Supercritical gas foaming using CO₂ was used to fabricate blank poly DL lactide-co-glycolide (PLGA) micro-porous foams. Paclitaxel-loaded PLGA foams were also produced for the first time using a modification of the supercritical gas foaming technique whereby pacltitaxel-loaded PLGA microparticle powders obtained from spray drying was foamed. In this study, it was found that using polymer powders, more compact foams and smaller pores foams may be achieved with lower saturation pressures and time which is due to the much higher surface area to volume ratio of the microparticle powders. Experiments were carried out with varying lactide to glycolide ratio of the copolymer PLGA and it was shown that the pore size, in vitro swelling behavior and drug release profiles may be altered by changing the copolymer composition used. The foams fabricated also have good mechanical strength which makes it suitable to be applied as an implantation material for the post-surgical controlled delivery of chemotherapeutic drugs. The residual organic solvent content of the paclitaxel-foams were well below the allowable limit set by the US Pharmacopeia as shown in the present study. The in vitro release profiles over a period of 5 weeks showed close to linear release.Singapore-MIT Alliance (SMA

Lee, Lai Yeng

Smith, Kenneth A.

Wang, Chi Hwa

DSpace@MIT

       Abstract Supercritical gas foaming using CO2 was used to fabricate blank poly DL lactide-co-glycolide (PLGA) micro-porous foams. Paclitaxel-loaded PLGA foams were also produced for the first time using a modification of the supercritical gas foaming technique whereby pacltitaxel-loaded PLGA microparticle powders obtained from spray drying was foamed. In this study, it was found that using polymer powders, more compact foams and smaller pores foams may be achieved with lower saturation pressures and time which is due to the much higher surface area to volume ratio of the microparticle powders. Experiments were carried out with varying lactide to glycolide ratio of the copolymer PLGA and it was shown that the pore size, in vitro swelling behavior and drug release profiles may be altered by changing the copolymer composition used. The foams fabricated also have good mechanical strength which makes it suitable to be applied as an implantation material for the post-surgical controlled delivery of chemotherapeutic drugs. The residual organic solvent content of the paclitaxel-foams were well below the allowable limit set by the US Pharmacopeia as shown in the present study. The in vitro release profiles over a period of 5 weeks showed close to linear release.   Index Terms - Keywords: Supercritical gas foaming technique, Poly DL lactide-co-glycolide (PLGA), Micro-porous foams, Controlled release, Paclitaxel  I. INTRODUCTION   icro-porous polymer foams have important applications in tissue engineering as scaffolds [1-3] drug delivery systems [4-5]. PLGA is one of the most popular biodegradable polymers used in porous foam fabrication as it is biocompatible and FDA approved [1,3-5]. The degradation rate of the foam may also be controlled by careful selection of the copolymer composition and   molecular weight [3]. Conventional methods of micro-porous foam formation such as solvent-casting, particulate leaching technique [3] are usually associated with the use of large amounts of organic solvents which may require extensive purification steps to remove the residual solvent. Using supercritical CO2 as the foaming agent, the use of organic solvent may be minimized or even eliminated in the production of PLGA foams as illustrated in previous studies [5].  The mechanism of supercritical gas foaming technique is as follows [1]: Under high pressure CO2, the glass transitin temperature (Tg) of PLGA is depressed and a polymer melt may be formed at lower temperatures with large amounts of dissolved CO2. Upon rapid depressurization, thermodynamic instability takes place and the CO2 molecules cluster and diffuse to form nuclei in the polymer matrix. As CO2 leaves the polymer and pressure is lowered, the Tg of polymer increases to above the operating temperature and vitrifies to form a solid polymeric foam The micro-porous foams can potentially be used in the post-surgical treatment of cancer cells by controlled release of chemotherapeutic agent. After a surgical removal of a tumor, not all the tumor cells will be removed. A controlled release device may be implanted immediately after the surgery to target the leftover cells to prevent proliferation of the leftover cells [6].  Paclitaxel is a promising chemotherapeutic agent against a wide spectrum of carcinomas [6-12]. It is a highly hydrophobic molecule with very low aqueous solubility. However, it was found to be associated with problems of extremely slow release when encapsulated in compressed discs [12]. In the present study, fabrication and characterization of blank and paclitaxel-loaded micro-porous foams as implantation material for post-surgical chemotherapy treatment of gliomas as well as other forms of carcinomas Micro-porous Paclitaxel-loaded PLGA foams - a new implant material for controlled release of chemotherapeutic agents  Lai Yeng Leea,b, Chi Hwa Wanga,b, Kenneth A. Smitha,c  aMolecular Engineering of Biological and Chemical Systems (MEBCS), Singapore-MIT Alliance, Singapore 117576 bDepartment of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117576 cDepartment of Chemical Engineering, Massachusetts Institute of Technology, Cambridge MA 02139  M    were studied and the operating conditions were optimized to achieve foams with good mechanical strength and controllable pore sizes.   II. EXPERIMENTAL A. Materials Polymer Poly DL lactide-co-glycolide (PLGA) for a range of copolymer composition was used in this study. PLGA 5050 Low IV was purchased from Lakeshore Biomaterials (Birmingham, AL). PLGA 5050 (Cat No. P2191 MW = 40,000 – 75,000 Da), PLGA 6535 (Cat No. 2066 MW = 40,000 – 75,000 Da), PLGA 7525 (Cat No. P1941 MW = 66,000 – 107,000 Da) and PLGA 8515 (Cat No. 430,471 MW = 50,000 – 75,000 Da) were purchased from Sigma Aldrich (St Louis, MO, USA). Phosphate Buffered Saline (PBS, pH = 7.4) was purchased from Sigma Aldrich (St Louis, MO, USA). Paclitaxel was a generous gift from Bristol Myers Squibb. Compressed CO2 (Airliquide Paris, France) was purchased from Soxal (Singapore Oxygen Air Liquide Pte Ltd). Dichloromethane (DCM) (DS1432, HPLC/Spectro Grade) and acetonitrile (ACN) (AS1122, HPLC/Spectro Grade) were purchased from Tedia (Farfield, OH, USA). Millipore water (Millipore Corporation, Billerica, MA) was used throughout the study.  B. Experimental Setup and foam fabrication A custom-made mold with height and diameters of 10mm was machined from a ½” MNPT plug as shown in Figure 1. For each experiment, approximately 100 mg of polymer was loaded into the mold and sealed with a porous stainless steel wire mesh. The mold was then fitted onto the high pressure vessel and placed in a circulating water bath maintained at 35 oC. Saturation pressures from 8 – 10MPa and saturation times of 1 – 4 hrs was used in this study. The depressurization step was maintained consistently for all the experiments at 1/5 opening of the needle valve of the automatic back pressure regulator. C. Spray Drying of paclitaxel-loaded microparticles Paclitaxel-loaded PLGA powders were fabricated using a spray drying technique (Buchi Mini Spray Dryer) using as a solvent for paclitaxel and PLGA. For all the spray drying experiments, 2w/v% polymer solution, airflow rate of 800NL/min at 70oC, 100% aspirator ratio and 20% liquid feed rate was used.  D. Characterization and in vitro studies Scanning Electron Microscopy (SEM, JEOL JSM-5600 LV, Japan) was used to study the porous structure and surface morphology of the polymeric foams fabricated in this study.  Differential Scanning Calorimetry (DSC, 2920 modulated, Universal V2.6D TA instruments) was carried out to determine the glass transition temperatures  of the PLGA foams Approximately 2-10 mg of particles was loaded onto standard aluminum pans (40mg) with lids. The samples were purged with pure dry nitrogen at a flow rate of 20 ml/min. A blank aluminum pan was used as a reference in all of the analyses. The analysis was carried out using a temperature ramp of 10 oC/min from 20 – 200 oC for blank PLGA foams and from 20 – 250 oC for paclitaxel-loaded PLGA foams.  Gas Chromatography with Mass Spectrometer detector (GC-MSD) was used to determine the residual DCM content of the foams. High pressure liquid chromatography (HPLC with UV-visible detectors, Shimadzu) was used to determine the encapsulation efficiencies and in vitro release profile of the paclitaxel-loaded particles. Discs of geometry 3mm diameter by 1mm height were punched from the paclitaxel-loaded foams and placed in PBS solution in a shaking water bath at 37 oC and 120 rpm. At predetermined time intervals, PBS solution was removed for sampling and fresh buffer was replaced. An ACN: water (50:50 v/v) solution was used as the mobile phase for HPLC analysis. FIGURE 1. Experimental Setup for supercritical gas foaming technique  VentCO2 P1V1V2C1C210mm10mm10mm diameter x 10mm height moldHPC1: Refrigerating Circulator  C2: Circulating water bath  P1: High Pressure Liquid Pump  HP: High Pressure Vessel V1: On/Off Ball Valve  V2: Automatic Back Pressure Regulator    III. RESULTS AND DISCUSSION A. Micro-porous PLGA foams Figure 2 shows the interior pore structure of blank PLGA foams with different lactide to glycolide content. A saturation pressure of 12MPa and saturation time of 4hrs was used to generate the foams. From Figure 2, it was observed that as the lactide content in the foams increase, the mean pore size decrease and which could be explained by the increased crystallinity of the polymer. It was also observed that PLGA 5050 yield open cell structures with interconnecting pores whereas PLGA 6535, PLGA 7525 and PLGA 8515 generally produced mostly closed cell structures.  The Tg of the foams were studied and Figure 3a and 3b shows the exotherm plot of the 4 different copolymers of PLGA before and after foaming respectively. The Tg of PLGA generally increases as lactide content increases. Comparing the Tg of PLGA before and after foaming, for all the 4 different copolymers, the Tg increases after foaming. This is due to the rapid solidification of the polymer during the depressurization step of the foaming process. The in vitro swelling behavior of the 4 PLGA copolymers in PBS maintained in physiological conditions was studied. As shown in Figure 4, PLGA 5050 swelled by approximately 15% in the first 3 weeks. As the glycolide content in PLGA increase, its hydrophillicity increase. Therefore, PLGA 5050 is the most hydrophobic polymer which attributed to its most pronounced swelling.  PLGA 6535 and PLGA 8515 showed little changes in volume. PLGA 7525 is observed to shrink and this may be due to the higher molecular weight (MW 66,000 – 107,000 Da) of the polymer as compared to the PLGA 5050, PLGA 6535 and PLGA 8515 used.    A: PLGA 5050B: PLGA 6535C: PLGA 7525D: PLGA 8515BC DA13.2847.49D11.8649.45C15.1954.05B18.2872.54AS.D (μm)Mean (μm)FIGURE 2. SEM observation of the interior pore structure of various blank PLGA foams obtained from polymer pellet (as obtained from Sigma Aldrich) using a saturation pressure of 12MPa and saturation time of 4hrs  -10-8-6-4-200 50 100 150 200Temperature (deg Celcius)Relative Exotherm (mW)PLGA 50:50PLGA 63:35PLGA 75:25PLGA 85:1552.0951.6547.6646.13MidPoint52.5752.2348.5148.08OnsetPLGA 85:15PLGA 75:25PLGA 65:35PLGA 50:50Tg (oC)-10-9-8-7-6-5-4-3-2-100 50 100 150 200Temperature (deg Celcius)Relative Exotherm (mW)PLGA 50:50PLGA 65:35PLGA 75:25PLGA 85:1548.7242.5542.2337.34MidPoint48.4642.1842.3736.50OnsetPLGA 85:15PLGA 75:25PLGA 65:35PLGA 50:50Tg (oC)FIGURE 3a. Glass transition temperatures of PLGA 5050, PLGA 6535, PLGA 7525 and PLGA 8515 before foaming FIGURE 3b. Glass transition temperatures of PLGA 5050, PLGA 6535, PLGA 7525 and PLGA 8515 after foaming  -0.3-0.2-0.100.10.20.30 5 10 15 20 25Time (days)Deviation from original diameterPLGA 5050PLGA 6535PLGA 7525PLGA 8515FIGURE 4. In vitro swelling behavior of PLGA foams    Hom ogeneous so lution of PLGA and paclitaxel in D ichloromethanePaclitaxel-loaded PLGA powders fabricated using spray drying m ethodPaclitaxel-loaded PLGA foams fabricated using supercritical gas foam ingFIGURE 5. . 2-Step Fabrication process for paclitaxel-loaded PLGA foams using combined spray-drying and supercritical gas foaming technique  B. Paclitaxel-loaded PLGA foams Paclitaxel-loaded PLGA foams were fabricated using a two-step process of spray drying followed by supercritical gas foaming of the microparticles as illustrated in figure 5. The purpose of the spray drying step was to achieve a molecular dispersion of paclitaxel in PLGA in the final foam product. The drug-loaded formulations studied were summarized in table 1 and the 5% drug loaded microparticle powders achieved from spray drying and the interior pore structures of 5% drug loaded foams are illustrated in figure 6a and 6b respectively. Figure 6a shows the spray dried powders with diameters ranging from 1 – 10 μm. From figure 6b, PLGA 5050 yield open cell structures and PLGA 8515 yield closed cell structures which are consistent with the results obtained for blank PLGA foams. The encapsulation efficiencies of the paclitaxel-loaded microparticles and foams were studied using HPLC and The encapsulation efficiency of paclitaxel from the spray dried powders to the final foamed product was determined as:                       -- (1)  From table 2, the drug encapsulation using the supercritical gas foaming technique has very high efficiency between 90 – 100%. High encapsulation efficiency is a very important factor in most pharmaceutical processes as the active ingredient is normally very rare and expensive.   To determine whether paclitaxel encapsulated was molecularly dispersed in the polymer matrix within the foam [13-14], a thermogram analysis was carried out on the 10% paclitaxel-loaded powders and foams as shown in figure 6. Crystalline paclitaxel has an endothermic melting peak at 223 oC [15] and from figure 7, no paclitaxel melting peak was observed for both the powders and foams. This showed that no crystalline paclitaxel was present in both formulations and the drug was molecularly dispersed in the PLGA polymer matrix.   Figure 8 shows the release profile of paclitaxel from the 5% formulations. The release from 5% drug-loaded PLGA 5050 (Low IV) has the fastest release rate which can be explained by its high affinity for water. Using a more hydrophobic polymer PLGA 8515, a much slower release rate was achieved. This showed that by selection of the copolymer used, the in vitro release of drug from the foams may be altered.    As DCM was used as a solvent during the spray drying step, it is important to ensure that the residual DCM content in the foams was low enough within the limits of 500ppm as stated by the US Pharmacopeia.  From Figure 9 it was shown that the spray dried microparticles has a high residual DCM content of approximately 300ppm. After freeze drying, the DCM content reduced by approximately 100ppm. Foaming with supercritical CO2 removed approximately 150 ppm of residual DCM from the powders which shows the supercritical CO2 removes DCM more effectively in much shorter times as compared to freeze drying.   TABLE  1. Composition of paclitaxel-loaded foams fabricated Sample Polymer Drug loading (%) A*-5% PLGA 5050 (Low IV) 5 D-5% PLGA 8515 (Sigma) 5 A-10% PLGA 5050 (Sigma) 10  TABLE 2. Encapsulation efficiencies of paclitaxel loaded powders and foams  IV. CONCLUSION Paclitaxel-loaded PLGA foams were fabricated for controlled delivery applications. These foams provide an alternative to conventional methods of making implants Sample A*-5% A-10% D-5% Drug loading in powders (mg/mg) 0.0478 0.1072 0.0490 Drug loading in foam (mg/mg) 0.0442 0.1072 0.0487 Encapsulation efficiency (%) 92.6 ~100 99.4 ( ) ( )( )% 100%mgdrug loading in foam mgEncapsulation efficiencymgdrug loading in powders mg= ×   using compression discs. Micro-porous foams have higher  surface-to-volume ratios, allowing more efficient drug release. The release profiles for the micro-porous foams fabricated as 3mm discs showed continuous and relatively linear release profile up to 5 weeks in vitro. The foams are mechanically strong enough to be used as an implant in surgical experiments. In addition, by altering the co-polymer ratio of the PLGA used, the release rate of paclitaxel may be modified to address therapeutic requirements.  ACKNOWLEDGMENT Authors would like to thank Singapore-MIT Alliance (C382-427-003-091) and the Agency for Science, Technology and Research (R279-000-201-305) for their financial support in this project. Authors would also like to thank Jingwei Xie, Hemin Nie and Benjamin, Yung Sheng Ong for their useful discussion and technical help rendered in this research.   REFERENCES [1] L. Singh, V. Kumar and B.D. Ratner, “Generation of porous micro-cellular 85/15 poly (DL-lactide-co-glycolide) foams for biomedical applications,”  Biomaterials, v25, pp. 2611–2617, 2004 FIGURE 9. Residual DCM content in paclitaxel-loaded powders and foams measured using GC-MSD -5-4.5-4-3.5-3-2.5-2-1.5-1-0.500 50 100 150 200 250Temperature (deg Celcius)Relative Exotherm (mW)Spray Dried Powders (10% drug loading)PLGA 50:50 Foams (10% drug loading)PLGA 50:50 Pellets0100200300400500600Powder Powder wFreeze DryFoam Foam wFreeze DryResidual DCM content (ppm)Maximum Allowable Residual DCM content051015202530350 10 20 30 40Time (Days)% Cumulative release PLGA 50:50 (A*-5%)PLGA 85:15 (D-5%)FIGURE 7. In vitro release of paclitaxel from PLGA 5050 (Low IV) and PLGA 8515 (Sigma Aldrich) FIGURE 8. Thermogram properties of 10% paclitaxel-loaded PLGA 5050 powders and foams and blank PLGA before foaming FIGURE 6b. SEM observation of the interior pore structure of 5% paclitaxel-loaded PLGA foams obtained powders using a saturation pressure of 8MPa and saturation time of 1hr  A*-5% D-5%A*-5% D-5%FIGURE 6a. 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Micro-porous Paclitaxel-Loaded PLGA Foams -- a New Implant Material for Controlled Release of Chemotherapeutic Agents

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Micro-porous Paclitaxel-Loaded PLGA Foams -- a New Implant Material for Controlled Release of Chemotherapeutic Agents

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