Protein Stabilization in Aqueous Solutions of Polyphosphazene

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Biomacromolecules 2010, 11, 2268–2273

Protein Stabilization in Aqueous Solutions of Polyphosphazene Polyelectrolyte and Non-Ionic Surfactants Alexander Marin, Daniel P. DeCollibus, and Alexander K. Andrianov* Apogee Technology, Inc., 129 Morgan Drive, Norwood, Massachusetts 02062 Received March 22, 2010; Revised Manuscript Received July 12, 2010

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. Chart 1.

Introduction Polyelectrolytes, water-soluble macromolecules containing ionic groups, have become increasingly important components of pharmaceutical formulations. Some of these polymers found uses as inactive excipients in Federal Drug Administration (FDA) approved drug products,1,2 whereas others have been investigated as constituents of drug delivery systems, such as microspheres, multilayered nanofilms, and microneedles.3-8 As these polyelectrolytes are formulated to share the same milieu with proteins, surfactants and other excipients, unique challenges arise due to the possibility of macromolecular interactions in such systems.9 This appears to be especially relevant to formulations of immunostimulating polyelectrolytes, an important class of biodegradable polyelectrolytes, which attracted attention due to the ability to stimulate immune responses to vaccine antigens.10-12 It has been shown that at least in some of these systems, the formation of protein-polyelectrolyte complexes can have a major impact on the biological performance of the formulation.13 Although the attention of research efforts has been almost entirely focused on the effect, which macromolecular interactions can engender on biological activity of the system, it can be hypothesized that other important properties of the formulation can be affected too. Thermal stability of pharmaceutical formulations, especially those containing protein components, represents one of the major parameters critical for the development of successful drug or vaccine products.14-17 Enhancement of thermal stability of vaccines and protein therapeutics can lead to a reduced dependence of their distribution and storage on cold-chain facilities, resulting in enormous savings.18,19 Therefore, in-depth investigation of factors affecting protein stability, including the role of various excipients and macromolecular delivery systems, has been one of the major focuses of pharmaceutical research.20 However, the potential effect of biologically important polyelectrolytes, such as immunostimulating macromolecules, on the stability of protein formulations still remains largely unclear. * To whom correspondence should be addressed. E-mail: aandrianov@ apogeebio.com.

Molecular Formula of PCPP

Moreover, little is known on the influence of macromolecular interactions in these systems and the role of other formulation excipients. The present Article investigates the effect of biologically a relevant polyelectrolyte, poly[di(carboxylatophenoxy)phosphazene], disodium salt (PCPP), and its formulations with surfactants on thermal stability of protein in aqueous solutions. PCPP is a well-defined synthetic macromolecule21 with phosphorus and nitrogen backbone and organic side groups containing carboxylic acid functionalities (Chart 1). Its biological significance as an immunostimulating compound has been demonstrated in vivo,11,22-25 including clinical trials,26,27 and although its somewhat unusual solution behavior21 warrants further investigation, preliminary studies, including those on interactions with proteins, have already been conducted.13,21 Horseradish peroxidase (HRP) was used as a protein component of the formulation because thermal inactivation of this enzyme can be conveniently monitored using a chromogenic substrate system providing a reliable correlate with protein stability, as defined by thermally induced conformation changes.28,29 Tween 20 (Tween) and Pluronic F68 (Pluronic) have been employed as nonionic surfactants because they are frequently used constituents of pharmaceutical and vaccine formulations capable of modifying protein stability.30,31 Thermal inactivation of HRP in these formulations was investigated in aqueous solutions at 50 °C, and the results are discussed in context of findings on macromolecular interactions in the system, which were studied using laser light scattering and turbidimetric titration methods.

10.1021/bm100603p  2010 American Chemical Society Published on Web 08/04/2010

Protein Stabilization in Aqueous Solutions

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Table 1. Relative Initial Activity of HRP in Mixtures Containing PCPP and Various Additivesa system

relative initial activity

HRP HRP+PCPP HRP+Tween HRP+PCPP+Tween HRP+ PCPP+Pluronic HRP+PCPP+PEG HRP+PCPP+dextran

1.00 1.28 1.35 1.43 1.45 1.26 1.23

a Measured in 0.1 M phosphate buffer, pH 7.4 at the following concentrations of components: HRP, 0.001 mg/mL; PCPP, 1 mg/mL; Tween, Pluronic, PEG, and dextran, 0.2 mg/mL each.

Experimental Section Materials. Poly[bis(carboxyatophenoxy)phosphazene], disodium salt (PCPP) (Sigma, St. Louis, MO), was purified and fractionated by multiple precipitations using sodium chloride32 to yield a fraction with MW 900 000 g/mol. Poly(ethylene glycol) (PEG), MW 400 (Sigma, St. Louis, MO); horseradish peroxidase (HRP) (Pierce, Rockford, IL); dextran, clinical grade, MW 60 000-90 000 (MP Biomedicals, Inc., Solon, OH); Poloxamer 188 (Pluronic F68), block copolymer of ethylene oxide and propylene oxide (75/30), MW 8400 (Spectrum Chemicals MFG Corp., Gardena, CA); polyoxyethylene (20) sorbitan monolaurate (Tween 20) (TCI America, Portland, OR); Albumin from bovine serum; 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), ABTS; sodium phosphate dibasic heptahydrate; sodium phosphate monobasic; potassium phosphate monobasic (Sigma, St. Louis, MO); and Dulbecco’s phosphate buffered saline, DPBS (sterile, without calcium or magnesium, Lonza, Walkersville, MD) were used as received. Kinetics of Thermal Inactivation of HRP. Kinetics of thermal inactivation of HRP was studied at 50 °C in 0.1 M phosphate buffer at pH 7.4. Stock solutions of HRP, PCPP, and other components were filtered before mixing (0.45 µm pore size). PCPP-HRP formulations were prepared by simple mixing of corresponding stock solutions at a desired ratio. Formulations, which also contained other components, were prepared by adding a component, such as surfactant, to PCPP solution, vortexing it for 15 s, incubating it at an ambient temperature without agitation for 10 min, adding stock solution of HRP, and vortexing the mixture for an additional 15 s. We prepared a series of samples for each formulation by placing 150 µL of the mixture in plastic vials. To maintain accurate temperature control, vials were placed in a water bath (Precision, model 182, Chicago, IL) and kept there until removed for the analysis of enzymatic activity. Thermal stability was evaluated by plotting residual activity of the enzyme versus time. Halflife was defined as a period of time at which 50% of enzymatic activity was lost. Enzymatic activity of HRP was determined using 2,2′-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) as a substrate by measuring UV absorbance at 405 nm (UV/vis spectrophotometer, Hitachi U-2810, Hitachi, San Jose, CA). Maximum linear rate ∆A405nm/minute was used to measure the activity (enzymatic assay of peroxidase from horseradish, EC 1.11.1.7, Sigma Prod. no. P-6782). Relative activity of HRP in the system was calculated as a ratio between its activity in the presence and the absence of additives. Relative initial activity of HRP was defined as a relative activity of enzyme not subjected to degradation experiments. An increase in the relative initial activity of the enzyme was observed in the presence of all additives (Table 1). Modulation of the initial activity of HRP by PCPP and additives (Table 1) also suggests modifications in the microenvironment of its active site. Typically, such improvements in catalytic properties of the enzyme may result from its conjugation or immobilization using PEG, polyelectrolytes, and polyelectrolyte complexes.33-36 Characterization Methods. Molecular weight analysis of polymers as well as determination of PCPP, HRP, and Tween concentrations, was performed using Hitachi LaChrom Elite high-performance liquid chromatography (HPLC) system, equipped with L-2I30 pump and

Figure 1. Effect of PCPP, Tween, and their mixture on the kinetics of thermal inactivation of HRP (0.001 mg/mL of HRP, 1.0 mg/mL of PCPP, 0.2 mg/mL of Tween, 50 °C, 0.1 M phosphate buffer, pH 7.4, n (number of experiments) ) 3).

degasser, L-2200 autosampler, L-2455 diode array detector, and L-2490 refractive index detector (Hitachi High Technologies America, Inc., San Jose, CA). Ultrahydrogel 250 size exclusion column (Waters Corporation, Milford, MA) was used for separation, and 0.1× PBS (pH 7.4) containing 10% of acetonitrile was employed as a mobile phase with a flow rate set to 0.75 mL/min. Data collection and processing were performed using EZChrom Elite Software (Hitachi High Technologies America, Inc., San Jose, CA). We performed turbidimetric titration at an ambient temperature by measuring the transmittance (T) of the mixture at 420 nm (UV/vis spectrophotometer, Hitachi U-2810, Hitachi, San Jose, CA) in 2 mL cuvettes with 1 cm path length using deionized water or phosphate buffer solution as a reference. In the case of HRP titration, the same concentration of HRP was in the sample and reference cell. The initial solutions were filtered using 0.2 µm Millex filters prior to titration. The solution was vortexed for 15 s after the addition of the titrant and monitored in the spectrophotometer until a stable turbidity reading ((0.1% T) was obtained. Dynamic light scattering measurements were performed using Zetasizer Nano-ZS (Malvern Instrument, Inc., Centerville, MA) in 0.1 M phosphate buffer (pH 7.4). Samples were filtered using 0.2 µm Millex filter before and after the mixing. The deconvolution of the measured correlation curve to an intensity size distribution was accomplished using a non-negative least-squares algorithm (NNLS).

Results and Discussion Thermal Inactivation of HRP in the Presence of PCPP and Surfactants. Advancement of PCPP as a potent immunostimulating compound creates an inciting moment for further investigation of its formulations with proteins, especially because it relates to such practically important characteristics as thermal stability. The present Article studies the effect of PCPP on the kinetics of thermal inactivation of HRP in aqueous solutions near physiological pH (7.4) at 50 °C. Because the loss of catalytical activity of HRP generally correlates with denaturation of the enzyme,28,37 the results can be broadly interpreted in terms of protein stability. As seen from Figure 1, PCPP displayed a significant stabilizing effect resulting in an approximately three-fold increase in the enzyme’s half-life. Conversely, the addition of Tween led to a substantially faster loss of catalytic activity in polymer-free

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Figure 2. Enzyme stabilizing effects (SE) of PCPP and its mixtures with various additives (Tween, Pluronic, PEG, and dextran) at 50 °C (The corresponding half-life values are presented next to each column in minutes; half-life for HRP was 9 min. 0.001 mg/mL of HRP, 1 mg/ mL of PCPP, other additives were used at a concentration of 0.2 mg/ mL, 0.1 M phosphate buffer, pH 7.4).

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Figure 3. Enzyme stabilizing effect as a function of PCPP concentration in the absence of additives and in a PCPP-Tween mixture (50 °C; 0.001 mg/mL of HRP, 0.01 mg/mL of Tween, 0.1 M phosphate buffer, pH 7.4, n ) 2).

solutions. However, when both polymer and surfactant were added to HRP as a mixture, the stabilizing effect superseded the one of PCPP alone and resulted in an almost 10-fold increase of the enzyme’s half-life (Figure 1). To compare the effects of various excipients on the stability of HRP, a stabilizing effect (SE) was defined as a logarithm of the ratio between the enzyme’s half-life in their presence (τ) and absence (τ0)

SE ) log(τ/τ0) Figure 2 shows that nonionic surfactants (Tween and Pluronic) used in combination with PCPP amplified the stabilizing effect of the polymer dramatically; however, the addition of water-soluble polymers, such as dextran or PEG, to PCPP did not result in any changes. Further, for PCPP-surfactant combinations, the phenomenon clearly cannot be explained via simple addition of effects produced by individual components because both Tween and Pluronic were rather destabilizing to HRP under conditions studied (Figure 2). Effects of PCPP and Surfactant’s Concentration. Figure 3 shows that the stabilizing effect of PCPP can be observed in a broad range of polymer concentrations. Furthermore, the degradation half-life tends to increase as the concentration of PCPP raises; however, the slope of the dependence is much steeper at low polymer content (Figure 3). The stabilizing effect was detected at a PCPP concentration as low as 0.005 mg/mL, at which the half-life of HRP was almost doubled compared with polymer-free enzyme solution. The same trends were observed in PCPP-Tween mixtures, although the amplitude appeared to always be higher than in the presence of PCPP alone (Figure 3). The effect of surfactant concentration on the enzyme inactivation kinetics was studied in mixtures of PCPP with Tween or Pluronic (Figure 4). Both systems showed an initial increase in the stabilizing effect as surfactant concentration raised, followed by dramatic decrease at their higher content. The latter was more pronounced for the Tween system, in which the highest concentration of Tween produced an enzyme destabilizing effect even in the presence of PCPP overwhelming the effect of polymer.

Figure 4. Effect of surfactant concentration on the enzyme stabilizing effect of PCPP-surfactant mixtures (50 °C, 0.1 M phosphate buffer, pH 7.4; 0.001 mg/mL of HRP, and 1.0 mg/mL of PCPP, n ) 2).

To understand the mechanism of observed stabilization, it is important to emphasize that stabilization of proteins in the presence of polymers is generally attributed to preferential exclusion, surface activity, steric hindrance of protein-protein interactions, or restriction of protein structural movement.15 Moreover, the ability of polyelectrolyte solutions to selfassemble is perhaps the main cause of some of the most unique and important functional properties displayed by ionic macromolecules.38 Synthetic polyanions have been reported to suppress thermoaggregation of oligomeric enzymes through the formation of polyelectrolyte complexes, although complexing with polyanions did not prevent their thermodenaturation.39 Other polyelectrolytes, such as polycations, have also been observed to have a slight thermal stabilization effect on proteins due to complex formation.40 Amphipols, polymeric surfactants containing hydrophilic polymer backbone and grafted hydrophobic chains, have been shown to solubilize and stabilize


Figure 5. Turbidimetric titration of HRP (1) and HRP-Tween mixture (2) with PCPP (0.2 mg/mL of HRP in each solution, 10 mg/mL of PCPP, 0.2 mg/mL of Tween, 0.005 M phosphate buffer, initial volume: 0.5 mL, pH 7.4).

membrane proteins.41,42 The proposed mechanism includes the formation of a protein-polymer complex through interactions of hydrophobic groups of the polymer with the hydrophobic surface of the protein so that such multipoint attachment damps collective movements and stabilizes protein.42 In accordance with these findings, we analyzed the stabilizing effect of PCPP in the context of its known ability to form noncovalent watersoluble complexes with proteins, in which polyphosphazene can carry a significant number of protein ligands.13 Studies of PCPP-HRP Interactions in Aqueous Solutions. Interactions between HRP and PCPP in solution were studied for potential phase separation using a turbidimetric method similar to the technique previously developed for protein-polyelectrolyte complexation.9,43,44 Titration of HRP with PCPP in aqueous solutions (pH 7.4) resulted in dependence characterized with a maximum (Figure 5, curve 1). Assuming that this maximum corresponds to the formation of a water-insoluble electrically neutral complex and represents a “stoichiometric end point”,43 the number of enzyme molecules bound to one polyelectrolyte chain can be estimated as 440. These results appear to be in line with previously observed data on polyelectrolyte complexes containing a large number of protein molecules43 and may be also reasonable to expect on the basis of large molecular weight and high charge density of PCPP. Titration of HRP in the presence of Tween resulted in a similar profile (Figure 5, curve 2); however, the turbidity appears to be significantly higher, and the end point was observed at a dramatically lower HRP/PCPP molar ratio: 103. Although further studies are warranted as to the physical state of insoluble complexes and characteristics of their water-soluble counterparts using approaches developed recently,9,45 it is clear that significant protein-polymer interactions occur in the system, and the surfactant appears to modulate formation and structure of the complex. Interactions in PCPP-surfactant systems were also studied using turbidimetric titration with aqueous solutions of sodium chloride and hydrochloric acid because both of them are known to induce precipitation of PCPP.21 Typical turbidimetric titration of PCPP with sodium chloride is shown in Figure


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Figure 6. Turbidimetric titration of PCPP and PCPP-Tween mixtures with sodium chloride at (1) 0, (2) 0.04, (3) 5, and (4) 20 mg/mL of Tween (1 mg/mL of PCPP; 22% (w/v) of sodium chloride, 0.05 M phosphate buffer, initial volume: 1 mL, pH 6.8).

Figure 7. Turbidimetric titration of PCPP and PCPP-Tween mixtures with hydrochloric acid at (1) 0, (2) 0.02, (3) 0.04, (4) 1.0, and (5) 5 mg/mL of Tween (1 mg/mL of PCPP; 0.05 N of hydrochloric acid; initial volume: 1 mL, deionized water).

6. The addition of Tween to the polymer resulted in a faster threshold of precipitation, which was directly dependent on the amount of added surfactant, indicating gradually decreasing solubility of PCPP in the presence of surfactant. The addition of Tween also had a substantial effect on the turbidimetric titration of PCPP with hydrochloric acid, which was dependent on the concentration of surfactant (Figure 7). At a low Tween concentration, the threshold of phase separation was slightly lower than that for PCPP alone (curves 2 and 3), which was consistent with data on the titration with sodium chloride. When the surfactant was added at a concentration of 1 and 5 mg/mL (curves 4 and 5), PCPP solubility at high acid content (low degree of dissociation) was greatly improved. Therefore, PCPP solubilizing effect under highly acidic conditions can be potentially attributed to the formation of micelles around hydrophobic patches of highly protonated polyacid. Similar effects were also observed for the PCPP-Pluronic system (data not shown).

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Table 2. Dynamic Light Scattering Studies of PCPP-HRP-Tween Systemsa sample

composition

D1 (nm)

1 2 3 4 5 6

HRP PCPP Tween PCPP+Tween PCPP+ HRP PCPP+HRP+Tween

8

D2 (nm) 84

10 11 11 12

81 81 80

a 0.1 mg/mL HRP, 0.2 mg/mL Tween, 1.0 mg/mL PCPP, and their mixtures in 0.1 M phosphate buffer, pH 7.4, standard deviation (5%, n ) 2.

The results of thermoinactivation studies clearly demonstrate the amplifying role of surfactant on the stabilizing effect of PCPP, whereas turbidimetric titrations suggest that surfactant has an ability to alter the properties of PCPP-HRP complexes (Figure 5) as well as modulate phase separation behavior of PCPP (Figures 6 and 7). Although the results appear to suggest the possibility of interactions between nonionic surfactants and PCPP through hydrogen bonding of carboxyl groups of the polymer and hydrophilic part of the surfactant molecule,38,44,46,47 it is important to emphasize that further research is needed to better understand the mechanism of stabilization as well as solution behavior of PCPP, its interactions with proteins and surfactants, and the morphology of complexes. The importance of amphiphilic component in our studies is emphasized with the fact that both protein stability and macromolecular interactions were enhanced only in the presence of surfactants, whereas the addition of other excipients hardly had any effect on these properties. Because studies are in progress to characterize such multicomponent assemblies further, it may be important to mention previously reported polymer-enzyme micelles formed through the complexation of PEG-poly(aspartic acid) block copolymer and lysozyme, resulting in the entrapment of enzyme in the hydrophobic core;48 however, stability studies on such systems have not been performed. Finally, soluble PCPP-HRP-Tween formulations were characterized by dynamic light scattering studies (Table 2). Intensity-size distributions for multicomponent mixtures displayed pronounced bimodality, whereas all distributions for individual components displayed single peaks. The addition of HRP or Tween to PCPP solution appears to have a minimal impact on the size; however, some reduction in polydispersity and increase in intensity of PCPP-related population is observed (data not shown). The presence of some small size population in both PCPP-HRP systems may indicate the existence of some unbound enzyme in the system. Zeta potential distribution of the HRP-PCPP-Tween system displayed a major peak (92.7% of the total area) at -68 mV, suggesting the stability of the system against aggregation, which can be important for its practical development.

Conclusions PCPP, a promising immunoadjuvant, displays a potent stabilizing effect on aqueous protein solutions at elevated temperatures. Nonionic surfactants, which are often present in vaccine formulations, amplify this phenomenon. There appears to be a correlation between stabilizing properties of PCPP-surfactant formulations and molecular interactions in these systems including complex formation. These findings can have a profound impact on the development of PCPP as vaccine adjuvant because improved thermal stability of vaccines can bring about an additional benefit of a better shelf life of the product and its reduced dependence on temperature-controlled supply chains.

Acknowledgment. We thank Professor P. L. Dubin for valuable comments and helpful discussions during the preparation of this manuscript. Supporting Information Available. Structures of Tween 20 and dextran and molecular formulas of Poloxamer 188 and poly(ethylene glycol). This material is available free of charge via the Internet at http://pubs.acs.org.

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Protein Stabilization in Aqueous Solutions of Polyphosphazene

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