Experiment with dual 5-FU and Cisplatin loading in PAMAM dendrimer G3.5-PNIPAM G3.5-PNIPAM was synthesized based on the reaction between PAMAM dendrimer G3.0 –PNIAM which was further mo[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 R2ajust (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)
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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 (a)(-CH2-) 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 CH3 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
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3.2.2 FT-IR spectrum of complex PAMAM Dendrimer G3.0-Cisplatin, G4.0-Cisplatin
Figure 3.2 FT-IR spectrum of PAMAM dendrimer G2.5, G3.5, G4.5 and complex G2.5-Cisplatin, G3.5-Cisplatin, G4.5-Cisplatin
FT-IR of PAMAM dendrimer G3.0 and G3.0-Cisplatin; G4.0 and G4.0-Cisplatin showcased the spectra shifting for –NH viberation at 1643 cm-1 to 1639 cm-1 (G3.0, G3.0-Cisplatin); 1643 cm-1 to 1642 cm-1 (G4.0, G4.0-Cisplatin) This sugguests the formation of the coordinative bond between cation Pt2+ and NH2 groups on the surface of PAMAM dendrimer G3.0 Furthermore, the reduction of intensity and the shifting of symmetric/ asymmetric vibration of aliphatic -CH2 at 2944 cm-1 and 2839 cm-1 in FT-IR spectrum of PAMAM dendrimer G3.0 to 2975 cm-1 and 2884 cm-1 in the complex G3.0-Cisplatin along with the aborption peaks at 3437 cm-1 (G3.0-Cisplatin) and 3427 cm-1 (G4.0-Cisplatin) corresponding to the N-H viberation on the cisplatin spectrum
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Figure 3.3 FT-IR spectrum of PAMAM dendrimer G3.0, G4.0 and the complexc G3.0-Cisplatin, G4.0-Cisplatin
(13)12 3.4 FT-IR spectrum of complex G4.0-PAA-Cisplatin
Figure 3.5 FT-IR spectrum of G4.0-PAA and complex G4.0-PAA-Cisplatin
FT-IR spectrum exhibits the slight shifting of asymmetric –COO viberation and the overlap of amide peak -NH in G3.0-PAA at 1572 cm-1 cm-1 to 1564 cm-1 and 1635 cm-1 in respected to G4.0-PAA-Cisplatin The weak intensity peaks contributing the stretching and viberation of -CH2 and CH-CO for G4.0-PAA at 1454 cm-1 and 1407 cm-1 are shifted to 1447 cm-1 1400 cm-1, respectively, in case of G4.0-PAA-Cisplatin A viberation at 3619 cm-1 is assigned to the stretching –OH of –COOH on 0-PAA This phenomina proposes the interaction of cation Pt2+ and -COO- on the surface of G4.0-PAA
3.5 1H-NMR result of PAMAM G3.0 and G 3.5 modififed with PNIPAM
As shown in the 1H-NMR spectrum of G3-PNIPAM (mole ratio 1:8), beside the typical proton peak for PAMAM G3.0, some the proton signals are originated from PNIPAM-COOH such as –CH3 (f) at 1,10-1,26 ppm, -(CH3)2CHNH- (l) at 3,99 ppm In addition, the proton of –CH2CH2CONH (c) shifts from 2.0 to 2.68 ppm, confirming the formation of linkage between NH2 of PAMAM G3.0 and–COOH of PNIPAM-COOH This results show the successful of synthesis nanocarrier based thermal responsive dendrimer
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Figure 3.6 1H-NMR spectrum of nanocarrier based on G3.0-PNIPAM (mol ratio 1:8)
From 1H-NMR spectrum of G3-PNIPAM, the grafting degree as well as the number PNIPAM-COOH conjugated on PAMAM G3.0 following the below formula:
In which:
%X =
SH(-CH (f)3) SH(-CH (a)2-) ∑H(-CH (f)3) ∑H(-CH (a) 2-)
.100%
SH(-CH (a)2-),SH(-CH (f)3) : the peak areas of peak (a) and peak (f) in 1 H-NMR
∑H(-CH (a)2-), ∑H(-CH (f)3) : the sums of protons at the peak (a) and (f) in the derivative dendrimer as theory
%X : Amidation degree
Regarding the formula, % X is 15,12 % and about 4,84 PNIPAM-COOH groups are successful conjugated on the PAMAM G3.0 (yield 96.8%) In the same maner, based on the 1H-NMR spectrum, these parameters of two mol ratio G3.0: PNIPAM = 1:5 and 1:10, were calculated and presented in table 3.2
Table 3.2 The numer PNIPAM groups conjugating on G3.0 and their estimated molecular weight
Sample PNIPAM groups Molecular
weight based on1H-NMR
Phase-transition temperature
G3.0-PNIPAM (1:5) 3.34 30,605 37,5 oC
G3.0-PNIPAM (1:8) 4.84 40,776 34 oC
G3.0-PNIPAM (1:10) 7.00 55,880 33 oC
The molecular weight of G3.0-PNIPAM (1:8) is 39,600 using GPC method, which is similar as the calculation from 1H-NMR spectrum
For G3.5-PNIPAM, beside the typical proton signals of PNIPAM-COOH, other proton signals originating from PAMAM dendrimer generation 3.5 such as –COOCH3 (h)
(3,73-3,78 ppm); –CONHCH2CH2N- (e) (3,26-3,36 ppm); –CH2CH2N
(a) (2,57-2,63 ppm) are also exhibited on the 1H-NMR spectrum of G3.5-PNIPAM This results provide the evidence for the linakage between –COOCH3 and amine groups on the surface of G3.0-PNIPAM In other world, the nanocarrier based on thermal sensitive G3.5-PNIPAM is well-established in this study
3.6 1H-NMR spectrum of PAA modified PAMAM G3.0
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A long with the typical proton signals for PAMAM dendrimer G3.0 such as peak –CH2CH2N (a)
(2.63ppm), peak –CONHCH2CH2N- (e) (3.30 ppm), the characterized peak for acid polyacrylic, including
>CHCOOH (b) (2.07 ppm) >CHCH2CH< (c) (1.61 ppm) exposes in the 1H-NMR spectrum of PAA modified
PAMAM G3.0 (G3.0-PAA) This revels the formation of the linkage -CO-NH between -NH2 groups on the surface of PAMAM dendrimer G3.0 and –COOH on PAA chains This observation can help to confirm the success of the G3.0-PAA synthesis process Regarding 1H-NMR spectrum of G3.0-PAA, the number PAA groups attacked PAMAM dendrimer G3.0 is 6,01 (yield: 50,1%) When mol rate PAMAM dendrimer G3.0: PAA was 1:6, the number PAA groups conjugated on the PAMAM dendrimer G3.0 is (yield: 83,3%)
3.7 1H-NMR spectrum of PAA modified PAMAM G4.0
In the same maner to G3.0-PAA, the 1H-NMR spectrum reveals the successful synthesis of carrier based on G4.0-PAA From the 1H-NMR spectrum of G4.0-PAA, the number of PAA attached on PAMAM dendrimer G4.0 is 15.16 (yield: 94.7%) With mole ratios PAMAM dendrimer G4.0: PAA is 1:8, the number PAA conjugating on the surface of PAMAM dendrimer G4.0 is 7.28 (yield: 91.0%) Further increase the mol of PAA in the ratio upto 1:24; however, the reaction was unscessfull (the solidification in reaction bath)
3.8 Amount of Pt from complexes
3.8.1 Amount of Pt from complex full generation PAMAM dendrimer -cisplatin
Figure 3.9 1H-NMR spectrum of PAA modified PAMAM dendrimer G3.0 (mol ratio 1:12)
(16)2 Table 3.3 Amount of Pt from complex full generation PAMAM dendrimer –cisplatin
(non-aqueous cisplatin)
No Sample %Cisplatin
1 G3.0-Cisplatin 9.63 1.47 G4.0-Cisplatin 16.95 1.29 Data was presented under average SD (standard
deviation), number of trials n=3
3.8.2 Amount of Pt from complex half-generation PAMAM dendrimer (non-aqueous cisplatin)
Table 3.4 Amount Pt from complex half-generation PAMAM- Cisplatin (nom-aqueos cisplatin)
No Sample %Cisplatin
1 G2.5-Cisplatin 15.89 ± 1.41 G3.5-Cisplatin 7.90 ± 1.92 G4.5-Cisplatin 5.90 ± 0.68 Data was presented under average SD (standard deviation), number of trials n=3
For half-generation PAMAM dendrimer, the amount of cisplatin was reduced with the increase of dendrimer generation The diminution of loading effectiveness of higher half-generation PAMAM may due to the steric hindrance of the carboxylate groups on the surface, which are tended to closely packed leading to the difficulty in accepting further cisplatin In the contrary, the amount of cisplatin was increased about 1.7 times following the growth of dendrimer generations from G3.0 to G4.0
3.8.3 Amount of Pt from complex half-generation PAMAM – cisplatin (aqueous cisplatin)
Table 3.5 Amount of Pt from complex G2.5-Cisplatin, G3.5-Cisplatin ang G4.5-Cisplatin
No Sample %Cisplatin
1 G2.5-Cisplatin 28.99 2.01 G2.5-Cisplatin (SA) 31.82 1.39 G3.5-Cisplatin 30.23 1.29 G3.5-Cisplatin (SA) 33.01 1.56 G4.5-Cisplatin 31.11 1.48 G4.5-Cisplatin (SA) 34.03 1.96 Data was presented under average SD (standard deviation), number of trials n=3; SA: Sonication
The results show that efficacy of conjugating cisplatin is higher than that of previous studies in
which cisplatin encapsulating in G2.5-Cisplatin and G2.5-Cisplatin were 10,33% and 2,3%, respectively The significant difference was because the form of cisplatin, in this study, cisplatin was first hydrolyzed with AgNO3 By this way, cisplatin was diaquated in form of cation [Pt(NH3)2(H2O)]2+ leading to increase the potency of cisplatin that conjugated on half-generation PAMAM dendrimer Increase of PAMAM generation induces the increase the number of functional groups on the surface and the retention of cisplatin was also increase However, the increase of surface functional groups, the encapsulation of cisplatin would decrease following the growth of PAMAM generation Kirkpatrick sugguested this observation is due to the difficulty in the convertion of the surface function groups into carboxylate groups when the generation PAMAM increased and due to through-space effects resulting the reduction of the possible binding between cisplatin and amine/ amide inside dendrimer
3.8.4 Amount of Pt from complex G3.0-PAA-Cisplatin and G4.0-G3.0-PAA-Cisplatin (aqueous cisplatin)
Table 3.6 Amount of Pt from complex G3.0-PAA-Cisplatin (aqueous cisplatin) and
G4.0-PAA-Cisplatin (aqueous cisplatin)
No Sample %Cisplatin
1 G3.0-PAA-Cisplatin
(1:6) 12.93 1.60
2 G3.0-PAA-Cisplatin
(1:12) 13.89 1.39
3 G4.0-PAA-Cisplatin
(1:8) 20.22 1.44
4 G4.0-PAA-Cisplatin
(1:16) 40.44 1.29
Data was presented under average SD (standard deviation), number of trials n=3
(17)3 carboxylic groups of PAA, the probability of the complex formation Pt-COO- increase
3.9 Comparison of cisplatin encapsulating in various carrier using either aqueous cisplatin or non-aqueous cisplatin
Table 3.7 Cisplatin (non-aqueous) loading in PAMAM dendrimer derivative N
o
Sample Number of binding group
% Cisplatin NH2 COOH
1 G2.5-COOH 32 15.89 1.41 G3.5-COOH 64 7.90 1.92 G4.5-COOH 128 5.90 0.68 G4.0-PAA
(1:16)
49 405 19.06 1.44 G3.0-NH2 32 9.63 1.47 G4.0-NH2 64 16.95 1.29 The effectiveness of PAMAM dendrimer in cisplatin delivery was summarized in table 3.7 The results show that cisplatin loading efficiency is lower than previous publiscations However, using AgNO3 to make full aqueous cisplatin in form [Pt(NH3)2(H2O)n]2+, the potency of the complexation reaction between Pt2+ from cisplatin and surface functional groups –COOH from PAMAM dendrimer carrier increase leading to the increase of amount cisplatin loading in carrier (table 3.8) ) Because the strong interaction between platinum and amino
groups on full generation PAMAM dendrimer (G3.0, G4.0) induces the stable complex which are difficult to release in dose of drug; consequently, full generation PAMAM dendrimer was not examined in this study From table 3.13, aqueous cisplatin could be easy to form the complex with carboxylate – COOH groups on the surface of carrier PAMAM dendrimer G4.0-PAA (1:16) consisting of a greater number carboxylate groups on the surface (405 groups) could encapsulate higher amount cisplatin, about 40.44% cisplatin, as compared to other carriers
Table 3.8 Cisplatin (aqueous) loading in PAMAM dendrimer derivative
N o
Sample Number of binding
group
% Cisplatin
NH2 COO
H
1 G2.5-COOH 32 31.82 1.39 G3.5-COOH 64 33.01 1.56 G4.5-COOH 128 34.03 1.96 G3.0-PAA
(1:6)
27 75 12.93 1.60 G3.0-PAA
(1:12)
26 90 13.89 1.39 G4.0-PAA
(1:8)
57 189 20.22 1.44 G4.0-PAA
(1:16) 49 405 40.44 1.29 3.10 Experiment with dual 5-FU and Cisplatin loading in PAMAM dendrimer G3.5-PNIPAM G3.5-PNIPAM was synthesized based on the reaction between PAMAM dendrimer G3.0 –PNIAM which was further modified the outer groups -COOCH3 to -COO- that could form complex with cisplatin In addition, thermal responsive PNIPAM on the surface of carrier is thermal responsive polymer with lower critical solution temperature (LCST, 320C) At the temperature under LCST, PNIPAM swells in maximum in drug solution and can encapsulate these drug inside their network At the temperature above LCST, polymer chains become to shrink and then release drug to inviroment Based on this phenomina, 5-FU can be loaded into PAMAM dendrimer G3.5-PNIPAM-Cisplatin
Result for 30mg 5FU encapsulating in 100mg copolymer PAMAM dendrimer G3.5-PNIPAM and complex PAMAM dendrimer G3.5-PNIPAM-Cisplatin are presented below:
Table 3.9 5-FU loading into complex G3.5-PNIPAM-Cisplatin
Free 5FU 5-FU loading
(18)4 PAMAM dendrimer
G3.5-PNIPAM-5FU-CisPt 4.32 0.26 25.68 0.26 20.43 0.17 85.61 0.88 PAMAM dendrimer
G3.5-PNIPAM-5FU 3.73 0.29 26.27 0.29 20.81 0.18 87.57 0.97 PNIPAM-CisPt-5FU 11.90 0.27 18.10 0.27 15.32 0.20 60.33 0.91 Data was presented under average SD (standard deviation), number of trials n=3
The complex PAMAM dendrimer G3.5-PNIPAM-Cisplatin shows the potency of 5FU encapsulation forming thermal sensitive nanogel containing dual anticancer drug, 5-FU and cisplatin Clinical protocol for cancer with cisplatin is usually combinated with other drug to increase the therapeutic value as wel as reduce the side effect of drug Therefore, PAMAM dendrimer G3.5-PNIPAM loading dual anticancer drug, 5-FU and cisplatin can be considered as the potential candidates in cancer treatment
3.11 TEM, DLS and zeta potential
The size of complex half generation PAMAM dendrimer nanoparticles with cisplatin are quite homogenous and size is in range 5-10 nm (fig 3.11) TEM images expose that the size of PAMAM dendrimer G3.0-PNIPAM is 190nm (fig 3.12) Compared to the initial size of PAMAM dendrimer G3.0 (3-4 nm), PAMAM dendrimer G3.0-PNIPAM procees the growth of size; consequently, increase the amount of drug encapsulation of nanoparticles Size of PAMAM dendrimer G3.5-PNIPAM-Cisplatin in aqueous is 184 nm which is bigger than the original one because of the PNIPAM covering the surface of PAMAM dendrimer G3.5 nanoparticles TEM images (fig 3.13) of PAMAM dendrimer G3.5-PNIPAM-Cisplatin exhibites the cross-linking between cisplatin and PAMAM dendrimer G3.5-PNIPAM
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TEM images of PAMAM G4.0 demonstrated the formation of uniform spherical nanoparticles with a Hình 3.13 TEM image of PAMAM dendrimer PNIPAM-Cisplatin and DLS result of
G3.5-PNIPAM-Cisplatin and G3.5-PNIPAM-Cisplatin
Hình 3.12 TEM image of G3.0, PAMAM dendrimer G3.0-PNIPAM and DLS result of PAMAM dendrimer G3.0-PNIPAM
Figure 3.14 TEM image of PAMAM dendrimer G4.0 (A), G4.0-PAA (C) and DLS of PAMAM dendrimer G4.0 (B), G4.0-PAA (D)
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diameter of 4.1±1.2 nm, which nearly matched the hydrodynamic diameter of 7.8±2.4 nm as measured by DLS The size of G4.0-PAA (28 nm) increases 3.6 times as compared to PAMAM dendrimer G4.0 Also, the increase in size of nanoparticles were recored after cisplatin encapsulation
To investigate the stability of carrier, zeta potential values of carrier in different conditions were measured The ζ value of G3.0-PAA (mol ratio 1:12); G4.0-PAA (mol ratio 1:8) and G4.0-PAA (mol ratio 1:16) exhibit the dependent on the pH of solution All carriers expose the moderate stability at neutral and pH 7.4 PAA-G4.0 with the mol ratio 1:16 shows the potential aggregation at pH 5.5 with ζ = 7.3mV This result reveals that the unstability of particles induces the aggregation and due to the aggregation lead to increase size of particles; thus, the particles cannot pass through lymp while promoting the accumulative in tumor site inducing the higher amount of cisplatin in tumor and higher anti-cancer feature At pH 7.4, the ζ value of PAA-G4.0 with mole ratio 1:16 is -58.6 mV, suggesting that the good stability of nanoparticles in plasma inviroment which helps to prolong drug action in circulation and to increase the possibility drug entering the target site 20 40 60 80 100
2 10
Tr an sm itt an ce [% ] pH
G3PAA 1:12 G4PAA 1:16 G4PAA 1:8
-13,9 mV -14,6 mV
19,0 mV b)
c)
a) a) b)
c)
-58,6 mV -19,2 mV
7,3 mV
Figure 3.16 Zeta potential of G3.0-PAA (mol ratio 1:12) at a pH 7,4; b pH 7,0 and c pH 5,5
Figure 3.17 Zeta potential of G4.0-PAA (mol ratio 1:16) at: a pH 7,4; b pH 7,0 and c pH 5,5
a) b)
c)
-23,2 mV -17,2 mV
14,1 mV
Figure 3.18 Zeta potential of G4.0-PAA (mol ratio 1:8) at: a pH 7,4; b pH 7,0 and c pH 5,5
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The effect of pH solution on turbidity of carriers was investigated As shown in figure 3.19, the solubility of G3.0-PAA 1:12, G4.0-PAA 1:8 and G4.0-PAA 1:16 depend on the pH and can be divided into areas: lower pH, precipitation (intermedia area) and high pH At intermedia area pH 3.5-5.5 (G4.0-PAA 1:16), pH 4,0 - 6,0 (G3.0-PAA 1:12, G4.0-PAA 1:8), the charge on the surface of carriers are neutral, pH solution ~ isoelectric point resulting in the precipitation
3.12 Result and dissusion about the invitro drug release
3.12.1 The drug release from the complex half-generation PAMAM- cisplatin (aqueous)
Table 3.10 in vitro drug release results for complex PAMAM dendrimer: G2.5-Cisplatin, G3.5-Cisplatin and G4.5-Cisplatin
Time (h)
G2.5-Cisplatin G3.5-Cisplatin G4.5 - Cisplatin
%Cisplatin %Cisplatin %Cisplatin
pH 5,5 pH 7,4 pH 5,5 pH 7,4 pH 5,5 pH 7,4
0 0 0 0
6 30.42 1.20 22.78 0.81 31.72 1.00 27.17 0.91 34.96 1.08 28.23 0.81 12 37.18 1.48 28.35 1.01 39.04 1.25 35.12 1.12 45.52 1.30 33.12 1.03 24 43.10 1.70 31.84 1.14 44.94 1.41 38.64 1.16 47.86 1.04 43.48 1.20 48 45.98 1.82 34.61 1.23 47.93 1.50 41.35 1.18 53.18 1.47 48.03 1.51 60 48.18 1.90 36.29 1.29 50.24 1.62 43.76 1.28 55.07 1.51 48.91 1.54 72 51.05 2.02 36.54 1.30 53.24 1.67 45.69 1.37 55.49 1.54 49.19 1.54 ANOVA results proclaim that the amount cisplatin release from PAMAM dendrimer denpend on time, pH media and the generation of PAMAM dendrimer In case of same generation, the cisplatin release at pH 5.5 is higher than that of pH 7.4 in the statistic maner After 60h, the amount of cisplatin release is non-remarkable different between each carrier
Figure 3.20 In vitro cisplatin release from PAMAM G2.5, PAMAM G3.5 and PAMAM G4.5 in phosphate-buffered saline (PBS) pH7.4 and acetate buffers saline (ABS) pH 5.5
The higher amount cisplatin release in ABS buffer pH 5.5 than in PBS buffer pH 7.4 could be due to the protonation of -COO- on the surface of PAMAM dendrimer in acid condition leading to the formation of – COOH; consequently, loss of labile ligand between half-generation PAMAM dendrimer and cisplatin Amount
0% 10% 20% 30% 40% 50% 60%
0 12 24 36 48 60 72
% C um ul at iv e C is pl at in Time (h)
pH 5.5 (G2.5cis) pH 7.4 (G2.5cis) pH 5.5 (G3.5cis)
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of cisplatin release in 72h from PAMAM G2.5, PAMAM G3.5 and PAMAM G4.5 are 51.05%, 53.24% and 55.49%, respectively
3.12.2 Potency of the 5-FU and Cisplatin release from dual drug system G3.5-PNIPAM-Cispaltin – 5FU
Bảng 3.11 The potency of 5FU release from G3.5-PNIPAM-CisPt and PNIPAM-CisPt
3.12.3 The potency of cisplatin relase from PAMAM dendrimer G4.0-PAA Table 3.12 Cisplatin release result of PAMAM dendrimer G4.0-PAA
Time (h) Cisplatin release (%) pH 5,5 pH 7,4
0 0
4 25.17 1.08 11.42 0.75 12 34.42 1.51 21.40 0.62 24 40.97 0.92 28.75 0.84 32 44.96 0.88 33.02 0.90 48 52.02 0.94 42.72 1.29 55 56.51 0.84 45.46 1.38 72 59.13 2.10 48.32 1.48
In case of G4.5, the active platinum compounds were smoothly released over a period of 24 h (reaching 43.48%) The cumulative cisplatin in PBS 7.4 (49.19%) was nearly similar as in ABS pH 5.5 at 72h, whereas the cisplatin from PAMAM dendrimer G4.0-PAA was was gradually increased from 11.42% to 32.02% in 32h It was believed that the action of two carriers according the functional groups on the surface induces the different trend of release of PAMAM dendrimer G4.and PAMAM dendrimer G4.0-PAA
3.13 Cisplatin release kinetic
The most suitbale release kinetic model is based on Akaike information criterion AIC and R2adj (Adjusted R-squared) calculating by R program
Table 3.13 AIC values and R2adj of each release kinetic model of different Cisplatin carriers at pH 5,5 and pH 7,4
G2.5-CisPt G3.5-CisPt G4.5-CisPt G4.0-PAA-CisPt
pH 5,5 zero order R2 adj 0.527 0.523 0.465 0.759
AIC 58.397 59.032 60.893 62.509
Time (h)
% 5-FU release
G3.5-PNIPAM-CisPt PNIPAM-CisPt pH 5,5 pH 7,4 pH 5,5 pH 7,4
0 0 0
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1st order R2adj 0.638 0.641 0.585 0.875
AIC -15.041 -14.256 -11.745 -22.971
Higuchi R2adj 0.804 0.801 0.756 0.95
AIC 52.236 52.915 55.397 49.911
Korsmeyer-Peppas R2adj 0.964 0.961 0.9056 0.992
AIC -29.183 -28.682 -24.469 -38.636
Hixson Crowell R2adj 0.601 0.602 0.544 0.840
AIC 1.827 2.561 4.835 -3.071
pH 7,4
Zero order R2adj 0.493 0.506 0.577 0.892
AIC 54.611 57.251 57.843 54.069
1st order R2adj 0.562 0.5986 0.675 0.943
AIC -19.490 -16.389 -15.440 -32.894
Higuchi R
2adj 0.781 0.787 0.846 0.994
AIC 48.749 51.354 50.776 30.847
Korsmeyer-Peppas R2adj 0.953 0.929 0.948 0.994
AIC -28.110 -25.232 -24.412 -32.765
Hixson Crowell R2adj 0.539 0.5674 0.643 0.928
AIC -2.398 0.546 1.358 -12.494
The fitting data indicase the Korsmeyer-Peppas model is the best suitable to explain the release mechanism from carrier This is the specific model for drug release from carriers based on polymer
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Figure 3.22 expresses the change of drug concentration (prediction) in blood plasma in the function of time, which reveals the relationship between amount of active drug injecting in circulation after t time The area under the curve (AUC) reflex the actual body exposure to drug after administration of a dose of the drug and is expressed in mg.h/L Mathematical results for AUC of each cisplatin carriers (table 3.14) sugguests that all carriers could be suitanable release; thus, the theurapectic value of drug is improved while the toxic level of drug is reduced leading to the diminishing the side effect of drug
Table 3.14 Phamacokinetic parameters of cisplatin carriers
G4.0-PAA-CisPt G4.5-CisPt G3.5-CisPt G2.5-CisPt pH 5.5 pH 7.4 pH 5.5 pH 7.4 pH 5.5 pH 7.4 pH 5.5 pH 7.4 Cmax (ng/mL) 15.92 13.75 19.19 16.46 16.51 14.80 15.86 11.92
AUC (mg.h/mL) 1.79 1.47 1.73 1.55 1.65 1.42 1.59 1.14
Tmax (h) 48-55 48-55 12 24 12-24 12 12-24 12
Figure 3.22 Prediction of cisplatin concentration in Plasma 3.15 Result and discussion about the cytotoxicity
3.15.1 Cytotoxicity of PAMAM dendrimer G4.5 against lung cancer cell NCI-H460
PAMAM dendrimer with carboxylate groups on the surface is safety to cell while free cisplatin induces the high toxic level and its IC50 (inihibit 50% cell growth) is 1,00 ± 0,11 μg /mL However, after conjugating with PAMAM dendrimer carrier, the toxicity of cisplatin reduces times, IC50 value is 3,23 ± 0,06 μg/mL for NCI-H460
Table 3.15 In vitro cytotoxicity of Carboxylate PAMAM dendrimer G4.5, Cisplatin and PAMAM G4.5-Cisplatin complex
Sample Concentration
(g/mL) Cell growth (NCI-H460 cell line) Carboxylate PAMAM dendrimer G4.5 100 112.25 0.87% cell growth
Cisplatin 1.00 ± 0.11 Inhibit 50% cell growth
PAMAM G4.5-Cisplatin (34.01 %
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Table 3.16 In vitro cytotoxicity of PAMAM G4.0-PAA, cisplatin and complex PAMAM G4.0-PAA-Cisplatin
Sample concentration
(g/mL) Cell growth (NCI-H460 cell line)
PAMAM G4.0-PAA 100 115.40 3.57% cell growth
Cisplatin 0.98 ± 0.09 Inhibit 50% cell growth
Complex G4.0-PAA-Cisplatin
(40.44 % Cisplatin) 2.88 0.08 Inhibit 50% cell growth 3.15.2 The result and the discussion of cytotoxicity of carrier based G4.0-PAA
Fig 3.23 shows that the cytotoxic effects of cationic PAMAM G4.0 were remarkable higher than PAMAM G4.5 as the amine groups (-NH2) on the surface of PAMAM dendrimer G4.0 are more toxic than that of carboxylate (-COO-) groups on the surface of PAMAM G4.5 Generally, anionic PAMAM G4.5 was relatively harmless toward MCF7 cells line in a concentration range of 30-150ppm Contrary, the percentage of inhibition of MCF-7 cell growth increased linearly with the increase of PAMAM G4.0 concentration PAMAM dendrimer G4.0 at 92.15±0.15 ppm could induce the diminishing of the growth of cells by 50% Image of MCF7 cell lines taken after 48h incubation period with cationic PAMAM G4.0 had the orange cells with dots in the nuclei and red cells, which was absented in wells cultured with modified PAMAM G4.0, anionic PAMAM G4.5, and PBS 1X despite the same applying concentration (100ppm)
Figure 3.23 In vitro cytotoxic effects on MCF-7 cells’ response to various types carries (PAMAM G4.0, PAMAM G4.5 and G4.0-PAA) with reference to untreated cells (A) MCF7 cell viability assessment in cells treated with unmodified and modified PAMAM G4.0, PAMAM G.5 and DMEM at 100ppm for 48h, by fluorescence microscopy (B) Dual staining (AO/EB) was applied to determinate viable cells from those in early (E)/ later (L) apoptosis (bright green and orange/yellow, respectively) and necrosis (N, red)
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fibroblast cell The cell viability drastically reduced to 9.32 ± 1.07% and 100 % mortality was recorded at 168h Contrarily, fibroblast cell in the culture medium with PAA-G4.0 and PAMAM G4.5 were able to maintain a good cell viability (≥100%)
Figure 3.24 Percentage cell viability of Human fibroblast treated with PAMAM G4.0, PAMAM G4.5, G4.0-PAA, Camptothecin (positive control) and PBS 1X (negative control) at the same concentration
(100ppm)
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3.15.3 Result and discussion about the cytotoxicity of dual drug cisplatin and 5-FU encapsulated PAMAM dendrimer G3.5-PNIPAM
Table 3.17 Inhibited perecentage of the growth of MCF7 cells
Sample Testing
concentration
Cell inhibition percentage (%)
Trail Trail Trail Average ± SD G3.5-NIPAM-5FU 100 µg/mL 68.17 69.57 68.41 68.72 ± 0.75 G3.5-NIPAM-CisPt-5FU 100 µg/mL 78.78 79.57 76.59 78.32 ± 1.55 Figure 3.25 Fluorescent microscopy—staining the fibroblast cells with acridine orange (AO)
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PAMAM dendrimer G3.5-PNIPAM-5-FU shows the high cytotoxicity level, about 68.72% The dual drug encapsulated PAMAM dendrimer G3.5-PNIPAM-cisPt-5FU helps to increase the inhibition of the cell growth (78.78%) due to high amount of cisplatin in PAMAM (35.22%) This reveals that PAMAM dendrimer G3.5-PNIPAM-cisPt-5FU has the high anticancer efficacy
Table 3.18 In vitro cytotoxicicity of PAMAM dendrimer G3.5-PNIPAM, 5-FU and PAMAM dendrimer G3.5-PNIPAM-CisPt-5FU
Sample Concentration
(g/mL)
Cell growth (NCI-H460 cell line)
PAMAM dendrimer G3.5 100 110.22 0.85% cell growth
PAMAM G3.5-PNIPAM 100 115.22 0.98% cell growth
5-FU 2.6 ± 0.31 Inhibit 50% cell growth
G3.5-PNIPAM-CisPt-5FU 1.625 ± 0.419 Inhibit 50% cell growth CONCLUSION AND SUGGESTION
CONCLUSION This dissertation draws conclusions from what has happened
- Generation (0.5–4.5 G) PAMAM dendrimers were successfully synthesized The obtained PAMAM dendrimer of generations G0.5 to G4.5 were chemical structure identified using 1H-NMR, from which the molecular mass of these dendrimer was calculated
- The complex PAMAM dendrimer G3.0-Cisplatin (9,63% Cisplatin) and PAMAM dendrimer G4.0-Cisplatin (16,95% G4.0-Cisplatin) were successfully synthesized
- The complex PAMAM dendrimer G2.5-Cisplatin (28,99% Cisplatin), PAMAM dendrimer G3.5-Cisplatin (30,23% G3.5-Cisplatin) and PAMAM dendrimer G4.5-G3.5-Cisplatin (31,11% G3.5-Cisplatin) were successful synthesized From ICP-MS method, loading method with the help of sonication improves encapsulation efficiency, for example, the amount of cisplatin loading in complex using sonication was higher than that of non-sonication method, PAMAM dendrimer G2.5-Cisplatin (31,82% Cisplatin), PAMAM dendrimer G3.5-Cisplatin (33,01% G3.5-Cisplatin) complex PAMAM dendrimer G4.5-G3.5-Cisplatin (34,03% G3.5-Cisplatin) The utilization of AgNO3 to hydrolyze cisplatin before performing complexation reaction with carboxylate groups on the surface of half generation PAMAM dendrimer also improves the Pt loading efficacy than that of non-aqueous cisplatin The obtained products are pale yellow powder
- PAMAM dendrimer G3.0 was successful modified with Poly N-isopropylacrylamide (PNIPAM-COOH) with different mole ratio, and also G3.5-PNIPAM was successful synthesized The calculation of number PNIPAM attaching on PAMAM dendrimer G3.0 and its molecular weight were based on 1H-NMR spectrum G3.0-PNIPAM showed the ability for dual drug encapsulation, 5-FU (20.43%) and Cisplatin (35,22%), and could be further applicated for another drugs
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with lung cancer cells NCI-H460, this complex expresses the potency in cancer cell inhibition and has the good release maner in acid environment pH 5.5
- TEM and DLS results reveals that these synthesis carriers were nano-size and homogenous distribution in aqueous
- Carboxylate PAMAM dendrimer G2.5, G3,5, G4.5, G3.5-PNIPAM and G4PAA showcase the sustained release of drugs in vitro
- These carriers and the complex PAMAM dendrimer G3.5-Cisplatin, G3.5-PNIPAM-Cisplatin-5FU and G4.0-PAA-Cisplatin reduce the toxic level of cisplatin and express the anti-cancer peroperty of cisplatin in the suitable way
SUGGESTION
The theurapetic value of Carboxylate PAMAM G3.5-PNIPAM, G4.0-PAA encapsulated anticancer drug cisplatin should be investigated on mice model which has the lung tumor Other experiment with another cancer cells should be carried in both in vitro and in vivo, specialized on the drug-resistant cell lines The pharmacokinetic of thuốc PAMAM dendrimer G4.0-PAA-Cisplatin in vivo should be determined The dual-drug ability of PAMAM dendrimer G3.0-PNIPAM, PAMAM dendrimer G3.5-PNIPAM, PAMAM dendrimer G4.0-PNIPAM and PAMAM dendrimer G4.5-PNIPAM for anticancer, 5-FU and Pt, should be identified Further, the effect of pH solution on the grafting degree of PAA on PAMAM dendrimer G4.0 should be noticed
THE NEW CONTRIBUTED POINTS OF DISSERTATION
- The complex PAMAM dendrimer G3.0-Cisplatin and PAMAM dendrimer G4.0-Cisplatin were successfully synthesized
- The complex PAMAM dendrimer G2.5-Cisplatin, PAMAM dendrimer G3.5-Cisplatin and PAMAM dendrimer G4.5-Cisplatin were successfully synthesized by using either sonication or non-sonication The hydrolysis of cisplatin by AgNO3 before doing the complexation reaction with carboxylate on the surface of half-generation PAMAM denderimer and combined with sonification help to improve the retention of Pt in the compex as compared to non-aqueous cisplatin
- The modification of PAMAM dendrimer G3.0 with Poly N-isopropylacrylamide (PNIPAM-COOH) was successful established with different mol ratio; and success in the synthesis of G3.5-PNIPAM was also involved The calculation of number PNIPAM attaching on PAMAM dendrimer G3.0 and its molecular weight were based on 1H-NMR spectrum Preliminary results show that G3.5-PNIPAM could carry both anticancer drugs, 5-FU (20.43%) and Cisplatin (35,22%), and could be used to delivery other kinds of drug
- The modification of PAMAM dendrimer G3.0 and G4.0 with poly acrylic (PAA) acid were successfully synthesized with different mol ratio The calculation of number PAA attaching on PAMAM dendrimer and its molecular weight were based on 1H-NMR G4.0-PAA containing 15 PAA groups attaching on the surface of PAMAM dendrimer G4.0 exhibits the best cisplatin encapsulation efficacy (40,44% Cisplatin) The cytotoxicity of the complex G4.0-PAA-Cisplatin was lower than that of free cisplatin (IC50 was times of free cisplatin) testing with lung cancer cells NCI-H460, this complex expresses the potency in cancer cell inhibition and has the good release maner in acid environment pH 5.5
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- These carriers and the complex PAMAM dendrimer G3.5-Cisplatin, G3.5-PNIPAM-Cisplatin-5FU and G4.0-PAA-Cisplatin reduce the toxic level of cisplatin and express the anti-cancer peroperty of cisplatin in the suitable way
PUBLISHCATION LIST
1 Ngoc Hoa Nguyen, Le Hang Dang, Dang Nam Nguyen, Cuu Khoa Nguyen, Ngoc Quyen Tran,
Polyacrylic-conjugated polyamidoamine G4.0 dendrimer as a potetial nanocarrier for effectively delivery of Cisplatin, Bulletin of materials science (accept: Nov 2020)
2 Phung Ngan Le, Ngoc Hoa Nguyen, Cuu Khoa Nguyen, Ngoc Quyen Tran, Smart dendrimer-based nanogel for enhancing 5-fluorouracil loading efficiency against MCF7 cancer cell growth, Bulletin of Materials Science 39(6):1493-1500, 2016
3 Hoang Nguyen, Ngoc Hoa Nguyen, Ngoc Quyen Tran, Cuu Khoa Nguyen, Improved loading method for Cisplatin in dendrimer carriers and behavior of the complex nanoparticles against NCI-H460 lung cancer cell, Int J Nanotechnology, Vol 15, N0 6, june 2015, pp 4106-4110 (5), 2015