Juan José Valle-Delgado

A simple process for lignin nanoparticle preparation

A lack of renewable resources and their inefficient use is a major challenge facing the society. Lignin is a natural biopolymer obtained mainly as a by-product from the pulp- and paper-making industries, and is primarily burned to produce energy. However, interest for using lignin in more advanced applications has increased rapidly. In particular, lignin based nanoparticles could find potential use in functional surface coatings, nanoglue, drug delivery, and microfluidic devices. In this work, a straightforward method to produce lignin nanoparticles from waste lignin obtained from kraft pulping is introduced. Spherical lignin nanoparticles were obtained by dissolving softwood kraft lignin in tetrahydrofuran (THF) and subsequently introducing water into the system through dialysis. No chemical modification of lignin was needed. Water acts as a non-solvent reducing lignins degrees of freedom causing the segregation of hydrophobic regions to compartments within the forming nanoparticles. The final size of the nanoparticles depended on the pre-dialysis concentration of dissolved lignin. The stability of the nanoparticle dispersion as a function of time, salt concentration and pH was studied. In pure water and at room temperature the lignin nanoparticle dispersion was stable for over two months, but a very low pH or high salt concentration induced aggregation. It was further demonstrated that the surface charge of the particles could be reversed and stable cationic lignin nanoparticles were produced by adsorption of poly(diallyldimethylammonium chloride) (PDADMAC).

Sulfated Polysaccharides Promote the Assembly of Amyloid β1–42 Peptide into Stable Fibrils of Reduced Cytotoxicity

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid β peptide (Aβ) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, Aβ1–42. Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant β-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for Aβ1–42 fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different Aβ1–42 species on Ca2+ homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca2+ that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca2+ increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

Modulation of Aβ42 fibrillogenesis by glycosaminoglycan structure

The role of amyloid β (Aβ) peptide in the onset and progression of Alzheimers disease is linked to the presence of soluble Aβ species. Sulfated glycosaminoglycans (GAGs) promote Aβ fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42‐residue fragment Aβ42 with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable Aβ42 fibrils is promoted by polymeric GAGs with negative charges placed in‐frame with the 4.8‐Å separating Aβ42 monomers within protofibrillar β‐sheets. Incubation of Aβ42 with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2‐ to 5‐fold, whereas in the presence of the cationic polysaccharide chitosan, Aβ42 fibrillar species were reduced by 25% and sensitivity to protease degradation increased ∼3‐fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between Aβ42 fibrils induced by PVS and Aβ42 fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from −11.2 to −13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of −7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble Aβ42 fibrils trapping soluble toxic forms.—Valle‐Delgado, J. J., Alfonso‐Prieto, M., de Groot, N. S., Ventura, S., Samitier, J., Rovira, C., Fernàndez‐Busquets, X. Modulation of Aβ42 fibrillogenesis by glycosaminoglycan structure. FASEB J. 24, 4250–4261 (2010). www.fasebj.org

Use of poly(amidoamine) drug conjugates for the delivery of antimalarials to Plasmodium.

Current malaria therapeutics demands strategies able to selectively deliver drugs to Plasmodium-infected red blood cells (pRBCs) in order to limit the appearance of parasite resistance. Here, the poly(amidoamines) AGMA1 and ISA23 have been explored for the delivery of antimalarial drugs to pRBCs. AGMA1 has antimalarial activity per se as shown by its inhibition of the in vitro growth of Plasmodium falciparum, with an IC₅₀ of 13.7 μM. Fluorescence-assisted cell sorting data and confocal fluorescence microscopy and transmission electron microscopy images indicate that both polymers exhibit preferential binding to and internalization into pRBCs versus RBCs, and subcellular targeting to the parasite itself in widely diverging species such as P. falciparum and Plasmodium yoelii, infecting humans and mice, respectively. AGMA1 and ISA23 polymers with hydrodynamic radii around 7 nm show a high loading capacity for the antimalarial drugs primaquine and chloroquine, with the final conjugate containing from 14.2% to 32.9% (w/w) active principle. Intraperitoneal administration of 0.8 mg/kg chloroquine as either AGMA1 or ISA23 salts cured P. yoelii-infected mice, whereas control animals treated with twice as much free drug did not survive. These polymers combining into a single chemical structure drug carrying capacity, low unspecific toxicity, high biodegradability and selective internalization into pRBCs, but not in healthy erythrocytes for human and rodent malarias, may be regarded as promising candidates deserving to enter the antimalarial therapeutic arena.

Nanotools for the Delivery of Antimicrobial Peptides

Antimicrobial peptide drugs are increasingly attractive therapeutic agents as their roles in physiopathological processes are being unraveled and because the development of recombinant DNA technology has made them economically affordable in large amounts and high purity. However, due to lack of specificity regarding the target cells, difficulty in attaining them, or reduced half-lives, most current administration methods require high doses. On the other hand, reduced specificity of toxic drugs demands low concentrations to minimize undesirable side-effects, thus incurring the risk of having sublethal amounts which favour the appearance of resistant microbial strains. In this scenario, targeted delivery can fulfill the objective of achieving the intake of total quantities sufficiently low to be innocuous for the patient but that locally are high enough to be lethal for the infectious agent. One of the major advances in recent years has been the size reduction of drug carriers that have dimensions in the nanometer scale and thus are much smaller than -and capable of being internalized by- many types of cells. Among the different types of potential antimicrobial peptide-encapsulating structures reviewed here are liposomes, dendritic polymers, solid core nanoparticles, carbon nanotubes, and DNA cages. These nanoparticulate systems can be functionalized with a plethora of biomolecules providing specificity of binding to particular cell types or locations; as examples of these targeting elements we will present antibodies, DNA aptamers, cell-penetrating peptides, and carbohydrates. Multifunctional Trojan horse-like nanovessels can be engineered by choosing the adequate peptide content, encapsulating structure, and targeting moiety for each particular application.

The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils.

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar β-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, β-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.

Direct measurements of non-ionic attraction and nanoscaled lubrication in biomimetic composites from nanofibrillated cellulose and modified carboxymethylated cellulose.

There is a growing interest to design biomimetic self-assembled composite films from renewable resources aimed at a combination of high toughness, strength and stiffness. However, the relationship between interfacial interactions of the components and the mechanical performance of the composite is still poorly understood. In this work we present evidence of the link between mechanical performance of carbohydrate-based composites with nanolubrication and with direct surface forces between the hard and soft domain in the system. Our approach was to use nanofibrillated cellulose (NFC) as the major reinforcing domain and to modify it by adsorption of a small amount of soft polyethylene glycol grafted carboxymethyl cellulose (CMC-g-PEG). The effect of the soft polymer on direct normal and friction forces in air between cellulose surfaces was evaluated using colloidal probe microscopy. The fibrillar structure of the NFC thin film affected the frictional behaviour; when decreasing load, the friction between pure cellulose surfaces increased, suggesting partial pull-out of fibrils, a phenomenon not observed for non-fibrillar cellulose substrates. Adsorption of CMC-g-PEG on both surfaces decreased the friction considerably but adhesion was still high. The symmetric system, having both cellulose substrates covered with the polymer, was compared to asymmetric systems where only one surface was covered with the polymer. Furthermore, a free standing composite film was prepared by non-ionic self-assembly of NFC and CMC-g-PEG with 99 : 1 weight-ratio; the mechanical properties of the macroscopic films were related to the nanoscaled interactions between the components. The composition studied showed excellent mechanical properties which do not follow the simple rule of mixture. Thus, a synergy in the direct surface forces and mechanical properties was found. This approach offers a robust path to aid in the efficient design of next generation biomimetic composites.

Biomimetic collagen I and IV double layer Langmuir-Schaefer films as microenvironment for human pluripotent stem cell derived retinal pigment epithelial cells

The environmental cues received by the cells from synthetic substrates in vitro are very different from those they receive in vivo. In this study, we applied the Langmuir-Schaefer (LS) deposition, a variant of Langmuir-Blodgett technique, to fabricate a biomimetic microenvironment mimicking the structure and organization of native Bruchs membrane for the production of the functional human embryonic stem cell derived retinal pigment epithelial (hESC-RPE) cells. Surface pressure-area isotherms were measured simultaneously with Brewster angle microscopy to investigate the self-assembly of human collagens type I and IV on air-subphase interface. Furthermore, the structure of the prepared collagen LS films was characterized with scanning electron microscopy, atomic force microscopy, surface plasmon resonance measurements and immunofluorescent staining. The integrity of hESC-RPE on double layer LS films was investigated by measuring transepithelial resistance and permeability of small molecular weight substance. Maturation and functionality of hESC-RPE cells on double layer collagen LS films was further assessed by RPE-specific gene and protein expression, growth factor secretion, and phagocytic activity. Here, we demonstrated that the prepared collagen LS films have layered structure with oriented fibers corresponding to architecture of the uppermost layers of Bruchs membrane and result in increased barrier properties and functionality of hESC-RPE cells as compared to the commonly used dip-coated controls.

Carbohydrate-Carbohydrate Interactions Mediated by Sulfate Esters and Calcium Provide the Cell Adhesion Required for the Emergence of Early Metazoans

Early metazoans had to evolve the first cell adhesion mechanism addressed to maintain a distinctive multicellular morphology. As the oldest extant animals, sponges are good candidates for possessing remnants of the molecules responsible for this crucial evolutionary innovation. Cell adhesion in sponges is mediated by the calcium-dependent multivalent self-interactions of sulfated polysaccharides components of extracellular membrane-bound proteoglycans, namely aggregation factors. Here, we used atomic force microscopy to demonstrate that the aggregation factor of the sponge Desmapsamma anchorata has a circular supramolecular structure and that it thus belongs to the spongican family. Its sulfated polysaccharide units, which were characterized via nuclear magnetic resonance analysis, consist preponderantly of a central backbone composed of 3-α-Glc1 units partially sulfated at 2- and 4-positions and branches of Pyr(4,6)α-Gal1→3-α-Fuc2(SO3)1→3-α-Glc4(SO3)1→3-α-Glc→4-linked to the central α-Glc units. Single-molecule force measurements of self-binding forces of this sulfated polysaccharide and their chemically desulfated and carboxyl-reduced derivatives revealed that the sulfate epitopes and extracellular calcium are essential for providing the strength and stability necessary to sustain cell adhesion in sponges. We further discuss these findings within the framework of the role of molecular structures in the early evolution of metazoans.

Adaptation of targeted nanocarriers to changing requirements in antimalarial drug delivery

The adaptation of existing antimalarial nanocarriers to new Plasmodium stages, drugs, targeting molecules, or encapsulating structures is a strategy that can provide new nanotechnology-based, cost-efficient therapies against malaria. We have explored the modification of different liposome prototypes that had been developed in our group for the targeted delivery of antimalarial drugs to Plasmodium-infected red blood cells (pRBCs). These new models include: (i) immunoliposome-mediated release of new lipid-based antimalarials; (ii) liposomes targeted to pRBCs with covalently linked heparin to reduce anticoagulation risks; (iii) adaptation of heparin to pRBC targeting of chitosan nanoparticles; (iv) use of heparin for the targeting of Plasmodium stages in the mosquito vector; and (v) use of the non-anticoagulant glycosaminoglycan chondroitin 4-sulfate as a heparin surrogate for pRBC targeting. The results presented indicate that the tuning of existing nanovessels to new malaria-related targets is a valid low-cost alternative to the de novo development of targeted nanosystems.