the invention is further described below by several illustrative examples . these examples are added to the preceding instruction for the sole purpose of further enabling the artisan of ordinary skill to make and practice the applicants &# 39 ; best mode of the invention . they are not intended to limit the scope of the claims appended hereto . the ability of polyphosphazenes to form coacervate systems in the presence of organic amines was demonstrated using aqueous solutions of pcpp and spermine tetrahydrochloride . a phase diagram of a polyphosphazene - spermine - water system was prepared as follows . sodium salt of pcpp ( weight average molecular weight 8 . 4 × 10 5 g / mol ) was dissolved in deionized water to prepare a series of solutions ranging in concentration from 0 . 002 to 3 . 6 % ( w / v ). solutions of spermine in deionized water were prepared ranging in concentration from 0 . 02 to 12 % ( w / v ). the polymer solutions were then mixed with the spermine solutions in the ratio of 1 . 0 ml to 0 . 2 ml , so that the concentration of pcpp and spermine in the resulting solutions varied in the 0 to 2 % ( w / v ) range . the solutions or dispersions were agitated by gentle shaking and then examined by microscope to determine the presence of coacervate droplets or precipitate . the phase diagram was then established by plotting the physical state of the system versus composition of the tertiary system — spermine , pcpp , and water ( fig1 ). the diagram contains three regions — coacervate , precipitate , and homogeneous solution . pcpp microspheres were prepared in a single step coacervation process using the physiologically acceptable organic amine , spermine , as both the coacervating and the cross - linking agent . 0 . 07 ml of 7 % solution of spermine in pbs ( ph 7 . 4 ) were added to 5 ml of 0 . 19 % aqueous pcpp solution ( pbs , ph 7 . 4 ) and were agitated gently by shaking . the mixture was then incubated at ambient temperature for 60 minutes . the suspension of microspheres was then diluted with a three - fold excess of pbs buffer ( ph 6 . 5 ), was let to stand for additional 30 minutes , and was thereafter examined for the presence of particulates using a mastersizer s ( malvern instrument ltd .). fig2 shows differential percentages of microspheres by number ( 1 ) and by volume ( 2 ) demonstrating narrow particle size distribution . the mean diameters were 0 . 41 μm and 1 . 52 μm by number and by volume respectively . the effect of polymer and spermine concentration on microsphere size was investigated . 0 . 19 % ( w / v ) and 0 . 38 % ( w / v ) aqueous pcpp solutions ( pbs buffer , ph 7 . 4 ) were prepared in the amount of 3 . 8 ml of each . to these solutions 0 . 04 ml and 0 . 08 ml of 7 % ( w / v ) spermine solution in pbs ( 7 . 4 ) were added respectively , so that the molar concentration of pcpp to spermine was kept the same for both mixtures ( 3 . 5 : 1 ). the mixtures were then incubated at ambient temperature for 60 minutes and particle size distribution of the resulting microspheres was analyzed using a mastersizer s ( malvern instrument ltd .). the results demonstrated the formation of particulates with a sub - micron size for the lower pcpp - spermine concentration ( 0 . 51 μm by volume ) and larger microspheres ( 1 . 79 μm by volume ) for a mixture with higher concentration . thus , varying total pcpp : spermine concentration in the reaction mixture allows for an effective control of microsphere - nanosphere size distribution .
methods of producing polyphosphazene microspheres comprising admixing aqueous solutions of a water - soluble polyphosphazene and an organic amine , or salt thereof , are disclosed .