Pharmaceutical Potential of Gelatin as a pH-responsive Porogen for Manufacturing Porous Poly(d,l-lactic-co-glycolic acid) Microspheres

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Hyunuk Kim

¹

, Hongil Park

¹

, Ju-Ho Lee

¹

, Eun Seong Lee

²

, Kyung Taek Oh

³

, Jeong-Hyun Yoon

¹

, Eun-Seok Park

⁴

, Kang Choon Lee

⁴

and Yu Seok Youn

^1†

1College of Pharmacy, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea

2Division of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea

3College of Pharmacy, Chung-Ang University, 221 Heukseok dong, Dongjak-gu, Seoul 155-756, Republic of Korea

4College of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Republic of Korea

(Received July 13, 2010·Revised July 30, 2010·Accepted July 31, 2010)

ABSTRACT−Porous poly(lactic-co-glycolic acid) microspheres (PLGA MS) have been utilized as an inhalation delivery system and a matrix scaffold system for tissue engineering. Here, gelatin (type A) is introduced as an extractable pH-responsive porogen, which is capable of controlling the porosity and pore size of PLGA microspheres. Porous PLGA microspheres were prepared by a water-in-oil-in-water (w1/o/w2) double emulsification/solvent evaporation method. The surface morphology of these microspheres was examined by varying pH (2.0~11.0) of water phases, using scanning electron microscopy (SEM). Also, their porosity and pore size were monitored by altering acidification time (1~5 h) using a phosphoric acid solution. Results showed that the pore-forming capability of gelatin was optimized at pH 5.0, and that the surface pore-formation was not significantly observed at pHs of < 4.0 or > 8.0. This was attributable to the balance between gel-formation by electrostatic repulsion and dissolution of gelatin. The appropriate time-selection between PLGA hardening and gelatin-washing out was considered as a second significant factor to control the porosity. Delaying the acidification time to ~5 h after emulsification was clearly effective to make pores in the microspheres. This finding suggests that the porosity and pore size of porous microspheres using gelatin can be significantly controlled depending on water phase pH and gelatin-removal time.

The results obtained in this study would provide valuable pharmaceutical information to prepare porous PLGA MS, which is required to control the porosity.

Key words−porous microspheres, gelatin, PLGA, porogen, pH-responsive

Poly(lactic-co-glycolic acid) (PLGA) microspheres have been utilized to deliver peptides, proteins or genes (Cohen et al., 1991). This PLGA matrix system provides satisfactory sustained effect and acceptable safety owing to the biocompat- ibility and biodegradability (Gombotz et al., 1995). In general, plain PLGA microspheres are spherical, dense, compact particles with smooth surface without significant pores. Recently, highly porous PLGA microspheres have been developed as an effective inhalation tool or polymeric three-dimensional scaf- folds for tissue engineering. First, porous microparticles with a low mass density of < 0.4 g/cm³are considered to be quite suit- able for inhalation because they are very light due to high porosity (Edwards et al., 1997). Therefore, these particles are likely to deposit onto the deep alveoli regions through airways after inhalation. Furthermore, the size-increase of porous microspheres to > 5^µm can escape severe phagocytosis by

lung macrophages (Edwards et al., 1998; Youn et al., 2009).

Second, porous microspheres can be an effective matrix scaf- folds appropriate for culturing various cells, and thus these porous microspheres on which cells are cultured can play as an in vitro tissue model (Kang et al., 2009; Kim et al., 2006).

Likewise, in addition to the original use of plain microspheres, highly porous microspheres can be utilized as various pharmaceutical purposes.

Many techniques have been developed to make highly porous PLGA microspheres. Most methods use unique pore- forming agents, which are called as porogens. These include osmosis-inducing agents, extractable porogens, and gas-foaming agents. Osmogens (salts or cyclodextrins etc.) create pores due to the osmotic pressure differences between the internal and external phases (Kwon et al., 2007; Lee et al., 2007;

Ungaro et al., 2006). Extractable porogens (Pluronics or fatty acid salts etc.) make pores using time difference between PLGA hardening and porogen-withdrawal from oil-phase to water phase (Chung et al., 2006; Kim et al., 2006; Sun et al., 2009). Finally, gas-foaming (effervescent) agents (ammonium

†Corresponding Author :

Tel : +82-51-510-2800, E-mail : ysyoun@pusan.ac.kr DOI : 10.4333/KPS.2010.40.4.245

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246 Hyunuk Kim et al.

bicarbonate or hydrogen peroxide etc.) form many gas bubbles to make surface pores (Kim et al., 2006; Yang et al., 2009).

Although quite many methods and materials for this pore- forming purpose are already used, most methods just make pores and can’t control the porosity itself, irrespective of load- ing amount. For this reason, gelatin was newly utilized as a porosity-controlling agent in this study. Gelatin is a well- known biocompatible protein and has non-toxic and safe fea- ture to human body. This modified protein has many different types due to origin source, pI values, and molecular weights.

Especially, its thixotropic property of sol-to-gel transition can be altered by pH control due to its pI value (Young et al., 2005). Here, porous PLGA microspheres were prepared using gelatin (type A) as a pH-responsive porogen. Their mor- phologies on the basis of the pH of water phase and gelatin- removal time were investigated by scanning electron microscopy (SEM).

Experimetal

Materials

Poly(d, l-lactic-co-glycolic acid) (PLGA) (lactic acid: glycolic acid = 50:50; Mw: approx. 10,000 Da) was purchased from Wako Pure Chemical Industries (Tokyo, Japan). Poly- vinyl alcohol (PVA, Mw: 30,000~70,000 Da) and gelatin (type A) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methylene chloride and all other reagents, unless oth- erwise specified, were obtained from Sigma-Aldrich.

Preparation of Porous PLGA Microspheres

Porous PLGA microspheres (MS) were prepared by a slight modification of a previously described procedure, a w1/ o/w2

double emulsification/solvent evaporation method using gelatin (type A) as an extractable porogen (Park et al., 2009).

Briefly, the water parts (w1) of dispersed phase were a variety of 1 mL buffer solutions with different pH values of 2.0~11.0 containing 100 mg gelatin, and the solutions were heated at 60^oC if required. PLGA (150 mg) was dissolved in 3 mL dichloromethane, poured into the w1 solutions, mixed thor- oughly, and then sonicated in ice bath using a Sonics Vibra- Cell Ultrasonic Processor (Sonics & Materials Inc. Newtown, CT, USA) for 30 s at an amplitude of 15%. The primary emul- sions prepared were added to the continuous phase (w2) of a 0.5% PVA solutions, which were pre-adjusted to the same pHs as their respective w1 solutions, and then emulsified at 3,000 rpm and room temperature using a Silverson Laboratory Mixer (model L4RT) with a 3/4-inch head (Silverson Machines, Inc.

East Longmeadow, MA, USA) for 2 min. The resultant emul-

sion solutions were further allowed to evaporate the solvent under gentle magnetic stirring under an air current at 40^oC for 1~5 h. Finally, a portion (1 mL) of phosphoric acid was added to each emulsion at predetermined time points. The resultant microspheres were centrifuged at 700 rpm, washed 3 times with deionized water, and freeze-dried overnight.

Morphology of Porous Microspheres Using Scanning Electron Microscopy (SEM)

The surface morphology of porous MS was examined by scanning electron microscopy (SEM, Hitachi S3500N, Japan). Dry powder of PLGA microspheres was attached to specimen stubs using double-side tape and sputter-coated with gold-palladium in an argon atmosphere using a Hummer I sputter coater (Anatech Ltd. St. Alexandria, VA, USA). The surface morphology of porous microspheres was observed by SEM.

Results and Discussion

Preparation of Porous PLGA Microspheres Using a Gelatin Porogen

Porous PLGA microspheres were prepared using a double w/o/w emulsification/solvent evaporation method using a gelatin porogen, varying the pH of dispersed- and continuous- phases (Figure 1). Gelatin is apt to swell and absorb 5~10 times its weight of water to form a gel below 35~40ôC, and thus it was temporarily heated at 60ôC in order to become soft and to get a sol property. It was then cooled down to around 35ôC before being mixed with a PLGA solution. In an attempt to briefly check the pore-forming capability of gelatin on the basis of pH, the pH of water phase was determined to be 2.0, 5.0, 8.0, and 11.0, considering the gelatin pI value (7.0~9.0).

As shown in Figure 2A, the microspheres prepared at pH 2.0 were found to have no significant pore on its surface. On the contrary, the microspheres prepared at pH 5.0 were shown to have a spherical shape and clearly notable pore-formation on its surface, and moreover its particle size seemed to be around 5~20 ^µm, which is appropriate for inhalation (Figure 2B). At pH 8.0, the microspheres had fairly many pores, but its shape seemed to be not spherical and distorted (Figure 2C). Also, as seen in Figure 2D, the microspheres prepared at a strong basic pH of 11.0 did not show any pores and spherical surface morphology. This fact is attributable to the gel formation ability of gelatin based on its pI value (7.0~9.0). At a strong acidic pH, gelatin was definitely dissolved as if a sol and did not have enough time to make pores before PLGA hardening. The sim- ilar situation seemed to occur at a strong basic pH condition,

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and moreover the alkalinity was likely to severely hydrolyze the PLGA polymer backbone and to adhere to one another. By contrast, gelatin tends to induce favorable gel formation at pH

5.0, partly due to the electrostatic repulsion, and thus gelatin seemed to have enough time to place on the surface of PLGA microspheres before PLGA solidification.

Figure 1. Preparation scheme of porous PLGA microspheres using gelatin as an extractable porogen.

Figure 2. SEM morphology of porous PLGA MS prepared under different pH conditions: A - pH 2.0, B - pH 5.0, C - pH 8.0, and D - pH 11.0.

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Optimization of Porous PLGA Microspheres Depending on pH of Water Phase

Porous PLGA MS was prepared as the same method described above, but the pH conditions of water phase were set to be acidic to neutral. As shown in Figure 3 and Table I, the surface morphology of microspheres was quite different according to the pH of water phase, although other preparation or formulation factor, i.e. volumes of each phase, gelatin load- ing amount, and hardening temperature etc., were constant. For the careful investigation, one of respective microspheres was focused on by SEM. Increasing the water phase pH from 2.0 to 5.0 resulted in the increased pore formation and pore size of PLGA microspheres. At pH 2.0~4.0, significant pore formation was not observed, and their surfaces were smooth. The microspheres prepared at pH 6.0 did not show acceptable pore formation, and those at pH 7.0 did not have overall spherical shape, although some pores on them. Contrary to such obser- vations, however, the microspheres prepared at pH 5.0 were found to have considerable pores with relatively large size.

Especially, as shown in Figure 3D, the particle size of this batch of such microspheres was really small, ~10 ^µm. From such findings, gelatin did not possess enough gel formation capability due to electrostatic repulsion at pHs < 4.0 or > 8.0.

Especially, gelatin seems to be quite well dissolved and leached out from the microspheres below pH below 4.0. Con- sequently, the PLGA porous microspheres prepared at pH 5.0 was considered to be optimal in terms of porosity, surface shape, and particle size.

Porosity Control of Gelatin PLGA Microspheres by Acidification Time

The surface morphology of PLGA microspheres prepared using a gelatin porogen at pH 5.0 was investigated as a func- tion of acidification time. Gelatin is freely soluble in a low pH derived by some acids, and thus phosphoric acid was added to

the microspheres to wash out the residual gelatin at predetermined times (1~5 h). As shown in Figure 4A, the microspheres acidified with phosphoric acid immediately after emulsification was found to have no pores on their surface or inner network. However, the microspheres acidified at 1 h after emulsification showed the traces of surface pores, albeit not much. The acidification at 2 h or 3 h after emulsification resulted in significant surface pore formation, and this formation reached the maximum at 4~5 h. This finding shows that the removal time of gelatin from the PLGA microspheres is a critical factor to control its porosity because gelatin gel

Fgure 3. Surface morphology of focused porous PLGA MS prepared under different pH conditions: A - pH 2.0, B - pH 3.0, C - pH 4.0, D - pH 5.0, E - pH 6.0, and F - pH 7.0.

Table I. Formulation and Preparation Condition of Porous PLGA Microspheres Polymer

(Da, amount) Porogen

(amount) w1 phase (volume) Disperse phase

(o) (volume) Continuous (w2)

phase (volume) Acidification time point

after preparation (h) Hardening time/

temperature PLGA 5010

(~10,000, 150 mg)

Gelatin type A

(100 mg) pH 2.0 acetate buffer (1 mL) Methylene chloride

(3 mL)

0.5% PVA (100 mL) adjusted to

the same pH as w1

phase

0, 1, 2, 3,

4, 5 ~5 h/40^oC

pH 3.0 acetate buffer (1 mL) pH 4.0 acetate buffer (1 mL) pH 5.0 acetate buffer (1 mL) pH 6.0 phosphate buffer (1 mL) pH 7.0 phosphate buffer (1 mL) pH 8.0 phosphate buffer (1 mL) pH 11.0 NaOH (1 mL)

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formed competes with PLGA frame in terms of space occu- pancy inside the microspheres. The gelatin removal by acidification is likely to give no room to pore formation before PLGA hardening, whereas the presence of gelatin gel occupies considerable room to convert to pores until PLGA hardening.

Conclusion

In summary, gelatin displayed great potential for the pore- forming capability, and the porosity and pore size were significantly controlled by altering the pH of both water phases and acidification time. The presence of gelatin gel formed inside the microspheres directly influences the surface pore- formation. Furthermore, the porogen-ability of gelatin was maximized at pH 5.0, due to the optimal balance between the gel-formation by electrostatic repulsion and acidity-based dissolution. Also, the appropriate time-selection between PLGA hardening and gelatin-washing out is considered as a critical factor to control the porosity. This finding would give mean- ingful pharmaceutical information to prepare porous PLGA MS, which is required to control the porosity.

Acknowledgments

This work was supported by the National Research Foun- dation of Korea (NRF) by the Korea government (MEST) (no.

2009-0083962), and by a grant of the Korean Health Tech- nology R&D Project, Ministry for Health, Welfare & Family

Affairs (A092018).

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