Single-Administration Long-Acting Microarray Patch with Ultrahigh Loading Capacity and Multiple Releases of Thermally Stable Antibodies.

Current antibody (Ab) therapies require development of stable formulations and an optimal delivery system. Here, we present a new strategy to create a single-administration long-lasting Ab-delivery microarray (MA) patch, which can carry high doses of thermally stabilized Abs. The MA fabricated by an additive three-dimensional manufacturing technology can be fully embedded into the skin via a single application to deliver doses of Abs at multiple programmable time points, thus sustaining Ab concentrations in systemic circulation. We developed an MA formulation that stabilized and delivered human immunoglobulins (hIg) in a time-controlled manner while maintaining their structure and functionality. As an example, the b12 Ab─a broadly neutralizing Ab against HIV-1─maintained antiviral activity in vitro after MA manufacturing and heat exposure. Pharmacokinetic studies of MA patch-delivered hIg in rats successfully provided a proof of concept for concurrent and time-delayed Ab delivery. These MA patches codeliver different Abs, providing a tool for expanded protection against viral infections or combination HIV therapy and prevention.

Artificial Taste Buds: Bioorthogonally Ligated Gustatory-Neuronal Multicellular Hybrids Enabling Intercellular Taste Signal Transmission.

Heterogeneous tissue models require the assembly and co-culture of multiple types of cells. Our recent work demonstrated taste signal transmission from gustatory cells to neurons by grafting single-stranded DNA into the cell membrane to construct multicellular assemblies. However, the weak DNA linkage and low grafting density allowed the formation of large gustatory cell self-aggregates that cannot communicate with neurons efficiently. This article presents the construction of artificial taste buds exhibiting active intercellular taste signal transmission through the hybridization of gustatory-neuronal multicellular interfaces using bioorthogonal click chemistry. Hybrid cell clusters were formed by the self-assembly of neonatal gustatory cells displaying tetrazine with a precultured embryonic hippocampal neuronal network displaying trans-cyclooctene. A bitter taste signal transduction was provoked in gustatory cells using denatonium benzoate and transmitted to neurons as monitored by intracellular calcium ion sensing. In the multicellular hybrids, the average number of signal transmissions was five to six peaks per cell, and the signal transmission lasted for ∼5 min with a signal-to-signal gap time of 10-40 s. The frequent and extended intercellular signal transmission suggests that the cell surface modification by the bioorthogonal click chemistry is a promising approach to fabricating functional multicellular hybrid clusters potentially useful for cell-based biosensors, toxicity assays, and tissue regeneration.

Hydrogel Skin-Covered Neurons Self-Assembled with Gustatory Cells for Selective Taste Stimulation.

Many technical challenges exist in the co-culture of multiple types of cells, including medium optimization, cell-to-cell connection, and selective data acquisition of cellular responses. Particularly, mixed cellular responses limit the precise interpretation of intercellular signal transduction. Here, we report the formation of an agarose gel skin on neurons closely assembled with gustatory cells to selectively stimulate gustatory cells by retarding the diffusion of tastants to neurons. The signal transmission, triggered by denatonium benzoate, from gustatory cells to neurons was monitored using intracellular calcium ion concentrations. The agarose gel skin efficiently suppressed the direct transfer of tastants to neurons, decreasing the number of responsive neurons from 56 to 13% and the number of calcium ion signals per neuron from multiple to single. The assembly of neurons with gustatory cells induced the high level of neuronal responses through taste signal transduction from gustatory cells to neurons. However, the calcium ion signal peaks of free neurons coated with agarose gel were much shorter and weaker than those of neurons closely assembled with gustatory cells. This work demonstrated that agarose gel skin is a simple, fast, and effective means to increase the signal selectivity of cellular responses in the co-culture of multiple types of cells.

Bioinspired Synthesis of Mesoporous Gold-silica Hybrid Microspheres as Recyclable Colloidal SERS Substrates.

Noble metal nanostructures have been intensively investigated as active substrates for surface-enhanced Raman spectroscopy (SERS) from visible to near-IR wavelengths. However, metal nanoparticle-based SERS analysis in solutions is very challenging due to uncontrollable and irreproducible colloid aggregation. Here we report the templated synthesis of porous gold-silica hybrid microspheres and their application as reusable colloidal SERS substrates. Mesoporous polymer microspheres are synthesized and used as templates for the synthesis of non-aggregated gold nanoparticles, followed by polydopamine-mediated silicification to fabricate mesoporous gold-silica hybrid microspheres. The mesoporous hybrid particles detect crystal violet in the order of 10-8 M and provide the structural durability of the immobilized gold nanoparticles, allowing them to be recycled for repeated SERS analyses for analytes in a solution with the similar sensitivity. This work suggests that the mesoporous gold-silica hybrid microspheres are attractive SERS substrates in terms of reusability, sensitivity, and stability.

Lipiodol nanoemulsions stabilized with polyglycerol-polycaprolactone block copolymers for theranostic applications.

BACKGROUND: Polyglycerol is an attractive hydrophilic building block of amphiphilic copolymers for biomedical and pharmaceutical applications due to its biocompatibility, facile chemical modification, and anti-fouling activity. Herein we introduce theranostic nanoemulsions incorporating anti-cancer therapeutic and contrast agents using linear polyglycerol-poly(ε-caprolactone) diblock copolymers (PG-b-PCL). Lipiodol is used as a core oil that dissolves paclitaxel and serves as a contrast agent for computer tomography (CT). METHODS: PG-b-PCL is synthesized by three-step processes: polymerization of ethoxyethyl glycerol ether; ring-opening polymerization of ε-caprolactone; and deprotection of the PEEGE block. In vitro cytotoxicity of the polyglycerolated lipiodol nanoemulsions is demonstrated using HeLa ovarian cancer cells. The applicability of the prepared nanoemulsions as a contrast agent for CT imaging is also evaluated using micro-CT. RESULTS: Three compositions of PG-b-PCL with different block lengths are synthesized to prepare nanoemulsions. The polyglycerolated lipiodol nanoemulsions exhibit excellent anti-cancer activities, while placebo nanoemulsions have no significant cytotoxicity under the same condition. Micro-CT imaging of the nanoemulsions confirms the ability of nanoemulsions as a contrast agent. CONCLUSIONS: This study suggests that PG-b-PCL is a promising polymeric emulsifier for effective stabilization and surface functionalization of drug delivery nanocarriers for therapeutic and imaging agents.

Polyglycerolated nanocarriers with increased ligand multivalency for enhanced in vivo therapeutic efficacy of paclitaxel.

Despite the excellent biocompatibility and antifouling effect of poly(ethylene glycol) (PEG), the high steric hindrance, limited chemical functionality, and low ligand multivalency of PEGylated nanocarriers often lead to inefficient cell targeting and intracellular trafficking. Hence, a new structure of hydrophilic corona allowing a higher ligand density without loss of excellent biocompatibility is highly desirable. Here we introduce tumor-targeted polyglycerolated (PGylated) nanocarriers that dramatically enhance the in vivo therapeutic efficacy of incorporated paclitaxel simply by increasing the surface density of hydrophobic tumor-targeting ligands. Linear polyglycerol-poly (ε-caprolactone) block copolymer (PG-b-PCL) is used to prepare PGylated lipiodol nanoemulsions, where PG serves as a corona conjugated with a large number of folic acid (FA) for efficient tumor targeting. Unlike FA-PEGylated nanoemulsions, FA-PGylated nanoemulsions can display a larger number of FA without structural destabilization. This property enables excellent anti-cancer activities and effective tumor regression in a cervical cancer xenograft murine model at a cumulative drug dose of ∼5 mg kg-1, which is about four fold smaller than that of commercial Taxol formulation. This study highlights the importance of surface chemistry of nanocarriers that enable multivalent ligand functionalization and high tolerance to the conjugation of hydrophobic ligands, which make PG as a very effective hydrophilic corona for in vivo drug delivery.

Importance of crystallinity of anchoring block of semi-solid amphiphilic triblock copolymers in stabilization of silicone nanoemulsions.

Polymer emulsifiers solidified at the interface between oil and water can provide exceptional dispersion stability to emulsions due to the formation of unique semi-solid interphase. Our recent works showed that the structural stability of paraffin-in-water emulsions highly depends on the oil wettability of hydrophobic block of methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-b-PCL). Here we investigate the effects of the crystallinity of hydrophobic block of triblock copolymer-based emulsifiers, PCLL-b-PEG-b-PCLL, on the colloidal properties of silicone oil-in-water nanoemulsions. The increased ratio of l-lactide to ε-caprolactone decreases the crystallinity of the hydrophobic block, which in turn reduces the droplet size of silicone oil nanoemulsions due to the increased chain mobility at the interface. All of the prepared nanoemulsions are very stable for a month at 37°C. However, the exposure to repeated freeze-thaw cycles quickly destabilizes the nanoemulsions prepared using the polymer with the reduced crystallinity. This work demonstrates that the anchoring chain crystallization in the semi-solid interphase is critically important for the structural robustness of nanoemulsions under harsh physical stresses.