The Rac1-inhibitor EHop-016 attenuates AML cell migration and enhances the efficacy of daunorubicin in MOLM-13 transplanted zebrafish larvae.

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a GTPase implicated in cell migration and homing of hematopoietic cells to the hematopoietic niche, and is commonly overexpressed in acute myeloid leukemia (AML). This can lead to quiescence of leukemic blasts in the niche and reduced response to therapy. We investigated the Rac1 inhibitor EHop-016 on AML by assessing its effects on MOLM-13 cells in vitro and in zebrafish larvae, regarding cell motility and therapeutic potential in combination with daunorubicin (DNR). In vitro assessment of proliferation and viability was by measurement of 3H-thymidine incorporation and detection of Annexin V/PI positive cells. Cell motility was evaluated by measurement of migration in a transwell system. Fluorescently stained MOLM-13 cells were injected into zebrafish larvae, and individual cells followed by confocal microscopy. Cell accumulation in the caudal hematopoietic tissue (CHT) was studied using a 12-hour timelapse, while in vivo efficacy of DNR, EHop-016 or a combination was investigated over 24 h. The in vitro results showed that EHop-016 acted synergistically in combination with DNR in reducing the viability of MOLM-13 cells (Bliss synergy score above 10 %). Non-toxic concentrations of EHop-016 reduced cell migration. These findings were reproduced in zebrafish larvae: larvae receiving both DNR and EHop-016 had significantly reduced tumor burden compared to the untreated control or single treatments. The accumulation of MOLM-13 cells in the CHT was reduced in larvae receiving EHop-016 treatment. Our findings demonstrate that targeting Rac1 in AML holds promise as a complementary treatment to established chemotherapy and should be further investigated.

Development of nanoparticles based on amphiphilic cyclodextrins for the delivery of active substances.

Although amphiphilic cyclodextrin derivatives (ACDs) serve as valuable building blocks for nanomedicine formulations, their widespread production still encounters various challenges, limiting large-scale manufacturing. This work focuses on a robust alternative pathway using mineral base catalysis to transesterify ß-cyclodextrin with long-chain vinyl esters, yielding ACD with modular and controlled hydrocarbon chain grafting. ACDs with a wide range of degrees of substitution (DS) were reliably synthesized, as indicated by extensive physicochemical characterization, including MALDI-TOF mass spectrometry. The influence of various factors, including the type of catalyst and the length of the hydrocarbon moiety of the vinyl ester, was studied in detail. ACDs were assessed for their ability to form colloidal suspensions by nanoprecipitation, with or without PEGylated phospholipid. Small-angle X-ray scattering and cryo-electron microscopy revealed the formation of nanoparticles with distinct ultrastructures depending on the DS: an onion-like structure for low and very high DS, and reversed hexagonal organization for DS between 4.5 and 6.1. We confirmed the furtivity of the PEGylated versions of the nanoparticles through complement activation experiments and that they were well tolerated in-vivo on a zebrafish larvae model after intravenous injection. Furthermore, a biodistribution experiment showed that the nanoparticles left the bloodstream within 10 h after injection and were phagocytosed by macrophages.

Ocrelizumab and ofatumumab, but not rituximab, trigger complement induction in vitro.

The clinical and adverse effects of the therapeutic monoclonal antibodies (mAb) ocrelizumab, ofatumumab and rituximab in multiple sclerosis (MS) are presently subject to extensive study. While the two former are approved for MS, the older and less costly rituximab is used off label, and adverse effect profiles are important in their evaluation. The three mAbs all induce B cell depletion, with complement-dependent cytotoxicity (CDC) as one of several mechanisms of action. Complement activation is also postulated to underlie adverse reactions related to infusion/injection. Such administration-related reactions are associated with all three mAbs, but comparisons have so far been indirect, resting on incidence reports from separate clinical trials. The objective of this study was to perform head-to-head comparison of complement activation by ofatumumab, ocrelizumab and rituximab. In vitro experiments were performed in whole blood from healthy donors. The complement-activating potential of the three mAbs was analyzed after 30 min of exposure to 0.3 mg/mL or 0.9 mg/mL of each drug, and compared with those of the well-known TNF inhibitory mAbs adalimumab and infliximab, the latter with recognized potential for infusion reactions. Ofatumumab, ocrelizumab, and infliximab, but not rituximab and adalimumab, triggered statistically significant complement activation measured as increased levels of terminal C5b-9 complement complex (TCC), a sensitive marker of such activation. While results demand careful interpretation, they provide an indication of distinct complement-inducing potential among anti-CD20 mAbs currently used to treat MS.

A new software tool for computer assisted in vivo high-content analysis of transplanted fluorescent cells in intact zebrafish larvae.

Acute myeloid leukemia and myelodysplastic syndromes are cancers of the bone marrow with poor prognosis in frail and older patients. To investigate cancer pathophysiology and therapies, confocal imaging of fluorescent cancer cells and their response to treatments in zebrafish larvae yields valuable information. While zebrafish larvae are well suited for confocal imaging, the lack of efficient processing of large datasets remains a severe bottleneck. To alleviate this problem, we present a software tool that segments cells from confocal images and track characteristics such as volume, location in the larva and fluorescent intensity on a single-cell basis. Using this software tool, we were able to characterise the responses of the cancer cell lines Molm-13 and MDS-L to established treatments. By utilizing the computer-assisted processing of confocal images as presented here, more information can be obtained while being less time-consuming and reducing the demand of manual data handling, when compared to a manual approach, thereby accelerating the pursuit of novel anti-cancer treatments. The presented software tool is available as an ImageJ java-plugin at https://zenodo.org/10.5281/zenodo.7383160 and the source code at https://github.com/Jfo004/ConfocalCellSegmentation.

Graphene-based phenformin carriers for cancer cell treatment: a comparative study between oxidized and pegylated pristine graphene in human cells and zebrafish.

Graphene is an attractive choice for the development of an effective drug carrier in cancer treatment due to its high adsorption area and pH-responsive drug affinity. In combination with the highly potent metabolic drug phenformin, increased doses could be efficiently delivered to cancer cells. This study compares the use of graphene oxide (GO) and polyethylene glycol stabilized (PEGylated) pristine graphene nanosheets (PGNSs) for drug delivery applications with phenformin. The cytotoxicity and mitotoxicity of the graphene-based systems were assessed in human cells and zebrafish larvae. Targeted drug release from GO and PGNSs was evaluated at different pH levels known to arise in proliferating tumor microenvironments. PGNSs were less cytotoxic and mitotoxic than GO, and showed an increased release of phenformin at lower pH in cells, compared to GO. In addition, the systemic phenformin effect was mitigated in zebrafish larvae when bound to GO and PGNSs compared to free phenformin, as measured by flavin metabolic lifetime imaging. These results pave the way for improved phenformin-based cancer therapy using graphene nano-sheets, where PGNSs were superior to GO.

Repurposing chlorpromazine for anti-leukaemic therapy by nanoparticle encapsulation.

Treatment of acute myeloid leukaemia (AML) relies on decades-old drugs, and while recent years have seen some breakthroughs, AML is still characterised by poor prognosis and survival rate. Drug repurposing can expedite the preclinical development of new therapies, and by nanocarrier encapsulation, the number of potentially viable drug candidates can be further expanded. The anti-psychotic drug chlorpromazine (CPZ) has been identified as a candidate for repurposing for AML therapy. Nanoencapsulation may improve the suitability of CPZ for the treatment of AML by reducing its effect on the central nervous system. Using the emulsion-evaporation technique, we have developed PEGylated PLGA nanoparticles loaded with CPZ for AML therapy. The nanoparticles were characterised to be between 150 and 300 nm by DLS, of spherical morphology by TEM, with a drug loading of at least 6.0% (w/w). After an initial burst release of adsorbed drug, the remaining 80% of the drug was retained in the PLGA nanoparticles for at least 24 h. The CPZ-loaded nanoparticles had equal cytotoxic potential towards AML cells to free CPZ, but acted more slowly, in line with the protracted drug release. Crucially, nanoparticles injected intravenously into zebrafish larvae did not accumulate in the brain, and nanoencapsulation also prevented CPZ from crossing an artificial membrane model. This demonstrates that the purpose for nanoencapsulation of CPZ is fulfilled, namely avoiding effects on the central nervous system while retaining the anti-AML activity of the drug.

Improved pH-Responsive Release of Phenformin from Low-Defect Graphene Compared to Graphene Oxide.

Graphene-based drug carriers provide a promising addition to current cancer drug delivery options. Increased accessibility of high-quality graphene made by plasma-enhanced chemical vapor deposition (PE-CVD) makes it an attractive material to revisit in comparison to the widely studied graphene oxide (GO) in drug delivery. Here, we show the potential of repurposing the metabolic drug phenformin for cancer treatment in terms of stability, binding, and pH-responsive release. Using covalent attachment of poly(ethylene glycol) (PEG) onto pristine (PE-CVD) graphene, we show that PEG stabilized graphene nanosheets (PGNS) are stable in aqueous solutions and exhibit higher binding affinity toward phenformin than GO. Moreover, we experimentally demonstrate an improved drug release from PGNS than GO at pH levels lower than physiological conditions, yet comparable to that found in tumor microenvironments.