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1.
Int J Biol Macromol ; 211: 639-652, 2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35569680

RESUMO

Polymeric hydrogels are increasingly considered as scaffolds for tissue engineering due to their extraordinary resemblance with the extracellular matrix (ECM) of many tissues. As cell adhesion is a key factor in regulating important cell functions, hydrogel scaffolds are often functionalized or loaded with a variety of bioactive molecules that can promote adhesion. Interesting biomimetic approaches exploit the properties of mussel-inspired recombinant adhesive proteins. In this work, we prepared hydrogel scaffolds with a 50%w mixture of k-carrageenan (kC) and polyvinyl alcohol (PVA), by a two-step physical gelation process, and we coated them with Perna viridis foot protein-5ß (Pvfp5ß). The mechanical and morphological properties of hydrogels were investigated both after conditioning with typical cell culture media and also after coating with the Pvfp5ß. The protein resulted strongly adsorbed onto the surface of the hydrogel and also able to penetrate in its interiors to a certain depth, mainly interacting with the kC component of the scaffold as resulted from the confocal analysis. Mouse embryonic fibroblasts NIH-3T3 were seeded on top of the hydrogels and cultured up to two weeks. The role of Pvfp5ß in promoting cell adhesion, spreading and colonization of the scaffold was demonstrated.


Assuntos
Fibroblastos , Álcool de Polivinil , Animais , Carragenina/metabolismo , Adesão Celular/fisiologia , Fibroblastos/metabolismo , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Camundongos , Álcool de Polivinil/metabolismo , Proteínas Recombinantes/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte
2.
Mater Sci Eng C Mater Biol Appl ; 131: 112545, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34857257

RESUMO

Cartilage or bone regeneration approaches based on the direct injection of mesenchymal stem cells (MSCs) at the lesion site encounter several challenges, related to uncontrolled cell spreading and differentiation, reduced cell viability and poor engrafting. This work presents a simple and versatile strategy based on the synergic combination of in-situ forming hydrogels and spheroids of adipose stem cells (SASCs) with great potential for minimally invasive regenerative interventions aimed to threat bone and cartilage defects. Aqueous dispersions of partially degalactosylated xyloglucan (dXG) are mixed with SASCs derived from liposuction and either a chondroinductive or an osteoinductive medium. The dispersions rapidly set into hydrogels when temperature is brought to 37 °C. The physico-chemical and mechanical properties of the hydrogels are controlled by polymer concentration. The hydrogels, during 21 day incubation at 37 °C, undergo significant structural rearrangements that support cell proliferation and spreading. In formulations containing 1%w dXG cell viability increases up to 300% for SASCs-derived osteoblasts and up to 1000% for SASCs-derived chondrocytes if compared with control 2D cultures. The successful differentiation into the target cells is supported by the expression of lineage-specific genes. Cell-cell and cell-matrix interactions are also investigated. All formulations resulted injectable, and the incorporated cells are fully viable after injection.


Assuntos
Hidrogéis , Xilanos , Regeneração Óssea , Cartilagem , Diferenciação Celular , Glucanos , Células-Tronco
3.
Regen Biomater ; 8(5): rbab040, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34386265

RESUMO

Hydrogel wound dressings can play critical roles in wound healing protecting the wound from trauma or contamination and providing an ideal environment to support the growth of endogenous cells and promote wound closure. This work presents a self-assembling hydrogel dressing that can assist the wound repair process mimicking the hierarchical structure of skin extracellular matrix. To this aim, the co-assembly behaviour of a carboxylated variant of xyloglucan (CXG) with a peptide amphiphile (PA-H3) has been investigated to generate hierarchical constructs with tuneable molecular composition, structure, and properties. Transmission electron microscopy and circular dichroism at a low concentration shows that CXG and PA-H3 co-assemble into nanofibres by hydrophobic and electrostatic interactions and further aggregate into nanofibre bundles and networks. At a higher concentration, CXG and PA-H3 yield hydrogels that have been characterized for their morphology by scanning electron microscopy and for the mechanical properties by small-amplitude oscillatory shear rheological measurements and compression tests at different CXG/PA-H3 ratios. A preliminary biological evaluation has been carried out both in vitro with HaCat cells and in vivo in a mouse model.

4.
Int J Biol Macromol ; 164: 2818-2830, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853619

RESUMO

Hydrogels for complex and chronic wound dressings must be conformable, absorb and retain wound exudates and maintain hydration. They can incorporate and release bioactive molecules that can accelerate the healing process. Wound dressings have to be in contact with the wound and epidermis, even for long periods, without causing adverse effects. Hydrogel dressing formulations based on biopolymers derived from terrestrial or marine flora can be relatively inexpensive and well tolerated. In the present article hydrogel films composed by agarose (1.0 wt%), κ-carrageenan at three different concentrations (0.5, 1.0 and 1.5 wt%) and glycerol (3.0 wt%) were prepared without recourse to crosslinking agents, and characterized for their mechanical properties, morphology, swelling and erosion behavior. The films resulted highly elastic and able to absorb and retain large amounts of fluids without losing their integrity. One of the films was loaded with the aqueous extract from Cryphaea heteromalla (Hedw.) D. Mohr for its antioxidant properties. Absence of cytotoxicity and ability to reduce the oxidative stress were demonstrated on NIH-3T3 fibroblast cell cultures. These results encourage further biological evaluations to assess their impact on the healing process.


Assuntos
Antioxidantes/farmacologia , Bryopsida/química , Carragenina/química , Fibroblastos/citologia , Extratos Vegetais/farmacologia , Sefarose/química , Animais , Antioxidantes/química , Bandagens , Fenômenos Biomecânicos , Sobrevivência Celular , Elasticidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Metilgalactosídeos , Camundongos , Células NIH 3T3 , Extratos Vegetais/química
5.
Int J Biol Macromol ; 149: 309-319, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31987942

RESUMO

Injectable, in-situ forming kefiran gels have been developed for potential applications as implantable drug delivery devices or scaffolds for tissue regeneration. Concentrated solutions (4, 5 and 6%w) of kefiran, extracted from kefir grains, have been assessed in term of viscosity and injectability through G26 syringe needles, and for their ability to undergo gelation upon mixing with different alcohols. Propylene glycol (PG) has been selected as gelling agent because it ensures homogenous gelation in relatively short times (from few minutes up to 6 h). The investigation of the rheological behavior of kefiran/PG gels varying polymer concentration and temperature (25 °C and 37 °C) has provided interesting hints to support a possible gelation mechanism that accounts also for the observed influence of the alcohol type. Finally, the study of kefiran/PG gels has been complemented with the investigation on selected formulations of the swelling/degradation behavior upon immersion in isotonic buffer solution for up to 40 days at 37 °C; of the ability of the gels to retain and/or release two model molecules; and within vitro cell viability and cytotoxicity tests, to support the absence of toxic effects on cells induced by direct contact with the gels or by leached components from these gels.


Assuntos
Sistemas de Liberação de Medicamentos , Hidrogéis/química , Polissacarídeos/química , Álcoois/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Hidrogéis/farmacologia , Propilenoglicol/química , Reologia
6.
Int J Biol Macromol ; 121: 784-795, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30342149

RESUMO

Crosslinked xyloglucan-poly(vinyl alcohol) based hydrogel films are interesting materials for wound healing applications. This work focuses on the hydrolytic degradation and consequent morphological modification of a XG-PVA film and on its interaction with cells, blood, bacteria. Biocompatibility of the film was assessed in vitro by investigating different aspects, such as cell viability, oxidative stress level, mitochondrial dysfunction and specific stress biomarkers. Partial adhesiveness was demonstrated by performing different attaching assays and phalloidin staining. Hemocompatibility of XG-PVA film after interaction with blood was evaluated by using a multi-parametric approach, including human Red Blood Cells (RBC) count, hemolytic response and platelets activation. Thrombin and fibrinogen concentrations were examined as marker of the coagulation cascade. After direct contact with human blood and peripheral blood mononuclear cells (PBMC), no evidence of cell defense response was observed. Antimicrobial activity of XG-PVA film was tested against Escherichia coli (E.coli). XG-PVA film promotes bacterial retentivity and provides mechanical protection against bacterial infiltration. After loading the film with ampicillin, an inhibitory E. coli growth zone was observed. All together these results indicate that the XG-PVA system is a promising material to be tested in vivo for wound healing applications.


Assuntos
Glucanos/química , Glucanos/farmacologia , Metilgalactosídeos/química , Cicatrização/efeitos dos fármacos , Xilanos/química , Xilanos/farmacologia , Células A549 , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Hemostasia/efeitos dos fármacos , Humanos , Hidrólise , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos
7.
Data Brief ; 21: 1950-1953, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30510982

RESUMO

In wound dressing applications, exudate absorption and retention are important properties. The data presented here assess the ability of the crosslinked xyloglucan-poly(vinyl alcohol) hydrogel films (XG-PVA), described in "Xyloglucan-based hydrogel films for wound dressing: Structure-property relationships" (Ajovalasit et al., 2018) [1] and "Biocompatibility, hemocompatibility and antimicrobial properties of xyloglucan-based hydrogel film for wound healing application" (Picone et al., 2019), to absorb and retain proteins. These properties were investigated by Comassie blue staining and electrophoresis of Fetal Serum Proteins.

8.
J Control Release ; 270: 23-36, 2018 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-29196041

RESUMO

Recent evidences suggest that insulin delivery to the brain can be an important pharmacological therapy for some neurodegenerative pathologies, including Alzheimer disease (AD). Due to the presence of the Blood Brain Barrier, a suitable carrier and an appropriate route of administration are required to increase the efficacy and safety of the treatment. Here, poly(N-vinyl pyrrolidone)-based nanogels (NG), synthetized by e-beam irradiation, alone and with covalently attached insulin (NG-In) were characterized for biocompatibility and brain delivery features in a mouse model. Preliminarily, the biodistribution of the "empty" nanocarrier after intraperitoneal (i.p.) injection was investigated by using a fluorescent-labeled NG. By fluorescence spectroscopy, SEM and dynamic light scattering analyses we established that urine clearance occurs in 24h. Histological liver and kidneys inspections indicated that no morphological alterations of tissues occurred and no immunological response was activated after NG injection. Furthermore, after administration of the insulin-conjugated nanogels (NG-In) through the intranasal route (i.n.) no alteration or immunogenic response of the nasal mucosa was observed, suggesting that the formulation is well tolerated in mouse. Moreover, an enhancement of NG-In delivery to the different brain areas and of its biological activity, measured as Akt activation levels, with reference to free insulin administration was demonstrated. Taken together, these results indicate that the synthesized NG-In enhances brain insulin delivery upon i.n. administration and strongly encourage its further evaluation as therapeutic agent against some neurodegenerative diseases.


Assuntos
Encéfalo/metabolismo , Portadores de Fármacos/administração & dosagem , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Acrilatos/administração & dosagem , Acrilatos/farmacocinética , Administração Intranasal , Animais , Portadores de Fármacos/farmacocinética , Géis , Hipoglicemiantes/farmacocinética , Insulina/farmacocinética , Masculino , Camundongos Endogâmicos C57BL , Mucosa Nasal/metabolismo , Povidona/administração & dosagem , Povidona/farmacocinética
9.
Carbohydr Polym ; 179: 262-272, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29111050

RESUMO

Thin xyloglucan-based hydrogel films have been synthetized and characterized in the prospect of producing wound dressings. Polyvinyl alcohol (PVA) and glycerol (Gro) were added to have an optimal combination of softness, conformability and resilience. Physical hydrogels have been transformed into permanent covalent hydrogels by reaction with glutaraldehyde (GA). Network structure-process-property relationships are discussed on the account of the results of several complementary characterizations: FTIR, rheology, thermal analysis, morphological analysis, moisture retention and swelling measurements. Selected formulations were also subjected to preliminary in vitro cytotoxicity tests. The physical and mechanical properties of some of the xyloglucan-based hydrogel films produced, combined with absence of cytotoxicity, make them suitable candidates for integration with sensors to monitor the wound healing process and further biological investigations in animal models.


Assuntos
Curativos Hidrocoloides , Glucanos/química , Hidrogéis/química , Hidrogéis/farmacologia , Cicatrização/efeitos dos fármacos , Xilanos/química , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Glutaral/química , Glicerol/química , Humanos , Hidrogéis/síntese química , Álcool de Polivinil/química , Reologia , Relação Estrutura-Atividade , Termogravimetria
10.
Pharmaceutics ; 9(3)2017 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-28672810

RESUMO

Administered by an oral route, Furosemide (FUR), a diuretic used in several edematous states and hypertension, presents bioavailability problems, reported as a consequence of an erratic gastrointestinal absorption due to various existing polymorphic forms and low and pH-dependent solubility. A mucoadhesive sublingual fast-dissolving FUR based film has been developed and evaluated in order to optimize the bioavailability of FUR by increasing solubility and guaranteeing a good dissolution reproducibility. The Differential Scanning Calorimetry (DSC) analyses confirmed that the film prepared using the solvent casting method entrapped FUR in the amorphous state. As a solid dispersion, FUR increases its solubility up to 28.36 mg/mL. Drug content, thickness, and weight uniformity of film were also evaluated. The measured Young's Modulus, yield strength, and relative elongation of break percentage (EB%) allowed for the classification of the drug-loaded film as an elastomer. Mucoadhesive strength tests showed that the force to detach film from mucosa grew exponentially with increasing contact time up to 7667 N/m². FUR was quickly discharged from the film following a trend well fitted with the Weibull kinetic model. When applied on sublingual mucosa, the new formulation produced a massive drug flux in the systemic compartment. Overall, the proposed sublingual film enhances drug solubility and absorption, allowing for the prediction of a rapid onset of action and reproducible bioavailability in its clinical application.

11.
Biol Chem ; 398(2): 277-287, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27508963

RESUMO

Poly(N-vinyl pyrrolidone)-based-nanogels (NGs), produced by e-beam irradiation, are conjugated with monoclonal antibodies (mAb) for active targeting purposes. The uptake of immuno-functionalized nanogels is tested in an endothelial cell line, ECV304, using confocal and epifluorescence microscopy. Intracellular localization studies reveal a faster uptake of the immuno-nanogel conjugate with respect to the 'bare' nanogel. The specific internalization pathway of these immuno-nanogels is clarified by selective endocytosis inhibition experiments, flow cytometry and confocal microscopy. Active targeting ability is also verified by conjugating a monoclonal antibody which recognizes the αvß3 integrin on activated endothelial cells. Epifluorescence images of the 'wound healing assay' on ECV304 cells provide evidence of nanogels localization only in the target cells. Therefore, the immuno-nanogels produced have the potential to recognize specific cell types in heterogeneous systems, which makes them promising candidates for targeted drug delivery applications.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Portadores de Fármacos/química , Nanoestruturas/química , Anticorpos Monoclonais/metabolismo , Transporte Biológico , Linhagem Celular , Géis , Humanos , Povidona/química
12.
Mater Sci Eng C Mater Biol Appl ; 70(Pt 1): 745-752, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27770950

RESUMO

The development of growth factors is very promising in the field of tissue regeneration but specifically designed formulations have to be developed in order to enable such new biological entities (NBEs). In particular, the range of therapeutic concentrations is usually very low compared to other active proteins and the confinement in the target site can be of crucial importance. In-situ forming scaffolds are very promising solutions for minimally invasive intervention in cartilage reconstruction and targeting of NBEs. In this work injectable, in-situ forming gels of a temperature responsive partially degalactosylated xyloglucan (Deg-XG) incorporating the growth factor FGF-18 are formulated and characterized. In particular, injectability and shear viscosity at room temperature, time-to-gel at body temperature, morphology and mechanical properties of gels are investigated. The highly hydrophobic growth factor is favorably incorporated and retained by the gel. Gels undergo a slow erosion process when immersed in PBS at 37°C that opens up their porous structure. The prolonged hydrothermal treatment leads to structural rearrangements towards tougher networks with increased dynamic shear modulus. Preliminary biological evaluations confirm absence of cytotoxicity and the ability of these scaffolds to host cells and promote their proliferation.


Assuntos
Cartilagem/fisiologia , Fenômenos Químicos , Fatores de Crescimento de Fibroblastos/farmacologia , Géis/química , Glucanos/química , Fenômenos Mecânicos , Xilanos/química , Animais , Cartilagem/efeitos dos fármacos , Bovinos , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Glucanos/síntese química , Injeções , Peso Molecular , Tecidos Suporte/química , Viscosidade , Xilanos/síntese química
13.
Molecules ; 21(11)2016 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-27886088

RESUMO

(1) Background: A new family of nanosystems able to discern between normal and tumor cells and to release a therapeutic agent in controlled way were synthetized by e-beam irradiation. This technique permits to obtain biocompatible, sterile, carboxyl-functionalized polyvinylpyrrolidone (PVP-co-acrylic acid) nanogels (NGs); (2) Methods: Here, we performed a targeting strategy based on the recognition of over-expressed proteins on tumor cells, like the folate receptor. The selective targeting was demonstrated by co-culture studies and flow cytometry analysis, using folate conjugated NGs. Moreover, nanoparticles were conjugated to a chemotherapeutic drug or to a pro-apoptotic siRNA through a glutathione sensitive spacer, in order to obtain a controlled release mechanism, specific for cancer cells. The drug efficiency was tested on tumor and healthy cells by flow cytometric analysis, confocal and epifluorescence microscopy and cytotoxicity assay; the siRNA effect was investigated by RNAi experiment; (3) Results: The data obtained showed that the use of NGs permits a faster cargo release in cancer cells, in response to high cytosolic glutathione level, also improving their efficacy; (4) Conclusion: The possibility of releasing biological molecules in a controlled way and to recognize a specific tumor target allows overcoming the typical limits of the classic cancer therapy.


Assuntos
Antioxidantes/farmacologia , Doxorrubicina/farmacologia , Neoplasias/metabolismo , Polietilenoglicóis/química , Polietilenoimina/química , RNA Interferente Pequeno/farmacologia , Animais , Antioxidantes/química , Linhagem Celular Tumoral , Ácido Fólico/química , Ácido Fólico/metabolismo , Transportadores de Ácido Fólico/antagonistas & inibidores , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Nanogéis , Nanopartículas/química , Neoplasias/tratamento farmacológico , Oxirredução/efeitos dos fármacos , Tamanho da Partícula , Polietilenoglicóis/farmacologia , Polietilenoimina/farmacologia , Povidona/química , Povidona/farmacologia
14.
Carbohydr Polym ; 152: 382-390, 2016 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-27516285

RESUMO

Partially degalactosylated xyloglucan from tamarind seeds (Deg-XG) is a very appealing biopolymer for the production of in situ gelling systems at physiological temperature. In this work, we observe that the morphology of hydrogels evolves towards high degrees of structural organization with time, yielding to dense stacks of thin membranes within 24h of incubation at 37°C. We also explore the possibility offered by gamma irradiation of controlling the time scale of this phenomenon, the final morphology and mechanical properties of the system. Structural and molecular modifications of Deg-XG with dose are investigated by FTIR, dynamic light scattering (DLS) and rotational viscosimetry. The impact on gelation ability and gel strength is studied by rheological analysis. The morphology evolution is investigated by SEM analysis, and absence of cytotoxicity verified by MTS assay and optical microscopy of neuroblastoma cells.


Assuntos
Biopolímeros , Raios gama , Glucanos/química , Glucanos/farmacocinética , Hidrogéis/síntese química , Hidrogéis/farmacocinética , Xilanos/química , Xilanos/farmacocinética , Biopolímeros/química , Biopolímeros/farmacocinética , Biopolímeros/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glucanos/síntese química , Glucanos/efeitos da radiação , Humanos , Hidrogéis/química , Teste de Materiais , Neuroblastoma/patologia , Polimerização/efeitos da radiação , Resistência ao Cisalhamento , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Fatores de Tempo , Viscosidade , Xilanos/síntese química , Xilanos/efeitos da radiação
15.
Biomaterials ; 80: 179-194, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26708643

RESUMO

A growing body of evidence shows the protective role of insulin in Alzheimer's disease (AD). A nanogel system (NG) to deliver insulin to the brain, as a tool for the development of a new therapy for Alzheimer's Disease (AD), is designed and synthetized. A carboxyl-functionalized poly(N-vinyl pyrrolidone) nanogel system produced by ionizing radiation is chosen as substrate for the covalent attachment of insulin or fluorescent molecules relevant for its characterization. Biocompatibility and hemocompatibility of the naked carrier is demonstrated. The insulin conjugated to the NG (NG-In) is protected by protease degradation and able to bind to insulin receptor (IR), as demonstrated by immunofluorescence measurements showing colocalization of NG-In(FITC) with IR. Moreover, after binding to the receptor, NG-In is able to trigger insulin signaling via AKT activation. Neuroprotection of NG-In against dysfunction induced by amyloid ß (Aß), a peptide mainly involved in AD, is verified. Finally, the potential of NG-In to be efficiently transported across the Blood Brain Barrier (BBB) is demonstrated. All together these results indicate that the synthesized NG-In is a suitable vehicle system for insulin deliver in biomedicine and a very promising tool to develop new therapies for neurodegenerative diseases.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Portadores de Fármacos/química , Insulina/administração & dosagem , Povidona/química , Doença de Alzheimer/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Insulina/farmacocinética , Insulina/farmacologia , Camundongos Endogâmicos C57BL , Nanoestruturas/química , Nanoestruturas/efeitos da radiação , Nanotecnologia/métodos , Radiação Ionizante , Receptor de Insulina/metabolismo
16.
J Nanosci Nanotechnol ; 15(5): 3445-67, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26504965

RESUMO

Controlled synthesis of nanoscalar and nanostructured materials enables the development of novel functional materials with fine-tuned optical, mechanical, electronic, magnetic, conductive and catalytic properties that are of use in numerous applications. These materials have also found their potential use in medicine as vehicles for drug delivery, in diagnostics or in combinations thereof. In principle, nanoparticles can be divided into two broad categories, organic and inorganic nanoparticles. For both types of nanoparticles there are numerous possible synthetic routes. Considering the large difference in nature of these materials and the elementary reactions involved in the synthetic routes, most manufacturing techniques are complex and only suitable for one type of particle. Interestingly, radiation chemistry, i.e., the use of ionizing radiation from radioisotopes and accelerators to induce nanomaterials or chemical changes in materials, has proven to be a versatile tool for controlled manufacturing of both organic and inorganic nanoparticles. The advantages of using radiation chemistry for this purpose are many, such as low energy consumption, minimal use of potentially harmful chemicals and simple production schemes. For medical applications one more advantage is that the material can be sterile as manufactured. Radiation-induced synthesis can be carried out in aqueous systems, which minimizes the use of organic solvents and the need for separation and purification of the final product. The radiation chemistry of water is well known, as are the various ways of fine-tuning the reactivity of the system towards a desired target by adding different solutes. This, in combination with the controllable and adjustable irradiation process parameters, makes the technique superior to most other chemical methods. In this review, we discuss the fundamentals of radiation chemistry and radiation-induced synthesis of nanoparticles in aqueous solutions. The impact of dose and dose rate as well as of controlled addition of various solutes on the final particle composition, size and size distribution are described in detail and discussed in terms of reaction mechanism and kinetics.

17.
J Tissue Eng Regen Med ; 9(12): 1394-403, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23225781

RESUMO

Co-culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small-scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold-based constructs generated by combining primary human ACs with human bone marrow MSCs (BM-MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM-MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co-culture of ACs (25%) with BM-MSCs (75%) in scaffolds resulted in up to 1.4-fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8-fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM-MSCs with a relatively low fraction of non-expanded ACs in porous scaffolds. The proposed co-culture strategy is directly relevant towards a single-stage surgical procedure for cartilage repair.


Assuntos
Células da Medula Óssea/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Engenharia Tecidual/métodos , Adulto , Idoso , Animais , Células da Medula Óssea/citologia , Cartilagem Articular/citologia , Condrócitos/citologia , Condrócitos/transplante , Técnicas de Cocultura , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Células Estromais/citologia , Células Estromais/metabolismo
18.
Epigenetics ; 8(6): 656-65, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23771052

RESUMO

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Citidina/análogos & derivados , Metilação de DNA , Glutationa Transferase/genética , Guanidinas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Pirróis/uso terapêutico , Animais , Linhagem Celular Tumoral , Citidina/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Glutationa Transferase/metabolismo , Humanos , Masculino , Camundongos , Neoplasias da Próstata/patologia , Distribuição Aleatória , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Biomacromolecules ; 13(6): 1805-17, 2012 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-22571354

RESUMO

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly(N-vinyl pyrrolidone)-grafted-(aminopropyl)methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to both quantify the available functional groups for bioconjugation and follow nanogels localization in cell cultures. Finally, a model protein, bovine serum albumin, was conjugated to the nanogels to demonstrate the attachment of biologically relevant molecules for targeting purposes in drug delivery. The described approach provides a novel strategy to fabricate biohybrid nanogels with a very promising potential in nanomedicine.


Assuntos
Materiais Biocompatíveis/síntese química , Elétrons , Géis/síntese química , Nanopartículas/química , Animais , Materiais Biocompatíveis/química , Células Cultivadas , Géis/química , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Propriedades de Superfície
20.
Mol Cancer ; 9: 259, 2010 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-20868484

RESUMO

BACKGROUND: Drug resistance is one of the major obstacles limiting the activity of anticancer agents. Activation of DNA repair mechanism often accounts for increase resistance to cancer chemotherapy. RESULTS: We present evidence that nemorubicin, a doxorubicin derivative currently in clinical evaluation, acts through a mechanism of action different from classical anthracyclines, requiring an intact nucleotide excision repair (NER) system to exert its activity. Cells made resistant to nemorubicin show increased sensitivity to UV damage. We have analysed the mechanism of resistance and discovered a previously unknown mechanism resulting from methylation-dependent silencing of the XPG gene. Restoration of NER activity through XPG gene transfer or treatment with demethylating agents restored sensitivity to nemorubicin. Furthermore, we found that a significant proportion of ovarian tumors present methylation of the XPG promoter. CONCLUSIONS: Methylation of a NER gene, as described here, is a completely new mechanism of drug resistance and this is the first evidence that XPG gene expression can be influenced by an epigenetic mechanism. The reported methylation of XPG gene could be an important determinant of the response to platinum based therapy. In addition, the mechanism of resistance reported opens up the possibility of reverting the resistant phenotype using combinations with demethylating agents, molecules already employed in the clinical setting.


Assuntos
Antineoplásicos/uso terapêutico , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Endonucleases/genética , Endonucleases/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Metilação de DNA , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias/terapia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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