Preclinical Efficacy of Cabazitaxel Loaded Poly(2-alkyl cyanoacrylate) Nanoparticle Variants.

Background: Biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs) are receiving increasing attention in anti-cancer nanomedicine development not only for targeted cancer chemotherapy, but also for modulation of the tumor microenvironment. We previously reported promising results with cabazitaxel (CBZ) loaded poly(2-ethylbutyl cyanoacrylate) NPs (PEBCA-CBZ NPs) in a patient derived xenograft (PDX) model of triple-negative breast cancer, and this was associated with a decrease in M2 macrophages. The present study aims at comparing two endotoxin-free PACA NP variants (PEBCA and poly(2-ethylhexyl cyanoacrylate); PEHCA), loaded with CBZ and test whether conjugation with folate would improve their effect. Methods: Cytotoxicity assays and cellular uptake of NPs by flow cytometry were performed in different breast cancer cells. Biodistribution and efficacy studies were performed in PDX models of breast cancer. Tumor associated immune cells were analyzed by multiparametric flow cytometry. Results: In vitro studies showed similar NP-induced cytotoxicity patterns despite difference in early NP internalization. On intravenous injection, the liver cleared the majority of NPs. Efficacy studies in the HBCx39 PDX model demonstrated an enhanced effect of drug-loaded PEBCA variants compared with free drug and PEHCA NPs. Furthermore, the folate conjugated PEBCA variant did not show any enhanced effects compared with the unconjugated counterpart which might be due to unfavorable orientation of folate on the NPs. Finally, analyses of the immune cell populations in tumors revealed that treatment with drug loaded PEBCA variants affected the myeloid cells, especially macrophages, contributing to an inflammatory, immune activated tumor microenvironment. Conclusion: We report for the first time, comparative efficacy of PEBCA and PEHCA NP variants in triple negative breast cancer models and show that CBZ-loaded PEBCA NPs exhibit a combined effect on tumor cells and on the tumor associated myeloid compartment, which may boost the anti-tumor response.

Lipids in Extracellular Vesicles: What Can Be Learned about Membrane Structure and Function?

Extracellular vesicles, such as exosomes, can be used as interesting models to study the structure and function of biological membranes as these vesicles contain only one membrane (i.e., one lipid bilayer). In addition to lipids, they contain proteins, nucleic acids, and various other molecules. The lipid composition of exosomes is here compared to HIV particles and detergent-resistant membranes, which also have a high content of sphingolipids, cholesterol, and phosphatidylserine (PS). We discuss interactions between the lipids in the two bilayers, and especially those between PS 18:0/18:1 in the inner leaflet and the very-long-chain sphingolipids in the outer leaflet, and the importance of cholesterol for these interactions. We also briefly discuss the involvement of ether-linked phospholipids (PLs) in such lipid raft-like structures, and the possible involvement of these and other lipid classes in the formation of exosomes. The urgent need to improve the quality of quantitative lipidomic studies is highlighted.

Modulation of Ricin Intoxication by the Autophagy Inhibitor EACC.

The compound EACC (ethyl (2-(5-nitrothiophene-2-carboxamido) thiophene-3-carbonyl) carbamate) was recently reported to inhibit fusion of autophagosomes with lysosomes in a reversible manner by inhibiting recruitment of syntaxin 17 to autophagosomes. We report here that this compound also provides a strong protection against the protein toxin ricin as well as against other plant toxins such as abrin and modeccin. The protection did not seem to be caused by inhibition of endocytosis and retrograde transport, but rather by inhibited release of the enzymatically active A-moiety to the cytosol. The TANK-binding kinase 1 (TBK1) has been reported to phosphorylate syntaxin 17 and be required for initiation of autophagy. The inhibitor of TBK1, MRT68601, induced in itself a strong sensitization to ricin, apparently by increasing transport to the Golgi apparatus. Importantly, MRT68601 increased Golgi transport of ricin even in the presence of EACC, but EACC was still able to inhibit intoxication, supporting the idea that EACC protects at a late step along the retrograde pathway. These results also indicate that phosphorylation of syntaxin 17 is not required for the protection observed.

Biodistribution, pharmacokinetics and excretion studies of intravenously injected nanoparticles and extracellular vesicles: Possibilities and challenges.

There is a large interest in developing nanoparticles and extracellular vesicles for delivery of therapeutics or imaging agents. Regulatory approval of such products requires knowledge about their biodistribution, metabolism and excretion. We here discuss possibilities and challenges of methods used for such studies, which most often are performed after labelling with radioactive isotopes or fluorescent molecules. It is important to evaluate if the labelled and unlabeled products can be expected to behave similarly in the body. Furthermore, one needs to critically consider whether the labels are still associated with the product at the time of analyses. We discuss advantages and disadvantages of different imaging modalities such as PET, SPECT, MRI, CT, ultrasound and optical imaging for whole-body biodistribution, and describe how to estimate the amount of labelled product in harvested organs and tissue. Microscopy of cells and tissues and various mass spectrometry methods are also discussed in this review.

Need for more focus on lipid species in studies of biological and model membranes.

The lipid composition of cellular membranes can impact a number of physiological processes such as signaling, cell migration, endocytosis and intracellular transport. In this article we focus on some aspects concerning analysis of lipids and research on lipid structure and function in mammalian cells that in our opinion have not obtained sufficient attention. This includes interleaflet coupling between the two layers of the membrane, and the role of lipid species, i.e. the role of the complete structure of the lipids, including lipid chain length and the position of double bonds. We highlight the role of PS species for membrane function. We also discuss the large diversity of PS species in different biological samples and the possible functional consequences, and we provide an overview of PS species from 40 different samples. Furthermore, recent studies show that there seems to be a coregulation concerning the levels of sphingolipids and ether lipids. We review and discuss the published data indicating such a coregulation. Moreover, we point to some of the pitfalls in the field of lipidomics and present suggestions for improvement. Finally, we discuss the importance of using asymmetric membrane models with a composition of lipid species that are common in biological membranes.

Are doping tests in sports trustworthy?: Athletes suffer from insufficiently defined criteria for doping tests: Athletes suffer from insufficiently defined criteria for doping tests.

The lack of clearly defined criteria for doping tests carries a great risk of punishing innocent athletes and undermines the fight against doping in international sports.

The Protein Toxins Ricin and Shiga Toxin as Tools to Explore Cellular Mechanisms of Internalization and Intracellular Transport.

Protein toxins secreted by bacteria and found in plants can be threats to human health. However, their extreme toxicity can also be exploited in different ways, e.g., to produce hybrid toxins directed against cancer cells and to study transport mechanisms in cells. Investigations during the last decades have shown how powerful these molecules are as tools in cell biological research. Here, we first present a partly historical overview, with emphasis on Shiga toxin and ricin, of how such toxins have been used to characterize processes and proteins of importance for their trafficking. In the second half of the article, we describe how one can now use toxins to investigate the role of lipid classes for intracellular transport. In recent years, it has become possible to quantify hundreds of lipid species using mass spectrometry analysis. Thus, it is also now possible to explore the importance of lipid species in intracellular transport. The detailed analyses of changes in lipids seen under conditions of inhibited toxin transport reveal previously unknown connections between syntheses of lipid classes and demonstrate the ability of cells to compensate under given conditions.

Cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles: toxicity and changes in the proteome of breast, colon and prostate cancer cells.

Nanoparticles composed of poly(alkyl cyanoacrylate) (PACA) have shown great promise due to their biodegradability and high drug loading capacity. Development of optimal PACA nanocarriers requires detailed analysis of the overall cellular impact exerted by PACA variants. We here perform a comprehensive comparison of cabazitaxel (CBZ)-loaded nanocarriers composed of three different PACA monomers, i.e. poly(n-butyl cyanoacrylate) (PBCA), poly(2-ethylbutyl cyanoacrylate) (PEBCA) and poly(octyl cyanoacrylate) (POCA). The cytotoxicity of drug-loaded and empty PACA nanoparticles were compared to that of free CBZ across a panel of nine cancer cell lines by assessing cellular metabolism, proliferation and protein synthesis. The analyses revealed that the cytotoxicity of all CBZ-loaded PACAs was similar to that of free CBZ for all cell lines tested, whereas the empty PACAs exerted much lower toxicity. To increase our understanding of the toxic effects of these treatments comprehensive MS-based proteomics were performed with HCT116, MDA-MB-231 and PC3 cells incubated with PACA-CBZ variants or free CBZ. Interestingly, PACA-CBZ specifically led to decreased levels of proteins involved in focal adhesion and stress fibers in all cell lines. Since we recently demonstrated that encapsulation of CBZ within PEBCA nanoparticles significantly improved the therapeutic effect of CBZ on a patient derived xenograft model in mice, we investigated the effects of this PACA variant more closely by immunoblotting. Interestingly, we detected several changes in the protein expression and degree of phosphorylation of SRC-pathway proteins that can be relevant for the therapeutic effects of these substances.

Biodistribution of Poly(alkyl cyanoacrylate) Nanoparticles in Mice and Effect on Tumor Infiltration of Macrophages into a Patient-Derived Breast Cancer Xenograft.

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects.

Mechanism of cellular uptake and cytotoxicity of paclitaxel loaded lipid nanocapsules in breast cancer cells.

Lipid nanocapsules (LNCs) have proven their efficacy in delivering different drugs to various cancers, but no studies have yet described their uptake mechanisms, paclitaxel (PTX) delivery or resulting cytotoxicity towards breast cancer cells. Herein, we report results concerning cellular uptake of LNCs and cytotoxicity studies of PTX-loaded LNCs (LNCs-PTX) on the three breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468. LNCs-PTX of sizes 50 ± 2 nm, 90 ± 3 nm and 120 ± 4 nm were developed by the phase inversion method. Fluorescence microscopy and flow cytometry were used to observe the uptake of fluorescently labeled LNCs and cellular uptake of LNCs-PTX was measured using HPLC analyses of cell samples. These studies revealed a higher uptake of LNCs-PTX in MDA-MB-468 cells than in the other two cell lines. Moreover, free PTX and LNCs-PTX exhibited a similar pattern of toxicity towards each cell line, but MDA-MB-468 cells appeared to be more sensitive than the other two cell lines, as evaluated by the MTT cytotoxicity assay and a cell proliferation assay based upon [3H]thymidine incorporation. Studies with inhibitors of endocytosis indicate that the cellular uptake is mainly via the Cdc42/GRAF-dependent endocytosis as well as by macropinocytosis, whereas dynamin-dependent processes are not required. Furthermore, our results indicate that endocytosis of LNCs-PTX is important for the toxic effect on cells. Western blot analysis revealed that LNCs-PTX induce cytotoxicity by means of apoptosis in all the three cell lines. Altogether, the results demonstrate that LNCs-PTX exploit different mechanisms of endocytosis in a cell-type dependent manner, and subsequently induce apoptotic cell death in the breast cancer cells here studied. The article also describes biodistribution studies following intravenous injection of fluorescently labeled LNCs in mice.

Diacylglycerol kinase and phospholipase D inhibitors alter the cellular lipidome and endosomal sorting towards the Golgi apparatus.

The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.

Physicochemical Characterization, Toxicity and In Vivo Biodistribution Studies of a Discoidal, Lipid-Based Drug Delivery Vehicle: Lipodisq Nanoparticles Containing Doxorubicin.

Many promising pharmaceutically active compounds have low solubility in aqueous environments and their encapsulation into efficient drug delivery vehicles is crucial to increase their bioavailability. Lipodisq nanoparticles are approximately 10 nm in diameter and consist of a circular phospholipid bilayer, stabilized by an annulus of SMA (a hydrolysed copolymer of styrene and maleic anhydride). SMA is used extensively in structural biology to extract and stabilize integral membrane proteins for biophysical studies. Here, we assess the potential of these nanoparticles as drug delivery vehicles, determining their cytotoxicity and the in vivo excretion pathways of their polymer and lipid components. Doxorubicin-loaded Lipodisqs were cytotoxic across a panel of cancer cell lines, whereas nanoparticles without the drug had no effect on cell proliferation. Intracellular doxorubicin release from Lipodisqs in HeLa cells occurred in the low-pH environment of the endolysosomal system, consistent with the breakdown of the discoidal structure as the carboxylate groups of the SMA polymer become protonated. Biodistribution studies in mice showed that, unlike other nanoparticles injected intravenously, most of the Lipodisq components were recovered in the colon, consistent with rapid uptake by hepatocytes and excretion into bile. These data suggest that Lipodisqs have the potential to act as delivery vehicles for drugs and contrast agents.

Structural Variants of poly(alkylcyanoacrylate) Nanoparticles Differentially Affect LC3 and Autophagic Cargo Degradation.

Nanoparticle drug carriers trigger a variety of cellular stress responses, including ER stress and antioxidant responses, but may also affect the intracellular degradative pathway autophagy. This can impose profound effects on drug delivery, cellular treatment responses, and nanoparticle cytotoxicity. We recently demonstrated that even small variations in the alkyl side chains of poly(alkylcyanoacrylate) (PACA) drug carrier nanoparticles, namely butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), differentially induce ER stress and redox imbalance in human cell lines. Here, we systematically investigate how these PACA variants affect autophagy. Interestingly, treatment with PEBCA or POCA particles led to intracellular accumulation of the autophagosome marker LC3-II, but via different mechanisms. PEBCA induced an integrated stress response-and ATF4-mediated increase in LC3B mRNA, whereas POCA blocked autophagic degradation of LC3-II and long-lived proteins in bulk. PBCA also increased LC3B mRNA via the integrated stress response and ATF4, but unlike PEBCA, it inhibited LC3 lipidation and autophagic cargo degradation. Our data demonstrate that even subtle variations in NP structure can have profoundly different impacts on autophagy, and that careful monitoring of autophagic flux and cargo degradation is critical for drawing accurate conclusions. Our findings have important implications for the choice of PACA monomer in different therapeutic settings.

Mass spectrometry-based measurements of cyclic adenosine monophosphate in cells, simplified using reversed phase liquid chromatography with a polar characterized stationary phase.

3', 5' - Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1-500 ng/mL cell extract.

The role of lipid species in membranes and cancer-related changes.

Several studies have demonstrated interactions between the two leaflets in membrane bilayers and the importance of specific lipid species for such interaction and membrane function. We here discuss these investigations with a focus on the sphingolipid and cholesterol-rich lipid membrane domains called lipid rafts, including the small flask-shaped invaginations called caveolae, and the importance of such membrane structures in cell biology and cancer. We discuss the possible interactions between the very long-chain sphingolipids in the outer leaflet of the plasma membrane and the phosphatidylserine species PS 18:0/18:1 in the inner leaflet and the importance of cholesterol for such interactions. We challenge the view that lipid rafts contain a large fraction of lipids with two saturated fatty acyl groups and argue that it is important in future studies of membrane models to use asymmetric membrane bilayers with lipid species commonly found in cellular membranes. We also discuss the need for more quantitative lipidomic studies in order to understand membrane function and structure in general, and the importance of lipid rafts in biological systems. Finally, we discuss cancer-related changes in lipid rafts and lipid composition, with a special focus on changes in glycosphingolipids and the possibility of using lipid therapy for cancer treatment.

An emerging focus on lipids in extracellular vesicles.

Extracellular vesicles contain a lipid bilayer membrane that protects the encapsulated material, such as proteins, nucleic acids, lipids and metabolites, from the extracellular environment. These vesicles are released from cells via different mechanisms. During recent years extracellular vesicles have been studied as possible biomarkers for different diseases, as biological nanoparticles for drug delivery, and in basic studies as a tool to understand the structure of biological membranes and the mechanisms involved in vesicular trafficking. Lipids are essential molecular components of extracellular vesicles, but at the moment our knowledge about the lipid composition and the function of lipids in these vesicles is limited. However, the interest of the research community in these molecules is increasing as their role in extracellular vesicles is starting to be acknowledged. In this review, we will present the status of the field and describe what is needed to bring it forward.

Drug-Loaded Photosensitizer-Chitosan Nanoparticles for Combinatorial Chemo- and Photodynamic-Therapy of Cancer.

In this study we have developed biodegradable polymeric nanoparticles (NPs) containing the cytostatic drugs mertansine (MRT) or cabazitaxel (CBZ). The NPs are based on chitosan (CS) conjugate polymers synthesized with different amounts of the photosensitizer tetraphenylchlorin (TPC). These TPC-CS NPs have high loading capacity and strong drug retention due to π-π stacking interactions between the drugs and the aromatic photosensitizer groups of the polymers. CS polymers with 10% of the side chains containing TPC were found to be optimal in terms of drug loading capacity and NP stability. The TPC-CS NPs loaded with MRT or CBZ displayed higher cytotoxicity than the free form of these drugs in the breast cancer cell lines MDA-MB-231 and MDA-MB-468. Furthermore, light-induced photochemical activation of the NPs elicited a strong photodynamic therapy effect on these breast cancer cells. Biodistribution studies in mice showed that most of the TPC-CS NPs accumulated in liver and lungs, but they were also found to be localized in tumors derived from HCT-116 cells. These data suggest that the drug-loaded TPC-CS NPs have a potential in combinatory anticancer therapy and as contrast agents.

Biological response and cytotoxicity induced by lipid nanocapsules.

BACKGROUND: Lipid nanocapsules (LNCs) are promising vehicles for drug delivery. However, since not much was known about cellular toxicity of these nanoparticles in themselves, we have here investigated the mechanisms involved in LNC-induced intoxication of the three breast cancer cell lines MCF-7, MDA-MD-231 and MDA-MB-468. The LNCs used were made of Labrafac™ Lipophile WL1349, Lipoid® S75 and Solutol® HS15. RESULTS: High resolution SIM microscopy showed that the DiD-labeled LNCs ended up in lysosomes close to the membrane. Empty LNCs, i.e. without encapsulated drug, induced not only increased lysosomal pH, but also acidification of the cytosol and a rapid inhibition of protein synthesis. The cytotoxicity of the LNCs were measured for up to 72 h of incubation using the MTT assay and ATP measurements in all three cell lines, and revealed that MDA-MB-468 was the most sensitive cell line and MCF-7 the least sensitive cell line to these LNCs. The LNCs induced generation of reactive free oxygen species and lipid peroxidation. Experiments with knock-down of kinases in the near-haploid cell line HAP1 indicated that the kinase HRI is essential for the observed phosphorylation of eIF2α. Nrf2 and ATF4 seem to play a protective role against the LNCs in MDA-MB-231 cells, as knock-down of these factors sensitizes the cells to the LNCs. This is in contrast to MCF-7 cells where the knock-down of these factors had a minor effect on the toxicity of the LNCs. Inhibitors of ferroptosis provided a large protection against LNC toxicity in MDA-MB-231 cells, but not in MCF-7 cells. CONCLUSIONS: High doses of LNCs showed a different degree of toxicity on the three cell lines studied, i.e. MCF-7, MDA-MD-231 and MDA-MB-468 and affected signaling factors and the cell fate differently in these cell lines.

Paclitaxel-loaded biodegradable ROS-sensitive nanoparticles for cancer therapy.

BACKGROUND: Reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, trigger biodegradation of polymer-based nanoparticles (NPs) bearing pinacol-type boronic ester groups. These NPs may selectively release their cargo, in this case paclitaxel (PTX), at the high levels of ROS present in the intracellular environment of inflamed tissues and most tumors. PURPOSE: The main objective was to determine anti-tumor efficacy of PTX-loaded ROS-sensitive NPs and to examine whether macrophage infiltration had any impact on treatment efficacy. METHODS: NPs were synthesized and their characteristics in the presence of H2O2 were demonstrated. Both confocal microscopy as well as flow cytometry approaches were used to determine degradation of ROS-sensitive NPs. HeLa cells were cultured in vitro and used to establish tumor xenografts in nude mice. In vivo experiments were performed to understand toxicity, biodistribution and anti-tumor efficacy of the NPs. Moreover, we performed immunohistochemistry on tumor sections to study infiltration of M1 and M2 subsets of macrophages. RESULTS: We demonstrated that PTX delivered in NPs containing a ROS-sensitive polymer exhibits a better anti-tumor efficacy than PTX in NPs containing ROS-non-sensitive polymer, free PTX or Abraxane® (nab-PTX). The biodistribution revealed that ROS-sensitive NPs exhibit retention in liver, spleen and lungs, suggesting a potential to target cancer metastasizing to these organs. Finally, we demonstrated a correlation between infiltrated macrophage subsets and treatment efficacy, possibly contributing to the efficient anti-tumor effects. CONCLUSION: Treatment with ROS-sensitive NPs containing PTX gave an improved therapeutic effect in HeLa xenografts than their counterpart, free PTX or nab-PTX. Our data revealed a correlation between macrophage infiltration and efficiency of the different antitumor treatments, as the most effective NPs resulted in the highest infiltration of the anti-tumorigenic M1 macrophages.