Enhancing adoptive T cell therapy for solid tumor with cell-surface anchored immune checkpoint inhibitor nanogels.

The efficacy of Adoptive Cell Therapy (ACT) for solid tumor is still mediocre. This is mainly because tumor cells can hijack ACT T cells' immune checkpoint pathways to exert immunosuppression in the tumor microenvironment. Immune Checkpoint Inhibitors such as anti-PD-1 (aPD1) can counter the immunosuppression, but the synergizing effects of aPD1 to ACT was still not satisfactory. Here we demonstrate an approach to safely anchor aPD1-formed nanogels onto T cell surface via bio-orthogonal click chemistry before adoptive transfer. The spatial-temporal co-existence of aPD1 with ACT T cells and the responsive drug release significantly improved the treatment outcome of ACT in murine solid tumor model. The average tumor weight of the group treated by cell-surface anchored aPD1 was only 18 % of the group treated by equivalent dose of free aPD1 and T cells. The technology can be broadly applicable in ACTs employing natural or Chimeric Antigen Receptor (CAR) T cells.

Study on Enhanced Dissolution of Azilsartan-Loaded Solid Dispersion, Prepared by Combining Wet Milling and Spray-Drying Technologies.

The purpose of this study was to develop a combination method of wet milling and spray-drying technologies to prepare the solid dispersion and improve the dissolution rate of poorly water-soluble drug candidates. Azilsartan (AZL) was selected as the model drug for its poor water solubility. In the study, AZL-loaded solid dispersion was prepared with polyethylene glycol 6000 (PEG6000) and hydroxypropyl cellulose with super low viscosity (HPC-SL) as stabilizers by using combination of wet grinding and spray-drying methods. The high AZL loading solid dispersion was then characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and Fourier transform infrared spectroscopy (FTIR). Besides, dissolution test was carried out by the paddle method and stability investigation was also conducted. As a result, the dissolution rate of the solid dispersion tablets was found to be greater than conventional tablets, but in close agreement with market tablets. Furthermore, the formulation was shown to be stable at 40 ± 2°C and 75 ± 5% for at least 6 months, owing to its decreased particle size, morphology, and its crystal form. It was concluded that the combination of wet milling and spray-drying approaches to prepare solid dispersion would be a prospective method to improve the dissolution rate of poorly water-soluble drugs.

Biomimetic production of silk-like recombinant squid sucker ring teeth proteins.

The sucker ring teeth (SRT) of Humboldt squid exhibit mechanical properties that rival those of robust engineered synthetic polymers. Remarkably, these properties are achieved without a mineral phase or covalent cross-links. Instead, SRT are exclusively made of silk-like proteins called "suckerins", which assemble into nanoconfined ß-sheet reinforced supramolecular networks. In this study, three streamlined strategies for full-length recombinant suckerin protein production and purification were developed. Recombinant suckerin exhibited high solubility and colloidal stability in aqueous-based solvents. In addition, the colloidal suspensions exhibited a concentration-dependent conformational switch, from random coil to ß-sheet enriched structures. Our results demonstrate that recombinant suckerin can be produced in a facile manner in E. coli and processed from mild aqueous solutions into materials enriched in ß-sheets. We suggest that recombinant suckerin-based materials offer potential for a range of biomedical and engineering applications.

Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics.

Recent advancements in biopharmaceutical industry have facilitated the development of novel bioactive macromolecular therapeutics. One of the challenges towards the clinical use of these biomacromolecules lies in the selection of appropriate carriers to protect, deliver and release them in vivo to maximize their pharmacological effects. Micro/nanoparticles made from biodegradable poly (d,l-lactic-co-glycolic acid) (PLGA) have been explored as delivery vehicles for therapeutics. Due to their excellent biocompatibility and controllable biodegradability, PLGA micro/nanoparticles could protect macromolecules from instant degradation in vivo while allowing tunable release rate and profile. In this review, recent progress in the design, fabrication/formulation and application of PLGA based micro/nanoparticles for the controlled delivery of biomacromolecules are discussed. Special focuses will be on the novel loading methods and releasing mechanisms of macromolecules as well as the in vivo applications of therapeutic macromolecule-loaded PLGA micro/nanoparticles.

Squid suckerin microneedle arrays for tunable drug release.

Microneedles are increasingly used in transdermal delivery of therapeutic agents due to the elimination of first-pass metabolism, simplicity of operation, and lack of pain, which collectively lead to improved patient compliance. However, microneedles are still met by challenges with regard to the choice of biocompatible materials and the control of drug release profiles. Herein, we tackle these limitations by producing microneedles from a biocompatible robust biopolymer, namely squid sucker ring teeth (SRT) proteins (suckerins), using a soft lithography method. Taking advantage of the modular sequence design of suckerins leading to their self-assembly into ß-sheet enriched structures, suckerin microneedles display an accurate replication of their templates with robust mechanical properties, endowing them with a high skin penetration capability. Critically, the ß-sheet content in the microneedles can be modulated by varying the solvent conditions, which allows tuning of the mechanical response, and in turn the drug release rates by more than one order of magnitude. In vitro skin permeation studies of suckerin microneedles using human cadaver skin samples suggest a fast onset and enhanced skin permeation of drugs compared to flat patches. The skin permeation can also be tailored 10-fold by applying hydrogen bond disruptor solutions. As a proof-of-concept, the anti-bacterial drug kanamycin is encapsulated within the microneedles, leading to efficient anti-bacterial activity and offering an additional benefit to further minimize the risk of infections caused by microneedle-based drug delivery systems. Lastly, suckerin microneedles are found to be biocompatible in cell culture studies, opening the door to further clinical applications.

Supramolecular ß-Sheets Stabilized Protein Nanocarriers for Drug Delivery and Gene Transfection.

Suckerin proteins, recently discovered in the sucker ring teeth of squids, represent a family of promising structural biomacromolecules that can form supramolecular networks stabilized by nanoconfined ß-sheets. Exploiting this feature as well as their specific amino acid composition, we demonstrate that artificial suckerin-19 (S-19) can be engineered into nanocarriers for efficient drug delivery and gene transfection in vitro and in vivo. First, we demonstrate that S-19 self-assembles into ß-sheet stabilized nanoparticles with controlled particle sizes of 100-200 nm that are able to encapsulate hydrophobic drugs for pH-dependent release in vitro, and that can effectively inhibit tumor growth in vivo. We also show that S-19 can complex and stabilize plasmid DNA, with the complexes stabilized by hydrophobic interactions of the ß-sheet domains as opposed to electrostatic interactions commonly achieved with cationic polymers, thus lowering cytotoxicity. The elevated Histidine content of S-19 appears critical to trigger endosomal escape by the proton sponge effect, thereby ensuring efficient gene transfection both in vitro and in vivo. Our study demonstrates that S-19 represents a promising functional protein nanocarrier that could be used for various drug and gene delivery applications.

From Soft Self-Healing Gels to Stiff Films in Suckerin-Based Materials Through Modulation of Crosslink Density and ß-Sheet Content.

Suckerins are block-copolymer-like structural proteins constituting the building blocks of the strong squid sucker-ring teeth. Here, recombinant suckerin-19 is processed into biomaterials spanning a wide range of elasticity, from very soft hydrogels to stiff films with elastic modulus in the gigapascal range. The elasticity is controlled by the interplay between the ß-sheet content and induced di-tyrosine crosslinking.

Multi-scale thermal stability of a hard thermoplastic protein-based material.

Although thermoplastic materials are mostly derived from petro-chemicals, it would be highly desirable, from a sustainability perspective, to produce them instead from renewable biopolymers. Unfortunately, biopolymers exhibiting thermoplastic behaviour and which preserve their mechanical properties post processing are essentially non-existent. The robust sucker ring teeth (SRT) from squid and cuttlefish are one notable exception of thermoplastic biopolymers. Here we describe thermoplastic processing of squid SRT via hot extrusion of fibres, demonstrating the potential suitability of these materials for large-scale thermal forming. Using high-resolution in situ X-ray diffraction and vibrational spectroscopy, we elucidate the molecular and nanoscale features responsible for this behaviour and show that SRT consist of semi-crystalline polymers, whereby heat-resistant, nanocrystalline ß-sheets embedded within an amorphous matrix are organized into a hexagonally packed nanofibrillar lattice. This study provides key insights for the molecular design of biomimetic protein- and peptide-based thermoplastic structural biopolymers with potential biomedical and 3D printing applications.

Nanoconfined ß-sheets mechanically reinforce the supra-biomolecular network of robust squid Sucker Ring Teeth.

The predatory efficiency of squid and cuttlefish (superorder Decapodiformes) is enhanced by robust Sucker Ring Teeth (SRT) that perform grappling functions during prey capture. Here, we show that SRT are composed entirely of related structural "suckerin" proteins whose modular designs enable the formation of nanoconfined ß-sheet-reinforced polymer networks. Thirty-seven previously undiscovered suckerins were identified from transcriptomes assembled from three distantly related decapodiform cephalopods. Similarity in modular sequence design and exon­intron architecture suggests that suckerins are encoded by a multigene family. Phylogenetic analysis supports this view, revealing that suckerin genes originated in a common ancestor ~350 MYa and indicating that nanoconfined ß-sheet reinforcement is an ancient strategy to create robust bulk biomaterials. X-ray diffraction, nanomechanical, and micro-Raman spectroscopy measurements confirm that the modular design of the suckerins facilitates the formation of ß-sheets of precise nanoscale dimensions and enables their assembly into structurally robust supramolecular networks stabilized by cooperative hydrogen bonding. The suckerin gene family has likely played a key role in the evolutionary success of decapodiform cephalopods and provides a large molecular toolbox for biomimetic materials engineering.

Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science.

Efforts to engineer new materials inspired by biological structures are hampered by the lack of genomic data from many model organisms studied in biomimetic research. Here we show that biomimetic engineering can be accelerated by integrating high-throughput RNA-seq with proteomics and advanced materials characterization. This approach can be applied to a broad range of systems, as we illustrate by investigating diverse high-performance biological materials involved in embryo protection, adhesion and predation. In one example, we rapidly engineer recombinant squid sucker ring teeth proteins into a range of structural and functional materials, including nanopatterned surfaces and photo-cross-linked films that exceed the mechanical properties of most natural and synthetic polymers. Integrating RNA-seq with proteomics and materials science facilitates the molecular characterization of natural materials and the effective translation of their molecular designs into a wide range of bio-inspired materials.

Sustained delivery of endostatin improves the efficacy of therapy in Lewis lung cancer model.

The purpose of this work was to develop an effective delivery system for antiangiogenic therapy. Endostatin was microencapsulated into poly(lactic-co-glycolic acid) (PLGA) microspheres by using a w/o/o multiple emulsification-evaporation technique. Endostatin microspheres showed the encapsulation efficiency 100% with mean particle size about 25 microm. Endostatin released in vitro from PLGA microspheres were biologically active and significantly inhibited the migration of endothelial cells. In rats, endostatin microspheres produced a sustained release process in which the steady-state concentration was reached from day 5 to day 27 with the steady-state levels of endostatin between 174.8+/-33.3 and 351.3+/-126.3 ng/ml. In Lewis lung cancer model, a dose of 10 mg/kg endostatin microspheres was just as effective in suppressing tumor growth as a dose of 2 mg/kg/day free endostatin for 35 days (total dose 70 mg/kg). These results indicated PLGA microspheres further reduced the amount of endostatin needed to achieve significant tumor inhibition in mice when compared with systemic administration.