Nuova ricerca

JASON THOMAS DUSKEY

Assegnista di ricerca presso: Dipartimento di Scienze della Vita sede ex-Scienze Farmaceutiche


Home |


Pubblicazioni

2021 - Glioblastoma: State of the Art of Treatments and Applications of Polymeric and Lipidic Nanomedicines [Capitolo/Saggio]
Sgarbi, V.; Duskey, J. T.; Ottonelli, I.; Da Ros, F.; Oddone, N.; Vandelli, M. A.; Forni, F.; Tosi, G.; Ruozi, B.
abstract

Glioblastoma multiforme (GBM) is one of the most devastating tumors affecting more than 5 in 100,000 people. Unfortunately, its diagnosis is often discovered in late stages and is normally deadly, having a life expectancy of 12–15 months and a mere 3% of the affected patients living 3 years or more independent of race, sex, and age. Sadly, current treatments (i.e., chemotherapy, radiation, surgery) are extremely aggressive and extend the patient’s life by little more than a year on average. Even when treatment appears successful, relapse is often experienced. These extreme treatments, combined with their lack of long-term success, call for new innovations. Among them, nanomedicine becomes one of the most promising approaches regarding possible applications in advancing or ameliorating GBM management. In this chapter, we will therefore analyze the state of the art and the most novel and outstanding innovation in terms of diagnosis and treatment options.


2021 - Insights into kinetics, release, and behavioral effects of brain-targeted hybrid nanoparticles for cholesterol delivery in Huntington's disease [Articolo su rivista]
Birolini, Giulia; Valenza, Marta; Ottonelli, Ilaria; Passoni, Alice; Favagrossa, Monica; Duskey, Jason T; Bombaci, Mauro; Vandelli, Maria Angela; Colombo, Laura; Bagnati, Renzo; Caccia, Claudio; Leoni, Valerio; Taroni, Franco; Forni, Flavio; Ruozi, Barbara; Salmona, Mario; Tosi, Giovanni; Cattaneo, Elena
abstract

Supplementing brain cholesterol is emerging as a potential treatment for Huntington's disease (HD), a genetic neurodegenerative disorder characterized, among other abnormalities, by inefficient brain cholesterol biosynthesis. However, delivering cholesterol to the brain is challenging due to the blood-brain barrier (BBB), which prevents it from reaching the striatum, especially, with therapeutically relevant doses. Here we describe the distribution, kinetics, release, and safety of novel hybrid polymeric nanoparticles made of PLGA and cholesterol which were modified with an heptapeptide (g7) for BBB transit (hybrid-g7-NPs-chol). We show that these NPs rapidly reach the brain and target neural cells. Moreover, deuterium-labeled cholesterol from hybrid-g7-NPs-chol is released in a controlled manner within the brain and accumulates over time, while being rapidly removed from peripheral tissues and plasma. We confirm that systemic and repeated injections of the new hybrid-g7-NPs-chol enhanced endogenous cholesterol biosynthesis, prevented cognitive decline, and ameliorated motor defects in HD animals, without any inflammatory reaction. In summary, this study provides insights about the benefits and safety of cholesterol delivery through advanced brain-permeable nanoparticles for HD treatment.


2020 - Development and biocompatibility assessments of poly(3-hydroxybutyrate-co-ε-caprolactone) microparticles for diclofenac sodium delivery [Articolo su rivista]
Musumeci, T.; Bonaccorso, A.; Carbone, C.; Impallomeni, G.; Ballistreri, A.; Duskey, J. T.; Puglisi, G.; Pignatello, R.
abstract

Osteoarthritis (OA) is a debilitating disease affecting joints and impairing the ability to perform everyday tasks. Current treatment regimens tend to provide little to no relief. Therefore, there is a huge need for alternative strategies to manage this painful condition. The delivery of anti-inflammatory drugs into an injured joint with the aim of eliminating articular inflammation and modulate cartilage damage could be a useful strategy to treat OA. Accordingly, the aim of this study is to prepare microparticles (MPs) from new biodegradable poly (3-hydroxybutyrate-co-ε-caprolactone) copolymers (PHBCL) for the potential intra-articular injection of Diclofenac sodium for OA treatment. MPs were prepared starting from copolymers having different molecular weights and an HB/CL molar ratio and studied for their morphologies and size distribution by scanning electron microscopy. Drug loading and encapsulation efficiency were also determined. The in vitro release profile was assessed by the dialysis bag technique and the kinetic profile was evaluated by using several mathematical models revealing a diffusion release mechanism. A1 polymer and related MPs, as representative of the group, were selected for further biological investigation. In vitro studies performed on CaCo-2 and Balb/3T3 cells showed no toxic effects at the desired concentrations as revealed by MTT, CFE and Comet assays.


2020 - Drug delivery across the blood–brain barrier: recent advances in the use of nanocarriers [Articolo su rivista]
Mulvihill, John JE; Cunnane, Eoghan M; Ross, Aisling M; Duskey, Jason T; Tosi, Giovanni; Grabrucker, Andreas M
abstract

The blood-brain barrier (BBB) has a significant contribution to homeostasis and protection of the CNS. However, it also limits the crossing of therapeutics and thereby complicates the treatment of CNS disorders. To overcome this limitation, the use of nanocarriers for drug delivery across the BBB has recently been exploited. Nanocarriers can utilize different physiological mechanisms for drug delivery across the BBB and can be modified to achieve the desired kinetics and efficacy. Consequentially, several nanocarriers have been reported to act as functional nanomedicines in preclinical studies using animal models for human diseases. Given the rapid development of novel nanocarriers, this review provides a comprehensive insight into the most recent advancements made in nanocarrier-based drug delivery to the CNS, such as the development of multifunctional nanomedicines and theranostics.


2020 - Enzyme Stability in Nanoparticle Preparations Part 1: Bovine Serum Albumin Improves Enzyme Function [Articolo su rivista]
Duskey, Jason Thomas; da Ros Ilaria Ottonelli, Federica; Zambelli, Barbara; Vandelli, Maria Angela; Tosi, Giovanni; Ruozi, Barbara
abstract


2020 - Investigating Novel Syntheses of a Series of Unique Hybrid PLGA-Chitosan Polymers for Potential Therapeutic Delivery Applications [Articolo su rivista]
Duskey, Jason Thomas; Baraldi, Cecilia; Gamberini, Maria Cristina; Ottonelli, Ilaria; Da Ros, Federica; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela; Ruozi, Barbara
abstract

Discovering new materials to aid in the therapeutic delivery of drugs is in high demand. PLGA, a FDA approved polymer, is well known in the literature to form films or nanoparticles that can load, protect, and deliver drug molecules; however, its incompatibility with certain drugs (due to hydrophilicity or charge repulsion interactions) limits its use. Combining PLGA or other polymers such as polycaprolactone with other safe and positively-charged molecules, such as chitosan, has been sought after to make hybrid systems that are more flexible in terms of loading ability, but often the reactions for polymer coupling use harsh conditions, films, unpurified products, or create a single unoptimized product. In this work, we aimed to investigate possible innovative improvements regarding two synthetic procedures. Two methods were attempted and analytically compared using nuclear magnetic resonance (NMR), fourier-transform infrared spectroscopy (FT-IR), and dynamic scanning calorimetry (DSC) to furnish pure, homogenous, and tunable PLGA-chitosan hybrid polymers. These were fully characterized by analytical methods. A series of hybrids was produced that could be used to increase the suitability of PLGA with previously non-compatible drug molecules


2020 - Nanoparticles as carriers for drug delivery of macromolecules across the blood-brain barrier [Articolo su rivista]
Tosi, Giovanni; Duskey, J. T.; Kreuter, Jörg
abstract

Introduction: Current therapies of neurodegenerative or neurometabolic diseases are, to a large extent, hampered by the inability of drugs to cross the blood-brain barrier (BBB). This very tight barrier severely restricts the entrance of molecules from the blood into the brain, especially macromolecular substances (i.e. neurotrophic factors, enzymes, proteins, as well as genetic materials). Due to their size, physicochemical properties, and instability, the delivery of these materials is particularly difficult.Areas covered: Recent research showed that biocompatible and biodegradable nanoparticles possessing tailored surface properties can enable a delivery of drugs and specifically of macromolecules across the blood-brain barrier by using carrier systems of the brain capillary endothelium (Trojan Horse strategy). In the present review, the state-of-art of nanoparticle-mediated drug delivery of different macromolecular substances into the brain following intravenous injection is summarized, and different nanomedicines that are used to enable the transport of neurotrophic factors and enzymes across the blood-brain barrier into the CNS are critically analyzed.Expert opinion: Brain delivery of macromolecules by an intravenous application using nanomedicines is now a growing area of interest which could be really translated into clinical application if dedicated effort will be given to industrial scale-up production.


2020 - Novel peptide-conjugated nanomedicines for brain targeting: In vivo evidence [Articolo su rivista]
Duskey, J. T.; Ottonelli, I.; Da Ros, F.; Vilella, A.; Zoli, M.; Kovachka, S.; Spyrakis, F.; Vandelli, M. A.; Tosi, G.; Ruozi, B.
abstract

Central nervous system (CNS) compartments remain one of the most difficult districts for drug delivery. This is due to the presence of the blood–brain barrier (BBB) that hampers 90% of drug passage, dramatically requiring non-invasive treatment strategies. Here, for the first time, the use of opioid-derived deltorphin-derivative peptides to drive biodegradable and biocompatible polymeric (i.e. poly-lactide-co-glycolide, PLGA) nanomedicines delivery across the BBB was described. Opioid-derived peptides were covalently conjugated to furnish activated polymers which were further used for fluorescently tagged nanoformulations. Beyond reporting production, formulation methodology and full physico-chemical characterization, in vivo tests generated clear proof of BBB crossing and CNS targeting by engineered nanomedicines opening the research to further applications of drug delivery and targeting in CNS disease models.


2020 - PLGA-PEG-ANG-2 Nanoparticles for Blood-Brain Barrier Crossing: Proof-of-Concept Study [Articolo su rivista]
Hoyos-Ceballos, Gina P; Ruozi, Barbara; Ottonelli, Ilaria; Da Ros, Federica; Vandelli, Maria Angela; Forni, Flavio; Daini, Eleonora; Vilella, Antonietta; Zoli, Michele; Tosi, Giovanni; Duskey, Jason T; López-Osorio, Betty L
abstract

The treatment of diseases that affect the central nervous system (CNS) represents a great research challenge due to the restriction imposed by the blood-brain barrier (BBB) to allow the passage of drugs into the brain. However, the use of modified nanomedicines engineered with different ligands that can be recognized by receptors expressed in the BBB offers a favorable alternative for this purpose. In this work, a BBB-penetrating peptide, angiopep-2 (Ang-2), was conjugated to poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles through pre- and post-formulation strategies. Then, their ability to cross the BBB was qualitatively assessed on an animal model. Proof-of-concept studies with fluorescent and confocal microscopy studies highlighted that the brain-targeted PLGA nanoparticles were able to cross the BBB and accumulated in neuronal cells, thus showing a promising brain drug delivery system.


2020 - Synthesis, Characterization, and In Vitro Studies of an Reactive Oxygen Species (ROS)-Responsive Methoxy Polyethylene Glycol-Thioketal-Melphalan Prodrug for Glioblastoma Treatment [Articolo su rivista]
Oddone, N.; Boury, F.; Garcion, E.; Grabrucker, A. M.; Martinez, M. C.; Da Ros, F.; Janaszewska, A.; Forni, F.; Vandelli, M. A.; Tosi, G.; Ruozi, B.; Duskey, J. T.
abstract

Glioblastoma (GBM) is the most frequent and aggressive primary tumor of the brain and averages a life expectancy in diagnosed patients of only 15 months. Hence, more effective therapies against this malignancy are urgently needed. Several diseases, including cancer, are featured by high levels of reactive oxygen species (ROS), which are possible GBM hallmarks to target or benefit from. Therefore, the covalent linkage of drugs to ROS-responsive molecules can be exploited aiming for a selective drug release within relevant pathological environments. In this work, we designed a new ROS-responsive prodrug by using Melphalan (MPH) covalently coupled with methoxy polyethylene glycol (mPEG) through a ROS-cleavable group thioketal (TK), demonstrating the capacity to self-assembly into nanosized micelles. Full chemical-physical characterization was conducted on the polymeric-prodrug and proper controls, along with in vitro cytotoxicity assayed on different GBM cell lines and “healthy” astrocyte cells confirming the absence of any cytotoxicity of the prodrug on healthy cells (i.e. astrocytes). These results were compared with the non-ROS responsive counterpart, underlining the anti-tumoral activity of ROS-responsive compared to the non-ROS-responsive prodrug on GBM cells expressing high levels of ROS. On the other hand, the combination treatment with this ROS-responsive prodrug and X-ray irradiation on human GBM cells resulted in an increase of the antitumoral effect, and this might be connected to radiotherapy. Hence, these results represent a starting point for a rationale design of innovative and tailored ROS-responsive prodrugs to be used in GBM therapy and in combination with radiotherapy.


2019 - Nanomedicine against Aβ aggregation by β–sheet breaker peptide delivery: In vitro evidence [Articolo su rivista]
Pederzoli, F.; Ruozi, B.; Duskey, J.; Hagmeyer, S.; Sauer, A. K.; Grabrucker, S.; Coelho, R.; Oddone, N.; Ottonelli, I.; Daini, E.; Zoli, M.; Vandelli, M. A.; Tosi, G.; Grabrucker, A. M.
abstract

The accumulation of amyloid β (Aβ) triggers a cascade of toxic events in Alzheimer’s disease (AD). The KLVFF peptide can interfere with Aβ aggregation. However, the peptide suffers from poor bioavailability and the inability to cross the blood–brain barrier. In this work, we study the possibility of adopting nanomedicine to overcome KLVFF limits in biodistribution. We produced new engineered polymeric nanoparticles (NPs), and we evaluated the cellular toxicity of these NPs and validated that KVLFF peptides released by NPs show the same promising effects on AD pathology. Our results revealed the successful generation of KVLFF loaded NPs that, without significant effects on cell heath, are even more potent in reversing Aβ-induced pathologies compared to the free peptide. Therefore, NPs will significantly advance KVLFF treatment as a therapeutic option for AD.


2019 - Nanomedicine in Alzheimer's disease: Amyloid beta targeting strategy [Capitolo/Saggio]
Tosi, Giovanni; Pederzoli, Francesca; Belletti, Daniela; Vandelli, Maria Angela; Forni, Flavio; Duskey, Jason Thomas; Ruozi, Barbara
abstract

The treatment of Alzheimer's disease (AD) is up to today one of the most unsuccessful examples of biomedical science. Despite the high number of literature evidences detailing the multifactorial and complex etiopathology of AD, no cure is yet present on the market and the available treatments are only symptomatic. The reasons could be ascribed on two main factors: (i) lack of ability of the majority of drugs to cross the blood-brain barrier (BBB), thus excluding the brain for any successful therapy; (ii) lack of selectivity and specificity of drugs, decreasing the efficacy of even potent anti-AD drugs. The exploitation of specifically engineered nanomedicines planned to cross the BBB and to target the most “hot” site of action (i.e., β-amyloid) is one of the most interesting innovations in drug delivery and could reasonably represent an promising choice for possible treatments and even early-diagnosis of AD. In this chapter, we therefore outline the most talented approaches in AD treatment with a specific focus on the main advantages/drawbacks and future possible translation to clinic application.


2019 - ROS-responsive “smart” polymeric conjugate: Synthesis, characterization and proof-of-concept study [Articolo su rivista]
Oddone, N.; Pederzoli, F.; Duskey, J. T.; De Benedictis, C. A.; Grabrucker, A. M.; Forni, F.; Vandelli, M. A.; Ruozi, B.; Tosi, G.
abstract

New approaches integrating stimuli-responsive linkers into prodrugs are currently emerging. These “smart” prodrugs can enhance the effectivity of conventional prodrugs with promising clinical applicability. Oxidative stress is central to several diseases, including cancer. Therefore, the design of prodrugs that respond to ROS stimulus, allowing a selective drug release in this condition, is fairly encouraging. Aiming to investigate the ROS-responsiveness of prodrugs containing the ROS-cleavable moiety, Thioketal (TK), we performed proof-of-concept studies by synthesizing ROS-responsive conjugate, namely mPEG-TK-Cy5, through exploiting Cy5 fluorescent dye. We demonstrated that, differently to non-ROS-responsive control conjugate (mPEG-Cy5), mPEG-TK-Cy5 shows a selective release of Cy5 in response to ROS in both, ROS-simulated conditions and in vitro on glioblastoma cells. Our results confirm the applicability of TK-technology in the design of ROS-responsive prodrugs, which constitutes a promising approach in cancer treatment. The translatability of this technology for other diseases treatment makes this a highly relevant and promising approach.


2019 - Targeting Brain Disease in MPSII: Preclinical Evaluation of IDS-Loaded PLGA Nanoparticles [Articolo su rivista]
Rigon, Laura; Salvalaio, Marika; Pederzoli, Francesca; Legnini, Elisa; Duskey, Jason Thomas; D'Avanzo, Francesca; De Filippis, Concetta; Ruozi, Barbara; Marin, Oriano; Vandelli, Maria Angela; Ottonelli, Ilaria; Scarpa, Maurizio; Tosi, Giovanni; Tomanin, Rosella
abstract

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the enzyme iduronate 2-sulfatase (IDS), which leads to the accumulation of glycosaminoglycans in most organ-systems, including the brain, and resulting in neurological involvement in about two-thirds of the patients. The main treatment is represented by a weekly infusion of the functional enzyme, which cannot cross the blood-brain barrier and reach the central nervous system. In this study, a tailored nanomedicine approach based on brain-targeted polymeric nanoparticles (g7-NPs), loaded with the therapeutic enzyme, was exploited. Fibroblasts from MPSII patients were treated for 7 days with NPs loaded with the IDS enzyme; an induced IDS activity like the one detected in healthy cells was measured, together with a reduction of GAG content to non-pathological levels. An in vivo short-term study in MPSII mice was performed by weekly administration of g7-NPs-IDS. Biochemical, histological, and immunohistochemical evaluations of liver and brain were performed. The 6-weeks treatment produced a significant reduction of GAG deposits in liver and brain tissues, as well as a reduction of some neurological and inflammatory markers (i.e., LAMP2, CD68, GFAP), highlighting a general improvement of the brain pathology. The g7-NPs-IDS approach allowed a brain-targeted enzyme replacement therapy. Based on these positive results, the future aim will be to optimize NP formulation further to gain a higher efficacy of the proposed approach.


2018 - Hybrid nanoparticles as a new technological approach to enhance the delivery of cholesterol into the brain [Articolo su rivista]
Belletti, Daniela; Grabrucker, Andreas Martin; Pederzoli, Francesca; Menerath, Isabel; Vandelli, Maria Angela; Tosi, Giovanni; Duskey, Thomas Jason; Forni, Flavio; Ruozi, Barbara
abstract

Restoration of the Chol homeostasis in the Central Nervous System (CNS) could be beneficial for the treatment of Huntington's Disease (HD), a progressive, fatal, adult-onset, neurodegenerative disorder. Unfortunately, Chol is unable to cross the blood–brain barrier (BBB), thus a novel strategy for a targeted delivery of Chol into the brain is highly desired. This article aims to investigate the production of hybrid nanoparticles composed by Chol and PLGA (MIX-NPs) modified with g7 ligand for BBB crossing. We described the impact of ratio between components (Chol and PLGA) and formulation process (nanoprecipitation or single emulsion process) on physico-chemical and structural characteristics, we tested MIX-NPs in vitro using primary hippocampal cell cultures evaluating possible toxicity, uptake, and the ability to influence excitatory synaptic receptors. Our results elucidated that both formulation processes produce MIX-NPs with a Chol content higher that 40%, meaning that Chol is a structural particle component and active compound at the same time. The formulation strategy impacted the architecture and reorganization of components leading to some differences in Chol availability between the two types of g7 MIX-NPs. Our results identified that both kinds of MIX-NPs are efficiently taken up by neurons, able to escape lysosomes and release Chol into the cells resulting in an efficient modification in expression of synaptic receptors that could be beneficial in HD.


2017 - Current Strategies for the Delivery of Therapeutic Proteins and Enzymes to Treat Brain Disorders [Capitolo/Saggio]
Duskey, Jason T.; Belletti, Daniela; Pederzoli, Francesca; Vandelli, Maria Angela; Forni, Flavio; Ruozi, Barbara; Tosi, Giovanni
abstract

Brain diseases and injuries are growing to be one of the most deadly and costly medical conditions in the world. Unfortunately, current treatments are incapable of ameliorating the symptoms let alone curing the diseases. Many brain diseases have been linked to a loss of function in a protein or enzyme, increasing research for improving their delivery. This is no easy task due to the delicate nature of proteins and enzymes in biological conditions, as well as the many barriers that exist in the body ranging from those in circulation to the more specific barriers to enter the brain. Several main techniques are being used (physical delivery, protein/enzyme conjugates, and nanoparticle delivery) to overcome these barriers and create new therapeutics. This review will cover recently published data and highlights the benefits and deficits of possible new protein or enzyme therapeutics for brain diseases.


2016 - Chaperonin–Dendrimer Conjugates for siRNA Delivery [Articolo su rivista]
Nussbaumer, M. G.; Duskey, J. T.; Rother, M.; Renggli, K.; Chami, M.; Bruns, N.
abstract

The group II chaperonin thermosome (THS) is a hollow protein nanoparticle that can encapsulate macromolecular guests. Two large pores grant access to the interior of the protein cage. Poly(amidoamine) (PAMAM) is conjugated into THS to act as an anchor for small interfering RNA (siRNA), allowing to load the THS with therapeutic payload. THS–PAMAM protects siRNA from degradation by RNase A and traffics KIF11 and GAPDH siRNA into U87 cancer cells. By modification of the protein cage with the cell-penetrating peptide TAT, RNA interference is also induced in PC-3 cells. THS–PAMAM protein–polymer conjugates are therefore promising siRNA transfection reagents and greatly expand the scope of protein cages in drug delivery applications.


2016 - Engineered non-toxic cationic nanocarriers with photo-triggered slow-release properties [Articolo su rivista]
Dinu, I. A.; Duskey, J. T.; Car, A.; Palivan, C. G.; Meier, W.
abstract

The charge density of polymers involved in drug delivery is a key parameter during cellular uptake; moreover, the nature of charged groups determines the encapsulation efficiency and nanocarrier stability. Unfortunately, the high toxicity and the burst release of loaded cargo are their major drawbacks. We have developed here a versatile strategy to design photo-responsive nanocarriers showing high stability, slow-release properties and low cytotoxicity. These delivery vehicles are intended to prolong the drug effect, reducing the dose frequency, decreasing the side effects and maintaining a proper level of the drug. Diblock copolymers based on poly(dimethylsiloxane) and poly(2-dimethylaminoethyl methacrylate) containing pendant photo-cleavable 2-nitrobenzyl moieties were synthesized by atom transfer radical polymerization and post-polymerization modification, and then self-assembled into nanoparticles. Dynamic light scattering and transmission electron microscopy showed that the size and morphology of nanoparticles were not affected by UV exposure. Nanoparticle cytotoxicity was evaluated in relation to the number and nature of positively charged units. The nanocarriers containing copolymers with a longer charged block were successfully taken up by cells and were non-toxic both before and after irradiation up to 300 μg mL-1. The slow photo-induced release of a negatively charged molecule, sulforhodamine B, reveals that the delivery is controlled not only by the photo-triggered transformation of hydrophilic blocks from cationic to zwitterionic, but also by a combination of forces that induce the self-assembly but do not allow the disruption of nanoparticles. Results suggest that this polymeric system plays a promising role as a nanocarrier for sustained, triggered drug delivery, preserving the non-toxicity after release.


2016 - Stimuli-Responsive Codelivery of Oligonucleotides and Drugs by Self-Assembled Peptide Nanoparticles [Articolo su rivista]
Sigg, S. J.; Postupalenko, V.; Duskey, J. T.; Palivan, C. G.; Meier, W.
abstract

Ever more emerging combined treatments exploiting synergistic effects of drug combinations demand smart, responsive codelivery carriers to reveal their full potential. In this study, a multifunctional stimuli-responsive amphiphilic peptide was designed and synthesized to self-assemble into nanoparticles capable of co-bearing and -releasing hydrophobic drugs and antisense oligonucleotides for combined therapies. The rational design was based on a hydrophobic l-tryptophan-d-leucine repeating unit derived from a truncated sequence of gramicidin A (gT), to entrap hydrophobic cargo, which is combined with a hydrophilic moiety of histidines to provide electrostatic affinity to nucleotides. Stimuli-responsiveness was implemented by linking the hydrophobic and hydrophilic sequence through an artificial amino acid bearing a disulfide functional group (H3SSgT). Stimuli-responsive peptides self-assembled in spherical nanoparticles in sizes (100-200 nm) generally considered as preferable for drug delivery applications. Responsive peptide nanoparticles revealed notable nucleotide condensing abilities while maintaining the ability to load hydrophobic cargo. The disulfide cleavage site introduced in the peptide sequence induced responsiveness to physiological concentrations of reducing agent, serving to release the incorporated molecules. Furthermore, the peptide nanoparticles, singly loaded or coloaded with boron-dipyrromethene (BODIPY) and/or antisense oligonucleotides, were efficiently taken up by cells. Such amphiphilic peptides that led to noncytotoxic, reduction-responsive nanoparticles capable of codelivering hydrophobic and nucleic acid payloads simultaneously provide potential toward combined treatment strategies to exploit synergistic effects.


2015 - Analysis of Molecular Parameters Determining the Antimalarial Activity of Polymer-Based Nanomimics [Articolo su rivista]
Najer, A.; Thamboo, S.; Duskey, J. T.; Palivan, C. G.; Beck, H. -P.; Meier, W.
abstract

Malaria and other infectious diseases are major global public health problems, which need to be tackled using new technologies to cope with the lack of efficacious vaccines and emerging drug resistance. A recently developed anti-infectious concept based on nanomimics tested with Plasmodium falciparum is analyzed for the molecular parameters determining its applicability. Nanomimics - nanoscaled polymer-based mimics of host cell membranes - are designed with a reduced number of surface-exposed malaria parasite receptor molecules (heparin), resulting in less potent invasion inhibition as determined in antimalarial assays. In contrast, when shorter receptor molecules are used to form nanomimics, more molecules are needed to obtain nanomimic potency similar to nanomimics with longer receptor molecules. The interaction of heparin on nanomimics with the processed Plasmodium falciparum merozoite surface protein 1-42 (PfMSP142) have a high affinity, Kd = 12.1 ± 1.6 × 10-9 m, as measured by fluorescence cross-correlation spectroscopy (FCCS). This detailed characterization of nanomimics and their molecular variants are an important step towards defining and optimizing possible nanomimic therapies for infectious diseases. Modified nanomimics are tested for their invasion inhibition potency against malaria parasites. Polymer-based host cell membrane mimics (nanomimics) are designed with variable length and number of surface-exposed malaria receptor molecules (heparin). Furthmore, the interaction of nanomimics with a responsible parasite ligand is characterized in detail to define nanomimic activity.


2015 - Miniaturization of gene transfection assays in 384- and 1536-well microplates [Articolo su rivista]
Li, J.; Crowley, S. T.; Duskey, J.; Khargharia, S.; Wu, M.; Rice, K. G.
abstract

The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)-DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35 μl and in 1536-well plates in a total assay volume of 8 μl. A luciferase assay performed in 384-well plates produced a Z′ score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250 cells per well in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.


2015 - New concepts to fight oxidative stress: Nanosized three-dimensional supramolecular antioxidant assemblies [Articolo su rivista]
Richard, P. U.; Duskey, J. T.; Stolarov, S.; Spulber, M.; Palivan, C. G.
abstract

Introduction: Misregulation of reactive oxygen species and reactive nitrogen species by the bodys antioxidant system results in oxidative stress, which is known to be associated with aging, and involved in various pathologies including cancer, neurodegenerative and cardiovascular diseases. A large variety of low-molecular-weight (LMW) antioxidant compounds and antioxidant enzymes have been proposed to alleviate oxidative stress, but their therapeutic efficacy is limited by their solubility, stability or bioavailability. In this respect, nanoscience-based systems are expected to provide more efficient mitigation of oxidative stress. Areas covered: The main nanoscience-based three-dimensional (3D) supramolecular assemblies, decorated with, or entrapping antioxidant compounds, or which possess intrinsic antioxidant activity are discussed and illustrated with recent examples. Assemblies with different architectures and sizes in the nanometer range serve: i) to deliver LMW antioxidant compounds or enzymes; ii) as antioxidant systems due to their intrinsic activity; and recently iii) to provide a confined space where catalytic antioxidant reactions take place in situ (nanoreactors and artificial organelles). A few insights into the role of antioxidants in mitigating oxidative stress caused by therapeutic compounds or drug carriers are also discussed.Expert opinion: Several challenges must still be overcome in the development of 3D supramolecular assemblies to efficiently fight oxidative stress. First, an improvement of the assemblies properties and stability in biological conditions has to be addressed. Second, new systems based on the combination of biomolecules or mimics in supramolecular assemblies should provide multifunctionality, stimuli-responsiveness and targeting properties for a more efficient therapeutic effect. Third, comparative studies are necessary to evaluate these systems in a standardized manner both in vitro and in vivo.


2015 - PH-Triggered Reversible Multiple Protein-Polymer Conjugation Based on Molecular Recognition [Articolo su rivista]
Liu, J.; Postupalenko, V.; Duskey, J. T.; Palivan, C. G.; Meier, W.
abstract

Polymer conjugation for protein-based therapeutics has been developed extensively, but it still suffers from conjugation leading to decrease in protein activity and generates complexes with limited diversity due to general classical systems only incorporating one protein per each complex. Here we introduce a site-specific noncovalent protein-polymer conjugation, which can reduce the heterogeneity of the conjugates without disrupting protein function, while allowing for the modulation of binding affinity and stability, affecting the pH dependent binding of the number of proteins per polymer. We compared classical one protein-polymer conjugates with multiple protein-polymer conjugates using His-tagged enhanced yellow fluorescence protein (His6-eYFP) and metal-coordinated tris-nitrilotriacetic acid (trisNTA-Men+) in a site-specific way. trisNTA-Men+-His6 acts as a reversible linker with pH-triggered release of functional protein from the trisNTA-functionalized copolymers. The nature of the selected Men+ and number of available trisNTA-Men+ on poly(N-isopropylacrylamide-co-tris-nitrilotriacetic acid acrylamide) (PNTn) copolymers enables predictable modulation of the conjugates binding affinity (0.09-1.35 μM), stability, cell toxicity, and pH responsiveness. This represents a promising platform that allows direct control over the properties of multiple protein-polymer conjugates compared to the classical single protein-polymer conjugates.


2015 - Structure-Activity Relationship of PEGylated Polylysine Peptides as Scavenger Receptor Inhibitors for Non-Viral Gene Delivery [Articolo su rivista]
Baumhover, N. J.; Duskey, J. T.; Khargharia, S.; White, C. W.; Crowley, S. T.; Allen, R. J.; Rice, K. G.
abstract

PEGylated polylysine peptides of the general structure PEG30 kDa-Cys-Trp-LysN (N = 10 to 30) were used to form fully condensed plasmid DNA (pGL3) polyplexes at a ratio of 1 nmol of peptide per μg of DNA (ranging from N:P 3:1 to 10:1 depending on Lys repeat). Co-administration of 5 to 80 nmols of excess PEG-peptide with fully formed polyplexes inhibited the liver uptake of 125I-pGL3-polyplexes. The percent inhibition was dependent on the PEG-peptide dose and was saturable, consistent with inhibition of scavenger receptors. The scavenger receptor inhibition potency of PEG-peptides was dependent on the length of the Lys repeat, which increased 10-fold when comparing PEG30 kDa-Cys-Trp-Lys10 (IC50 of 20.2 μM) with PEG30 kDa-Cys-Trp-Lys25 (IC50 of 2.1 μM). We hypothesize that PEG-peptides inhibit scavenger receptors by spontaneously forming small 40 to 60 nm albumin nanoparticles that bind to and saturate the receptor. Scavenger receptor inhibition delayed the metabolism of pGL3-polyplexes, resulting in efficient gene expression in liver hepatocytes following delayed hydrodynamic dosing. PEG-peptides represent a new class of scavenger inhibitors that will likely have broad utility in blocking unwanted liver uptake and metabolism of a variety of nanoparticles.


2014 - Nanoparticle ligand presentation for targeting solid tumors [Articolo su rivista]
Duskey, J. T.; Rice, K. G.
abstract

Among the many scientific advances to come from the study of nanoscience, the development of ligand-targeted nanoparticles to eliminate solid tumors is predicted to have a major impact on human health. There are many reports describing novel designs and testing of targeted nanoparticles to treat cancer. While the principles of the technology are well demonstrated in controlled lab experiments, there are still many hurdles to overcome for the science to mature into truly efficacious targeted nanoparticles that join the arsenal of agents currently used to treat cancer in humans. One of these hurdles is overcoming unwanted biodistribution to the liver while maximizing delivery to the tumor. This almost certainly requires advances in both nanoparticle stealth technology and targeting. Currently, it continues to be a challenge to control the loading of ligands onto polyethylene glycol (PEG) to achieve maximal targeting. Nanoparticle cellular uptake and subcellular targeting of genes and siRNA also remain a challenge. This review examines the types of ligands that have been most often used to target nanoparticles to solid tumors. As the science matures over the coming decade, careful control over ligand presentation on nanoparticles of precise size, shape, and charge will likely play a major role in achieving success.


2014 - PH-responsive PDMS-b-PDMAEMA micelles for intracellular anticancer drug delivery [Articolo su rivista]
Car, A.; Baumann, P.; Duskey, J. T.; Chami, M.; Bruns, N.; Meier, W.
abstract

A series of poly(dimethysiloxane)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMS-b-PDMAEMA) block copolymers were synthesized with atom transfer radical polymerization (ATRP). In aqueous solution the polymers self-assembled into micelles with diameters between 80 and 300 nm, with the ability to encapsulate DOX. The polymer with the shortest PDMAEMA block (5 units) displayed excellent cell viability, while micelles containing longer PDMAEMA block lengths (13 and 22 units) led to increased cytotoxicity. The carriers released DOX in response to a decrease in pH from 7.4 to 5.5. Confocal laser scanning microscopy (CLSM) revealed that nanoparticles were taken up by endocytosis into acidic cell compartments. Furthermore, DOX-loaded nanocarriers exhibited intracellular pH-response as changes in cell morphology and drug release were observed within 24 h.


2014 - The uptake mechanism of PEGylated DNA polyplexes by the liver influences gene expression [Articolo su rivista]
Khargharia, S.; Baumhover, N. J.; Crowley, S. T.; Duskey, J.; Rice, K. G.
abstract

Two uptake mechanisms were identified for PEGylated DNA polyplex biodistribution to the liver. At a low polyplex dose, a rapid-uptake mechanism dominates, resulting in 60% capture by liver in 5 min, due to a saturable receptor-mediated process. Rapid-uptake led to the fast metabolism of polyplexes by liver (t 1/2 =2.1 h), correlating with a 1-μg pGL3 polyplex dose losing full transfection competency after 4 h in the liver. Dose escalation of either polyplex or poly(ethylene glycol) (PEG) peptide led to the saturation of rapid-uptake and revealed a delayed-uptake mechanism for polyplexes by liver. Delayed-uptake was characterized by the slower liver accumulation of 40% of the polyplex dose over 40 min, followed by slow metabolism (t 1/2 =15 h) and an extended time (12 h) for a 1-μg pGL3 polyplex dose, remaining fully transfection competent in the liver. The delayed-uptake mechanism is consistent with polyplexes crossing liver fenestrated endothelial cells to reach steady state in the space of Disse. The results describe how to control polyplex biodistribution to liver to avoid rapid-uptake and metabolism, in favor of delayed-uptake, to preserve polyplex transfection competency in the liver for up to 12 h.


2011 - Metabolically stabilized long-circulating PEGylated polyacridine peptide polyplexes mediate hydrodynamically stimulated gene expression in liver [Articolo su rivista]
Fernandez, C. A.; Baumhover, N. J.; Duskey, J. T.; Khargharia, S.; Kizzire, K.; Ericson, M. D.; Rice, K. G.
abstract

A novel class of PEGylated polyacridine peptides was developed that mediate potent stimulated gene transfer in the liver of mice. Polyacridine peptides, (Acr-X) n-Cys-polyethylene glycol (PEG), possessing 2-6 repeats of Lys-acridine (Acr) spaced by either Lys, Arg, Leu or Glu, were Cys derivatized with PEG (PEG 5000 kDa) and evaluated as in vivo gene transfer agents. An optimal peptide of (Acr-Lys) 6-Cys-PEG was able to bind to plasmid DNA (pGL3) with high affinity by polyintercalation, stabilize DNA from metabolism by DNAse and extend the pharmacokinetic half-life of DNA in the circulation for up to 2 h. A tail vein dose of PEGylated polyacridine peptide pGL3 polyplexes (1 g in 50 l), followed by a stimulatory hydrodynamic dose of normal saline at times ranging from 5 to 60 min post-DNA administration, led to a high level of luciferase expression in the liver, equivalent to levels mediated by direct hydrodynamic dosing of 1 g of pGL3. The results establish the unique properties of PEGylated polyacridine peptides as a new and promising class of gene delivery peptides that facilitate reversible binding to plasmid DNA, protecting it from DNase in vivo resulting in an extended circulatory half-life, and release of transfection-competent DNA into the liver to mediate a high-level of gene expression upon hydrodynamic boost. © 2011 Macmillan Publishers Limited All rights reserved.