Microfluidic-assisted preparation of RGD-decorated nanoparticles: exploring integrin-facilitated uptake in cancer cell lines.

This study is about fine tuning the targeting capacity of peptide-decorated nanoparticles to discriminate between cells that express different integrin make-ups. Using microfluidic-assisted nanoprecipitation, we have prepared poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles with a PEGylated surface decorated with two different arginine-glycine-aspartic acid (RGD) peptides: one is cyclic (RGDFC) and has specific affinity towards αvß3 integrin heterodimers; the other is linear (RGDSP) and is reported to bind equally αvß3 and α5ß1. We have then evaluated the nanoparticle internalization in two cell lines with a markedly different integrin fingerprint: ovarian carcinoma A2780 (almost no αvß3, moderate in α5ß1) and glioma U87MG (very high in αvß3, moderate/high in α5ß1). As expected, particles with cyclic RGD were heavily internalized by U87MG (proportional to the peptide content and abrogated by anti-αvß3) but not by A2780 (same as PEGylated particles). The linear peptide, on the other hand, did not differentiate between the cell lines, and the uptake increase vs. control particles was never higher than 50%, indicating a possible low and unselective affinity for various integrins. The strong preference of U87MG for cyclic (vs. linear) peptide-decorated nanoparticles was shown in 2D culture and further demonstrated in spheroids. Our results demonstrate that targeting specific integrin make-ups is possible and may open the way to more precise treatment, but more efforts need to be devoted to a better understanding of the relation between RGD structure and their integrin-binding capacity.

"Tandem" Nanomedicine Approach against Osteoclastogenesis: Polysulfide Micelles Synergically Scavenge ROS and Release Rapamycin.

We show the first example of a synergic approach of oxidant (ROS) scavenging carrier and ROS-responsive drug release in the context of a potential therapy against osteoporosis, aiming to inhibit the differentiation of inflammatory cells into osteoclasts. In our "tandem" approach, a branched amphiphilic, PEGylated polysulfide (PPSES-PEG) was preferred over a linear analogue, because of improved homogeneity in the aggregates (spherical micelles vs mixture of wormlike and spherical), increased stability, and higher drug loading (up to ∼22 wt % of antiosteoclastic rapamycin). These effects are ascribed to the branching inhibiting crystallization in the polysulfide blocks. The ROS-scavenging micelles alone were already able to reduce osteoclastogenesis in a RAW 264.7 model, but the "drug" combination (the polymer itself + rapamycin released only under oxidation) completely abrogated the process. An important take-home message is that the synergic performance depended very strongly on the oxidant:oxidizable group molar ratio, a parameter to carefully tune in the perspective of targeting specific diseases.

Binding and Internalization in Receptor-Targeted Carriers: The Complex Role of CD44 in the Uptake of Hyaluronic Acid-Based Nanoparticles (siRNA Delivery).

CD44 is an endocytic hyaluronic acid (HA) receptor, and is overexpressed in many carcinomas. This has encouraged the use of HA to design CD44-targeting carriers. This paper is about dissecting the mechanistic role of CD44. Here, HA-decorated nanoparticles are used to deliver siRNA to both tumoral (AsPC-1, PANC-1, HT-29, HCT-116) and non-tumoral (fibroblasts, differently polarized THP-1 macrophages, HUVEC) human cell lines, evaluating the initial binding of the nanoparticles, their internalization rate, and the silencing efficiency (cyclophilin B (PPIB) gene). Tumoral cells internalize faster and experience higher silencing than non-tumoral cells. This is promising as it suggests that, in a tumor, HA nanocarriers may have limited off-target effects. More far-reaching is the inter-relation between the four parameters of the study: CD44 expression, HA binding on cell surfaces, internalization rate, and silencing efficiency. No correlation is found between binding (an early event) and any of the other parameters, whereas silencing correlates both with speed of the internalization process and CD44 expression. This study confirms on one hand that HA-based carriers can perform a targeted action, but on the other it suggests that this may not be due to a selective binding event, but rather to a later recognition leading to selective internalization.

Evaluating the Efficiency of Hyaluronic Acid for Tumor Targeting via CD44.

The development of delivery systems capable of tumor targeting represents a promising strategy to overcome issues related to nonspecific effects of conventional anticancer therapies. Currently, one of the most investigated agents for cancer targeting is hyaluronic acid (HA), since its receptor, CD44, is overexpressed in many cancers. However, most of the studies on CD44/HA interaction have been so far performed in cell-free or genetically modified systems, thus leaving some uncertainty regarding which cell-related factors influence HA binding and internalization (collectively called "uptake") into CD44-expressing cells. To address this, the expression of CD44 (both standard and variants, designated CD44s and CD44v, respectively) was evaluated in human dermal fibroblasts (HDFs) and a large panel of cancer cell lines, including breast, prostate, head and neck, pancreatic, ovarian, colorectal, thyroid, and endometrial cancers. Results showed that CD44 isoform profiles and expression levels vary across the cancer cell lines and HDF and are not consistent within the cell origin. Using composite information of CD44 expression, HA binding, and internalization, we found that the expression of CD44v can negatively influence the uptake of HA, and, instead, when cells primarily expressed CD44s, a positive correlation was observed between expression and uptake. In other words, CD44shigh cells bound and internalized more HA compared to CD44slow cells. Moreover, CD44shigh HDFs were less efficient in uptaking HA compared to CD44shigh cancer cells. The experiments described here are the first step toward understanding the interplay between CD44 expression, its functionality, and the underlying mechanism(s) for HA uptake. The results show that factors other than the amount of CD44 receptor can play a role in the interaction with HA, and this represents an important advance with respect to the design of HA-based carriers and the selection of tumors to treat according to their CD44 expression profile.

The different ways to chitosan/hyaluronic acid nanoparticles: templated vs direct complexation. Influence of particle preparation on morphology, cell uptake and silencing efficiency.

This study is about linking preparative processes of nanoparticles with the morphology of the nanoparticles and with their efficiency in delivering payloads intracellularly. The nanoparticles are composed of hyaluronic acid (HA) and chitosan; the former can address a nanoparticle to cell surface receptors such as CD44, the second allows both for entrapment of nucleic acids and for an endosomolytic activity that facilitates their liberation in the cytoplasm. Here, we have systematically compared nanoparticles prepared either A) through a two-step process based on intermediate (template) particles produced via ionotropic gelation of chitosan with triphosphate (TPP), which are then incubated with HA, or B) through direct polyelectrolyte complexation of chitosan and HA. Here we demonstrate that HA is capable to quantitatively replace TPP in the template process and significant aggregation takes place during the TPP-HA exchange. The templated chitosan/HA nanoparticles therefore have a mildly larger size (measured by dynamic light scattering alone or by field flow fractionation coupled to static or dynamic light scattering), and above all a higher aspect ratio (R g/R H) and a lower fractal dimension. We then compared the kinetics of uptake and the (antiluciferase) siRNA delivery performance in murine RAW 264.7 macrophages and in human HCT-116 colorectal tumor cells. The preparative method (and therefore the internal particle morphology) had little effect on the uptake kinetics and no statistically relevant influence on silencing (templated particles often showing a lower silencing). Cell-specific factors, on the contrary, overwhelmingly determined the efficacy of the carriers, with, e.g., those containing low-MW chitosan performing better in macrophages and those with high-MW chitosan in HCT-116.

Assessing global range expansion in a cryptic species complex: insights from the red seaweed genus Asparagopsis (Florideophyceae).

The mitochondrial genetic diversity, distribution and invasive potential of multiple cryptic operational taxonomic units (OTUs) of the red invasive seaweed Asparagopsis were assessed by studying introduced Mediterranean and Hawaiian populations. Invasive behavior of each Asparagopsis OTU was inferred from phylogeographic reconstructions, past historical demographic dynamics, recent range expansion assessments and future distributional predictions obtained from demographic models. Genealogical networks resolved Asparagopsis gametophytes and tetrasporophytes into four A. taxiformis and one A. armata cryptic OTUs. Falkenbergia isolates of A. taxiformis L3 were recovered for the first time in the western Mediterranean Sea and represent a new introduction for this area. Neutrality statistics supported past range expansion for A. taxiformis L1 and L2 in Hawaii. On the other hand, extreme geographic expansion and an increase in effective population size were found only for A. taxiformis L2 in the western Mediterranean Sea. Distribution models predicted shifts of the climatically suitable areas and population expansion for A. armata L1 and A. taxiformis L1 and L2. Our integrated study confirms a high invasive risk for A. taxiformis L1 and L2 in temperate and tropical areas. Despite the differences in predictions among modelling approaches, a number of regions were identified as zones with high invasion risk for A. taxiformis L2. Since range shifts are likely climate-driven phenomena, future invasive behavior cannot be excluded for the rest of the lineages.

Nanomanufacturing through microfluidic-assisted nanoprecipitation: Advanced analytics and structure-activity relationships.

We have employed microfluidics (cross-shaped chip) for the preparation of drug-loaded poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles. The polymer precipitates from an acetone solution upon its controlled laminar mixing (flow focusing) with an aqueous solution of a surfactant, allowing for an operator-independent, up-scalable and reproducible preparative process of nanoformulations. Firstly, using PEGylated surfactants we have compared batch and microfluidic processes, and showed the superior reproducibility of the latter and its strong dependency on the acetone/water ratio (flow rate ratio). We have then focused on the issue of purification from free surfactant, and employed advanced characterization techniques such as flow-through dynamic light scattering as the in-line quality control technique, and field flow fractionation (FFF) with dynamic and static light scattering detection, which allowed the detection of surfactant micelles in mixture with nanoparticles (hardly possible with stand-alone dynamic light scattering). Finally, we have shown that the choice of polymer and surfactant affects the release behaviour of a model drug (paclitaxel), with high molecular weight PLGA (RG756) and low molecular weight surfactant (tocopheryl poly(ethylene glycol) 1000 succinate, TPGS) apparently showing higher burst and accelerated release.

Chitosan/Hyaluronic Acid Nanoparticles: Rational Design Revisited for RNA Delivery.

Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver an RNA/DNA cargo to cells overexpressing HA receptors such as CD44. For these systems, unequivocal links have not been established yet between chitosan macromolecular (molecular weight; degree of deacetylation, i.e., charge density) and nanoparticle variables (complexation strength, i.e., stability; nucleic acid protection; internalization rate) on one hand, and transfection efficiency on the other hand. Here, we have focused on the role of avidity on transfection efficiency in the CD44-expressing HCT-116 as a cellular model; we have employed two differently sized payloads (a large luciferase-encoding mRNA and a much smaller anti-Luc siRNA), and a small library of chitosans (variable molecular weight and degree of deactylation). The RNA avidity for chitosan showed-as expected-an inverse relationship: higher avidity-higher polyplex stability-lower transfection efficiency. The avidity of chitosan for RNA appears to lead to opposite effects: higher avidity-higher polyplex stability but also higher transfection efficiency. Surprisingly, the best transfecting particles were those with the lowest propensity for RNA release, although this might be a misleading relationship: for example, the same macromolecular parameters that increase avidity can also boost chitosan's endosomolytic activity, with a strong enhancement in transfection. The performance of these nonviral vectors appears therefore difficult to predict simply on the basis of carrier- or payload-related variables, and a more holistic consideration of the journey of the nanoparticle, from cell uptake to cytosolic bioavailability of payload, is needed. It is also noteworthy that the nanoparticles used in this study showed optimal performance under slightly acidic conditions (pH 6.4), which is promising for applications in a tumoral extracellular environment. It is also worth pointing out that under these conditions we have for the first time successfully delivered mRNA with chitosan/HA nanoparticles.

The CD44-Mediated Uptake of Hyaluronic Acid-Based Carriers in Macrophages.

CD44 is a potentially rewarding target in cancer therapy, although its mechanisms of ligand binding and internalization are still poorly understood. In this study, we have established quantitative relationships between CD44 expression in differently polarized macrophages (M0, M1, and M2-polarized THP-1 human macrophages) and the uptake of hyaluronic acid (HA)-based materials, which are potentially usable for CD44 targeting. We have validated a robust method for macrophage polarization, which sequentially uses differentiating and polarizing factors, and allows to show that CD44 expression depends on polarization (M1 > M0 ≥ M2). It is noteworthy that THP-1 M2 expressed CD44v6, suggesting their suitability as a model of tumor-associated macrophages. In the uptake of HA, both as a soluble polymer and in the form of (siRNA-loaded) nanoparticles, CD44 expression correlated positively with binding, but negatively with internalization. Counterintuitively, it appears that a higher presence of CD44 (in M1) allows a more efficient capture of HA materials, but a lower expression (in M2) is conducive to better internalization. Although possibly cell-specific, this unexpected relationship indicates that the common paradigm "higher CD44 expression = better targetability" is too simplistic; mechanistic details of both receptor presentation and association still need to be elucidated for a predictable targeting behavior.

Displasia fibrosa de seno maxilar / Fibrous Dysplasia of Maxillary Sinus

La displasia fibrosa fue descrita por Lichtenstein en 1938, es una enfermedad caracterizada por el reemplazo progresivo de tejido óseo normal por una proliferación de tejido conectivo fibroso, por su forma de presentación se clasifica en monostótica o poliostótica. La forma monostótica, localizada en la región craneofacial constituye solo el 10 % de los casos, se presenta con mayor frecuencia en el maxilar superior y puede afectar a huesos adyacentes como el cigomático, esfenoides y occipital. La degeneración sarcomatosa puede ocurrir en un 0,5 %. Se presenta el caso de una paciente femenina, de 37 años de edad, operada ocho años antes de displasia fibrosa en el Servicio de Cirugía Máxilofacial del Hospital Vladimir I. Lenin, que luego de su embarazo y parto presenta un aumento de volumen en área malar y maxilar izquierdos, a la que se le realiza biopsia que da como resultado una recidiva. Se le realiza cirugía remodelativa de pared anterior de seno maxilar, a través de una incisión de Weber-Ferguson, con buenos resultados estéticos y funcionales.

Fibrous dysplasia was described by Lichtenstein in 1938, is a progressive disease characterized by replacement of normal bone tissue by proliferation of fibrous connective tissue, its presentation is classified in monostotic or polyostotic. The monostotic form, located in the craniofacial region constitutes only 10 % of cases, it occurs most frequently in the maxilla and adjacent bones can affect as the zygomatic, sphenoid and occipital. The sarcomatous degeneration can occur in 0.5 %. A 37-year-old female patient, who underwent fibrous dysplasia surgery eight years ago at Maxillofacial Surgery Department of Vladimir I. Lenin Hospital, which after the pregnancy and delivery presented a volume increase in malar and left area is presented in this paper, biopsy is performed whose results showed a recurrence. Remodeling surgery was performed of anterior wall of the maxillary sinus through a Weber-Ferguson incision with good cosmetic and functional results.

Displasia fibrosa de seno maxilar / Fibrous Dysplasia of Maxillary Sinus

La displasia fibrosa fue descrita por Lichtenstein en 1938, es una enfermedad caracterizada por el reemplazo progresivo de tejido óseo normal por una proliferación de tejido conectivo fibroso, por su forma de presentación se clasifica en monostótica o poliostótica. La forma monostótica, localizada en la región craneofacial constituye solo el 10 por ciento de los casos, se presenta con mayor frecuencia en el maxilar superior y puede afectar a huesos adyacentes como el cigomático, esfenoides y occipital. La degeneración sarcomatosa puede ocurrir en un 0,5 por ciento. Se presenta el caso de una paciente femenina, de 37 años de edad, operada ocho años antes de displasia fibrosa en el Servicio de Cirugía Máxilofacial del Hospital Vladimir I. Lenin, que luego de su embarazo y parto presenta un aumento de volumen en área malar y maxilar izquierdos, a la que se le realiza biopsia que da como resultado una recidiva. Se le realiza cirugía remodelativa de pared anterior de seno maxilar, a través de una incisión de Weber-Ferguson, con buenos resultados estéticos y funcionales(AU)

Fibrous dysplasia was described by Lichtenstein in 1938, is a progressive disease characterized by replacement of normal bone tissue by proliferation of fibrous connective tissue, its presentation is classified in monostotic or polyostotic. The monostotic form, located in the craniofacial region constitutes only 10 percent of cases, it occurs most frequently in the maxilla and adjacent bones can affect as the zygomatic, sphenoid and occipital. The sarcomatous degeneration can occur in 0.5 percent. A 37-year-old female patient, who underwent fibrous dysplasia surgery eight years ago at Maxillofacial Surgery Department of Vladimir I. Lenin Hospital, which after the pregnancy and delivery presented a volume increase in malar and left area is presented in this paper, biopsy is performed whose results showed a recurrence. Remodeling surgery was performed of anterior wall of the maxillary sinus through a Weber-Ferguson incision with good cosmetic and functional results(AU)

Presentación de un caso clínico con diagnóstico de diente ectópico intranasal / Presentation of a Patient with Intranasal Ectopic Tooth Diagnosis

Se estudió paciente 54 años de edad, masculino que desde los 10 años presentaba rinorrea unilateral derecha, blanco amarillenta, no fétida y obstrucción nasal ipsilateral. A la rinoscopia anterior se visualizó un rinolito de mediano tamaño que ocupaba el piso de la fosa nasal derecha, observándose entonces en la zona posterior del rinolito un aumento de volumen blanco, duro, de aproximadamente ocho milímetros, fijo, no doloroso, que se proyectaba a la cavidad nasal de forma puntiaguda. Fue valorado de conjunto por Otorilaringolo-Máxilo-Facial. Se indicó rayo x donde se apreció imagen radiopaca ubicada en zona supralveolar a nivel de piso de fosa nasal derecha, de forma conoide. Se realizó cirugía exploratoria intraoral confirmándose el diagnóstico de diente ectópico intranasal y se procedió a su exéresis. El post-operatorio fue satisfactorio. Se evoluciona el paciente actualmente por consulta externa encontrándose asintomático...(AU)

A male patient of 54 years old, who since he was 10 years had right unilateral rhinorrhea, white yellowish, no fetid and ipsilateral. A rhinoscopy was done and a rhinolite of mild size was observed in the inferior wall of the nasal fossa. The rhinolith had a volume enhacement, hard , of eight millimeters and without pain. The case was assesed by the specialists and an X-ray was done. The results showed an opaque image of cone form. An exploratory surgical treatment was given in order to diagnose the case. The results revealed the diagnosis of an ectopic intranasal tooth and the exeresis procedure was applied. The postoperative results were satisfactory as well as the patient´s progress...(AU)