On-chip production of nanometer sized 'Ultra fine' bubble populations

Microbubble (MB) contrast agents have been used for many years as image enhancers for medical Ultrasound (US). Ultra-Fine bubble (UFB) populations of bubbles <1 µm in diameter are a relatively new technology that has found use as highly effective ‘eco’ cleaning agents. High-resolution US imaging is another potentially exciting area for UFB. This paper reports the on-chip production of UFB populations with a diameter of ~ 500 – 700 nm at a concentration of 10¹⁰ bub / mL. These UFB showed US scattering at higher frequency fields and enhanced contrast when imaging in in vivo mouse models

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

The Role of Csmd1 during Mammary Gland Development

The Cub Sushi Multiple Domains-1 (CSMD1) protein is a tumour suppressor which has been shown to play a role in regulating human mammary duct development in vitro. CSMD1 knockdown in vitro demonstrated increased cell proliferation, invasion and motility. However, the role of Csmd1 in vivo is poorly characterised when it comes to ductal development and is therefore an area which warrants further exploration. In this study a Csmd1 knockout (KO) mouse model was used to identify the role of Csmd1 in regulating mammary gland development during puberty. Changes in duct development and protein expression patterns were analysed by immunohistochemistry. This study identified increased ductal development during the early stages of puberty in the KO mice, characterised by increased ductal area and terminal end bud number at 6 weeks. Furthermore, increased expression of various proteins (Stat1, Fak, Akt, Slug/Snail and Progesterone receptor) was shown at 4 weeks in the KO mice, followed by lower expression levels from 6 weeks in the KO mice compared to the wild type mice. This study identifies a novel role for Csmd1 in mammary gland development, with Csmd1 KO causing significantly more rapid mammary gland development, suggesting an earlier adult mammary gland formation

Translating Biomarkers of Cholangiocarcinoma for Theranosis: A Systematic Review

Cholangiocarcinoma (CCA) is a rare disease with poor outcomes and limited research efforts into novel treatment options. A systematic review of CCA biomarkers was undertaken to identify promising biomarkers that may be used for theranosis (therapy and diagnosis). MEDLINE/EMBASE databases (1996–2019) were systematically searched using two strategies to identify biomarker studies of CCA. The PANTHER Go-Slim classification system and STRING network version 11.0 were used to interrogate the identified biomarkers. The TArget Selection Criteria for Theranosis (TASC-T) score was used to rank identified proteins as potential targetable biomarkers for theranosis. The following proteins scored the highest, CA9, CLDN18, TNC, MMP9, and EGFR, and they were evaluated in detail. None of these biomarkers had high sensitivity or specificity for CCA but have potential for theranosis. This review is unique in that it describes the process of selecting suitable markers for theranosis, which is also applicable to other diseases. This has highlighted existing validated markers of CCA that can be used for active tumor targeting for the future development of targeted theranostic delivery systems. It also emphasizes the relevance of bioinformatics in aiding the search for validated biomarkers that could be repurposed for theranosis

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

Morphological Control of Seedlessly-Synthesised Gold Nanorods using Binary Surfactants

High purity gold nanorods (AuNRs) with tunable morphology have been synthesized through a binary-surfactant seedless method, which enables the formation of monocrystalline AuNRs with diameters between 7 and 35 nm. The protocol has high shape yield and monodispersity, demonstrating good reproducibility and scalability allowing synthesis of batches 0.5 l in volume. Morphological control has been achieved through the adjustment of the molar concentrations of cetyltrimethylammonium bromide and sodium oleate in the growth solution, providing fine tuning of the optical scattering and absorbance properties of the AuNRs across the visible and NIR spectrum. Sodium oleate was found to provide greatest control over the aspect ratio (and hence optical properties) with concentration changes between 10 and 23 mM leading to variation in the aspect ratio between 2.8 and 4.8. Changes in the geometry of the end-caps were also observed as a result of manipulating the two surfactant concentrations

Cyclooxygenase activity mediates colorectal cancer cell resistance to the omega-3 polyunsaturated fatty acid eicosapentaenoic acid

Purpose The naturally-occurring omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) is safe, well-tolerated and inexpensive, making it an attractive anti-cancer intervention. However, EPA has only modest anti-colorectal cancer (CRC) activity, when used alone. Both cyclooxygenase (COX) isoforms metabolise EPA and are over-expressed in CRC cells. We investigated whether COX inhibition increases the sensitivity of CRC cells to growth inhibition by EPA. Methods A panel of 18 human and mouse CRC cell lines was used to characterize the differential sensitivity of CRC cells to the growth inhibitory effects of EPA. The effect of CRISPR-Cas9 genetic deletion and pharmacological inhibition of COX-1 and COX-2 on the anti-cancer activity of EPA was determined using in vitro and in vivo models. Results Genetic ablation of both COX isoforms increased sensitivity of CT26 mouse CRC cells to growth inhibition by EPA in vitro and in vivo. The non-selective COX inhibitor aspirin and the selective COX-2 inhibitor celecoxib increased sensitivity of several human and mouse CRC cell lines to EPA in vitro. However, in a MC38 mouse CRC cell tumour model, with dosing that mirrored low-dose aspirin use in humans, thereby producing significant platelet COX-1 inhibition, there was ineffective intra-tumoral COX-2 inhibition by aspirin and no effect on EPA sensitivity of MC38 cell tumours. Conclusion Cyclooxygenase inhibition by non-steroidal anti-inflammatory drugs represents a therapeutic opportunity to augment the modest anti-CRC activity of EPA. However, intra-tumoral COX inhibition is likely to be critical for this drug-nutrient interaction and careful tissue pharmacodynamic profiling is required in subsequent pre-clinical and human studies

Nested-Nanobubbles for Ultrasound Triggered Drug Release

Due to their size (1-10 μm) microbubble-based drug delivery agents suffer from confinement to the vasculature, limiting tumour penetration and potentially reducing drug efficacy. Nanobubbles (NBs) have emerged as promising candidates for ultrasound triggered drug delivery, due to their small size allowing drug delivery complexes to take advantage of the enhanced permeability and retention effect. In this study we describe a simple method for production of Nested-NBs, by encapsulation of NBs (~ 100 nm) within drug loaded liposomes. This method combines the efficient and well-established drug loading capabilities of liposomes, whilst utilizing NBs as an acoustic trigger for drug release. Encapsulation was characterized using Transmission Electron Microscopy with an encapsulation efficiency of 22 ± 2 %. Nested-NBs demonstrated echogenicity using diagnostic B-mode imaging and acoustic emissions were monitored during high intensity focused ultrasound (HIFU) in addition to monitoring of model drug release. Results showed that although the encapsulated NBs were destroyed by pulsed HIFU (peak negative pressure 1.54 – 4.83 MPa), signified by loss of echogenicity and detection of inertial cavitation, no model drug release was observed. Changing modality to continuous wave (CW) HIFU produced release across a range of peak negative pressures (2.01 – 3.90 MPa), likely due to a synergistic effect of mechanical and increased thermal stimuli. Due to this, we predict that our NBs contain a mixed population of both gaseous and liquid core particles, which upon CW HIFU undergo rapid phase conversion, triggering liposomal drug release. This hypothesis was investigated using previously described models to predict the existence of droplets and their phase change potential and the ability of this phase change to induce liposomal drug release

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

Tumour associated vasculature-on-a-chip for the evaluation of microbubble-mediated delivery of targeted liposomes

The vascular system is the primary route for the delivery of therapeutic drugs throughout the body and is an important barrier at the region of disease interest, such as a solid tumour. The development of complex 3D tumour cultures has progressed significantly in recent years however, the generation of perfusable vascularised tumour models still presents many challenges. This study presents a microfluidic-based vasculature system that can be induced to display properties of tumour-associated blood vessels without direct incorporation of tumour cells. Conditioning healthy endothelial–fibroblast cell vasculature co-cultures with media taken from tumour cell cultures was found to result in the formation of disorganised, tortuous networks which display characteristics consistent with those of tumour-associated vasculature. Integrin αvβ3, a cell adhesion receptor associated with angiogenesis, was found to be upregulated in vasculature co-cultures conditioned with tumour cell media (TCM) – consistent with the reported αvβ3 expression pattern in angiogenic tumour vasculature in vivo. Increased accumulation of liposomes (LSs) conjugated to antibodies against αvβ3 was observed in TCM networks compared to non-conditioned networks, indicating αvβ3 may be a potential target for the delivery of drugs specifically to tumour vasculature. Furthermore, the use of microbubbles (MBs) and ultrasound (US) to further enhance the delivery of LSs to TCM-conditioned vasculature was investigated. Quantification of fluorescent LS accumulation post-perfusion of the vascular network showed 3-fold increased accumulation with the use of MBs and US, suggesting that targeted LS delivery could be further improved with the use of locally administered MBs and US