Co-encapsulation of granzyme B and perforin in nanocapsules for tumour therapy: biomimicking immune cells.

Granzyme B (GrB)-based immunotherapy is of interest for cancer treatment. However, insufficient cellular uptake and a lack of targeting remain challenges to make use of GrB for solid tumour therapy. As GrB induced cell death requires the help of perforin (PFN), we designed a system (nGPM) for the co-delivery of GrB and PFN. Therefore, GrB and PFN were loaded in a porous polymeric nanocapsule rich in acetylcholine analogues and matrix metalloproteinase-2 (MMP-2) responsive peptides. The neutrally charged nGPM nanocapsules showed as long circulating time and accumulated at the tumour sites. Once in the tumour the outside shell of nanocapsules became degraded by overexpressed MMP-2 proteases, resulting in the release of GrB and PFN. We found that the PFN complex formed small pores on the surface of tumour cells which allow GrB to enter the cytoplasm of tumour cells inducing cell apoptosis and tumour suppression significantly.

Multistage-Responsive Dual-Enzyme Nanocascades for Synergistic Radiosensitization-Starvation Cancer Therapy.

Radiotherapy is a common cancer treatment approach in clinical practice, yet its efficacy has been restricted by tumor hypoxia. Nanomaterials-mediated systemic delivery of glucose oxidase (GOx) and catalase (CAT) or CAT-like nanoenzymes holds the potential to enhance tumor oxygenation. However, they face the challenge of intermediate (hydrogen peroxide [H2 O2 ]) escape during systemic circulation if the enzyme pair is not closely placed to largely decompose H2 O2 , leading to oxidative stress on normal tissues. In the present study, a oxygen-generating nanocascade, n(GOx-CAT)C7A , constructed by strategically placing an enzymatic cascade (GOx and CAT) within a polymeric coating rich in hexamethyleneimine (C7A) moieties, is reported. During blood circulation, C7A remains predominantly non-protonated , achieving prolonged blood circulation due to its low-fouling surface. Once n(GOx-CAT)C7A reaches the tumor site, the acidic tumor microenvironment (TME) induces protonation of C7A moieties, resulting in a positively charged surface for enhanced tumor transcytosis. Moreover, GOx and CAT are covalently conjugated into close spatial proximity (<10 nm) for effective H2 O2  elimination. As demonstrated by the in vivo results, n(GOx-CAT)C7A achieves effective tumor retention and oxygenation, potent radiosensitization and antitumor effects. Such a dual-enzyme nanocascade for smart O2  delivery holds great potential for enhancing the hypoxia-compromised cancer therapies.

Surfaces Coated with Polymer Brushes Work as Carriers for Histidine Ammonia Lyase.

The immobilization of enzymes on solid supports is an important challenge in biotechnology and biomedicine. In contrast to other methods, enzyme deposition in polymer brushes offers the benefit of high protein loading that preserves enzymatic activity in part due to the hydrated 3D environment that is available within the brush structure. The authors equipped planar and colloidal silica surfaces with poly(2-(diethylamino)ethyl methacrylate)-based brushes to immobilize Thermoplasma acidophilum histidine ammonia lyase, and analyzed the amount and activity of the immobilized enzyme. The poly(2-(diethylamino)ethyl methacrylate) brushes are attached to the solid silica supports either via a "grafting-to" or a "grafting-from" method. It is found that the grafting-from method results in higher amounts of deposited polymer and, consequently, higher amounts of Thermoplasma acidophilum histidine ammonia lyase. All polymer brush-modified surfaces show preserved catalytic activity of the deposited Thermoplasma acidophilum histidine ammonia lyase. However, immobilizing the enzyme in polymer brushes using the grafting-from method resulted in twice the enzymatic activity from the grafting-to approach, illustrating a successful enzyme deposition on a solid support.

A novel Granzymes and miRNA nanocapsules co-delivery system for tumor suppression.

Granzymes-based immunotherapy for the treatment of solid tumors has gained great success and played more and more important effect in clinical studies. However, the antitumor effect of Granzymes still requires improvements owing to the cell evasion and metastasis of cancer. To overcome these limitations, synergistic combinatorial anti-tumor effect of Granzyme B (GrB) and miR-21 inhibitor (miR-21i) for breast cancer therapy through a new co-delivery system was investigated in present study. GrB was covalently bonded with miR-21i by disulfide bond and encapsulated in a nanocapsule formed byin situpolymerization of N -(3-aminopropyl) methacrylamide (APM), ethylene glycol dimethacrylate (EGDMA) and 2-Methacryloyloxyethyl phosphorylcholine (MPC). The nanocapsules possessed spherical and uniform diameter size as well as pH responsiveness in various environments. MTT and flow cytometry analysis showed that a synergistic anti-proliferation and promoting apoptosis effect was achieved when the nanocapsules were added into breast cancer cell lines. More importantly, the cell evasion ability was markedly inhibited using the nanocapusles detected through transwell invasion assay. Also thein vivoanti-tumor therapeutic efficacy of GrB-miR-21i nanocapusles was evaluated in a mouse tumor model. In conclusion, the nanocapsules for simultaneously delivery of GrB and miR-21i produce a synergistic effect in human breast cancer therapy.

An ultrasensitive electrochemiluminescent sensing platform for oxygen metabolism based on bioactive magnetic beads.

Reactive oxygen species (ROS), produced during oxygen metabolism, participate in and regulate various life processes. It is of great significance to monitor ROS in biological organs to further study oxygen metabolism. Herein, an ultrasensitive sensing platform is developed with electrochemiluminescent (ECL) signalling by integrating bioactive magnetic beads (BMBs) on indium tin oxide (ITO) coated glass using a magnet. For the first time, AuNPs were successfully deposited on Fe3O4 NPs in situ by reduction of α-ketoglutaric acid (α-KG), therefore the electroactive protein, haemoglobin (Hb) or cytochrome C (Cyt C), was assembled on via covalent bonds. The protein can realize direct electron transfer (DET) and catalyse the redox of ROS, reaching a detection limit of 6.21 µM or 0.6 µM of H2O2. Also Au@Fe3O4 NPs efficiently enhanced the ECL of luminol, promoting the sensing ability for ROS. This simultaneous effect endows the platform with low LOD of ROS for 7.69 nM (Hb), or 1.97 nM (Cyt C). Finally, the feasibility and practicality of the sensing platform were verified by monitoring the ROS released from mouse myocardial tissue.

Polymer Micelles vs Polymer-Lipid Hybrid Vesicles: A Comparison Using RAW 264.7 Cells.

Bottom-up synthetic biology aims to integrate artificial moieties with living cells and tissues. Here, two types of structural scaffolds for artificial organelles were compared in terms of their ability to interact with macrophage-like murine RAW 264.7 cells. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) was used to assemble micelles and polymer-lipid hybrid vesicles together with 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids in the latter case. In addition, the pH-sensitive fusogenic peptide GALA was conjugated to the carriers to improve their lysosomal escape ability. All assemblies had low short-term toxicity toward macrophage-like murine RAW 264.7 cells, and the cells internalized both the micelles and hybrid vesicles within 24 h. Assemblies containing DOPE lipids or GALA in their building blocks could escape the lysosomes. However, the intracellular retention of the building blocks was only a few hours in all the cases. Taken together, the provided comparison between two types of potential scaffolds for artificial organelles lays out the fundamental understanding required to advance soft material-based assemblies as intracellular nanoreactors.

Locomotion of micromotors in paper chips.

Locomotion of nano/micromotors in non-aqueous environments remains a challenging task. We assembled magnetic micromotors with different surface coatings and explored their locomotion in paper chips. Poly(L-lysine) deposition resulted in positively charged micromotors. Immobilized cellulase was used to increase the micromotors' paper penetration depth while a polyethylene glycol (PEG) coating was employed to limit the interaction between the micromotors and the cellulose fibers. All micromotors were able to move in the top layers of the paper chips with velocities dependent on the magnetic forces used to induce their locomotion, their sizes and the types of employed paper chips. Maximum speeds of up to ∼25 µm s-1 were observed for PEGylated micromotors in the fibrous cellulose environment. This type of micromotors has the potential to be considered in the area of paper microfluidics to facilitate distribution, or collection of moieties for biosensing or cell culture.

Enzyme Mimic Facilitated Artificial Cell to Mammalian Cell Signal Transfer.

Catalyzing biochemical reactions with enzymes and communicating with neighboring cells via chemical signaling are two fundamental cellular features that play a critical role in maintaining the homeostasis of organisms. Herein, we present an artificial enzyme (AE) facilitated signal transfer between artificial cells (ACs) and mammalian HepG2 cells. We synthesize metalloporphyrins (MPs) based AEs that mimic cytochrome P450 enzymes (CYPs) to catalyze a dealkylation and a hydroxylation reaction, exemplified by the conversion of resorufin ethyl ether (REE) to resorufin and coumarin (COU) to 7-hydroxycoumarin (7-HC), respectively. The AEs are immobilized in hydrogels to produce ACs that generate the two diffusive fluorophores, which can diffuse into HepG2 cells and result in dual intracellular emissions. This work highlights the use of AEs to promote AC to mammalian signal transfer, which opens up new opportunities for integrating the synthetic and living world with a bottom-up strategy.

Mitochondria Encapsulation in Hydrogel-Based Artificial Cells as ATP Producing Subunits.

Artificial cells (ACs) aim to mimic selected structural and functional features of mammalian cells. In this context, energy generation is an important challenge to be addressed when self-sustained systems are desired. Here, mitochondria isolated from HepG2 cells are employed as natural subunits that facilitate chemically driven adenosine triphosphate (ATP) synthesis. The successful mitochondria isolation is confirmed by monitoring the preserved inner membrane potential, the respiration, and the ATP production ability. The encapsulation of the isolated mitochondria in gelatin-based hydrogels results in similar initial ATP production compared to mitochondria in solution with a sustained ATP production over 24 h. Furthermore, luciferase is coencapsulated with the mitochondria in gelatin-based particles to create ACs and employ the in situ produced ATP to drive the catalytic conversion of d-luciferin. The coencapsulation of luciferase-loaded liposomes with mitochondria in gelatin-based hydrogels is additionally explored where the encapsulation of mitochondria and liposomes resulted in clustering effects that are likely contributing to the functional performance of the active entities. Taken together, mitochondria show potential in cell mimicry to facilitate energy-dependent processes.

Cell mimicry as a bottom-up strategy for hierarchical engineering of nature-inspired entities.

Artificial biology is an emerging concept that aims to design and engineer the structure and function of natural cells, organelles, or biomolecules with a combination of biological and abiotic building blocks. Cell mimicry focuses on concepts that have the potential to be integrated with mammalian cells and tissue. In this feature article, we will emphasize the advancements in the past 3-4 years (2017-present) that are dedicated to artificial enzymes, artificial organelles, and artificial mammalian cells. Each aspect will be briefly introduced, followed by highlighting efforts that considered key properties of the different mimics. Finally, the current challenges and opportunities will be outlined. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Giant Concentric Metallosupramolecule with Aggregation-Induced Phosphorescent Emission.

Fluorescent metallosupramolecules have received considerable attention due to their precisely controlled dimensions as well as the tunable photophysical and photochemical properties. However, phosphorescent analogues are still rare and limited to small structures with low-temperature phosphorescence. Herein, we report the self-assembly and photophysical studies of a giant, discrete metallosupramolecular concentric hexagon functionalized with six alkynylplatinum(II) bzimpy moieties. With a size larger than 10 nm and molecular weight higher than 26 000 Da, the assembled terpyridine-based supramolecule displayed phosphorescent emission at room temperature. Moreover, the supramolecule exhibited enhanced aggregation-induced phosphorescent emission compared to the ligand by tuning the aggregation states through intermolecular interactions and significant enhancement of emission to CO2 gas.

A novel Granzyme B nanoparticle delivery system simulates immune cell functions for suppression of solid tumors.

Cell-based immunotherapy for the treatment of hematologic malignancies, such as leukemia and lymphoma, has seen much success and played an increasingly important role in clinical studies. Nevertheless, the efficacy of immunotherapy in solid tumors still needs improvements due to the immunosuppressive properties of tumor cells and the microenvironment. To overcome these limitations, we prepared a novel tumor-targeting delivery system based on the underlying mechanism of immune-targeted cell death that encapsulated granzyme B protein within a porous polymeric nanocapsule. Methods: A cell-penetrating peptide TAT was attached onto granzyme B (GrB) to enhance its transmembrane transport efficiency and potency to induce cell apoptosis. The endocytosis and internalization pathways of GrB-TAT (GrB-T) were analyzed in comparison with perforin by confocal microscopy and flow cytometry. Furthermore, the positively charged GrB-T was wrapped into nanoparticles by p-2-methacryloyloxy ethyl phosphorylcholine (PMPC)-modified HA (hyaluronic acid). The nanoparticles (called TCiGNPs) were characterized in terms of zeta potential and by transmission electron microscopy (TEM). The in vitro anti-tumor effects of GrB-T were examined by cell apoptosis assay and Western blotting analysis. The in vivo anti-tumor therapeutic efficacy of TCiGNPs was evaluated in a mouse tumor model. Results: The TAT peptide could play a role similar to perforin to mediate direct transmembrane transfer of GrB and improve GrB-induced cell apoptosis. The TCiGNPs were successfully synthesized and accumulated in the solid tumor through enhanced permeability and retention (EPR) effect. In the tumor microenvironment, TCiGNPs could be degraded by hyaluronidase and triggered the release of GrB-T. The TAT peptide enabled the translocation of GrB across the plasma membrane to induce tumor cell apoptosis in vivo.Conclusion: We successfully developed a granzyme B delivery system with a GrB-T core and a PMPC/HA shell that simulated CTL/NK cell-mediated cancer immunotherapy mechanism. The GrB delivery system holds great promise for cancer treatment analogous to the CTL/NK cell-induced immunotherapy.

Disintegrating polymer multilayers to jump-start colloidal micromotors.

Colloidal systems with autonomous mobility are attractive alternatives to static particles for diverse applications. We present a complementary approach using pH-triggered disintegrating polymer multilayers for self-propulsion of swimmers. It is illustrated both experimentally and theoretically that homogenously coated swimmers exhibit higher velocity in comparison to their Janus-shaped counterparts. These swimmers show directional and random motion in microfluidic channels with a steep and shallow pH gradient, respectively. Further, a higher number of deposited polymer multilayers, steeper pH gradients and lower mass of the swimmers result in higher self-propulsion velocities. This new self-propulsion mechanism opens up unique opportunities to design, for instance, fast and yet biocompatible swimmers using the diverse tools of polymer chemistry to custom-synthesise the polymeric building blocks to assemble multilayers.

Supramolecular Kandinsky circles with high antibacterial activity.

Nested concentric structures widely exist in nature and designed systems with circles, polygons, polyhedra, and spheres sharing the same center or axis. It still remains challenging to construct discrete nested architecture at (supra)molecular level. Herein, three generations (G2-G4) of giant nested supramolecules, or Kandinsky circles, have been designed and assembled with molecular weight 17,964, 27,713 and 38,352 Da, respectively. In the ligand preparation, consecutive condensation between precursors with primary amines and pyrylium salts is applied to modularize the synthesis. These discrete nested supramolecules are prone to assemble into tubular nanostructures through hierarchical self-assembly. Furthermore, nested supramolecules display high antimicrobial activity against Gram-positive pathogen methicillin-resistant Staphylococcus aureus (MRSA), and negligible toxicity to eukaryotic cells, while the corresponding ligands do not show potent antimicrobial activity.

Hard Fusion Based Spectrum Sensing over Mobile Fading Channels in Cognitive Vehicular Networks.

An explosive growth in vehicular wireless applications gives rise to spectrum resource starvation. Cognitive radio has been used in vehicular networks to mitigate the impending spectrum starvation problem by allowing vehicles to fully exploit spectrum opportunities unoccupied by licensed users. Efficient and effective detection of licensed user is a critical issue to realize cognitive radio applications. However, spectrum sensing in vehicular environments is a very challenging task due to vehicle mobility. For instance, vehicle mobility has a large effect on the wireless channel, thereby impacting the detection performance of spectrum sensing. Thus, gargantuan efforts have been made in order to analyze the fading properties of mobile radio channel in vehicular environments. Indeed, numerous studies have demonstrated that the wireless channel in vehicular environments can be characterized by a temporally correlated Rayleigh fading. In this paper, we focus on energy detection for spectrum sensing and a counting rule for cooperative sensing based on Neyman-Pearson criteria. Further, we go into the effect of the sensing and reporting channel conditions on the sensing performance under the temporally correlated Rayleigh channel. For local and cooperative sensing, we derive some alternative expressions for the average probability of misdetection. The pertinent numerical and simulating results are provided to further validate our theoretical analyses under a variety of scenarios.

MiR-340 Inhibits Triple-Negative Breast Cancer Progression by Reversing EZH2 Mediated miRNAs Dysregulated Expressions.

The anti-tumor efficacy of miR-340 has been recently characterized in cancers. However, the underlying mechanisms of miR-340 inhibited cell growth and invasion in triple-negative breast cancer (TNBC) have not been well elucidated. In this study, we found that miR-340 expression was negatively correlated with EZH2 (Enhancer of zeste homolog 2) expression in TNBC tissues and cell lines. Subsequent luciferase reporter assay confirmed that EZH2 was a novel molecule target of miR-340. Upregulated miR-340 levels by mimics transfection significantly inhibited the MDA-MB-231 and MDA-MB-468 breast cancer cells proliferation, invasion and migration, and induced more cell apoptosis. Meanwhile, miR-340 inhibited the tumor growth in an orthotopic MDA-MB-231 breast cancer mouse model. Furthermore, we found the reduced EZH2 expression by miR-340 mimics transfection decreased the DNMT1, H3K27me3, ß-catenin and P-STAT3 expressions, which ultimately resulted in miR-21 activity blockage and miR-200a/b expression upregulation. The results of rescue experiments further confirmed that miR-340 inhibited triple-negative breast cancer progression through targeting EZH2. Taken together, our results identified miR-340 as a tumor suppressor in TNBC, moreover, an EZH2 medicated regulatory loop was established. Post-transcriptional suppression of EZH2 expression not only blocked STAT3 mediated miR-21 trans-activation, but also reversed the miR-200a/b silencing via reducing DNMT1 and H3K27me3 expressions. MiR-21 inhibition and miR-200a/b expression triggered by miR-340 ultimately cooperated in the TNBC progression.

Fluorescent Cross-Linked Supramolecular Polymer Constructed by Orthogonal Self-Assembly of Metal-Ligand Coordination and Host-Guest Interaction.

A new host molecule consists of four terpyridine groups as the binding sites with zinc(II) ion and a copillar[5]arene incorporated in the center as a spacer to interact with guest molecule was designed and synthesized. Due to the 120 ° angle of the rigid aromatic segment, a cross-linked dimeric hexagonal supramolecular polymer was therefore generated as the result of the orthogonal self-assembly of metal-ligand coordination and host-guest interaction. UV/Vis spectroscopy, (1) H NMR spectroscopy, viscosity and dynamic light-scattering techniques were employed to characterize and understand the cross-linking process with the introduction of zinc(II) ion and guest molecule. More importantly, well-defined morphology of the self-assembled supramolecular structure can be tuned by altering the adding sequence of the two components, that is, the zinc(II) ion and the guest molecule. In addition, introduction of a competitive ligand suggested the dynamic nature of the supramolecular structure.

Blood Exosomes Endowed with Magnetic and Targeting Properties for Cancer Therapy.

Exosomes are a class of naturally occurring nanoparticles that are secreted endogenously by mammalian cells. Clinical applications for exosomes remain a challenge because of their unsuitable donors, low scalability, and insufficient targeting ability. In this study, we developed a dual-functional exosome-based superparamagnetic nanoparticle cluster as a targeted drug delivery vehicle for cancer therapy. The resulting exosome-based drug delivery vehicle exhibits superparamagnetic behavior at room temperature, with a stronger response to an external magnetic field than individual superparamagnetic nanoparticles. These properties enable exosomes to be separated from the blood and to target diseased cells. In vivo studies using murine hepatoma 22 subcutaneous cancer cells showed that drug-loaded exosome-based vehicle delivery enhanced cancer targeting under an external magnetic field and suppressed tumor growth. Our developments overcome major barriers to the utility of exosomes for cancer application.

Two bent-shaped π-organogelators: synthesis, fluorescence, self-assembly and detection of volatile acid vapours in gel films and in gel-gel states.

Two novel bent-shaped π-organogelators 6a and 6b having different terminal pyridine rings as responsive sites were designed, synthesized and fully characterized. A subtle difference in the position of the N atom at the pyridine ring greatly affected their fluorescence and gelation properties. 6b showed remarkably stronger fluorescence both in solution and in the solid state as compared to 6a. Theoretical calculation revealed a clear discrepancy in the electron distribution between them. Furthermore, driven by π­π stacking interaction and hydrophobic interaction, both 6a and 6b can gelate several organic solvents with different polarities. Rheological studies, spectroscopic tests and powder X-ray diffraction showed that 6a displayed a closer stacking mode leading to stronger gel robustness. The xerogel films of 6a and 6b were prepared and utilized to detect acid vapours. Both of them can fulfil the detection of acid vapours through a distinct fluorescence change which could be seen by the naked eye under a UV lamp, but with different sensing modes. A rare gel to gel transformation was also observed upon exposure to acid vapours accompanied by a morphological change.

Efficient delivery of therapeutic miRNA nanocapsules for tumor suppression.

miRNA nanocapsules are synthesized with enhanced stability for miRNA delivery with high transduction efficiency, offering a novel class of miRNA vectors for cancer therapy.