Maleimide-Thiol Linkages Alter the Biodistribution of SN38 Therapeutic Microbubbles Compared to Biotin-Avidin While Preserving Parity in Tumoral Drug Delivery.

Therapeutic microbubbles (thMBs) contain drug-filled liposomes linked to microbubbles and targeted to vascular proteins. Upon the application of a destructive ultrasound trigger, drug uptake to tumour is improved. However, the structure of thMBs currently uses powerful non-covalent bonding of biotin with avidin-based proteins to link both the liposome to the microbubble (MB) and to bind the targeting antibody to the liposome-MB complex. This linkage is not currently FDA-approved, and therefore, an alternative, maleimide-thiol linkage, that is currently used in antibody-drug conjugates was examined. In a systematic manner, vascular endothelial growth factor receptor 2 (VEGFR2)-targeted MBs and thMBs using both types of linkages were examined for their ability to specifically bind to VEGFR2 in vitro and for their ultrasound imaging properties in vivo. Both showed equivalence in the production of the thMB structure, in vitro specificity of binding and safety profiles. In vivo imaging showed subtle differences for thMBs where biotin thMBs had a faster wash-in rate than thiol thMBs, but thiol thMBs were longer-lived. The drug delivery to tumours was also equivalent, but interestingly, thiol thMBs altered the biodistribution of delivery away from the lungs and towards the liver compared to biotin thMBs, which is an improvement in biosafety.

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 αvß3, 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 αvß3 expression pattern in angiogenic tumour vasculature in vivo. Increased accumulation of liposomes (LSs) conjugated to antibodies against αvß3 was observed in TCM networks compared to non-conditioned networks, indicating αvß3 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.

Structural and mechanical properties of folded protein hydrogels with embedded microbubbles.

Globular folded proteins are powerful building blocks to create biomaterials with mechanical robustness and inherent biological functionality. Here we explore their potential as advanced drug delivery scaffolds, by embedding microbubbles (MBs) within a photo-activated, chemically cross-linked bovine serum albumin (BSA) protein network. Using a combination of circular dichroism (CD), rheology, small angle neutron scattering (SANS) and microscopy we determine the nanoscale and mesoscale structure and mechanics of this novel multi-composite system. Optical and confocal microscopy confirms the presence of MBs within the protein hydrogel, their reduced diffusion and their effective rupture using ultrasound, a requirement for burst drug release. CD confirms that the inclusion of MBs does not impact the proportion of folded proteins within the cross-linked protein network. Rheological characterisation demonstrates that the mechanics of the BSA hydrogels is reduced in the presence of MBs. Furthermore, SANS reveals that embedding MBs in the protein hydrogel network results in a smaller number of clusters that are larger in size (∼16.6% reduction in number of clusters, 17.4% increase in cluster size). Taken together, we show that MBs can be successfully embedded within a folded protein network and ruptured upon application of ultrasound. The fundamental insight into the impact of embedded MBs in protein scaffolds at the nanoscale and mesoscale is important in the development of future platforms for targeted and controlled drug delivery applications.

The Influence of Nanobubble Size and Stability on Ultrasound Enhanced Drug Delivery.

Lipid-shelled nanobubbles (NBs) are emerging as potential dual diagnostic and therapeutic agents. Similar to their micron-scale counterparts, microbubbles (1-10 µm), they can act as ultrasound contrast agents as well as locally enhance therapeutic uptake. Recently, it has been shown that the reduced size of NBs (<1 µm) promotes increased uptake and accumulation in tumor interstitial space, which can enhance their diagnostic and therapeutic performance. However, accurate characterization of NB size and concentration is challenging and may limit their translation into clinical use. Their submicron nature limits accuracy of conventional microscopy techniques, while common light scattering techniques fail to distinguish between subpopulations present in NB samples (i.e., bubbles and liposomes). Due to the difficulty in the characterization of NBs, relatively little is known about the influence of size on their therapeutic performance. In this study, we describe a novel method of using a commercially available nanoparticle tracking analysis system, to distinguish between NBs and liposomes based on their differing optical properties. We used this technique to characterize three NB populations of varying size, isolated via centrifugation, and subsequently used this to assess their potential for enhancing localized delivery. Confocal fluorescence microscopy and image analysis were used to quantify the ultrasound enhanced uptake of fluorescent dextran into live colorectal cancer cells. Our results showed that the amount of localized uptake did not follow the expected trends, in which larger NB populations out-perform smaller NBs, at matched concentration. To understand this observed behavior, the stability of each NB population was assessed. It was found that dilution of the NB samples from their stock concentration influences their stability, and it is hypothesized that both the total free lipid and interbubble distance play a role in NB lifetime, in agreement with previously proposed theories and models.

Controlling the Optical Properties of Gold Nanorods in One-Pot Syntheses.

We present the characterization of the CTAB-oleate controlled synthesis of gold nanorods (AuNRs). Concentrations of key compounds in the synthetic system were varied in the presence of oleate, including HCl, borohydride, silver nitrate, and ascorbic acid. The longitudinal surface plasmon resonance peak was sensitive to changes in all concentrations. Reducing the concentration of Ag ions below 66 µM led to slower reaction kinetics and incomplete Au reduction. Variation of the ascorbic acid concentration revealed that oleate is responsible for around 44% of reduction of Au3+ to Au+ before nucleation in these experiments. Increasing the oleate concentration significantly slows the growth kinetics and leads to much longer synthesis times of above 12 h for reaction completion. These observations will enable the design of better methods of synthesizing of AuNRs using binary surfactants.

AI-enabled, implantable, multichannel wireless telemetry for photodynamic therapy.

Photodynamic therapy (PDT) offers several advantages for treating cancers, but its efficacy is highly dependent on light delivery to activate a photosensitizer. Advances in wireless technologies enable remote delivery of light to tumors, but suffer from key limitations, including low levels of tissue penetration and photosensitizer activation. Here, we introduce DeepLabCut (DLC)-informed low-power wireless telemetry with an integrated thermal/light simulation platform that overcomes the above constraints. The simulator produces an optimized combination of wavelengths and light sources, and DLC-assisted wireless telemetry uses the parameters from the simulator to enable adequate illumination of tumors through high-throughput (<20 mice) and multi-wavelength operation. Together, they establish a range of guidelines for effective PDT regimen design. In vivo Hypericin and Foscan mediated PDT, using cancer xenograft models, demonstrates substantial suppression of tumor growth, warranting further investigation in research and/or clinical settings.

A Single Short 'Tone Burst' Results in Optimal Drug Delivery to Tumours Using Ultrasound-Triggered Therapeutic Microbubbles.

Advanced drug delivery systems, such as ultrasound-mediated drug delivery, show great promise for increasing the therapeutic index. Improvements in delivery by altering the ultrasound parameters have been studied heavily in vitro but relatively little in vivo. Here, the same therapeutic microbubble and tumour type are used to determine whether altering ultrasound parameters can improve drug delivery. Liposomes were loaded with SN38 and attached via avidin: biotin linkages to microbubbles. The whole structure was targeted to the tumour vasculature by the addition of anti-vascular endothelial growth factor receptor 2 antibodies. Tumour drug delivery and metabolism were quantified in SW480 xenografts after application of an ultrasound trigger to the tumour region. Increasing the trigger duration from 5 s to 2 min or increasing the number of 5 s triggers did not improve drug delivery, nor did changing to a chirp trigger designed to stimulate a greater proportion of the microbubble population, although this did show that the short tone trigger resulted in greater release of free SN38. Examination of ultrasound triggers in vivo to improve drug delivery is justified as there are multiple mechanisms at play that may not allow direct translation from in vitro findings. In this setting, a short tone burst gives the best ultrasound parameters for tumoural drug delivery.

Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5-7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

Evaluation of the Targeting and Therapeutic Efficiency of Anti-EGFR Functionalised Nanoparticles in Head and Neck Cancer Cells for Use in NIR-II Optical Window.

Gold nanoparticles have been indicated for use in a diagnostic and/or therapeutic role in several cancer types. The use of gold nanorods (AuNRs) with a surface plasmon resonance (SPR) in the second near-infrared II (NIR-II) optical window promises deeper anatomical penetration through increased maximum permissible exposure and lower optical attenuation. In this study, the targeting and therapeutic efficiency of anti-epidermal growth factor receptor (EGFR)-antibody-functionalised AuNRs with an SPR at 1064 nm was evaluated in vitro. Four cell lines, KYSE-30, CAL-27, Hep-G2 and MCF-7, which either over- or under-expressed EGFR, were used once confirmed by flow cytometry and immunofluorescence. Optical microscopy demonstrated a significant difference (p < 0.0001) between targeted AuNRs (tAuNRs) and untargeted AuNRs (uAuNRs) in all four cancer cell lines. This study demonstrated that anti-EGFR functionalisation significantly increased the association of tAuNRs with each EGFR-positive cancer cell. Considering this, the MTT assay showed that photothermal therapy (PTT) significantly increased cancer cell death (>97%) in head and neck cancer cell line CAL-27 using tAuNRs but not uAuNRs, apoptosis being the major mechanism of cell death. This successful targeting and therapeutic outcome highlight the future use of tAuNRs for molecular photoacoustic imaging or tumour treatment through plasmonic photothermal therapy.

Targeted microbubbles carrying lipid-oil-nanodroplets for ultrasound-triggered delivery of the hydrophobic drug, combretastatin A4.

The hydrophobicity of a drug can be a major challenge in its development and prevents the clinical translation of highly potent anti-cancer agents. We have used a lipid-based nanoemulsion termed Lipid-Oil-Nanodroplets (LONDs) for the encapsulation and in vivo delivery of the poorly bioavailable combretastatin A4 (CA4). Drug delivery with CA4 LONDs was assessed in a xenograft model of colorectal cancer. LC-MS/MS analysis revealed that CA4 LONDs, administered at a drug dose four times lower than drug control, achieved equivalent concentrations of CA4 intratumorally. We then attached CA4 LONDs to microbubbles (MBs) and targeted this construct to VEGFR2. A reduction in tumor perfusion was observed in CA4 LONDs-MBs treated tumors. A combination study with irinotecan demonstrated a greater reduction in tumor growth and perfusion (P = 0.01) compared to irinotecan alone. This study suggests that LONDs, either alone or attached to targeted MBs, have the potential to significantly enhance tumor-specific hydrophobic drug delivery.

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 ß-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±1.39×107 [n=4] at 33 days of age vs. 1.06±0.24×107 [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.

Evaluating Phospholipid-Functionalized Gold Nanorods for In Vivo Applications.

Gold nanorods (AuNRs) have attracted a great deal of attention due to their potential for use in a wide range of biomedical applications. However, their production typically requires the use of the relatively toxic cationic surfactant cetyltrimethylammonium bromide (CTAB) leading to continued demand for protocols to detoxify them for in vivo applications. In this study, a robust and facile protocol for the displacement of CTAB from the surface of AuNRs using phospholipids is presented. After the displacement, CTAB is not detectable by NMR spectroscopy, surface-enhanced Raman spectroscopy, or using pH-dependent ζ-potential measurements. The phospholipid functionalized AuNRs demonstrated superior stability and biocompatibility (IC50  > 200 µg mL-1 ) compared to both CTAB and polyelectrolyte functionalized AuNRs and are well tolerated in vivo. Furthermore, they have high near-infrared (NIR) absorbance and produce large amounts of heat under NIR illumination, hence such particles are well suited for plasmonic medical applications.

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.

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.

Exploring High Aspect Ratio Gold Nanotubes as Cytosolic Agents: Structural Engineering and Uptake into Mesothelioma Cells.

The generation of effective and safe nanoagents for biological applications requires their physicochemical characteristics to be tunable, and their cellular interactions to be well characterized. Here, the controlled synthesis is developed for preparing high-aspect ratio gold nanotubes (AuNTs) with tailorable wall thickness, microstructure, composition, and optical characteristics. The modulation of optical properties generates AuNTs with strong near infrared absorption. Surface modification enhances dispersibility of AuNTs in aqueous media and results in low cytotoxicity. The uptake and trafficking of these AuNTs by primary mesothelioma cells demonstrate their accumulation in a perinuclear distribution where they are confined initially in membrane-bound vesicles from which they ultimately escape to the cytosol. This represents the first study of the cellular interactions of high-aspect ratio 1D metal nanomaterials and will facilitate the rational design of plasmonic nanoconstructs as cytosolic nanoagents for potential diagnosis and therapeutic applications.

Ultrasound-triggered therapeutic microbubbles enhance the efficacy of cytotoxic drugs by increasing circulation and tumor drug accumulation and limiting bioavailability and toxicity in normal tissues.

Most cancer patients receive chemotherapy at some stage of their treatment which makes improving the efficacy of cytotoxic drugs an ongoing and important goal. Despite large numbers of potent anti-cancer agents being developed, a major obstacle to clinical translation remains the inability to deliver therapeutic doses to a tumor without causing intolerable side effects. To address this problem, there has been intense interest in nanoformulations and targeted delivery to improve cancer outcomes. The aim of this work was to demonstrate how vascular endothelial growth factor receptor 2 (VEGFR2)-targeted, ultrasound-triggered delivery with therapeutic microbubbles (thMBs) could improve the therapeutic range of cytotoxic drugs. Methods: Using a microfluidic microbubble production platform, we generated thMBs comprising VEGFR2-targeted microbubbles with attached liposomal payloads for localised ultrasound-triggered delivery of irinotecan and SN38 in mouse models of colorectal cancer. Intravenous injection into tumor-bearing mice was used to examine targeting efficiency and tumor pharmacodynamics. High-frequency ultrasound and bioluminescent imaging were used to visualise microbubbles in real-time. Tandem mass spectrometry (LC-MS/MS) was used to quantitate intratumoral drug delivery and tissue biodistribution. Finally, 89Zr PET radiotracing was used to compare biodistribution and tumor accumulation of ultrasound-triggered SN38 thMBs with VEGFR2-targeted SN38 liposomes alone. Results: ThMBs specifically bound VEGFR2 in vitro and significantly improved tumor responses to low dose irinotecan and SN38 in human colorectal cancer xenografts. An ultrasound trigger was essential to achieve the selective effects of thMBs as without it, thMBs failed to extend intratumoral drug delivery or demonstrate enhanced tumor responses. Sensitive LC-MS/MS quantification of drugs and their metabolites demonstrated that thMBs extended drug exposure in tumors but limited exposure in healthy tissues, not exposed to ultrasound, by persistent encapsulation of drug prior to elimination. 89Zr PET radiotracing showed that the percentage injected dose in tumors achieved with thMBs was twice that of VEGFR2-targeted SN38 liposomes alone. Conclusions: thMBs provide a generic platform for the targeted, ultrasound-triggered delivery of cytotoxic drugs by enhancing tumor responses to low dose drug delivery via combined effects on circulation, tumor drug accumulation and exposure and altered metabolism in normal tissues.

Nested Nanobubbles for Ultrasound-Triggered Drug Release.

Because of their size (1-10 µm), microbubble-based drug delivery agents suffer from confinement to the vasculature, limiting tumor penetration and potentially reducing the drug efficacy. Nanobubbles (NBs) have emerged as promising candidates for ultrasound-triggered drug delivery because of 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-nanobubbles (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 while 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 (PNP) 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 PNPs (2.01-3.90 MPa), likely because of a synergistic effect of mechanical and increased thermal stimuli. Because of 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.

High-throughput microfluidics for evaluating microbubble enhanced delivery of cancer therapeutics in spheroid cultures.

Drug penetration into solid tumours remains a major challenge in the effective treatment of cancer. Microbubble (MB) mediated sonoporation offers a potential solution to this by enhancing the uptake of drugs into cells. Additionally, in using an ultrasound (US) trigger, drug delivery can be localised to the tumour, thus reducing the off-site toxicity associated with systemic delivery. The majority of in vitro studies involving the observation of MB-enhanced drug efficacy have been conducted on 2D monolayer cell cultures, which are known to be poor models for in vivo tumours. 3D spheroid cultures allow for the production of multicellular cultures complete with extracellular matrix (ECM) components. These cultures effectively recreate many of the physiological features of the tumour microenvironment and have been shown to be far superior to previous 2D monolayer models. However, spheroids are typically handled in well-plates in which the fluid environment is static, limiting the physiological relevance of the model. The combination of 3D cultures and microfluidics would allow for the production of a dynamic system in which spheroids are subjected to in vivo like fluid flow and shear stresses. This study presents a microfluidic device containing an array of spheroid traps, into which multiple pre-grown colorectal cancer (CRC) spheroids were loaded. Reservoirs interfaced with the chip use hydrostatic pressure to passively drive flow through the system and subject spheroids to capillary like flow velocities. The use of reservoirs also enabled multiple chips to be run in parallel, allowing for the screening of multiple therapeutic treatments (n = 690 total spheroids analysed). This microfluidic platform was used to investigate MB enhanced drug delivery and showed that co-delivery of 3 µM doxorubicin (DOX) + MB + US reduced spheroid viability to 48 ± 2%, compared to 75 ± 5% observed with 3 µM DOX alone. Delivery of drug loaded MBs (DLMBs), in which DOX-loaded liposomes (DOX-LS) were conjugated to MBs, reduced spheroid viability to 62 ± 3%, a decrease compared to the 75 ± 3% viability observed with DOX-LS in the absence of MBs + US.

CD105 is a prognostic marker and valid endothelial target for microbubble platforms in cholangiocarcinoma.

PURPOSE: The current treatment outcomes in cholangiocarcinoma are poor with cure afforded only by surgical extirpation. The efficacy of targeting the tumoural endothelial marker CD105 in cholangiocarcinoma, as a basis for potential microbubble-based treatment, is unknown and was explored here. METHODS: Tissue expression of CD105 was quantified using immunohistochemistry in 54 perihilar cholangiocarcinoma samples from patients who underwent resection in a single centre over a ten-year period, and analysed against clinicopathological data. In vitro flow assays using microbubbles functionalised with CD105 antibody were conducted to ascertain specificity of binding to murine SVR endothelial cells. Finally, CD105-microbubbles were intravenously administered to 10 Balb/c nude mice bearing heterotopic subcutaneous human extrahepatic cholangiocarcinoma (TFK-1 and EGI-1) xenografts after which in vivo binding was assessed following contrast-enhanced destruction replenishment ultrasound application. RESULTS: Though not significantly associated with any examined clinicopathological variable, we found that higher CD105 expression was independently associated with poorer patient survival (median 12 vs 31 months; p = 0.002). In vitro studies revealed significant binding of CD105-microbubbles to SVR endothelial cells in comparison to isotype control (p = 0.01), as well as in vivo to TFK-1 (p = 0.02) and EGI-1 (p = 0.04) mouse xenograft vasculature. CONCLUSION: Our results indicate that CD105 is a biomarker eminently suitable for cholangiocarcinoma targeting using functionalised microbubbles.