Biomimetic Hemostatic Powder Derived from Coacervate-Immobilized Thermogelling Copolymers.

Intrinsic hemostasis is an innate body response to prevent bleeding based on the sol-gel transition of blood. However, it is often inadequate for exceptional situations, such as acute injury and coagulation disorders, which typically require immediate medical intervention. Herein, we report the preparation of an efficient hemostatic powder, composed of tannic acid (TA), poly(ethylene glycol) (PEG), and poly(d,l-lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) triblock copolymer (TB), for biomimetic hemostasis at the bleeding sites. TA has a high affinity for biomolecules and cells and can form coacervates with PEG driven by hydrogen bonding. TB enhances the mechanical strength and provides thermoresponsiveness. The hemostatic powder can rapidly transit into a physical and biodegradable seal on wet substrates under physiological conditions, demonstrating its promise for the generation of instant artificial clots. Importantly, this process is independent of the innate blood clotting process, which could benefit those with blood clotting disorders. This biomimetic hemostatic powder is an adaptive topical sealing agent for noncompressible and irregular wounds, which is promising for biomedical applications.

Engineering Poly(ethylene glycol) Nanoparticles for Accelerated Blood Clearance Inhibition and Targeted Drug Delivery.

Surface modification with poly(ethylene glycol) (PEGylation) is an effective strategy to improve the colloidal stability of nanoparticles (NPs) and is often used to minimize cellular uptake and clearance of NPs by the immune system. However, PEGylation can also trigger the accelerated blood clearance (ABC) phenomenon, which is known to reduce the circulation time of PEGylated NPs. Herein, we report the engineering of stealth PEG NPs that can avoid the ABC phenomenon and, when modified with hyaluronic acid (HA), show specific cancer cell targeting and drug delivery. PEG NPs cross-linked with disulfide bonds are prepared by using zeolitic imidazolate framework-8 NPs as templates. The reported templating strategy enables the simultaneous removal of the template and formation of PEG NPs under mild conditions (pH 5.5 buffer). Compared to PEGylated liposomes, PEG NPs avoid the secretion of anti-PEG antibodies and the presence of anti-PEG IgM and IgG did not significantly accelerate the blood clearance of PEG NPs, indicating the inhibition of the ABC effect for the PEG NPs. Functionalization of the PEG NPs with HA affords PEG NPs that retain their stealth properties against macrophages, target CD44-expressed cancer cells and, when loaded with the anticancer drug doxorubicin, effectively inhibit tumor growth. The innovation of this study lies in the engineering of PEG NPs that can circumvent the ABC phenomenon and that can be functionalized for the improved and targeted delivery of drugs.

Hot Melt Super Glue: Multi-Recyclable Polyphenol-Based Supramolecular Adhesives.

The rapid and facile synthesis of hot melt super glue (HMSG) via the formation of adhesive supramolecular networks between catechol or pyrogallol hydroxyl groups (-OH) of polyphenols and repeat units (-CH2 CH2 O-) of poly(ethylene glycol) (PEG) based on hydrogen bonds is reported. The adhesion strength of HMSG, processed by heating-cooling of polyphenols and PEG without additional solvents, can be tuned up to 8.8 MPa via changing the molecular weight of PEG and the ratio of hydrogen bonding donors and receptors. The advantages of the reported HMSG lie in the ease and scalability of the assembly process, rapid adhesion on various substrates with excellent processability, resistance of low temperature and organic solvents, and recyclable adhesion strength. The solvent-free HMSG represents a promising adhesive supramolecular network to expand the versatility and application of polyphenol-based materials.

Polypeptide Nanoparticles with pH-Sheddable PEGylation for Improved Drug Delivery.

The variation of tumor microenvironments provides a tool for the construction of stimulus-responsive nanomedicines to enhance drug delivery efficacy. Herein, the assembly of drug-loaded polypeptide nanoparticles (NPs) with pH-sheddable modification of poly(ethylene glycol) (PEG) is prepared to enhance therapeutic efficiency. Poly(l-lysine) and poly(l-glutamic acid) were self-assembled to fabricate polypeptide NPs by electrostatic interactions, followed by PEGylation based on amidation reaction. The NP sizes can be controlled by tuning the molecular weight or the ratio of polypeptides. The PEG coating is cleavable at the tumor acid microenvironment to reverse the surface charge and reduce the NP size, which effectively enhances cell uptake. In addition, the presence of reducing reagent (e.g., glutathione) in cancer cells induces the drug (i.e., cisplatin) release from the polypeptide NPs and subsequently results in the cell toxicity. This reported method highlights the engineering of transformable polypeptide drug carriers, which provides a promising way for enhanced drug delivery efficacy.

Ligand-Functionalized Poly(ethylene glycol) Particles for Tumor Targeting and Intracellular Uptake.

Drug carriers typically require both stealth and targeting properties to minimize nonspecific interactions with healthy cells and increase specific interaction with diseased cells. Herein, the assembly of targeted poly(ethylene glycol) (PEG) particles functionalized with cyclic peptides containing Arg-Gly-Asp (RGD) (ligand) using a mesoporous silica templating method is reported. The influence of PEG molecular weight, ligand-to-PEG molecule ratio, and particle size on cancer cell targeting to balance stealth and targeting of the engineered PEG particles is investigated. RGD-functionalized PEG particles (PEG-RGD particles) efficiently target U-87 MG cancer cells under static and flow conditions in vitro, whereas PEG and cyclic peptides containing Arg-Asp-Gly (RDG)-functionalized PEG (PEG-RDG) particles display negligible interaction with the same cells. Increasing the ligand-to-PEG molecule ratio improves cell targeting. In addition, the targeted PEG-RGD particles improve cell uptake via receptor-mediated endocytosis, which is desirable for intracellular drug delivery. The PEG-RGD particles show improved tumor targeting (14% ID g-1) when compared with the PEG (3% ID g-1) and PEG-RDG (7% ID g-1) particles in vivo, although the PEG-RGD particles show comparatively higher spleen and liver accumulation. The targeted PEG particles represent a platform for developing particles aimed at balancing nonspecific and specific interactions in biological systems.

Nanoengineering of Poly(ethylene glycol) Particles for Stealth and Targeting.

The assembly of particles composed solely or mainly of poly(ethylene glycol) (PEG) is an emerging area that is gaining increasing interest within bio-nano science. PEG, widely considered to be the "gold standard" among polymers for drug delivery, is providing a platform for exploring fundamental questions and phenomena at the interface between particle engineering and biomedicine. These include the targeting and stealth behaviors of synthetic nanomaterials in biological environments. In this feature article, we discuss recent work in the nanoengineering of PEG particles and explore how they are enabling improved targeting and stealth performance. Specific examples include PEG particles prepared through surface-initiated polymerization, mesoporous silica replication via postinfiltration, and particle assembly through metal-phenolic coordination. This particle class exhibits unique in vivo behavior (e.g., biodistribution and immune cell interactions) and has recently been explored for drug delivery applications.

Tuning the Properties of Polymer Capsules for Cellular Interactions.

Particle-cell interactions are governed by, among other factors, the composition and surface properties of the particles. Herein, we report the preparation of various polymer capsules with different compositions and properties via atom transfer radical polymerization mediated continuous assembly of polymers (CAPATRP), where the cellular interactions of these capsules, particularly fouling and specific targeting, are examined by flow cytometry and deconvolution microscopy. Acrylated eight-arm poly(ethylene glycol) (8-PEG) and poly(N-(2-hydroxypropyl)-methacrylamide) (PHPMA) as well as methacrylated hyaluronic acid (HA), poly(glutamic acid) (PGA), and poly(methacrylic acid) (PMA) are used as macro-cross-linkers to obtain a range of polymer capsules with different compositions (PEG, PHPMA, HA, PGA, and PMA). Capsules composed of low-fouling polymers, PEG and PHPMA, show negligible association with macrophage Raw 264.7, monocyte THP-1, and HeLa cells. HA capsules, although moderately low-fouling (<22%) to HeLa, BT474, Raw 264.7, and THP-1 cells, exhibit high targeting specificity to CD44-over-expressing MDA-MB-231 cells. In contrast, PGA and PMA capsules show high cellular association toward phagocytic Raw 264.7 and THP-1 cells. These findings demonstrate the capability of the CAPATRP technique in preparing polymer capsules with specific cellular interactions.

Nanoengineered Templated Polymer Particles: Navigating the Biological Realm.

Nanoengineered materials offer tremendous promise for developing the next generation of therapeutics. We are transitioning from simple research questions, such as "can this particle eradicate cancer cells?" to more sophisticated ones like "can we design a particle to preferentially deliver cargo to a specific cancer cell type?" These developments are poised to usher in a new era of nanoengineered drug delivery systems. We primarily work with templating methods for engineering polymer particles and investigate their biological interactions. Templates are scaffolds that facilitate the formation of particles with well-controlled size, shape, structure, stiffness, stability, and surface chemistry. In the past decade, breakthroughs in engineering new templates, combined with advances in coating techniques, including layer-by-layer (LbL) assembly, surface polymerization, and metal-phenolic network (MPN) coordination chemistry, have enabled particles with specific physicochemical properties to be engineered. While materials science offers an ever-growing number of new synthesis techniques, a central challenge of therapeutic delivery has become understanding how nanoengineered materials interact with biological systems. Increased collaboration between chemists, biologists, and clinicians has resulted in a vast research output on bio-nano interactions. Our understanding of cell-particle interactions has grown considerably, but conventional in vitro experimentation provides limited information, and understanding how to bridge the in vitro/in vivo gap is a continuing challenge. As has been demonstrated in other fields, there is now a growing interest in applying computational approaches to advance this area. A considerable knowledge base is now emerging, and with it comes new and exciting opportunities that are already being capitalized on through the translation of materials into the clinic. In this Account, we outline our perspectives gained from a decade of work at the interface between polymer particle engineering and bio-nano interactions. We divide our research into three areas: (i) biotrafficking, including cellular association, intracellular transport, and biodistribution; (ii) biodegradation and how to achieve controlled, responsive release of therapeutics; and (iii) applications, including drug delivery, controlling immunostimulatory responses, biosensing, and microreactors. There are common challenges in these areas for groups developing nanoengineered therapeutics. A key "lesson-learned" has been the considerable challenge of staying informed about the developments relevant to this field. There are a number of reasons for this, most notably the interdisciplinary nature of the work, the large numbers of researchers and research outputs, and the limited standardization in technique nomenclature. Additionally, a large body of work is being generated with limited central archiving, other than vast general databases. To help address these points, we have created a web-based tool to organize our past, present, and future work [Bio-nano research knowledgebase, http://bionano.eng.unimelb.edu.au/knowledge_base/ (accessed May 2, 2016)]. This tool is intended to serve as a first step toward organizing results in this large, complex area. We hope that this will inspire researchers, both in generating new ideas and also in collecting, collating, and sharing their experiences to guide future research.

An Enzyme-Coated Metal-Organic Framework Shell for Synthetically Adaptive Cell Survival.

A bioactive synthetic porous shell was engineered to enable cells to survive in an oligotrophic environment. Eukaryotic cells (yeast) were firstly coated with a ß-galactosidase (ß-gal), before crystallization of a metal-organic framework (MOF) film on the enzyme coating; thereby producing a bioactive porous synthetic shell. The ß-gal was an essential component of the bioactive shell as it generated nutrients (that is, glucose and galactose) required for cell viability in nutrient-deficient media (lactose-based). Additionally, the porous MOF coating carried out other vital functions, such as 1) shielding the cells from cytotoxic compounds and radiation, 2) protecting the non-native enzymes (ß-gal in this instance) from degradation and internalization, and 3) allowing for the diffusion of molecules essential for the survival of the cells. Indeed, this bioactive porous shell enabled the survival of cells in simulated extreme oligotrophic environments for more than 7 days, leading to a decrease in cell viability less than 30 %, versus a 99 % decrease for naked yeast. When returned to optimal growth conditions the bioactive porous exoskeleton could be removed and the cells regained full growth immediately. The construction of bioactive coatings represents a conceptually new and promising approach for the next-generation of cell-based research and application, and is an alternative to synthetic biology or genetic modification.

Shape-Dependent Activation of Cytokine Secretion by Polymer Capsules in Human Monocyte-Derived Macrophages.

Particles with tailored geometries have received significant attention due to their specific interactions with biological systems. In this work, we examine the effect of polymer capsule shape on cytokine secretion by human monocyte-derived macrophages. Thiolated poly(methacrylic acid) (PMASH) polymer capsules with different shapes (spherical, short rod-shaped, and long rod-shaped) were prepared by layer-by-layer assembly. The effect of PMASH capsule shape on cellular uptake and cytokine secretion by macrophages differentiated from THP-1 monocytes (dTHP-1) was investigated. PMASH capsules with different shapes were internalized to a similar extent in dTHP-1 cells. However, cytokine secretion was influenced by capsule geometry: short rod-shaped PMASH capsules promoted a stronger increase in TNF-α and IL-8 secretion compared with spherical (1.7-fold in TNF-α and 2.1-fold in IL-8) and long rod-shaped (2.8-fold in TNF-α and 2.0-fold in IL-8) PMASH capsules in dTHP-1 cells (capsule-to-cell ratio of 100:1). Our results indicate that the immunological response based on the release of cytokines is influenced by the shape of the polymer capsules, which could be potentially exploited in the rational design of particle carriers for vaccine delivery.

Engineered Metal-Phenolic Capsules Show Tunable Targeted Delivery to Cancer Cells.

We engineered metal-phenolic capsules with both high targeting and low nonspecific cell binding properties. The capsules were prepared by coating phenolic-functionalized hyaluronic acid (HA) and poly(ethylene glycol) (PEG) on calcium carbonate templates, followed by cross-linking the phenolic groups with metal ions and removing the templates. The incorporation of HA significantly enhanced binding and association with a CD44 overexpressing (CD44+) cancer cell line, while the incorporation of PEG reduced nonspecific interactions with a CD44 minimal-expressing (CD44-) cell line. Moreover, high specific targeting to CD44+ cells can be balanced with low nonspecific binding to CD44- cells simply by using an optimized feed-ratio of HA and PEG to vary the content of HA and PEG incorporated into the capsules. Loading an anticancer drug (i.e., doxorubicin) into the obtained capsules resulted in significantly higher cytotoxicity to CD44+ cells but lower cytotoxicity to CD44- cells.

Engineering Polymer Hydrogel Nanoparticles for Lymph Node-Targeted Delivery.

The induction of antigen-specific adaptive immunity exclusively occurs in lymphoid organs. As a consequence, the efficacy by which vaccines reach these tissues strongly affects the efficacy of the vaccine. Here, we report the design of polymer hydrogel nanoparticles that efficiently target multiple immune cell subsets in the draining lymph nodes. Nanoparticles are fabricated by infiltrating mesoporous silica particles (ca. 200 nm) with poly(methacrylic acid) followed by disulfide-based crosslinking and template removal. PEGylation of these nanoparticles does not affect their cellular association in vitro, but dramatically improves their lymphatic drainage in vivo. The functional relevance of these observations is further illustrated by the increased priming of antigen-specific T cells. Our findings highlight the potential of engineered hydrogel nanoparticles for the lymphatic delivery of antigens and immune-modulating compounds.

Flow-Based Assembly of Layer-by-Layer Capsules through Tangential Flow Filtration.

Layer-by-layer (LbL) assembly on nano- and microparticles is of interest for a range of applications, including catalysis, optics, sensors, and drug delivery. One current limitation is the standard use of manual, centrifugation-based (pellet/resuspension) methods to perform the layering steps, which can make scalable, highly controllable, and automatable production difficult to achieve. Here, we develop a fully flow-based technique using tangential flow filtration (TFF) for LbL assembly on particles. We demonstrate that multilayered particles and capsules with different sizes (from micrometers to submicrometers in diameter) can be assembled on different templates (e.g., silica and calcium carbonate) using several polymers (e.g., poly(allylamine hydrochloride), poly(styrenesulfonate), and poly(diallyldimethylammonium chloride)). The full system only contains fluidic components routinely used (and automated) in industry, such as pumps, tanks, valves, and tubing in addition to the TFF filter modules. Using the TFF LbL system, we also demonstrate the centrifugation-free assembly, including core dissolution, of drug-loaded capsules. The well-controlled, integrated, and automatable nature of the TFF LbL system provides scientific, engineering, and practical processing benefits, making it valuable for research environments and potentially useful for translating LbL assembled particles into diverse applications.

Engineering low-fouling and pH-degradable capsules through the assembly of metal-phenolic networks.

Metal-phenolic coordination chemistry provides a simple and rapid way to fabricate ultrathin films. Here, we report a facile strategy for the preparation of low-fouling and pH-degradable metal-phenolic network (MPN) capsules using a synthetic polyphenol derivative, poly(ethylene glycol) (PEG)-polyphenol, as a building block. PEG-MPN capsules exhibit reduced nonspecific protein adsorption and cell association compared with tannic acid (TA)-MPN capsules. In addition, they show faster disassembly at a biologically relevant pH (5) than TA-MPN capsules (80% in 5 h vs 30% in 10 days). PEG-MPN capsules combine both the low fouling properties of PEG and the advantages of the MPN-driven assembly process (e.g., fast assembly and pH-degradability).

Redox-Sensitive PEG-Polypeptide Nanoporous Particles for Survivin Silencing in Prostate Cancer Cells.

We report the engineering of intracellular redox-responsive nanoporous poly(ethylene glycol)-poly(l-lysine) particles (NPEG-PLLs). The obtained particles exhibit no toxicity while maintaining the capability to deliver a small interfering RNA sequence (siRNA) targeting the anti-apoptotic factor, survivin, in prostate cancer cells. The redox-mediated cleavage of the disulfide bonds stabilizing the NPEG-PLL-siRNA complex results in the release of bioactive siRNA into the cytosol of prostate cancer PC-3 cells, which, in turn, leads to the effective silencing (∼59 ± 8%) of the target gene. These findings, obtained under optimal conditions, indicate that NPEG-PLLs may protect the therapeutic nucleic acid in the extracellular and intracellular environments, thus preventing the occurrence of competitive interactions with serum and cytosolic proteins as well as degradation by RNase. The intracellular trafficking and final fate of the NPEG-PLLs were investigated by a combination of deconvolution microscopy, fluorescence lifetime imaging microscopy, and super-resolution structured illumination microscopy. A significant impairment of cell survival was observed in cells concomitantly exposed to paclitaxel and siRNA-loaded NPEG-PLLs. Overall, our findings indicate that NPEG-PLLs represent a highly loaded depot for the delivery of therapeutic nucleic acids to cancer cells.

Engineering enzyme-cleavable hybrid click capsules with a pH-sheddable coating for intracellular degradation.

The engineering of layer-by-layer (LbL) hybrid click capsules that are responsive to biological stimuli is reported. The capsules comprise a pH-sheddable, non cross-linked outer coating that protects enzyme-cleavable inner layers. Upon cellular uptake, the outer coating is released and the capsules are enzymatically degraded. In vitro cell degradation results in rapid capsule degradation (10 min) upon cellular internalization.

Surface-initiated polymerization within mesoporous silica spheres for the modular design of charge-neutral polymer particles.

We report a templating approach for the preparation of functional polymer replica particles via surface-initiated polymerization in mesoporous silica templates. Subsequent removal of the template resulted in discrete polymer particles. Furthermore, redox-responsive replica particles could be engineered to disassemble in a reducing environment. Particles, made of poly(methacryloyloxyethyl phosphorylcholine) (PMPC) or poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA), exhibited very low association to human cancer cells (below 5%), which renders the reported charge-neutral polymer particles a modular and versatile class of highly functional carriers with potential applications in drug delivery.

Fluidized bed layer-by-layer microcapsule formation.

Polymer microcapsules can be used as bioreactors and artificial cells; however, preparation methods for cell-like microcapsules are typically time-consuming, low yielding, and/or involve custom microfluidics. Here, we introduce a rapid (∼30 min per batch, eight layers), scalable (up to 500 mg of templates), and efficient (98% yield) microcapsule preparation technique utilizing a fluidized bed for the layer-by-layer (LbL) assembly of polymers, and we investigate the parameters that govern the formation of robust capsules. Fluidization in water was possible for particles of comparable diameter to mammalian cells (>5 µm), with the experimental flow rates necessary for fluidization matching well with the theoretical values. Important variables for polymer film deposition and capsule formation were the concentration of polymer solution and the molecular weight of the polymer, while the volume of the polymer solution had a negligible impact. In combination, increasing the polymer molecular weight and polymer solution concentration resulted in improved film deposition and the formation of robust microcapsules. The resultant polymer microcapsules had a thickness of ∼5.5 nm per bilayer, which is in close agreement with conventionally prepared (quiescent (nonflow) adsorption/centrifugation/wash) LbL capsules. The technique reported herein provides a new way to rapidly generate microcapsules (approximately 8 times quicker than the conventional means), while being also amenable to scale-up and mass production.

Tuning particle biodegradation through polymer-peptide blend composition.

We report the preparation of polymer-peptide blend replica particles via the mesoporous silica (MS) templated assembly of poly(ethylene glycol)-block-poly(2-diisopropylaminoethyl methacrylate-co-2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethyl methacrylate) (PEG45-b-P(DPA55-co-PgTEGMA4)) and poly(l-histidine) (PHis). PEG45-b-P(DPA55-co-PgTEGMA4) was synthesized by atom transfer radical polymerization (ATRP), and was coinfiltrated with PHis into poly(methacrylic acid) (PMA)-coated MS particles assembled from different peptide-to-polymer ratios (1:1, 1:5, 1:10, or 1:15). Subsequent removal of the sacrificial templates and PMA resulted in monodisperse, colloidally stable, noncovalently cross-linked polymer-peptide blend replica particles that were stabilized by a combination of hydrophobic interactions between the PDPA and the PHis, hydrogen bonding between the PEG and PHis backbone, and π-π stacking of the imidazole rings of PHis side chains at physiological pH (pH ∼ 7.4). The synergistic charge-switchable properties of PDPA and PHis, and the enzymatic degradability of PHis, make these particles responsive to pH and enzymes. In vitro studies, in simulated endosomal conditions and inside cells, demonstrated that particle degradation kinetics could be engineered (from 2 to 8 h inside dendritic cells) based on simple adjustment of the peptide-to-polymer ratio used.

Nanoscale engineering of low-fouling surfaces through polydopamine immobilisation of zwitterionic peptides.

We report a versatile approach for the design of substrate-independent low-fouling surfaces via mussel-inspired immobilisation of zwitterionic peptides. Using mussel-inspired polydopamine (PDA) coatings, zwitterionic glutamic acid- and lysine-based peptides were immobilised on various substrates, including noble metals, metal oxides, polymers, and semiconductors. The variation of surface chemistry and surface wettability upon surface treatment was monitored with X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. Following peptide immobilisation, the surfaces became more hydrophilic due to the strong surface hydration compared with PDA-coated surfaces. The peptide-functionalised surfaces showed resistance to human blood serum adsorption and also effectively prevented the adhesion of gram-negative and gram-positive bacteria (i.e., Escherichia coli and Staphylococcus epidermidis) and mammalian cells (i.e., NIH 3T3 mouse embryonic fibroblast cells). The versatility of mussel-inspired chemistry combined with the unique biological nature and tunability of peptides allows for the design of low-fouling surfaces, making this a promising coating technique for various applications.