Capsule-like Safe Genetic Vectors-Cell-Penetrating Core-Shell Particles Selectively Release Functional Small RNA and Entrap Its Encoding DNA.

The breakthrough of genetic therapy is set back by the lack of suitable genetic vector systems. We present the development of permeability-tunable, capsule-like, polymeric, micron-sized, core-shell particles for delivery of recombinant nucleic acids into target cells. These particles were demonstrated to effectively release rod-shaped small hairpin RNA and to selectively retain the RNA-encoding DNA template, which was designed to form a bulky tripartite structure. Thus, they can serve as delivery vectors preloaded with cargo RNA or alternatively as RNA-producing micro-bioreactors. The internalization of particles by human tissue culture cells inversely correlated with particle size and with the cell to particle ratio, although at a higher than stoichiometric excess of particles over cells, cell viability was impaired. Among primary human peripheral blood mononuclear cells, up to 50% of the monocytes displayed positive uptake of particles. Finally, these particles efficiently delivered siRNA into HEK293T cells triggering functional knockdown of the target gene lamin A/C. Particle-mediated knockdown was superior to that observed after conventional siRNA delivery via lipofection. Core-shell particles protect encapsulated nucleic acids from degradation and target cell genomes from direct contact with recombinant DNA, thus representing a promising delivery vector system that can be explored for genetic therapy and vaccination.

Learning-Based Cell Injection Control for Precise Drop-on-Demand Cell Printing.

Drop-on-demand (DOD) printing is widely used in bioprinting for tissue engineering because of little damage to cell viability and cost-effectiveness. However, satellite droplets may be generated during printing, deviating cells from the desired position and affecting printing position accuracy. Current control on cell injection in DOD printing is primarily based on trial-and-error process, which is time-consuming and inflexible. In this paper, a novel machine learning technology based on Learning-based Cell Injection Control (LCIC) approach is demonstrated for effective DOD printing control while eliminating satellite droplets automatically. The LCIC approach includes a specific computational fluid dynamics (CFD) simulation model of piezoelectric DOD print-head considering inverse piezoelectric effect, which is used instead of repetitive experiments to collect data, and a multilayer perceptron (MLP) network trained by simulation data based on artificial neural network algorithm, using the well-known classification performance of MLP to optimize DOD printing parameters automatically. The test accuracy of the LCIC method was 90%. With the validation of LCIC method by experiments, satellite droplets from piezoelectric DOD printing are reduced significantly, improving the printing efficiency drastically to satisfy requirements of manufacturing precision for printing complex artificial tissues. The LCIC method can be further used to optimize the structure of DOD print-head and cell behaviors.

Hand-Held Photometer for Instant On-Spot Quantification of Nucleic Acids, Proteins, and Cells.

This paper presents a novel hand-held photometer, termed "Photopette", for on-spot absorbance measurements of biochemical analytes. The Photopette is a multicomponent, highly portable device with an overall weight of 160 g, which fits within 202 mm × 47 mm × 42 mm. Designed in the form factor of a micropipette, Photopette integrates a photodiode detector with light emitting diodes (LEDs) to form a highly customizable photometer which supports a wide variety of applications within the wavelengths between 260 and 1050 nm. A dual-purpose disposable reflective tip was designed to act as a sample holder and a light-reflecting system, which is in stark contrast to the operation of mainstream spectrophotometers and photometers. Small volume analytes may be measured with low sample loss using this proprietary CuveTip. A user-friendly software application running on smart devices was developed to control and read the values from Photopette via a low-energy Bluetooth link. This one-step strategy allows measurements on-spot without sample transfer, minimizing cross-contamination and human error. The results reported in this paper demonstrate Photopette's great potential to quantify DNA, direct protein, and cell density directly within the laminar flow hood. Results are compared with a Nanodrop 2000c spectrophotometer, a mainstream spectrophotometer for small-volume measurements.

Engineering and Design of Polymeric Shells: Inwards Interweaving Polymers as Multilayer Nanofilm, Immobilization Matrix, or Chromatography Resins.

Hydrogels with complex internal structures are required for advanced drug delivery systems and tissue engineering or used as inks for 3D printing. However, hydrogels lack the tunability and diversity of polymeric shells and require complicated postsynthesis steps to alter its structure or properties. We report on the first integrated approach to assemble and design polymeric shells to take on various complex structures and functions such as multilayer nanofilms, multidensity immobilization matrix, or multiadhesive chromatography resins via the tuning of four assembly parameters: (a) poly(allylamine) (PA) concentration, (b) number of poly(allylamine)/poly(styrenesulfonic acid) (PA/PSSA) incubations, (c) poly(allylamine) (PA) to poly(ethylene glycol) (PEG) grafting ratio, and (d) % H2O present during assembly. Our approach combines the complex 3D structures of hydrogels with the versatility of self-assembled polymeric layers. Polymeric shells produced from our method have a highly uniform material distribution and well-defined shell boundaries. Shell thickness, density, and adhesive properties are easily tunable. By virtue of such unique material features, we demonstrate that polymeric shells can be designed to expand beyond its conventional function as thin films and serve as immobilization matrix, chromatography resins, or even reaction compartments. This technique could also uncover interesting perspectives in the development of novel multimaterials for 3D printing to synthesize scaffolds at a higher order of complexity.

PEG-based autonomous capillary system with integrated microbead array for immunoassay.

Surface-tension-driven capillary systems (CSs) enable self-powered delivery of samples and reagents for bioassays and thus are especially suitable for point-of-care applications. Current silicon and polymer based CSs require extensive work in professional cleanroom for the fabrication of either the silicon device itself or the micromold for polymer processing. In this work, we fabricated a PEG-based CS in a one-step photopolymerization process without the requirement of any cleanroom work. Water, buffer and serum can flow autonomously in this CS and the liquid flow rate can be tuned by modification with surfactant solution of different concentrations. We further integrated an antibody-coated microbead array into this CS for the autonomous immunoassay of two tumor markers, prostate specific antigen (PSA) and human chorionic gonadotropin (hCG), in serum samples with a total assay time of less than 10min. The detection limits for the two tumor markers were at sub-nanogram per milliliter range which is lower than their clinical threshold concentrations for cancer diagnosis. Moreover, simultaneous and multiplex detection of the two tumor markers was also achieved by using spatially encoded microbeads. This low cost and easy-fabricated CS enables fast, multiplex and autonomous immunoassay for protein markers and has the potential to be applied for point-of-care diagnostics on real clinical samples.

Tuning the Mechanical Properties of Hydrogel Core-Shell Particles by Inwards Interweaving Self-Assembly.

Mechanical properties of hydrogel particles are of importance for their interactions with cells or tissue, apart from their relevance to other applications. While so far the majority of works aiming at tuning particle mechanics relied on chemical cross-linking, we report a novel approach using inwards interweaving self-assembly of poly(allylamine) (PA) and poly(styrenesulfonic acid) (PSSA) on agarose gel beads. Using this technique, shell thicknesses up to tens of micrometers can be achieved from single-polymer incubations and accurately controlled by varying the polymer concentration or incubation period. We quantified the changes in mechanical properties of hydrogel core-shell particles. The effective elastic modulus of core-shell particles was determined from force spectroscopy measurements using the colloidal probe-AFM (CP-AFM) technique. By varying the shell thickness between 10 and 24 µm, the elastic modulus of particles can be tuned in the range of 10-190 kPa and further increased by increasing the layer number. Through fluorescence quantitative measurements, the polymeric shell density was found to increase together with shell thickness and layer number, hence establishing a positive correlation between elastic modulus and shell density of core-shell particles. This is a valuable method for constructing multidensity or single-density shells of tunable thickness and is particularly important in mechanobiology as studies have reported enhanced cellular uptake of particles in the low-kilopascal range (<140 kPa). We anticipate that our results will provide the first steps toward the rational design of core-shell particles for the separation of biomolecules or systemic study of stiffness-dependent cellular uptake.

Incorporation of a prolyl hydroxylase inhibitor into scaffolds: a strategy for stimulating vascularization.

Clinical applications of tissue engineering are constrained by the ability of the implanted construct to invoke vascularization in adequate extent and velocity. To overcome the current limitations presented by local delivery of single angiogenic factors, we explored the incorporation of prolyl hydroxylase inhibitors (PHIs) into scaffolds as an alternative vascularization strategy. PHIs are small molecule drugs that can stabilize the alpha subunit of hypoxia-inducible factor-1 (HIF-1), a key transcription factor that regulates a variety of angiogenic mechanisms. In this study, we conjugated the PHI pyridine-2,4-dicarboxylic acid (PDCA) through amide bonds to a gelatin sponge (Gelfoam(®)). Fibroblasts cultured on PDCA-Gelfoam were able to infiltrate and proliferate in these scaffolds while secreting significantly more vascular endothelial growth factor than cells grown on Gelfoam without PDCA. Reporter cells expressing green fluorescent protein-tagged HIF-1α exhibited dose-dependent stabilization of this angiogenic transcription factor when growing within PDCA-Gelfoam constructs. Subsequently, we implanted PDCA-Gelfoam scaffolds into the perirenal fat tissue of Sprague Dawley rats for 8 days. Immunostaining of explants revealed that the PDCA-Gelfoam scaffolds were amply infiltrated by cells and promoted vascular ingrowth in a dose-dependent manner. Thus, the incorporation of PHIs into scaffolds appears to be a feasible strategy for improving vascularization in regenerative medicine applications.

Gel pad array chip for high throughput and multi-analyte microbead-based immunoassays.

We present here a gel pad array chip for high-throughput and multi-analyte microbead-based immunoassays. The chip is fabricated by photo-patterning of two polymeric gels, polyacrylamide gel and polyethylene glycol (PEG) gel, on a glass slide. The resulting chip consists of 40 polyacrylamide gel pad array units for the immobilization of microbeads and each gel pad array is surrounded with a PEG micropillar ring to confine the samples within the microarray. As a proof of concept, this chip was tested for quantitative immunoassays for two model cancer markers, human chorionic gonadotropin (hCG) and prostate specific antigen (PSA), in serum samples. Detection limits below the physiological threshold level for cancer diagnosis were achieved with good inter- and intra-chip reproducibility. Moreover, by using spatial encoded microbeads, simultaneous detection of both hCG and PSA on each gel pad array is achieved with single filter fluorescence imaging. This gel pad array chip is easy to use, easy to fabricate with low cost materials and minimal equipment and reusable. It could be a useful tool for common biolabs to customize their own microbead array for multi-analyte immunoassays.

Mitochondrial routing of glucose and sucrose polymers after pinocytotic uptake: avenues for drug delivery.

Mitochondria are key organelles organizing cellular metabolic flux. Therefore, a targeted drug delivery to mitochondria promises the advancement of medicine in fields that are associated with mitochondrial dysfunction. However, successful mitochondrial drug delivery is limited by complex transport steps across organelle membranes and fast drug efflux in cases of multidrug resistance. Strategies to deliver small-molecular-weight drugs to mitochondria are very limited, while the use of complex polymeric carriers is limited by a lack of clinical feasibility. We show here that clinically established macromolecules such as a sucrose copolymer (Ficoll 70/400 kDa) and polyglucose (dextran 70/500 kDa) are micropinocytosed swiftly by mesenchymal stem cells and subsequently routed to mitochondria. The intracellular level of Ficoll appears to decrease over time, suggesting that it does not persist within cells. After coupling to polysucrose, the low-molecular-weight photodynamic drug Rose Bengal reached mitochondria and thus exhibited an increased destructive potential after laser excitation. These findings support new opportunities to deliver already clinically approved drugs to mitochondria.

Multiplex detection platform for tumor markers and glucose in serum based on a microfluidic microparticle array.

We present a multiplex detection platform based on a microfluidic microparticle array to detect proteins and glucose in serum simultaneously. Multiplex detection of proteins and glucose was performed using biofunctionalized microparticles arrayed on gel-based microstructures integrated in microfluidics. The microparticles immobilized on these microstructures showed high stability under microfluidic flow conditions. With arrays of antibody-coated microbeads, microfluidic quantitative immunoassays for two protein tumor markers, human chorionic gonadotropin (hCG) and prostate specific antigen (PSA) were performed in serum samples with detection limits bellow the cut-off values for cancer diagnosis. Parallel to the immunoassays, quantitative enzymatic assays for glucose in the physiological concentration range were performed. Multiplex detection was achieved by using a spatially encoded microarray. By patterning antibody-coated microbeads and enzyme-containing microparticles on a novel mixed structure array, we successfully demonstrated simultaneous immunoassays (binding based assay) for proteins and an enzymatic assay (reaction kinetic based assay) for glucose. Our microparticle arrays could be potentially used for the detection of multiple categories of biomolecules (proteins, small metabolites and DNA) for clinical diagnostics and other biological applications.

Utilizing microfluidics to synthesize polyethylene glycol microbeads for Förster resonance energy transfer based glucose sensing.

Here, we utilize microfluidic droplet technology to generate photopolymerizeable polyethylene glycol (PEG) hydrogel microbeads incorporating a fluorescence-based glucose bioassay. A microfluidic T-junction and multiphase flow of fluorescein isothiocyanate dextran, tetramethyl rhodamine isothiocyanate concanavalin A, and PEG in water were used to generate microdroplets in a continuous stream of hexadecane. The microdroplets were photopolymerized mid-stream with ultraviolet light exposure to form PEG microbeads and were collected at the outlet for further analysis. Devices were prototyped in PDMS and generated highly monodisperse 72 ± 2 µm sized microbeads (measured after transfer into aqueous phase) at a continuous flow rate between 0.04 ml/h-0.06 ml/h. Scanning electron microscopy analysis was conducted to analyze and confirm microbead integrity and surface morphology. Glucose sensing was carried out using a Förster resonance energy transfer (FRET) based assay. A proportional fluorescence intensity increase was measured within a 1-10 mM glucose concentration range. Microfluidically synthesized microbeads encapsulating sensing biomolecules offer a quick and low cost method to generate monodisperse biosensors for a variety of applications including cell cultures systems, tissue engineering, etc.

A 'microfluidic pinball' for on-chip generation of Layer-by-Layer polyelectrolyte microcapsules.

Inspired by the game of "pinball" where rolling metal balls are guided by obstacles, here we describe a novel microfluidic technique which utilizes micropillars in a flow channel to continuously generate, encapsulate and guide Layer-by-Layer (LbL) polyelectrolyte microcapsules. Droplet-based microfluidic techniques were exploited to generate oil droplets which were smoothly guided along a row of micropillars to repeatedly travel through three parallel laminar streams consisting of two polymers and a washing solution. Devices were prototyped in PDMS and generated highly monodisperse and stable 45±2 µm sized polyelectrolyte microcapsules. A total of six layers of hydrogen bonded polyelectrolytes (3 bi-layers) were adsorbed on each droplet within <3 minutes and a fluorescent intensity measurement confirmed polymer film deposition. AFM analysis revealed the thickness of each polymer layer to be approx. 2.8 nm. Our design approach not only provides a faster and more efficient alternative to conventional LbL deposition techniques, but also achieves the highest number of polyelectrolyte multilayers (PEMs) reported thus far using microfluidics. Additionally, with our design, a larger number of PEMs can be deposited without adding any extra operational or interfacial complexities (e.g. syringe pumps) which are a necessity in most other designs. Based on the aforementioned advantages of our device, it may be developed into a great tool for drug encapsulation, or to create capsules for biosensing where deposition of thin nanofilms with controlled interfacial properties is highly required.

A portable generic DNA bioassay system based on in situ oligonucleotide synthesis and hybridization detection.

In this study, we present a portable and generic DNA bioassay system based on in situ oligonucleotide synthesis followed by hybridization based detection. The system include two main parts, an oligonucleotide synthesizer and a fluorescence detection system. The oligonucleotide synthesizer is based on microfluidic technology and capable of synthesizing any desired oligonucleotide which can be either used as a primer for PCR based detection (external) or a probe for hybridization based detection (integrated) of a target DNA analyte. The oligonucleotide sequence can be remotely sent to the system. The integrated fluorescence detection system is based on a photodiode to detect Texas Red fluorophore as low as 0.5 fmol. The complete system, integrating the oligonucleotide synthesizer and fluorescence detection system, was successfully used to distinguish DNA from two different bacteria strains. The presented generic portable instrument has the potential to detect any desired DNA target sequence in the field. Potential applications are for homeland security and fast responses to emerging bio-threats.

In-situ measurement of cellular microenvironments in a microfluidic device.

We report on the integration of optical microsensors into a cell culture microchannel device. We demonstrate the possibility of measuring the glucose and oxygen concentrations in the microenvironment of the mammalian cells cultured in a microchannel device. Furthermore, cell proliferation and morphology could be monitored microscopically while these measurements were being made. Through the use of multiple sensors along the length of the microchannel, concentration gradients of various metabolites, such as oxygen, as well as the effects of cell uptake and perfusion rate of growth medium on these gradients could be studied. As such, the system allowed real-time observations of the cells' response to their chemical microenvironment. Our approach allows cell culture and cell assays to be performed simultaneously in an integrated microchannel system with potential applications as a research tool or drug screening method.

Matrix-assisted colloidosome reverse-phase layer-by-layer encapsulating biomolecules in hydrogel microcapsules with extremely high efficiency and retention stability.

The layer-by-layer (LbL) polyelectrolyte self-assembly encapsulation method has attracted much interest because of its versatility to use various polymers for capsule formation, ability to encapsulate different templates, and capability to control capsule permeability. Traditionally, the LbL method was performed in water as solvent and limited to poorly or non-water-soluble templates. Using the matrix-assisted LbL method, complex mixtures of water-soluble proteins or DNA could be encapsulated within agarose microbeads templates but leakage of biomolecules into the water phase during the LbL process results in low encapsulation efficiency. Recently, the reverse-phase LbL (RP-LbL) method was introduced to perform LbL and encapsulation of water-soluble templates in organic solvents, thus preventing the templates from dissolving and allowing high encapsulation efficiency. However, encapsulation of complex mixtures of biomolecules or other substances with quantitative encapsulation efficiency remained impossible. Here we present a new approach for encapsulation of biomolecules or complex mixtures thereof with almost 100% encapsulation efficiency. The ability of our method to achieve high encapsulation efficiency arises from the combination of two strategies. (1) Using microparticles as surface stabilizer to create stable biomolecule-loaded hydrogel microbeads, termed matrix-assisted colloidosome (MAC), that are able to disperse in oil and organic solvents. (2) Using the RP-LbL method to fabricate polymeric capsule "membranes", thereby preventing diffusion of the highly water-soluble biomolecules. Using an oil phase during emulsification and an organic solvent phase during encapsulation could completely prevent leakage of water-soluble biomolecules and almost 100% encapsulation efficiency is achieved. Microcapsules fabricated with our method retained nearly 100% of encapsulated proteins during a 7 day incubation period in water. The method was demonstrated on model proteins and may be extended to other biomolecules or mixtures. Our method is a valuable addition to the family of encapsulation techniques and can significantly contribute to the fields of bioreactors and bioanalytical microcapsules.

Reusable optical bioassay platform with permeability-controlled hydrogel pads for selective saccharide detection.

A reusable optical bioassay platform using permeability-controlled hydrogel pads for selective saccharide detection has been developed. An optical glucose detection assay based on fluorescence resonance energy transfer (FRET) between dye-labeled dextran and Concanavalin A (ConA) was incorporated into hydrogel pads by entrapment. The hydrogel pads are constructed from hemispherical hydrogel attached onto hydrophobic surfaces of a microtiter plate. The resulted hemispherical hydrogel pads entrapping the sensing biological materials were further surface coated with polyelectrolyte multilayers through a Layer-by-Layer (LbL) self-assembly process to create a permeability-controlled membrane with nanometer thickness. The selective permeable LbL film deposited on the hydrogel surface allows small molecular weight analytes to diffuse into the hydrogel pads while the large molecular weight sensing biological molecules are immobilized. An encapsulation efficiency of 75% for the ConA/Dextran complex within the coated hydrogel pads was achieved and no significant leakage of the complex was observed. Glucose calibration curve with linear range from 0 to 10mM glucose was obtained. Selective permeability of the hydrogel pads has been demonstrated by measurement of saccharides with various molecular weights. The LbL hydrogel pads could selectively detect monosaccharides (glucose, MW=180) and disaccharides (sucrose, MW=342) while polysaccharides (dextran, MW approximately 70kDa) cannot diffuse through the LbL layer and are excluded. LbL hydrogel pads allow regeneration of the FRET system with good signal reproducibility of more than 90% to construct a reusable and reagentless optical bioassay platform.

Real time observation of diffusion and bioaffinity binding processes in single polyelectrolyte-coated microcapsules: a fluorescence-based approach.

We report on using fluorescence microscopy to study, visualize and determine the diffusion phenomena into and bioaffinity binding within single microcapsules in real time by using biotin-fluorescein as diffusive species and encapsulated avidin as binding partner. Microcapsules were constructed by entrapment of avidin within an agarose matrix and encapsulated with polyelectrolyte layers by Layer-by-Layer (LbL) polyelectrolyte self assembly. A "ring" of high fluorescence intensity advancing with time towards the capsule centre was observed during incubation of capsules with fluorescent-labeled biotin. Fluorescence intensity was build up in capsule areas where binding to avidin occurred and was visualized in real time. A model for the diffusion process in microcapsules was developed and experimental data was plotted and fitted well with trends predicted by the model. The value of the diffusion coefficient for biotin-fluorescein was determined to be 3.5x10(-8)cm(2)/s, which is comparable to literature values of similar sized molecules.

Reverse-phase LbL-encapsulation of highly water soluble materials by layer-by-layer polyelectrolyte self-assembly.

We report on a novel method for the encapsulation of highly water soluble materials by using layer-by-layer (LbL) polyelectrolyte self-assembly. State of the art polyelectrolyte self-assembly LbL coating and encapsulation methods are only applicable to insoluble or poorly water soluble template materials, because the process is performed in water causing dissolution of the solid template. Our method extends the material spectrum to highly water soluble template materials by using non-ionized polyelectrolytes in an organic phase (reverse-phase) instead of polyelectrolyte salts in an aqueous environment. By using the reverse-phase layer-by-layer (RP-LbL) technique, we have demonstrated the direct encapsulation of proteins, glucose, vitamin C, and inorganic salts in the solid state. Multilayer deposition was proven, layer thickness was determined by AFM, and the advantage of the method to prepare powders of encapsulated materials was demonstrated. The method is simple, robust, and applicable to a broad range of substances with potential applications in several industries.