Junfei Xia

Top-down fabrication of polyelectrolyte-thermoplastic hybrid microparticles for unidirectional drug delivery to single cells.

Micro/nanoparticles are widely used for drug delivery and traditionally synthesized via the bottom-up approach. Recent years have seen considerable efforts in developing novel methods for fabricating micro/nanoparticles through the top-down approach, which offers superb capabilities for controlling the size, geometry, internal structure, and composition of the particles. [ 1 , 2 ] Particles produced by this approach can thus possess functionalities that cannot be attained by those produced by the bottom-up methods, e.g., unidirectional dug release. [ 3 ]

Neural patterning of human induced pluripotent stem cells in 3-D cultures for studying biomolecule-directed differential cellular responses

INTRODUCTION Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells/tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capacity of signaling factors that regulate 3-D neural tissue patterning in vitro and differential responses of the resulting neural populations to various biomolecules have not yet been fully understood. METHODS By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog (SHH) signaling, this study generated different 3-D neuronal cultures that were mainly comprised of either cortical glutamatergic neurons or motor neurons. RESULTS Abundant glutamatergic neurons were observed following the treatment with an antagonist of SHH signaling, cyclopamine, while Islet-1 and HB9-expressing motor neurons were enriched by an SHH agonist, purmorphamine. In neurons derived with different neural patterning factors, whole-cell patch clamp recordings showed similar voltage-gated Na(+)/K(+) currents, depolarization-evoked action potentials and spontaneous excitatory post-synaptic currents. Moreover, these different neuronal populations exhibited differential responses to three classes of biomolecules, including (1) matrix metalloproteinase inhibitors that affect extracellular matrix remodeling; (2) N-methyl-d-aspartate that induces general neurotoxicity; and (3) amyloid β (1-42) oligomers that cause neuronal subtype-specific neurotoxicity. CONCLUSIONS This study should advance our understanding of hiPSC self-organization and neural tissue development and provide a transformative approach to establish 3-D models for neurological disease modeling and drug discovery. STATEMENT OF SIGNIFICANCE Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells, tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capability of sonic hedgehog-related small molecules to tune different neuronal subtypes in 3-D differentiation from hiPSCs and the differential cellular responses of region-specific neuronal subtypes to various biomolecules have not been fully investigated. By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog signaling, this study provides knowledge on the differential susceptibility of region-specific neuronal subtypes derived from hiPSCs to different biomolecules in extracellular matrix remodeling and neurotoxicity. The findings are significant for understanding 3-D neural patterning of hiPSCs for the applications in brain organoid formation, neurological disease modeling, and drug discovery.

Asymmetric biodegradable microdevices for cell-borne drug delivery.

Use of live cells as carriers for drug-laden particulate structures possesses unique advantages for drug delivery. In this work, we report on the development of a novel type of particulate structures called microdevices for cell-borne drug delivery. The microdevices were fabricated by soft lithography with a disklike shape. Each microdevice was composed of a layer of biodegradable thermoplastic such as poly(lactic-co-glycolic acid). One face of the thermoplastic layer was covalently grafted with a cell-adhesive polyelectrolyte such as poly-l-lysine. This asymmetric structure allowed the microdevices to bind to live cells through bulk mixing without causing cell aggregation. Moreover, the cell-microdevice complexes were largely stable, and the viability and proliferation ability of the cells were not affected by the microdevices over a week. In addition, sustained release of a mock drug from the microdevices was demonstrated. This type of microdevice promises to be clinically useful for sustained intravascular drug delivery.

Enhanced Radiation Therapy with Multilayer Microdisks Containing Radiosensitizing Gold Nanoparticles

A challenge of X-ray radiation therapy is that high dose X-rays at therapeutic conditions damage normal cells. This paper describes the use of gold nanoparticle-loaded multilayer microdisks to enhance X-ray radiation therapy, where each microdisk contains over 10(5) radiosensitizing nanoparticles. The microdisks are attached on cell membranes through electrostatic interaction. Upon X-ray irradiation, more photoelectrons and Auger electrons are generated in the vicinity of the nanoparticles, which cause water ionization and lead to the formation of free radicals that damage the DNA of adjacent cancer cells. By attaching a large amount of gold nanoparticles on cancer cells, the total X-ray dose required for DNA damage and cell killing can be reduced. Due to their controllable structure and composition, multilayer microdisks can be a viable choice for enhanced radiation therapy with nanoparticles.

Facile functionalization and assembly of live cells with microcontact-printed polymeric biomaterials.

The functionalization and assembly of live cells with microfabricated polymeric biomaterials have attracted considerable interest in recent years, but the conventional methods suffer from high cost, high complexity, long processing time or inadequate capability. The present study reports on the development of a novel method for functionalizing and assembling live cells by integrating microcontact printing of polymeric biomaterials with a temperature-sensitive sacrificial layer prepared by spin-coating. This method has been used not only to functionalize live cells with microscopic polyelectrolyte and thermoplastic structures of various sizes and shapes, but also to assemble the cells into macroscopic stripes and sheets. The method is applicable to multiple types of cells, including human leukemic cells, mouse embryonic stem cells and human mesenchymal stem cells in the forms of single cells and cell aggregates. In addition, the microcontact-printed structures can be prepared using biodegradable and biocompatible polyelectrolytes and thermoplastic. The unique combination of low cost, ease of use and high versatility renders this method potentially useful for diverse biomedical applications, including drug delivery, cell tracking and tissue engineering.

Versatile surface micropatterning and functionalization enabled by microcontact printing of poly(4-aminostyrene).

Microcontact printing (μCP) of polyelectrolytes is a facile and powerful method for surface micro/nanopatterning and functionalization. Poly(4-aminostyrene) (PAS) is a polyelectrolyte that can be converted to aryldiazonium salt and exhibits pH-dependent hydrophobicity. Here we demonstrate μCP of PAS and the expansion of this technique in various directions. First, the microcontact-printed PAS can be diazotized to micropattern biomolecules including DNA and protein and nanomaterials including single-walled carbon nanotubes and gold nanoparticles. Second, the diazotized PAS enables μCP of a metallic structure on a carbon surface. Third, the hydrophobic nature of PAS at the neutral pH allows the microcontact-printed PAS-based polyelectrolyte multilayer to be used as masks for wet etching. Lastly, this technique allows facile fabrication of highly engineered microparticles with a unique structure. Overall, this work has established a novel μCP platform with various potential applications.

Single cell patterning for high throughput sub-cellular toxicity assay

Cell population based toxicity assays cannot distinguish responses of single cells and sub-cellular organelles; while single cell assays are limited by low statistical power due to small number of cells examined. This article reports a new single cell array based toxicity assay, in which cell responses at population level, single cell level and sub-cellular level can be obtained simultaneously at high throughput. The single cell array was produced by microcontact printing and selected area cell attachment, and exposed to damaging X-ray radiation, which was followed by fluorescence imaging after staining. Two image processing softwares written in Python and MATLAB were used to determine the expressions of proteins associated with cell migration and invasion, and production of reactive oxygen species (ROS), respectively. The results showed significant differences in responses at single cell level and distinctive molecular heterogeneity at sub-cellular level in a large population of cells upon exposure to radiation.

Generation of Well-Defined Micro/Nanoparticles via Advanced Manufacturing Techniques for Therapeutic Delivery

Micro/nanoparticles have great potentials in biomedical applications, especially for drug delivery. Existing studies identified that major micro/nanoparticle features including size, shape, surface property and component materials play vital roles in their in vitro and in vivo applications. However, a demanding challenge is that most conventional particle synthesis techniques such as emulsion can only generate micro/nanoparticles with a very limited number of shapes (i.e., spherical or rod shapes) and have very loose control in terms of particle sizes. We reviewed the advanced manufacturing techniques for producing micro/nanoparticles with precisely defined characteristics, emphasizing the use of these well-controlled micro/nanoparticles for drug delivery applications. Additionally, to illustrate the vital roles of particle features in therapeutic delivery, we also discussed how the above-mentioned micro/nanoparticle features impact in vitro and in vivo applications. Through this review, we highlighted the unique opportunities in generating controllable particles via advanced manufacturing techniques and the great potential of using these micro/nanoparticles for therapeutic delivery.