Molecular-Level Interactions of Polyphosphazene Immunoadjuvants

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Molecular-Level Interactions of Polyphosphazene Immunoadjuvants and Their Potential Role in Antigen Presentation and Cell Stimulation Alexander K. Andrianov, Alexander Marin, and Thomas R. Fuerst Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01251 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016

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Molecular-Level Interactions of Polyphosphazene Immunoadjuvants and Their Potential Role in Antigen Presentation and Cell Stimulation Alexander K. Andrianov,* Alexander Marin, and Thomas R. Fuerst Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850, United States

ABSTRACT:

Two

macromolecular

poly[di(carboxylatophenoxy)phosphazene],

immunoadjuvants PCPP

and

poly[di(carboxylatoethylphenoxy)phosphazene], PCEP have been investigated for their molecular interactions with model and bio-pharmaceutically 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

1 ACS Paragon Plus Environment

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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 non-specific 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.

INTRODUCTION Immunoadjuvants, which are capable of enhancing and modulating antigen specific immune responses, serve as key components in the development of modern vaccines.1-5 This term may generally include either specific immunostimulating molecules, which directly activate innate immune receptors, or delivery systems, which consist of nonimmunostimulating components, but still function as adjuvants by providing more effective antigen presentation to the immune system.1 Whereas the first group is typically represented by well-defined compounds, such as MPL (monophosphoryl lipid A) or CpG (oligodeoxynucleotide), the delivery systems generally rely on particulate formulations, such

as

insoluble aluminum

salts

or oil-in-water

emulsions.1 Water-soluble

polyphosphazene polyelectrolytes, a family of large synthetic macromolecules, present an attractive alternative to such bi-phasic delivery systems.6, 7 These macromolecules have already demonstrated potent ability to enhance immune responses to various bacterial and viral antigens in several animal models.8-11 Ionic polyphosphazenes, which consist of 2 ACS Paragon Plus Environment

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phosphorus and nitrogen backbone and pendant organic groups with carboxylic acid moieties, are hydrolytically degradable and are capable of spontaneous self-assembly with

other

macromolecules

under

physiological

Poly[di(carboxylatophenoxy)phosphazene],

PCPP

conditions.12-14 and

poly[di(carboxylatoethylphenoxy)phosphazene], PCEP (Chart 1) are probably the most investigated representatives of this class of vaccine adjuvants to date. Despite the welldocumented activity in vivo, their mechanism of action is still under discussion. Although it has been long assumed that antigen delivery and presentation, including in the multimeric form, is an important function of these compounds, previous studies on protein – polymer interactions were limited to their interactions with a single model protein, BSA.13 There have been no reports on the selectivity of such interactions, functional properties of proteins in these non-covalent complexes, or even data on selfassembly in formulations with vaccine antigens. In addition, recent findings on the ability of PCPP to induce activation and maturation of dendritic cells15 and PCEP to activate adjuvant core response genes16 in the absence of antigen suggest that the role of such macromolecules may not be limited to the delivery and presentation of the antigen. However, the molecular-level mechanisms of the effects induced by these synthetic polyelectrolytes remain unclear. The objective of the present paper is to investigate interactions of PCPP and PCEP with model proteins, vaccine antigens, and immune receptors on the molecular level in solutions at near physiological conditions, which is conducted using Asymmetric Field Flow Fractionation (AF4) and Dynamic Light Scattering (DLS) methods. For the first time we report on the ability of PCPP and PCEP to bind vaccine antigens and


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immunologically important receptors, including Toll-Like Receptors (TLR) proteins. Furthermore, both polyphosphazenes are investigated in cellular assays for their ability to activate TLR signaling pathways and display pH-dependent membrane disruptive activity, which is a cornerstone in elucidating the mechanism of action of these important polyphosphazene immunoadjuvants. Chart 1. Chemical structures of PCPP and PCEP in their sodium salt forms.

O

O

O

O

O

O O

O

Na Na

Na

CH 2 CH 2

CH 2 CH 2

Na

O

O

O

O P P N

N n

n

PCEP

PCPP

EXPERIMENTAL SECTION Materials. Poly[di(carboxyatophenoxy)phosphazene], sodium salt (PCPP) and poly[di(carboxyatoethylphenoxy)phosphazene], sodium salt (PCEP) were synthesized as described previously.11, 17 Both polymers were analyzed for bacterial endotoxin by kinetic turbidimetric tests and were found essentially endotoxin free. Hepatitis C Recombinant protein (HCV E2) was kindly provided by Dr. Roy A. Mariuzza and Ebolavirus (EBOV) glycoprotein (GP) was kindly provided by Dr. Gilad A. Ofek. Subtilisin A (Protease, Bacillus licheniformis Type VIII, lyophilized powder, 7-15 units/mg solid, EC 3.4.21.62, Molecular Weight (MW) 27,287 Da, Isoelectric point (pI 9.4), Lectin from Lens culinaris


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(lentil, lyophilized powder, MW 49,000 Da, pI 8.2, 8.6, 8.8), Cytochrome C (equine heart, lyophilized powder, MW 12,384 Da, pI 10.0-10.5, 95%), Myoglobin (equine skeletal muscle, lyophilized powder, 95-100%, MW 17,600 Da, pI 7.3, 6.8), Bovine serum albumin (BSA) (lyophilized powder, 98%, MW 66,000 Da, pI 4.8), Ovalbumin (Albumin, chicken egg white, lyophilized powder, 98%, MW 44,287 Da, pI 4.54, 4.9), Avidin (egg white, lyophilized powder, 98%, MW 66,000 Da, pI 10.5), Avidin-FITC (egg white, lyophilized powder, ≥60%, MW 66,000 Da, pI 10.5), Biotin (5-fluorescein) conjugate (BIO-FITC), Diammonium salt of 2,2'-azinobis-(3-ethylbenzthiazoline-6sulfonate) (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish Peroxidase (HRP) (300 units/mg, lyophilized powder, EC 1.11.1.7, pI 3-9, MW 44,000), Green Fluorescent Protein (GFP) (lyophilized powder, 98%, pI 6.2, MW 28,700 Da), Phosphate Buffered Saline (PBS, pH 7.4) were purchased from ThermoFisher Scientific (Grand Island, NY, USA). Asparaginase (lyophilized powder, ≥98%, MW 32,000 Da, pI 3.3-9.7, specific activity 225 IU/mg), Natural Chicken Deglycosylated Avidin (≥95%, MW 60,000 Da), HIV-1 gp120 (ADA) (Clade B) (solution in PBS, ≥95%, MW 70,000-100,000 Da), Influenza hemagglutinin, HA (A/California/07/2009)(H1N1) (solution in PBS, ≥95%) were purchased from eEnzyme (Gaithersburg, MD, USA). Hepatitis B Surface Antigen (Adw) (recombinant protein, ≥95%) was purchased from Abcam (Cambridge, MA, USA). Human TLR3 (recombinant, lyophilized powder, ≥95%, MW 110,000-120,000 Da), Human TLR4 (recombinant, lyophilized powder, ≥95%, MW 90,000-95,000 Da), Mouse TLR9 Fc Chimera (recombinant, lyophilized powder, ≥95%, MW 140,000-170,000 Da), Human


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MMR/CD206 (recombinant, lyophilized powder, ≥90%, MW 180,000 Da) were purchased from R&D System Inc. (Minneapolis, MN, USA). Asymmetrical Flow Field Flow Fractionation (AF4). Studies of protein – polymer interactions were conducted by Asymmetrical Flow Field Flow Fractionation (AF4), which offers the possibility of a separation under shear free conditions and without unwanted interactions with the surface of a stationary phase.18 Formulations were dissolved in PBS (pH 7.4) and separated in a channel, to which an external cross-flow was applied, which acted perpendicular to the carrier-flow resulting in sample separation. The size-dependent diffusion abilities of molecules lead to an arrangement in different layers of the parabolic flow profile inside the channel with small molecules eluting first and the larger molecules or particles eluting later.18 The AF4 system, a Postnova AF2000 MT series (Postnova Analytics GmbH, Landsberg, Germany), was equipped with two PN1130 isocratic pumps, PN7520 solvent degasser, PN5120 injection bracket and UVVis detector (SPD-20A/20AV, Shimadzu Scientific Instruments, Columbia, MD 21046). A regenerated cellulose membrane with molecular weight cutoff of 10 kDa (Postnova Analytics GmbH, Landsberg, Germany) and a 350 µm spacer were used in a separation micro-channel employing both laminar and cross flows of an eluent - PBS (pH 7.4). The collected data was processed using AF2000 software (Postnova Analytics GmbH). Dynamic Light Scattering (DLS). Hydrodynamic diameters of polymers and their complexes with proteins in aqueous solutions were carried out using ZetaSizer Nano series, ZEN3500, (Malvern Instruments Ltd., Worcestershire, UK). Formulation components were filtered using 0.22 µm Millex syringe filters (EMD Millipore, Billerica, MA) before mixing.


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Human Toll-Like Receptor (TLR) Screening in Cellular Assays. PCPP and PCEP were screened for their ability to stimulate TLR signaling in cells. The assay (InvivoGen, San Diego, CA, USA), utilizes HEK293 derived cells overexpressing a given human TLR gene. Monitoring of signaling through the TLR is based on the activation of a transcription factor - nuclear factor-kappa B (NF-kB). The secreted embryonic alkaline phosphatase (SEAP) reporter is used, which is under the control of a promoter inducible by the transcription factor NF-κB. In a 96-well plate (200 µL total volume) containing the appropriate cells (50,000-75,000 cells/well), 20 µL of the polymer solution or the positive control ligand is added to the wells. The media added to the wells is designed for the detection of NF-κB induced SEAP expression. After a 16-24 hr incubation the optical density (OD) is read at 650 nm on a Molecular Devices SpectraMax 340PC absorbance detector. All experiments were performed in triplicates. The following control ligands were used: hTLR2: HKLM (heat-killed Listeria monocytogenes) at 1x108 cells/mL, hTLR3: Poly(I:C) HMW at 1 µg/mL, hTLR4: E. coli K12 LPS at 100 ng/mL, hTLR5: S. typhimurium flagellin at 100 ng/mL, hTLR7: CL307 at 1 µg/mL, hTLR8: CL075 at 1 µg/mL, hTLR9: CpG ODN2006 at 100 ng/mL. TLR- Negative Control Cell Lines were as follows: HEK293/Null1: TNFα at 100 ng/mL (control for human TLR2, 3, 5, 8 and 9), HEK293/Null1-k: TNFα at 100 ng/mL (control for humanTLR7), HEK293/Null2: TNFα at 100 ng/mL (control for human TLR4). Evaluation of Membrane Disruptive Activity. The membrane disruptive activity of multifunctional carriers, which can be correlated to the ability of the carrier to facilitate endosomal escape and cytosolic delivery of pharmaceutical agent, was tested as described previously.19-21 100 µL of fresh Porcine Red Blood Cells (RBC) as a 10% suspension in


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phosphate buffered saline (PBS) (Innovative Technology Inc., Novi, MI) was resuspended in 900 µL of PBS. 50 µL of re-suspended RBC was added to 950 µL of the PCPP-PEG or PCPP formulation in PBS at the appropriate pH, inverted several times for mixing, and incubated in a 37 °C for 60 min. Cells were then centrifuged at 14,000 rpm for 5 min, and the absorbance of the supernatant was then measured at 540 nm using Multiskan Spectrum microplate spectrophotometer (ThermoFisher Scientific, Waltham, MA). To determine 100% hemolysis, RBCs were suspended in distilled water and lysed by ultrasound (Branson Sonifier, Model 450). All hemolysis experiments were done in triplicate.

RESULTS AND DISCUSSION Analysis of Protein-Polyphosphazene Interactions in Solution Using Field Flow Fractionation and Dynamic Laser Light Scattering. The biological role of polyphosphazene polyelectrolytes as vaccine delivery and immunostimulatory agents has been typically linked to antigenic proteins, with which these macromolecules are typically co-formulated. Although proteins and polyphosphazenes do not require chemical conjugation and are physically mixed in vaccine formulations, the possibility of non-covalent association between antigens and these macromolecular adjuvants has been discussed as a major factor, which can potentially affect biological activity of the vaccine.13 To investigate interactions between polyphosphazene adjuvants, PCPP and PCEP, and a broad range of model and pharmaceutically important proteins, we employed a method of Asymmetric Flow Field Flow Fractionation (AF4). AF4 is an elution-based method, in


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which the separation is carried out in a single liquid phase and an external flow of the mobile phase is applied perpendicularly to the direction of sample flow through a channel equipped with semi-permeable membrane.18 Similar to size-exclusion HPLC, the materials are separated by size, however, as opposed to chromatographic methods, the upper size limit for the analyte can reach as high as 100 µm.18 The method minimizes or even completely eliminates one of the main experimental challenges in a direct investigation of molecular interactions, such as potential interference with stationary phase (HPLC or Surface Plasmon resonance methods), or the influence of the electric field (electrophoretic methods).18 All formulations were also monitored for changes in molecular sizes using Dynamic Laser Light Scattering (DLS) – a conventional technique for studying self-assembly processes between polyelectrolytes and proteins.22 Phosphate buffered saline (PBS) with pH 7.4, which is a common buffer for modeling near physiological conditions, was employed as a medium. The methodology is exemplified in Figure 1A, which displays AF4 fractograms of PCPP, a model protein - Avidin, and their mixture. The formation of the complex can be typically detected by measuring the decrease or even disappearance of a protein peak in the fractogram of the complex. As seen in Figure 1A, the absence of Avidin peak in its formulation

with

PCPP

demonstrates

complete

binding

of

this

protein

to

polyphosphazene. This finding can be further confirmed by employing fluorescently labeled Avidin, which allows monitoring complex formation by AF4 with detection in the visible part of the spectrum – 495 nm (Figure 1B). At this wavelength, PCPP alone cannot be detected. However, the addition of fluorescently labeled protein to PCPP results not only in the disappearance of the protein peak as observed above, but also leads


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to a ‘stained’ complex peak, which is practically identical in shape and position to a PCPP peak observed at 210 nm (Figure 1A). This unambiguously provides a proof of Avidin-PCPP complex formation in the system. DLS results for the Avidin – PCPP system are shown in Figure 1C. Addition of Avidin to PCPP (Figure 1C, trace 1) in small quantities typically does not produce significant conformation changes in the polymer (Figure 1C, trace 2). However, significant changes in DLS profiles can be observed at a higher protein to polymer molar ratios, which can also result in a significant aggregation (Figure 1C, trace 3-5) or even formation of insoluble precipitate. Protein - Polyphosphazene Binding Maps and Isotherms. We investigated PCPP and PCEP for their ability to interact with eleven well-characterized model proteins and five vaccine antigens at near physiological conditions. The percent of protein binding was calculated on the basis of unbound protein in the formulation using AF4 methodology described above. The resulting protein-polyphosphazene ‘interaction map’, in which proteins are arranged in accordance with their isoelectric point, is shown in Figure 2. Both polyphosphazenes exhibited strongest affinity to proteins with high isoelectric points, such as Lysozyme, Avidin, and Cytochrome C, which demonstrates the importance of ionic interactions in the system, since polyphosphazene polyelectrolytes are negatively charged. However, no direct correlation of binding to isoelectric point was observed. The most notable exception is a strong interaction detected between both polyphosphazene polyelectrolytes and BSA, a protein with a relatively low isoelectric point of 4.8.23 Since hydrophobic cleft of this protein is a major determinant in interactions of BSA with other proteins and polyelectrolytes,24-26 this may suggest the


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importance of hydrophobic interactions in such systems. However, the reduction of positive charges on the protein through covalent coupling of fluorescein isothiocyanate to aminogroups of lysine residues resulted in a complete elimination of interactions. The absence of binding between polyphosphazenes and FITC-BSA (data not shown), underlines the dominating role of electrostatic forces in this system despite the relatively low isoelectric point of the protein and possibility of hydrophobic interactions. Importantly, it is the presence and characteristics of the ‘charge patches’ – anionic or cationic domains on the protein, not the overall charge, that plays a critical role in protein-polyelectrolyte interactions.22 Typical protein-polyphosphazene binding isotherms are shown in Figure 3A. Interactions of PCPP or PCEP with Cytochrome C are expressed as a molar ratio of bound protein molecules per polyphosphazene chain (fractional occupancy) versus concentration of free protein. From the formulation prospective, binding isotherm can be employed as a guide to select systems with optimal ratio of bound and unbound protein. Although, PCPP appears to be somewhat superior to PCEP in terms of saturation with protein molecules, the curves show similar profiles. Figure 3B displays binding isotherms of two proteins, Cytochrome C and Avidin, to PCPP. Despite the fact, that isoelectric points for both proteins are similar, they show a dramatic difference in affinity to PCPP. The value of protein concentration at half-saturation of polyphosphazene, which corresponds to a dissociation constant for a simplified binding model,27 was utilized for a crude, semi-quantitative comparison of two curves. The two orders of magnitude difference in such apparent constants (approximately 2x10-7 for Avidin versus 2x10-5 for Cytochrome C) may indicate significant differences in the mechanism of binding for two


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proteins. Although it is not possible to make an accurate comparison of the above apparent dissociation constants with values for other biological systems due to the simplified nature of the approximation, it still may be worth mentioning that the micromolar range of dissociation constants is typical for binding of signaling protein to a biological target.27 A distinct feature of Avidin, which differentiates this glycoprotein from Cytochrome C, is a significant, about 10 % of the molecular weight, content of carbohydrate (Figure 3D).28 Since PCPP has previously demonstrated the ability to form hydrogen bonds at pH 7.4, which led to self-assembly of this polymer with macromolecules, such as poly(ethylene oxide),29 it was important to explore the effect of glycan on interactions of Avidin with polyphosphazenes. To this end, we investigated the potential binding of a non-glycosylated Avidin to PCPP. Strikingly, a carbohydrate free protein showed a dramatically lower affinity to PCPP (Figure 3C). This, for the first time, provides evidence pointing to a major role, which hydrogen bonds can play in interactions between polyelectrolytes and proteins at near physiological conditions. The differences in binding between a polyelectrolyte and proteins with similar isoelectric points have been also explained previously in view of relevant protein charge anisotropies.26 Surface electrostatics for Avidin and Cytochrome C are visualized in Figure 3D using PyMOL molecular graphics system and structural data provided by Protein Data Bank. Although both proteins display distinct positive charge patches, Cytochrome C appears to exhibit more defined electronegative domains raising the possibility of more intense repulsive interactions with the polymer. However, the more quantitative analysis of electrostatic


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potential surfaces is needed to further understand the significance of such potential contribution. Interactions of Polyphosphazenes with Vaccine Antigens. Since biomedical applications of polyphosphazene polyelectrolytes involve their formulation with vaccine antigens, we investigated a number of antigenic proteins, which have been previously studied with polyphosphazenes, for their potential interactions with PCPP and PCEP (Figure 4). As seen in the Figure, all five viral antigens studied showed strong affinity to both polyelectrolytes as measured by AF4 method (Figure 4A). Addition of all tested antigens to PCPP or PCEP also resulted in a significant (1.5-4 fold) increase in the size of polyphosphazene as determined by DLS under the conditions studied (Figure 4B). Such dramatic increase in size may suggest intermolecular aggregation and, with the exception of Avidin, was not characteristic for formulations of polyphosphazenes with model proteins shown in Figure 2, for which the diameter of polymer remained unchanged or underwent only minor alterations upon addition of proteins (data not shown). It is important to mention, that unlike other proteins studied for their interactions with polyphosphazenes, Avidin and vaccine antigens of this study are glycoproteins containing a significant amount of carbohydrates, with the latter capable of highly cooperative hydrogen bonding.30 This provides further support for the previously mentioned observation on the role of hydrogen bonds in interactions between polyphosphazenes and proteins and emphasizes the need for comprehensive physicochemical studies of vaccine formulations. Interactions of PCPP and PCEP with Immune Receptor Proteins in Solution. Targeting of host innate immune system through activation of immune receptors


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constitutes an important approach in the development of immunoadjuvants. To this end, pattern-recognition receptors (PRRs), which recognize pathogen associated molecular patterns (PAMPs) from microorganisms or danger-associated molecular patterns (DAMPs) from damaged tissue, present particular interest. Two main classes of PRRs have been described in mammalian cells: membrane-bound receptors, such as Toll-like receptors (TLRs)31-34 and C-type Lectin receptors (CLRs), such as Mannose receptor (MR).35 TLRs, which are a family of PRRs, recognize conserved molecular motifs that are shared by infectious agents, but which are absent in the host, function as primary sensors of the innate immune system to recognize microbial pathogens.31-34 TLR and CLRs can be typically expressed on members of the innate and adaptive immune system, such as dendritic cells (DC) or macrophages. It has been previously established that PCPP treatment can induce DC activation as assessed by upregulation of co-stimulatory molecules and cytokine production.15 In addition, PCEP induced potent expression of ‘adjuvant core response genes’, including upregulation of TLR-4 and TLR-9 gene expression.16 However, the molecular basis of such phenomena remains unknown. Immune receptor proteins, similarly to other membrane proteins, are characterized with regions of high lipophilic character and significant content of carbohydrates.36 The observed higher affinity of polyphosphazenes to glycosylated and amphipathic proteins encouraged us to investigate interactions of these polymers with immune receptors. We have explored the ability of PCPP and PCEP to bind TLR3, -4, 9 and MR in solution using AF4 method. As seen from Figure 5, both polyphosphazenes demonstrate strong affinity to soluble receptors under physiological conditions. Similar effects were observed for polyphosphazenes, which were already formulated with a representative


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antigen – HA. These findings may suggest a potential direct activation of immune cells by PCPP and PCEP through TLR signaling pathway, both on the external cell surface (TLR4) and endosomal (TLR3 and -9) levels.1,

32

Synthetic polymers have been

extensively studied as vaccine excipients and have been traditionally viewed as particulate delivery vehicles capable of facilitating physical uptake of the antigen by antigen presenting cells.37 Present results may broaden our views on their functionality and trigger further research on their interactions with cellular receptors. Interestingly, it has been recently reported that another polymer carrier, poly(methyl vinyl ether-comaleic anhydride) nanoparticles with an average size of 230 nm and electronegative surface charge, can also act as an agonist of TLR2, -4, and -5, triggering a Th1-profile cytokine release.37 Binding of PCPP and PCEP to MR, which is also demonstrated in Figure 5, may reveal a potential targeting utility of polyphosphazenes. It has been previously demonstrated that targeting of MR of antigen presenting cells can facilitate effective induction of potent cellular and humoral immune responses and therefore is a highly desirable property for an immunoadjuvant.1, 38 TLR Stimulatory Effects of Polyphosphazenes in Cellular Assays. Binding interactions of PCPP and PCEP with soluble TLR receptors observed on the molecular level prompted us to evaluate the ability of polyphosphazenes to activate TLR signaling in cellular assays. As mentioned above, despite the previously discovered ability of polyphosphazenes to stimulate DCs15 or induce changes in the gene expression of some “adjuvant core response genes”,16 there has been no reports on the activation of TLR signaling pathways in cells by polyphosphazenes.


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To this end, it was interesting to investigate polyphosphazenes in cellular assays featuring engineered HEK293 cells, which overexpress human TLR genes, specifically TLR2, -3, -4, -5, -7, -8, and -9. These cells utilize an NF-kB-inducible secreted embryonic alkaline phosphatase reporter gene as the read-out. Ligand-induced activation of the NF-kB pathway is then monitored using enzymatic detection assays. Figure 6 shows the results of these studies for PCPP (Figure 6A) and PCEP (Figure 6B). Cellular responses are presented as a percent of the positive control, which is individual for each given TLR. Empty bars show data for the same polymers and cell lines, which do not express any TLR, but still have an NF-kB inducible reporter (TLR- negative control cell lines). Although it is clear that both PCPP and PCEP were able to stimulate cellular responses, they do not appear specific for most TLRs as TLR- data also show some activation. The strongest responses for TLR overexpressed cells with minimal TLRstimulation was observed in case of TLR 8 and TLR 9 for PCPP and the same receptors, plus TLR 3 for PCEP (Figure 6). Interestingly, all of the above receptors are typically associated with nucleic acid agonists, which bear some formal structural similarities to polyphosphazenes - polyacids with phosphorus containing backbone. The results of the above cellular assays, along with demonstrated polyphosphazene – TLR binding on the molecular level, necessitate further investigation, especially TLR 8 and TLR 9 signaling pathways for polyphosphazene adjuvants. Interestingly, these receptors represent an endolysosomal type of TLRs,32 and the results may suggest that modulation of cellular uptake of PCEP to increase its accumulation into endosomes may present an important way of optimizing this polyphosphazene adjuvant.


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pH-Dependent Membrane Disruptive Activity of Polyphosphazenes. Crosspresentation of antigens by dendritic cells plays central role in the induction of efficient immune responses, especially CD8+ T-cell responses, which are critical for the immunological control of tumors and infectious diseases. It has been shown that vaccine delivery systems can enhance the efficacy of presentation of soluble protein antigen directing it into the major histocompatibility complex (MHC) class I antigen presentation pathway.39 This has been attributed to the ability of nanoparticulates or liposomes to increase the amount of antigen that escapes from endosomes into the cytoplasm.39-41 Some polyacids also have a potential to facilitate endosomal escape of proteins through pH sensitive membrane active behavior, typically through the pH triggered conformational changes and formation of hydrophobic aggregates during the acidification in an early endosomal environment.19 However, the membrane disruptive properties, which can be displayed by these macromolecules, do not always correlate with the desirable physiological range. We investigated the ability of PCPP and PCEP to disrupt eukaryotic cell membranes in the pH range of 6.0-7.5. These studies were conducted employing a standard test utilizing red blood cells (RBC) as endosomal membrane models.19,

42

As seen from Figure 7,

PCPP does not possess pH dependent membrane disruptive activity in the pH range investigated and is essentially insoluble at more acidic solutions. Somewhat unexpectedly, it was found that PCEP displays significant pH dependent membrane destabilizing activity within a pH range of approximately pH 6.0 - 6.9, which includes the pH environment of early endosomes.43 It can be hypothesized that the more hydrophobic structure of PCEP compared to PCPP plays critical role in interactions with cellular


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membranes. The finding of pH dependent membrane disruptive activity PCEP can provide new insights for better understanding differences in immunoadjuvant activities of these polyphosphazenes. CONCLUSIONS Investigation of mechanisms, by which polyphosphazene polyacids amplify an immune response to vaccine antigens, necessitates an in-depth exploration of their molecular level interactions with antigenic proteins and key immune receptors. However the potential existence of such interactions has been largely ignored in the majority of studies conducted to date. The results of our investigation suggest that every time the vaccine is formulated with polyphosphazene for immunological evaluation, there is a substantial probability of non-covalent antigen-adjuvant interactions being established in the system. In the present study both polyphosphazene adjuvants, PCPP and PCEP, displayed avidity to multiple proteins, including all vaccine antigens studied, when they were mixed in aqueous solutions at near physiological conditions. High isoelectric point and glycosylation of protein appear to favor their binding to polyphosphazenes, which emphasizes the role of ionic interactions and hydrogen bonding. These findings provide the basis for further development of polyphosphazenes as vaccine delivery systems and also underline the need for a comprehensive physico-chemical analysis of polyphosphazene formulations, which can open pathways to a more efficient delivery of the antigen and rational design of polyphosphazene formulations. For the first time we have demonstrated that polyphosphazene adjuvants are capable of direct binding to soluble immune receptors, such as various Human TLRs and mannose receptor, MR. This, along with equally significant data, which suggest the ability of


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polyphosphazenes to activate TLR signaling pathways in cellular assays, provide support for positioning polyphosphazenes as macromolecules with dual, antigen carrier – immunostimulant, functionality, rather than just vaccine delivery vehicles. Finally, the distinct difference between PCPP and PCEP molecules has been uncovered in their potential capacity to facilitate endosomal escape of the antigen, which will require further studies on the cross-presentation of the antigen formulated with these two adjuvants.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax 1 240-314-6225. Phone 1 240-314-6456.

ACKNOWLEDGEMENTS Authors are grateful to Dr. Peter C. Fusco for valuable discussions and Dr. Brian G. Pierce for expert technical assistance with 3-D molecular visualization of proteins. ABBREVIATIONS AF4, asymmetric flow field flow fractionation; DLS, dynamic light scattering; PCPP, poly[di(carboxyatophenoxy)phosphazene],

sodium

salt;

PCEP,

poly[di(carboxyatoethylphenoxy)phosphazene], sodium salt; TLR, toll-like receptor; PBS, phosphate buffered saline; FITC, fluorescein isothiocyanate; MW, molecular weight; pI, isoelectric point; RBC, red blood cells; DC, dendritic cells; HEK293, human embryonic kidney 293 cells; HCV E2, hepatitis C virus protein; EBOV, Ebolavirus; HIV,


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human immunodeficiency virus; GP, glycoprotein; BSA, bovine serum albumin; BIOFITC, Biotin (5-fluorescein) conjugate; ABTS, diammonium salt of 2,2'-azinobis-(3ethylbenzthiazoline-6-sulfonate); HRP, horseradish peroxidase; HBsAg, hepatitis B surface antigen; HA, Influenza hemagglutinin; MR, mannose receptor; SEAP, secreted embryonic alkaline phosphatase; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; DAMP, danger-associated molecular pattern; PRR, pattern-recognition receptor, CLR, C-type Lectin receptor; TNF, tumor necrosis factor.

REFERENCES 1.

Reed, S. G.; Orr, M. T.; Fox, C. B., Key roles of adjuvants in modern vaccines.

Nat Med 2013, 19, (12), 1597-1608. 2.

De Gregorio, E.; Rappuoli, R., From empiricism to rational design: a personal

perspective of the evolution of vaccine development. Nature Reviews Immunology 2014, 14, (7), 505-514. 3.

Rappuoli, R.; Pizza, M.; Del Giudice, G.; De Gregorio, E., Vaccines, new

opportunities for a new society. Proceedings of the National Academy of Sciences 2014, 111, (34), 12288-12293. 4.

Wu, T. Y.-H.; Singh, M.; Miller, A. T.; De Gregorio, E.; Doro, F.; D’Oro, U.;

Skibinski, D. A.; Mbow, M. L.; Bufali, S.; Herman, A. E., Rational design of small


Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

molecules as vaccine adjuvants. Science translational medicine 2014, 6, (263), 263ra160263ra160. 5.

Brito, L. A.; Malyala, P.; O’Hagan, D. T., Vaccine adjuvant formulations: A

pharmaceutical perspective. Seminars in Immunology 2013, 25, (2), 130-145. 6.

Powell, B. S.; Andrianov, A. K.; Fusco, P. C., Polyionic vaccine adjuvants:

another look at aluminum salts and polyelectrolytes. Clinical and experimental vaccine research 2015, 4, (1), 23-45. 7.

Andrianov, A. K., Polyphosphazene vaccine delivery vehicles: state of

development and perspectives. In Polyphosphazenes for biomedical applications. , Andrianov, A. K., Ed. Hoboken: Wiley: 2009; pp 47-63. 8.

Andrianov, A. K.; DeCollibus, D. P.; Gillis, H. A.; Kha, H. H.; Marin, A.;

Prausnitz,

M.

R.;

Babiuk,

L.

A.;

Townsend,

H.;

Mutwiri,

G.,

Poly[di(carboxylatophenoxy)phosphazene] is a potent adjuvant for intradermal immunization. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, (45), 18936-18941. 9.

Mutwiri, G.; Benjamin, P.; Soita, H.; Townsend, H.; Yost, R.; Roberts, B.;

Andrianov, A. K.; Babiuk, L. A., Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) is a potent enhancer of mixed Th1/Th2 immune responses in mice immunized with influenza virus antigens. Vaccine 2007, 25, (7), 1204-1213. 10. Andrianov, A. K.; Decollibus, D. P.; Marin, A.; Webb, A.; Griffin, Y.; Webby, R. J., PCPP-formulated H5N1 influenza vaccine displays improved stability and dose-


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 38

sparing effect in lethal challenge studies. Journal of Pharmaceutical Sciences 2011, 100, (4), 1436-1443. 11. Andrianov, A. K.; Marin, A.; Chen, J., Synthesis, properties, and biological activity of Poly[di(sodium carboxylatoethylphenoxy)phosphazene]. Biomacromolecules 2006, 7, (1), 394-399. 12. DeCollibus, D. P.; Marin, A.; Andrianov, A. K., Effect of Environmental Factors on Hydrolytic Degradation of Water-Soluble Polyphosphazene Polyelectrolyte in Aqueous Solutions. Biomacromolecules 2010, 487-492. 13. Andrianov, A. K.; Marin, A.; Roberts, B. E., Polyphosphazene polyelectrolytes: A link between the formation of noncovalent complexes with antigenic proteins and immunostimulating activity. Biomacromolecules 2005, 6, (3), 1375-1379. 14. Andrianov, A. K.; Marin, A.; Fuerst, T. R., Self-assembly of polyphosphazene immunoadjuvant with poly(ethylene oxide) enables advanced nanoscale delivery modalities and regulated pH-dependent cellular membrane activity. Heliyon 2016, 2, (4), Article e00102. 15. Palmer, C. D.; Ninković, J.; Prokopowicz, Z. M.; Mancuso, C. J.; Marin, A.; Andrianov, A. K.; Dowling, D. J.; Levy, O., The effect of stable macromolecular complexes of ionic polyphosphazene on HIV Gag antigen and on activation of human dendritic cells and presentation to T-cells. Biomaterials 2014, 35, (31), 8876-8886.


Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

16. Awate, S.; Wilson, H. L.; Lai, K.; Babiuk, L. A.; Mutwiri, G., Activation of adjuvant core response genes by the novel adjuvant PCEP. Molecular Immunology 2012, 51, (3–4), 292-303. 17. Andrianov, A. K.; Svirkin, Y. Y.; LeGolvan, M. P., Synthesis and biologically relevant properties of polyphosphazene polyacids. Biomacromolecules 2004, 5, (5), 1999-2006. 18. Messaud, F. A.; Sanderson, R. D.; Runyon, J. R.; Otte, T.; Pasch, H.; Williams, S. K. R., An overview on field-flow fractionation techniques and their applications in the separation and characterization of polymers. Progress in Polymer Science 2009, 34, (4), 351-368. 19. Yessine, M.-A.; Leroux, J.-C., Membrane-destabilizing polyanions: interaction with lipid bilayers and endosomal escape of biomacromolecules. Advanced drug delivery reviews 2004, 56, (7), 999-1021. 20. Lackey, C. A.; Murthy, N.; Press, O. W.; Tirrell, D. A.; Hoffman, A. S.; Stayton, P. S., Hemolytic activity of pH-responsive polymer-streptavidin bioconjugates. Bioconjugate chemistry 1999, 10, (3), 401-405. 21. Rozema, D. B.; Ekena, K.; Lewis, D. L.; Loomis, A. G.; Wolff, J. A., Endosomolysis by masking of a membrane-active agent (EMMA) for cytoplasmic release of macromolecules. Bioconjugate chemistry 2003, 14, (1), 51-57. 22. Kayitmazer, A. B.; Seeman, D.; Minsky, B. B.; Dubin, P. L.; Xu, Y., Protein– polyelectrolyte interactions. Soft Matter 2013, 9, (9), 2553-2583.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 38

23. Shi, Q.; Zhou, Y.; Sun, Y., Influence of pH and Ionic Strength on the Steric Mass Action Model Parameters around the Isoelectric Point of Protein. Biotechnology progress 2005, 21, (2), 516-523. 24. Chang, B.; Mahoney, R., Enzyme thermostabilization by bovine serum albumin and other proteins: evidence for hydrophobic interactions. Biotechnology and applied biochemistry 1995, 22, 203-214. 25. Li, Y.; Mattison, K. W.; Dubin, P. L.; Havel, H. A.; Edwards, S. L., Light scattering studies of the binding of bovine serum albumin to a cationic polyelectrolyte. Biopolymers 1996, 38, (4), 527-533. 26. Silva, R. A.; Urzúa, M. D.; Petri, D. F. S.; Dubin, P. L., Protein Adsorption onto Polyelectrolyte Layers: Effects of Protein Hydrophobicity and Charge Anisotropy. Langmuir 2010, 26, (17), 14032-14038. 27. Kuriyan, J.; Konforti, B.; Wemmer, D., The molecules of life: Physical and chemical principles. Garland Science: 2012. 28. Hiller, Y.; Gershoni, J. M.; Bayer, E. A.; Wilchek, M., Biotin binding to avidin. Oligosaccharide side chain not required for ligand association. Biochemical journal 1987, 248, (1), 167-171. 29. Linhardt, A.; König, M.; Schöfberger, W.; Brüggemann, O.; Andrianov, A. K.; Teasdale, I., Biodegradable Polyphosphazene Based Peptide-Polymer Hybrids. Polymers 2016, 8, (4), 161.


Page 25 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

30. Jeffrey, G. A.; Saenger, W., Hydrogen Bonding in Carbohydrates. In Hydrogen Bonding in Biological Structures, Springer Berlin Heidelberg: Berlin, Heidelberg, 1991; pp 169-219. 31. O'Neill, L. A. J.; Golenbock, D.; Bowie, A. G., The history of Toll-like receptors redefining innate immunity. Nat Rev Immunol 2013, 13, (6), 453-460. 32. Sasai, M.; Yamamoto, M., Pathogen Recognition Receptors: Ligands and Signaling Pathways by Toll-Like Receptors. International Reviews of Immunology 2013, 32, (2), 116-133. 33. Broz, P.; Monack, D. M., Newly described pattern recognition receptors team up against intracellular pathogens. Nature Reviews Immunology 2013, 13, (8), 551-565. 34. Bhardwaj, N.; Gnjatic, S.; Sawhney, N. B., TLR agonists: Are they good adjuvants? Cancer journal (Sudbury, Mass.) 2010, 16, (4), 382. 35. Azad, A. K.; Rajaram, M. V. S.; Schlesinger, L. S., Exploitation of the Macrophage Mannose Receptor (CD206) in Infectious Disease Diagnostics and Therapeutics. Journal of cytology & molecular biology 2014, 1, (1), 1000003. 36. Hughes, R. C., Membrane glycoproteins: a review of structure and function. Elsevier: 2014. 37. Tamayo, I.; Irache, J. M.; Mansilla, C.; Ochoa-Repáraz, J.; Lasarte, J. J.; Gamazo, C., Poly (anhydride) nanoparticles act as active Th1 adjuvants through Toll-like receptor exploitation. Clinical and Vaccine Immunology 2010, 17, (9), 1356-1362.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 38

38. Keler, T.; Ramakrishna, V.; Fanger, M. W., Mannose receptor-targeted vaccines. Expert Opinion on Biological Therapy 2004, 4, (12), 1953-1962. 39. Shen, H.; Ackerman, A. L.; Cody, V.; Giodini, A.; Hinson, E. R.; Cresswell, P.; Edelson, R. L.; Saltzman, W. M.; Hanlon, D. J., Enhanced and prolonged cross presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles. Immunology 2006, 117, (1), 78-88. 40. Stier, E. M.; Mandal, M.; Lee, K.-D., Differential cytosolic delivery and presentation of antigen by listeriolysin O-liposomes to macrophages and dendritic cells. Molecular pharmaceutics 2005, 2, (1), 74-82. 41. Lin, M.-L.; Zhan, Y.; Villadangos, J. A.; Lew, A. M., The cell biology of crosspresentation and the role of dendritic cell subsets. Immunology and cell biology 2008, 86, (4), 353-362. 42. Yessine, M.-A.; Lafleur, M.; Meier, C.; Petereit, H.-U.; Leroux, J.-C., Characterization of the membrane-destabilizing properties of different pH-sensitive methacrylic acid copolymers. Biochimica et Biophysica Acta (BBA)-Biomembranes 2003, 1613, (1), 28-38. 43. Schmaljohann, D., Thermo-and pH-responsive polymers in drug delivery. Advanced drug delivery reviews 2006, 58, (15), 1655-1670.


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Chart 1. Chemical structures of PCPP and PCEP in their sodium salt forms.

Figure Captions Figure 1. AF4 fractograms of (A) PCPP, Avidin, and their mixture at 210 nm; (B) PCPP, FITC-Avidin, and their mixture at 495 nm (0.25 mg/mL PCPP, 0.2 mg/mL Avidin, PBS, pH 7.4); and (C) DLS profiles of PCPP (1) and PCPP with 0.05 (2), 0.1 (3), 0.15 (4), and 0.25 (5) mg/mL Avidin (0.25 mg/mL PCPP, PBS, pH 7.4).


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Figure 2. Protein binding by PCPP and PCEP as determined by AF4 (proteins are arranged according to their isoelectric point, 0.25 mg/mL PCPP or PCEP, 0.1 mg/mL protein, PBS, pH 7.4). Figure 3. Binding isotherms (molar protein – polyphosphazene ratio in the complex versus concentration of unbound protein) for (A) PCPP and PCEP with Cytochrome C, (B) Cytochrome C and Avidin with PCPP, and (C) Cytochrome C, Avidin and Deglycosylated Avidin with PCPP - initial parts of binding isotherms are shown (0.25 mg/mL PCPP or PCEP, 0.07 – 0.5 mg/mL Avidin, 0.025 – 1.5 mg/mL Cytochrome C, PBS, pH 7.4). (D) Schematic presentations of electrostatic potential surfaces for Avidin and Cytochrome C and glycosylated regions of Avidin. Figure 4. (A) Antigen binding by PCPP and PCEP as determined by AF4 and (B) Fold increase in the molecular diameter of polyphosphazene upon addition of antigens (0.25 mg/mL PCPP or PCEP, 0.1 mg/mL Antigen, PBS, pH 7.4). Figure 5. Binding of soluble TLRs and MR with PCPP or PCEP as determined by AF4 (0.1 mg/mL PCPP or PCEP, 0.05 mg/mL protein, PBS, pH 7.4). Figure 6. TLR stimulation by PCPP and PCEP as assessed by NF-kB activation in HEK293 cells, which utilize secreted embryonic alkaline phosphatase (SEAP) reporter gene as a read-out. Cell stimulation was measured as a ‘Fold Induction’ factor – the ratio of optical densities (alkaline phosphatase activities) measured in the presence and absence of the polymer. The ‘Fold Induction’ factor for each polymer is then expressed in a graph as a percent of the same factor for the positive control. Filled bars represent values for cells expressing given TLRs for (A) PCPP and (B) PCEP. Empty bars show 28 ACS Paragon Plus Environment

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data for the same polymers and cell lines, which do not express any TLR, but still have an NF-kB inducible reporter (TLR- negative control cell lines). The values on the graph represent averages of three screenings and expressed in percent of cells activated by a positive control. Figure 7. Hemolysis of Red Blood Cells as a function of pH for PCPP and PCEP (polymer concentration - 0.025 mg/mL, 10 mM phosphate buffer, 0.9 % of sodium chloride).


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O

O

O

O

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Na

Na

O

O

O

O

Na

CH 2 CH 2

CH 2 CH 2

O

O

O

O

P

P N N

n

n

PCEP

PCPP

ACS Paragon Plus Environment

Na

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C

B

A

DLS

FFF - 495 nm

FFF - 210 nm

5 Complex

Avidin

Rela(ve Intensity, %

Intensity, 495 nm

Complex

Intensity, 210 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

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FITC-Avidin

4

3

2

PCPP

1

PCPP 0

0

6

10

14

18

Time, min

22

6

10

14 18 Time, mim

22

ACS Paragon Plus Environment

10

100

Diameter, nm

1000

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Isoelectric Point

Protein Binding, %

PCPP

10

PCEP

Isoelectric point

100

8

6

50

4

2

0 e in rome sin A m d i y v z A 6li ch Lyso Sub Cyto

0 n in se Lec6 yoglob ragina a M Asp ACS Paragon Plus Environment

GFP

HRP

BSA lbumin Ova

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Biomacromolecules 180

A

200

PCPP

B

Avidin

Protein/PCPP, mol/mol

Cytochrome C/ PP, mol/mol

150

120

PCEP

90

60

150

Cytochrome C 100

50

30

0 0

20

40

60

80

Cytochrome C, μmol/L

100

0 0

20

40

Protein, μmol/L

Electrosta

Molecular-Level Interactions of Polyphosphazene Immunoadjuvants

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