Alexander Marin

Poly[di(carboxylatophenoxy)phosphazene] is a potent adjuvant for intradermal immunization

Intradermal immunization using microfabricated needles represents a potentially powerful technology, which can enhance immune responses and provide antigen sparing. Solid vaccine formulations, which can be coated onto microneedle patches suitable for simple administration, can also potentially offer improved shelf-life. However the approach is not fully compatible with many vaccine adjuvants including alum, the most common adjuvant used in the vaccine market globally. Here, we introduce a polyphosphazene immuno adjuvant as a biologically potent and synergistic constituent of microneedle-based intradermal immunization technology. Poly[di(carboxylatophenoxy)phosphazene], PCPP, functions both as a vaccine adjuvant and as a key microfabrication material. When used as part of an intradermal delivery system for hepatitis B surface antigen, PCPP demonstrates superior activity in pigs compared to intramascular administration and significant antigen sparing potential. It also accelerates the microneedle fabrication process and reduces its dependence on the use of surfactants. In this way, PCPP-coated microneedles may enable effective intradermal vaccination from an adjuvanted patch delivery system.

Mechanism of the ultrasonic activation of micellar drug delivery

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.

Acoustic activation of drug delivery from polymeric micelles: effect of pulsed ultrasound

The effect of a continuous wave (CW) and pulsed 20-kHz ultrasound on the Doxorubicin (DOX) uptake by HL-60 cells from the phosphate buffered saline solution (PBS) and Pluronic micellar solutions was studied. Both CW and pulsed ultrasound enhanced DOX uptake from PBS and Pluronic micelles. The main factor that effected drug uptake was ultrasound power density; however, with increasing power, the enhanced drug uptake was accompanied by the extensive cell sonolysis. For PBS, no significant effect of duration of the ultrasound pulse or inter-pulse interval on the drug uptake was observed. For Pluronic micelles, the uptake increased with increasing pulse duration in the range 0.1-2 s, overall sonication time being the same. For 2-s pulses, the uptake was close to that under CW ultrasound. There was no significant effect of the duration of the inter-pulse interval on the drug uptake from Pluronic micelles. The data on the effect of pulse duration on drug uptake suggest that the characteristic times of drug release from micelles and drug uptake by the cells are comparable. The results point to two independent mechanisms controlling acoustic activation of drug uptake from Pluronic micelles. Both mechanisms work in concert. The first one is related to the acoustically-triggered drug release from micelles that results in higher concentration of the free drug in the incubation medium. The second mechanism is based on the perturbation of cell membranes that results in the increased uptake of the micellar-encapsulated drug. The intracellular uptake of Pluronic micelles was confirmed by fluorescence microscopy.

Intracellular uptake of Pluronic copolymer: effects of the aggregation state

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.

Microneedles with Intrinsic Immunoadjuvant Properties: Microfabrication, Protein Stability, and Modulated Release

ABSTRACTPurposeIntradermal 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.MethodsCoated microneedles were fabricated by depositing formulations containing Poly[di(carboxylatophenoxy)phosphazene], PCPP, on metal shafts, and their physico-chemical characterization was conducted.ResultsMicrofabrication 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.ConclusionsMicroneedles containing PCPP coatings provide improved protein stability, modulated release, and protein-friendly microfabrication process.

Effect of Environmental Factors on Hydrolytic Degradation of Water-Soluble Polyphosphazene Polyelectrolyte in Aqueous Solutions

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.

Protein stabilization in aqueous solutions of polyphosphazene polyelectrolyte and non-ionic surfactants.

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.

PCPP-formulated H5N1 influenza vaccine displays improved stability and dose-sparing effect in lethal challenge studies.

The potential impact of an influenza pandemic can be mitigated through the realization of a successful vaccination program. The implementation of antigen stabilization and dose-sparing technologies is an important step in improving availability of vaccines at the time of a pandemic outbreak. We investigated poly[di(carboxylatophenoxy)phosphazene] (PCPP) as a potential stabilizing and immunostimulating agent for H5N1 influenza vaccine. Physicochemical characterization of PCPP-formulated H5N1 influenza vaccine revealed macromolecular complexation in the system, whereas single radial immunodiffusion assay verified antigenicity of the formulation in vitro. PCPP-enhanced formulation displayed a fourfold increase in the half-life at 40°C compared with a nonadjuvanted vaccine. Lethal challenge studies in ferrets demonstrated 100% protection for low-antigen dose PCPP-adjuvanted formulations (1 μg of hemagglutinin) and at least a 10-fold antigen-sparing effect. Therefore, PCPP demonstrated an ability to improve thermal stability of H5N1 influenza vaccine in solutions and provide for a substantial dose-sparing effect in vivo.

Self-assembly of polyphosphazene immunoadjuvant with poly(ethylene oxide) enables advanced nanoscale delivery modalities and regulated pH-dependent cellular membrane activity

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.

PCPP-Adjuvanted Respiratory Syncytial Virus (RSV) sF Subunit Vaccine: Self-Assembled Supramolecular Complexes Enable Enhanced Immunogenicity and Protection

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.