Dose-dependent Uptake Research Articles

Multiple Myeloma Derived Extracellular Vesicle Uptake by Monocyte Cells Stimulates IL-6 and MMP-9 Secretion and Promotes Cancer Cell Migration and Proliferation.

Multiple Myeloma (MM) is an incurable haematological malignancy caused by uncontrolled growth of plasma cells. MM pathogenesis is attributed to crosstalk between plasma cells and the bone marrow microenvironment, where extracellular vesicles (EVs) play a role. In this study, EVs secreted from a panel of MM cell lines were isolated from conditioned media by ultracentrifugation and fluorescently stained EVs were co-cultured with THP-1 monocyte cells. MM EVs from three cell lines displayed a differential yet dose-dependent uptake by THP-1 cells, with H929 EVs displaying the greatest EV uptake compared to MM.1s and U266 EVs suggesting that uptake efficiency is dependent on the cell line of origin. Furthermore, MM EVs increased the secretion of MMP-9 and IL-6 from monocytes, with H929 EVs inducing the greatest effect, consistent with the greatest uptake efficiency. Moreover, monocyte-conditioned media collected following H929 EV uptake significantly increased the migration and proliferation of MM cells. Finally, EV proteome analysis revealed differential cargo enrichment that correlates with disease progression including a significant enrichment of spliceosome-related proteins in H929 EVs compared to the U266 and MM.1s EVs. Overall, this study demonstrates that MM-derived EVs modulate monocyte function to promote tumour growth and metastasis and reveals possible molecular mechanisms involved.

Deep dive into the chronic toxicity of tyre particle mixtures and their leachates

Particles from the tread of vehicle tyres are a global pollutant, which are emitted into the environment at an approximate rate of 1.4 kg.year−1 for an average passenger-car. In this study, popular tyre brands were used to generate a tyre tread microparticle mixture. The chronic toxicity of both particles and chemical leachates were compared on a planktonic test species (Daphnia magna). Over 21 days of exposure, pristine tyre tread microparticles were more toxic (LC50 60 mg.L−1) than chemical lechates alone (LC50 542 mg.L−1). Microparticles and leachates showed distinct effects on reproduction and morphological development at environmentally relevant concentrations, with dose-dependent uptake of particles visible in the digestive tract. Chemical characterization of leachates revealed a metal predominance of zinc, titanium, and strontium. Of the numerous organic chemicals present, at least 54 were shared across all 5 tyre brands, with many classified to be very toxic. Our results provide a critically needed information on the toxicity of tyre tread particles and the associated chemicals that leach from them to inform future mitigation measures. We conclude that tyre particles are hazardous pollutants of particular concern that are close to or possibly above chronic environmental safety limits in some locations.

Large-scale production of superparamagnetic iron oxide nanoparticles by flame spray pyrolysis: In vitro biological evaluation for biomedical applications

Despite the large number of synthesis methodologies described for superparamagnetic iron oxide nanoparticles (SPIONs), the search for their large-scale production for their widespread use in biomedical applications remains a mayor challenge. Flame Spray Pyrolysis (FSP) could be the solution to solve this limitation, since it allows the fabrication of metal oxide nanoparticles with high production yield and low manufacture costs. However, to our knowledge, to date such fabrication method has not been upgraded for biomedical purposes. Herein, SPIONs have been fabricated by FSP and their surface has been treated to be subsequently coated with dimercaptosuccinic acid (DMSA) to enhance their colloidal stability in aqueous media. The final material presents high quality in terms of nanoparticle size, homogeneous size distribution, long-term colloidal stability and magnetic properties. A thorough in vitro validation has been performed with peripheral blood cells and mesenchymal stem cells (hBM-MSCs). Specifically, hemocompatibility studies show that these functionalized FSP-SPIONs-DMSA nanoparticles do not cause platelet aggregation or impair basal monocyte function. Moreover, in vitro biocompatibility assays show a dose-dependent cellular uptake while maintaining high cell viability values and cell cycle progression without causing cellular oxidative stress. Taken together, the results suggest that the FSP-SPIONs-DMSA optimized in this work could be a worthy alternative with the benefit of a large-scale production aimed at industrialization for biomedical applications.

Development and characterization of lung surfactant-coated polymer nanoparticles for pulmonary drug delivery

Lung cancer is often diagnosed at an advanced stage where tumors are usually inoperable and first-line therapies are inefficient and have off-targeted adverse effects, resulting in poor patient survival. Here, we report the development of an inhalable poly lactic-co-glycolic acid polymer-based nanoparticle (PLGA-NP) formulation with a biomimetic Infasurf® lung surfactant (LS) coating, for localized and sustained lung cancer drug delivery. The nanoparticles (188 ± 7 nm) were stable in phosphate buffered saline, serum and Gamble's solution (simulated lung fluid), and demonstrated cytocompatibility up to 1000 μg/mL concentration and dose-dependent uptake by lung cancer cells. The LS coating significantly decreased nanoparticle (NP) uptake by NR8383 alveolar macrophages in vitro compared to uncoated NPs. The coating, however, did not impair NP uptake by A549 lung adenocarcinoma cells. The anti-cancer drug gemcitabine hydrochloride encapsulated in the PLGA core was released in a sustained manner while the paclitaxel loaded in the LS shell demonstrated a rapid or burst release profile over 21 days. The drug-loaded NPs significantly decreased cancer cell survival and colony formation in vitro compared to free drugs and single drug-loaded NPs. In vivo studies confirmed greater retention of LS-coated NPs in the lungs of C57BL/6 WT mice compared to uncoated NPs, at 24 h and 72 h following intranasal administration. The overall results confirm that LS coating is a unique strategy for cloaking polymeric NPs to potentially prevent their rapid lung clearance and facilitate prolonged pulmonary drug delivery.

Abstract P4-07-18: FerOX, an anthracyclines nanoformulation to lead toward immuno-mediated cancer remission, preserving T-cell immune competence

Abstract Background Chemotherapy with anthracyclines, such as doxorubicin (DOX), still remains a mainstay in cancer treatment. DOX kills tumor cells by direct interference with DNA replication, thus it is very effective against highly proliferating cells. Since DOX is not cell-specific drug, it displays severe side effects that limit its clinical use. Besides its direct cytotoxicity, it enhances also tumor immunogenicity, since DOX-induced immunogenic cancer cell death facilitates tumor antigens presentation. However, the generation of an adaptive immune response is one of the most highly- proliferative processes, thus it may be affected by DOX cytotoxicity, paradoxically preventing the establishment of a proper adaptive antitumor immune response. Therefore, the effect of DOX on dividing T-cells urges investigation. Methods We explored the phenotypic and functional effects of DOX on lymphocytes from healthy donors and from breast cancer (BC) patients. Purified peripheral blood mononuclear cells (PBMC) were subjected to an uptake assay and to a CFSE-based proliferation assay in vitro. Characterization of lymphocytes was made by flow cytometry (FC) and confocal microscopy. Innovative DOX nanoformulation with proved efficacy in cancer cell line, BC Patient-derived Organoids (PDO) and murine model of BC, has been assessed in parallel to liposomal DOX, evaluating both DOX uptake and their capability to preserve proliferation potential in different PBMC subpopulation. Results PBMC incubated in vitro with DOX displayed a dose-dependent DOX uptake that results in a dramatic proliferative impairment. The same behavior has been observed in BC patient-derived PBMC, isolated immediately after the end of first cycle of chemotherapy, which show a prompt DOX internalization coupled with a dramatic impairment of proliferative potential in comparison to the match-paired PMBC isolated before the treatment. Interestingly, FC characterization showed that DOX internalization is higher in CD8+ than CD4+ T-cells and mainly affects central memory, effector memory and terminal differentiated effector. Since pivotal to every adaptive immune response is the activation and massive proliferation of T cells from their resting state, we have exploited a DOX formulation in ferritin nanocages (FerOX), that displays a TfR1-mediated tumor specific homing in order to circumvent drug internalization in those immune cells. FerOX efficacy has been proved in BC-PDO, evidencing an inverse correlation between TfR1 expression and HFn-DOX IC50. HFn nanoformulation protects immune cells from DOX toxic effects, reducing DOX uptake and proliferative impairment, mainly preserving Naïve T-cells. The capability of FerOX to preserve T-cells competence to generate an immune response has been corroborated by an in vitro experiment aimed to mimic what could happens to patient’s PBMCs if they could be treated with FerOX. Conclusions As a whole, our data indicate that preventing T- cells chemotherapy-related toxicity through FerOX could allow the host adaptive immune system to fully benefit from the DOX-induced immunogenic cell death to vaccinate against cancer. This strategy could be promising for complete tumor eradication and might generate a long- term immunosurveillance of recurrences and metastases. Citation Format: Serena Mazzucchelli, Francesco Mainini, Francesco Andreata, Marta Truffi, Arianna Bonizzi, Francesca Piccotti, Marta Sevieri, Fabio Corsi. FerOX, an anthracyclines nanoformulation to lead toward immuno-mediated cancer remission, preserving T-cell immune competence [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-18.

New Insights into the Biological Response Triggered by Dextran-Coated Maghemite Nanoparticles in Pancreatic Cancer Cells and Their Potential for Theranostic Applications

Iron oxide nanoparticles are one of the most promising tools for theranostic applications of pancreatic cancer due to their unique physicochemical and magnetic properties making them suitable for both diagnosis and therapy. Thus, our study aimed to characterize the properties of dextran-coated iron oxide nanoparticles (DIO-NPs) of maghemite (γ-Fe2O3) type synthesized by co-precipitation and to investigate their effects (low-dose versus high-dose) on pancreatic cancer cells focusing on NP cellular uptake, MR contrast, and toxicological profile. This paper also addressed the modulation of heat shock proteins (HSPs) and p53 protein expression as well as the potential of DIO-NPs for theranostic purposes. DIO-NPs were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering analyses (DLS), and zeta potential. Pancreatic cancer cells (PANC-1 cell line) were exposed to different doses of dextran-coated ɣ-Fe2O3 NPs (14, 28, 42, 56 μg/mL) for up to 72 h. The results revealed that DIO-NPs with a hydrodynamic diameter of 16.3 nm produce a significant negative contrast using a 7 T MRI scanner correlated with dose-dependent cellular iron uptake and toxicity levels. We showed that DIO-NPs are biocompatible up to a concentration of 28 μg/mL (low-dose), while exposure to a concentration of 56 μg/mL (high-dose) caused a reduction in PANC-1 cell viability to 50% after 72 h by inducing reactive oxygen species (ROS) production, reduced glutathione (GSH) depletion, lipid peroxidation, enhancement of caspase-1 activity, and LDH release. An alteration in Hsp70 and Hsp90 protein expression was also observed. At low doses, these findings provide evidence that DIO-NPs could act as safe platforms in drug delivery, as well as antitumoral and imaging agents for theranostic uses in pancreatic cancer.

Investigating the Role of Classical Ayurveda-Based Incineration Process on the Synthesis of Zinc Oxide Based Jasada Bhasma Nanoparticles and Zn2+ Bioavailability.

Jasada bhasma (JB) is a zinc oxide-based Indian traditional Ayurveda-based herbo-metallic nanoparticle used for the treatment of zinc (Zn) deficiency and autoimmune and inflammatory disorders. JB is made by following the Ayurveda-based guidelines using zinc oxide (ZnO) as a raw material and going through 17 cycles of the high-temperature incineration and trituration process known as "Ma̅raṇa" in the presence of herbal decoctions prepared from the leaves ofAzadirachta indica andAloe vera gel. These cycles improve the purity of the parent material and transform its physicochemical properties, converting it into nanoparticles. However, there still exists a knowledge gap regarding the role of incineration in the physicochemical transformation of the Zn raw material into JB nanoparticles and the biological interaction of the final product. In the present study, the JB samples obtained during different Ma̅raṇa cycles were carefully studied for their physicochemical transformation using analytical methods such as powdered X-ray diffraction (XRD), small-angle X-ray scattering (SAXS), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy, Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and dynamic light scattering (DLS). According to the XRD results, the Zn and oxygen molecules in hexagonal ZnO wurtzite crystals gradually realigned as a result of repeated heat treatments that caused lattice tension and crystal size reduction from 53.14 to 42.40 nm. A morphological transition from 1.5 μm rod shape to 31 nm in the JB particles can be seen using FESEM and SAXS analyses. The existence of 10 nm-sized nanoparticles in the finished product was confirmed by HRTEM. The presence of ZnO was confirmed in all samples by FTIR and Raman spectroscopies. Cell viability analysis showed an inhibitory concentration 50% of >1000 μg/mL for JB nanoparticles, revealing no adverse effects in human colon Caco-2 cells. A dose-dependent uptake and intracellular accumulation of JB nanoparticles were observed in Caco-2 cells using inductively coupled plasma-based mass spectroscopy (ICP-MS). Bioavailability of Zn2+ ions (6% w/w) through JB dissolution in acidic pH 4.0 was observed, representing the stomach and intracellular lysosomal physiological conditions. Therefore, the study showed that the repeated incineration cycles produced biocompatible JB nanoparticles through the physicochemical transformation at molecular levels capable of delivering bioavailable Zn2+ ions under physiological conditions. In conclusion, the medicinal properties of JB nanoparticles described in Ayurveda were found to originate from their small size and dissolution properties, formed through the classical incineration-based synthesis process.

Intracellular accumulation and immunological response of NIR-II polymeric nanoparticles

Polymeric nanoparticles (NPs) are extremely promising for theranostic applications. However, their interest depends largely on their interactions with immune system, including the capacity to activate inflammation after their capture by macrophages. In the present study, we generated monodisperse poly(ethyl methacrylate) (PEMA) NPs loaded with hydrophobic photoluminescent gold nanoclusters (Au NCs) emitting in the NIR-II optical windows and studied their interaction in vitro with J774.1A macrophages. PEMA NPs showed an efficient time and dose dependent cellular uptake with up to 70 % of macrophages labelled in 24 h without detectable cell death. Interestingly, PEMA and Au-PEMA NPs induced an anti-inflammatory response and a strong down-regulation of nitric oxide level on lipopolysacharides (LPS) activated macrophages, but without influence on the levels of reactive oxygen species (ROS). These polymeric NPs may thus present a potential interest for the treatment of inflammatory diseases.

Multicompartment Nanoparticles by Crystallization-Driven Self-Assembly of Star Polymers: Combining High Stability and Loading Capacity.

Herein novel multicompartment nanoparticles (MCNs) that combine high stability and cargo loading capacity are developed. The MCNs are fabricated by crystallization-driven self-assembly (CDSA) of a tailor-made 21 arm star polymer, poly(L-lactide)[poly(tert-butyl acrylate)-block-poly(ethylene glycol)]20 [PLLA(PtBA-b-PEG)20 ]. Platelet-like or spherical MCNs containing a crystalline PLLA core and hydrophobic PtBA subdomains are formed and stabilized by PEG. Hydrophobic cargos, such as Nile Red and chemotherapeutic drug doxorubicin, can be successfully encapsulated into the collapsed PtBA subdomains with loading capacity two orders of magnitude higher than traditional CDSA nanoparticles. Depolarized fluorescence measurements of the Nile Red loaded MCNs suggest that the free volume of the hydrophobic chains in the nanoparticles may be the key for regulating their drug loading capacity. In vitro study of the MCNs suggests excellent cytocompatibility of the blank nanoparticles as well as a dose-dependent cellular uptake and cytotoxicity of the drug-loaded MCNs.

Biodegradable and Inherently Fluorescent pH-Responsive Nanoparticles for Cancer Drug Delivery.

The development of two novel pH-only and pH- and thermo-responsive theranostic nanoparticle (NP) formulations to deliver an anticancer drug and track the accumulation and therapeutic efficacy of the formulations through inherent fluorescence. A pH-responsive formulation was synthesized from biodegradable photoluminescent polymer (BPLP) and sodium bicarbonate (SBC) via an emulsion technique, while a thermoresponsive BPLP copolymer (TFP) and SBC were used to synthesize a dual-stimuli responsive formulation via free radical co-polymerization. Cisplatin was employed as a model drug and encapsulated during synthesis. Size, surface charge, morphology, pH-dependent fluorescence, lower critical solution temperature (LCST; TFP NPs only), cytocompatibility and in vitro uptake, drug release kinetics and anticancer efficacy were assessed. While all BPLP-SBC and TFP-SBC combinations produced spherical nanoparticles of a size between 200-300 nm, optimal polymer-SBC ratios were selected for further study. Of these, the optimal BPLP-SBC formulation was found to be cytocompatible against primary Type-1 alveolar epithelial cells (AT1) up to 100 μg/mL, and demonstrated sustained drug release over 14 days, dose-dependent uptake, and marked pH-dependent A549 cancer cell killing (72 vs. 24% cell viability, at pH 7.4 vs. 6.0). The optimal TFP-SBC formulation showed excellent cytocompatibility against AT1 cells up to 500 μg/mL, sustained release characteristics, dose-dependent uptake, pH-dependent (78% at pH 7.4 vs. 64% at pH 6.0 at 37°C) and marked temperature-dependent A549 cancer cell killing (64% at 37°C vs. 37% viability at pH 6.0, 41°C). In all, both formulations hold promise as inherently fluorescent, stimuli-responsive theranostic platforms for passively targeted anti-cancer therapy.

A Small Peptide Increases Drug Delivery in Human Melanoma Cells.

Melanoma is the most fatal type of skin cancer and is notoriously resistant to chemotherapies. The response of melanoma to current treatments is difficult to predict. To combat these challenges, in this study, we utilize a small peptide to increase drug delivery to melanoma cells. A peptide library array was designed and screened using a peptide array-whole cell binding assay, which identified KK-11 as a novel human melanoma-targeting peptide. The peptide and its D-amino acid substituted analogue (VPWxEPAYQrFL or D-aa KK-11) were synthesized via a solid-phase strategy. Further studies using FITC-labeled KK-11 demonstrated dose-dependent uptake in human melanoma cells. D-aa KK-11 significantly increased the stability of the peptide, with 45.3% remaining detectable after 24 h with human serum incubation. Co-treatment of KK-11 with doxorubicin was found to significantly enhance the cytotoxicity of doxorubicin compared to doxorubicin alone, or sequential KK-11 and doxorubicin treatment. In vivo and ex vivo imaging revealed that D-aa KK-11 distributed to xenografted A375 melanoma tumors as early as 5 min and persisted up to 24 h post tail vein injection. When co-administered, D-aa KK-11 significantly enhanced the anti-tumor activity of a novel nNOS inhibitor (MAC-3-190) in an A375 human melanoma xenograft mouse model compared to MAC-3-190 treatment alone. No apparent systemic toxicities were observed. Taken together, these results suggest that KK-11 may be a promising human melanoma-targeted delivery vector for anti-melanoma cargo.

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies.

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington’s disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.

AUPHOS, A “FIRST-IN CLASS“ ORAL AGENT FOR CORRECTING METABOLIC DYSFUNCTION IN IBD

Abstract BACKGROUND Mucosal repair is the goal of medical therapy in IBD. Prospective inception cohort pediatric studies suggest that suppression of mitochondrial genes lead to unfavorable clinical outcomes in ulcerative colitis (UC) and Crohn’s disease (CD). We posit that the inability to restore epithelial mitochondrial respiration impairs ulcer healing in IBD. In the current study, we tested whether a novel compound (AuPhos) that improves mitochondrial function promotes ulcer healing in IBD. METHODS Biodistribution and pharmacokinectic (PK) studies were performed (6h, 12h, 24h) after single oral dose (25mg/kg) of AuPhos using C57/BL/6 (WT) mice. Mitochondrial uptake of AuPhos was determined by measuring gold content in normal colonocytes (NCM460) using graphite furnace atomic absorption spectroscopy. Oxygen consumption rate (OCR) in isolated crypt-IECs from AuPhos-fed WT mice were examined using high-resolution respirometry. The effect of AuPhos in colitis was tested in DSS (2%; 7d) WT (B6) mice. Disease activity index (DAI) (weight loss, diarrhea, and hematochezia), intestinal permeability (FITC-dextran), histological scoring and the frequency of fissioning crypts were determined throughout the 21d time course. Tissue mRNA levels for mitochondrial enzyme, cytokine, chemokine and intestinal stem cell (ISC) marker genes were assessed by RT-qPCR. RESULTS Biodistribution and PK studies revealed that higher levels of orally administered AuPhos in small intestine and colon tissue, with limited systemic absorption. AuPhos displayed a dose-dependent mitochondrial uptake in IEC where respirometry showed >30% increase in OCR after oral dosing in vivo. AuPhos treatment decreased DAI (**; p<0.01), histologic colitis scores D11-15 (*; p<0.05) and FITC-dextran permeability in DSS colitis mice. Notably, AuPhos increased fissioning crypt numbers near healing ulcers in DSS-colitis mice (**; p<0.01) while it reduced mRNA for pro-inflammatory cytokines/chemokines (IL-1β, IL-6, IFN-γ, IP10). AuPhos induced mRNA for ISC markers [Lgr4 (*; p<0.05), Lgr5, Lrig1 (***; p<0.001)] and mitochondrial complexes [mtCO1 (**; p<0.01) and COX2 (***; p<0.001)]. These findings suggest that AuPhos-induced mitochondrial respiration drives transcriptomic regulation of IEC genes involved in colitis ulcer healing. CONCLUSION Our data suggests that AuPhos compound improves ulcer healing under acute colitis conditions by increasing mucosal OxPHOS metabolism. Specifically, we find that AuPhos increases mitochondrial respiration-induced crypt fissioning and ulcer healing associated with reduced mucosal inflammation and increased barrier integrity. Overall, AuPhos may be an important “first-in class” oral therapeutic for correcting the metabolic dysfunction central to IBD pathogenesis.

Mechanistic studies on the adverse effects of manganese overexposure in differentiated LUHMES cells

Manganese (Mn) is an essential trace element, but overexposure is associated with toxicity and neurological dysfunction. Accumulation of Mn can be observed in dopamine-rich regions of the brain in vivo and Mn-induced oxidative stress has been discussed extensively. Nevertheless, Mn-induced DNA damage, adverse effects of DNA repair, and possible resulting consequences for the neurite network are not yet characterized. For this, LUHMES cells were used, as they differentiate into dopaminergic-like neurons and form extensive neurite networks. Experiments were conducted to analyze Mn bioavailability and cytotoxicity of MnCl2, indicating a dose-dependent uptake and substantial cytotoxic effects. DNA damage, analyzed by means of 8-oxo-7,8-dihydro-2′-guanine (8oxodG) and single DNA strand break formation, showed significant dose- and time-dependent increase of DNA damage upon 48 h Mn exposure. Furthermore, the DNA damage response was increased which was assessed by analytical quantification of poly(ADP-ribosyl)ation (PARylation). Gene expression of the respective DNA repair genes was not significantly affected. Degradation of the neuronal network is significantly altered by 48 h Mn exposure. Altogether, this study contributes to the characterization of Mn-induced neurotoxicity, by analyzing the adverse effects of Mn on genome integrity in dopaminergic-like neurons and respective outcomes.

A novel β-glucan–oligonucleotide complex selectively delivers siRNA to APCs via Dectin-1

Delivering therapeutic nucleic acids to targeted cells and organs has been a challenge for decades. A novel technology to deliver oligonucleotide therapeutics to immune cells is here described. In this approach, a macromolecular complex of oligonucleotides and the β-1,3-glucan schizophyllan (SPG) is selectively delivered to cells expressing a lectin receptor, Dectin-1, via SPG–Dectin-1 interaction. Detailed investigation of Dectin-1-expressing cells revealed that Dectin-1 is expressed in all subsets of monocytes as well as dendritic cell (DC) populations, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs), in humans. The expression patterns in mice and humans are comparable, except for the expression in pDCs. The results indicate that Dectin-1 is expressed on cells capable of professional antigen presentation, except for B cells. We chose CD40 as a target gene for small interfering RNA (siRNA) as CD40 expression in antigen-presenting cells (APCs), particularly in DCs, plays critical roles in regulating immune responses. Dose-dependent cellular uptake of siCD40–SPG complexes was confirmed in cells expressing Dectin-1. Gene silencing activity was confirmed in vitro by the reduction of CD40 mRNA and by the site-specific cleavage of CD40 mRNA as determined by the 5’ RNA ligase-mediated rapid amplification of cDNA ends (5’RLM-RACE) technique. In vivo activity of siCD40–SPG complexes was demonstrated as the reduced CD40 protein expression in monocytes and DCs in mice. Furthermore, the in vivo activity of siCD40–SPG targeting human CD40 was confirmed in cynomolgus monkeys by the 5’RLM-RACE technique. In conclusion, we have demonstrated the receptor-ligand binding-mediated delivery of siRNA targeting immune-regulating monocytes and DCs via the interaction of SPG and its receptor, Dectin-1. As monocytes and DCs play central roles in inducing and controlling immune responses, Dectin-1-targeted delivery of nucleic acids should provide a useful tool for developing drugs to treat a wide range of diseases, including autoimmune diseases, allergy, and cancer, as well as transplantation.

Low efficiency of leucocyte plugging-based drug delivery to cancer in mice

Cells of the immune system were proposed for use as Trojan horse for tumour-specific drug delivery. The efficacy of such cell-based drug delivery depends on the site-specific cell homing. This present study was aimed to investigate the potential of leucocytes for intratumoural site-specific enrichment using a locoregional application route in experimental liver tumours. Human neutrophils were isolated from peripheral blood and directly labelled with calcein AM or loaded with doxorubicin. The neutrophil loading and release of doxorubicin and the migration and adhesion to ICAM-1 were analysed in vitro. Macrophages were isolated and activated in vitro. Leucocyte plugging and the distribution pattern in the liver microvasculature were studied ex vivo, and the efficacy of leucocyte plugging in tumour blood vessels was analysed in vivo after superselective intra-arterial injection in mouse liver tumour models. Neutrophils were characterised by the high dose-dependent uptake and rapid release of doxorubicin. Doxorubicin loading did not affect neutrophil migration function. Neutrophil plugging in liver microvasculature was very high (> 90%), both after ex vivo perfusion and after injection in vivo. However, neutrophils as well as activated macrophages plugged insufficiently in tumour blood vessels and passed through the tumour microvasculture with a very low sequestration rate in vivo. Neutrophils possess several properties to function as potentially effective drug carriers; however, the tumour site-specific drug delivery after selective locoregional injection was observed to be insufficient owing to low intratumoural microvascular plugging.Graphical abstract

Targeting the Mechanism of c-Mpl Exon-Inclusion through Antisense Oligonucleotides Yields a Dominant Negative Isoform

▪Activating mutations of the Thrombopoietin (THPO) receptor, c-MPL, have been revealed as drivers in myeloproliferative diseases (MPDs).In addition, wildtype c-MPL signaling has been shown to be required for the pathogenesis of Calreticulin-mutated MPDs and implicated in leukemic stem cell survival in CML and AML. Therefore, the ability to impair c-MPL signaling has the potential to provide a novel, targeted approach toward these difficult to treat diseases. Alternative splicing of c-MPL gives rise to a truncated dominant negative isoform, MPL-TR, which skips exons 9 and 10 and is conserved between species. Our prior work using a knockout mouse model identified the spliceosomal protein, Ott1(Rbm15), as a key regulator of alternative splicing between Mpl-TR and the full length isoform, Mpl-FL. Loss of Ott1 increased the fraction of Mpl-TR from 33% to 73% in hematopoietic stem cells (HSCs) and abolished Thpo response. Furthermore, overexpression of Mpl-TR impaired HSC function in vivo. To further delineate the global effects of Ott1 on alternative splicing within the HSC compartment, we performed RNA-seq on sorted Ott1-deleted Lin-Sca1+Kit+ (LSK) cells. DEXseq analysis of significant differential exon expression identified 1821 exon exclusion and 158 exon inclusion events in the Ott1-deleted cells compared to wildtype controls, establishing Ott1 as primarily an exon-inclusion factor. The splicing factor Srsf2 has been observed in the same complex as Ott1 and also functions primarily as a cassette exon inclusion factor, therefore we compared previously identified Srsf2 exon targets and found 15-19% overlapped with Ott1-affected exons, suggesting a functional link between the proteins in the spliceosome.Srsf2 utilizes the exon splicing enhancer (ESE) sequence to promote exon inclusion. Scanning the c-Mpl sequence revealed an ESE within exon 10 that is conserved between mice and humans. We hypothesized blocking the exon 10 ESE with an anti-sense oligonucleotide (AON) would interfere with the spliceosomal machinery responsible for c-Mpl exon-inclusion and lead to exon skipping, thus resulting in production of the dominant negative Mpl-TR isoform. An advantage of AON-targeted therapy is the ability to produce highly specific agents based on sequence complementarity. We synthesized chemically modified AONs with phosphorothioate backbones, either fully modified with 2'- O -methoxyethyl RNA (MOE) and thus designed to induce splice-switching, or designed as gapmers to elicit cleavage of their targets by RNase H. MOE-based AONs targeted towards the exon 10 ESE were the most effective at increasing the Mpl-TR isoform fraction, increasing them from 30-40% to 60-70% in murine cell lines and primary bone marrow/fetal liver cells. AONs directed toward the splice acceptors and donors of exons 9 and 10 were less effective, suggesting a key role for the exon 10 ESE in regulating c-Mpl splicing decision. To test the ability of AONs to successfully target in vivo, fluorophore-labelled AONs were used in a dose-escalation study in mice. 18 hours after tail vein injection with 1-6 mg/kg un-encapsulated AON dose, bone marrow and spleen were harvested and lineage labelled. High levels of dose-dependent uptake were observed, particularly within the myeloid compartment (granulocyte >99%; megakaryocyte 55%; macrophage 20.9%; erythroid 44.7%; B cell 5% and CD4 T 45.6% and CD8 T 77%). To assess uptake and intracellular localization of AONs within the HSC compartment, AMNIS flow cytometry was performed and showed robust nuclear localization within 64% of LSK cells and 42% of myeloid progenitors. The high levels of uptake in a wide range of hematopoietic lineages, including HSCs, confirms IV injection of naked AONs can efficiently target the hematopoietic compartment. In summary, through uncovering the pivotal cis- and trans-factors controlling splicing at the c-Mpl locus, we were able to develop a specific, effective agent for impairing the Thpo/Mpl axis. DisclosuresNo relevant conflicts of interest to declare.

Malic acid grafted Fe3O4 nanoparticles for controlled drug delivery and efficient heating source for hyperthermia therapy

Tailoring surface features is essential for creating specific functional properties on superparamagnetic Fe3O4 nanoparticles for biomedical applications. In this regard, we explored the use of malic acid as a surface passivating agent for designing biocompatible, highly water-dispersible Fe3O4 magnetic nanocarriers (MMNCs) for high payload of anticancer drug, doxorubicin hydrochloride (DOX). The efficacious grafting of malic acid onto the surface of Fe3O4 was apparent from infrared spectroscopy, dynamic light scattering, zeta-potential and thermogravimetric measurements. XRD and TEM analyses revealed the formation of highly crystalline single-phase Fe3O4 nanoparticles. They showed good aqueous colloidal stability, pH dependent surface charge characteristics and superparamagnetic behavior at room temperature. The electrostatic conjugation of drug onto the surface of MMNCs was optimized by varying the ratio of DOX to MMNCs, and a maximum loading efficiency of 72% was achieved at their 1:10 ratio. The DOX conjugated MMNCs (DOX-MMNCs) exhibited pH dependent controlled release characteristics. These DOX-MMNCs demonstrated dose dependent cellular uptake and retained considerable toxicity of DOX towards breast cancer (MCF-7) cell line. Further, our magnetic hyperthermia studies showed excellent heating efficiency of these MMNCs within the permissible limit of field strength and frequency reported for a safe application of hyperthermia to patients. Specifically, a water-dispersible surface decorated magnetic formulation was developed for pH-responsive controlled release of chemotherapeutic drug and efficient heating source for hyperthermia therapy.

(-)-Englerin-A Has Analgesic and Anti-Inflammatory Effects Independent of TRPC4 and 5.

Recently, we found that the deletion of TRPC5 leads to increased inflammation and pain-related behaviour in two animal models of arthritis. (-)-Englerin A (EA), an extract from the East African plant Phyllanthus engleri has been identified as a TRPC4/5 agonist. Here, we studied whether or not EA has any anti-inflammatory and analgesic properties via TRPC4/5 in the carrageenan model of inflammation. We found that EA treatment in CD1 mice inhibited thermal hyperalgesia and mechanical allodynia in a dose-dependent manner. Furthermore, EA significantly reduced the volume of carrageenan-induced paw oedema and the mass of the treated paws. Additionally, in dorsal root ganglion (DRG) neurons cultured from WT 129S1/SvIm mice, EA induced a dose-dependent cobalt uptake that was surprisingly preserved in cultured DRG neurons from 129S1/SvIm TRPC5 KO mice. Likewise, EA-induced anti-inflammatory and analgesic effects were preserved in the carrageenan model in animals lacking TRPC5 expression or in mice treated with TRPC4/5 antagonist ML204.This study demonstrates that while EA activates a sub-population of DRG neurons, it induces a novel TRPC4/5-independent analgesic and anti-inflammatory effect in vivo. Future studies are needed to elucidate the molecular and cellular mechanisms underlying EA’s anti-inflammatory and analgesic effects.

Use of C. elegans as a 3R-compliant in vivo model for the chemoprevention of cisplatin-induced neurotoxicity

Anticancer therapeutics can provoke severe side effects that impair the patient's quality of life. A frequent dose-limiting side effect of platinum-based anticancer therapy is neurotoxicity. Its pathophysiology is poorly understood, and effective preventive or therapeutic measures are missing. Therefore, elucidation of the molecular mechanism of platinating drug-induced neurotoxicity and the development of preventive strategies is urgently needed. To this end, we aim to use C. elegans as a 3R-compliant in vivo model. The 3R principles were conceived for animal welfare in science concerning animal experiments, which should be replaced, reduced or refined. We can analytically demonstrate dose-dependent uptake of cisplatin (CisPt) in C. elegans, as well as genotoxic and cytotoxic effects based on DNA adduct formation (i.e., 1,2-GpG intrastrand crosslinks), induction of apoptosis, and developmental toxicity. Measuring the impairment of pharyngeal pumping as a marker of neurotoxicity, we found that especially CisPt reduces the pumping frequency at concentrations where basal and touch-provoked movement were not yet affected. CisPt causes glutathione (GSH) depletion and RNAi-mediated knockdown of the glutamate-cysteine ligase GCS-1 aggravates the CisPt-induced inhibition of pharyngeal pumping. Moreover, N-acetylcysteine (NAC) mitigated CisPt-triggered toxicity, indicating that GSH depletion contributes to the CisPt-induced pharyngeal damage. In addition to NAC, amifostine (WR1065) also protected the pharynx of C. elegans from the toxic effects of CisPt. Measuring pharyngeal activity by the electrophysiological recording of neurotransmission in the pharynx, we confirmed that CisPt is neurotoxic in C. elegans and that NAC is neuroprotective in the nematode. The data support the hypothesis that monitoring the pharyngeal activity of C. elegans is a useful surrogate marker of CisPt-induced neurotoxicity. In addition, a low GSH pool reduces the resistance of neurons to CisPt treatment, and both NAC and WR1065 are capable of attenuating platinum-induced neurotoxicity during post-incubation in C. elegans. Overall, we propose C. elegans as a 3R-compliant in vivo model to study the molecular mechanisms of platinum-induced neurotoxicity and to explore novel neuroprotective therapeutic strategies to alleviate respective side effects of platinum-based cancer therapy.