Ramon Novoa-Carballal

Controlling Cancer Cell Fate Using Localized Biocatalytic Self-Assembly of an Aromatic Carbohydrate Amphiphile

We report on a simple carbohydrate amphiphile able to self-assemble into nanofibers upon enzymatic dephosphorylation. The self-assembly can be triggered by alkaline phosphatase (ALP) in solution or in situ by the ALP produced by osteosarcoma cell line, SaOs2. In the latter case, assembly and localized gelation occurs mainly on the cell surface. The gelation of the pericellular environment induces a reduction of the SaOs2 metabolic activity at an initial stage (≤7 h) that results in cell death at longer exposure periods (≥24 h). We show that this effect depends on the phosphatase concentration, and thus, it is cell-selective with prechondrocytes ATDC5 (that express ∼15-20 times lower ALP activity compared to SaOs2) not being affected at concentrations ≤1 mM. These results demonstrate that simple carbohydrate derivatives can be used in an antiosteosarcoma strategy with limited impact on the surrounding healthy cells/tissues.

A Nanomedicine Transports a Peptide Caspase-3 Inhibitor across the Blood–Brain Barrier and Provides Neuroprotection

Caspases play an important role as mediators of cell death in acute and chronic neurological disorders. Although peptide inhibitors of caspases provide neuroprotection, they have to be administered intracerebroventricularly because they cannot cross the blood–brain barrier (BBB). Herein, we present a nanocarrier system that can transfer chitosan nanospheres loaded with N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK), a relatively specific caspase-3 inhibitor, across BBB. Caspase-3 was chosen as a pharmacological target because of its central role in cell death. Polyethylene glycol-coated nanospheres were conjugated to an anti-mouse transferrin receptor monoclonal antibody (TfRMAb) that selectively recognizes the TfR type 1 on the cerebral vasculature. We demonstrate with intravital microscopy that this nanomedicine is rapidly transported across the BBB without being measurably taken up by liver and spleen. Pre- or post-treatment (2 h) with intravenously injected Z-DEVD-FMK-loaded nanospheres dose dependently decreased the infarct volume, neurological deficit, and ischemia-induced caspase-3 activity in mice subjected to 2 h of MCA occlusion and 24 h of reperfusion, suggesting that they released an amount of peptide sufficient to inhibit caspase activity. Similarly, nanospheres inhibited physiological caspase-3 activity during development in the neonatal mouse cerebellum on postnatal day 17 after closure of the BBB. Neither nanospheres functionalized with TfRMAb but not loaded with Z-DEVD-FMK nor nanospheres lacking TfRMAb but loaded with Z-DEVD-FMK had any effect on either paradigm, suggesting that inhibition of caspase activity and subsequent neuroprotection were due to efficient penetration of the peptide into brain. Thus, chitosan nanospheres open new and exciting opportunities for brain delivery of biologically active peptides that are useful for the treatment of CNS disorders.

Structural analysis of fructans produced by acetic acid bacteria reveals a relation to hydrocolloid function

Some strains of acetic acid bacteria (Gluconobacter frateurii TMW 2.767, Gluconobacter cerinus DSM 9533T, Neoasaia chiangmaiensis NBRC 101099, Kozakia baliensis DSM 14400) produce high amounts of fructans, which can be exploited in food applications as previously demonstrated empirically for dough systems. In order to get insight into the structure and functionality of these polymers, we investigated the fructans isolated from these strains with respect to their linkage types and molecular weights/shapes using NMR spectroscopy and AF4-MALS-RI. Each fructan was identified as levan. The isolated levan fractions were highly similar according to their basic linearity and linkage types, but differed significantly in terms of their individual molecular weight distributions. In aqueous solutions the size of levan molecules present in all isolated levans continuously increased with their molecular weight and they tended to adopt a more compact molecular shape. Our data suggest that the increasing molecular weight of a levan particle enforces intramolecular interactions to reach the structural compactness of a microgel with hydrocolloid properties.

Systemically Administered Brain-Targeted Nanoparticles Transport Peptides across the Blood—Brain Barrier and Provide Neuroprotection

Although growth factors and anti-apoptotic peptides have been shown to be neuroprotective in stroke models, translation of these experimental findings to clinic is hampered by limited penetration of peptides to the brain. Here, we show that a large peptide like the basic fibroblast growth factor (bFGF) and a small peptide inhibitor of caspase-3 (z-DEVD-FMK) can effectively be transported to the brain after systemic administration by incorporating these peptides to brain-targeted nanoparticles (NPs). Chitosan NPs were loaded with peptides and then functionalized by conjugating with antibodies directed against the transferrin receptor-1 on brain endothelia to induce receptor-mediated transcytosis across the blood—brain barrier (BBB). Pre-ischemic systemic administration of bFGF- or z-DEVD-FMK-loaded NPs significantly decreased the infarct volume after 2-hour middle cerebral artery occlusion and 22-hour reperfusion in mice. Co-administration of bFGF- or z-DEVD-FMK-loaded NPs reduced the infarct volume further and provided a 3-hour therapeutic window. bFGF-loaded NPs were histologically detected in the brain parenchyma and also restored ischemia-induced Akt dephosphorylation. The neuroprotection was not observed when receptor-mediated transcytosis was inhibited with imatinib or when bFGF-loaded NPs were not conjugated with the targeting antibody, which enables them to cross the BBB. Nanoparticles targeted to brain are promising drug carriers to transport large as well as small BBB-impermeable therapeutics for neuroprotection against stroke.

Anti-tumor efficacy of chitosan-g-poly(ethylene glycol) nanocapsules containing docetaxel: anti-TMEFF-2 functionalized nanocapsules vs. non-functionalized nanocapsules.

The development and evaluation of PEGylated chitosan (CS) nanocapsules (NCs) conjugated to a monoclonal antibody anti-TMEFF-2 (CS-PEG-anti-TMEFF-2 mAb NCs) for targeted delivery of docetaxel (DCX) is presented. CS-PEG-Biotin NCs, displaying biotin tags at their surface, were obtained and efficiently functionalized with an anti-TMEFF-2 mAb through a convenient avidin-biotin approach. Cell cycle analysis after treatment with different DCX-loaded CS-PEG NC formulations indicated that the encapsulated drug remained fully active, showing a similar functional behavior to free DCX. In vivo efficacy studies using a non-small cell lung carcinoma xenograft revealed that CS-PEG-anti-TMEFF-2 NCs resulted as effective as free DCX (Taxotere®). Interestingly, differences on the pharmacodynamic behavior among the different DCX formulations were observed. Thus, while free DCX exhibited a fast and short effect on tumor volume reduction, CS-PEG-anti-TMEFF-2 mAb NCs showed a delayed and prolonged action, with no significant side effects of treatments.

Dynamics of Chitosan by 1H NMR Relaxation

The dynamics of chitosan (CS) in solution have been studied by (1)H NMR relaxation [longitudinal (T(1)) and transverse (T(2)) relaxation times and NOE] as a function of the degrees of acetylation (DA, 1-70) and polymerization (DP, 10-1200), temperature (278-343 K), concentration (0.1-30 g/L), and ionic strength (50-400 mM). This analysis points to CS as a semirigid polymer with increased flexibility at higher DA in agreement with reduced electrostatic repulsions between protonated amino groups.

The dynamics of GATG glycodendrimers by NMR diffusion and quantitative 13C relaxation

The dynamics of GATG glycodendrimers have been investigated by NMR translational diffusion and quantitative (13)C relaxation studies (Lipari-Szabo model-free), allowing the determination of the correlation times describing the dendrimer segmental orientational mobility.

Interpolyelectrolyte complexes based on hyaluronic acid-block-poly(ethylene glycol) and poly-L-lysine

The preparation of spherical, nanosized interpolyelectrolyte complexes by the interaction of hyaluronic acid-block-poly(ethylene glycol) (HA-b-PEG) with poly-L-lysine (PLL) at a stoichiometric charge-to-charge ratio is described. The complexation was studied by dynamic light scattering and cryogenic transmission electron microscopy for HA-b-PEG with different lengths of the HA and PEG blocks. A minimal molecular weight of the HA block (ca. 9 kDa) is necessary for efficient complexation, while a minimal molecular weight of the PEG block (ca. 5 kDa) is needed to prevent macroscopic aggregation. The formed nanoassemblies have hydrodynamic radii ranging from 45 to 150 nm with very low dispersity indices (D.I. = 0.02–0.05). They appear as soft objects with a nanogel structure and pronouncedly swell on addition of NaCl. The increasing ionic strength leads to disruption of the complexes above ca. 120–160 mM of NaCl. Decreasing the ionic strength by dialysis reforms the nanogels, demonstrating the reversibility of nanogel formation. Crosslinking with carbodiimide leads to nanoassemblies with a very well defined structure and high stability against ionic strength.

Tunable nano-carriers from clicked glycosaminoglycan block copolymers

Oxime click reaction is used for the synthesis of diblock copolymers of polyethylene glycol (PEG) and glycosaminoglycans (GAGs) with different molecular weights (Mw) and sulfation degrees. The ability of these copolymers to carry positively charged proteins is evidenced by their assembly with poly-l-lysine as a model: interpolyelectrolyte complexes with tunable size at the nanometric scale (radius of 25-90 nm) and narrow distribution are described. We demonstrate that there is a critical Mw of GAGs for the formation of stable complexes and that the sulfation degree determines the size of the nano-assemblies. Highly sulfated GAGs form the smallest complexes that are stable to at least 500 mM ionic strength: a property that is not usual for GAG interpolyelectrolyte complexes. The feasibility of the synthesised block copolymers as protein carriers is further evidenced by their complexation with fibroblast growth factor (FGF-2). The described assets of GAG block copolymers together with the intrinsic GAG properties such as biorecognition and biodegradability open up new opportunities in the design of selective encapsulation/release nanosystems with a stealth PEG corona.

GATG dendrimers and PEGylated block copolymers: from synthesis to bioapplications.

Dendrimers are synthetic macromolecules composed of repetitive layers of branching units that emerge from a central core. They are characterized by a tunable size and precise number of peripheral groups which determine their physicochemical properties and function. Their high multivalency, functional surface, and globular architecture with diameters in the nanometer scale makes them ideal candidates for a wide range of applications. Gallic acid-triethylene glycol (GATG) dendrimers have attracted our attention as a promising platform in the biomedical field because of their high tunability and versatility. The presence of terminal azides in GATG dendrimers and poly(ethylene glycol) (PEG)-dendritic block copolymers allows their efficient functionalization with a variety of ligands of biomedical relevance including anionic and cationic groups, carbohydrates, peptides, or imaging agents. The resulting functionalized dendrimers have found application in drug and gene delivery, as antiviral agents and for the treatment of neurodegenerative diseases, in diagnosis and as tools to study multivalent carbohydrate recognition and dendrimer dynamics. Herein, we present an account on the preparation and recent applications of GATG dendrimers in these fields.