A Microfluidics-Assisted Double-Barreled Nanobioconjugate Synthesis Introducing Aprotinin as a New Moonlight Nanocarrier Protein: Tested toward Physiologically Relevant 3D-Spheroid Models.

Proteins are promising substances for introducing new drug carriers with efficient blood circulation due to low possibilities of clearance by macrophages. However, such natural biopolymers have highly sophisticated molecular structures, preventing them from being assembled into nanoplatforms with manipulable payload release profiles. Here, we report a novel anticancer nanodrug carrier moonlighting protein, Aprotinin, to be used as a newly identified carrier for cytotoxic drugs. The Aprotinin-Doxorubicin (Apr-Dox) nanobioconjugate was prepared via a single-step microfluidics coflow mixing technique, a feasible and simple way to synthesize a carrier-based drug design with a double-barreled approach that can release and actuate two therapeutic agents simultaneously, i.e., Apr-Dox in 1:11 ratio (the antimetastatic carrier drug aprotinin and the chemotherapeutic drug DOX). With a significant stimuli-sensitive (i.e., pH) drug release ability, this nanobioconjugate achieves superior bioperformances, including high cellular uptake, efficient tumor penetration, and accumulation into the acidic tumor microenvironment, besides inhibiting further tumor growth by halting the urokinase plasminogen activator (uPA) involved in metastasis and tumor progression. Distinctly, in healthy human umbilical vein endothelial (HUVEC) cells, drastically lower cellular uptake of nanobioconjugates has been observed and validated compared to the anticancer agent Dox. Our findings demonstrate an enhanced cellular internalization of nanobioconjugates toward breast cancer, prostate cancer, and lung cancer both in vitro and in physiologically relevant biological 3D-spheroid models. Consequently, the designed nanobioconjugate shows a high potential for targeted drug delivery via a natural and biocompatible moonlighting protein, thus opening a new avenue for proving aprotinin in cancer therapy as both an antimetastatic and a drug-carrying agent.

One-Step Synthesis of Ultrasmall Nanoparticles in Glycerol as a Promising Green Solvent at Room Temperature Using Omega-Shaped Microfluidic Micromixers.

Despite innovations in the synthesis protocol of nanoparticles (NPs), the size distribution and uniformity of particles still remain as crucial attributes. Homogeneous and rapid nucleation is a critical phenomenon to obtain monodisperse nanoparticles. Herein, we have carried out the synthesis of metal nanoparticles in a customized microfluidic (MF) chip, with 18 omega-shaped micromixers, by using glycerol as a promising green solvent and reducing agent at various concentrations (10-80%), and simultaneous comparison of the results from batch synthesis. Initially, mixing characterization for 10-80% glycerol was obtained by adjusting the Peclet (Pe) number. Further, the effect of the Pe number, time, and concentrations of polyvinylpyrrolidone, metal source, and glycerol on the NP size was investigated. Interestingly, the experimental findings depicted that by varying different parameters, the spherical nanoparticles with an average ultrasmall particle diameter of <2 nm were obtained at all glycerol concentrations (10-80%), as compared to batch synthesis (giving a yield of ∼10-fold larger particles). The mixing efficiency in this MF chip design was analyzed by using a fluorescent dye in glycerol, while the particle morphology and size were characterized by using dynamic light scattering, transmission electron microscopy, and ultraviolet-visible spectroscopy. Hence, compared to the conventional benchtop-assisted NP synthesis, this study unveils the significant effect of the microfluidic technique on the synthesis of ultrasmall and homogeneous nanoparticles in a single step, using an environmentally friendly solvent.

Cholesterol biogenesis is a PTEN-dependent actionable node for the treatment of endocrine therapy-refractory cancers.

PTEN and PIK3CA mutations are the most prevalent PI3K pathway alterations in prostate, breast, colorectal, and endometrial cancers. p110ß becomes the prominent PI3K isoform upon PTEN loss. In this study, we aimed to understand the molecular mechanisms of PI3K dependence in the absence of PTEN. Using online bioinformatical tools, we examined two publicly available microarray datasets with aberrant PI3K activation. We found that the rate-limiting enzyme of cholesterol biogenesis, SQLE, was significantly upregulated in p110ß-hyperactivated or PTEN-deficient mouse prostate tumors. Concomitantly, the expression of cholesterol biosynthesis pathway enzymes was directly correlated with PI3K activation status in microarray datasets and diminished upon PTEN re-expression in PTEN-null prostate cancer cells. Particularly, PTEN re-expression decreased SQLE protein levels in PTEN-deficient prostate cancer cells. We performed targeted metabolomics and detected reduced levels of cholesteryl esters as well as free cholesterol upon PTEN re-expression. Notably, PTEN-null prostate and breast cancer cell lines were more sensitive to pharmacological intervention with the cholesterol pathway than PTEN-replete cancer cells. Since steroid hormones use sterols as structural precursors, we studied whether cholesterol biosynthesis may be a metabolic vulnerability that enhances antihormone therapy in PTEN-null castration-resistant prostate cancer cells. Coinhibition of cholesterol biosynthesis and the androgen receptor enhanced their sensitivity. Moreover, PTEN suppression in endocrine therapy-resistant luminal-A breast cancer cells leads to an increase in SQLE expression and a corresponding sensitization to the inhibition of cholesterol synthesis. According to our data, targeting cholesterol biosynthesis in combination with the hormone receptor signaling axis can potentially treat hormone-resistant prostate and breast cancers.

Novel Size-Tunable and Straightforward Ultra-Small Nanoparticle Synthesis in a Varying Concentration Range of Glycerol as a Green Reducing Solvent.

Despite all the possibilities available so far for the synthesis of nanoparticles (NPs), synthesizing ultra-small (<10 nm) monodispersed particles is still demanding. Getting a particular size with a straightforward method is a trial-and-error approach. To explore this prospective, in the current study, we have introduced a protocol which offers a varying concentration range of glycerol to successfully generate the NPs of repeatable and consistent particle size in each synthesis, thus giving an alternative from lengthy tentative preparations and/or testing protocols. Since synthesizing controlled sized nanoparticles in aqueous medium is somewhat difficult as the balance of particle growth and nucleation is challenging to control, herein, we used a polyol method with glycerol both as a solvent medium as well as reducing species for silver nitrate, as an example model ion source, to execute the nanoparticle synthesis. In order to maintain the stability of the synthesized NPs, polyvinylpyrolidone (PVP) was added as a stabilizer. The synthesis, monodispersity, and stability were confirmed using techniques such as UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and X-ray powder diffraction (XRD), while morphological analysis and ultra-small size validation were conducted using TEM, SEM, and AFM. Interestingly, in the various concentrations of glycerol solution used (10-100%), we have observed a tunable linear size range to obtain ultra-small nanoparticles (<10 nm) up to 60% glycerol, while further increasing the glycerol component increased the size approximately to ∼160 nm, providing tunable properties in this synthesis procedure. Hence, this study provides a distinct possibility to obtain ultra-small nanoparticles with a tunable size feature for further applications in numerous fields.

Microwave Heating Induced On-Demand Droplet Generation in Microfluidic Systems.

In this note, we report a simple, new method for droplet generation in microfluidic systems using integrated microwave heating. This method enables droplet generation on-demand by using microwave heating to induce Laplace pressure change at the interface of the two fluids. The distance between the interface and junction and microwave excitation power have been found to influence droplet generation. Although this method is limited in generating droplets with a high rate, the fact that it can be integrated with microwave sensing that can be used as the feedback to tune the supply flow of materials presents unique advantages for applications that require dynamic tuning of material properties in droplets.

Passive microinjection within high-throughput microfluidics for controlled actuation of droplets and cells.

Microinjection is an effective actuation technique used for precise delivery of molecules and cells into droplets or controlled delivery of genes, molecules, proteins, and viruses into single cells. Several microinjection techniques have been developed for actuating droplets and cells. However, they are still time-consuming, have shown limited success, and are not compatible with the needs of high-throughput (HT) serial microinjection. We present a new passive microinjection technique relying on pressure-driven fluid flow and pulsative flow patterns within an HT droplet microfluidic system to produce serial droplets and manage rapid and highly controlled microinjection into droplets. A microneedle is secured within the injection station to confine droplets during the microinjection. The confinement of droplets on the injection station prevents their movement or deformation during the injection process. Three-dimensional (3D) computational analysis is developed and validated to model the dynamics of multiphase flows during the emulsion generation. We investigate the influence of pulsative flows, microneedle parameters and synchronization on the efficacy of microinjection. Finally, the feasibility of implementing our microinjection model is examined experimentally. This technique can be used for tissue engineering, cells actuation and drug discovery as well as developing new strategies for drug delivery.

Effective Thermo-Capillary Mixing in Droplet Microfluidics Integrated with a Microwave Heater.

In this study, we present a microwave-based microfluidic mixer that allows rapid mixing within individual droplets efficiently. The designed microwave mixer is a coplanar design with a small footprint, which is fabricated on a glass substrate and integrated with a microfluidic chip. The mixer works essentially as a resonator that accumulates an intensive electromagnetic field into a spiral capacitive gap (around 200 µm), which provides sufficient energy to heat-up droplets that pass through the capacitive gap. This microwave actuation induces nonuniform Marangoni stresses on the interface, which results in three-dimensional motion inside the droplet and thus fast mixing. In order to evaluate the performance of the microwave mixer, droplets with highly viscous fluid, 75% (w/w) glycerol solution, were generated, half of which were seeded with fluorescent dye for imaging purposes. The relative importance of different driving forces for mixing was evaluated qualitatively using magnitude analysis, and the effect of the applied power on mixing performance was also investigated. Mixing efficiency was quantified using the mixing index, which shows as high as 97% mixing efficiency was achieved within the range of milliseconds. This work demonstrates a very unique approach of utilizing microwave technology to facilitate mixing in droplet microfluidics systems, which can potentially open up areas for biochemical synthesis applications.

Microwave temperature measurement in microfluidic devices.

In spite of various existing thermometry methods for microfluidic applications, it remains challenging to measure the temperature of individual droplets in segmented flow since fast moving droplets do not allow sufficient exposure time demanded by both fluorescence based techniques and resistance temperature detectors. In this contribution, we present a microwave thermometry method that is non-intrusive and requires minimal external equipment. This technique relies on the correlation of fluid temperature with the resonance frequency of a microwave sensor that operates at a GHz frequency range. It is a remote yet direct sensing technique, eliminating the need for mixing fluorescent dyes with the working fluid. We demonstrated that the sensor operates reliably over multiple tests and is capable of both heating and sensing. It measures temperature to within ±1.2 °C accuracy and can detect the temperature of individual droplets.

Label-free high-throughput detection and content sensing of individual droplets in microfluidic systems.

This study reports a microwave-microfluidics integrated approach capable of performing droplet detection at high-throughput as well as content sensing of individual droplets without chemical or physical intrusion. The sensing system consists of a custom microwave circuitry and a spiral-shaped microwave resonator that is integrated with microfluidic chips where droplets are generated. The microwave circuitry is very cost effective by using off-the-shelf components only. It eliminates the need for bulky benchtop equipment, and provides a compact, rapid and sensitive tool compatible for Lab-on-a-Chip (LOC) platforms. To evaluate the resonator's sensing capability, it was first applied to differentiate between single-phase fluids which are aqueous solutions with different concentrations of glucose and potassium chloride respectively by measuring its reflection coefficient as a function of frequency. The minimum concentration assessed was 0.001 g ml(-1) for potassium chloride and 0.01 g ml(-1) for glucose. In the droplet detection experiments, it is demonstrated that the microwave sensor is able to detect droplets generated at as high throughput as 3.33 kHz. Around two million droplets were counted over a period of ten minutes without any missing. For droplet sensing experiments, pairs of droplets that were encapsulated with biological materials were generated alternatively in a double T-junction configuration and clearly identified by the microwave sensor. The sensed biological materials include fetal bovine serum, penicillin antibiotic mixture, milk (2% mf) and d-(+)-glucose. This system has significant advantages over optical detection methods in terms of its cost, size and compatibility with LOC settings and also presents significant improvements over other electrical-based detection techniques in terms of its sensitivity and throughput.