The thesis consists of 135 pages, including 21 tables of data, 50 figures and 144 references. The structure of the thesis consists of: Introduction: 4 pages; Overview: 32 pages; Experimental part: 13 pages; Results and discussion: 63 pages; Recent contributions of the thesis: 1 page; The list of published works: 2 pages; References: 18 pages. There is also an appendix containing the spectrums and diagrams that measure the characteristics and properties of components and AG/CS/LOV, AG/CS/LOV/ginsenoside Rb1 composites with 11 pages.
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THACH THI LOC
RESEARCHES ABOUT FABRICATION, CHARACTERIZATION, PROPERTIES OF ALGINATE/CHITOSAN POLYMER COMPOSITE WITH GINSENOSIDE RB1 AND LOVASTATIN
Specialization: Organic Chemistry
Code: 9440114
SUMMARY OF DOCTORAL THESIS IN CHEMISTRY
NGHE AN - 2020
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THE PROJECT WAS COMPLETED AT
The School of Natural Sciences Education - Vinh University and the Institute
for Tropical Technology - Vietnam Academy of Science and Technology
Supervisors: Prof Dr Thai Hoang
Assoc Prof Dr Le Duc Giang
The thesis can be found at:
- Nguyen Thuc Hao Library, Vinh University
- Vietnam National Library
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PREFACE
1 Reasons for the subject choice:
Chitosan (CS) and sodium alginate (AG) are natural polymers that are applied widely in various fields CS is a natural polysaccharide formed during the deacetylation of chitin from shells of shrimp and other crustaceans in alkaline condition It comprises an unbranched chain consisting of poly-(1, 4)-2-amino-2-deoxy-D-glucopyranose, and it is a unique basic linear polysaccharide Chitosan polymer having hydroxide and amine groups in most repeat units and the protonation of the amine groups makes the polymer soluble in dilute acid solution CS is widely used in food and pharmaceutical industry, also in biotechnological fields Furthermore, CS has been extensively studied on biomaterials due to its biodegradability and biocompatibility However, the disadvantage of CS is very sensitive to moisture, which limits the use of this natural polymer To overcome its disadvantage, CS is often combined with relatively stable moisture-proof polymers such as alginate (AG), polylactic acid (PLA), polyethylene glycol fumarate, poly (vinyl alcohol), etc AG is dissolved in water to form a highly viscous solution, so it is used to increase storage life and retain original quality of foods Therefore, synthesis and application of AG/CS blend with different active substances have been researched by many scientists
According to the review documents, the use of AG/CS polymer composites carrying drugs has certain effects, so this research direction has been attracting the attention of many scientists over the world However, so far, no work has been published on the characteristics and properties of the combination of AG/CS polymers with ginsenoside Rb1 (extracted from Panax Pseudo-Ginseng in Vietnam) and lovastatin as a controlled release drug (cholesterol reduction, heart-related diseases treatment) Therefore,
I/PhD student chose the topic: “Researches about fabrication,
characterization, properties of alginate/chitosan polymer composite with ginsenoside Rb1 and lovastatin”
2 Subjects for study
The AG/CS composites which contain LOV for treatment of cardiovascular diseases, lowering cholesterol and active ingredient ginsenoside Rb1 found in Panax Pseudo-Ginseng Wall’s powder with their characteristics, properties and applications
3 Research Tasks
- Prepare natural polymer composites of AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 in micrometer and nanometer sizes
Trang 4in different pH buffer solutions
- Set-up different kinetic models for drug release from AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composites in different pH buffer solutions
- Determine toxicity of AG/CS/LOV nanoparticles on rat
4 Thesis structure
The thesis consists of 135 pages, including 21 tables of data, 50 figures and 144 references The structure of the thesis consists of: Introduction: 4 pages; Overview: 32 pages; Experimental part: 13 pages; Results and discussion: 63 pages; Recent contributions of the thesis: 1 page; The list of published works: 2 pages; References: 18 pages There is also an appendix containing the spectrums and diagrams that measure the characteristics and properties of components and AG/CS/LOV,
AG/CS/LOV/ginsenoside Rb1 composites with 11 pages
CHAPTER 1 OVERVIEW
In chapter 1, we presented an overview of the following:
1 Chitosan (CS): Introduction, composition, structure, properties and main applications of CS
2 Alginate (AG): Introduction, structure, classification, physical and chemical properties and applications of AG
3 Polymer composite materials based on alginate/chitosan (AG/CS) carrying drugs, pharmaceuticals: Domestic and foreign studies on preparing methods and applications of AG/CS/drug composites in film and particle forms
4 Overview of lovastatin (LOV): General introduction about the structure, properties, pharmacokinetics of LOV and research results on the polymers carrying LOV in the world
5 Overview of Panax Pseudo-ginseng and ginsenoside Rb1: General introduction about the structure, characteristics, properties and applications of Ginsenoside Rb1 and polymers carrying Panax Pseudo-ginseng and ginsenoside Rb1 in the world
6 Current update on researching polymer composites carrying drugs in Vietnam
From the overview, it can be seen that the the use of AG/CS polymer composites carrying drugs has certain effects, so this research
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direction has been attracting the attention of scientists in the world However, so far, no work has been published on the characteristics and properties of the combination of AG/CS polymers with ginsenoside Rb1 and LOV to a controlled release drug (cholesterol reduction, heart-related diseases treatment)
CHAPTER 2 EXPERIMENTS AND METHODS 2.1 Raw materials, chemicals, and tools
2.1.1 Raw materials, chemicals
Alginate (AG), chitosan (CS), lovastatin (LOV) are produced by Sigma Aldrich; Ginsenoside Rb1 (Rb1) is provided by the Institute of Medicinal Materials, Ministry of Health
Sodium tripolyphosphate (STPP), polyethylene oxide (PEO) and polycaprolactone (PCL) are commercial products manufactured by Sigma Aldrich
Potassium chloride (KCl, solid), sodium hydroxide (NaOH, solid), calcium chloride (CaCl2, solid), monopotassium phosphate (KH2PO4, solid), 37% chlorhydric acid (HCl) solution, ethanol, acetic acid solution (CH3COOH) 1% formulated with 99.5% acetic acid: commercial products
of China
2.1.2 Experimental tools and devices
- Magnetic stirrer, analytical balance, dryer, ultrasonic machine, centrifuge, etc
- Glassware: measuring cylinders, pipettes, beakers, conical flasks, burettes, glass chopsticks, etc
2.1.3 Research devices
- Fourier Nexus 670 transformative infrared spectrometer (United States) at the Institute for Tropical Technology - Vietnam Academy of Science and Technology (VAST) ; Zetasizer Ver 620 device at the Institute
of Materials Science - VAST; Scanning field scanning electron microscope (FESEM) (FESEM S- 4800, Hitachi, Japan) at the Institute of Hygiene and Epidemiology and the Institute of Materials Science - VAST; Differential scanning thermal analyzer DSC DSC-60 (Japan) at Department of Chemistry, Hanoi National University of Education; UV-Vis Spectrometer (Cintra 40, GBC, USA) at the Institute for Tropical Technology - VAST
2.2 Preperation of alginate/chitosan (AG/CS) composites carrying LOV
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2.2.1 Preperation of alginate/chitosan/lovastatin (AG/CS/LOV) composite films by solution method
120 mg of AG was dissolved in 20 ml of distilled water and 30 mg
of CS was dissolved in 20 ml of 1% acetic acid before mixing together to obtain solution A (ratio of AG/CS is 80/20) 15 mg of LOV (10% in comparison with total weight of AG-CS) was dissolved in 10 ml of ethanol
to obtain solution B Solution B was added into solution A and this mixture was sonicated for 15 minutes to obtain a uniform solution Then, this solution was poured into the petri dish and naturally evaporated solvent about 48 hours The obtained AG/CS/LOV film is abbreviated AC82-L10 Similary, the content of LS was varied from 0 to 30 % and ratio of AG/CS
is fixed to prepare other samples The obtained AG/CS/LOV films are abbreviated AC82Lx (AG/CS 80/20 –LOV 10-30) where x is LOV content (10-30%)
2.2.2 Preperation of AG/CS/LOV nanoparticles by ionic gelation method
The AG/CS/LOV nanoparticles were prepared by ionic gelation according to the following steps: First, AG was dissolved in distilled water until a solution was formed before the addition of CaCl2 to increase the viscosity of the solution (solution 1) In addition, CS was dissolved in 1% acetic acid solution (solution 2), while LOV was dissolved in ethanol (solution 3) Next, solution 1 was added dropwise to solution 2 and stirred
in an ultrasonic bath to form a uniform solution Thereafter, solution 3 was poured into the mixture of solution 1 and solution 2 and then ultrasonicated five times for 5 mins Finally, the mixed solution was centrifuged at 4°C before lyophilization in a FreeZone 2.5 machine (Labconco, USA) The ratios of AG, CS, LOV, and the coding of prepared samples are presented
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of AG and CS was fixed at 0.8 gram and 0.2 gram, respectively The mass
of LOV and ginsenoside Rb1 was changed to make AG/CS/LOV/Rb1 composite films
2.3.2 Preperation of AG/CS/LOV/ginsenoside Rb1 nanoparticles
by ionic gelation method
General procedure: 50 mg of STPP and 11 mg of CaCl2 were dissolved in 50 ml and 20 ml of distilled water, respectively 20 mg (10%)
of LOV and ginsenoside Rb1 (1 -5%) in were dissolved in 10 ml of ethanol (drug solution) 100 mg CS was dissolved in 50 ml of 1% acetic acid solution (CS solution) and 100 mg of AG in 50 ml of distilled water (AG solution) 5 ml of CaCl2 solution was slowly added to STPP solution and the mixed solution was ultrasonicated three times at 18000-20000 rpm for
15 minutes before mixing them with LOV solution The mixed solution was slowly added to AG solution and they were stirred by sonication for 30 minutes The CS was poured into the mixed solution and this solution was ultrasonicated three times at 18000-20000 rpm for 15 minutes Finally, the mixed solution was centrifuged at 4°C before lyophilization in a FreeZone 2.5 machine (Labconco, USA) Products after centrifuging was dried on FreeZone 2.5 freeze-drying equipment (Labconco, USA) at the Institute of Natural Products Chemistry - VAST to evaporate the remaining solvent in the product After that, the solid mixture is finely ground into a powder with agate mortar and stored in a sealed PE bag
2.4 Research methods
Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) method; Scanning field emission electron microscopy (FESEM), Differential scanning calorimetric method (DSC), ultraviolet-visible spectroscopy (UV-Vis)
2.5 In vitro release studies of alginate/chitosan/lovastatin
(AG/CS/LOV) and alginate/chitosan/lovatstain/ginsenoside Rb1
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a beaker containing 200 ml of different pH buffer solutions and stirring continuously for 48 hours at 400 rpm After 48 hours, removing the insoluble LOV and recording the UV - Vis spectrum of the LOV solution at different concentrations by dilution method at the maximum absorption wavelength of LOV in each buffer Setting – up the calibration equation
for ginsenoside Rb1 is quite similar to LOV
Processing data obtained by Excel software, find the calibration equations of LOV in different pH media/solutions with corresponding regression coefficients
2.5.2 Determining drug carrying efficiency of AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composites
Similar to the calibration of LOV and ginsenoside Rb1 in different
pH buffer solutions, the calibration equations of LOV and ginsenoside Rb1 was also set – up in ethanol solvent to determine the content of LOV and ginsenoside Rb1 carried by the AG/CS composites
Steps to take: drying AG/CS/LOV/ginsenoside Rb1 composites in
a vacuum drying device at 25 - 30oC for 6 hours Dissolve an exact mass of the sample in a suitable volume of ethanol for 2 hours so that the LOV in the sample dissolves completely into ethanol Filter the solution and record UV-Vis spectra at the maximum wavelengths corresponding to LOV and ginsenoside Rb1 The volume of LOV and ginsenoside Rb1 carried by the AG/CS compossite was processed by Excel software using the calibration equations of LOV and ginsenoside Rb1 in ethanol LOV and ginsenoside Rb1 carrying capacity of AG/CS composite materials is calculated by the following formula:
Medicines carrying performance (%) = The amount of medication carried The initial medication volume x
100%
2.5.3 In vitro drug release studies
In vitro LOV and ginsenoside Rb1 release process from
AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composites were carried out in different pH solutions An exact amount of the composite material was put into a 200 ml container containing a buffer solution at 37°C Stir
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the mixture with a magnetic stirrer at 400 rpm Every hour from stirring, draw exactly 10ml of solution and compensate 10ml buffer solution to maintain the volume of solution The filtered solution was measured optical density at λmax determined from the calibration curve equation for each different pH solution The drug release test was conducted for 32 consecutive hours and the percentage of LOV and ginsenoside Rb1 released at time t was calculated using the following formula:
% Lov gp = The amount of Lov released at t The initial Lov volume x 100%
% Rb1gp = The amount of Rb1 released at t The initial Rb1 volume x 100%
(100 – W)1/3 = 1001/3 – k3t (Eq.3) Higuchi’s square root of time equation (diffusion model) (HG):
Power law equation or Korsmeyer-Peppas model (KMP):
Mt/M∞ = k5tn (Eq.5)
Where k is drug release constant; C t and C0 is concentration of drug at
initial time and testing time; W t and W0 is weight of drug at 0 and t hour;
M t /M ∞ is the fractional drug release into dissolution medium; and n is the
diffusional constant that characterizes the drugrelease transport mechanism
With n ≤ 0.5, the drug diffusion from the polymer matrix corresponds to a
Fickian diffusion and a quasi-Fickian diffusion mechanism,
respectively With 0.5 < n < 1, an anomalous, non-Fickian drug diffusion occurs With n = 1, a non-Fickian, case of II (relaxational) transport or zero-order release kinetics could be observed, and n > 1 to super case II
transport
To find the most suitable kinetic models for the release process of LOV and ginsenoside Rb1 from the AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composites (in film and particle forms), the data of drug release content were calculated according to Eq.1-Eq.5 equations
2.6 Toxicity test of AG/CS/LOV nanoparticles
Acute and subchronic toxicities of LOV-carrying nanoparticles was carried out in vivo in adult healthy Swiss mice The procedure was strictly
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performed in a laboratory at the Military Medical Academy following the guidance of the Organization for Economic Co-operation and Development (OECD)
CHAPTER 3 RESULTS AND DISCUSSIONS
3.1 Investigation of conditions for manufacturing alginate/chitosan/lovastatin (AG/CS/LOV)
After investigating some conditions for making alginate/chitosan (AG/CS) composite film, the results are as follows:
Used
Evenly Didn’t
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The results showed that when using an AG: CS ratio of 6: 4 or 7: 3
or stirring the mixture at high temperatures (500C and 900C), the polymer solution mixture had agglomeration phenomenon and using ultrasonic stirring at high speed, the mixed solution is more homogeneous Therefore, suitable conditions for creating AG/CS composite film are: AG: CS ratio = 8: 2; temperature: 250C; concentration of substances: [AG] = 0.32 g/ml; [CS] = 0.1 g/ml; stirring time: 1 hour; using ultrasonicator
3.2 Characteristics and properties of alginate/chitosan/lovastatin composite material (AG/CS/LOV)
3.2.1 Characteristics and properties of AG/CS/LOV composite film
3.2.1.1 Fourier transform infrared spectroscopy (FTIR) of AG/CS/LOV composite film
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Table 3.1 Wavenumbers corresponding to peaks of specific functional
groups in AC82Lx nanocomposite films
3.2.1.2 Morphology of AG/CS/LOV composite film
The SEM images of AC82 film, LOV and AC82L10, AC82L30 films in figure 3.4, figure 3.5 and figure 3.6, respectively It is clear that LOV (bar shape) and CS (circle shape) were dispersed into AG matrix For AC82L10 film, the size of LOV and CS phases about 5-10 µm and 0.5-3
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µm, respectively The CS particles and LOV bars were agglomerated together due to interactions between polymer-polymer and drug-drug The SEM image of AC82L30 film appeared CS and LOV phases with bigger size, the size of CS phase in range 1 to 6 µm and LOV phase from 5 to 20
µm The size of dispersion phases of blend film was increased at high LOV content can be caused by affinity of drug-drug stronger than affinity of drug-polymer leading to agglomeration of drug to bigger size At LOV content of 10%, dispersion phases in blend film had smaller size due to good interaction of CS, AG and LOV by hydrogen bonding and dipole –
dipole interactions as mentioned above
Figure 3.4 FESEM images of the
AC82 composite films
Figure 3.5 FESEM images of LOV
Figure 3.6 Ảnh FESEM images of AC82L10 (A) and AC82L30 (B)
composite films
3.2.2 Characteristics and properties of AG/CS/LOV composite particles
3.2.2.1 FTIR spectra of AG/CS/LOV nanoparticles
Figure 3.7 is FTIR spectra of AG/CS/LOV nanoparticles prepared with different content of LOV It can observe clearly the slight shift of some characteristic peaks when comparing the FTIR spectra of AG/CS/LOV nanoparticles with the FTIR spectrum of LOV, CS and AG as was stated above
B
A
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Figure 3.7 FTIR spectra of AG/CS/LOV nanoparticles prepared
with different content of LOV
Clearly, it can be descried the difference in wavenumber of C=O groups in the FTIR spectra of AG/CS/LOV nanoparticles in comparison with that of CS,
AG and LOV In particular, the peaks corresponding to NH2, C-O, OH groups
in FTIR spectra of ACL nanoparticles also shifted significantly (3-105 cm-1) in comparison with similar peaks in FTIR spectra of AG, CS and LOV This change can be caused by interactions between C=O, C-O, OH groups in LOV with C-O, NH, OH groups in CS and C=O, C-O, OH groups in AG through dipole-dipole interactions and hydrogen bond In addition, the change in LOV content is less effect on the structure of AG/CS/LOV nanoparticles becausse the wavenumber of some characterized groups shift slightly, only 1-7 cm-1
3.2.2.2 Size distribution of AG/CS/LOV composite particles
To investigate the influence of AG/CS ratio on the size distribution of AG/CS/LOV nanoparticles, the AG/CS/LOV samples were prepared with different AG/CS ratio Figure 3.8 performed the size distribution diagrams
of AC6/3-L10, AC6.5/3-L10 and AC7/3-L10 samples It can be seen that the size particles of all samples changed in the range from 68 nm to 1718
nm Among all prepared samples, the average diameter particles reached minimum size at 86.2 ± 3.7 nm corresponding to AG/CS ratio of 6.5/3 The different size distribution of tested samples may be caused by the interaction diverse between solvent and drug, drug and drug, solvent and polymer, polymer and polymer, solvent and solvent, drug and polymer, and
so on In general, smaller the size of particles is, better distribution of drug
in solution is So, the suitable AG/CS ratio for preparation AG/CS/LOV nanoparticles is 6.5/3
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Figure 3.8 The size distribution
diagrams of AG/CS/LOV
nanoparticles prepared with
different AG/CS ratio
Figure 3.9 The size distribution
diagrams of AG/CS /LOV nanoparticles
The AG/CS/LOV nanoparticles were prepared with the LOV content changed in the range of 10 to 30 wt.% in comparison with the total weight
of AG and CS to investigate the effect of LOV on the particle size of AG/CS/LOV nanoparticles It is clearly observed that the size of AG/CS/LOV nanoparticles with LOV content of 10 wt.% and 20 wt.% was much smaller than that of AG/CS/LOV nanopaticles containing 30 wt.% of LOV This means that using LOV low weight in the AG/CS/LOV nanoparticles can affected more effectively to particle size nanoparticles than LOV large weight in the nanoparticles It can suggest an explaintion:
at a small weight of LOV, the interactions between drug and polymer are stronger than that between drug and drug Therefore, polymers can absord a large amount of drug and drug can distribute more regularly in the struture of AG/CS/LOV nanoparticles and AG/CS/LOV nanoparticles have structure closer and smaller size In contrast, using a large of LOV, the interactions between drug-drug predominate more than interactions between drug-polymer Thus, it can occur the agglomeration of drug together in the struture
of AG/CS/LOV nanoparticles causing the AG/CS/LOV nanoparticles have bigger size
3.2.2.3 Morphology of AG/CS/LOV composite particles
The FESEM images of AC6.5/3 and AG/CS/LOV nanoparticles with different content of LOV are demonstrated in figure 3.10 The AC6.5/3-L20 and AC6.5/3-L30 nanoparticles had a tendency of agglomeration together from 100-200nm basical particles to form larger particles with micromet size (Figures 3.10 C,D) Interestingly, the AC6.5/3-L10 nanoparticles were spherical, separate and uniform size, only 50-80 nm
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(Fig.3.10B) This result was similar to the size distribution of AC6.5/3-L10 nanoparticles presented above Predictably, at 10 wt.% of LOV, it can interact stronger and distribute better in CS and AG than other content of LOV
Figure 3.10 FESEM image of AG/CS nanocomposites without LOV: AC6.5/3 (A) and with LOV: AC6.5/3-L10 (B); AC6.5/3-L20(C); AC6.5/3-
L30 (D)
3.2.2.4 Thermal behavior of AG/CS/LOV nanoparticles
Table 3.2 showed some thermal characteristics of LOV, CS, AG and AG/CS/LOV nanoparticles with different LOV content The melting temperature of LOV, CS and AG was performed at 174.6oC, 106.8oC and 119.7oC, respectively while the melting temperature of AG/CS/LOV nanoparticles was exhibited from 107.2oC - 113.4oC depending on the LOV content Noticealy, the melting temperature of AG/CS/LOV nanoparticles was higher than that of CS and lower than that of AG It can be confirmed that CS and AG were partly combatibility through some physical interactions as mentioned Therefore, the structure of AG/CS/LOV nanoparticles became closer and drug release from nanoparticles can
be controlled more easily
Table 3.2 Melting temperature and enthalpy of AG/CS/LOV nanoparticles