A Synergistic Approach Therapy for Colorectal Cancer Based on Exosomes and Exploitation of Metabolic Pathways.

In order to reduce the side effects of traditional chemotherapy in the treatment of colorectal cancer (CRC), a new drug delivery system has been developed in this work, based on exosomes that can host two drugs that act synergistically: farnesol (that stops the cell cycle) and paclitaxel (prevents microtubule system depolymerization). Firstly, exosomes were isolated from different cell cultures (from colorectal cancer and from fibroblast as example of normal cell line) by different methods and characterized by western blot, TEM and DLS, and results showed that they express classical protein markers such as CD9 and HSP-70 and they showed spherical morphology with sizes from 93 nm to 129 nm depending on the source. These exosomes were loaded with both drugs and its effect was studied in vitro. The efficacy was studied by comparing the viability of cell cultures with a colorectal cancer cell line (HCT-116) and a normal cell line (fibroblast HS-5). Results showed that exosomes present a specific effect with more reduction in cell viability in tumour cultures than healthy ones. In summary, exosomes are presented in this work as a promising strategy for colorectal cancer treatment.

Development of a thermosensitive hydrogel based on Polaxamer 407 and gellan gum with inclusion complexes (Sulfobutylated-ß-cyclodextrin-Farnesol) as a local drug delivery system.

This work proposes the development of a thermosensitive local drug release system based on Polaxamer 407, also known as Pluronic® F-127 (PF-127), Gellan Gum (GG) and the inclusion complex Sulfobutylated-ß-cyclodextrin (CD) with Farnesol (FOH). Rheological properties of the hydrogels and their degradation were studied. According to the rheological results, a solution of 20% w/v of PF-127 forms a strong gel with a gelling temperature of about 25 °C (storage modulus of 15,000 Pa). The addition of the GG increased the storage modulus (optimal concentration of 0.5 % w/v) twofold without modifying the gelling temperature. Moreover, including 0.5% w/v of GG also increased 6 times the degradation time of the hydrogel. Regarding the inclusion complex, the addition of free CD decreased the viscosity and the gel strength since polymer chains were included in CD cavity without affecting the gelling temperature. Contrarily, the inclusion complex CD-FOH did not significantly modify any property of the formulation because the FOH was hosted in the CD. Furthermore, a mathematical model was developed to adjust the degradation time. This model highlights that the addition of the GG decreases the number of released chains from the polymeric network (which coincides with an increase in the storage modulus) and that the free CD reduces the degradation rate, protecting the polymeric chains. Finally, FOH release was quantified with a specific device, that was designed and printed for this type of system, observing a sustainable drug release (similar to FOH aqueous solubility, 8 µM) dependent on polymer degradation.

Supercritical impregnation of starch aerogels with quercetin: Fungistatic effect and release modelling with a compartmental model.

This work proposes the use of supercritical CO2 to impregnate starch (potato and corn) aerogels with quercetin for a potential fungistatic application. Starch aerogels were successfully produced with supercritical drying, but different results were found depending on the amylose/amylopectin ratio. A higher amount of amylose increases aerogels' specific surface area (with a structure with nanofibrils and nodes) due to the linear and amorphous character of this polymer, whereas a higher amount of amylopectin decreases this property until values of only 25 m2·g-1, obtaining an aerogel with a rough surface. These results were explained with XRD, thermogravimetric, and rheological results (triple step with two temperature sweeps and a time sweep and steady state analysis) concerning hydrogel formation. In fact, retrogradation step plays a more important role in hydrogel formation for a starch source with a higher amount of amylopectin due to an increase in the different polymers' interactions. Supercritical impregnation of quercetin on the aerogels was successfully performed (a loading around 0.30 % with respect to the amount of polymer), and in vitro results indicated that the aerogels produced a fungistatic effect on different types of fungi, but only in the first 12 h because the microorganisms adapted to the surrounding environment. Finally, a compartmental model was used to fit the drug release, which is controlled by quercetin aqueous solubility, indicating the main mass transfer resistances (mass transfer through aerogels was always around 500 min-1 and dissolution process mass transfer from 5·10-3 to 1.65·10-3 s-1) and how an increase in the specific surface area of the aerogels (in the case of corn aerogel) provided a stronger initial burst (70-80 % in 20 min). In fact, this initial burst release was mathematically related to a parameter, that varies from 0.178 to 0.036 depending on the aerogel composition. This study shows that starch aerogels can be impregnated with a hydrophobic compound with fungistatic effect by using supercritical CO2, modifying in addition the drug release by changing the native starch.

Validation of a compartmental model to predict drug release from porous structures produced by ScCO2 techniques.

A global release model is proposed to study the drug release from porous materials for pharmaceutical applications. This model is defined by implementing a compartmental model where the release profile could be explained as the combination of mass transfer phenomena through three compartments as well as a desorption process or dissolution process from the support. This model was validated with five different systems produced with supercritical CO2 (aerogels, membranes, and fibers), showing different release processes. Numerical results indicate that this compartmental approach can be useful to determine adsorption and desorption constants as well as mass transfer resistances within the material. Likewise, this model can predict lag phases and imbibition phenomena. Therefore, the development of compartmental models can be an alternative to traditional models to successfully predict the drug profile of porous materials, achieving a complete understanding of the involved phenomena regardless of the material characteristics.

Engineering Alginate-Based Dry Powder Microparticles to a Size Suitable for the Direct Pulmonary Delivery of Antibiotics.

The inhaled route is regarded as one of the most promising strategies as a treatment against pulmonary infections. However, the delivery of drugs in a dry powder form remains challenging. In this work, we have used alginate to form microparticles containing an antibiotic model (colistin sulfate). The alginate microparticles were generated by atomization technique, and they were characterized by antimicrobial in vitro studies against Pseudomonas aeruginosa. Optimization of different parameters allowed us to obtain microparticles as a dry powder with a mean size (Feret diameter) of 4.45 ± 1.40 µm and drug loading of 8.5 ± 1.50%. The process developed was able to concentrate most of the colistin deposits on the surface of the microparticles, which could be observed by SEM and a Dual-Beam microscope. This produces a fast in vitro release of the drug, with a 100% release achieved in 4 h. Physicochemical characterization using the FTIR, EDX and PXRD techniques revealed information about the change that occurs from the amorphous to a crystalline form of colistin. Finally, the cytotoxicity of microparticles was tested using lung cell lines (A549 and Calu-3). Results of the study showed that alginate microparticles were able to inhibit bacterial growth while displaying non-toxicity toward lung cells.

An Approach to Minimize Tumour Proliferation by Reducing the Formation of Components for Cell Membrane.

Isoprenoids are natural compounds essential for a great number of cellular functions. One of them is farnesol (FOH), which can reduce cell proliferation, but its low solubility in aqueous solvents limits its possible clinical use as a pharmacological tool. One alternative is the use of cyclodextrins (CDs) which house hydrophobic molecules forming inclusion complexes. To assess FOH potential application in anticancer treatments, Sulfobutylated ß-cyclodextrin Sodium Salt (SBE-ß-CD) was selected, due to it has high solubility, approbation by the FDA, and numerous studies that ensure its safety to be administered parenterally or orally without nephrotoxicity associated. The therapeutic action of farnesol and complex were studied in different carcinoma cells, compared with a normal cell line. Farnesol showed selectivity, affecting the viability of colon and liver cancer cells more than in breast cancer cells and fibroblasts. All cells suffered apoptosis after being treated with 150 µM of free FOH, but the complex reduced their cell viability between 50 and 75%. Similar results were obtained for both types of isomers, and the addition of phosphatidylcholine reverses this effect. Finally, cell cycle analysis corroborates the action of FOH as inducer of a G0/G1 phase; when the cells were treated using the complex form, this viability was reduced, reaching 50% in the case of colon and liver, 60% in fibroblasts, and only 75% in breast cancer.

Tuning Alginate Microparticle Size via Atomization of Non-Newtonian Fluids.

A new approach based on the atomization of non-Newtonian fluids has been proposed to produce microparticles for a potential inhalation route. In particular, different solutions of alginate were atomized on baths of different crosslinkers, piperazine and barium chloride, obtaining microparticles around 5 and 40 microns, respectively. These results were explained as a consequence of the different viscoelastic properties, since oscillatory analysis indicated that the formed hydrogel beads with barium chloride had a higher storage modulus (1000 Pa) than the piperazine ones (20 Pa). Pressure ratio (polymer solution-air) was identified as a key factor, and it should be from 0.85 to 1.00 to ensure a successful atomization, obtaining the smallest particle size at intermediate pressures. Finally, a numerical study based on dimensionless numbers was performed to predict particle size depending on the conditions. These results highlight that it is possible to control the microparticles size by modifying either the viscoelasticity of the hydrogel or the experimental conditions of atomization. Some experimental conditions (using piperazine) reduce the particle size up to 5 microns and therefore allow their use by aerosol inhalation.

Steady and Oscillatory Shear Flow Behavior of Different Polysaccharides with Laponite®.

The rheological behavior, in terms of steady and oscillatory shear flow, of Laponite® with different polysaccharides (alginate, chitosan, xanthan gum and levan) in salt-free solutions was studied. Results showed that a higher polymer concentration increased the zero-rate viscosity and decreased the critical strain rate (Cross model fit) as well as increasing the elastic and viscous moduli. Those properties (zero-rate viscosity and critical strain rate) can be a suitable indicator of the effect of the Laponite® on the shear flow behavior for the different solutions. Specifically, the effect of the Laponite® predominates for solutions with large critical strain rate and low zero-rate viscosity, modifying significantly the previous parameters and even the yield stress (if existing). On the other hand, larger higher polymeric concentration hinders the formation of the platelet structure, and polymer entanglement becomes predominant. Furthermore, the addition of high concentrations of Laponite® increases the elastic nature, but without modifying the typical mechanical spectra for polymeric solutions. Finally, Laponite® was added to (previously crosslinked) gels of alginate and chitosan, obtaining different results depending on the material. These results highlight the possibility of predicting qualitatively the impact of the Laponite® on different polymeric solutions depending on the solutions properties.

A comprehensive study on levan nanoparticles formation: Kinetics and self-assembly modeling.

Levan nanoparticles formation is a complicated phenomenon involving simultaneously polymeric reaction kinetics and nanoparticles self-assembly theory. These phenomena are studied in this work with experimental and computational methodologies. Specifically, the effect of different parameters on levan kinetics and nanoparticles production in a cell-free system environment have been studied. Results point out that 37 °C is the best temperature for synthesizing levan as well as the existence of a substrate inhibition effect for polymeric reaction. This work also highlights that raffinose can be used for producing and that an increase on the ratio enzyme-substrate increases the velocity of conversion. However, the previous experimental conditions did not produce an important effect on self-assembly formed levan nanoparticles (always 110 nm) as long as the required levan concentration (CAC) for nanoparticles reorganization is achieved. To have a better understanding of these results, a model was developed to explain numerically levan kinetics and nanoparticle self-assembly. This model was built by taking into account enzyme poisoning effect (also demonstrated experimentally) and a diffusion limited cluster model for the aggregation phenomenon. Simulation results fit properly experimental data and catalytic parameters as well as predicting accurately the value of CAC for producing its reorganization into nanoparticles by self-assembly.

Differences in levan nanoparticles depending on their synthesis route: Microbial vs cell-free systems.

Differences between the levan obtained from bacteria and from cell-free systems were studied in this work. Results showed that both polymers are non-porous solids (type II isotherm with 20 m2/g) with a main thermal decomposition at 200 °C and a negligible value of protein adsorption. Microbial levan produced nanoparticles of 90 nm in diameter whereas nanoparticles of 110 nm were obtained with the polymer obtained from a cell-free system. Both polymers behave as aggregates depending on the critical aggregation concentration. At the same time, that concentration depends on the technique used for the polymer synthesis. Cell-free system aggregation concentration is 0.24 mg/mL whereas a concentration of 0.05 mg/mL was found for the microbial system. In both cases, the average molecular weight of the aggregate is higher than 2000 kDa. These results highlight the existence of aggregation equilibrium for both polymers that has to be taken into account for future applications.

Levan-Capped Silver Nanoparticles for Bactericidal Formulations: Release and Activity Modelling.

An environmentally friendly technique was used to produce levan-capped silver nanoparticles of about 30 nm (with a loading of 30%) that showed bactericide effect, for E. coli and B. subtilis. That effect was mathematically studied with a dose-response model (lethal dose of 12.4 ppm and 6.8 ppm respectively). These silver nanoparticles were subsequently introduced in a gel to create a silver release system with bacteria inhibition activity. Silver release from the gel and its bactericidal activity was theoretically studied to develop a unique model that is able to predict accurately both silver release and lethal dose for any type of bacteria. This model will be useful for performing predictions for future silver in gel applications.

Effect of bacteria type and sucrose concentration on levan yield and its molecular weight.

BACKGROUND: Levan has been traditionally produced from microorganism. However, there is a continuous effort in looking for new strains that improve levan production yield and uses alternative sugar sources for growth. Despite having a wide range of data about levan yield, there are not papers which allow controlling molecular weight, and that plays an essential role for further applications. RESULTS: The effect of the sucrose concentration on levan yield (and its molecular weight) from Bacillus atrophaeus and Acinetobacter nectaris (Gram positive and Gram negative respectively) was studied in this work. It was found that A. nectaris growth (from 3 to 1.5 g L-1 in 40 h) and its levan production (from 3 to 1.5 g L-1) decreases by increasing sucrose concentration (best results at a concentration of 120 g L-1) whereas B. atrophaeus growth (3.5 g L-1 in 30 h) and its levan production (also 3.5 g L-1) were not affected by modifying that parameter. Levan molecular weight from A. nectaris decreases by increasing sucrose concentration (from 8000 to 2000 kDa) whereas levan molecular weight from B. Atrophaeus remains always around 50 kDa. By performing a kinetic study, it was shown that A. nectaris growth follows a substrate-inhibition model, whereas Monod equation provided a good fit for B. atrophaeus growth. Finally, wastes from orange juice industry were used as a medium culture to cultivate those microorganism, obtaining good results with B. atrophaeus (growth 3 g L-1 in 30 h). CONCLUSIONS: Levan production kinetics was determined and compared between different bacteria types.

Development of a nanoparticle system based on a fructose polymer: Stability and drug release studies.

New drug delivery systems (DDSs) with levan or its carboxymethylated form, as carriers, and 5-fluorouracil as a drug, are produced in this work. Levan is obtained after cultivating A. nectaris and polymer nanoparticles are created in water by a self-assembled process. The effect of pH and the ionic strength on polymer nanoparticles aggregation is studied. Basic pHs produces a particle size between 300 and 400nm with a Z-potential around -20mV because a basic medium promotes repulsion forces. DDSs of 300-400nm and a Z-potential about -25mV are prepared by taking advantage of the amphiphilic properties of the levan. The drug is bound to either levan or carboxymethyllevan surfaces by electrostatic interactions, obtaining the best results at basic pHs. 45-70% of the drug is released from the levan in 23h depending on the pH preparation, whereas only a low percentage of the drug is released from the carboxymethyllevan.

Understanding and optimizing the addition of phytohormones in the culture of microalgae for lipid production.

Some studies have described the use of phytohormones in microalgal culture for the production of biodiesel or selected fatty acids. However, no study has determined the amount of phytohormones that maximizes lipid yield. We determined the optimal concentration of auxins and gibberellins (which is between 40 and 60 µM) in two strains (Scenedemus abundans and Chlorella ellipsoidea) suitable for biodiesel production. More than 3-fold increment was reached with S. abundans and near 7-fold increment with C. ellipsoidea. Furthermore, this work suggests that the improved growth of the microalgae in the presence of the phytohormones was due to a reduction in the level of reactive oxygen species (ROS) in the cells. An economic analysis showed that, due to its low cost, auxin offers a positive cost-benefit balance and therefore could be used at large scale. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1203-1211, 2016.

Effect of nitrogen source on growth and lipid accumulation in Scenedesmus abundans and Chlorella ellipsoidea.

Discovering microalgae strains containing a high lipid yield and adequate fatty acid composition is becoming a crucial fact in algae-oil factories. In this study, two unknown strains, named Scenedesmus abundans and Chlorella ellipsoidea, have been tested for their response to different nitrogen sources, in order to determine its influence in the production of lipids. For S. abundans, autotrophic culture with ammonium nitrate offers the maximum lipid yield, obtaining up to 3.55 mg L(-1) d(-1). For C. ellipsoidea, heterotrophic culture with ammonium nitrate has been shown to be the best condition, reaching a lipid production of 9.27 mg L(-1) d(-1). Moreover, fatty acid composition obtained from these cultures meets international biodiesel standards with an important amount of C18:1, achieving 70% of total fatty acids and thus representing a potential use of these two strains at an industrial scale.