RESEARCH POLYAMIDOAMINE DENDRIMERS MODIFIED BY BIOCOMPATIBLE POLYMERS (PEG AND PLURONIC) APPLICATION TO DRUG

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY THI TRAM CHAU NGUYEN RESEARCH POLYAMIDOAMINE DENDRIMERS MODIFIED BY BIOCOMPATIBLE POLYMERS (PEG AND PLURONIC) APPLICATION TO DRUG CARRIER Specialization : POLYMER AND COMPOSITE MATERIALS Code: 62.44.01.25 DOCTORAL THESIS ABSTRACTS’ MATERIAL SCIENCES Ho Chi Minh City – 2016 The work was completed at: Chamber of pharmaceutical materials, Institute for Materials Science Applications, Vietnam Academy of Science and Technology Scientific guidance : Ass Prof Dr CUU KHOA NGUYEN Dr NGOC QUYEN TRAN 1st Peer Reviewer: 2nd Peer Reviewer: The theeesis dissertation will be defended in front of doctoral thesis judgement, held at the Academy of Sciences Institute of Applied Materials, Graduate University of Science and Technology , No , Mac Dinh Chi , District 1, HCMC city, Viet Nam At , ……………, 2016 Can learn dissertation at the library: National Library of Vietnam, Library of Vietnam Academy of Science and Technology INTRODUCTION EXORDIUM Along with the development of science and technology, man has constantly researching to find new materials to serve the needs of life Nanotechnology was born to meet this critical need In recent years, the focus nanopolymer is applied research in the field of medicine Dendrimer is one of the most widely studied nanopolymer by spherical structures with plenty of space inside can be applied as drug carriers, protein and gene distribution More and more drugs are being used to face the problems of solubility, biological effects, poor absorption and short shelf life In addition, the cure has many side effects Particularly anti-cancer drugs are not only toxic to cancer cells but also toxic to the healthy cells Many scientific reports indicate that the encapsulation of drugs to treat cancer or nano polymer delivery system has advanced significantly in the water solubility and stability of the drug store , which enhances the activity against the block u and reduce the side effects of the drug PAMAM dendrimers as carriers nanopolymer one can work as a useful tool for the delivery of drugs , as well as gene therapy and chemotherapy [ 14 , 22 , 35 , 44 , 53 , 68 , 7476 ] However, one drawback of PAMAM dendrimer is causing toxicity in the blood and cell lysis by the strong interaction of the -NH2 groups on the surface PAMAM with the cell membrane, leading to cell membrane disruption which means that cell death [ 44 , 53 , 79 , 88 ] To solve this problem, the group -NH2 PAMAM dendrimer surface was modified by the biocompatible polymer, which eliminate the positive charge on the amine groups or prevent contact between the groups with -NH2 membrane reduces toxicity, interoperability creates high biological carriers, thereby improving the efficiency with medicines and therapy [ 14 , 35 , 80 , 83] Besides the PAMAM surface modification can also increase the drug 's ability to carry PAMAM On that basis, we propose the topic "Research polyamidoamine dendrimers modified by biocompatible polymers ( PEG and Pluronic ) application to drug carrier " GOAL OF THE THESIS Drug carrier research based on PAMAM dendrimer modified biocompatible polymer ( Pluronic, polyethylene glycol ), with the goal of reducing the toxicity of PAMAM ( biocompatible increase ) and increased ability to carry drugs of PAMAM CONTENT OF RESEARCH THESIS Page Synthesis of dendrimer G5.0 PAMAM generation from center of ethylenediamine (EDA) Study generation dendrimer modified PAMAM G2.0, G3.0, G4.0, G5.0 with Polyethylene glycol 4000 (PEG4K), Polyethylene glycol 6000 (PEG6K), Polyethylene glycol 10000 (PEG10K), Polyethylene glycol 12000 (PEG12K) Denatured Research G2.0 PAMAM dendrimer generation 4, G3.0, G4.0, G5.0 with p123 Pluronic, Pluronic F68, Pluronic F127 and Pluronic F108 Research G4.0 PAMAM dendrimer synthesis-F127 with the molar ratio of PAMAM / F127 different Research encapsulated effective anticancer drugs 5-Fluorouracil (5-FU) of the nano carrier-PEG PAMAM and PAMAM-Pluronic Surveys drug release rate of 5-Fluorouracil-generation dendrimer G4.0 PAMAM PEG6K / 5-FU in vitro environment PBS buffer (pH = 7.4) Surveys drug release rate of 5-Fluorouracil-G4.0 PAMAM dendrimer system p123 / 5-FU and PAMAM G4.0-F127 / 5-FU in vitro environment PBS buffer (pH = 7.4) Research cytotoxicity MCF-7 breast cancer and fibroblast (Fibroblast) for PAMAM dendrimer nano delivery system, PAMAM-PEG, PAMAM-Pluronic, PAMAM-PEG / 5-FU and PAMAM-Pluronic / 5FU MEANING OF SCIENCE AND PRACTICE OF THE THESIS - Synthesis and modified PAMAM dendrimer generations with the biocompatible polymer PEG and Pluronic to create drug delivery system taking into biocompatible and capable of carrying high medication - The study by the polymer modified PAMAM and Pluronic PEG with different molecular weight as the scientific basis for the binding reaction conditions or Pluronic number of different circuit to PEG molecules PAMAM PAMAM create variable systems nanoscale properties different and capable of carrying various drugs, particularly anti-cancer drug FU - The outcome of the subject is the scientific basis for further studies in order to bring the drug system created " smart " towards the destination on the basis of PAMAM NEW CONTRIBUTIONS OF THE THESIS (1) With terms of PAMAM - Pluronic synthesis: - Researched synthesized and denatured dendrimers successful generation PAMAM G2.0; G3.0; G4.0; G5.0 with Pluronic have different molecular weights ( P123; F68; F127; F108) - In particular , as long as Pluronic circuit associated with PAMAM more difficult Page ( ) With terms of comparing drugs encapsulated in PAMAM-PEG carrier and G4.0 PAMAM-Pluronic are PAMAM G4.0-PEG and PAMAM G4.0-Pluronic likely better encapsulated drug In particular, PAMAM-Pluronic encapsulated hydrophobic drug 5-FU better corresponding PAMAM-PEG ( ) The ability to bring medicines PAMAM-P123 > PAMAM-F127 > PAMAM-F108 > PAMAM-F68 influenced HLB value rather than the influence of the circuit structure LAYOUT OF THE THESIS The thesis has 169 pages with 30 tables, 102 diagrams, graphs Besides the introduction ( pages ), conclusions ( pages ), list of publications ( pages ) and references ( 14 pages ), Annex ( 15 pages ), the thesis is divided into chapters as follows: Chapter : Overview 41 pages Chapter : Experimental 26 pages Chapter : Results and argumentation 66 Pages Page CHAPTER OVERVIEW About the specific dendrimer and dendrimer polyamidoamine ( PAMAM ), the method of synthesis, properties and their applications in the field of medicine and pharmacy Introduction of methods of PAMAM drug carries the meaning of bringing drugs, particularly anti-cancer drugs bearing ( - fluorouracil for example ) by passively and actively The method of surface modification agents PAMAM with biocompatible as polyethyleneglycol ( PEG ) or Pluronic to increase the biocompatibility, increased ability to bring medicines PAMAM Overview some research in the field of synthesis, modified PAMAM make us carry anticancer drugs In recent years, many studies of modified PAMAM dendrimer biocompatible polymer However, most studies have used methods of activating Pluronic, PEG by NPC and controlled by mole ratio of 1: (Pluronic / PEG: NPC) and using catalysts (pyridine) or solvent (benzene ) during the reaction The downside of this method is prone ability was activated PEG Pluronic or both ends by the NPC and the result can Pluronic or PEG Link input PAMAM both make covered membrane surface and prevents PAMAM part of the drug does not enter the space in the structure PAMAM [67, 110, 124] On the other hand, have not seen any published using a molecular weight Pluronic different generations of modified PAMAM chain, to build a system of structures affected PAMAM dendrimer generations, structured Pluronic kinds to the extent modified, cell toxicity and effectiveness encapsulated drug So the research of this thesis, we focus modified PAMAM dendrimer generation G2.0, G3.0, G4.0, G5.0 and by types of PEG type with a molecular weight Pluronic different to build on the systemic level and the expected modification of the nanopolymer drug Page CHAPTER EXPERIMENTAL RESEARCH 2.1 CHEMICALS , EQUIPMENT AND LABORATORY INSTRUMENTS Chemicals used in the research of the thesis is the chemical purity of Sigma - Aldrich brands , Acros Organics (USA ) , Merck ( Germany ), Amresco ( South Korea) Major equipment and instruments used as vacuum freeze dryer Eyela FDU -2100 ( Japan ) at the Institute of Chemical Technology, Institute HLKH & T VN; JEOL JEM 1400 machine ( Japan ) TEM imaging, at the University of Technology, HCMC HCM; analyzer infrared spectroscopy FTIR Bruker Equinox 55 ( Germany ), at the Institute of Materials Science Applications , and Technology Institute HLKH VN; Agilent GPC gel chromatography 1260 ( US ); HPLC liquid chromatography measured by the Agilent 1260 ( US ); Analysis flow measurement in laboratory cell - SHPT- BM Genetics in Ho Chi Minh City University of Science HCMCity 2.2 METHOD OF STUDY Using the method to synthesize nano - Divergent PAMAM dendrimer generations Using 1H-NMR spectrum, FTIR and GPC to determine the structural components and molecular weight of generations PAMAM and PAMAM modified with biocompatible polymers Using transmission electron microscopy TEM to examine the morphology of the PAMAM - PEG and PAMAM - Pluronic Use the SRB staining method , dyed fluorescent dye MTT and FDA / EB to assess cytotoxicity in vitro Using UV-Vis and HPLC to assess the ability to carry drugs and release of PAMAMPEG/5-FU and PAMAM-Pluronic/5- FU Using cell culture techniques to assess the biocompatibility of the hydrogel and hydrogel composite types synthesis 2.3 EXPERIMENTAL METHOD 2.3.1 PAMAM dendrimer synthesis G5.0 generation from core to ethylenediamine ( EDA) PAMAM dendrimer synthesis process G5.0 generation through 12 stages, starting phase synthesis G-0.5 generation derived by core ethylenediamine (EDA) turn to the next generation G0, G0.5, G1.0, G1.5, G2.0, G2.5, G3.0, G3.5, G4.0, G4.5 and G5.0 (Figure 2.1) Page Figure 2.1 PAMAM dendrimer synthesis diagram generation 2.3.2 PAMAM dendrimer synthesis modified G2.0; G3.0; G4.0; and G5.0 generations with PEG 4000 ( PEG4K ); PEG 6000 ( PEG6K ); PEG 10000 (PEG10K ) and PEG 12000 (PEG12K) To connect PAMAM dendrimer PEG molecules on the surface NH2 groups through three stages, need to use p - nitrophenyl chloroformate (NPC) and tyramine ( TA ) produces intermediates The structure of intermediate products NPC-PEG-NPC, NPC-PEG-TA and PAMAM-PEG product may be determined by the results of 1H-NMR spectroscopic analysis , FTIR , GPC and TEM images 2.3.3 PAMAM dendrimer synthesis modified G2.0; G3.0; G4.0; and G5.0 generations with Pluronic P123 ; F68 ; F127 and F108 Similarly PEG and the structure of the intermediate product NPC-Plu-NPC , the NPCPlu-TA and PAMAM-Pluronic product may be determined by the results of 1H-NMR spectroscopic analysis , FTIR , GPC and TEM images 2.3.4 Synthetic nano carrier PAMAM G4.0-F127 with PAMAM G4.0:F127 molar ratio different 2.3.5 Encapsulated anti-cancer drug 5-Fluorouracil (5-FU) to the type of dendrimer PAMAM-PEG and PAMAM-Pluronic 2.3.6 Survey release rate the 5-FU drug of PAMAM-PEG/5-FU , PAMAM-Pluronic/5-FU and 5-FU control 2.3.7 Determination of cell toxicity of the nano carrier The toxicity of nano delivery system is determined on breast cancer cells MCF-7 and fibroblasts ( Fibroblast ) Page CHAPTER RESULTS AND ARGUMENT 3.1 SYNTHESIS PAMAM G-0.5 FROM GENERATION TO GENERATION G5.0 3.1.1 Determining the structure of PAMAM dendrimer based on mass spectrometry MS MS is mass spectrometry method to determine effective molecular weight polymer Figure 3.1 MS spectra of dendrimer PAMAM from G-0.5 and G2.0 MS spectra demonstrate products from G-0.5 and G 2.0 true for structured products , consistent with the theory ( Figure 3.1 and Table 3.1 ) Table 3.1 Molercular weight of dendrimer PAMAM based on MS spectra Genration PAMAM G-0.5 G0.0 G0.5 G1.0 G1.5 G2.0 G2.5 G3.0 G3.5 G4.0 G5.0 ideal MLT C18H32O8N2 407 C22H48O4N10 517 C54H96O20N10 1212 C62H128O12N26 1430 C126H224O44N26 2823 C142H288O28N58 3256 C270H480O92N58 6045 C302H608O60N122 6909 C558H992O188N122 12489 C622H1248O124N250 14215 C1262H2528O252N506 28826 (*: Indeterminable) Molecular formulation MMS 405 517 1206 1428 2808 3259 * * * * * MS Deviations (%) 0,02 0,00 0,06 0,02 0,15 0,03 Although MS is a powerful method to determine molecular weights, but with the dendrimer has a molecular weight greater from G2.5 ( M = 6049 ) onwards, the MS can not Page be identified This result is consistent with the group's research Schwartz [ 98 ] and Hood [ 46 ] So 1H-NMR can be effective ways to monitor and assess molecular weight and the metabolism of dendrimer and especially the dendrimer [ 25 , 29 , 49 , 58 , 73 , 112-113 ] and especially the generation dendrimer ( G ) large [ 47 , 69 ] 3.1.2 Determining the structure of PAMAM dendrimer based on 1H-NMR spectrum Chemical shifts for proton featured in PAMAM dendrimer was noted in many previous reports [ 38 , 88-89 , 98 , 102 , 106 , 119 , 124 ] In the 1H-NMR spectrum results corresponding to the typical proton dendrimer structure : -CH2CH2N< (a) at δH = 2.6 ppm; -CH2CH2CO- (b) at δH = 2.8-2.9 ppm; - CH2CH2CONH- (c) at δH = 2.3-2.4 ppm; -CH2CH2NH2 (d) at δH = 2.7-2.8 ppm; CONHCH2CH2N- (e) at δH = 3.2-3.4 ppm; -CH2CH2COOCH3- (g) at δH = 2.4-2.5 ppm and -COOCH3 (h) at δH = 3,7 ppm H-NMR Analysis result of dendrimer PAMAM of generation (Figure 3.2) [74] H-NMR PAMAM G-0.5: at δH = 2.497 ppm (a), δH = 2.756-2.784 ppm (b), δH = 2.386-2.454 ppm (g) and δH = 3.628-3.702 ppm (h) H-NMR PAMAM G0.0: at δH = 2.561-2.573 ppm (a), δH = 2.771-2.815 ppm (b), δH = 2.373-2.400 ppm (c), δH = 2.728-2.753 ppm (d) and δH = 3.246-3.336 ppm (e) H-NMR PAMAM G0.5: at δH = 2.536-2.560 ppm (a), δH = 2.730-2.783 ppm (b), δH = 2.338-2.394 ppm (c), δH = 3.255-3.312 ppm (e), δH = 2.423-2.496 ppm (g) δH = 3.6313.674 ppm (h) H-NMR PAMAM G1.0: at δH = 2.588-2.601 ppm (a), δH = 2.802-2.829 ppm (b), δH = 2.375-2.402 ppm (c), δH = 2.733-2.758 ppm (d) δH = 3.258-3.270 ppm (e) H-NMR PAMAM G1.5: at δH = 2.567-2.654 ppm (a), δH = 2.778-2.848 ppm (b), δH = 2.391-2.419 ppm (c), δH = 3.266-3.368 ppm (e), δH = 2.472-2.499 ppm (g) δH = 3.688 ppm (h) H-NMR PAMAM G2.0: at δH = 2.582-2.608 ppm (a), δH = 2.795-2.822 ppm (b), δH = 2.368-2.394 ppm (c), δH = 2.699-2.741 ppm (d) and δH = 3.250-3.328 ppm (e) H-NMR PAMAM G2.5: at δH = 2.536-2.631 ppm (a), δH = 2.748-2.858 ppm (b), δH = 2.390-2.417 ppm (c), δH = 3.261-3.331 ppm (e), δH = 2.473-2.499 ppm (g) and δH = 3.683-3.688 ppm (h) H-NMR PAMAM G3.0: at δH = 2.605-2.618 ppm (a), δH = 2.804-2.831 ppm (b), δH = 2.379-2.404 ppm (c), δH = 2.735-2.760 ppm (d) and δH = 3.261-3.334 ppm (e) H-NMR PAMAM G3.5: at δH = 2.570-2.634 ppm (a), δH = 2.780-2.846 ppm (b), δH = 2.393-2.419 ppm (c), δH = 3.268-3.369 ppm (e), δH = 2.475-2.501 ppm (g) and δH = 3.6313.689 ppm (h) Page 10 Figure 3.3 Synthetic scheme NPC-PEG-NPC(a) , NPC-PEG-TA(b) , PAMAM-PEG(c) Reaction modified PAMAM ( G2.0 , G3.0 , G4.0 , G5.0 ) with PEG carbon chain length difference ( PEG4K , PEG6K , PEG10K , PEG12K ) has the step response, the spectrum 1H chart 1H-NMR similar, so here we use the spectrum of reactions PAMAM map with PEG4K as an example, that street of PEG remaining in appendix , , H-NMR spectrum results of NPC-PEG-NPC, NPC-PEG-TA , PAMAM-PEG measured in CDCl3 solvent Stage : Results of analysis of the structure of NPC-PEG-NPC was demonstrated by nuclear magnetic resonance spectrum (Figure 3.4 and Appendix ) Map with the signal spectrum of the protons in PEG as follows : 3.40 to 3.79 ppm signal is δH = proton of the methylene group ( -OCH2 - CH2O- ) on EO and methylene proton signal linked directly with groups at δH = 4.44 ppm carbonate ( -CH2-O-NPC ) The arrival of the two signals at δH = 7.39 ppm double δH = 8.29 ppm and are two characteristics of proton signals NPC group ( CH = CH ) PEG activation level over 90% is calculated from the ratio of the proton integral aromatic ( NPC ) and methylene protons ( PEG ) This result is consistent with studies Park [ 64 ] and Nguyen [ 26 ] Figure 3.4 1H-NMR spectrum of the NPC-PEG4K-NPC H-NMR spectra of the map NPC-PEG -NPC others are shown in appendix and also for similar results in 1H-NMR spectrum of map NPC-PEG4K-NPC Page 14 Stage : Results of analysis of the structure of NPC-PEG-TA is expressed through nuclear magnetic resonance spectrum (Figure 3.5 and Appendix ) Map with the signal spectrum of the protons in PEG as follows : 3.40 to 3.79 ppm signal is δH = proton of the methylene group ( -OCH2 - CH2O- ) on EO of PEG Methylene protons directly linked with the carbonate group at δH = 4.44 ppm NPC ( -CH2-O-NPC ) In addition, the methylene protons appear more direct link with the TA in the carbonate group δH = 4.2 ppm ( -CH2-OTA ) This result is consistent with research by Park [ 64 ] and Nguyen [ 26 ] Figure 3.5 1H-NMR spectrum of the NPC-PEG4K-TA The arrival of the two signals at δH = 7.40 ppm double and δH = 8:29 ppm is characteristic of the two signals of the aromatic protons NPC (-CH=CH-) Map signal spectrum appearing at δH = 6.78 ppm and 7.02 ppm δH = signal conjugated aromatic protons (CH = CH) group of tyramine This result is consistent with research by Nguyen [26] About 50% of the original NPC has been replaced by TA This result is obtained from the integral calculation rate of proton and proton NPC's on TA H-NMR spectrum chart of the NPC-PEG-TA, as shown in Appendix 5, and also for similar results in 1H-NMR spectrum of map NPC-PEG4K-TA Stage 3: Results of analysis of the structure of PAMAM and PAMAM-PEG is expressed through nuclear magnetic resonance spectrum (Figure 3.2, Figure 3.6 and Appendix 6) The PAMAM-PEG after synthetic dialysis membrane MWCO days Map signal spectrum proton of PAMAM in PAMAM-PEG (Figure 3.6 and Appendix 6) as: -CH2CH2N [...]... after PAMAM modified by PEG or Pluronic be encapsulated drug 5-FU ( PAMAM G4.0-PEG6K/5-FU, PAMAM G4.0-P123/5-FU and PAMAM G4.0-F127/5-FU ) is capable of powerful cytotoxic concentrations cytotoxic IC50 2-3 times compared to 5-FU control Graph 3.5 Results cytotoxic IC50 of nano carriers 3.7.2 The result is toxic fibroblasts ( Fibroblast ) of nanomaterials transmission system 5 -FU a According to MTT dyeing... successfully modified PAMAM dendrimer generation G2.0, G3.0, G4.0 and G5.0 with biocompatible polymer PEG with different chain lengths (PEG4 K, PEG6K, PEG10K and PEG12K) Molecular weight of the dendrimer PAMAM-PEG product and quantity mounted branch PEG polymer PAMAM be determined and calculated by 1H-NMR and GPC (3) Has successfully modified PAMAM dendrimer generation G2.0, G3.0, G4.0 and G5.0 with biocompatible. .. modified by PEG or Pluronic (G4.0-PEG, G4.0 -Pluronic) did not cytotoxic (with a biocompatible) Page 24 CONCLUDE Based on the results of research completed thesis content, has drawn the following conclusions : (1) Good as successful synthesis Da dendrimer synthesis to generation PAMAM G5.0 Products PAMAM dendrimer generations from G-0.5 and G5.0 is determined by structural methods 1H-NMR and MS and calculation... % and DL % ) is determined indirectly by measuring the results of HPLC and following formula, in which 5FU is Wtotal of the initial 5-FU drug and the amount of 5-FU is not loaded in the carrier is Wfree - 5-FU [ 9 , 68 ] Page 20 The dendrimer PAMAM G2.0 , G3.0 , G4.0 and G5.0 after being modified by the PEG with different molecular weights ( PEG 4K, PEG6K, PEG 10K, PEG 12K ) is used to hydrophobic drug. .. PEG and Pluronic) the more difficult the longer the denaturation (the number of branches fell PAMAM polymer tied up) (5) PAMAM dendrimers with amine groups on the surface of potentially cytotoxic, however all product-PEG modified PAMAM and PAMAM-Pluronic were not cytotoxic (cell disease and healthy cells ), which PAMAM-PEG biocompatible taking better PAMAMPluronic (6) inability PEG encapsulated drug 5-FU... about 2-3 times compared to 5 -FU controls PROPOSAL Research PAMAM dendrimer modified with other polymers such as dextran , chitosan , gelatin , alginate Then proceed to test the ability to carry and release of nano drug carriers with multiple anticancer drugs like doxorubicin other ( DOX ), methotrexate ( MTX ) , paclitaxel ( Taxol ), cis- platinum Vivo studies of PAMAM-PEG/5-FU and PAMAM-Pluronic/5-FU... the PAMAM-PEG and the corresponding PAMAM-Pluronic PAMAM-Pluronic are encapsulated hydrophobic drug 5-FU better Page 25 (7) or PAMAM dendrimers PAMAM-PEG-encapsulated Pluronic after the drug 5-FU, the drug release rate is much slower than the control sample 5-FU (8) When encapsulated in PAMAM modified (PAMAM G4.0-PEG6K, PAMAM and PAMAM G4.0-P123 G.4.0-F127), the drug 5-FU toxicity reduced by about 2-3... to affect the ability to carry the drug 5-FU and the molecular weight PEG bigger bulky space hinders drugs 5-FU is difficult to go into the space inside the PAMAM dendrimer structure should effectively bring relievers with increasing carbon chain lengths of PEG molecules 3.5.3 The survey 's ability to carry drugs PAMAM-Pluronic Statistics obtained HPLC results in graph 3.4 bring efficiency nano drug. .. shows that at a concentration of 100 micrograms / ml : PAMAM without denaturation ( PAMAM G4.0 ) cytotoxic , the Page 22 PAMAM after modified by PEG or Pluronic (PAMAM G4.0-PEG and PAMAM G4.0Pluronic ) significantly reduces cell cytotoxicity of PAMAM Table 3.3 Toxicological results MCF-7 cells according to the SRB staining method Sample concentration (μg/ ml) Antimicrobial activity ( % cell growth )... of the peak area of protons at the position b and 1H-NMR spectrum a ( peak b and a is 8.000 and 4.000 , respectively :NMR = 8.000 / 4.000 = 2 ); the total number of protons in the molecular structure of the dendrimer Page 12 PAMAM G-0.5 ( b and a peak of 8 and 4 , respectively  LT = 8/4 = 2 ) MNMR is calculated as follows: Table 3.2 Molecular weight 1H-NMR based on the G-0.5 and G5.0 PAMAM Generation