Ana Carina Loureiro Mendes

Self-assembly in nature: using the principles of nature to create complex nanobiomaterials

Self-assembly is a ubiquitous process in biology where it plays numerous important roles and underlies the formation of a wide variety of complex biological structures. Over the past two decades, materials scientists have aspired to exploit natures assembly principles to create artificial materials, with hierarchical structures and tailored properties, for the fabrication of functional devices. Toward this goal, both biological and synthetic building blocks have been subject of extensive research in self-assembly. In fact, molecular self-assembly is becoming increasingly important for the fabrication of biomaterials because it offers a great platform for constructing materials with high level of precision and complexity, integrating order and dynamics, to achieve functions such as stimuli-responsiveness, adaptation, recognition, transport, and catalysis. The importance of peptide self-assembling building blocks has been recognized in the last years, as demonstrated by the literature available on the topic. The simple structure of peptides, as well as their facile synthesis, makes peptides an excellent family of structural units for the bottom-up fabrication of complex nanobiomaterials. Additionally, peptides offer a great diversity of biochemical (specificity, intrinsic bioactivity, biodegradability) and physical (small size, conformation) properties to form self-assembled structures with different molecular configurations. The motivation of this review is to provide an overview on the design principles for peptide self-assembly and to illustrate how these principles have been applied to manipulate their self-assembly across the scales. Applications of self-assembling peptides as nanobiomaterials, including carriers for drug delivery, hydrogels for cell culture and tissue repair are also described.

Hybrid electrospun chitosan-phospholipids nanofibers for transdermal drug delivery

Chitosan (Ch) polysaccharide was mixed with phospholipids (P) to generate electrospun hybrid nanofibers intended to be used as platforms for transdermal drug delivery. Ch/P nanofibers exibithed average diameters ranging from 248±94nm to 600±201nm, depending on the amount of phospholipids used. Fourier Transformed Infra-Red (FTIR) spectroscopy and Dynamic Light Scattering (DLS) data suggested the occurrence of electrostatic interactions between amine groups of chitosan with the phospholipid counterparts. The nanofibers were shown to be stable for at least 7days in Phosphate Buffer Saline (PBS) solution. Cytotoxicity studies (WST-1 and LDH assays) demonstrated that the hybrid nanofibers have suitable biocompatibility. Fluorescence microscopy, also suggested that L929 cells seeded on top of the CH/P hybrid have similar metabolic activity comparatively to the cells seeded on tissue culture plate (control). The release of curcumin, diclofenac and vitamin B12, as model drugs, from Ch/P hybrid nanofibers was investigated, demonstrating their potential utilization as a transdermal drug delivery system.

NMR Derivatives for Quantification of 2H and 13C‐Enrichment of Human Glucuronide from Metabolic Tracers

Quantification of 2H and 13C enrichment distributions in human urinary glucuronide following ingestion of 2H2O and 13C gluconeogenic tracers was achieved by NMR spectroscopy of the 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone and 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivatives. The derivatization process is simple and can be applied to any glucuronide species. The derivatives are highly soluble in acetonitrile and generate well‐resolved and narrow 2H and 13C NMR signals. The 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided resolution of the six glucuronide 13C signals and numerous 13C isotopomer populations through one‐ and two‐bond 13C‐13C‐coupling, while the 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided complete resolution of the 2H NMR signals for the five glucuronide hydrogens. The isopropylidene methyl signals were also resolved and provided an internal 2H enrichment standard following the acetonation of glucuronolactone with deuterated acetone.

Encapsulation and survival of a chondrocyte cell line within xanthan gum derivative

A chemical derivative of xanthan gum polysaccharide is investigated as a new artificial matrix for the encapsulation of chondrocytic cells. Toward this goal, a novel micro-droplet generator is developed to produce microcapsules. Microcapsules with an average diameter of 500 µm, smooth surface, and homogeneous size distribution are obtained. ATDC5 cells encapsulated in carboxymethyl xanthan (CMX) microcapsules remain viable and are observed to proliferate for prolonged culture periods with enhanced metabolic activity. Furthermore, retention of the chondrogenic phenotype is exhibited by the cells within CMX, suggesting the ability of this material to be applied in cell-delivery therapies.

Effects of electrospun chitosan wrapping for dry-ageing of beef, as studied by microbiological, physicochemical and low-field nuclear magnetic resonance analysis

The effects of using electrospun chitosan fibres as a wrapping material for dry-ageing beef was studied and compared to traditional dry-ageing and wet-ageing of beef for up to 21 days. The chitosan treatment showed improved results in terms of yield, reduction of microbial counts, yeasts and moulds, and lighter appearance compared to traditional dry-ageing. Weight and trimming losses were minimal in the wet-ageing beef. However, significant growth of lactic acid bacteria was observed in this group. Transverse relaxation times indicated a lower degree of muscle denaturation during ageing in the chitosan dry-ageing beef compared to the traditional dry-ageing meat. A principal component analysis furthermore indicated that 60.6% of the variation between samples and ageing treatments could be described by differences in the water content and distribution in the muscle. The study showed that electrospun chitosan fibre mats have potential as a wrapping material for improved quality during dry-ageing of beef.

Microfluidic Fabrication of Self-Assembled Peptide-Polysaccharide Microcapsules as 3D Environments for Cell Culture

We report a mild cell encapsulation method based on self-assembly and microfluidics technology. Xanthan gum, an anionic polysaccharide, was used to trigger the self-assembly of a positively charged multidomain peptide. The self-assembly resulted in the formation of a nanofibrous matrix and using a microfluidic device, microcapsules with homogeneous size were fabricated. The properties and performance of xanthan-peptide microcapsules were optimized by changing peptide/polysaccharide ratio and their effects on the microcapsule permeability and mechanical stability were analyzed. The effect of microcapsule formulation on viability and proliferation of encapsulated chondrocytic (ATDC5) cells was also investigated. The encapsulated cells were metabolically active, showing an increased viability and proliferation over 21 days of in vitro culture, demonstrating the long-term stability of the self-assembled microcapsules and their ability to support and enhance the survival of encapsulated cells over a prolonged time. Self-assembling materials combined with microfluidics demonstrated to be an innovative approach in the fabrication of cytocompatible matrix for cell microencapsulation and delivery.

Hepatic UDP-glucose 13C isotopomers from [U-13C]glucose : A simple analysis by 13C NMR of urinary menthol glucuronide

Menthol glucuronide was isolated from the urine of a healthy 70‐kg female subject following ingestion of 400 mg of peppermint oil and 6 g of 99% [U‐13C]glucose. Glucuronide 13C‐excess enrichment levels were 4–6% and thus provided high signal‐to‐noise ratios (SNRs) for confident assignment of 13C‐13C spin‐coupled multiplet components within each 13C resonance by 13C NMR. The [U‐13C]glucuronide isotopomer derived via direct pathway conversion of [U‐13C]glucose to [U‐13C]UDP‐glucose was resolved from [1,2,3‐13C3]‐ and [1,2‐13C2]glucuronide isotopomers derived via Cori cycle or indirect pathway metabolism of [U‐13C]glucose. In a second study, a group of four overnight‐fasted patients (63 ± 10 kg) with severe heart failure were given peppermint oil and infused with [U‐13C]glucose for 4 hr (14 mg/kg prime, 0.12 mg/kg/min constant infusion) resulting in a steady‐state plasma [U‐13C]glucose enrichment of 4.6% ± 0.6%. Menthol glucuronide was harvested and glucuronide 13C‐isotopomers were analyzed by 13C NMR. [U‐13C]glucuronide enrichment was 0.6% ± 0.1%, and the sum of [1,2,3‐13C3] and [1,2‐13C2]glucuronide enrichments was 0.9% ± 0.2%. From these data, flux of plasma glucose to hepatic UDPG was estimated to be 15% ± 4% that of endogenous glucose production (EGP), and the Cori cycle accounted for at least 32% ± 10% of GP. Magn Reson Med, 2006.

Fabrication of phospholipid–xanthan microcapsules by combining microfluidics with self-assembly

We report the synthesis of an amphiphilic polysaccharide, a phospholipid (1,2-dioleoyl-sn-glycero-phosphoetilamine, DOPE) conjugated with the anionic xanthan gum, and its ability to spontaneously self-assemble under mild aqueous conditions. This work also aimed to apply a microfluidic platform that can precisely fabricate microsized and monodispersed capsules for cell encapsulation. Stable hollow capsular structures were obtained by the generation of homogeneous spherical droplets of the self-assembled polymer in the microfluidic device through the formation of a water-in-oil emulsion, followed by the stabilization of the polymer aggregates in a separate collection vessel containing phosphate-buffered saline (physiological ionic strength and pH). The properties (size, morphology, permeability) and performance (stability) of the obtained microcapsules were studied, as well their ability to support the viability, function and proliferation of encapsulated cells. ATDC5 cells were encapsulated within the capsules and shown to remain viable, evidencing increased cellular metabolic activity over 21 days of in vitro culture. By combining microfluidic droplet generation and self-assembly of xanthan-DOPE, we were able to fabricate microcapsules that provided an adequate environment for cells to survive and proliferate.

Palmitoylation of xanthan polysaccharide for self-assembly microcapsule formation and encapsulation of cells in physiological conditions

Hydrophobized polysaccharides have emerged as a promising strategy in the biomedical field due to the versatility to design functional structures through the spontaneous self-assembly in cell-friendly conditions. Based on this concept, xanthan, a bacterial extracellular polysaccharide with potential as encapsulating matrix, was conjugated with hydrophobic palmitoyl groups to obtain an amphiphilic system able to form capsules by self-assembly processes. The conjugation of xanthan was performed at different xanthan/palmitoyl chloride ratios and Fourier transformed infrared, 1H nuclear magnetic resonance spectroscopies, as well as wide angle X-ray diffraction, differential scanning calorimetry were performed to characterize the obtained conjugates. The results showed that the increase in the hydrophobic reactant promoted higher hydrophobic interaction and consequently higher molecular organization. At certain palmitoyl concentrations and through a proper balance between charge repulsion and hydrophobic interaction, the amphiphilic molecules self-assembled into stable capsular hollow structures in the presence of physiological ion concentration and pH. Poly-L-lysine coated microcapsules with an average diameter of 576.6 ± 74 μm and homogenous size distribution were obtained. The morphology revealed by scanning electron microscopy showed microcapsules with two distinct layers. The ability of palmitoyl-xanthan microcapsules to sustain viability and proliferation of encapsulated cells was confirmed by AlamarBlue and DNA assays. These findings suggest the application of palmitoyl-xanthan microcapsules as a potential material for cell encapsulation in cell-based therapies.

Nanomechanics of electrospun phospholipid fiber

Electrospun asolectin phospholipid fibers were prepared using isooctane as a solvent and had an average diameter of 6.1 ± 2.7 μm. Their mechanical properties were evaluated by nanoindentation using Atomic Force Microscopy, and their elastic modulus was found to be approximately 17.2 ± 1 MPa. At a cycle of piezo expansion-retraction (loading-unloading) of a silicon tip on a fiber, relatively high adhesion was observed during unloading. It is proposed that this was primarily due to molecular rearrangements at the utmost layers of the fiber caused by the indentation of the hydrophilic tip. The phospholipid fibers were shown to be stable in ambient conditions, preserving the modulus of elasticity up to 24 h.