Nanobiotechnology and Nanomaterials for Gene Expression and Bacterial Growth Control

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 11-01-2017Esta tesis tiene embargado el acceso al texto completo hasta el 11-07-2017In this thesis, we have initiated four research projects. Three of them related to the development and evaluation of nanomaterials as antimicrobials. The fourth one is devoted to the assessment of gold nanoparticles modified with oligonucleotides as regulators of p21 expression. The first project has been carried out at the Universidad Autónoma de Madrid and deals with the preparation of silver nanoparticles (AgNPs) using broths from bacteria and their evaluation as antimicrobials. Particularly, four psychrophilic bacteria isolated from Antarctica have been evaluated. Different conditions were assessed, including the addition of NaCl and incubation temperatures. The AgNPs obtained were characterized and their antimicrobial properties evaluated against Gram-negative and Gram-positive bacteria. We also have evaluated the antimicrobial properties of silver nanoclusters stabilized by DNA (DNA-AgNCs), which are novel fluorescent materials. After testing 9 oligonucleotides with different sequence and length we found that the antibacterial activity depends on the sequence of the oligonucleotides employed. The sequences tested yielded fluorescent AgNCs, which can be grouped in blue, yellow and red emitters. Interestingly, blue emitters yielded poor antibacterial activity whereas yellow and red emitters afforded an activity similar to AgNO3. Finally, we prepared a trimeric structure containing the sequence that afforded the best antimicrobial activity, which inhibited the growth of Gram positive and negative bacteria in the sub-micromolar range. In the third project, we demonstrated that oligonucleotides modified with a fluorescent dye (HEX) can be used in photodynamic therapy to kill bacteria. Interestingly, the sequence of the oligonucleotide employed modulates the antimicrobial activity of the system. The last project has been done in collaboration with the Centro Nacional de Biotecnología (CNB) and the preliminary results of the regulation of p21 with modified gold nanoparticles are presented. P21 is involved in cell senescence and replication, and its regulation can be done using different oligonucleotides, such as antisense and siRNAs. However, the translocation into the cells of nucleic acids is not easy and transfection systems are required. In this case, oligonucleotides were conjugated to gold nanoparticles (AuNPs) and used in T-cells. The results suggest that the approach employed is valid although further optimization is required

Metal-Based Nanomaterials Photodynamic Action with a Focus on Au and Ag Nanomaterials

Photodynamic action is the interaction between cells and oxygen, light, and chemical reagent (photosensitizers). Photodynamic techniques include photodynamic diagnosis (PDD), fluorescence-guided tumor resection, and photodynamic therapy (PDT). PDD and PDT have the exact mechanism. They are based on light and tissue interaction with a difference. PDT is along with the destruction of the lesion against PDD that the diagnosis is made without destruction. Photosensitizers (PSs) could be organic and inorganic. Metal-based PSs were considered, due to the disadvantages of organic PSs such as low quantum yield and small stock shift, and high toxicity. We have examined the metal-based nanomaterials PDT in recent years. The titles considered are including the introduction that consists of explanations about photodynamic action, PDD, PDT and history of PDT, PDT mechanism, PDT effects on the immune system, photosensitizers, and metal-based nanomaterials in the photodynamic application, which this section addresses along with the application of metal nanomaterials (with a focus on gold and silver nanomaterials) in photodynamic techniques

Molecular detection of Brucella species in patients suspicious of Brucellosis from Zanjan, Iran

Abstract Brucella is an intracellular pathogen capable of infecting animals and humans. The aim of this study was to identify Brucella spp in sera of high risk individuals by a polymerase chain reaction (PCR)-based method. A total of 180 patients suspected to have Brucellosis were examined by serological tests. To establish a PCR protocol for diagnosis of active brucellosis, DNA was extracted from the serum samples by using a commercial kit. PCR amplification was done for detection of Brocella DNA using BCSP31 target gene and IS711 locus. The PCR assay showed that an amplicon of 223 bp was obtained in 73.8% (133/180) of the tested sera using primers (B 4 /B 5 ) derived from a gene encoding the 31-kDa Brucella abortus antigen. In another PCR, an amplicon of 498 bp was obtained in 63.8% (115/180) of the samples using Brucella abortus-specific primers derived from a locus adjacent to the 3'-end of IS711, and also an amplicon of 731 bp was produced in 4.4% (8/180) of the tested samples using Brucella melitensis-specific primers. When the Wright method was used as a gold standard, the sensitivity and specificity of the PCR technique for genus identification were found to be 96 and 80.7%, respectively. However, the sensitivity value obtained with the species-specific PCR method was 82%, and specificity was similar to that previous reported. This is the first report of a high frequency of Brucella abortus in patients suspicious of Brucellosis from the Zanjan province

Molecular detection of Brucella species in patients suspicious of Brucellosis from Zanjan, Iran

Brucella is an intracellular pathogen capable of infecting animals and humans. The aim of this study was to identify Brucella spp in sera of high risk individuals by a polymerase chain reaction (PCR)-based method. A total of 180 patients suspected to have Brucellosis were examined by serological tests. To establish a PCR protocol for diagnosis of active brucellosis, DNA was extracted from the serum samples by using a commercial kit. PCR amplification was done for detection of Brocella DNA using BCSP31 target gene and IS711 locus. The PCR assay showed that an amplicon of 223 bp was obtained in 73.8% (133/180) of the tested sera using primers (B4/B5) derived from a gene encoding the 31-kDa Brucella abortus antigen. In another PCR, an amplicon of 498 bp was obtained in 63.8% (115/180) of the samples using Brucella abortus-specific primers derived from a locus adjacent to the 3'-end of IS711, and also an amplicon of 731 bp was produced in 4.4% (8/180) of the tested samples using Brucella melitensis-specific primers. When the Wright method was used as a gold standard, the sensitivity and specificity of the PCR technique for genus identification were found to be 96 and 80.7%, respectively. However, the sensitivity value obtained with the species-specific PCR method was 82%, and specificity was similar to that previous reported. This is the first report of a high frequency of Brucella abortus in patients suspicious of Brucellosis from the Zanjan province

Photothermal and radiotherapy with alginate-coated gold nanoparticles for breast cancer treatment

Abstract Radiation therapy and phototherapy are commonly used cancer treatments that offer advantages such as a low risk of adverse effects and the ability to target cancer cells while sparing healthy tissue. A promising strategy for cancer treatment involves using nanoparticles (NPs) in combination with radiation and photothermal therapy to target cancer cells and improve treatment efficacy. The synthesis of gold NPs (AuNPs) for use in biomedical applications has traditionally involved toxic reducing agents. Here we harnessed dopamine (DA)-conjugated alginate (Alg) for the facile and green synthesis of Au NPs (Au@Alg-DA NPs). Alg-DA conjugate reduced Au ions, simultaneously stabilized the resulting AuNPs, and prevented aggregation, resulting in particles with a narrow size distribution and improved stability. Injectable Au@Alg-DA NPs significantly promoted ROS generation in 4T1 breast cancer cells when exposed to X-rays. In addition, their administration raised the temperature under a light excitation of 808 nm, thus helping to destroy cancer cells more effectively. Importantly, no substantial cytotoxicity was detected in our Au@Alg-DA NPs. Taken together, our work provides a promising route to obtain an injectable combined radio enhancer and photothermally active nanosystem for further potential clinic translation

Antibacterial Activity of DNA-Stabilized Silver Nanoclusters Tuned by Oligonucleotide Sequence

Silver nanoclusters (AgNCs) stabilized by DNA are promising materials with tunable fluorescent properties, which have been employed in a plethora of sensing systems. In this report, we explore their antimicrobial properties in Gram-positive and Gram-negative bacteria. After testing 9 oligonucleotides with different sequence and length, we found that the antibacterial activity depends on the sequence of the oligonucleotide employed. The sequences tested yielded fluorescent AgNCs, which can be grouped in blue, yellow, and red emitters. Interestingly, blue emitters yielded poor antibacterial activity, whereas yellow and red emitters afforded an activity similar to silver nitrate. Furthermore, structural studies using circular dichroism indicate the formation of complexes with different stability and structure, which might be one of the factors that modulate their activity. Finally, we prepared a trimeric structure containing the sequence that afforded the best antimicrobial activity, which inhibited the growth of Gram-positive and negative bacteria in the submicromolar range

Antibacterial Activity of DNA-Stabilized Silver Nanoclusters Tuned by Oligonucleotide Sequence

Silver nanoclusters (AgNCs) stabilized by DNA are promising materials with tunable fluorescent properties, which have been employed in a plethora of sensing systems. In this report, we explore their antimicrobial properties in Gram-positive and Gram-negative bacteria. After testing 9 oligonucleotides with different sequence and length, we found that the antibacterial activity depends on the sequence of the oligonucleotide employed. The sequences tested yielded fluorescent AgNCs, which can be grouped in blue, yellow, and red emitters. Interestingly, blue emitters yielded poor antibacterial activity, whereas yellow and red emitters afforded an activity similar to silver nitrate. Furthermore, structural studies using circular dichroism indicate the formation of complexes with different stability and structure, which might be one of the factors that modulate their activity. Finally, we prepared a trimeric structure containing the sequence that afforded the best antimicrobial activity, which inhibited the growth of Gram-positive and negative bacteria in the submicromolar range

Facile preparation of silver based radiosensitizers via biomineralization method for enhanced in vivo breast cancer radiotherapy

Abstract To solve the traditional radiotherapy obstacles, and also to enhance the radiation therapy efficacy various radiosensitizers have been developed. Radiosensitizers are promising agents that under X-ray irradiation enhance injury to tumor tissue by accelerating DNA damage. In this report, silver-silver sulfide nanoparticles (Ag-Ag2S NPs) were synthesized via a facile, one-pot and environmentally friendly biomineralization method. Ag-Ag2S was coated with bovine serum albumin (BSA) in situ and applied as an X-ray sensitizer to enhance the efficiency of radiotherapy. Also, folic acid (FA) was conjugated to Ag-Ag2S@BSA to impart active targeting capability to the final formulation (Ag-Ag2S@BSA-FA). Prepared NPs were characterized by transmission electron microscopes (TEM), scanning electron microscope (SEM), dynamic light scattering (DLS), ultraviolet–visible spectroscopy (UV–Vis), X-ray diffraction analysis (XRD), and X-ray photoelectron spectroscopy (XPS) techniques. Results show that most of the NPs have well-defined uniform Janus structures. The biocompatibility of the NPs was then evaluated both in vitro and in vivo. A series of in vitro assays were performed on 4T1 cancer cells to evaluate the therapeutic efficacy of the designed NPs. In addition, the radio-enhancing ability of the NPs was tested on the 4T1 breast cancer murine model. MTT, live and dead cell staining, apoptosis, ROS generation, and clonogenic in vitro assays demonstrated the efficacy of NPs as radiosensitizers in radiotherapy. In vivo results as well as H&E staining tumor tissues confirmed tumor destruction in the group that received Ag-Ag2S@BSA-FA NPs and exposed to X-ray. The results showed that prepared tumor-targeted Ag-Ag2S@BSA-FA NPs could be potential candidates as radiosensitizers for enhanced radiotherapy