Polymersome production on a microfluidic platform using pH sensitive block copolymers

The Application of Microfluidics in Preparing Nano Drug Delivery Systems

Design of polymeric micelles, formed by self-assembly of amphiphilic block copolymers, is crucial for encapsulation of poorly soluble drugs, leading to the development of promising carrier systems. Herein, we synthesized amphiphilic diblock copolymers of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) and methacrylate R-polyhedral oligomeric silsesquioxanes (POSS) (vertex R-groups of POSS cage modified with ethyl (C2H5), hexayl (C6H13), octayl (C8H17) alkyl chain) via RAFT polymerization technique. Polymeric micelle was formed in aqueous environment, and the absolute size was calculated to be around 26–43 nm. The increased alkyl chain length of the R-groups of POSS led to loosely packed association of the micelles. The polymeric micelles encapsulated the hydrophobic model drugs (paclitaxel and α-tocopherol), and the encapsulation efficiency was strongly dependent on the structure of drug molecules. The drug-loaded micelles were stable for 5 days at 25 °C. The release % of both the drugs from the micelles...

Novel applications of poly(ethylene glycol)-bl-poly(propylene sulfide) block copolymers

The potential of a novel pH-triggered block copolymer as a promising drug delivery platform for the cancer therapy has been explored. The block copolymer poly(ethylene oxide)-b-poly(glycerol monomethacrylate)-b-poly[2-(diisopropylamino)ethyl methacrylate] herein referred to as PEO113-b-PG2MA30-b-PDPA50 upon dissolution in ethanol followed by single-step nanoprecipitation in phosphate buffered saline (PBS) self-assembled into highly regular spherical micelles whose structure was characterized in detail by static (SLS), dynamic (DLS) and electrophoretic (ELS) light scattering, small angle X-ray scattering (SAXS), fluorescence spectroscopy and transmission electron microscopy (TEM). The micellar size (2RH = 42 nm) and micellar molecular weight (Mw(micelles) > 106 kDa) were found to be in the range to avoid renal clearance providing a long blood circulation time. Their size is below the cut-off size of the leaky pathological vasculature (DH < 200 nm), making them candidates for the use in cancer therapy based on the EPR effect. The pH-responsive PDPA core could be loaded with the poorly water-soluble anti-cancer drug paclitaxel (PTX) with encapsulation efficiency ∼70% and drug loading content ∼7% wdrug/wpolymer. The pKa of the diisopropylamino group of the PDPA block was determined as pKa = 6.8 in the simulated physiological condition, which is remarkably close to the pH microenvironment of tumoral cells. The release experiments evidenced that approximately 90% of the encapsulated PTX was sustained at the PDPA micellar core within the first 9 h at pH 7.4 whilst only 18 h were required for complete drug release at pH 5.0. These results suggest that the micellar dissociation might be triggered at the slightly acid tumoral extracellular environments (pH < pKa(PDPA)). The nanostructures were further placed in contact with human plasma or human serum albumin (HSA) diluted in PBS. The DLS experiments revealed that the micelles are especially stable for up to at least 48 h in such conditions, attesting the possibly long blood circulation time of the nanoparticles at serum environments which is a pre-requisite for the drug delivery applications. The cell viability experiments demonstrated that the drug-free block copolymer micelles are non-toxic and the number of viable cells is always greater than 85% compared to the survival number of a control group.

We developed the microfluidic light emitting diode (LED) sensor for methanol detection. The linear gradient-generating microfluidic device consists of two inlet and four outlet microchannels. The concentration gradients of methanol were stably generated in the microfluidic platform in a temporal and spatial manner. The methanol harvested from microfluidic platforms was analyzed by measuring electrical conductivity, showing that currents were decreased with the methanol content. The methanol in the microfluidic device was also observed by LED sensor. Therefore, this microfluidic LED device could be a powerful platform for methanol sensor applications.

ABSTRACTA well‐defined amphiphilic block copolymer consisting of a hydrophobic block poly(methyl methacrylate) (PMMA) and a hydrophilic block poly[N,N–2‐(dimethylamino) ethyl methacrylate] (PDMAEMA) was synthesized by activator generated by the electron transfer for atom transfer radical polymerization method (AGET ATRP). Kinetics study revealed a linear increase in the graph concentration of PMMA‐b‐PDMAEMA with the reaction time, indicating that the polymer chain growth was consistent with a controlled process. The gel permeation chromatography results indicated that the block copolymer had a narrow molecular weight distribution (Mw/Mn = 1.42) under the optimal reaction conditions. Then, poly(vinylidene fluoride) (PVDF)/PMMA‐b‐PDMAEMA blend membranes were prepared via the standard immersion precipitation phase inversion process, using the block copolymer as additive to improve the hydrophilicity of the PVDF membrane. The presence and dispersion of PMMA‐b‐PDMAEMA clearly affected the morphology and improved the hydrophilicity of the as‐synthesized blend membranes as compared to the pristine PVDF membranes. By incorporating 15 wt % of the block copolymer, the water contact angle of the resulting blend membranes decreased from pure PVDF membrane 98° to 76°. The blend membranes showed good stability in the 20 d pure‐water experiment. The bovine serum albumin (BSA) absorption experiment revealed a substantial antifouling property of the blend membranes in comparison with the pristine PVDF membrane. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 42080.

Persistent cells, representing a small fraction of a bacterial population, are refractory to antibiotic therapy. It is a phenotypic transient state. The underlying mechanisms of persistence remain largely unknown, but even divergent hypotheses agree on a fundamental component: persistence arises from phenotypic heterogeneity. Novel approaches are needed to elucidate which factors allow some bacteria to persist but not others. High-content screening (HCS), which uses microscopy as a screening tool, enables the visualization of single-cell phenotypes and is thus particularly suited for studying bacterial persistence. Traditionally performed with eukaryotic cells in micro-well plate format, executing HCS with prokaryotic cells required the development of a screening novel strategy due to their much smaller size. Microfluidics has proved to be a remarkable toolbox for bacterial studies. In combination with time-lapse microscopy, it permits the study of dynamic processes with single-cell resolution in a tightly controlled microenvironment. Though numerous microfluidic platforms for prokaryotes imaging have been developped, the current state of the art does not allow the imaging of thousands of genotypes in parallel, as is required for high-content screening. The focus of this thesis is to develop a high-throughput microfluidic screening platform enabling high-content imaging of prokaryotic cells. This platform allows 1152 bacterial strains to be imaged in parallel on both the single-cell and sub-cellular level with precise control over the growth environment. First, we demonstrate the ability of the platform to effectively screen for both altered cell division phenotypes through subcellular markers imaging in a static environment and for phenotypes emerging in complex medium profiles, such as bacterial persistence. Second, we employed our platform in a genetic screen, exposing a mutant library of Mycobacterium smegmatis to the antibiotic Isoniazid (INH), and identified a total of 31 mutants with altered persistence rates during antibiotic exposure as compared to the wild-type. Eight of these mutants had decreased persistence rates while 23 mutants displayed increased persistence. Third, using the same platform we performed in depth analysis of mutants with altered rates of persistence in order to identify the cell division and cell death dynamics underlying persistence. This single-cell analysis revealed unexpected dynamics where a mutant with decreased persistence displayed a lower killing rate compared to the wild-type. This high-throughput microfluidic screening platform is highly versatile and can be used in a wide range of bacterial studies, as it is capable of imaging not only single cells, but also subcellular markers, such as those based on the cell cycle or bacterial septa. This novel technology thus opens new doors for microbiologists.

Intracellular protein delivery may provide a safe and non-genome integrated strategy for targeting abnormal or specific cells for applications in cell reprogramming therapy. Thus, highly efficient intracellular functional protein delivery would be beneficial for protein drug discovery. In this study, we generated a cationic polyethyleneimine (PEI)-modified gelatin nanoparticle and evaluated its intracellular protein delivery ability in vitro and in vivo. The experimental results showed that the PEI-modified gelatin nanoparticle had a zeta potential of approximately +60 mV and the particle size was approximately 135 nm. The particle was stable at different biological pH values and temperatures and high protein loading efficiency was observed. The fluorescent image results revealed that large numbers of particles were taken up into the mammalian cells and escaped from the endosomes into the cytoplasm. In a mouse C26 cell-xenograft cancer model, particles accumulated in cancer cells. In conclusion, the PEI-modified gelatin particle may provide a biodegradable and highly efficient protein delivery system for use in regenerative medicine and cancer therapy.

Biodegradable polymer nano‐ and microparticles are of interest as drug carriers due to their capability to modulate drug release. Two different methods, including solvent displacement and spray drying, are used, resulting in the fabrication of Artemisone (ART)‐loaded polymeric nano‐ and microparticles, respectively. Scanning electron microscopy, transmission electron microscopy, dynamic light scattering, and asymmetric flow field flow fractionation (AF4) are employed to evaluate the morphology and size of the particles. The encapsulation and loading efficiency of the drug as well as cumulative release are characterized using high‐performance liquid chromatography (HPLC). HPLC results confirm that the spray drying method provides higher encapsulation efficiencies of about 97%, whereas, in nanoparticles prepared by solvent displacement, the highest encapsulation efficiency is 71%. Using these production methods, round‐shaped particles with homogeneous size distribution are fabricated. Moreover, the preparation procedure is optimized to obtain particles with the highest encapsulation and drug loading efficiency. Using the solvent displacement method, stable dispersions in water are obtained without adding any surfactant, whereas, for spray‐dried microparticles, tween 80 is needed to redisperse particles. The micro‐ and nanoparticles show different release properties, which are of interest for drug delivery application of hydrophobic drugs like ART.

The importance of the hydrodynamic cavitation phenomenon in small domains has been increasing during recent decades along with the global demand for microfluidic devices involving small-scale cavitation applications. Different characteristics of microscale hydrodynamic cavitation relative to the conventional size can be exploited in futuristic applications and improvements in the performances of new-generation microfluidic devices. Therefore, in-depth studies on the fundamentals of microscale hydrodynamic cavitation are required to reveal new physics of small-scale hydrodynamic cavitation. In this study, microfluidic devices with rough surfaces and micro restrictive elements are fabricated so that a basic study on ‘Hydrodynamic Cavitation on Chip’ is performed. Cavitating flows are investigated under transient and fully developed turbulent conditions within the Reynolds number range between 2962 and 8620 and cavitation number range between 2.025 and 0.72. The microfluidic devices have short restrictive elements with hydraulic diameters of 75, 66.6 and 50 µm and lengths of 2 mm, which are connected to a bigger microchannel with a width of 900 µm and length of 2 mm, called an ‘extended channel’. Different upstream pressures up to 900 Psi are applied at the inlet. The hydrodynamic cavitation inception is recorded and analyzed for each microfluidic device. Flow patterns are characterized inside the microfluidic devices from cavitation inception to chocked flow conditions. Accordingly, it is observed that the transition from inception to choked occurs slowly in contrast to microscale hydrodynamic cavitation results in the literature under laminar flow conditions. Moreover, the comparison between microfluidic devices with roughened and plane (smooth) surfaces reveals that the roughened surface results in more intense cavitating flows, especially at higher upstream pressures relative to the plane surface.

The present study describes the development of novel block copolymer nanocarriers of the phytocannabinoid cannabidiol (CBD), designed to enhance the solubility of the drug in water while achieving high encapsulation efficiency and prolonged drug release. Firstly, a well-defined amphiphilic block copolymer consisting of two outer hydrophilic polyglycidol (PG) blocks and a middle hydrophobic block of poly(ε-caprolactone) bearing pendant cinnamyl moieties (P(CyCL-co-CL)) were synthesized by the click coupling reaction of PG-monoalkyne and P(CyCL-co-CL)-diazide functional macroreagents. A non-modified polyglycidol/poly(ε-caprolactone) amphiphilic block copolymer was obtained as a referent system. Micellar carriers based on the two block copolymers were formed via the solvent evaporation method and loaded with CBD following two different protocols-loading during micelle formation and loading into preformed micelles. The key parameters/characteristics of blank and CBD-loaded micelles such as size, size distribution, zeta potential, molar mass, critical micelle concentration, morphology, and encapsulation efficiency were determined by using dynamic and static multiangle and electrophoretic light scattering, transmission electron microscopy, and atomic force microscopy. Embedding CBD into the micellar carriers affected their hydrodynamic radii to some extent, while the spherical morphology of particles was not changed. The nanoformulation based on the copolymer bearing cinnamyl moieties possessed significantly higher encapsulation efficiency and a slower rate of drug release than the non-modified copolymer. The comparative assessment of the antiproliferative effect of micellar CBD vs. the free drug against the acute myeloid leukemia-derived HL-60 cell line and Sezary Syndrome HUT-78 demonstrated that the newly developed systems have pronounced antitumor activity.

Efficient encapsulation of proteins in submicro liposomes using a supercritical fluid assisted continuous process

Complexation ability of poly(2‐(dimethylamino)ethyl methacrylate)‐b‐poly(hydroxy propyl methacrylate) (PDMAEMA‐b‐PHPMA) amphiphilic doubly thermo‐responsive block copolymers, and their quaternized counterparts QPDMAEMA‐b‐PHPMA, toward bovine serum albumin (BSA) is studied in aqueous solutions. The PDMAEMA‐b‐PHPMA amphiphilic block copolymers self‐assemble in nanostructured aggregates with PDMAEMA coronas having different inner structure and micro‐polarity depending on the solubilization protocol utilized when inserted in aqueous media. By incorporating different BSA concentrations, we investigate the copolymer–protein interactions by light scattering measurements in aqueous solutions in a broad temperature range, utilizing different solubilization protocols for the copolymers. Fluorescence spectroscopy and ζ‐potential measurements were also utilized to investigate the structure and properties of the copolymer/protein complexes formed in each case. Such knowledge may lead to a better understanding of the inner structure and micro polarity of the nanostructured aggregates formed by the novel (Q)PDMAEMA‐b‐PHPMA copolymers, along with their potential abilities in nanocarrier formation, protein complexation, stabilization, and delivery.

Objective To prepare PLGA/PEG microsphcres for sustained release of bovine serum albumin (BSA), and evaluate the effect of the micrnsphere composition, proportion of the components and integration of PEG on the size, BSA encapsulation rate and in vitro BSA release properties of the microspheres. Methods BSA-loaded PLGA/PEG microspheres were prepared by water-in-oil-in-water double emulsions-solvent evaporation method (W/O/W). The morphology of the microspheres was observed with scanning elect'on microscope, the diameter was determined by Mastersizer 2000, and the encapsulation efficiency and in vitro BSA release were evaluated by UV spectrophotometry. Results Increased GA to LA ratio was associated with increased size of the microspheres and accelerated BSA release. The incorporation of PEG obviously increased the BSA encapsulation rate to as much as 88.22% with also increased BSA release rate. The BSA-PLGA/PEG biodegradable microspheres prepared was characterized by high encapsulation rate, low burst release, and slow BSA release for over 3 weeks at high drug concentrations. Conclusion BSA-PLGA/PEG microspheres with relatively high encapsulation efficiency and proper drug loading can be prepared by adjusting the parameters during the preparation process. Key words: PLGA/PEG microspheres; Bovine serum albumin; Double emulsious-solvent evaporation technique; Controlled release

Main observation and conclusionThe intracellular delivery of metal‐organic frameworks (MOFs) encapsulated with functional biomolecules represents a promising avenue in the field of biomedicine and biosensing. To improve the cellular uptake efficiency of MOFs, here we report the fabrication of cancer cell‐derived exosome membrane (EM)‐coated zeolitic imidazolate framework‐8 (EM‐ZIF‐8) nanoparticles by using a microfluidic sonication device. EM‐ZIF‐8 nanoparticles loaded with FITC‐labeled bovine serum albumin (BSA) can be taken up by cancer cells and evade phagocytosis more efficiently than their counterparts (ZIF‐8 nanoparticles). Moreover, we use EM‐ZIF‐8 loaded with Rhodamine B (RhB) for in situ imaging of cellular adenosine triphosphate (ATP). The shield effect of ZIF‐8 suppresses the fluorescence of RhB, and the presence of ATP disrupts the ZIF‐8 structure based on the competitive coordination between ATP and Zn2+, leading to the restoration of RhB fluorescence. This method allows accurate detection of the fluctuation of ATP in A549 cells induced by Ca2+ or 2‐DDG treatment. The devised biomimetic EM‐ZIF‐8 nanoparticles thus provide an efficient platform for intracellular drug delivery and ATP sensing.