Novel biodegradable cationic core shell nanoparticles for codelivery of drug and DNA 4

Chapter Results and Discussion 4.1 Polymer Synthesis 4.1.1 Synthesis and characterization of N-(2-bromoethyl) carbarmoyl cholesterol (Be-chol) N-(2-bromoethyl) carbarmoyl cholesterol (Be-chol) has a bromoethyl group that was used to quaternize the main chain at the amino group and produce positive charges at the same sites. Be-chol was also designed as the random dispersed hydrophobic pendant chains. It was synthesized by connecting bromoethylamine onto the cholesterol molecule through the amidation reaction with cholesteryl chloroformate as showed in Scheme 1. Be-chol was obtained in yield of ~ 78% after twice consecutive-purification by recrystallization with ethanol and acetone, respectively. TLC analysis showed one point at Rf of 0.68 in the mixture of toluene, hexane and methanol (8:8:1), indicating that Be-chol was pure. Figure 4.1 and Figure 4.2 display the 1H-NMR and FTIR spectra of purified Be-chol, respectively. As showed in Figure 4.1, the 1H peak at δ 5.10 (Signal HN) was due to the amide groups (CONH) (See Fig. 4.1). δ 3.60 (Signal H4) and 3.61 (Signal H5) were attributed to the 2-bromoethyl groups. δ 4.52 (H1) and 5.40 (H2) were associated with the cholesterol units. The ratio of the H1, H2, HN, H4 and H5 peak areas was determined to be 1:1:1:2:2, confirming the successful synthesis of Be-chol. The FTIR spectrum of Be-chol also evidenced its successful synthesis. The IR peak at 3325 cm-1 68 was due to -NH- stretching (see Figure 4.2). Peaks from -C=O stretching and -NHbending overlapped at 1685 cm-1. The peak at 1536 cm-1 was attributed to -C-N- stretching. In summary, pure Be-chol was successfully synthesized. The purity of Be-chol is important for its further grafting onto the main chain. For instance, the impurity, cholesteryl formic acid, may act as a catalyst to promote the hydrolysis of the main chain (PMDS or PMDA). H3C CH3 H3 C H3C CH3 H O d Br e H NH b O c f H a Figure 4.1 1H-NMR spectrum of N-(2-bromoethyl) carbarmoyl cholesterol (Be-chol). 69 Figure 4.2 FTIR spectrum of N-(2-bromoethyl) carbarmoyl cholesterol (Be-chol). 4.1.2 Synthesis and characterization of PMDS and PMDA Poly(N-methyldiethylamine sebacate) (PMDS) and Poly(N-methyldiethylamine adipate) (PMDA) are the main chains of the designed polymers. The successful synthesis of PMDS was verified by 1H-NMR and FTIR spectra as shown in Figures 4.3 and Figure 4.4, respectively. NMR peaks at δ 2.71–2.73 (signal a), δ 1.62 (signal b) and δ 1.32 (signals c and d) were attributed to the protons of four different -CH2- groups from the sebacate units (see Figures 4.3). Peaks at δ 4.17–4.19 (signal e) and δ 2.30-2.37 (signals f and g) were due to protons of two different -CH2- groups and the -CH3 group linked to the nitrogen atom. IR spectrum also confirmed the polyester formation (see Figure 4.4). The -C=O stretching shifted to a lower wavenumber (1736 cm-1) compared to carbonyl halide (1805 cm-1) due to the inductive effect of halide. The peak at 1172 cm-1 was attributed to C-O. 70 The 1H-NMR and FTIR spectra of PMDA confirmed its successful synthesis (Figure 4.5 and Figure 4.6). NMR peak at δ 1.63 (signal b) was attributed to the -CH2- groups of adipate, which were connected to another two -CH2- groups. Peaks at δ 2.67-2.76 (signal a) were attributed to the -CH2- groups of adipate (Figure 4.5), which were connected to carboxyl group and another -CH2- group. Peaks at δ 2.27-2.37 (signals d and e) came from the protons of the -CH2- and -CH3 groups connected with the nitrogen. Peaks at δ 4.1-4.2 (signal c) were attributed to the -CH2- groups connected to the oxygen. The FTIR spectrum of PMDA also evidenced its successful synthesis (Figure 4.6). Stretching vibration of C-O at 1174 cm-1 and stretching vibration of C=O at 1732 cm-1 were observed, indicating the existence of ester group in the polymer. From the 1H-NMR spectra of PMDS and PMDA, no impurity peaks were observed, especially the peaks of triethylamine that may influence the subsequent reaction. In addition, it indicates that the purification method applied was suitable and effective. O b C a d c O C f O e N O n CH3 g Figure 4.3 1H-NMR spectrum of PMDS. 71 Figure 4.4 FTIR spectrum of PMDS. O b C a O C d O c N O n CH3 e Figure 4.5 1H-NMR spectrum of PMDA. 72 Figure 4.6 FTIR spectrum of PMDA. 4.1.3 Synthesis and characterization of P(MDS-co-CES) and P(MDA-co-CEA) The synthesis of P(MDS-co-CES) and P(MDA-co-CEA) was performed by grafting Be-chol onto PMDS and PMDA through quaternization reaction. This reaction needs to be performed at a relatively high temperature when alkyl bromide is used as the reagent for quaternization. The purposes to introduce the cholesteryl group onto PMDS and PMDA are to use the cholesteryl group as the core-forming block and to produce positive charges on the main chain. The successful synthesis of P(MDS-co-CES) and P(MDA-coCEA) was evidenced by 1H-NMR and FTIR spectra. Figure 4.7 and Figure 4.8 show the H-NMR and FTIR spectra of P(MDS-co-CES), respectively. The 1H-NMR spectrum of P(MDS-co-CES) illustrates peaks at δ 2.7–2.8 (signal a), 1.5–1.7 (signal b), 1.2–1.4 (signals c and d), 4.0–4.2 (signal e) and 2.2–2.4 (signals f and g) due to protons from the PMDS main chain (Figure 4.7). Various peaks at δ 0.7–1.2 were attributed to the cholesterol groups. The peak at δ 5.38 arose from the proton of =CH- in the cholesterol 73 groups (signal h). The peak at δ 0.7 was from the methyl group directly linked to the cyclic hydrocarbon (signal i). The information provided by the 1H-NMR spectrum of P(MDS-co-CES) proved that the cholesteryl group was successfully grafted onto the PMDS main chain. Figure 4.8 shows the FTIR spectrum of P(MDS-co-CES), which also evidenced the successful quaternization. The peak at 1252 cm-1 was attributed to C-N stretching of amine. The shift and increased intensity of this peak compared with that of PMDS (1240 cm-1) illustrated the formation of a quaternary ammonium salt. The 1H-NMR and FTIR spectra of P(MDA-co-CEA) also gave similar results as P(MDS-co-CES). As shown in Figure 4.9, the wide peak at δ 2.66 (signal a) and the peak at δ 1.67 (signal b) were from the protons of the methylene groups (-CH2-) in the adipate segments. The multiple peaks at δ 4.0-4.2 (signal c) came from the methylene group (CH2-) in the N-methyldiethanolamine segments. Signal d of another methylene group linked to the nitrogen atom of N-methyldiethanolamine was overlapped with signal e of the methyl group directly linked to the nitrogen at δ 2.3-2.4. The peaks at δ 0.7-1.2 were from the cholesterol group. In particular, the peaks at δ 5.37 (signal f) and δ 0.69 (signal g) came from the protons linked to the carbon with double bond (=CH-) and the methyl group linked to the cyclic hydrocarbon, respectively. Moreover, Figure 4.10 illustrates the peak at 1251 cm-1 from the C-N stretching of amine. The shift and increased intensity of this peak compared with that of PMDA (1240 cm-1) illustrated the formation of a quaternary ammonium salt. The degree of grafting (Rg), defined as the ratio of the amines quaternized by N-(2bromoethyl) carbarmoyl cholesterol to the total number of amines on the PMDS main chain, can be estimated as follows, 74 Rg = (∆ApNHm/∆AmNHp) × 100%, Where ∆Ap is the area of the selected peak from the pendant chain, ∆Am is the area of the selected peak from the main chain, NHp is the number of hydrogen atoms in the selected group from the pendant chain, and NHm is the number of hydrogen atoms in the selected group from the main chain. Only suitable protons from the pendant chain and the main chain of the polymers were selected in the calculation. The proton signal selected should not overlap with signals from other protons. Furthermore, those protons affected by the quaternized amines should not be used. For P(MDS-co-CES), the proton of the methylene group linked to the carbonyl group of the main chain (signal a), as well as the proton of the methylidyne group (-CH=) linked to the double bond (signal h) and the proton of the methyl group linked to the hexane and pentane cycles of the pendant chain (signal i) were considered suitable for use in the estimation of Rg. For the proton of methylene linked to the carbonyl group overlapped with other signals. The proton of methylene linked to the ester (O=C-O-CH2-, signal c) on the main chain was chosen since this proton is far from the nitrogen atom of the quaternary ammonium. The inductive effect of the quaternary ammonium on the proton was neglected. For the pendant chain, the same protons were used as P(MDS-co-CES) (i.e. signal f and signal g). Based on the peak areas of signal a and signal i, Rg for P(MDS-co-CES) (Batch No. 120902b) was estimated to be 27.0% (i.e. Rg=∆AHi×4×100%/3×∆AHa=2.046×4×100%/3×10.1=27.0%). Based on the peak areas of signal c and signal g, Rg for P(MDA-co-CEA) (Batch No. 110102b) was estimated to be 56.0% (i.e. Rg=∆AHg×4×100%/3×∆AHc=2.59×4×100%/6.17×3=56.0%). By changing the molar ratio of the pendant chain to the PMDS or PMDA main chain, Rg 75 of the cholesterol moiety and the positive charge of P(MDS-co-CES) could be modulated. The polymers with different cholesteryl grafting degree were synthesized by changing the amount of Be-chol precursor. The purity of PMDS and PMDA may influence the grafting degree of Be-chol onto PMDS and PMDA. For example, the residue of triethylamine added to absorb HCl could undergo quaternization reaction with Be-chol. Therefore, even a small amount of triethylamine and its salt form can affect the grafting reaction significantly. CH3 H3C i H3C CH3 H H3C H H O h O g O C a c b d O C O NH CH e f N O q O O C C O Br + N CH3 O p Figure 4.7 1H-NMR spectrum of P(MDS-co-CES). 76 Figure 4.8 FTIR spectrum of P(MDS-co-CES). CH3 H3C g H3C CH3 H H3C H H O O C O a b C O NH CH c d N f O e O q O O C C O Br + N CH3 O p Figure 4.9 1H-NMR spectrum of P(MDA-co-CEA). 77 1.0E+10 1.0E+09 1.0E+08 RLU/mg protein 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00 Control 10 15 20 25 30 N/P ratio Figure 4.84 Luciferase expression level in HEK293 cells transfected with P(MDS-coCES)/DNA complexes of N/P ratio = 15 (Control, Batch No. 120902b), PMDS nanoparticles fabricated via dialysis at the N/P ratios specified. (Polymer Batch No. 310803) 1.0E+09 1.0E+08 RLU/mg protein 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00 Control 10 15 20 25 30 N/P ratio Figure 4.85 Luciferase expression level in HepG2 cells transfected with P(MDS-co-CES) /DNA complexes of N/P ratio = 15 (Control, Batch No. 120902b), PMDS nanoparticles fabricated via dialysis at the N/P ratios specified. (Polymer Batch No. 310803) 162 1.0E+09 1.0E+08 RLU/mg protein 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00 Control 10 15 20 25 30 N/P ratio Figure 4.86 Luciferase expression level in HeLa cells transfected with P(MDS-co-CES) micelles/DNA complexes of N/P ratio = 25 (Control, Batch No. 010704), PMDS nanoparticles fabricated via dialysis at the N/P ratios specified. (Polymer Batch No. 310803) 4.7.3 In vitro gene transfection of PEG-P(MDS-co-CES) Pegylation of P(MDS-co-CES) causes another great change in polymer structure. Since PEG is excellent hydrophilic polymer, the conjugated PEG is expected to arrange at the surface of the micelles and serve as the shell at the outmost. As discussed in the previous sections of this chapter, PEG conjugation improved the stability of the micelles but weakened the DNA binding ability of the micelles. In order to study the influence of PEG on in vitro gene transfection, PEG2000-P(MDS-co-CES) micelles were used as a vector. PEG2000-P(MDS-co-CES) (Batch No. 051103a) had Mw of 3900 Da, Mn of 2600 Da and cholesterol grafting degree of 16% and PEG content of 7.6% (see Table 4.1). The 163 micelles were prepared by dialysis in 0.02M sodium acetate buffer with pH 4.6. The particle size was 161 nm before filtration using 0.22 µm filter. Figure 4.87, Figure 4.88 and Figure 4.89 show luciferase expression level in HEK293, HepG2 and HeLa cells respectively. The luciferase expression level increased with increasing the N/P ratio and did not reach the highest level at the N/P ratio of 30. This indicates that the highest gene transfection level may occur at an N/P ratio higher than 30. At the same N/P ratio, PEG2000-P(MDS-co-CES) yielded lower luciferase expression level than P(MDS-co-CES) in all the three cell lines. The results are in agreement with the difference in the DNA binding ability between PEG2000-P(MDS-co-CES) and P(MDS-co-CES). In addition, PEG2000-P(MDS-co-CES) also showed cell specificity in gene transfection in comparison with P(MDS-co-CES). For example, the luciferase expression level was much higher in HEK293 and HepG2 cells than in HeLa cells. In summary, P(MDS-co-CES) micelles provided high gene transfection efficiency in various cell lines. PEG2000-P(MDS-co-CES) micelles also yielded reasonably high gene transfection efficiency. Coupling with their good stability in the blood and lower toxicity compared to PEI, PEG2000-P(MDS-co-CES) micelles may make a promising carrier for systemic delivery of drug and gene. 164 1.0E+10 1.0E+09 1.0E+08 RLU/mg protein 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00 Control 10 15 20 25 30 N/P ratio Figure 4.87 Luciferase expression level in HEK293 cells transfected with P(MDS-coCES)/DNA complexes of N/P ratio = 15 (Control, Batch No. 120902b), PEG2000P(MDS-co-CES) micelles fabricated via dialysis at the N/P ratios specified. (Polymer Batch No. 051103a) 1.0E+09 1.0E+08 RLU.mg protein 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00 Control 10 15 20 25 30 N/P ratio Figure 4.88 Luciferase expression level in HepG2 cells transfected with P(MDS-coCES)/DNA complexes of N/P ratio = 15(Control, Batch No. 120902b), PEG2000P(MDS-co-CES) micelles fabricated via dialysis at the N/P ratios specified. (Polymer Batch No. 051103a) 165 1.0E+09 1.0E+08 RLU/mg protein 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00 Control 10 15 20 25 30 N/P ratio Figure 4.89 Luciferase expression level in HeLa cells transfected with P(MDS-coCES)/DNA complexes of N/P ratio = 25 (Control, Batch No. 010704), PEG2000P(MDS-co-CES) micelles fabricated via dialysis at the N/P ratios specified. (Polymer Batch No. 051103a) 4.8 In vitro synergistic effect 4.8.1 Synergistic effect of cyclosporin A and luciferase gene in KB-31-MA cells Cyclosporin A [Parkar M. H. et. al., 2004], an immunosuppressive agent, was reported to suppress the efflux pump function of P-glycoprotein in cancer cells [Saito T. et. al., 2004]. In this experiment, both cyclosporin A and luciferase-encoded plasmid were incorporated into P(MDS-co-CES) micelles (Polymer Batch No. 010704) and delivered to MB-31-MA cells to improve gene transfection. Loading of cyclosporin A (5mg) into the core of polymeric micelles (15mg) were performed by dialysis against 0.02M sodium acetate buffer with pH 4.6. The dialysis process was similar to loading of pyrene and indomethacin. The encapsulation efficiency measured by HPLC was 50.4%. The gene expression level of codelivery system was compared with blank micelles/DNA 166 complexes. PEI/DNA complexes at N/P 10 and the naked DNA were used as controls. The results were shown in Figure 4.90. With increasing the N/P ratio from to 10, the cyclosporin A-loaded micelles/DNA complexes mediated an increased luciferase expression level, which reached the highest at the N/P ratio of 15. The highest transfection level achieved by the cyclosporin A loaded micelles was even higher than that of PEI. However, for the blank micelles, the tansfection level maintained almost as low as that of naked DNA. This indicates that the P-glycoprotein in the membrane seriously prevented the micelles from being taken up by the cells. Therefore, the micelles can be a very successful codelivery system of cyclosporin A to inhibit the efflux effect of cells. The results obtained in this section show again that the gene expression level is dominated by the uptake efficiency of the cells. Moreover, putting cyclosporin A and the plasmid in one single system (i.e. the cationic micelles) improved water-solubility of cyclosporin A and protected it against degradation caused by enzymes existing in the blood. By doing so, it is also possible to target both cyclosporin A and the plasmid to the same cell type. 167 Luciferase expression (RLU/mg) 1.E+08 1.E+07 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 Control 10 15 20 25 N/P Ratio Blank micelles Cyclosporin A loaded micelles Figure 4.90 Luciferase expression level in MB-31-MA cells transfected with PEI/DNA complexes of N/P ratio = 10 (black bar, control), naked DNA (white bar, control) P(MDS-co-CES) micelles loaded with and without cyclosporin A fabricated via dialysis at the N/P ratios specified. (Cyclosporin A concentration: 1.0, 5.0, 10.0, 15.0, 20.0 and 25 µg/ml with N/P ratio increasing from to 25 Polymer Batch No. 010704) 4.8.2 Synergistic effect of paclitaxel and luciferase gene/GFP gene in 4T1 cells Paclitaxel, a kind of anti-mitotic drug was reported to be able to enhance gene expression in some cell lines [Son K. et. al., 1996; Margit Maria Janat-Amsbury et. al., 2004]. Specially, Margit Maria Janat-Amsbury et. al. has reported the combined effect of interleukin 12 gene and paclitaxel in a metastatic breast cancer model (4T1 cells)[Maria Janat-Amsbury et. al., 2004]. In this section, the paclitaxel will be applied as the co-agent to study synergistic effect between the drug and the luciferase/GFP gene expression using P(MDS-co-CES) micelles as codelivery system in 4T1 cells. The paclitaxel was incorporated into micelles via dialysis. The procedure of loading paclitaxel is similar to that for loading cyclosporin A. The loading level was measured by UV (see section 3.4.3). The luciferase gene expression level and GFP expression efficiency of blank 168 micelles and paclitaxel loaded micelles were shown in figure 4.91. The histogram statistics and the fluorescent photos of the GFP transfected cells are shown in figure 4.92 and figure 4.93 as side evidences. Figure 4.91 shows that the paclitaxel loaded micelles has much higher luciferase gene expression level than the blank micelles (left). The highest level achieved by paclitaxel loaded micelles was 13 times higher than that achieved by blank micelles. This shows that the enhancement effect was very obvious in 4T1 cells. Similarly, the percentages of GFP gene transfected cells have also been improved abruptly (figure 4.91, right). The highest value was improved from 10% to 30%. The histogram statistics picture and fluorescent photos clearly evidenced this result. Summarily, the results obtained above shows that the micelles is a kind of efficient vehicle to codeliver hydrophobic drug and gene. The synergistic effect can be simply 1.E+10 1.E+08 1.E+06 1.E+04 1.E+02 1.E+00 10 15 20 25 N/P ratio Polymeric micelles+PCT Polymeric micelles eGFP tranfection efficiency (%) L u cife s e e x p re ss io n (R L U /m g ) realized by codelivery of the drug and gene. 35 30 25 20 15 10 10 15 20 25 N/P ratio Blank micelles paclitexal loaded micelles Figure 4.91 Luciferase expression level (left) and GFP expression efficiency (right) in 4T1 cells transfected with P(MDS-co-CES) micelles/DNA complexes loaded with and without paclitaxel fabricated via dialysis at the N/P ratios specified. (Paclitaxel concentration: 1.1, 5.6, 11.2, 16.8, 22.4 and 28.0 µg/ml for luciferase expression, and 1.5, 7.8, 15.7, 23.5, 31.4, 39.2μg/ml for GFP expression with N/P ratio increasing from to 25, Polymer Batch No. 010704) 169 Blank cell N/P 15 20 Blank nanoparticles 25 N/P 15 20 25 Paclitaxel loaded nanoparticles Figure 4.92 Histogram statistics pictures of GFP expression efficiency in 4T1 cells transfected with P(MDS-co-CES) micelles/DNA complexes loaded with and without paclitaxel obtained by flowcytometer. 170 N/P N/P 15 20 Blank Nanoparticles 25 15 20 25 Paclitaxel loaded nanoparticles Figure 4.93 Fluorescent photographs of 4T1 cells transfected by GFP gene enhanced by paclitaxel obtained by fluorescent microscope. 4.9 In vivo gene transfection of P(MDS-co-CES) Since PEG-grafted P(MDS-co-CES) assembled the feature of P(MDS-co-CES) in in vitro gene transfection. In vivo gene transfection studies were performed for proof of the principle using P(MDS-co-CES) in the cochlea of guinea pigs and a mouse breast tumor model. 4.9.1 Gene delivery to cochlea of guinea pig P(MDS-co-CES) (Batch No. 260304) used in the cochlea of guinea pigs had Mw of 7900 Da, Mn of 3300 Da and cholesterol grafting degree of 27%. The micelles prepared in 0.02M sodium acetate buffer with pH 4.6 had an average diameter of 267 nm before filtration using 0.2 µm filter. The micelles/DNA complexes were fabricated at the N/P 171 ratio of 10. The dose of DNA was µg per animal. The cochlea was transfected positively with the micelles/DNA complexes in six of nine animals and with naked DNA in one of seven animals. In five of six guinea pigs transfected with the micelles/DNA complexes, luciferase expression was found in both ears. This means that the left ear of guinea pig was treated with the DNA complexes but gene expression was found in both the left and right ears. Figure 4.94 shows luciferase expression level in both the left and right inner ears of five guinea pigs after 24 hours transfection of naked DNA and the micelles/DNA complexes. The inner ears of five animals without any treatment were used as the background. The luciferase level obtained from the animals treated with naked DNA and the micelles/DNA complexes was subtracted with the background to get the final readings. As shown in Figure 4.94, the luciferase expression level induced by the micelles/DNA complexes was much higher compared to naked DNA. It was 2.1×105 RLU/mg protein for the micelles/DNA complexes and 8.9×103 RLU/mg for naked DNA. Figure 4.95 displays positive GFP expression in the cochlea of guinea pigs after 24 hours transfection by GFP-plasmid complexed to the micelles. This means that the complexes were able to cross the round window membrane, achieving positive luciferase and GFP expression. The cationic micelles may make a promising vector for in vivo gene delivery to the cochlea for correcting hearing loss. 172 3.5E+05 RLU/mg protein 3.0E+05 2.5E+05 2.0E+05 1.5E+05 1.0E+05 5.0E+04 0.0E+00 polymeric micelles/DNA Naked DNA Figure 4.94 Luciferase expression level in the cochlea of guinea pigs after 24 hours transfection. (Polymer Batch No. 260304, n=5) Figure 4.95 GFP expression in the cochlea of guinea pigs, observed under a fluorescent microscope. (Polymer Batch No. 151002) 4.9.2 Gene delivery to tumor P(MDS-co-CES) micelles (Batch No. 010704) were used as a vector to deliver luciferase-encoded plasmid to tumor-bearing balb/C mice by local injection at the tumor 173 site and i.v. injection through tail vein. The micelles prepared in 0.02M sodium acetate buffer with pH 4.6 had an average diameter of 110 nm after filtration using 0.2 µm filter. The micelles/DNA complexes were fabricated at different N/P ratio. The dose of DNA was µg per animal for the local injection and 20 µg per animal for the i.v. injection. Figure 4.96 displays that luciferase expression level at the tumor site changed as a function of N/P ratio after 48 hours post local injection. An increased N/P ratio resulted in greater gene transfection efficiency. In addition, the micelles/DNA complexes mediated higher gene expression level than PEI/DNA complexes. Figure 4.97 shows organ distribution of luciferase expression after 48 hours post i.v. injection (upper) and the percentage expression in different organ (lower left) and the total expression level (lower right). The luciferase expression level mediated by PEI/DNA complexes was found to be the highest in the lung, which is consistent with the data reported by T. Merdan et. al. (2002). Unlike PEI, the gene expression level induced by the micelles/DNA complexes was distributed quite evenly in the various organs. This indicates that P(MDS-co-CES)/DNA complexes were more stable in the blood than PEI/DNA complexes. Furthermore, the luciferase expression level of P(MDS-coCES)/DNA complexes at the tumor site was higher than that of PEI/DNA complexes at the N/P ratios tested. This may be because of their greater stability in the blood and/or EPR effect at tumor. The calculation of the percentage expression in different organs has proved the serious entrapment of the PEI/DNA complexes in the lung (figure 4.97, lower left). As shown in the figure, 90% of the gene expression occurred in the lung for PEI complexes while for the micelles complexes, except N/P ratio 1, the expression occurred in the lung is less than 24%. At the same time, the expression of micelles in tumor was 174 much higher than that of PEI. Considering that the total expression level of micelles was lower than PEI (figure 4.97, lower right), the relatively high expression level in tumor is especially significant. This indicates that the micelles complexes possess much higher passive targeting ability than PEI complexes. According to the studies performed previously, this could be relevant with the high stability of the micelles in blood. In summary, compared to PEI, the polymeric micelles/DNA complexes can avoid serious entrapment by the lung and possess strong passive targeting ability. These properties are very possibly related with the stability of the complexes provided by the stable core-shell structure of the micelles. If DNA binding ability of the micelles can be improved further by introducing secondary and primary amine, the micelles can be a very promising gene vector. 8000 RLU/mg protein 7000 6000 5000 4000 3000 2000 1000 PEI 10 15 20 25 N/P ratio Figure 4.96 Luciferase expression level at the tumor site after 48 hours post local injection. (Polymer Batch No. 010704, n=5) 175 1.0E+06 1.0E+05 RLU/org 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00 Tumor Heart Lung N/P=1 Liver N/P=5 N/P=10 Kidney PEI 1.4E+05 100% 90% 1.2E+05 80% 1.0E+05 70% Total RLU/aminal Percentage expression in different organ Spleen 60% 50% 40% 8.0E+04 6.0E+04 4.0E+04 30% 20% 2.0E+04 10% 0.0E+00 0% Tumor Heart Lung Liver Spleen kidney 10 PEI N/P ratio N/P1 N/P5 N/P10 PEI Figure 4.97 Luciferase expression level (upper), percentage expression (lower left) in various organs and total expression level (lower right) after 48 hours post tail vein injection (Polymer Batch No. 010704, n=5) 4.10 In vivo synergistic effect Similar to the in vitro synergistic effect realized in 4T1 cells by codelivery of paclitaxel and luciferase gene, the in vivo synergistic effect was studied by local injection of the paclitaxel loaded micelles/DNA complexes into the 4T1 tumor model established subcutaneously in the side belly of balb/C mice. Into each mouse 30μl of micelles complexes carrying 2μg of DNA was injected. The results were shown in figure 4.98. As shown in this figure, the expression level in tumor of paclitaxel loaded micelles was 16 times higher than that of blank micelles. The enhancement of the gene expression by 176 paclitaxel was very obvious. This shows that the micelles are an excellent vehicle for codelivery of hydrophobic drug and gene to realize the synergistic effect not only in vitro but also in vivo. 1.0E+06 RLU/tumor 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00 10 15 20 N/P ratio PCT loaded nanoparticles Blank nanoparticles 25 Figure 4.98 In vivo luciferase gene expression enhancement by paclitaxel obtained by local injection into 4T1 tumor established in balb/C mice. [P value ( student’s t test) between PCT loaded and blank micelles at N/P 5, 10, 15, 20, 25 were 0.0011, 0.038, 0.038, 0.0062, 0.05 respectively, P[...]... 240 0 1.3 / 46 .7 300603 PMDA 49 00 3200 1.5 / / 120803 PMDA 5100 3200 1.6 / / 310803 PMDS 7300 3700 2.0 / / 191003a P(MDS-co-CES) (310803)* 48 00 340 0 1 .4 / 13.6 191003b P(MDS-co-CES) (310803)* 46 00 2900 1.6 / 9 .4 90 301003 PMDS 12500 5800 2.1 / / 110204a P(MDS-co-CES) (301003) 540 0 2900 1.8 110204b P(MDA-co-CEA) (120803)* 1700 1600 1.0 / 34. 4 040 3 04 PMDS 11200 48 00 2.3 / / 2603 04 P(MDS-co-CES) ( 040 3 04) *... 0.001g/L 600 40 0 300 200 40 0 300 200 100 100 0 0 360 370 380 390 40 0 41 0 42 0 43 0 λ (nm) 300 310 320 330 340 350 360 λ (nm) Figure 4. 34 Fluorescence spectra of pyrene in aqueous solutions of P(MDA-co-CEA) at different concentrations (left, λem=339 nm for the emission spectra; right, λex=390 nm for the excitation spectra) 4. 2.2 Size and stability of the polymeric micelles Size and zeta potential of the micelles... The results are shown in Figure 4. 31 and Figure 4. 32 From Figure 4. 31, the CMC (I1/I3) of PEG5000-P(MDS-co-CES) in deionized water, the acetate buffer (0.02M) with pH of 5.6 and 4. 6 was 42 .7, 74. 7 and 74. 3 mg/L respectively The CMC (I338/I333) in deionized water, the acetate buffer (0.02M) with pH of 5.6 and 4. 6 was 13.2, 18.8 and 13.2 mg/L respectively Similar with that of P(MDS-co-CES), pH did not pose... temperature of P(MDS-co-CES) and P(MDA-co-CEA) was 73.5ºC and 24. 3ºC, respectively The melting point of P(MDS-co-CES) was also observed at 138.2ºC Figure 4. 21 TGA spectrum of PMDA (Batch No 110902) Figure 4. 22 TGA spectrum of P(MDA-co-CEA) (Batch No 111002a) 86 Figure 4. 23 TGA spectrum of PMDS (Batch No 120902a) Figure 4. 24 TGA spectrum of P(MDS-co-CES) (Batch No 120902b) Figure 4. 25 DSC spectrum of P(MDS-co-CES)... spectrum of PEG5000-PMDS 80 Figure 4. 15 1H-NMR spectrum of PEG5000-PMDA Figure 4. 16 1H-NMR spectrum of PEG550-P(MDS-co-CES) 81 Figure 4. 17 1H-NMR spectrum of PEG1100-P(MDS-co-CES) Figure 4. 18 1H-NMR spectrum of PEG2000-P(MDS-co-CES) 82 Figure 4. 19 1H-NMR spectrum of PEG5000-P(MDS-co-CES) Figure 4. 20 1H-NMR spectrum of PEG5000-P(MDA-co-CEA) 4. 1.5 Molecular weight, grafting degree as well as PEG contents of. .. properties of PMDA, P(MDA-co-CEA), PMDS and P(MDS-co-CES) polymers were studied using TGA and DSC Figure 4. 21 to Figure 4. 24 show the TGA spectra of PMDA and P(MDAco-CEA), PMDS and P(MDS-co-CES), respectively The maximal degradation temperature of P(MDS-co-CES) and P(MDA-co-CEA) appeared at 339.7ºC and 339.8ºC, respectively while those of PMDS and PMDA occurred at temperatures higher than 40 0ºC The... because of the greater hydrophilictity of the shell- forming segment Figures 4. 33 and 4. 34 provide direct observation of the spectra change of pyrene with the change of P(MDS-co-CES) and P(MDA-co-CEA) concentration in deionized water From these figures, the I3 peak of the emission spectra can be observed to increase greatly with the increase of concentration while in the excitation spectra the slope of the... conjugation of PEG onto PMDS and PMDA changed the polarity of the polymer and the compatibility of the polymer with toluene, which restricted fully stretching of the polymer chain in toluene and thus provide steric hindrance to the quaternization reaction Figure 4. 11 1H-NMR spectrum of PEG550-PMDS 79 Figure 4. 12 1H-NMR spectrum of PEG1100-PMDS Figure 4. 13 1H-NMR spectrum of PEG2000-PMDS Figure 4. 14 1H-NMR... out of the body through the renal system during the degrading process and prevent the accumulation of the foreign materials in the body The in vitro degradation tests of P(MDS-co-CES) and P(MDA-co-CEA) were performed in PBS (pH 7 .4) Figure 4. 27 shows the weight loss of the polymers as function of incubation time On the third day, the weight of P(MDS-co-CES) slightly increased because of water uptake and. .. with pH of 4. 6 and 5.6 was similar (1.5 mg/L versus 1.9 mg/L) In addition, the CMC of P(MDS-co-CES) in the sodium acetate buffer (0.1M) was similar to that in the buffer (0.02M) with a lower ionic strength (see Figure 4. 28 and Figure 4. 29) These findings suggest that pH and ionic strength did not affect the CMC significantly This property is important for DNA binding of the polymer at low pH and for in . 73 Figure 4. 6 FTIR spectrum of PMDA. 4. 1.3 Synthesis and characterization of P(MDS-co-CES) and P(MDA-co-CEA) The synthesis of P(MDS-co-CES) and P(MDA-co-CEA) was performed by grafting. stretching of amine. The shift and increased intensity of this peak compared with that of PMDS (1 240 cm -1 ) illustrated the formation of a quaternary ammonium salt. The 1 H-NMR and FTIR spectra of. Figure 4. 11 1 H-NMR spectrum of PEG550-PMDS. 80 Figure 4. 12 1 H-NMR spectrum of PEG1100-PMDS. Figure 4. 13 1 H-NMR spectrum of PEG2000-PMDS. Figure 4. 14 1 H-NMR spectrum of PEG5000-PMDS.