Augmenting Neutrophil Extracellular Traps with Carbonized Polymer Dots: A Potential Treatment for Bacterial Sepsis.

Sepsis is a life-threatening condition that can progress to septic shock as the body's extreme response to pathogenesis damages its own vital organs. Staphylococcus aureus (S. aureus) accounts for 50% of nosocomial infections, which are clinically treated with antibiotics. However, methicillin-resistant strains (MRSA) have emerged and can withstand harsh antibiotic treatment. To address this problem, curcumin (CCM) is employed to prepare carbonized polymer dots (CPDs) through mild pyrolysis. Contrary to curcumin, the as-formed CCM-CPDs are highly biocompatible and soluble in aqueous solution. Most importantly, the CCM-CPDs induce the release of neutrophil extracellular traps (NETs) from the neutrophils, which entrap and eliminate microbes. In an MRSA-induced septic mouse model, it is observed that CCM-CPDs efficiently suppress bacterial colonization. Moreover, the intrinsic antioxidative, anti-inflammatory, and anticoagulation activities resulting from the preserved functional groups of the precursor molecule on the CCM-CPDs prevent progression to severe sepsis. As a result, infected mice treated with CCM-CPDs show a significant decrease in mortality even through oral administration. Histological staining indicates negligible organ damage in the MRSA-infected mice treated with CCM-CPDs. It is believed that the in vivo studies presented herein demonstrate that multifunctional therapeutic CPDs hold great potential against life-threatening infectious diseases.

Alkaloids and Polyacetylenes from Hippobroma longiflora with Antiangiogenesis Properties.

Four new alkaloids, hippobrines A-D (1-4), along with three new polyacetylenes, hippobrenes A-C (5-7), were isolated from Hippobroma longiflora. Compounds 1-3 possess an unprecedented carbon skeleton. All of the new structures were determined by analyzing their mass and NMR spectroscopic data. The absolute configurations of 1 and 2 were confirmed by single-crystal X-ray analyses, and the absolute configurations of 3 and 7 were deduced using their ECD spectra. Plausible biogenetic pathways of 1 and 4 were proposed. In regard to bioactivities, all compounds (1-7) exhibited weak antiangiogenic activity against human endothelial progenitor cells, with IC50 values ranging from 21.1 ± 1.1 to 44.0 ± 2.3 µg/mL.

Use of cilomilast-loaded phosphatiosomes to suppress neutrophilic inflammation for attenuating acute lung injury: the effect of nanovesicular surface charge.

BACKGROUND: Cilomilast is a phosphodiesterase 4 (PDE4) inhibitor for treating inflammatory lung diseases. This agent has a narrow therapeutic index with significant adverse effects on the nervous system. This study was conducted to entrap cilomilast into PEGylated phosphatidylcholine-rich niosomes (phosphatiosomes) to improve pulmonary delivery via the strong affinity to pulmonary surfactant film. Neutrophils were used as a cell model to test the anti-inflammatory activity of phosphatiosomes. In an in vivo approach, mice were given lipopolysaccharide to produce acute lung injury. The surface charge in phosphatiosomes that influenced the anti-inflammatory potency is discussed in this study. RESULTS: The average diameter of the phosphatiosomes was about 100 nm. The zeta potential of anionic and cationic nanovesicles was - 35 and 32 mV, respectively. Cilomilast in both its free and nanocapsulated forms inhibited superoxide anion production but not elastase release in activated neutrophils. Cationic phosphatiosomes mitigated calcium mobilization far more effectively than the free drug. In vivo biodistribution evaluated by organ imaging demonstrated a 2-fold ameliorated lung uptake after dye encapsulation into the phosphatiosomes. The lung/brain distribution ratio increased from 3 to 11 after nanocarrier loading. The intravenous nanocarriers deactivated the neutrophils in ALI, resulting in the elimination of hemorrhage and alveolar wall damage. Only cationic phosphatiosomes could significantly suppress IL-1ß and TNF-α in the inflamed lung tissue. CONCLUSIONS: These results suggest that phosphatiosomes should further be investigated as a potential nanocarrier for the treatment of pulmonary inflammation.

Monovalent antibody-conjugated lipid-polymer nanohybrids for active targeting to desmoglein 3 of keratinocytes to attenuate psoriasiform inflammation.

To improve the treatment of psoriasiform inflammation, we developed actively targeted nanocarriers loaded with the phosphodiesterase 4 inhibitor AN2728. Methods: Phospholipid-poly(lactic-co-glycolic acid) nanohybrids were prepared and conjugated with monovalent anti-desmoglein 3 antibody to bind keratinocytes. Results: The actively targeted nanohybrids were 229 nm in mean size with a nearly neutral surface charge. Flow cytometry and confocal microscopy showed a 9-fold increase in keratinocyte uptake of targeted nanohybrids relative to non-targeted nanoparticles. The nanoparticles localized mainly in lysosomes after internalization. AN2728-loaded antibody-conjugated nanocarriers inhibited cytokine/chemokine overexpression in activated keratinocytes without affecting cell viability. The targeted nanohybrids also suppressed neutrophil migration by reducing CXCL1 and CXCL2 release from keratinocytes. Following subcutaneous administration in mice, the nanohybrids distributed to the epidermis and hair follicles. In a psoriasis-like skin mouse model, the actively targeted nanoparticles were superior to free drug and non-targeted nanoparticles in mitigating skin inflammation. Intervention with the targeted nanosystem reduced the epidermal thickness of the psoriasiform lesion from 191 to 42 µm, decreased the Psoriasis Area Severity Index by 74%, restored barrier function, and returned chemokine levels to baseline. Conclusions: Our developed nanosystem was safe and demonstrated efficient targeting properties for the treatment of cutaneous inflammation.

Mechanism of Nanoformulated Graphene Oxide-Mediated Human Neutrophil Activation.

Understanding the molecular mechanisms of graphene oxide (GO)-based biomaterials is important for logical biomedical applications. Previous studies have revealed biointeractions between GO and immune effector cells, but the effects on neutrophils, crucial cells in the immune system, have not been thoroughly discussed. In this study, GO nanoformulations were synthesized with different functional groups, including GO, GO-carboxylated (GO-COOH), and PEGylated GO (GO-PEG), with different surface features, which were elucidated using imaging methods and surface-sensitive quantitative spectroscopic techniques, including atomic force microscopy (AFM), transmission electron microscopy (TEM), and X-ray photoemission spectroscopy (XPS). The GO-based nanoformulations elicited reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation in human neutrophils. Nanoformulated GO stimulates NET development via the formation of ROS. An endocytosis study revealed that nanoformulated GO facilitated internalization by neutrophils via macropinocytosis and actin-dependent phagocytosis. Importantly, calcium mobilization and phosphorylation proteins such as mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) and AKT were involved in the activation of neutrophils. These findings offer the first verification that nanoformulated GO exhibits direct effects on human neutrophils.

Cisplatin encapsulated in phosphatidylethanolamine liposomes enhances the in vitro cytotoxicity and in vivo intratumor drug accumulation against melanomas.

BACKGROUND: Cisplatin is a potent anticancer drug for treating melanoma. OBJECTIVE: The aim of this study was to evaluate the possibility of using liposomes, for intratumoral distribution in a melanoma, composed of phosphatidylethanolamine (PE), for its cytotoxicity. METHOD: The in vitro drug release, in vitro cytotoxicity against melanoma, and in vivo residence time in the tumor of liposome-encapsulated cisplatin were investigated. The liposomes were prepared and characterized in terms of their morphology, size, zeta potential, and drug loading. RESULT: The size of the PE liposomes attained a level of approximately 100 nm. The concentration of cisplatin encapsulated in PE liposomes was 50-70% dependent on the presence or absence of polyethylene glycol (PEG) derivatives. On the other hand, no or negligible cisplatin molecules were encapsulated in egg phosphatidylcholine (EPC) liposomes. PE liposomes had higher cytotoxicity than classic liposomes or free cisplatin. Images of confocal laser scanning microscopy confirmed the great potency of PE liposomes to deliver cisplatin into cells. The incorporation of PEG derivatives completely inhibited the proliferation of melanoma cells. With in vivo intratumoral administration, the cisplatin concentration in the tumor tissue was maintained at a high level for 72 h after application of the PE liposomes. The PE liposomes delivered cisplatin into the tumor approximately 3.6 times more efficiently than the free drug. CONCLUSION: These results demonstrate that PE liposomes represent a potentially useful strategy for targeting cisplatin delivery into melanomas.

Enhancement of the transdermal delivery of catechins by liposomes incorporating anionic surfactants and ethanol.

The aim of this study was to develop and evaluate liposomal formulations encapsulating tea catechins, which possess antioxidant and chemopreventive activities. Liposomes were characterized for size, zeta potential, and entrapment efficiency. Both in vitro and in vivo skin permeation were examined using nude mouse skin as a model. The results suggested that the liposomal composition plays an important role in affecting the efficiency of transdermal catechin delivery. Incorporation of anionic surfactants such as deoxycholic acid (DA) and dicetyl phosphate (DP) in the liposomes in the presence of 15% ethanol increased the (+)-catechin permeation by five to seven-fold as compared to the control. The flexibility of bilayers is suggested as an important factor governing the enhancing effect of liposomes. Intercellular spaces within the stratum corneum but not shunt routes are the major pathways for catechin delivery from liposomes. (+)-Catechin and (-)-epicatechin are isomers which showed similar encapsulation efficiencies and skin permeation in liposomes. (-)-Epigallocatechin-3-gallate showed the highest encapsulation rate and in vivo skin deposition level in liposomes among all catechins tested. The stability and in vitro tranepidermal water loss test indicated the safety of the practical use of liposomes developed in this study.

Physicochemical characteristics and in vivo deposition of liposome-encapsulated tea catechins by topical and intratumor administrations.

Tea polyphenols, including (+)-catechin, (-)-epicatechin, and (-)-epigallocatechin-3-gallate (EGCG), have been shown to possess potent antioxidant and anticancer activities. The aim of this study was to evaluate the possibility of using liposomes for the local delivery, including skin and tumor deposition, of these polyphenols. Liposomes containing egg phosphatidylcholine, cholesterol, or anionic species were prepared by a solvent evaporation method and then were subjected to a probe sonicator. The size, zeta potential and entrapment efficiency of these liposomal formulations were determined to provide correlations with results from a subsequent in vivo study. The release rate study showed that inclusion of an anionic species, such as deoxycholic acid (DA) or dicetyl phosphate (DP), increased the permeability of the lipid bilayers, leading to the rapid release of these formulations. No significant increase in skin deposition of catechins was observed after topical application of liposomes. On the other hand, a greater amount of catechins were delivered into the solid tumor by liposomes than by the aqueous solution. The drug release rate and vesicle size of liposomes may influence drug deposition in tumor tissues. The isomers, (+)-catechin and (-)-epicatechin, showed different physicochemical properties in liposomes and for local deposition in the skin and tumor. Finally, the presence of gallic acid ester in the structure of EGCG significantly increased the tissue uptake of catechins.

Cationic additives in nanosystems activate cytotoxicity and inflammatory response of human neutrophils: lipid nanoparticles versus polymeric nanoparticles.

This report compares the effect of lipid and polymeric nanoparticles upon human neutrophils in the presence of cationic surfactants. Nanostructured lipid carriers and poly(lactic-co-glycolic) acid nanoparticles were manufactured as lipid and polymeric systems, respectively. Some cytotoxic and proinflammatory mediators such as lactate dehydrogenase (LDH), elastase, O2(•-), and intracellular Ca(2+) were examined. The nanoparticles showed a size of 170-225 nm. Incorporation of cetyltrimethylammonium bromide or soyaethyl morpholinium ethosulfate, the cationic surfactant, converted zeta potential from a negative to a positive charge. Nanoparticles without cationic surfactants revealed a negligible change on immune and inflammatory responses. Cationic surfactants in both nanoparticulate and free forms induced cell death and the release of mediators. Lipid nanoparticles generally demonstrated a greater response compared to polymeric nanoparticles. The neutrophil morphology observed by electron microscopy confirmed this trend. Cetyltrimethylammonium bromide as the coating material showed more significant activation of neutrophils than soyaethyl morpholinium ethosulfate. Confocal microscope imaging displayed a limited internalization of nanoparticles into neutrophils. It is proposed that cationic nanoparticles interact with the cell membrane, triggering membrane disruption and the following Ca(2+) influx. The elevation of intracellular Ca(2+) induces degranulation and oxidative stress. The consequence of these effects is cytotoxicity and cell death. Caution should be taken when selecting feasible nanoparticulate formulations and cationic additives for consideration of applicability and toxicity.

Cationic liposomes evoke proinflammatory mediator release and neutrophil extracellular traps (NETs) toward human neutrophils.

Cationic liposomes are widely used as nanocarriers for therapeutic and diagnostic purposes. The cationic components of liposomes can induce inflammatory responses. This study examined the effect of cationic liposomes on human neutrophil activation. Cetyltrimethylammonium bromide (CTAB) or soyaethyl morpholinium ethosulfate (SME) was incorporated into liposomes as the cationic additive. The liposomes' cytotoxicity and their induction of proinflammatory mediators, intracellular calcium, and neutrophil extracellular traps (NETs) were investigated. The interaction of the liposomes with the plasma membrane triggered the stimulation of neutrophils. CTAB liposomes induced complete leakage of lactate dehydrogenase (LDH) at all concentrations tested, whereas SME liposomes released LDH in a concentration-dependent manner. CTAB liposomes proved to more effectively activate neutrophils compared with SME liposomes, as indicated by increased superoxide anion and elastase levels. Calcium influx increased 9-fold after treatment with CTAB liposomes. This influx was not changed by SME liposomes compared with the untreated control. Scanning electron microscopy (SEM) and immunofluorescence images indicated the presence of NETs after treatment with cationic liposomes. NETs could be quickly formed, within minutes, after CTAB liposomal treatment. In contrast to this result, NET formation was slowly and gradually increased by SME liposomes, within 4h. Based on the data presented here, it is important to consider the toxicity of cationic liposomes during administration in the body. This is the first report providing evidence of NET production induced by cationic liposomes.

Non-invasive synergistic treatment of brain tumors by targeted chemotherapeutic delivery and amplified focused ultrasound-hyperthermia using magnetic nanographene oxide.

The combination of chemo-thermal therapy is the best strategy to ablate tumors, but how to heat deep tumor tissues effectively without side-damage is a challenge. Here, a systemically delivered nanocarrier is designed with multiple advantages, including superior heat absorption, highly efficient hyperthermia, high drug capacity, specific targeting ability, and molecular imaging, to achieve both high antitumor efficacy and effective amplification of hyperthermia with minimal side effects.

Development and evaluation of perfluorocarbon nanobubbles for apomorphine delivery.

Apomorphine is a dopamine receptor agonist for treating Parkinson's disease. However, its clinical application is limited by its instability and the need for frequent injections. The aim of the present work was to develop acoustically active perfluorocarbon nanobubbles (PNs) for encapsulation of both apomorphine HCl and base forms to circumvent these delivery problems. The PNs were prepared using coconut oil and perfluoropentane as the inner phase, which was emulsified by phospholipids and cholesterol. The morphology, size, zeta potential, and drug release of the PNs were characterized. The particle size ranged from 150 to 380 nm, with differences in the oil or perfluorocarbon ratio in the formulations. Atomic force microscopy confirmed oval- or raisin-shaped particles and a narrow size distribution of these systems (polydispersity index = 0.25-0.28). The stability experimental results indicated that PNs could protect apomorphine from degradation. Evaporation of the PNs at 37 degrees C was also limited. Apomorphine HCl and base in PNs showed retarded and sustained release profiles. Ultrasound imaging confirmed the echogenic activity of PNs developed in this study. The apomorphine HCl release by insonation at 1 MHz showed enhancements of two- to fourfold compared to the non-ultrasound group, illustrating a possible drug-targeting effect. On the contrary, apomorphine base showed a decreased release profile with ultrasound application. Apomorphine-loaded PNs showed promising stability and safety. They were successful in sustaining apomorphine delivery.

Relationship between unstimulated salivary flow rate and saliva composition of healthy children in Taiwan.

BACKGROUND: Saliva is one of the most important factors in regulating oral health, with flow rate and composition changing throughout development and during disease. In view of the shortage of data, the present study aimed to shed light on the relationship between unstimulated salivary flow rate and saliva composition of healthy children in Taiwan. METHODS: Forty-four normal, healthy children from 3-14 years of age were divided into three age groups: pre-school, elementary school and junior-high school. All participants received salivary flow rate, pH and saliva composition analysis under unstimulated conditions. One-way ANOVA and Pearson's correlation were used. Statistical significance was set at p < 0.05. RESULTS: Our results suggest that, under unstimulated conditions, the salivary flow rate of the elementary school group was greater than that of the pre-school group (p < 0.05). No difference in pH was found among the three groups. Intergroup salivary calcium, phosphorus and amylase did not reach statistical difference. As the flow rate increased, the pH increased (r = 0.364, p < 0.05) but the protein level decreased (r = -0.473, p < 0.05). In addition, salivary protein was positively correlated to age (r = 0.479, p < 0.05) and negatively correlated to pH (r = -0.361, p < 0.01). CONCLUSION: Age-related increase in the unstimulated salivary flow rate of pre-school and elementary school groups was noted. As the flow rate increased, the pH increased but the protein level decreased. The information obtained may serve as reference values for the growing interest in saliva as a diagnostic tool, especially monitoring those with neurological or oral motor dysfunction.