The influence of intercalating perfluorohexane into lipid shells on nano and microbubble stability

Microbubbles are potential diagnostic and therapeutic agents. In vivo stability is important as the bubbles are required to survive multiple passages through the heart and lungs to allow targeting and delivery. Here we have systematically varied key parameters affecting microbubble lifetime to significantly increase in vivo stability. Whilst shell and core composition are found to have an important role in improving microbubble stability, we show that inclusion of small quantities of C6F14 in the microbubble bolus significantly improves microbubble lifetime. Our results indicate that C6F14 inserts into the lipid shell, decreasing surface tension to 19 mN m-1, and increasing shell resistance, in addition to saturating the surrounding medium. Surface area isotherms suggest that C6F14 incorporates into the acyl chain region of the lipid at a high molar ratio, indicating ∼2 perfluorocarbon molecules per 5 lipid molecules. The resulting microbubble boluses exhibit a higher in vivo image intensity compared to commercial compositions, as well as longer lifetimes

One-step fabrication of hollow-channel gold nanoflowers with excellent catalytic performance and large single-particle SERS activity

Hollow metallic nanostructures have shown potential in various applications including catalysis, drug delivery and phototherapy, owing to their large surface areas, reduced net density, and unique optical properties. In this study, novel hollow gold nanoflowers (HAuNFs) consisting of an open hollow channel in the center and multiple branches/tips on the outer surface are fabricated for the first time, via a facile one-step synthesis using an auto-degradable nanofiber as a bifunctional template. The one-dimensional (1D) nanofiber acts as both a threading template as well as a promoter of the anisotropic growth of the gold crystal, the combination of which leads to the formation of HAuNFs with a hollow channel and nanospikes. The synergy of favorable structural/surface features, including sharp edges, open cavity and high-index facets, provides our HAuNFs with excellent catalytic performance (activity and cycling stability) coupled with large single-particle SERS activity (including ∼30 times of activity in ethanol electro-oxidation and ∼40 times of single-particle SERS intensity, benchmarked against similar-sized solid gold nanospheres with smooth surfaces, as well as retaining 86.7% of the initial catalytic activity after 500 cycles in ethanol electro-oxidation). This innovative synthesis gives a nanostructure of the geometry distinct from the template and is extendable to fabricating other systems for example, hollow-channel silver nanoflowers (HAgNFs). It thus provides an insight into the design of hollow nanostructures via template methods, and offers a versatile synthetic strategy for diverse metal nanomaterials suited for a broad range of applications

Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model

BACKGROUND: Both cell-associated and cell-free HIV virions are present in semen and cervical secretions of HIV-infected individuals. Thus, topical microbicides may need to inactivate both cell-associated and cell-free HIV to prevent sexual transmission of HIV/AIDS. To determine if the mild acidity of the healthy vagina and acid buffering microbicides would prevent transmission by HIV-infected leukocytes, we measured the effect of pH on leukocyte motility, viability and intracellular pH and tested the ability of an acidic buffering microbicide (BufferGel(®)) to prevent the transmission of cell-associated HIV in a HuPBL-SCID mouse model. METHODS: Human lymphocyte, monocyte, and macrophage motilities were measured as a function of time and pH using various acidifying agents. Lymphocyte and macrophage motilities were measured using video microscopy. Monocyte motility was measured using video microscopy and chemotactic chambers. Peripheral blood mononuclear cell (PBMC) viability and intracellular pH were determined as a function of time and pH using fluorescent dyes. HuPBL-SCID mice were pretreated with BufferGel, saline, or a control gel and challenged with HIV-1-infected human PBMCs. RESULTS: Progressive motility was completely abolished in all cell types between pH 5.5 and 6.0. Concomitantly, at and below pH 5.5, the intracellular pH of PBMCs dropped precipitously to match the extracellular medium and did not recover. After acidification with hydrochloric acid to pH 4.5 for 60 min, although completely immotile, 58% of PBMCs excluded ethidium homodimer-1 (dead-cell dye). In contrast, when acidified to this pH with BufferGel, a microbicide designed to maintain vaginal acidity in the presence of semen, only 4% excluded dye at 10 min and none excluded dye after 30 min. BufferGel significantly reduced transmission of HIV-1 in HuPBL-SCID mice (1 of 12 infected) compared to saline (12 of 12 infected) and a control gel (5 of 7 infected). CONCLUSION: These results suggest that physiologic or microbicide-induced acid immobilization and killing of infected white blood cells may be effective in preventing sexual transmission of cell-associated HIV

Reduction in the resident intestinal myelomonocytic cell population occurs during ApcMin/+ mouse intestinal tumorigenesis

With its significant contribution to cancer mortality globally, advanced colorectal cancer (CRC) requires new treatment strategies. However, despite recent good results for mismatch repair (MMR)‑deficient CRC and other malignancies, such as melanoma, the vast majority of MMR‑proficient CRCs are resistant to checkpoint inhibitor (CKI) therapy. MMR‑proficient CRCs commonly develop from precursor adenomas with enhanced Wnt‑signalling due to adenomatous polyposis coli (APC) mutations. In melanomas with enhanced Wnt signalling due to stabilized β‑catenin, immune anergy and resistance to CKI therapy has been observed, which is dependent on micro‑environmental myelomonocytic (MM) cell depletion in melanoma models. However, MM populations of colorectal adenomas or CRC have not been studied. To characterize resident intestinal MM cell populations during the early stages of tumorigenesis, the present study utilized the ApcMin/+ mouse as a model of MMR‑proficient CRC, using enhanced green fluorescent protein (EGFP) expression in the mouse lysozyme (M‑lys)lys‑EGFP/+ mouse as a pan‑myelomonocytic cell marker and a panel of murine macrophage surface markers. Total intestinal lamina propria mononuclear cell (LPMNC) numbers significantly decreased with age (2.32±1.39x107 [n=4] at 33 days of age vs. 1.06±0.24x107 [n=8] at 109 days of age) during intestinal adenoma development in ApcMin/+ mice (P=0.05; unpaired Student's t‑test), but not in wild‑type littermates (P=0.35). Decreased total LPMNC numbers were associated with atrophy of intestinal lymphoid follicles and the absence of MM/lymphoid cell aggregates in ApcMin/+ mouse intestine, but not spleen, compared with wild‑type mice. Furthermore, during the early stage of intestinal adenoma development, there was a two‑fold reduction of M‑lys expressing cells (P=0.05) and four‑fold reduction of ER‑HR3 (macrophage sub‑set) expressing cells (P=0.05; two tailed Mann‑Whitney U test) in mice with reduced total intestinal LPMNCs (n=3). Further studies are necessary to determine the relevance of these findings to immune‑surveillance of colorectal adenomas or MMR‑proficient CRC CKI therapy resistance

Antimicrobial and Efflux Pump Inhibitory Activity of Caffeoylquinic Acids from Artemisia absinthium against Gram-Positive Pathogenic Bacteria

Background: Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria. Methodology/Principal Findings: In this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology. Conclusions/Significance: These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4′,5′-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.National Institutes of Health (U.S.) (grant R01GM59903)National Institutes of Health (U.S.) (grant R01AI050875)Netherlands Organization for Scientific Research (VICI grant 700.56.442)Massachusetts Technology Transfer Center (MTTC)National Institutes of Health (U.S.) (grant 5U54MH084690-02

One‐Step Preparation of Biocompatible Gold Nanoplates with Controlled Thickness and Adjustable Optical Properties for Plasmon‐Based Applications

The ability to synthesize plasmonic nanomaterials with well‐defined structures and tailorable size is crucial for exploring their potential applications. Gold nanoplates (AuNPLs) exhibit appealing structural and optical properties, yet their applications are limited by difficulties in thickness control. Other challenges include a narrow range of tunability in size and surface plasmon resonance, combined with a synthesis conventionally involving cytotoxic cetyltrimethylammonium (CTA) halide surfactant. Here, a one‐step, high‐yield synthesis of single‐crystalline AuNPLs is developed, based on the combined use of two structure‐directing agents, methyl orange and FeBr3, which undergo preferential adsorption onto different crystalline facets of gold. The obtained AuNPLs feature high shape homogeneity that enables mesoscopic self‐assembly, broad‐range tunability of dimensions (controlled thickness from ≈7 to ≈20 nm, accompanied by modulation of the edge length from ≈150 nm to ≈2 µm) and plasmonic properties. These merits, coupled with a preparation free of CTA‐halide surfactants, have facilitated the exploration of various uses, especially in bio‐related areas. For example, they are demonstrated as biocompatible photothermal agents for cell ablation in NIR I and NIR II windows. This work paves the way to the innovative fabrication of anisotropic plasmonic nanomaterials with desired attributes for wide‐ranging practical applications

Evaluation of Lipid-Stabilised Tripropionin Nanodroplets as a Delivery Route for Combretastatin A4

Lipid-based nanoemulsions are a cheap and elegant route for improving the delivery of hydrophobic drugs. Easy and quick to prepare, nanoemulsions have promise for the delivery of different therapeutic agents. Although multiple studies have investigated the effects of the oil and preparation conditions on the size of the nanoemulsion nanodroplets for food applications, analogous studies for nanoemulsions for therapeutic applications are limited. Here we present a study on the production of lipid-stabilised oil nanodroplets (LONDs) towards medical applications. A number of biocompatible oils were used to form LONDs with phospholipid coatings, and among these, squalane and tripropionin were chosen as model oils for subsequent studies. LONDs were formed by high pressure homogenisation, and their size was found to decrease with increasing production pressure. When produced at 175 MPa, all LONDs samples exhibited sizes between 100 − 300 nm, with polydispersity index PI between 0.1 − 0.3. The LONDs were stable for over six weeks, at 4 °C, and also under physiological conditions, showing modest changes in size (<10%). The hydrophobic drug combretastatin A4 (CA4) was encapsulated in tripropionin LONDs with an efficiency of approximately 76%, achieving drug concentration of approximately 1.3 mg/ml. SVR mouse endothelial cells treated with CA4 tripropionin LONDs showed the microtubule disruption, characteristic of drug uptake for all tested doses, which suggests successful release of the CA4 from the LONDs

The use of high-frequency ultrasound imaging and biofluorescence for in vivo evaluation of gene therapy vectors

Background: Non-invasive imaging of the biodistribution of novel therapeutics including gene therapy vectors in animal models is essential. Methods: This study assessed the utility of high-frequency ultrasound (HF-US) combined with biofluoresence imaging (BFI) to determine the longitudinal impact of a Herpesvirus saimiri amplicon on human colorectal cancer xenograft growth. Results: HF-US imaging of xenografts resulted in an accurate and informative xenograft volume in a longitudinal study. The volumes correlated better with final ex vivo volume than mechanical callipers (R = 0.7993, p = 0.0002 vs. R = 0.7867, p = 0.0014). HF-US showed that the amplicon caused lobe formation. BFI demonstrated retention and expression of the amplicon in the xenografts and quantitation of the fluorescence levels also correlated with tumour volumes.Conclusions: The use of multi-modal imaging provided useful and enhanced insights into the behaviour of gene therapy vectors in vivo in real-time. These relatively inexpensive technologies are easy to incorporate into pre-clinical studies

Developing Hollow-Channel Gold Nanoflowers as Trimodal Intracellular Nanoprobes

Gold nanoparticles-enabled intracellular surface-enhanced Raman spectroscopy (SERS) provides a sensitive and promising technique for single cell analysis. Compared with spherical gold nanoparticles, gold nanoflowers, i.e., flower-shaped gold nanostructures, can produce a stronger SERS signal. Current exploration of gold nanoflowers for intracellular SERS has been considerably limited by the difficulties in preparation, as well as background signal and cytotoxicity arising from the surfactant capping layer. Recently, we have developed a facile and surfactant-free method for fabricating hollow-channel gold nanoflowers (HAuNFs) with great single-particle SERS activity. In this paper, we investigate the cellular uptake and cytotoxicity of our HAuNFs using a RAW 264.7 macrophage cell line, and have observed effective cellular internalization and low cytotoxicity. We have further engineered our HAuNFs into SERS-active tags, and demonstrated the functionality of the obtained tags as trimodal nanoprobes for dark-field and fluorescence microscopy imaging, together with intracellular SERS

A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development.

Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself