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Cocaine is a highly addictive drug that has seen a steady uptrend causing severe health problems worldwide. Currently, there are no approved therapeutics for treating cocaine use disorder; hence, there is an urgent need to identify new medications. Immunopharmacotherapeutics is a promising approach utilizing endogenous antibodies generated through active vaccination, and if properly programmed, can blunt a drug's psychoactive and addictive effects. However, drug vaccine efficacy has largely been limited by the modest levels of antibodies induced. Herein, we explored an adjuvant system consisting of a polyphosphazene macromolecule, specifically poly[di(carboxylatoethylphenoxy)-phosphazene] (PCEP), a biocompatible synthetic polymer that was solicited for improved cocaine conjugate vaccine delivery performance. Our results demonstrated PCEP's superior assembling efficiency with a cocaine hapten as well as with the combined adjuvant CpG oligodeoxynucleotide (ODN). Importantly, this combination led to a higher titer response, balanced immunity, successful sequestering of cocaine in the blood, and a reduction in the drug in the brain. Moreover, a PCEP-cocaine conjugate vaccine was also found to function well via intranasal administration, where its efficacy was demonstrated through the antibody titer, affinity, mucosal IgA production, and a reduction in cocaine's locomotor activity. Overall, a comprehensive evaluation of PCEP integrated within a cocaine vaccine established an advance in the use of synthetic adjuvants in the drugs of abuse vaccine field.

Often inspired by nature, techniques for precise droplet manipulation have found applications in microfluidics, microreactors, and water harvesting. However, a widely applicable strategy for surface modification combining simultaneous hydrophobicity and pH-sensitivity has not yet been achieved by employing environmentally friendly assembly conditions. The introduction of pH-responsive groups to an otherwise fluorinated polyphosphazene (PPZ) unlocks pH-selective droplet capture and transfer. Here, an all-aqueous layer-by-layer (LbL) deposition of polyelectrolytes is used to create unique hydrophobic coatings, endowing surfaces with the ability to sense environmental pH. The high hydrophobicity of these coatings (ultimately reaching a contact angle120° on flat surfaces) is enabled by the formation of hydrophobic nanoscale domains and controllable by the degree of fluorination of PPZs, polyamine-binding partners, deposition pH, and coating thickness. Inspired by the hierarchical structure of rose petals, these versatile coatings reach a contact angle150° when deposited on structured surfaces while introducing a tunable adhesivity that enables precise droplet manipulation. The films exhibited a strongly pronounced parahydrophobic rose petal behavior characterized through the contact angle hysteresis. Depositing as few as five bilayers (∼25 nm) on microstructured rather than smooth substrates resulted in superhydrophobicity with water contact angles150° and the attenuation of the contact angle hysteresis, enabling highly controlled transfer of aqueous droplets. The pH-selective droplet transfer was achieved between surfaces with either the same microstructure and LbL film building blocks, which were assembled at different pH, or between surfaces with different microstructures coated with identical films. The demonstrated capability of these hydrophobic LbL films to endow surfaces with controlled hydrophobicity through adsorption from aqueous solutions and control the adhesion and transfer of water droplets between surfaces can be used in droplet-based microfluidics applications and water collection/harvesting.

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.

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.

espanolLa industria textil en Colombia es fuente de empleo para mas de 200000 personas y en la ciudad de Medellin se encuentra mas del 50% de esta produccion. La necesidad de modelar ymejorar procesos textiles permite a este renglon de la economia ser competitivo a nivel internacional. En este documento serealiza una descripcion acerca del uso de la simulacion discreta en una empresa de serviciosde acabados textiles, a traves de la interpretacionde cuatro escenarios;lo que se traduce en demostrarel potencial de la simulacion discretaen entornos productivosde servicios y su alto impacto almodelar sin necesidad de experimentar con el sistema real. El metodo utilizado en el presente escritose resumeen tres etapas,la primera comprendela metodologia de simulacion, la segunda los datos que soportanla simulacion y por ultimoun analisis de resultadoscon la comparativa de los escenarios. La simulacion fue validada estadisticamente y verificada con los comportamientos reales de la empresay seejecuta por medio de herramientas de software como EasyFit®, Microsoft Excel® y Simul8® EnglishThe textile industry in Colombia is a source of employment for more than 200.000 people and more than 50% of this production is undertaken in Medellin. Modeling and improving textile processes allow this economic line to be competitive internationally. In this paper, we make a description about the use of discrete event simulation in a textile finishing company through the presentation of the results of four scenarios, which finally shows the potential of discrete simulations in productive environments and its high impact when modelling part of reality without the necessity of experimenting with the real system. The method used in this paper is summarized in three major stages: the first one is the simulation methodology, the second one is the data to support the simulation, and the final stage is an analysis of the results with the comparison of the four scenarios. The simulation was statistically validated and verified with the real behaviors of the company and it is executed by using software tools such as EasyFit®, Microsoft Excel® and Simul8®

Current human papillomavirus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain for which vaccine coverage is lacking. In addition, all current HPV vaccines rely on aluminum salt-based adjuvant formulations that function through unclear mechanisms with few substitutes available. In an effort to expand the toolbox of available adjuvants suitable for HPV vaccines, we compared the immunogenicity of the RG1-VLP (virus-like particle) vaccine in BALB/c mice when formulated with either the aluminum hydroxide adjuvant Alhydrogel or the novel polyphosphazene macromolecular adjuvant poly[di (carboxylatoethylphenoxy) phosphazene] (PCEP). PCEP-formulated RG1-VLPs routinely outperformed VLP/Alhydrogel in several measurements of VLP-specific humoral immunity, including consistent improvements in the magnitude of antibody (Ab) responses to both HPV16-L1 and the L2 RG1 epitope as well as neutralizing titers to HPV16 and cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39. Dose-sparing studies indicated that RG1-VLPs could be reduced in dose by 75% and the presence of PCEP ensured activity comparable to a full VLP dose adjuvanted by Alhydrogel. In addition, levels of HPV16-L1 and -L2-specific Abs were achieved after two vaccinations with PCEP as adjuvant that were equivalent to or greater than levels achieved with three vaccinations with Alhydrogel alone, indicating that the presence of PCEP resulted in accelerated immune responses that could allow for a decreased dose schedule. Given the extensive clinical track record of polyphosphazenes, these data suggest that substitution of alum-based adjuvants with PCEP for the RG1-VLP vaccine could lead to rapid seropositivity requiring fewer boosts, the dose-sparing of commercial VLP-based vaccines, and the establishment of longer-lasting humoral responses to HPV.

Clinically, the use of photo-activated polymer-based dental composites is the preferred method to restore carious teeth due to their esthetics and ease of application. However, the longevity of these resin-based composite tooth restorations can be compromised by the sensitivity of the bonded interface between the composite and the tooth surface. The objective of the current study was to modify the tooth surface with novel fluorinated polyphosphazenes (PPZs), thereby improving the stability of the interface. Binding isotherms of PPZs with collagen (CLG) and hydroxyapatite (HA), two of the primary components of teeth, were established and indicate significant and stable adsorption to the surfaces of these materials. PPZs were also shown to protect CLG against acidic dissolution in a model system. A composite material consisting of the fluorinated polymer and CLG demonstrated three-dimensional stability and significant hydrophobicity. Additionally, no hemolytic activity was observed when evaluated using a porcine red blood cells (RBC) assay. Bovine dentin treated with PPZs demonstrated increased contact angle (hydrophobicity) compared with control samples and resisted fluid penetration when assessed using a dye penetration study. Finally, microhardness evaluation of bovine dentin treated with PPZs and exposed to an acidic challenge showed that treated dentin resisted demineralization. The hardness of the untreated control was significantly reduced after exposure when compared with the PPZ-treated samples. This study represents a novel approach to overcoming the current limitations of composite restorations. These results are promising to improve the longevity of composite dental restorations and may have wider use in sealants, varnishes, and other dental applications. Tooth decay remains a prevalent problem worldwide. Polymer-based composites are the most frequently used tooth restorative used in the clinic. The longevity of these fillings is limited due to conditions in the mouth that can weaken the adhesive used to bond the composite to the natural tooth. The current study uses novel polyphosphazenes (PPZs), hybrid organic-inorganic macromolecules with tunable hydrophilic-hydrophobic properties to coat the tooth surface to achieve better compatibility with the adhesive, thereby improving the longevity of the restoration. Results indicate that PPZs have significant and stable adsorption onto teeth, which may lead to a more stable bonded interface.

Two well-defined synthetic polyphosphazene immunoadjuvants, PCPP and PCEP, were studied for their ability to potentiate the immune response to the hepatitis C virus (HCV) E2 glycoprotein antigen in vivo. We report that PCEP induced significantly higher serum neutralization and HCV-specific IgG titers in mice compared to other adjuvants used in the study: PCPP, Alum, and Addavax. PCEP also shifted the response toward the desirable balanced Th1/Th2 immunity, as evaluated by the antibody isotype ratio (IgG2a/IgG1). The in vivo results were analyzed in the context of antigen-adjuvant molecular interactions in the system and in vitro immunostimulatory activity of formulations. Asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) analysis showed that both PCPP and PCEP spontaneously self-assemble with the E2 glycoprotein with the formation of multimeric water-soluble complexes, which demonstrates the role of polyphosphazene macromolecules as vaccine delivery vehicles. Intrinsic in vitro immunostimulatory activity of polyphosphazene adjuvants, which was assessed using a mouse macrophage cell line, revealed comparable activities of both polymers and did not provide an explanation of their in vivo performance. However, PCEP complexes with E2 displayed greater stability against agglomeration and improved in vitro immunostimulatory activity compared to those of PCPP, which is in line with superior in vivo performance of PCEP. The results emphasize the importance of often neglected antigen-polyphosphazene self-assembly mechanisms in formulations, which can provide important insights on their in vivo behavior and facilitate the establishment of a structure-activity relationship for this important class of immunoadjuvants.

Resiquimod or R848 (RSQD) is a Toll-like receptor (TLR) 7/8 agonist which shows promise as vaccine adjuvant due to its potential to promote highly desirable cellular immunity. The development of this small molecule in the field to date has been largely impeded by its rapid in vivo clearance and lack of association with vaccine antigens. Here, we report a multimeric TLR 7/8 construct of nano-scale size, which results from a spontaneous self-assembly of RSQD with a water-soluble clinical-stage polymer - poly[di(carboxylatophenoxy)phosphazene] (PCPP). The formation of ionically paired construct (PCPP-R) and a ternary complex, which also includes Hepatitis C virus (HCV) antigen, has been demonstrated by dynamic lights scattering (DLS), turbidimetry, fluorescence spectroscopy, asymmetric flow field flow fractionation (AF4), and 1H NMR spectroscopy methods. The resulting supramolecular assembly PCPP-R enabled superior immunostimulation in cellular assays (mouse macrophage reporter cell line) and displayed improved in vitro hemocompatibility (human erythrocytes). In vivo studies demonstrated that PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response towards desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). The non-covalent multimerization approach stands in contrast to previously suggested RSQD delivery methods, which involve covalent conjugation or encapsulation, and offers a flexible methodology that can be potentially integrated with other parenterally administered drugs.

Significance Hepatitis C virus chronically infects approximately 1% of the world’s population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

In this work, novel sulfo-fluoro polyphosphazenes (PPzs) were synthesized via macromolecular substitution of polydichlorophosphazene utilizing "non-covalent protection" methodology by converting acid functionalities into hydrophobic alkylammonium salts. Resulting PPzs showed excellent solubility in aqueous solutions over a broad pH range and contained ∼25% sulfo- groups and 20% either trifluoroethoxy- (FESP) or trifluoromethylphenoxy- (FPSP) side groups, as determined by NMR spectroscopy. Their polyelectrolyte behavior was evaluated by binding with an oppositely charged polyion, branched polyethylenimine (PEI), which resulted in the formation of interpolymer complexes as shown by dynamic light scattering (DLS). Contrary to a sulfonated, nonfluorinated PPz homopolymer (SP), fluorinated macromolecules effectively bound human serum albumin (HSA) as revealed by dynamic light scattering and asymmetric flow field flow fractionation (AF4) studies. Moreover, FESP and FPSP both displayed low hemolytic activity as evaluated in solution using porcine red blood cells. Using the layer-by-layer (LbL) technique, FESP and FPSP were assembled into nanocoatings with PEI. Both fluorinated and nonfluorinated sulfo PPzs showed linear growth with PEI because of strong ionic pairing between sulfo and amino groups. However, films of fluorinated PPzs displayed higher hydrophobicity, lower swelling, and improved stability in high ionic strength environment when compared to coatings formed by a sulfonated, nonfluorinated SP, or a carbon-chain polymer poly(styrene sulfonic acid). Hemocompatibility of FESP and FPSP nanofilms was demonstrated

Resiquimod or R848 (RSQD) is a Toll-like receptor (TLR) 7/8 agonist which shows promise as vaccine adjuvant due to its potential to promote highly desirable cellular immunity. The development of this small molecule in the field to date has been largely impeded by its rapid in vivo clearance and lack of association with vaccine antigens. Here, we report a multimeric TLR 7/8 construct of nano-scale size, which results from a spontaneous self-assembly of RSQD with a water-soluble clinical-stage polymer - poly[di(carboxylatophenoxy)phosphazene] (PCPP). The formation of ionically paired construct (PCPP-R) and a ternary complex, which also includes Hepatitis C virus (HCV) antigen, has been demonstrated by dynamic lights scattering (DLS), turbidimetry, fluorescence spectroscopy, asymmetric flow field flow fractionation (AF4), and (1)H NMR spectroscopy methods. The resulting supramolecular assembly PCPP-R enabled superior immunostimulation in cellular assays (mouse macrophage reporter cell line) and displayed improved in vitro hemocompatibility (human erythrocytes). In vivo studies demonstrated that PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response towards desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). The non-covalent multimerization approach stands in contrast to previously suggested RSQD delivery methods, which involve covalent conjugation or encapsulation, and offers a flexible methodology that can be potentially integrated with other parenterally administered drugs.

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Nonionic fluorinated polyphosphazenes, such as poly[bis(trifluoroethoxy)phosphazene] (PTFEP), display superb biocompatibility, yet their deposition to surfaces has been limited to solution casting from organic solvents or thermal molding. Herein, hydrophobic coatings of fluorinated polyphosphazenes are demonstrated through controlled deposition of ionic fluorinated polyphosphazenes (iFPs) from aqueous solutions using the layer-by-layer (LbL) technique. Specifically, the assemblies included poly[(carboxylatophenoxy)(trifluoroethoxy)phosphazenes] with varied content of fluorine atoms as iFPs (or poly[bis(carboxyphenoxy)phosphazene] (PCPP) as a control nonfluorinated polyphosphazene) and a variety of polycations. Hydrophobic interactions largely contributed to the formation of LbL films of iFPs with polycations, leading to linear growth and extremely low water uptake. Hydrophobicity-enhanced ionic pairing within iFP/BPEI assemblies gave rise to large-amplitude oscillations in surface wettability as a function of capping layer, which were the largest for the most fluorinated iFP, while control PCPP/polycation systems remained hydrophilic regardless of the film top layer. Neutron reflectometry (NR) studies indicated superior layering and persistence of such layering in salt solution for iFP/BPEI films as compared to control PCPP/polycation systems. Hydrophobicity of iFP-capped LbL coatings could be further enhanced by using a highly porous polyester surgical felt rather than planar substrates for film deposition. Importantly, iFP/polycation coatings displayed biocompatibility which was similar to or superior to that of solution-cast coatings of a clinically validated material (PTFEP), as demonstrated by the hemolysis of the whole blood and protein adsorption studies.

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.

PCPP, a well-defined polyphosphazene macromolecule, has been studied as an immunoadjuvant for a soluble form of the postfusion glycoprotein of respiratory syncytial virus (RSV sF), which is an attractive vaccine candidate for inducing RSV-specific immunity in mice and humans. We demonstrate that RSV sF-PCPP formulations induce high neutralization titers to RSV comparable to alum formulations even at a low PCPP dose and protect animals against viral challenge both in the lung and in the upper respiratory tract. PCPP formulations were also characterized by Th1-biased responses, compared to Th2-biased responses that are more typical for RSV sF alone or RSV sF-alum formulations, suggesting an inherent immunostimulating activity of the polyphosphazene adjuvant. We defined these immunologically active RSV sF-PCPP formulations as self-assembled water-soluble protein-polymer complexes with distinct physicochemical parameters. The secondary structure and antigenicity of the protein in the complex were fully preserved during the spontaneous aqueous self-assembly process. These findings further advance the concept of polyphosphazene immunoadjuvants as unique dual-functionality adjuvants integrating delivery and immunostimulating modalities in one water-soluble molecule.

Poly[di(carboxylatomethylphenoxy)phosphazene] (PCMP), a new member of polyphosphazene immunoadjuvant family, is synthesized. In vitro assessment of a new macromolecule revealed hydrolytic degradation profile and immunostimulatory activity comparable to its clinical stage homologue PCPP; however, PCMP was characterized by a beneficial reduced sensitivity to the ionic environment. In vivo evaluation of PCMP potency was conducted with human papillomavirus (HPV) virus-like particles (VLPs) based RG1-VLPs vaccine. In contrast with previously reported self-assembly of polyphosphazene adjuvants with proteins, which typically results in the formation of complexes with multimeric display of antigens, PCMP surface modified VLPs in a composition dependent pattern, which at a high polymer-to VLPs ratio led to stabilization of antigenic particles. Immunization experiments in mice demonstrated that PCMP adjuvanted RG1-VLPs vaccine induced potent humoral immune responses, in particular, on the level of highly desirable protective cross-neutralizing antibodies, and outperformed PCPP and Alhydrogel adjuvanted formulations.

Biocompatible antibacterial coatings are highly desirable to prevent bacterial colonization on a wide range of medical devices from hip implants to skin grafts. Traditional polyelectrolytes are unable to directly form coatings with cationic antibiotics at neutral pH and suffer from high degrees of antibiotic release upon exposure to physiological concentrations of salt. Here, novel inorganic-organic hybrid polymer coatings based on direct layer-by-layer assembly of anionic polyphosphazenes (PPzs) of various degrees of fluorination with cationic antibiotics (polymyxin B, colistin, gentamicin, and neomycin) are reported. The coatings displayed low levels of antibiotic release upon exposure to salt and pH-triggered response of controlled doses of antibiotics. Importantly, coatings remained highly surface active against Escherichia coli and Staphylococcus aureus, even after 30 days of pre-exposure to physiological conditions (bacteria-free) or after repeated bacterial challenge. Moreover, coatings displayed low (

This paper contains results of numerical and experimental investigation of glow discharge plasma created in a chamber of a new-type large-volume coaxial gridded hollow cathode. The discharge is created in argon at 25 Pa by applying time-varying power with frequency 20 kHz on electrodes. A 2-D model of the discharge was built using COMSOL Multiphysics. Self-consistent description of the discharge was obtained using the extended fluid approach, which couples continuity equations for charged particles and electron energy balance with Poisson's equation for electric potential. Electron transport coefficients and rates of electron-impact reactions were calculated using the electron energy distribution function. The spatial and radial distributions of plasma potential (V p ), electron density (n e ), and electron temperature (T e ) were obtained. It is shown that the plasma inside the chamber is similar to the negative glow of a dc glow discharge. Comparison of numerical results with the Langmuir probe measurements of electron density and electron temperature is presented and showed a good agreement.

Novel oppositely charged polyphosphazene polyelectrolytes containing grafted poly(ethylene glycol) (PEG) chains were synthesized as modular components for the assembly of biodegradable PEGylated protein delivery vehicles. These macromolecular counterparts, which contained either carboxylic acid or tertiary amino groups, were then formulated at near physiological conditions into supramolecular assemblies of nanoscale level, below 100 nm. Nanocomplexes with electroneutral surface charge, as assessed by zeta potential measurements, were stable in aqueous solutions, which suggests their compact polyelectrolyte complex "core"-hydrophilic PEG "shell" structure. Investigation of PEGylated polyphosphazene nanocomplexes as agents for noncovalent PEGylation of the therapeutic protein l-asparaginase (L-ASP) in vitro demonstrated their ability to dramatically reduce protein antigenicity, as measured by antibody binding using enzyme linked immunosorbent assay (ELISA). Encapsulation in nanocomplexes did not affect enzymatic activity of L-ASP, but improved its thermal stability and proteolytic resistance. Gel permeation chromatography (GPC) experiments revealed that all synthesized polyphosphazenes exhibited composition controlled hydrolytic degradability in aqueous solutions at neutral pH and showed greater stability at lower temperatures. Overall, novel hydrolytically degradable polyphosphazene polyelectrolytes capable of spontaneous self-assembly into PEGylated nanoparticulates in aqueous solutions can potentially enable a simple and effective approach to modifying therapeutic proteins without the need for their covalent modification.

Neonates and infants are susceptible to infection due to distinct immune responses in early life. Therefore, development of vaccine formulation and delivery systems capable of activating human newborn leukocytes is of global health importance. Poly[di(carboxylatophenoxy)phosphazene] (PCPP) belongs to a family of ionic synthetic polyphosphazene polyelectrolyte compounds that can form non-covalent interactions with protein antigens and demonstrate adjuvant activity in animals and in human clinical trials. However, little is known about their ability to activate human immune cells. In this study, we characterized the effects of PCPP alone or in combination with a model antigen (recombinant HIV-Gag (Gag)), on the maturation, activation and antigen presentation by human adult and newborn dendritic cells (DCs) in vitro . PCPP treatment induced DC activation as assessed by upregulation of co-stimulatory molecules and cytokine production. Studies benchmarking PCPP to Alum, the most commonly used vaccine adjuvant, demonstrated that both triggered cell death and release of danger signals in adult and newborn DCs. When complexed with Gag antigen, PCPP maintained its immunostimulatory characteristics while permitting internalization and presentation of Gag by DCs to HIV-Gag-specific CD4 + T cell clones. The PCPP vaccine formulation outlined here has intrinsic adjuvant activity, can facilitate effective delivery of antigen to DCs, and may be advantageous for induction of beneficial T cell-mediated immunity. Moreover, polyphosphazenes can further reduce cost of vaccine production and distribution through their dose-sparing and antigen-stabilizing properties, thus potentially eliminating the need for cold chain distribution.

Two macromolecular immunoadjuvants, poly[di(carboxylatophenoxy)phosphazene], PCPP, and poly[di(carboxylatoethylphenoxy)phosphazene], PCEP, have been investigated for their molecular interactions with model and biopharmaceutically important proteins in solutions, as well as for their TLR stimulatory effects and pH-dependent membrane disruptive activity in cellular assays. Solution interactions between polyphosphazenes and proteins, including antigens and soluble immune receptor proteins, have been studied using Asymmetric Flow Field Flow Fractionation (AF4) and Dynamic Light Scattering (DLS) at near physiological conditions: phosphate buffered saline, pH 7.4. Polyphosphazenes demonstrated selectivity in their molecular interactions with various proteins, but displayed strong binding with all vaccine antigens tested in the present study. It was found that both PCPP and PCEP showed strong avidity to soluble immune receptor proteins, such as Mannose Receptor (MR) and certain Toll-Like Receptor (TLR) proteins. Studies on TLR stimulation in vitro using HEK293 cells with overexpressed human TLRs revealed activation of TLR7, TLR8, and TLR9 signaling pathways, albeit with some nonspecific stimulation, for PCPP and the same pathways plus TLR3 for PCEP. Finally, PCEP, but not PCPP, demonstrated pH-dependent membrane disruptive activity in the pH range corresponding to the pH environment of early endosomes, which may play a role in a cross-presentation of antigenic proteins.

Microneedles containing sodium carboxymethylcellulose (CMC) formulations were fabricated to include an external chitosan (CS) layer to modulate their hydration profile, an important parameter affecting their application as intradermal delivery devices and their storage. The microfabrication process was carried out under conditions that enabled the formation of polyelectrolyte complexes between these oppositely charged macromolecules. CMC–CS microneedles were characterized by water uptake in a humid environment, contact angle measurements, dissolution in aqueous solutions, and protein-release profiles. The results demonstrate that the microneedles containing CMC–CS formulations displayed suppressed moisture sensitivity in water vapors compared to their unmodified CMC counterparts while the maintaining quick protein-release characteristics required for their uses. This approach also showed the potential for sustained protein-release applications, as the CMC–CS formulations could be combined in layers to fabricate multicompartment microneedle coatings with delayed release characteristics. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Applications of polyelectrolytes as pharmaceutical excipients or biologically active agents generated an increased interest in formulations, in which ionic macromolecules share the same milieu with protein drugs or vaccine antigens. Macromolecular interactions, which often take place in such systems, can potentially impact formulation activity and stability. The present article reports that poly[di(carboxylatophenoxy)phosphazene], disodium salt (PCPP), which has been previously shown to be a potent vaccine adjuvant, also displays a strong protein stabilizing effect in aqueous solutions that can be significantly amplified in the presence of nonionic surfactants. The phenomenon is studied in the context of macromolecular interactions in the system and is linked to the formation of PCPP-protein and PCPP-protein-surfactant complexes.

Polyphosphazenes with amino acid ester side groups show potential as hydrolytically degradable materials for biomedical applications. This study focuses on practical aspects of their use as biodegradable materials, such as effects of the hydrolytic environment and sample processing. Poly[di(ethyl glycinato)phosphazene], PEGP, and poly[di(ethyl alaninato)phosphazene], PEAP, were prepared by macromolecular substitution reaction, ensuring the absence of the residual chlorine atoms to avoid their influence on the hydrolysis. The kinetics of polymer degradation was studied by simultaneously measuring polymer mass loss, molecular weight decrease, and the release of phosphates and ammonia. The effect of pH, buffer composition, temperature, casting solvents, and film thickness were investigated.

Polyphosphazene polyelectrolytes containing various amounts of hydrophobic fluorinated moieties and ionic carboxylic acid groups were synthesized. Polymer compositions and molecular weights were characterized by NMR and gel permeation chromatography. Interestingly, poly[(carboxylatophenoxy)(trifluoroethoxy)phosphazene] containing 60 mol % fluorinated groups was found to be soluble in aqueous solutions. The behavior of fluorinated polyelectrolytes in reactions of ionic complexation with multivalent and monovalent salts was studied in aqueous solutions and ethanol–water mixtures. Such reactions led to the formation of ionotropic hydrogels under mild conditions and, thus, are of importance to the development of microencapsulation processes and controlled release formulations. All of the synthesized polymers underwent phase separation in the presence of multivalent ionic crosslinkers, such as spermine and calcium chloride. This included a water-soluble polyelectrolyte containing 40 mol % ionic groups and hydrophobic polymer with only 3 mol % carboxylic acid groups. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 53–58, 2007

Novel water-soluble biodegradable polyphosphazenes are described. Polymers are constructed on the basis of a biodegradable polyphosphazene backbone to mimic the structure of poly(vinylpyrrolidone), PVP. Poly{di[2-(2-oxo-1-pyrrolidinyl)ethoxy]phosphazene}, PYRP, and its copolymers containing biologically active carboxylatophenoxy side groups have been synthesized. The ability of these polymers to degrade in aqueous environment at 37 °C is demonstrated by light scattering-GPC and NMR studies. The degradation is manifested by the gradual decrease in the molecular weight and release of the side group −1-(2-hydroxyethyl)-2-pyrrolidone. It has been shown that the incorporation of N-ethylpyrrolidone containing side groups in the polymer structure can modulate degradation profiles of phosphazene copolymers. The kinetics of hydrolytic degradation and the effect of pH on the degradation rate have been investigated.

A single-step approach for the synthesis of polyphosphazenes containing sulfonic acid functionalities is developed. Polyphosphazene “sulfonation” is conducted via the direct replacement of chlorine...

The effect of the Pluronic P-105 aggregation state on its uptake by HL-60 cells was studied by flow cytometry, fluorescence spectroscopy, and confocal and fluorescence microscopy using a fluorescently labeled Pluronic P105. In the low concentration region below the critical micelle concentration (CMC), Pluronic uptake was proportional to the concentration in the incubation medium. The proportionality broke sharply above the CMC, revealing a less efficient intracellular uptake of Pluronic micelles than that of unimers. The data suggested that Pluronic micelles were internalized via fluid-phase endocytosis while unimers were internalized via diffusion through plasma membranes. Based on the above findings, the shielding effect of Pluronic micelles on drug intracellular uptake was explained.

Irganox®HP-136(lactone), which is a mixture of 90% of 5,7-di- tert -butyl-3-(3,4-dimethylphenyl)3 H -benzofuran-2-one and 10% of 5,7-di- tert -butyl-3-(2,3-dimethylphenyl)3 H -benzofuran-2-one behaves as a medium strength chain-breaking antioxidant during polypropylene oxidation at 180 and 200 °C. The critical concentration (vide infra) required to inhibit oxidation is higher than that of the phenolic antioxidant 2246. The lactone is slowly consumed during the induction period and then much faster when the critical concentration is reached. Phosphites, sulphides and phenols increase the efficiency of the lactone during polymer oxidation. The use of lactone allows the amount of phenolic and phosphorous containing stabilizers to decrease without decreasing the thermooxidative stability of the polymer.

The solubility and diffusion of Irganox ® HP-136 (lactone), which is a mixture of 90% of 5,7-di- tert -butyl-3-(3,4-dimethylphenyl)3 H -benzofuran-2-one and 10% of 5,7-di- tert -butyl-3-(2,3-dimethylphenyl)3 H -benzofuran-2-one (lactone) in polypropylene (PP) were studied at temperatures ranging from 50 to 100 °C. The lactone is characterized by a good solubility and relatively low migration in the polymer. The rate of washing out of lactone with heptane at room temperature is considerably higher than that with dodecane and ethanol, increases with initial concentration of lactone in the polymer and decreases with time. The physical behavior of lactone in PP is similar to that of 2,2′-methylene-bis(4-methyl-6- tert -butylphenol) having similar molecular weight.

The mechanism of the ultrasonic enhancement of the uptake of cytotoxic drugs, doxorubicin (DOX) and ruboxyl (Rb) by HL-60 cells from Pluronic micelles was studied. DOX and Rb sorption from either PBS or micellar Pluronic solutions is described by Langmuir-type isotherms characteristic of substrates with limited number of sorption centers. The sorption limits for Rb from PBS and Pluronic were considerably higher than those for DOX, presumably due to much higher Rb partitioning into cell membranes. The overall number of drug sorption centers for both drugs decreased in the presence of Pluronic implying the effect of Pluronic on the DNA conformation, which was confirmed by the electron paramagnetic resonance (EPR) experiments using Rb as a spin probe. Ultrasound increased drug uptake by the cells from PBS and Pluronic solutions. The fluorescence microscopy and flow cytometry experiments using fluorescently-labeled Pluronic showed that ultrasound enhanced both the intracellular uptake of Pluronic micelles and Pluronic trafficking into cell nuclei. A scheme is suggested that describes various equilibria controlling drug/cell interactions and effect of ultrasound on these equilibria. Under the action of ultrasound, the equilibrium between the micellar-encapsulated and free drug is shifted in the direction of free drug due to micelle perturbation; the equilibrium between extracellular and internalized drug is shifted to the intracellular drug due to the ultrasound-induced cellular changes that enhance the accessibility of various cellular structures to drug. An important advantage offered by ultrasound is that the same degree of the intracellular drug uptake may be achieved at a substantially lower drug concentration in the incubation medium.

2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) and its bis-1-oxyl analogue react with triethyl and triisopropyl phosphite forming tetramethylpiperidine phosphite which is the main product of the reaction together with dialkyl phosphates. Minor products are tetramethylpiperidinyldialkyl phosphate and tetramethylpiperidine. The reaction mechanism was elucidated by observing the intermediate formation of the phosphoranyl radical which evolves forming the isolated products. The rate of this reaction is considerably lower compared to the stabilization reaction observed between phosphites and hydroperoxides during polymer oxidation. Thus the former reaction could be considered negligible in terms of polymer stabilization under the conditions studied.

The oxidation of PE precipitated from decane or chlorobenzene was studied in the presence of tetrakis[methylene-3-(3′,5′-di- tert -butyl-4′-hydroxy-phenyl) propionate]methane at 180 °C by measuring the oxygen consumption. The efficiency of the antioxidant is higher on PE precipitated from chlorobenzene than from decane and, in both solvents, from the most diluted solutions. In addition, it has been seen that the decrease of the rate of PE precipitation gives rise to an increase in the stability of the polymer to oxidation. The changes in short-range-order arrangement of the macromolecules during precipitation are discussed.

Water-soluble polyphosphazene polyacids, such as poly[di(carboxylatophenoxy)phosphazene] (PCPP), have been of significant interest due to their unique immunoadjuvant and vaccine delivery properties. We report that PCPP can spontaneously self-assemble into intermolecular complexes with common formulation excipients − polyethers in aqueous solutions at neutral pH through the establishment of hydrogen bonds. The resulting advanced PCPP delivery modalities can range from macromolecular assemblies at the nanoscale level to physically cross-linked hydrogels and the physical state can be modulated through varying polymer ratios and molecular weight of polyether. It has been demonstrated that such macromolecular complexes maintain protein-binding ability − a key characteristics of the delivery system. Importantly, the non-covalent modification of PCPP immunoadjuvant with polyethers introduces pH dependent membrane disruptive activity, which is not characteristic for PCPP itself, and is typically correlated to the ability of macromolecular carrier to facilitate endosomal escape. This can potentially affect the mechanism of immunoadjuvant action displayed by PCPP, afford means for its fine-tuning, as well as provide important insights for understanding the relationship between fundamental physico-chemical characteristics of polyphosphazene immunoadjuvants and their activity in vivo.

The thermoxidation of polypropylene (PP) at 200 °C in the presence of phenothiazine ( 1 ) and 3,7-di- tert -butylphenothiazine ( 2 ) was studied by measuring the oxygen consumption. The rate of oxygen consumption during the induction period passes through a minimum when the antioxidant concentration increases. Both compounds studied have the same critical concentrations but at high concentrations 2 is more efficient than 1 . The results obtained may be explained by the differences in the evaporation rate of 1 and 2 during oxidation. ESR signals of radicals originating from 1 and 2 after polymer oxidation at 160 °C were observed.

A series of five acridan derivatives was studied as antioxidants in the thermoxidation of polypropylene. None induced major retardation in the polymer oxidation. Two of them, 9,9-diphenyl- and 9,9-dimethyl-acridans, showed synergism when used in the presence of dilauryl thiodipropionate (DLTP). In order to understand the role of the intermediate nitroxides during the oxidation, 9,9-diphenylacridan-1-oxyl was prepared and studied in the presence and in the absence of DLTP. The main reactivity of the nitroxide is explained through disproportionation and hydrogen abstraction reactions and formation of alkylated hydroxylamine, where the alkyl group is the polymer chain.

The formation of a charge-transfer complex between tetracyanoethylene (TCNE) and hexamethylbenzene (HMB) in isotactic polypropylene (PP) has been studied by using UV-VIS spectroscopy. The effective equilibrium constant of complex formation K e increases with the concentration of TCNE or HMB in the polymer. Hexadecane affects the K e in PP, while it has no influence on the complex in solution. The theory of non-uniform additives distribution in the polymer medium and the reactivity are discussed.

The kinetics of nitroxide formation on a polypropylene (PP) chain by reaction between carbonyl groups of γ-irradiated PP and 2-amino-2-methyl-1-propanol has been studied. During the reaction the total concentration of carbonyl and amine groups passes through a maximum. At the end of the reaction a weak ESR signal due to nitroxide groups was detected and its intensity was strongly enhanced by peroxidation with m-chloroperbenzoic acid. The presence of nitroxides after the reaction with the aminoalcohol has been explained by the interaction of an oxazolidine moiety with singlet oxygen coming from the decomposition of PP-hydroperoxides or by reaction of the latter compounds with the oxazolidine nucleus.

Degradation of a water-soluble polyphosphazene, poly[di(carboxylatophenoxy)phosphazene], disodium salt (PCPP) has been studied in aqueous solutions at elevated temperature. This synthetic polyelectrolyte is of interest as vaccine adjuvant and its degradability constitutes an important component of its safety and formulation stability profiles. The degradation process is manifested by a gradual reduction in the molecular weight of the polymer and cleavage of side groups, which is consistent with previously reported data on hydrolytical breakdown of water-soluble polyphosphazenes. The kinetics of hydrolytical degradation exhibits distinct pH dependence and the process is faster in solutions with lower pH. Remarkably, a number of hydrogen bond forming additives, such as polyethylene glycol and Tween displayed a dramatic accelerating effect on the degradation of PCPP, whereas inorganic salts, such as sodium chloride and potassium chloride, showed a trend for its retardation. The results can be potentially explained on the basis of acid promoted hydrolysis mechanism and macromolecular interactions in the system.

Intradermal immunization using microneedles requires compatible immunoadjuvant system. To address this challenge, we investigated microneedles coated with polyphosphazene polyelectrolyte, which served both as microfabrication material and an immunoadjuvant compound.Coated microneedles were fabricated by depositing formulations containing Poly[di(carboxylatophenoxy)phosphazene], PCPP, on metal shafts, and their physico-chemical characterization was conducted.Microfabrication of PCPP-coated microneedles exhibited strong dependence on protein-PCPP interactions in solutions and allowed for high efficiency of protein encapsulation. 70°C thermal inactivation studies demonstrated a remarkable increase in functional stability of protein in coated microneedles compared to solution formulation. A potential for modulation of protein release from coated microneedles has been demonstrated through ionic complexation of PCPP with small ions.Microneedles containing PCPP coatings provide improved protein stability, modulated release, and protein-friendly microfabrication process.

A new water-soluble polyphosphazene polyelectrolyte containing carboxylate functionalities, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) was synthesized via reaction of macromolecular substitution. The polymer was characterized using (1)H, (31)P NMR, and gel permeation chromatography with multiangle laser light scattering detection. PCEP was shown to undergo hydrolytic degradation in aqueous solutions, as indicated by the decrease in the molecular weight and the release of side groups. A series of incompletely substituted copolymers of PCEP containing varying amounts of residual chlorine atoms was also prepared. The rate of degradation for such copolymers increased with the rise in the content of chlorine atoms. In vivo studies demonstrated high potency of PCEP as a vaccine immunoadjuvant. The new polyphosphazene was also shown to be capable of forming microspheres in aqueous solutions via reactions of ionic complexation with physiologically occurring amines, such as spermine.

Polyphosphazene polyelectrolytes are potent immunostimulants. Their in vivo performance has been demonstrated for various antigens in a number of animal models. To improve understanding of the mechanism of action, we performed a comparative study in a model system: bovine serum albumin, BSA−poly[di(carboxylatophenoxy)phosphazene], PCPP, in vitro and in vivo. Multi-angle laser light scattering (MALLS) and size-exclusion HPLC methods were used to investigate polyphosphazene−protein formulations in an attempt to establish correlations between their physicochemical behavior and immunostimulating activity. These studies revealed the formation of water-soluble noncovalent protein−polymer complexes in the system. It was shown that both the amount of bound protein and the complex conformation could play an important role in the in vivo performance of the polyphosphazene polyelectrolytes.