Suitability of Spongelike PLGA Microspheres as Depot Injection of Hydrophobic Drugs

Abstract

The purpose of the study is developing a sustained release depot injection of hydrophobic drugs by use of porous PLGA microspheres with spongelike skeleton. Isopropyl formate was used as a dispersed solvent for PLGA (lactide:glycolide ratio = 85:15; Mw = 45,942). A dispersed phase consisting of isopropyl formate, PLGA and progesterone (4 ml/0.25 g/40, 80, or 120 mg) was emulsified in 40 ml of a 0.5% polyvinyl alcohol aqueous solution by use of a digital hotplate stirrer. After 3 min-stirring, 28% ammonia solution (3, 5, or 7 ml) was added into the oil-in-water (o/w) emulsion and stirred for 15 min. Ammonia, partitioning into the dispersed phase, reacted with isopropyl formate to yield water-soluble isopropanol and formamide. Since they were anti-solvents toward PLGA, oil droplets were transformed into solid microspheres. Also, their leaching into the aqueous phase paved the way for the formation of numerous pores across microsphere matrices. The microsphere suspension was poured into 150 ml of a 0.1% polyvinyl alcohol solution and stirred for 2 hrs. The microspheres were collected by filtration and dried overnight under vacuum. The major quality attributes of the spongelike PLGA microspheres were analyzed by various methods. As control, non-porous PLGA microspheres with a compact matrix were prepared following a traditional solvent evaporation method. Figure 1 illustrates a scanning electron microscopic image showing the external and internal morphology of our typical porous microspheres. Their internal morphology was characterized with a spongelike skeleton and numerous pores. Compared to typical nonporous PLGA microspheres, the porous microspheres showed very interesting quality attributes. In the solvent evaporation method, since the microsphere hardening took a long time, the tendency of progesterone in emulsion droplets to diffuse into the aqueous phase and crystallize was increased. Therefore, the encapsulation efficiency (EE)% of progesterone was determined as low as 41.2 to 52.2%. By contrast, the ammonolysis-based microencapsulation process delivered a very fast rate of microsphere hardening, so progesterone crystallization could be minimized. As a result, depending upon microsphere formulations, the microencapsulation technique showed high EE% values ranging from 72.7 to 85.1%. The level of a residual organic solvent in the spongelike microspheres was measured to be very low. For example, a residual solvent of 0.26±0.09% was observed toward the porous microspheres preparing using 0.25 g PLGA, 120 mg progesterone, and 7 ml of the ammonia solution. In contrast, the non-porous microspheres with a compact matrix contained a residual solvent content of 3.26 to 4.07%. The Tg of porous PLGA microspheres also was measured to be lower than the Tg of non-porous microspheres. Finally, the dissolution rate of progesterone from the porous microspheres was at least two times faster than that from the non-porous microspheres. Hydrophobic drugs tend to crystallize in the aqueous phase during a typical emulsion-template microencapsulation process. A slow rate of microsphere hardening might be accountable for this phenomenon. In addition, due to a non-porous nature of the microsphere matrix, a special drying technique is required to meet a compendia limit set for the organic solvent used for microencapsulation. Not only there is a lag time before drug release takes place, but also the rate of polymer degradation is not constant due to autocatalysis. These limitations are expected to be solved by using spongelike microspheres. Compared to conventional non-porous PLGA microspheres, the spongelike microspheres reported in this study displayed better quality attributes.;본 연구의 목적은 스폰지형 골격을 갖는 다공성 PLGA (Poly-d,l-lactide-co-glycolide) 미립구를 사용하여 소수성 약물의 서방형 약물 방출을 개발하는 데 있다. 소수성 약물결정은 유제기반 봉입과정 동안 미립자 표면이나 수상에서 결정화되는 경향이 있다. 이는 O/W 에멀젼 기반의 용매 증발법으로 미립자에 약물을 봉입했을 때, 분산상으로 사용한 용매제거 속도가 느리기 때문에 생성된다. 완성된 미립구를 회수하기 위해 여과하는 과정에서, 봉입 되지 않은 약물 결정은 미립구와 함께 얻어진다. 이러한 현상은 미립구를 전자 주사 현미경으로 관찰하여 확인할 수 있었다. 또한 미립구 입도 분포를 측정 하였을 때, 약물결정이 함께 측정되어 분포 편차가 큰 것도 알 수 있었다. 0.25 % SDS가 녹아있는 인산완충용액을 사용하여 다공성, 비다공성 미립구의 In vitro release를 진행하였다. 이 과정에서 비다공성 미립구의 경우, 약물 방출이 일어나기 전의 lag time이 있을 뿐 아니라, 자동 촉매 작용으로 인해 폴리머의 분해속도가 일정하지 않음을 확인할 수 있었다. TGA(Thermogravimetric Analysis)와 DSC(Differential scanning calorimetry)를 이용하여 미립구 내 잔류유기용매와 열적거동양상도 확인하였으며, 미립구에 봉입된 프로게스테론이 어떤 물리적 성상을 띠는지를 규명하였다. 미립구의 비다공성 특성으로 인해 봉입과정에서 사용되는 유기용매가 높은 수준으로 잔류함으로써 특수 건조 기술이 필요하였다. 이러한 모든 한계는 암모니아 분해반응을 사용하여 해결될 것으로 예상된다. 암모니아 분해기반 PLGA 미립구를 제조하기 위하여, 프로게스테론을 고분자와 함께 IPF(Isopropyl formate)에 녹여 PVA(Polyvinyl alcohol)용액에 분산시킨 후 암모니아를 첨가시켰다. 결과적으로 IPF는 친수성물질로 바뀌면서 물에 녹을 수 있게 되었고, 이 과정에서 에멀젼은 빠르게 경화되었다. 위 과정에서 제조된 미립구는 84.69 에서 96.03%의 높은 봉입률을 가졌다. 본 연구결과를 종합하여 볼 때, PLGA 미립구에 소수성 약물을 봉입할 경우, 종래의 에멀젼 기반 용매 증발법으로 제조된 미립구와 비교하여볼 때, 암모니아 분해반응을 이용한 미립구 제조기법은 기존의 방법들을 대체할 수 있는 가능성을 지닌 것으로 보인다.