Omar González-Ortega

Gold nanoparticles and vaccine development

Mucosal vaccines constitute an advantageous immunization approach to achieve broad immunization against widespread diseases; however, improvements in this field are still required to expand their exploitation. As gold nanoparticles are biocompatible and can be easily functionalized with antigens, they have been proposed as carriers for the delivery of vaccines. The study of gold nanoparticles (AuNPs) in vaccinology has been of interest for a number of research groups in recent years and important advances have been made. This review provides a summary of the AuNPs synthesis methodologies and an updated overview of the current AuNPs-based vaccines under development. The implications of these advances for the development of new mucosal vaccines as well as future prospects for the field are discussed.

Adsorption of peptides and small proteins with control access polymer permeation to affinity binding sites. Part I: Polymer permeation-immobilized metal ion affinity chromatography separation adsorbents with polyethylene glycol and immobilized metal ions.

Despite the many efforts to develop efficient protein purification techniques, the isolation of peptides and small proteins on a larger than analytical scale remains a significant challenge. Recovery of small biomolecules from diluted complex biological mixtures, such as human serum, employing porous adsorbents is a difficult task mainly due to the presence of concentrated large biomolecules that can add undesired effects in the system such as blocking of adsorbent pores, impairing diffusion of small molecules, or competition for adsorption sites. Adsorption and size exclusion chromatography (AdSEC) controlled access media, using polyethylene glycol (PEG) as a semi-permeable barrier on a polysaccharide matrix, have been developed and explored in this work to overcome such effects and to preferentially adsorb small molecules while rejecting large ones. In the first part of this work, adsorption studies were performed with small peptides and proteins from synthetic mixtures using controlled access polymer permeation adsorption (CAPPA) media created by effectively grafting PEG on an immobilized metal affinity chromatography (IMAC) agarose resin, where chelating agents and immobilized metal ions were used as the primary affinity binding sites. Synthetic mixtures consisted of bovine serum albumin (BSA) with small proteins, peptides, amino acids (such as histidine or Val⁴-Angiotensin III), and small molecules-spiked human serum. The synthesized hybrid adsorbent consisted of agarose beads modified with iminodiacetic (IDA) groups, loaded with immobilized Cu(II) ions, and PEG. These CAPPA media with grafted PEG on the interior and exterior surfaces of the agarose matrix were effective in rejecting high molecular weight proteins. Different PEG grafting densities and PEG of different molecular weight were tested to determine their effect in rejecting and controlling adsorbent permeation properties. Low grafting density of high molecular weight PEG was found to be as effective as high grafting density of low molecular weight PEG in the rejecting properties of the semi-permeable synthesized media.

Adsorption of peptides and small proteins with control access polymer permeation to affinity binding sites. Part II : Polymer permeation-ion exchange separation adsorbents with polyethylene glycol and strong anion exchange groups

In chromatographic separations, the most general problem in small biomolecule isolation and purification is that such biomolecules are usually found in extremely low concentrations together with high concentrations of large molecular weight proteins. In the first part of this work, adsorption and size exclusion chromatography (AdSEC) controlled access media, using polyethylene glycol (PEG) as a semi-permeable barrier on a polysaccharide Immobilized Metal Affinity Chromatography (IMAC) matrix was synthesized and used to develop chromatographic adsorbents that preferentially adsorb and separate low molecular weight biomolecules while rejecting large molecular weight proteins. In this second part, we expand the concept of controlled access polymer permeation adsorption (CAPPA) media by grafting polyethylene glycol (PEG) on a high capacity polysaccharide ion exchange (IEX) chromatographic resin where PEG acts as a semi-permeable barrier that preferentially allows the permeation of small molecules while rejecting large ones. The IEX resin bearing quaternary ammonium groups binds permeated biomolecules according to their ion exchange affinity while excluding large biomolecules by the PEG barrier and thus cannot compete for the binding sites. This new AdSEC media was used to study the retention of peptides and proteins covering a wide range of molecular weights from 1 to 150 kDa. The effect of protein molecular weight towards retention by ion exchange was performed using pure protein solutions. Recovery of insulin from insulin-spiked human serum and insulin-spiked human urine was evaluated under polymer controlled permeation conditions. The CAPPA media consisted of agarose beads modified with amino-PEG-methoxy and with trimethyl ammonium groups, having chloride capacities between 20 and 40 μeq/mL and were effective in rejecting high molecular weight proteins while allowing the preferential adsorption of small proteins and peptides.

Plant-Made Vaccines in the Fight Against Cancer

Immunotherapies constitute an important trend in developing new cancer treatments, and several promising candidates are under evaluation in clinical trials. Plants have entered the fight against cancer because they constitute low-cost and efficient hosts for biopharmaceutical production that can also serve as oral delivery vehicles. This review is focused on the knowledge gained through the development of anticancer plant-made vaccines reported thus far, and highlights the potential of this technology - its success being reflected in the number of candidates that are close to market. Future prospects for anticancer plant-made vaccines are also identified.

Bioactivity of an antihypertensive peptide expressed in Chlamydomonas reinhardtii

In this study, we developed a transplastomic C. reinhardtii strain that accumulates anti-hypertensive peptides. Tandem repeats of VLPVP peptide were included. PCR analysis confirmed the presence of the transgene in the modified strains. After in vitro digestion of biomass of a recombinant C. reinhardtii strain the VLVPV peptide was identified and quantified by HPLC. The highest expression line produced 0.292mg of recombinant protein per mg of freeze-dried biomass. Intragastric administration of the genetically modified strain to spontaneous hypertensive rats at a dose of 30mg/kg of body weight of recombinant protein significantly reduced systolic blood pressure. At the same dose, the recombinant protein exerts an ACE-inhibitory effect. This is the first study that indicates the potential of this microalga producing an antihypertensive peptide as a dietary supplement for hypertension patients.

Algevir: An Expression System for Microalgae Based on Viral Vectors

The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus) and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin), which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture), which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins.

Synthesis of silica-core gold nanoshells and some modifications/variations

Gold nanoshells are particles usually composed of a spherical silica core coated with a thin gold layer. Their chemical and optical properties make them suitable and attractive for medical applications, namely cancer treatment and diagnosis, as they have been studied for biosensing, imaging, and photothermal ablation. For their synthesis, most of the reported methods are based on the first reported by Oldenburg et al. In this method, silica nanoparticles are first produced and then modified to incorporate amino groups aimed to adsorb small gold clusters, which in turn act as the nucleation sites for the reduction of additional gold until complete gold shells are formed. In this review, we examine some common conditions to synthesize gold nanoshells based on this process, along with important aspects that need to be followed to ensure the production of gold nanoshells having homogeneous size and shape that render suspensions with consistent properties in the near-infrared region of the electromagnetic spectrum. Since the customary method is laborious and time-consuming, three additional processes intended to simplify or reduce some steps are described as well. Finally, fundamental aspects on the chemistry of the synthesis and their variations involved in all the revised processes are also presented. Figures from our own findings are included to support these descriptions. Please notice that this review focuses on the synthesis; other reviews focus in optical properties and applications.

Purification of Human Serum Immunoglobulins Using Immobilized Metal Affinity Chromatography with Ethylenediamine Triacetic Acid as Chelating Agent

Purified immunoglobulins are required for a wide range of applications in biotechnology such as laboratory and clinical reagents, novel therapeutics, and immunoaffinity ligands in chromatography. The present work describes the effective separation of immunoglobulins from mixtures of synthetic immunoglobulin solutions and human serum using immobilized metal affinity chromatography and specified pH and imidazole gradients. Ethylenediamine triacetic acid was used as the chelating agent and Cu(II) as the immobilized ion on an agarose matrix. The modified agarose gel presented Cu(II) and immunoglobulin capacities of 65 µmol/mL and 46 mg/mL, respectively. pH and imidazole gradients were performed in order to find the best conditions for the affinity of immunoglobulins toward the immobilized Cu(II) ions. The chelating systems were able to effectively retain immunoglobulins and showed no measurable affinity towards human serum albumin under all working conditions. In the purification of immunoglobulins from human serum, a pH gradient was able to release immunoglobulins (1.3 mg) with a total purity of 60% (and a purity of 84% for 65% recovery of all adsorbed protein), while an imidazole gradient allowed the recovery of 0.8 mg of immunoglobulins with a total purity of 75% (and a purity of 83% for 70% recovery of all adsorbed protein).

Lipid accumulation during nitrogen and sulfur starvation in Chlamydomonas reinhardtii overexpressing a transcription factor

Algal triacylglycerol biosynthesis is of increasing interest for potential biodiesel production. In this study, we investigated the lipids content and gene expression changes in Chlamydomonas reinhardtii strain overexpressing a Dof-type transcription factor in response to nutrient deficiency. The untransformed strain grew in TAP complete medium and accumulated only 4.1% dry cell weight of fatty acids; while in the transgenic strain, the total fatty acids content increased to 15.58 and 17.02% in medium without sulfur or nitrogen, respectively. The major fatty acids found in the stressed microalgae were palmitic, oleic (all strains), and linoleic (transgenic strain). Additionally, we observed that the transgenic strain does not have a defect in carbon partitioning but redirect the carbon flux from starch to lipids. Among the most interesting results in the transcriptional profile, we found an overexpression of the enzymes lecithin cholesterol acyltransferase, glycerol-3-phosphatase acyltransferase, and phosphatidylcholine-sterol O-aciltransferase. As observed in this study, the combination of strategies (overexpression of a transcription factor and nutrient deficit) increases not only the content of total lipids in transgenic cells compared to an untransformed strain but, also the proportion of specific fatty acids that are desirable for biodiesel production.

Calcium sustained release, pH changes and cell viability induced by chitosan-based pastes for apexification

We explored chitosan-based sustained release pastes for apexification. The study aimed to formulate chitosan-based pastes loaded with calcium hydroxide (CH) or with calcium chloride (CC), and to evaluate the sustained release of Ca2+ and pH changes in deionized water as well as the effect of the pastes on cell viability. The pastes were formulated by dissolution of the chitosan in 1% or 2% acetic acid (AAC) plus the addition of CH or CC, then were suspended in deionized water for 50 days; the released Ca(II) and pH were measured with an electrode probe. The effect of the pastes on viability of human dental pulp cells was evaluated with a MTS assay. The results showed that the pastes prepared with 1% and 2% AAC and loaded with CH released a 74.9% and a 76.1% of the Ca2+ content, respectively, while the pastes prepared with 1% and 2% AAC loaded with CC released a content of Ca2+ of 90.8% and 76.6%, respectively. A control paste (CH and polyethylene glycol) released a 95.4%; significant statistical differences were found between the percentage of the experimental pastes and the control. The CH-loaded pastes caused an alkaline pH at the starting of the study, but the pH became neutral at the ending. The pH of the CC-loaded pastes was neutral at the starting and was acid at the ending. The pastes no affected on the cell viability. The chitosan-based pastes showed a suitable sustained release profile and cytocompatibility.