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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.

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.

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.

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.

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.

Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non-toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non-covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ-PEG) and a pyrrolidone containing linear derivative (PZ-PYR) for their ability to intracellularly deliver FITC-avidin, a model protein. In endothelial cells, PZ-PYR/protein exhibited both faster internalization and higher uptake levels than PZ-PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ-PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ-PEG/avidin colocalized more profusely with endo-lysosomes, and PZ-PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ-PYR-delivered anti-F-actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell-impermeable Bax-BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ-PYR, demonstrating endo-lysosomal escape. These biodegradable PZs were non-toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

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.

Copyright © 2020 American Society for Microbiology. An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.IMPORTANCE Hepatitis C virus infects approximately 1% of the world's population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested in vitro and in vivo, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target.

The development of successful vaccines has been increasingly reliant on the use of immunoadjuvants - additives, which can enhance and modulate immune responses to vaccine antigens. Immunoadjuvants of the polyphosphazene family encompass synthetic biodegradable macromolecules, which attain in vivo activity via antigen delivery and immunostimulation mechanisms. Over the last decades, the technology has witnessed evolvement of next generation members, expansion to include various antigens and routes of administration, and progression to clinical phase. This was accompanied by gaining important insights into the mechanism of action and the development of a novel class of virus-mimicking nano-assemblies for antigen delivery. The present review evaluates in vitro and in vivo data generated to date in the context of latest advances in understanding the primary function and biophysical behavior of these macromolecules. It also provides an overview of relevant synthetic and characterization methods, macromolecular biodegradation pathways, and polyphosphazene-based multi-component, nanoparticulate, and microfabricated formulations.

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.

HIV-1 broadly neutralizing antibodies (bNAbs) are being explored as passively administered therapeutic and preventative agents. However, the extensively diversified HIV-1 envelope glycoproteins (Env) rapidly acquire mutations to evade individual bNAbs in monotherapy regimens. The use of a “single” agent to simultaneously target distinct Env epitopes is desirable to overcome viral diversity. Here, we report the use of tandem single-chain variable fragment (ScFv) domains of two bNAbs, specific for the CD4-binding site and V3 glycan patch, to form anti-HIV-1 bispecific ScFvs (Bi-ScFvs). The optimal Bi-ScFv crosslinks adjacent protomers within one HIV-1 Env spike and has greater neutralization breadth than its parental bNAbs. Furthermore, the combination of this Bi-ScFv with a third bNAb recognizing the Env membrane proximal external region (MPER) results in a trispecific bNAb, which has nearly pan-isolate neutralization breadth and high potency. Thus, multispecific antibodies combining functional moieties of bNAbs could achieve outstanding neutralization capacity with augmented avidity., Broadly neutralizing antibodies targeting HIV Env could potentially be utilized as therapeutics. Here, Steinhardt et al. engineer a trispecific antibody with specificity for the receptor-binding site, a conserved Env glycan patch and the Env membrane proximal region with nearly pan-isolate neutralization breadth and high potency.

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.

Nanoparticulate and water-soluble formulations of ionic polyphosphazenes and protein cargo - lysozyme (LYZ) were prepared by their self-assembly in aqueous solutions at near physiological pH (pH 7.4) in the presence and absence of an ionic cross-linker – spermine tetrahydrochloride. Efficiency of LYZ encapsulation, physico-chemical characteristics of formulations, and the effect of reaction parameters were investigated using asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) methods. The effect of both polymer formulations on encapsulated LYZ was evaluated using soluble oligosaccharide substrate, whereas their ability to present the protein to cellular surfaces was assessed by measuring enzymatic activity of encapsulated LYZ against Micrococcus lysodeikticus cells. It was found that both soluble and cross-linked polymer matrices reduce lysis of bacterial cells by LYZ, whereas activity of encapsulated protein against oligosaccharide substrate remained practically unchanged indicating no adverse effect of polyphosphazene on protein integrity. Moreover, nanoparticulate formulations display distinctly different behavior in cellular assays when compared to their soluble counterparts. LYZ encapsulated in polyphosphazene nanoparticles shows approximately 2.5-fold higher activity in its ability to lyse cells as compared with water-soluble LYZ-PCPP formulations. A new approach to PEGylation of polyphosphazene nanoparticles was also developed. The method utilizes a new ionic polyphosphazene derivative, which contains graft (polyethylene glycol) chains. PEGylation allows for an improved control over the size of nanoparticles and broader modulation of their cross-linking density, while still permitting for protein presentation to cellular substrates.

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 (

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.

A series of biodegradable drug delivery polymers with intrinsic multifunctionality have been designed and synthesized utilizing polyphosphazene macromolecular engineering approach. Novel water-soluble polymers, which contain carboxylic acid and pyrrolidone moieties attached to inorganic phosphorus-nitrogen backbone, were characterized by a suite of physico-chemical methods to confirm their structure, composition, and molecular sizes. All synthesized polyphosphazenes displayed composition dependent hydrolytic degradability in aqueous solutions at neutral pH. Their formulations were stable at lower temperatures, potentially indicating adequate shelf life, but were characterized by accelerated degradation kinetics at elevated temperatures, including 37°C. It was found that synthesized polyphosphazenes are capable of environmentally triggered self-assembly to produce nanoparticles with narrow polydispersity in the size range between 150 and 700 nm. Protein loading capacity of copolymers has been validated via their ability to non-covalently bind avidin without altering its biological functionality. Acid induced membrane disruptive activity of polyphosphazenes has been established with an onset corresponding to endosomal pH range and being dependent on polymer composition. The synthesized polyphosphazenes facilitated cell-surface interaction followed by time-dependent, vesicular mediated, and saturable internalization of a model protein cargo into cancer cells, demonstrating potential for intracellular delivery.

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.

A vast number of studies explore the potential of intradermal immunization, its role in the developing of new and improved vaccines and providing access to them globally. The advancement of microneedle technology offers new avenues for the practical realization of this approach, but, on certain instances, introduces new limitations and challenges in the formulation development, which involve the concern over its compatibility with existing and emerging immunoadjuvants. The present paper attempts to review various aspects of immunoadjuvant enhanced microneedle immunization and focuses on the potential of synthetic biomaterials that can play a multifunctional role in such approach. PCPP, a synthetic polyphosphazene macromolecular compound, is discussed as an example of such material that can potentially enable the technology as a microfabrication agent and a potent intradermal immunoadjuvant.

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.

Water-soluble polyphosphazenes are an emerging class of biologically important synthetic polymers. Rapid advancements in the field have been facilitated with the discovery of potent polyphosphazene immunoadjuvants, use of hydrophilic polyphosphazenes in mild microencapsulation processes, and synthesis of water-soluble biodegradable polyphosphazenes – analogs of the well known pharmaceutical carriers. Present review summarizes the most recent advances in the synthesis of water-soluble polyphosphazenes, studies on their degradation and biological performance.