Desenvolvimento de nanomagnetogéis para aplicações biomédicas

Dissertação de mestrado em Micro e Nano TecnologiasO desenvolvimento de nanosistemas inteligentes tem suscitado grande interesse na área da nanomedicina devido às suas potenciais aplicações biomédicas. Estes são sistemas de libertação controlada capazes de direccionar o agente activo para o local de interesse e aí controlar o seu perfil de libertação. No presente trabalho, nanopartículas magnéticas de óxido de ferro, γ-Fe2O3 (maguemite), foram estabilizadas com um nanogel de dextrino, que foi previamente sintetizado e caracterizado. A maguemite apresenta propriedades superparamagnéticas, ou seja, as nanopartículas permanecem dispersas e estáveis após remoção do campo magnético, uma vez que não retêm o magnetismo, podendo ser direccionadas através da aplicação de um campo magnético externo. Estas nanopartículas superparamagnéticas têm ainda a capacidade de diminuir os tempos de relaxação dos protões das moléculas de água presentes nos tecidos, actuando como agente de contraste em imagiologia de ressonância magnética (IRM). A sua aplicação sem revestimento, em organismos, encontra limitações devido ao seu tamanho e estabilidade, assim como ao reconhecimento por parte do sistema fagocitário mononuclear, que reduz o seu tempo de permanência no organismo, impedindo que as nanopartículas atinjam o alvo. Uma forma de ultrapassar este problema é revestindo as nanopartículas de óxido de ferro com polímeros que lhes confiram estabilidade e protecção in vivo. Os revestimentos poderão também servir de agentes de transporte de moléculas activas, como fármacos. O principal objectivo deste trabalho foi estudar a incorporação/estabilização de nanopartículas de óxido de ferro superparamagnéticas num nanogel de dextrino, obtendo-se um nanomagnetogel. Foram preparadas diferentes formulações, usando uma quantidade constante de nanogel e variando a quantidade de óxido de ferro. As formulações foram caracterizadas física e quimicamente tendo sido verificado que o tamanho do complexo γ- Fe2O3@nanogel é aproximadamente o mesmo para todas as formulações preparadas (140 nm), assim como a estabilidade é mantida até ao final do tempo de observação (4 semanas). O tamanho foi analisado por DLS, crio- MEV e MET. Verificou-se que para a concentração de nanogel estudada (0,5 mg/mL) a saturação com óxido de ferro ocorre quando se adiciona 2,93 mM de ferro tendo-se estabilizado 1,21 mM de ferro. Estudos de relaxividade apresentaram tempos de relaxação transversal (T2) muito inferiores aos tempos de relaxação longitudinal (T1). Os valores de relaxividade são por isso bastante elevados, característicos de agentes de contraste à base de óxido de ferro. Esta particularidade é especialmente importante quando se pretende uma aplicação como agentes de contraste para IRM. O complexo foi ainda estudado em ensaios de biocompatibilidade. Os ensaios de MTT revelaram que o nanomagnetogel não comprometeu a viabilidade dos fibroblastos 3T3. Os macrófagos internalizaram o nanomagnetogel 3 h após incubação. O nanomagnetogel desenvolvido demonstrou possuir características interessantes para aplicação como agente de contraste em IRM, podendo ser direccionado após administração intravenosa, por aplicação de um campo magnético externo. O nanomagnetogel foi funcionalizado com um agente quelante que complexa um metal trivalente, como o samário 153 (radioactivo), e caracterizado. Demonstrou-se que será possível realizar estudos de biodistribuição por emissão de raios-γ, após administração intravenosa, com e sem aplicação de um campo magnético numa região específica (por exemplo tumor).The development of nanosystems has created great interest in nanomedicine due to their diverse biomedical applications. Nanosystems are controlled drug delivery carriers capable of directing the targeting agent to a specific site. In the present study, superparamagnetic iron oxide nanoparticles, γ-Fe2O3 (maghemite) were stabilized in a previously synthesized and characterized, dextrin nanogel. Maghemite is a superparamagnetic material that through application of an external magnetic field can be directed to a desired location. By removal of the externally applied magnetic field they do not retain significant magnetization, thus remaining stable and dispersed. They can also be directed through an external magnetic field. Superparamagnetic nanoparticles are also able to shorten the relaxation times of neighboring protons (water molecules) present in tissues, acting as contrast agents in magnetic resonance imaging (MRI). Their in vivo application in the absence of a stabilizer (coating agent) presents limitations, for they are easily taken up by the reticuloendothelial system, decreasing the circulation time of the nanoparticles, hindering their purpose. A commitment between size and stability must therefore be achieved to overcome such obstacles. Polymer stabilizers are used as coatings for iron oxide nanoparticles in order to promote stability and in vivo protection. The coatings can also serve as carriers for active molecules such as drugs. The main purpose of this work was to study the loading of superparamagnetic iron oxide nanoparticles inside a dextrin nanogel, thus obtaining a nanomagnetogel. Different formulations were prepared using different volumes of iron oxide and maintaining the nanogel concentration. These formulations were characterized in size and stability. The average size of the nanomagnetogels was approximately 140 nm, according to studies with DLS, cryo-SEM and TEM. For 0.5 mg/mL of nanogel, the maximum iron concentration stabilized was 1.21 mM. Relaxivity studies presented very short transverse relaxation times, which is a particular characteristic of superparamagnetic nanoparticles, and an extremely important feature of contrast agents for MRI. Their biocompatibility was also studied and MTT assays showed that the γ-Fe2O3@nanogel complex does not affect cell viability in 3T3 fibroblasts. The uptake of the complex by macrophages can be inferred by the blue staining inside the cells, after the cells were incubated in the presence of the formulations for 3 hours. The studied nanomagnetogels demonstrated to be good contrast agents for MRI. Nonetheless, it should be interesting to study a higher iron loading inside the nanogel, by increasing the nanogel concentration. Also, the incorporation of a hydrophobic drug inside the hydrophobic domains of the nanomagnetogel should be considered as a possible study subject, as should the biodistribution of the complex through intravenous administration, with or without the appliance of an external magnetic field in the area of interest (e.g. tumour)

Antimicrobial performance of lignin embedded in bacterial nanocellulose membranes

The development of bio-based antimicrobial polymeric composites has never been so urgent. Novel antimi- crobial fibrous-based biocomposites will certainly allow the development of important solutions to fight the present and future Pandemics, while reducing the dependence of petrochemical based polymers and fibers. Lignin has a pivotal function in preventing the invasion of phytopathogens, thus, this work explores the anti- microbial potential of lignin when embedded in a biosynthesized fibrous nanomatrix with superior mechanical properties: bacterial nanocellulose (BNC). Lignin was subjected to alkali treatment to promote the inclusion of lignin within BNC which comprises pores ranging from 20 to 300 nm. Both alkali treatment efficiency, bac- tericidal and antiviral activities were investigatedThe authors would like to acknowledge the project PLASMAMED - PTDC/CTM-TEX/28295/2017 fnanced by FCT, FEDER and POCI in the frame of the Portugal 2020 program, the project UID/CTM/00264/2019 of 2C2T under the COMPETE and FCT/MCTES (PIDDAC) co-fnanced by FEDER through the PT2020 program. Liliana Melro acknowledges her Doctoral grant awarded by FCT (2020.04919.BD)

Bio-synthesised fibrous-based meshes for abdominal hernia with enhanced mechanical and antimicrobial properties

Abdominal hernia (AH) encompasses the most prevalent types of hernia: inguinal, umbilical and incisional. Notwithstanding current hernia complications represent a low death toll (nearly 0.001 % in developed countries), non-reducible hernias are the most severe cases, which require urgent surgical intervention due to their life-threatening nature. In a single year, at the United States of America, more than 800 thousand surgeries are performed to repair inguinal hernias. Abdominal hernia ubiquitous symptoms include pain, which may represent a mild discomfort or even an impairing morbidity. Nevertheless, some patients suffer from morbidity in the post-operative period. Recurrence was reduced when the application of a propylene mesh replaced primary suture repair more than 60 years ago. Surprisingly, currently the most prevalent hernia mesh materials are based on petrochemical plastics such as polypropylene, polyester, polystyrene and expanded polytetrafluoroethylene. Unfortunately, despite the plethora of commercial hernia meshes, an improvement of the hernia meshes is still warranted, since petrochemical materials exhibit a deterioration over time which generate complications and recurrence. This project envisages the complete replacement of the conventional plastic-based material of hernia meshes by a fully bio- based material with superior mechanical properties: bacterial nanocellulose (BNC). BNC is synthesized by bacteria and is composed of a 3D matrix of 100 % nanofibrils of cellulose, each with a diameter ranging between 20 to 100 nm. When BNC producing bacterium are cultured in static culture, the BNC is formed as membrane (nanoporous mesh comprising pores of 100 to 300 nm in diameter) at the surface of the culture medium and adopts the shape of the available surface. Therefore, it is easy to control the membrane surface shape, as well as its thickness, which can be controlled by the incubation time (longer incubation time will result in a larger thickness). The selection of the most adequate bacterium for the production of the hernia mesh will be performed Nevertheless, for a hernia mesh to be viable it requires pores with a specific diameter to allow the permeability of leukocytes, fibroblasts, and permit the arrangement of collagen and blood vessels. Per se, the BNC mesh does not possess such large specific pores with the required diameter (> 75 μm), thus it is proposed the design and development of a template to achieve a AH mesh that meets the necessary requirements. Furthermore, due to the high complexity of hernia mesh infection, which is extremely difficult to adequately treat without removing the mesh, this project envisages the functionalization of BNC AH mesh with antimicrobial properties. Two approaches will be considered for the BNC AH mesh functionalization, namely: in situ synthesis and adsorption through filtration. NPs optimal concentration and functionalization process will be examined and tailored to obtain a BNC AH mesh with effective antimicrobial activity and negligible cytotoxicity. According the AH implantation site, three different hernia meshes classes are usually applied: low, medium and high weight, thus the optimal antimicrobial BNC meshes of each class will be patented to represent a viable commercial alternative, by displaying superior mechanical properties, biocompatibility and low infection propensity, to considerably improve AH patience overall wellbeing.FEDER funds under the COMPETE program and by National Funds through Fundação para a Ciência e Tecnologia (FCT) under the project POCI-01- 0145-FEDER-007136 and UID/CTM/00264/2013. PLASMAMED project PTDC/CTMTEX/28295/2017 funded by FCT, FEDER e POCI through the program Portugal 202

Deposition of silver nanocomposite on textiles for controllable antibacterial activity using atmospheric pressure dielectric barrier discharge (DBD) plasma

Conventional antibacterial coatings by wet chemistry, low-pressure plasma and sputtering have several drawbacks but the most important is their uncontrollable antibacterial activity. The main objective of this work is to produce, by dielectric barrier discharge (DBD) plasma-assisted deposition at atmospheric pressure, a new generation of coatings containing silver nanoparticles (AgNPs) with controllable antibacterial activity on medical textiles. AgNPs was deposited by ultrasound-assisted, dip-coating, exhaustion at 30 ̊C and spray deposition methods. The deposited NPs were tuned using a “sandwich” coating structure where a 1st antibacterial nanocomposite layer is covered by a 5-50 nm thick 2nd polymeric layer in order to prolong antibacterial effect. A broad range of deposition parameters were investigated including plasma power, plasma gas, discharge gap, NPs type and size, types of precursor and textile substrate. The main advantage of this plasma-assisted deposition is its capability to produce, in a continuous process, coatings with strong bonding, high deposition rate and precise release of antimicrobial agent by variation of coating composition and thickness. The ultrasound method displays an irregular distribution despite its local good deposition. The dip coating and spray methods did not reach the minimum amount of AgNPs on the fabric surface and showed high AgNPs agglomeration. The exhaustion method showed the best results for both NPs distribution and reduced agglomeration. The best antibacterial performance was exhaustion at 30 °C, which exhibited less agglomeration and the best antibacterial efficacy against S. aureus (4 log reduction). For E. coli, the antimicrobial effect showed good results in all the exhaustion samples (5 log reduction). Atmospheric plasma is an alternative and cost-competitive method to low-pressure plasma and wet chemical treatments for medical textiles, avoiding the need of expensive vacuum equipment, allowing continuous and uniform processing of fibers surfaces and providing intrinsic sterility of the treated surfaces. New insights in mechanisms of activation, degradation and functionalization of NPs, and polymers will have a huge potential for long-term exploitation of biomedical applications and will contribute to better quality of life and health.This work was funded by European Regional Development funds (FEDER) through the Competitiveness and Internationalization Operational Program (POCI) – COMPETE and by National Funds through Fundação para a Ciência e Tecnologia (FCT) under the project UID/CTM/00264/2019, Investigator FCT Research contract (IF/00071/2015) and the project PTDC/CTM-TEX/28295/2017 financed by FCT, FEDER and POCI in the frame of the Portugal 2020 program

Antibacterial hydrogel dressings and their applications in wound treatment

Antimicrobial hydrogels, both in semi-stiff sheets and amorphous form, have been extensively studied for wound management mainly owing to their high-water content, lower wound adherence, promoted autolysis debridement, epithelial migration, and granulation growth. Benefiting from the recent advances in materials science, biotechnology, and a growing understanding of wound microbiology, an extensive variety of antimicrobial hydrogels have been developed. These novel antimicrobial hydrogels can prevent and control microbial infection. In addition, they possess wound healing functions for improved wound management. This chapter will provide a comprehensive summary of the current studied antimicrobial hydrogels in literature and available hydrogel dressings in the market, including their design, fabrication method, and wound management efficacy in vitro or in vivo. The detailed and critical discussion of the advantages and disadvantages of each type of hydrogel dressing will provide insights into the future design of antimicrobial hydrogels for better management of wounds in clinical application

Antibacterial properties of bacterial nanocellulose functionalized with metal nanoparticles via in situ synthesis

[Excerpt] Wound infections are generally caused by pathogens and multidrug-resistant (MDR) strains that render the administration of antibiotics ineffective. An alternative is to treat infected wounds at the initial stage using a fibrous bionanopolymer, bacterial nanocellulose (BNC), functionalized with antimicrobial metal nanoparticles (MNPs). BNC is a highly promising wound dressing due to its very high-water retention capacity (> 99 %) and high porosity. Such properties enable the absorbance of exudates, whilst maintaining the environment moist allowing the exchange of air. However, BNC is absent of antibacterial properties, thus gold (Au), copper (Cu), and copper oxide (Cu2O) NPs were incorporated within the nanofibrous structure of the biopolymer via in situ synthesis

Antimicrobial activity of bacterial nanocellulose modified with chestnut extract

Chestnut wood extracts are rich in tannins that exhibit numerous health-promoting properties. The incorporation of 5% (w/v) chestnut extract within the nanofibrous structure of bacterial nanocellulose (BNC) produced by Gluconacetobacter hansenii ATCC 53582 was obtained through exhaustion. This simple processing methodology resulted in a flexible (upon addition of 2% (w/v) glycerol), biodegradable, biocompatible nanocomposite for potential application in medical appliances.Portuguese Foundation for Science and Technology (FCT), FEDER funds through Portugal 2020 Competitive Factors Operational Program (POCI), and thePortuguese Government (PG) for the projects: UID/CTM/00264/2021 of Centre for Textil e Science and Technology (2C2T) and PTDC/CTM-TEX/1213/2020;FCT, the Ministry of Science, Technology and Higher Education (MCTES), the European Social Fund (FSE) and the European Union (UE)for her Ph.D. funding via scholarship 2020.04919.BD;FCT, FEDER, POCI, and PG for her research grant POCI-01-0247-ERDF-047124

Modification of nanocellulose

Nanocellulose (NC) represents a pivotal material for the sustainable strategies of the future. NC comprises cellulose nanofibrils (CNFs), cellulose nanocrystals (CNCs), and bacterial nanocellulose (BNC), each exhibiting unique and exceptional physicochemical properties. These properties encompass high specific surface area, high tensile strength, lightweight, biodegradability, good barrier properties, and high processing versatility. However, the range of properties and applications can be significantly expanded through the modification of NC, involving both chemical and physical methodologies, which introduce a plethora of functional groups to the densely populated hydroxyl groups present in pristine NC. The modification processes discussed in this chapter encompass chemical and physical modifications that were reported mostly within the last 5 years. The described methodologies emphasize the potential of NC as a substrate for advanced functional and sustainable material

Chapter 34 - Biocompatibility of nanocellulose: Emerging biomedical applications

Nanocellulose already proved to be a highly relevant material for biomedical applications, ensued by its outstanding mechanical properties and, more importantly, its biocompatibility. Nevertheless, despite their previous intensive research, a notable number of emerging applications are still being developed. Interestingly, this drive is not solely based on the nanocellulose features, but also heavily dependent on sustainability. The three core nanocelluloses encompass cellulose nanocrystals (CNCs), cellulose nanofibrils (CNFs), and bacterial nanocellulose (BNC). All these different types of nanocellulose display highly interesting biomedical properties per se, after modification and when used in composite formulations. Novel applications that use nanocellulose includewell-known areas, namely, wound dressings, implants, indwelling medical devices, scaffolds, and novel printed scaffolds. Their cytotoxicity and biocompatibility using recent methodologies are thoroughly analyzed to reinforce their near future applicability. By analyzing the pristine core nanocellulose, none display cytotoxicity. However, CNF has the highest potential to fail long-term biocompatibility since it tends to trigger inflammation. On the other hand, neverdried BNC displays a remarkable biocompatibility. Despite this, all nanocelluloses clearly represent a flag bearer of future superior biomaterials, being elite materials in the urgent replacement of our petrochemical dependence

The assessment of bacterial nanocellulose functionalized with metal nanoparticles

[Excerpt] Introduction Biocompatibility is one of the mandatory requirements of indwelling medical devices to avoid foreign body reactions and consequential surgical removal.This research was funded by FEDER funds through the Operational Competitiveness Program–COMPETE, under the project POCI-01-0247-FEDER-068924 and by National Funds through Fundação para a Ciência e Tecnologia (FCT), under the project UID/CTM/00264/2020. Cátia Alves, Liliana Melro, Behnaz Mehravani, and Ana Isabel Ribeiro acknowledge FCT, MCTES, FSE, and UE PhD grants 2022.10454.BD, 2020.04919.BD, 2022.13094.BD, and SFRH/BD/137668/2018