Publications by authors named "Francesca Mastrotto"

37 Publications

Characterization of a Nanovaccine Platform Based on an α1,2-Mannobiose Derivative Shows Species-non-specific Targeting to Human, Bovine, Mouse, and Teleost Fish Dendritic Cells.

Mol Pharm 2021 Jun 9. Epub 2021 Jun 9.

Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, United States.

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 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-PEG-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 , 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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.1c00048DOI Listing
June 2021

Pullulan Based Bioconjugates for Ocular Dexamethasone Delivery.

Pharmaceutics 2021 May 26;13(6). Epub 2021 May 26.

Faculty of Health Sciences, School of Pharmacy, University of Eastern Finland, Yliopistonranta 1C, 70211 Kuopio, Finland.

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% / 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.
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http://dx.doi.org/10.3390/pharmaceutics13060791DOI Listing
May 2021

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

J Control Release 2021 May 11;335:21-37. Epub 2021 May 11.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, 35131 Padova, Italy.

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 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-b-(hydGlu-r-Leu). 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 slightly higher than those of free Doxo. The carrier assembled with the polymer containing 6:10:1 hydGlu/Leu/PEG molar ratio {mPEG-b-[(Doxo-hydGlu)-r-Leu]} 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-b-[(Doxo-hydGlu)-r-Leu] displayed a better safety profile when compared to commercially available Caelyx®.
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http://dx.doi.org/10.1016/j.jconrel.2021.05.010DOI Listing
May 2021

Combining Inducible Lectin Expression and Magnetic Glyconanoparticles for the Selective Isolation of Bacteria from Mixed Populations.

ACS Appl Mater Interfaces 2021 Apr 14;13(16):19230-19243. Epub 2021 Apr 14.

Division of Molecular Therapeutics and Formulation, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, U.K.

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 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 . 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) mannosylated glycopolymers found to perform better than those assembled from a longer Dopa-(ManAA) analogue. The extraction efficiency of fimbriated 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.
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http://dx.doi.org/10.1021/acsami.1c00907DOI Listing
April 2021

Screening of chemical linkers for development of pullulan bioconjugates for intravitreal ocular applications.

Eur J Pharm Sci 2021 Jun 2;161:105785. Epub 2021 Mar 2.

University of Padova, Department of Pharmaceutical and Pharmacological Sciences, Via F. Marzolo 5, 35131 Padova, Italy. Electronic address:

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 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.
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http://dx.doi.org/10.1016/j.ejps.2021.105785DOI Listing
June 2021

Preliminary Study of a 1,5-Benzodiazepine-Derivative Labelled with Indium-111 for CCK-2 Receptor Targeting.

Molecules 2021 Feb 9;26(4). Epub 2021 Feb 9.

Radiopharmaceutical Chemistry Section, Nuclear Medicine Unit, AUSL-IRCCS di Reggio Emilia, Viale Risorgimento 80, 42122 Reggio Emilia, Italy.

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 [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.
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http://dx.doi.org/10.3390/molecules26040918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916174PMC
February 2021

Glabrescione B delivery by self-assembling micelles efficiently inhibits tumor growth in preclinical models of Hedgehog-dependent medulloblastoma.

Cancer Lett 2021 02 26;499:220-231. Epub 2020 Nov 26.

Department of Molecular Medicine, University La Sapienza, Roma, Italy; Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Molecular Medicine, University La Sapienza, Roma, Italy. Electronic address:

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-cholane. mPEG-cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG-cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG-cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG-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-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.
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http://dx.doi.org/10.1016/j.canlet.2020.11.028DOI Listing
February 2021

Thermosensitive "Smart" Surfaces for Biorecognition Based Cell Adhesion and Controlled Detachment.

Macromol Biosci 2021 02 4;21(2):e2000277. Epub 2020 Nov 4.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, Padova, 35131, Italy.

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 -N-hydroxysuccinimide (NHS) and p(NIPam-co-Am) -bis-NHS with the surface aminopropyl groups and conjugation of GRGDS to the carboxylic acid termini of p(NIPam-co-Am) -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 /p(NIPam-co-Am) -GRGDS molar ratio. These data suggest the system can be exploited to modulate cell integrin/GRGDS binding for controlled cell capture and release.
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http://dx.doi.org/10.1002/mabi.202000277DOI Listing
February 2021

Phagocytosis and inflammation in crystal-induced arthritis: a synovial fluid and in vitro study.

Clin Exp Rheumatol 2021 May-Jun;39(3):494-500. Epub 2020 Jul 21.

Department of Medicine - DIMED, Campus Biomedico Pietro D'Abano, University of Padova, Italy.

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.
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May 2021

Preliminary evaluation of the production of non-carrier added Ag as core of a therapeutic radiopharmaceutical in the framework of ISOLPHARM_Ag experiment.

Appl Radiat Isot 2020 Oct 6;164:109258. Epub 2020 Jul 6.

Legnaro National Laboratories, National Institute of Nuclear Physics, 35020, Legnaro (Padua), Italy.

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 Ag, a β emitter potentially interesting for therapeutic applications. Thus, in the framework of ISOLPHARM, INFN supported a two-years experiment, called ISOLPHARM_Ag, aimed at evaluating the feasibility of the production of aAg 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 Ag in-target yields, whereas experiments with stable Ag were performed to test the ionization, transport and collection of Ag beams.
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http://dx.doi.org/10.1016/j.apradiso.2020.109258DOI Listing
October 2020

Recellularized Colorectal Cancer Patient-derived Scaffolds as in vitro Pre-clinical 3D Model for Drug Screening.

Cancers (Basel) 2020 Mar 13;12(3). Epub 2020 Mar 13.

Fondazione Istituto di Ricerca Pediatrica Città della Speranza, 35129 Padua, Italy.

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 () 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) 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 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.
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http://dx.doi.org/10.3390/cancers12030681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140024PMC
March 2020

Dexamethasone Loaded Liposomes by Thin-Film Hydration and Microfluidic Procedures: Formulation Challenges.

Int J Mol Sci 2020 Feb 26;21(5). Epub 2020 Feb 26.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy.

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.
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http://dx.doi.org/10.3390/ijms21051611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084920PMC
February 2020

In Vitro and in Vivo Behavior of Liposomes Decorated with PEGs with Different Chemical Features.

Mol Pharm 2020 Apr 21;17(4):1444. Epub 2020 Feb 21.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, 35131 Padova, Italy.

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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00149DOI Listing
April 2020

In Vitro and in Vivo Behavior of Liposomes Decorated with PEGs with Different Chemical Features.

Mol Pharm 2020 02 22;17(2):472-487. Epub 2020 Jan 22.

Department of Pharmaceutical and Pharmacological Sciences , University of Padova , via F. Marzolo 5 , 35131 Padova , Italy.

The colloidal stability, in vitro toxicity, cell association, and in vivo pharmacokinetic behavior of liposomes decorated with monomethoxy-poly(ethylene glycol)-lipids (mPEG-lipids) with different chemical features were comparatively investigated. Structural differences of the mPEG-lipids used in the study included: (a) surface-anchoring moiety [1,2-distearoyl--glycero-3-phosphoethanolamine (DSPE), cholesterol (Chol), and cholane (Chln)]; (b) mPEG molecular weight (2 kDa mPEG and 5 kDa mPEG); and (c) mPEG shape (linear and branched PEG). In vitro results demonstrated that branched (mPEG)-DSPE confers the highest stealth properties to liposomes (∼31-fold lower cell association than naked liposomes) with respect to all PEGylating agents tested. However, the pharmacokinetic studies showed that the use of cholesterol as anchoring group yields PEGylated liposomes with longer permeance in the circulation and higher systemic bioavailability among the tested formulations. Liposomes decorated with mPEG-Chol had 3.2- and ∼2.1-fold higher area under curve (AUC) than naked liposomes and branched (mPEG)-DSPE-coated liposomes, respectively, which reflects the high stability of this coating agent. By comparing the PEGylating agents with same size, namely, linear 5 kDa PEG derivatives, linear mPEG-DSPE yielded coated liposomes with the best in vitro stealth performance. Nevertheless, the in vivo AUC of liposomes decorated with linear mPEG-DSPE was lower than that obtained with liposomes decorated with linear mPEG-Chol. Computational molecular dynamics modeling provided additional insights that complement the experimental results.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00887DOI Listing
February 2020

Real-Time Label-Free Targeting Assessment and in Vitro Characterization of Curcumin-Loaded Poly-lactic--glycolic Acid Nanoparticles for Oral Colon Targeting.

ACS Omega 2019 Oct 1;4(16):16878-16890. Epub 2019 Oct 1.

Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00014 Helsinki, Finland.

The exploitation of curcumin for oral disease treatment is limited by its low solubility, poor bioavailability, and low stability. Surface-functionalized poly-lactic--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 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.
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http://dx.doi.org/10.1021/acsomega.9b02086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796886PMC
October 2019

Physical PEGylation to Prevent Insulin Fibrillation.

J Pharm Sci 2020 01 19;109(1):900-910. Epub 2019 Oct 19.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, 35131 Padova, Italy.

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-cholane and mPEG-cholesterol, respectively. Microcalorimetric analyses showed that mPEG-cholane and mPEG-cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 10 and 1.14 10 M, 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-cholesterol has superior stabilizing effect of the protein conformation than mPEG-cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG-cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG-cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG-cholane and mPEG-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.
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http://dx.doi.org/10.1016/j.xphs.2019.10.020DOI Listing
January 2020

H-NMR metabolomics reveals the Glabrescione B exacerbation of glycolytic metabolism beside the cell growth inhibitory effect in glioma.

Cell Commun Signal 2019 08 28;17(1):108. Epub 2019 Aug 28.

IRCCS Neuromed, Pozzilli, IS, Italy.

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 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-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 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.
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http://dx.doi.org/10.1186/s12964-019-0421-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712882PMC
August 2019

Oligo-guanidyl targeted bioconjugates forming rod shaped polyplexes as a new nanoplatform for oligonucleotide delivery.

J Control Release 2019 09 7;310:58-73. Epub 2019 Aug 7.

Departement of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo, 5, 35131 Padova, Italy.

Novel bioconjugates (Agm-M-PEG-FA) for active oligonucleotide (ON) delivery have been developed by conjugating a cationic oligo-guanidyl star-like shaped "head" (Agm-M) to a polymeric "tail" (PEG) terminating with folic acid (FA) as targeting agent or methoxy group (Agm-M-PEG-FA and Agm-M-PEG-OCH, 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-M-PEG-OCH:Agm-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-M-PEG-OCH 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.
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http://dx.doi.org/10.1016/j.jconrel.2019.08.005DOI Listing
September 2019

Novel Oligo-Guanidyl-PEG Carrier Forming Rod-Shaped Polyplexes.

Mol Pharm 2019 04 21;16(4):1678-1693. Epub 2019 Mar 21.

Department of Pharmaceutical and Pharmacological Sciences , University of Padova , Via F. Marzolo 5 35131 Padova , Italy.

A novel unconventional supramolecular oligo-cationic structure (Agm-M-PEG-OCH) has been synthesized to yield high efficiency therapeutic oligonucleotide (ON) delivery. Agm-M-PEG-OCH was obtained by a multistep protocol that included the conjugation of agmatine (Agm) moieties to maltotriose (M), which was further derivatized with one poly(ethylene glycol) (PEG) chain. Gel electrophoresis analysis showed that the 19 base pairs dsDNA model ON completely associates with Agm-M-PEG-OCH at 3 N/P molar ratio, which is in agreement with the in silico molecular predictions. Isothermal titration calorimetry (ITC) analyses showed that the Agm-M-PEG-OCH/ON association occurs through a combination of mechanisms depending on the N/P ratios resulting in different nanostructures. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the Agm-M-PEG-OCH/ON polyplexes have rod-shape structure with a mean diameter of 50-75 nm and aspect ratio depending on the N/P ratio. The polyplexes were stable over time in buffer, while a slight size increase was observed in the presence of serum proteins. Cell culture studies showed that neither Agm-M-PEG-OCH nor polyplexes displayed cytotoxic effects. Cellular uptake depended on the cell line and polyplex composition: cellular internalization was higher in the case of MCF-7 and KB cells compared to MC3T3-E1 cells and polyplexes with smaller aspect ratio were taken-up by cells more efficiently than polyplexes with higher aspect ratio. Finally, preliminary studies showed that our novel carrier efficiently delivered ONs into cells providing gene silencing.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00014DOI Listing
April 2019

Control of Peptide Aggregation and Fibrillation by Physical PEGylation.

Biomacromolecules 2018 10 4;19(10):3958-3969. Epub 2018 Sep 4.

Department of Pharmaceutical and Pharmacological Sciences , Università degli Studi di Padova , via F. Marzolo 5 , 35131 Padova , Italy.

Peptide therapeutics have the potential to self-associate, leading to aggregation and fibrillation. Noncovalent PEGylation offers a strategy to improve their physical stability; an understanding of the behavior of the resulting polymer/peptide complexes is, however, required. In this study, we have performed a set of experiments with additional mechanistic insight provided by in silico simulations to characterize the molecular organization of these complexes. We used palmitoylated vasoactive intestinal peptide (VIP-palm) stabilized by methoxy-poly(ethylene glycol)-cholane (PEG-cholane) as our model system. Homogeneous supramolecular assemblies were found only when complexes of PEG-cholane/VIP-palm exceeded a molar ratio of 2:1; at and above this ratio, the simulations showed minimal exposure of VIP-palm to the solvent. Supramolecular assemblies formed, composed of, on average, 9-11 PEG-cholane/VIP-palm complexes with 2:1 stoichiometry. Our in silico results showed the structural content of the helical conformation in VIP-palm increases when it is complexed with the PEG-cholane molecule; this behavior becomes yet more pronounced when these complexes assemble into larger supramolecular assemblies. Our experimental results support this: the extent to which VIP-palm loses helical structure as a result of thermal denaturation was inversely related to the PEG-cholane:VIP-palm molar ratio. The addition of divalent buffer species and increasing the ionic strength of the solution both accelerate the formation of VIP-palm fibrils, which was partially and fully suppressed by 2 and >4 mol equivalents of PEG-cholane, respectively. We conclude that the relative freedom of the VIP-palm backbone to adopt nonhelical conformations is a key step in the aggregation pathway.
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http://dx.doi.org/10.1021/acs.biomac.8b00887DOI Listing
October 2018

Chitosans as new tools against biofilms formation on the surface of silicone urinary catheters.

Int J Biol Macromol 2018 Oct 19;118(Pt B):2193-2200. Epub 2018 Jul 19.

Department of Biomolecular Sciences, University of Urbino, 61029 Urbino, PU, Italy.

Urinary catheters contamination by microorganisms is a major cause of hospital acquired infections and represents a limitation for long-term use. In this work, biofilms of Klebsiella pneumoniae and Escherichia coli clinical isolates were developed on urinary catheters for 48 and 72 h in artificial urine medium (AUM) with different molecular weight chitosans (AUM-CS solutions) at pH 5.0. The number of viable bacteria was determined by standard plate count agar while crystal violet (CV) staining was carried out to assess biomass production (optical density at 570 nm) in the mentioned conditions. Re-growth of each strain was also evaluated after 24 h re-incubation of the treated catheters. Significant decreases of log CFU/catheter and biomass production were observed for all the biofilms developed in AUM-CS compared with the controls in AUM. The percentages of biofilm removal were slightly higher for E. coli biofilms (up to 90.4%) than those of K. pneumoniae (89.7%); in most cases, the complete inhibition of bacterial re-growth on treated catheter pieces was observed. Contact time influenced chitosan efficacy rather than its molecular weight or the biofilms age. The results confirmed the potentiality of chitosans as a biomacromolecule tool to contrast biofilm formation and reduce bacterial re-growth on urinary catheters.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.07.088DOI Listing
October 2018

pH-Controlled Liposomes for Enhanced Cell Penetration in Tumor Environment.

ACS Appl Mater Interfaces 2018 May 15;10(21):17646-17661. Epub 2018 May 15.

Department of Pharmaceutical and Pharmacological Sciences , University of Padova , Via F. Marzolo 5 , Padova 35131 , Italy.

An innovative pH-switchable colloidal system that can be exploited for site-selective anticancer drug delivery has been generated by liposome decoration with a new novel synthetic non-peptidic oligo-arginine cell-penetration enhancer (CPE) and a quenching PEGylated counterpart that detaches from the vesicle surface under the acidic conditions of tumors. The CPE module ( Arg- DAG) is formed by four arginine units conjugated to a first-generation (G1) 2,2-bis(hydroxymethyl)propionic acid (bis-MPA)/2,2-bis(aminomethyl)propionic acid (bis-AMPA) polyester dendron terminating with 1,2-distearoyl-3-azidopropane for liposome bilayer insertion. The zeta potential of the Arg- DAG-decorated liposomes increased up to +32 mV as the Arg- DAG/lipids molar ratio increased. The Arg- DAG liposome shielding at pH 7.4 was provided by methoxy-PEG-polymethacryloyl sulfadimethoxine (mPEG-SDM) with 7.1 apparent p K. Zeta potential, surface plasmon resonance and synchrotron small-angle X-ray scattering analyses showed that at pH 7.4 mPEG-SDM associates with polycationic Arg- DAG-decorated liposomes yielding liposomes with neutral zeta potential. At pH 6.5, which mimics the tumor environment, mPEG-SDM detaches from the liposome surface yielding Arg- DAG exposure. Flow cytometry and confocal microscopy showed a 30-fold higher HeLa cancer cell association of the Arg- DAG-decorated liposomes compared to non-decorated liposomes. At pH 7.4, the mPEG-SDM-coated liposomes undergo low cell association while remarkable cell association occurred at pH 6.5, which allowed for the controlled intracellular delivery of model macromolecules and small molecules loaded in the liposome under tumor conditions.
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http://dx.doi.org/10.1021/acsami.8b03469DOI Listing
May 2018

Enhanced uptake in 2D- and 3D- lung cancer cell models of redox responsive PEGylated nanoparticles with sensitivity to reducing extra- and intracellular environments.

J Control Release 2018 05 10;277:126-141. Epub 2018 Mar 10.

Division of Molecular Therapeutics and Formulation, School of Pharmacy, University of Nottingham, UK. Electronic address:

In the treatment of lung cancer, there is an urgent need of innovative medicines to optimize pharmacological responses of conventional chemotherapeutics while attenuating side effects. Here, we have exploited some relatively unexplored subtle differences in reduction potential, associated with cancer cell microenvironments in addition to the well-known changes in intracellular redox environment. We report the synthesis and application of novel redox-responsive PLGA (poly(lactic-co-glycolic acid)) -PEG (polyethylene glycol) nanoparticles (RR-NPs) programmed to change surface properties when entering tumor microenvironments, thus enhancing cell internalization of the particles and their drug cargo. The new co-polymers, in which PEG and PLGA were linked by 'anchiomeric effector' dithiylethanoate esters, were synthesized by a combination of ring-opening polymerization and Michael addition reactions and employed to prepare NPs. Non redox-responsive nanoparticles (nRR-NPs) based on related PLGA-PEG copolymers were also prepared as comparators. Spherical NPs of around 120 nm diameter with a low polydispersity index and negative zeta potential as well as good drug loading of docetaxel were obtained. The NPs showed prolonged stability in relevant simulated biological fluids and a high ability to penetrate an artificial mucus layer due to the presence of the external PEG coating. Stability, FRET and drug release studies in conditions simulating intracellular reductive environments demonstrated a fast disassembly of the external shell of the NPs, thus triggering on-demand drug release. FACS measurements and confocal microscopy showed increased and faster uptake of RR-NPs in both 2D- and 3D- cell culture models of lung cancer compared to nRR-NPs. In particular, the 'designed-in' reductive instability of RR-NPs in conditioned cell media, the fast PEG release in the extracellular compartment, as well as a diminution of uptake rate in control experiments where extracellular thiols were neutralized, suggested a partial extracellular release of the PEG fringe that promoted rapid internalization of the residual NPs into cells. Taken together, these results provide further evidence of the effectiveness of PEGylated reducible nanocarriers to permeate mucus layer barriers, and establish a new means to enhance cancer cell uptake of drug carriers by extra-and intra-cellular cleavage of protein- and cell-shielding hydrophilic blocks.
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http://dx.doi.org/10.1016/j.jconrel.2018.03.011DOI Listing
May 2018

Switching of Macromolecular Ligand Display by Thermoresponsive Polymers Mediates Endocytosis of Multiconjugate Nanoparticles.

Bioconjug Chem 2018 04 14;29(4):1030-1046. Epub 2018 Mar 14.

School of Pharmacy , University of Nottingham , University Park , Nottingham NG72RD , United Kingdom.

Ligand-mediated targeting and internalization of plasma membrane receptors is central to cellular function. These types of receptors have accordingly been investigated as targets to facilitate entry of diagnostic and therapeutic constructs into cells. However, there remains a need to characterize how receptor targeting agents on nanoparticles interact at surface receptors and whether it is possible to control these interactions via exogenous stimuli. Here, we describe the switchable display of the iron-transporting protein, transferrin (Tf), at the surface of thermoresponsive polymer-coated gold nanoparticles and show that internalization of the coated nanoparticles into target cells changes across temperature ranges over which transferrin is expected to be sterically "hidden" by an extended polymer chain and then "revealed" by polymer chain collapse. The switching process is dependent on the numbers of transferrin molecules and thermoresponsive polymer chains attached and whether the assay temperature is above or below the transition temperatures of the responsive polymers at the nanoparticle surfaces. Significantly, however, the control of internalization is critically reliant on overall nanoparticle colloidal stability while the thermoresponsive component of the surface undergoes conformational change. The data show that the cell entry function of complex and large biomolecule ligands can be modulated by polymer-induced accessibility change but that a simple "hide and reveal" mechanism for ligand display following polymer chain collapse is insufficient to account for nanoparticle uptake and subsequent intracellular trafficking.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00704DOI Listing
April 2018

Chitosan Loaded into a Hydrogel Delivery System as a Strategy to Treat Vaginal Co-Infection.

Pharmaceutics 2018 Feb 3;10(1). Epub 2018 Feb 3.

Department of Biomolecular Sciences, University of Urbino, Piazza del Rinascimento n° 6, 61029 Urbino (PU), Italy.

Polymeric hydrogels are common dosage forms designed for the topical administration of antimicrobial drugs to treat vaginal infections. One of the major advantages of using chitosan in these formulations is related to the intrinsic and broad antimicrobial activity exerted on bacteria and fungi by this natural polymer. Most vaginal yeast infections are caused by the pathogenic fungus . However, despite the anti-Candida activity towards and strains susceptibility to low molecular weight chitosan being documented, no information is available regarding the antimicrobial efficacy of mixed hydrogels in which chitosan is dispersed in a polymeric matrix. Therefore, the aim of the study is to evaluate the anti-Candida activity against eight different albicans and non-albicans strains of a mixed hydroxypropyl methylcellulose (HPMC)/chitosan hydrogel. Importantly, chitosan was dispersed in HPMC matrix either assembled in nanoparticles or in a monomolecular state to eventually correlate any variation in terms of rheological and mucoadhesive properties, as well as anti-Candida activity, with the chitosan form. Hydrogels containing 1% / chitosan, either as free polymer chain or assembled in nanoparticles, showed an improved mucoadhesiveness and an anti-Candida effect against all tested albicans and non-albicans strains. Overall, the results demonstrate the feasibility of preparing HPMC/CS mixed hydrogels intended for the prevention and treatment of Candida infections after vaginal administration.
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http://dx.doi.org/10.3390/pharmaceutics10010023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874836PMC
February 2018

New molecular targets for functionalized nanosized drug delivery systems in personalized therapy for hepatocellular carcinoma.

J Control Release 2017 12 16;268:184-197. Epub 2017 Oct 16.

Department of Medicine, University of Padova, Padova, Italy.

Hepatocellular carcinoma, the most frequent solid tumor of the liver, has a very poor prognosis, being the second most common cause of death from cancer worldwide. The incidence and mortality of this liver tumor are increasing in most areas of the world as a consequence of aging and the emerging of new risk factors such as the metabolic syndrome, beside the recognized role of hepatitis B and C viral infections and alcohol abuse. Despite the increasing knowledge on the molecular mechanisms underlying hepatic carcinogenesis, effective therapeutic strategies are still an unmet clinical need. Efforts have been made to develop selective drugs as well as effective targeted drug delivery systems. The development of novel drug carriers for therapeutic molecules can indeed offer a valuable strategy to ameliorate the efficacy of HCC treatment. In this review, we discuss recent drug delivery strategies for HCC treatment based on the exploitation of targeted nanoparticles (NPs). Indeed, a few of these platforms have achieved an advanced stage of preclinical development. Here, we review the most promising drug nanovehicles based on both synthetic and natural polymers, including polysaccharides that have emerged for their biocompatibility and biodegradability. To maximize site-selectivity and therapeutic efficacy, drug delivery systems should be functionalized with ligands which can specifically recognize and bind targets expressed by HCC, namely cell membrane associated antigens, receptors or biotransporters. Cell surface and intracellular molecular targets are exploited either to selectively deliver drug-loaded nanovehicles or to design novel selective therapeutics. In conclusion, the combination of novel and safe drug delivery strategies based on site-specific targeted drug nanovehicles with therapeutic molecular targets may significantly improve the pharmacological efficacy for the treatment of HCC.
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http://dx.doi.org/10.1016/j.jconrel.2017.10.027DOI Listing
December 2017

Control of aggregation temperatures in mixed and blended cytocompatible thermoresponsive block co-polymer nanoparticles.

Soft Matter 2017 Oct;13(40):7441-7452

School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, UK.

A small library of thermoresponsive amphiphilic copolymers based on polylactide-block-poly((2-(2-methoxyethoxy)ethyl methacrylate)-co-(oligoethylene glycol methacrylate)) (PLA-b-P(DEGMA)-co-(OEGMA)), was synthesised by copper-mediated controlled radical polymerisation (CRP) with increasing ratios of OEGMA : DEGMA. These polymers were combined in two ways to form nanoparticles with controllable thermal transition temperatures as measured by particle aggregation. The first technique involved the blending of two (PLA-b-P(DEGMA)-co-(OEGMA)) polymers together prior to assembling nanoparticles (NPs). The second method involved mixing pre-formed nanoparticles of single (PLA-b-P(DEGMA)-co-(OEGMA)) polymers. The observed critical aggregation temperature T did not change in a linear relationship with the ratios of each copolymer either in the nanoparticles blended from different copolymers or in the mixtures of pre-formed nanoparticles. However, where co-polymer mixtures were based on (OEG)MA ratios within 5-10 mole%, a linear relationship between (OEG)MA composition in the blends and T was obtained. The data suggest that OEGMA-based copolymers are tunable over a wide temperature range given suitable co-monomer content in the linear polymers or nanoparticles. Moreover, the thermal transitions of the nanoparticles were reversible and repeatable, with the cloud point curves being essentially invariant across at least three heating and cooling cycles, and a selected nanoparticle formulation was found to be readily endocytosed in representative cancer cells and fibroblasts.
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http://dx.doi.org/10.1039/c7sm00920hDOI Listing
October 2017

Polymers for binding of the gram-positive oral pathogen Streptococcus mutans.

PLoS One 2017 3;12(7):e0180087. Epub 2017 Jul 3.

School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.

Streptococcus mutans is the most significant pathogenic bacterium implicated in the formation of dental caries and, both directly and indirectly, has been associated with severe conditions such as multiple sclerosis, cerebrovascular and peripheral artery disease. Polymers able to selectively bind S. mutans and/or inhibit its adhesion to oral tissue in a non-lethal manner would offer possibilities for addressing pathogenicity without selecting for populations resistant against bactericidal agents. In the present work two libraries of 2-(dimethylamino)ethyl methacrylate (pDMAEMA)-based polymers were synthesized with various proportions of either N,N,N-trimethylethanaminium cationic- or sulfobetaine zwitterionic groups. These copolymers where initially tested as potential macromolecular ligands for S. mutans NCTC 10449, whilst Escherichia coli MG1655 was used as Gram-negative control bacteria. pDMAEMA-derived materials with high proportions of zwitterionic repeating units were found to be selective for S. mutans, in both isolated and S. mutans-E. coli mixed bacterial cultures. Fully sulfobetainized pDMAEMA was subsequently found to bind/cluster preferentially Gram-positive S. mutans and S. aureus compared to Gram negative E. coli and V. harveyi. A key initial stage of S. mutans pathogenesis involves a lectin-mediated adhesion to the tooth surface, thus the range of potential macromolecular ligands was further expanded by investigating two glycopolymers bearing α-mannopyranoside and β-galactopyranoside pendant units. Results with these polymers indicated that preferential binding to either S. mutans or E. coli can be obtained by modulating the glycosylation pattern of the chosen multivalent ligands without incurring unacceptable cytotoxicity in a model gastrointestinal cell line. Overall, our results allowed to identify a structure-property relationship for the potential antimicrobial polymers investigated, and suggest that preferential binding to Gram-positive S. mutans could be achieved by fine-tuning of the recognition elements in the polymer ligands.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180087PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495209PMC
October 2017

Engineered Polymer-Transferrin Conjugates as Self-Assembling Targeted Drug Delivery Systems.

Biomacromolecules 2017 May 11;18(5):1532-1543. Epub 2017 Apr 11.

School of Pharmacy, University of Nottingham , University Park, Nottingham NG7 2RD, United Kingdom.

Polymer-protein conjugates can be engineered to self-assemble into discrete and well-defined drug delivery systems, which combine the advantages of receptor targeting and controlled drug release. We designed specific conjugates of the iron-binding and transport protein, transferrin (Tf), to combine the advantages of this serum-stable protein as a targeting agent for cancer cells with self-assembling polymers to act as carriers of cytotoxic drugs. Tf variants were expressed with cysteine residues at sites spanning different regions of the protein surface, and the polymer conjugates grown from these variants were compared with polymer conjugates grown from nonselectively derivatized sites on native Tf. The resulting synthetic biopolymer hybrids were evaluated for self-assembly properties, size and topology, ability to carry an anticancer drug (paclitaxel), and cytotoxicity with and without a drug payload in a representative human colon cancer cell line. The results demonstrated that the engineered Tf variant polymer conjugates formed better-defined self-assembled nanoparticles than the nonselectively derivatized conjugates and showed greater efficacy in paclitaxel delivery. A polymer conjugate grown from a specific Tf variant, S415C was found to be taken up rapidly into cancer cells expressing the Tf-receptor, and, while tolerated well by cells in the absence of drugs, was as cytotoxic as free paclitaxel, when loaded with the drug. Importantly, the S415C conjugate polymer was not the most active variant in Tf-receptor binding, suggesting that the nanoscale self-assembly of the polymer-protein hybrid is also a key factor in delivery efficacy. The data overall suggest new design rules for polymer-biopolymer hybrids and therapeutic delivery systems, which include engineering specific residues for conjugation that mediate nanoscale assembly as well as control of ligand-receptor interactions to target specific cell types.
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http://dx.doi.org/10.1021/acs.biomac.7b00101DOI Listing
May 2017