Delineating the tumour microenvironment response to a lipid nanoparticle formulation.

Nanoparticles can reduce cytotoxicity, increase circulation time and increase accumulation in tumours compared to free drug. However, the value of using nanoparticles for carrying small molecules to treat tumours at the cellular level has been poorly established. Here we conducted a cytodistribution analysis on Doxorubicin-treated and Doxil-treated tumours to delineate the differences between the small molecule therapeutic Doxorubicin and its packaged liposomal formulation Doxil. We found that Doxil kills more cancer cells, macrophages and neutrophils in the 4T1 breast cancer tumour model, but there is delayed killing compared to its small molecule counterpart Doxorubicin. The cellular interaction with Doxil has slower uptake kinetics and the particles must be degraded to release the drug and kill the cells. We also found that macrophages and neutrophils in Doxil-treated tumours repopulated faster than cancer cells during the relapse phase. While researchers conventionally use tumour volume and animal survival to determine a therapeutic effect, our results show diverse cell killing and a greater amount of cell death in vivo after Doxil liposomes are administered. We conclude that the fate and behaviour of the nanocarrier influences its effectiveness as a cancer therapy. Further investigations on the interactions between different nanoparticle designs and the tumour microenvironment components will lead to more precise engineering of nanocarriers to selectively kill tumour cells and prolong the therapeutic effect.

Macrophages Actively Transport Nanoparticles in Tumors After Extravasation.

Nanoparticles need to navigate a complex microenvironment to target cells in solid tumors after extravasation. Diffusion is currently the accepted primary mechanism for nanoparticle distribution in tumors. However, the extracellular matrix can limit nanoparticle diffusion. Here, we identified tumor-associated macrophages as another key player in transporting and redistributing nanoparticles in the tumor microenvironment. We found tumor-associated macrophages actively migrate toward nanoparticles extravasated from the vessels, engulfing and redistributing them in the tumor stroma. The macrophages can carry the nanoparticles 2-5 times deeper in the tumor than passive diffusion. The amount of nanoparticles transported by the tumor-associated macrophages is size-dependent. Understanding the nanoparticle behavior after extravasation will provide strategies to engineer them to navigate the microenvironment for improved intratumoral targeting and therapeutic effectiveness.

Impact of Tumor Barriers on Nanoparticle Delivery to Macrophages.

The delivery of therapeutic nanoparticles to target cells is critical to their effectiveness. Here we quantified the impact of biological barriers on the delivery of nanoparticles to macrophages in two different tissues. We compared the delivery of gold nanoparticles to macrophages in the liver versus those in the tumor. We found that nanoparticle delivery to macrophages in the tumor was 75% less than to macrophages in the liver due to structural barriers. The tumor-associated macrophages took up more nanoparticles than Kupffer cells in the absence of barriers. Our results highlight the impact of biological barriers on nanoparticle delivery to cellular targets.

The Design, Synthesis and Evaluation of Rho-kinase Inhibitory Activity of 4-aryl-thiazole-2-amines.

Rho-associated kinases (ROCK) are a class of serine/threonine kinases that play important roles in various biological processes. ROCK are becoming attractive targets for drug designing. A novel scaffold was designed according to molecular hybridization strategy, then a series of 4-aryl-5-aminomethyl-thiazole-2-amines were synthesized, and their inhibitory activities on ROCK were screened by enzyme-linked immunosorbent assay (ELISA). The results showed that 4-aryl-5-aminomethyl-thiazole-2-amines derivatives displayed certain ROCK II inhibitory activities. The IC50 value of the most potent compound 4v was found to be 20 nM. The preliminary structure-activity-relationship investigation showed that compounds with 4-pyridine substitution were generally found to be more potent than compounds with 3-pyridine substitution. The molecular docking studies indicated that more optimization work needs to conduct to obtain more potent ROCK inhibitors.

Specific Endothelial Cells Govern Nanoparticle Entry into Solid Tumors.

The successful delivery of nanoparticles to solid tumors depends on their ability to pass through blood vessels and into the tumor microenvironment. Here, we discovered a subset of tumor endothelial cells that facilitate nanoparticle transport into solid tumors. We named these cells nanoparticle transport endothelial cells (N-TECs). We show that only 21% of tumor endothelial cells located on a small number of vessels are involved in transporting nanoparticles into the tumor microenvironment. N-TECs have an increased expression of genes related to nanoparticle transport and vessel permeability compared to other tumor endothelial cells. The N-TECs act as gatekeepers that determine the entry point, distribution, cell accessibility, and number of nanoparticles that enter the tumor microenvironment.

A framework for designing delivery systems.

The delivery of medical agents to a specific diseased tissue or cell is critical for diagnosing and treating patients. Nanomaterials are promising vehicles to transport agents that include drugs, contrast agents, immunotherapies and gene editors. They can be engineered to have different physical and chemical properties that influence their interactions with their biological environments and delivery destinations. In this Review Article, we discuss nanoparticle delivery systems and how the biology of disease should inform their design. We propose developing a framework for building optimal delivery systems that uses nanoparticle-biological interaction data and computational analyses to guide future nanomaterial designs and delivery strategies.

Design, synthesis and biological evaluation of 4-aryl-5-aminoalkyl-thiazole-2-amines derivatives as ROCK II inhibitors.

A series of 4-aryl-5-aminoalkyl-thiazole-2-amines were designed and synthesized, and their inhibitory activity on ROCK II was screened by enzyme-linked immunosorbent assay (ELISA). The results showed that 4-aryl-5-aminomethyl-thiazole-2-amines derivatives had certain ROCK II inhibitory activities. Compound 10l showed ROCK II inhibitory activity with IC50 value of 20 nM.

Synthesis and Evaluation of Biological Properties of 2-Amino-thiazole-4-carboxamides: Amide Linkage Analogues of Pretubulysin.

Pretubulysin is a bio-precursor of highly toxic tetrapeptide tubulysins. Although pretubulysin has a much simpler chemical structure, it has similar anti-mitotic potency. A series of 2-amino-thiazole-4-carboxamides were designed and synthesized based on the structure of cemadotin. These are all novel compounds and their structures are characterized by 1H-NMR, 13C-NMR, and high resolution (HR)MS. The antitumor activities of these compounds were screened using the methyl thiazolyl tetrazolium colorimetric (MTT) cell viability method in MCF7 (breast cancer) and NCI-H1650 (lung cancer) cells. All the synthesized compounds 6a-n showed moderate anti-proliferation activities. Compounds 6m exhibited antitumor activity with the IC50 value of 0.47 and 1.1 µM in MCF7 and NCI-H1650 cells, respectively.

The dose threshold for nanoparticle tumour delivery.

Nanoparticle delivery to solid tumours over the past ten years has stagnated at a median of 0.7% of the injected dose. Varying nanoparticle designs and strategies have yielded only minor improvements. Here we discovered a dose threshold for improving nanoparticle tumour delivery: 1 trillion nanoparticles in mice. Doses above this threshold overwhelmed Kupffer cell uptake rates, nonlinearly decreased liver clearance, prolonged circulation and increased nanoparticle tumour delivery. This enabled up to 12% tumour delivery efficiency and delivery to 93% of cells in tumours, and also improved the therapeutic efficacy of Caelyx/Doxil. This threshold was robust across different nanoparticle types, tumour models and studies across ten years of the literature. Our results have implications for human translation and highlight a simple, but powerful, principle for designing nanoparticle cancer treatments.

Nanoparticle Uptake in a Spontaneous and Immunocompetent Woodchuck Liver Cancer Model.

There is a tremendous focus on the application of nanomaterials for the treatment of cancer. Nonprimate models are conventionally used to assess the biomedical utility of nanomaterials. However, these animals often lack an intact immunological background, and the tumors in these animals do not develop spontaneously. We introduce a preclinical woodchuck hepatitis virus-induced liver cancer model as a platform for nanoparticle (NP)-based in vivo experiments. Liver cancer development in these out-bred animals occurs as a result of persistent viral infection, mimicking human hepatitis B virus-induced HCC development. We highlight how this model addresses key gaps associated with other commonly used tumor models. We employed this model to (1) track organ biodistribution of gold NPs after intravenous administration, (2) examine their subcellular localization in the liver, (3) determine clearance kinetics, and (4) characterize the identity of hepatic macrophages that take up NPs using RNA-sequencing (RNA-seq). We found that the liver and spleen were the primary sites of NP accumulation. Subcellular analyses revealed accumulation of NPs in the lysosomes of CD14+ cells. Through RNA-seq, we uncovered that immunosuppressive macrophages within the woodchuck liver are the major cell type that take up injected NPs. The woodchuck-HCC model has the potential to be an invaluable tool to examine NP-based immune modifiers that promote host anti-tumor immunity.

The entry of nanoparticles into solid tumours.

The concept of nanoparticle transport through gaps between endothelial cells (inter-endothelial gaps) in the tumour blood vessel is a central paradigm in cancer nanomedicine. The size of these gaps was found to be up to 2,000 nm. This justified the development of nanoparticles to treat solid tumours as their size is small enough to extravasate and access the tumour microenvironment. Here we show that these inter-endothelial gaps are not responsible for the transport of nanoparticles into solid tumours. Instead, we found that up to 97% of nanoparticles enter tumours using an active process through endothelial cells. This result is derived from analysis of four different mouse models, three different types of human tumours, mathematical simulation and modelling, and two different types of imaging techniques. These results challenge our current rationale for developing cancer nanomedicine and suggest that understanding these active pathways will unlock strategies to enhance tumour accumulation.

Liposome Imaging in Optically Cleared Tissues.

Three-dimensional (3D) optical microscopy can be used to understand and improve the delivery of nanomedicine. However, this approach cannot be performed for analyzing liposomes in tissues because the processing step to make tissues transparent for imaging typically removes the lipids. Here, we developed a tag, termed REMNANT, that enables 3D imaging of organic materials in biological tissues. We demonstrated the utility of this tag for the 3D mapping of liposomes in intact tissues. We also showed that the tag is able to monitor the release of entrapped therapeutic agents. We found that liposomes release their cargo >100-fold faster in tissues in vivo than in conventional in vitro assays. This allowed us to design a liposomal formulation with enhanced ability to kill tumor associated macrophages. Our development opens up new opportunities for studying the chemical properties and pharmacodynamics of administered organic materials in an intact biological environment. This approach provides insight into the in vivo behavior of degradable materials, where the newly discovered information can guide the engineering of the next generation of imaging and therapeutic agents.

Design and Biological Evaluation of 3-Aryl-4-alkylpyrazol-5-amines Based on the Target Fishing.

BACKGROUND: Pyrazol-5-amine derivatives are an important class of heterocyclic compounds. However, there are less 4-alkyl substituted pyrazoles reported. OBJECTIVE: Here reported are the design, synthesis and biological evaluation of 3-aryl-4- alkylpyrazol-5-amines derivatives. METHODS: A serials of 3-aryl-4-alkylpyrazol-5-amines were designed and the biological action targets were screened by target fishing function of Discovery Studio software. The synthesis route involved 3-oxo-3-arylpropanenitrile formation, alkylation, pyrazole formation, and amides formation. The antitumor activities of these compounds were carried out by thiazolyl blue tetrazolium bromide (MTT) method using U-2 OS (osteosarcoma) and A549 (lung cancer) tumor cells. RESULTS: Eight 3-aryl-4-alkylpyrazol-5-amines were synthesized, and their structures were verified by 1H NMR, 13C NMR, and HRMS. Thirteen pharmacophores were mapped out by target fishing. Compound 5h showed anti-proliferation activities against U-2 OS and A549 tumor cell with IC50 value of 0.9 µM and 1.2 µM, respectively. CONCLUSION: Compound 5h might represent a promising scaffold for the further development of novel antitumor drugs.

Nanoparticle Size Influences Antigen Retention and Presentation in Lymph Node Follicles for Humoral Immunity.

Lymph node follicles capture and retain antigens to induce germinal centers and long-lived humoral immunity. However, control over antigen retention has been limited. Here we discovered that antigen conjugated to nanoparticle carriers of different sizes impacts the intralymph node transport and specific cell interaction. We found that follicular dendritic cell (FDC) networks determine the intralymph node follicle fate of these nanoparticles by clearing smaller ones (5-15 nm) within 48 h and retaining larger ones (50-100 nm) for over 5 weeks. The 50-100 nm-sized nanoparticles had 175-fold more delivery of antigen at the FDC dendrites, 5-fold enhanced humoral immune responses of germinal center B cell formation, and 5-fold more antigen-specific antibody production over 5-15 nm nanoparticles. Our results show that we can tune humoral immunity by simply manipulating the carrier size design to produce effectiveness of vaccines.

The Design, Synthesis and Preliminary Pharmacokinetic Evaluation of d3-Poziotinib Hydrochloride.

To establish a synthetic route to d3-poziotinib hydrochloride. Treatment of 4-chloro-7-hydroxyquinazolin-6-yl pivalate (1) with d3-methyliodide afforded the etherization product, which reacted with 3,4-dichloro-2-fluoroaniline to generate the key intermediate d3-4-(3,4-dichloro-2-fluorophenylamino)-7-methoxyquinazolin-6-yl pivalate (3). Followed the de-protection reaction, the nucleophilic substitution (SN2) reaction with tert-butyl 4-(tosyloxy)piperidine-1-carboxylate (TSP), and the de-protection reaction of t-butoxycarbonyl (Boc) group, and the amide formation reaction with acrylyl chloride, d3-poziotinib was obtained, which was converted to hydrochloride salt by treatment with concentrated hydrochloric acid (HCl). Starting from a known compound 4-chloro-7-hydroxyquinazolin-6-yl pivalate (1), after 7 steps transformation, d3-poziotinib hydrochloride was obtained with a total yield of 9.02%. The structure of d3-poziotinib hydrochloride was confirmed by 1H-NMR, 13C-NMR, and high resolution (HR)-MS. Meanwhile, the in vitro microsomal stability experiment showed that d3-poziotinib had a longer half time (t1/2 = 4.6 h) than poziotinib (t1/2 = 3.5 h).

Characterizing the protein corona of sub-10 nm nanoparticles.

Studies into the interactions of serum proteins with nanoparticles are typically performed using nanoparticles that are larger than the size of proteins. Due to this size discrepancy, adsorbed proteins are commonly depicted as a globular structure surrounding a nanoparticle. Here, we asked how we should view nanoparticle-protein complexes when the nanoparticles are of similar size or smaller than the proteins with which they interact. We showed that nanoparticles can serve as a cargo on a protein rather than as a carrier of the protein in a size-dependent manner. This can occur when nanoparticles are below 10 nm in diameter. We discovered that when the nanoparticle is a cargo on the protein, the binding of the protein to the receptor target is minimally affected in contrast to the nanoparticle serving as a carrier. Our study should change how we view and describe nanoparticle-protein complexes when the nanoparticles involved are equal in size or smaller than proteins.

Elimination Pathways of Nanoparticles.

Understanding how nanoparticles are eliminated from the body is required for their successful clinical translation. Many promising nanoparticle formulations for in vivo medical applications are large (>5.5 nm) and nonbiodegradable, so they cannot be eliminated renally. A proposed pathway for these nanoparticles is hepatobiliary elimination, but their transport has not been well-studied. Here, we explored the barriers that determined the elimination of nanoparticles through the hepatobiliary route. The route of hepatobiliary elimination is usually through the following pathway: (1) liver sinusoid, (2) space of Disse, (3) hepatocytes, (4) bile ducts, (5) intestines, and (6) out of the body. We discovered that the interaction of nanoparticles with liver nonparenchymal cells ( e. g., Kupffer cells and liver sinusoidal endothelial cells) determines the elimination fate. Each step in the route contains cells that can sequester and chemically or physically alter the nanoparticles, which influences their fecal elimination. We showed that the removal of Kupffer cells increased fecal elimination by >10 times. Combining our results with those of prior studies, we can start to build a systematic view of nanoparticle elimination pathways as it relates to particle size and other design parameters. This is critical to engineering medically useful and translatable nanotechnologies.

Effect of removing Kupffer cells on nanoparticle tumor delivery.

A recent metaanalysis shows that 0.7% of nanoparticles are delivered to solid tumors. This low delivery efficiency has major implications in the translation of cancer nanomedicines, as most of the nanomedicines are sequestered by nontumor cells. To improve the delivery efficiency, there is a need to investigate the quantitative contribution of each organ in blocking the transport of nanoparticles to solid tumors. Here, we hypothesize that the removal of the liver macrophages, cells that have been reported to take up the largest amount of circulating nanoparticles, would lead to a significant increase in the nanoparticle delivery efficiency to solid tumors. We were surprised to discover that the maximum achievable delivery efficiency was only 2%. In our analysis, there was a clear correlation between particle design, chemical composition, macrophage depletion, tumor pathophysiology, and tumor delivery efficiency. In many cases, we observed an 18-150 times greater delivery efficiency, but we were not able to achieve a delivery efficiency higher than 2%. The results suggest the need to look deeper at other organs such as the spleen, lymph nodes, and tumor in mediating the delivery process. Systematically mapping the contribution of each organ quantitatively will allow us to pinpoint the cause of the low tumor delivery efficiency. This, in effect, enables the generation of a rational strategy to improve the delivery efficiency of nanoparticles to solid tumors either through the engineering of multifunctional nanosystems or through manipulation of biological barriers.

Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood.

A significant challenge to delivering therapeutic doses of nanoparticles to targeted disease sites is the fact that most nanoparticles become trapped in the liver. Liver-resident macrophages, or Kupffer cells, are key cells in the hepatic sequestration of nanoparticles. However, the precise role that the macrophage phenotype plays in nanoparticle uptake is unknown. Here, we show that the human macrophage phenotype modulates hard nanoparticle uptake. Using gold nanoparticles, we examined uptake by human monocyte-derived macrophages that had been driven to a "regulatory" M2 phenotype or an "inflammatory" M1 phenotype and found that M2-type macrophages preferentially take up nanoparticles, with a clear hierarchy among the subtypes (M2c > M2 > M2a > M2b > M1). We also found that stimuli such as LPS/IFN-γ rather than with more "regulatory" stimuli such as TGF-ß/IL-10 reduce per cell macrophage nanoparticle uptake by an average of 40%. Primary human Kupffer cells were found to display heterogeneous expression of M1 and M2 markers, and Kupffer cells expressing higher levels of M2 markers (CD163) take up significantly more nanoparticles than Kupffer cells expressing lower levels of surface CD163. Our results demonstrate that hepatic inflammatory microenvironments should be considered when studying liver sequestration of nanoparticles, and that modifying the hepatic microenvironment might offer a tool for enhancing or decreasing this sequestration. Our findings also suggest that models examining the nanoparticle/macrophage interaction should include studies with primary tissue macrophages.

Incidence and characteristics of needlestick injuries among medical trainees at a community teaching hospital: A cross-sectional study.

OBJECTIVES: This field study aimed to determine the incidence and distribution of needlestick injuries among medical trainees at a community teaching hospital in Toronto, Canada. METHODS: The study was performed during the 2013-2015 academic years at Toronto East General Hospital (TEGH), a University of Toronto-affiliated community-teaching hospital during the 2013-2015 academic years. Eight-hundred and forty trainees, including medical students, residents, and post-graduate fellows, were identified and invited via email to participate in an anonymous online fluidsurveys.com survey of 16 qualitative and quantitative questions. RESULTS: Three-hundred and fifty trainees responded (42% response rate). Eighty-eight (25%) respondents reported experiencing at least one injury at TEGH. In total, our survey identified 195 total injuries. Surgical trainees were significantly more likely to incur injuries than non-surgical trainees (IRR = 3.03, 95% CI 1.80-5.10). Orthopaedic surgery trainees had the highest risk of a needlestick injury, being over 12 times more likely to be injured than emergency medicine trainees (IRR = 12.4, 95% CI 2.11-72.32). Only 28 of the 88 most recent needlestick injuries were reported to occupational health. Trainees reported a perception of insignificant risk, lack of resources and support for reporting, and injury stigmatization as reasons for not reporting needlestick injuries. CONCLUSIONS: Needlestick injuries were a common underreported risk to medical trainees at TEGH. Future research should investigate strategies to reduce injury and improve reporting among the high-risk and reporting-averse trainees.