Voxelated bioprinting of modular double-network bio-ink droplets.

Analogous of pixels to two-dimensional pictures, voxels-in the form of either small cubes or spheres-are the basic building blocks of three-dimensional objects. However, precise manipulation of viscoelastic bio-ink voxels in three-dimensional space represents a grand challenge in both soft matter science and biomanufacturing. Here, we present a voxelated bioprinting technology that enables the digital assembly of interpenetrating double-network hydrogel droplets made of polyacrylamide/alginate-based or hyaluronic acid/alginate-based polymers. The hydrogels are crosslinked via additive-free and biofriendly click reaction between a pair of stoichiometrically matched polymers carrying norbornene and tetrazine groups, respectively. We develop theoretical frameworks to describe the crosslinking kinetics and stiffness of the hydrogels, and construct a diagram-of-state to delineate their mechanical properties. Multi-channel print nozzles are developed to allow on-demand mixing of highly viscoelastic bio-inks without significantly impairing cell viability. Further, we showcase the distinctive capability of voxelated bioprinting by creating highly complex three-dimensional structures such as a hollow sphere composed of interconnected yet distinguishable hydrogel particles. Finally, we validate the cytocompatibility and in vivo stability of the printed double-network scaffolds through cell encapsulation and animal transplantation.

3D Printable Modular Soft Elastomers from Physically Cross-linked Homogeneous Associative Polymers.

Three-dimensional (3D) printing of elastomers enables the fabrication of many technologically important structures and devices. However, there remains a critical need for the development of reprocessable, solvent-free, soft elastomers that can be printed without the need for post-treatment. Herein, we report modular soft elastomers suitable for direct ink writing (DIW) printing by physically cross-linking associative polymers with a high fraction of reversible bonds. We designed and synthesized linear-associative-linear (LAL) triblock copolymers; the middle block is an associative polymer carrying amide groups that form double hydrogen bonding, and the end blocks aggregate to hard glassy domains that effectively act as physical cross-links. The amide groups do not aggregate to nanoscale clusters and only slow down polymer dynamics without changing the shape of the linear viscoelastic spectra; this enables molecular control over energy dissipation by varying the fraction of the associative groups. Increasing the volume fraction of the end linear blocks increases the network stiffness by more than 100 times without significantly compromising the extensibility. We created elastomers with Young's moduli ranging from 8 kPa to 8 MPa while maintaining the tensile breaking strain around 150%. Using a high-temperature DIW printing platform, we transformed our elastomers to complex, highly deformable 3D structures without involving any solvent or post-print processing. Our elastomers represent the softest melt reprocessable materials for DIW printing. The developed LAL polymers synergize emerging homogeneous associative polymers with a high fraction of reversible bonds and classical block copolymer self-assembly to form a dual-cross-linked network, providing a versatile platform for the modular design and development of soft melt reprocessable elastomeric materials for practical applications.

Multi Stimuli-Responsive Aggregation-Induced Emission Active Polymer Platform Based on Tetraphenylethylene-Appended Maleic Anhydride Terpolymers.

Multi stimuli-responsive aggregation-induced emission (AIE) active polymers have great application prospects in high-tech innovations. Herein, three types of tetraphenylethylene (TPE)-containing monomers were synthesized and utilized in preparing TPE-appended maleic anhydride terpolymers. After hydrolysis, the produced TPE-appended maleic acid terpolymers have identical linear charge densities but different "primary" structures, which created widely varied microenvironments around the carboxylate and TPE groups. Benefiting from the synergistic interaction of the TPE moiety and the terpolymer conformation change, the TPE-appended maleic acid terpolymers exhibited fluorescence changes in response to multi stimuli, including pH, ionic strength, Ca2+, and bovine serum albumin. On both the "signaling" and the "stimuli acceptor" sides, the multi stimuli-responsive fluorescence behavior was influenced markedly by the terpolymer primary structure. The fundamental insights gained in the present work are important for developing an efficient and versatile stimuli-responsive AIE-active polymer platform for chemo-sensing, bioimaging, and so on.

Prolonged melittin release from polyelectrolyte-based nanocomplexes decreases acute toxicity and improves blood glycemic control in a mouse model of type II diabetes.

Gating modifier toxins (GMTs) from animal venom have shown great potential in controlling blood glucose levels in type II diabetes (T2D), but their high acute toxicity and quick clearance in the body hamper their potential therapeutic use. Inspired by their highly positive charge, we have developed a nanocomplex system based on polyelectrolytes, in which strong interactions form between positively charged GMTs and negatively charged dextran sulfate (DS). Using melittin as a model GMT and adapting flash nanocomplexation (FNC) technology for complex preparation, uniform nanocomplexes (polydispersity index: ~0.1) with high melittin encapsulation efficiency (~100%), high payload capacity (~30%), and tunable release profiles were formulated. In contrast to the high acute liver toxicity and low survival rate (60% after 8 days) observed after a single intraperitoneal (i.p.) injection of 3 mg/kg free melittin, melittin-loaded nanocomplexes displayed improved safety (100% survival after 8 days) due to prolonged melittin release. In a mouse model of T2D, a single i.p. injection of nanocomplexes decreased the blood glucose level to 12 mmol/L within 12 h and maintained it within the therapeutic range (<15 mmol/L) for 48 h. In addition, body weight decreased following treatment. This GMT/DS binary system shows great promise due to its simple components, facile preparation method, and enhanced potential druggability, including a decreased dosing frequency, decreased acute toxicity, and improved pathological indicators.

A polyphenol-metal nanoparticle platform for tunable release of liraglutide to improve blood glycemic control and reduce cardiovascular complications in a mouse model of type II diabetes.

Liraglutide is a GLP-1 receptor agonist recently approved for Type-II diabetes (T2D) treatment with superior hypoglycemic effect while also improving cardiovascular function for the patients. However, its application has been limited by its short half-life (~13 h), which requires daily injections to maintain effective drug concentrations in blood, thus increasing the risk of poor patient compliance and complications. In this study, we developed a ternary liraglutide/tannic acid (TA)/Al3+ nanoparticle system based on hydrogen bond formation between liraglutide and TA and stabilized by complex coordination interaction between TA and Al3+. This ternary nanoparticle formulation offers sustained release of liraglutide for >8 days by optimizing the concentration of TA during nanoparticle assembly. A flash nanocomplexation (FNC) process was adopted to confer homogeneous mixing of the three components and control the assembly kinetics, thus enabling efficient encapsulation, a tunable drug release profile, improved nanoparticle size uniformity, and a high degree of colloidal stability. Upon a single intraperitoneal (i.p.) administration, the optimized formulation effectively lowered the high blood glucose level in a T2D db/db mice model to the normal range (8-10 mmol/L) within 6 h, maintained it for 60 more hours, and kept it lower than the original level for >6 days. In a 30-day treatment study, the nanoparticle formulation with a dosage frequency of once every 5 days exhibited similar or better control of blood sugar level (20% reduction in HbA1c) and weight control than daily injection of free liraglutide at the same treatment dose. The extended glycemic control led to distinctive improvements on reducing cardiomyopathy, including inhibition in lipo-toxicity by decreasing 40% of triglyceride, 30% of diacylglycerol and 50% of PKC level in the heart, as well as ameliorating oxidative stress and cell apoptosis activities through positive regulation on superoxidase, malondialdehyde, caspase-3 and Bax. This nanoparticle system demonstrates improved therapeutic potential owing to its long-acting glycemic control with improved cardiovascular function and reduced tissue toxicity in multiple organs.

Kinetic Control in Assembly of Plasmid DNA/Polycation Complex Nanoparticles.

Polyelectrolyte complex (PEC) nanoparticles assembled from plasmid DNA (pDNA) and polycations such as linear polyethylenimine (lPEI) represent a major nonviral delivery vehicle for gene therapy tested thus far. Efforts to control the size, shape, and surface properties of pDNA/polycation nanoparticles have been primarily focused on fine-tuning the molecular structures of the polycationic carriers and on assembly conditions such as medium polarity, pH, and temperature. However, reproducible production of these nanoparticles hinges on the ability to control the assembly kinetics, given the nonequilibrium nature of the assembly process and nanoparticle composition. Here we adopt a kinetically controlled mixing process, termed flash nanocomplexation (FNC), that accelerates the mixing of pDNA solution with polycation lPEI solution to match the PEC assembly kinetics through turbulent mixing in a microchamber. This achieves explicit control of the kinetic conditions for pDNA/lPEI nanoparticle assembly, as demonstrated by the tunability of nanoparticle size, composition, and pDNA payload. Through a combined experimental and simulation approach, we prepared pDNA/lPEI nanoparticles having an average of 1.3 to 21.8 copies of pDNA per nanoparticle and average size of 35 to 130 nm in a more uniform and scalable manner than bulk mixing methods. Using these nanoparticles with defined compositions and sizes, we showed the correlation of pDNA payload and nanoparticle formulation composition with the transfection efficiencies and toxicity in vivo. These nanoparticles exhibited long-term stability at -20 °C for at least 9 months in a lyophilized formulation, validating scalable manufacture of an off-the-shelf nanoparticle product with well-defined characteristics as a gene medicine.

Surface Coating Approach to Overcome Mucosal Entrapment of DNA Nanoparticles for Oral Gene Delivery of Glucagon-like Peptide 1.

Oral delivery of nucleic acid therapy is a promising strategy in treating various diseases because of its higher patient compliance and therapeutic efficiency compared to parenteral routes of administration. However, its success has been limited by the low transfection efficiency resulting from nucleic acid entrapment in the mucus layer and epithelial barrier of the gastrointestinal (GI) tract. Herein, we describe an approach to overcome this phenomenon and improve oral DNA delivery in the context of treating type II diabetes (T2D). Linear PEI (lPEI) was used as a carrier to form complexes with plasmid DNA encoding glucagon-like peptide 1 (GLP-1), a common target in T2D treatments. These nanoparticles were then coated with a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol)-2000 (DMG-PEG) to render the nanoparticle surface hydrophilic and electrostatically neutral. The surface-modified lPEI/DNA nanoparticles showed higher diffusivity and transport in the mucus layer of the GI tract and mediated high levels of transfection efficiency in vitro and in vivo. Moreover, these modified nanoparticles demonstrated high levels of GLP-1 expression for more than 24 h in the liver, lungs, and intestine in a T2D murine model after a single dose, as well as controlled blood glucose levels within a normal range for at least 18 h with repeatable therapeutic effects upon multiple dosages. Taken together, this work demonstrates the feasibility of an oral plasmid DNA delivery approach in the treatment of T2D through a facile surface modification to improve the mucus permeability and delivery efficiency of the nanoparticles.

Sustained release of exendin-4 from tannic acid/Fe (III) nanoparticles prolongs blood glycemic control in a mouse model of type II diabetes.

Exendin-4 has been clinically adopted as an effective drug for treating type 2 diabetes (T2D), but its short circulation half-life in the blood requires two injections per day to maintain effective glycemic control. This significantly limits its clinical application. In this study, we developed a tannic acid/exendin-4/Fe3+ ternary nanoparticle system to provide sustained release of exendin-4 in vivo. The formation of these nanoparticles relies on TA/exendin-4 complexation and stabilization through TA-Fe3+ coordination, where the rapid reaction kinetics can benefit from efficient mixing of all three components. Adapting our recently developed flash nanocomplexation (FNC) method, we formulated nanoparticles with high encapsulation efficiency (~ 100%) of exendin-4, high payload capacity, and high degrees of uniformity and stability because the rapid turbulent mixing facilitated a homogeneous distribution of all three components in the complexation process. Intraperitoneal injection in mice showed that exendin-4 released from the nanoparticles had an AUC 7.2-fold higher than the free exendin-4 injection. Efficacy study in a T2D mouse model showed that the optimized formulation achieved a rapid reduction of the blood glucose level to the normal range within <12 h and maintained the same level for 72 h following a single intraperitoneal dose. The blood glucose level was maintained to below the therapeutic level (< 15 mmol/L) for 6 days, and the treatment led to reduced body weight with pathological and functional improvements in the kidney and liver. This tannic acid/exendin-4/Fe3+ ternary nanoparticle system holds translational potential in treating T2D, due to its improved treatment outcomes in terms of extended release of exendin-4, prolonged control of blood glucose level, reduced dosing frequency, and improved pathological indicators.

Size-controlled lipid nanoparticle production using turbulent mixing to enhance oral DNA delivery.

Lipid-based nanoparticles (LNPs) have been developed to address the transport and uptake barriers to enhance the delivery efficiency of plasmid DNA therapeutics. In these systems, plasmid DNA can be encapsulated through condensation by a cationic lipid to form lipo-complexes, or polycation following complexation into cationic liposomes to form lipo-polyplexes. Conventional methods for achieving these two DNA-delivering LNP vehicles suffer from significant batch-to-batch variation, poor scalability and complicated multi-step preparation procedures. Resultant nanoparticles often have uncontrollable size and surface charge with wide distribution, and poor stability when exposed to physiological media. Here we report a single-step flash nanocomplexation (FNC) process using turbulent mixing to prepare uniform lipo-complex or lipo-polyplex LNPs in a scalable manner, demonstrating excellent control over the nanoparticle size (from 40 to several hundred nm) and surface charge, with narrow size distribution. The FNC-produced LNPs could be purified and concentrated using a tangential flow filtration (TFF) process in a scalable manner. An optimized formulation of purified lipo-complex LNPs (DOTAP/Chol/DNA, 45 nm) showed significantly higher (5-fold in the lungs and 4-fold in the liver) transgene expression activity upon oral dosage than lipo-polyplex LNPs (DPPC/Chol/lPEI/DNA, 75 nm) or lPEI/DNA nanoparticles (43 nm). Repeated dosing (4 days, 150 µg/day) of the lipo-complex LNPs sustained the transgene activity over a period of one week without detectable toxicity in major organs, suggesting its potential for clinical translation. STATEMENT OF SIGNIFICANCE: We report a new method to prepare uniform size-controlled lipid-based DNA-loaded nanoparticles by turbulent mixing delivered by a multi-inlet vortex mixer. Two distinct compositions were successfully prepared: (1) lipo-complexes, through condensation of the plasmid DNA by cationic lipids; (2) lipo-polyplexes, by encapsulation of DNA/PEI together with neutral lipids. Comparing with conventional methods, which use multi-step processes with high batch-to-batch variations and poor control over nanoparticle characteristics, this method offers a single-step, continuous and reproducible assembly methodology that would promote the translation of such gene medicine products. Effective purification and concentration of nanoparticles were achieved by adopted tangential flow filtration method. Following oral gavage in mice, the lipo-complex nanoparticles showed the highest level of transgene expression in the lung and liver.

[Change of eustachian tube surfactant of guinea pigs suffering from secretory otitis media].

OBJECTIVE: To observe the change of surface active agents of eustachian tube of guinea pigs suffering from secretory otitis media, to explore the effects of surface active agents on secretory otitis media. METHODS: To establish the animal model of guinea pigs suffering from secretory otitis media. To analyse the changes of biochemical component and activity of eustachian tube's surfactant, meanwhile; to observe surfactant change after ectogenic surfactant treatment. RESULTS: The main components of the surfactant decrease in the model group of guinea pigs suffering from secretory otitismedia, phosphatidylcholine (PC) is (19.9 +/- 1.7)%, phosphatidylethanolamine(PE) is (36.8 +/- 2.7)%, minimum surface tension (gamma min) is (18.5 +/- 2.4) mN/m, and PC is (25.7 +/- 2.1)%, PE is (43.7 +/- 3.8)%, (P < 0.001), and gamma min is (7.6 +/- 0.8) mN/m in normal group (P < 0.001). After giving Ectogenic surfactant treatment, PC and PE increase, PC is (23.3 +/- 2.2)%, PE is (42.5 +/- 3.6)%; gamma min reduce, it is (11.8 +/- 2.3) mN/m. CONCLUSIONS: The main biochemical components of the surfactant obviously decrease, the activity of the surfactant weakens in secretory otitis media of guinea pig. This study showed the treatment of Ectogenic surfactant is effective.