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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 (â¼50% 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 methionine-mimetic PTGG chains. Both in mice and rats, PTGG-ovalbumin displayed circulation half-lives up to twice as long as PEG-ovalbumin, but most importantlyâand differently from PEGâwithout 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.
Assuntos
Polietilenoglicóis , Polímeros , Ratos , Camundongos , Humanos , Animais , Polietilenoglicóis/metabolismo , Polímeros/farmacologia , Glicerol , Espécies Reativas de Oxigênio , Ovalbumina , Estabilidade ProteicaRESUMO
The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a "domino" mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.
Assuntos
Proteína BRCA2 , Rad51 Recombinase , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Reparo do DNA , Recombinação Homóloga , Peptídeos/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismoRESUMO
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.
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Neoplasias , Pró-Fármacos , Animais , Sistemas de Liberação de Medicamentos , Ácido Hialurônico/uso terapêutico , Masculino , Camundongos , Camundongos SCID , Micelas , Neoplasias/tratamento farmacológico , Pró-Fármacos/farmacologiaRESUMO
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 increases in accordance with CXCR4 expression in FBS/medium. We demonstrated that CXCL12-decorated nanoparticles do not induce cell migration on their own, but their pre-incubation with THP-1 significantly decreases CXCR4+-cell migration, thereby antagonizing the chemotactic action of CXCL12. The use of biodegradable and immune-compatible chemokine-mimetic nanoparticles to reduce cell migration opens the way to novel antagonists with potential application in cancer treatments and inflammation.
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This study is about fine tuning the targeting capacity of peptide-decorated nanoparticles to discriminate between cells that express different integrin make-ups. Using microfluidic-assisted nanoprecipitation, we have prepared poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles with a PEGylated surface decorated with two different arginine-glycine-aspartic acid (RGD) peptides: one is cyclic (RGDFC) and has specific affinity towards αvß3 integrin heterodimers; the other is linear (RGDSP) and is reported to bind equally αvß3 and α5ß1. We have then evaluated the nanoparticle internalization in two cell lines with a markedly different integrin fingerprint: ovarian carcinoma A2780 (almost no αvß3, moderate in α5ß1) and glioma U87MG (very high in αvß3, moderate/high in α5ß1). As expected, particles with cyclic RGD were heavily internalized by U87MG (proportional to the peptide content and abrogated by anti-αvß3) but not by A2780 (same as PEGylated particles). The linear peptide, on the other hand, did not differentiate between the cell lines, and the uptake increase vs. control particles was never higher than 50%, indicating a possible low and unselective affinity for various integrins. The strong preference of U87MG for cyclic (vs. linear) peptide-decorated nanoparticles was shown in 2D culture and further demonstrated in spheroids. Our results demonstrate that targeting specific integrin make-ups is possible and may open the way to more precise treatment, but more efforts need to be devoted to a better understanding of the relation between RGD structure and their integrin-binding capacity.
Assuntos
Integrinas/metabolismo , Microfluídica/métodos , Nanopartículas/química , Neoplasias/metabolismo , Oligopeptídeos , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Feminino , Glioma/metabolismo , Humanos , Modelos Lineares , Espectroscopia de Ressonância Magnética , Microscopia Confocal , Microscopia de Fluorescência , Neoplasias Ovarianas/metabolismo , Poloxâmero , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Rodaminas/químicaRESUMO
The mechanical properties of the cellular microenvironment play a crucial role in modulating cell function, and many pathophysiological processes are accompanied by variations in extracellular matrix (ECM) stiffness. Lysyl oxidase (LOx) is one of the enzymes involved in several ECM-stiffening processes. Here, we engineered poly(ethylene glycol) (PEG)-based hydrogels with controlled mechanical properties in the range typical of soft tissues. These hydrogels were functionalized featuring free primary amines, which allows an additional chemical LOx-responsive behavior with increase in crosslinks and hydrogel elastic modulus, mimicking biological ECM-stiffening mechanisms. Hydrogels with elastic moduli in the range of 0.5-4 kPa were obtained after a first photopolymerization step. The increase in elastic modulus of the functionalized and enzyme-responsive hydrogels was also characterized after the second-step enzymatic reaction, recording an increase in hydrogel stiffness up to 0.5 kPa after incubation with LOx. Finally, hydrogel precursors containing HepG2 (bioinks) were used to form three-dimensional (3D) in vitro models to mimic hepatic tissue and test PEG-based hydrogel biocompatibility. Hepatic functional markers were measured up to 7 days of culture, suggesting further use of such 3D models to study cell mechanobiology and response to dynamic variation of hydrogels stiffness. The results show that the functionalized hydrogels presented in this work match the mechanical properties of soft tissues, allow dynamic variations of hydrogel stiffness, and can be used to mimic changes in the microenvironment properties of soft tissues typical of inflammation and pathological changes at early stages (e.g., fibrosis, cancer).
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CD44 is an endocytic hyaluronic acid (HA) receptor, and is overexpressed in many carcinomas. This has encouraged the use of HA to design CD44-targeting 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 inter-relation 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.
Assuntos
Receptores de Hialuronatos/química , Ácido Hialurônico/química , Nanopartículas/química , Linhagem Celular , Linhagem Celular Tumoral , Quitosana/química , Sistemas de Liberação de Medicamentos/métodos , Difusão Dinâmica da Luz , Células HCT116 , Células HT29 , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , RNA Interferente Pequeno/química , Células THP-1RESUMO
This study is about (1) nanomanufacturing (focusing on microfluidic-assisted nanoprecipitation), (2) advanced colloid characterization (focusing on field flow fractionation), and (3) the possible restructuring of surface disulfides. Disulfides are dynamic and exchangeable groups, and here we specifically focus, first, on their use to introduce biofunctional groups and, second, on their re-organization, 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(ε-caprolactone) 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 coupled with DLS, static light scattering (SLS), and refractive index detectors, we show that relatively small amounts of >100 nm aggregates (cryogenic transmission electron microscopy 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, for example, a cell-targeting effect.
Assuntos
Dissulfetos/química , Microfluídica , Nanopartículas/química , Cisteína/química , Dissulfetos/síntese química , Etilenoglicóis/química , Etilenoglicóis/farmacologia , Glutationa/química , Células HCT116 , Humanos , Cinética , Nanopartículas/administração & dosagem , Peptídeos/química , Poliésteres/química , Poliésteres/farmacologia , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia , Propriedades de SuperfícieRESUMO
The development of delivery systems capable of tumor targeting represents a promising strategy to overcome issues related to nonspecific effects of conventional anticancer therapies. Currently, one of the most investigated agents for cancer targeting is hyaluronic acid (HA), since its receptor, CD44, is overexpressed in many cancers. However, most of the studies on CD44/HA interaction have been so far performed in cell-free or genetically modified systems, thus leaving some uncertainty regarding which cell-related factors influence HA binding and internalization (collectively called "uptake") into CD44-expressing cells. To address this, the expression of CD44 (both standard and variants, designated CD44s and CD44v, respectively) was evaluated in human dermal fibroblasts (HDFs) and a large panel of cancer cell lines, including breast, prostate, head and neck, pancreatic, ovarian, colorectal, thyroid, and endometrial cancers. Results showed that CD44 isoform profiles and expression levels vary across the cancer cell lines and HDF and are not consistent within the cell origin. Using composite information of CD44 expression, HA binding, and internalization, we found that the expression of CD44v can negatively influence the uptake of HA, and, instead, when cells primarily expressed CD44s, a positive correlation was observed between expression and uptake. In other words, CD44shigh cells bound and internalized more HA compared to CD44slow cells. Moreover, CD44shigh HDFs were less efficient in uptaking HA compared to CD44shigh cancer cells. The experiments described here are the first step toward understanding the interplay between CD44 expression, its functionality, and the underlying mechanism(s) for HA uptake. The results show that factors other than the amount of CD44 receptor can play a role in the interaction with HA, and this represents an important advance with respect to the design of HA-based carriers and the selection of tumors to treat according to their CD44 expression profile.
Assuntos
Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/uso terapêutico , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Citometria de Fluxo , Humanos , Imuno-HistoquímicaRESUMO
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 mildly larger size (measured by dynamic light scattering alone or by field flow fractionation coupled to static or dynamic light scattering), and above all a higher aspect ratio (R g/R H) and a lower fractal dimension. We then compared the kinetics of uptake and the (antiluciferase) siRNA delivery performance in murine RAW 264.7 macrophages and in human HCT-116 colorectal tumor cells. The preparative method (and therefore the internal particle morphology) had little effect on the uptake kinetics and no statistically relevant influence on silencing (templated particles often showing a lower silencing). Cell-specific factors, on the contrary, overwhelmingly determined the efficacy of the carriers, with, e.g., those containing low-MW chitosan performing better in macrophages and those with high-MW chitosan in HCT-116.
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In this work we evaluate the effect of polymer composition and architecture of (PEGylated) polyesters on particle size and paclitaxel (PTX) loading for particles manufactured via microfluidic-assisted, continuous-flow nanoprecipitation using two microfluidic chips with different geometries and mixing principles. We have prepared poly (d,l-lactic acid-co-caprolactone) (PLCL) from ring-opening polymerization (ROP) of LA and CL mixtures and different (macro) initiators (namely, 1-dodecanol, a MeO-PEG-OH, and a 4-armed star PEG-OH), rendering polyesters that vary in monomer composition (i.e. LA/CL ratios) and architecture (i.e. linear vs 4-armed star). Continuous-flow nanoprecipitation was assayed using two microfluidic chips: a cross-flow chip with a X-shaped mixing junction (2D laminar flow focusing) and a micromixer featuring a Y-shaped mixing junction and a split and recombine path (2D laminar flow focusing convinced with stream lamination for faster mixing). Nanoparticle formulations were produced with Z-average sizes in the range of 30-160â¯nm, although size selectivity could be seen for different polymer/chip combinations; for instance, smaller particles were obtained with Y-shaped micromixer (30-120â¯nm), specially for the PEGylated polyesters (30-50â¯nm), whereas the cross-flow chip systematically produced larger particles (80-160â¯nm). Loading of the anti-cancer drug paclitaxel (PTX) was also heavily influenced not only by the nature of the polyester, but also by the geometry of the microfluidic chip; higher drug loadings were obtained with the cross-flow reactor and the star block copolymers. Finally, decreasing the LA/CL ratio generally had a positive effect on drug loading.
Assuntos
Antineoplásicos Fitogênicos/química , Microfluídica , Nanopartículas/química , Paclitaxel/química , Poliésteres/química , Polietilenoglicóis/química , Precipitação Química , Composição de Medicamentos/métodos , Tamanho da PartículaRESUMO
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 their biological properties: for example, only after tyrosinase treatment ovalbumin directly induced dendritic cell maturation, either alone or as a HyA conjugate.
Assuntos
Substâncias Macromoleculares/química , Monofenol Mono-Oxigenase/química , Ácidos Borônicos/química , Catecóis/química , Estudos de Viabilidade , Ácido Hialurônico/química , Peptídeos/química , Quinonas/químicaRESUMO
Polysaccharides (PSs) have been extensively studied in healthcare applications; here, we focus our attention on their use as components of nanomaterials in the management of cancer and inflammatory pathologies. Key advantages of PSs are easy availability, general biodegradability and biocompatibility, low or negligible toxicity, often a low immunogenicity and finally an ease of chemical modification. Here, we pay particular attention to the large family of amphiphilic PS derivatives (AMPDs); they are synthesized by modifying hydrophilic PSs with a variety of hydrophobic groups, which allow the constructs to self-assemble into various nanostructures in aqueous solution. This review focuses on AMPD-based self-assembled nanoparticles, from the chemical synthesis of AMPDs, through nanoparticle preparative strategies, to the most recent applications in cancer and inflammation management, including therapeutics, imaging and theranostics. We also offer an overview, which we feel lacks in the current literature, of the relation between the nature of the hydrophilic PSs and that of the hydrophobic components, of linkers, targeting groups and cross-linkers, and of the actual properties and in vivo fate of AMPD-based nanoparticles. Finally, we believe that this comprehensive insight into the possible effects of AMPDs' structural components on the performance of nanosystems, can provide criteria for a rational and molecular level-based design of AMPDs.
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Inflamação/tratamento farmacológico , Nanoestruturas/administração & dosagem , Neoplasias/tratamento farmacológico , Polissacarídeos/administração & dosagem , Animais , Desenho de Fármacos , Humanos , Nanoestruturas/química , Polissacarídeos/químicaRESUMO
Fetal growth restriction (FGR) in pregnancy is commonly caused by impaired uteroplacental blood flow. Vasodilators enhance uteroplacental perfusion and fetal growth in humans and animal models; however, detrimental maternal and fetal side effects have been reported. We hypothesised that targeted uteroplacental delivery of a vasodilator would enhance drug efficacy and reduce the risks associated with drug administration in pregnancy. Phage screening identified novel peptides that selectively accumulated in the uteroplacental vasculature of pregnant mice. Following intravenous injection, the synthetic peptide CNKGLRNK selectively bound to the endothelium of the uterine spiral arteries and placental labyrinth in vivo; CNKGLRNK-decorated liposomes also selectively bound to these regions. The nitric oxide donor 2-[[4-[(nitrooxy)methyl]benzoyl]thio]-benzoic acid methyl ester (SE175) induced significant relaxation of mouse uterine arteries and human placental arteries in vitro; thus, SE175 was encapsulated into these targeted liposomes and administered to healthy pregnant C57BL/6J mice or endothelial nitric oxide synthase knockout (eNOS-/-) mice, which exhibit impaired uteroplacental blood flow and FGR. Liposomes containing SE175 (0.44mg/kg) or PBS were administered on embryonic (E) days 11.5, 13.5, 15.5 and 17.5; fetal and placental weights were recorded at term and compared to mice injected with free PBS or SE175. Targeted uteroplacental delivery of SE175 had no effect on fetal weight in C57BL/6J mice, but significantly increased fetal weight and mean spiral artery diameter, and decreased placental weight, indicative of improved placental efficiency, in eNOS-/- mice; free SE175 had no effect on fetal weight or spiral artery diameter. Targeted, but not free SE175 also significantly reduced placental expression of 4-hydroxynonenal, cyclooxygenase-1 and cyclooxygenase-2, indicating a reduction in placental oxidative stress. These data suggest that exploiting vascular targeting peptides to selectively deliver SE175 to the uteroplacental vasculature may represent a novel treatment for FGR resulting from impaired uteroplacental perfusion.
Assuntos
Circulação Sanguínea/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Retardo do Crescimento Fetal/tratamento farmacológico , Peptídeos/farmacocinética , Placenta/efeitos dos fármacos , Útero/efeitos dos fármacos , Vasodilatadores/farmacocinética , Animais , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Feminino , Humanos , Injeções Intravenosas , Lipossomos/administração & dosagem , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peptídeos/administração & dosagem , Gravidez , Resultado do Tratamento , Vasodilatadores/administração & dosagem , Vasodilatadores/farmacologiaRESUMO
Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver an 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 luciferase-encoding 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 expected-an 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 tumoral 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.
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Quitosana/química , Ácido Hialurônico/química , Nanopartículas/química , Difusão Dinâmica da Luz , Células HCT116 , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Peso Molecular , Peptídeos Cíclicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: The object of this study was to develop a thermally and reactive oxygen species-responsive nanocarrier system for cancer therapy. RESULTS: PPS-PNIPAm block copolymer was designed and synthesised using a combination of living anionic ring-opening polymerization and atom transfer radical polymerization. The synthesized polymer formed micellar aggregates in water and demonstrated dual responsiveness towards temperature and oxidants. Using doxorubicin (DOX) as a model drug, encapsulation and in vitro release of the drug molecules in PPS-PNIPAm nanocarriers confirmed the responsive release properties of such system. Cell uptake of the DOX loaded micelles was investigated with human breast cancer cell line (MCF-7). The results showed Dox-loaded micelles were able to be taken by the cells and mainly reside in the cytoplasma. In the stimulated cells with an elevated level of ROS, more released DOX was observed around the nuclei. In the cytotoxicity experiments, the Dox-loaded micelles demonstrated comparable efficacy to free DOX at higher concentrations, especially on ROS stimulated cells. CONCLUSIONS: These results demonstrated that PPS-PNIPAm nanocarriers possess the capability to respond two typical stimuli in inflammatory cells: temperature and oxidants and can be used in anticancer drug delivery.
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Resinas Acrílicas/metabolismo , Antibióticos Antineoplásicos/administração & dosagem , Preparações de Ação Retardada/metabolismo , Doxorrubicina/administração & dosagem , Polietilenoglicóis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sulfetos/metabolismo , Resinas Acrílicas/química , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Feminino , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Células MCF-7 , Micelas , Polietilenoglicóis/química , Sulfetos/química , TemperaturaRESUMO
This study presents a quantitative assessment of the complexation between boronic acids and diols as a reversible and double-stimulus (oxidation and acidification)-responsive bioconjugation reaction. First, by using a competition assay, we have evaluated the equilibrium constants (water, pH 7.4) of 34 boronate/diol pairs, using diols of both aliphatic and aromatic (catechols) nature; in general, catechols were characterized by constants 3 orders of magnitude higher than those of aliphatic diols. Second, we have demonstrated that successful complexation with diols generated in situ via enzymatic reactions, and the boronate complexation was also employed to calculate the Michaelis-Menten parameters for two catechol-producing reactions: the demethylation of 3-methoxytyramine and the 2-hydroxylation of estradiol, respectively, mediated by P4502D6 and P4501A2. Third, we have prepared phenylboronic acid-functionalized hyaluronic acid (HA) and demonstrated the pH and H2O2-responsive character of the adducts that it formed with Alizarin Red S (ARS) used as a model catechol. The versatility and selectivity of the complexation and the mild character of the chemical species involved therefore make the boronate/catechol reaction an interesting candidate for bioconjugation purposes.
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Antraquinonas/química , Ácidos Borônicos/química , Catecóis/química , Dopamina/análogos & derivados , Estradiol/química , Antraquinonas/metabolismo , Ácidos Borônicos/metabolismo , Catecóis/metabolismo , Cromatografia de Afinidade , Dopamina/química , Dopamina/metabolismo , Estradiol/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Oxirredução , Água/química , Água/metabolismoRESUMO
CD44 is a potentially rewarding target in cancer therapy, although its mechanisms of ligand binding and internalization are still poorly understood. In this study, we have established quantitative relationships between CD44 expression in differently polarized macrophages (M0, M1, and M2-polarized THP-1 human macrophages) and the uptake of hyaluronic acid (HA)-based materials, which are potentially usable for CD44 targeting. We have validated a robust method for macrophage polarization, which sequentially uses differentiating and polarizing factors, and allows to show that CD44 expression depends on polarization (M1 > M0 ≥ M2). It is noteworthy that THP-1 M2 expressed CD44v6, suggesting their suitability as a model of tumor-associated macrophages. In the uptake of HA, both as a soluble polymer and in the form of (siRNA-loaded) nanoparticles, CD44 expression correlated positively with binding, but negatively with internalization. Counterintuitively, it appears that a higher presence of CD44 (in M1) allows a more efficient capture of HA materials, but a lower expression (in M2) is conducive to better internalization. Although possibly cell-specific, this unexpected relationship indicates that the common paradigm "higher CD44 expression = better targetability" is too simplistic; mechanistic details of both receptor presentation and association still need to be elucidated for a predictable targeting behavior.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico , Macrófagos/metabolismo , Nanopartículas/química , Linhagem Celular Tumoral , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/farmacocinética , Ácido Hialurônico/farmacologia , Macrófagos/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
Most current vaccines are either inactivated pathogen-derived or protein/peptide-based, although attenuated and vector vaccines have also been developed. The former induce at best moderate protection, even as multimeric antigen, due to limitations in antigen loads and therefore capacity for inducing robust immune defense. While attenuated and vector vaccines offer advantages through their replicative nature, drawbacks and risks remain with potential reversion to virulence and interference from preexisting immunity. New advances averting these problems are combining self-amplifying replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (12-15 kb) derived from viral genomes defective in at least one structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitation with RepRNA is RNase-sensitivity and inefficient uptake by dendritic cells (DCs)-absolute requirements for efficacious vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Encapsulating RepRNA into chitosan nanoparticles, as well as condensing RepRNA with polyethylenimine (PEI), cationic lipids, or chitosans, has proven effective for delivery to DCs and induction of immune responses in vivo.
Assuntos
Células Dendríticas/imunologia , RNA/imunologia , Replicon/imunologia , Vacinas/imunologia , Animais , Sistemas de Liberação de Medicamentos/métodos , Técnicas de Transferência de Genes , Humanos , RNA/genética , Replicon/genética , Vacinas/genéticaRESUMO
Nanoparticles based on hyaluronic acid (HA) are designed to deliver tannic acid (TA) as an antimicrobial agent. The presence of HA makes these particles potentially useful to target bacteria that colonize cells presenting HA membrane receptors (e.g. CD44), such as macrophages. HA bearing 3-aminophenyl boronic acid groups (HA-APBA) is reacted with TA, yielding nanoparticles with a size that decreases with decreasing HA molecular weight (e.g. 200 nm for 44 kDa, 400 nm for 737 kDa). The boronate esters make the nanoparticles stable at physiological pH, but their hydrolysis in an acidic environment (pH = 5) leads to swelling/solubilization, therefore potentially allowing TA release in endosomal compartments. We have assessed the nanoparticle toxicity profile (on RAW 264.7 macrophages) and their antimicrobial activity (on E. coli and on both methicillin-sensitive and -resistant S. aureus). The antibacterial effect of HA-APBA/TA nanoparticles was significantly higher than that of TA alone, and has very similar activity to TA coformulated with a reducing agent (ascorbic acid), which indicates both the nanoparticles to protect TA catechols from oxidation, and the effective release of TA after nanoparticle internalization. Therefore, there is potential for these nanoparticles to be used in stable, effective, and potentially targetable nanoparticle-based antimicrobial formulations.