Ultrafast and Controlled Capturing, Loading, and Release of Extracellular Vesicles by a Portable Microstructured Electrochemical Fluidic Device.

Extracellular vesicles (EVs) are secreted by all living cells and are found in body fluids. They exert numerous physiological and pathological functions and serve as cargo shuttles. Due to their safety and inherent bioactivity, they have emerged as versatile therapeutic agents, biomarkers, and potential drug carriers. Despite the growing interest in EVs, current progress in this field is, in part, limited by relatively inefficient isolation techniques. Conventional methods are indeed slow, laborious, require specialized laboratory equipment, and may result in low yield and purity. This work describes an electrochemically controlled "all-in-one" device enabling capturing, loading, and releasing of EVs. The device is composed of a fluidic channel confined within antibody-coated microstructured electrodes. It rapidly isolates EVs with a high level of purity from various biofluids. As a proof of principle, the device is applied to isolate EVs from skin wounds of healthy and diabetic mice. Strikingly, it is found that EVs from healing wounds of diabetic mice are enriched in mitochondrial proteins compared to those of healthy mice. Additionally, the device improves the loading protocol of EVs with polyplexes, and may therefore find applications in nucleic acid delivery. Overall, the electrochemical device can greatly facilitate the development of EVs-based technologies.

Improving extracellular vesicles production through a Bayesian optimization-based experimental design.

With the growing demand and diversity of biological drugs, developing optimal processes for their accelerated production with minimal resource utilization is a pressing challenge. Typically, such optimization involves multiple target properties, such as production yield, biological activity, and product purity. Therefore, strategic experimental design techniques that can characterize the parameter space while simultaneously arriving at the optimal process satisfying multiple target properties are required. To achieve this, we propose the use of a multi-objective batch Bayesian optimization (MOBBO) algorithm and illustrate its successful application for the production of extracellular vesicles (EVs) from a 3D culture of mesenchymal stem cells (MSCs) considering three objectives, namely to maximize the vesicle-to-protein ratio, maximize the enzymatic activity of the MSC-EV protein CD73, and minimize the amount of calregulin impurities. We show that the optimal combination of the process parameters to address the intended objectives could be achieved with only 32 experiments. For the four parameters considered (i.e., microcarrier concentration, seeding density, centrifugation time, and impeller speed), this number of experiments is comparable to or lower than the classical design of experiments (DoE) and the traditional one-factor-at-a-time (OFAT) approach. We illustrate how the algorithm adaptively samples in the process parameter space, selectively excluding unfavorable regions, thus minimizing the number of experiments required to reach optimal conditions. Finally, we compare the obtained solutions to the literature data and present possible applications of the collected data for other modeling activities such as Quality by Design, process monitoring, control, and scale-up.

Encapsulation of Hydrophilic Compounds in Small Extracellular Vesicles: Loading Capacity and Impact on Vesicle Functions.

Their natural functions in intercellular communication render extracellular vesicles (EV) highly attractive for drug delivery applications. However, the loading efficiency of present methods to incorporate particularly hydrophilic low molecular weight drugs of biomedical interest is largely unexplored, as is the impact these methods may have on the intrinsic structural and biological vesicle properties. Here, different methods are exploited to incorporate hydrophilic non-membrane permeable compounds into stem cell-derived small EV, and to assess the vesicle characteristics after the different loading processes. When comparing several methods head-to-head, the loading capacity increases in the order saponin ≤ sonication < fusion < freeze-thawing ≤ osmotic shock. Interestingly, the structural and biological functions of small EV are dependent on the applied encapsulation process, with the functional properties being altered at a greater extent. Therefore, the importance of including additional characterization parameters to probe alterations of the biological functionality of small EV is clearly demonstrated. Here, freeze-thawing and particularly the osmotic shock have proven to be the most appropriate methods for EV loading, as they achieve a high drug encapsulation and yet preserve the investigated structural and biological vesicle characteristics.

Stromal Modulation and Treatment of Metastatic Pancreatic Cancer with Local Intraperitoneal Triple miRNA/siRNA Nanotherapy.

Nanomedicines achieve tumor-targeted delivery mainly through enhanced permeability and retention (EPR) effect following intravenous (IV) administration. Unfortunately, the EPR effect is severely compromised in pancreatic cancer due to hypovascularity and dense desmoplastic stroma. Intraperitoneal (IP) administration may be an effective EPR-independent local delivery approach to target peritoneal tumors. Besides improved delivery, effective combination delivery strategies are needed to improve pancreatic cancer therapy by targeting both cancer cells and cellular interactions within the tumor stroma. Here, we described simple cholesterol-modified polymeric CXCR4 antagonist (PCX) nanoparticles (to block cancer-stroma interactions) for codelivery of anti-miR-210 (to inactivate stroma-producing pancreatic stellate cells (PSCs)) and siKRASG12D (to kill pancreatic cancer cells). IP administration delivered the nanoparticles to an orthotopic syngeneic pancreatic tumors as a result of preferential localization to the tumors and metastases with disrupted mesothelium and effective tumor penetration. The local IP delivery resulted in nearly 15-fold higher tumor accumulation than delivery by IV injection. Through antagonism of CXCR4 and downregulation of miR-210/KRASG12D, the triple-action nanoparticles favorably modulated desmoplastic tumor microenvironment via inactivating PSCs and promoting the infiltration of cytotoxic T cells. The combined therapy displayed improved therapeutic effect when compared with individual therapies as documented by the delayed tumor growth, depletion of stroma, reduction of immunosuppression, inhibition of metastasis, and prolonged survival. Overall, we present data that a local IP delivery of a miRNA/siRNA combination holds the potential to improve pancreatic cancer therapy.

Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats.

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here. An emulsification strategy, without any mechanical or chemical pre-treatment, was developed to increase the bioavailability of solid, poorly-consumable fats. Wild type R. eutropha strain H16 produced 79-82% (w/w) polyhydroxybutyrate (PHB) per cell dry weight (CDW) when cultivated on various fats. A productivity of 0.3g PHB/(L × h) with a total PHB production of 24 g/L was achieved using tallow as carbon source. Using a recombinant strain of R. eutropha that produces poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], 49-72% (w/w) of PHA per CDW with a HHx content of 16-27 mol% were produced in shaking flask experiments. The recombinant strain was grown on waste animal fat of the lowest quality available at lab fermenter scale, resulting in 45 g/L CDW with 60% (w/w) PHA per CDW and a productivity of 0.4 g PHA/(L × h). The final HHx content of the polymer was 19 mol%. The use of low quality waste animal fats as an inexpensive carbon feedstock exhibits a high potential to accelerate the commercialization of PHAs.

Control of cell morphology of probiotic Lactobacillus acidophilus for enhanced cell stability during industrial processing.

The viability of bacteria during industrial processing is an essential quality criterion for bacterial preparations, such as probiotics and starter cultures. Therefore, producing stable microbial cultures during proliferation is of great interest. A strong correlation between the culture medium and cellular morphology was observed for the lactic acid bacterium Lactobacillus acidophilus NCFM, which is commonly used in the dairy industry as a probiotic supplement and as a starter culture. The cell shapes ranged from single short rods to long filamentous rods. The culture medium composition could control this phenomenon of pleomorphism, especially the use of peptone in combination with an adequate heating of the medium during preparation. Furthermore, we observed a correlation between the cell size and stability of the microorganisms during industrial processing steps, such as freeze-drying, extrusion encapsulation and storage following dried preparations. The results revealed that short cells are more stable than long cells during each of the industrially relevant processing steps. As demonstrated for L. acidophilus NCFM, the adaptation of the medium composition and optimized medium preparation offer the possibility to increase the concentration of viable cells during up- and survival rate during down-stream processing.

Stability of creatine derivatives during simulated digestion in an in vitro model.

Newly developed forms of creatine are often claimed to exhibit improved bioavailability and efficacy. They are of great interest for sports nutrition and therapeutic uses. However, for most newer creatine forms stability after ingestion under physiological conditions is insufficiently documented, relevant data are inconsistent or even missing. Therefore, we developed a controlled simulated digestion system for testing different creatine derivatives in specific simulated parts of the human digestive system. All derivatives showed high stability with negligible formation of creatinine.

Recovery of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Ralstonia eutropha cultures with non-halogenated solvents.

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA-based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non-halogenated solvents. Several non-halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17-30 mol%) remained completely in solution, while polymer with a lower HHx content (11-16 mol%) formed a gel-like phase. All PHA in solution could be precipitated by addition of threefold volumes of n-hexane or n-heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non-halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA.

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by Ralstonia eutropha in high cell density palm oil fermentations.

Improved production costs will accelerate commercialization of polyhydroxyalkanoate (PHA) polymer and PHA-based products. Plant oils are considered favorable feedstocks, due to their high carbon content and relatively low price compared to sugars and other refined carbon feedstocks. Different PHA production strategies were compared using a recombinant strain of Ralstonia eutropha that produces high amounts of P(HB-co-HHx) when grown on plant oils. This R. eutropha strain was grown to high cell densities using batch, extended batch, and fed batch fermentation strategies, in which PHA accumulation was triggered by nitrogen limitation. While extended batch culture produced more biomass and PHA than batch culture, fed batch cultivation was shown to produce the highest levels of biomass and PHA. The highest titer achieved was over 139 g/L cell dry weight (CDW) of biomass with 74% of CDW as PHA containing 19 mol% HHx. Our data suggest that the fermentation process is scalable with a space time yield (STY) better than 1 g PHA/L/h. The achieved biomass concentration and PHA yield are among the highest reported for the fermentation of recombinant R. eutropha strains producing P(HB-co-HHx).

HypE: an algorithm for fast hypervolume-based many-objective optimization.

In the field of evolutionary multi-criterion optimization, the hypervolume indicator is the only single set quality measure that is known to be strictly monotonic with regard to Pareto dominance: whenever a Pareto set approximation entirely dominates another one, then the indicator value of the dominant set will also be better. This property is of high interest and relevance for problems involving a large number of objective functions. However, the high computational effort required for hypervolume calculation has so far prevented the full exploitation of this indicator's potential; current hypervolume-based search algorithms are limited to problems with only a few objectives. This paper addresses this issue and proposes a fast search algorithm that uses Monte Carlo simulation to approximate the exact hypervolume values. The main idea is not that the actual indicator values are important, but rather that the rankings of solutions induced by the hypervolume indicator. In detail, we present HypE, a hypervolume estimation algorithm for multi-objective optimization, by which the accuracy of the estimates and the available computing resources can be traded off; thereby, not only do many-objective problems become feasible with hypervolume-based search, but also the runtime can be flexibly adapted. Moreover, we show how the same principle can be used to statistically compare the outcomes of different multi-objective optimizers with respect to the hypervolume--so far, statistical testing has been restricted to scenarios with few objectives. The experimental results indicate that HypE is highly effective for many-objective problems in comparison to existing multi-objective evolutionary algorithms. HypE is available for download at http://www.tik.ee.ethz.ch/sop/download/supplementary/hype/.

Dual feeding strategy for the production of alpha-amylase by Bacillus caldolyticus using complex media.

In this study, the objective was to investigate an exponential feeding strategy for fed-batch production of thermostable alpha-amylase (E.C. 3.2.1.1.) from the Bacillus caldolyticus (DSM405). The parameters for establishing compositions of feed media and feeding rate were obtained by statistical analysis of batch and continuous shake flask experiments. These parameters were casitone to starch ratio of 2.67g(casitone)g(starch)(-1), maintenance coefficient 0.174g(casitone)g(DW)(-1)h(-1), cell yield 0.62g(DW)g(casitone)(-1) and mu(opt)=0.2h(-1). The exponentially fed fermentation resulted in yield of 120Uml(-1) alpha-amylase that was thermostable up to 105 degrees C. Results of the exponentially fed fermentation have been discussed in the light of a feed-back controlled fed-batch fermentation reported earlier by the authors. A comparison of the temperature and pH effects on amylase produced by B. caldolyticus and on several other commercially available amylases has also been presented.