Arianna Gennari - Academia.edu (original) (raw)
Papers by Arianna Gennari
Journal of the American Chemical Society
This study addresses well-known shortcomings of poly(ethylene glycol) (PEG)-based conjugates. PEG... more This study addresses well-known shortcomings of poly(ethylene glycol) (PEG)-based conjugates. PEGylation is by far the most common method employed to overcome immunogenicity and suboptimal pharmacokinetics of, for example, therapeutic proteins but has significant drawbacks. First, PEG offers no protection from denaturation during lyophilization, storage, or oxidation (e.g., by biological oxidants, reactive oxygen species); second, PEG's inherent immunogenicity, leading to hypersensitivity and accelerated blood clearance (ABC), is a growing concern. We have here developed an 'active-stealth' polymer, poly(thioglycidyl glycerol)(PTGG), which in human plasma is less immunogenic than PEG (35% less complement activation) and features a reactive oxygen species-scavenging and anti-inflammatory action (∼50% less TNF-α in LPS-stimulated macrophages at only 0.1 mg/mL). PTGG was conjugated to proteins via a one-pot process; molar mass-and grafting density-matched PTGG-lysozyme conjugates were superior to their PEG analogues in terms of enzyme activity and stability against freeze-drying or oxidation; the latter is due to sacrificial oxidation of methioninemimetic PTGG chains. Both in mice and rats, PTGG-ovalbumin displayed circulation half-lives up to twice as long as PEGovalbumin, but most importantly�and differently from PEG�without any associated ABC effect seen either in the time dependency of blood concentration, in the liver/splenic accumulation, or in antipolymer IgM/IgG titers. Furthermore, similar pharmacokinetic results were obtained with PTGGylated/PEGylated liposomal nanocarriers. PTGG's 'active-stealth' character therefore makes it a highly promising alternative to PEG for conjugation to biologics or nanocarriers.
Materials Advances
Keratin based electrospun fibres containing cinnamon essential oil are highly antioxidant and ant... more Keratin based electrospun fibres containing cinnamon essential oil are highly antioxidant and antibacterial, and promote reduced tissue inflammation after skin burns.
RAD51, a key player in the homologous recombination (HR) mechanism, is a critical protein to pres... more RAD51, a key player in the homologous recombination (HR) mechanism, is a critical protein to preserve genomic stability. BRCA2, upon DNA damage, promotes RAD51 fibrils disassembly and its nuclear recruitment.Here, we use BRC4, a peptide derived from the fourth BRC repeat of BRCA2; BRC4 induces RAD51 defibrillation through a ‘domino’ effect, eroding fibrils from their termini, and yielding monomeric RAD51. The congruence among several techniques (static and dynamic light scattering, negative staining transmission electron microscopy (TEM), and microscale thermophoresis) allows an accurate estimation of the kinetic and thermodynamic parameters of this process. BRC4 lacks, however, a nuclear localization sequence; therefore, it cannot transport RAD51 into the nucleus, thus behaving as a RAD51 inhibitor. Cellular assays (BxPC-3, pancreatic cancer cells) indeed show that BRC4 efficiently inhibits HR and enhances the cytotoxic effect of cisplatin, a DNA-damaging drug.The present study she...
CD44 is the major cell surface receptor of hyaluronic acid (HA) and mediates the response of cell... more CD44 is the major cell surface receptor of hyaluronic acid (HA) and mediates the response of cells to their microenvironment. Whereas the standard isoform (CD44s) is ubiquitously expressed throughout the body, variant isoforms (CD44v) are aberrantly overexpressed in certain tumours, thus CD44 has become a potential target for cancer chemotherapy. However, CD44 is also one of the most abundantly expressed macrophage receptors, rendering this cellular type a potential competitor for the uptake of targeted nanocarriers in vivo. An in vitro human macrophage model is described herein. This model would allow to evaluate CD44 offtargeting. In particular, we have developed a procedure for the differentiation and activation of THP-1 cells. The CD44 expression of THP-1 macrophages was compared to that of the tumoural cell line HT-29 in terms of amount and isoform composition. The internalisation kinetics of HA nanoparticles was also evaluated.
CHEMICAL MODIFICATION OF THE HYALURONIC ACID 2.1 INTRODUCTION 2.1.1 Modification of the carboxyli... more CHEMICAL MODIFICATION OF THE HYALURONIC ACID 2.1 INTRODUCTION 2.1.1 Modification of the carboxylic acid group 2.1.2 Modification of the hydroxy group 2.2 AMIDATION 2.2.1 INTRODUCTION 2.2.2 EXPERIMENTAL PROCEDURES 2.2.3 RESULTS AND DISCUSSION 2.2.4 CONCLUSION 2.3 DIISIOCYANATES AS CROSS-LINKER AGENTS FOR HA Contents ______________________________________________________________________________ 2.3.1 INTRODUCTION 2.3.2 EXPERIMENTAL PROCEDURES 2.3.3 RESULTS AND DISCUSSION 2.3.4 CONCLUSION 2.4 HA MODIFICATION VIA Cu I CATALYSED 1,3-CYCLOADDITION 2.4.1 INTRODUCTION 2.4.2 EXPERIMENTAL PROCEDURES 2.4.3 RESULTS AND DISCUSSION 2.4.4 CONCLUSION 2.5 PROLIFERATION AND DIFFERENTIATION OF MESOANGIOBLASTS ON HYALURONAN DERIVATIVES 2.5.1 INTRODUCTION 2.5.2 EXPERIMENTAL PROCEDURES 2.5.3 RESULTS AND DISCUSSION 2.5.4 CONCLUSION 2.6 ACKNOWLEDGEMENTS 2.7 REFERENCES 2.8 SUPPORTING INFORMATION
Materials Science and Engineering: C, 2021
Hyaluronic acid (HA)-based prodrugs bearing double-responsive (acid pH or oxidation) boronates of... more Hyaluronic acid (HA)-based prodrugs bearing double-responsive (acid pH or oxidation) boronates of catechol-containing drugs were used to treat xenografted human prostate tumours (LNCaP) in SCID mice. The HA prodrugs accumulated significantly only in tumours (impressively, up to 40% of the injected dose after 24 h) and in liver, with negligible - actually anti-inflammatory - consequences in the latter. A quercetin-HA prodrug significantly slowed down tumour growth, in a dose-dependent fashion and with a much higher efficacy (up to 4 times) than equivalent doses of free quercetin. In short, boronated HA appears to be a very promising platform for targeted chemotherapy.
Macromolecules, 2021
We describe how the organocatalytic, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-based lactide ring-... more We describe how the organocatalytic, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-based lactide ring-opening polymerization can be effectively performed in a very polar solvent, N-methylpyrrolidone (NMP). Due to a low ceiling temperature, this "living" mechanism has been unreported to date, but we here demonstrate that through a combination of low temperature and repeated monomer additions (starve-fed process), this mechanism enables the generation of a plethora of multifunctional homo-and (stereo)block-poly(lactide)s (PLAs) with exquisite control of the molecular weight dispersity (typically Đ < 1.1) and topology (from linear through 4-, 6-, or 8-armed stars and up to ∼140 armed combs). They are scarcely obtainable or inaccessible through more classical synthetic methods due to the poor solubility of multifunctional initiators (polyols) in most organic solvents and monomer melts. In these precisely designed structures, branching significantly altered the nature of the materials' hydrolytic degradation, allowing them to acquire a pronounced surface character (as opposed to the bulk degradation of linear polymers). Finally, we have assessed the amenability of this method to in situ block copolymerization by using the tacticity of PLLA blocks in PLLA-b-PDLLA versus PDLLA-b-PLLA (L-LA polymerized before or after DL-LA) as a sensitive method to detect (stereochemical) defects.
Tetrahedron, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nanomaterials, 2020
Chemokine-induced chemotaxis mediates physiological and pathological immune cell trafficking, as ... more Chemokine-induced chemotaxis mediates physiological and pathological immune cell trafficking, as well as several processes involving cell migration. Among them, the role of CXCL12/CXCR4 signaling in cancer and metastasis is well known, and CXCR4 has been often targeted with small molecule-antagonists or short CXCL12-derived peptides to limit the pathological processes of cell migration and invasion. To reduce CXCR4-mediated chemotaxis, we adopted a different approach. We manufactured poly(lactic acid-co-glycolic acid) (PLGA)/Pluronic F127 nanoparticles through microfluidics-assisted nanoprecipitation and functionalized them with streptavidin to docking a biotinylated CXCL12 to be exposed on the nanoparticle surface. Our results show that CXCL12-decorated nanoparticles are non-toxic and do not induce inflammatory cytokine release in THP-1 monocytes cultured in fetal bovine and human serum-supplemented media. The cell internalization of our chemokine receptor-targeting particles incre...
Beilstein Journal of Nanotechnology, 2019
This study is about linking preparative processes of nanoparticles with the morphology of the nan... more This study is about linking preparative processes of nanoparticles with the morphology of the nanoparticles and with their efficiency in delivering payloads intracellularly. The nanoparticles are composed of hyaluronic acid (HA) and chitosan; the former can address a nanoparticle to cell surface receptors such as CD44, the second allows both for entrapment of nucleic acids and for an endosomolytic activity that facilitates their liberation in the cytoplasm. Here, we have systematically compared nanoparticles prepared either A) through a two-step process based on intermediate (template) particles produced via ionotropic gelation of chitosan with triphosphate (TPP), which are then incubated with HA, or B) through direct polyelectrolyte complexation of chitosan and HA. Here we demonstrate that HA is capable to quantitatively replace TPP in the template process and significant aggregation takes place during the TPP–HA exchange. The templated chitosan/HA nanoparticles therefore have a mi...
Biomacromolecules, 2019
We show the first example of a synergic approach of oxidant (ROS) scavenging carrier and ROSrespo... more We show the first example of a synergic approach of oxidant (ROS) scavenging carrier and ROSresponsive drug release in the context of a potential therapy against osteoporosis, aiming to inhibit the differentiation of inflammatory cells into osteoclasts. In our 'tandem' approach, a branched amphiphilic, PEGylated polysulfide (PPSES-PEG) was preferred over a linear analogue, because of improved homogeneity in the aggregates (spherical micelles vs. mixture of worm-like and spherical), increased stability and higher drug loading (up to ~22%wt. of anti-osteoclastic rapamycin). These effects are ascribed to the branching inhibiting crystallization in the polysulfide blocks. The ROSscavenging micelles alone were already able to reduce osteoclastogenesis in a RAW 264.7 model, but the 'drug' combination (the polymer itself + rapamycin released only under oxidation) completely abrogated the process. An important take-home message is that the synergic performance depended very strongly on the oxidant:oxidizable group molar ratio, a parameter to carefully tune in the perspective of targeting specific diseases.
Advanced Healthcare Materials, 2019
often, while we may have some mechanistic knowledge of how a delivery vehicle initially binds to ... more often, while we may have some mechanistic knowledge of how a delivery vehicle initially binds to such a group, we hardly know what happens during later phases. These phases, however, are equally critical for the efficacy of the treatment: for example, whether a nucleic acid-loaded particle remains on a cell surface or is transported intracellularly can make the difference between therapeutic success and failure. In short, the rational development of a targeted delivery approach should require the understanding of both surface binding and internalization processes, and the interplay between them. Here, we have focused on these points, using nanoparticles where hyaluronic acid (HA) acts as a targeting element and siRNA is the payload (i.e., the drug). Rather than for cell targeting, HA is better known as a major component of extracellular matrices, [1] and for its use as a matrix component in regenerative medicine, [2] as a dermal filler, [3] or a viscosupplementation agent. [4] However, besides its physical properties, HA is also capable of interacting with a number of biomolecules. [5] Most, but not all HA-binding proteins (also referred to as hyaladherins) share a homologous, HA-binding Link domain, whose CD44 is an endocytic hyaluronic acid (HA) receptor, and is overexpressed in many carcinomas. This has encouraged the use of HA to design CD44targeting carriers. This paper is about dissecting the mechanistic role of CD44. Here, HA-decorated nanoparticles are used to deliver siRNA to both tumoral (AsPC-1, PANC-1, HT-29, HCT-116) and non-tumoral (fibroblasts, differently polarized THP-1 macrophages, HUVEC) human cell lines, evaluating the initial binding of the nanoparticles, their internalization rate, and the silencing efficiency (cyclophilin B (PPIB) gene). Tumoral cells internalize faster and experience higher silencing than non-tumoral cells. This is promising as it suggests that, in a tumor, HA nanocarriers may have limited off-target effects. More far-reaching is the interrelation between the four parameters of the study: CD44 expression, HA binding on cell surfaces, internalization rate, and silencing efficiency. No correlation is found between binding (an early event) and any of the other parameters, whereas silencing correlates both with speed of the internalization process and CD44 expression. This study confirms on one hand that HA-based carriers can perform a targeted action, but on the other it suggests that this may not be due to a selective binding event, but rather to a later recognition leading to selective internalization.
Langmuir, 2019
Hybrids composed of liposomes (L) and metallic nanoparticles (NP) hold great potential for imagin... more Hybrids composed of liposomes (L) and metallic nanoparticles (NP) hold great potential for imaging and drug delivery purposes. However, the efficient incorporation of metallic nanoparticles into liposomes using conventional methodologies has so far proved to be challenging. In this study, we report the fabrication of hybrids of liposomes and hydrophobic gold nanoparticles of size 2-4 nm (Au) using a microfluidic assisted self-assembly process. The incorporation of increasing amounts of Au nanoparticles into liposomes was examined using microfluidics and compared to L-AuNP hybrids prepared by the reverse-phase evaporation method. Our microfluidics strategy produced L-AuNP hybrids with a homogeneous size distribution, smaller polydispersity index, and a 3-fold increase in loading efficiency when compared to those hybrids prepared using the reverse-phase method of production. Quantification of the loading efficiency was determined by ultraviolet spectroscopy, inductively coupled plasma mass spectroscopy and centrifugal field flow fractionation and confirmed qualitatively by transmission electron microscopy. The higher loading of gold nanoparticles into the liposomes achieved using microfluidics produced a slightly thicker and more rigid bilayer as determined with small angle neutron scattering. These observations were confirmed using fluorescent anisotropy and atomic force microscopy, respectively. Structural characterization of the liposomal-nanoparticle hybrids with cryo-electron microscopy revealed the coexistence of membrane-embedded and interdigitated nanoparticle-rich domains suggesting AuNP incorporation through hydrophobic interactions. The microfluidic technique that we describe in this study allows for the automated production of monodisperse liposomal-nanoparticle hybrids with high loading capacity highlighting the utility of microfluidics to improve the payload of metallic nanoparticles within liposomes, thereby enhancing their application for imaging and drug delivery.
ACS Applied Materials & Interfaces, 2019
This study is about 1) nanomanufacturing (focusing on microfluidic-assisted nanoprecipitation), 2... more This study is about 1) nanomanufacturing (focusing on microfluidic-assisted nanoprecipitation), 2) advanced colloid characterization (focusing on field flow fractionation, FFF), 3) the possible restructuring of surface disulfides. Disulfides are dynamic and exchangeable groups, and here we specifically focus firstly on their use to introduce biofunctional group, and secondly on their reorganization, which may lead to variable surface chemistries and uncontrolled cell interactions. The particles were obtained via microfluidic-assisted (flow-focused) nanoprecipitation of poly(ethylene glycol)-b-poly(-caprolactone) (PEG-PCL) bearing or not a 2-pyridyl disulfide (PDS) terminal group, which quantitatively exchanges with thiols in solution. In this study, we have paid specific attention to size characterization, thereby also demonstrating the limitations of dynamic light scattering (DLS) as a stand-alone technique. By using Asymmetric Flow Field Flow Fractionation (AF4) coupled with DLS, static light scattering (SLS) and refractive index detectors, we show that relatively small amounts of >100 nm aggregates (cryo-TEM and SLS/DLS comparison suggesting them to be wormlike micelles) dominated the stand-alone DLS results, whereas the 'real' size distributions picked < 50 nm. Our key result is that the kinetics of the conjugation based on PDS-thiol exchange was controlled by the thiol pKa, and this also determined the rate of the exchange between the resulting disulfides and glutathione (GSH). In particular, more acidic thiols (e.g. peptides where a cysteine is flanked by cationic residues) react faster with PDS, but their disulfides hardly exchange with GSH; the reverse applies to thiols with a higher pKa. Disulfides that resist against restructuring via thiol-disulfide exchange allow for a stable bioconjugation, although they may be bad news for payload release under reducing conditions. However, experiments of both thiol release and nanoparticles uptake in cells (HCT116) show that also the disulfides formed from less acidic, and therefore less reactive and more exchangeable thiols, were stable for at least a few hours even in a GSH-rich (10 mM) environment; this suggests a sufficiently long stability of surface groups to achieve e.g. a cell targeting effect.
Bioconjugate chemistry, Jan 19, 2018
We present a method for tyrosine-selective and reversible bioconjugation; tyrosines are enzymatic... more We present a method for tyrosine-selective and reversible bioconjugation; tyrosines are enzymatically converted into catechols and in situ "clicked" onto boronic acids. Importantly, our process selectively produces catechols and avoids quinones, thereby improving the control over the chemical identity of the products. We have conjugated boronic acid-containing hyaluronic acid (HyA) to peptides bearing tyrosines in variable number and position; the use of tagging peptides for the provision of well exposed tyrosine residues-in our case the hemagglutinin-derived HA-tag-makes our approach applicable to virtually any protein; we have demonstrated this concept by conjugating HA-tagged ovalbumin to HyA, thereby also showing the feasibility of producing chimeric proteoglycans. A caveat of this appproach is that, although the formation of boronic esters does not affect the biological recognition of substrates (ovalbumin and HyA), the introduction of catechols may alter some of thei...
International journal of pharmaceutics, Jan 20, 2017
We have employed microfluidics (cross-shaped chip) for the preparation of drug-loaded poly(lactic... more We have employed microfluidics (cross-shaped chip) for the preparation of drug-loaded poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles. The polymer precipitates from an acetone solution upon its controlled laminar mixing (flow focusing) with an aqueous solution of a surfactant, allowing for an operator-independent, up-scalable and reproducible preparative process of nanoformulations. Firstly, using PEGylated surfactants we have compared batch and microfluidic processes, and showed the superior reproducibility of the latter and its strong dependency on the acetone/water ratio (flow rate ratio). We have then focused on the issue of purification from free surfactant, and employed advanced characterization techniques such as flow-through dynamic light scattering as the in-line quality control technique, and field flow fractionation (FFF) with dynamic and static light scattering detection, which allowed the detection of surfactant micelles in mixture with nanoparticles (hardly pos...
Molecular Pharmaceutics, 2017
Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver a RNA/DNA cargo to cells overe... more Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver a RNA/DNA cargo to cells overexpressing HA receptors such as CD44. For these systems, unequivocal links have not been established yet between chitosan macromolecular (molecular weight; degree of deacetylation, i.e. charge density) and nanoparticle variables (complexation strength, i.e. stability; nucleic acid protection; internalization rate) on one hand, and transfection efficiency on the other hand. Here, we have focused on the role of avidity on transfection efficiency in the CD44-expressing HCT-116 as a cellular model; we have employed two differently sized payloads (a large luciferaseencoding mRNA and a much smaller anti-luc siRNA), and a small library of chitosans (variable molecular weight and degree of deactylation). The RNA avidity for chitosan showed-as expectedan inverse relationship: higher avidity-higher polyplex stability-lower transfection efficiency. The avidity of chitosan for RNA appears to lead to opposite effects: higher avidity-higher polyplex stability but also higher transfection efficiency. Surprisingly, the best transfecting particles were those with the lowest propensity for RNA release, although this might be a misleading relationship: for example, the same macromolecular parameters that increase avidity can also boost chitosan's endosomolytic activity, with a strong enhancement in transfection. The performance of these nonviral vectors appears therefore difficult to predict simply on the basis of carrier-or payload-related variables and a more holistic consideration of the journey of the nanoparticle, from cell uptake to cytosolic bioavailability of payload, is needed. It is also noteworthy that the nanoparticles used in this study showed optimal performance under slightly acidic conditions (pH 6.4), which is promising for applications in a tumoural extracellular environment. It is also worth pointing out that under these conditions we have for the first time successfully delivered mRNA with chitosan/HA nanoparticles.
Advanced Healthcare Materials, 2016
Macromolecular Rapid Communications, 2016
Families of amphiphilic oxidation-responsive polymers (poly(ethylene glycol)-polysulfides) with d... more Families of amphiphilic oxidation-responsive polymers (poly(ethylene glycol)-polysulfides) with different architectures (linear, 4, 6, and 8-armed stars and 10, 15, and 20-armed combs) and compositions (variable ethylene sulfide/propylene sulfide ratio) are prepared. In water, all the polymers assemble in spherical micelles, with critical micellar concentrations <0.01 mg mLfor all the branched polymers. Triple-detection gel permeation chromatography (GPC) and asymmetric field flow fractionation (AFFF) with dynamic and static light scattering detection, respectively, show an increasing compaction of the polymeric coil and a strong reduction of the aggregation number with increasing degree of branching. The key finding of this study is that the kinetics of the oxidative response sharply depend on the branching; in particular, it is highlighted that the degree of branching influences the lag time before a response can be observed rather than the speed of the response itself, a phenomenon that is attributed to a branching-dependent solubility of the oxidant in the polysulfide matrix.
Applied Radiation and Isotopes, 2017
Journal of the American Chemical Society
This study addresses well-known shortcomings of poly(ethylene glycol) (PEG)-based conjugates. PEG... more This study addresses well-known shortcomings of poly(ethylene glycol) (PEG)-based conjugates. PEGylation is by far the most common method employed to overcome immunogenicity and suboptimal pharmacokinetics of, for example, therapeutic proteins but has significant drawbacks. First, PEG offers no protection from denaturation during lyophilization, storage, or oxidation (e.g., by biological oxidants, reactive oxygen species); second, PEG's inherent immunogenicity, leading to hypersensitivity and accelerated blood clearance (ABC), is a growing concern. We have here developed an 'active-stealth' polymer, poly(thioglycidyl glycerol)(PTGG), which in human plasma is less immunogenic than PEG (35% less complement activation) and features a reactive oxygen species-scavenging and anti-inflammatory action (∼50% less TNF-α in LPS-stimulated macrophages at only 0.1 mg/mL). PTGG was conjugated to proteins via a one-pot process; molar mass-and grafting density-matched PTGG-lysozyme conjugates were superior to their PEG analogues in terms of enzyme activity and stability against freeze-drying or oxidation; the latter is due to sacrificial oxidation of methioninemimetic PTGG chains. Both in mice and rats, PTGG-ovalbumin displayed circulation half-lives up to twice as long as PEGovalbumin, but most importantly�and differently from PEG�without any associated ABC effect seen either in the time dependency of blood concentration, in the liver/splenic accumulation, or in antipolymer IgM/IgG titers. Furthermore, similar pharmacokinetic results were obtained with PTGGylated/PEGylated liposomal nanocarriers. PTGG's 'active-stealth' character therefore makes it a highly promising alternative to PEG for conjugation to biologics or nanocarriers.
Materials Advances
Keratin based electrospun fibres containing cinnamon essential oil are highly antioxidant and ant... more Keratin based electrospun fibres containing cinnamon essential oil are highly antioxidant and antibacterial, and promote reduced tissue inflammation after skin burns.
RAD51, a key player in the homologous recombination (HR) mechanism, is a critical protein to pres... more RAD51, a key player in the homologous recombination (HR) mechanism, is a critical protein to preserve genomic stability. BRCA2, upon DNA damage, promotes RAD51 fibrils disassembly and its nuclear recruitment.Here, we use BRC4, a peptide derived from the fourth BRC repeat of BRCA2; BRC4 induces RAD51 defibrillation through a ‘domino’ effect, eroding fibrils from their termini, and yielding monomeric RAD51. The congruence among several techniques (static and dynamic light scattering, negative staining transmission electron microscopy (TEM), and microscale thermophoresis) allows an accurate estimation of the kinetic and thermodynamic parameters of this process. BRC4 lacks, however, a nuclear localization sequence; therefore, it cannot transport RAD51 into the nucleus, thus behaving as a RAD51 inhibitor. Cellular assays (BxPC-3, pancreatic cancer cells) indeed show that BRC4 efficiently inhibits HR and enhances the cytotoxic effect of cisplatin, a DNA-damaging drug.The present study she...
CD44 is the major cell surface receptor of hyaluronic acid (HA) and mediates the response of cell... more CD44 is the major cell surface receptor of hyaluronic acid (HA) and mediates the response of cells to their microenvironment. Whereas the standard isoform (CD44s) is ubiquitously expressed throughout the body, variant isoforms (CD44v) are aberrantly overexpressed in certain tumours, thus CD44 has become a potential target for cancer chemotherapy. However, CD44 is also one of the most abundantly expressed macrophage receptors, rendering this cellular type a potential competitor for the uptake of targeted nanocarriers in vivo. An in vitro human macrophage model is described herein. This model would allow to evaluate CD44 offtargeting. In particular, we have developed a procedure for the differentiation and activation of THP-1 cells. The CD44 expression of THP-1 macrophages was compared to that of the tumoural cell line HT-29 in terms of amount and isoform composition. The internalisation kinetics of HA nanoparticles was also evaluated.
CHEMICAL MODIFICATION OF THE HYALURONIC ACID 2.1 INTRODUCTION 2.1.1 Modification of the carboxyli... more CHEMICAL MODIFICATION OF THE HYALURONIC ACID 2.1 INTRODUCTION 2.1.1 Modification of the carboxylic acid group 2.1.2 Modification of the hydroxy group 2.2 AMIDATION 2.2.1 INTRODUCTION 2.2.2 EXPERIMENTAL PROCEDURES 2.2.3 RESULTS AND DISCUSSION 2.2.4 CONCLUSION 2.3 DIISIOCYANATES AS CROSS-LINKER AGENTS FOR HA Contents ______________________________________________________________________________ 2.3.1 INTRODUCTION 2.3.2 EXPERIMENTAL PROCEDURES 2.3.3 RESULTS AND DISCUSSION 2.3.4 CONCLUSION 2.4 HA MODIFICATION VIA Cu I CATALYSED 1,3-CYCLOADDITION 2.4.1 INTRODUCTION 2.4.2 EXPERIMENTAL PROCEDURES 2.4.3 RESULTS AND DISCUSSION 2.4.4 CONCLUSION 2.5 PROLIFERATION AND DIFFERENTIATION OF MESOANGIOBLASTS ON HYALURONAN DERIVATIVES 2.5.1 INTRODUCTION 2.5.2 EXPERIMENTAL PROCEDURES 2.5.3 RESULTS AND DISCUSSION 2.5.4 CONCLUSION 2.6 ACKNOWLEDGEMENTS 2.7 REFERENCES 2.8 SUPPORTING INFORMATION
Materials Science and Engineering: C, 2021
Hyaluronic acid (HA)-based prodrugs bearing double-responsive (acid pH or oxidation) boronates of... more Hyaluronic acid (HA)-based prodrugs bearing double-responsive (acid pH or oxidation) boronates of catechol-containing drugs were used to treat xenografted human prostate tumours (LNCaP) in SCID mice. The HA prodrugs accumulated significantly only in tumours (impressively, up to 40% of the injected dose after 24 h) and in liver, with negligible - actually anti-inflammatory - consequences in the latter. A quercetin-HA prodrug significantly slowed down tumour growth, in a dose-dependent fashion and with a much higher efficacy (up to 4 times) than equivalent doses of free quercetin. In short, boronated HA appears to be a very promising platform for targeted chemotherapy.
Macromolecules, 2021
We describe how the organocatalytic, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-based lactide ring-... more We describe how the organocatalytic, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-based lactide ring-opening polymerization can be effectively performed in a very polar solvent, N-methylpyrrolidone (NMP). Due to a low ceiling temperature, this "living" mechanism has been unreported to date, but we here demonstrate that through a combination of low temperature and repeated monomer additions (starve-fed process), this mechanism enables the generation of a plethora of multifunctional homo-and (stereo)block-poly(lactide)s (PLAs) with exquisite control of the molecular weight dispersity (typically Đ < 1.1) and topology (from linear through 4-, 6-, or 8-armed stars and up to ∼140 armed combs). They are scarcely obtainable or inaccessible through more classical synthetic methods due to the poor solubility of multifunctional initiators (polyols) in most organic solvents and monomer melts. In these precisely designed structures, branching significantly altered the nature of the materials' hydrolytic degradation, allowing them to acquire a pronounced surface character (as opposed to the bulk degradation of linear polymers). Finally, we have assessed the amenability of this method to in situ block copolymerization by using the tacticity of PLLA blocks in PLLA-b-PDLLA versus PDLLA-b-PLLA (L-LA polymerized before or after DL-LA) as a sensitive method to detect (stereochemical) defects.
Tetrahedron, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nanomaterials, 2020
Chemokine-induced chemotaxis mediates physiological and pathological immune cell trafficking, as ... more Chemokine-induced chemotaxis mediates physiological and pathological immune cell trafficking, as well as several processes involving cell migration. Among them, the role of CXCL12/CXCR4 signaling in cancer and metastasis is well known, and CXCR4 has been often targeted with small molecule-antagonists or short CXCL12-derived peptides to limit the pathological processes of cell migration and invasion. To reduce CXCR4-mediated chemotaxis, we adopted a different approach. We manufactured poly(lactic acid-co-glycolic acid) (PLGA)/Pluronic F127 nanoparticles through microfluidics-assisted nanoprecipitation and functionalized them with streptavidin to docking a biotinylated CXCL12 to be exposed on the nanoparticle surface. Our results show that CXCL12-decorated nanoparticles are non-toxic and do not induce inflammatory cytokine release in THP-1 monocytes cultured in fetal bovine and human serum-supplemented media. The cell internalization of our chemokine receptor-targeting particles incre...
Beilstein Journal of Nanotechnology, 2019
This study is about linking preparative processes of nanoparticles with the morphology of the nan... more This study is about linking preparative processes of nanoparticles with the morphology of the nanoparticles and with their efficiency in delivering payloads intracellularly. The nanoparticles are composed of hyaluronic acid (HA) and chitosan; the former can address a nanoparticle to cell surface receptors such as CD44, the second allows both for entrapment of nucleic acids and for an endosomolytic activity that facilitates their liberation in the cytoplasm. Here, we have systematically compared nanoparticles prepared either A) through a two-step process based on intermediate (template) particles produced via ionotropic gelation of chitosan with triphosphate (TPP), which are then incubated with HA, or B) through direct polyelectrolyte complexation of chitosan and HA. Here we demonstrate that HA is capable to quantitatively replace TPP in the template process and significant aggregation takes place during the TPP–HA exchange. The templated chitosan/HA nanoparticles therefore have a mi...
Biomacromolecules, 2019
We show the first example of a synergic approach of oxidant (ROS) scavenging carrier and ROSrespo... more We show the first example of a synergic approach of oxidant (ROS) scavenging carrier and ROSresponsive drug release in the context of a potential therapy against osteoporosis, aiming to inhibit the differentiation of inflammatory cells into osteoclasts. In our 'tandem' approach, a branched amphiphilic, PEGylated polysulfide (PPSES-PEG) was preferred over a linear analogue, because of improved homogeneity in the aggregates (spherical micelles vs. mixture of worm-like and spherical), increased stability and higher drug loading (up to ~22%wt. of anti-osteoclastic rapamycin). These effects are ascribed to the branching inhibiting crystallization in the polysulfide blocks. The ROSscavenging micelles alone were already able to reduce osteoclastogenesis in a RAW 264.7 model, but the 'drug' combination (the polymer itself + rapamycin released only under oxidation) completely abrogated the process. An important take-home message is that the synergic performance depended very strongly on the oxidant:oxidizable group molar ratio, a parameter to carefully tune in the perspective of targeting specific diseases.
Advanced Healthcare Materials, 2019
often, while we may have some mechanistic knowledge of how a delivery vehicle initially binds to ... more often, while we may have some mechanistic knowledge of how a delivery vehicle initially binds to such a group, we hardly know what happens during later phases. These phases, however, are equally critical for the efficacy of the treatment: for example, whether a nucleic acid-loaded particle remains on a cell surface or is transported intracellularly can make the difference between therapeutic success and failure. In short, the rational development of a targeted delivery approach should require the understanding of both surface binding and internalization processes, and the interplay between them. Here, we have focused on these points, using nanoparticles where hyaluronic acid (HA) acts as a targeting element and siRNA is the payload (i.e., the drug). Rather than for cell targeting, HA is better known as a major component of extracellular matrices, [1] and for its use as a matrix component in regenerative medicine, [2] as a dermal filler, [3] or a viscosupplementation agent. [4] However, besides its physical properties, HA is also capable of interacting with a number of biomolecules. [5] Most, but not all HA-binding proteins (also referred to as hyaladherins) share a homologous, HA-binding Link domain, whose CD44 is an endocytic hyaluronic acid (HA) receptor, and is overexpressed in many carcinomas. This has encouraged the use of HA to design CD44targeting carriers. This paper is about dissecting the mechanistic role of CD44. Here, HA-decorated nanoparticles are used to deliver siRNA to both tumoral (AsPC-1, PANC-1, HT-29, HCT-116) and non-tumoral (fibroblasts, differently polarized THP-1 macrophages, HUVEC) human cell lines, evaluating the initial binding of the nanoparticles, their internalization rate, and the silencing efficiency (cyclophilin B (PPIB) gene). Tumoral cells internalize faster and experience higher silencing than non-tumoral cells. This is promising as it suggests that, in a tumor, HA nanocarriers may have limited off-target effects. More far-reaching is the interrelation between the four parameters of the study: CD44 expression, HA binding on cell surfaces, internalization rate, and silencing efficiency. No correlation is found between binding (an early event) and any of the other parameters, whereas silencing correlates both with speed of the internalization process and CD44 expression. This study confirms on one hand that HA-based carriers can perform a targeted action, but on the other it suggests that this may not be due to a selective binding event, but rather to a later recognition leading to selective internalization.
Langmuir, 2019
Hybrids composed of liposomes (L) and metallic nanoparticles (NP) hold great potential for imagin... more Hybrids composed of liposomes (L) and metallic nanoparticles (NP) hold great potential for imaging and drug delivery purposes. However, the efficient incorporation of metallic nanoparticles into liposomes using conventional methodologies has so far proved to be challenging. In this study, we report the fabrication of hybrids of liposomes and hydrophobic gold nanoparticles of size 2-4 nm (Au) using a microfluidic assisted self-assembly process. The incorporation of increasing amounts of Au nanoparticles into liposomes was examined using microfluidics and compared to L-AuNP hybrids prepared by the reverse-phase evaporation method. Our microfluidics strategy produced L-AuNP hybrids with a homogeneous size distribution, smaller polydispersity index, and a 3-fold increase in loading efficiency when compared to those hybrids prepared using the reverse-phase method of production. Quantification of the loading efficiency was determined by ultraviolet spectroscopy, inductively coupled plasma mass spectroscopy and centrifugal field flow fractionation and confirmed qualitatively by transmission electron microscopy. The higher loading of gold nanoparticles into the liposomes achieved using microfluidics produced a slightly thicker and more rigid bilayer as determined with small angle neutron scattering. These observations were confirmed using fluorescent anisotropy and atomic force microscopy, respectively. Structural characterization of the liposomal-nanoparticle hybrids with cryo-electron microscopy revealed the coexistence of membrane-embedded and interdigitated nanoparticle-rich domains suggesting AuNP incorporation through hydrophobic interactions. The microfluidic technique that we describe in this study allows for the automated production of monodisperse liposomal-nanoparticle hybrids with high loading capacity highlighting the utility of microfluidics to improve the payload of metallic nanoparticles within liposomes, thereby enhancing their application for imaging and drug delivery.
ACS Applied Materials & Interfaces, 2019
This study is about 1) nanomanufacturing (focusing on microfluidic-assisted nanoprecipitation), 2... more This study is about 1) nanomanufacturing (focusing on microfluidic-assisted nanoprecipitation), 2) advanced colloid characterization (focusing on field flow fractionation, FFF), 3) the possible restructuring of surface disulfides. Disulfides are dynamic and exchangeable groups, and here we specifically focus firstly on their use to introduce biofunctional group, and secondly on their reorganization, which may lead to variable surface chemistries and uncontrolled cell interactions. The particles were obtained via microfluidic-assisted (flow-focused) nanoprecipitation of poly(ethylene glycol)-b-poly(-caprolactone) (PEG-PCL) bearing or not a 2-pyridyl disulfide (PDS) terminal group, which quantitatively exchanges with thiols in solution. In this study, we have paid specific attention to size characterization, thereby also demonstrating the limitations of dynamic light scattering (DLS) as a stand-alone technique. By using Asymmetric Flow Field Flow Fractionation (AF4) coupled with DLS, static light scattering (SLS) and refractive index detectors, we show that relatively small amounts of >100 nm aggregates (cryo-TEM and SLS/DLS comparison suggesting them to be wormlike micelles) dominated the stand-alone DLS results, whereas the 'real' size distributions picked < 50 nm. Our key result is that the kinetics of the conjugation based on PDS-thiol exchange was controlled by the thiol pKa, and this also determined the rate of the exchange between the resulting disulfides and glutathione (GSH). In particular, more acidic thiols (e.g. peptides where a cysteine is flanked by cationic residues) react faster with PDS, but their disulfides hardly exchange with GSH; the reverse applies to thiols with a higher pKa. Disulfides that resist against restructuring via thiol-disulfide exchange allow for a stable bioconjugation, although they may be bad news for payload release under reducing conditions. However, experiments of both thiol release and nanoparticles uptake in cells (HCT116) show that also the disulfides formed from less acidic, and therefore less reactive and more exchangeable thiols, were stable for at least a few hours even in a GSH-rich (10 mM) environment; this suggests a sufficiently long stability of surface groups to achieve e.g. a cell targeting effect.
Bioconjugate chemistry, Jan 19, 2018
We present a method for tyrosine-selective and reversible bioconjugation; tyrosines are enzymatic... more We present a method for tyrosine-selective and reversible bioconjugation; tyrosines are enzymatically converted into catechols and in situ "clicked" onto boronic acids. Importantly, our process selectively produces catechols and avoids quinones, thereby improving the control over the chemical identity of the products. We have conjugated boronic acid-containing hyaluronic acid (HyA) to peptides bearing tyrosines in variable number and position; the use of tagging peptides for the provision of well exposed tyrosine residues-in our case the hemagglutinin-derived HA-tag-makes our approach applicable to virtually any protein; we have demonstrated this concept by conjugating HA-tagged ovalbumin to HyA, thereby also showing the feasibility of producing chimeric proteoglycans. A caveat of this appproach is that, although the formation of boronic esters does not affect the biological recognition of substrates (ovalbumin and HyA), the introduction of catechols may alter some of thei...
International journal of pharmaceutics, Jan 20, 2017
We have employed microfluidics (cross-shaped chip) for the preparation of drug-loaded poly(lactic... more We have employed microfluidics (cross-shaped chip) for the preparation of drug-loaded poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles. The polymer precipitates from an acetone solution upon its controlled laminar mixing (flow focusing) with an aqueous solution of a surfactant, allowing for an operator-independent, up-scalable and reproducible preparative process of nanoformulations. Firstly, using PEGylated surfactants we have compared batch and microfluidic processes, and showed the superior reproducibility of the latter and its strong dependency on the acetone/water ratio (flow rate ratio). We have then focused on the issue of purification from free surfactant, and employed advanced characterization techniques such as flow-through dynamic light scattering as the in-line quality control technique, and field flow fractionation (FFF) with dynamic and static light scattering detection, which allowed the detection of surfactant micelles in mixture with nanoparticles (hardly pos...
Molecular Pharmaceutics, 2017
Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver a RNA/DNA cargo to cells overe... more Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver a RNA/DNA cargo to cells overexpressing HA receptors such as CD44. For these systems, unequivocal links have not been established yet between chitosan macromolecular (molecular weight; degree of deacetylation, i.e. charge density) and nanoparticle variables (complexation strength, i.e. stability; nucleic acid protection; internalization rate) on one hand, and transfection efficiency on the other hand. Here, we have focused on the role of avidity on transfection efficiency in the CD44-expressing HCT-116 as a cellular model; we have employed two differently sized payloads (a large luciferaseencoding mRNA and a much smaller anti-luc siRNA), and a small library of chitosans (variable molecular weight and degree of deactylation). The RNA avidity for chitosan showed-as expectedan inverse relationship: higher avidity-higher polyplex stability-lower transfection efficiency. The avidity of chitosan for RNA appears to lead to opposite effects: higher avidity-higher polyplex stability but also higher transfection efficiency. Surprisingly, the best transfecting particles were those with the lowest propensity for RNA release, although this might be a misleading relationship: for example, the same macromolecular parameters that increase avidity can also boost chitosan's endosomolytic activity, with a strong enhancement in transfection. The performance of these nonviral vectors appears therefore difficult to predict simply on the basis of carrier-or payload-related variables and a more holistic consideration of the journey of the nanoparticle, from cell uptake to cytosolic bioavailability of payload, is needed. It is also noteworthy that the nanoparticles used in this study showed optimal performance under slightly acidic conditions (pH 6.4), which is promising for applications in a tumoural extracellular environment. It is also worth pointing out that under these conditions we have for the first time successfully delivered mRNA with chitosan/HA nanoparticles.
Advanced Healthcare Materials, 2016
Macromolecular Rapid Communications, 2016
Families of amphiphilic oxidation-responsive polymers (poly(ethylene glycol)-polysulfides) with d... more Families of amphiphilic oxidation-responsive polymers (poly(ethylene glycol)-polysulfides) with different architectures (linear, 4, 6, and 8-armed stars and 10, 15, and 20-armed combs) and compositions (variable ethylene sulfide/propylene sulfide ratio) are prepared. In water, all the polymers assemble in spherical micelles, with critical micellar concentrations <0.01 mg mLfor all the branched polymers. Triple-detection gel permeation chromatography (GPC) and asymmetric field flow fractionation (AFFF) with dynamic and static light scattering detection, respectively, show an increasing compaction of the polymeric coil and a strong reduction of the aggregation number with increasing degree of branching. The key finding of this study is that the kinetics of the oxidative response sharply depend on the branching; in particular, it is highlighted that the degree of branching influences the lag time before a response can be observed rather than the speed of the response itself, a phenomenon that is attributed to a branching-dependent solubility of the oxidant in the polysulfide matrix.
Applied Radiation and Isotopes, 2017