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 M

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 :

1 Ass Prof Dr CUU KHOA NGUYEN

2 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 1 , 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

1 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 , 74-

76 ]

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 "

2 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

3 CONTENT OF RESEARCH THESIS

1 Synthesis of dendrimer G5.0 PAMAM generation from center of ethylenediamine (EDA)

2 Study 4 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)

3 Denatured Research G2.0 PAMAM dendrimer generation 4, G3.0, G4.0, G5.0 with p123 Pluronic, Pluronic F68, Pluronic F127 and Pluronic F108

4 Research G4.0 PAMAM dendrimer synthesis-F127 with the molar ratio of PAMAM / F127 different

5 Research encapsulated effective anticancer drugs 5-Fluorouracil (5-FU) of the nano carrier-PEG PAMAM and PAMAM-Pluronic

6 Surveys drug release rate of 5-Fluorouracil-generation dendrimer G4.0 PAMAM PEG6K / 5-FU in vitro environment PBS buffer (pH = 7.4)

7 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)

8 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

4 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 5 -

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

5 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

( 2 ) 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

( 3 ) The ability to bring medicines PAMAM-P123 > PAMAM-F127 > PAMAM-F108

> PAMAM-F68 influenced HLB value rather than the influence of the circuit structure

6 LAYOUT OF THE THESIS

The thesis has 169 pages with 30 tables, 102 diagrams, 8 graphs Besides the introduction ( 2 pages ), conclusions ( 3 pages ), list of publications ( 2 pages ) and references ( 14 pages ), Annex ( 15 pages ), the thesis is divided into 3 chapters as follows:

Chapter 1 : Overview 41 pages

Chapter 2 : Experimental 26 pages

Chapter 3 : Results and argumentation 66 Pages

CHAPTER 1 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 ( 5 - 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: 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 2 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 4 by 4 types of PEG type with a molecular weight Pluronic different to build on the systemic level and the expected modification of the

nanopolymer drug

CHAPTER 2 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 PEG/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)

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 Plu-NPC , the Plu-TA and PAMAM-Pluronic product may be determined by the results of 1H-NMR

NPC-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 )

CHAPTER 3 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

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 1 H-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 = 2.72.8 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

-1H-NMR Analysis result of dendrimer PAMAM of generation (Figure 3.2) [74]

1H-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)

1H-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)

1H-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) và δH = 3.631-3.674 ppm (h)

1H-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) và δH = 3.258-3.270 ppm (e)

1H-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) và δH = 3.688 ppm (h)

1H-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)

1H-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)

1H-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)

1H-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 = 3.631-3.689 ppm (h)

1H-NMR PAMAM G4.0: at δH = 2.550 ppm (a), δH = 2.770 ppm (b), δH = 2.352 ppm (c), δH = 2.746-2.758 ppm (d) and δH = 3.225-3.259 ppm (e)

1H-NMR PAMAM G5.0: tại δH = 2.544 ppm (a), δH = 2.849 ppm (b), δH = 2.340 ppm (c), δH = 2.761 ppm (d) và δH = 3.239-3.251 ppm (e)

Figure 3.2 dendrimer PAMAM 1H-NMR spectrum from G-0.5 to G5.0

According to 1H-NMR spectrum result , characteristic peaks of protons at the position ( a) and ( e ) always appear clearly and do not overlap with any other peak, so this peak selected two used to calculate PAMAM dendrimer evaluate the formulas, molecular weight dendrimer 1H - NMR spectrum through the following :

( e ) 2

( a ) 2

( e ) 2

( a ) 2

H( C H )

H( C H ) NMR

H H

H( C H )

S

  ,

( a ) 2

MLT: Molecular weight of dendrimer PAMAM in theoritical calculation based on molecular formulation

Applying the above formula, molecular weight ( KLPT ) PAMAM dendrimers are calculated based on 1H-NMR spectrum is not much different than KLPT calculated based on the molecular formula ( Table 3.1 )

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

Molecular PAMAM generation ≤ G5.0 was successfully synthesized and relatively complete structure and stability should be applied in the medical - pharmaceutical field

G2.0-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 4 , 5 , 6

1H-NMR spectrum results of NPC-PEG-NPC, NPC-PEG-TA , PAMAM-PEG measured

in CDCl3 solvent

Stage 1 : Results of analysis of the structure of NPC-PEG-NPC was demonstrated by nuclear magnetic resonance spectrum (Figure 3.4 and Appendix 4 ) 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

1H-NMR spectra of the map NPC-PEG -NPC others are shown in appendix 4 and also for similar results in 1H-NMR spectrum of map NPC-PEG4K-NPC