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.
MINISTRY OF EDUCATION AND TRAINING VINH UNIVERSITY 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 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 Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended at the Council of doctoral thesis examiners of Vinh University at…… on … of … , 2020 The thesis can be found at: - Nguyen Thuc Hao Library, Vinh University - Vietnam National Library PREFACE 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” 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 Research Tasks - Prepare natural polymer composites of AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 in micrometer and nanometer sizes - Identify characteristics and properties of AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composites - Study on release LOV from AG/CS/LOV composites, release LOV and ginsenoside Rb1 from AG/CS/LOV/ginsenoside Rb1 composites in 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 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: pages; Overview: 32 pages; Experimental part: 13 pages; Results and discussion: 63 pages; Recent contributions of the thesis: page; The list of published works: 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 OVERVIEW In chapter 1, we presented an overview of the following: Chitosan (CS): Introduction, composition, structure, properties and main applications of CS Alginate (AG): Introduction, structure, classification, physical and chemical properties and applications of AG 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 Overview of lovastatin (LOV): General introduction about the structure, properties, pharmacokinetics of LOV and research results on the polymers carrying LOV in the world Overview of Panax Pseudo-ginseng and ginsenoside Rb1: General introduction about the structure, characteristics, properties and applications of Ginsenoside Rb1 and polymers carrying Panax Pseudoginseng and ginsenoside Rb1 in the world 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 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 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 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 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 was added dropwise to solution and stirred in an ultrasonic bath to form a uniform solution Thereafter, solution was poured into the mixture of solution and solution and then ultrasonicated five times for 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 in Table 2.1 Table 2.1 Ratios of AG, CS, LOV and the coding of prepared samples AG (wt.%) CS (wt.%) LOV (wt.%) Signature of samples 60.6 30.4 9.0 AC6/3-L10 62.2 28.8 9.0 AC6.5/3-L10 63.6 27.3 9.0 AC7/3-L10 57.0 26.3 16.7 AC6.5/3-L20 52.6 24.3 23.1 AC6.5/3-L30 2.3 Preperation of alginate/chitosan/lovavstati/ginsenoside Rb1 (AG/CS/LOV/ginsenoside Rb1) composites 2.3.1 Preperation of AG/CS/LOV/ginsenoside Rb1 composite films by solution method Firstly, AG and CS with calculated weights were dissolved in distilled water and 1% acetic acid solution, respectively whereas LOV and ginsenoside Rb1 were dissolved in ethanol solvent (drug solution) Next, the drug solution was dropped into the solution of AG which was added CaCl2 and stirred on a magnetic stirrer After that, the CS solution was dropped to mixture of AG and drug, and the mixed solution was ultrasonicated three times for 15 minutes Then, the composite mixture was poured into the petri dish and the solvent was been naturally evaporated for 24 hours Finally, film production was dried at 500C for hours The mass 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) 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), ultravioletvisible spectroscopy (UV-Vis) 2.5 In vitro release studies of alginate/chitosan/lovastatin (AG/CS/LOV) and alginate/chitosan/lovatstain/ginsenoside Rb1 (AG/CS/LOV/ginsenoside Rb1) from composite materials in various pH buffers 2.5.1 Setting - up calibration equations of LOV and ginsenoside Rb1 in different pH buffers Research simulating drug release process similar to the typical digestive organs in the human body in environments with pH 2.0; pH 4.5; pH 6.8 and pH 7.4 as the following process: weighing 0.01g LOV, put into 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 hours Dissolve an exact mass of the sample in a suitable volume of ethanol for 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: The amount of medication carried Medicines carrying performance (%) = x The initial medication volume 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 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: The amount of Lov released at t % Lovgp = x 100% The initial Lov volume The amount of Rb1 released at t % Rb1gp = x 100% The initial Rb1 volume 2.5.4 Kinetic studies The drug release mechanism from polymer matrix usually is calculated according to some popular kinetics as depicted below: Zero-orderer kinetic (ZO): Wt = W0 + k1t (Eq.1) First-orderer kinetic (FO): log Ct = log C0 - k2t/2.303 (Eq.2) Hixson – Crowell’s cube-root equation (HCW): (100 – W)1/3 = 1001/3 – k3t (Eq.3) Higuchi’s square root of time equation (diffusion model) (HG): Wt = k4t (Eq.4) Power law equation or Korsmeyer-Peppas model (KMP): Mt/M∞ = k5tn (Eq.5) Where k is drug release constant; Ct and C0 is concentration of drug at initial time and testing time; Wt and W0 is weight of drug at and t hour; Mt/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 > 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 performed in a laboratory at the Military Medical Academy following the guidance of the Organization for Economic Co-operation and Development (OECD) CHAPTER 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: Condition Result Condition Result AG: CS Ratio :4 250C Temperature 7:3 500C 900C 8:2 9:1 Evenly Using Used ultrasonicator Didn’t Frontloaded use The results showed that when using an AG: CS ratio of 6: or 7: 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: 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 14 The AG/CS/LOV nanoparticles with different LOV content had a difference in melting temperature and melting enthalpy as listed in Table This result can explained by LOV interacts weakly with CS, AG at the large content of LOV, leading to structure of AG/CS/LOV nanoparticles less tightly and easier for melting As a result, AC6.5/3-L20 and AC6.5/3L30 nanoparticles have melting temperature and melting enthalpy lower than the AC6.5/3-L10 nanoparticles 3.3 Characteristics and properties of alginate/chitosan/lovastatin/ginsenoside Rb1 (AG/CS/LOV/ginsenoside Rb1) composite materials 3.3.1 Characteristics and properties of AG/CS/LOV/ginsenoside Rb1 composite films 3.3.1.1 FTIR spectra of AG/CS/LOV/ginsenoside Rb1 composite films Figures 3.11 and 3.12 presented the FTIR spectra of AC82L10Rx composite films It can be seen the characteristic peaks of AG, CS, LOV, and ginsenoside Rb1 were appeared in the FTIR spectra of AC82L10Rx composite films The peaks of -NH3OC group which were formed by the electrostatic interaction between the protonated amino groups of CS and the carboxylate groups of AG dissociated to COO− groups were located at 2167 cm-1 and 2360 cm-1 Figure 3.11 FTIR spectrum of Figure 3.12 FTIR spectrum of AC82L10Rx composite film AC82-Lx-R5 composite film As adding ginsenoside Rb1 into the AG/CS/LOV composite films, it was recognized a strong shift of NH3OC and the hydroxyl group in the FTIR spectra of CS, AG, LOV, ginsenoside Rb1 and AC82L10Rx composite films This proved that the presence of ginsenoside Rb1 could lead to the stronger electrostatic interaction between AG and CS as well as increase the intermolecular hydrogen bond between ginsenoside Rb1, LOV, AG and CS 3.3.1.2 Morphology of AG/CS/LOV/ginsenoside Rb1 composite films 15 Figure 3.13 displayed the FESEM images of the AC82L10Rx composite films at different content of ginsenoside Rb1 It can be seen that the presence of ginsenoside Rb1 in the composite film helped the dispersion of LOV to become more evenly in AG/CS blend and the size of LOV bars were significantly decreased For instance, LOV had bar and rod shape with size in the range from 30 µm to 40 µm in AG/CS/10% LOV (AC82L10R0) film and LOV size was reduced (5 – 10 µm) when adding wt.% of ginsenoside Rb1 This result exhibited that ginsenoside Rb1 can play an important role of auxiliary dispersion and a compatibilizer in the AC82L10Rx composite films thanks to the increase in intermolecular hydrogen bond of components in the film As a result, the agglomeration of LOV in the composite films was decreased Similarly, the FESEM images of AG/CS/LOV/5% ginsenoside Rb1 composite films with variable LOV content (AC82-Lx-R5) were expressed in Figure 3.14 AC82L10 AC82L10R1 AC82L10R3 AC82L10R5 Figure 3.13 FESEM image of the AC82L10Rx composite film AC82 (a) AC82R5 (b) AC82L5R5 (c) AC82L10R5 (d) AC82L15R5 (e) AC82L20R5 (f) Figure 3.14 FESEM images of composite films: AC82 (a), AC82R5 (b), AC82L5R5 (c), AC82L10R5 (d), AC82L15R5 (e) and AC82L20R5 (f) 3.3.1.3 Thermal behavior of AG/CS/LOV/ginsenoside Rb1 composite films From data in table 3.3, the temperature melting of AC82L10R0 composite film was significantly lower than that of AG, CS and LOV The AC82L10R0 film had two endothermic peaks at close to 1300C and 1800C characterized for the dehydration and melting of polymer blend The decomposition of the biopolymers took place represented by an exothermic peak at close 2400C similar to the decomposition of AG When adding 16 ginsenoside Rb1 into AC82L10R0 film, the melting temperature of the AC82L10Rx composite films was fixed but their melting enthalpy had a great change The decrease in the melting enthalpy as increasing the ginsenoside Rb1 content in the composite films can confirm the reduction in the relative crystal degree of the composite films It can affect on the drug release from AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composite films as discussed below Table 3.3 Thermal parameters obtained from DSC diagrams of AG, CS, LOV, ginsenoside Rb1 and AG/CS/LOV/ginsenoside Rb1 composite films Exothermic Endothermic peak DSC peak (oC) Melting Enthalpy Sample temperature melting (oC) (J/g) AG 119.7 358.6 240 - 2600C CS 106.8 130.6 LOV 174.6 Ginsenoside Rb1 98.9 186.7 134.1 444.6 AC82L10 240.6 181.6 133.7 401.6 AC82L10R1 241.4 178.8 130.1 415.8 AC82L10R3 239.7 180.7 133.5 383.1 AC82L10R5 243.9 178.4 AC82R5 131.5 520.7 241.6 AC82L5R5 120.9 293.2 241.4 136.1 371.1 AC82L15R5 244.7 179.4 1334.0 444.8 AC82L20R5 241.4 181.2 3.3.2 Characteristics and properties of AG/CS/LOV/ginsenoside Rb1 composite particles 3.3.2.1 FTIR spectra of AG/CS/LOV/ginsenoside Rb1 nanoparticles FTIR spectra of AG/CS/LOV/ginsenoside Rb1 nanocomposites using sodium tripolyphosphate (STPP) as a cross-linking agent were indicated in Figure 3.15 17 Figure 3.15 FTIR spectra of AC11L10Rx composite particles When changing the content of ginsenoside Rb1 in AG/CS/LOV/ginsenoside Rb1 composite particles (AC11L10Rx), the positions of wave number corresponding to the characteristic peaks for functional groups in AC11L10Rx composite particles was similar Thus, the content of ginsenoside Rb1 did not affect on the interaction between the components in AC11L10Rx composite particles 3.3.2.2 Morphology of AG/CS/LOV/Rb1 composite particles The FESEM image of AC11L10R0 composite particles (ratio of AG/CS was fixed at 1/1 (wt.%/wt.%), the content of LOV was 10 wt %) at magnifications of 10,000 times and 30,000 times was represented in Figure 3.16 Observing SEM images, it can be seen that the AC11L10R0 composite particles had an uneven structure, the LOV bars and LOV particles dispersed in AG/CS polymer blend with a size of about 1.5 μm had a tendency to agglomeration to form bigger – size particles Figure 3.16 FESEM image of Figure 3.17 FESEM image of AC11L10 sample AC11L10R1 sample The dispersion ability of LOV in AG/CS polymer blend was improved when ginsenoside Rb1 was added to the AC11L10R0 nanoparticles Observing the SEM image of the AC11L10Rx nanoparticles (Figures 3.17 - 3.19), it is clear that the 18 composites particles had tended to be separated when using a small amount of wt.% ginsenoside Rb1 However, the LOV bars had not been completely broken in the AG/CS polymer blend Figure 3.18 FESEM image of Figure 3.19 FESEM image of AC11L10R3 sample AC11L10R5 sample Observing the FESEM image of AC11L10R3 nanoparticles (Figure 3.18), it can be seen the nanoparticles were formed quite uniformly and separated with a size of about 100 - 300 nm By increasing the content of ginsenoside Rb1 to wt.%, the particle size of the nanoparticles were greatly reduced, even reached to several tens of nanometers (Figure 3.19) However, LOV bars and particles tended to coalesce forming larger blocks, about 200 nm - μm in AG/CS polymer blend This can be explained by the increase in the content of ginsenoside Rb1 which can increase the internal molecular linkage between the ginsenoside Rb1 molecules, leading to the agglomeration of ginsenoside Rb1 particles As a result, the AC11L10Rx nanoparticles having ginsenoside content Rb1, x > wt.%) were obtained with uneven structures 3.3.2.3 Size distribution of AG/CS/LOV/ginsenoside Rb1 composite particles Table 3.4 performed particle size, peak width of the AC11L10 composite particles with different content of ginsenoside Rb1 Table 3.4 Average particle size of AC11L10Rx composite particles Peak Average particle Particle size Particle size width size (nm) range (nm) d (nm) % (nm) (r) D = d ± r/2 AC11L10R0 480 – 1053 586.80 24.30 123.70 586.80 ± 61.85 76 – 150 78.20 2.30 9.78 78.20 ± 4.89 AC11L10R1 250 – 800 369.10 22.90 76.92 369.10 ± 38.46 AC11L10R3 95 – 950 328.50 12.70 136.90 328.50 ± 68.45 19 4000 – 6500 5274.00 1.70 424.60 5274.00 ± 212.30 55 – 90 58.40 0.40 10.36 58.40 ± 5.18 AC11L10R5 90 – 1050 333.50 10.80 158.50 333.50 ± 79.25 4500 – 8500 5118.00 2.70 537.00 5118.00 ± 268.50 It was clear that the AC11L10 composite particles without ginsenoside Rb1 had the largest average particle size The remaining samples with different ginsenoside Rb1 content had relatively uniform average particle size (328.5 ± 68.45 nm - 369.1 ± 38.46 nm) and smaller than the AC11L10 composite particles The AC11L10R5 nanoparticles had average particle sizes smaller than the other two samples This can be due to ginsenoside Rb1 acting as a size stabilizer for AG/CS/LOV nanoparticles In the presence of ginsenoside Rb1, thanks to the interaction of ginsenoside Rb1 with AG, CS and LOV, the dispersion ability of LOV bars into AG/CS polymer blend was improved 3.3.2.4 Thermal bahavior analysis of AG/CS/LOV/ginsenoside Rb1 nanoparticles DSC diagrams of the AG/CS/LOV/ginsenoside Rb1 composite particles using sodium tripolyphosphate (STPP) as a crosslinking agent were demonstrated in Figure 3.20 Figure 3.20 DSC diagram of AC11L10Rx composite particles Observing the DSC diagram of the composite particle AC11L10R0, it can be seen that peaks were corresponding to phase transition processes of the composite particles Endothermic and exothermic peaks of the composite particles reflected the melting and decomposition processes of the components in the composite particle to form peaks larger than those corresponding peak on the DSC diagrams of original AG, CS, and LOV 3.3.2.5 Drug carrying efficiency of AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composite particles LOV and ginsenoside Rb1 carrying efficiency of the composite particles depends on AG/CS ratio, LOV content and ginsenoside Rb1 content, method of preparing composite particles Tables 3.5 and 3.6 20 presented the LOV and ginsenoside Rb1 carrying efficiency of AC11L10Rx composite particles The LOV and ginsenoside Rb1 carrying efficiency was determined by the ultraviolet-visible spectroscopy method using the maximum wavelengths corresponding to LOV and ginsenoside Rb1 The obtained results showed that the AC11L10R3 composite particles had the highest both LOV and ginsenoside Rb1 carrying capacity Table 3.5 LOV carrying efficiency of AC11L10Rx composite particles Optical LOV carrying LOV amount Sample density efficiency (%) (g) AC11L10R0 0.57 62.81 0.0126 AC11L10R1 0.58 61.38 0.0123 AC11L10R3 0.63 77.69 0.0155 AC11L10R5 0.59 70.64 0.0141 Table 3.6 Ginsenoside Rb1 carrying efficiency of AC11L10Rx nanoparticles Ginsenoside Rb1 Optical Ginsenoside Rb1 Sample carrying density amount(g) efficiency (%) AC11L10R1 0.0039 71.22 0.00142 AC11L10R3 0.0074 76.80 0.00461 AC11L10R5 0.0103 73.31 0.00733 3.4 In vitro drug release and kinetic studies from AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composites in different pH buffer solutions 3.4.1 Study on release of LOV and ginsenoside Rb1 from AG/CS/LOV composite films and AG/CS/LOV/ginsenoside Rb1 Figure 3.21 and 3.22 displayed the content of LOV released from the AC82Lx composite films with the various content of LOV from 10 to 30 wt.% in pH and pH 7.4 buffer solutions Figure 3.21 Content of LOV released from AC82Lx composite films with various initial LOV content in pH 2.0 solution Figure 3.22 Content of LOV released from AC82Lx composite films with various initial LOV content in pH 7.4 solution 21 It can be seen that the LOV content significantly influenced on content of LOV released from the composite films The content of LOV released from AC82Lx nanocomposite films was decreased with rising initial content of LOV in the nanocomposite films at the same pH solution and testing time In the pH 2.0 and pH 7.4 solutions, process of LOV release from the AC82Lx nanocomposite films had two stages: rapid release stage at the first 12 hours and then the slow release (controlled) stage This results were similar to the release of LOV and ginsenoside Rb1 from AG/CS/LOV/ginsenoside Rb1 composite films Figure 3.23 Content of ginsenoside Rb1 released from AC82L10Rx nanocomposite films in pH solution Figure 3.24 Content of ginsenoside Rb1 released from AC82L10Rx nanocomposite films in pH 7.4 solution 3.4.2 Kinetics of LOV and Rb1 release from AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composite films Figure 3.25 presented the dynamic lines for LOV releasing from AG/CS/LOV nanocomposite films containing wt.% PEO as compatibilizer in pH 2.0 solution according to fast and slow release stages The Kosmeyer - Peppas (KMP) dynamic model had the highest regression coefficient which was always higher 0.9 for all of composite films Observing the n values in the equation, it was clearly regcognize that the slow release process of both of LOV and Rb1 was non-Fickian transport (n < 0.45) while the slow release process of LOV and ginsenoside Rb1 follow Fickian diffusion in both of the acid and base enviroment 22 Figure 3.25 Kinetic models of LOV released from AG/CS/LOV nanocomposite films containing wt.% PEO in pH 2.0 solution 3.4.3 Study on release of LOV and ginsenoside Rb1 from AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composite particles Figures 3.26 and 3.27 displayed the content of LOV released from the AC6.5/3-Lx composite particles with the various content of LOV from 10 to 30 wt.% in pH and pH 7.4 solutions It is clearly that the process of releasing LOV from AG/CS/LOV nanoparticles also included stages: fast release and slow release The content of LOV released from AC6.5/3-Lx composite particles was decreased with rising initial content of LOV in the composite particles at the same pH solution and testing time 23 Figure 3.26 Content of LOV Figure 3.27 Content of LOV released from AG/CS/LOV released from AG/CS/LOV composite particles with various composite particles with various initial LOV content in pH 2.0 initial LOV content in pH 7.4 solution solution 3.4.4 Kinetics of LOV and Rb1 release from AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 nanoparticles The kinetics of LOV release process from AC6.5/3-L10 composite particles in different pH solutions (7.4, 6.5, 4.5 and 2.0) were statistically calculated in table 3.7 It is clear that the KMP dynamic model had the highest regression coefficient which were always higher 0.9 for all of samples The slow release process of both of LOV and Rb1 was non-Fickian transport (n < 0.45) while the slow release process of LOV and ginsenoside Rb1 follow Fickian diffusion in both of the acid and base environment Table 3.7 Regression coefficient (R2) of kinetic equations reflects LOV released from AC6.5/3-L10 composite particles in different pH solutions LOV quick release stage pH of solution ZO FO HG HCW KMP pH = 7.4 0.99 0.96 0.98 0.95 0.99 pH = 6.5 0.96 0.98 0.94 0.96 0.99 pH = 4.5 0.97 0.99 0.91 0.90 0.99 pH = 2.0 0.98 0.96 0.93 0.92 0.99 LOV slow release stage pH of solution ZO FO HG HCW KMP pH = 7.4 0.94 0.90 0.99 0.99 0.97 pH = 6.5 0.99 0.92 0.92 0.93 0.98 pH = 4.5 0.95 0.97 0.97 0.93 0.95 pH = 2.0 0.96 0.97 0.97 0.91 0.98 3.5 Study on toxicity of AG/CS/LOV nanoparticles on rat Table 3.8 - 3.11 displayed mean body weight, hematological and biochemical parameters, rats’ organs mean weights of rats treated by AG/CS/LOV nanoparticles with two doses of 100, 300 mg/kg body and 24 figure 3.28 indicated histological changes in kidneys of rats after 28 treatment days Table 3.8 Mean body weights of rats after 28 days treated by AG/CS/LOV nanoparticles Group using AG/CS/LOV nanoparticles (Mean ±SEM) (g, ±SEM) Survey Bodyweight Dose of week (g, ±SEM) Dose of 300mg/kg 100mg/kg body body weight weight 185.16 ± 13.68 176.00 ± 14.20 198.33 ± 13.78 187.16 ± 13.78 176.66 ± 14.30 199.66 ± 13.77 195.00 ± 15.66 188.83 ± 16.26 206.50 ± 15.89 198.33 ± 16.53 196.66 ± 17.12 213.83 ± 16.65 205.16 ± 17.73 205.00 ± 18.32 223.00 ± 17.74 Table 3.9 Hematological parameters of rats after 28 days treated by AG/CS/LOV nanoparticles Parameter WBC (103/µl) RBC (106/µl) Hemoglobin (g/dL) Red cell mass (%) MCV (fl) MCH (pg) MCHC (%) Platelets (103/µl) Mean ± SEM 8.01 ± 0.86 6.66 ± 0.19 11.88 ± 0.29 36.48 ± 0.98 54.02 ± 0.88 17.88 ± 0.19 32.66 ± 0.28 481.80 ± 46.49 Group using AG/CS/LOV nanoparticles (Mean ±SEM) Dose of 100mg/kg Dose of 300mg/kg body weight body weight 8.24 ± 0.92 8.19 ± 0.87 6.83 ± 0.13 6.19 ± 0.31 12.09 ± 0.28 10.88 ± 0.47 36.68 ± 1.04 33.08 ± 1.35 53.27 ± 0.89 53.67 ± 0.69 17.55 ± 0.24 17.67 ± 0.18 33.05 ± 0.30 32.94 ± 0.25 368.90 ± 65.18 468.90 ± 68.12 Table 3.10 Biochemical parameters of rats after 28 days treated by AG/CS/LOV nanoparticles Group using AG/CS/LOV nanoparticles (Average ±SEM) Average ± Index Dose of Dose of SEM 100mg/kg body 300mg/kg body weight weight AST (U/L) 153.31 ± 11.92 198.03 ± 46.84 158.10 ± 19.82 ALT (U/L) 38.22 ± 3.85 53.95 ± 12.63 45.94 ± 4.71 Creatinine (mg/dL) 65.42 ± 2.77 71.16 ± 1.91 60.31 ± 4.63 Concentration of red 4.65 ± 0.39 4.16 ± 0.23 4.98 ± 0.39 blood cells (%) 25 Table 3.11 Mean weights of rats’ organs after 28 days treated by AG/CS/LOV nanoparticles Group using AG/CS/LOV nanoparticles (g, ±SEM) Weight Organ (g, ±SEM) Dose of 100mg/kg Dose of 300mg/kg body weight body weight Liver 6.43 ± 0.28 6.32 ± 0.44 6.53 ± 0.58 Kidney 1.29 ± 0.09 1.25 ± 0.06 1.40 ± 0.12 Spleen 0.33 ± 0.02 0.34 ± 0.02 0.35 ± 0.03 Figure 3.28 Histological changes in kidneys of rats after 28 days treated by AG/CS/LOV nanoparticles The obtained showed the above values of rats treated by AG/CS/LOV nanoparticles were normal in comparison of rats no-treated by AG/CS/LOV nanoparticles (control group, only treated by saline solution) The rat’s liver and kidney images were also normal and there was no significant change in liver and kidney structure of the rat groups treated by AG/CS/LOV nanoparticles Thus, the AG/CS/LOV nanoparticles is safe or non-toxic and might be applied to lower serum cholesterol in animal models as well as humans CONCLUSIONS Alginate/chitosan (AG/CS) polymer blends carrying lovastatin (LOV) and ginsenoside Rb1 were successfully prepared in film and paricle forms having micrometer and nanometer sizes The results of analysis of Fourier transform infrared spectroscopy (FTIR) spectra, thermal behavior of AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composites showed that AG, CS, LOV, ginsenoside Rb1 and compabilizers (polycaprolactone, polyethylene oxide) and sodium tripolyphosphate (STPP) cross-linked interacted through hydrogen bonds and bipolar-bipolar interactions (between drug functional groups of drugs, drugs/polymers and polymerpolymer) The average particle size of AG/CS/LOV and AG/CS/10% 26 LOV/1-5% ginsenoside Rb1 particles (with AG/CS ratio of 1/1) were 340 ± 23.5 nm, 328.5 ± 68.45 nm and 369.1 ± 38.46 nm The AC11L10Rx composite particles (ratio of AG/CS fixed at 1/1 (wt.%/wt.%), content of LOV of 10 wt % and the content of ginsenoside Rb1 changed from to wt.% had LOV and ginsenoside Rb1 carring efficiency were 62.71 - 70.64% and 71.22 - 73.31%, respectively The process of LOV and ginsenoside Rb1 release from AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 composite materials in various pH solutions includes stages: rapid release stage and slow release stage as controlled The KMP dynamic models had the highest regression coefficient which were always higher 0.9 for two stages of LOV and ginsenoside Rb1 release from AG/CS/LOV and AG/CS/LOV/ginsenoside Rb1 nanoparticles The rapid release process of both of LOV and ginsenoside Rb1 was non-Fickian transport (n > 0.5) while the slow release process of LOV and ginsenoside Rb1 (n < 0.5) followed Fickian diffusion in both of the acid and base environment The mean body weight, hematological and biochemical parameters, rats’ organs mean weights of rats treated by AG/CS/LOV nanoparticles with two doses of 100, 300 mg/kg body were normal in comparison of rats no-treated by AG/CS/LOV nanoparticles (control group) The rat’s liver and kidney images were also normal and there was no significant change in liver and kidney structure of the rat group treated by AG/CS/LOV nanoparticles Thus, the AG/CS/LOV nanoparticles is safe or non-toxic and might be applied to lower serum cholesterol in animal models as well as humans 27 PUBLISHED WORKS Nguyen Thuy Chinh, Nguyen Thi Hien Ly, Tran Thi Mai, Nguyen Thi Thu Trang, Thach Thi Loc, Le Duc Giang, Nguyen Quang Tung, Thai Hoang Characteristics and properties of chitosan/alginate polymer blend carrying lovastatin drug, Vietnam Journal of Science and Technology, Vol 54 (2B), 118-124 (2016) (ACI) Thach Thi Loc, Thai Hoang, Nguyen Thuy Chinh, Le Duc Giang, Study on the effect of some compatible substances on the ability to release lovastatin from alginate/chitosan/lovastatin conjugate composite films, Journal of Chemistry, Vol 56, No 3, 389-395 (2018) Nguyen Thuy Chinh, Thach Thi Loc, Le Duc Giang, Nguyen Thi Thu Trang, Tran Thi Mai, Thai Hoang, Effect of polyethylene oxide on properties of chitosan/alginate/lovastatin composites, Vietnam Journal of Science and Technology, Vol 56 (2A), 156-162 (2018) (ACI) Nguyen Thuy Chinh, Thach Thi Loc, Le Duc Giang, Ngo Phuong Thuy, Vu Thi Hien, Thai Hoang, Effect of polycaprolactone on characteristics and drug release of alginate/chitosan/lovastatin composite films, Vietnam Journal of Science and Technology, Vol 56 (4A), 13-21 (2018) (ACI) Thai Hoang, Kavitha Ramadass, Thach Thi Loc, Tran Thi Mai, Le Duc Giang, Vu Viet Thang, Tran Minh Tuan, Nguyen Thuy Chinh, Novel Drug Delivery System Based on Ginsenoside Rb1 Loaded to Chitosan/Alginate Nanocomposite Films, Journal of Nanoscience and Nanotechnology, Vol 19, 3293-3300 (2019) (ISI) Thai Hoang, Tran Dai Lam, Thach Thi Loc, Le Duc Giang, Tran Do Mai Trang, Vu Quoc Trung, Nguyen Tuan Anh, Nguyen Duy Trinh, Nguyen Thuy Chinh, Effect of Both Lovastatin and Ginsenoside Rb1 on Some Properties and In-Vitro Drug Release of Alginate/Chitosan/Lovastatin/Ginsenoside Rb1 Composite Films, Journal of Polymers and the Environment, Vol 27, 2728-2738 (2019) (ISI) Thach Thi Loc, Nguyen Thuy Chinh, Vu Thi Diu, Le Duc Giang, Ha Van Hang, Thai Hoang, Effect of calcium chloride concentration on characteristics and drug release of alginate/chitosan/ginsenoside Rb1/lovastatin composite particles, Vietnam Journal of Chemistry, Vol 57 (6E1,2), 347 – 353 (2019) (ACI) Hoang Thai, Chinh Thuy Nguyen, Loc Thi Thach, Mai Thi Tran, Huynh Duc Mai, Trang Thi Thu Nguyen, Giang Duc Le, Mao Van Can, Lam Dai Tran, Giang Long Bach, Kavitha Ramadass, C I Sathish, Quan Van Le, Characterization of chitosan/alginate/lovastatin 28 nanoparticles and investigation of their toxic effects in vitro and in vivo, Scientific Reports 10(1):909 (2020) (ISI) Thi Loc Thach, Thuy Chinh Nguyen, An Quan Vo, Minh Thanh Do, Quang Tung Nguyen, Anh Nguyen, Long Giang Bach, and Hoang Thai, Assessment of the Role of Ginsenoside RB1 Active Substance in Alginate/Chitosan/Lovastatin Composite Films, International Journal of Polymer Science (2020), ID 5807974, https://doi.org/10.1155/2020/5807974 (ISI) ... Trinh, Nguyen Thuy Chinh, Effect of Both Lovastatin and Ginsenoside Rb1 on Some Properties and In- Vitro Drug Release of Alginate/Chitosan/ Lovastatin/ Ginsenoside Rb1 Composite Films, Journal of. .. Characteristics and properties of alginate/chitosan/ lovastatin/ ginsenoside Rb1 (AG/CS/LOV/ginsenoside Rb1) composite materials 3.3.1 Characteristics and properties of AG/CS/LOV/ginsenoside Rb1 composite. .. topic: ? ?Researches about fabrication, characterization, properties of alginate/chitosan polymer composite with ginsenoside Rb1 and lovastatin? ?? Subjects for study The AG/CS composites which contain