Gold nanoparticles functionalised with fast water exchanging Gd3+ chelates: Linker effects on the relaxivity

The relaxivity displayed by Gd3+ chelates immobilized onto gold nanoparticles is the result of complex interplay between nanoparticle size, water exchange rate and chelate structure. In this work we study the effect of the length of -thioalkyl linkers, anchoring fast water exchanging Gd3+ chelates onto gold nanoparticles, on the relaxivity of the immobilized chelates. Gold nanoparticles functionalized with Gd3+ chelates of mercaptoundecanoyl and lipoyl amide conjugates of the DO3A-N-(-amino)propionate chelator were prepared and studied as potential CA for MRI. High relaxivities per chelate, of the order of magnitude 28-38 mM-1s-1 (30 MHz, 25 ºC) were attained thanks to simultaneous optimization of the rotational correlation time and of the water exchange rate. Fast local rotational motions of the immobilized chelates around connecting linkers (internal flexibility) still limit the attainable relaxivity. The degree of internal flexibility of the immobilized chelates seems not to be correlated with the length of the connecting linkers. Biodistribution and MRI studies in mice suggest that the in vivo behavior of the gold nanoparticles is determined mainly by size. Small nanoparticles (HD= 3.9 nm) undergo fast renal clearance and avoidance of the RES organs while larger nanoparticles (HD= 4.8 nm) undergo predominantly hepatobiliary excretion. High relaxivities, allied to chelate and nanoparticle stability and fast renal clearance in vivo suggests that functionalized gold nanoparticles hold great potential for further investigation as MRI Contrast Agents. This study contributes to understand the effect of linker length on the relaxivity of gold nanoparticles functionalized with Gd3+ complexes. It is a relevant contribution towards “design rules” for nanostructures functionalized with Gd3+ chelates as Contrast Agents for MRI and multimodal imaging.

A systematic study of the structural and magnetic properties of Mn-, Co-, and Ni-Doped Colloidal Fe3O4 nanoparticles

A series of colloidal MxFe3-xO4 (M = Mn, Co, Ni; x = 0–1) nanoparticles with diameters ranging from 6.8 to 11.6 nm was synthesized by hydrothermal reaction in aqueous medium at low temperature (200 °C). Energy-dispersive X-ray microa-nalysis and inductively coupled plasma spectrometry confirms that the actual elemental compositions agree well with the nominal ones. The structural properties of obtained nanoparticles were investigated by using powder X-ray diffraction, Raman scattering, Mössbauer spectroscopy, and electron microscopy. The results demonstrate that our synthesis technique leads to the formation of chemically uniform single-phase solid solution nanoparticles with cubic spinel structure, confirming the intrinsic doping. Magnetic studies showed that, in comparison to Fe3O4, the saturation magnetization of MxFe3-xO4 (M = Mn, Ni) decreases with increasing dopant concentration, while Co-doped samples showed similar saturation magnetizations. On other hand, whereas Mn- and Ni-doped nanoparticles exhibits superparamagnetic behavior at room temperature, ferromagnetism emerges for CoxFe3-xO4 nanoparticles, which can be tuned by the level of Co doping.

Chitosan-based scaffolds for tissue regeneration: preparation and microstructure characterization

Scaffolds are porous three-dimensional supports, designed to mimic the extracellular environment and remain temporarily integrated into the host tissue while stimulating, at the molecular level, specific cellular responses to each type of body tissues. The major goal of the research work entertained herein was to study the microstructure of scaffolds made from chitosan (Ch), blends of chitosan and sodium alginate (Ch/NaAlg), blends of chitosan, sodium alginate and calcium chloride (Ch/NaAlg/CaCl2) and blends of chitosan, sodium alginate and hydroxyapatite (Ch/NaAlg/HA). Scaffolds possessing ideal physicochemical properties facilitate cell proliferation and greatly increase the rate of recovery of a damaged organ tissue. Using CT three-dimensional images of the scaffolds, it was observed that all scaffolds had a porosity in the range 64%-92%, a radius of maximum pore occurrence in the range 95m-260m and a permeability in the range 1×10-10-18×10-10 m2. From the results obtained, the scaffolds based on Ch, Ch/NaAlg and Ch/NaAlg/CaCl2 would be most appropriate both for the growth of osteoid and for bone tissue regeneration, while the scaffold made with a blend of Ch/NaAlg/HA, by possessing larger pores size, might be used as a support for fibrovascular tissue.

Delivery as nanoparticles reduces imatinib mesylate-induced cardiotoxicity and improves anticancer activity

Clinical effectiveness of imatinib mesylate in cancer treatment is compromised by its off-target cardiotoxicity. In the present study, we have developed physically stable imatinib mesylate-loaded poly(lactide-co-glycolide) nanoparticles (INPs) that could sustainably release the drug, and studied its efficacy by in vitro anticancer and in vivo cardiotoxicity assays. MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay revealed that INPs are more cytotoxic to MCF-7 breast cancer cells compared to the equivalent concentration of free imatinib mesylate. Wistar rats orally administered with 50 mg/kg INPs for 28 days showed no significant cardiotoxicity or associated changes. Whereas, increased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels, and reduced white blood cell, red blood cell, and hemoglobin content were observed in the animals administered with free drug. While the histological sections from hearts of animals that received INPs did not show any significant cardiotoxic symptoms, loss of normal architecture and increased cytoplasmic vacuolization were observed in the heart sections of animals administered with free imatinib mesylate. Based on these results, we conclude that nano-encapsulation of imatinib mesylate increases its efficacy against cancer cells, with almost no cardiotoxicity.

PEGylated ofloxacin nanoparticles render strong antibacterial activity against many clinically important human pathogens.

The rise of bacterial resistance against important drugs threatens their clinical utility. Fluoroquinones, one of the most important classes of contemporary antibiotics has also reported to suffer bacterial resistance. Since the general mechanism of bacterial resistance against fluoroquinone antibiotics (e.g. ofloxacin) consists of target mutations resulting in reduced membrane permeability and increased efflux by the bacteria, strategies that could increase bacterial uptake and reduce efflux of the drug would provide effective treatment. In the present study, we have compared the efficiencies of ofloxacin delivered in the form of free drug (OFX) and as nanoparticles on bacterial uptake and antibacterial activity. Although both poly(lactic-co-glycolic acid) (OFX-PLGA) and methoxy poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (OFX-mPEG-PLGA) nanoformulations presented improved bacterial uptake and antibacterial activity against all the tested human bacterial pathogens, namely, Escherichia coli, Proteus vulgaris, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus, OFX-mPEG-PLGA showed significantly higher bacterial uptake and antibacterial activity compared to OFX-PLGA. We have also found that mPEG-PLGA nanoencapsulation could significantly inhibit Bacillus subtilis resistance development against OFX.

Effect of chitosan-Aloe vera coating on postharvest quality of blueberry (Vaccinium corymbosum) fruit

The present study was carried out to evaluate the effect of chitosan-based edible coatings with Aloe vera extract on the postharvest blueberry fruit quality during storage at 5 °C. Firstly, A. vera fractions (pulp and liquid) were extracted from leaves and evaluated in terms of antifungal and antioxidant capacities. The choice of the most adequate chitosan and A. vera fraction concentrations to be incorporated in coating formulation was made based on the wettability of the corresponding coating solutions. Coatings with 0.5% (w/v) chitosan + 0.5% (w/v) glycerol + 0.1% (w/v) Tween 80 + 0.5% (v/v) A. vera liquid fraction presented the best characteristics to uniformly coat blueberry surface. Physico-chemical (i.e., titratable acidity, pH, weight loss) and microbiological analyses of coated blueberries (non-inoculated or artificially inoculated with Botrytis cinerea) were performed during 25 d. Microbiological growth and water loss levels were approximately reduced by 50% and 42%, respectively, in coated blueberries after 25 d compared to uncoated blueberries. After 15 d, weight loss values were 6.2% and 3.7% for uncoated and chitosanA. vera coated blueberries, respectively. Uncoated fruits presented mold contamination after 2 d of storage (2.0 ± 0.32 log CFU g1), whilst fruits with chitosan-based coatings with A. vera presented mold contamination only after 9 d of storage (1.3 ± 0.35 log CFU g1). Overall, coatings developed in this study extend blueberries shelf-life for about 5 d, demonstrating for the first time that the combination of chitosan and A. vera liquid fraction as edible coating materials has great potential in expanding the shelf-life of fruits.

Magnetoliposomes based on manganese ferrite nanoparticles as nanocarriers for antitumor drugs

Manganese ferrite nanoparticles with a size distribution of 26 ± 7 nm (from TEM measurements) were synthesized by the coprecipitation method. The obtained nanoparticles exhibit a superparamagnetic behaviour at room temperature with a magnetic squareness of 0.016 and a coercivity field of 6.3 Oe. These nanoparticles were either entrapped in liposomes (aqueous magnetoliposomes, AMLs) or covered with a lipid bilayer, forming solid magnetoliposomes (SMLs). Both types of magnetoliposomes, exhibiting sizes below or around 150 nm, were found to be suitable for biomedical applications. Membrane fusion between magnetoliposomes (both AMLS and SMLs) and GUVs (giant unilamellar vesicles), the latter used as models of cell membranes, was confirmed by F¨orster Resonance Energy Transfer (FRET) assays, using a NBD labeled lipid as the energy donor and Nile Red or rhodamine B-DOPE as the energy acceptor. A potential antitumor thienopyridine derivative was successfully incorporated into both aqueous and solid magnetoliposomes, pointing to a promising application of these systems in oncological therapy, simultaneously as hyperthermia agents and nanocarriers for antitumor drugs.

Magnetoliposomes based on manganese ferrite nanoparticles as nanocarriers for antitumor drugs

Publicado em "NanoPT2016 book of abstracts"

Development of polymeric nanoparticles containing neuroprotective compounds of Hypericum perforatum

Tese de Doutoramento em Biologia das Plantas - MAP BIOPLANT

Production of elastic Chitosan/Chondroitin sulphate multilayer films for tissue regeneration

Membrane-like scaffolds are suitable to induce regeneration in many and different anatomic sites, such as periodontal membrane, skin, liver and cardiac tissues. In some circumstances, the films should adapt to geometrical changes of the attached tissues, such as in cardiac or blood vessel tissue engineering applications. In this context, we developed stretchable two-dimensional multilayer constructs through the assembling of two natural-based polyelectrolytes, chitosan (CHT) and chondroitin sulphate (CS), using the layer-by-layer methodology. The morphology, topography and the transparency of the films were evaluated. The in- fluence of genipin, a natural-derived cross-linker agent, was also investigated in the control of the mechanical properties of the CHT/CS films. The water uptake ability can be tailored by changing the cross-linker concentration, which influenced the young modulus and ultimate tensile strength. The maximum extension tends to decrease with the increase of genipin concentration, compromising the elastic properties of CHT/CS films: nevertheless using lower cross-linker contents, the ultimate tensile stress is similar to the films not cross-linked but exhibiting a significant higher modulus. The in vitro biological assays showed better L929 cell adhesion and proliferation when using the crosslinked membranes and confirmed the non-cytotoxicity of the CHT/CS films. The developed free-standing biomimetic multilayer could be designed to fulfill specific therapeutic requirements by tuning properties such as swelling, mechanical and biological performances.

Adhesive bioactive coatings inspired by sea life

Inspired by nature, in particular by the marine mussels adhesive proteins (MAPs) and by the tough brick-and-mortar nacre-like structure, novel multilayered films are prepared in the present work. Organic-inorganic multilayered films, with an architecture similar to nacre based on bioactive glass nanoparticles (BG), chitosan and hyaluronic acid modified with catechol groups, which are the main responsible for the outstanding adhesion in MAPs, are developed for the first time. The biomimetic conjugate is prepared by carbodiimide chemistry and analyzed by ultraviolet-visible spectrophotometry. The build-up of the multilayered films is monitored with a quartz crystal microbalance with dissipation monitoring and their topography is characterized by atomic force microscopy. The mechanical properties reveal that the films containing catechol groups and BG present an enhanced adhesion. Moreover, the bioactivity of the films upon immersion in a simulated body fluid solution for 7 days is evaluated by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction. It was found that the constructed films promote the formation of bone-like apatite in vitro. Such multifunctional mussel inspired LbL films, which combine enhanced adhesion and bioactivity, could be potentially used as coatings of a variety of implants for orthopedic applications.

Highly robust chitosan hydrogels via a fast, simple and biocompatible dual crosslinking-based process

Load-bearing soft tissues such as cartilage, blood vessels and muscles are able to withstand a remarkable compressive stress of several MPa without fracturing. Interestingly, most of these structural tissues are mainly composed of water and in this regard, hydrogels, as highly hydrated 3D-crosslinked polymeric networks, constitute a promising class of materials to repair lesions on these tissues. Although several approaches can be employed to shape the mechanical properties of artificial hydrogels to mimic the ones found on biotissues, critical issues regarding, for instance, their biocompatibility and recoverability after loading are often neglected. Therefore, an innovative hydrogel device made only of chitosan (CHI) was developed for the repair of robust biological tissues. These systems were fabricated through a dual-crosslinking process, comprising a photo- and an ionic-crosslinking step. The obtained CHIbased hydrogels exhibited an outstanding compressive strength of ca. 20 MPa at 95% of strain, which is several orders of magnitude higher than those of the individual components and close to the ones found in native soft tissues. Additionally, both crosslinking processes occur rapidly and under physiological conditions, enabling cellsâ encapsulation as confirmed by high cell survival rates (ca. 80%). Furthermore, in contrast with conventional hydrogels, these networks quickly recover upon unloading and are able to keep their mechanical properties under physiological conditions as result of their non-swell nature.

Influence of chitosan coating on protein-based nanohydrogels properties and in vitro gastric digestibility

Chitosan coating was applied in Lactoferrin (Lf)-Glycomacropeptide (GMP) nanohydrogels by layer-by-layer coating process. A volume ratio of 0.1 of Lf-GMP nanohydrogels (0.2 mg.mL-1, at pH 5.0) to chitosan (1 mg.mL-1, at pH 3) demonstrated to be the optimal condition to obtain stable nanohydrogels with size of 230 ± 12 nm, a PdI of 0.22 ± 0.02 and a -potential of 30.0 ± 0.15 mV. Transmission electron microscopy (TEM) images showed that the application of chitosan coating in Lf-GMP did not affect the spherical shape of nanohydrogels and confirmed the low aggregation of nanohydrogels in solution. The analysis of chemical interactions between chitosan and Lf-GMP nanohydrogels were performed by Fourier transform infrared spectroscopy (FTIR) and by circular dichroism (CD) that revealed that a specific chemical interaction occurring between functional groups of protein-based nanohydrogels and active groups of the chitosan was established. The effect of chitosan coating on release mechanisms of Lf-GMP nanohydrogels at acid conditions (pH 2, 37 ºC) was evaluated by the encapsulation of a model compound (caffeine) in these systems. Linear Superposition Model was used to fit the experimental data and revealed that Fick and relaxation mechanisms are involved in caffeine release. It was also observed that the Fick contribution increase with the application of chitosan coating. In vitro gastric digestion was performed with Lf-GMP nanohydrogels and Lf-GMP nanohydrogels with chitosan coating and it was observed that the presence of chitosan improve the stability of Lf and GMP (proteins were hydrolysed at a slower rate and were present in solution by longer time). Native electrophoreses revealed that the nanohydrogels without coating remained intact in solution until 15 min and with chitosan coating remained intact until 60 min, during gastric digestion.

In vivo imaging of glycol chitosan-based nanogel biodistribution

The preclinical development of nanomedicines raises several challenges and requires a comprehensive characterization. Among them is the evaluation of the biodistribution following systemic administration. In previous work, the biocompatibility and in vitro targeting ability of a glycol chitosan (GC) based nanogel have been validated. In the present study, its biodistribution in the mice is assessed, using near-infrared (NIR) fluorescence imaging as a tool to track the nanogel over time, after intravenous administration. Rapid whole body biodistribution of both Cy5.5 labeled GC nanogel and free polymer is found at early times. It remains widespreadly distributed in the body at least up to 6 h postinjection and its concentration then decreases drastically after 24 h. Nanogel blood circulation half-life lies around 2 h with the free linear GC polymer presenting lower blood clearance rate. After 24 h, the blood NIR fluorescence intensity associated with both samples decreases to insignificant values. NIR imaging of the organs shows that the nanogel had a body clearance time of 48 h, because at this time point a weak signal of NIR fluorescence is observed only in the kidneys. Hereupon it can be concluded that the engineered GC nanogel has a fairly long blood circulation time, suitable for biomedical applications, namely, drug delivery, simultaneously allowing efficient and quick body clearance.

Folate-targeted nanoparticles for rheumatoid arthritis therapy

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease, affecting almost 1% of the world population. Although the cause of RA remains unknown, the complex interaction between immune mediators (cytokines and effector cells) is responsible for the joint damage that begins at the synovial membrane. Activated macrophages are critical in the pathogenesis of RA and have been shown to specifically express a receptor for the vitamin folic acid (FA), folate receptor (FR). This particular receptor allows internalization of FA-coupled cargo. In this review we will address the potential of nanoparticles as an effective drug delivery system for therapies that will directly target activated macrophages. Special attention will be given to stealth degree of the nanoparticles as a strategy to avoid clearance by macrophages of the mononuclear phagocytic system (MPS). This review summarizes the application of FA-target nanoparticles as drug delivery systems for RA and proposes prospective future directions.