Summary of Material science doctoral thesis: Improving the effective delivery of cisplatin anti cancer drug of dendrimer nanocarrier

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Research purpose: Preparation and characterization of nanocarrier systems for drug delivery system based on the modification of dendrimer (PAMAM) with biocompatible surfaces such as PNIPAM and PAA to improve the capping cisplatin.

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Nội dung Text: Summary of Material science doctoral thesis: Improving the effective delivery of cisplatin anti cancer drug of dendrimer nanocarrier

MINISTRY OF EDUCATION AND VIETNAM ACADEMY OF SCIENCE AND TRAINING TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY NGUYEN NGOC HOA IMPROVING THE EFFECTIVE DELIVERY OF CISPLATIN ANTI CANCER DRUG OF DENDRIMER NANOCARRIER Field of Study: Polymer and Composite Code: 9 44 01 25 SUMMARY OF MATERIAL SCIENCE DOCTORAL THESIS HO CHI MINH, 2020
The thesis was completed at Institute of Applied Materials Science - Graduate University of Science and Technology Vietnam Academy of Science and Technology Supervisor 1: Prof., Dr. Nguyen Cuu Khoa Supervisor 2: Assoc., Prof., Dr. Tran Ngoc Quyen Reviewer 1: … Reviewer 2: … Reviewer 3: …. The thesis shall be defended in front of the Thesis Committee at Academy Level at Institute of Applied Materials Science - Vietnam Academy of Science and Technology At...... hour....... date...... month....., 2021 The thesis can be found at: - The National Library of Vietnam - The Library of Graduate University of Science and Technology 1
INTRODUCTION 1. The necessity of the thesis Denndrimers were first introduced during the period 1970–1990 by two different groups : Buhleier et al and Tomalia et al. Dendrimers are nano-sized, radially symmetric molecules with well-defined, homogeneous, and monodisperse structure consisting of tree-like arms or branches. Dendrimers are nearly mono-disperse macromolecules that contain symmetric branching units built around a small molecule or a linear polymer core. Dendrimers are hyperbranched macromolecules with a carefully tailored architecture, the end-groups (i.e., the groups reaching the outer periphery), which can be functionalized, thus modifying their physicochemical or biological properties. Dendrimers are designed to drugs delivery to enhance the pharmacokinetics and biological distribution of the drug and to enhance its target ability. Due to their exquisite structure, drug molecules are instantly capped with dendrimer molecules by means of physical adsorption, electrostatic interaction, covalent binding with the peripheral functional groups, or encapsulating inside the dendrimeric crevices. The dendrimeric crevices are usually hydrophobic, which can encapsulate the drug molecule by means of hydrophobic. Further, the high density of peripheral groups of multifunctional nature (amine, NH2 or carboxylate COO-) allows to establish electrostatic interaction with drug and then bring them to the target site. Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It has been extensively used for the cure of different types of neoplasms including head and neck, lung, ovarian, leukemia, breast, brain, kidney and testicular cancers. In general, cisplatin and other platinum-based compounds are considered as cytotoxic drugs which kill cancer cells by damaging DNA, inhibiting DNA synthesis and mitosis, and inducing apoptotic cell death. However, because of drug resistance and numerous undesirable side effects such as severe kidney problems, allergic reactions, decrease immunity to infections, gastrointestinal disorders, hemorrhage, and hearing loss especially in younger patients, other platinum- containing anti-cancer drugs such as carboplatin, oxaliplatin and others, have also been used. Furthermore, combination therapies of cisplatin with other drugs have been highly considered to overcome drug-resistance and reduce toxicity. In the last decade, an alternative strategy following the revolution of nanotechnology has been a shift in focus from platinum complex design to Cisplatin carriers in order to enhance anticancer activity and reduce its side-effects. Among numerous Cisplatin delivery methods, Cisplatin conjugated carriers have been proven as a promising option. Cisplatin can be attached appropriately to the nano-devices containing ester or amide linkages or carboxylate connectivity. These interactions can later be hydrolyzed inside the cell allowing drugs to accumulate in the tumor site. Generally, the conjugate between Cisplatin and carriers revealed an improved efficacy of the platinum drug in cancer treatment compared to physical encapsulation. In this thesis, we modify the surface functional groups of PAMAM dendrimers to enhance the drug delivery capacity of these carriers. 2. Research purpose Preparation and characterization of nanocarrier systems for drug delivery system based on the modification of dendrimer (PAMAM) with biocompatible surfaces such as PNIPAM and PAA to improve the capping cisplatin 3. Research content 2
- Synthesizing the derivative PAMAM dendrimer (PAMAM dendrimer - Poly(N- isopropylacrylamide), PAMAM dendrimer - Poly acrylic acid). - Evaluating their chemical structure and grafting degree - Evaluating the capping cisplatin ability of PAMAM dendrimer and their derivative such as PAMAM dendrimer - Poly(N-isopropylacrylamide), PAMAM dendrimer - Poly acrylic acid. - Analyzing the structure of the complex carrier – drug and evaluating the release of cisplatin from carrier. - Identifying the cytotoxicity of PAMAM dendrimer and their derivative CHAPTER 1. OVERVIEW 1.1. Introduction to dendrimer and biocompatibility of dendrimer 1.1.1. Introduction The term “dendrimer” was first mentioned by Donald A. Tomalia in 1985s. The word “dendrimer” is Greek in origin, “Dendron”, by means of tree branch. Up to now, various studies have been published about structure of dendrimer molecule, dendrimer synthesis and application of dendrimer in difference fields. In general, dendrimers are nano-polymer with spherical morphology and branched structure and have more advantages than that of linear polymer. Structure of dendrimers include three part as illustrating in figure 1.1. Figure 1.1. A typical structure of dendrimer - A dendrimer is comprised of three different parts: (i) central core consisting of atom or the molecule with at least two similar functional groups, (ii) branches, arising from the central atom/molecules core composed by repeat units and the brigde between the terminal functional groups and their core, (iii) numerous terminal functional groups (anion, cation, neutral, hydrophobic or hydrophilic groups) located at the edge of the moleculer which are also called peripheral functional groups. Dendrimer, specialized on PAMAM dendrimer with open open structure, various internal cavities and amine/ester-terminated surface functional groups, have been a tremendous motivator for multi-drug delivery nanocarriers to kill cancer cells following passive targeting or active targeting mechanism. 1.1.2. Biocompatibility of dendrimer 3
Dendrimer has been considered as smart carrier because they can help drug to enter to cytoplasm, escape biological barriers, take a longer blood circulation time that enable to create the clinical effect and allow drugs to reach their target sites. The primary source of cytotoxicity of PAMAM dendrimers is due to their surface groups. Surface groups with amine (-NH2) of PAMAM and PPI dendrimer induce the risk of cell hemolysis depending on the concentration while the charge neutrality terminated dendrimers or anionic terminated surface are found to lower toxicity or non-toxic. To increase the biocompability, the possible for target therapy, as well as diminishing their toxic, mainting their exquisite drug delivery feature, the surface of PAMAM dendrimer should be modified with biocompabile and targeting molecules. 1.2. Cisplatin anticancer drugs 1.2.1. Properties of Cisplatin Figure 1.2. Cisplatin drug molecule. Cisplatin (CAS no. 15663-27-1, MF-Cl2H6N2Pt; NCF-119875), cisplatinum, also called cis- diamminedichloroplatinum (II), is a metallic (platinum) coordination compound with a square planar geometry. Cisplatin was first synthesized by M. Peyrone in 1844 and its chemical structure was first elucidated by Alfred Werner in 1893. However, the compound did not gain scientific investigations until the 1960s when the initial observations of Rosenberg et al. (1965) at Michigan State University pointed out that certain electrolysis products of platinum mesh electrodes were capable of inhibiting cell division in Escherichia coli created much interest in the possible use of these products in cancer chemotherapy. Cisplatin has been especially interesting since it has shown anticancer activity in a variety of tumors including cancers of the ovaries, testes, and solid tumors of the head and neck. It was discovered to have cytotoxic properties in the 1960s, and by the end of the 1970s it had earned a place as the key ingredient in the systemic treatment of germ cell cancers. Among many chemotherapy drugs that are widely used for cancer, cisplatin is one of the most compelling ones. It was the first FDA-approved platinum compound for cancer treatment in 1978. This has led to interest in platinum (II)—and other metal—containing compounds as potential anticancer drugs. CHAPTER 2. Materials and Methods 2.1. Materials Chemical agents were purchased from Acros, Sigma Aldrich, Merck with high purity, suitable for synthetic organic chemistry and for analytical specifications. Equipment: desiccator, sonication, magnetic Stirrer and hot plate, vacuum oven, vacuum rotary evaporator Eyala, water bath memmert, freeze dryer at German Vietnamese Technology Center, Ho Chi Minh City University of Food Industry. Morphology and size of dried particles was taken by TEM at 140kV (JEOL JEM 140, Japan). Fourier-transform infrared spectroscopy (FTIR) was analysed by Equinox 55 Bruker. HPLC was done by Agilent 1260 (USA). 1H-NMR spectrum was obtained from Bruker Avance 500. Amount of Pt was determined using ICP-MS-7700x/Agilent (VILAS). The cytotoxic assay was investigated following the help of Molecular Lab, Genetics Department, University of Science, HCM. 2.2. Methods 2.2.1. Synthesis of PAMAM dendrimer of generation G4.5 from the ethylenediamine (EDA) core 4
The synthetic route of PAMAM dendrimer of generation G4.5 was employed 11 steps (figure 2.1), starting from the reaction between ethylenediamine (EDA) and methyl acrylate (MA) to form G-0.5 to which the next generation G0, G0.5, G1.0, G1.5, G2.0, G2.5, G3.0, G3.5, G4.0 và G4.5 were expanded. The chemical structure and the molecular mass of the obtained products were identified by 1H-NMR. Figure 2.1. Synthetic route of PAMAM dendrimer 2.2.2. Synthesis of PAMAM dendrimer G3.0, G4.0 conjugated Cisplatin Cisplatin was dissolved in water and stirred at room temperature under N2 inviroment. The solution of PAMAM dendrimer G3.0, G4.0 in water was adjusted pH to 7-8 using HCl. PAMAM dendrimer solution was drop-wised into prepared cisplatin solution and stirred for 24h following 1 h with sonication at room temperature under N2 gas. The unbound cisplatin was removed via dialysis. The obtained product was then freeze dried to get powder. 2.2.3. Synthesis PAMAM dendrimer G2.5, G3,5, G 4.5 conjugated cisplatin PAMAM dendrimer G2.5, G3.5, G4.5 were hydrolyzed by NaOH to form carboxylated groups COO - on the surface and were then used to perform the complex compound with cisplatin as section 2.2.2 2.2.4. Synthesis PAMAM dendrimer G2.5, G3,5, G 4.5 conjugated aqueous cisplatin Hydrolyzed cisplatin was prepared using AgNO3 to withdraw the choloride ion on cisplatin leading to the formation of monoaqua [cis-(NH2)2PtCl(H2O)] and diaqua [cis-(NH2)2Pt(H2O)2]. The reaction was taken place at room temperature, under N2 and continuous stirring. The hydrolyzed PAMAM dendrimer G2.5, G3.5, G4.5 by NaOH was drop-wised into aqueous cisplatin, stirring for 24h following the sonication in 1 hours under N2 at room temperature. The obtained product was then freeze dried to get powder. 2.2.5. Modification of PAMAM dendrimer G 3.0 with Poly(N-isopropylacrylamide) (PNIPAM) Carboxylated (-COOH) terminated PNIPAM was activated by pnitrophenyl chloroformate (NPC) and N-Hydroxysuccinimide (NHS) following the reaction with NH2 groups on the surface of PAMAM dendrimer G 3.0 under stirring condition for 24h. The obtained products were purified by dialysis membrane and then free-dried to get powder. The chemical structure and grafting degree were estimated by 1H-NMR. 2.2.6. Syntheis of PAMAM dendrimer G 3.5-PNIPAM 5
The remained amino groups (-NH2) on PAMAM dendrimer G3.0- PNIPAM were reacted with methyl acrylate in 96h under N2 condition to form PAMAM dendrimer G 3.5-PNIPAM. The chemical structure and grafting degree were estimated by 1H-NMR. 2.2.7. Synthesis of the complex PAMAM dendrimer G3.5-PNIPAM and Cisplatin The complexation reaction between PAMAM dendrimer G3.5-PNIPAM and cisplatin was similar to the description in section 2.2.4 2.2.8. Modification of PAMAM dendrimer G3.0, G4.0 with poly (acrylic acid) (PAA) PAA was activated using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) before reacting with NH2-terminal surface function groups of PAMAM dendrimer G3.0, G4.0. The obtained products were purified by dialysis membrane and then free-dried to get powder. The chemical structure and grafting degree were estimated by 1H-NMR. 2.2.9. Synthesis the complex PAMAM dendrimer G3.0-PAA, PAMAM dendrimer G4.0-PAA and cisplatin The complexation reaction between PAMAM dendrimer G3.0-PAA, PAMAM dendrimer G4.0-PAA and cisplatin was similar to the description in section 2.2.4 2.2.10. Evaluation the encapsulation and release of 5FU from the complex PAMAM dendrimer G3.5-PNIPAM-Cisplatin 5-FU was dissolved into deionized water (DI) and then drop-wised into PAMAM dendrimer G3.5- PNIPAM-Cisplatin solution. Sonication was applied for 1 h and then the reaction was under regular stirred for 24h at room temperature. The obtained products were purified by dialysis membrane and then free-dried to get powder. The encapsulation efficacy and the amount of 5-FU release from carrier were analysized by HPLC. 2.2.11. Determine amount of cisplatin in products using ICP-MS ICP was performed with ICP-MS-7700x/Agilent. Amount of Pt was calculated based on Pt 195 and Lutetium 175 as internal standard. 2.2.12. Evaluation of in vitro drug release In vitro release study was investigated with 2 type buffer (pH 7,4 and pH 5,5) as the function of time. 2.2.13. Kinetic and pharmacokinetic drug release The first screening the selection of release kinetic model for cisplatin was come from the common models such as zero-order, first-order, Higuchi, Kormeyer-Peppas and Hixson-Crowell. The right model for kinetic release was based on the AIC criteria (Akaike information criterion) and R 2ajust (Adjusted R2), calculating by R program. From the in vitro release and their kinetic model, the pharmacokinetic parameters for cisplatin from nanocarriers were identified. 2.2.14. In vitro cytotoxicity Cytotoxicity against lung cancer cells NCI-H460 and breat cancer cells MCF-7 were determined using SRB assay. CHAPTER 3: RESULT AND DISCUSION 3.1. Synthesis of PAMAM dendrimer of generations G0.5 to G4.5 Evaluation of the chemical structure of dendrimer PAMAM using 1H-NMR 6
The chemical shift of specific proton signals on dendrimer PAMAM were recored in various previous reports. The resultant 1H –NMR spectrum showcased the typical protron siginals of dendrimer structure such as: -CH2CH2N< (a) at δH = 2.60 ppm; -CH2CH2CO- (b) at δH = 2.80-2.90 ppm; -CH2CH2CONH- (c) at δH = 2.30 - 2.40 ppm; -CH2CH2NH2 (d) at δH = 2.70 -2.80 ppm; -CONHCH2CH2N- (e) at δH = 3.20 - 3.40 ppm; -CH2CH2COOCH3- (g) at δH = 2.40 - 2.50 ppm and -COOCH3 (h) at δH = 3.70 ppm. The 1H-NMR spectrum of various dendrimer PAMAM generation was presented below: 1 H-NMR PAMAM G-0.5: at δH = 2.47 - 2.50 ppm (a), δH = 2.77-2.80 ppm (b), δH = 2.54 ppm (g) and δH = 3,68 ppm (h). 1 H -NMR PAMAM G0.0: at δH = 2.56 - 2.57 ppm (a), δH = 2.77 - 2.82 ppm (b), δH = 2.37 - 2.40 ppm (c), δH = 2.71 -2.75 ppm (d) and δH = 3.25 - 3.27 ppm (e). 1 H -NMR PAMAM G0.5: at δH = 2.54 -2.57 ppm (a), δH = 2.76 - 2.82 ppm (b), δH = 2.37 - 2.40 ppm (c), δH = 3.24 - 3.26 ppm (e), δH = 2.45 - 2.48 ppm (g) and δH = 3.66 ppm (h). 1 H -NMR PAMAM G1.0: at δH = 2.59 - 2.60 ppm (a), δH = 2.80 -2.82 ppm (b), δH = 2.38 - 2.40 ppm (c), δH = 2.73 - 2.76 ppm (d) and δH = 3.26 - 3.28 ppm (e). 1 H -NMR PAMAM G1.5: at δH = 2.58 - 2.59 ppm (a), δH = 2.78 - 2.86 ppm (b), δH = 2.39 - 2.42 ppm (c), δH = 3.27 - 3.29 ppm (e), δH = 2.47 -2.50 ppm (g) and δH = 3.69 ppm (h). 1 H -NMR PAMAM G2.0: at δH = 2.57 - 2.59 ppm (a), δH = 2.77 -2.81 ppm (b), δH = 2.36 -2.38 ppm (c), δH = 2.68 -2.74 ppm (d) and δH = 3.24 - 3.27 ppm (e). 1 H -NMR PAMAM G2.5: at δH = 2.57 - 2.64 ppm (a), δH = 2.84 - 2.86 ppm (b), δH = 2.40 -2.42 ppm (c), δH = 3.27 -3.30 ppm (e), δH = 2.48 - 2.46 ppm (g) and δH = 3.68 - 3.69 ppm (h). 1 H -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). 1 H -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). 1 H -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). 1 H -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). 7
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: In which: (e) (a) (e) SH(-CH2 -) SH(-CH2 -) , SH(-CH2 -) : the peak areas of protons 1 (a) at (a) and (e) in H-NMR χNMR SH(-CH2 -) (e) (a) M(NMR) = .MLT = (e) .MLT ∑ H(-CH2 -) , ∑ H(-CH2 -): the sums of protons at the (e) χLT ∑ H(-CH2 -) and (a) position s in the molecular formula of (a) PAMAM dendrimer. ∑ H(-CH2 -) 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. (e) (a) Different H(-CH2-) H(-CH2-) χLT M(LT) χNMR M(NMR) (%) 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% 8
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 (ν C=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 Pt 2+ and carboxylate -COO- groups on the surface of PAMAM dendrimer. 9
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 10
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 -CH 2 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. 11
3.4. FT-IR spectrum of complex G4.0-PAA-Cisplatin Figure 3.4. FTIR spectrum of G3.0-PAA and complex G3.0-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. 12
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: (a) (f) SH(-CH2 -) , SH(-CH3 ) : the peak areas of peak (a) and peak (f) in 1H- (f) SH(-CH3 ) NMR (a) (a) (f) SH(-CH2 -) ∑ H(-CH2 -), ∑ H(-CH3 ) : the sums of protons at the peak (a) and (f) in the %X = (f) .100% derivative dendrimer as theory ∑ H(-CH3 ) (a) %X : Amidation degree ∑ H(-CH2 -) 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 Phase- weight based transition on1H-NMR 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. Figure 3.7. MW of G3.0-PNIPAM (1:8) using GPC method. 3.6. 1H-NMR spectrum of PAA modified PAMAM G3.0 13
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%) Figure 3.8. 1H-NMR spectrum of G3.5-PNIPAM 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) Figure 3.9. 1H-NMR spectrum of PAA modified Figure 3.10. 1H-NMR spectrum of PAA modified PAMAM dendrimer G3.0 (mol ratio 1:12) PAMAM dendrimer G 4.0 (mole ratio 1:16) 3.8. Amount of Pt from complexes 3.8.1. Amount of Pt from complex full generation PAMAM dendrimer -cisplatin 14
Table 3.3. Amount of Pt from complex full which cisplatin encapsulating in G2.5-Cisplatin and generation PAMAM dendrimer –cisplatin (non- G2.5-Cisplatin were 10,33% and 2,3%, respectively. aqueous cisplatin) The significant difference was because the form of No. Sample %Cisplatin cisplatin, in this study, cisplatin was first hydrolyzed 1 G3.0-Cisplatin 9.63  1.47 with AgNO3. By this way, cisplatin was diaquated in 2 G4.0-Cisplatin 16.95  1.29 Data was presented under average  SD (standard form of cation [Pt(NH3)2(H2O)]2+ leading to increase deviation), number of trials n=3 the potency of cisplatin that conjugated on half- 3.8.2. Amount of Pt from complex half- generation PAMAM dendrimer. Increase of generation PAMAM dendrimer (non-aqueous PAMAM generation induces the increase the number cisplatin) of functional groups on the surface and the retention Table 3.4. Amount Pt from complex half- of cisplatin was also increase. However, the increase generation PAMAM- Cisplatin (nom-aqueos of surface functional groups, the encapsulation of cisplatin) cisplatin would decrease following the growth of No. Sample %Cisplatin PAMAM generation. Kirkpatrick sugguested this 1 G2.5-Cisplatin 15.89 ± 1.41 2 G3.5-Cisplatin 7.90 ± 1.92 observation is due to the difficulty in the convertion 3 G4.5-Cisplatin 5.90 ± 0.68 of the surface function groups into carboxylate Data was presented under average  SD (standard groups when the generation PAMAM increased and deviation), number of trials n=3 due to through-space effects resulting the reduction For half-generation PAMAM dendrimer, the of the possible binding between cisplatin and amine/ amount of cisplatin was reduced with the increase of amide inside dendrimer. dendrimer generation. The diminution of loading 3.8.4. Amount of Pt from complex G3.0- effectiveness of higher half-generation PAMAM PAA-Cisplatin and G4.0-PAA-Cisplatin (aqueous may due to the steric hindrance of the carboxylate cisplatin) groups on the surface, which are tended to closely Table 3.6. Amount of Pt from complex G3.0-PAA- packed leading to the difficulty in accepting further Cisplatin (aqueous cisplatin) and G4.0-PAA- cisplatin. In the contrary, the amount of cisplatin was Cisplatin (aqueous cisplatin) increased about 1.7 times following the growth of No. Sample %Cisplatin dendrimer generations from G3.0 to G4.0. 1 G3.0-PAA-Cisplatin 12.93  1.60 3.8.3. Amount of Pt from complex half- (1:6) generation PAMAM – cisplatin (aqueous 2 G3.0-PAA-Cisplatin 13.89  1.39 (1:12) cisplatin) 3 G4.0-PAA-Cisplatin 20.22  1.44 Table 3.5. Amount of Pt from complex G2.5- (1:8) Cisplatin, G3.5-Cisplatin ang G4.5-Cisplatin 4 G4.0-PAA-Cisplatin 40.44  1.29 (1:16) No. Sample %Cisplatin Data was presented under average  SD (standard 1 G2.5-Cisplatin 28.99  2.01 deviation), number of trials n=3 2 G2.5-Cisplatin (SA) 31.82  1.39 The amount of cisplatin increases when 3 G3.5-Cisplatin 30.23  1.29 4 G3.5-Cisplatin (SA) 33.01  1.56 increase the mol ratio of pH sensitive polymer Poly 5 G4.5-Cisplatin 31.11  1.48 acrylic acid (PAA). As compare to cisplatin 6 G4.5-Cisplatin (SA) 34.03  1.96 incapsulating in G3.0-Cisplatin, G4.0-Cisplatin and Data was presented under average  SD (standard G3.0-PAA-Cisplatin, G4.0-PAA-Cisplatin, the deviation), number of trials n=3; SA: Sonication presentation of PAA induces the increase of cisplatin The results show that efficacy of conjugating loading. This could be explained due to large amount cisplatin is higher than that of previous studies in 2
carboxylic groups of PAA, the probability of the groups on full generation PAMAM dendrimer (G3.0, - complex formation Pt-COO increase. G4.0) induces the stable complex which are difficult 3.9. Comparison of cisplatin to release in dose of drug; consequently, full encapsulating in various carrier using either generation PAMAM dendrimer was not examined in aqueous cisplatin or non-aqueous cisplatin this study. From table 3.13, aqueous cisplatin could Table 3.7. Cisplatin (non-aqueous) loading in be easy to form the complex with carboxylate – PAMAM dendrimer derivative COOH groups on the surface of carrier. PAMAM dendrimer G4.0-PAA (1:16) consisting of a greater N Sample Number of % number carboxylate groups on the surface (405 o. binding group Cisplatin groups) could encapsulate higher amount cisplatin, NH2 COOH 1 G2.5-COOH 0 32 15.89  1.41 about 40.44% cisplatin, as compared to other 2 G3.5-COOH 0 64 7.90  1.92 carriers. 3 G4.5-COOH 0 128 5.90  0.68 Table 3.8. Cisplatin (aqueous) loading in PAMAM 4 G4.0-PAA 49 405 19.06  1.44 dendrimer derivative (1:16) N Sample Number of % 5 G3.0-NH2 32 0 9.63  1.47 o. binding Cisplatin 6 G4.0-NH2 64 0 16.95  1.29 group NH2 COO The effectiveness of PAMAM dendrimer in H cisplatin delivery was summarized in table 3.7. The 1 G2.5-COOH 0 32 31.82  1.39 results show that cisplatin loading efficiency is lower 2 G3.5-COOH 0 64 33.01  1.56 than previous publiscations. However, using AgNO3 3 G4.5-COOH 0 128 34.03  1.96 4 G3.0-PAA 27 75 12.93  1.60 to make full aqueous cisplatin in form (1:6) 2+ [Pt(NH3)2(H2O)n] , the potency of the complexation 5 G3.0-PAA 26 90 13.89  1.39 reaction between Pt 2+ from cisplatin and surface (1:12) 6 G4.0-PAA 57 189 20.22  1.44 functional groups –COOH from PAMAM dendrimer (1:8) carrier increase leading to the increase of amount 7 G4.0-PAA 49 405 40.44  1.29 cisplatin loading in carrier (table 3.8). ). Because the (1:16) strong interaction between platinum and amino 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 3
PAMAM dendrimer G3.5-PNIPAM-5FU- 4.32  0.26 25.68  0.26 20.43  0.17 85.61  0.88 CisPt PAMAM dendrimer 3.73  0.29 26.27  0.29 20.81  0.18 87.57  0.97 G3.5-PNIPAM-5FU 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. 4
Hình 3.12. TEM image of G3.0, PAMAM dendrimer G3.0-PNIPAM and DLS result of PAMAM dendrimer G3.0-PNIPAM. Hình 3.13. TEM image of PAMAM dendrimer G3.5-PNIPAM-Cisplatin and DLS result of G3.5- PNIPAM-Cisplatin and PNIPAM-Cisplatin. Figure 3.14. TEM image of PAMAM dendrimer G4.0 (A), G4.0-PAA Figure 3.15. TEM image of (C) and DLS of PAMAM dendrimer G4.0 (B), G4.0-PAA (D) PAMAM dendrimer G3.0-PAA- Cisplatin and PAMAM dendrimer G4.0-PAA-Cisplatin TEM images of PAMAM G4.0 demonstrated the formation of uniform spherical nanoparticles with a 5
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. a) -13,9 mV b) -14,6 mV a) -58,6 mV b) -19,2 mV c) 19,0 mV c) 7,3 mV Figure 3.16. Zeta potential of G3.0-PAA (mol ratio Figure 3.17. Zeta potential of G4.0-PAA (mol 1:12) at a. pH 7,4; b. pH 7,0 and c. pH 5,5 ratio 1:16) at: a. pH 7,4; b. pH 7,0 and c. pH 5,5 100 a) -23,2 mV b) -17,2 mV Transmittance [%] 80 60 40 20 c) 14,1 mV 0 2 4 6 8 10 pH G3PAA 1:12 G4PAA 1:16 G4PAA 1:8 Figure 3.18. Zeta potential of G4.0-PAA (mol ratio Figure 3.19. The solubility of carriers depending on 1:8) at: a. pH 7,4; b. pH 7,0 and c. pH 5,5 pH solution 6