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MINISTRY OF EDUCATION AND
TRAINING VIETNAM ACADEMY OF SCIENCE AND
TECHNOLOGY
Field of Study: Polymer and Composite
Code: 9 44 01 25
1
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: …
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
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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
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- 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
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.
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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
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). 1<sub>H-NMR spectrum was obtained from Bruker Avance 500. Amount of Pt </sub>
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
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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 1<sub>H-NMR. </sub>
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-<sub> </sub>
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,
2.2.5. Modification of PAMAM dendrimer G 3.0 with Poly(N-isopropylacrylamide) (PNIPAM)
Carboxylated (-COOH) terminated PNIPAM was activated by pnitrophenyl 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 1<sub>H-NMR. </sub>
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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 1<sub>H-NMR. </sub>
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
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 R2
ajust (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
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The chemical shift of specific proton signals on dendrimer PAMAM were recored in various previous
reports. The resultant 1<sub>H –NMR spectrum showcased the typical protron siginals of dendrimer structure such </sub>
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 1<sub>H-NMR spectrum of various dendrimer PAMAM generation was presented below: </sub>
1<sub>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) </sub>
and δH = 3,68 ppm (h).
1<sub>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 </sub>
(c), δH = 2.71 -2.75 ppm (d) and δH = 3.25 - 3.27 ppm (e).
1<sub>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 </sub>
(c), δH = 3.24 - 3.26 ppm (e), δH = 2.45 - 2.48 ppm (g) and δH = 3.66 ppm (h).
1<sub>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 </sub>
(c), δH = 2.73 - 2.76 ppm (d) and δH = 3.26 - 3.28 ppm (e).
1<sub>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 </sub>
(c), δH = 3.27 - 3.29 ppm (e), δH = 2.47 -2.50 ppm (g) and δH = 3.69 ppm (h).
1<sub>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 </sub>
(c), δH = 2.68 -2.74 ppm (d) and δH = 3.24 - 3.27 ppm (e).
1<sub>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 </sub>
(c), δH = 3.27 -3.30 ppm (e), δH = 2.48 - 2.46 ppm (g) and δH = 3.68 - 3.69 ppm (h).
1<sub>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 </sub>
(c), δH = 2.74 - 2.76 ppm (d) and δH = 3.26 -3.29 ppm (e).
1<sub>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 </sub>
(c), δH = 3.27 -3.37 ppm (e), δH = 2.48 -2.51 ppm (g) and δH = 3.69 ppm (h).
1<sub>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 </sub>
(c), δH = 2.74 – 2.76 ppm (d) and δH = 3.26 -3.28 ppm (e).
1<sub>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 </sub>
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Figure 3.1.1<sub>H-NMR spectrum of various PAMAM Dendrimer generation </sub>
Thoughout the integral ratios of 2 peaks of protons at (a) and (e) on the 1<sub>H-NMR of dendrimer </sub>
molecules (χ<sub>NMR</sub>) and the intergal ratio of the number of the protons at (a) and (e) in the theorical dendrimer
structure (χ<sub>L.T</sub>), the molecular weight of dendrimers can be established following the below equation:
M<sub>(NMR)</sub> = χNMR
χ<sub>LT</sub> .MLT =
S<sub>H(-CH</sub> (e)<sub>2</sub><sub>-)</sub>
S<sub>H(-CH</sub> (a)<sub>2</sub><sub>-)</sub>
∑H<sub>(-CH</sub>
2-)
(e)
∑H<sub>(-CH</sub>
2-)
(a)
.M<sub>LT</sub>
In which:
S<sub>H(-CH</sub> (e)<sub>2</sub><sub>-)</sub>, S<sub>H(-CH</sub> (a)<sub>2</sub><sub>-)</sub> : the peak areas of protons
at (a) and (e) in 1<sub>H-NMR </sub>
∑H<sub>(-CH</sub> (e)<sub>2</sub><sub>-)</sub>, ∑H<sub>(-CH</sub> (a)<sub>2</sub><sub>-)</sub>: the sums of protons at the (e)
and (a) position s in the molecular formula of
PAMAM dendrimer.
MLT : the theoretical molecular weight of
PAMAM dendrimer.
The results were calculated according to:
Table 3.1. Calculated molecular mass of Dendrimer following 1<sub>H-NMR. </sub>
H<sub>(-CH</sub> (e)<sub>2</sub><sub>-)</sub> H<sub>(-CH</sub> (a)<sub>2</sub><sub>-)</sub> χ<sub>LT</sub> M(LT) χ<sub>NMR</sub> M(NMR) Different <sub>(%) </sub>
G-0.5 8 (b) 4 2 404 2.01 405.62 0.40%
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G0.5 8 12 0.67 1205 0.67 1205.42 0.06%
G1.0 24 12 2.00 1430 1.95 1396.18 2.36%
G1.5 24 28 0.86 2808 0.81 2668.19 4.96%
G2.0 56 28 2.00 3257 1.95 3181.78 2.30%
G2.5 56 60 0.93 6012 0.90 5774.30 3.95%
G3.0 120 60 2.00 6910 1.90 6556.70 5.11%
G3.5 120 124 0.97 12420 0.92 11809.71 4.91%
G4.0 248 124 2.00 14216 1.90 13510.97 4.96%
G4.5 248 252 0.98 25237 0.90 23103.55 8.45%
A series of generation PAMAM dendrimers from G-0.5 to G-4.5 were successfully achieved; these
dendrimers had the regular and high stability in structure; consequently, they could be effective drug drug
delivery system.
3.2. FT-IR spectrum of the complex PAMAM dendrimer and cisplatin
3.2.1. FTIR PAMAM dendrimer G2.5, G3.5, G4.5 and complex G2.5-CisPt, G3.5-CisPt,
G4.5-CisPt
Both FT-IR spectrum of PAMAM G2.5, G3.5 contain strong absorption peak (νC=O) and moderate
absorption peak (νC-O) at 1731 cm-1, 1045 cm-1 (G2.5); 1736 cm-1, 1646 cm-1 (G3.5), respectively,
corresponding to the vibiration of ester functional group. A broad band with strong viberation corresponds to
the stretching –OH groups at 3294 cm-1<sub> (G2.5); 3302 cm</sub>-1<sub> (G3.5); 3426 cm</sub>-1<sub> (G4.5), which hinder the </sub>
viberation of amide bonding. FT-IR also presents the assymetric stretching –CH2, CH3, –CH3 at 2952 cm-1,
2832 cm-1<sub> (G2.5); 2952 cm</sub>-1<sub>, 2830 cm</sub>-1 <sub>(G3.5) and out-of-plane stretching </sub><sub></sub><sub>CH</sub>
3 at 1360 cm-1 (G2.5), 1359
cm-1<sub> (G3.5), 1399 cm</sub>-1 <sub>(G4.5). The vibrational modes of the obtained FT-IR of various PAMAM dendrimer </sub>
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-<sub> leading to the intensity of viberation of ester groups (ν</sub>
C=O, νC-O) are reduced. Also, due to
the overlap of asymmetrical/symetrical stretching of COO-<sub> on viberation of amide band I, amide band II and </sub>
vibration of aliphatic CH3, the intensity of these peaks are increased, confirming the presentation of the
viberation of N-H bonding in cisplatin. Together, the change in the intensity of these peaks provide the
evidence for the formation of coordinative bond between Pt2+<sub> and carboxylate -COO</sub>-<sub> groups on the surface of </sub>