Single-Particle Plasmon Sensor to Monitor Proteolytic Activity in Real Time.

We have established a label-free plasmonic platform that monitors proteolytic activity in real time. The sensor consists of a random array of gold nanorods that are functionalized with a design peptide that is specifically cleaved by thrombin, resulting in a blueshift of the longitudinal plasmon. By monitoring the plasmon of many individual nanorods, we determined thrombin's proteolytic activity in real time and inferred relevant kinetic parameters. Furthermore, a comparison to a kinetic model revealed that the plasmon shift is dictated by a competition between peptide cleavage and thrombin binding, which have opposing effects on the measured plasmon shift. The dynamic range of the sensor is greater than two orders of magnitude, and it is capable of detecting physiologically relevant levels of active thrombin down to 3 nM in buffered conditions. We expect these plasmon-mediated label-free sensors to open the window to a range of applications stretching from the diagnostic and characterization of bleeding disorders to fundamental proteolytic and pharmacological studies.

Maximizing mRNA vaccine production with Bayesian optimization.

Messenger RNA (mRNA) vaccines are a new alternative to conventional vaccines with a prominent role in infectious disease control. These vaccines are produced in in vitro transcription (IVT) reactions, catalyzed by RNA polymerase in cascade reactions. To ensure an efficient and cost-effective manufacturing process, essential for a large-scale production and effective vaccine supply chain, the IVT reaction needs to be optimized. IVT is a complex reaction that contains a large number of variables that can affect its outcome. Traditional optimization methods rely on classic Design of Experiments methods, which are time-consuming and can present human bias or based on simplified assumptions. In this contribution, we propose the use of Machine Learning approaches to perform a data-driven optimization of an mRNA IVT reaction. A Bayesian optimization method and model interpretability techniques were used to automate experiment design, providing a feedback loop. IVT reaction conditions were found under 60 optimization runs that produced 12 g · L-1 in solely 2 h. The results obtained outperform published industry standards and data reported in literature in terms of both achievable reaction yield and reduction of production time. Furthermore, this shows the potential of Bayesian optimization as a cost-effective optimization tool within (bio)chemical applications.

mRNA vaccines manufacturing: Challenges and bottlenecks.

Vaccines are one of the most important tools in public health and play an important role in infectious diseases control. Owing to its precision, safe profile and flexible manufacturing, mRNA vaccines are reaching the stoplight as a new alternative to conventional vaccines. In fact, mRNA vaccines were the technology of choice for many companies to combat the Covid-19 pandemic, and it was the first technology to be approved in both United States and in Europe Union as a prophylactic treatment. Additionally, mRNA vaccines are being studied in the clinic to treat a number of diseases including cancer, HIV, influenza and even genetic disorders. The increased demand for mRNA vaccines requires a technology platform and cost-effective manufacturing process with a well-defined product characterisation. Large scale production of mRNA vaccines consists in a 1 or 2-step in vitro reaction followed by a purification platform with multiple steps that can include Dnase digestion, precipitation, chromatography or tangential flow filtration. In this review we describe the current state-of-art of mRNA vaccines, focusing on the challenges and bottlenecks of manufacturing that need to be addressed to turn this new vaccination technology into an effective, fast and cost-effective response to emerging health crises.

Monitoring Proteolytic Activity in Real Time: A New World of Opportunities for Biosensors.

Proteases play a pivotal role in several biological processes, from digestion, cell proliferation, and differentiation to fertility. Deregulation of protease metabolism can result in several pathological conditions (i.e., cancer, neurodegenerative disorders, and others). Therefore, monitoring proteolytic activity in real time could have a fundamental role in the early diagnosis of these diseases. Herein, the main approaches used to develop biosensors for monitoring proteolytic activity are reviewed. A comparison of the advantages and disadvantages of each approach is provided along with a discussion of their importance and promising opportunities for the early diagnosis of severe diseases. This new era of biosensors can be characterized by the ability to control and monitor biological processes, ultimately improving the potential of personalized medicine.

Fluorescent dye nano-assemblies by thiol attachment directed to the tips of gold nanorods for effective emission enhancement.

The conjugation of dye-labelled DNA oligonucleotides with gold nanorods has been widely explored for the development of multifunctional fluorescent nanoprobes. Here, we show that the functionalization route is crucial to achieve enhanced emission in dye nano-assemblies based on gold nanorods. By using a tip-selective approach for thiol attachment of dye molecules onto gold nanorods, it was possible to effectively increase the emission by more than 10-fold relatively to that of a free dye. On the other hand, a non-selective approach revealed that indiscriminate surface functionalization has a detrimental effect on the enhancement. Simulations of discrete dipole approximation gave further insight into the surface distribution of plasmon-enhanced emission by confirming that tip regions afford an effective enhancement, while side regions exhibit a negligible effect or even emission quenching. The contrast between dye nano-assemblies obtained from tip- and non-selective functionalization was further characterized by single-particle fluorescence emission. These studies showed that tip-functionalized gold nanorods with an average of only 30 dye molecules have a comparable to or even stronger emission than non-selectively functionalized particles with approximately 10 times more dye molecules. The results herein reported could significantly improve the performance of dye nano-assemblies for imaging or sensing applications.

Ionic Liquid-Polymer Nanoparticle Hybrid Systems as New Tools to Deliver Poorly Soluble Drugs.

The use of functional excipients such as ionic liquids (ILs) and the encapsulation of drugs into nanocarriers are useful strategies to overcome poor drug solubility. The aim of this work was to evaluate the potential of IL-polymer nanoparticle hybrid systems as tools to deliver poorly soluble drugs. These systems were obtained using a methodology previously developed by our group and improved herein to produce IL-polymer nanoparticle hybrid systems. Two different choline-based ILs and poly (lactic-co-glycolic acid) (PLGA) 50:50 or PLGA 75:25 were used to load rutin into the delivery system. The resulting rutin-loaded IL-polymer nanoparticle hybrid systems presented a diameter of 250-300 nm, with a low polydispersity index and a zeta potential of about -40 mV. The drug association efficiency ranged from 51% to 76%, which represents a good achievement considering the poor solubility of rutin. No significant particle aggregation was obtained upon freeze-drying. The presence of the IL in the nanosystem does not affect its sustained release properties, achieving about 85% of rutin released after 72 h. The cytotoxicity studies showed that the delivery system was not toxic to HaCat cells. Our findings may open a new paradigm on the therapy improvement of diseases treated with poorly soluble drugs.

Density Gradient Selection of Colloidal Silver Nanotriangles for Assembling Dye-Particle Plasmophores.

A simple method based on sucrose density gradient centrifugation is proposed here for the fractionation of colloidal silver nanotriangles. This method afforded particle fractions with surface plasmon resonances, spanning from red to infrared spectral ranges that could be used to tune optical properties for plasmonic applications. This feature was exemplified by selecting silver nanotriangle samples with spectral overlap with Atto-655 dye's absorption and emission in order to assemble dye-particle plasmophores. The emission brightness of an individual plasmophore, as characterized by fluorescence correlation spectroscopy, is at least 1000-fold more intense than that of a single Atto-655 dye label, which renders them as promising platforms for the development of fluorescence-based nanosensors.

Colorimetric Detection of DNA Strands on Cellulose Microparticles Using ZZ-CBM Fusions and Gold Nanoparticles.

Nucleic acid testing requires skilled personnel and expensive instrumentation. A method for the colorimetric detection of oligonucleotides that combines cellulose microparticles with biomolecular recognition is presented. DNA sequences from Trypanosoma brucei and dengue are used as model targets. Cellulose microparticles (≈20 µm) are bioactived by anchoring anti-biotin antibodies via fusions that combine a carbohydrate-binding module (CBM) with the ZZ fragment of protein A. Samples are prepared by incubating DNA probes immobilized on ≈14 nm gold nanoparticles (AuNPs) with biotin-labeled targets and mixed with bioactive microparticles. The presence of unlabeled targets could also be probed by introducing a second, biotinylated DNA probe. The target:probe-AuNP hybrids are mixed with and captured by the microparticles, which change color from white to red. Depletion of AuNPs from the liquid is also signaled by a decrease in absorbance at 525 nm. It was possible to detect targets with concentrations as low as 50 n m. In the presence of noncomplementary targets, microparticles remain white and the liquid remains red. The system is able to discriminate targets with a high degree of homology (≈53%). Overall, it is demonstrated that simple systems for the visual detection of nucleic acids can be set up by combining cellulose microparticles with biomolecular recognition agents based on CBMs and AuNPs.

Plasmid Copy Number of pTRKH3 in Lactococcus lactis is Increased by Modification of the repDE Ribosome-Binding Site.

Plasmids for DNA vaccination are exclusively produced in the Gram-negative Escherichia coli. One important drawback of this system is the presence of lipopolysaccharides. The generally recognized as safe Lactococcus lactis (L. lactis) would constitute a safer alternative for plasmid production. A key requirement for the establishment of a cost-effective L. lactis-based plasmid manufacturing is the availability of high-copy number plasmids. Unfortunately, the highest copy number reported in Gram-positive bacteria for the pAMß1 replicon is around 100 copies. The purpose of this work is to engineer the repDE ribosome-binding site (RBS) of the pTRKH3 plasmid by site-directed mutagenesis in order to increase the plasmid copy number in L. lactis LMG19460 cells. The pTRKH3-b mutant is the most promising candidate, achieving 215 copies of plasmid per chromosome, a 3.5-fold increase when compared to the nonmodified pTRKH3, probably due to a stronger RBS sequence, a messenger RNA secondary structure that promotes the RepDE expression, an ideal intermediate amount of transcriptional repressors and the presence of a duplicated region that added an additional RBS sequence and one new in-frame start codon. pTRKH3-b is a promising high-copy number shuttle plasmid that will contribute to turn lactic acid bacteria into a safer and economically viable alternative as DNA vaccines producers.

Extreme Enhancement of Single-Molecule Fluorescence from Porphyrins Induced by Gold Nanodimer Antennas.

Porphyrins are typically weak emitters, which presents challenges to their optical detection by single-molecule fluorescence microscopy. In this contribution, we explore the enhancement effect of gold nanodimer antennas on the fluorescence of porphyrins in order to enable their single-molecule optical detection. Four meso-substituted free-base porphyrins were evaluated: two cationic, one neutral, and one anionic porphyrin. The gold nanodimer antennas are able to enhance the emission from these porphyrins by a factor of 105-106 increase in the maximum detected photon rates. This extreme enhancement is due to the combination of an antenna effect on the excitation rate that is estimated to be above 104-fold and an emission efficiency that corresponds to an increase of 2-10 times in the porphyrin's fluorescence quantum yield.

Colorimetric detection of D-dimer in a paper-based immunodetection device.

A microfluidic paper-based analytical device (µPADs) immunoassay for detection of the blood native biomarker D-dimer is reported. The µPAD is created by wax printing on a single piece of chromatographic paper and combined with an anti-D-dimer capture antibody and conjugates of anti-D-dimer antibody with 40 nm gold nanoparticles. The presence of D-dimer in buffer/simulated plasma samples is successfully reported for concentrations as low as 15 ng D-dimer/mL. Linearity between signal intensity and D-dimer concentration is observed up to 100 ng/mL. Using an appropriate dilution, the test could be used to yield positive results only for those samples with a D-dimer concentration above the clinically relevant threshold range of 250-500 ng/mL. Finally, the merits and pitfalls of using µPADs as compared to lateral flow devices in immunoassays are discussed.

Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase.

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5'-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6-14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48-817-fold) translation initiation rate. No effect of the 5'-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5'-UTR. Interestingly, the amount of this transcript was 2.6-3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5'-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5'-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5'-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.

Towards effective non-viral gene delivery vector.

Despite very good safety records, clinical trials using plasmid DNA failed due to low transfection efficiency and brief transgene expression. Although this failure is both due to poor plasmid design and to inefficient delivery methods, here we will focus on the former. The DNA elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats showed detrimental effects on plasmids' performance as biopharmaceuticals. On the other hand, careful selection of promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences for therapeutic protein expression can enhance the clinical efficacy. Minimal vectors, which are devoid of the bacterial backbone and consist exclusively of the eukaryotic expression cassette, demonstrate better performance in terms of expression levels, bioavailability, transfection rates and increased therapeutic effects. Although the results are promising, minimal vectors have not taken over the conventional plasmids in clinical trials due to challenging manufacturing issues.

Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing.

The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼141 mg/L) than MG1655ΔendAΔrecA (∼48 mg/L) and DH5α (∼40 mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200 mg/L.

Evidence that the insertion events of IS2 transposition are biased towards abrupt compositional shifts in target DNA and modulated by a diverse set of culture parameters.

Insertion specificity of mobile genetic elements is a rather complex aspect of DNA transposition, which, despite much progress towards its elucidation, still remains incompletely understood. We report here the results of a meta-analysis of IS2 target sites from genomic, phage, and plasmid DNA and find that newly acquired IS2 elements are consistently inserted around abrupt DNA compositional shifts, particularly in the form of switch sites of GC skew. The results presented in this study not only corroborate our previous observations that both the insertion sequence (IS) minicircle junction and target region adopt intrinsically bent conformations in IS2, but most interestingly, extend this requirement to other families of IS elements. Using this information, we were able to pinpoint regions with high propensity for transposition and to predict and detect, de novo, a novel IS2 insertion event in the 3' region of the gfp gene of a reporter plasmid. We also found that during amplification of this plasmid, process parameters such as scale, culture growth phase, and medium composition exacerbate IS2 transposition, leading to contamination levels with potentially detrimental clinical effects. Overall, our findings provide new insights into the role of target DNA structure in the mechanism of transposition of IS elements and extend our understanding of how culture conditions are a relevant factor in the induction of genetic instability.

On the dual effect of glucose during production of pBAD/AraC-based minicircles.

Minicircles are promising vectors for DNA vaccination, gene or cell therapies due to their increased transfection efficacy and transgene expression. The in vivo production of these novel vectors involves the arabinose inducible excision of a parental molecule into a minicircircle and a miniplasmid bacterial backbone. Tight control of recombination is crucial to maximize minicircle yields and purity. In this work, a minicircle production system was constructed that relies on the enzymatic activity of ParA resolvase, a recombinase that is expressed under the transcription control of the arabinose inducible expression system pBAD/AraC, and on Escherichia coli BWAA, a strain improved for arabinose uptake. Undesired recombination already after 4h of incubation in Luria-Bertani broth at 37 °C was observed due to the leaky expression from pBAD/AraC. While addition of glucose to the growth media repressed this leaky expression, it triggered a pH drop to 4.5 during exponential phase in shake flasks, which suppressed growth and plasmid production. The quantitative PCR analysis confirmed only few copies of high-copy number plasmid inside of the E. coli cells. To ensure the stability of minicircle-producing system, seed cultures should be grown at 30 °C with glucose overnight whereas cells for minicircle production should be grown in shake flasks at 37 °C without glucose up to early stationary phase when the recombination is induced by addition of arabinose.

Engineering of Escherichia coli strains for plasmid biopharmaceutical production: scale-up challenges.

Plasmid-based vaccines and therapeutics have been making their way into the clinic in the last years. The existence of cost-effective manufacturing processes capable of delivering high amounts of high-quality plasmid DNA (pDNA) is essential to generate enough material for trials and support future commercialization. However, the development of pDNA manufacturing processes is often hampered by difficulties in predicting process scale performance of Escherichia coli cultivation on the basis of results obtained at lab scale. This paper reports on the differences observed in pDNA production when using shake flask and bench-scale bioreactor cultivation of E. coli strains MG1655ΔendAΔrecA and DH5α in complex media with 20 g/L of glucose. MG1655ΔendAΔrecA produced 5-fold more pDNA (9.8 mg/g DCW) in bioreactor than in shake flask (1.9 mg/g DCW) and DH5α produced 4-fold more pDNA (8 mg/g DCW) in bioreactor than in shake flask (2 mg/g DCW). Accumulation of acetate was also significant in shake flasks but not in bioreactors, a fact that was attributed to a lack of control of pH.

Metabolic viability of Escherichia coli trapped by dielectrophoresis in microfluidics.

The spatial and temporal control of biological species is essential in complex microfluidic biosystems. In addition, if the biological species is a cell, microfluidic handling must ensure that the cell's metabolic viability is maintained. The use of DEP for cell manipulation in microfluidics has many advantages because it is remote and fast, and the voltages required for cell trapping scale well with miniaturization. In this paper, the conditions for bacterial cell (Escherichia coli) trapping using a quadrupole electrode configuration in a PDMS microfluidic channel were developed both for stagnant and for in-flow fluidic situations. The effect of the electrical conductivity of the fluid, the applied electric field and frequency, and the fluid-flow velocity were studied. A dynamic exchange between captured and free-flowing cells during DEP trapping was demonstrated. The metabolic activity of trapped cells was confirmed by using E. coli cells genetically engineered to express green fluorescent protein under the control of an inducible promoter. Noninduced cells trapped by negative DEP and positive DEP were able to express green fluorescent protein minutes after the inducer was inserted in the microchannel system immediately after DEP trapping. Longer times of trapping prior to exposure to the inducer indicated first a degradation of the cell metabolic activity and finally cell death.

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production.

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.

Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing.

Plasmid DNA (pDNA) has become very attractive as a biopharmaceutical, especially for gene therapy and DNA vaccination. Currently, there are a few products licensed for veterinary applications and numerous plasmids in clinical trials for use in humans. Recent work in both academia and industry demonstrates a need for technological and economical improvement in pDNA manufacturing. Significant progress has been achieved in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on engineering of Escherichia coli strains for plasmid DNA production, understanding the differences between the traditional use of pDNA for recombinant protein production and its role as a biopharmaceutical. We will present recent developments in engineering of E. coli strains, highlight essential genes for improvement of pDNA yield and quality, and analyze the impact of various process strategies on gene expression in pDNA production strains.