Priming with oncolytic adenovirus followed by anti-PD-1 and paclitaxel treatment leads to improved anti-cancer efficacy in the 3D TNBC model.

Triple-negative breast cancer (TNBC) is considered one of the most incurable malignancies due to its clinical characteristics, including high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Therefore, it remains a critical unmet medical need. On the other hand, poor delivery efficiency continues to reduce the efficacy of anti-cancer therapeutics developed against solid tumours using various strategies, such as genetically engineered oncolytic vectors used as nanocarriers. The study was designed to evaluate the anti-tumour efficacy of a novel combinatorial therapy based on oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with an anti-PD-1 (pembrolizumab) and paclitaxel (PTX). Here, we first tested the antineoplastic effect in two-dimensional (2D) and three-dimensional (3D) breast cancer models in MDA-MB-231, MDA-MB-468 and MCF-7 cells. Then, to further evaluate the efficacy of combinatorial therapy, including immunological aspects, we established a three-dimensional (3D) co-culture model based on MDA-MB-231 cells with peripheral blood mononuclear cells (PBMCs) to create an integrated system that more closely mimics the complexity of the tumour microenvironment and interacts with the immune system. Treatment with OV as a priming agent, followed by pembrolizumab and then paclitaxel, was the most effective in reducing the tumour volume in TNBC co-cultured spheroids. Further, T-cell phenotyping analyses revealed significantly increased infiltration of CD8+, CD4+ T and Tregs cells. Moreover, the observed anti-tumour effects positively correlated with the level of CD4+ T cell infiltrates, suggesting the development of anti-cancer immunity. Our study demonstrated that combining different immunotherapeutic agents (virus, pembrolizumab) with PTX reduced the tumour volume of the TNBC co-cultured spheroids compared to relevant controls. Importantly, sequential administration of the investigational agents (priming with the vector) further enhanced the anti-cancer efficacy in 3D culture over other groups tested. Taken together, these results support further evaluation of the virus in combination with anti-PD-1 and PTX for the treatment of triple-negative breast cancer patients. Importantly, further studies with in vivo models should be conducted to better understand the translational aspects of tested therapy.

Novel combinatorial therapy of oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with anti PD-1 exhibits enhanced anti-cancer efficacy through promotion of intratumoral T-cell infiltration and modulation of tumour microenvironment in mesothelioma mouse model.

Introduction: Malignant mesothelioma is a rare and aggressive form of cancer. Despite improvements in cancer treatment, there are still no curative treatment modalities for advanced stage of the malignancy. The aim of this study was to evaluate the anti-tumor efficacy of a novel combinatorial therapy combining AdV5/3-D24-ICOSL-CD40L, an oncolytic vector, with an anti-PD-1 monoclonal antibody. Methods: The efficacy of the vector was confirmed in vitro in three mesothelioma cell lines - H226, Mero-82, and MSTO-211H, and subsequently the antineoplastic properties in combination with anti-PD-1 was evaluated in xenograft H226 mesothelioma BALB/c and humanized NSG mouse models. Results and discussion: Anticancer efficacy was attributed to reduced tumour volume and increased infiltration of tumour infiltrating lymphocytes, including activated cytotoxic T-cells (GrB+CD8+). Additionally, a correlation between tumour volume and activated CD8+ tumour infiltrating lymphocytes was observed. These findings were confirmed by transcriptomic analysis carried out on resected human tumour tissue, which also revealed upregulation of CD83 and CRTAM, as well as several chemokines (CXCL3, CXCL9, CXCL11) in the tumour microenvironment. Furthermore, according to observations, the combinatorial therapy had the strongest effect on reducing mesothelin and MUC16 levels. Gene set enrichment analysis suggested that the combinatorial therapy induced changes to the expression of genes belonging to the "adaptive immune response" gene ontology category. Combinatorial therapy with oncolytic adenovirus with checkpoint inhibitors may improve anticancer efficacy and survival by targeted cancer cell destruction and triggering of immunogenic cell death. Obtained results support further assessment of the AdV5/3-D24-ICOSL-CD40L in combination with checkpoint inhibitors as a novel therapeutic perspective for mesothelioma treatment.

Feeding Next-Generation Nanomedicines to Europe: Regulatory and Quality Challenges.

New and innovative nanomedicines have been developed and marketed over the past half-century, revolutionizing the prognosis of many human diseases. Although a univocal regulatory definition is not yet available worldwide, the term "nanomedicines" generally identifies medicinal products that use nanotechnology in their design or production. Due to the intrinsic high structural complexity of these products, the scientific and regulatory communities are reflecting on how to revise the regulatory framework to provide a more appropriate benefit/risk balance to authorize them on the market, considering the impact of their peculiar physicochemical features in the evaluation of efficacy and safety patterns. Herein, a critical perspective is provided on the current open issues regarding regulatory qualification and physicochemical characterization of nanosystems considering the current European regulatory framework on nanomedicine products. Practicable paths for improving their quality assurance and predicting their fate in vivo are also argued. Strengthening the multilevel alliance among academic institutions, industrial stakeholders, and regulatory authorities seems strategic to support innovation by standard approaches (e.g., qualification, characterization, risk assessment), and to expand current knowledge, also benefiting from the new opportunities offered by artificial intelligence and digitization in predictive modelling of the impact of nanomedicine characteristics on their fate in vivo.

Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery.

Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.

Curcumin Administration Improves Force of mdx Dystrophic Diaphragm by Acting on Fiber-Type Composition, Myosin Nitrotyrosination and SERCA1 Protein Levels.

The vegetal polyphenol curcumin displays beneficial effects against skeletal muscle derangement induced by oxidative stress, disuse or aging. Since oxidative stress and inflammation are involved in the progression of muscle dystrophy, the effects of curcumin administration were investigated in the diaphragm of mdx mice injected intraperitoneally or subcutaneously with curcumin for 4-12-24 weeks. Curcumin treatment independently of the way and duration of administration (i) ameliorated myofiber maturation index without affecting myofiber necrosis, inflammation and degree of fibrosis; (ii) counteracted the decrease in type 2X and 2B fiber percentage; (iii) increased about 30% both twitch and tetanic tensions of diaphragm strips; (iv) reduced myosin nitrotyrosination and tropomyosin oxidation; (v) acted on two opposite nNOS regulators by decreasing active AMP-Kinase and increasing SERCA1 protein levels, the latter effect being detectable also in myotube cultures from mdx satellite cells. Interestingly, increased contractility, decreased myosin nitrotyrosination and SERCA1 upregulation were also detectable in the mdx diaphragm after a 4-week administration of the NOS inhibitor 7-Nitroindazole, and were not improved further by a combined treatment. In conclusion, curcumin has beneficial effects on the dystrophic muscle, mechanistically acting for the containment of a deregulated nNOS activity.

Influence of Folate-Targeted Gold Nanoparticles on Subcellular Localization and Distribution into Lysosomes.

The cell interaction, mechanism of cell entry and intracellular fate of surface decorated nanoparticles are known to be affected by the surface density of targeting agents. However, the correlation between nanoparticles multivalency and kinetics of the cell uptake process and disposition of intracellular compartments is complicated and dependent on a number of physicochemical and biological parameters, including the ligand, nanoparticle composition and colloidal properties, features of targeted cells, etc. Here, we have carried out an in-depth investigation on the impact of increasing folic acid density on the kinetic uptake process and endocytic route of folate (FA)-targeted fluorescently labelled gold nanoparticles (AuNPs). A set of AuNPs (15 nm mean size) produced by the Turkevich method was decorated with 0-100 FA-PEG3.5kDa-SH molecules/particle, and the surface was saturated with about 500 rhodamine-PEG2kDa-SH fluorescent probes. In vitro studies carried out using folate receptor overexpressing KB cells (KBFR-high) showed that the cell internalization progressively increased with the ligand surface density, reaching a plateau at 50:1 FA-PEG3.5kDa-SH/particle ratio. Pulse-chase experiments showed that higher FA density (50 FA-PEG3.5kDa-SH molecules/particle) induces more efficient particle internalization and trafficking to lysosomes, reaching the maximum concentration in lysosomes at 2 h, than the lower FA density of 10 FA-PEG3.5kDa-SH molecules/particle. Pharmacological inhibition of endocytic pathways and TEM analysis showed that particles with high folate density are internalized predominantly by a clathrin-independent process.

Tailoring surface properties of liposomes for dexamethasone intraocular administration.

Diseases of the posterior eye segment are often characterized by intraocular inflammation, which causes, in the long term, severe impairment of eye functions and, ultimately, vision loss. Aimed at enhancing the delivery of anti-inflammatory drugs to the posterior eye segment upon intravitreal administration, we developed liposomes with an engineered surface to control their diffusivity in the vitreous and retina association. Hydrogenated soybean phosphatidylcholine (HSPC)/cholesterol liposomes were coated with (agmatinyl)6-maltotriosyl-acetamido-N-(octadec-9-en-1-yl)hexanamide (Agm6-M-Oleate), a synthetic non-peptidic cell penetration enhancer (CPE), and/or 5% of mPEG2kDa-DSPE. The zeta potential of liposomes increased, and the mobility in bovine vitreous and colloidal stability decreased with the Agm6-M-Oleate coating concentration. Oppositely, mPEG2kDa-DSPE decreased the zeta potential of liposomes and restored both the diffusivity and the stability in vitreous. Liposomes with 5 mol% Agm6-M-Oleate coating were well tolerated by ARPE-19 retina cells either with or without mPEG2kDa-DSPE, while 10 mol% Agm6-M-Oleate showed cytotoxicity. Agm6-M-Oleate promoted the association of liposomes to ARPE-19 cells with respect to plain liposomes, while mPEG2kDa-DSPE slightly reduced the cell interaction. Dexamethasone hemisuccinate (DH) was remotely loaded into liposomes with a loading capacity of ∼10 wt/wt%. Interestingly, mPEG2kDa-DSPE coating reduced the rate of DH release and enhanced the disposition of Agm6-M-Oleate coated liposomes in the ARPE-19 cell cytosol resulting in a more efficient anti-inflammatory effect. Finally, mPEG2kDa-DSPE enhanced the association of DH-loaded Agm6-M-Oleate coated liposomes to explanted rat retina, which reflected in higher viability of inner and outer nuclear layer cells.

Mannosylated Polycations Target CD206+ Antigen-Presenting Cells and Mediate T-Cell-Specific Activation in Cancer Vaccination.

Immunotherapy is deemed one of the most powerful therapeutic approaches to treat cancer. However, limited response and tumor specificity are still major challenges to address. Herein, mannosylated polycations targeting mannose receptor- are developed as vectors for plasmid DNA (pDNA)-based vaccines to improve selective delivery of genetic material to antigen-presenting cells and enhance immune cell activation. Three diblock glycopolycations (M15A12, M29A25, and M58A45) and two triblock copolymers (M29A29B9 and M62A52B32) are generated by using mannose (M), agmatine (A), and butyl (B) derivatives to target CD206, complex nucleic acids, and favor the endosomal escape, respectively. All glycopolycations efficiently complex pDNA at N/P ratios <5, protecting the pDNA from degradation in a physiological milieu. M58A45 and M62A52B32 complexed with plasmid encoding for antigenic ovalbumin (pOVA) trigger the immune activation of cultured dendritic cells, which present the SIINFEKL antigenic peptide via specific major histocompatibility complex-I. Importantly, administration of M58A45/pOVA elicits SIINFEKL-specific T-cell response in C56BL/6 mice bearing the melanoma tumor model B16-OVA, well in line with a reduction in tumor growth. These results qualify mannosylation as an efficient strategy to target immune cells in cancer vaccination and emphasize the potential of these glycopolycations as effective delivery vehicles for nucleic acids.

From Immunosuppression to Immunomodulation - Turning Cold Tumours into Hot.

Cancer cells employ various mechanisms to evade and suppress anti-cancer immune responses generating a "cold" immunosuppressive tumour microenvironment. Oncolytic viruses are a promising tool to convert tumour immunosuppression to immunomodulation and improve the efficacy of cancer treatment. Emerging preclinical and clinical findings confirm that oncolytic viruses act in a multimodal scheme, triggering lyses, immunogenic cell death and finally inducing anti-cancer immune responses. In this paper, we tested the local administration of a novel oncolytic adenovirus AdV-D24-ICOSL-CD40L expressing co-stimulatory molecules ICOSL and CD40L to induce the production of tumour infiltrating lymphocytes to the site of injection. Subsequently, in immunocompetent mouse models, we studied possible correlation between tumour infiltrates and anti-cancer efficacy. Described results showed that the delivery of oncolytic viruses encoding immunomodulatory transgenes in combination with anti-PD1 resulted in synergistic inhibition of both melanoma and mesothelioma tumours. Importantly anti-cancer effect positively correlated with cytotoxic CD8+ tumour-infiltrating lymphocytes exerting a central role in the tumour volume control thus generating beneficial outcomes that will undoubtedly provide new insights into possible future treatment strategies to combat cancer. Altogether our findings highlight the importance of oncolytic vectors able to modulate anti-cancer immune responses that can correlate with efficacy in solid malignancies.

Novel Insights Into Mesothelioma Therapy: Emerging Avenues and Future Prospects.

Malignant mesothelioma is a rare and aggressive cancer that develops in the thin layer surrounding the mesothelium and is mainly caused by asbestos exposure. Despite improvements in patient prognosis with conventional cancer treatments, such as surgery, chemotherapy, and radiotherapy, there are still no curative treatment modalities for advanced disease. In recent years, new therapeutic avenues have been explored. Improved understanding of the mechanisms underlying the dynamic tumor interaction with the immune system has led to the development of immunotherapeutic approaches. Numerous recent clinical trials have shown a desire to develop more effective treatments that can be used to fight against the disease. Immune checkpoint inhibitors, oncolytic adenoviruses, and their combination represent a promising strategy that can be used to synergistically overcome immunosuppression in the mesothelioma tumor microenvironment. This review provides a synthesized overview of the current state of knowledge on new therapeutic options for mesothelioma with a focus on the results of clinical trials conducted in the field.

Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells.

Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of "omics" techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.

Optimization of Biomimetic, Leukocyte-Mimicking Nanovesicles for Drug Delivery Against Colorectal Cancer Using a Design of Experiment Approach.

The development of biomimetic nanoparticles (NPs) has revolutionized the concept of nanomedicine by offering a completely new set of biocompatible materials to formulate innovative drug delivery systems capable of imitating the behavior of cells. Specifically, the use of leukocyte-derived membrane proteins to functionalize nanovesicles (leukosomes) can enable their long circulation and target the inflamed endothelium present in many inflammatory pathologies and tumors, making them a promising and versatile drug delivery system. However, these studies did not elucidate the critical experimental parameters involved in leukosomes formulation. In the present study, we approached the preparation of leukosomes using a design of experiment (DoE) method to better understand the influence of experimental parameters on leukosomes features such as size, size distribution, and protein loading. We also validated this formulation technologically and tested its behavior in in vitro colorectal cancer (CRC) models, including CRC patient-derived tumor organoids (PDOs). We demonstrated leukosomes biocompatibility, endothelium adhesion capability, and tumor target in three-dimensional (3D) settings using CRC cell lines. Overall, our study offers a novel conceptual framework for biomimetic NPs using a DoE strategy and consolidates the high therapeutic potential of leukosomes as a viable drug delivery system for anti-inflammatory and antineoplastic applications.

Latest Advances in Biomimetic Cell Membrane-Coated and Membrane-Derived Nanovectors for Biomedical Applications.

In the last decades, many nanovectors were developed for different diagnostic or therapeutic purposes. However, most nanosystems have been designed using a "bottom-up" approach, in which the basic components of the nanovector become assembled to achieve complex and specific behaviors. Despite the fine control of formulative conditions, the complexity of these systems often results cumbersome and difficult to scale-up. Recently, biomimetic materials emerged as a complementary or alternative design approach through a "top-down strategy", using cell-derived materials as building blocks to formulate innovative nanovectors. The use of cell membranes as nanoparticle coatings endows nanomaterials with the biological identity and some of the functions of the cells they are derived from. In this review, we discuss some of the latest examples of membrane coated and membrane-derived biomimetic nanomaterials and underline the common general functions offered by the biomaterials used. From these examples, we suggest a systematic classification of these biomimetic materials based on their biological sources and formulation techniques, with their respective advantages and disadvantages, and summarize the current technologies used for membranes isolation and integration on nanovectors. We also discuss some current technical limitations and hint to future direction of the improvement for biomimetics.

Tobramycin-loaded complexes to prevent and disrupt Pseudomonas aeruginosa biofilms.

Carbohydrate-based materials are increasingly investigated for a range of applications spanning from healthcare to advanced functional materials. Synthetic glycopolymers are particularly attractive as they possess low toxicity and immunogenicity and can be used as multivalent ligands to target sugar-binding proteins (lectins). Here, we utilised RAFT polymerisation to synthesize two families of novel diblock copolymers consisting of a glycopolymers block containing either mannopyranose or galactopyranose pendant units, which was elongated with sodium 2-acrylamido-2-methyl-1-propanesulfonate (AMPS) to generate a polyanionic block. The latter enabled complexation of cationic aminoglycoside antibiotic tobramycin through electrostatic interactions (loading efficiency in the 0.5-6.3 wt% range, depending on the copolymer). The resulting drug vectors were characterized by dynamic light scattering, zeta-potential, and transmission electron microscopy. Tobramycin-loaded complexes were tested for their ability to prevent clustering or disrupt biofilm of the Pseudomonas aeruginosa Gram-negative bacterium responsible for a large proportion of nosocomial infection, especially in immunocompromised patients. P. aeruginosa possesses two specific tetrameric carbohydrate-binding adhesins, LecA (PA-IL, galactose/N-acetyl-D-galactosamine-binding) and LecB (PA-IIL, fucose/mannose-binding), and the cell-associated and extracellular adhesin CdrA (Psl/mannose-binding) thus ideally suited for targeted drug delivery using sugar-decorated tobramycin-loaded complexes here developed. Both aliphatic and aromatic linkers were utilised to link the sugar pendant units to the polyacrylamide polymer backbone to assess the effect of the nature of such linkers on bactericidal/bacteriostatic properties of the complexes. Results showed that tobramycin-loaded complexes efficiently suppressed (40 to 60% of inhibition) in vitro biofilm formation in PAO1-L P. aeruginosa and that preferential targeting of PAO1-L biofilm can be achieved using mannosylated glycopolymer-b-AMPSm.

Tyrosine kinase inhibitor prodrug-loaded liposomes for controlled release at tumor microenvironment.

Tyrosine kinase inhibitors (TKIs) represent one of the most advanced class of therapeutics for cancer treatment. Most of them are also cytochrome P450 (CYP) inhibitors and/or substrates thereof. Accordingly, their efficacy and/or toxicity can be affected by CYP-mediated metabolism and by metabolism-derived drug-drug interactions. In order to enhance the therapeutic performance of these drugs, we developed a prodrug (Pro962) of our TKI TK962 specifically designed for liposome loading and pH-controlled release in the tumor. A cholesterol moiety was linked to TK962 through pH-sensitive hydrazone bond for anchoring to the liposome phospholipid bilayer to prevent leakage of the prodrug from the nanocarrier. Bioactivity studies performed on isolated target kinases showed that the prodrug maintains only partial activity against them and the release of TK962 is required. Biopharmaceutical studies carried out with prodrug loaded liposomes showed that the prodrug was firmly associated with the vesicles and the drug release was prevented under blood-mimicking conditions. Conversely, conventional liposome loaded with TK962 readily released the drug. Flow cytometric studies showed that liposomes efficiently provided for intracellular prodrug delivery. The use of the hydrazone linker yielded a pH-controlled drug release, which resulted in about 50% drug release at pH 4 and 5 in 2 h. Prodrug, prodrug loaded liposomes and active lead compound have been tested against cancer cell lines in either 2D or 3D models. The liposome formulation showed higher cytotoxicity than the unformulated lead TK962 in both 2D and 3D models. The stability of prodrug, prodrug loaded liposomes and active lead compound in human serum and against human, mouse, and rat microsomes was also assessed, demonstrating that liposome formulations impair the metabolic reactions and protect the loaded compounds from catabolism. The results suggest that the liposomal formulation of pH releasable TKI prodrugs is a promising strategy to improve the metabolic stability, intracellular cancer cell delivery and release, and in turn the efficacy of this class of anticancer drugs.

Chronic Systemic Curcumin Administration Antagonizes Murine Sarcopenia and Presarcopenia.

Curcumin administration attenuates muscle disuse atrophy, but its effectiveness against aging-induced, selective loss of mass or force (presarcopenia or asthenia/dynopenia), or combined loss (sarcopenia), remains controversial. A new systemic curcumin treatment was developed and tested in 18-month-old C57BL6J and C57BL10ScSn male mice. The effects on survival, liver toxicity, loss of muscle mass and force, and satellite cell responsivity and commitment were evaluated after 6-month treatment. Although only 24-month-old C57BL10ScSn mice displayed age-related muscle impairment, curcumin significantly increased survival of both strains (+20-35%), without signs of liver toxicity. Treatment prevented sarcopenia in soleus and presarcopenia in EDL of C57BL10ScSn mice, whereas it did not affect healthy-aged muscles of C57BL6J. Curcumin-treated old C57BL10ScSn soleus preserved type-1 myofiber size and increased type-2A one, whereas EDL maintained adult values of total myofiber number and fiber-type composition. Mechanistically, curcumin only partially prevented the age-related changes in protein level and subcellular distribution of major costamere components and regulators. Conversely, it affected satellite cells, by maintaining adult levels of myofiber maturation in old regenerating soleus and increasing percentage of isolated, MyoD-positive satellite cells from old hindlimb muscles. Therefore, curcumin treatment successfully prevents presarcopenia and sarcopenia development by improving satellite cell commitment and recruitment.

Retinal neuroprotection by controlled release of a VCP inhibitor from self-assembled nanoparticles.

Mutations in rhodopsin lead to its misfolding resulting in autosomal dominant retinitis pigmentosa (adRP). Pharmacological inhibition of the ATP-driven chaperone valosin-containing protein (VCP), a molecular checkpoint for protein quality control, slows down retinal degeneration in animal models. However, poor water-solubility of VCP inhibitors poses a challenge to their clinical translation as intravitreal injections for retinal treatment. In order to enable the delivery of VCP inhibitors, we have developed and investigated two formulations for the VCP inhibitor ML240. Nanoformulations of ML240 were obtained by using amphiphilic polymers methoxy-poly (ethylene glycol)5kDa-cholane (mPEG5kDa-cholane) and methoxy-poly (ethylene glycol)5kDa-cholesterol (mPEG5kDa-cholesterol). Both formulations increased the water-solubility of ML240 by two orders of magnitude and prolonged the drug released over ten days. In addition, encapsulation of ML240 in mPEG5kDa-cholane showed superior photoreceptor protection at lower drug concentrations, normalized rhodopsin localization, and alleviated inflammatory microglial responses in an ex vivo rat model of retinal degeneration. The study demonstrates the potential of VCP inhibitor nanoformulations to treat adRP, a pharmacologically orphan disease.

Polymer Coated Oncolytic Adenovirus to Selectively Target Hepatocellular Carcinoma Cells.

Despite significant advances in chemotherapy, the overall prognosis of hepatocellular carcinoma (HCC) remains extremely poor. HCC targeting strategies were combined with the tumor cell cytotoxicity of oncolytic viruses (OVs) to develop a more efficient and selective therapeutic system. OVs were coated with a polygalactosyl-b-agmatyl diblock copolymer (Gal32-b-Agm29), with high affinity for the asialoglycoprotein receptor (ASGPR) expressed on the liver cell surface, exploiting the electrostatic interaction of the positively charged agmatine block with the negatively charged adenoviral capsid surface. The polymer coating altered the viral particle diameter (from 192 to 287 nm) and zeta-potential (from -24.7 to 23.3 mV) while hiding the peculiar icosahedral symmetrical OV structure, as observed by TEM. Coated OVs showed high potential therapeutic value on the human hepatoma cell line HepG2 (cytotoxicity of 72.4% ± 4.96), expressing a high level of ASGPRs, while a lower effect was attained with ASPGR-negative A549 cell line (cytotoxicity of 54.4% ± 1.59). Conversely, naked OVs showed very similar effects in both tested cell lines. Gal32-b-Agm29 OV coating enhanced the infectivity and immunogenic cell death program in HepG2 cells as compared to the naked OV. This strategy provides a rationale for future studies utilizing oncolytic viruses complexed with polymers toward effective treatment of hepatocellular carcinoma.

Pullulan Based Bioconjugates for Ocular Dexamethasone Delivery.

Posterior segment eye diseases are mostly related to retinal pathologies that require pharmacological treatments by invasive intravitreal injections. Reduction of frequent intravitreal administrations may be accomplished with delivery systems that provide sustained drug release. Pullulan-dexamethasone conjugates were developed to achieve prolonged intravitreal drug release. Accordingly, dexamethasone was conjugated to ~67 kDa pullulan through hydrazone bond, which was previously found to be slowly cleavable in the vitreous. Dynamic light scattering and transmission electron microscopy showed that the pullulan-dexamethasone containing 1:20 drug/glucose unit molar ratio (10% w/w dexamethasone) self-assembled into nanoparticles of 461 ± 30 nm and 402 ± 66 nm, respectively. The particles were fairly stable over 6 weeks in physiological buffer at 4, 25 and 37 °C, while in homogenized vitreous at 37 °C, the colloidal assemblies underwent size increase over time. The drug was released slowly in the vitreous and rapidly at pH 5.0 mimicking lysosomal conditions: 50% of the drug was released in about 2 weeks in the vitreous, and in 2 days at pH 5.0. In vitro studies with retinal pigment epithelial cell line (ARPE-19) showed no toxicity of the conjugates in the cells. Flow cytometry and confocal microscopy showed cellular association of the nanoparticles and intracellular endosomal localization. Overall, pullulan conjugates showed interesting features that may enable their successful use in intravitreal drug delivery.

PEG-polyaminoacid based micelles for controlled release of doxorubicin: Rational design, safety and efficacy study.

A library of amphiphilic monomethoxypolyethylene glycol (mPEG) terminating polyaminoacid co-polymers able to self-assemble into colloidal systems was screened for the delivery and controlled release of doxorubicin (Doxo). mPEG-Glu/Leu random co-polymers were generated by Ring Opening Polymerization from 5 kDa mPEG-NH2 macroinitiator using 16:0:1, 8:8:1, 6:10:1, 4:12:1 γ-benzyl glutamic acid carboxy anhydride monomer/leucine N-carboxy anhydride monomer/PEG molar ratios. Glutamic acid was selected for chemical conjugation of Doxo, while leucine units were introduced in the composition of the polyaminoacid block as spacer between adjacent glutamic repeating units to minimize the steric hindrance that could impede the Doxo conjugation and to promote the polymer self-assembly by virtue of the aminoacid hydrophobicity. The benzyl ester protecting the γ-carboxyl group of glutamic acid was quantitatively displaced with hydrazine to yield mPEG5kDa-b-(hydGlum-r-Leun). Doxo was conjugated to the diblock co-polymers through pH-sensitive hydrazone bond. The Doxo derivatized co-polymers obtained with a 16:0:1, 8:8:1, 6:10:1 Glu/Leu/PEG ratios self-assembled into 30-40 nm spherical nanoparticles with neutral zeta-potential and CMC in the range of 4-7 µM. At pH 5.5, mimicking endosome environment, the carriers containing leucine showed a faster Doxo release than at pH 7.4, mimicking the blood conditions. Doxo-loaded colloidal formulations showed a dose dependent cytotoxicity on two cancer cell lines, CT26 murine colorectal carcinoma and 4T1 murine mammary carcinoma with IC50 slightly higher than those of free Doxo. The carrier assembled with the polymer containing 6:10:1 hydGlu/Leu/PEG molar ratio {mPEG5kDa-b-[(Doxo-hydGlu)6-r-Leu10]} was selected for subsequent in vitro and in vivo investigations. Confocal imaging on CT26 cell line showed that intracellular fate of the carrier involves a lysosomal trafficking pathway. The intratumor or intravenous injection to CT26 and 4T1 subcutaneous tumor bearing mice yielded higher antitumor activity compared to free Doxo. Furthermore, mPEG5kDa-b-[(Doxo-hydGlu)6-r-Leu10] displayed a better safety profile when compared to commercially available Caelyx®.