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The efficient encapsulation of small molecule active ingredients has been a challenge for many decades across many commercial applications. Recently, successful attempts to address this issue have included deposition of thin metal shells onto liquid filled polymer microcapsules or emulsion droplets to provide an impermeable barrier to diffusion. In this work we have developed a novel method to protect small molecule active ingredients by deposition of thin mineral shells. Platinum nanoparticles are used to catalyse and direct growth of a calcium phosphate shell onto liquid filled polymer microcapsules under various reaction conditions. Findings indicate that a non-porous protective shell is formed on the majority of the microcapsule population, with small concentrations of the core material being released only from those microcapsules with defects, over a 7 days period, when conducting forced release studies into a solvent for the core oil. The resulting microcapsules show no significant cell toxicity when exposed to HEK 293 cells for 72 hours.

The implementation of accurate and sensitive molecular detection for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is paramount to effectively control the ongoing coronavirus disease 2019 (COVID-19) pandemic. In this regard, we herein propose the specific and highly sensitive SARS-CoV-2 detection based on nanoyeast single-chain-variable fragment (scFv) and ultrasensitive plasmonic nanobox-integrated nanomixing microassay. Importantly, this designed platform showcases the utility of nanoyeast-scFvs as specific capture reagents targeting the receptor-binding domain (RBD) of the virus and as monoclonal antibody alternatives suitable for cost-effective mass production and frequent testing. By capitalizing on single-particle active nanoboxes as plasmonic nanostructures for surface-enhanced Raman scattering (SERS), the microassay utilizes highly sensitive Raman signals to indicate virus infection. The developed microassay further integrated nanomixing for accelerating molecular collisions. Through the synergistic working of nanoyeast-scFv, plasmonic nanoboxes, and nanomixing, the highly specific and sensitive SARS-CoV-2 detection is achieved as low as 17 virus/μL without any molecular amplification. We successfully demonstrate SARS-CoV-2 detection in saliva samples of simulated patients at clinically relevant viral loads, suggesting the possibility of this platform for accurate and noninvasive patient screening.

At diagnosis, 22% of colorectal cancer (CRC) patients have metastases, and 50% later develop metastasis. Peptide receptor radionuclide therapy (PRRT), such as 177Lu-PSMA-617, is used to treat metastatic prostate cancer. 177Lu-PSMA-617 targets prostate-specific membrane antigen (PSMA), a cell-surface protein enriched in prostate cancer and the neovasculature of other solid tumors, including CRC. We performed 68Ga-PSMA-11 PET/CT imaging of 10 patients with metastatic CRC to assess metastasis avidity. Eight patients had lesions lacking avidity, and 2 had solitary metastases exhibiting very low avidity. Despite expression of PSMA in CRC neovasculature, none of the patients exhibited tumor avidity sufficient to be considered for 177Lu-PSMA-617 PRRT.

The clinical translation of new nanoparticle-based therapies for high-grade glioma (HGG) remains extremely poor. This has partly been due to the lack of suitable preclinical mouse models capable of replicating the complex characteristics of recurrent HGG (rHGG), namely the heterogeneous structural and functional characteristics of the blood-brain barrier (BBB). The goal of this study is to compare the characteristics of the tumor BBB of rHGG with two different mouse models of HGG, the ubiquitously used U87 cell line xenograft model and a patient-derived cell line WK1 xenograft model, in order to assess their suitability for nanomedicine research. Method: Structural MRI was used to assess the extent of BBB opening in mouse models with a fully developed tumor, and dynamic contrast enhanced MRI was used to obtain values of BBB permeability in contrast enhancing tumor. H&E and immunofluorescence staining were used to validate results obtained from the in vivo imaging studies. Results: The extent of BBB disruption and permeability in the contrast enhancing tumor was significantly higher in the U87 model than in rHGG. These values in the WK1 model are similar to those of rHGG. The U87 model is not infiltrative, has an entirely abnormal and leaky vasculature and it is not of glial origin. The WK1 model infiltrates into the non-neoplastic brain parenchyma, it has both regions with intact BBB and regions with leaky BBB and remains of glial origin. Conclusion: The WK1 mouse model more accurately reproduces the extent of BBB disruption, the level of BBB permeability and the histopathological characteristics found in rHGG patients than the U87 mouse model, and is therefore a more clinically relevant model for preclinical evaluations of emerging nanoparticle-based therapies for HGG.

INTRODUCTION: Pretargeting strategies that do not rely on the expression of molecular targets have expanded imaging and therapy options for cancer patients. Nanostars with designed multivalency and which highly accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect may therefore be the ideal vectors for the development of a passive pretargeting approach. METHODS: Nanostars were synthesized, consisting of 7–8 centre-cross-linked arms that were modified with trans-cyclooctene (TCO) using poly(ethylene glycol) (PEG) linkers of 12 or 106 monomer units or without linker. The bioorthogonal click reaction with radiofluorinated 2,2’-(7-(2-(tetrazine-poly(ethyleneglycol)(11)-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([(18)F]F-Tz-PEG(11)-NODA) or 2,2’-(7-(2-(tetrazine-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([(18)F]F-Tz-NODA) was measured by ex vivo biodistribution studies and positron emission tomography (PET) in mice bearing tumors with high EPR characteristics. Bioorthogonal masking was performed using a tetrazine-functionalized dextran polymer (Tz-DP). RESULTS: Highest tumor accumulation of [(18)F]F-Tz-PEG(11)-NODA was observed for nanostars functionalized with TCO without linker, with a tumor uptake of 3.2 ± 0.4 %ID/g and a tumor-to-muscle ratio of 12.8 ± 4.2, tumor-to-large intestine ratio of 0.5 ± 0.3 and tumor-to-kidney ratio of 2.0 ± 0.3, being significantly higher than for nanostars functionalized with TCO-PEG(12) (P < 0.05) or TCO-PEG(106) (P < 0.05). Tumor uptake and tumor-to-tissue ratios did not improve upon bioorthogonal masking with Tz-DP or when using a smaller, more lipophilic tetrazine([(18)F]F-Tz-NODA). CONCUSIONS: A pretargeting strategy was developed based on the passive delivery of TCO-functionalized nanostars. Such a strategy would allow for the imaging and treatment of tumors with apparent EPR characteristics, with high radioactive tumor doses and minimal doses to off-target tissues.

Increasing accumulation and retention of nanomedicines within tumor tissue is a significant challenge, particularly in the case of brain tumors where access to the tumor through the vasculature is restricted by the blood–brain barrier (BBB). This makes the application of nanomedicines in neuro-oncology often considered unfeasible, with efficacy limited to regions of significant disease progression and compromised BBB. However, little is understood about how the evolving tumor–brain physiology during disease progression affects the permeability and retention of designer nanomedicines. We report here the development of a modular nanomedicine platform that, when used in conjunction with a unique model of how tumorigenesis affects BBB integrity, allows investigation of how nanomaterial properties affect uptake and retention in brain tissue. By combining different in vivo longitudinal imaging techniques (including positron emission tomography and magnetic resonance imaging), we have evaluated the retention of nanomedicines with predefined physicochemical properties (size and surface functionality) and established a relationship between structure and tissue accumulation as a function of a new parameter that measures BBB leakiness; this offers significant advancements in our ability to relate tumor accumulation of nanomedicines to more physiologically relevant parameters. Our data show that accumulation of nanomedicines in brain tumor tissue is better correlated with the leakiness of the BBB than actual tumor volume. This was evaluated by establishing brain tumors using a spontaneous and endogenously derived glioblastoma model providing a unique opportunity to assess these parameters individually and compare the results across multiple mice. We also quantitatively demonstrate that smaller nanomedicines (20 nm) can indeed cross the BBB and accumulate in tumors at earlier stages of the disease than larger analogues, therefore opening the possibility of developing patient-specific nanoparticle treatment interventions in earlier stages of the disease. Importantly, these results provide a more predictive approach for designing efficacious personalized nanomedicines based on a particular patient’s condition., Nanomedicines show potential to treat brain tumors using simultaneous PET-MRI as a tool to directly correlate physiological conditions with tumor accumulation in the more human relevant mouse model.

Background: CUB domain-containing protein 1 (CDCP1) is a cell surface receptor regulating key signalling pathways in malignant cells. CDCP1 has been proposed as a molecular target to abrogate oncogenic signalling pathways and specifically deliver anti-cancer agents to tumors. However, the development of CDCP1-targeting agents has been questioned by its frequent proteolytic processing which was thought to result in shedding of the CDCP1 extracellular domain limiting its targetability. In this study, we investigated the relevance of targeting CDCP1 in the context of pancreatic ductal adenocarcinoma (PDAC) and assess the impact of CDCP1 proteolysis on the effectiveness of CDCP1 targeting agents. Methods: The involvement of CDCP1 in PDAC progression was assessed by association analysis in several PDAC cohorts and the proteolytic processing of CDCP1 was evaluated in PDAC cell lines and patient-derived cells. The consequences of CDCP1 proteolysis on its targetability in PDAC cells was assessed using immunoprecipitation, immunostaining and biochemical assays. The involvement of CDCP1 in PDAC progression was examined by loss-of-function in vitro and in vivo experiments employing PDAC cells expressing intact or cleaved CDCP1. Finally, we generated antibody-based imaging and therapeutic agents targeting CDCP1 to demonstrate the feasibility of targeting this receptor for detection and treatment of PDAC tumors. Results: High CDCP1 expression in PDAC is significantly associated with poorer patient survival. In PDAC cells proteolysis of CDCP1 does not always result in the shedding of CDCP1-extracellular domain which can interact with membrane-bound CDCP1 allowing signal transduction between the different CDCP1-fragments. Targeting CDCP1 impairs PDAC cell functions and PDAC tumor growth independently of CDCP1 cleavage status. A CDCP1-targeting antibody is highly effective at delivering imaging radionuclides and cytotoxins to PDAC cells allowing specific detection of tumors by PET/CT imaging and superior anti-tumor effects compared to gemcitabine in in vivo models. Conclusion: Independent of its cleavage status, CDCP1 exerts oncogenic functions in PDAC and has significant potential to be targeted for improved radiological staging and treatment of this cancer. Its elevated expression by most PDAC tumors and lack of expression by normal pancreas and other major organs, suggest that targeting CDCP1 could benefit a significant proportion of PDAC patients. These data support the further development of CDCP1-targeting agents as personalizable tools for effective imaging and treatment of PDAC.

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter

Expression levels of biomarkers are generally unknown at initial diagnosis. The development of theranostic probes that do not rely on biomarker availability would expand therapy options for cancer patients, improve patient selection for nanomedicine and facilitate treatment of inoperable patients or patients with acquired therapy resistance. Herein, we report the development of star polymers, also known as nanostars, that allow for molecular imaging and/or endoradiotherapy based on passive targeting via the enhanced permeability and retention (EPR) effect.Methods: We synthesised a star copolymer, consisting of 7-8 centre-cross-linked arms that were modified with Gd3+ for magnetic resonance imaging (MRI), and functionalised either with Zr-89 for in vivo quantification and positron emission tomography (PET) imaging, or with Lu-177 for endoradiotherapy. H-1 longitudinal relaxivities were determined over a continuum of magnetic field strengths ranging from 0.24 mT - 0.94 T at 37 degrees C (nuclear magnetic relaxation dispersion (NMRD) profile) and T-1-weighted MRI contrast enhancement was visualized at 3 T and 7 T. PET imaging and ex vivo biodistribution studies were performed in mice bearing tumours with high EPR (CT26) or low EPR (BxPC3) characteristics. Therapy studies were performed in mice with high EPR tumours and mean absorbed organ doses were estimated for a standard human model.Results: The star copolymer with Gd3+ displayed a significantly superior contrast enhancement ability (T-1 = 0.60 s) compared to the standard clinical contrast agent Gadovist (T-1 = 1.0 s). Quantification of tumour accumulation using the radiolabelled nanostars in tumour-bearing mice demonstrated an exceptionally high uptake in tumours with high EPR characteristics (14.8 - 21.7 %ID/g). Uptake of the star polymers in tumours with low EPR characteristics was significantly lower (P

Survival outcomes for patients with glioblastoma multiforme (GBM) have remained poor for the past 15 years, reflecting a clear challenge in the development of more effective treatment strategies. The efficacy of systemic therapies for GBM is greatly limited by the presence of the blood-brain barrier (BBB), which prevents drug penetration and accumulation in regions of infiltrative tumour, as represented in a consistent portion of GBM lesions. Focused ultrasound (FUS) - a technique that uses low-frequency ultrasound waves to induce targeted temporary disruption of the BBB - promises to improve survival outcomes by enhancing drug delivery and accumulation to infiltrating tumour regions. In this review we discuss the current state of preclinical investigations using FUS to enhance delivery of systemic therapies to intracranial neoplasms. We highlight critical methodological inconsistencies that are hampering clinical translation of FUS and we provide guiding principles for future preclinical studies. Particularly, we focus our attention on the importance of the selection of clinically relevant animal models and to the standardization of methods for FUS delivery, which will be paramount to the successful clinical translation of this promising technology for treatment in GBM patients. We also discuss how preclinical FUS research can benefit the development of GBM immunotherapies.

Colorectal cancer (CRC) is the third most common malignancy in the world, with 22% of patients presenting with metastatic disease and a further 50% destined to develop metastasis. Molecular imaging uses antigen-specific ligands conjugated to radionuclides to detect and characterise primary cancer and metastases. Expression of the cell surface protein CDCP1 is increased in CRC, and here we sought to assess whether it is a suitable molecular imaging target for the detection of this cancer. CDCP1 expression was assessed in CRC cell lines and a patient-derived xenograft to identify models suitable for evaluation of radio-labelled 10D7, a CDCP1-targeted, high-affinity monoclonal antibody, for preclinical molecular imaging. Positron emission tomography-computed tomography was used to compare zirconium-89 (89Zr)-10D7 avidity to a nonspecific, isotype control 89Zr-labelled IgGκ1 antibody. The specificity of CDCP1-avidity was further confirmed using CDCP1 silencing and blocking models. Our data indicate high avidity and specificity for of 89Zr-10D7 in CDCP1 expressing tumors at. Significantly higher levels than normal organs and blood, with greatest tumor avidity observed at late imaging time points. Furthermore, relatively high avidity is detected in high CDCP1 expressing tumors, with reduced avidity where CDCP1 expression was knocked down or blocked. The study supports CDCP1 as a molecular imaging target for CRC in preclinical PET-CT models using the radioligand 89Zr-10D7.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Multicentre clinical trials evaluating the role ofCompared to the gCS method, the MI method showed significantly lower intra- and inter-reader variability both in background activity and in BTV definition.The proposed semiautomated MI method minimises intra- and inter-reader variability, providing a valuable approach for standardisation of

The functionalization of proteins with different cargo molecules is highly desirable for a broad range of applications. However, the reproducible production of defined conjugates with multiple functionalities is a significant challenge. Herein, we report the dual site-specific labeling of an antibody fragment, utilizing the orthogonal Sortase A and π-clamp conjugation methods, and demonstrate that binding of the antibody fragment to its target receptor is retained after dual labeling.

Systemic therapies have limited efficacy against brain metastases, largely because passive delivery of naked compounds via the bloodstream does not achieve sufficiently high or evenly dispersed intratumoural concentrations. Heterogeneous tissue architecture, abnormal perfusion, hypoxic zones and high interstitial fluid pressure are key factors limiting drug delivery, compounded by patchy blood-tumour-barrier permeability. Also, brain metastases are usually detected late, once patients become symptomatic. We are investigating whether engineered biopharmaceuticals might improve diagnostic sensitivity for earlier detection, as well as therapeutic efficacy and side-effect profiles of existing agents through active tumour targeting, delayed clearance and microenvironment-mediated activation. This study is proceeding with parallel preclinical and clinical tracks. Preclinical aims: (1) Develop and characterise monoclonal antibody (mAb) fragments (scFvs) that target the brain metastasis markers HER2 and HER3; (2) Functionalise polyethylene glycol (PEG)-based nanocarriers with the scFvs, along with imaging agents to facilitate in vivoand ex vivoanalysis of tissue distribution; (3) Functionalise HER2/3-targeted carriers with doxorubicin via an acid-labile hydrazone bond for release in hypoxic environments, or the endosome compartment after internalization. Results to date. His-tagged HER2- and HER3-targeted scFvs based on ligand-binding sequences of clinically-approved mAbs were expressed and purified from Expi293 suspension cultures. Binding affinities are an order of magnitude stronger than parent mAbs (KD 2-8x10E-11M), determined using surface plasmon resonance analysis. The scFvs are cytostatic and moderately cytotoxic in vitro, with IC50s in order of 0.4-1.0μM. HER2 and HER3 scFvs exhibited dose-dependent, additive growth inhibition when used in combination, and induced internalisation of their receptor ligands within 4 hours in SKBr3 cells. Conclusions.The scFvs are strong carrier-tethering candidates in terms of both extracellular and intracellular payload release. Carrier synthesis is currently underway and preliminary in vivo data will be presented. Clinical aims: (1) Develop and characterise 89Zirconium-labelled HER2-targeted PET tracers based on parent mAb and scFv; (2) Compare uptake and retention of the tracers in breast cancer patients with brain metastases; (3) Computationally relate tumour uptake to the administered dose, perfusion, tumour size and HER2 expression; (4) Determine the uptake range within and between patients, and the minimum size for reliable detection. Results to date. The mAb tracer has been synthesised, characterised and labelling processes scaled for clinical production. It is stable in physiologic conditions, retains HER2-binding activity and has a favourable biodistribution profile in NOD-SCID mice bearing BT474 xenografts. Conclusions. Australian regulatory approvals are in place and recruitment for the mAb imaging trial ("BoNSAI") has begun. Preliminary data will be presented. Citation Format: Saunus JM, Lim M, Puttick S, Kalita-de Croft P, Houston ZH, Jones ML, Latter MJ, Campbell LC, Thomas P, Jeffree RL, Rose SE, Mahler SM, Thurecht KJ, Scott AM, Lakhani SR. Innovation in diagnosis and treatment of brain metastases using multifunctional nanomedicines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-19-02.

BackgroundNovel targeted therapies for children diagnosed with medulloblastoma (MB), the most common malignant pediatric brain tumor, are urgently required. A major hurdle in the development of effective therapies is the impaired delivery of systemic therapies to tumor cells due to a specialized endothelial blood-brain barrier (BBB). Accordingly, the integrity of the BBB is an essential consideration in any preclinical model used for assessing novel therapeutics. This study sought to assess the functional integrity of the BBB in several preclinical mouse models of MB.MethodsDynamic contrast enhancement magnetic resonance imaging (MRI) was used to evaluate blood-brain-tumor barrier (BBTB) permeability in a murine genetically engineered mouse model (GEMM) of Sonic Hedgehog (SHH) MB, patient-derived orthotopic xenograft models of MB (SHH and Gp3), and orthotopic transplantation of GEMM tumor cells, enabling a comparison of the direct effects of transplantation on the integrity of the BBTB. Immunofluorescence analysis was performed to compare the structural and subcellular features of tumor-associated vasculature in all models.ResultsContrast enhancement was observed in all transplantation models of MB. No contrast enhancement was observed in the GEMM despite significant tumor burden. Cellular analysis of BBTB integrity revealed aberrancies in all transplantation models, correlating to the varying levels of BBTB permeability observed by MRI in these models.ConclusionsThese results highlight functional differences in the integrity of the BBTB and tumor vessel phenotype between commonly utilized preclinical models of MB, with important implications for the preclinical evaluation of novel therapeutic agents for MB.

In this paper, we investigate anomalous diffusion models in a mouse model of glioblastoma, a grade IV brain tumour, and study how the anomalous diffusion model parameters reflect the change in tumour tissue microstructure. Diffusion-weighted MRI data with multiple b-values at 9.4 T was acquired from mice bearing U87 brain tumour cells at four time points. Voxel-level fitting of the MRI data was performed on the classical mono-exponential model, and four anomalous diffusion models, namely, the stretched exponential model, the sub-diffusion model, the continuous time random walk model and the fractional Bloch-Torrey equation. The performance of the anomalous diffusion parameters for differentiating the three-concentric layers of tumour tissue (i.e., core; intermediate zone; peripheral and hyper-vascularised tumour layer) was evaluated with multinomial logistic regression and multi-class classification analysis. We found that parameter \(\alpha \) from the stretched exponential model, parameter \(\upbeta \) from the sub-diffusion model and parameter \(\upbeta \) from the continuous time random walk model provide a clear delineation of the three layers of tumour tissue. The analysis revealed that the combination of diffusion coefficient D and anomalous diffusion parameter (\(\alpha \) and/or \(\upbeta \)) greatly improved the classification power in terms of F1-scores compared with the current approach in clinics, in which D is used alone. Hence, our mouse brain tumour study demonstrated that anomalous diffusion model parameters are useful for differentiating different tumour layers and normal brain tissue.

Background High-grade glioma (HGG) remains a recalcitrant clinical problem despite many decades of research. A major challenge in improving prognosis is the inability of current therapeutic strategies to address a clinically significant burden of infiltrating tumor cells that extend beyond the margins of the primary tumor mass. Such cells cannot be surgically excised nor efficiently targeted by radiation therapy. Therapeutic targeting of this tumor cell population is significantly hampered by the presence of an intact blood–brain barrier (BBB). In this study, we performed a preclinical investigation of the efficiency of MR-guided Focused Ultrasound (FUS) to temporarily disrupt the BBB to allow selective delivery of a tumor-targeting antibody to infiltrating tumor. Methods Structural MRI, dynamic-contrast enhancement MRI, and histology were used to fully characterize the MR-enhancing properties of a patient-derived xenograft (PDX) orthotopic mouse model of HGG and to develop a reproducible, robust model of nonenhancing HGG. PET–CT imaging techniques were then used to evaluate the efficacy of FUS to increase 89Zr-radiolabeled antibody concentration in nonenhancing HGG regions and adjacent non-targeted tumor tissue. Results The PDX mouse model of HGG has a significant tumor burden lying behind an intact BBB. Increased antibody uptake in nonenhancing tumor regions is directly proportional to the FUS-targeted volume. FUS locally increased antibody uptake in FUS-targeted regions of the tumor with an intact BBB, while leaving untargeted regions unaffected. Conclusions FUS exposure successfully allowed temporary BBB disruption, localized to specifically targeted, nonenhancing, infiltrating tumor regions and delivery of a systemically administered antibody was significantly increased.

There is currently intense interest in new methods for understanding the fate of therapeutically-relevant cells, such as mesenchymal stem cells (MSCs). The absence of a confounding background signal and consequent unequivocal assignment makes 19F MRI one of the most attractive modalities for the tracking of injected cells in vivo. We describe here the synthesis of novel partly-fluorinated polymeric nanoparticles with small size and high fluorine content as MRI agents. The polymers, constructed from perfluoropolyether methacrylate (PFPEMA) and oligo(ethylene glycol) methacrylate (OEGMA) have favourable cell uptake profiles and excellent MRI performance. To facilitate cell studies the polymer was further conjugated with a fluorescent dye creating a dual-modal imaging agent. The efficacy of labelling of MSCs was assessed using 19F NMR, flow cytometry and confocal microscopy. The labelling efficiency of 2.6 ± 0.1 × 1012 19F atoms per cell, and viability of >90% demonstrates high uptake and good tolerance by the cells. This loading translates to a minimum 19F MRI detection sensitivity of ∼7.4 × 103 cells per voxel. Importantly, preliminary in vivo data demonstrate that labelled cells can be readily detected within a short acquisition scan period (12 minutes). Hence, these copolymers show outstanding potential for 19F MRI cellular tracking and for quantification of non-phagocytic and therapeutically-relevant cells in vivo.

Thermal decomposition (TD) products of the ionic liquids (ILs) [CnC1Im][BF4] and [CnC1Im][PF6] ([CnC1Im]+ = 1-alkyl-3-methylimidazolium, [BF4]- = tetrafluoroborate, and [PF6]- = hexafluorophosphate) were prepared, ex situ, by bulk heating experiments in a bespoke setup. The respective products, CnC1(C3N2H2)BF3 and CnC1(C3N2H2)PF5 (1-alkyl-3-methylimidazolium-2-trifluoroborate and 1-alkyl-3-methylimidazolium-2-pentafluorophosphate), were then vaporized and analyzed by direct insertion mass spectrometry (DIMS) in order to identify their characteristic MS signals. During IL DIMS experiments we were subsequently able, in situ, to identify and monitor signals due to both IL vaporization and IL thermal decomposition. These decomposition products have not been observed in situ during previous analytical vaporization studies of similar ILs. The ex situ preparation of TD products is therefore perfectly complimentary to in situ thermal stability measurements. Experimental parameters such as sample surface area to volume ratios are consequently very important for ILs that show competitive vaporization and thermal decomposition. We have explained these experimental factors in terms of Langmuir evaporation and Knudsen effusion-like conditions, allowing us to draw together observations from previous studies to make sense of the literature on IL thermal stability. Hence, the design of experimental setups are crucial and previously overlooked experimental factors.

Metal microcapsules have recently received attention and are being developed as improved carrier materials when compared to polymer microcapsules. In this work we have developed a novel, simplified method by which polymeric microcapsules can be synthesised using a combination of poly(vinyl pyrrolidone)-stabilised platinum nanoparticles (PVP-Pt) and poly(vinyl pyrrolidone) (PVP) as stabilisers, to allow for a secondary metal shell to be grown. We investigate the relationship between the molar ratio of reducing agent to platinum salt and the size of the resulting NPs and seek to develop further fundamental understanding of the factors governing the secondary metal shell thickness and quality, to allow production of cost-effective metal microcapsules without sacrificing core retention efficiency. We found that the size of the nanoparticles had no significant effect on secondary shell thickness, but did affect the quality of the resulting gold shells. Gold salt concentration was found to be a limiting factor in the electroless deposition of the metal shell. In this work, we have successfully produced PLGA microcapsules with more cost-effective gold shells, as thin as 56 ± 13 nm and capable of complete core retention of volatile actives.

Continuing our research efforts in developing mesoporous silicon nanoparticle-based biomaterials for cancer therapy, we employed here porous silicon nanoparticles as a nanocarrier to deliver contrast agents to diseased cells. Nanoconfinement of small molecule Gd-chelates (L1-Gd) enhanced the T 1 contrast dramatically compared to distinct Gd-chelate (L1-Gd) by virtue of its slow tumbling rate, increased number of bound water molecules, and their occupancy time. The newly synthesized Gd-chelate (L1-Gd) was covalently grafted on silicon nanostructures and conjugated to an antibody specific for epidermal growth factor receptor (EGFR) via a hydrazone linkage. The salient feature of this nanosized contrast agent is the capability of EGFR targeted delivery to cancer cells. Mesoporous silicon nanoparticles were chosen as the nanocarrier because of their high porosity, high surface area, and excellent biodegradability. This type of nanosized contrast agent also performs well in high magnetic fields. Refereed/Peer-reviewed

19F magnetic resonance imaging (MRI) is a powerful noninvasive imaging technique with demonstrated potential for the detection of important diseases. The major challenge in the design of 19F MRI agents is signal attenuation caused by the reduced solubility and segmental mobility of probes with high numbers of fluorine atoms. Careful choice of the fluorinated moiety is required to maintain image quality at the fluorine contents required for high imaging sensitivity. Here we report the synthesis of perfluoropolyether (PFPE) end-functionalized homopolymers of oligo(ethylene glycol) methyl ether acrylate (poly(OEGA)m-PFPE) as highly sensitive 19F MRI contrast agents (CAs). The structural characteristics, conformation and aggregation behavior, 19F NMR relaxation properties, and 19F MR imaging were studied in detail. Dynamic light scattering and molecular dynamics (MD) simulations were conducted and demonstrated that poly(OEGA)m-PFPE with the longest poly(OEGA)m segments (m = 20) undergoes single-chain folding ...

Poly (1,6-bis- (p-carboxyphenoxy hexane)-co- (sebacic anhydride)) (PANH), is a polyanhydride copolymer which has good biocompatibility, and degrades to non-toxic products with a predictable rate of degradation. Nano-sized hydroxyapatite (HAP) is a well-known biomaterial which has been applied in bone regeneration due to its osteoconductivity. However, nano-sized HAP has poor colloidal stability which leads to agglomeration when incorporated into polymeric composites. In this work we describe the surface grafting of poly(ɛ-caprolactone) to HAP (PCL-gHAP) to improve the interfacial adhesion and dispersion of HAP particles in a PANH matrix to form a composite material. The use of scanning electron microscopy-backscattered electron (SEM-BSE) detector and the combination of focused ion beam (FIB)/ transmission electron microscopy (TEM) provided a powerful approach for observing the dispersion of HAP particles in the polymer matrix. We show that surface modification of HAP with PCL improved the homogeneity of the dispersion of HAP particles in the composites and affected the composite morphology during hydrolytic degradation. Composites with high HAP content displayed high compressive strength and a fast rate of degradation. The PCL-gHAP/PANH composites showed superior maintenance of mechanical properties compared with HAP/PANH composites during degradation. A preliminary in vivo study on rat calvaria repair, demonstrated the superior performance of PCL-gHAP/PANH composites. The results suggest that the newly developed PCL-gHAP/PANH composite materials have great potential of serving as a new substrate for bone tissue engineering.

At diagnosis, 22% of colorectal cancer (CRC) patients have metastases, and 50% later develop metastasis. Peptide receptor radionuclide therapy (PRRT), such as

Metal shell microcapsules have been shown to completely retain their core until its release is triggered, making them a promising candidate for use as a controllable drug delivery vehicle due to their superior retention properties as compared to polymer shell microcapsules. Focused ultrasound (FUS) has been successfully utilised to trigger release of lipophilic drugs from polymer microcapsules, and in this work the response of gold shell microcapsules with and without an inner polymeric shell, to FUS and standard ultrasound is explored. The results show that gold shell microcapsules with an inner polymer shell rupture when exposed to standard ultrasound and that there is a linear correlation between the gold shell thickness and the extent of shell rupture. When FUS is applied to these microcapsules, powers as low as 0.16 W delivered in bursts of 10 ms/s over a period of 120 s are sufficient to cause rupture of 53 nm gold shell microcapsules. Additional findings suggest that gold shell microcapsules without the polymer layer dispersed in a hydrogel matrix, as opposed to aqueous media, rupture more efficiently when exposed to FUS, and that thicker gold shells are more responsive to ultrasound-triggered rupture regardless of the external environment. Release of dye from all successfully ruptured capsules was sustained over a period of between 7 and 35 days. These findings suggest that emulsion-templated gold shell microcapsules embedded in a hydrogel matrix would be suitable for use as an implantable drug delivery vehicle with FUS used to externally trigger release.

Stimuli-responsive contrast agents (CAs) show great promise for the early detection and understanding of cancer. In particular, CAs that respond to ion content may allow monitoring of both the development of cancer and the efficacy of novel therapies. Understanding the responsive behavior of stimuli-responsive CAs in different ionic environments requires development of more advanced characterization methods. Here we report a high-resolution NMR and molecular dynamics (MD) study of conformational changes of ion-responsive 19F copolymers of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and 2,2,2-trifluoroethyl acrylate (TFEA) (poly(OEGMA-co-TFEA)) in the presence of salt. 1H 2D NOESY spectra were collected with and without the presence of NaCl and demonstrated that further separation of OEGMA and TFEA segments and closer association of OEGMA side chain were observed with the addition of NaCl. MD simulations provided additional information on the changes in conformation and were consistent with th...

Thermoresponsive dendronized polymers, displaying remarkable phase behavior, are currently being studied for their potential exploitation as polymeric sensors and biomaterials. Understanding the conformational transitions occurring at the LCST is essential for improved design and translation of these polymers. The combination of NMR and molecular dynamics simulations opens a unique window onto the thermal behavior, showing that the peripheries of the dendrons, while driving the thermal properties, largely retain mobility above the critical temperature. The cores of the dendrons and the polymeric main chain are highly rigid below the thermal transition and increasingly so above the LCST. Both the experimental and computational studies reveal stretching of the interior segments of the dendrons with associated changes in spatial arrangements of the structural units. Furthermore, diffusion-ordered NMR and DLS below and above the LCST show a further hierarchy of dynamics within different size aggregates. The c...

To develop novel contrast agents for bimodal molecular imaging, we report here the design and synthesis of multifunctional hyperbranched polymers containing iodine and fluorine and their application as CT/19F MRI bimodal imaging contrast agents. A hyperbranched iodopolymer (HBIP), which was composed of 2-(2′,3′,5′-triiodobenzoyl)ethyl methacrylate (TIBMA), poly(ethylene glycol) methyl ether methacrylate (PEGMA) and a degradable crosslinker, was first synthesised by reversible addition–fragmentation chain transfer (RAFT) polymerization. Then the HBIP was chain extended with PEGMA and 2,2,2-trifluoroethyl acrylate (TFEA) to form hyperbranched iodopolymers containing 19F (HBIPFs). A series of HBIPFs with different contents of iodine and fluorine were prepared. Nanoparticles with diameters of 10–15 nm were formed by direct dissolution of HBIPFs in water, and the biodegradability was revealed by the treatment of reducing agents. The radio-opacity of these nanoparticles in aqueous solution was confirmed by in vitro CT experiments, and solutions of the nanoparticles were visualised by 19F MRI. These results suggest that the HBIPFs are attractive candidates for CT/19F MRI bimodal imaging.

The field of precision oncology is rapidly progressing toward integrated “multiomics” analysis of multiple molecular species (such as DNA, RNA, or proteins) to provide a more complete profile of tumor heterogeneity. Micro/nanomaterial-based systems, which leverage the unique properties of miniature materials, are currently well positioned to expand beyond rudimentary biomarker detection toward multiomics signature analysis. To enable clinical translation, the rational design and implementation of miniaturized systems should be driven by the unique clinical challenges present at various crucial cancer stages. This review features micro/nanomaterial-based systems that are robustly tested on real patient samples for molecular biomarker detection at i) initial cancer screening and/or diagnosis, ii) cancer prognosis and risk stratification, and iii) longitudinal treatment/recurrence monitoring. Furthermore, this review discusses the use of micro/nanomaterials to facilitate sample preparation for different molecular biomarker species. Finally, this review deliberates on the recent paradigm shift of micro/nanomaterial-based system innovation toward integrated multiomics cancer signature analysis and puts forth insights and perspectives on existing challenges. It is anticipated that this review could stimulate the propagation of new concepts and approaches to kick-start a new generation of clinically translational technologies that capitalize on multiomics cancer signatures.

The EphA3 receptor has recently emerged as a functional tumour-specific therapeutic target in glioblastoma (GBM). EphA3 is significantly elevated in recurrent disease, is most highly expressed on glioma stem cells (GSCs), and has a functional role in maintaining self-renewal and tumourigenesis. An unlabelled EphA3-targeting therapeutic antibody is currently under clinical assessment in recurrent GBM patients. In this study, we assessed the efficacy of EphA3 antibody drug conjugate (ADC) and radioimmunotherapy (RIT) approaches using orthotopic animal xenograft models. Brain uptake studies, using positron emission tomography/computed tomography (PET/CT) imaging, show EphA3 antibodies are effectively delivered across the blood-tumour barrier and accumulate at the tumour site with no observed normal brain reactivity. A robust anti-tumour response, with no toxicity, was observed using EphA3, ADC, and RIT approaches, leading to a significant increase in overall survival. Our current research provides evidence that GBM patients may benefit from pay-loaded EphA3 antibody therapies.

Despite significant improvements in the clinical management of glioblastoma, poor delivery of systemic therapies to the entire population of tumour cells remains one of the biggest challenges in the achievement of more effective treatments. On the one hand, the abnormal and dysfunctional tumour vascular network largely limits blood perfusion, resulting in an inhomogeneous delivery of drugs to the tumour. On the other hand, the presence of an intact blood-brain barrier (BBB) in certain regions of the tumour prevents chemotherapeutic drugs from permeating through the tumour vessels and reaching the diseased cells. In this review we analyse in detail the implications of the presence of a dysfunctional vascular network and the impenetrable BBB on drug transport. We discuss advantages and limitations of the currently available strategies for remodelling the tumour vasculature aiming to ameliorate the above mentioned limitations. Finally we review research methods for visualising vascular dysfunction and highlight the power of DCE- and DSC-MRI imaging to assess changes in blood perfusion and BBB permeability.

There is currently intense interest in new methods for understanding the fate of therapeutically-relevant cells, such as mesenchymal stem cells (MSCs). The absence of a confounding background signal and consequent unequivocal assignment makes 19F MRI one of the most attractive modalities for the tracking of injected cells in vivo. We describe here the synthesis of novel partly-fluorinated polymeric nanoparticles with small size and high fluorine content as MRI agents. The polymers, constructed from perfluoropolyether methacrylate (PFPEMA) and oligo(ethylene glycol) methacrylate (OEGMA) have favourable cell uptake profiles and excellent MRI performance. To facilitate cell studies the polymer was further conjugated with a fluorescent dye creating a dual-modal imaging agent. The efficacy of labelling of MSCs was assessed using 19F NMR, flow cytometry and confocal microscopy. The labelling efficiency of 2.6 ± 0.1 × 1012 19F atoms per cell, and viability of90% demonstrates high uptake and good tolerance by the cells. This loading translates to a minimum 19F MRI detection sensitivity of ∼7.4 × 103 cells per voxel. Importantly, preliminary in vivo data demonstrate that labelled cells can be readily detected within a short acquisition scan period (12 minutes). Hence, these copolymers show outstanding potential for 19F MRI cellular tracking and for quantification of non-phagocytic and therapeutically-relevant cells in vivo.

High-resolution NMR measurements and molecular dynamics (MD) simulations have been applied to the study of thermoresponsive copolymers of poly(ethylene glycol) methyl ether methacrylate (OEGMA) and 2,2,2-trifluoroethyl acrylate (TFEA) (poly(OEGMA-co-TFEA)) synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization. The detailed chemical microstructure of poly(OEGMA-co-TFEA) was investigated by means of various high-resolution NMR techniques. The polymer in aqueous solution possesses a lower critical solution temperature (LCST) at which significant changes in conformation are apparent. 1H 2D NOESY spectra were collected at temperatures below and above the LCST and demonstrated closer association of the exterior segments of the OEGMA side chains with the TFEA units above the LCST. MD simulations provided additional information on the changes in conformation and were consistent with the experimental findings. The combination of MD simulations with a detailed experimental study of ...

Imaging using more than one biological process using PET could be of great utility, but despite previously proposed approaches to dual-tracer imaging, it is seldom performed. The alternative of performing multiple scans is often infeasible for clinical practice or even in research studies. Dual-tracer PET scanning allows for multiple PET radiotracers to be imaged within the same imaging session. In this paper we describe our approach to utilise the basis pursuit method to aid in the design of dual-tracer PET imaging experiments, and later in separation of the signals. The advantage of this approach is that it does not require a compartment model architecture to be specified or even that both signals are distinguishable in all cases. This means the method for separating dual-tracer signals can be used for many feasible and useful combinations of biology or radiotracer, once an appropriate scanning protocol has been decided upon. Following a demonstration in separating the signals from two consecutively injected radionuclides in a controlled experiment, phantom and list-mode mouse experiments demonstrated the ability to test the feasibility of dual-tracer imaging protocols for multiple injection delays. Increases in variances predicted for kinetic macro-parameters V

Background Over the past decade, molecular imaging has played a key role in the progression of drug delivery platforms from concept to commercialisation. Of the molecular imaging techniques commonly utilised, positron emission tomography (PET) can yield a breadth of information not easily accessible by other methodologies and when combined with other complementary imaging modalities, is a powerful tool for pre- and clinical development of therapeutics. However, very little research has focussed on the information available from complimentary imaging modalities. This paper reports on the data-rich methodologies of contrast enhanced PET/CT and PET/MRI for probing efficacy of polymer drug delivery platforms. Results The information available from an ExiTron nano 6000 contrast enhanced PET/CT and a gadolinium (Gd) enhanced PET/MRI image of a 64Cu labeled HBP in the same mouse was qualitatively compared. Conclusions Gd contrast enhanced PET/MRI offers a powerful methodology for investigating the distribution of polymer drug delivery platforms in vivo and throughout a tumour volume. Furthermore, information about depth of penetration away from primary blood vessels can be gleaned, potentially leading to development of more efficacious delivery vehicles for clinical use.

With the aim of developing stimuli-responsive imaging agents, we report here the synthesis of core crosslinked star (CCS) polymers and their evaluation as pH-sensitive 19F magnetic resonance imaging (19F MRI) contrast agents. Block copolymers consisting of poly(ethylene glycol)methyl ether methacrylate (PPEGMA) as the first block and a copolymer of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and 2,2,2-trifluoroethyl methacrylate (TFEMA) as the second block were synthesised using RAFT polymerisation. The polymerisation kinetics were studied in detail. The block copolymers were then used as arm precursors for the arm-first synthesis of CCS polymers through RAFT dispersion polymerisation. The synthetic conditions were investigated and optimised. CCS polymers with a degradable core were also synthesised and evaluated as 19F MRI contrast agents. The degradation of the core was confirmed by treatment with various reducing agents. The particle size, 19F NMR signal and relaxation times as well as 19F MRI imaging performance of the CCS polymers were studied at a range of value of solution pH. Significant enhancement of the image intensity was observed when the pH was decreased from 8 to 5, indicating that the CCS nanoparticles could be used as 19F MRI contrast agents for the detection of the acidic environment within tumour tissue.

Background: Increasingly, evidence from studies in both animal models and patients suggests that cardiovascular dysfunction is important in HD. Previous studies measuring function of the left ventricle (LV) in the R6/2 mouse model have found a clear cardiac abnormality, albeit with preserved LV systolic function. It was hypothesized that an impairment of RV function might play a role in this condition via mechanisms of ventricular interdependence.Objective: To investigate RV function in the R6/2 mouse model of Huntington's disease (HD).Methods: Cardiac cine- magnetic resonance imaging (MRI) was used to determine functional parameters in R6/2 mice. In a first experiment, these parameters were derived longitudinally to determine deterioration of cardiac function with disease progression. A second experiment compared the response to a stress test (using dobutamine) of wildtype and early-symptomatic R6/2 mice. Results: There was progressive deterioration of RV systolic function with age in R6/2 mice. Furthermore, beta-adrenergic stimulation with dobutamine revealed RV dysfunction in R6/2 mice before any overt symptoms of the disease were apparent.Conclusions: This work adds to accumulating evidence of cardiovascular dysfunction in R6/2 mice, describing for the first time the involvement of the right ventricle. Cardiovascular dysfunction should be considered, both when treatment strategies are being designed, and when searching for biomarkers for HD.

Brain metastases are the most prevalent of intracranial malignancies. They are associated with a very poor prognosis and near 100% mortality. This has been the case for decades, largely because we lack effective therapeutics to augment surgery and radiotherapy. Notwithstanding improvements in the precision and efficacy of these life-prolonging treatments, with no reliable options for adjunct systemic therapy, brain recurrences are virtually inevitable. The factors limiting intracranial efficacy of existing agents are both physiological and molecular in nature. For example, heterogeneous permeability, abnormal perfusion and high interstitial pressure oppose the conventional convective delivery of circulating drugs, thus new delivery strategies are needed to achieve uniform drug uptake at therapeutic concentrations. Brain metastases are also highly adapted to their microenvironment, with complex cross-talk between the tumor, the stroma and the neural compartments driving speciation and drug resistance. New strategies must account for resistance mechanisms that are frequently engaged in this milieu, such as HER3 and other receptor tyrosine kinases that become induced and activated in the brain microenvironment. Here, we discuss molecular and physiological factors that contribute to the recalcitrance of these tumors, and review emerging therapeutic strategies, including agents targeting the PI3K axis, immunotherapies, nanomedicines and MRI-guided focused ultrasound for externally controlling drug delivery.

Recent evidence has suggested that the solvent environment in ionic liquids is dynamic and composed of polar and non-polar domains governed by the summation of weak interactions and self-assembly. Consequently, the effect of a nano-structured solvent environment on chemistries conducted in ionic liquids is coming under increased scrutiny. In this work we investigate how the domain-like structure of ionic liquids affects the kinetics and products of the reversible addition fragmentation chain transfer (RAFT) controlled free radical polymerisation (FRP) of methyl methacrylate in a number of room temperature ionic liquids. By utilising rotating frame Overhauser effect spectroscopy (ROESY) to probe the solvation environment of the 2-cyano prop-2-yl dithiobenzoate (CPDB) RAFT agent, we show that in almost all cases preferential partitioning of the dithiobenzoate-moiety of the RAFT agent into the ionic domain of the ionic liquid occurs.