Effect of Plasmid DNA Size on Chitosan or Polyethyleneimine Polyplexes Formulation.

Gene therapy could be simply defined as a strategy for the introduction of a functional copy of desired genes in patients, to correct some specific mutation and potentially treat the respective disorder. However, this straightforward definition hides very complex processes related to the design and preparation of the therapeutic genes, as well as the development of suitable gene delivery systems. Within non-viral vectors, polymeric nanocarriers have offered an ideal platform to be applied as gene delivery systems. Concerning this, the main goal of the study was to do a systematic evaluation on the formulation of pDNA delivery systems based on the complexation of different sized plasmids with chitosan (CH) or polyethyleneimine (PEI) polymers to search for the best option regarding encapsulation efficiency, surface charge, size, and delivery ability. The cytotoxicity and the transfection efficiency of these systems were accessed and, for the best p53 encoding pDNA nanosystems, the ability to promote protein expression was also evaluated. Overall, it was showed that CH polyplexes are more efficient on transfection when compared with the PEI polyplexes, resulting in higher P53 protein expression. Cells transfected with CH/p53-pDNA polyplexes presented an increase of around 54.2% on P53 expression, while the transfection with the PEI/p53-pDNA polyplexes resulted in a 32% increase.

Non-coding RNAs: Emerging from the discovery to therapeutic applications.

The knowledge about non-coding RNAs (ncRNAs) is rapidly increasing with new data continuously emerging, regarding their diverse types, applications, and roles. Particular attention has been given to ncRNA with regulatory functions, which may have a critical role both in biological and pathological conditions. As a result of the diversity of ncRNAs and their ubiquitous involvement in several biologic processes, ncRNA started to be considered in the biomedical field, with immense potential to be exploited either as biomarkers or as therapeutic agents in certain pathologies. Indeed, ncRNA-based therapeutics have been proposed in many disorders and some even reached clinical trials. However, to prepare an RNA product suitable for pharmacological applications, certain criteria must be fulfilled, and it has to be guaranteed RNA purity, stability, and bioactivity. So, in this review, the different types of ncRNAs are identified and characterized, by describing their biogenesis, functions, and applications. A perspective on the main challenges and innovative approaches for the future and broad therapeutic application of RNA is also presented.

Applications of gellan natural polymer microspheres in recombinant catechol-O-methyltransferase direct capture from a Komagataella pastoris lysate.

The present work shows the application of nickel- and magnesium-crosslinked gellan microspheres in ionic and affinity capture strategies to directly extract hSCOMT from the complex Komagataella pastoris lysate through a simple batch method. Both formulations present similar morphology, but nickel-crosslinked microspheres present higher crosslinker content and smaller diameters. Four different capture strategies were established, by manipulating the ionic strength, pH, temperature and competing agents' presence. The most promising results for hSCOMT capture and clarification were obtained employing an ionic strategy with nickel-crosslinked microspheres and an affinity strategy with magnesium-crosslinked microspheres at 4 °C. The bioactivity results (200%) and purification degree (70%) of hSCOMT captured by the ionic strategy were more satisfactory probably due to the soft ionic conditions used (100 mM NaCl). For the first time, the gellan polysaccharide versatility was demonstrated in the microsphere application for the direct capture of hSCOMT from a complex lysate, simplifying isolation biotechnological procedures.

Dilemma on plasmid DNA purification: binding capacity vs selectivity.

Plasmid DNA chromatography is a powerful field in constant development and evolution. The use of this technique is considered mandatory in the production of an efficient and safe formulation to be applied for plasmid-mediated gene therapy. Concerning this, the search for an ideal chromatographic support/ligand combination motivated scientist to pursue a continuous improvement on the plasmid chromatography performance, looking for a progression on the ligands and supports used. The present review explores the different approaches used over time to purify plasmid DNA, ambitioning both high recovery and high purity levels. Overall, it is presented a critical discussion relying on the relevance of the binding capacity versus selectivity of the supports.

Improved ionic-liquid-functionalized macroporous supports able to purify nucleic acids in one step.

Nucleic acids are relevant biopolymers in therapy and diagnosis, for which their purity and biological activity are of crucial relevance. However, these features are difficult to achieve by cost-effective methods. Herein, we report the functionalization of a macroporous chromatographic support functionalized with an ionic liquid (IL) with remarkable performance to purify nucleic acids. An initial screening with distinct IL chemical structures supported in silica was carried out, allowing to identify the IL 1-methyl-3-propylimidazolium chloride as the most promising ligand. A chromatographic macroporous matrix able to be used in preparative liquid chromatography was then functionalized and binding/elution studies were performed. The IL 1-methyl-3-propylimidazolium chloride acts as a multimodal ligand with a remarkable dynamic binding capacity. This macroporous support allows the (one-step) purification of nucleic acids, namely small RNAs, ribosomal RNA, and genomic DNA, from a bacterial lysate, and can be regenerated and reused without compromising its separation performance.

Purification of supercoiled p53-encoding plasmid using an arginine-modified macroporous support.

p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alternative to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA purification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macroporous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic feasibility in case of scale-up.

The use of size-exclusion chromatography in the isolation of supercoiled minicircle DNA from Escherichia coli lysate.

Minicircle DNA (mcDNA) is the new cutting-edge technology which researchers have been exploring for gene therapy and DNA vaccination. Although it presents enormous advantages in comparison to conventional plasmid DNA regarding bioactivity and safety, its challenging isolation from parental plasmid and miniplasmid has been setting back its launching in biomedical sciences. In this work, it is demonstrated the use of a simple size exclusion chromatographic method for the isolation of supercoiled mcDNA. Sephacryl S-1000 SF matrix was explored under different conditions (flow, peak fractionation volume and sample loading) to achieve the best performance and retrieve a mcDNA sample devoid of other bacterial contaminants or plasmid species resultant from the recombination process. This isolation methodology resulted in 66.7% of mcDNA recovery with 98.1% of purity. In addition, to show the robustness of the method, the potential of using this matrix for the isolation of a larger mcDNA was also evaluated. Upon adjusting the flow or the column volume, the larger mcDNA molecule was also successfully isolated. Overall, a simple and effective strategy has been established for the isolation of supercoiled mcDNA, underlining the potential of size exclusion chromatography in mcDNA separation.

Quality assessment of supercoiled minicircle DNA by cadaverine-modified analytical chromatographic monolith.

Minicircle DNA (mcDNA) is the ultimate non-viral DNA vector, presenting higher biosafety and therapeutic effect than conventional plasmid DNA (pDNA). However, given the similarity between mcDNA and its precursor, the parental plasmid (PP), analytical methodologies established for pDNA are unable to distinguish mcDNA from PP. Thus, a new need emerged for the implementation of suitable, rapid and non-expensive analytical methodologies for the characterization of mcDNA samples. Recently, our research group was able to develop a purification strategy for the isolation of supercoiled (sc) mcDNA resorting to cadaverine-modified monolith. Considering the promising results obtained with this strategy, a cadaverine-modified analytical monolith was prepared and explored for mcDNA quantification. Thus, a strategy of three-step increasing NaCl gradient was considered to first elute RNA/protein content, then isolate sc mcDNA and finally eliminate PP and other impurities still bounded to the matrix. A calibration curve was constructed with different sc mcDNA standards within a range of 1-25 µg/mL. Linearity, accuracy, precision and selectivity of this method were validated according to the international guidelines and the limit of detection and the lower limit of quantification were determined as 1 µg/mL. For the first time, to the best of our knowledge, an analytical method for mcDNA quantification is described. Besides ensuring the safety of mcDNA application by assessing the product purity, such methodology can be used in the future to control industrial mcDNA production and purification, perhaps aiding in the establishment of optimized and less expensive biotechnological operations.

Cancer gene therapy mediated by RALA/plasmid DNA vectors: Nitrogen to phosphate groups ratio (N/P) as a tool for tunable transfection efficiency and apoptosis.

Cancer gene therapy based on p53 tumor suppressor gene supplementation emerges as one of the most challenging and promising strategies. The development of a suitable gene delivery system is imperative to ensure the feasibility and viability of cancer gene therapy in a clinical setting. The conception of delivery systems based on cell- penetrating peptides may deeply contribute for the evolution of therapy efficacy. In this context, the present work explores the p53 encoding plasmid DNA (pDNA) condensation ability of RALA peptide to produce a suitable intracellular delivery platform. These carriers, formed at several nitrogen to phosphate groups (N/P) ratio, were characterized in terms of morphology, size, surface charges, loading and complexation capacity and the fine structure has been analyzed by Fourier-transformed infrared (FTIR) spectroscopy. Confocal microscopy studies confirmed intracellular localization of nanoparticles, resulting in enhanced sustained pDNA uptake. Moreover, in vitro transfection of HeLa cells mediated by RALA/pDNA vectors allows for gene release and p53 protein expression. From these progresses, apoptosis in cancer cells has been investigated. It was found that N/P ratio strongly tailors gene transfection efficiency and, thus, it can be fine-tuned for desired degree of both protein expression and apoptosis. The great asset of the proposed system relies precisely on the use of N/P ratio as a tailoring parameter that can not only modulate vector´s properties but also the extent of pDNA delivery, protein expression and, consequently, the efficacy of p53 mediated cancer therapy.

Minicircle DNA purification: Performance of chromatographic monoliths bearing lysine and cadaverine ligands.

Minicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy, since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However, the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid purification. In this work, two monolithic supports were modified with lysine and its decarboxylated derivative, cadaverine, to explore their performance in the sc mcDNA purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general, cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith, at acidic pH. For instance, at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59, respectively, while for cadaverine modified monolith was 20.32 and 27.12, respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However, recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith, achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample, obtained by the column that showed the best performance, was characterized by real-time PCR, presenting 98.4% purity and 78.6% recovery yield. The impurities content, namely genomic DNA, proteins and endotoxins, was found within the criteria established by regulatory agencies. Overall, a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column, with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.

DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatography.

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ±â€¯0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.

Bacteriocin production of the probiotic Lactobacillus acidophilus KS400.

In the last years, the use of probiotics, including Lactobacillus species, has received much attention to prevent and treat vaginal disorders. These species have been described as having the ability to colonize the epithelial surface and produce antimicrobial metabolites that are able to control the remaining vaginal microflora. This study aimed to identify and characterize, for the first time, a bacteriocin natively produced by Lactobacillus acidophilus KS400 (probiotic strain from Gynoflor®-Medinova AG, Switzerland) and its antimicrobial activity against relevant urogenital pathogens. After organic acids and hydrogen peroxide neutralization in the fermented Lactobacillus acidophilus KS400 culture medium, bacteriocin activity was tested against the indicator microorganism Lactobacillus delbrueckii ATCC9649. The fermentation of Lactobacillus acidophilus KS400 for bacteriocin production was carried out in batch mode, and its antimicrobial activity, optical density and pH were monitored. After production and extraction, the bacteriocin molecular weight was estimated by electrophoresis and tested against vaginal pathogenic microorganisms. As described for other bacteriocins, batch fermentation profiles indicated that bacteriocin production occurs during the exponential growth phase of the lactobacilli, and declines during their stationary growth phase. The molecular weight of the bacteriocin is approximately 7.5 kDa. The bacteriocin containing protein extract was shown to inhibit the growth of Gardnerella vaginalis, Streptococcus agalactiae, Pseudomonas aeruginosa and the indicator strain Lactobacillus delbrueckii ATCC9649. We conclude that L. acidophilus KS400 produces bacteriocin with antimicrobial activity against relevant urogenital pathogens.

L-tryptophan and dipeptide derivatives for supercoiled plasmid DNA purification.

The present study focus on the preparation of chromatography supports for affinity-based chromatography of supercoiled plasmid purification. Three l-tryptophan based supports are prepared through immobilization on epoxy-activated Sepharose and characterized by HR-MAS NMR. The SPR is employed for a fast screening of l-tryptophan derivatives, as potential ligands for the biorecognition of supercoiled isoform, as well as, to establish the suitable experimental conditions for the chromatography. The results reveal that the overall affinity is high (KD=10(-9) and 10(-8)M) and the conditions tested show that the use of HEPES 100mM enables the separation and purification of supercoiled at T=10°C. The STD-NMR is performed to accomplish the epitope mapping of the 5'-mononucleotides bound to l-tryptophan derivatives supports. The data shows that the interactions between the three supports and the 5'-mononucleotides are mainly hydrophobic and π-π stacking. The chromatography experiments are performed with l-tryptophan support and plasmids pVAX-LacZ and pPH600. The supercoiled isoform separation is achieved at T=10°C by decreasing the concentration of (NH4)2SO4 from 2.7 to 0M in HEPES for pVAX-LacZ and 2.65M to 0M in HEPES for pPH600. Overall, l-tryptophan derivatives can be a promising strategy to purify supercoiled for pharmaceutical applications.

Purification of influenza deoxyribonucleic acid-based vaccine using agmatine monolith.

Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation.

Preparative isolation of polymerase chain reaction products using mixed-mode chromatography.

The polymerase chain reaction (PCR) has become one of the most useful techniques in molecular biology laboratories around the world. The purification of the target DNA product is often challenging, however, and most users are restricted to employing available commercial kits. The recent developments in mixed-mode chromatography have shown higher selectivity for a variety of nucleic acid-containing samples. Capto Adhere is a mixed-mode chromatography resin that offers a high-selectivity ligand and is here applied for the purification of amplified DNAs from PCR mixtures in a 10-min single step, with yields above 95%, high linearity, and high precision for different concentrations.

Incorporation of antimicrobial peptides on functionalized cotton gauzes for medical applications.

A large group of low molecular weight natural compounds that exhibit antimicrobial activity has been isolated from animals and plants during the past two decades. Among them, peptides are the most widespread resulting in a new generation of antimicrobial agents with higher specific activity. In the present study we have developed a new strategy to obtain antimicrobial wound-dressings based on the incorporation of antimicrobial peptides into polyelectrolyte multilayer films built by the alternate deposition of polycation (chitosan) and polyanion (alginic acid sodium salt) over cotton gauzes. Energy dispersive X ray microanalysis technique was used to determine if antimicrobial peptides penetrated within the films. FTIR analysis was performed to assess the chemical linkages, and antimicrobial assays were performed with two strains: Staphylococcus aureus (Gram-positive bacterium) and Klebsiella pneumonia (Gram-negative bacterium). Results showed that all antimicrobial peptides used in this work have provided a higher antimicrobial effect (in the range of 4 log-6 log reduction) for both microorganisms, in comparison with the controls, and are non-cytotoxic to normal human dermal fibroblasts at the concentrations tested.

Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.

Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

Performance of an anthropometric assessment method as a predictor of low birthweight and being small for gestational age.

BACKGROUND: The present study aimed to evaluate the performance of the method proposed in 2009 by the Institute of Medicine for the anthropometric assessment of pregnant women, predicting perinatal outcomes: adequacy of birthweight, adequacy of birthweight according to gestational age [small for gestational age (SGA), large for gestational age (LGA)] and gestational and neonatal complications. METHODS: The study comprised a cross-sectional study involving 827 post-partum women (>20 years) who were treated in a public maternity hospital in the city of Rio de Janeiro. Data collection occurred by interviews and record consultation. Adequacy of weight gain during pregnancy was determined as being insufficient and excessive for the recommended range according to nutritional status category. Odds ratios (OR) and 95% confidence intervals (CI) were estimated for logistic regression. RESULTS: Some 36.2% of women initiated gestation with some weight deviation. The prevalence of the outcomes studied was 4.6% (n = 35) for low birthweight, 3.7% (n = 31) for SGA, 5.7% (n = 47) for LGA, 45.2% (n = 374) for pregnancy complications and 15.2% (n = 85) for neonatal complications. For women with insufficient weight gain, the new recommendation indicated a correlation in the prediction of low birthweight (OR = 3.76, 95% CI = 1.53-9.21), SGA newborns (OR = 5.77, 95% CI = 2.10-15.8) and gestational complications (OR = 1.72, 95% CI = 1.20-2.48). CONCLUSIONS: The method evaluated demonstrated a better sensitivity and specificity for the main outcomes related to insufficient weight gain (low birthweight and SGA). Regarding excessive foetal growth (LGA), gestational and neonatal complications demonstrated sensitivity for an intermediate value.

Histamine monolith versatility to purify supercoiled plasmid deoxyribonucleic acid from Escherichia coli lysate.

Preparation of high quantities of supercoiled plasmid DNA of pharmaceutical grade purity is a research area where intensive investigation is being performed. From this standpoint, several downstream methods have been proposed, among them the monolithic chromatographic strategies owing to excellent mass transfer properties of monolithic supports and their high binding capacity for large biomolecules. The present study explores the physicochemical properties of histamine ligand in a supercoiled plasmid DNA purification process from an Escherichia coli clarified lysate, where the emphasis is given to the elution strategy that allows higher selectivity and efficient removal of other impurities besides the open circular isoform. The combination of high NaCl concentration and acidic pH allowed the elimination of 89% of RNA during the preparative loading of the lysate sample. The results of the purification strategy with ascending sodium chloride gradient revealed that 97% of supercoiled plasmid DNA was recovered with a purity degree of 99%. In addition, using a combined purification strategy with ascending sodium chloride (capture step) and then descending ammonium sulfate (polishing step) gradient, it was achieved a lower supercoiled plasmid DNA recovery yield of 79% with a purity degree of 92%, although the dynamic binding capacity under these conditions was higher than in the previous strategy. A significant reduction of host contents, such as proteins, RNA and genomic DNA, was obtained in both purification strategies. Accordingly, histamine is a useful and versatile ligand that allows the desirable supercoiled plasmid purification with high yield and purity level.