155 Chapter CHAPTER Lysine-Based Peptide Functionalized PLGA Foams for Controlled DNA Delivery† 8.1 Introduction Over recent years, DNA delivery research has become increasingly popular due to its potential therapeutic and medicinal applications. Among various delivery devices, microspheres and nanoparticles are the most widely used carriers in DNA delivery due to their uniform morphology and efficacy in cell transfection (Leong et al., 1998; MacLaughlin et al., 1998; Mao et al., 2001; Roy et al., 1997; Mao et al., 1996). There is also growing interest in the use of porous materials as DNA delivery matrices. The stable and uniform porous structures, tunable pore size and well-defined surface properties of these materials allows the incorporation and release of a diversity of proteins and DNAs in a more reproducible and predictable manner (Thomson et al., 1998; Chen et al., 2001; Torres et al., 2006; Song et al., 2005). Furthermore, the three-dimensional porous structures can be easily molded into a desired shape, which can hold both DNA and cells simultaneously, and provide an appropriate platform for the formation and remodeling of new tissue with the degradation of the mold (Goldstein et al., 2001). Thus, porous foams appear to have significant advantages over microspheres and nanoparticles towards † This chapter highlights the work published in H. Nie, L.Y. Lee, H. Tong and C.H. Wang. Lysine-Based Peptide Functionalized PLGA Foams for Controlled Gene Delivery. J. Control. Release 2009. Chapter 156 creating a DNA delivery and tissue engineering dual system. Micro-porous PLGA foams engineered by supercritical carbon dioxide foaming technique with large, controlled pore size and highly ordered morphology offer an intriguing channel structure for DNA delivery and cell adhesion (Mikos et al., 1994). Furthermore, the sorption capacity and characteristics of micro-porous PLGA foam could be substantially altered by anchoring a variety of functional groups onto the external and internal pore surfaces. Porous PLGA foam has been frequently used in drug and protein delivery (Hsu et al., 1996; Kim et al., 2006). However, its application in DNA delivery has been limited, mainly due to its negative surface charges, resulting in a strong charge repulsion that hinders the adsorption of DNA and attachment of normal cells onto the foams. Therefore, surface functionalization of the PLGA foam is essential to convert it to an effective DNA carrier to hold DNA and subsequently release it in a sustained manner. PLGA/chitosan composite foams developed in Chapter show promising results in controlled release of DNA, but the release rate of DNA and subsequent expression of target protein is too low, especially in the initial stage (Figures 7.5a and 7.7b). Therefore, an initial and significant release of DNA is demanded in order to optimize this kind of devices. Lysine, an α-amino acid of chemical formula HO2CCH(NH2)(CH2)4NH2, pKa 10.54 and hydrophobility of -3.9 (Civitelli et al., 1992), is a potentially good candidate as supplement for PLGA to fine-tune its charge property and hydrophilicity for DNA delivery purposes. The primary amine side groups of lysine can interact and form complexes with DNA molecules. In this study, the functionalization of PLGA porous foam matrix was accomplished using Lysine-based peptides. It was speculated that the Chapter 157 functionalized foams may have different DNA loading and release profiles and thus cell transfection level, depending on the molecular properties of the peptides being used. Particularly in this study, PLGA porous foams were functionalized using K4 and K20 peptides and the surface physical properties of the foams were investigated using a series of state-of-the-art techniques, such as SEM and XPS. BMP-2 plasmid was used as a model DNA and loaded onto foams with and without surface modification. The adsorption capacities of the foams and in vitro release of the model DNA in phosphatebuffered saline (PBS) were studied. In addition, cell proliferation on the foams and in vitro DNA expression were also investigated. 8.2 Materials and methods 8.2.1 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, USA). Dichloromethane (DCM) (Cat. No. DR-0440) was purchased from Tedia Company Inc. (Fairfield, OH, US.). Fmoc-Lys (Boc)-OH and 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). PreMix WST-1 cell proliferation assay system, Thermo Scientific NanoDropTM 1000 Spectrophotometer and BMP-2 ELISA Kit were procured from Takara Bio Inc. (Otsu, Shiga, Japan), Thermo Fisher Scientific Inc. (Wilmington, DE, US) and R&D Systems (Minneapolis, MN, US), respectively. 158 Chapter 8.2.2 Preparation of foams and Lysine peptides Blank PLGA foams were engineered based on a gas foaming method using supercritical CO2 as the blowing agent. All the procedures are the same as explained in Chapter (see Figure 7.1). Both peptides K-K-K-K-G (K4) and K-K-K-K-K-K-K-K-K-K-K-K-K-K-KK-K-K-K-K-G (K20) (where K and G represents Lysine and glycine residue, respectively) were synthesized in-house on an automated Multipep peptide synthesizer (Intavis, Germany). All peptides were assembled on Fmoc-Glycine resin (substitution level = 0.66 mmole/g resin) at 50 μmole scale. Stepwise couplings of amino acids were accomplished using double coupling method with 5-fold excesses of amino acids, equivalent activator reagents, 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and N-Hydroxybenzotriazole, and two equivalents of base, N-methylmorpholine. The removal of Fmoc was completed using 20% piperidine in dimethylformamide (DMF). Cycles of deprotection, washings, double couplings, and washings were repeated until the desired chain length was achieved. The dried peptidyl-resin was cleaved by a cocktail solution composed of 95% trifluoroacetic acid (TFA), 2.5% deionized water, and 2.5% triethylsilane (v/v). The crude peptide was purified using an Agilent 1100 semipreparative high performance liquid chromatography (HPLC) (Santa Clara, CA). The purification was performed on an Agilent Zorbax 300SB-C18 reverse phase (RP) column (5 μm particle size, 300Ǻ pore size, 25 x 1.0 cm) with a linear gradient of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in acentonitrile) from 10% B to 45% B in 30 at a flow rate of mL/min. The purity of all peptides was greater than 95% by analytical RP-HPLC and matrix-assisted laser desorption/ionization-time of flight mass Chapter 159 spectroscopy (MALDI-TOF MS) on a Bruker AutoFlex II MALDI-TOF MS (Bruker, Bremen, Germany) (data not shown). 8.2.3 Peptides conjugation K4 and K20 were employed to study the effects of chain length and surface charges on the adsorption and release patterns of plasmid DNA. Blank PLGA foams were sterilized with 70% ethanol and washed thrice with excess sterilized water. To functionalize the PLGA foams, the peptides (K4 or K20) were incorporated covalently onto the surface of the PLGA foams using a condensation coupling method (Li et al., 1998). Briefly, the carboxyl groups on the foam surface were first activated by 10mM of N-Ethyl-N'-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride, Sigma-Aldrich)/ N-Hydroxysulfosuccinimide (NHS, Aldrich) sterilized by filtering through 0.22 μm filter for h with occasional shaking at room temperature. The foams were then washed times with sterilized water to eliminate excess EDC/NHS. The peptides (K4 or K20) were covalently immobilized onto the activated foams by immersing the foams in the peptide solutions (0.1mM) at room temperature overnight (Khew et al., 2007). After that, the unbound K4 or K20 was desorbed in copious amounts of PBS for h at room temperature. The resultant foams were thoroughly washed with DI water and dried in air. 8.2.4 Characterization of morphology and porosity The morphology of samples (blank foams, F0; K4-functionalized foams, F1; K20functionalized foams, F2) was examined using scanning electron microscopy (SEM) (JSM 5600LV, JEOL). The porosity of the porous pure PLGA foam and the modified Chapter 160 PLGA foams was measured using a mercury intrusion porosimeter AutoPore III 9420 (Micromeritics, Norcross, GA) (Zhang et al., 2009). 8.2.5 Atomic composition of foam surface The chemical structure and atomic composition of the blank and surface-modified foams were characterized using X-ray photoelectron spectroscopy (XPS) (VG ESCALAB 220IXL; Thermo VG Scientific, UK), with the data processing performed using XPSPEAK (Version 4.1) software. Wide scan (0-1000 eV) and high-resolution (C1s, O1s, and N1s) spectra were acquired, respectively. 8.2.6 Plasmid preparation and loading procedures A pT7T3D-PacI encoding BMP-2 was used in this study. The plasmid DNA was amplified in a transformant of Escherichia coli bacteria and isolated from the bacteria by PureLinkTM HiPure Plasmid DNA Purification Kit-Maxiprep K2100-07 (Invitrogen Corporation, MD, USA). The DNA concentration was determined using a Thermo Scientific NanoDropTM 1000 Spectrophotometer. For saturation loading of foams with plasmid DNA, different kinds of foams (F0, F1 and F2) were introduced into 0.5 mL of TE buffered solution of DNA (200 μg/mL) and soaked for 24 h under constant stirring. The foams were then dried under vacuum after quick and through washes by DI water. 8.2.7 DNA adsorption capacity on foams Besides the atomic composition analysis, the densities of DNA attracted on different foams were quantified. Briefly, 15 mg of each scaffold was dissolved in 0.5 mL of DCM Chapter 161 and mL of PBS (pH 5.0) was introduced to scaffold/DCM solution, vortexed, and centrifuged (Hettich Zentrifugen, Universal 32R, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany) at 14,000 rpm for min. The aqueous layer was collected, and two more extraction cycles were performed to maximize DNA recovery. The water phases were kept frozen at -20 °C until they were analyzed for DNA concentrations using Thermo Scientific NanoDropTM 1000 Spectrophotometer. 8.2.8 In vitro DNA release studies Foams were sterilized using Co-60 gamma irradiation at a dose of 15 kGy before using for DNA adsorption, and following DNA in vitro release and cell culture studies. The in vitro release of plasmid DNA was carried out over a period of 20 days and the cumulative release curve was plotted. Foams of mg each loaded with plasmid was added to mL of PBS (pH=7.4) and the resultant solution was then placed in an orbital shaker bath (GFL® 1092) maintained at 37 °C and 120 rpm. The sample (0.1 mL) was extracted from the solution at specific intervals and then topped up with 0.1 mL of fresh media. Each study group (F0, F1 and F2) was tested in triplicate and all the collected samples were stored at -20 °C until the release assay. The DNA concentration in each sample was determined by Thermo Scientific NanoDropTM 1000 Spectrophotometer. To evaluate the effects of charge interaction on the molecular integrity of plasmid DNA, agarose DNA gel electrophoresis was used to determine the integrity of plasmid DNA released from the foams in vitro after days. Release samples were diluted six-fold in Blue/Orange Loading Dye (Promega, Madison, WI, US). A 12 μL of loading Chapter 162 buffer/sample was loaded into each well of 1% agarose gel. Electrophoresis was conducted using a Bio-Rad Mini-PROTEAN III electrophoresis system (Bio-Rad Laboratories, CA, US) at a constant voltage (60V) for 120 with native plasmid DNA as control. SYBR Gold staining (Molecular Probes, Invitrogen) was employed to stain plasmid in samples/control and Gene Genius Bio Imaging system (Syngene, Cambridge, UK) was used to image the gels. 8.2.9 Preparation and culture of rat marrow stromal cells The seeds of rat marrow stromal cells (rMSCs) used in the current study were donated from the orthopaedic research center, Kaohsiung Medical University as a gift. They were cultured in DMEM supplemented with 4mM-glutamine (Biological Industries, Kibbutz Beit Haemek, Israel), 25 mM HEPES buffer, 10% fetal bovine serum (Gibco), 10U/mL penicillin G sodium and 10 mg/mL streptomycin as Fungizone (Gibco) and incubated at 37 °C and 5% CO2 humid atmosphere in 75cm2 cell culture flasks. The cells were extracted with PBS solution containing 0.25% trypsin-EDTA (Biological Industries, Kibbutz Beit Haemek, Israel) and normally subcultured at a density of x 104 cells/cm2. 8.2.10 Cell viability assay 100 μL of rMSCs suspension (1 x 105 cells/mL) along with different foams were added into wells of 96-well plates (NunclonTM, Roskilde, Denmark) and incubated at 37 °C and 5% CO2 humid atmosphere. 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, second and third day), cell viability was measured using a standard cell proliferation 163 Chapter assay (PreMix WST-1 cell proliferation assay system, Takara Bio Inc, Shiga, Japan). The cell viability was calculated as following (Takashima et al., 2007): Cell viability (%) = (Abs test cells/Abs control cells) x 100% (8.1) Where “Abs test cells” represents the amount of formazan produced by cells treated with the different formulations and “Abs control cells” represents the amount of formazan produced by cells in the control. 8.2.11 In vitro experiment of cells transfection 100 μL of rMSC suspension (1 x 105 cells/mL) was added into wells of 96-well plates (NunclonTM, Roskilde, Denmark) and incubated for 16 h for adherence. Afterwards, the media was aspirated from the wells and the wells were washed once with DMEM before 100 μL of new DMEM was added to each well along with different foams as described in the cytotoxicity experiment. To measure the level of gene transfection of rMSC cultured, the cells were washed three times with PBS, and homogenized in the lysis buffer (0.1M Tris-HCl, 2mM EDTA, 0.1% Triton X-100). After staying in ice for 10 mins, the sample lysate (100 μL) was centrifuged at 12,000 rpm for at °C, and the supernatant was carefully collected and kept in the ice. To measure the expression level of BMP-2 gene, 50 µl of the supernatant was collected and the BMP-2 protein was determined by a BMP2 ELISA Kit (R&D Systems, US). All transfection experiments were performed at predetermined intervals and assayed in triplicate (Nie and Wang, 2007; Li et al., 2003). 8.2.12 Statistic 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. Chapter Figure 8.1 SEM images of blank foam (F0) and functionalized foams (F1 and F2). 164 Chapter 165 8.3 Results and discussion 8.3.1 Surface characterization and porosity measurement of foams As a dual system for tissue engineering and DNA delivery, the porous structure can provide both sufficient space for blood circulation and also extended surface area for the entrapment of large amount of DNA. Figure 8.1 shows typical SEM morphologies of F0, F1 and F2 and the 3D inter-connected porous structures are evident. Three foams from the same batch were measured and the average value (with a sampling size of 100 pores) was used to indicate the diameter. From the SEM images of F0, the pore diameters of the foams are relatively uniform and they all fall within the range of 20.8-59.5 µm. After conjugation of peptides, the pore shapes become irregular and the inter-connected porous structures are modified as well. Some pores are isolated and not connected to other pores in F0. In contrast, all pores in F1 and F2 are open and interconnected. The changes in pore structures are ascribed to the activation of the carboxyl groups by EDC/NHS, as similar changes in pore structures are also observed in foams treated by EDC/NHS alone, prior to the incorporation of K4/K20. Actually the interconnectivity of blank foams is not so good and many pores are blocked by thin membranes, as shown by the arrows in Figure 8.1. However, the membranes are very weak and easy to be damaged by the harsh environment imposed by EDC/NHS. The destruction of pores modifies the structures and increases the interconnectivity of foams. Table 8.1 shows the initial porosity of F0 and also the foams after going through the surface modifications by K4 or K20. As an evidence of structural changes after the conjugations of peptides, the porosities of F1 and F2 are slightly higher than F0. This result confirmed that the process of lysine Chapter 166 modifications on foams did slightly change the interconnectivity of pores and create more channels in the three-dimensional structure. 8.3.2 XPS spectra of modified surfaces Figure 8.2a illustrates the C1s high-resolution XPS spectra of the foams before DNA loading. The C1s spectrum of F0 shows the characteristic peak of C–C/C–H, C-O and C=O bonds with binding energies of 284.8 eV, 287 eV and 289.1 eV respectively. In contrast, the spectrum of F1 conjugated with K4 (Figure 8.2b) showed that the two C1s peaks at around 284.8 eV and 287 eV were perturbed by other peaks. After peakdeconvolution, one peak corresponding to C-N centered at 286.4 eV was observed. When the conjugation peptide was changed from K4 to K20, significant increase of C-N peak was detected (Figure 8.2c). The presence of C-N peak displays the successful conjugation of peptides on both F1 and F2. Furthermore, the successful grafting of peptides on foam surface was also verified by the presence of nitrogen (N1s) peaks at 397.9 eV from the N1s high-resolution XPS spectra. As shown in Figure 8.3a, significantly higher peaks of N1s were detected in F1 and F2 than that in F0. The N1s signal (before DNA adsorption) is directly associated with lysine peptides, so the percentages of C-N peak areas were well correlated with the nitrogen atomic concentration as indicated in Table 8.2. Similarly, the P2p signal is directly corresponding to the DNA on foams (after DNA adsorption), so an additional element P was detected on all foams after DNA loading process as shown in Figure 8.3b. The atomic percentage of P in F0, F1 and F2 are 0.48, 2.23 and 1.89 respectively, which are 167 Chapter consistent with the results of surface density analysis. As shown in Table 8.1, the most of DNA was bound to F1 but the least of DNA was attracted onto F0. Table 8.1 Porosity and DNA adsorption capacity of different foams Samples Porosity (% ± S.D., n=3) DNA adsorption capacity (µg DNA/mg foam) F0: Blank PLGA 66.6 ± 3.1 1.03±0.17 F1: K4 conjugated PLGA 76.3 ± 2.9 4.32±0.13 F2: K20 conjugated PLGA 78.8 ± 2.5 3.81±0.12 Table 8.2 Atomic composition (C1s, O1s, N1s and P2p) and percentage of C1s in XPS spectra of different foams before and after DNA adsorption Atomic Conc. (%) Peak Ratio (%) of C1s Samples C1s O1s N1s P2p C-C at 284.8 ± 0.1 eV C-N at 286.4 ± 0.1 eV C-O at 287.0 ± 0.1 eV O-C=O at 289.1 ± 0.1 eV F0 66.19 33.62 0.19 --- 46.67 --- 27.27 26.06 F1 65.47 33.19 1.34 --- 21.78 20.63 33.01 24.58 F2 60.54 37.56 1.90 --- 27.42 27.38 26.88 18.32 F0/DNA 59.20 39.13 1.19 0.48 29.34 32.83 22.34 15.49 F1/DNA 59.85 32.93 4.99 2.23 49.60 13.48 23.03 13.89 F2/DNA 57.61 35.68 4.82 1.89 49.27 13.11 22.51 15.11 168 Chapter F0 C 1s C-C C-O O-C=O 292 290 288 286 284 282 280 Binding energy (eV) (a) C 1s F1 C-C C-O C-N O-C=O 292 290 288 286 284 282 280 Binding energy (eV) (b) F2 C 1s C-N C-C C-O O-C=O 292 290 288 286 284 282 280 Binding energy (eV) (c) Figure 8.2 C1s high-resolution XPS spectra for F0, F1 and F2 before DNA adsorption. 169 Chapter F0 F1 F2 408 406 N 1s 404 402 400 398 396 394 392 Binding energy (eV) (a) F0 F1 F2 140 138 P 2p 136 134 132 130 128 126 124 Binding energy (eV) (b) Figure 8.3 (a) N1s high-resolution XPS spectra for F0, F1 and F2 before DNA adsorption. (b) P2p high-resolution XPS spectra for F0, F1 and F2 after DNA adsorption. 170 Chapter 8.3.3 In vitro release studies Cumulative release percentage (100%) 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 F0 F1 F2 0.2 0.1 0.0 10 12 14 16 18 20 Time (days) (a) (b) Figure 8.4 (a) DNA cumulative release curves for F0, F1 and F2 (mean ± S.D., n=3). (b) Electrophoretic mobility analysis of naked DNA and in vitro samples released after days. All samples were run on a 1% agarose gel and stained with SYBR® Gold nucleic acid gel stain. Lane 1: native DNA; lane 2: DNA released from F0; lane 3: DNA released from F1; lane 4: DNA released from F2. Chapter 171 Figure 8.4a shows the in vitro DNA release profiles of different samples. It can be observed that the release of DNA from F0 was the fastest which completed in only days. The initial burst release can be attributed to the following reasons: (1) DNA adsorbed on the outer surface can be easily desorbed; (2) The large concentration gradient between DNA in F1 or F2 and in buffer solution would prompt the fast release of DNA; (3) The loosely trapped DNA in the micro pores could be easily released. As the channels in the foam are interconnected, some interior structures within the foam could entrap and retain DNA even after times of washing. However, the release profiles of DNA from F1 and F2 were evidently different from that of F0. While 60% of the DNA loaded on F0 was released in a huge burst lasting for a period of two days, there were two small release bursts observed for the case of F1 and F2, each burst release was followed by a period of slow release profile. Compared with the release profile of F0, the release profiles of F1 and F2 are relatively linear and sustainable. The constant rate of the in vitro release processes suggested that there exist some interactions between the entrapped DNA molecules and the micro-porous PLGA matrix supplemented with -NH2 functional groups. Otherwise, the percentage release rates for F1 and F2 should be higher than F0 as F1 and F2 have higher porosity and DNA from F1 and F2 would meet lower resistance to diffuse due to higher interconnectivity as compared with F0. Chapter 172 8.3.4 Plasmid integrity check Following the characterization of release kinetics, the structural integrity of the released DNA was examined. As shown from the agarose gel electrophoresis results, the released DNA retained its structural integrity as evidenced by the distinct bands present on the gel (Figure 8.4b). In summary, the released DNA survived both the adsorbing and releasing processes. Moreover, it was shown that the plasmid DNA encapsulated in the different foams was released in a supercoiled form within the time scale of days, and was independent of the types of foams (Figure 8.4b). This condensation of DNA in size may trigger the interactions between DNA and peptides. Consequently, DNA molecules may even penetrate into the deeper layer of K20 on foam F2 due to the long chain of K20. For the DNA molecules penetrated into deep layer of K20, their release was hindered by both structural entrapment and charge interactions, thus leading to the observation of the most sustainable release of DNA for the case of F2. 8.3.5 Cell viability study Figure 8.5 shows the cytotoxicity of different foams with the blank tissue culture plate well as the blank and the well with free DNA as control. Generally, no significant change was detected on all the three kinds of foams throughout the whole testing period of days, but an obvious reduction in cell viability was detected in the control group and F0 on day and day 3. This finding is similar to the observation in other research group (Chun et al., 2004). One of the reasons for the cytotoxicity in control and F0 may be attributed to the high initial concentration in the control group and a burst release of DNA during the initial hours from F0. Intense transfection of DNA may impose damage on cell 173 Chapter membrane and lead to cell death in serious cases. As the amounts and released rates of DNA from F1 and F2 are much lower than F0 during the first days, no such kind of negative effect on cell viability was observed. Indeed, it is reported in literature that PolyLysine is toxic to cells (Fischer et al., 2003; Ahn et al., 2004). However, the lysine peptides (K4 and K20) used in the current study are short in length and are covalently immobilized to the foam matrix. Therefore, they are not easy to detach from the foam surfaces. Furthermore, it is difficult to encapsulate DNA into particles and impose obvious toxicity to cells. This statement is clearly supported by the toxicity data shown in Figure 8.5. From all these results, it can be deduced that surface modification using Lysine peptides does not impose significant cytotoxicity on cells. 1.6 1.4 blank F0 F1 F2 Cell viability (100%) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Time (days) Figure 8.5 Cytotoxicity analysis of pure and functionalized foams, with tissue culture plate as control (mean ± S.D., n=3). Chapter 174 8.3.7 In vitro cell transfection testing The release device is considered ineffective if the released DNA fails to transfect the cells or to be expressed. The foams were assessed for in vitro transfection in rMSCs and their individual sustainability in expression of BMP-2 protein. It is shown in Figure 8.6 that the cells cultured on all foams expressed BMP-2 in the period of days. On day 1, control and F0 demonstrated slightly higher BMP-2 concentrations than other groups, but all the groups (control, F0, F1 and F2) showed comparable BMP-2 concentrations over the first days. This phenomenon indirectly demonstrates the existence of a saturation state of DNA transfection. As a result, a high initial DNA concentration or a burst release of DNA does not necessarily lead to high DNA transfection and expression. After days, the advantages of control group and F0 in BMP-2 expression vanished and the corresponding concentration was even significantly lower than those of F1 and F2 on day 5. The decrease in BMP-2 expression for the case of the control group could be ascribed to the cytotoxicity imposed by the high concentration of DNA as demonstrated in Fig. 8.5. In contrast, the drop of BMP-2 expression in F0 samples should be attributed to the synergic effects of cytotoxicity of F0 samples and their poor sustainability of DNA release. Around 70% of the DNA entrapped in F0 is released within the first days and the following release after days is negligible. Moreover, as similar to the case of control, it might be hard for cells to survive in the environments with high concentration of DNA, resulting in low viability and subsequent low expression efficiency. As a result, lower BMP-2 concentrations were detected in F0 than F1 and F2 on day and day 7, respectively. Particularly, F2 presented a sustained expression of BMP-2 over the testing period of days. On day 7, the expression level in F2 was almost 1-fold higher than those 175 Chapter displayed in F0 and F1. This significant enhancement and sustainability in expression level should be attributed to the higher DNA adsorption capacity of F2 and its controlled release lasting for longer than two weeks. The above-mentioned expression experiments suggest that the peptide-functionalized PLGA foams could be used as a potential DNA carrier in gene therapy, as the DNA released from the functionalized scaffolds appeared to be stable and produced lasting gene expression in mammalian cells. BMP-2 concentration (pg/mL) 300 F0 F1 F2 250 *+ 200 * 150 * * * * + 100 50 Time (days) Figure 8.6 In vitro expression of DNA released from pure and functionalized foams over days period (mean ± S.D., n=3). * Statistically different from F0 (α[...]... within the foam could entrap and retain DNA even after 3 times of washing However, the release profiles of DNA from F1 and F2 were evidently different from that of F0 While 60% of the DNA loaded on F0 was released in a huge burst lasting for a period of two days, there were two small release bursts observed for the case of F1 and F2, each burst release was followed by a period of slow release profile... expression of DNA, which is favorable for DNA delivery and tissue engineering applications Furthermore, the fast release of DNA could be a good supplement to the PLGA/chitosan foams developed in Chapter 7 In future study, the two techniques investigated in Chapters 7 and 8 can be well coupled and utilized to develop PLGA/chitosan/lysine composite foams in three steps (freeze drying, supercritical CO2 foaming, ... in foams was in a supercoiled form This kind of DNA conformation may trigger the penetration of DNA molecules into the deeper layer of K20 coating on foam F2 due to the long chain of K20, instead of just being anchored on the top of K20 coating This is likely to be the reason for slower release of DNA in F2 compared with F1 In summary, two main factors determine the adsorption efficacy of DNA onto PLGA... cytotoxicity in control and F0 may be attributed to the high initial concentration in the control group and a burst release of DNA during the initial hours from F0 Intense transfection of DNA may impose damage on cell 173 Chapter 8 membrane and lead to cell death in serious cases As the amounts and released rates of DNA from F1 and F2 are much lower than F0 during the first 3 days, no such kind of negative effect... charge interactions and conformational structure of the peptides These two factors determine the amount of DNA adsorbed and the subsequent release profiles Three-dimensional foams have been widely investigated regarding their applications in tissue engineering and it has been proved that polymeric foams are promising candidates for tissue engineering (Chun et al., 2004; Nie et al., 2008a; Takahashi and. .. more sustained in comparison to the blank foam F0 The positively charged surface of the functionalized foams appeared to be favorable for loading DNA and displayed sustained release of DNA, possibly due to a balance of electrostatic interaction and hydrophilic interaction between DNA and the surface of F1 or F2 The sustained release of DNA from F1 and F2 led to negligible cytotoxicity and sustained expression... detected in F0 than F1 and F2 on day 5 and day 7, respectively Particularly, F2 presented a sustained expression of BMP-2 over the testing period of 7 days On day 7, the expression level in F2 was almost 1-fold higher than those 175 Chapter 8 displayed in F0 and F1 This significant enhancement and sustainability in expression level should be attributed to the higher DNA adsorption capacity of F2 and its controlled. .. distinct bands present on the gel (Figure 8.4b) In summary, the released DNA survived both the adsorbing and releasing processes Moreover, it was shown that the plasmid DNA encapsulated in the different foams was released in a supercoiled form within the time scale of 5 days, and was independent of the types of foams (Figure 8.4b) This condensation of DNA in size may trigger the interactions between DNA and. .. Schmieder et al., 20 07) However, few have reported about Lysine-based peptides modified foams for tissue engineering and DNA delivery Toward realizing an effective dual system for tissue engineering and DNA delivery, our work presents intriguing findings which may have significant impact for tissue engineers and scientists for improving the surface properties of PLGA, one of the most promising biomaterials,... with the release profile of F0, the release profiles of F1 and F2 are relatively linear and sustainable The constant rate of the in vitro release processes suggested that there exist some interactions between the entrapped DNA molecules and the micro-porous PLGA matrix supplemented with -NH2 functional groups Otherwise, the percentage release rates for F1 and F2 should be higher than F0 as F1 and F2 . expression of target protein is too low, especially in the initial stage (Figures 7. 5a and 7. 7b). Therefore, an initial and significant release of DNA is demanded in order to optimize this kind of. DNA and subsequently release it in a sustained manner. PLGA/chitosan composite foams developed in Chapter 7 show promising results in controlled release of DNA, but the release rate of DNA and. the release profile of F0, the release profiles of F1 and F2 are relatively linear and sustainable. The constant rate of the in vitro release processes suggested that there exist some interactions