An injectable, nanostructured implant for the delivery of adenosine triphosphate: towards long-acting formulations of small, hydrophilic drugs.

While long-acting injectable treatments are gaining increasing interest in managing chronic diseases, the available drug delivery systems almost exclusively rely on hydrophobic matrixes, limiting their application to either hydrophobic drugs or large and hydrophilic molecules such as peptides. To address the technological lock for long-acting delivery systems tailored to small, hydrophilic drugs such as anticancer and antiviral nucleoside/nucleotide analogues, we have synthesized and characterized an original approach with a multi-scale structure: (i) a nucleotide (adenosine triphosphate, ATP) is first incorporated in hydrophilic chitosan-Fe(III) nanogels; (ii) these nanogels are then transferred by freeze-drying and resuspension into a water-free, hydrophobic medium containing PLGA and an organic solvent, N-methyl-2-pyrrolidone. We show that this specific association allows an injectable and homogeneous dispersion, able to form in situ implants upon injection in physiological or aqueous environments. This system releases ATP in vitro without any burst effect in a two-step mechanism, first as nanogels acting as an intermediate reservoir over a week, then as free drug over several weeks. In vivo studies confirmed the potential of such nanostructured implants for sustained drug release following subcutaneous injection to mice hock, opening perspectives for sustained and targeted delivery through the lymphatic system.

Thermosensitive polymer prodrug nanoparticles prepared by an all-aqueous nanoprecipitation process and application to combination therapy.

Despite their great versatility and ease of functionalization, most polymer-based nanocarriers intended for use in drug delivery often face serious limitations that can prevent their clinical translation, such as uncontrolled drug release and off-target toxicity, which mainly originate from the burst release phenomenon. In addition, residual solvents from the formulation process can induce toxicity, alter the physico-chemical and biological properties and can strongly impair further pharmaceutical development. To address these issues, we report polymer prodrug nanoparticles, which are prepared without organic solvents via an all-aqueous formulation process, and provide sustained drug release. This was achieved by the "drug-initiated" synthesis of well-defined copolymer prodrugs exhibiting a lower critical solution temperature (LCST) and based on the anticancer drug gemcitabine (Gem). After screening for different structural parameters, prodrugs based on amphiphilic diblock copolymers were formulated into stable nanoparticles by all-aqueous nanoprecipitation, with rather narrow particle size distribution and average diameters in the 50-80 nm range. They exhibited sustained Gem release in human serum and acetate buffer, rapid cellular uptake and significant cytotoxicity on A549 and Mia PaCa-2 cancer cells. We also demonstrated the versatility of this approach by formulating Gem-based polymer prodrug nanoparticles loaded with doxorubicin (Dox) for combination therapy. The dual-drug nanoparticles exhibited sustained release of Gem in human serum and acidic release of Dox under accelerated pathophysiological conditions. Importantly, they also induced a synergistic effect on triple-negative breast cancer line MDA-MB-231, which is a relevant cell line to this combination.

Assessment of Squalene-Adenosine Nanoparticles in Two Rodent Models of Cardiac Ischemia-Reperfusion.

Reperfusion injuries after a period of cardiac ischemia are known to lead to pathological modifications or even death. Among the different therapeutic options proposed, adenosine, a small molecule with platelet anti-aggregate and anti-inflammatory properties, has shown encouraging results in clinical trials. However, its clinical use is severely limited because of its very short half-life in the bloodstream. To overcome this limitation, we have proposed a strategy to encapsulate adenosine in squalene-based nanoparticles (NPs), a biocompatible and biodegradable lipid. Thus, the aim of this study was to assess, whether squalene-based nanoparticles loaded with adenosine (SQAd NPs) were cardioprotective in a preclinical cardiac ischemia/reperfusion model. Obtained SQAd NPs were characterized in depth and further evaluated in vitro. The NPs were formulated with a size of about 90 nm and remained stable up to 14 days at both 4 °C and room temperature. Moreover, these NPs did not show any signs of toxicity, neither on HL-1, H9c2 cardiac cell lines, nor on human PBMC and, further retained their inhibitory platelet aggregation properties. In a mouse model with experimental cardiac ischemia-reperfusion, treatment with SQAd NPs showed a reduction of the area at risk, as well as of the infarct area, although not statistically significant. However, we noted a significant reduction of apoptotic cells on cardiac tissue from animals treated with the NPs. Further studies would be interesting to understand how and through which mechanisms these nanoparticles act on cardiac cells.

Phase I Study of Androgen Deprivation Therapy in Combination with Anti-PD-1 in Melanoma Patients Pretreated with Anti-PD-1.

PURPOSE: Androgen deprivation regenerates the thymus in adults, expanding of T-cell receptor V ß repertoire in blood and lymphoid organs and tumor-infiltrating lymphocytes in human prostate tumors. In melanoma murine models, androgen receptor promotes metastases and androgen blockade potentiates antitumor vaccine efficacy. This phase I study evaluated the safety, efficacy, and pharmocodynamics of androgen deprivation with the gonadotropin releasing hormone (GnRH) agonist triptorelin combined with nivolumab in male patients with melanoma resistant to anti-PD-1. PATIENTS AND METHODS: Adult male patients with advanced melanoma who progressed under anti-PD-1 containing regimens received triptorelin 3.75 mg every 4 weeks, nivolumab 3 mg/kg every 2 weeks, and bicalutamide 50 mg once daily during the first 28 days. Tumor response was first assessed after 3 months; adverse events (AE) were monitored throughout the study. T-cell receptor excision circles (TREC), a biomarker of thymus activity, were explored throughout the study. RESULTS: Of 14 patients, 4 were locally advanced and 10 had distant metastases. There were no grade 4 or 5 AEs. Five grade three AEs were reported in 4 patients. According to RECIST v1.1, best overall response was partial response (PR) in one patient with a pancreas metastasis, stable disease (SD) in 5 patients, and progressive disease in 8 patients. According to iRECIST, a second PR occurred after an initial pseudoprogression, TRECs increased in 2 patients, one with PR who also had an increase in TILs, and the second with SD. CONCLUSIONS: This combination was well tolerated. Disease control was obtained in 42.8% (RECIST) and 50% (iRECIST). The evidence for thymus rejuvenation was limited.

Before in vivo studies: In vitro screening of sphingomyelin nanosystems using a relevant 3D multicellular pancreatic tumor spheroid model.

Sphingomyelin nanosystems have already shown to be promising carriers for efficient delivery of anticancer drugs. For further application in the treatment of pancreatic tumor, the investigation on relevant in vitro models able to reproduce its physio-pathological complexity is mandatory. Accordingly, a 3D heterotype spheroid model of pancreatic tumor has been herein constructed to investigate the potential of bare and polyethylene glycol-modified lipid nanosystems in terms of their ability to penetrate the tumor mass and deliver drugs. Regardless of their surface properties, the lipid nanosystems successfully diffused through the spheroid without inducing toxicity, showing a clear safety profile. Loading of the bare nanosystems with a lipid prodrug of gemcitabine was used to evaluate their therapeutic potential. While the nanosystems were more effective than the free drug on 2D cell monocultures, this advantage, despite their efficient penetration capacity, was lost in the 3D tumor model. The latter, being able to mimic the tumor and its microenvironment, was capable to provide a more realistic information on the cell sensitivity to treatments. These results highlight the importance of using appropriate 3D tumor models as tools for proper in vitro evaluation of nanomedicine efficacy and their timely optimisation, so as to identify the best candidates for later in vivo evaluation.

Identification of Small Molecules Inhibiting Cardiomyocyte Necrosis and Apoptosis by Autophagy Induction and Metabolism Reprogramming.

Improvement of anticancer treatments is associated with increased survival of cancer patients at risk of cardiac disease. Therefore, there is an urgent need for new therapeutic molecules capable of preventing acute and long-term cardiotoxicity. Here, using commercial and home-made chemolibraries, we performed a robust phenotypic high-throughput screening in rat cardiomyoblast cell line H9c2, searching for small molecules capable of inhibiting cell death. A screen of 1600 compounds identified six molecules effective in preventing necrosis and apoptosis induced by H2O2 and camptothecin in H9c2 cells and in rat neonatal ventricular myocytes. In cells treated with these molecules, we systematically evaluated the expression of BCL-2 family members, autophagy progression, mitochondrial network structure, regulation of mitochondrial fusion/fission, reactive oxygen species, and ATP production. We found that these compounds affect autophagy induction to prevent cardiac cell death and can be promising cardioprotective drugs during chemotherapy.

The macrophage microtubule network acts as a key cellular controller of the intracellular fate of Leishmania infantum.

The parasitophorous vacuoles (PVs) that insulate Leishmania spp. in host macrophages are vacuolar compartments wherein promastigote forms differentiate into amastigote that are the replicative form of the parasite and are also more resistant to host responses. We revisited the biogenesis of tight-fitting PVs that insulate L. infantum in promastigote-infected macrophage-like RAW 264.7 cells by time-dependent confocal laser multidimensional imaging analysis. Pharmacological disassembly of the cellular microtubule network and silencing of the dynein gene led to an impaired interaction of L. infantum-containing phagosomes with late endosomes and lysosomes, resulting in the tight-fitting parasite-containing phagosomes never transforming into mature PVs. Analysis of the shape of the L. infantum parasite within PVs, showed that factors that impair promastigote-amastigote differentiation can also result in PVs whose maturation is arrested. These findings highlight the importance of the MT-dependent interaction of L. infantum-containing phagosomes with the host macrophage endolysosomal pathway to secure the intracellular fate of the parasite.

Key role of the macrophage microtubule network in the intracellular lifestyle of Leishmania amazonensis.

We conducted a study to decipher the mechanism of the formation of the large communal Leishmania amazonensis-containing parasitophorous vacuole (PV) and found that the macrophage microtubule (MT) network dynamically orchestrates the intracellular lifestyle of this intracellular parasite. Physical disassembly of the MT network of macrophage-like RAW 264.7 cells or silencing of the dynein gene, encoding the MT-associated molecular motor that powers MT-dependent vacuolar movement, by siRNA resulted in most of the infected cells hosting only tight parasite-containing phagosome-like vacuoles randomly distributed throughout the cytoplasm, each insulating a single parasite. Only a minority of the infected cells hosted both isolated parasite-containing phagosome-like vacuoles and a small communal PV, insulating a maximum of two to three parasites. The tight parasite-containing phagosome-like vacuoles never matured, whereas the small PVs only matured to a small degree, shown by the absence or faint acquisition of host-cell endolysosomal characteristics. As a consequence, the parasites were unable to successfully complete promastigote-to-amastigote differentiation and died, regardless of the type of insulation.

Dynamic organization of Herpesvirus glycoproteins on the viral envelope revealed by super-resolution microscopy.

The processes of cell attachment and membrane fusion of Herpes Simplex Virus 1 involve many different envelope glycoproteins. Viral proteins gC and gD bind to cellular receptors. Upon binding, gD activates the gH/gL complex which in turn activates gB to trigger membrane fusion. Thus, these proteins must be located at the point of contact between cellular and viral envelopes to interact and allow fusion. Using super-resolution microscopy, we show that gB, gH/gL and most of gC are distributed evenly round purified virions. In contrast, gD localizes essentially as clusters which are distinct from gB and gH/gL. Upon cell binding, we observe that all glycoproteins, including gD, have a similar ring-like pattern, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. We also observe that contrary to cell-free particles, gD mostly colocalizes with other glycoproteins on cell-bound particles. The differing patterns of localization of gD between cell-free and cell-bound viruses indicates that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. This redistribution of glycoproteins upon cell attachment could contribute to initiate the cascade of activations leading to membrane fusion.

Hybrid Lipid Polymer Nanoparticles for Combined Chemo- and Photodynamic Therapy.

Retinoblastoma is a malignant tumor of the retina in infants. Conventional therapies are associated to severe side effects and some of them induce secondary tumors. Photodynamic therapy (PDT) thus appears as a promising alternative as it is nonmutagenic and generates minimal side effects. The effectiveness of PDT requires the accumulation of a photosensitizer (PS) in the tumor. However, most porphyrins are hydrophobic and aggregate in aqueous medium. Their incorporation into a nanocarrier may improve their delivery to the cell cytoplasm. In this work, we designed biodegradable liponanoparticles (LNPs) consisting of a poly(d,l)-lactide (PDLLA) nanoparticle coated with a phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-3-trimethylammonium-propane) bilayer. An anticancer drug, beta-lapachone (ß-Lap) and a PS, m-THPC, were co-encapsulated for combined chemo- and PDT because it has been suggested that they may have a synergistic effect based on the activation of ß-Lap by PDT-induced over-expression of the enzyme NQO1. Using dynamic light scattering measurements, cryogenic transmission electron microscopy, and fluorescence confocal microscopy, we selected the appropriate conditions for the encapsulation of the compounds. LNPs were internalized in retinoblastoma cells within few hours. No obvious synergistic effect related to the activation of ß-Lap by PDT was observed. Conversely, the LNPs were cytotoxic at lower doses of the two encapsulated compounds as compared to the single therapies. Analysis of the combinatorial treatment showed that PDT and chemotherapy had an additive effect on the viability of retinoblastoma cells.

Light sheet fluorescence microscopy versus confocal microscopy: in quest of a suitable tool to assess drug and nanomedicine penetration into multicellular tumor spheroids.

We recently constructed a multicellular spheroid model of pancreatic tumor based on a triple co-culture of cancer cells, fibroblasts and endothelial cells and characterized by the presence of fibronectin, an important component of the tumor extracellular matrix. By combining cancer cells and stromal components, this model recreates in vitro the three-dimensional (3D) architecture of solid tumors. In this study, we used these hetero-type spheroids as a tool to assess the penetration of doxorubicin (used as a model drug) through the whole tumor mass either in a free form or loaded into polymer nanoparticles (NPs), and we investigated whether microscopy images, acquired by Confocal Laser Scanning Microscopy (CLSM) and Light Sheet Fluorescence Microscopy (LSFM), would be best to provide reliable information on this process. Results clearly demonstrated that CLSM was not suitable to accurately monitor the diffusion of small molecules such as the doxorubicin. Indeed, it only allowed to scan a layer of 100 µm depth and no information on deeper layers could be available because of a progressive loss of the fluorescence signal. On the contrary, a complete 3D tomography of the hetero-type multicellular tumor spheroids (MCTS) was obtained by LSFM and multi-view image fusion which revealed that the fluorescent molecule was able to reach the core of spheroids as large as 1 mm in diameter. However, no doxorubicin-loaded polymer nanoparticles were detected in the spheroids, highlighting the challenge of nanomedicine delivery through biological barriers. Overall, the combination of hetero-type MCTS and LSFM allowed to carry out a highly informative microscopic assessment and represents a suitable approach to precisely follow up the drug penetration in tumors. Accordingly, it could provide useful support in the preclinical investigation and optimization of nanoscale systems for drug delivery to solid tumors.

PDE2 regulates membrane potential, respiration and permeability transition of rodent subsarcolemmal cardiac mitochondria.

Cyclic adenosine monophosphate (cAMP) production regulates certain aspects of mitochondria function in rodent cardiomyocytes, such as ATP production, oxygen consumption, calcium import and mitochondrial permeability transition (MPT), but how this cAMP pool is controlled is not well known. Here, expression, localization and activity of several cAMP-degrading enzymes, i.e. phosphodiesterases (PDEs), were investigated in isolated rodent cardiac mitochondria. In contrast to the heart ventricle where PDE4 is the major PDE, in cardiac mitochondria, cGMP-stimulated PDE2 activity was largest than PDE3 and PDE4 activities. PDE2 expression was mainly detected in subsarcolemmal mitochondria in association with the inner membrane rather than in interfibrillar mitochondria. PDE2, 3 and 4 activities were further confirmed in neonatal rat cardiomyocytes by real time FRET analysis. In addition, the pharmacological inhibition or the cardiac-specific overexpression of PDE2 modulated mitochondrial membrane potential loss, MPT and calcium import. In mitochondria isolated from PDE2 transgenic mice with a cardiac selective PDE2 overexpression, the oxidative phosphorylation (OXPHOS) was significantly lower than in wild-type mice, but stimulated by cGMP. Thus, cAMP degradation by PDEs represents a new regulatory mechanism of mitochondrial function.

The chaperone-like protein CDC48 regulates ascorbate peroxidase in tobacco.

There is increasing evidence that the chaperone-like protein CDC48 (cell division cycle 48) plays a role in plant immunity. Cytosolic ascorbate peroxidase (cAPX), which is a major regulator of the redox status of plant cells, has previously been shown to interact with CDC48. In this study, we examined the regulation of cAPX by the ATPase NtCDC48 during the cryptogein-induced immune response in tobacco cells. Our results not only confirmed the interaction between the proteins but also showed that it occurs in the cytosol. cAPX accumulation was modified in cells overexpressing NtCDC48, a process that was shown to involve post-translational modification of cAPX. In addition, cryptogein-induced increases in cAPX activity were suppressed in cells overexpressing NtCDC48 and the abundance of the cAPX dimer was below the level of detection. Furthermore, the levels of both reduced (GSH) and oxidized glutathione (GSSG) and the GSH/GSSG ratio decreased more rapidly in response to the elicitor in these cells than in controls. A decrease in cAPX activity was also observed in response to heat shock in the cells overexpressing NtCDC48, indicating that the regulation of cAPX by NtCDC48 is not specific to the immune response.

Biodegradable Pickering emulsions of Lipiodol for liver trans-arterial chemo-embolization.

Water-in-oil (W/O) Lipiodol emulsions remain the preferable choice for local delivery of chemotherapy in the treatment of hepatocellular carcinoma. However, their low stability severely hampers their efficiency. Here, remarkably stable W/O Lipiodol emulsion stabilized by biodegradable particles was developed thanks to Pickering technology. The addition of poly(lactide-co-glycolide) nanoparticles (NPs) into the aqueous-phase of the formulation led to W/O Pickering emulsion by a simple emulsification process through two connected syringes. Influence of nanoparticles concentration and water/oil ratio on emulsion stability and droplet size were studied. All formulated Pickering emulsions were W/O type, stable for at least one month and water droplets size could be tuned by controlling nanoparticle concentration from 24 µm at 25 mg/mL to 69 µm at 5 mg/mL. The potential of these emulsions to efficiently encapsulate chemotherapy was studied through the internalization of doxorubicin (DOX) into the aqueous phase with a water/oil ratio of 1/3 as recommended by the medical community. Loaded-doxorubicin was released from conventional emulsion within a few hours whereas doxorubicin from stable Pickering emulsion took up to 10 days to be completely released. In addition, in vitro cell viability evaluations performed on the components of the emulsion and the Pickering emulsion have shown no significant toxicity up to relatively high concentrations of NPs (3 mg/mL) on two different cell lines: HUVEC and HepG2. STATEMENT OF SIGNIFICANCE: We present an original experimental research in the field of nanotechnology for biomedical applications. In particular, we have formulated, thanks to Pickering technology, a new therapeutic emulsion stabilized with biodegradable PLGA nanoparticles. As far as we know, this is the first therapeutic Pickering emulsion reported in the literature for hepatocellular carcinoma. Such a new emulsion allows to easily prepare a predictable and stable lipiodolized emulsion having all the required characteristics for optimum tumor uptake. As demonstrated throughout our manuscript, emulsions stabilized with these nanoparticles have the advantage of being biodegradable, biocompatible and less toxic compared to usual emulsions stabilized with synthetic surfactants. These findings demonstrate the plausibility of the use of Pickering emulsions for chemoembolization as a therapeutic agent in extended release formulations.

CSRP3 mediates polyphenols-induced cardioprotection in hypertension.

Berries contain bioactive polyphenols, whose capacity to prevent cardiovascular diseases has been established recently in animal models as well in human clinical trials. However, cellular processes and molecular targets of berries polyphenols remain to be identified. The capacity of a polyphenol-enriched diet (i.e., blueberries, blackberries, raspberries, strawberry tree fruits and Portuguese crowberries berries mixture) to promote animal survival and protect cardiovascular function from salt-induced hypertension was evaluated in a chronic salt-sensitive Dahl rat model. The daily consumption of berries improved survival of Dahl/salt-sensitive rats submitted to high-salt diet and normalized their body weight, renal function and blood pressure. In addition, a prophylactic effect was observed at the level of cardiac hypertrophy and dysfunction, tissue cohesion and cardiomyocyte hypertrophy. Berries also protected the aorta from fibrosis and modulated the expression of aquaporin-1, a channel involved in endothelial water and nitric oxide permeability. Left ventricle proteomics analysis led to the identification of berries and salt metabolites targets, including cystein and glycin-rich protein 3 (CSRP3), a protein involved in myocyte cytoarchitecture. In neonatal rat ventricular cardiomyocytes, CSRP3 was validated as a target of a berries-derived polyphenol metabolite, 4-methylcatechol sulfate, at micromolar concentrations, mimicking physiological conditions of human plasma circulation. Accordingly, siRNA silencing of CSRP3 and 4-methylcatechol sulfate pretreatment reversed cardiomyocyte hypertrophy and CSRP3 overexpression induced by phenylephrine. Our systemic study clearly supports the modulation of CSRP3 by a polyphenol-rich berries diet as an efficient cardioprotective strategy in hypertension-induced heart failure.

Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro.

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of supramolecular structures with multifunctional properties, required for efficient cancer therapy by photodynamic therapy (PDT). The synthesis of two new lipid-porphyrin conjugates coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to either chemically modified lyso-phosphatidylcholine (PhLPC) or egg lyso-sphingomyelin (PhLSM) is reported. The impact of the lipid backbone of these conjugates on their self-assembling properties, as well as on their physicochemical properties, including interfacial behavior at the air/buffer interface, fluorescence and absorption properties, thermotropic behavior, and incorporation rate in the membrane of liposomes were studied. Finally, their photodynamic activity was evaluated on esophageal squamous cell carcinoma (ESCC) and normal esophageal squamous epithelium cell lines. The liposome-like vesicles resulting from self-assembly of the pure conjugates were unstable and turned into aggregates with undefined structure within few days. However, both lipid-porphyrin conjugates could be efficiently incorporated in lipid vesicles, with higher loading rates than unconjugated Pheo-a. Interestingly, phototoxicity tests of free and liposome-incorporated lipid-porphyrin conjugates demonstrated a better selectivity in vitro to esophageal squamous cell carcinoma relative to normal cells.

Multicellular spheroid based on a triple co-culture: A novel 3D model to mimic pancreatic tumor complexity.

The preclinical drug screening of pancreatic cancer treatments suffers from the absence of appropriate models capable to reproduce in vitro the heterogeneous tumor microenvironment and its stiff desmoplasia. Driven by this pressing need, we describe in this paper the conception and the characterization of a novel 3D tumor model consisting of a triple co-culture of pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC), which assembled to form a hetero-type multicellular tumor spheroid (MCTS). By histological analyses and Selective Plain Illumination Microscopy (SPIM) we have monitored the spatial distribution of each cell type and the evolution of the spheroid composition. Results revealed the presence of a core rich in fibroblasts and fibronectin in which endothelial cells were homogeneously distributed. The integration of the three cell types enabled to reproduce in vitro with fidelity the influence of the surrounding environment on the sensitivity of cancer cells to chemotherapy. To our knowledge, this is the first time that a scaffold-free pancreatic cancer spheroid model combining both tumor and multiple stromal components has been designed. It holds the possibility to become an advantageous tool for a pertinent assessment of the efficacy of various therapeutic strategies. STATEMENT OF SIGNIFICANCE: Pancreatic tumor microenvironment is characterized by abundant fibrosis and aberrant vasculature. Aiming to reproduce in vitro these features, cancer cells have been already co-cultured with fibroblasts or endothelial cells separately but the integration of both these essential components of the pancreatic tumor microenvironment in a unique system, although urgently needed, was still missing. In this study, we successfully integrated cellular and acellular microenvironment components (i.e., fibroblasts, endothelial cells, fibronectin) in a hetero-type scaffold-free multicellular tumor spheroid. This new 3D triple co-culture model closely mimicked the resistance to treatments observed in vivo, resulting in a reduction of cancer cell sensitivity to the anticancer treatment.

Fluorescent polymer prodrug nanoparticles with aggregation-induced emission (AIE) properties from nitroxide-mediated polymerization.

Aggregation-induced emission (AIE)-active polymer prodrug nanoparticles were readily prepared by growing short, well-defined polymer chains from an AIE dye by nitroxide-mediated polymerization, followed by co-nanoprecipitation of the resulting conjugates with similarly constructed anticancer polymer prodrugs. The nanoparticles had sharp fluorescence signal offering excellent imaging ability in living cells and their intra cellular localization to be accurately monitored.

Anticancer copper(II) phosphorus dendrimers are potent proapoptotic Bax activators.

A multivalent phosphorus dendrimer 1G3 and its corresponding Cu-complex, 1G3-Cu have been recently identified as agents retaining high antiproliferative potency. This antiproliferative capacity was preserved in cell lines overexpressing the efflux pump ABC B1, whereas cross-resistance was observed in ovarian cancer cell lines resistant to cisplatin. Theoretical 3D models were constructed: the dendrimers appear as irregularly shaped disk-like nano-objects of about 22 Å thickness and 49 Å diameter, which accumulated in cells after penetration by endocytosis. To get insight in their mode of action, cell death pathways have been examined in human cancer cell lines: early apoptosis was followed by secondary necrosis after multivalent phosphorus dendrimers exposure. The multivalent plain phosphorus dendrimer 1G3 moderately activated caspase-3 activity, in contrast with the multivalent Cu-conjugated phosphorus dendrimer 1G3-Cu which strikingly reduced the caspase-3 content and activity. This decrease of caspase activity is not related to the presence of copper, since inorganic copper has no or little effect on caspase-3. Conversely the potent apoptosis activation could be related to a noticeable translocation of Bax to the mitochondria, resulting in the release of AIF into the cytosol, its translocation to the nucleus and a severe DNA fragmentation, without alteration of the cell cycle. The multivalent Cu-conjugated phosphorus dendrimer is more efficient than its non-complexed analog to activate this pathway in close relationship with the higher antiproliferative potency. Therefore, this multivalent Cu-conjugated phosphorus dendrimer 1G3-Cu can be considered as a new and promising first-in-class antiproliferative agent with a distinctive mode of action, inducing apoptosis tumor cell death through Bax activation pathway.

In Vitro Modulation of TrkB Receptor Signaling upon Sequential Delivery of Curcumin-DHA Loaded Carriers Towards Promoting Neuronal Survival.

PURPOSE: To in vitro investigate the capacity of carrier-free and lipid-nanoparticle (NP)-encapsulated phytochemical compounds to prevent neuronal damage through neurotrophin potentiating activities. Delivery of molecules promoting the neurotrophin receptor signaling in the central nervous system (CNS) present ongoing interest for combination therapy development. METHODS: Super-resolution Stimulated Emission Depletion (STED) microscopy imaging and flow cytometry analysis were employed to study the expression of the neurotrophin TrkB receptor in a neuronal cell model, which is highly responsive to binding of brain-derived neurotrophic factor (BDNF). Dual drug-loaded nanoparticle formulations, prepared by self-assembly of lyotropic lipids and PEGylated amphiphile derivatives, were delivered to differentiated human neuroblastoma SH-SY5Y cells subjected to degenerative conditions. RESULTS: The expression of BDNF in the intra and extracellular domains was quantified by ELISA and flow cytometry after sequential treatment of the degenerating SH-SY5Y cells by neurotherapeutic formulations. Flow cytometry was also used to assess the phosphorylation of the transcription factor cAMP response element-binding protein (CREB) in the intracellular domain as a result of the treatment by nanoformulations. CONCLUSION: Over time, dual drug formulations (curcumin and docosahexaenoic acid (DHA)) promoted the neuronal survival and repair processes through enhanced BDNF secretion and increased phosphorylation of CREB as compared to untreated degenerating cells.