130 Chapter CHAPTER PLGA/Chitosan Composites from a Combination of Spray Drying and Supercritical Fluid Foaming Techniques: New carriers for DNA delivery † 7.1 Introduction In recent decades gene delivery research has grown very rapidly due to its huge potential as a future therapeutic strategy for clinical applications. Non-viral delivery device constructed from biodegradable polymer is preferred considering the associated safety issues. Among the various non-viral dosage forms employed in gene delivery, microspheres and nanoparticles are widely used because of their uniform morphology and high transfection of cells. However, there has been growing interest recently in the use of porous materials since they offer several attractive features, such as stable and uniform porous structures, tunable pore size and well defined surface properties (Thomson et al., 1996; Chen et al., 2001; Torres et al., 2007; Song et al., 2005). These desired properties allow the encapsulation or adsorption of particular gene and releasing it in a more reproducible and predictable manner. Moreover, porous structures can support cells’ attachment and hold/release DNA to induce the formation of new tissue. † This chapter highlights the work published in H. Nie, S.T. Khew, L.Y. Lee, K.L. Poh, Y.W. Tong and C.H. Wang. PLGA/Chitosan Composites from a Combination of Spray Drying and Supercritical Fluid Foaming Techniques: New Carriers for Gene Delivery. J. Control. Release 129, 207-214. 2008. Chapter 131 Consequently, it will degrade gradually and give 3-dimensional structural guide in the formation and regeneration of tissue (Goldstein et al., 2001). Therefore, combining the concept of gene delivery and tissue engineering, porous foam has an obvious advantage over microspheres and nanoparticles. The potential of chitosan as a polycationic gene carrier has been explored widely in recent years (Roy et al., 1999; Leong et al., 1998; MacLaughlin et al., 1998; Mao et al., 2001; Roy et al., 1997; Mao et al., 1996; Richardson et al., 1999). Chitosan is a biodegradable polysaccharide (Onishi and Machida, 1999) extracted from crustaceans and has been shown to be non-toxic in animals (Rao and Sharma, 1997) and humans (Aspden et al., 1997). Due to its good biocompatibility and cytotoxicity performance, it has been widely used in pharmaceutical research and in industry as a carrier for drug delivery. Previously, it was used as an encapsulation shell in most cases. In contrast, in the present work, its polycationic property will be used as an additive for controlling DNA release and adhering cells. On the other hand, supercritical CO2 technique is a versatile foaming tool to produce micro-porous structures with particular pore sizes and shapes. Here, the morphology and porosity of foams produced by supercritical CO2 foaming can be easily altered by changing operation parameters like pressure, temperature and gas release rate (Mikos and Temenoff, 2000; Kim et al., 2006). In this study, the surface physical and chemical properties of pure PLGA and PLGA/chitosan porous foams fabricated by supercritical CO2 foaming were investigated by SEM, XRD, FTIR, and XPS. A model DNA plasmid, Chapter 132 encoding luciferase, was encapsulated into the foams and the in vitro release in phosphate-buffered saline (PBS) medium was performed. Furthermore, the cell adhesion, cytotoxicity and in vitro gene expression in different composition of foams were investigated. 7.2 Materials and methods 7.2.1 Materials Poly (D,L lactic-co-glycolic acid) (PLGA) containing a free carboxyl end group (uncapped) with L/G molar ratio of 50:50 (PLGA 4A, MW=63k, IV=0.44) was purchased from Lakeshore Biomaterials (Cat. W3066-603, AL, US). Chitosan (medium molecular weight and 75-85% deacetylated), Phosphate-buffered saline (PBS) buffer containing 0.1 M sodium phosphate and 0.15 M sodium chloride, pH 7.4, (used for in vitro study) were purchased from Sigma Aldrich (St. Louis, MO, US). Dichloromethane (DCM) (Cat. No. DR-0440) was purchased from Tedia Company Inc. (Fairfield, OH, US.). PreMix WST-1 cell proliferation assay system, PicoGreen dsDNA quantitation kit and Steady-GloTM luciferase assay system were purchased from Takara Bio Inc. (Otsu, Shiga, Japan), Invitrogen (Carlsbad, CA, US) and Promega (Madison, WI, US), respectively. 7.2.2 Plasmid preparation and loading procedure The pIRES2-EGFP-hRluc vector expressing both enhanced green fluorescence protein (EGFP) and Renilla luciferase reporter (hRluc) was constructed by grafting hRluc sequence from a commercial pGL4.75[hRluc/CMV] vector (Promega, Madison, WI) onto the backbone of a commercial pIRES2-EGFP vector with CMV promoter (BD Chapter 133 Biosciences, San Jose, CA). The engineered plasmid was amplified in a transformant of Escherichia coli and isolated from the bacteria by PureLinkTM HiPure plasmid DNA purification kit-Maxiprep K2100-07 (Invitrogen Corporation, US). PLGA microparticles encapsulated with plasmid DNA were fabricated by a spray drying method. A 10% wt/vol PLGA polymer solution using DCM as the solvent was prepared by dissolving 1g PLGA into 10 mL of DCM. The resultant mixture was agitated by applying vortex until a clear and homogeneous organic phase was formed. Meanwhile a specified amount of plasmid was dissolved in DI water to form aqueous phase. After adding the aqueous and organic phases together, the mixture was sonicated for about 10 seconds and the resultant emulsion was transferred to a Buchi 191 Mini Spray Drier (Flawil, Switzerland). The temperature and air flow rate for spray drier were set to 70 °C and 700 L/h. PLGA foams were obtained by a gas foaming method using supercritical CO2 as the blowing agent. A mold (interior dimensions 10mm diameter by 10mm height) were designed, and Teflon gaskets and aluminum tape was used to seal the mold during the foaming process. The experimental setup is shown in Figure 7.1. Specified weight of PLGA microparticles loaded with plasmid from the spray drier was first loaded into the mold and sealed with aluminum tape. Several small holes were made on the top of the mold to allow CO2 to equilibrate with the PLGA during the foaming process. Pressure of approximately 120 bars was used for all the experiments and the equilibrating time was 134 Chapter hours. At the end of the experiment, the CO2 pressure was reduced to ambient pressure at a rate of approximately 0.05 MPa.s-1, and the foam was then removed from the mold. For the PLGA/chitosan composite porous foams, the procedure is similar to the abovementioned PLGA foams, but with the addition of specified percentages of chitosan crystals to plasmid loaded microparticles (pre-fabricated by spraying drying) to obtain a uniform mixture before the foaming process. Throughout the present study, scaffolds F0, F1 and F2 correspond to the foams with 0%, 5% and 10% of chitosan, respectively. C2 V2 Vent 10mm CO2 V1 C1 HP 10mm P1 M Figure 7.1 Schematic of experimental setup for the supercritical gas foaming system. (C1) Refrigerating Circulator; (C2) Circulating water bath; (P1) High pressure liquid pump; (HP) High pressure view cell; (V1) On/Off Ball Valve; (V2) Automatic back pressure regulator with needle valve; (M) Custom-made mold inserted at the high pressure view cell for holding the foaming samples. 7.2.3 Characterization methods The morphology of pure PLGA foams and PLGA/chitosan composite foams was analyzed by scanning electron microscopy (SEM) (JSM 5600LV, JEOL). The porosity of the porous pure and modified PLGA foams was calculated by the liquid displacement method (Zhang et al., 2008). Briefly, the samples were cut into a disk shape (6mm 135 Chapter diameter by 0.5mm height), and ethanol was used as a liquid medium. The foam sample was submerged in a known volume (V1) of ethanol and a series of brief evacuation repressurization cycles was conducted to force the liquid into the pores of the sample. After these cycles the volume of the liquid and liquid-impregnated sample is V2. When the liquid-impregnated sample was removed, the remaining liquid volume is V3. The porosity was expressed as Porosity = (V1 – V3) / (V2 - V3) x 100% (7.1) The X-Ray Diffraction (XRD) patterns were recorded under ambient conditions on a Shimadzu XRD-6000 with Cu Kα radiation. A diffraction range of 5-25° (2θ) was selected and the XRD analysis was performed at 2°/min. Differential scanning calorimetry (DSC) was employed to determine the effects of chitosan concentration on the glass transition temperature and the decomposition temperature of PLGA. The sample was heated from 30 °C to 400 °C at a constant temperature increment of 10 °C/minute and purged with nitrogen gas at 30 mL/min. X-ray photoelectron spectroscopy (XPS) was performed on a Kratos Axis His instrument to characterize surface nitrogen and phosphate species. A Mg Kα X-ray source (hv = 1253.6 eV) with an analyzer pass energy of 40 eV was operated at 10 mA and 15 kV. All experiments presented here were performed in an ultra-high-vacuum (UHV) chamber with a base pressure of less than 10-9 Torr. The encapsulation efficiency (EE) of plasmid DNA in scaffolds is defined as the percentage of actual plasmid DNA loading to the theoretical plasmid DNA loading (Xie and Wang, 2005) as shown in Equation (7.2): 136 Chapter EE = C plasmid × V water W sample × W plasmid + W PLGA + W chitosan W plasmid × 100 (7.2) Where Cplasmid is the plasmid concentration in the water phase of extraction; Vwater is the volume of water phase of extraction; Wsample is the weight of each foam sample used for EE analysis; Wplasmid, WPLGA and Wchitosan are the weights of plasmid DNA, PLGA and chitosan used in the foam fabrication process, respectively. In the analysis for encapsulation efficiency, 5mg of each scaffold was dissolved in mL of DCM and mL of PBS (pH 7.4) was then introduced to extract DNA. In the process of foaming samples F1 and F2, DNA may be encapsulated into chitosan due to charge interactions. To ensure all DNA was released from PLGA and chitosan, chitosanase from Streptomyces griseus [lyophilized powder, Sigma Aldrich (St. Louis, MO, US)] was utilized to degrade chitosan for the cases of scaffolds F1 and F2. The resultant emulsion was then centrifuged at 9000rpm and 20 °C for 20min (Hettich Zentrifugen, Universal 32R, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany) to separate the water and oil phases. The water phase was carefully collected and kept frozen at -20 °C before the analysis of DNA concentration using PicoGreen dsDNA quantitation kit. 7.2.4 In vitro gene release studies The in vitro release of plasmid was carried out over a period of 65 days and the cumulative release curve was plotted. The foams were cut into cylindrical sections (6mm diameter by 0.5mm height), and each section was incubated at 37 °C with 10 mL PBS buffer in 15mL tubes. The resultant mixture was placed in an orbital shaker bath (GFL® Chapter 137 1092) at 37 °C, 120rpm. mL of sample mixture was extracted at specific intervals and mL of fresh media was replaced. Each sample group was tested with triplicate samples at each interval and all the collected samples were stored at -20 °C until the end of the release assay. DNA concentrations in all samples were measured by PicoGreen dsDNA quantitation kit. Similarly, to ensure that all DNA was released from PLGA and chitosan, chitosanase from Streptomyces griseus was utilized to degrade chitosan for the cases of scaffolds F1 and F2. To evaluate the effects of the fabrication process (including spray drying, supercritical CO2 foaming, and handling of samples) on the molecular integrity of plasmid DNA, agarose DNA gel electrophoresis was utilized to determine the integrity of plasmid DNA released out from foams in vitro after days and 30 days. DNA samples were diluted sixfold in Blue/Orange loading dye (Promega, Madison, WI, US). An 24 μL volume of loading buffer/sample was loaded into each well of a 1.0% agarose gel and electrophoresis was conducted using a Bio-Rad Mini-PROTEAN III electrophoresis system (Cat No: 165-3301 and 165-3302, Bio-Rad Laboratories, California, US) at a constant voltage (60V) for 100 minutes with native plasmid DNA as control and high DNA mass laddar (Invitrogen Corporation, Maryland, US) as marker. A Bio Imaging system, Gene Genius (Syngene, UK) was used to image the gels. 7.2.5 Preparation and culture of fibroblast cells Fibroblast cells were cultured using Dulbecco’s modified Eagle’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (Gibco) at 37 °C in a 95% Chapter 138 air-5% CO2 atmosphere. The medium was changed on the 4th day of culture and every days thereafter. When the cells of the first passage became sub-confluent (usually 7-10 days after seeding), cells were detached from the flask by treatment for at 37 °C with PBS solution of 0.25wt% trypsin and 0.02wt% ethylenediaminetetraacetic acid (EDTA). Cells were normally sub-cultured at a density of x 104 cells/cm2. Cells of the second passage at sub-confluence were used for the subsequent experiments. 7.2.6 Cell adhesion to foams Prior to cell seeding, all foams were sterilized by UV for hours. Cells were seeded into all the foams according to an agitated seeding method because the method was shown to be effective in seeding cells homogenously into porous 3-D structures (Takahashi and Tabata, 2003). Briefly, 0.5 mL of cell suspension (1 x 106 cells/mL) and the foam were placed in 15mL tubes (Greiner Bio-one, Monroe, NC, US) on an orbital shaker (GFL® 1092) and agitated at 37 °C at 300 rpm for 1-4 h. The cell-seeded foams were thoroughly washed with PBS to exclude non-adherent cells and subjected to the subsequent experiment. To determine the number of cells seeded into each foam over the period of hours, the foam was washed three times with PBS, cut down with a scissors, and homogenized in the lysis buffer (0.1M Tris-HCl, 2mM EDTA, 0.1% Triton X-100). The sample lysate (2 mL) was centrifuged at 12,000 rpm for at °C, and the supernatant was carefully collected and kept in the ice. The total DNA intensities in all foams were determined by 139 Chapter PicoGreen dsDNA quantitation kit and compared to get the relative adhesion ability of cells to each type of foam. 7.2.7 Cytotoxicity assay of foams Prior to carrying out any cell culture assay, foams were sterilized under UV light for h and then placed in 24-well plates (NunclonTM, Roskilde, Denmark). One mL of fibroblast cell suspension (1 x 104 cells/mL) was added into each well and incubated in a humid atmosphere at 37 °C and 5% CO2 up to days. A blank well culturing the same number of cells under the same conditions (without foam) was denoted as a control. At specific intervals (on the first, third, fifth and seventh day), cell viability was measured using a standard cell proliferation assay (PreMix WST-1 cell proliferation assay system, Takara Bio Inc, Shiga, Japan). The cell viability can be calculated by (Xie and Wang, 2006): Cell viability (%) = (Abs test cells/Abs control cells) x 100% (7.3) Where “Abs test cells” represents the amount of formazan determined for cells treated with the different formulations and “Abs control cells” represents the amount of formazan determined for untreated control cells. 7.2.8 In vitro experiment of cells transfection The cells were seeded onto 24-well plate containing different foams at a density of x 104 cells/well in mL of culture medium. To measure the level of gene transfection of fibroblast cells cultured at specific time after foam introduction, the cells were removed from the incubator and treated with Steady-GloTM luciferase assay system (Promega, Madison, WI). Assay of the luciferase activity in the lysate of transfectants were Chapter 140 performed by the instructions of the manufacturer and the luciferase activity was measured with a spectrophotometer (Tecan Trading AG, Switzerland) (Li et al., 2003). The protein concentration was determined by a Micro-BCA protein assay kit (Nie and Wang, 2007). All transfection experiments were carried out over days and performed in triplicate. 7.2.9 Statistical analysis All data are presented as mean ± S.D. throughout this study. Statistical analysis of the experimental data was performed and α < 0.05 is considered as significantly different. 7.3 Results and discussion 7.3.1 DNA purity and concentration To ensure DNA purity isolated from Escherichia coli, the absorbance ratio at the wavelength of 260-280nm has to be maintained between 1.8 and 2.0 (Hosseinkhani et al., 2006). The ratio for the DNA after purification by PureLinkTM HiPure Plasmid DNA Purification Kit was determined to be 1.9, which demonstrated that DNA purity agreed with requirement. 7.3.2 Characterization of functionalized foams As a dual system for tissue engineering and gene delivery, the porous structure can provide both sufficient space for blood circulation and also large surface area for the entrapment of large amount of gene. Figure 7.2 shows the typical SEM morphologies of the sprayed PLGA powder, F0, F1 and F2. As shown in Figure 7.2a, sprayed particles loaded with plasmid are round and smooth with a diameter of 2-10 µm. After the Chapter 141 particles was sprayed through gas forming, they fused and formed a porous scaffold F0 and the corresponding 3D inter-connected porous structures were evident, but the pores are not uniform and random in all directions (Figure 7.2b). After the incorporation of 5% of chitosan, the pores were relatively more uniform and aligning in a particular direction than F0 (Figure 7.2c). When the chitosan content was increased to 10%, the foam seemed to be flower-like with all pores interconnected (Figure 7.2d). Triplicate samples for each types of foam with a sampling size of 100 pores were measured and the average value was used to indicate the diameter. The pore diameters of F0 and F1 fall in the range of 20.8-59.5 µm but the diameter of F2 is difficult to determine as the morphology is highly different. Moreover, in F0 and F1, some pores are isolated from other pores. But in F2, all pores are open and interconnected in structure. Table 7.1 shows the initial porosity of F0 and also the foams after going through the modification of different percentages of chitosan. The porosities of F1 and F2 are slightly higher than F0. The results confirmed that the process of chitosan modifications on foams did slightly change the interconnectivity of pores and create more channels in the 3-D structures. This modification may be good for cell attachment as higher porosity may be capable of producing larger surface area for cell adherence. Figure 7.3a shows the DSC analysis performed for different composition of foams along with pure chitosan powders. The DSC thermogram of pure chitosan powders exhibited an exothermic peak centered at 300 °C. In contrast, pure PLGA foam F0 showed an obvious endothermic decomposition peak centered at 345 ºC. F1 had a slight shift of lowered decomposition temperature, and the decomposition peaks of F2 were obviously 142 Chapter compensated by the exothermic peaks of chitosan powders. This finding proved that chitosan powders were physically blended with PLGA in the micro-porous foam samples after CO2 foaming. Figure 7.2 Morphology of plasmid loaded spray dried PLGA particles (a), pure PLGA scaffold F0 (b), and PLGA/chitosan scaffolds F1 and F2 (c and d respectively). Table 7.1 Elemental composition and DNA encapsulation efficiency of sprayed powders and foams with different chitosan concentrations and porosities of foams C O P N Encapsulation Porosity (%) (%) (%) (%) Efficiency (% ± SD) (% ± SD) 44.91 55.09 0.00 0.00 75 ± _ F0 44.91 55.09 0.00 0.00 70 ± 79.5 ± 2.3 F1 61.57 37.30 0.00 1.13 70 ± 82.0 ± 3.2 F2 60.59 37.78 0.00 1.63 71 ± 86.7 ± 3.1 Sprayed powders 143 Chapter F0 F1 F2 Chitosan crystals 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 o Tem perature ( C ) (a) F0 F1 F2 Chitosan crystals 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 -1 Frequency ( cm ) (b) Figure 7.3 DSC thermograms (a) and X-ray diffraction patterns (b) of chitosan crystals and PLGA/chitosan scaffolds with different chitosan concentrations. X-ray diffraction measurement was employed to examine the chitosan distribution on the surface of foams. As shown in Figure 7.3b, the powder x-ray diffraction pattern of Chapter 144 PLGA/chitosan scaffolds displayed peaks at ~10° (2θ) and 20° (2θ). The medium molecular weight chitosan was in crystalline state and the intensity of the peak at ~20° (2θ) was much higher than that at ~10° (2θ). When processing chitosan powder and PLGA microparticles into foams, none of the two peaks at ~10° (2θ) and 20° (2θ) was observed in F1 and F2. This result demonstrated that chitosan was poorly crystallized on the surface of F1 and F2 after incorporation. Subsequent XPS study would confirm whether chitosan was present on the surface of matrix. Table 7.1 also shows the encapsulation efficiency results of DNA in sprayed particles and three types of foams. It was found that supercritical CO2 foaming and chitosan blending had little influence on the encapsulation efficiency of DNA. This result also indicates that in the process of foaming, the DNA pre-encapsulated inside sprayed PLGA particles can remain encapsulated in foams. As shown in Figure 7.4, significant differences in the intensity of N1s signal were detected in sprayed particles and the types of foams. No nitrogen signals were obtained in sprayed particles and blank PLGA foam (F0). This is reasonable as there is no nitrogen in the co-polymer of PLGA. Although there should be nitrogen in DNA, its trace amount may not be detected by XPS. For the cases of scaffolds F1 and F2, obvious N1s peaks were present at the binding energy of 298 eV, and the intensity increased with increasing chitosan percentages. This observation further confirmed that chitosan was coated on the surface of foam and this content could be enhanced by simply increasing the weight percentage of chitosan crystals before the foaming process. 145 Chapter Intensity (OPS) Sprayed powder F0 F1 F2 392 394 396 398 400 402 404 406 408 Binding Energy (eV) N-1s-3 Figure 7.4 N1s high-resolution XPS spectra for chitosan crystals and PLGA/chitosan scaffolds with different contents of chitosan. 7.3.3 In vitro release studies Figure 7.5a shows the in-vitro DNA release profiles of the three types of scaffolds samples F0, F1 and F2. It was observed that foam F0 released DNA plasmid faster than the other two with a complete release in 40 days. The initial burst is attributed to the following possible reasons: Firstly, a very small amount of DNA, adsorbed on the outer surface of foams but undetectable by XPS, can be easily desorbed and released from the surface; Secondly, at early times the driving force (concentration difference) of plasmid DNA is high since its concentration in buffer solution is nearly zero at the early stage of the release process. This leads to a fast release of plasmid DNA in the initial stage. Lastly, some DNA loosely trapped in the mesopores could be easily released at the beginning. The subsequent slow release following the initial burst till the end could be Chapter 146 attributed to the gradual degradation of PLGA matrix. The remaining DNA entrapped inside foams diffused out along with the degradation of PLGA matrix gradually. This type of release profile is typical for PLGA-based delivery devices, such as microparticles and microfibers (Xie and Wang, 2007; Nie and Wang, 2007). In contrast, the release profiles of F1 and F2 are significantly different from that of F0. They released DNA at a lower rate in the initial stage than F0, but a second release burst was observed at around day 28, which could be sustained till the end of release. For the cases of F1, it released about 60% and 30% of DNA during the first and second burst respectively. While for the case of F2, it released about 30% and 50% of DNA during the first and second burst, respectively. Interestingly, except the presence of the two-burst characteristic, the release profile of F2 is relatively similar to a zero-order or linear release. This pseudo-zero-order or linear release can’t be obtained by conventional surface modification methods (Nafee et al., 2007; Petri et al., 2007; Kumar et al., 2004; Garcia-Fuentes et al., 2005), in which polycationic agents are just utilized to modify pre-fabricated surface of particles, so the polycationic agents can’t penetrate into the interior structure of matrix and consequently the polycationic properties can’t sustain with the degradation of the matrix. As a result, the DNA encapsulated inside particles is released as a burst. The present method ensures that chitosan is uniformly dispersed inside the foams F1 and F2, therefore the foams can release DNA in a controlled manner. This is advantageous over that of F0 since high intrusion rate of plasmid DNA would not guarantee a high transfection efficiency or expression due to the limitation of “copy number” of a particular cell line. Moreover, it might also damage the cell membrane and thus results in a higher toxicity level. Chapter 147 (a) (b) Figure 7.5 Accumulative in vitro release profiles of DNA from F0, F1 and F2 (a) and electrophoretic mobility analysis of native DNA and in vitro samples released after days and 30 days (b). All samples were performed on a 1.0% agarose gel and stained with ethidium bromide. Lane 1: High DNA mass ladder; lane 2: Native plasmid DNA; lane 3: DNA released from F0 after days; lane 4: DNA released from F1 after days; lane 5: DNA released from F2 after days; lane 6: DNA released from F0 after 30 days; lane 7: DNA released from F1 after 30 days; lane 8: DNA released from F2 after 30 days. Chapter 148 7.3.4 Plasmid integrity check Following the characterization of release kinetics, it is also important to verify the structural integrity of the released DNA. Results from agarose gel electrophoresis demonstrated that the released DNA after and 30 days retained its original structural integrity as evidenced by the distinct bands present on the gel (Figure 7.5b). This shows that the plasmid DNA integrity could be maintained in the course of the spray drying process, high-pressure supercritical CO2 foaming process, and post-processing conditions (handling of scaffold, incubations, and lyophilization). Comparing the results for plasmid DNA released from foams F0, F1, and F2 with the native plasmid DNA, it was also confirmed that the addition of chitosan on PLGA matrix posed no observable side effect on its structural integrity. 7.3.5 Cell attachment testing Figure 7.6 demonstrates that cells prefer to attach to scaffolds F1 and F2 rather than F0. After the first hour, number of cells in attached on F0 was comparable to those in F1 and F2. At the following three hours, the number of cells attached on F0 did not show significant increase. In contrast, those attached in F1 and F2 changed significantly in the same three-hour period. The number of cells attached on F1 increased by 100%, 60%, and 10% after 2, 3, and hours of cell seeding, respectively as compared to that after just one-hour incubation. Similarly, the number of cells attached on F2 increased by 56%, 60% and 12.5% after 2, 3, and hours of cell seeding, respectively as compared to that after just one-hour incubation. This suggests that chitosan coating on PLGA surface Chapter 149 could enhance cell adherence dramatically, which is advantageous for cell growth, gene expression, and subsequent tissue formation. Figure 7.6 Comparison of cell attachment on F0, F1 and F2 over hours (mean ± S.D., n=3). * Statistically different from F0 and § statistically different from F1 (α[...]... microparticles and microfibers (Xie and Wang, 2007; Nie and Wang, 2007) In contrast, the release profiles of F1 and F2 are significantly different from that of F0 They released DNA at a lower rate in the initial stage than F0, but a second release burst was observed at around day 28, which could be sustained till the end of release For the cases of F1, it released about 60 % and 30% of DNA during the first and. .. released DNA after 3 and 30 days retained its original structural integrity as evidenced by the distinct bands present on the gel (Figure 7.5b) This shows that the plasmid DNA integrity could be maintained in the course of the spray drying process, high-pressure supercritical CO2 foaming process, and post-processing conditions (handling of scaffold, incubations, and lyophilization) Comparing the results... surface of matrix Table 7.1 also shows the encapsulation efficiency results of DNA in sprayed particles and three types of foams It was found that supercritical CO2 foaming and chitosan blending had little influence on the encapsulation efficiency of DNA This result also indicates that in the process of foaming, the DNA pre-encapsulated inside sprayed PLGA particles can remain encapsulated in foams... the binding energy of 298 eV, and the intensity increased with increasing chitosan percentages This observation further confirmed that chitosan was coated on the surface of foam and this content could be enhanced by simply increasing the weight percentage of chitosan crystals before the foaming process 145 Chapter 7 Intensity (OPS) Sprayed powder F0 F1 F2 392 394 3 96 398 400 402 404 4 06 408 Binding... expression of F1 and F2 are comparable to the blank foam F0, which may demonstrate significant potential for clinical applications 7.4 Conclusions In this study, PLGA/chitosan composite porous foams were fabricated by the combination of spray drying and supercritical CO2 foaming techniques and the effects of chitosan incorporation on the following factors was investigated: in vitro release of DNA, cell... F0, F1 and F2 after day 5 because the release rates of DNA from them are significantly different after 5 days as shown in Figure 7.5a The correlations between in vitro release profiles and in vitro expression data demonstrate that the bioactivity, not just the integrity of the DNA (shown in Figure 7.5b) released from all foams are maintained after going through the processes of spray drying, supercritical. .. diameters of F0 and F1 fall in the range of 20.8-59.5 µm but the diameter of F2 is difficult to determine as the morphology is highly different Moreover, in F0 and F1, some pores are isolated from other pores But in F2, all pores are open and interconnected in structure Table 7.1 shows the initial porosity of F0 and also the foams after going through the modification of different percentages of chitosan... growth of surrounding cells This fast degradation rate demonstrated the negative effects after day 5 and they showed significant decrease of cell viability in day 5 and 7 In contrast, the high viscosity and strength of chitosan coated on the surface of F1 and F2 might hinder and slow down the degradation of -COOH groups Therefore, the foam structure could be maintained for a longer period of time and. .. cell attachment and subsequent cell viability The sustained release of DNA from F1 or F2 led to lower cytotoxicity and more sustained expression of gene than F0 This property is favorable for gene delivery and tissue engineering In brief, although this type of devices require further improvement on their standardization of pore structures and release profile, their easy fabrication and modification... first and second burst respectively While for the case of F2, it released about 30% and 50% of DNA during the first and second burst, respectively Interestingly, except the presence of the two-burst characteristic, the release profile of F2 is relatively similar to a zero-order or linear release This pseudo-zero-order or linear release can’t be obtained by conventional surface modification methods (Nafee . cases of scaffolds F1 and F2. To evaluate the effects of the fabrication process (including spray drying, supercritical CO 2 foaming, and handling of samples) on the molecular integrity of. could be maintained in the course of the spray drying process, high-pressure supercritical CO 2 foaming process, and post-processing conditions (handling of scaffold, incubations, and lyophilization) sustained till the end of release. For the cases of F1, it released about 60 % and 30% of DNA during the first and second burst respectively. While for the case of F2, it released about 30% and