Applications of electrospinning and supercritical carbon dioxide foaming techniques in controlled release and bone regeneration 6

In this study, the surface physical and chemical properties of pure PLGA and PLGA/chitosan porous foams fabricated by supercritical... Furthermore, the cell adhesion, cytotoxicity and in

CHAPTER 7

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

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

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

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

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

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

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 2

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 mentioned 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

above-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

diameter by 0.5mm height), and ethanol was used as a liquid medium The foam sample

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 V 2 When

porosity was expressed as

Porosity = (V 1 – V 3 ) / (V 2 - V 3) x 100% (7.1) The X-Ray Diffraction (XRD) patterns were recorded under ambient conditions on a

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):

WW

WW

VC

EE

plasmid

chitosan PLGA

plasmid sample

In the analysis for encapsulation efficiency, 5mg of each scaffold was dissolved in 1 mL

of DCM and 5 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

buffer in 15mL tubes The resultant mixture was placed in an orbital shaker bath (GFL®

1092) at 37 °C, 120rpm 1 mL of sample mixture was extracted at specific intervals and 1

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

agarose DNA gel electrophoresis was utilized to determine the integrity of plasmid DNA

released out from foams in vitro after 3 days and 30 days DNA samples were diluted

six-fold 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

air-5% CO2 atmosphere The medium was changed on the 4th day of culture and every 3 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 5 min 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 2 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 6 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 4 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 5 min at 4 °C, and the supernatant was carefully collected and kept in the ice The total DNA intensities in all foams were determined by

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 6 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 7 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 1 x

104 cells/well in 1 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

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 7 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.,

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

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