Effect of Environmental Factors on Hydrolytic Degradation of Water

Jul 21, 2010 - Biomacromolecules , 2010, 11 (8), pp 2033–2038. DOI: 10.1021/ ... (17, 18, 20-23). One of the most significant representatives of thi...
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Biomacromolecules 2010, 11, 2033–2038

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Effect of Environmental Factors on Hydrolytic Degradation of Water-Soluble Polyphosphazene Polyelectrolyte in Aqueous Solutions Daniel P. DeCollibus, Alexander Marin, and Alexander K. Andrianov* Apogee Technology, Inc., 129 Morgan Drive, Norwood, Massachusetts 02062 Received April 13, 2010; Revised Manuscript Received June 3, 2010

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. Chart 1.

Introduction Emerging applications of synthetic polymers in drug delivery systems, tissue engineering, and bionanotechnology frequently require their biodegradability.1-6 Therefore, in-depth understanding of polymer degradation mechanisms and factors affecting them has become an important prerequisite for achieving a desired biological response and safety profile. Moreover, shelf life of biomaterials and pharmaceutical formulations, along with their tolerance to various micro- and nanofabrication processes are greatly affected by the kinetics of polymer breakdown.7,8 Although degradation of biologically relevant polymers can occur through a number of mechanisms including enzymatic, microbial, oxidative, and thermal rearrangement, hydrolytic degradation remains one of the most practically important and common pathways.8-14 Hydrolysis of chemical bonds in an aqueous environment leading to the cleavage of polymer backbone is a prevailing mechanism for many families of biodegradable synthetic polymers including polyesters, polyanhydrides, polyphosphoesters, and polyphosphazenes.3,8,9,12,15-18 The absolute majority of these polymers are not soluble in aqueous solutions and undergo surface or bulk erosion, so that their erosion profiles are defined not only by hydrolytic sensitivity of chemical links, but also by physical characteristics of the material, such as hydrophobicity and morphology, which have an effect on water diffusion in the matrix.3,8,19 Water soluble hydrolytically degradable systems are less common and present a particular interest as the impact of such macroscopic factors on their degradation is limited and polymer breakdown is controlled mainly on the molecular level by chemical reactions.17,18,20-23 One of the most significant representatives of this subclass, poly[di(carboxylatophenoxy)phosphazene], disodium salt (PCPP) gained considerable attention as a vaccine adjuvant and dem* To whom correspondence should be addressed. E-mail: aandrianov@ apogeebio.com.

Molecular Formula of PCPP

onstrated an impressive performance in vivo with multiple bacterial and viral antigens.24,25 This synthetic polyelectrolyte with a molecular formula comprised of a phosphorus and nitrogen backbone and benzoic acid side groups (Chart 1) is generally soluble in neutral and basic aqueous solutions.26 Its ability to undergo molecular breakdown in aqueous solution has been reported previously and it has also been emphasized that the kinetics can be affected by certain structural irregularities, demonstrating the need for their control.26 However, the mechanism of degradation, its main characteristics, including degradation products, as well as the impact of various environmental parameters on the degradation kinetics remain largely unknown. The latter can be of significant interest as PCPP displays high pH and ionic sensitivity, and exceptional ability to form complexes with proteins and other biologically important molecules.26-28 Moreover, as a potential component of pharmaceutical formulations, PCPP can share the same milieu with various excipients, such as nonionic surfactants, salts, viscosity enhancers, and byproducts of the degradation processes. This is especially relevant to evolving applications of PCPP as a microfabrication material for transdermal devices and microencapsulation systems.28,29 The present paper investigates degradation of PCPP in aqueous solutions by integrating the results of molecular weight monitoring and release of polymer side groups, the main degradation product, on various stages of the process. It also explores the effect of environmental factors, such as pH, addition

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of salts, and hydrogen bond forming compounds, interpreting the results in the context of macromolecular interactions in the system and suggested degradation mechanism.

Experimental Section Materials. PCPP (Sigma-Aldrich, St. Louis, MO) was purified and fractionated by multiple precipitations using sodium chloride as described previously,26 resulting in a polymer with weight average molecular weight of 855000 g/mol. Sodium 4-hydroxybenzoate (HBA); hydrochloric acid (37%); potassium phosphate, monobasic; sodium phosphate heptahydrate, dibasic (Sigma-Aldrich, St. Louis, MO); glycerol, 99.5+% (Alfa-Aesar, Ward Hill, MA); polyoxyethylene(20) sorbitan monolaurate, Tween-20 (Tween; Tokyo Chemical Industry, Japan); polyethylene glycol 400 (PEG); phosphate buffered saline (PBS), 10×; sodium chloride; potassium chloride; sodium borate decahydrate (EMD Chemicals, Gibbstown, NJ); and HPLC grade acetonitrile (Fisher Schientific, Pittsburgh, PA) were all used as received. Analytical Methods. Size exclusion-high performance liquid chromatography (SEC-HPLC) was performed using a Hitachi HPLC system equipped with an Ultrahydrogel Linear column (Waters Corporation, Milford, MA) and L-2450 diode array detector (Hitachi LaChrome Elite system, Hitachi, San Jose, CA). Samples were filtered using 0.45 µm syringe filters and 50 µL aliquots were used for the analysis. PBS (0.1×) containing 10% acetonitrile was employed as a mobile phase at a flow rate of 0.75 mL/min. Molecular weight (Mw) calculations were preformed using EZ-Chrome Elite software (Agilent Technologies, Santa Clara, CA). Calibration curve was obtained using narrow poly(acrylic acid) standards ranging from 900 Da to 1500 kDa (American Polymer Standards Corporation, Mentor, OH). Molar concentrations of polyphosphazene macromolecules during the degradation process were calculated as a ratio between mass concentration of PCPP in the sample and its molecular weight at each time point. The rate of new chain formation (VN) was determined at initial stages of polymer degradation as the slope of the least-squares regression line resulting from the plot of molar PCPP concentration versus time. The content of released HBA in degradation samples was determined by SEC-HPLC at 245 nm using solutions containing HBA and PCPP of known concentrations as standards. Rate of HBA release (VHBA) was determined similar to PCPP chain formation described above. Degradation Studies. Degradation studies were carried out using aqueous 1 mg/mL solutions of PCPP. Each formulation was dosed in a glass vial, sealed, and placed in the incubator equipped with orbital shaker (Max Q Mini 4450, Thermo Fisher Scientific, Waltham, MA). The temperature was maintained at 70 °C for the duration of the experiment. Preliminary studies on the temperature-time dependence of PCPP degradation indicate that the rate of PCPP degradation at this temperature can be expected to be approximately 7-fold higher than polymer degradation at physiological temperature (37 °C).30 Aliquots were taken periodically for SEC-HPLC analysis and pH determination as described above. Solutions were formulated in one of the following media: deionized water, 1× PBS (pH 7.4), 0.02 M phosphate buffers (pH 7.5, pH 8.2), or 0.02 M borate buffers (pH 9.4, pH 10.0). Sodium chloride, potassium chloride, PEG, Tween, glycerol, and HBA were added to some formulations to evaluate their effect on the degradation profile of PCPP. Potentiometric Titration. Potentiometric titration of 0.1% (w/v) PCPP solutions in deionized water was carried out at ambient temperature under stirring. Some formulations also contained Tween at 0.5% (w/v), PEG at 0.5% (w/v), and sodium chloride at 0.8% (w/v) concentrations. Calculated aliquots of 0.1 N sodium hydroxide solution were added to each sample to achieve complete ionization of PCPP and then final solutions were titrated with 0.1 N aqueous hydrochloric acid. pH measurements were taken approximately 2 min after addition of the titrant using a Mettler Toledo InLab Micro Pro combination electrode with thermocouple attached to a Mettler Toledo SevenEasy

Figure 1. Changes in molecular weight of PCPP (open symbols) and release of HBA (filled symbols) in deionized water (triangles) and PBS (circles) as a function of time (70 °C, 0.1% solutions of PCPP, Mw0: molecular weight of PCPP before degradation experiment).

pH reader (Mettler Toledo, Greifensee, Switzerland). The electrode was calibrated with three buffer solutions (pH 4.01, pH 7.00, pH 10.01) prior to use. The first inflection point (degree of dissociation, R equals 100%) was determined by creating first derivative plots of the titration curves. The second inflection point, at which PCPP was fully protonated (R equals 0%), was calculated due to insolubility of highly protonated polymer. Henderson-Hasselbalch equation was used to calculate the apparent negative logarithm of the dissociation constant (pKa).31

Results and Discussion Degradation of PCPP in Aqueous Solutions. Hydrolysis of water-soluble and hydrophobic polyphosphazenes typically occurs via cleavage of side groups followed by the breakdown of polymer chain.15,16,21 Therefore, degradation of PCPP was investigated by simultaneous monitoring of the system for changes in the molecular weight of the polymer and the release of the potential degradation product: cleaved side group, HBA. Degradation studies were carried out both in deionized water and phosphate buffer (pH 7.4) and monitoring was performed using HPLC-SEC system coupled with a UV/vis diode array detector. The experiments were conducted at 70 °C to accelerate the process and achieve high levels of polymer decomposition in a reasonable time period. The results, presented in Figure 1, demonstrate dramatic changes in polymer molecular weight (decrease of up to 85%) accompanied with some release of HBA (less than 1% of the total content of hydroxyl benzoic acid side groups in PCPP). Kinetic profiles were similar for both solutions, although degradation in water was somewhat faster and was accompanied by some decrease in pH (from pH 8.7 to 7.9), which can indicate the release of small quantities of acidic products. A narrowing of the molecular weight distribution during the degradation was observed in both systems (parameter of polydispersity changed from 2.6 to 2.0), which is in agreement with random scission model of polymer degradation reported previously.32 The observed molecular weight and side groups release profiles were generally consistent with results for the hydrolysis of water-soluble polyphosphazene containing N-ethylpyrrolidone groups, which suggests degradation pathway (Scheme 1) similar to the previously proposed.21 Main features of such a mechanism

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Scheme 1. Schematic Representation of PCPP Degradation Pathway in Aqueous Solutions

Figure 2. Kinetics of PCPP molecular weight changes in buffer solutions of various pH (70 °C, 0.1% solutions of PCPP, 0.02 M phosphate (pH 7.5 and 8.2) or borate (pH 9.4 and 10.0) buffer solutions, Mw0: molecular weight of PCPP before degradation experiment).

include hydrolytical cleavage of the side group leading to the experimentally observed release of HBA and generation of macromolecular hydroxyl derivative, stage I, formation of the unstable phosphazane structure, stage II, and ultimately the breakdown of the polymer chain, stage III. Recently, a pathway for a protonation of substituted polyphosphazene resulting in a P+-NH bond was reported,13 which is also included in the scheme (stages IV and V). As has been previously reported, the polyphosphazene degradation products can also include ammonium and phosphate ions.13,21,33 Effect of pH. The effect of pH on the rate of polyphosphazene degradation is one of the most discussed in the literature and it has been long suggested that either intramolecular or intermolecular acid catalysis constitutes an important stage in the degradation pathway.33 The hypothesis has been later confirmed for a number of water-soluble derivatives,21,23 as well as for some less hydrophobic water-insoluble polyphosphazenes,21 and protonation of nitrogen of the phosphazene backbone has also been a focus of several studies.13,33,34 These findings warranted studies on the effect of pH on the degradation of PCPP. Because polyphosphazene polyelectrolyte is only soluble at basic and neutral pH, the experiments were limited to buffer solutions in pH range of 7.5-10. As seen from Figure 2, a decrease in solution pH generally led to an increase of the reaction rate and basic conditions dramatically inhibited degradation of PCPP. This is in line with the majority of abovementioned reports for hydrolytically degradable polyphosphazenes and suggests a catalytic effect of an acid in the system. To better understand the effect of pH on degradation of PCPP, the ratios between the rates of new polymer chain formation and HBA generation in the system were calculated and plotted as a function of pH (Figure 3). The rates of new chain formation were calculated on the basis of molecular weights of PCPP using initial stages of degradation. As seen from Figure 3, the ratios

Figure 3. Effect of pH on the ratio of new chain formation (VN) and HBA release (VHBA) rates.

were in the range of 0.003 to 0.03 indicating that new polymer chains were generated at rates approximately 100-fold lower than those of HBA release. Therefore, it appears that if the release of side group precedes rupture of the main chain, only a small number of side group cleavages lead to the breakdown of the polymer chain, which suggests that the rate of I is at least initially higher than the rates of II or III (Scheme 1). Importantly, the ratio increases as pH declines indicating a shift toward higher incidence of chain breakdowns. This clearly suggests that protonation of nitrogen in the backbone leading to phosphazane structure (stage II of Scheme 1) can be a rate limiting step of the degradation process and accelerates polymer breakdown. It is also noteworthy that the rate of HBA release also increases as pH decreases (data not shown), suggesting that acid catalysis also plays a role in the hydrolysis of the link between the side group and the main chain (I).

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Figure 4. Kinetics of PCPP molecular weight changes in deionized water (1), 2% (w/v) aqueous potassium chloride (2), and 2% (w/v) aqueous sodium chloride (3) solutions (70 °C), 0.1% solutions of PCPP, Mw0: molecular weight of PCPP before degradation experiment).

Effect of Inorganic Salts. One of the fundamental features of PCPP as a polyelectrolyte lies its ability to undergo phase separation in the presence of inorganic salts in aqueous solutions.26 In fact, formation of coacervate systems upon addition of sodium chloride is a basis for applications of PCPP as a microencapsulation agent.35 Thus, it was imperative to investigate hydrolytical behavior of PCPP in formulations containing inorganic salts. Sodium and potassium chlorides were used as their physiological relevance, presence in pharmaceutical formulation, and notably dissimilar effects on phase separation behavior of PCPP were of importance.26 The experiments were limited to concentrations of salts, which are sufficiently low to preserve homogeneity of PCPP solutions. The results of degradation studies are shown in Figure 4. Interestingly, both sodium and potassium chloride demonstrated the ability to substantially repress hydrolytic breakdown of polyphosphazene polyelectrolyte, which can have significant practical implications on the development of stable pharmaceutical formulations. The effect was similar for both salts and manifested in an at least 4-fold increase in the time of 25% reduction in the molecular weight of the polymer. Although seemingly unexpected, the results can be easier to understand taking into account the effect of salts on the acidic and conformational properties of PCPP in aqueous solutions. Figure 5 shows dependences of the dissociation constant on the degree of PCPP ionization both in the presence of sodium chloride (squares) and in salt-free aqueous solutions (circles). As expected, addition of a salt led to a substantial increase in the dissociation constant, which is typical for polyelectrolytes and generally the result of the screening of electrostatic interactions in the presence of salts.36,37 Based on PCPP dissociation constant in sodium chloride solutions, it can be assumed that polyelectrolyte is completely ionized at neutral pH. However, the values in salt-free solutions suggest presence of some undissociated carboxylic acid groups under the conditions of degradation experiments. These polymer based acidic side groups can potentially participate in the protonation of nitrogen in the backbone (reaction II or IV of Scheme 1) in the intramolecular mechanism, similar to previously described catalyzed hydrolysis reactions in polyelectrolytes.36 To this end,

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Figure 5. Negative logarithm of PCPP dissociation constant (pKa) vs degree of ionization of PCPP in deionized water (circles), 0.5% (w/v) PEG (triangles), 0.5% (w/v) Tween (crosses), and 0.8% (w/v) sodium chloride (squares) solutions (0.1 N hydrochloric acid was used for titration of 0.1% PCPP at 25 °C; Henderson-Hasselbach equation and neutralization values were used to calculate the dissociation constant).

the lack or decrease in the rate of intramolecular catalysis in the presence of salt can be responsible for the retardation effect observed in Figure 4. Furthermore, concentration of protons in the polyion domain can be also reduced due to repressed attractive interactions between them and anionic macromolecule in the presence of salt.36 Finally, the impact of conformational changes cannot be also ruled out, as addition of salt is capable of reducing the size of macromolecule,26,36,37 which can have a substantial negative effect on both efficiency of intramolecular catalysis and accessibility of the polyphosphazene backbone. Effect of Hydrogen Bond Forming Compounds. The role of intermolecular hydrogen bonding in biological interactions and environmental fate of pharmaceutical compounds is well recognized.27,38 Furthermore, as part of drug or vaccine formulations, pharmaceuticals frequently share the same milieu with nonionic surfactants and other excipients capable of forming hydrogen bonds.29,39-41 To this end, it was important to investigate if such excipients can modify the degradation profile of PCPP. Figure 6 shows the effect of PEG on the kinetics of PCPP molecular weight decline in water at various concentrations of additive. As seen from the Figure, this polyether showed the ability to dramatically boost the hydrolysis rate of PCPP (up to a 20-fold decrease in half-life) in a concentration-dependent manner. Such accelerating effect of PEG was also confirmed in a phosphate buffer at pH 7.4, although the effect was somewhat less pronounced (data not shown). Nonionic surfactant, containing short polyether segments (Tween), and alcohols, such as glycerol, and sodium salt of HBA were also tested for a potential influence on the degradation of PCPP. Figure 7 shows reduction in half-life of PCPP in the presence of various concentrations of Tween, expressed as a percent of polymer’s half-life in the absence of an additive. Remarkable effects, with up to 70-fold decreases in the half-life, were observed for such systems both in phosphate buffer and in deionized water. Reductions (5- and 10-fold) were detected for glycerol and sodium salt of HBA in phosphate buffers. Thus, water-soluble compounds containing ether or hydroxyl groups, regardless of

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intramolecular catalysis by carboxylic acid groups can play an important role in the degradation of PCPP, and both the effects of salt and hydrogen bond forming excipients appear to support this mechanism. Interestingly, previously reported copolymers of PCPP containing polyether side groups also appeared to undergo degradation at a higher rate than PCPP, which can further support this mechanism.45

Conclusions

Figure 6. Effect of PEG on the kinetics of PCPP molecular weight changes in aqueous solutions at 0% (1), 0.1% (2), and 1% of PEG (70 °C), 0.1% solutions of PCPP, deionized water, Mw0: molecular weight of PCPP before degradation experiment).

Degradation of PCPP in aqueous solution at elevated temperature is characterized by the gradual decrease in the polymer molecular weight, narrowing molecular weight distribution, and is accompanied by the release of the side group HBA, which is in general agreement with previously described hydrolytical degradation profiles of water-soluble polyphosphazenes. The rate of degradation appears to be pH-dependent, and the process accelerates as the pH of solution decreases, which supports the mechanism of acid-catalyzed hydrolysis with the protonation of nitrogen in the backbone. Inorganic salts appear to retard the rate of degradation, whereas some hydrogen bonding watersoluble additives impart a dramatic acceleration effect. Both phenomena can be potentially explained on the basis of macromolecular complexation and intramolecular catalysis involving carboxylic acid groups of the polymer.

References and Notes

Figure 7. Reduction in half-life of PCPP in the presence of various concentrations of Tween in water and PBS, expressed as a percent of the polymer’s half-life in the absence of an additive (70 °C, 0.1% solutions of PCPP).

significant differences in the molecular weight, have demonstrated strong catalytic effects on the degradation of PCPP. Previously reported studies of PCPP-Tween and PCPP-PEG solutions using turbidimetric titration and dynamic light scattering methods suggested macromolecular complexation in the system.42 To understand PCPP degradation results, acidic properties of PCPP in the presence of these excipients were also investigated using potentiometric titration (Figure 5). As opposed to the effect of salt discussed above, both PEG and Tween decreased the dissociation constant of PCPP. This further supports the formation of macromolecular complexes in the system involving nonionized carboxylic acid groups. Thus, a higher concentration of acidic groups and some potential changes in reactogenicity in the complex38,43,44 may increase the rate of intramolecular catalysis and bear responsibility for the observed acceleration effect. We therefore hypothesize that

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