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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 [r]
(1)MINISTRY OF EDUCATION AND
TRAINING VIETNAM ACADEMY OF SCIENCE AND
TECHNOLOGY
GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY
NGUYEN NGOC HOA
IMPROVING THE EFFECTIVE DELIVERY OF CISPLATIN ANTI CANCER DRUG OF
DENDRIMER NANOCARRIER
Field of Study: Polymer and Composite Code: 44 01 25
SUMMARY OF MATERIAL SCIENCE DOCTORAL THESIS
(2)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: … Reviewer 2: … Reviewer 3: …
The thesis shall be defended in front of the Thesis Committee at Academy Level at Institute of Applied Materials Science - Vietnam Academy of Science and Technology
At hour date month , 2021
The thesis can be found at: - The National Library of Vietnam
(3)2 INTRODUCTION 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 OVERVIEW 1.1 Introduction to dendrimer and biocompatibility of dendrimer
1.1.1 Introduction
The term “dendrimer” was first mentioned by Donald A Tomalia in 1985s The word “dendrimer” is Greek in origin, “Dendron”, by means of tree branch Up to now, various studies have been published about structure of dendrimer molecule, dendrimer synthesis and application of dendrimer in difference fields In general, dendrimers are nano-polymer with spherical morphology and branched structure and have more advantages than that of linear polymer Structure of dendrimers include three part as illustrating in figure 1.1
Figure 1.1 A typical structure of dendrimer
- A dendrimer is comprised of three different parts: (i) central core consisting of atom or the molecule with at least two similar functional groups, (ii) branches, arising from the central atom/molecules core composed by repeat units and the brigde between the terminal functional groups and their core, (iii) numerous terminal functional groups (anion, cation, neutral, hydrophobic or hydrophilic groups) located at the edge of the moleculer which are also called peripheral functional groups
Dendrimer, specialized on PAMAM dendrimer with open open structure, various internal cavities and amine/ester-terminated surface functional groups, have been a tremendous motivator for multi-drug delivery nanocarriers to kill cancer cells following passive targeting or active targeting mechanism
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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 by Alfred Werner in 1893 However, the compound did not gain scientific investigations until the 1960s when the initial observations of Rosenberg et al (1965) at Michigan State University pointed out that certain electrolysis products of platinum mesh electrodes were capable of inhibiting cell division in Escherichia coli created much interest in the possible use of these products in cancer chemotherapy Cisplatin has been especially interesting since it has shown anticancer activity in a variety of tumors including cancers of the ovaries, testes, and solid tumors of the head and neck It was discovered to have cytotoxic properties in the 1960s, and by the end of the 1970s it had earned a place as the key ingredient in the systemic treatment of germ cell cancers Among many chemotherapy drugs that are widely used for cancer, cisplatin is one of the most compelling ones It was the first FDA-approved platinum compound for cancer treatment in 1978 This has led to interest in platinum (II)—and other metal—containing compounds as potential anticancer drugs
CHAPTER Materials and Methods 2.1 Materials
Chemical agents were purchased from Acros, Sigma Aldrich, Merck with high purity, suitable for synthetic organic chemistry and for analytical specifications
Equipment: desiccator, sonication, magnetic Stirrer and hot plate, vacuum oven, vacuum rotary evaporator Eyala, water bath memmert, freeze dryer at German Vietnamese Technology Center, Ho Chi Minh City University of Food Industry Morphology and size of dried particles was taken by TEM at 140kV (JEOL JEM 140, Japan) Fourier-transform infrared spectroscopy (FTIR) was analysed by Equinox 55 Bruker HPLC was done by Agilent 1260 (USA) 1H-NMR spectrum was obtained from Bruker Avance 500 Amount of Pt
was determined using ICP-MS-7700x/Agilent (VILAS) The cytotoxic assay was investigated following the help of Molecular Lab, Genetics Department, University of Science, HCM
2.2 Methods
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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 G4.5 were expanded The chemical structure and the molecular mass of the obtained products were identified by 1H-NMR
Figure 2.1 Synthetic route of PAMAM dendrimer
2.2.2 Synthesis of PAMAM dendrimer G3.0, G4.0 conjugated Cisplatin
Cisplatin was dissolved in water and stirred at room temperature under N2 inviroment The solution of
PAMAM dendrimer G3.0, G4.0 in water was adjusted pH to 7-8 using HCl PAMAM dendrimer solution was drop-wised into prepared cisplatin solution and stirred for 24h following h with sonication at room temperature under N2 gas The unbound cisplatin was removed via dialysis The obtained product was then
freeze dried to get powder
2.2.3 Synthesis PAMAM dendrimer G2.5, G3,5, G 4.5 conjugated cisplatin
PAMAM dendrimer G2.5, G3.5, G4.5 were hydrolyzed by NaOH to form carboxylated groups COO-
on the surface and were then used to perform the complex compound with cisplatin as section 2.2.2
2.2.4 Synthesis PAMAM dendrimer G2.5, G3,5, G 4.5 conjugated aqueous cisplatin
Hydrolyzed cisplatin was prepared using AgNO3 to withdraw the choloride ion on cisplatin leading to
the formation of monoaqua [cis-(NH2)2PtCl(H2O)] and diaqua [cis-(NH2)2Pt(H2O)2] The reaction was taken
place at room temperature, under N2 and continuous stirring The hydrolyzed PAMAM dendrimer G2.5, G3.5, G4.5 by NaOH was drop-wised into aqueous cisplatin, stirring for 24h following the sonication in hours under N2 at room temperature The obtained product was then freeze dried to get powder
2.2.5 Modification of PAMAM dendrimer G 3.0 with Poly(N-isopropylacrylamide) (PNIPAM)
Carboxylated (-COOH) terminated PNIPAM was activated by 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 1H-NMR
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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 1H-NMR
2.2.7 Synthesis of the complex PAMAM dendrimer G3.5-PNIPAM and Cisplatin
The complexation reaction between PAMAM dendrimer G3.5-PNIPAM and cisplatin was similar to the description in section 2.2.4
2.2.8 Modification of PAMAM dendrimer G3.0, G4.0 with poly (acrylic acid) (PAA)
PAA was activated using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) before reacting with NH2-terminal surface function groups of PAMAM dendrimer G3.0, G4.0 The obtained products were
purified by dialysis membrane and then free-dried to get powder The chemical structure and grafting degree were estimated by 1H-NMR
2.2.9 Synthesis the complex PAMAM dendrimer G3.0-PAA, PAMAM dendrimer G4.0-PAA and cisplatin
The complexation reaction between PAMAM dendrimer G3.0-PAA, PAMAM dendrimer G4.0-PAA and cisplatin was similar to the description in section 2.2.4
2.2.10 Evaluation the encapsulation and release of 5FU from the complex PAMAM dendrimer G3.5-PNIPAM-Cisplatin
5-FU was dissolved into deionized water (DI) and then drop-wised into PAMAM dendrimer G3.5-PNIPAM-Cisplatin solution Sonication was applied for 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 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 1H –NMR spectrum showcased the typical protron siginals of dendrimer structure such
as: -CH2CH2N< (a) at δH = 2.60 ppm; -CH2CH2CO- (b) at δH = 2.80-2.90 ppm; -CH2CH2CONH- (c) at δH
= 2.30 - 2.40 ppm; -CH2CH2NH2 (d) at δH = 2.70 -2.80 ppm; -CONHCH2CH2N- (e) at δH = 3.20 - 3.40 ppm;
-CH2CH2COOCH3- (g) at δH = 2.40 - 2.50 ppm and -COOCH3 (h) at δH = 3.70 ppm
The 1H-NMR spectrum of various dendrimer PAMAM generation was presented below:
1H-NMR PAMAM G-0.5: at δH = 2.47 - 2.50 ppm (a), δH = 2.77-2.80 ppm (b), δH = 2.54 ppm (g)
and δH = 3,68 ppm (h)
1H -NMR PAMAM G0.0: at δH = 2.56 - 2.57 ppm (a), δH = 2.77 - 2.82 ppm (b), δH = 2.37 - 2.40 ppm
(c), δH = 2.71 -2.75 ppm (d) and δH = 3.25 - 3.27 ppm (e)
1H -NMR PAMAM G0.5: at δH = 2.54 -2.57 ppm (a), δH = 2.76 - 2.82 ppm (b), δH = 2.37 - 2.40 ppm
(c), δH = 3.24 - 3.26 ppm (e), δH = 2.45 - 2.48 ppm (g) and δH = 3.66 ppm (h)
1H -NMR PAMAM G1.0: at δH = 2.59 - 2.60 ppm (a), δH = 2.80 -2.82 ppm (b), δH = 2.38 - 2.40 ppm
(c), δH = 2.73 - 2.76 ppm (d) and δH = 3.26 - 3.28 ppm (e)
1H -NMR PAMAM G1.5: at δH = 2.58 - 2.59 ppm (a), δH = 2.78 - 2.86 ppm (b), δH = 2.39 - 2.42 ppm
(c), δH = 3.27 - 3.29 ppm (e), δH = 2.47 -2.50 ppm (g) and δH = 3.69 ppm (h)
1H -NMR PAMAM G2.0: at δH = 2.57 - 2.59 ppm (a), δH = 2.77 -2.81 ppm (b), δH = 2.36 -2.38 ppm
(c), δH = 2.68 -2.74 ppm (d) and δH = 3.24 - 3.27 ppm (e)
1H -NMR PAMAM G2.5: at δH = 2.57 - 2.64 ppm (a), δH = 2.84 - 2.86 ppm (b), δH = 2.40 -2.42 ppm
(c), δH = 3.27 -3.30 ppm (e), δH = 2.48 - 2.46 ppm (g) and δH = 3.68 - 3.69 ppm (h)
1H -NMR PAMAM G3.0: at δH = 2.61 - 2.62 ppm (a), δH = 2.80 -2.83 ppm (b), δH = 2.38 - 2.40 ppm
(c), δH = 2.74 - 2.76 ppm (d) and δH = 3.26 -3.29 ppm (e)
1H -NMR PAMAM G3.5: at δH = 2.57 -2.64 ppm (a), δH = 2.84-2.85 ppm (b), δH = 2.38 -2.43 ppm
(c), δH = 3.27 -3.37 ppm (e), δH = 2.48 -2.51 ppm (g) and δH = 3.69 ppm (h)
1H -NMR PAMAM G4.0: at δH = 2.59 -2.62 ppm (a), δH = 2.80 -2.83 ppm (b), δH = 2.39 – 2.40 ppm
(c), δH = 2.74 – 2.76 ppm (d) and δH = 3.26 -3.28 ppm (e)
1H -NMR PAMAM G4.5: at δH = 2.57 - 2.65 ppm (a), δH = 2.84 – 2.85 ppm (b), δH = 2.39 – 2.42
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Figure 3.1.1H-NMR spectrum of various PAMAM Dendrimer generation
Thoughout the integral ratios of peaks of protons at (a) and (e) on the 1H-NMR of dendrimer
molecules (χNMR) and the intergal ratio of the number of the protons at (a) and (e) in the theorical dendrimer structure (χL.T), the molecular weight of dendrimers can be established following the below equation:
M(NMR) = χNMR
χLT MLT =
SH(-CH (e)2-) SH(-CH (a)2-) ∑H(-CH
2-)
(e) ∑H(-CH
2-)
(a)
.MLT
In which:
SH(-CH (e)2-), SH(-CH (a)2-) : the peak areas of protons at (a) and (e) in 1H-NMR
∑H(-CH (e)2-), ∑H(-CH (a)2-): 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 1H-NMR
H(-CH (e)2-) H(-CH (a)2-) χLT M(LT) χNMR M(NMR) Different (%)
G-0.5 (b) 404 2.01 405.62 0.40%
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G0.5 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 (G2.5); 3302 cm-1 (G3.5); 3426 cm-1 (G4.5), which hinder the
viberation of amide bonding FT-IR also presents the assymetric stretching –CH2, CH3, –CH3 at 2952 cm-1,
2832 cm-1 (G2.5); 2952 cm-1, 2830 cm-1 (G3.5) and out-of-plane stretching CH
3 at 1360 cm-1 (G2.5), 1359
cm-1 (G3.5), 1399 cm-1 (G4.5) The vibrational modes of the obtained FT-IR of various PAMAM dendrimer
generation were similar to PAMAM dendrimer G2.5, 3.5, 4.5
The FT-IR spectrum of all complex PAMAM G2.5-Cisplatin, G3.5-Cisplatin, G4.5-Cisplatin also have similar signal as compared to PAMAM G2.5, 3.5, 4.5 However, the absorption of these peaks are quite difference Due to the formation of complex, the ester functional groups at the surface of PAMAM are converted to COO- leading to the intensity of viberation of ester groups (ν
C=O, νC-O) are reduced Also, due to
the overlap of asymmetrical/symetrical stretching of COO- on viberation of amide band I, amide band II and
vibration of aliphatic CH3, the intensity of these peaks are increased, confirming the presentation of the
viberation of N-H bonding in cisplatin Together, the change in the intensity of these peaks provide the evidence for the formation of coordinative bond between Pt2+ and carboxylate -COO- groups on the surface of