The introduction of molecules with biological properties on textile materials is essential for a number of biotechnological applications. With the purpose of testing new processes applied to textiles, in this study, we present the first results on the feasibility of using the Layerby-Layer (LbL) deposition process in natural fibers such as cotton, with natural polyelectrolytes like chitosan (CH) and alginic acid sodium salt (ALG), the durability of CH/ALG multilayer on cotton were evaluated. The increase of negative charges to the substrate cotton was made with NaBr and TEMPO, to ensure the success of the process of LbL. Three characterization methods to assess electrostatic LbL deposition were performed: the contact angle between a liquid (water) and the sample surface, in order to characterize the wettability of the samples with the different layers of CH and ALG; dyeing of the CH/ALG assembled cotton fabric with cationic methylene blue that shows regular changes in terms of color depth (K/S value), which indicate that the surface were alternately deposited with CH and ALG layers and, finally, the analysis by infrared spectroscopy using Fourier Transform with Attenuated Total Reflection (ATR-FTIR), to assess the changes in the interaction between CH and ALG deposited on cotton samples.

The combination of photothermal and chemo- therapies displays a high potential to increase the efficacy of the cancer treatments or even promote their eradication. In this study, the micromoulding and electrospraying techniques were combined to produce polyvinylpyrrolidone microneedles coated with chitosan and poly (vinyl alcohol) for mediating the delivery of doxorubicin and AuMSS nanorods (Dox@MicroN) to cancer cells. The microneedles' physicochemical characterization demonstrated that the electrospraying technique can be used to produce a layer-by-layer coating consisting of layers of doxorubicin-loaded chitosan and AuMSS enriched poly (vinyl alcohol). Further, the Dox@MicroN patches presented a good photothermal capacity leading to a temperature increase of 12 °C under near-infrared irradiation (808 nm, 1.7 W/cm-2 for 5 min), which in conjugation with the chitosan' pH sensitivity could be used to control the doxorubicin release. Moreover, the microneedles were able to penetrate the tumor-mimicking agarose gel and promote a layer dependent drug release. Additionally, the Dox@MicroN patches' capacity to simultaneously mediate the chemo- and photothermal-therapies rendered a superior cytotoxic effect against the cervical cancer cells. Overall, the Dox@MicroN patches demonstrated to be a simple macroscale delivery device that can be used to mediate the local administration of new drug-photothermal combinations, avoiding all the issues related to the systemic administration of anti-cancer therapeutics.

The design of poly(ionic liquids) (PILs) and their application as solid phase microextraction (SPME) fibers has been attracting enormous attention mainly due to the need for new SPME coating materials with improved analytical sensitivity. In this work, the tunability of PILs is explored by preparing different imidazolium monomers bearing benzyl, naphtylmethyl or pentyl pending groups that were subsequently co-polymerized, by reversible addition–fragmentation chain transfer (RAFT) polymerization with styrene. The obtained co-polymers showed excellent thermal stability up to 275 °C, with no melting point up to 250 °C. SPME fibers were prepared by an innovative approach based on layer-by-layer spray coating. The thin (<10 μm) SPME coatings were tested in GC-FID for the detection of volatile aromatic compounds such as benzene (B), toluene (T), ethylbenzene (E) and xylene (X) present in aqueous samples and the extraction parameters optimized. Superior results were obtained when comparing these LbL PILS-based SPME fibers with a commercial fiber composed of poly(dimethylsiloxane), with an increase in the detectable areas of 83%, 69%, 57% and 58% for B, T, E and X, respectively. Low relative standard deviations were obtained for the same fiber (< 5.6%) and also for different fibers (< 9.8%). Furthermore, a spiked soil sample was used to mimic a real contaminated soil sample and excellent recovery results, ranging from 67.0% to 102.2%, were obtained.

Surfaces with biological functionalities are of great interest for biomaterials, tissue engineering, biophysics, and for controlling biological processes. The layer-by-layer (LbL) assembly is a highly versatile methodology introduced 30 years ago, which consists of assembling complementary polyelectrolytes or biomolecules in a stepwise manner to form thin self-assembled films. In view of its simplicity, compatibility with biological molecules, and adaptability to any kind of supporting material carrier, this technology has undergone major developments over the past decades. Specific applications have emerged in different biomedical fields owing to the possibility to load or immobilize biomolecules with preserved bioactivity, to use an extremely broad range of biomolecules and supporting carriers, and to modify the film's mechanical properties via crosslinking. In this review, the focus is on the recent developments regarding LbL films formed as 2D or 3D objects for applications in drug delivery and tissue engineering. Possible applications in the fields of vaccinology, 3D biomimetic tissue models, as well as bone and cardiovascular tissue engineering are highlighted. In addition, the most recent technological developments in the field of film construction, such as high-content liquid handling or machine learning, which are expected to open new perspectives in the future developments of LbL, are presented.

Food smart packaging has emerged as a promising technology to address consumer concerns regarding food conservation and food safety. In this context, we report the rational design of azide-containing pyranoflavylium-based pH-sensitive dye for subsequent click chemistry conjugation toward a chitosan-modified alkyne. The chitosan-pyranoflavylium conjugate was characterized by infrared (ATR-FTIR), ultraviolet−visible (UV−vis), nuclear magnetic resonance (NMR) spectroscopies, and dynamic light scattering (DLS), as well as its thermodynamic parameters related to their pH-dependent chromatic features. The fabrication of thin-films through electrostatic-driven layer-by-layer (LbL) assembly technology was first screened by quartz crystal microbalance with dissipation monitoring (QCM-D) onto gold substrates, and then free-standing (FS) multilayered membranes from polypropylene substrate were obtained using a homemade automatic dipping robot. The membranes’ characterization included morphology analysis and thickness evaluation, assessed by scanning electron microscopy (SEM), pH-responsive color change performance tests using buffer solutions at different pH levels, and biogenic amines-enriched model solutions, demonstrating the feasibility and effectiveness of the chitosan-pyranoflavylium/alginate biomembranes for food spoilage monitoring. This work provides insights toward the development of innovative pH-responsive smart biomaterials for advanced and sustainable technological packaging solutions, which could significantly contribute to ensuring food safety and quality, while reducing food waste.

The layer-by-layer (LbL) deposition technique is widely used to develop multilayered films based on the directed assembly of complementary materials. In the last decade, thin multilayers prepared by LbL deposition have been applied in biological fields, namely, for cellular encapsulation, due to their versatile processing and tunable properties. Their use was suggested as an alternative approach to overcome the drawbacks of bulk hydrogels, for endocrine cells transplantation or tissue engineering approaches, as effective cytoprotective agents, or as a way to control cell division. Nanostructured multilayered materials are currently used in the nanomodification of the surfaces of single cells and cell aggregates, and are also suitable as coatings for cell-laden hydrogels or other biomaterials, which may later be transformed to highly permeable hollow capsules. In this Focus Review, we discuss the applications of LbL cell encapsulation in distinct fields, including cell therapy, regenerative medicine, and biotechnological applications. Insights regarding practical aspects required to employ LbL for cell encapsulation are also provided.

Membranes have been explored as patches in tissue repair and regeneration, most of them presenting a flat geometry or a patterned texture at the nano/micrometer scale. Herein, a new concept of a flexible membrane featuring well arrays forming pore-like environments to accommodate cell culture is proposed. The processing of such membranes using polysaccharides is based on the production of multilayers using the layer-by-layer methodology over a patterned PDMS substrate. The detached multilayered membrane exhibits a layer of open pores at one side and a total thickness of 38±2.2µm. The photolithography technology used to produce the molds allows obtaining wells on the final membranes with a tuned shape and micro-scale precision. The influence of post-processing procedures over chitosan/alginate films with 100 double layers, including crosslinking with genipin or fibronectin immobilization, on the adhesion and proliferation of human osteoblast-like cells is also investigated. The results suggest that the presence of patterned wells affects positively cell adhesion, morphology and proliferation. In particular, it is seen that cells colonized preferentially the well regions. The geometrical features with micro to sub-millimeter patterned wells, together with the nano-scale organization of the polymeric components along the thickness of the film will allow to engineer highly versatile multilayered membranes exhibiting a pore-like microstructure in just one of the sides, that could be adaptable in the regeneration of multiple tissues.

O objetivo deste trabalho consistiu na síntese e caracterização de sistemas inteligentes para encapsular bactérias lácteas probióticas, designadamente Lactobacillus acidophilus, de modo a aumentar a sua estabilidade e viabilidade quando atravessam o trato gastrointestinal (consequente da ingestão oral dos alimentos). Os sistemas de encapsulamento basearam-se num complexo polímero natural-copolímero sintético, Quitosano-(Poli(ácido acrílico)-Pluronic-Poli(ácido acrílico)) e estudou-se a influência de diferentes peso moleculares do copolímero acrílico na manutenção da viabilidade celular das bactérias. Os copolímeros Poli(ácido acrílico)-Pluronic-Poli(ácido acrílico) (PAA-PLU-PAA) foram sintetizados através de uma reação de polimerização por emulsão e apresentaram um peso molecular de 24 kDa e 12 kDa, sendo ambos constituídos pela mesma fração de segmentos PAA e Pluronic (50/50).O encapsulamento das bactérias foi realizado através da técnica layer-by-layer (LbL) e as condições de encapsulamento (concentração e pH da solução tampão e dos polielectrólitos, tempo de centrifugação nas lavagens, tempo de incubação para a deposição de cada camada) foram primeiramente estudadas e otimizadas. As condições ótimas foram alcançadas para pH=6, uma concentração de solução tampão igual a 0,15M, uma concentração molar de Quitosano e de PAA-PLU-PAA igual a 4,11׿10¿^(-8) mol/mL, centrifugação a 8000 rpm durante 5 min e 1h de incubação, a 37°C e 100 rpm, para a deposição de cada camada de polielectrólito.Os resultados demonstraram que para o copolímero acrílico de peso molecular 24 kDa, os sistemas de revestimento constituídos por 2 e por 4 camadas foram os que melhor permitiram a difusão de nutrientes, assegurando o crescimento das bactérias encapsuladas próximo do crescimento obtido para as bactérias livres. A taxa de sobrevivência dos probióticos revestidos com 2 camadas, após a passagem em fluido gástrico simulado (FGS) e em fluido intestinal simulado (FIS), foi de 84 % e a taxa de sobrevivência das bactérias revestidas com 4 camadas foi de 67%. Para o copolímero de peso molecular 12 kDa, apenas o sistema constituído por 2 camadas de revestimento permitiu que as bactérias tivessem acesso à quantidade necessária de nutrientes para exercerem a sua atividade metabólica e, neste caso, a taxa de sobrevivência das bactérias após o processo de digestão simulado foi de 83 %.Relativamente à informação existente na literatura, os aspetos inovadores deste trabalho compreendem o desenvolvimento de um sistema inteligente para o encapsulamento dos probióticos, a combinação dos polímeros usados (polímero orgânico-polímero inorgânico) e a avaliação de diferentes pesos moleculares poliméricos na estrutura de encapsulamento.

Tissue engineering is an important branch of regenerative medicine that uses cells, materials (scaffolds), and suitable biochemical and physicochemical factors to improve or replace specific biological functions. In particular, the control of cell behavior (namely, of cell adhesion, proliferation and differentiation) is a key aspect for the design of successful therapeutical approaches. In this study, poly(lactic-co-glycolic acid) (PLGA) fiber mats were prepared using the electrospinning technology (the fiber diameters were in the micrometer range). Furthermore, the electrospun fiber mats thus formed were functionalized using the layer-by- layer (LbL) technique with chitosan and alginate (natural and biodegradable polyelectrolytes having opposite charges) as a mean for the immobilization of pDNA/dendrimer complexes. The polyelectrolyte multilayer deposition was confirmed by fluorescence spectroscopy using fluorescent-labeled polyelectrolytes. The electrospun fiber mats coated with chitosan and alginate were successfully loaded with complexes of pDNA and poly(amidoamine) (PAMAM) dendrimers (generation 5) and were able of releasing them in a controlled manner along time. In addition, these mats supported the adhesion and proliferation of NIH 3T3 cells and of human mesenchymal stem cells (hMSCs) in their surface. Transfection experiments using a pDNA encoding for luciferase showed the ability of the electrospun fiber mats to efficiently serve as gene delivery systems. When a pDNA encoding for bone morphogenetic protein-2 (BMP-2) was used, the osteoblastic differentiation of hMSCs cultured on the surface of the mats was promoted. Taken together, the results revealed that merging the electrospinning technique with the LbL technique, can be a suitable methodology for the creation of biological active matrices for bone tissue engineering.

In the past, most bone tissue engineering (TE) strategies were focused on the recapitulation of intramembranous ossification (IMO) process. Bone-like tissues were successfully obtained in vitro, however, after implantation the created tissues lack of a functional vascular supply, resulting in necrotic cores. During the last decade, approaches based in endochondral ossification (ECO) have been increasingly explored. The secretion of osteogenic and angiogenic factors by the cells present in the hypertrophic cartilage templates allows bone tissue repair but also helps tissue vascularization. In chapter I, the most recent ECO approaches used in bone TE are highlighted and discussed. Chapter II describes the methods used to develop an in vitro ECO approach. In chapter III, the results obtained related with the characterization of the obtained bone-like microtissues are presented and discussed. The developed in vitro ECO model relies in the co-culture of 3D micro-cartilaginous templates with umbilical cord-derived mesenchymal stem/stromal cells (UCMSCs) and human umbilical vein endothelial cells (HUVECs). Our hypothesis is that such engineered and privileged microenvironment would mimic the ECO process, leading to the in vitro production of vascularized bone-like microtissues. For that, MSCs-only 3D microtemplates were produced at high-rates and cultured in vitro for 21 days. Then, such microtemplates were co-cultured with UCMSCs and HUVECs within liquefied and multilayered microcapsules, in dynamic conditions, for another 21 days. Microcapsules with chondrogenically-primed 3D microtemplates were cultured with (ECO) and without (ECO control) osteogenic differentiation factors. Also, microcapsules with non-primed 3D microtemplates were cultured with (IMO) and without (negative control) osteogenic differentiation factors. Results show that both 3D microtemplates and microcapsules were able to maintain cell viability up to 21 days. The cartilaginous nature of the 3D templates cultured in chondrogenic medium was confirmed. After 21 days of encapsulation, the ECO microcapsules presented evidence of bone extracellular matrix production and higher matrix mineralization, being the only condition to present a calcium/phosphorous (Ca/P) ratio (1.71) close to the native hydroxyapatite ratio (1.67). Furthermore, both ECO and ECO control conditions successfully induced endothelial cell recruitment. These data show the relevance of using chondrogenically primed 3D microtemplates in bone repair, highlighting the advantage of ECO over IMO approach. In conclusion, this ECO bioencapsulation approach revealed to be a promising bone regeneration strategy. Ultimately, chapter IV discusses the main conclusions and future perspectives related to the potential of ECO approaches in tissue engineering and regenerative medicine.