed mice exhibited enhanced activation by agonists. Moreover, we also show that mice of both sexes exposed to THS have decreased tail bleeding as well as decreased thrombus occlusion time. In terms of the role of sex, intersex disparities in thrombus development and hemostasis as well as in platelet aggregation were, interestingly, observed. Together, our findings show that exposing mice to THS for 3 months is sufficient to predispose them to thrombosis; which seems to be driven, at least in part, by an increased activity in platelets, and that it does not manifest equally in both sexes.     

Radiolabeled Gold Nanoseeds Decorated with Substance P Peptides: Synthesis,Characterization and In Vitro Evaluation in Glioblastoma Cellular Models

Despite some progress, the overall survival of patients with glioblastoma (GBM) remains extremely poor. In this context, there is a pressing need to develop innovative therapy strategies for GBM, namely those based on nanomedicine approaches. Towards this goal, we have focused on nanoparticles (AuNP-SP and AuNP-SPTyr8) with a small gold core (ca. 4 nm), carrying DOTA chelators and substance P (SP) peptides. These new SP-containing AuNPs were characterized by a variety of analytical techniques, including TEM and DLS measurements and UV-vis and CD spectroscopy, which proved their high in vitro stability and poor tendency to interact with plasma proteins. Their labeling with diagnostic and therapeutic radionuclides was efficiently performed by DOTA complexation with the trivalent radiometals ⁶⁷Ga and ¹⁷⁷Lu or by electrophilic radioiodination with ¹²⁵I of the tyrosyl residue in AuNP-SPTyr8. Cellular studies of the resulting radiolabeled AuNPs in NKR1-positive GBM cells (U87, T98G and U373) have shown that the presence of the SP peptides has a crucial and positive impact on their internalization by the tumor cells. Consistently, ¹⁷⁷Lu-AuNP-SPTyr8 showed more pronounced radiobiological effects in U373 cells when compared with the non-targeted congener ¹⁷⁷Lu-AuNP-TDOTA, as assessed by cell viability and clonogenic assays and corroborated by Monte Carlo microdosimetry simulations.     

羟基磷灰石空心纳米球的制备及表征

采用W\O\W复乳法制备羟基磷灰石,内水相为(NH4)2HPO4水溶液,中间油相为易挥发的环己烷,外水相为Ca(NO3)2.4H2O水溶液。分析了反应中传质机理为Ca2+扩散到内水相,在碱性下与内水相的HPO42-反应生成羟基磷灰石。通过XRD、FT-IR、SEM、TEM分析了产物的成分和形貌,证实产物为300nm左右的羟基磷灰石空心球。讨论了反应温度对纳米球形貌的影响,当温度为10℃时发生界面反应,得到空心的纳米球。     

明胶纳米颗粒的制备及表征

以B型明胶为原料,戊二醛为交联剂,通过改进的两步去溶剂法制备了较小粒径（约100nm）的明胶纳米颗粒(Gelatin nanoparticles, GNPs);研究了分散体系浓度、去溶剂化试剂、交联剂用量的影响。结果表明：降低明胶在二次分散体系中的浓度,制得的GNPs的尺寸更小且分布均匀。残留的去溶剂化试剂丙酮必须去除,因为它会使GNPs分散性变差。在明胶质量浓度为8 g/L的分散液中,仅用质量分数为4.5%的戊二醛(与明胶质量比)即可制得稳定的GNPs,其粒径为50~150 nm,PDI （polydispersity index）约为0.14,Zeta电位绝对值< 30 mV。AFM和 TEM测试结果表明GNPs呈光滑球形,为边缘部分比核心更为疏松的非均质结构。     

Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

The purpose of the present work was to formulate and evaluate cationic poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency) and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells) of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95%) could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable.