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Targeted Radionuclide Therapy (TRT) is a medical technique exploiting radionuclides to combat cancer growth and spread. TRT requires a supply of radionuclides that are currently produced by either cyclotrons or nuclear research reactors. In this context, the ISOLPHARM project investigates the production of innovative radionuclides for medical applications. This production will be based on the forthcoming SPES facility at the Legnaro National Laboratories (LNL) of the National Institute for Nuclear Physics (INFN), an ISOL facility where high-purity radioactive beams will be used to produce carrier-free radiopharmaceuticals. Previous studies demonstrated that a significant amount of 111 Ag, an innovative β/γ emitter suitable for TRT with theranostic applications, can be obtained at the SPES facility. The present work describes the first imaging study on phantoms with 111 Ag performed by the ISOLPHARM collaboration. This is a fundamental step to pave the way for the upcoming in vivo studies on the 111 Ag-based radiopharmaceutical currently being developed. The imaging potential of this radionuclide was investigated by acquiring phantom images with Cerenkov Luminescence Imaging (CLI) and digital autoradiography (ARG)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The cholecystokinin type 2 receptor (CCK2-R) represents an ideal target for cancer therapy since it is overexpressed in several tumors and is associated with poor prognosis. Nastorazepide (Z-360), a selective CCK2-R antagonist, has been widely investigated as a CCK2-R ligand for targeted therapy; however, its high hydrophobicity may represent a limit to cell selectivity and optimal in vivo biodistribution. Here, we present three new fluorescent Z-360 derivatives ( IP-002 G -Rho, IP-002 L -Rho, and IP-002 M -Rho ) in which nastorazepide was linked, through spacers bearing different saccharides (glucose (G), lactose (L), and maltotriose (M)), to sulforhodamine B. A fourth compound ( IP-002 H -Rho ) with no pendant sugar was also synthesized as a control. Through two-dimensional (2D) and three-dimensional (3D) in vitro studies, we evaluated the compound association with and selectivity for CCK2-R-overexpressing cells (A431-CCK2-R + ) vs CCK2-R-underexpressing cells (A431 WT). 2D in vitro studies highlighted a progressive increase of IP-002 x -Rho association with A431-CCK2-R + cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002 M -Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002 G -Rho and IP-002 L -Rho , respectively. Unexpectedly, IP-002 H -Rho showed a similar cell association to that of IP-002 L -Rho but with no difference between the two tested cell lines. On the contrary, association with A431-CCK2-R + cells as compared to the A431 WT was found to be 1.08-, 1.14-, and 1.37-fold higher for IP-002 G -Rho , IP-002 L -Rho , and IP-002 M -Rho , respectively, proving IP-002 M -Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R + cells incubated with IP-002 M -Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002 M -Rho by A431-CCK2-R + cells only was observed on 3D scaffolds embedded with cells, underlining the importance of 3D models in in vitro preliminary evaluation.

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Author LK is an employee and/or shareholder of Valo Therapeutics. The other authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The aim of the present study consists of the evaluation of the biodistribution of a novel 68 Ga-labeled radiopharmaceutical, [ 68 Ga]Ga-NODAGA-Z360, injected into Balb/c nude mice through histopathological analysis on bioptic samples and radiomics analysis of positron emission tomography/computed tomography (PET/CT) images. The 68 Ga-labeled radiopharmaceutical was designed to specifically bind to the cholecystokinin receptor (CCK2R). This receptor, naturally present in healthy tissues such as the stomach, is a biomarker for numerous tumors when overexpressed. In this experiment, Balb/c nude mice were xenografted with a human epidermoid carcinoma A431 cell line (A431 WT) and overexpressing CCK2R (A431 CCK2R+), while controls received a wild-type cell line. PET images were processed, segmented after atlas-based co-registration and, consequently, 112 radiomics features were extracted for each investigated organ / tissue. To confirm the histopathology at the tissue level and correlate it with the degree of PET uptake, the studies were supported by digital pathology. As a result of the analyses, the differences in radiomics features in different body districts confirmed the correct targeting of the radiopharmaceutical. In preclinical imaging, the methodology confirms the importance of a decision-support system based on artificial intelligence algorithms for the assessment of radiopharmaceutical biodistribution.

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 (Arg 4 -DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCE free -LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCE free -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 OCE free -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 OCE free -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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium responsible for severe nosocomial infections and is considered a critical pulmonary pathogen for both immunocompromised and cystic fibrosis patients. Planktonic cells of P. aeruginosa possess intrinsic and acquired resistances, inactivating several classes of conventional antibiotics. Additionally, this bacterium can grow, forming biofilms, and complex structures, further hampering the action of multiple antibiotics. Here, we report the biological properties of D-Q53 CecB, an all-D enantiomer of the silkworm natural peptide Q53 CecB. Compared to the L-variant, D-Q53 CecB was resistant to in vitro degradation by humans and P. aeruginosa elastases and showed an enhanced bactericidal activity against P. aeruginosa planktonic bacteria. D-Q53 CecB was thermostable and maintained its antimicrobial activity at high salt concentrations and in the presence of divalent cations or fetal-bovine serum, although at reduced levels. Against different types of human cells, D-Q53 CecB showed cytotoxic phenomena at concentrations several folds higher compared to those active against P. aeruginosa. When L- and D-Q53 CecB were compared for their antibiofilm properties, both peptides were active in inhibiting biofilm formation. However, the D-enantiomer was extremely effective in inducing biofilm degradation, suggesting this peptide as a favorable candidate in an anti- Pseudomonas therapy.

Despite substantial improvements in breast cancer (BC) treatment there is still an urgent need to find alternative treatment options to improve the outcomes for patients with advanced-stage disease. Photodynamic therapy (PDT) is gaining a lot of attention as a BC therapeutic option because of its selectivity and low off-target effects. However, the hydrophobicity of photosensitizers (PSs) impairs their solubility and limits the circulation in the bloodstream, thus representing a major challenge. The use of polymeric nanoparticles (NPs) to encapsulate the PS may represent a valuable strategy to overcome these issues. Herein, we developed a novel biomimetic PDT nanoplatform (NPs) based on a polymeric core of poly(lactic-co-glycolic)acid (PLGA) loaded with the PS meso-tetraphenylchlorin disulfonate (TPCS 2a ). TPCS 2a @NPs of 98.89 ± 18.56 nm with an encapsulation efficiency percentage (EE%) of 81.9 ± 7.92% were obtained and coated with mesenchymal stem cells-derived plasma membranes (mMSCs) (mMSC-TPCS 2a @NPs, size of 139.31 ± 12.94 nm). The mMSC coating armed NPs with biomimetic features to impart long circulation times and tumor-homing capabilities. In vitro, biomimetic mMSC-TPCS 2a @NPs showed a decrease in macrophage uptake of 54% to 70%, depending on the conditions applied, as compared to uncoated TPCS 2a @NPs. Both NP formulations efficiently accumulated in MCF7 and MDA-MB-231 BC cells, while the uptake was significantly lower in normal breast epithelial MCF10A cells with respect to tumor cells. Moreover, encapsulation of TPCS 2a in mMSC-TPCS 2a @NPs effectively prevents its aggregation, ensuring efficient singlet oxygen ( 1 O 2 ) production after red light irradiation, which resulted in a considerable in vitro anticancer effect in both BC cell monolayers (IC 50 < 0.15 µM) and three-dimensional spheroids.

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-PEG 3.5kDa -SH molecules/particle, and the surface was saturated with about 500 rhodamine-PEG 2kDa -SH fluorescent probes. In vitro studies carried out using folate receptor overexpressing KB cells (KB FR-high ) showed that the cell internalization progressively increased with the ligand surface density, reaching a plateau at 50:1 FA-PEG 3.5kDa -SH/particle ratio. Pulse-chase experiments showed that higher FA density (50 FA-PEG 3.5kDa -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-PEG 3.5kDa -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.

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 (Agm 6 -M-Oleate), a synthetic non-peptidic cell penetration enhancer (CPE), and/or 5% of mPEG 2kDa -DSPE. The zeta potential of liposomes increased, and the mobility in bovine vitreous and colloidal stability decreased with the Agm 6 -M-Oleate coating concentration. Oppositely, mPEG 2kDa -DSPE decreased the zeta potential of liposomes and restored both the diffusivity and the stability in vitreous. Liposomes with 5 mol% Agm 6 -M-Oleate coating were well tolerated by ARPE-19 retina cells either with or without mPEG 2kDa -DSPE, while 10 mol% Agm 6 -M-Oleate showed cytotoxicity. Agm 6 -M-Oleate promoted the association of liposomes to ARPE-19 cells with respect to plain liposomes, while mPEG 2kDa -DSPE slightly reduced the cell interaction. Dexamethasone hemisuccinate (DH) was remotely loaded into liposomes with a loading capacity of ∼10 wt/wt%. Interestingly, mPEG 2kDa -DSPE coating reduced the rate of DH release and enhanced the disposition of Agm 6 -M-Oleate coated liposomes in the ARPE-19 cell cytosol resulting in a more efficient anti-inflammatory effect. Finally, mPEG 2kDa -DSPE enhanced the association of DH-loaded Agm 6 -M-Oleate coated liposomes to explanted rat retina, which reflected in higher viability of inner and outer nuclear layer cells., (Copyright © 2023. Published by Elsevier B.V.)

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO 4 -3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO 4 -3-Gal-glycopolymer-CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO 4 -3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies.

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 (M 15 A 12 , M 29 A 25 , and M 58 A 45 ) and two triblock copolymers (M 29 A 29 B 9 and M 62 A 52 B 32 ) 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. M 58 A 45 and M 62 A 52 B 32 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 M 58 A 45 /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.

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-AMPS m ., (© 2021. Controlled Release Society.)

The 64Cu-labeled chelator was analyzed in vivo by positron emission tomography (PET) imaging to evaluate its biodistribution in a murine model at different acquisition times. For this purpose, nine 6-week-old female Balb/C nude strain mice underwent micro-PET imaging at three different time points after 64Cu-labeled chelator injection. Specifically, the mice were divided into group 1 (acquisition 1 h after [64Cu] chelator administration, n = 3 mice), group 2 (acquisition 4 h after [64Cu]chelator administration, n = 3 mice), and group 3 (acquisition 24 h after [64Cu] chelator administration, n = 3 mice). Successively, all PET studies were segmented by means of registration with a standard template space (3D whole-body Digimouse atlas), and 108 radiomics features were extracted from seven organs (namely, heart, bladder, stomach, liver, spleen, kidney, and lung) to investigate possible changes over time in [64Cu]chelator biodistribution. The one-way analysis of variance and post hoc Tukey Honestly Significant Difference test revealed that, while heart, stomach, spleen, kidney, and lung districts showed a very low percentage of radiomics features with significant variations (p-value < 0.05) among the three groups of mice, a large number of features (greater than 60% and 50%, respectively) that varied significantly between groups were observed in bladder and liver, indicating a different in vivo uptake of the 64Cu-labeled chelator over time. The proposed methodology may improve the method of calculating the [64Cu]chelator biodistribution and open the way towards a decision support system in the field of new radiopharmaceuticals used in preclinical imaging trials.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of polysaccharides, nucleic acids and proteins. We hypothesised that carbohydrates could contribute to immune recognition of Pseudomonas aeruginosa biofilms by engaging C-type lectins. Here we show binding of Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2 to P. aeruginosa biofilms. We also demonstrate that DC-SIGN, unlike MR and Dectin-2, recognises planktonic P. aeruginosa cultures and this interaction depends on the presence of the common polysaccharide antigen. Within biofilms DC-SIGN, Dectin-2 and MR ligands appear as discrete clusters with dispersed DC-SIGN ligands also found among bacterial aggregates. DC-SIGN, MR and Dectin-2 bind to carbohydrates purified from P. aeruginosa biofilms, particularly the high molecular weight fraction (HMW; >132,000 Da), with K D s in the nM range. These HMW carbohydrates contain 74.9-80.9% mannose, display α-mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and inhibit Dectin-2-mediated cellular activation. In addition, biofilm carbohydrates reduce the association of the DC-SIGN ligand Lewis x , but not fucose, to human monocyte-derived dendritic cells (moDCs), and alter moDC morphology without affecting early cytokine production in response to lipopolysaccharide or P. aeruginosa cultures. This work identifies the presence of ligands for three important C-type lectins within P. aeruginosa biofilm structures and purified biofilm carbohydrates and highlights the potential for these receptors to impact immunity to P. aeruginosa infection., (© 2021. The Author(s).)

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-NH 2 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 mPEG 5kDa -b-(hydGlu m -r-Leu n ). 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 IC 50 slightly higher than those of free Doxo. The carrier assembled with the polymer containing 6:10:1 hydGlu/Leu/PEG molar ratio {mPEG 5kDa -b-[(Doxo-hydGlu) 6 -r-Leu 10 ]} 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, mPEG 5kDa -b-[(Doxo-hydGlu) 6 -r-Leu 10 ] displayed a better safety profile when compared to commercially available Caelyx®., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Dendritic cells serve as the main immune cells that trigger the immune response. We developed a simple and cost-effective nanovaccine platform based on the α1',2-mannobiose derivative for dendritic cell targeting. In previous work, we have formulated the α1,2-mannobiose-based nanovaccine platform with plasmid DNA and tested it in cattle against BoHV-1 infection. There, we have shown that the dendritic cell targeting using this nanovaccine platform in vivo can boost the immunogenicity, resulting in a long-lasting immunity. In this work, we aim to characterize the α1',2-mannobiose derivative, which is key in the nanovaccine platform. This DC-targeting strategy takes advantage of the specific receptor known as DC-SIGN and exploits its capacity to bind α1,2-mannobiose that is present at terminal ends of oligosaccharides in certain viruses, bacteria, and other pathogens. The oxidative conjugation of α1',2-mannobiose to NH 2 -PEG 2kDa -DSPE allowed us to preserve the chemical structure of the non-reducing mannose of the disaccharide and the OH groups and the stereochemistry of all carbons of the reducing mannose involved in the binding to DC-SIGN. Here, we show specific targeting to DC-SIGN of decorated micelles incubated with the Raji/DC-SIGN cell line and uptake of targeted liposomes that took place in human, bovine, mouse, and teleost fish DCs in vitro , by flow cytometry. Specific targeting was found in all cultures, demonstrating a species-non-specific avidity for this ligand, which opens up the possibility of using this nanoplatform to develop new vaccines for various species, including humans.

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 (Gal 32 - b -Agm 29 ), 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. Gal 32 - b -Agm 29 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.

The treatment of posterior segment disorders of the eye requires therapeutic strategies to achieve drug effects over prolonged times. Innovative colloidal delivery systems can be designed to deliver drugs to the retina and prolong their intravitreal permanence. In order to exploit pullulan (Pull) as polymeric drug carrier for intravitreal drug delivery, derivatives of hydrophobic model molecule rhodamine B (RhB) were conjugated to the pullulan backbone through linkers with different stability, namely ether (Et), hydrazone (Hy) or ester (Es) bond to obtain Pull-Et-RhB, Pull-Hy-RhB and Pull-Es-RhB, respectively. Dynamic light scattering and transmission electron microscopy analyses showed that the polymer conjugates self-assembled into 20-25 nm particles. Pull-Et-RhB was fairly stable at all tested pH values. At the vitreal pH of 7.4, 50% of RhB was released from Pull-Hy-RhB and Pull-Es-RhB in 11 and 6 days, respectively. At endosomal pH (5.5), 50% of RhB was released from Pull-Hy-RhB and Pull-Es-RhB in 4 and 1 days, respectively. Multiple particle tracking analyses in ex vivo porcine eye model showed that the diffusivity of the bioconjugates in the vitreous was about 10 3 times lower than in water. Flow cytometry and confocal microscopy analyses showed that bioconjugates are remarkably taken up by the retinal RPE cells. In vivo studies showed that after intravitreal injection to mice, the bioconjugates localize in the ganglion cell layer and in the inner and outer plexiform layers. Pull-Hy-RhB particles were detected also inside the retinal blood vessels. These results demonstrate that pullulan with tailored linkers for drug conjugation is a promising vehicle for long-acting intravitreal injectables that are capable to permeate to the retina., (Copyright © 2021. Published by Elsevier B.V.)

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.

Objectives: The aim of this study was to investigate the relationship between the degree of crystal phagocytosis and the magnitude of the local inflammatory process using fresh synovial fluid (SF) collected from patients with crystal-induced arthritis. In parallel, an in vitro model of crystal-induced inflammation was used to assess the effect of cell priming on crystal phagocytosis and IL-1ß production., Methods: SF was collected from 20 patients with gout and 20 with pyrophosphate crystal-induced arthritis and examined under ordinary and polarised light microscopy for total and differential white blood cell (WBC) count and crystal search. The total phagocytosis index was determined in SF along with IL-1β, IL-8, IL-10, and TGFβ levels. The in vitro studies were performed using primed or unprimed THP-1 cells stimulated with calcium pyrophosphate (CPP) crystals, monosodium urate (MSU) crystals and/or cytochalasin D., Results: We demonstrated that the phagocytosis index calculated on the total number of cells was independent from the inflammatory local indices such as WBC and the percentage of polymorphonuclear cells but showed a positive correlation with pro-inflammatory cytokines. By contrast, the local inflammatory indices (WBC and IL-1ß) showed a strong positive correlation with the percentage of polymorphonuclear cells with crystals internalised and a negative correlation with the percentage of mononuclear cells with crystals internalised. The in vitro study showed that phagocytosis represents a fundamental step in the induction of the inflammatory response to MSU and CPP crystals, but it also occurs in absence of cell priming., Conclusions: The results of this study indicate a possible role of non-inflammatory phagocytosis in limiting the acute attack of crystal-induced arthritis.

The selective isolation of bacteria from mixed populations has been investigated in varied applications ranging from differential pathogen identification in medical diagnostics and food safety to the monitoring of microbial stress dynamics in industrial bioreactors. Selective isolation techniques are generally limited to the confinement of small populations in defined locations, may be unable to target specific bacteria, or rely on immunomagnetic separation, which is not universally applicable. In this proof-of-concept work, we describe a novel strategy combining inducible bacterial lectin expression with magnetic glyconanoparticles (MGNPs) as a platform technology to enable selective bacterial isolation from cocultures. An inducible mutant of the type 1 fimbriae, displaying the mannose-specific lectin FimH, was constructed in Escherichia coli allowing for "on-demand" glycan-binding protein presentation following external chemical stimulation. Binding to glycopolymers was only observed upon fimbrial induction and was specific for mannosylated materials. A library of MGNPs was produced via the grafting of well-defined catechol-terminal glycopolymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization to magnetic nanoparticles. Thermal analysis revealed high functionalization (≥85% polymer by weight). Delivery of MGNPs to cocultures of fluorescently labeled bacteria followed by magnetic extraction resulted in efficient depletion of type 1 fimbriated target cells from wild-type or afimbriate E. coli . Extraction efficiency was found to be dependent on the molecular weight of the glycopolymers utilized to engineer the nanoparticles, with MGNPs decorated with shorter Dopa-(ManAA) 50 mannosylated glycopolymers found to perform better than those assembled from a longer Dopa-(ManAA) 200 analogue. The extraction efficiency of fimbriated E. coli was also improved when the counterpart strain did not harbor the genetic apparatus for the expression of the type 1 fimbriae. Overall, this work suggests that the modulation of the genetic apparatus encoding bacterial surface-associated lectins coupled with capture through MGNPs could be a versatile tool for the extraction of bacteria from mixed populations.

Aberrant activation of the Hedgehog (Hh) pathway leads to the development of several tumors, including medulloblastoma (MB), the most common pediatric brain malignancy. Hh inhibitors acting on GLI1, the final effector of Hh signaling, offer a valuable opportunity to overcome the pitfalls of the existing therapies to treat Hh-driven cancers. In this study, the toxicity, delivery, biodistribution, and anticancer efficacy of Glabrescione B (GlaB), a selective GLI1 inhibitor, were investigated in preclinical models of Hh-dependent MB. To overcome its poor water solubility, GlaB was formulated with a self-assembling amphiphilic polymer forming micelles, called mPEG 5kDa -cholane. mPEG 5kDa -cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG 5kDa -cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG 5kDa -cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG 5kDa -cholane micelles was delivered through the blood-brain barrier and drastically inhibited tumor growth in both allograft and orthotopic models of Hh-dependent MB. Our findings reveal that mPEG 5kDa -cholane/GlaB is a good candidate for the treatment of Hh-driven tumors and provide relevant implications for the translation of GlaB into clinical practice., (Copyright © 2020. Published by Elsevier B.V.)

The cholecystokinin-2 receptor (CCK-2R) is overexpressed in several human cancers but displays limited expression in normal tissues. For this reason, it is a suitable target for developing specific radiotracers. In this study, a nastorazepide-based ligand functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator (IP-001) was synthesized and labelled with indium-111. The radiolabeling process yielded >95% with a molar activity of 10 MBq/nmol and a radiochemical purity of >98%. Stability studies have shown a remarkable resistance to degradation (>93%) within 120 h of incubation in human blood. The in vitro uptake of [ 111 In]In-IP-001 was assessed for up to 24 h on a high CCK-2R-expressing tumor cell line (A549) showing maximal accumulation after 4 h of incubation. Biodistribution and single photon emission tomography (SPECT)/CT imaging were evaluated on BALB/c nude mice bearing A549 xenograft tumors. Implanted tumors could be clearly visualized after only 4 h post injection (2.36 ± 0.26% ID/cc), although a high amount of radiotracer was also found in the liver, kidneys, and spleen (8.25 ± 2.21%, 6.99 ± 0.97%, and 3.88 ± 0.36% ID/cc, respectively). Clearance was slow by both hepatobiliary and renal excretion. Tumor retention persisted for up to 24 h, with the tumor to organs ratio increasing over-time and ending with a tumor uptake (1.52 ± 0.71% ID/cc) comparable to liver and kidneys.

The biorecognition-based control of attachment/detachment of MCF-7 cancer cells from polymer-coated surfaces is demonstrated. A glass surface is coated with a thermoresponsive statistical copolymer of poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAm-co-Am)], which is end-capped with the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, and the hydrophilic polymer poly(ethylene glycol) (PEG). Below the lower critical solution temperature (LCST) of p(NIPAm-co-Am) (38 °C), the copolymers are in the extended conformation, allowing for accessibility of the GRGDS peptides to membrane-associated integrins thus enabling cell attachment. Above the LCST, the p(NIPAm-co-Am) polymers collapse into globular conformations, resulting in the shielding of the GRGDS peptides into the PEG brush with consequent inaccessibility to cell-surface integrins, causing cell detachment. The surface coating is carried out by a multi-step procedure that included: glass surface amination with 3-aminopropyltriethoxysilane; reaction of mPEG 5kDa -N-hydroxysuccinimide (NHS) and p(NIPam-co-Am) 15.1kDa -bis-NHS with the surface aminopropyl groups and conjugation of GRGDS to the carboxylic acid termini of p(NIPam-co-Am) 15.1kDa -COOH. A range of spectrophotometric, surface, and microscopy assays confirmed the identity of the polymer-coated substrates. Competition studies prove that MCF-7 cancer cells are attached via peptide recognition at the coated surfaces according to the mPEG 5kDa /p(NIPam-co-Am) 15.1kDa -GRGDS molar ratio. These data suggest the system can be exploited to modulate cell integrin/GRGDS binding for controlled cell capture and release., (© 2020 Wiley-VCH GmbH.)

Research in the field of radiopharmaceuticals is increasingly promoted by the widespread and growing interest in applying nuclear medicine procedures in both disease diagnosis and treatment. The production of radionuclides of medical interest is however a challenging issue. Along with the conventional techniques other innovative approaches are being investigated and, among those, the ISOLPHARM project is being developed at INFN-LNL (Istituto Nazionale di Fisica Nucleare - Laboratori Nazionali di Legnaro). Such technique foresees the employment of the SPES ISOL facility to produce isobarically pure Radioactive Ion Beams (RIBs), obtained thanks to electromagnetic mass separation and collected on appropriate substrates. The latter are successively recovered and dissolved, allowing thus the chemical separation and harvesting of the nuclides of interest, free from any isotopic contaminant. Although ISOLPHARM can be potentially employed for most of the routinely used medical radioisotopes, its innovation potential is better expressed considering its capability to provide carrier free unconventional nuclides, difficult to produce with state-of-art techniques, such as 111 Ag, a β - emitter potentially interesting for therapeutic applications. Thus, in the framework of ISOLPHARM, INFN supported a two-years experiment, called ISOLPHARM&#95;Ag, aimed at evaluating the feasibility of the production of a 111 Ag labelled radiopharmaceutical. The ISOL production yields are estimated by computing intensive Monte Carlo codes, that require an appropriate custom Information Technology infrastructure. The presented work is focused on the first part of the production chain including the capability to extract, ionize, and collect stable Ag beams with SPES technologies. MC calculations were used to estimate the expected 111 Ag in-target yields, whereas experiments with stable Ag were performed to test the ionization, transport and collection of Ag beams., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Colorectal cancer (CRC) shows highly ineffective therapeutic management. An urgent unmet need is the random assignment to adjuvant chemotherapy of high-risk stage II and stage III CRC patients without any predictive factor of efficacy. In the field of drug discovery, a critical step is the preclinical evaluation of drug cytotoxicity, efficacy, and efficiency. We proposed a patient-derived 3D preclinical model for drug evaluation that could mimic in vitro the patient's disease. Surgically resected CRC tissue and adjacent healthy colon mucosa were decellularized by a detergent-enzymatic treatment. Scaffolds were recellularized with HT29 and HCT116 cells. Qualitative and quantitative characterization of matched recellularized samples were evaluated through histology, immunofluorescences, scanning electron microscopy, and DNA amount quantification. A chemosensitivity test was performed using an increasing concentration of 5-fluorouracil (5FU). In vivo studies were carried out using zebrafish ( Danio rerio ) animal model. Permeability test and drug absorption were also determined. The decellularization protocol allowed the preservation of the original structure and ultrastructure. Five days after recellularization with HT29 and HCT116 cell lines, the 3D CRC model exhibited reduced sensitivity to 5FU treatments compared with conventional 2D cultures. Calculated the half maximal inhibitory concentration (IC 50 ) for HT29 treated with 5FU resulted in 11.5 µM in 3D and 1.3 µM in 2D, and for HCT116, 9.87 µM in 3D and 1.7 µM in 2D. In xenograft experiments, HT29 extravasation was detected after 4 days post-injection, and we obtained a 5FU IC 50 fully comparable to that observed in the 3D CRC model. Using confocal microscopy, we demonstrated that the drug diffused through the repopulated 3D CRC scaffolds and co-localized with the cell nuclei. The bioengineered CRC 3D model could be a reliable preclinical patient-specific platform to bridge the gap between in vitro and in vivo drug testing assays and provide effective cancer treatment., Competing Interests: The authors declare no potential conflicts of interest.

Liposomes have been one of the most exploited drug delivery systems in recent decades. However, their large-scale production with low batch-to-batch differences is a challenge for industry, which ultimately delays the clinical translation of new products. We have investigated the effects of formulation parameters on the colloidal and biopharmaceutical properties of liposomes generated with a thin-film hydration approach and microfluidic procedure. Dexamethasone hemisuccinate was remotely loaded into liposomes using a calcium acetate gradient. The liposomes produced by microfluidic techniques showed a unilamellar structure, while the liposomes produced by thin-film hydration were multilamellar. Under the same remote loading conditions, a higher loading capacity and efficiency were observed for the liposomes obtained by microfluidics, with low batch-to-batch differences. Both formulations released the drug for almost one month with the liposomes prepared by microfluidics showing a slightly higher drug release in the first two days. This behavior was ascribed to the different structure of the two liposome formulations. In vitro studies showed that both formulations are non-toxic, associate to human Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells, and efficiently reduce inflammation, with the liposomes obtained by the microfluidic technique slightly outperforming. The results demonstrated that the microfluidic technique offers advantages to generate liposomal formulations for drug-controlled release with an enhanced biopharmaceutical profile and with scalability.

Insulin is one of the most marketed therapeutic proteins worldwide. However, its formulation suffers from fibrillation, which affects the long-term storage limiting the development of novel devices for sustained delivery including portable infusion devices. We have investigated the effect of physical PEGylation on structural and colloidal stability of insulin by using 2 PEGylating agents terminating with polycyclic hydrophobic moieties, cholane and cholesterol: mPEG 5kDa -cholane and mPEG 5kDa -cholesterol, respectively. Microcalorimetric analyses showed that mPEG 5kDa -cholane and mPEG 5kDa -cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 10 4 and 1.14 10 5 M -1 , respectively. At room temperature, the 2 PEGylating agents yielded comparable structural stabilization of α-helix conformation and decreased dimerization of insulin. However, melting studies showed that mPEG 5kDa -cholesterol has superior stabilizing effect of the protein conformation than mPEG 5kDa -cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG 5kDa -cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG 5kDa -cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG 5kDa -cholane and mPEG 5kDa -cholesterol assemblies with comparable kinetic profiles. The physical PEGylation has a beneficial effect on the stabilization and shielding of the insulin structure into the monomeric form, which is not prone to fibrillation and aggregation., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The exploitation of curcumin for oral disease treatment is limited by its low solubility, poor bioavailability, and low stability. Surface-functionalized poly-lactic- co -glycolic acid (PLGA) nanoparticles (NPs) have shown promising results to ameliorate selective delivery of drugs to the gastro-intestinal tract. In this study, curcumin-loaded PLGA NPs (C-PLGA NPs) of about 200 nm were surface-coated with chitosan (CS) for gastro-intestinal mucosa adhesion, wheat germ agglutinin (WGA) for colon targeting or GE11 peptide for tumor colon targeting. Spectrometric and zeta potential analyses confirmed the successful functionalization of the C-PLGA NPs. Real-time label-free assessment of the cell membrane-NP interactions and NP cell uptake were performed by quartz crystal microbalance coupled with supported lipid bilayers and by surface plasmon resonance coupled with living cells. The study showed that CS-coated C-PLGA NPs interact with cells by the electrostatic mechanism, while both WGA- and GE11-coated C-PLGA NPs interact and are taken up by cells by specific active mechanisms. In vitro cell uptake studies corroborated the real-time label-free assessment by yielding a curcumin cell uptake of 7.3 ± 0.3, 13.5 ± 1.0, 27.3 ± 4.9, and 26.0 ± 1.3 μg per 10 4 HT-29 cells for noncoated, CS-, WGA-, and GE11-coated C-PLGA NPs, respectively. Finally, preliminary in vivo studies showed that the WGA-coated C-PLGA NPs efficiently accumulate in the colon after oral administration to healthy Balb/c mice. In summary, the WGA- and GE11-coated C-PLGA NPs displayed high potential for application as active targeted carriers for anticancer drug delivery to the colon., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

Novel bioconjugates (Agm 6 -M-PEG-FA) for active oligonucleotide (ON) delivery have been developed by conjugating a cationic oligo-guanidyl star-like shaped "head" (Agm 6 -M) to a polymeric "tail" (PEG) terminating with folic acid (FA) as targeting agent or methoxy group (Agm 6 -M-PEG-FA and Agm 6 -M-PEG-OCH 3 , respectively). Gel electrophoresis showed that the bioconjugates completely associated with ONs at 3 nitrogen/phosphate (N/P) ratio. Studies performed with folate receptor (FR)-overexpressing HeLa cells, showed that optimal cell up-take was obtained with the 75:25 w/w Agm 6 -M-PEG-OCH 3 :Agm 6 -M-PEG-FA mixture. Dynamic light scattering and transmission electron microscopy showed that the polyplexes had size <80 nm with narrow polydispersity and rod-shaped morphology. The polyplexes were stable for several hours in plasma while ON was released in the presence of heparin concentration 16-times higher than the physiological one. The polyplexes displayed negligible cytotoxicity, hemolysis and low pro-inflammatory TNF-α release. Studies performed with FR-overexpressing HeLa and MDA-MB-231 cells using siRac1 revealed that the folated polyplexes caused significantly higher gene silencing (86.1 ± 9.6%) and inhibition of cell migration (40%) than the non-folated polyplexes obtained with Agm 6 -M-PEG-OCH 3 only. Although cytofluorimetric analyses showed similar cell uptake for both folated and non-folated polyplexes, confocal, TEM and competition studies showed that the folated polyplexes were taken-up by lysosome escaping caveolin-mediated pathway with final polyplex localization within cytosol, while non-folated polyplexes were preferentially taken-up via clathrin-mediated pathway to localize in the lysosomes. Finally, preliminary in vivo studies carried out in mice revealed that the folated polyplexes dispose in the tumor mass., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway., Methods: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used 1 H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG 5kDa -Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice., Results: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted 1 H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition., Conclusions: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.