Nano-Assembled Polyphosphazene Delivery System Enables Effective Intranasal Immunization with Nipah Virus Subunit Vaccine.

The ultimate vaccine against infections caused by Nipah virus should be capable of providing protection at the respiratory tract─the most probable port of entry for this pathogen. Intranasally delivered vaccines, which target nasal-associated lymphoid tissue and induce both systemic and mucosal immunity, are attractive candidates for enabling effective vaccination against this lethal disease. Herein, the water-soluble polyphosphazene delivery vehicle assembles into nanoscale supramolecular constructs with the soluble extracellular portion of the Hendra virus attachment glycoprotein─a promising subunit vaccine antigen against both Nipah and Hendra viruses. These supramolecular constructs signal through Toll-like receptor 7/8 and promote binding interactions with mucin─an important feature of effective mucosal adjuvants. High mass contrast of phosphorus-nitrogen backbone of the polymer enables a successful visualization of nanoconstructs in their vitrified state by cryogenic electron microscopy. Here, we characterize the self-assembly of polyphosphazene macromolecule with biologically relevant ligands by asymmetric flow field flow fractionation, dynamic light scattering, fluorescence spectrophotometry, and turbidimetric titration methods. Furthermore, a polyphosphazene-enabled intranasal Nipah vaccine candidate demonstrates the ability to induce immune responses in hamsters and shows superiority in inducing total IgG and neutralizing antibodies when benchmarked against the respective clinical stage alum adjuvanted vaccine. The results highlight the potential of polyphosphazene-enabled nanoassemblies in the development of intranasal vaccines.

Engineering Degradation Rate of Polyphosphazene-Based Layer-by-Layer Polymer Coatings.

Degradable layer-by-layer (LbL) polymeric coatings have distinct advantages over traditional biomedical coatings due to their precision of assembly, versatile inclusion of bioactive molecules, and conformality to the complex architectures of implantable devices. However, controlling the degradation rate while achieving biocompatibility has remained a challenge. This work employs polyphosphazenes as promising candidates for film assembly due to their inherent biocompatibility, tunability of chemical composition, and the buffering capability of degradation products. The degradation of pyrrolidone-functionalized polyphosphazenes was monitored in solution, complexes and LbL coatings (with tannic acid), providing the first to our knowledge comparison of solution-state degradation to solid-state LbL degradation. In all cases, the rate of degradation accelerated in acidic conditions. Importantly, the tunability of the degradation rate of polyphosphazene-based LbL films was achieved by varying film assembly conditions. Specifically, by slightly increasing the ionization of tannic acid (near neutral pH), we introduce electrostatic "defects" to the hydrogen-bonded pairs that accelerate film degradation. Finally, we show that replacing the pyrrolidone side group with a carboxylic acid moiety greatly reduces the degradation rate of the LbL coatings. In practical applications, these coatings have the versatility to serve as biocompatible platforms for various biomedical applications and controlled release systems.

Immunopotentiating Polyphosphazene Delivery Systems: Supramolecular Self-Assembly and Stability in the Presence of Plasma Proteins.

Studies on the biological performance of nanomedicines have been increasingly focused on the paradigm shifting role of the protein corona, which is imminently formed once the formulation is placed in a complex physiological environment. This phenomenon is predominantly studied in the context of protein adsorption science, while such interactions for water-soluble systems remain virtually unexplored. In particular, the importance of plasma protein binding is yet to be understood for pharmaceuticals designed on the basis of supramolecular architectures, which generally lack well-defined surfaces. Water-soluble ionic polyphosphazenes, clinically proven immunoadjuvants and vaccine delivery vehicles, represent an example of a system that requires supramolecular coassembly with antigenic proteins to attain an optimal immunopotentiating effect. Herein, the self-assembly behavior and stability of noncovalently bound complexes on the basis of a model antigen─hen egg lysozyme─and polyphosphazene adjuvant are studied in the presence of plasma proteins utilizing isothermal calorimetry, asymmetric flow field flow fractionation, dynamic light scattering, and size exclusion chromatography methods. The results demonstrate that although plasma proteins, such as human serum albumin (HSA), show detectable avidity to polyphosphazene, the strength of such interactions is significantly lower than that for the model antigen. Furthermore, thermodynamic parameters indicate different models of binding: entropy driven, which is consistent with the counterion release mechanism for albumin versus electrostatic interactions for lysozyme, which are characterized by beneficial enthalpy changes. In vitro protein release experiments conducted in Franz diffusion cells using enzyme-linked immunoassay detection suggest that the antigen-adjuvant complex stability is not adversely affected by the presence of the most physiologically abundant protein, which confirms the importance of the delivery modality for this immunoadjuvant. Moreover, HSA shows an unexpected stabilizing effect on complexes with high antigen load─an important consideration for further development of polyphosphazene adjuvanted vaccine formulations and their functional assessment.

Polyphosphazene-derived carbon modified nanowires for high-performance electrochemical energy storage.

Two one-dimensional nanowires, Fe3O4and MnO2nanowires, were modified with polyphosphazene-derived carbon (PZSC) usingin situpolymerization and high-temperature calcination methods. PZSC coated with MnO2nanowire (MnO2/PZSCNW) was designed as the positive electrode, while PZSC coated with Fe3O4nanowire (Fe3O4/PZSCNW) was designed as the negative electrode. Both MnO2/PZSCNW (+) and Fe3O4/PZSCNW (-) exhibit much larger specific capacities than the corresponding MnO2and Fe3O4nanowires, reaching 75.5 mAh g-1and 75.9 mAh g-1, respectively. The maximum specific capacity, power and energy density of MnO2/PZSCNW (+)//Fe3O4/PZSCNW (-) in alkaline electrolyte are up to 63.2 mAh g-1, 429.6 W kg-1and 53.7 Wh kg-1, respectively. After 10 000 cycles, the cell maintains 100% capacity. The experimental results indicate that the polyphosphazene-derived carbon coating can significantly improve the electrochemical performance, providing a feasible solution for constructing high-performance supercapacitors.

Phosphorus and Silicon-Based Macromolecules as Degradable Biomedical Polymers.

Synthetic polymers are indispensable in biomedical applications because they can be fabricated with consistent and reproducible properties, facile scalability, and customizable functionality to perform diverse tasks. However, currently available synthetic polymers have limitations, most notably when timely biodegradation is required. Despite there being, in principle, an entire periodic table to choose from, with the obvious exception of silicones, nearly all known synthetic polymers are combinations of carbon, nitrogen, and oxygen in the main chain. Expanding this to main-group heteroatoms can open the way to novel material properties. Herein the authors report on research to incorporate the chemically versatile and abundant silicon and phosphorus into polymers to induce cleavability into the polymer main chain. Less stable polymers, which degrade in a timely manner in mild biological environments, have considerable potential in biomedical applications. Herein the basic chemistry behind these materials is described and some recent studies into their medical applications are highlighted.

Fluorine-Functionalized Polyphosphazene Immunoadjuvant: Synthesis, Solution Behavior and In Vivo Potency.

The inclusion of fluorine motifs in drugs and drug delivery systems is an established tool for modulating their biological potency. Fluorination can improve drug specificity or boost the vehicle's ability to cross cellular membranes. However, the approach has yet to be applied to vaccine adjuvants. Herein, the synthesis of fluorinated bioisostere of a clinical stage immunoadjuvant-poly[di(carboxylatophenoxy)phosphazene], PCPP-is reported. The structure of water-soluble fluoropolymer-PCPP-F, which contains two fluorine atoms per repeat unit-was confirmed using 1H, 31P and 19F NMR, and its molecular mass and molecular dimensions were determined using size-exclusion chromatography and dynamic light scattering. Insertion of fluorine atoms in the polymer side group resulted in an improved solubility in acidic solutions and faster hydrolytic degradation rate, while the ability to self-assemble with an antigenic protein, lysozyme-an important feature of polyphosphazene vaccine adjuvants-was preserved. In vivo assessment of PCPP-F demonstrated its greater ability to induce antibody responses to Hepatitis C virus antigen when compared to its non-fluorinated counterpart. Taken together, the superior immunoadjuvant activity of PCPP-F, along with its improved formulation characteristics, demonstrate advantages of the fluorination approach for the development of this family of macromolecular vaccine adjuvants.

Design and Synthesis of Amphiphilic Graft Polyphosphazene Micelles for Docetaxel Delivery.

The structural versatility of polydichlorophosphazene derived from the inestimable possibilities to functionalize the two halogens, attached to each phosphazene main chain unit, attracted increasing attention in the last decade. This uncountable chemical derivatization is doubled by the amphiphilic roleplay demonstrated by polyphosphazenes containing twofold side-chained hydrophilic and hydrophobic moieties. Thus, it is able to encapsulate specific bioactive molecules for various targeted nanomedicine applications. A new amphiphilic graft, polyphosphazenes (PPP/PEG-NH/Hys/MAB), was synthesized via the thermal ring-opening polymerization of hexachlorocyclotriphosphazene, followed by a subsequent two-step substitution reaction of chlorine atoms with hydrophilic methoxypolyethylene glycol amine/histamine dihydrochloride adduct (PEG-NH2)/(Hys) and hydrophobic methyl-p-aminobenzoate (MAB), respectively. Fourier transform infrared spectroscopy (FTIR) and 1H and 31P-nuclear magnetic resonance spectroscopy (NMR) have been used to validate the expected architectural assembly of the copolymer. Docetaxel loaded micelles based on synthesized PPP/PEG-NH/Hys/MAB were designed by dialysis method. The micelles size was evaluated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The drug release profiles from the PPP/PEG-NH/Hys/MAB micelles were established. In vitro cytotoxicity tests of PPP/PEG-NH/Hys/MAB micelles loaded with Docetaxel revealed that designed polymeric micelles exhibited an increased cytotoxic effect on MCF-7 cells.

Degradable Bottlebrush Polypeptides and the Impact of their Architecture on Cell Uptake, Pharmacokinetics, and Biodistribution In Vivo.

Bottlebrush polymers are highly promising as unimolecular nanomedicines due to their unique control over the critical parameters of size, shape and chemical function. However, since they are prepared from biopersistent carbon backbones, most known bottlebrush polymers are non-degradable and thus unsuitable for systemic therapeutic administration. Herein, we report the design and synthesis of novel poly(organo)phosphazene-g-poly(α-glutamate) (PPz-g-PGA) bottlebrush polymers with exceptional control over their structure and molecular dimensions (Dh ≈ 15-50 nm). These single macromolecules show outstanding aqueous solubility, ultra-high multivalency and biodegradability, making them ideal as nanomedicines. While well-established in polymer therapeutics, it has hitherto not been possible to prepare defined single macromolecules of PGA in these nanosized dimensions. A direct correlation was observed between the macromolecular dimensions of the bottlebrush polymers and their intracellular uptake in CT26 colon cancer cells. Furthermore, the bottlebrush macromolecular structure visibly enhanced the pharmacokinetics by reducing renal clearance and extending plasma half-lives. Real-time analysis of the biodistribution dynamics showed architecture-driven organ distribution and enhanced tumor accumulation. This work, therefore, introduces a robust, controlled synthesis route to bottlebrush polypeptides, overcoming limitations of current polymer-based nanomedicines and, in doing so, offers valuable insights into the influence of architecture on the in vivo performance of nanomedicines.

Polyphosphazene-Based Biomaterials for Biomedical Applications.

Recently, synthetic polymers have attracted great interest in the field of biomedical science. Among these, polyphosphazenes (PPZs) are regarded as one of the most promising materials, due to their structural flexibility and biodegradability compared to other materials. PPZs have been developed through numerous studies. In particular, multi-functionalized PPZs have been proven to be potential biomaterials in various forms, such as nanoparticles (NPs) and hydrogels, through the introduction of various functional groups. Thus, PPZs have been applied for the delivery of therapeutic molecules (low molecular weight drugs, genes and proteins), bioimaging, phototherapy, bone regeneration, dental liners, modifiers and medical devices. The main goal of the present review is to highlight the recent and the most notable existing PPZ-based biomaterials for aforementioned applications, with future perspectives in mind.

Cyclo- and Polyphosphazenes for Biomedical Applications.

Cyclic and polyphosphazenes are extremely interesting and versatile substrates characterized by the presence of -P=N- repeating units. The chlorine atoms on the P atoms in the starting materials can be easily substituted with a variety of organic substituents, thus giving rise to a huge number of new materials for industrial applications. Their properties can be designed considering the number of repetitive units and the nature of the substituent groups, opening up to a number of peculiar properties, including the ability to give rise to supramolecular arrangements. We focused our attention on the extensive scientific literature concerning their biomedical applications: as antimicrobial agents in drug delivery, as immunoadjuvants in tissue engineering, in innovative anticancer therapies, and treatments for cardiovascular diseases. The promising perspectives for their biomedical use rise from the opportunity to combine the benefits of the inorganic backbone and the wide variety of organic side groups that can lead to the formation of nanoparticles, polymersomes, or scaffolds for cell proliferation. In this review, some aspects of the preparation of phosphazene-based systems and their characterization, together with some of the most relevant chemical strategies to obtain biomaterials, have been described.

Supramolecular Protein-Polyelectrolyte Assembly at Near Physiological Conditions-Water Proton NMR, ITC, and DLS Study.

The in vivo potency of polyphosphazene immunoadjuvants is inherently linked to the ability of these ionic macromolecules to assemble with antigenic proteins in aqueous solutions and form physiologically stable supramolecular complexes. Therefore, in-depth knowledge of interactions in this biologically relevant system is a prerequisite for a better understanding of mechanism of immunoadjuvant activity. Present study explores a self-assembly of polyphosphazene immunoadjuvant-PCPP and a model antigen-lysozyme in a physiologically relevant environment-saline solution and neutral pH. Three analytical techniques were employed to characterize reaction thermodynamics, water-solute structural organization, and supramolecular dimensions: isothermal titration calorimetry (ITC), water proton nuclear magnetic resonance (wNMR), and dynamic light scattering (DLS). The formation of lysozyme-PCPP complexes at near physiological conditions was detected by all methods and the avidity was modulated by a physical state and dimensions of the assemblies. Thermodynamic analysis revealed the dissociation constant in micromolar range and the dominance of enthalpy factor in interactions, which is in line with previously suggested model of protein charge anisotropy and small persistence length of the polymer favoring the formation of high affinity complexes. The paper reports advantageous use of wNMR method for studying protein-polymer interactions, especially for low protein-load complexes.

Hetero and homo α,ω-chain-end functionalized polyphosphazenes.

The control of chain-ends is fundamental in modern macromolecular chemistry for directed one-to-one bioconjugation and the synthesis of advanced architectures such as block copolymers or bottlebrush polymers and the preparation of advanced soft materials. Polyphosphazenes are of growing importance as elastomers, biodegradable materials and in biomedical drug delivery due to their synthetic versatility. While controlled polymerization methods have been known for some time, controlling both chain-ends with high fidelity has proven difficult. We demonstrate a robust synthetic route to hetero and homo α,ω-chain-end functionalized polyphosphazenes via end-capping with easily accessible, functionalized triphenylphosphine-based phosphoranimines. A versatile thiol-ene "click"-reaction approach then allows for subsequent conversion of the end-capped polymers with various functional groups. Finally, we demonstrate the utility of this system to prepare gels based on homo α,ω-chain-end functionalized polyphosphazenes. This development will enhance their progress in various applications, particularly in soft materials and as degradable polymers.

Hollow Silica Microparticles Based on Amphiphilic Polyphosphazenes.

Hollow microparticles are important materials, offering a larger surface area and lower density than their solid counterparts. Furthermore, their inner void space can be exploited for the encapsulation and release of guest species in a variety of applications. Herein, we present phosphazene-based silica hollow microparticles prepared via a surfactant-free sol-gel process through self-assembly of the alkoxysilyl-containing polymer in water-ethanol solution. Solely, a silane-derived polyphosphazene was used as the precursor for the microparticle formation, without additional classical silica sources. These novel hollow silica-based microparticles were prepared without surfactant, using a designed amphiphilic polyphosphazene for the particle formation made by two components, a hydrophilic unit consisting of 3-mercaptopropyl(trimethoxysilane), and a hydrophobic unit (dodecanethiol) attached to the double bonds from the poly(allylamine)phosphazene backbone via a thiol-ene photoreaction. Due to these two functionalities, a "vesicle"-like self-assembled structure was formed in the reaction medium, which could be then utilized for the microparticle preparation. The influence of NaOH during the synthesis was shown to affect the size and the wall thickness of the microparticles. This effect may enhance the possibilities to tailor such microparticles for drug delivery purposes or for future controlled release of other substances, such as drugs, fragrances, or anticorrosive pigments.

A Computational Procedure for Atomistic Modelling of Polyphosphazenes towards Better Capturing Molecular-Level Structuring and Thermo-Mechanical Properties.

Poly(phosphazenes)(PZ) are versatile polymers due to their tunable properties that can be tailored for specific applications. Despite extensive experimental research, not all properties are tested, and the list of PZs studied via molecular simulations is limited. Further, a general procedure to generate and test PZ systems is lacking. We present an in situ polymerization procedure developed to make, test, and tune the thermo-mechanical properties of four PZs-poly(dichlorophosphazene)(PZ-DC), poly[bis(2,2,2-trifluoroethoxy)]phosphazene (PZ-TFE), poly(2,2,2-trifluoroethoxy-5,6-diazidohexanoxy) phosphazene (PZ-Azido), and poly(2,2,2-trifluoroethoxy-5,6-dinitratohexanoxy)phosphazene (PZ-Nitrato) via molecular dynamics simulations. The predicted thermo-mechanical properties (i.e., density and glass transition temperature) agreed with experimental values when a direct comparison of PZ systems was possible. This demonstrates the reproducibility and reliability of our procedure which will help understand the behaviour of PZs at the molecular scale.

(PNSiMe3 )4 (NMe)6 : A Robust Tetravalent Phosphaza-adamantane Scaffold for Molecular and Macromolecular Construction.

Tetraarylmethanes and adamantanes are important rigid covalent connectors that play a four-way scaffolding role in molecular and materials chemistry. We report the synthesis of a new tetravalent phosphaza-adamantane cage, (PNSiMe3 )4 (NMe)6 (2), that shows high thermal, air, and redox stability due to its geometry. It nevertheless participates in covalent four-fold functionalization reactions along its periphery. The combination of a robust core and reactive corona makes 2 a convenient inorganic scaffold upon which tetrahedral molecular and macromolecular chemistry can be constructed. This potential is demonstrated by the synthesis of a tetrakis(bis(phosphine)iminium) ion (in compound 3) and the first all P/N poly(phosphazene) network (5).

Cationic Fluoropolyphosphazenes: Synthesis and Assembly with Heparin as a Pathway to Hemocompatible Nanocoatings.

The development of state-of-the-art blood-contacting devices can be advanced through integrating hemocompatibility, durability, and anticoagulant functionalities within engineered nanoscale coatings. To enable all-aqueous assembly of nanocoatings combining omniphobic fluorinated features with the potent anticoagulant activity of hydrophilic heparin, two fluoropolymers containing cationic functionalities were synthesized─poly[(trifluoroethoxy)(dimethylaminopropyloxy)phosphazene], PFAP-O, and poly[(trifluoroethoxy)(dimethylaminopropylamino)phosphazene], PFAP-A. Despite a relatively high content of fluorinated pendant groups─approximately 50% (mol) in each─both polymers displayed solubility in aqueous solutions and were able to spontaneously form stable supramolecular complexes with heparin, as determined by dynamic light scattering and asymmetric flow field-flow fractionation methods. Heparin-containing coatings were then assembled by layer-by-layer deposition in aqueous solutions. Nanoassembled coatings were evaluated for potential thrombogenicity in three important categories of in vitro tests─coagulation by thrombin generation, platelet retention, and hemolysis. In all assays, heparin-containing fluoro-coatings consistently displayed superior performance compared to untreated titanium surfaces or fluoro-coatings assembled using poly(acrylic acid) in the absence of heparin. Short-term stability studies revealed the noneluting nature of these noncovalently assembled coatings.

Nano-Assembly of Quisinostat and Biodegradable Macromolecular Carrier Results in Supramolecular Complexes with Slow-Release Capabilities.

Self-assembly of ionically charged small molecule drugs with water-soluble biodegradable polyelectrolytes into nano-scale complexes can potentially offer a novel and attractive approach to improving drug solubility and prolonging its half-life. Nanoassemblies of quisinostat with water-soluble PEGylated anionic polyphosphazene were prepared by gradient-driven escape of solvent resulting in the reduction of solvent quality for a small molecule drug. A study of binding, analysis of composition, stability, and release profiles was conducted using asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) spectroscopy. Potency assays were performed with WM115 human melanoma and A549 human lung cancer cell lines. The resulting nano-complexes contained up to 100 drug molecules per macromolecular chain and displayed excellent water-solubility and improved hemocompatibility when compared to co-solvent-based drug formulations. Quisinostat release time (complex dissociation) at near physiological conditions in vitro varied from 5 to 14 days depending on initial drug loading. Multimeric complexes displayed dose-dependent potency in cell-based assays and the results were analyzed as a function of complex concentration, as well as total content of drug in the system. The proposed self-assembly process may present a simple alternative to more sophisticated delivery modalities, namely chemically conjugated prodrug systems and nanoencapsulation-based formulations.

Synthesis of a Reactive Template-Induced Core-Shell PZS@ZIF-67 Composite Microspheres and Its Application in Epoxy Composites.

Developing superior properties of epoxy resin composites with high fire resistance, light smoke, and low toxicity has been the focus of the research in the flame-retardant field. In particular, it is essential to decrease the emissions of toxic gases and smoke particles generated during the thermal decomposition of epoxy resin (EP) to satisfy the industrial requirements for environmental protection and safety. Consequently, the PZS@ZIF-67 composite was designed and synthesized by employing the hydroxyl group-containing polyphosphazene (poly(cyclotriphosphazene-co-4,4'-dihydroxydiphenylsulfone), PZS) as both the interfacial compatibility and an in situ template and the ZIF-67 nanocrystal as a nanoscale coating and flame-retardant cooperative. ZIF-67 nanocrystal with multidimensional nanostructures was uniformly wrapped on the surface of PZS microspheres. Subsequently, the acquired PZS@ZIF-67 composite was incorporated into the epoxy resin to prepare composite samples for the study of their fire safety, toxicity suppression, and mechanical performance. Herein, the EP/5% PZS@ZIF-67 passed the V-0 rating in a UL-94 test with a 31.9% limit oxygen index value. More precisely, it is endowed with a decline of 51.08%, 28.26%, and 37.87% of the peak heat release rate, the total heat release, and the total smoke production, respectively. In addition, the unique structure of PZS@ZIF-67 microsphere presented a slight impact on the mechanical properties of EP composites at low loading. The PZS@ZIF-67 possible flame-retardant mechanism was speculated based on the analysis of the condensed phase and the gas phase of EP composites.

In Vivo Evaluation of the Regenerative Capability of Glycylglycine Ethyl Ester-Substituted Polyphosphazene and Poly(lactic-co-glycolic acid) Blends: A Rabbit Critical-Sized Bone Defect Model.

In an effort to understand the biological capability of polyphosphazene-based polymers, three-dimensional biomimetic bone scaffolds were fabricated using the blends of poly[(glycine ethylglycinato)75(phenylphenoxy)25]phosphazene (PNGEGPhPh) and poly(lactic-co-glycolic acid) (PLGA), and an in vivo evaluation was performed in a rabbit critical-sized bone defect model. The matrices constructed from PNGEGPhPh-PLGA blends were surgically implanted into 15 mm critical-sized radial defects of the rabbits as structural templates for bone tissue regeneration. PLGA, which is the most commonly used synthetic bone graft substitute, was used as a control in this study. Radiological and histological analyses demonstrated that PNGEGPhPh-PLGA blends exhibited favorable in vivo biocompatibility and osteoconductivity, as the newly designed matrices allowed new bone formation to occur without adverse immunoreactions. The X-ray images of the blends showed higher levels of radiodensity than that of the pristine PLGA, indicating higher rates of new bone formation and regeneration. Micro-computed tomography quantification revealed that new bone volume fractions were significantly higher for the PNGEGPhPh-PLGA blends than for the PLGA controls after 4 weeks. The new bone volume increased linearly with increasing time points, with the new tissues observed throughout the defect area for the blend and only at the implant site's extremes for the PLGA control. Histologically, the polyphosphazene system appeared to show tissue responses and bone ingrowths superior to PLGA. By the end of the study, the defects with PNGEGPhPh-PLGA scaffolds exhibited evidence of effective bone tissue ingrowth and minimal inflammatory responses. Thus, polyphosphazene-containing biomaterials have excellent translational potential for use in bone regenerative engineering applications.