Controlled Drug Delivery - American Chemical Society

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Ionically Cross-Linked Polyphosphazene Microspheres Alexander K. Andrianov, Jianping Chen, Sameer S. Sule, and Bryan E. Roberts Avant Immunotherapeutics, Inc., 119 Fourth Avenue, Needham, MA 02494

Ionically cross-linked polyphosphazene microspheres were prepared under mild conditions by a simple aqueous coacervation process utilizing aluminum and calcium salts as cross-linking reagents. Hydrolytic degradability of the obtained hydrogel microspheres was shown to be sensitive to the type of the ionic cross-linker. It was demonstrated that microsphere size distribution can be controlled by the conditions of the coacervation step and is only slightly affected by the concentration of the ionic cross-linker under the conditions studied. The method potentially allows the use of designed polyphosphazene polyelectrolytes to prepare microspheres with desired physico-chemical and biological properties.

Polymeric hydrogel microspheres have been used extensively in the development of advanced drug delivery systems and controlled release technologies [1]. In particular, polyphosphazene hydrogel microspheres are of interest as carriers for a variety of prophylactic and therapeutic agents because of potential biocompatibility and the fact that phosphazene polymers can be designed to generate any combination of properties needed for specific biomedical applications [2]. Microspheres based on some phosphazene polyelectrolytes, such as poly[di(carboxylatophenoxy)phosphazene] (PCPP) also display powerful immunostimulatory properties, that makes them an ideal candidate as a vaccine delivery vehicle [3]. Recently we described a simple aqueous based method of preparing polyphosphazene hydrogel microspheres with controlled microsphere size distribution [4]. In this process aqueous polyphosphazene solutions are coacervated using solution of sodium chloride followed by cross-linking of the coacervated microdroplets with calcium ions to produce ionically cross-linked microspheres.

© 2000 American Chemical Society

Park and Mrsny; Controlled Drug Delivery ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

395

396 The advantages of this method are in its simplicity, reproducibility and that it avoids the use of organic solvents, heat, and also allows the microsphere size to be varied by a simple manipulation of the experimental conditions.


This paper reviews the potential of the coacervation method in the preparation of polyphosphazene hydrogel microspheres with specific characteristics, such as desired biological activity of the carrier polymer and controlled degradability. Particularly, we expand the scope of the microencapsulation method to include preparation, characterization and study of the hydrolytic stability of aluminum crosslinked polyphosphazene microspheres and discuss properties of some of the potential matrix forming polyphosphazenes.

Experimental Materials.

PCPP-sodium salt was prepared using the synthetic method described elsewhere [5, 6]. Poly(dichlorophosphazene) was reacted with sodium salt of propyl p-hydroxybenzoate and the obtained product was subjected to the alkaline hydrolysis. The weight-average molecular weight (M ) of the polymer was determined as described previously [7] and was equal to 860,000 g/mol. Fluorescein isothiocyanate labeled bovine albumin, FITC-BSA (Sigma Chemical Co., St. Louis, MO) contained 11.2 moles of FITC per 1 mole of BSA and was used as received. Dulbecco's phosphate buffered saline, PBS (pH 7.4) was purchased from Sigma Chemical Co., St. Louis, MO. A l l other chemicals were reagent grade. w

Analytical Methods.

The molecular weight of the polymer was determined by gel permeation chromatography (510 HPLC pump, Waters, Milford, MA) in an aqueous phase using Dawn DSP-F multi-angle laser light scattering detector, MALLS (Wyatt Technology, Santa Barbara, CA), Waters 410 refractive index detector (Waters, Milford, MA) and Ultrahydrogel Linear column (Waters, Milford, MA) as described previously [7]. Phosphate buffered saline PBS (pH 7.4) was employed as a mobile phase. The particle size distribution of the microspheres was analysed by a Malvern Mastersizer S Particle Sizer (Malvern Instruments, Southborough, MA) using a 300RF lens and a small volume dispersion unit. The coacervatemicrodroplets and the microspheres were studied using Olympus CK-2 inverted microscope (Olympus, Japan). Microspheres loaded with FITC-BSA were also investigated using an Olympus BH-2 fluorescent microscope (Olympus, Japan). Efficiency of microencapsulation was determined using a F-2000 Hitachi fluorescence spectrometer (Hitachi, Japan).


397


Microsphere Preparation.

Aqueous PCPP-salt solutions were prepared by dissolving the polymer in water and diluting with deionized water and PBS (pH 7.4) to a final concentration of 0.2% (w/v). Aluminum lactate (Aldrich Chemical Company, Milwaukee, WI) solution was prepared in deionized water, 8% (w/v) and adjusting the pH of the solution to 6.7 using NaOH. The coacervate of PCPP was prepared in sodium chloride solution as described elsewhere [4]. The droplets were cross-linked by the addition of the coacervate dispersion to 8% aluminum lactate or 8.8% calcium chloride solution. The suspension was stirred for one hour, and the microspheres were collected and washed with water on a 0.45 μπι Nalgene filter (Nalge Company, Rochester, NY).

Encapsulation of FITC-BSA. FITC-BSA was encapsulated in the aluminum cross-linked PCPP microspheres. 4 ml of 0.2% (w/v) PCPP-sodium salt solution was mixed with 0.3 ml of 0.5% (w/v) aqueous solution of FITC-BSA. To this solution, 7.4 ml of 6.22% (w/v) aqueous solution of sodium chloride was added and the mixture was shaken. After 8 minutes of incubation when a significant amount of microspheres was observed, the coacervate was poured slowly into a 500 ml of 8% (w/v) aluminum lactate solution and stirred for 30 minutes. The obtained microspheres were collected and washed with water on a 0.45 μπι filter. The efficiencyof encapsulation of FITC-BSA in the microspheres was then determined. The microspheres were filtered through a 0.45 μιη Millipore MillexHV hydrophilic PVDF filter (Millipore Corporation, Bedford, MA). The filtrate was then analyzed using a F-2000 Hitachi fluorescence spectrometer (λ 492nm, λ 514nm) to determine the concentration of FITC-BSA in solution. 6Χ

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Results and Discussion

Polyphosphazenes as Drug Delivery Vehicles.

Polyphosphazenes present a rare opportunity as polymers for biomedical and specialty applications due to the versatile molecular design, ease of structural manipulations for these macromolecules, and a sophisticated spectrum of physical, chemical, and biological properties. The term polyphosphazenes covers a large family of organometallic polymers containing a backbone consisting of alternating phosphorus and nitrogen atoms with two organic side groups attached to each phosphorus atom:


398 R I -—ρ

1


- R Fundamental developments in polyphosphazene synthesis yielded synthetic pathways allowing a choice of a tremendous variety of substituents to be introduced in the polymer to create macromolecular structures with versatility that cannot be achieved using traditional polymerization or polycondensation techniques [2]. Since physico-chemical and biological properties of polyphosphazenes are determined to a great extent by the structure of polymer side groups and their combinations, polyphosphazenes can be designed to display customized and potentially unique sets of characteristics. Furthermore the peculiarity of this class of polymers is not only in the tremendous number of structures that can be synthesized, but also in the phosphorus-nitrogen backbone, which has a remarkable skeletal flexibility manifested in specific polymers having some of the lowest glass transition temperatures known in polymer chemistry. Due to its hydrophilicity, the phosphazene backbone can also serve as an ideal starting point for designing watersoluble and hydrophilic polymers, or can be rendered hydrolytically degradable when combined with appropriate side groups. This structural versatility combined with the unique character of the phosphorus-nitrogen backbone make polyphosphazenes especially attractive for targeting specific applications, especially biological. Polyphosphazenes, for example, were investigated and showed potential as materials for matrices for microcapsules and microspheres, protein and drug delivery vehicles, immunostimulants, bioinert medical devices and implants [2]. Phosphazene polyelectrolytes (polyphosphazenes containing ionic or ionizable groups) are a new rapidly developing class of phosphazene polymers which display some remarkable biological and physico-chemical properties that differentiate these synthetic polymers from conventional polyelectrolytes. In particular, some phosphazene polyelectrolytes demonstrate the ability to form inter- and intramolecular hydrogen bonds under physiological conditions and exhibit unusual ionic selectivity. Polyelectrolytes have been designed and synthesized to have high charge density, high hydrophobicity, biodegradability and high pKa values. One of the most important functional properties of phosphazene polyelectrolytes uncovered to date is the ability of some of these polymers to form aqueous based coacervation systems and some peculiar gel forming characteristics mimicking those

PCCP


399 of natural products, such as alginate [4, 8]. These properties prompted the use of phosphazene polyelectrolytes, such as PCPP, in a number of patented aqueous microencapsulation techniques demonstrating the potential of these polymers as carriers for the delivery of vaccines, proteins, other biologies and drugs. The advantages of these methods are in the simplicity of preparation, mild conditions of encapsulation, and the ability to control microsphere size distribution.


Preparation of Polyphosphazene Microspheres by Aqueous Coacervation. It was reported previously that aqueous solutions of PCPP can form a coacervate phase when treated with solutions of sodium chloride at room temperature [4]. A phase diagram established for a PCPP - NaCl - water shows that the separation of a macromolecular solution into two immiscible liquid phases - a concentrated polyphosphazene coacervate phase and a polymer deficient phase, can take place in a relatively wide range of polymer and salt concentrations and results in the formation of polymer coacervate microdroplets of spherical shape in the broad size range [4]. Though such systems are in dynamic equilibrium and relatively unstable, the advantage of PCPP as a coacervate forming material is in the ability of this polyphosphazene polyelectrolyte to form stable ionotropic gels almost instantaneously when exposed to calcium ions in aqueous solutions (Scheme 1). Addition of multivalent ionic cross-linker usually does not change the coacervate microdroplet size and shape despite changes in the ionic composition of the solution. At the same time, presence of significant quantities of sodium ions does not affect ionic gelation of PCPP and does not have an adverse effect on already formed microspheres despite the possibility of gel destabilizing ion-exchange reactions. The microspheres have spherical shape, typically, with a narrow size distribution (Figure 1). Microspheres of different sizes can be prepared by varying the conditions of coacervation, such as polymer and salt concentration and time of incubation in sodium chloride solutions (Figure 2).

Preparation of Microspheres with Variable Hydrolytic Stability.

Hydrogel microspheres with degradable matrices present a significant interest in the delivery of high molecular weight peptide and protein drugs [1]. The release of the encapsulated agent in such systems can be controlled not only by the permeability of the matrix, but also by the rate of matrix erosion determined by the stability of the polymer backbone and cross-linker. Polyphosphazenes, including polyphosphazene polyelectrolytes, can be synthetically designed to degrade with a predetermined rate [9]. However, ionic cross-linking of polyelectrolyte chains with multivalent ions presents another simple and attractive technique for the development of bioerodible hydrogels [10], since different degrees of stability of the ionic bridges can be achieved using different multivalent cations [11]. Polyphosphazene ionotropic hydrogel microspheres is an example of a protein delivery system where the choice of a multivalent cross-linker can have a potentially dramatic impact on the release and erosion properties of the matrix. Originally, coacervation based microencapsulation process was developed for PCPP utilizing calcium chloride as a cross-linking reagent. The resulting calcium cross-linked


400

Microspheres

Aqueous Polyphosphazene Solution

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€ Coacervation

©

Ο Ο Cross-linking

Microdroplets Scheme 1. PREPARATION OF POLYPHOSPHAZENE MICROSPHERES Differential number and volume, % 20

0.1

1

10

100

Particle Dia meter, μιη

Figure 1. Particle size distribution of PCPP hydrogel microspheres by number (1) and by volume (2).



401

Figure 2. Effect of the coacervation conditions on the microsphere mean diameters (Dataarefromreference [4]).



402 microspheres are usually characterized by high sensitivity to the ionic environment and can readily erode and dissolve in the solutions with high concentrations of salts of monovalent ions. In order to develop microspheres with superior stability in the solutions with higher ionic strength a number of aluminum salts were investigated as cross-linking agents for the PCPP microspheres. As a result of this study aluminum lactate was shown to be an effective cross-linker producing microspheres with a narrow size distribution, both by volume and by number with a little effect of aluminum lactate concentration on the size distribution. The analysis of microspheres obtained under the same conditions and cross-linked with aluminum lactate solutions of differentconcentrations (1%, 4% and 6% (w/v)) demonstrated that the increase in the concentration of the cross-linker resulted in a somewhat more narrow particle size distribution of microspheres and complete prevention of the formation of aggregates (Figure 3).

Differential volume, % 20 _

0.1

1.0

10.0

100.0

1000.0

Particle diameter, μηι

Figure 3. Particle size distribution of PCPP hydrogel microspheres cross-linked with different concentrations of aluminum lactate (1)1% (2) 4% (3) 6% Stability of the aluminum cross-linked PCPP microspheres in PBS was studied by incubating the microspheres in PBS (pH 7.4). Contrary to calcium cross-linked PCPP microspheres, the aluminum based PCPP microspheres were found to be stable for at least 1 month. Figure 4 shows the particle size distribution of the microspheres before and after incubation in PBS for 24 hours. As seen from the figure the microsphere size distribution profiles are practically identical. These


403 results demonstrate the potential of this system to prepare ionic hydrogel microspheres stable in aqueous solutions at neutral pH. Studies are now ongoing to prepare microspheres with a combination of cross-linkers.


Differential volume, %

2

1.0

10.0

100.0

Particle diameter, μ m

Figure 4. Particle size distribution of PCPP hydrogel microspheres before (1) and after (2) incubation in PBS (pH 7.4) for 24 hrs.

Microsphere Matrix Properties.

The versatility of the matrix properties can be achieved via utilization of designed phosphazene polyelectrolytes. The realization of this potential can be especially dramatic in case of some phosphazene polyelectrolytes which were shown to be effective in modulating biological responses related to host defense mechanisms against a variety of disease causing agents. PCPP, for instance, was demonstrated to be a potent immunostimulant inducing high level of protective antibodies against a number of vaccine antigens [3, 12]. It was shown, however, that the immunostimulating activity of PCPP (Figure 5) can be significantly improved through the manipulation of the macromolecular structure and the synthesis of new polyphosphazene derivatives [13]. There is an indication that the utilization of such chemical modification approaches can lead not only to greatly enhanced antibody levels, but also to the stimulation of cell mediated responses. This expands the potential applications of the designed polyphosphazenes, and



Figure 5. Serum IgG titers after subcutaneous immunization of Balb/C mice with μg of Split X-31 influenza adjuvanted with various polyphosphazene polyelectrolytes (week 3 data).


405 polyphosphazene based microspheres, beyond vaccines to treatment of cancer and persistent infections.

immunotherapeutic


Conclusions

The elucidation of novel polymeric derivatives with superior biological activity, bioinert carrier properties, and advanced functional characteristics can be potentially achieved by the extension of conventional combinatorial chemistry approaches to the design of phosphazene polymers. Although polyphosphazene synthesis is somewhat more involved than that of common petro-chemical polymers, it offers a unique synthetic flexibility not available for other classes of polymers. Polyphosphazenes of almost unlimited structural varieties can be synthesized including mixed substituent copolymers in which several different side groups can be introduced in a polymer structure by simultaneous or sequential substitution. Significant advances were also made in the development of the synthetic methods with a focus on a precise control of macromolecular structure and molecular weight distribution, as well as monitoring of the molecular parameters during the polymer transformation [6]. The synthesized libraries of polyphosphazenes can be evaluated for functional and biological properties and further refined utilizing computational chemical modeling to rationalize the design of phosphazene polymers for specific applications.

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10. Prasad, M . P.; Kalyanasunddaram, M . J. Appl. Polym. Sci. 1993, 49, 20752079. 11. Axelos, M . Α. V.; Mestdagh, M . M.; Francois, J. Macromolecules 1994, 27, 6594-6602.


12. Payne, L. G.; Jenkins, S. Α.; Woods, A. L.; Grund, E. M.; Geribo, W. E.; Loebelenz, J. R.; Andrianov, A. K.; Roberts, Β. E. Vaccine 1998, 16, 92 - 98. 13. Andrianov, A. K.; Sargent, J. R.; Sule, S. S.; Le Golvan, M . P.; Woods, A. L.; Jenkins, S. Α.; Payne, L. G. Journal of Bioactive and Compatible Polymers 1998, 13, 243-256.


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