Improving the effective delivery of cisplatin anti cancer drug of dendrimer nanocarrier

e), δH = 2.48 - 2.46 ppm (g) and δH = 3.68 - 3.69 ppm (h). 1H -NMR PAMAM G3.0: at δH = 2.61 - 2.62 ppm (a), δH = 2.80 -2.83 ppm (b), δH = 2.38 - 2.40 ppm (c), δH = 2.74 - 2.76 ppm (d) and δH = 3.26 -3.29 ppm (e). 1H -NMR PAMAM G3.5: at δH = 2.57 -2.64 ppm (a), δH = 2.84-2.85 ppm (b), δH = 2.38 -2.43 ppm (c), δH = 3.27 -3.37 ppm (e), δH = 2.48 -2.51 ppm (g) and δH = 3.69 ppm (h). 1H -NMR PAMAM G4.0: at δH = 2.59 -2.62 ppm (a), δH = 2.80 -2.83 ppm (b), δH = 2.39 – 2.40 ppm (c), δH = 2.74 – 2.76 ppm (d) and δH = 3.26 -3.28 ppm (e). 1H -NMR PAMAM G4.5: at δH = 2.57 - 2.65 ppm (a), δH = 2.84 – 2.85 ppm (b), δH = 2.39 – 2.42 ppm (c), δH = 3.27 - 3.31 ppm (e), δH = 2.47 - 2.50 ppm (g) and δH = 3.69 ppm (h). 8 Figure 3.1. 1H-NMR spectrum of various PAMAM Dendrimer generation Thoughout the integral ratios of 2 peaks of protons at (a) and (e) on the 1H-NMR of dendrimer molecules (χNMR) and the intergal ratio of the number of the protons at (a) and (e) in the theorical dendrimer structure (χL.T), the molecular weight of dendrimers can be established following the below equation: M(NMR) = χNMR χLT .MLT = SH(-CH2-) (e) SH(-CH2-) (a) ∑ H(-CH2-) (e) ∑ H(-CH2-) (a) .MLT In which: SH(-CH2-) (e) , SH(-CH2-) (a) : the peak areas of protons at (a) and (e) in 1H-NMR ∑ H(-CH2-) (e) , ∑ H(-CH2-) (a) : the sums of protons at the (e) and (a) position s in the molecular formula of PAMAM dendrimer. MLT : the theoretical molecular weight of PAMAM dendrimer. The results were calculated according to: Table 3.1. Calculated molecular mass of Dendrimer following 1H-NMR. ෍ H(-CH2-) (e) ෍ H(-CH2-) (a) χLT M(LT) χNMR M(NMR) Different (%) G-0.5 8 (b) 4 2 404 2.01 405.62 0.40% G0 8 4 2.00 517 1.99 515.02 0.32% 9 G0.5 8 12 0.67 1205 0.67 1205.42 0.06% G1.0 24 12 2.00 1430 1.95 1396.18 2.36% G1.5 24 28 0.86 2808 0.81 2668.19 4.96% G2.0 56 28 2.00 3257 1.95 3181.78 2.30% G2.5 56 60 0.93 6012 0.90 5774.30 3.95% G3.0 120 60 2.00 6910 1.90 6556.70 5.11% G3.5 120 124 0.97 12420 0.92 11809.71 4.91% G4.0 248 124 2.00 14216 1.90 13510.97 4.96% G4.5 248 252 0.98 25237 0.90 23103.55 8.45% A series of generation PAMAM dendrimers from G-0.5 to G-4.5 were successfully achieved; these dendrimers had the regular and high stability in structure; consequently, they could be effective drug drug delivery system. 3.2. FT-IR spectrum of the complex PAMAM dendrimer and cisplatin 3.2.1. FTIR PAMAM dendrimer G2.5, G3.5, G4.5 and complex G2.5-CisPt, G3.5-CisPt, G4.5- CisPt Both FT-IR spectrum of PAMAM G2.5, G3.5 contain strong absorption peak (νC=O) and moderate absorption peak (νC-O) at 1731 cm-1, 1045 cm-1 (G2.5); 1736 cm-1, 1646 cm-1 (G3.5), respectively, corresponding to the vibiration of ester functional group. A broad band with strong viberation corresponds to the stretching –OH groups at 3294 cm-1 (G2.5); 3302 cm-1 (G3.5); 3426 cm-1 (G4.5), which hinder the viberation of amide bonding. FT-IR also presents the assymetric stretching –CH2, CH3, –CH3 at 2952 cm-1, 2832 cm-1 (G2.5); 2952 cm-1, 2830 cm-1 (G3.5) and out-of-plane stretching CH3 at 1360 cm-1 (G2.5), 1359 cm-1 (G3.5), 1399 cm-1 (G4.5). The vibrational modes of the obtained FT-IR of various PAMAM dendrimer generation were similar to PAMAM dendrimer G2.5, 3.5, 4.5. The FT-IR spectrum of all complex PAMAM G2.5-Cisplatin, G3.5-Cisplatin, G4.5-Cisplatin also have similar signal as compared to PAMAM G2.5, 3.5, 4.5. However, the absorption of these peaks are quite difference. Due to the formation of complex, the ester functional groups at the surface of PAMAM are converted to COO- leading to the intensity of viberation of ester groups (νC=O, νC-O) are reduced. Also, due to the overlap of asymmetrical/symetrical stretching of COO- on viberation of amide band I, amide band II and vibration of aliphatic CH3, the intensity of these peaks are increased, confirming the presentation of the viberation of N-H bonding in cisplatin. Together, the change in the intensity of these peaks provide the evidence for the formation of coordinative bond between Pt2+ and carboxylate -COO- groups on the surface of PAMAM dendrimer. 10 3.2.2. FT-IR spectrum of complex PAMAM Dendrimer G3.0-Cisplatin, G4.0-Cisplatin Figure 3.2. FT-IR spectrum of PAMAM dendrimer G2.5, G3.5, G4.5 and complex G2.5-Cisplatin, G3.5- Cisplatin, G4.5-Cisplatin FT-IR of PAMAM dendrimer G3.0 and G3.0-Cisplatin; G4.0 and G4.0-Cisplatin showcased the spectra shifting for –NH viberation at 1643 cm-1 to 1639 cm-1 (G3.0, G3.0-Cisplatin); 1643 cm-1 to 1642 cm-1 (G4.0, G4.0-Cisplatin). This sugguests the formation of the coordinative bond between cation Pt2+ and NH2 groups on the surface of PAMAM dendrimer G3.0. Furthermore, the reduction of intensity and the shifting of symmetric/ asymmetric vibration of aliphatic -CH2 at 2944 cm-1 and 2839 cm-1 in FT-IR spectrum of PAMAM dendrimer G3.0 to 2975 cm-1 and 2884 cm-1 in the complex G3.0-Cisplatin along with the aborption peaks at 3437 cm-1 (G3.0-Cisplatin) and 3427 cm-1 (G4.0-Cisplatin) corresponding to the N-H viberation on the cisplatin spectrum. 3.3. FT-IR spectrum of the complex G3.0-PAA and Cisplatin 11 Figure 3.3. FT-IR spectrum of PAMAM dendrimer G3.0, G4.0 and the complexc G3.0-Cisplatin, G4.0-Cisplatin FT-IR spectrum exhibits the slight shifting of asymmetric –COO viberation and amide peak -NH in G3.0-PAA at 1644 cm-1 and 1571 cm-1 to 1642 cm-1 and 1565 cm-1 in case of G3.0-PAA-Cisplatin. These peaks with weak intensity assigning to the stretching and bending of -CH2 and CH-CO in G3.0-PAA are at 1454 cm-1 and 1409 cm-1, which are shifting to 1453 cm-1 và 1406 cm-1 in term of G3.0-PAA-Cisplatin. The sharp peak at 3435 cm-1 regarding to the stretching N-H group in cisplatin. This behavior proposes the interaction of cation Pt2+ and -COO- on the surface of G3.0-PAA. 12 3.4. FT-IR spectrum of complex G4.0-PAA-Cisplatin Figure 3.5. FT-IR spectrum of G4.0-PAA and complex G4.0-PAA-Cisplatin FT-IR spectrum exhibits the slight shifting of asymmetric –COO viberation and the overlap of amide peak -NH in G3.0-PAA at 1572 cm-1 cm-1 to 1564 cm-1 and 1635 cm-1 in respected to G4.0-PAA-Cisplatin. The weak intensity peaks contributing the stretching and viberation of -CH2 and CH-CO for G4.0-PAA at 1454 cm-1 and 1407 cm-1 are shifted to 1447 cm-1 và 1400 cm-1, respectively, in case of G4.0-PAA-Cisplatin. A viberation at 3619 cm-1 is assigned to the stretching –OH of –COOH on 0-PAA. This phenomina proposes the interaction of cation Pt2+ and -COO- on the surface of G4.0-PAA. 3.5. 1H-NMR result of PAMAM G3.0 and G 3.5 modififed with PNIPAM As shown in the 1H-NMR spectrum of G3-PNIPAM (mole ratio 1:8), beside the typical proton peak for PAMAM G3.0, some the proton signals are originated from PNIPAM-COOH such as –CH3 (f) at 1,10- 1,26 ppm, -(CH3)2CHNH- (l) at 3,99 ppm. In addition, the proton of –CH2CH2CONH (c) shifts from 2.0 to 2.68 ppm, confirming the formation of linkage between NH2 of PAMAM G3.0 and–COOH of PNIPAM- COOH. This results show the successful of synthesis nanocarrier based thermal responsive dendrimer. Figure 3.4. FTIR spectrum of G3.0-PAA and complex G3.0-PAA-Cisplatin 13 Figure 3.6. 1H-NMR spectrum of nanocarrier based on G3.0-PNIPAM (mol ratio 1:8) From 1H-NMR spectrum of G3-PNIPAM, the grafting degree as well as the number PNIPAM-COOH conjugated on PAMAM G3.0 following the below formula: In which: %X = SH(-CH3) (f) SH(-CH2-) (a) ∑ H(-CH3) (f) ∑ H(-CH2-) (a) .100% SH(-CH2-) (a) , SH(-CH3) (f) : the peak areas of peak (a) and peak (f) in 1H- NMR ∑ H(-CH2-) (a) , ∑ H(-CH3) (f) : the sums of protons at the peak (a) and (f) in the derivative dendrimer as theory %X : Amidation degree Regarding the formula, % X is 15,12 % and about 4,84 PNIPAM-COOH groups are successful conjugated on the PAMAM G3.0 (yield 96.8%). In the same maner, based on the 1H-NMR spectrum, these parameters of two mol ratio G3.0: PNIPAM = 1:5 and 1:10, were calculated and presented in table 3.2. Table 3.2. The numer PNIPAM groups conjugating on G3.0 and their estimated molecular weight Sample PNIPAM groups Molecular weight based on1H-NMR Phase- transition temperature G3.0-PNIPAM (1:5) 3.34 30,605 37,5 oC G3.0-PNIPAM (1:8) 4.84 40,776 34 oC G3.0-PNIPAM (1:10) 7.00 55,880 33 oC The molecular weight of G3.0-PNIPAM (1:8) is 39,600 using GPC method, which is similar as the calculation from 1H-NMR spectrum. For G3.5-PNIPAM, beside the typical proton signals of PNIPAM-COOH, other proton signals originating from PAMAM dendrimer generation 3.5 such as –COOCH3 (h) (3,73- 3,78 ppm); –CONHCH2CH2N- (e) (3,26-3,36 ppm); –CH2CH2N (a) (2,57-2,63 ppm) are also exhibited on the 1H-NMR spectrum of G3.5-PNIPAM. This results provide the evidence for the linakage between –COOCH3 and amine groups on the surface of G3.0-PNIPAM. In other world, the nanocarrier based on thermal sensitive G3.5-PNIPAM is well-established in this study. 3.6. 1H-NMR spectrum of PAA modified PAMAM G3.0 Figure 3.7. MW of G3.0-PNIPAM (1:8) using GPC method. 14 A long with the typical proton signals for PAMAM dendrimer G3.0 such as peak –CH2CH2N (a) (2.63ppm), peak –CONHCH2CH2N- (e) (3.30 ppm), the characterized peak for acid polyacrylic, including >CHCOOH (b) (2.07 ppm) >CHCH2CH< (c) (1.61 ppm) exposes in the 1H-NMR spectrum of PAA modified PAMAM G3.0 (G3.0-PAA). This revels the formation of the linkage -CO-NH between -NH2 groups on the surface of PAMAM dendrimer G3.0 and –COOH on PAA chains. This observation can help to confirm the success of the G3.0-PAA synthesis process. Regarding 1H-NMR spectrum of G3.0-PAA, the number PAA groups attacked PAMAM dendrimer G3.0 is 6,01 (yield: 50,1%). When mol rate PAMAM dendrimer G3.0: PAA was 1:6, the number PAA groups conjugated on the PAMAM dendrimer G3.0 is 5 (yield: 83,3%) 3.7. 1H-NMR spectrum of PAA modified PAMAM G4.0 In the same maner to G3.0-PAA, the 1H-NMR spectrum reveals the successful synthesis of carrier based on G4.0-PAA. From the 1H-NMR spectrum of G4.0-PAA, the number of PAA attached on PAMAM dendrimer G4.0 is 15.16 (yield: 94.7%). With mole ratios PAMAM dendrimer G4.0: PAA is 1:8, the number PAA conjugating on the surface of PAMAM dendrimer G4.0 is 7.28 (yield: 91.0%). Further increase the mol of PAA in the ratio upto 1:24; however, the reaction was unscessfull (the solidification in reaction bath) 3.8. Amount of Pt from complexes 3.8.1. Amount of Pt from complex full generation PAMAM dendrimer -cisplatin Figure 3.9. 1H-NMR spectrum of PAA modified PAMAM dendrimer G3.0 (mol ratio 1:12) Figure 3.10. 1H-NMR spectrum of PAA modified PAMAM dendrimer G 4.0 (mole ratio 1:16) Figure 3.8. 1H-NMR spectrum of G3.5-PNIPAM 2 Table 3.3. Amount of Pt from complex full generation PAMAM dendrimer –cisplatin (non- aqueous cisplatin) No. Sample %Cisplatin 1 G3.0-Cisplatin 9.63  1.47 2 G4.0-Cisplatin 16.95  1.29 Data was presented under average  SD (standard deviation), number of trials n=3 3.8.2. Amount of Pt from complex half- generation PAMAM dendrimer (non-aqueous cisplatin) Table 3.4. Amount Pt from complex half- generation PAMAM- Cisplatin (nom-aqueos cisplatin) No. Sample %Cisplatin 1 G2.5-Cisplatin 15.89 ± 1.41 2 G3.5-Cisplatin 7.90 ± 1.92 3 G4.5-Cisplatin 5.90 ± 0.68 Data was presented under average  SD (standard deviation), number of trials n=3 For half-generation PAMAM dendrimer, the amount of cisplatin was reduced with the increase of dendrimer generation. The diminution of loading effectiveness of higher half-generation PAMAM may due to the steric hindrance of the carboxylate groups on the surface, which are tended to closely packed leading to the difficulty in accepting further cisplatin. In the contrary, the amount of cisplatin was increased about 1.7 times following the growth of dendrimer generations from G3.0 to G4.0. 3.8.3. Amount of Pt from complex half- generation PAMAM – cisplatin (aqueous cisplatin) Table 3.5. Amount of Pt from complex G2.5- Cisplatin, G3.5-Cisplatin ang G4.5-Cisplatin No. Sample %Cisplatin 1 G2.5-Cisplatin 28.99  2.01 2 G2.5-Cisplatin (SA) 31.82  1.39 3 G3.5-Cisplatin 30.23  1.29 4 G3.5-Cisplatin (SA) 33.01  1.56 5 G4.5-Cisplatin 31.11  1.48 6 G4.5-Cisplatin (SA) 34.03  1.96 Data was presented under average  SD (standard deviation), number of trials n=3; SA: Sonication The results show that efficacy of conjugating cisplatin is higher than that of previous studies in which cisplatin encapsulating in G2.5-Cisplatin and G2.5-Cisplatin were 10,33% and 2,3%, respectively. The significant difference was because the form of cisplatin, in this study, cisplatin was first hydrolyzed with AgNO3. By this way, cisplatin was diaquated in form of cation [Pt(NH3)2(H2O)]2+ leading to increase the potency of cisplatin that conjugated on half- generation PAMAM dendrimer. Increase of PAMAM generation induces the increase the number of functional groups on the surface and the retention of cisplatin was also increase. However, the increase of surface functional groups, the encapsulation of cisplatin would decrease following the growth of PAMAM generation. Kirkpatrick sugguested this observation is due to the difficulty in the convertion of the surface function groups into carboxylate groups when the generation PAMAM increased and due to through-space effects resulting the reduction of the possible binding between cisplatin and amine/ amide inside dendrimer. 3.8.4. Amount of Pt from complex G3.0- PAA-Cisplatin and G4.0-PAA-Cisplatin (aqueous cisplatin) Table 3.6. Amount of Pt from complex G3.0-PAA- Cisplatin (aqueous cisplatin) and G4.0-PAA- Cisplatin (aqueous cisplatin) No. Sample %Cisplatin 1 G3.0-PAA-Cisplatin (1:6) 12.93  1.60 2 G3.0-PAA-Cisplatin (1:12) 13.89  1.39 3 G4.0-PAA-Cisplatin (1:8) 20.22  1.44 4 G4.0-PAA-Cisplatin (1:16) 40.44  1.29 Data was presented under average  SD (standard deviation), number of trials n=3 The amount of cisplatin increases when increase the mol ratio of pH sensitive polymer Poly acrylic acid (PAA). As compare to cisplatin incapsulating in G3.0-Cisplatin, G4.0-Cisplatin and G3.0-PAA-Cisplatin, G4.0-PAA-Cisplatin, the presentation of PAA induces the increase of cisplatin loading. This could be explained due to large amount 3 carboxylic groups of PAA, the probability of the complex formation Pt-COO- increase. 3.9. Comparison of cisplatin encapsulating in various carrier using either aqueous cisplatin or non-aqueous cisplatin Table 3.7. Cisplatin (non-aqueous) loading in PAMAM dendrimer derivative N o. Sample Number of binding group % Cisplatin NH2 COOH 1 G2.5-COOH 0 32 15.89  1.41 2 G3.5-COOH 0 64 7.90  1.92 3 G4.5-COOH 0 128 5.90  0.68 4 G4.0-PAA (1:16) 49 405 19.06  1.44 5 G3.0-NH2 32 0 9.63  1.47 6 G4.0-NH2 64 0 16.95  1.29 The effectiveness of PAMAM dendrimer in cisplatin delivery was summarized in table 3.7. The results show that cisplatin loading efficiency is lower than previous publiscations. However, using AgNO3 to make full aqueous cisplatin in form [Pt(NH3)2(H2O)n]2+, the potency of the complexation reaction between Pt2+ from cisplatin and surface functional groups –COOH from PAMAM dendrimer carrier increase leading to the increase of amount cisplatin loading in carrier (table 3.8). ). Because the strong interaction between platinum and amino groups on full generation PAMAM dendrimer (G3.0, G4.0) induces the stable complex which are difficult to release in dose of drug; consequently, full generation PAMAM dendrimer was not examined in this study. From table 3.13, aqueous cisplatin could be easy to form the complex with carboxylate – COOH groups on the surface of carrier. PAMAM dendrimer G4.0-PAA (1:16) consisting of a greater number carboxylate groups on the surface (405 groups) could encapsulate higher amount cisplatin, about 40.44% cisplatin, as compared to other carriers. Table 3.8. Cisplatin (aqueous) loading in PAMAM dendrimer derivative N o. Sample Number of binding group % Cisplatin NH2 COO H 1 G2.5-COOH 0 32 31.82  1.39 2 G3.5-COOH 0 64 33.01  1.56 3 G4.5-COOH 0 128 34.03  1.96 4 G3.0-PAA (1:6) 27 75 12.93  1.60 5 G3.0-PAA (1:12) 26 90 13.89  1.39 6 G4.0-PAA (1:8) 57 189 20.22  1.44 7 G4.0-PAA (1:16) 49 405 40.44  1.29 3.10. Experiment with dual 5-FU and Cisplatin loading in PAMAM dendrimer G3.5-PNIPAM G3.5-PNIPAM was synthesized based on the reaction between PAMAM dendrimer G3.0 –PNIAM which was further modified the outer groups -COOCH3 to -COO- that could form complex with cisplatin. In addition, thermal responsive PNIPAM on the surface of carrier is thermal responsive polymer with lower critical solution temperature (LCST, 320C). At the temperature under LCST, PNIPAM swells in maximum in drug solution and can encapsulate these drug inside their network. At the temperature above LCST, polymer chains become to shrink and then release drug to inviroment. Based on this phenomina, 5-FU can be loaded into PAMAM dendrimer G3.5-PNIPAM-Cisplatin. Result for 30mg 5FU encapsulating in 100mg copolymer PAMAM dendrimer G3.5-PNIPAM and complex PAMAM dendrimer G3.5-PNIPAM-Cisplatin are presented below: Table 3.9. 5-FU loading into complex G3.5-PNIPAM-Cisplatin Free 5FU 5-FU loading mg mg %DL % EE 4 PAMAM dendrimer G3.5-PNIPAM-5FU- CisPt 4.32  0.26 25.68  0.26 20.43  0.17 85.61  0.88 PAMAM dendrimer G3.5-PNIPAM-5FU 3.73  0.29 26.27  0.29 20.81  0.18 87.57  0.97 PNIPAM-CisPt-5FU 11.90  0.27 18.10  0.27 15.32  0.20 60.33  0.91 Data was presented under average  SD (standard deviation), number of trials n=3 The complex PAMAM dendrimer G3.5-PNIPAM-Cisplatin shows the potency of 5FU encapsulation forming thermal sensitive nanogel containing dual anticancer drug, 5-FU and cisplatin. Clinical protocol for cancer with cisplatin is usually combinated with other drug to increase the therapeutic value as wel as reduce the side effect of drug. Therefore, PAMAM dendrimer G3.5-PNIPAM loading dual anticancer drug, 5-FU and cisplatin can be considered as the potential candidates in cancer treatment. 3.11. TEM, DLS and zeta potential The size of complex half generation PAMAM dendrimer nanoparticles with cisplatin are quite homogenous and size is in range 5-10 nm (fig 3.11). TEM images expose that the size of PAMAM dendrimer G3.0-PNIPAM is 190nm (fig 3.12). Compared to the initial size of PAMAM dendrimer G3.0 (3-4 nm), PAMAM dendrimer G3.0-PNIPAM procees the growth of size; consequently, increase the amount of drug encapsulation of nanoparticles. Size of PAMAM dendrimer G3.5-PNIPAM-Cisplatin in aqueous is 184 nm which is bigger than the original one because of the PNIPAM covering the surface of PAMAM dendrimer G3.5 nanoparticles. TEM images (fig 3.13) of PAMAM dendrimer G3.5-PNIPAM-Cisplatin exhibites the cross-linking between cisplatin and PAMAM dendrimer G3.5-PNIPAM. Figure 3.11. TEM image of the complex half-generation PAMAM dendrimer – Cisplatin. 5 TEM images of PAMAM G4.0 demonstrated the formation of uniform spherical nanoparticles with a Hình 3.13. TEM image of PAMAM dendrimer G3.5-PNIPAM-Cisplatin and DLS result of G3.5- PNIPAM-Cisplatin and PNIPAM-Cisplatin. Hình 3.12. TEM image of G3.0, PAMAM dendrimer G3.0-PNIPAM and DLS result of PAMAM dendrimer G3.0-PNIPAM. Figure 3.14. TEM image of PAMAM dendrimer G4.0 (A), G4.0-PAA (C) and DLS of PAMAM dendrimer G4.0 (B), G4.0-PAA (D) Figure 3.15. TEM image of PAMAM dendrimer G3.0-PAA- Cisplatin and PAMAM dendrimer G4.0-PAA-Cisplatin 6 diameter of 4.1±1.2 nm, which nearly matched the hydrodynamic diameter of 7.8±2.4 nm as measured by DLS. The size of G4.0-PAA (28 nm) increases 3.6 times as compared to PAMAM dendrimer G4.0. Also, the increase in size of nanoparticles were recored after cisplatin encapsulation. To investigate the stability of carrier, zeta potential values of carrier in different conditions were measured. The ζ value of G3.0-PAA (mol ratio 1:12); G4.0-PAA (mol ratio 1:8) and G4.0-PAA (mol ratio 1:16) exhibit the dependent on the pH of solution. All carriers expose the moderate stability at neutral and pH 7.4. PAA-G4.0 with the mol ratio 1:16 shows the potential aggregation at pH 5.5 with ζ = 7.3mV. This result reveals that the unstability of particles induces the aggregation and due to the aggregation lead to increase size of particles; thus, the particles cannot pass through lymp while promoting the accumulative in tumor site inducing the higher amount of cisplatin in tumor and higher anti-cancer feature. At pH 7.4, the ζ value of PAA-G4.0 with mole ratio 1:16 is -58.6 mV, suggesting that the good stability of nanoparticles in plasma inviroment which helps to prolong drug action in circulation and to increase the possibility drug entering the target site. 0 20 40 60 80 100 2 4 6 8 10 Tr an sm itt an ce [% ] pH G3PAA 1:12 G4PAA 1:16 G4PAA 1:8 -13,9 mV -14,6 mV 19,0 mV b) c) a) a) b) c) -58,6 mV -19,2 mV 7,3 mV Figure 3.16. Zeta potential of G3.0-PAA (mol ratio 1:12) at a. pH 7,4; b. pH 7,0 and c. pH 5,5 Figure 3.17. Zeta potential of G4.0-PAA (mol ratio 1:16) at: a. pH 7,4; b. pH 7,0 and c. pH 5,5 a) b) c) -23,2 mV -17,2 mV 14,1 mV Figure 3.18. Zeta potential of G4.0-PAA (mol ratio 1:8) at: a. pH 7,4; b. pH 7,0 and c. pH 5,5 Figure 3.19. The solubility of carriers depending on pH solution 7 The effect of pH solution on turbidity of carriers was investigated. As shown in figure 3.19, the solubility of G3.0-PAA 1:12, G4.0-PAA 1:8 and G4.0-PAA 1:16 depend on the pH and can be divided into 3 areas: lower pH, precipitation (intermedia area) and high pH. At intermedia area pH 3.5-5.5 (G4.0-PAA 1:16), pH 4,0 - 6,0 (G3.0-PAA 1:12, G4.0-PAA 1:8), the charge on the surface of carriers are neutral, pH solution ~ isoelectric point resulting in the precipitation. 3.12. Result and dissusion about the invitro drug release 3.12.1. The drug release from the complex half-generation PAMAM- cisplatin (aqueous) Table 3.10. in vitro drug release results for complex PAMAM dendrimer: G2.5-Cisplatin, G3.5-Cisplatin and G4.5-Cisplatin Time (h) G2.5-Cisplatin G3.5-Cisplatin G4.5 - Cisplatin %Cisplatin %Cisplatin %Cisplatin pH 5,5 pH 7,4 pH 5,5 pH 7,4 pH 5,5 pH 7,4 0 0 0 0 0 0 0 6 30.42  1.20 22.78  0.81 31.72  1.00 27.17  0.91 34.96  1.08 28.23  0.81 12 37.18  1.48 28.35  1.01 39.04  1.25 35.12  1.12 45.52  1.30 33.12  1.03 24 43.10  1.70 31.84  1.14 44.94  1.41 38.64  1.16 47.86 1.04 43.48  1.20 48 45.98  1.82 34.61  1.23 47.93  1.50 41.35  1.18 53.18  1.47 48.03  1.51 60 48.18  1.90 36.29  1.29 50.24  1.62 43.76  1.28 55.07  1.51 48.91  1.54 72 51.05  2.02 36.54  1.30 53.24  1.67 45.69  1.37 55.49  1.54 49.19  1.54 ANOVA results proclaim that the amount cisplatin release from PAMAM dendrimer denpend on time, pH media and the generation of PAMAM dendrimer. In case of same generation, the cisplatin release at pH 5.5 is higher than that of pH 7.4 in the statistic maner. After 60h, the amount of cisplatin release is non- remarkable different between each carrier. Figure 3.20. In vitro cisplatin release from PAMAM G2.5, PAMAM G3.5 and PAMAM G4.5 in phosphate- buffered saline (PBS) pH7.4 and acetate buffers saline (ABS) pH 5.5 The higher amount cisplatin release in ABS buffer pH 5.5 than in PBS buffer pH 7.4 could be due to the protonation of -COO- on the surface of PAMAM dendrimer in acid condition leading to the formation of – COOH; consequently, loss of labile ligand between half-generation PAMAM dendrimer and cisplatin. Amount 0% 10% 20% 30% 40% 50% 60% 0 12 24 36 48 60 72 % C um ul at iv e C is pl at in Time (h) pH 5.5 (G2.5cis) pH 7.4 (G2.5cis) pH 5.5 (G3.5cis) pH 7.4 (G3.5cis) pH 5.5 (G4.5cis) pH 7.4 (G4.5cis) 8 of cisplatin release in 72h from PAMAM G2.5, PAMAM G3.5 and PAMAM G4.5 are 51.05%, 53.24% and 55.49%, respectively. 3.12.2. Potency of the 5-FU and Cisplatin release from dual drug system G3.5-PNIPAM- Cispaltin – 5FU Bảng 3.11. The potency of 5FU release from G3.5-PNIPAM-CisPt and PNIPAM-CisPt 3.12.3. The potency of cisplatin relase from PAMAM dendrimer G4.0-PAA Table 3.12. Cisplatin release result of PAMAM dendrimer G4.0-PAA Time (h) Cisplatin release (%) pH 5,5 pH 7,4 0 0 0 4 25.17  1.08 11.42  0.75 12 34.42  1.51 21.40  0.6