Biomacromolecules 2004, 5, 1999-2006
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Synthesis and Biologically Relevant Properties of Polyphosphazene Polyacids Alexander K. Andrianov,* Yuri Y. Svirkin, and Mark P. LeGolvan Parallel Solutions Inc., 763D Concord Avenue, Cambridge, Massachusetts 02138 Received April 28, 2004; Revised Manuscript Received June 8, 2004
Polyphosphazene polyacids show potential as immunostimulating compounds and materials for microencapsulation. Their synthesis requires multistep chemical transition from a hydrolytically unstable macromolecular precursor, poly(dichlorophosphazene), to a water-soluble polyelectrolyte. Insufficient synthetic control in these reactions can lead to molecular weight variations and formation of macromolecules with “structural defects” resulting in significant variations in polymer performance. Simple and reproducible “one pot-one solvent” method is reported for the preparation of polyphosphazene polyacids-poly[di(carboxylatophenoxy)phosphazene] and its copolymers. Molecular weight characteristics and polymer compositions were studied as a function of reaction parameters. Macromolecular byproducts, incompletely substituted polymers containing hydroxyl groups and partially deprotected polymers containing propyl ester functionalities, were synthesized and characterized. It was demonstrated, that the presence of such groups can affect polymer characteristics, such as hydrolytic degradation profiles, immunostimulating activity, and microsphere forming properties. In vivo studies showed that the immunostimulating activity of polyphosphazene polyacids correlates with the content of acid functionalities in the polymer. Introduction Polyphosphazenes offer critical advantages for the design and synthesis of biologically functional macromolecules, such as broad structural diversity, high functional density, and tailored biodegradability of phosphorus-nitrogen backbone. In particular, polyphosphazene polyacids, such as poly[di(carboxylatophenoxy)phosphazene] (PCPP), present interest as water-soluble vaccine delivery systems and microencapsulation agents.1-7 The synthesis of polyphosphazene polyacids involves multistep transformation of a reactive macromolecular intermediate, poly(dichlorophosphazene) (PDCP), to a stable polymer with organic side groups (Scheme 1). Such a conversion of a hydrolytically unstable precursor to a polymer designed for applications in aqueous media allows limited control over polymer composition and molecular weight characteristics prompting the search for alternative compounds and methods.8 There are clear indications that biological performance of polyphosphazenes can be greatly affected by their molecular weight and composition.3,7 Thus, it was of critical importance to develop well-defined and simple synthetic methods allowing reliable control over the biologically relevant properties of these polymers. There was also a need to investigate the effect of various reaction byproducts, macromolecules containing “structural defects” on polymer performance. Our previous report focused on the synthesis and stabilization of macromolecular polyphosphazene precursor, PDCP.9 The present paper describes new synthetic methods allowing * To whom correspondence should be addressed. E-mail: aandrianov@ parallelsolutionsinc.com.
fast and controlled conversion of this reactive precursor to biologically active polyphosphazene polyacids. We investigated the effect of reaction conditions on polymer’s molecular weight characteristics. We also synthesized mixed substituent copolymers of PCPP containing “structural defects”, propyl ester functionalities and hydroxyl groups, and studied the influence of these groups on polymer degradation characteristics, microencapsulating properties, and biological activity. Experimental Section Materials. Hexachlorocyclotriphosphazene, trimer (Nippon Fine Chemicals, Japan) was used as received. Sodium propyl paraben, U. S. P./N. F. grade (Spectrum Quality Products, Inc., Gardena, California), and propyl 4-hydroxybenzoate (propyl paraben), 99.5+% (Aldrich Chemical Co., Inc., Milwaukee, Wisconsin), were dried prior to use in a vacuum oven at 80 °C for 2 h. 2-Methoxyethyl ether (diglyme), anhydrous, 99.5% (Aldrich Chemical Co., Inc., Milwaukee, Wisconsin), was used as received. Analytical Methods. 31P and 1H NMR spectra were recorded using Bruker AM 360 NMR spectrometer with an Oxford magnet operated at 145 and 360 MHz, respectively. D2O was used as a solvent. Aqueous GPC analysis was conducted as described previously.10 The chromatographic system included a Waters 510 HPLC pump with pulse dampener (Waters, Milford, MA), an Ultrahydrogel Linear column (Waters, Milford, MA) connected in series to a multi-angle laser light scattering (MALLS) detector (DAWN DSP-F, Wyatt Technology, Santa Barbara, CA), a Waters 486 tunable UV/visible
10.1021/bm049745d CCC: $27.50 © 2004 American Chemical Society Published on Web 08/24/2004
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Scheme 1. Synthetic Pathway to Polyphosphazene Polyacids
absorbance detector set at 254 nm, and a Waters 410 refractive index detector. The absolute molecular weights were determined by HPLC-MALLS using phosphate buffered saline, PBS (pH 7.4), as a mobile phase for PCPP samples and PBS/acetonitrile mixture, 5:1 (v/v), for PCPP copolymers. GPC analysis of PDCP was performed in diglyme as a mobile phase as reported previously.9 The chromatographic system was equipped with a Waters 510 HPLC pump (Waters, Milford, MA), one inline filter, 0.5 micron highpressure filter (Rainin, Woburn, MA), and a Waters model U6K Universal Liquid Chromatograph Injector. A Waters Styragel HR 5E column was used with a Waters Styragel guard column (Waters, Milford, MA). Turbidity measurements of polymer solutions were conducted using Hitachi U-2000 spectrophotometer at 400 nm. PCPP Synthesis. The macromolecular precursor, PDCP, was synthesized using ring-opening polymerization of hexachlorocyclotriphosphazene in the titanium pressure reactor as described previously.9 Sodium propyl paraben, nucleophilic reagent, was dissolved in diglyme using propyl 4-hydroxybenzoate as a cosolvent. 85 g of propyl paraben (0.48 mol) was dispersed in 21 mL of diglyme and the dispersion was heated with constant stirring until melted (110 °C). 96 g of sodium propyl paraben (0.48 mol) was then added to the melt and the heating was continued until the formation of a clear solution. This solution was then diluted with 190 mL of diglyme and added to the three neck reaction flask charged with 130 mL of 0.2 M polydichlorophosphazene solution in diglyme while stirring. The reaction mixture was refluxed for 2 h under nitrogen and then cooled to 95° C. 141 mL of aqueous 13 N potassium hydroxide solution (1.8 mol) was slowly added with vigorous stirring to the reaction mixture to bring about the hydrolysis and subsequent precipitation of PCPP. 20 mL of water was then added to facilitate an efficient phase separation. The liquid organic layer was decanted and the precipitate was dissolved in 300 mL of 15% (w/v) aqueous sodium chloride solution and then precipitated by addition of 600 mL of deionized water. The aqueous layer was decanted, the precipitate dissolved in 150 mL of deionized water, and finally precipitated by addition of 150 mL of ethanol. The PCPP precipitate was filtered and dried. The yield was 7.2 g (75% of theoretical). Based on the results of HPLC analysis, Karl Fisher titration, and elemental analysis, the purity of the polymer was 96.5%. Polymer structure was verified by 31P and 1H NMR, the molecular weight was determined by GPCMALLS as described below. 1H NMR: 6.4 ppm; 7.2 ppm. 31 P NMR: -18.2 ppm.
Synthesis of Mixed Substituent PCPP Copolymers Containing Propyl Ester Functionalities. The substitution reaction was carried out as described above, however deprotection reaction was conducted using reduced concentration of base as described below. After 2 h of reflux, the polymer solution was cooled to 90° C, and 47 mL of 13 N aqueous potassium hydroxide solution (0.3 mol) was added with constant stirring. Reaction was continued at 90 °C under vigorous stirring for an additional 120-230 min, depending on the desired degree of deprotection. Polymer was precipitated by addition of 300 mL of water and recovered by decantation. Precipitate was dissolved in water, purified by additional precipitations with 15% (w/v) aqueous sodium chloride solution and ethanol, and lyophilized. The yield varied in the range of 67-70% of theoretical. 1H NMR: 1.1 ppm; 1.9 ppm; 4.3 ppm; 6.4 ppm; 7.2 ppm. 31P NMR: -18.2 ppm. Synthesis of Mixed Substituent PCPP Copolymers Containing Hydroxyl Groups. The substitution and deprotection reactions were carried out as described above. Sodium propyl paraben was used in deficient amounts (polymer 6-90%, polymer 7-95%, and polymer 8-98% of the chlorine atom content in PDCP). The yields are as follows: polymer 6 (4.6 g; 48% of theoretical); polymer 7 (5.3 g; 56% of theoretical); polymer 8 (6.4 g; 67% of theoretical). 1 H NMR: 6.4 ppm; 7.2 ppm. 31P NMR: -18.2 ppm; -11.0 ppm. Microsphere Preparation. Ionically cross-linked microspheres were prepared as described previously.6 The following is the typical example of microsphere preparation. 4 mL of 0.2% (w/v) solution of PCPP in phosphate buffer solution (pH 7.4) and 7.4 mL of 6.2% (w/v) aqueous sodium chloride solution were mixed, shaken, and incubated at room temperature for 6 min or until coacervate droplets with a mean size approximately 4-6 µm were formed. The obtained coacervate dispersion was poured into 800 mL of 8.8% (w/ v) calcium chloride solution in water. The suspension was stirred for 1 h, and the microspheres were collected and washed with water on a 0.45 mm Nalgene filter (Nalge Company, Rochester, NY). The particle size distribution of the microspheres was analyzed by a Malvern Mastersizer S Particle Sizer (Malvern Instruments, Southborough, MA). The coacervate microspheres were also studied using Olympus CK-2 inverted microscope (Olympus, Japan). In Vivo Immunization Experiments. Split influenza virus X-31 was prepared, purified, and quantitated as described previously.3 Polymer solutions were prepared by dissolving the appropriate amount of polymer in phosphate buffered saline, pH 7.4 at 55 °C with agitation. The antigen solution
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Scheme 2. Potential Reaction Pathways in the Synthesis of PCPP
was then slowly added to the polymer solution so that 30 µg of polymer and 5 µg of total influenza protein were contained in 0.1 mL. Female 7-8 week old BALB/c mice were randomized into groups (5 mice per group). Mice were immunized with polymer/antigen formulations (5 groups) and with antigen alone (control group). Formulations (0.1 mL) were injected subcutaneously with a 25 g needle in the loose skin over the neck. Blood samples were taken from the retroorbital sinus of CO2 anesthetized mice. Antigen-specific antibodies in mouse serum were determined by ELISA as described previously.3 The endpoint titers were the reciprocal of the highest sample dilution producing a signal 2-fold greater than that of an antibody-negative sample at the same dilution. The average antibody titers for a group of mice were expressed as geometric mean titers (GMT). Results and Discussion Synthesis of PCPP. Synthetic route to polyphosphazene polyacids involves conversion of macromolecular precursor containing hydrolytically labile P-Cl bonds into polymers designed for aqueous environment. PCPP, the most studied phosphazene polyelectrolyte, is prepared via 2-step modification of PDCP, which involves (1) substitution of chlorine atoms with nucleophile containing ester of hydroxybenzoic acid and (2) hydrolysis of ester function under mild alkaline conditions (deprotection reaction).11 Reaction pathways in PCPP synthesis, as well as structures of potential byproducts resulting from incomplete substitution and deprotection reactions are shown in Scheme 2. The existing method is time-consuming and requires use of phase transfer agents, mild reaction conditions, multiple intermediate purifications
and solvent changes.11 However, it still fails to produce polymer with reproducible molecular weight parameters and degradation characteristics. To improve synthetic control we developed “single potsingle solvent approach” for the preparation of PCPP and its copolymers. The method utilizes diglyme stabilized PDCP solutions,9 forced conditions of the substitution reaction, aqueous based deprotection reaction, sodium propyl paraben as nucleophilic reagent, short reaction times, and purification based on salt precipitation of PCPP (phase diagram discussed later in the manuscript). A summary of the reaction conditions is shown in Table 1. 31 P and 1H NMR and GPC analysis of PCPP produced by this method did not reveal any “structural defects” in the polymer in detectable quantities. Based on NMR, GPC, and elemental analysis data, the purity of PCPP was determined to be in excess of 98%. No substituted trimer products were detected in any of the polymers described in this paper. Completion of the hydrolysis step in less than 30 min accompanied by the precipitation of deprotected polymer is an interesting peculiarity of this synthetic method. Typically, alkaline hydrolysis of esters in macromolecular systems is characterized by the pronounced “neighbor effect”: retardation of the reaction and incomplete conversion due to growing density of anionic groups on the polymer and repulsive interactions between them and hydroxide ions.12 Apparent absence of the retardation effect in the PCPP deprotection step can probably be explained by polymer solubility at high ionic strength (this property will be discussed in more detail below). As the hydrolysis reaction proceeds and polymer hydrophilicity increases, the polymer absorbs more water despite its high salt and base content. Due to high ionic strength, electrostatic repulsions between
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Table 1. Reaction Conditions for the Synthesis of PCPP reaction step substitution intermediate purifications deprotection/recovery
purification
reagents/reaction conditions (A) NaOPhCOOC3H7 (co-melted with HOPhCOOC3H7 1:1) (B) Diglyme,a 120 °C, 2 hb (C) 16 N aqueous potassium hydroxide, 90 °C, 30 minc PCPP (precipitate, remains in reactor), (D) hydrophobic contaminants (supernatant). (E) aqueous sodium chloride (phase diagram discussed below): (G) hydrophillic contaminants (supernatant), PCPP (precipitate)
process scheme
a “Stabilizing effect” of diglyme on PDCP solutions was reported previously.9 reaction confirmed by 1H NMR.
anionic groups on the polymer and hydroxide groups are suppressed. Thus, base is accumulating in the vicinity of nonhydrolyzed groups and further accelerates the reaction. Synthesis of Mixed Substituent PCPP Derivatives Containing Propyl Ester Side Groups (Partially Deprotected Polymers). Mixed substituent PCPP copolymers containing both carboxylic acid and propyl ester side groups (Scheme 2, PCPP copolymers 2-5) are potential byproducts of the PCPP synthesis and also an interesting class of hydrophobically modified polyelectrolytes. We were unable to synthesize these polymers by modulating time of the deprotection reaction under the conditions described above. However, degree of deprotection can be easily controlled by varying the hydrolysis time at the reduced (6-fold) concentration of the base (Figure 1). Polymer compositions calculated from 1H NMR data, the molecular weight averages determined by HPLC, i.e., static light scattering measurements, as well as refractive index increments of these polymers are presented in Table 2. Synthesis of PCPP Copolymers Containing Hydroxyl Groups: Incompletely Substituted Polymers. Incomplete replacement of chlorine atoms in polyphosphazenes in the substitution stage can potentially affect degradation characteristics of the end product. To investigate this effect, we attempted to synthesize PCPP derivatives containing variable amounts of residual chlorine atoms (Scheme 2, incomplete substitution pathway). Reactions were conducted using deficient quantities of nucleophiles (Table 3). After completion of the reaction, purified polymers were analyzed by 31P and 1H NMR, GPC, and elemental analysis. All 31P spectra contained a small peak at -11 ppm as well as PCPP peak at -18.2 ppm indicating the presence of additional side groups in these polymers. Unexpectedly, the content of these groups based on NMR data was practically constant for all polymers (approximately 2%). Elemental analysis showed chlorine levels in the samples to be below 0.1% for all polymers. These results apparently indicate a conversion of residual
b
Completion of the reaction confirmed by 31P NMR. c Completion of the
Figure 1. Effect of the hydrolysis reaction time on the content of propyl ester groups in hydrophobically modified PCPP copolymers.
chlorine atoms into hydroxyl side groups during the deprotection step (Scheme 2, incomplete substitution pathway). The presence of hydroxyl side groups in incompletely substituted macromolecules was also reported previously for other polyphosphazenes.13 A lack of correlation between the composition of the reaction mixture and the amount of hydroxyl groups in the polymer can be explained by the degradation of polymers 6 and 7 during the reaction, manifested by the decrease in weight average molecular weight (Table 3). As seen from the Table, polymer 8 did not show significant reduction in molecular weight compared to PCPP (8.5‚105 g/mol). Molecular Weight Changes in the Reaction. Reproducibility and control of molecular weight characteristics are prerequisites for the synthesis of biologically relevant
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Properties of Polyphosphazene Polyacids Table 2. Properties of Polymers 1-5
polymer
X, % mol
Y, % mol
dn/dc mL/g
1 2 3 4 5
100 92 78 52 30
0 8 22 48 70
0.250 0.234 0.224 0.207 0.162
Mw × 10-3 g/mol
Mn × 10-3 g/mol
850 927 990 1407 1725
590 611 730 1022 1259
Table 3. Synthesis and Properties of Polymers 6-8 reaction mixture, mol,
Mw × 10-3 g/mol
polymer 6 7 8
0.90 0.95 0.98
205 600 820
Figure 3. Logarithm of root-mean-square radius 〈r2〉1/2 versus logarithm of molecular weight for the PDCP and PCPP samples.
Figure 2. Change in the degree of polymerization (DP) during chemical transformation of four polyphosphazene samples (% of change ) DPPCPP/DPPDCP x 100; DP for starting PDCP samples varied from 1300 to 7000).
polymers. Molecular weight changes during the reaction were monitored by size-exclusion HPLC, light scattering analysis of polyphosphazene samples before and after conversion. Reproducibility of PCPP synthesis was confirmed for more than 100 samples. For PCPP prepared using the synthetic method described above, the weight average molecular weight fluctuations were not in excess of 10%. The extent of correlation between the molecular weights of derivatized polyphosphazene and its macromolecular precursor was of particular interest in our studies. This question is one of the least investigated in polyphosphazene chemistry and still remains under discussion in the literature.14 We prepared four samples of PDCP with variable degrees of polymerization (1300-7000 determined by light scattering analysis, Figure 2) and then converted them to PCPP using the process described above. Degree of polymerization values for PCPP samples were found to be at 60-70% of the level expected
based on PDCP analysis (Figure 2). These reduced values might indicate either the presence of hydrolytic reactions during the conversion or experimental error in the molecular weight determination. Importantly, the level of the molecular weight decrease was uniform across the investigated range and was practically independent of the molecular weight of the starting sample. These results indicate the absence of experimental factors capable of random scrambling of the molecular weights during polymer functionalization. Figure 3 shows conformational plots (root-mean-square radius versus molecular weight) for PCPP and corresponding PDCP sample. It is noteworthy that the profile obtained for PDCP in a nonaqueous GPC is similar to the corresponding aqueous PCPP profile. These results also confirm our previous reports on the presence of branching in polyphosphazene samples obtained by ring opening polymerization.9 We have also found that the presence of hexachlorocyclotriphosphazene in the substitution reaction can result in the decrease of the molecular weight of the final product. This chlorinated cyclic phosphazene compound used for the synthesis of PDCP is also one of the possible contaminants in the substitution reaction, which is capable of competing with PDCP for the reaction with organic nucleophiles. Figure 4 shows the effect of hexachlorocyclotriphosphazene: sodium propyl hydroxybenzoate molar ratio in the substitution reaction on the weight average molecular weight of PCPP. As seen from the figure, even small quantities of this
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Figure 4. Effect of hexachlorocyclotriphosphazene present in the substitution reaction on the molecular weight of PCPP (0.07 M PDCP; 1.4 M sodium propyl hydroxybenzoate; diglyme).
compound can result in the significant decrease of the molecular weight, probably through the change in nucleophile to chlorine atoms ratio in the system. These results suggest the possibility of post-polymerization molecular weights modulation in the synthesis of polyphosphazenes through the regulation of nucleophile-to-polymer ratio in the system. Biologically Relevant Properties. (i) Immunostimulating Properties. PCPP shows remarkable activity as an immunoadjuvant and was studied extensively in combination with various antigens.15 Nevertheless, the mechanism and structure - activity relationship in this system are not fully understood. Hydrophobically modified PCPP copolymers 2-5 (Scheme 2) present an interesting opportunity for the investigation of the effect of ionic density and hydrophobicity on polymer performance. In addition, a practical need for their investigation arises from the fact that they are potential synthetic PCPP byproducts resulting from the possibility of incomplete deprotection reaction. The immunoadjuvant activity of mixed substituent copolymers and PCPP was evaluated using influenza antigen in mice. Groups of mice were subcutaneously immunized with antigen, detergent split X-31 influenza virus particle suspension mixed with aqueous solutions of polymers. The immune responses, i.e., IgG titers, induced by these formulations are presented in Figure 5. As seen from the figure, all synthesized polymers enhanced the antibody response as compared to the levels elicited by the antigen alone. It is evident from the figure that the content of carboxylic acid groups determines the activity of the polymer as an immunomodulator. Reduction in the content of acid groups in the polymer gradually decreases the immune response. (ii) Degradation Characteristics. Hydrolytic degradation of biologically active polymers is an important property defining their performance, safety, and storage conditions. Degradation profiles of PCPP and PCPP with hydroxyl groups in aqueous solutions are shown in Figure 6. As seen from the figure, the molecular weight loss of the incompletely substituted sample was significantly faster than of the
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Figure 5. Serum IgG titers after subcutaneous immunization of Balb/C mice with Split X-31 influenza formulated with polymers containing variable amounts of carboxylic acid groups (polymers 1-5, week 3 data; antigen dose, 5 µg; control. Split X-31 influenza; all formulations are based on phosphate buffer saline, pH 7.4).
Figure 6. Weight average molecular weight loss of PCPP (1) and incompletely substituted PCPP (copolymer 8) (2) versus degradation time (aqueous PBS pH 7.4, 55 °C).
properly substituted sample and was characterized with wellpronounced initial rapid degradation phase. These results demonstrate the importance of substitution control in polyphosphazene chemistry and the need for reliable synthetic methods. Careful control of the hydroxyl groups, “structural defects” or “weak links” in polyphosphazene structures, can open a pathway to the tailoring of polymer degradation characteristics. (iii) Aqueous Solution Behavior and Microsphere Forming Properties. The ability to form aqueous coacervate systems in the presence of sodium ions (Figure 7) is one of the most captivating PCPP properties. Interestingly, this phase separation cannot be achieved using lithium or potassium ions, which suggests the importance of ionic radius
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Figure 7. Phase diagrams for aqueous PCPP-sodium chloride system. Shaded region denotes coacervate phase (22 °C).
and steric factors in polymer-ion interactions. It can be expected, that the introduction of other groups in PCPP structure, such as propyl ester groups in mixed substituent copolymers, can cause distortions in molecular configurations and have a profound effect on polymer behavior. “Sodium sensitivity” of PCPP finds practical applications, and previously we reported a simple microencapsulation method, which involves coacervation of PCPP solution with subsequent cross-linking of the coacervate microdroplets using calcium salts.6 This method allows effective control over microsphere size, uses mild conditions, and is applicable to a broad range of biological materials, including proteins and cells.1-2,16-17 From the application standpoint, the use of polyphosphazene polyelectrolytes with variable ionic density and hydrophobicity can provide effective means for the modulation of microsphere properties. We compared the ability of PCPP and its hydrophobically modified copolymers 2-5 (Table 2) to form coacervate systems in the presence of sodium chloride. All copolymers showed solubility in aqueous solutions at pH 7.4; however, only derivatives containing 78 and 92% mol of acid groups (copolymers 3 and 2) demonstrated PCPP-type behavior in the formation of coacervate phase. Copolymers with lower content of carboxylic acid groups showed no evidence of phase separation. The results of turbidimetric titration with aqueous sodium chloride demonstrated that the coacervation threshold is a function of carboxylic acid content in the polymer. As seen from Figure 8, polymers with higher content of carboxylic acid groups (lower content of hydrophobic groups) required less salt to induce phase separation (Figure 8). These results emphasize the importance of steric component in PCPP-sodium salt interactions, where even minimal distortions in PCPP structure lead to the loss of “sodium sensitivity”. The order of phase separation was reversed when polymers were titrated with aqueous hydrochloric acid, where according to the expectations, more hydrophobic polymers required less acid for their precipitation (Figure 9). Mixed substituent copolymers 2 and 3 were then successfully used to prepare microspheres using coacervation method. The results on their higher acid sensitivity shown in Figure 9 demonstrate their potential for the preparation
Figure 8. Turbidimetric titration of aqueous solutions of PCPP (1) and copolymers (2-3, for description see Table 2) with aqueous sodium chloride (polymer concentration, 0.08% (w/v); sodium chloride concentration, 7 N).
Figure 9. Turbidimetric titration of aqueous solutions of PCPP (1) and mixed substituent PCPP copolymers (2-4, see Table 2 for description) with an aqueous hydrochloric acid (polymer concentration, 0.08% (w/v); concentration of hydrochloric acid, 0.05 N).
of microspheres with “low pH protection” characteristics, which make them especially attractive for oral protein delivery. Conclusions A simple “single pot-single solvent” method for the synthesis of PCPP and its derivatives is developed. The method allows for high degree of reproducibility and effective control over polymer molecular weight characteristics and composition. Potential reaction byproducts, mixed substituent PCPP copolymers containing propyl ester and
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hydroxyl groups, were synthesized and their biologically relevant properties were studied. Immune stimulating activity of polyphosphazene polyacids is dependent on the content of carboxylic acid groups and can be modulated through the introduction of propyl ester functionalities. Addition of propyl ester groups to PCPP structure also results in a significant decrease of sodium binding compared to PCPP and thus affects polymer microencapsulation performance. Incompletely substituted PCPP samples containing hydroxyl groups show accelerated hydrolytic breakdown demonstrating potential for the development of materials with controlled degradation profiles. Acknowledgment. The authors thank Dr. U. Ramstedt for valuable discussions and help with conducting in vivo studies and Mrs. Jianping Chen for assistance with titration experiments. References and Notes (1) Andrianov, A. K.; Payne, L. G. AdV. Drug DeliVery ReV. 1998, 34, 155-170. (2) Andrianov, A. K.; Payne, L. G. AdV. Drug DeliVery ReV. 1998, 31, 185-196. (3) Andrianov, A. K.; Sargent, J. R.; Sule, S. S.; Le Golvan, M. P.; Woods, A. L.; Jenkins, S. A.; Payne, L. G. J. Bioact. Compat. Polym. 1998, 13, 243-256.
Andrianov et al. (4) Andrianov, A. K.; Cohen, S.; Visscher, K. B.; Payne, L. G.; Allcock, H. R.; Langer, R. J. Controlled Release 1993, 27, 69-77. (5) Andrianov, A. K.; Payne, L. G. In Microparticulate Systems for the DeliVery of Proteins and Vaccines; Cohen, S., Bernstein, H., Eds.; Marcel Dekker: New York, 1996; pp 127-147. (6) Andrianov, A. K.; Chen, J.; Payne, L. G. Biomaterials 1998, 19, 109-115. (7) Payne, L. G.; Jenkins, S. A.; Woods, A. L.; Grund, E. M.; Geribo, W. E.; Loebelenz, J. R.; Andrianov, A. K.; Roberts, B. E. Vaccine 1998, 16, 92-98. (8) Qiu, L. J. Appl. Polym. Sci. 2003, 87, 986-992. (9) Andrianov, A. K.; Chen, J.; LeGolvan, M. P. Macromolecules 2004, 37, 414-420. (10) Andrianov, A. K.; LeGolvan, M. P. J. of Appl. Polym. Sci. 1996, 60, 2289-2295. (11) Allcock, H. R.; Kwon, S. Macromolecules 1989, 22, 75-79. (12) Kudryavtsev, Y. V.; Litmanovich, A. D.; Plate´, N. A. Macromolecules 1998, 31, 4642-4644. (13) Tanigami, T.; Ohta, H.; Orii, R.; Yamaura, K.; Matsuzawa, S. J. Inorg. Organomet. Polym. 1995, 5, 135-153. (14) Klein, J. A.; Bell, A. T.; Soong, D. S. Macromolecules 1987, 20, 782-789. (15) Payne, L. G.; Jenkins, S. A.; Andrianov, A. K.; Roberts, B. E. In Vaccine Design; Powell, M. F. Newman, M. J., Eds.; Plenum Press: New York, 1995; pp. 473-493. (16) Cohen, S.; Ban˜o, M. C.; Visscher, K. B.; Chow, M.; Allcock, H. R.; Langer, R. J. Am. Chem. Soc. 1990, 112, 7832-7833. (17) Ban˜o, M. C.; Cohen, S.; Visscher, K. B.; Allcock, H. R.; Langer, R. BioTechnology 1991, 9, 468-471.
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