Reversible Self-Healing for Preserving Optical Transparency and Repairing Mechanical Damage in Composites.

This research concentrates on the healing of optical properties, roughness, contact angle hysteresis, and shallow scratches in polymer/nanoparticle composites. A series of ternary composite blends [epoxy/halloysite nanotubes (HNTs)/cellulose acetate butyrate (CAB)] with various CAB concentrations were fabricated and subjected to a series of mechanical damages. The optimized concentration of a nanoparticle is 1.0 vol %, and the CAB concentration is 3.0 vol % based on the mechanical reinforcement and wear resistance. Nanoscale scratching, microlevel falling-sand test, and macrolevel Taber abrasions were utilized to damage the surfaces. The induced damage (roughness and surface scratch up to hundreds of nanometers in depth) healed upon heating. At any temperatures above the softening transition of the semi-interpenetrating network structure of the polymer composites, CAB migrates into the microcracks, and the essential mechanical parameters (modulus, strength, strain to failure) are recovered; in our particular epoxy/HNTs/CAB system, optical transparency is also recovered efficiently. CAB also moves to the macroscopic air/specimen interface and favorably modifies the surface properties, reducing the roll-off angles of water droplets from ∼90° to ∼20°. Through an appropriate choice of CAB additives with different molecular weights, the healing temperature can be tailored.

Macrophages with cellular backpacks for targeted drug delivery to the brain.

Most potent therapeutics are unable to cross the blood-brain barrier following systemic administration, which necessitates the development of unconventional, clinically applicable drug delivery systems. With the given challenges, biologically active vehicles are crucial to accomplishing this task. We now report a new method for drug delivery that utilizes living cells as vehicles for drug carriage across the blood brain barrier. Cellular backpacks, 7-10 µm diameter polymer patches of a few hundred nanometers in thickness, are a potentially interesting approach, because they can act as drug depots that travel with the cell-carrier, without being phagocytized. Backpacks loaded with a potent antioxidant, catalase, were attached to autologous macrophages and systemically administered into mice with brain inflammation. Using inflammatory response cells enabled targeted drug transport to the inflamed brain. Furthermore, catalase-loaded backpacks demonstrated potent therapeutic effects deactivating free radicals released by activated microglia in vitro. This approach for drug carriage and release can accelerate the development of new drug formulations for all the neurodegenerative disorders.

Enhanced Wear Resistance of Transparent Epoxy Composite Coatings with Vertically Aligned Halloysite Nanotubes.

The influence of nanoparticle orientation on wear resistance of transparent composite coatings has been studied. Using a nozzle spray coating method, halloysite nanotubes (HNTs) were aligned in the in-plane and out-of-plane directions and in various randomly oriented states. Nanoscratching, falling sand, and Taber Abrasion tests were used to characterize the wear resistance at different length scales. Composites consistently displayed better wear resistance than pure epoxy. Samples with out-of-plane particle orientations exhibited better wear-resistant behavior than those with in-plane particle distributions. In nanoscratching tests, the out-of-plane orientation decreases the normalized scratch volume by as much as 60% compared to pure epoxy. In the falling sand and Taber Abrasion tests, out-of-plane aligned halloysite particles resulted in surfaces with smaller roughness based on stylus profilometry and SEM observations. The decrease in roughness values after these wear tests can be as large as 67% from pure epoxy to composites. Composites with higher out-of-plane particle orientation factors exhibited better light transmittance after sand impingements and other wear tests. This study suggests a useful strategy for producing material systems with enhanced mechanical durability and more durable optical properties.

Spray-Coated Halloysite-Epoxy Composites: A Means To Create Mechanically Robust, Vertically Aligned Nanotube Composites.

Halloysite nanotube-filled epoxy composites were fabricated using spray-coating methods. The halloysite nanotubes (HNTs) were aligned by the hydrodynamic flow conditions at the spray nozzle, and the polymer viscosity helped to preserve this preferential orientation in the final coatings on the target substrates. Electron microscopy demonstrated a consistent trend of higher orientation degree in the nanocomposite coatings as viscosity increased. The nanoindentation mechanical performances of these coatings were studied using a Hysitron TriboIndenter device. Composites showed improvements up to ∼50% in modulus and ∼100% in hardness as compared to pure epoxy, and the largest improvements in mechanical performance correlated with higher alignment of HNTs along the plane-normal direction. Achieving this nanotube alignment using a simple spray-coating method suggests potential for large-scale production of multifunctional anisotropic nanocomposite coatings on a variety of rigid and deformable substrates.

Liposome-Loaded Cell Backpacks.

Cell backpacks, or micron-scale patches of a few hundred nanometers in thickness fabricated by layer-by-layer (LbL) assembly, are potentially useful vehicles for targeted drug delivery on the cellular level. In this work, echogenic liposomes (ELIPs) containing the anticancer drug doxorubicin (DOX) are embedded into backpacks through electrostatic interactions and LbL assembly. Poly(allylamine hydrochloride)/poly(acrylic acid) (PAH/PAA)n , and poly(diallyldimethylammonium chloride)/poly(styrene sulfonate) (PDAC/SPS)n film systems show the greatest ELIP incorporation of the films studied while maintaining the structural integrity of the vesicles. The use of ELIPs for drug encapsulation into backpacks facilitates up to three times greater DOX loading compared to backpacks without ELIPs. Cytotoxicity studies reveal that monocyte backpack conjugates remain viable even after 72 h, demonstrating promise as drug delivery vehicles. Because artificial vesicles can load many different types of drugs, ELIP containing backpacks offer a unique versatility for broadening the range of possible applications for cell backpacks.

Optimization of amine-rich multilayer thin films for the capture and quantification of prostate-specific antigen.

It is demonstrated that poly(allylamine hydrochloride)/poly(styrenesulfonate) (PAH/SPS) multilayer films can be successfully tailored for the capture and detection of small biomolecules in dilute concentrations. Based on in vitro results, these films could be potentially applied for rapid and high-throughput diagnosis of dilute biomarkers in serum or tissue. PAH presents functional amino groups that can be further reacted with desired chemistries in order to create customizable and specific surfaces for biomolecule capture. A variety of film assembly characteristics were tested (pH, molecular weight of PAH, and ionic strength) to tune the biotinylation and swelling behavior of these films to maximize detection capabilities. The resultant optimized biotinylated PAH/SPS 9.3/9.3 system was utilized in conjunction with quantum dots (Qdots) to capture and detect a dilute biomarker for prostate cancer, prostate-specific antigen (PSA). Compared to previous work, our system presents a good sensitivity for PSA detection within the clinically relevant range of 0.4-100 ng/mL.

Sugar-mediated disassembly of mucin/lectin multilayers and their use as pH-Tolerant, on-demand sacrificial layers.

The layer-by-layer (LbL) assembly of thin films on surfaces has proven to be an extremely useful technology for uses ranging from optics to biomedical applications. Releasing these films from the substrate to generate so-called free-standing multilayer films opens a new set of applications. Current approaches to generating such materials are limited because they can be cytotoxic, difficult to scale up, or have undesirable side reactions on the material. In this work, a new sacrificial thin film system capable of chemically triggered dissolution at physiological pH of 7.4 is described. The film was created through LbL assembly of bovine submaxillary mucin (BSM) and the lectin jacalin (JAC) for a (BSM/JAC) multilayer system, which remains stable over a wide pH range (pH 3-9) and at high ionic strength (up to 5 M NaCl). This stability allows for subsequent LbL assembly of additional films in a variety of conditions, which could be released from the substrate by incubation in the presence of a competitive inhibitor sugar, melibiose, which selectively disassembles the (BSM/JAC) section of the film. This novel multilayer system was then applied to generate free-standing, 7 µm diameter, circular ultrathin films, which can be attached to a cell surface as a "backpack". A critical thickness of about 100 nm for the (BSM/JAC) film was required to release the backpacks from the glass substrate, after incubation in melibiose solution at 37 °C for 1 h. Upon their release, backpacks were subsequently attached to murine monocytes without cytotoxicity, thereby demonstrating the compatibility of this mucin-based release system with living cells.

Desenvolvimento e fabricação de filmes ultra-­finos, obtidos pela técnica layer-by-layer, para aplicações na entrega direcionada de fármacos e na captura seletiva de bio-­marcadores / Development and fabrication of ultrathin films obtained by layer­-by­-layer, aiming targeted drug delivery applications and the selective capture of biomarkers

O objetivo geral deste trabalho foi explorar a versatilidade de filmes multicamadas de polieletrólitos (PEM) e suas aplicações em sistemas de entrega de drogas e como filmes funcionais para aplicações biomédicas. Filmes PEM montados pela técnica de camada por camada (layer­-by­-layer, LbL), foram explorados em três aplicações principais. Na primeira, foi explorado o desenvolvimento de um protocolo de funcionalização em filmes de poli(alilamina)/poli (estireno sulfonato), PAH/SPS. Os parâmetros de construção do filme para biotinilação dos grupamentos amina do PAH foram otimizados e aplicados na captura e detecção do antígeno específico da próstata (PSA), na concentração de 100 a 0,1 ng/mL, usando pontos quânticos (Qdots). Em comparação com outros trabalhos, este sistema apresentou uma boa sensibilidade na detecção de PSA, dentro do limite de detecção clínica de 0,4 a 0,1 ng/mL. A segunda aplicação envolveu o desenvolvimento de filmes de sacrifício baseados nas interações naturais da mucina submandibular bovina e da lectina, jacalina (BSM/JAC). Filmes de BSM/JAC apresentaram estabilidade quando submetidos a uma ampla faixa de pH (pH 3-­-9) e em solução de alta força iônica (5 M NaCl). A dissolução dos filmes BSM/JAC pôde ser seletivamente desencadeada mediante à incubação em solução de melibiose, 37 °C, pH 7,4, sem apresentar citotoxicidade às células. Na última parte deste trabalho, a incorporação de lipossomos ecogênicos (ELIP) em mochilas celulares foi investigada. Mochilas celulares são "patches" de 7­-10 µm de diâmetro que podem ser fabricados por meio de deposição alternada de polímeros utilizando-­-se a técnica de LbL, sobre uma matriz pré­-moldada obtida por fotolitografia, a fim de criar um sistema composto por três multicamadas estratificadas: uma região de liberação, para promover o destacamento do substrato, uma região de carga de droga, e uma região adesiva às células. O uso de ELIP permitiu incorporação de até 9x mais doxorrubicina (DOX) se comparado com o fármaco livre em solução absorvido pelos dos filmes. A liberação de DOX pelos filmes foi monitorado por 25 dias. Mochilas contendo ELIP-­DOX foram então aderidos a monócitos, e sua viabilidade monitorados por 72h. Mochilas vazias mostraram diminuir a proliferação de monócitos ao longo das 72 horas, enquanto mochilas carregadas com ELIP-­DOX mostraram uma diminuição dramática na população celular, apontando uma potencialização dos efeitos da droga pela sua proximidade com as células

The overall goal of this thesis was to exploit the versatility of polyelectrolite multilayers (PEM) to be applied in drug delivery systems and biofunctionalizable films for biomedical applications. PEM films assembled by the layer-by­-layer technique were explored in three main applications. In the first part of this work, the development of a functionalization protocol of poly(allylamine)/poly(styrene sulfonate), PAH/SPS was explored. The optimal film parameters to the use of biotinylated multilayers were applied for the capture and detection of prostate specific antigen (PSA) protein in the range of 100 to 0.1 ng/mL, by using quantum dots. Compared to previous work, this system presented a good sensitivity for PSA detection that is within the clinical limit range of 0.4 to 0.1 ng/mL. The second application involved the creation of a novel sacrificial multilayer film. Films based in natural interactions of bovine submaxillary mucin and the lectin jacalin, BSM/JAC were assembled. BSM/JAC films showed stability when underwent a wide rage of pH (pH 3 to 9) and high ionic strength (5 M NaCl) solutions. BSM/JAC dissolution could be triggered released by incubation in melibiose at 37 °C in pH 7.4 buffer, without cytotoxicity. In the last part of this work the incorporation of echogenic liposomes (ELIP) into cell backpacks was investigated. Cell backpacks are 7-10 µm diameter patches that can be fabricated through LbL polymer deposition onto a photopatterned array to create a stacked composite of three stratified multilayer systems: a releasable region for easy detachment from the substrate, a drug payload region, and a cell adhesive region. The use of ELIP allowed up to 9x more doxorubicin (DOX) loading when compared to free drug in solution adsorbed through the films. DOX release from films was monitored for over 25 days. ELIP­-DOX backpacks were then attached to mouse monocytes and their viability monitored by 72h. Empty backpacks showed to decrease monocytes proliferation over the course of 72h, while ELIP­-DOX backpacks showed a dramatic decrease in cell population, showing that DOX effects were enhancement in drug potency by its proximity

Inhibition of calcification of bovine pericardium after treatment with biopolymers, E-beam irradiation and in vitro endothelization.

This work has investigated the in vitro calcification of bovine pericardium (BP) treated with chitosan (C), silk fibroin (SF) and electron beam irradiation after its endothelization in vitro. For this purpose, freeze-dried BP membranes treated with mixtures of C and SF (1:3, 1:1 and 3:1) and then irradiated by electron beam irradiation were seeded with human umbilical vein endothelial cells (HUVEC) in vitro. After 3 weeks of cultivation these membranes were submitted to in vitro calcification tests using simulated body fluid as the calcifying agent. Control membranes were also studied (without endothelial cells exposure). The results have shown that the membrane compatibility with HUVECs in vitro prevent such biomaterial from calcifying, showing a potential application in biomaterial area, such as cardiac valves and repair patches.

Cytotoxicity and endothelial cell adhesion of lyophilized and irradiated bovine pericardium modified with silk fibroin and chitosan.

Grafts of biological tissues have been used since the 1960s as an alternative to the mechanical heart prostheses. Nowadays, the most consolidated treatment to bovine pericardial (BP) bioprostheses is the crosslinking with glutaraldehyde (GA), although GA may induce calcification in vivo. In previous work, our group demonstrated that electron beam irradiation applied to lyophilized BP in the absence of oxygen promoted crosslinks among collagen fibers of BP tissue. In this work, the incorporation of silk fibroin (SF) and chitosan (CHIT) in the BP not treated with GA was studied. The samples were irradiated and then analyzed for their cytotoxicity and the ability of adhesion and growth of endothelial cells. Initially, all samples showed cytotoxicity. However, after a few washing cycles, the cytotoxicity due to acetic acid and ethanol residues was removed from the biomaterial making it suitable for the biofunctional test. The samples modified with SF/CHIT and electron beam irradiated favored the adhesion and growth of endothelial cells throughout the tissue.

Care during freeze-drying of bovine pericardium tissue to be used as a biomaterial: a comparative study.

Bovine pericardium (BP) tissue is widely used in the manufacture of bioprosthetics. The effects of freeze-drying on the BP tissue have been studied by some researchers in order to decrease their cytotoxicity due to preservation in formaldehyde solution, and to increase the lifetime of the product in storage. This study was undertaken in order to study the effect of freeze-drying in the structure of BP. To perform this study BP samples were freeze-dried in two different types of freeze-dryers available in our laboratory: a laboratory freeze-dryer, in which it was not possible to control parameters and a pilot freeze-dryer, wherein all parameters during freezing and drying were controlled. After freeze-drying processes, samples were analyzed by SEM, Raman spectroscopy, tensile strength, water uptake tests and TEM. In summary, it has been demonstrated that damages occur in collagen fibers by the loss of bulk water of collagen structure implicating in a drastic decreasing of BP mechanical properties due to its structural alterations. Moreover, it was proven that the collagen fibrils suffered breakage at some points, which can be attributed to the uncontrolled parameters during drying.