(pamam)- modified nano drug delivery system for targeting cancer

MINISTRY OF EDUCATION AND
VIETNAM ACADEMY OF SCIENCE AND
TRAINING
TECHNOLOGY
GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY
NGUYEN THI BICH TRAM
THE SYNTHETIC STUDY ON POLYAMIDOAMINE
(PAMAM)- MODIFIED NANO DRUG DELIVERY
SYSTEM FOR TARGETING CANCER CELLS
Subject: ORGANIC CHEMISTRY
Subject code: 62 44 01 14
CHEMISTRY PHD DISSERTATION ABSTRACT
HO CHI MINH CITY, 2016
The PhD’s thesis was completed at:
Department of Materials and Pharmaceutical Chemistry, Institute of Applied
Materials Science, Vietnam Academy of Science and Technology.
Primary supervisor : Assoc. Prof. PhD. NGUYEN CUU KHOA
Co-Supervisor : Prof. PhD. NGUYEN CONG HAO
Reviewer 1:
Reviewer 2:
Reviewer 3:
The dissertation defense will be evaluated by Graduate Academic Committee at
Institute of Applied Materials Science, Vietnam Academy of Science and
Technology on .........................
The PhD dissertation will be published at:
Library of the Academy of Science and Technology of Vietnam
LIST OF PUBLICATIONS RELATED TO THE
THESIS
1.
Nguyễn Thị Bích Trâm, Trần Ngọc Quyển, Nguyễn Cửu Khoa, Ứng
dụng phổ cộng hưởng từ hạt nhân trong phân tích đánh giá các
polyaminoamin dendrimer và dẫn xuất, Tạp chí Hóa học, 51(4AB), pp
276-279, 2013.
2.
Thị Bich Tram Nguyen, Ngoc Quyen Tran, Cuu Khoa Nguyen,
Biocompatible and cellular uptake enhancement of polyamidoamine
dendrimer via fatty alkyl conjugation, Tạp chí Hóa học, 51(4AB), pp
259-263, 2013.
3.
Ngoc Quyen Tran, Ngoc Hoa Nguyen, Thị Bich Tram Nguyen, Nguyen
Cuu Khoa, Positive effect of dendrimers nanocarriers on reducing
cytotoxicity of anticancer drugs, Tạp chí Hóa học, 51(4AB), pp 270-275,
2013.
4.
Thị Bich Tram Nguyen, Ngoc Quyen Tran, Cuu Khoa Nguyen,
Cytotoxic behaviors of pamam-based dendrimers loading platinium
compounds, Tạp chí Khoa học và Công nghệ, 51(5A), pp 334-341,
2013.
5.
Cuu Khoa Nguyen, Ngoc Quyen Tran, Thi Bich Tram Nguyen, Kim
Ngoc Phan, Dendrimer-based nanocarriers demonstrating a high
efficiency for loading and releasing anticancer drugs against cancer cells
in vitro and in vivo, Tạp chí Khoa học và Công nghệ, 51(5A), pp 224232, 2013.
6.
Thi Bich Tram Nguyen, Phuc Thinh Nguyen, Minh Nhật Hồ, Cuu Khoa
Nguyen, and Ngoc Quyen Tran, 5-Fluororacil loading and releasing
behavior from alkylated polyamidoamine G3.0, the 7th International
Workshop on Advanced Materials Science and nanotechnology, Hạ
Long City, VN, 2014.
7.
Nguyen Thi Bich Tram, Phuc Thinh Nguyen, Nguyen Đại Hải, Nguyen
Cuu Khoa, Tran Ngoc Quyen, 5-Fluororacil loading and releasing
behavior from alkylated polyamidoamine G3.0 dendrimer - folate, Tạp
chí Hóa học, 53(4e3), pp 168-173, 2015.
8.
Thi Bich Tram Nguyen, Thi Tram Chau Nguyen, Hoang Chinh Tran,
Cuu Khoa Nguyen, and Ngoc Quyen Tran, 1-H NMR Spectroscopy as
an
Effective
Method
for
Predicting
Molecular
Weight
of
Polyaminoamine Dendrimers and Their Derivatives, International
Journal of Polymer Analysis and Characterization, International Journal
of Polymer Anal. Charact., 20: 57–68, 2015.
ABSTRACT
Cancer is one of the leading killers in the world. The relentless progress
of science has brought on a better outcome for patients with cancer. With
effective treatment purposes, the current study focused on treatment at the
cancer cells and minimize the harm to healthy cells of the body, medicine
called targeted therapy. Targeted treatments open up a new direction in the
treatment of cancer. This method involves the use of these drugs have the
ability to attack specifically against cancer cells. Ability to distinguish cancer
cells and normal cells to help targeted therapies become a treatment method
chosen today.
The dendrimer is used as an interest bearing targeted drugs in cancer
treatment. Dendrimer is a polymer nano features more suitable for the role of a
carrier with molecular structures of spherical shape, with many branches inside
the empty space, there are many groups that operate on the surface. However,
the active functional groups at the dendrimer surface chemical interactions biological cells in the body so they are toxic to the cells. To overcome these
limitations and to improve some properties of support for bringing the right
place and effective drugs, the dendrimer surface was modified by an agent in
the biocompatible as alkyl, PEG (Polyethylene Glycol ), ... combining targeted
agents such as folic acid, peptide RGD (Arg-Gly-Asp), Anti-EGFR (epidermal
growth factor receptor) and antibodies Anti-HER-2 (human Growth Factor
receptor epidermal), biotin, ... through chemical bonds.
Because of these reasons, we have carried out to implement the project:
“The study synthesized polyamidoamine (PAMAM) modified nano drug
delivery system potentially targeted to cancer cells”.
1
The objective of the thesis
Synthesis of PAMAM dendrimer generation and PAMAM dendrimer
surface modification with alkylating agents and targeted agents (folic acid) for
the purpose of construction of nanomaterials carry anticancer drugs with
biocompatible properties and classification drug distribution to the correct
location of cancer cells.
To achieve these goals, the thesis has made the following
1. Successfully synthesized PAMAM dendrimer from generations G-0.5
to G3.0.
2. Determination of the structure and molecular weight of PAMAM
dendrimer synthesis.
3. PAMAM dendrimer surface modification G3.0 with agent alkyl (acyl
chloride, alcohols, carboxylic acids, alkylamine).
4. PAMAM dendrimer surface modification G3.0 or G3.0 PAMAM
dendrimer-alkyl with targeted agents (folic acid).
5. Identify structural components, metabolism, molecular weight, the
metabolic alkyl group of PAMAM G3.0 modified dendrimer
derivatives.
6. Survey of cell toxicity G3.0 PAMAM dendrimer and G3.0 PAMAM
dendrimer derivatives.
7. Survey of the nano-sized dendrimers PAMAM G3.0 and G3.0
PAMAM dendrimer derivatives.
8. Survey possibility of delivery system targeted anticancer drugs.
2
9. Surveying the ability to carry and release G3.0 PAMAM dendrimer
drug-drug system.
3
Meaning scientific and practical
The research results of the thesis shows some meaningful conclusions
and practical science follows:
-
Valuable topics in the study of the compound targeted anti-cancer
drugs, which are compounds anticancer drug delivery to the correct
location of cancer cells. This is a social problem is of current interest.
-
The new findings 1H-NMR spectral in evaluating molecular weight
dendrimer has important implications in the application and evaluation
process metabolize synthetic compounds, drugs, ...
4
The layout of the thesis
The thesis has 119 pages with 23 tables, 48 figure, 8 graphs and 21 charts.
Besides the introduction (3 pages), concluded (2 pages), list of publications (2
pages) and references (20 pages) thesis is divided into 3 chapters as follows:
Chapter 1: Overview (30 pages)
Chapter 2: Experiment (30 pages)
Chapter 3: Results and Discussion (59 pages)
5
New contributions of the thesis
1.
Successfully synthesized PAMAM dendrimer generations from G-0.5
and G3.0 (response performance 71-89%), with the aim of obtaining
PAMAM G2.5 dendrimer and PAMAM G3.0 dendrimer used for the
surface modification of PAMAM dendrimer.
2.
Building successful new calculation method based on spectral data 1 H
nuclear magnetic resonance and mass spectrometry NMR MS in the low
generation PAMAM dendrimer from G-0.5 and G2.0 to calculate the
molecular weight of the dendrimer high-generation PAMAM (G3.0
generation onwards). Effect of deviation from the theory of this method is
about 0-6%, equivalent to results using mass spectrometry MALDI-TOFMS.
3.
Successfully synthesized compounds of G3.0 PAMAM dendrimer
modified with alkylating ranges as acyl chloride, carboxylic acid, alcohol
and G2.5 PAMAM dendrimer with alkylamine.
4.
Has successfully built a new calculation method based on 1H-NMR
spectral data to determine metabolism (x%), the transformation group (z
group), molecular weight of PAMAM- alkyl dendrimer derivatives.
5.
Examined cytotoxicity in MCF-7 cells of the dendrimer PAMAM G3.0
and G3.0 PAMAM dendrimer derivatives, with the number of carbon
atoms of the alkyl chain increased from 2 to 14 (PAMAM G3.0-C2
dendrimer, G3.0-C6, G3.0- C10 and G3.0-C14). Results showed that
after the alkylation, cytotoxicity of
PAMAM G3.0-alkyl dendrimer
derivatives significantly reduced compared to G3.0 PAMAM dendrimer
6
has not modified. G3.0 PAMAM dendrimer toxicity decreases as long
alkyl carbon circuit.
6.
Has successfully mounted folic acid targeted agent to drug delivery
system of PAMAM G3.0 dendrimer and G3.0-C6 PAMAM dendrimer
derivatives forming PAMAM G3.0-FA dendrimer and PAMAM G3.0C6-FA dendrimer drug delivery systems.
7.
Microscopic laser scanning confocal have demonstrated the ability of the
ability of the system targeted initiative of the PAMAM G3.0-C6-FA
drug delivery system.
8.
Surveyed the size of PAMAM G3.0 dendrimer and PAMAM G3.0-C6;
PAMAM G3.0-FA; PAMAM G3.0-C6-FA dendrimer derivatives by
TEM images. The size of the delivery system is in the range 4-6nm.
9.
Successfully surveyed the ability to carry and release 5-FU anticancer
drugs of PAMAM G3.0 dendrimer and PAMAM G3.0-C6-FA dendrimer
derivatives. Results showed that the PAMAM G3.0 dendrimer molecule
loads 6 molecules of 5-FU, while PAMAM G3.0-C6-FA dendrimer
derivatives carries more (9 molecules of 5-FU). PBS environment, 5-FU
drug is released from the PAMAM G3.0-C6-FA/5FU drug delivery
system slower than the 5-FU reference.
7
Method of study
-
Using MS spectrum, 1H-NMR to analyze the structure of the product.
-
Using transmission electron microscopy (TEM) evaluation of product
shapes.
-
1
H-NMR spectrum used to predict PAMAM dendrimer molecular
weight and the metabolism of PAMAM modified products..
-
Using EZ-Cytox Cell Viability Assay Kit analytical methods to
evaluate the toxicity of the cell.
-
Using
laser
scanning
microscopy
confocal
to
evaluate
interoperability of the system to bring the drug to the cells.
8
the
RESULTS AND DISCUSSION
3.1 PAMAM G3.0 DENDRIMER SYNTHESIS
The PAMAM dendrimer products with ethylenediamine core from
generation
G-0.5 to G3.0 are synthesized, has viscid form, dark yellow
gradually from G-0.5 to G3.0. PAMAM dendrimer generations are identified
structural components and molecular weight through physical and chemical
methods of modern analysis.
Determining the structure of PAMAM dendrimer based on 1H-
3.1.1
MNR spectrum
1
H-NMR spectrum of PAMAM dendrimers from G-0.5 to G3.0 (500
MHz, MeOD, δ ppm) (Figure 3.1) appears proton signal characteristic of the
peak of the compound: -CH2CH2N< (a); -CH2CH2CO- (b); -CH2CH2CONH(c);
-CH2CH2NH2
(d,
even
generation);
-CONHCH2CH2N-
CH2CH2COOCH3 (g, odd generation) và -COOCH3 (h, odd generation).
9
(e);
-
Figure 3.1: 1H-NMR spectrum and molecular structure of PAMAM dendrimer
generations from G-0.5 and G3.0
10
1
H-NMR spectral data of the products from PAMAM dendrimer
generation from G-0.5 to G3.0 was statistically by table 3.1.
Table 3.1: 1H-NMR spectral data of the PAMAM dendrimer generations from G-0.5 to G3.0
Chemical shifts (, ppm)
H
location
H of group
a
-CH2CH2N<
b
c
d
e
g
h
G-0.5
G0.0
G0.5
G1.0
G1.5
G2.0
G2.5
G3.0
2.56-
2.53-
2.58-
2.56-
2.58-
2.53-
2.60-
2.57
2.56
2.60
2.65
2.60
2.63
2.61
2.75-
2.77-
2.73-
2.80-
2.77-
2.79-
2.74-
2.80-
2.78
2.81
2.78
2.82
2.84
2.82
2.85
2.83
2.37-
2.33-
2.37-
2.39-
2.36-
2.39-
2.37-
2.40
2.39
2.40
2.41
2.39
2.41
2.40
2.49
-CH2CH2CO-
-CH2CH2CONH2.72-
2.73-
2.69-
2.73-
2.75
2.75
2.74
2.76
-CH2CH2NH2
3.24-
3.25-
3.25-
3.26-
3.25-
3.26-
3.26-
3.33
3.31
3.27
3.36
3.32
3.33
3.33
-CONHCH2CH2N2.38-
2.42-
2.47-
2.47-
2.45
2.49
2.49
2.49
3.62-
3.63-
-CH2CH2COOCH3
-COOCH3
3.683.68
3.70
3.67
3.68
Through spectral data sheet of the dendrimer PAMAM generation 0.5 GG3.0 (table 3.1), again confirms the repeated signals of protons in PAMAM
dendrimer generations.
The proton signals in place a, b, c, e always appear in PAMAM
dendrimer molecule. Proton signals in place d appears only in even generation
(G0.0, G1.0, G3.0, …) and proton signals in place g, h appears only in odd
generation (G0.5, G1.5, G2.5, …).
Result analysis 1H-NMR spectra coincides with articles on past PAMAM
dendrimer synthesis [1, 4, 21, 23, 28, 56, 80, 89, 103, 104].
11
3.1.2
Determine the molecular weight of the PAMAM dendrimer based
on MS spectrum
When we analyze MS spectrum (Appendix 1), the results showed the
PAMAM dendrimer generation from G-0.5 to G2.0 with molecular weights
consistent with molecular mass in theory (Table 3.4) , false offsets 0-0.25%
compared with the theory.
The results in Table 3.4 show that MS mass spectrometry method to
determine effective molecular weight lower generation dendrimer PAMAM (G
≤ 2.0), false offsets 0-0.25% when compared with the theory. However with the
dendrimer has a molecular weight greater from G2.5 (Wt; 6045) or more, we
can not determine molecular weight by MS spectrum. This is a limitation of the
MS mass spectrometry when surveyed on compounds of high molecular
weight..
3.1.3
Determine the molecular weight of the dendrimer PAMAM based on 1HNMR spectra
The proton signals in 2 positions (e), (a) always appear and no overlap
1
on H-MNR spectra in each molecule dendrimer PAMAM (Figure 3.1 to 3.8)
should be chosen to calculate molecular weight PAMAM dendrimer.
The area of the proton signal at 2 positions (a), (e) shown on the 1HMNR spectra of PAMAM dendrimers from G-0.5 to G3.0 were we statistics in
Table 3.2.
Table 3.2: Proportion area of the proton signal at 2 positions (a), (e) shown on
the 1H-MNR spectra (NMR) of PAMAM dendrimers from G-0.5 to G3.0
G
G-0.5
S
(e)
H(  C H 2  )
8(H in position b)
S
(a )
H(  C H 2  )
 NMR 
2
4
12
S
S
(e)
H ( C H2 )
(a)
H ( C H2 )
G0.0
3,987
2,000
1,985
G0.5
0,778
1,168
0,6661
G1.0
1,000
0,532
1,8797
G1.5
1,864
2,186
G2.0
1,964
1,048
0,8527
1,8740
G2.5
2,058
2,335
0,8814
G3.0
1,923
1,018
1.8890
Applying the formula for calculating the molecular weight of the
dendrimer (WtNMR) are we propose through 1H-NMR spectra (Formula 2.1),
using the data in (Table 1.1, Table 2.1 and Table 3.2). We have calculated
molecular weight PAMAM dendrimers from generation G-0.5 to G3.0, the
results shown in Table 3.4.
Using the formula above for the dendrimer PAMAM G-0.5, we use the proton
signal (a) and (b) to calculate the volume of signal molecules by proton (e) has not
appeared in the molecular structure of these substances.
S (b)
H(  C H 2  )
Wt NMR

 NMR .Wt LT 
 LT
S
(a )
H(  C H 2  )
H
H
(b)
(  C H2  )
.Wt LT
8
 4 .407  407
8
4
(a )
(  C H2  )
Deviation of the molecular weight of the PAMAM G-0.5 dendrimer
compared to the theoretical value:
% 
M tt  M lt
407  407
.100% 
.100%  0%
M lt
407
With the PAMAM dendrimer from G0.0 to G3.0 generation, we use the
shift of the proton signals (a) and (e) to calculate their molecular weights.
13
S
(e)
S
(a )
H(  C H 2  )
Wt NMR

 NMR .Wt LT 
 LT
H(  C H 2  )
H
H
.Wt LT
(e)
(  C H2  )
3,987
2,000

.518  516
8
4
(a )
(  C H2  )
Deviation of the molecular weight of the PAMAM G0.0 dendrimer
compared to the theoretical value:
M  M lt
516  517
%  tt
.100% 
.100%  0,19%
M lt
517
Table 3.3: Molecular weight of PAMAM dendrimers from G-0.5 to G3.0 on
the basis of 1H-NMR and MS spectra
LT
NMR
MS
G
WtLT
WtNMR
Deviation (%)
WtMS
Deviation (%)
G-0.5
407
407
0
407
0
G0.0
517
516
0,19
518
0,19
G0.5
1204
1202
0,17
1207
0,25
G1.0
1430
1344
6,01
1429
0,14
G1.5
2830
2815
0,53
2809
0,14
G2.0
3256
3061
5,99
3260
0,09
G2.5
6045
5720
5,38
*
G3.0
6909
6529
5,50
*
(*:not measured by molecular weight by large molecular weight)
Looking at the results table 3.4, molecular weight PAMAM dendrimers
from G-0.5 to G3.0 completely determined by 1H-NMR spectra. Molecular
weight PAMAM dendrimers calculations are based on 1H-MNR spectra is not
14
much different than the theoretical molecular weight (average deviation from
the theory of 0-6%). Meanwhile, MS spectrum has the deviation smaller (from
0-0.25%), but this method can only be determined from PAMAM dendrimer
generations from G-0.5 to G2.0. This shows the advantages of 1H-NMR
spectrum, not only to identify the molecular structure but also identified the
molecular weight PAMAM dendrimer without being limited by the large
molecular weight. So we have used 1H-NMR spectrum to calculate molecular
weight PAMAM dendrimers for large molecular weight (from G2.5 generation
onwards) while MS spectrometry method can not be identified.
In addition, when comparing the PAMAM dendrimer molecular mass
values between 1H-NMR spectra experimental methods and theoretical value,
deviations from 0-6% (Table 3.3), the deviations of this method is equivalent to
deviations when using MALDI-TOF-MS spectra [77].
So we came up with the conclusion that can be trusted 1H-NMR spectra
using a convenient and efficient way to identify structural features of dendrimer
PAMAM and PAMAM dendrimer derivatives. Especially 1H-NMR spectra
used to determine the molecular weight of the PAMAM dendrimer and
PAMAM dendrimer derivatives without having to use additional MALDI-TOFMS or another molecule mass spectrometry. This is a new discovery of 1HNMR spectral studies of PAMAM dendrimer.
3.2 SYNTHESIS OF ALKYLATED G3.0 PAMAM DENDRIMER
3.2.1
PAMAM G3.0 dendrimer modified with acyl chloride
The product has viscid form, light
yellow, with general structural
formula: G3.0-(NH-CO-CH2(CH2)nCH3)z (Figure 3.9).
15
Figure 3.2: G3.0-(NH-CO-CH2(CH2)nCH3)z product structure
1
H-NMR spectrum of PAMAM G3.0-acyl chloride dendrimer derivatives
proton signal appeared characteristic peaks of PAMAM dendrimer: CH2CH2N< (a); -CH2CH2CO- (b); -CH2CH2CONH- (c); -CONHCH2CH2N(e); besides appearing proton signal characteristic of the group -CH3 (j) and
proton signals -CH2CH2NH2 (d) not losing shows some amino groups (of total
32 amino groups) of PAMAM G3.0 dendrimer surface was mounted through
linking amide.
3.2.1.1
Survey on time
PAMAM G3.0 dendrimer modified reaction surveyed over a period of 12
hours (G3.0-C10-12h), 24 hours (G3.0-C10-24h), 36 hours (G3.0-C10-36h) and
48 hours (G3.0-C10-48h).
16
1
H-NMR spectral data of product G3.0 PAMAM dendrimer modified
with decanoyl chloride over time (Appendix 2, 3,4,5) are statistically over Table
3.5.
Table 3.4: 1H-NMR spectral data of the survey PAMAM G3.0 dendrimer
modified with decanoyl chloride over time
H
Chemical shifts (, ppm)
H of group
location
G3.0-C10-12h
G3.0-C10-24h
G3.0-C10-36h
G3.0-C10-48h
a
-CH2CH2N(30 groups)
2, 53-2,54
2,68
2,57
2,62-2,64
b
-CH2CH2CO(60 groups)
2,73
2,75-2,77
2,77
2,84-2,85
c
-H2CH2CONH(60+z nhóm)
2,34
2,35-2,44
2,37-2,45
2,18-2,21
d
-CH2NH2
(32- z groups)
2,83-2,96
2,82-2,89
3,01
3,03
e
CONHCH2CH2(60+z groups)
3,21-3,34
3,32-3,46
3,24-3,40
3,30-3,45
j
-CH3
(z groups)
0.77
0,91
0,80
0,91
Based on the shift of the proton signal (a) and (j) on 1H-NMR spectrum
was statistically through table 3.5, apply formulas (Formulas 2.2) determine
conversion degree (x%), the conversion group (z groups) and molecular weight
(WtNMR) of PAMAM G3.0-C10-12h, G3.0-C10-24h, G3.0- C10-36h and G3.0C10-48h dendrimer derivatives (table 3.6).
Applying example with PAMAM G3.0-C10-36h dendrimer derivative.
The conversion degree of product is calculated on the basis of 1H-MNR
spectrum (Figure 3.10) according to the above formula:
17
S
( j)
H(  C H3 )
S
x% 
(a )
H(  C H 2  )
H
H
( j)
(  C H3 )
3,362
4,327
.100% 
.100%  48,56%
32x3
30x2
(a )
(  C H2  )
The conversion group:
z = x%.32 = 48,56%.32 = 15,5  16 groups
Molecular weight of PAMAM G3.0-C10-36h dendrimer derivative is
calculated as follows (using the data of table 1.2, table 2.3):
WtNMR = WtLT(PAMAM 3.0 dendrimer) + z. WtLT(dodecanoyl chloride) – z. WtLT(HCl)
= 6909 + 16.218,76 – 16.36,46084
= 9826
(with PAMAM G3.0 dendrimer = 6909, H = 1,00784; Cl = 35,453)
Figure 3.1: 1H-NMR spectra of PAMAM G3.0-C10-36h (G3.0-C10) derivative
18
Derivatives of PAMAM G3.0-C10-12h, G3.0-C10-24h and G3.0-C1048h are similar calculated and recorded in the following table (Table 3.6).
Table 3.1: Survey results of PAMAM G3.0 dendrimer modified with decanoyl
chloride over time
PAMAM G3.0
S
S
derivative
( j)
H(  C H3 )
(a )
H(  C H 2  )
H
H
( j)
(  C H3 )
(a )
(  C H2  )
conversion
degree (x%)
conversion
grou (z
nhóm)
molecular
weight (WtNMR)
G3.0-C10-12h
0,747
2,764
32x3
30x2
16,89
5
7820
G3.0-C10-24h
0,124
0,321
32x3
30x2
24,14
8
8367
3,362
4,327
32x3
30x2
48,56
16
9826
3,000
4,161
32x3
30x2
45,06
14
9461
G3.0-C10-36h
(G3.0-C10)
G3.0-C10-48h
Looking at the results Table 3.6 we can see, the reaction time is
surveyed in turn is 12 hours, 24 hours, 36 hours and 48 hours, the conversion
degree of the derivatives is the highest in 36 hours. In the period from 12-36
hours, the ability to react G3.0 PAMAM dendrimer modified between
ascending and decanoyl chloride. However, as we continue to extend the
reaction time to 48 hours, the conversion degree of the derivatives does not
increase anymore, but started to decline (Graph 3.1), due to prolonged reaction
time, the product is hydrolyzed affect conversional efficiency.
19
Graph 3.1: The reaction time urvey results of PAMAM G3.0 dendrimer
modified with decanoyl chloride
Thus, the conversion degree of derivatives at 36 hours is the best time in
the survey range. Based on this, we continue to conduct modified PAMAM
G3.0 dendrimer with acyl chloride another in the conditions of the time 36
hours.
3.2.1.2 PAMAM G3.0 dendrimer modified with acyl chloride (acetyl chloride,
hexanoyl chloride, myristoyl chloride) in 36 hours
The acyl chloride was also studied as acetyl chloride, hexanoyl chloride,
myristoyl chloride to modified PAMAM G3.0 in the same 36 hour time.
Similar calculations on the basis of 1-H NMR spectrum, the conversion degree
(x%), the conversion group (z) and molecular weight as with decanoyl chloride.
Results showed that the alkyl chain with longer carbon atoms from C2 to C10,
the numbers on the alkyl group (z) mounted on PAMAM increased from 10 to
16 group. However, when the alkyl chain increasing C14, substituted alkyl
groups attached to PAMAM number will decrease, due to the influence of
factors of space (Graph 3.2).
20
Graph 3. 2: The results of PAMAM G3.0 dendrimer modified with acyl chloride
3.2.2 PAMAM G3.0 dendrimer modified with carboxylic acid
The product of PAMAM G3.0 dendrimer modified with carboxylic acids
(acetic acid, hexanoic acid, decanoic acid and myristic acid) has viscid form,
light yellow, like PAMAM G3.0 dendrimer modified with acyl chloride, there
general structural formula is G3.0-(NH-CO-CH2(CH2)nCH3)z (Figure 3.3).
Calculated on the basis of 1H-NMR spectrum, the conversion degree
(x%), conversion groups (z) and molecular weight is calculated as above.
Results showed that increasing alkyl chain from 2 to 10 carbon alkyl group, the
alkyl numbers mounted on PAMAM increased from 10 to 15 groups. However,
when the 14 carbon alkyl chain increased, the alkyl groups attached to the
dendrimer PAMAM G3.0 will drop to 12 due to factors of space (Graph 3.3).
Graph 3.3: The results of PAMAM G3.0 dendrimer modified with carboxylic acid
21
PAMAM G3.0 dendrimer modified with carboxylic acid is also the basis
for reaction of PAMAM G3.0 dendrimer modified with folic acid targeted
agents.
3.2.3
PAMAM G3.0 dendrimer modified with alcohol
The product of PAMAM G3.0 dendrimer modified with alcohol has
viscid form, light yellow, with general structural formula is as follows G3.0(NH-COO-CH2(CH2)nCH3)z (Figure 3.4).
Figure 3.2: The structure of PAMAM G3.0-alcohol derivative
Calculated on the basis of 1H-NMR spectra, the conversion degree (x%),
conversion groups (z) and molecular weight is calculated as above. Results
showed the PAMAM G3.0 dendrimer when modifying with alcolhol used by
NPC activator for the high conversion degree. (Graph 3.4).
22
Graph 3.4: The results of PAMAM G3.0 dendrimer modified with alcohol
3.2.4
PAMAM G3.0 dendrimer modified with alkylamines
The product of PAMAM G2.5 dendrimer modified with alkylamines has
viscid form, light yellow, with general structural formula is as follows G2.5(CO-NH-CH2(CH2)nCH3)z (Figure 3.5).
Figure 3.3: The structure of PAMAM G2.5-alkylamine derivative
Calculated on the basis of 1H-NMR spectra, the conversion degree (x%),
conversion groups (z) and molecular weight is calculated as above. The results
show that:
23
-
At higher reaction temperatures, the conversion group (z) of
dodecylamine higher (Graph 3.5a).
-
With increased from 4 carbon alkyl (butylamine), 6 carbon
(hexylamine), 10 carbon (decylamine) and 12 carbon (dodecylamine),
at reaction temperature 80oC, the conversion group (z) increased from 4
to 11 (Graph 3.5b).
(a)
(b)
Graph 3.5: The results of PAMAM G3.0 dendrimer modified with dodecylamine
with temperature (a) and alkylamines (b)
3.3
PAMAM G3.0 DENDRIMER MODIFIED WITH TARGETTING
AGENTS - FOLIC ACID
The product of PAMAM G3.0 dendrimer and PAMAM G3.0-C6
modified with targetting agents - folic acid have get PAMAM G3.0-FA và
PAMAM G3.0-C6-FA products (Figure 3.6).
1
H-NMR spectra (Figure 3.16) of PAMAM G3.0-C6-FA dendrimer
derivative shows that besides the appearance of proton signal characteristic of
G3.0 PAMAM dendrimer, there are other proton signal characteristic of the
group of atoms in a molecule group of folic acid (k, m, r, p and q) and the alkyl
group proton signal (j) in hexanoyl chloride. This demonstrates that there are
24
link of the group of folate and hexanoyl group with PAMAM G3.0 dendrimer.
PAMAM G3.0-C6-FA dendrimer derivatives has yellow-orange. Structural
formula PAMAM G 3.0-C6-FA dendrimer derivative (Figure 3.16).
Figure 3.4: The structure and 1H-NMR spectra of PAMAM G 3.0-C6-FA derivative
Based on the shift of the proton signal (a) and (j) on 1H-NMR spectra,
apply formulas (Formulas 2.2) determine the conversion degree (x%),
conversion groups (z) and molecular weight (WtNMR) of PAMAM G3.0-FA and
PAMAM G3.0-C6-FA derivatives
Table 3.4: The conversion degree (x%), conversion groups (z), and molecular
weight (WtNMR) of PAMAM G3.0-FA and PAMAM G3.0-C6-FA derivatives on
the basis of 1H-NMR spectra
x%
Product
PAMAM G3.0-FA
z nhóm
WtNMR
Conclusion Product
3
8179
PAMAM G3.0-(FA)3
8768
PAMAMG3.0-(C6)6-(FA)3
folate
8,01
hexanoyl
20
6
folate
8,01
3
PAMAM G3.0-C6-FA
The researchers of Michigan Nanotechnology Institute and the University
of Michigan, they were attached to the PAMAM G5.0 dendrimer folic acid with
a ratio of 3: 1 and is effective targeted to cancer cells as expected [79]. From
the experimental results, we have also been attached 3 folic acid molecules to
25
the surface of PAMAM G 3.0 dendrimer. This is a results promising for the
study of the tagetting drug carriers.
3.4
TEST RESULTS CYTOTOXICITY
Ability to inhibit cell growth of PAMAM G3.0 dendrimer and PAMAM
G3.0-alkyl dendrimer derivatives were tested on MCF-7 cells (Frederick, MD,
USA) by EZ-Cytox Cell Viability Assay Kit, at Ajou University, Suwon City,
Korea.
In this test, we chose dendrimer PAMAM 3.0 and 4 alkyl derivatives of
PAMAM G3.0 dendrimer, with the number of alkyl carbon atoms increased
from 2 to 14 (PAMAM G3.0-C2, G3.0-C6, G3.0-C10 and G3.0-C14) to
examine cell cytotoxicity.
Graph 3.6: The result of cytotoxic survey
Test methods by EZ-Cytox Cell Viability Assay Kit on MCF-7 cells, test
results showed that PAMAM 3.0 cytotoxic, however alkyl derivatives of
PAMAM G3.0 sharply reduced the cytotoxicity of PAMAM G3.0. Cytotoxicity
of PAMAM-alkyl significantly reduced when the alkyl chain length extends
(Graph 3.6). The results coincide with the research of Rachaneekorn
Jevprasesphant [70]
26
3.5
CELL TRANSFECTION RESULTS
The ability cell transfection of G3.0-C6-FITC and G3.0-C6-FA-FITC
was tested on Hela cell line by Confocal laser scanning microscopy at at Ajou
University, Suwon City, Korea.
(a)
(b)
(c)
Figure 3.10: Results analyzed by Confocal laser scanning microscopy of
control cell (a), cells treated with PAMAM G3.0-C6-FITC (b) and cells treated
with PAMAM G3.0-C6- FA-FITC (c).
Through the cell transfection results, we have demonstrated the ability to
active targeted the PAMAM G3.0-C6-FA-FITC dendrimer derivatives and
passive abilities targeted PAMAM G3.0-C6-FITC dendrimer's derivatives.
Consistent with studies Kukowska-Latallo JF [86], Youngseon Choi [109] and
articles [44, 47-49, 79-83, 103, 111]. Research results show the potential of
cancer therapy using new drug delivery system linking folic acid targeted
agents, contributing to improving the effectiveness and safety of treatments.
27
3.6
NANO SIZE
DERIVATIVES
OF
PAMAM
G3.0
DENDRIMER
AND
Figure 3.5: TEM image of PAMAM G3.0 and PAMAM G3.0-C6 derivative
Figure 3.6: TEM image of PAMAM G3.0-FA and G3.0-C6-FA derivative
TEM images showed that the nanoparticles G3.0 PAMAM dendrimer
was formed with spherical shaped and the particle size ranging from 3 nm to 4
nm (Fig. 3.7).
After PAMAM G3.0 dendrimer modified with hexanoyl chloride and
folic acid to products (PAMAM G3.0-C6, G3.0-FA, G3.0-C6-FA) has a particle
size of 4 to 6 nm (Fig. 3.8). TEM images of PAMAM G3.0 dendrimer and
derivatives showed the compounds synthesized nanoscale matching molecular
theory. With this nano-sized dendrimers derivatives have huge potential in the
medical field - pharmaceutical, such as carrying drugs, transporting genes, …
[4, 5, 22, 90, 112-114]
28
3.7
THE SURVEY OF THE ABILITY CARRYING AND RELEASING
DRUG
3.7.1
The survey of the ability carrying 5-fluorouracil anti-cancer drug
Test results showed that the ability to carry PAMAM dendrimer drug's
effectiveness:
-
1 gram of PAMAM G3.0 dendrimer carriers can carry 112,97mg 5-FU
drug corresponding 6 molecules of 5-FU drug be carried within one
PAMAM G3.0 molecular structure.
-
1 gram of PAMAM G3.0-C6-FA dendrimer derivatives can carry
133,52mg 5-FU drug corresponding 9 molecules of 5-FU drug be
carried within one PAMAM G3.0-C6- FA derivative.
3.7.2
The survey of the ability releasing 5-fluorouracil anti-cancer drug
In vitro test results show the ability of the drug released slowly from the
system PAMAM G3-C6-FA/5-FU and release slower than 5-FU freedom. This
is a significant improvement in extending drug bioavailability, because anticancer 5-FU has been reported to be time very short circulating half-life [34].
29
CONCLUSION
Looking back at the original goals of the thesis“The study synthesized
polyamidoamine (PAMAM) modified nano drug delivery system potentially
targeted to cancer cells”. Some new results of the thesis can achieve
summarized as follows:
1.
Successfully synthesized PAMAM dendrimer generations from G-0.5 and
G3.0 (response performance 71-89%), with the aim of obtaining PAMAM
G2.5 dendrimer and PAMAM G3.0 dendrimer used for the surface
modification of PAMAM dendrimer.
2.
Building successful new calculation method based on spectral data 1 H
nuclear magnetic resonance and mass spectrometry NMR MS in the low
generation PAMAM dendrimer from G-0.5 and G2.0 to calculate the
molecular weight of the dendrimer high-generation PAMAM (G3.0
generation onwards). Effect of deviation from the theory of this method is
about 0-6%, equivalent to results using mass spectrometry MALDI-TOFMS.
3.
Successfully synthesized compounds of G3.0 PAMAM dendrimer
modified with alkylating ranges as acyl chloride, carboxylic acid, alcohol
and G2.5 PAMAM dendrimer with alkylamine.
4.
Has successfully built a new calculation method based on 1H-NMR
spectral data to determine metabolism (x%), the transformation group (z
group), molecular weight of PAMAM- alkyl dendrimer derivatives.
5.
Examined cytotoxicity in MCF-7 cells of the dendrimer PAMAM G3.0
and G3.0 PAMAM dendrimer derivatives, with the number of carbon
atoms of the alkyl chain increased from 2 to 14 (PAMAM G3.0-C2
dendrimer, G3.0-C6, G3.0- C10 and G3.0-C14). Results showed that after
30
the alkylation, cytotoxicity of PAMAM G3.0-alkyl dendrimer derivatives
significantly reduced compared to G3.0 PAMAM dendrimer has not
modified. G3.0 PAMAM dendrimer toxicity decreases as long alkyl
carbon circuit.
6.
Has successfully mounted folic acid targeted agent to drug delivery system
of PAMAM G3.0 dendrimer and G3.0-C6 PAMAM dendrimer derivatives
forming PAMAM G3.0-FA dendrimer and PAMAM G3.0-C6-FA
dendrimer drug delivery systems.
7.
Confocal laser scanning microscopy have demonstrated the ability of the
system targeted initiative of the PAMAM G3.0-C6-FA drug delivery
system.
8.
Surveyed the size of PAMAM G3.0 dendrimer and PAMAM G3.0-C6;
PAMAM G3.0-FA; PAMAM G3.0-C6-FA dendrimer derivatives by TEM
images. The size of the delivery system is in the range 4-6nm.
9.
Successfully surveyed the ability to carry and release 5-FU anticancer
drugs of PAMAM G3.0 dendrimer and PAMAM G3.0-C6-FA dendrimer
derivatives. Results showed that the PAMAM G3.0 dendrimer molecule
loads 6 molecules of 5-FU, while PAMAM G3.0-C6-FA dendrimer
derivatives carries more (9 molecules of 5-FU). PBS environment, 5-FU
drug is released from the PAMAM G3.0-C6-FA/5FU drug delivery system
slower than the 5-FU reference.
31
REQUEST
-
Continue this theme with higher PAMAM dendrimer generation to
achieve containing drugs better efficiency.
-
Research the modified PAMAM G3.0 dendrimer with other targeted
agents as Arg-Gly-Asp (RGD); epidermal growth factor receptor
(EGFR); và kháng thể HER-2, ...
32