THE ANTIATHEROGENIC EFFECT OF NEW BIOCOMPATIBLE
CATIONICALLY MODIFIED POLYSACCHARIDES: CHITOSAN
AND PULLULAN - THE COMPARISON STUDY

Chitosan and pullulan are biocompatible polysaccharides obtained from natural sources. They have many biomedical applications (1, 2). Particularly noteworthy are the numerous applications of chitosan in the pharmaceutical industry, in fields such as binder in wet granulation, diluents in direct compression of tablets (27), slow-release of drugs from granules and tablets, films controlling drug release, drug carrier in micro parcticle systems, preparation of hydrogels, agent for increasing viscosity in solutions, disintegrant, wetting agent, improvement of dissolution of poorly soluble drug substances, bioadhesive polimer, absorption enhancer, site-specific drug delivery, biodegradable polimer (microparticles, implants), carrier in relation to vaccine delivery or gene therapy (27). The simplest quaternarized derivative of chitosan is the trimethylammonium salt of chitosan that is water soluble at neutral pH (27, 28). As has been previously reported, pullulan and chitosan cationically modified by covalent attachment of glycidyltrimethylammonium chloride (GTMAC), had antiatherogenic properties and influenced lipid metabolism (1, 2). It seems that these polysaccharides act on the hematopoietic system like other polysaccharide of natural origin, dextran. It has been reported that dextran infusions caused a reduction of low density lipoproteins (LDL) in the plasma compartment in humans (29). I was shown that up to 40% of the LDL fraction was eliminated from the plasma compartment under the influence of dextran (29, 30-32). It was supposed that this phenomenon could be due to an interaction between dextrans and LDL particles. By laser light scattering it was show that dextrans particles interact with LDL (29). It was postulated that dextran-dependent formation of LDL associates detected in in vitro studies could be responsible for in vivo effect of dextran on the lipid metabolism.

It was reported that cationically-modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (chitosan-GTMAC, HTCC), exhibits some unique beneficial effects that have not been found in the parent polymer (1). What is more, pullulan after substitution with GTMAC (Pull-GTMAC) also showed antiatherosclerotic effects (2). Pull-GTMAC after oral administration to female apoE-knockout mice at a dose of 300 mg/kg b.w./day for 18 weeks reduced the area of atherosclerotic plaque. At the same dose, cationically modified pullulan also had influence on lipid metabolism and decreased raw fat content in the feces of the apoE-knockout mice (2). In this article, we have taken an attempt to evaluate and compare antiatherogenic properties of cationically modified pullulan and chitosan after cationic modification. Furthermore, we have described specific acting of these polymers on lipid metabolism in the model of atherosclerosis in vivo and in vitro on cell line.

Cationic modification of chitosan with different degree of substitution (DS) can be performed by reaction of chitosan molecule with GTMAC (1, 3). Native chitosan also has antiatherosclerotic properties and influences lipid metabolism, but it acts in higher dose than HTCC because of the lower biocompatibility of unmodified polymer resulting from its poor solubility at physiological pH (1, 4). What is interesting, it have been proven that unmodified chitosan administered orally with feed (at content of 5% of feed mass) to apoE-knockout mice for 20 weeks significantly decreased cholesterol level by about 36% compared to the control group of apoE(–/–) mice (i.e. fed without chitosan in a diet) (1, 4). What is more, studies of aortic plaque areas have shown that this polymer also significantly decreased atherogenesis process in whole aorta (by about 42% compared to the control group) (1, 4).

HTCC polymer with a substitution degree of 63.6% shows very good solubility in water at physiological pH (i.e, pH value of 7.4 corresponding to that of blood) (1, 3). HTCC (Mw = 205,000 Da) complexed and neutralized both low-molecular-weight heparin (LMWH) and unfractionated heparin (UFH) (1, 3). Heparin, an anionic anticoagulant, can be removed from body fluids using pH-sensitive genipin crosslinked chitosan microspheres (1, 5). Other beneficial effects of cationically modified chitosan are similar to those of unmodified chitosan such as inhibition of proliferation of cancer cells (1, 6), antibacterial (1, 7) and antifungal properties (1, 8), adsorption enhancement of hydrophilic drugs through mucous membranes (1, 9-11), and improvement of proliferation of human periodontal ligament cells (HPDLC) (1, 12).

Pullulan (Mw = 200 kDa) after cationic modification exhibits several unique properties that are not observed in the native polymer (2). Although, Pull-GTMAC had no influence on lipid profile in serum of female apoE-knockout mice after feeding with a feed containing cationically modified pullulan in a dose of 300 mg/kg b.w./day for 18 weeks, this polysaccharide had statistically significant impact on the area of atherosclerotic plaque (2). Both Pull-GTMAC and unmodified chitosan inhibit the atherosclerotic plaque development after oral administration of the polymers in the rodent model of atherosclerosis (2, 1). Pullulan-GTMAC reduced the area of atherosclerotic plaque by 13% in comparison to control group of apoE(–/–) mice (2). This effect might be due to the influence of Pull-GTMAC on the expression of the genes responsible for lipid metabolism (like in the case of HTCC), because it was shown that pull-GTMAC caused statistically significant increase of expression of LDL receptor in the liver of apoE(–/–) mice after treatment with cationically modified pullulan for 18 weeks in a dose of 300 mg/kg b.w./day (2). In comparison to HTCC, Pull-GTMAC additionally showed an anticancer potential in in vitro studies on HepG2 cell-line (2). An anti-proliferative activity of cationically modified pullulan was observed after 24-hour and 48-hour incubation of the cells with the polymer (2). In this paper we would like to focus on comparing the intensity of anti-atherosclerotic effect of cationically modified chitosan and cationically modified pullulan in the same and different doses after oral administration to apoE-knockout mice and show differences and similarities in the action mechanism of these polymers.

MATERIALS AND METHODS

Materials

Low molecular weight chitosan, LMWC, 75 – 85% deacetylated (Aldrich); glacial acetic acid, p.a., min. 99.5% (Chempur); glycidyltrimethylammonium chloride technical, ≥ 90%, GTMAC (Aldrich); acetone, p.a., (Chempur); Pullulan from Aureobasidium pullulans, Mw = 200 kDa (Sigma); sodium hydroxide G.R., NaOH (Lach:ner); RPMI Medium 1640 (1×) (Gibco); Fetal Bovine Serum (FBS) (Gibco); enoxaparin Clexane 40 mg/0.4 ml, Enoxaparinum natricum solution for injection (Sanofi Aventis); n-hexane, p. a. (Chempur, Poland), hexane Chromasol V®, for HPLC, ≥ 97.0% (GC) (N-Hexane) (Sigma-Aldrich, Germany); anhydrous sodium sulfate, Na2SO4, ≥ 99.0% (Sigma-Aldrich) dried at 100°C; acetone, p.a. (POCH, Poland); acetone p.a., CH3COCH3 (Chempur); phosphate buffered saline, 10 × concentrate, BioPerformance Certified (PBS at pH 7.4, concentrate dissolved 10 × in distilled water for using in experiments) (Sigma); Kilik cryostat embedding medium (Bio-Optica); glass microscope slides SuperFrost® (Menzel Glaser); poly-L-lysine solution 0.1% w/v in water; thimerosal 0.01%, added as preservative (Sigma-Aldrich); Oil Red O (Sigma); Rneasy® Fibrous Tissue Mini Kit (Qiagen); Rneasy® Lipid Tissue Mini Kit (Qiagen); RT² First Strand Kit (Qiagen); RT² qPCR SYBR Green/ROX MasterMix (Qiagen); PAMM080ZC: Mouse Lipoprotein Signaling and Cholesterol Metabolism RT² Profiler™ PCR Array (Qiagen); RevertAidTM Reverse Transcriptase (Fermentas); 10 mM dNTPs Ultrapure Mix (EURx); 5 × Reaction Buffer for M-MulVRT (Fermentas); RiboLockTM RNase Inhibitor (Fermentas); PPM24937F-200: RT² qPCR Primer Assay for Mouse Ldlr (Qiagen); PPM02946E-200: RT² qPCR Primer Assay for Mouse Gapdh (Qiagen); SybrGreen Jump StartTMTaq MixTM Ready for quantitative PCR, MgCl2 in buffer (Sigma); Water Biotechnology Performance Certified (Sigma-Aldrich), Qiazol, lysis reagent kit (Qiagen), DMEM medium (1 ×) (Gibco), Chloroform (99%, Sigma), isopropanol (99%, Sigma), RNase-free water (Sigma), human 3-hydroxy-3-methylglutaryl-CoA (human HMG-CoA) reductase sense and antisense primer (Oligo.pl), human glyceraldehyde 3-phosphate dehydrogenase sense and antisense primer (Sigma), Micro Amp Fast optical 96-well reaction plate with barcode, with a capacity of 0.1 ml well (Applied Biosystems), Micro Amp Optical adhesive film PCR compatible (Applied Biosystems).

Experimental rodent model of atherosclerosis (characterization and treatment)

All animal procedures were approved by the Local Ethics Committee for the Animal Experiments at Jagiellonian University.

In one of the experiments 12 homozygous male apoE-knockout mice were used (APOE-M B6.129P2-Apoetm1Unc N11, Taconic, Denmark) with the genetic background of C57BL/6J mice. The males were divided randomly into two groups: a control group in size of six mice (n = 6), not receiving the tested compound in the diet, and a research group (n = 6) fed with a feed containing cationically modified chitosan at a dose of 200 mg/kg/b.w./day for 16 weeks. In the second experiment 24 homozygous female apoE-knockout mice were used (Taconic, Denmark) with nomenclature: APOE-F B6.129P2-Apoetm1UncN11 homozygous mice at the age of seven to eight weeks, with the genetic background of C57BL/6J mice. Females were divided randomly into two groups: a control group with size of 14 mice (n = 14), not receiving the tested compound in the diet, and a research group of 10 mice (n = 10) fed with a diet containing HTCC at a dose of 300 mg/kg b.w./day, which has been given the tested compound for 18 weeks. In the third experiment also apolipoprotein E-deficient mice were used in number of 10 female B6.129P2-Apoetm1Unc/J homozygous mice (the Jackson Laboratory, USA) with a mixed C57BL/6 × 129 genetic background, at the age of eight weeks. The individuals were also divided into two groups: a research group of 5 mice (n = 5) fed with a diet containing Pull-GTMAC at a dose of 500 mg/kg b.w./day, which has been given for 18 weeks and a control group with size of 5 mice (n = 5), not receiving the tested polymer in the feed. The mice from all experiments were quarantined and fed with a Labofeed H fodder- using a diet called chow diet (containing 5% fat; ‘Morawski’ Works of Feed, Poland) mixed with cationically modified polysaccharides (chitosan or pullulan) for 16 – 18 weeks in the Animal House of the Department of Immunology at Faculty of Medicine at Jagiellonian University Medical College in the daily cycle of light and darkness (the length of the light corresponding to the natural day length in spring and summer) except the third experiment in which the animals were exposed to 1:1-h light-dark cycle (12 h of light and 12 h of darkness), in the conditions of temperature range from 21 – 22°C and humidity of 60%. All the animals were kept in ad libitum access to food and water.

Synthesis of cationically modified polysaccharides (HTCC, Pull-GTMAC)

HTCC was synthesized according the established procedure (3) with some modifications. Reaction of substitution of pullulan with glycidyltrimethylammonium chloride, that led to obtaining the cationically modified pullulan (Pull-GTMAC), was performed using the procedure described in the previous study (2).

Procedure of synthesis of N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC)

Thirty grams of chitosan (low molecular weight chitosan, Aldrich) was dispersed in 1200 ml of distilled water with constant stirring using magnetic stirrer. Then, 130 ml of 0.5% acetic acid was added and the mixture was stirred for 30 minutes. Next, 82.8 ml of 90% glycidyltrimethylammonium chloride (GTMAC) was added and the reaction mixture was heated for 18 h at 55°C with constant stirring. Then, the suspension was centrifuged at 9000 rpm (revolutions per minute) for 5 min at 23°C (Centrifuge MPW-351R, rotor no.: REF 11457) in order to remove suspended particles of chitosan. Product of the reaction was isolated from supernatant by precipitation with acetone. The excess of acetone was evaporated using a vacuum evaporator. Next, the precipitate was dissolved in distilled water. Then, the obtained solution was centrifuged again at 4000 rpm for 10 min and the polymer dissolved in supernatant was precipitated with new portion of acetone. The described procedure of dissolution and precipitation was repeated twice. The reaction product obtained in the last cycle of precipitation was dried in a vacuum oven for 24 hours and lyophilized under vacuum.

Synthesis of Pull-GTMAC

Nine grams of pullulan (Mw = 200 kDa) has been dissolved in 450 ml of distilled water with constant stirring using magnetic stirrer (2). Next, 130 ml of GTMAC and water mixture was added (the mixture contained 50 ml of distilled water and 80 ml of GTMAC). When the mixture of the polymer and water was mixed in a 1000 ml round-bottomed flask, 20 ml of 5 M NaOH was added. Than, the reaction mixture was heated for 14 h at 80°C with stirring on magnetic stirrer. Next, the mixture was cooled and dialyzed, the obtained solution was concentrated using rotary evaporator and freeze-dried.

Chemical analysis of the cationically modified polysaccharides

1. Chemical characterization of HTCC

HTCC (205 000 Da) was received with substitution degree (DS) equal to 63.6% and with very good solubility in water at neutral pH (pH = 7.4) (3). The zeta potential (ς potential) of 1 mg/ml HTCC solution in PBS buffer (at pH = 7.4) was measured in folded capillary cells using Zetasizer Nano-ZS (Malvern Instruments) at 25°C. In order to confirm the structure of the HTCC, the FT-IR (Fourier Transform infrared) spectra of parent chitosan and its cationic derivative were recorded using a Bruker IFS 48 526 spectrometer.

2. Chemical characterization of Pull-GTMAC chloride

In the FT-IR spectrum of Pull-GTMAC the band at a wavenumber of 1470 cm–1, characteristic for methyl groups of the quaternary amine, was shown, that has not been observed in the native polysaccharide (2). This band determined the cationic modification. The elemental analysis showed that the modified pullulan contained 3% of nitrogen and this element has not been present in unmodified pullulan (2). It was noticed that the corresponding degree of substitution of Pull-GTMAC was equal 71.25% (2). The zeta potential of Pull-GTMAC was measured at pH 7.4 and was equal +15.2 mV (2).

Plasma lipid analysis of apoE-knockout mice treated with the cationically modified polysaccharide. Influence of HTCC on plasma lipid profile

The analysis of plasma lipid profile was performed in male apoE-knockout mice fed for 16 weeks with a feed containing HTCC at a dose of 200 mg/kg/b.w./day (6 animals in the group). The control plasma samples were derived from the apoE(–/–) mice fed with a feed containing no cationically modified polysaccharide (6 mice).

Levels of various lipid fractions (triglycerides, total cholesterol, LDL, HDL) were determined for group of apoE-knockout mice fed with a feed containing cationically modified chitosan and for the control group. The results of marking of lipid faction concentrations were expressed in mmol/l of plasma and presented as a mean value ± S.E.M. for all individuals in a group. The plasma samples volume for measurements was estimated at 210 µl. The survey was performed in the liquid phase of the plasma using automatic biochemical analyzers. HDL cholesterol measurement by the direct enzymatic- colorimetric method by Abell-Kendall was conducted on Hitachi 917 and Modular P analysers, for total cholesterol- the CHOD-PAP method of analysis on Hitachi 917 and Modular P analyzers, for triglycerides - the GPO-PAP method on Hitachi 917 and Modular P analysers, for cholesterol LDL (DIRECT) - the enzymatic method on Modular P analyser.

Measurements of the area of atherosclerotic plaque under influence of the modified polymers. Description of the cross-section method for groups of mice on diet containing HTCC or Pull-GTMAC

Two experiments (with HTCC and Pull-GTMAC) were performed on female mice. The cross-sections of the aortic root were prepared using the standard protocol (2, 24-26). At the beginning, the series of 10 adjacent cryostat sections were cut using a cryostat (Jung cryostat CM 1800, Leica) every 100 µm from the proximal 1 mm long segment of aortic root at the temperature of

–20°C. Sections collection was started at starting point that was located in 100-µm distance from place where the aortic valves appeared (using microscope to inspect the frozen scraps) and the sections were gathered at 100-µm intervals. The cut sections were collected on glass microscope slides (special for cryostat) covered with 10 µl solution of poly-L-lysine per 1 glass and air dried. Next, all the sections were fixed and stained with oil red O dye (ORO), that is used in staining of lipoproteins, triglycerides and fats. The fixation of the frozen sections was based on incubation in buffered 4% formalin for 10 min (formaldehyde solution in phosphate buffer at pH = 7.4) and washing twice for 5 minutes in distilled water. The microscope slides with fixed sections stained with ORO were covered with cover slips in gelatin-glycerol mixture (glycerogelatin). Then, microscopic pictures of cross-sections of the aortic root were taken using Camedia 5050 digital camera (Olympus) and BX50 microscope (Olympus) with 4 times magnifying lens (in the experiment with HTCC total magnification amounted to 40 times). In the experiment with Pull-GTMAC the conditions were following: the pictures were taken using Axioskop 20 microscope (Zeiss), AxioCam ICc 5 digital camera (Zeiss) and the 2.5 times magnifying lens (under total magnification of 25 times). The collected pictures of preparations after treatment with HTCC were stored as .jpeg files with resolution of 2040 × 1536 pixels. The microscopic images connected with research with Pull-GTMAC were stored as .jpeg files with resolution of 2452 × 2056 pixels. A morphometric computer image analysis was performed in order to evaluate the areas of the atherosclerotic plaques using a LSM 5 Image Browser computer program (Zeiss). The results obtained for apoE(–/–) mice were presented as the average area of atherosclerotic plaques situated on consecutive 9 sections spaced about 100 µm. The average area of atherosclerotic plaque with SEM for all tested groups of the mice was calculated as the mean value counted from areas measured for all individuals in the group. In one of the experiments carried out on females, 10 mice were used in the method of cross-section of the aorta in the group receiving the feed with HTCC at a dose of 300 mg/kg/b.w./day for 18 weeks and 14 animals were used in the group obtaining no tested compound.

In the second experiment, five mice (n = 5) were used to perform the cross-section method in the group receiving a fodder with Pull-GTMAC at a dose of 500 mg/kg b.w./day for 18 weeks and 5 animals were used in the group receiving no tested substance.

Evaluation of the influence of HTCC on gene expression of lipid metabolism in apoE(–/–) mice tissues

The study of low density lipoprotein receptor gene (LDLR) expression was carried out using real-time PCR method on cDNA material from the liver of female apoE-knockout mice fed with a feed containing HTCC at a dose of 300 mg/kg b.w./day for 18 weeks. Isolation of RNA was performed by column-based method using RNA isolation kit for tissues. After isolation, one thousand nanograms of RNA was dissolved in RNase free water in final volume of 10.7 µl used for a sample prepared to the reaction of reverse transcription. Next, after addition to the sample 0.9 µl of 3 times diluted (in RNase-free water) solution of oligonucleotide primers, denaturation of RNA was carried out at 72°C for 10 min in T3 Thermocycler (Biometra). Then, 8.4 µl of a reaction mixture was added, which contained 2 µl of buffer of triphosphate deoxynucleotides mixture, 0.9 µl of reverse transcriptase in concentration of 200 U/µl, 4 µl of buffer for reverse transcriptase, and 1.5 µl of RNase inhibitor at a concentration of 40 U/µl. The prepared samples were incubated in a thermocycler with the following profile of temperature: annealing (step of hybridization of the primers): 42°C for 2 hours, elongation: 70°C for 10 min. To carry out the real-time PCR reaction, commercial mixtures of antisense and sense primers were used: for murine receptor for low density lipoprotein (mouse LDLR), and for mouse glyceraldehyde-3-phosphate (mouse GAPDH) as a reference gene. Primer assays (main solutions of the primer mixture) were used at a concentration of 10 µM for each reaction mixture prepared for examined genes. Then, cDNA material from each tested sample was 10 times diluted in RNase-free water. Thirteen microliters of reaction mixture was added to the wells of 96-well plate, which contained: 1 µl of a mixture of primers [10 µM], 7.5 µl of a mixture containing the SYBR Green dye, 4.5 µl of RNase-free water. Next, 2 µl of diluted cDNA was added to the wells in 3 replicates for each of the tested genes (LDLR and GAPDH). The reaction plate was transferred to the thermocycler for real-time PCR (7900 HT Fast Real-Time PCR System with 96-Well Fast Block, Applied Biosystems). The following temperature profile was used in the real-time PCR method: Step 1 – 95.0°C for 10 min, Step 2 – (40 cycles) 95.0°C for 30 s, 60.0°C for 1 min (annealing), 72.0°C for 45 s, Step 3 – (dissociation stage): 95.0°C for 15 s, 60.0°C for 15 s, 95.0°C for 15 s. The real-time PCR reaction results were initially analyzed using the Sequence Detection Systems Version 2.4 computer program (Applied Biosystems). Then, analysis of the results was carried out using ΔΔCt method. The number of the animals in groups for the reaction was 3 individuals (n = 3) in both the tested and control group. The results after optimisation for performed real-time PCR reaction were shown as the mean value of an expression change (in relative units) for 3 mice ± SEM.

In vitro incubation of cationically modified polysaccharides with HepG2 cells

The cells of human liver hepatocellular carcinoma cell line (HepG2 cells) were incubated with Pull-GTMAC and HTCC in varied concentrations for 24 hours. The experiments were repeated several times (incubation of cells in 3 – 4 wells of cell culture plates for each concentration of the compound). The tests were performed on the cells of the HepG2 cell line cultured in RPMI cell culture medium with 5% (v/v) of FBS (fetal bovine serum). Starvation of the cells for 24 hours was performed in DMEM cell culture medium with 0.5% FBS (for incubation with Pull-GTMAC) or in RPMI cell culture medium with 0.5% FBS (for experiments with HTCC). Incubation of the cells was performed with HTCC at 10 and 30 µg/ml of cell culture medium for 24 hours. In experiments with Pull-GTMAC the concentrations were 10, 30 and 100 µg/ml of cell culture medium and the incubation time was 24 hours. The solutions with given substance dissolved in NaClaq were administered in a volume of 50 µl per one well in a 6-well plate. In the control sample (relative to the incubation with the cationic polysaccharide) 50 µl of 0.9% NaCl-solution was used per well with HepG2 cells.

Procedure of incubation of HepG2 cells with HTCC and Pull-GTMAC

The HepG2 cells were dropped on 96-well plates (TPP Company, Switzerland) in the number of 1000,000 cells per well containing 2000 µl of RPMI medium with 5% FBS (for experiments with HTCC) or with 2000 µl of DMEM medium with 5% FBS (for incubation with Pull-GTMAC). The tested cells were incubated for 24 hours with cationically modified pullulan or chitosan in an incubator (incubator Heracell 240, Heraeus Instruments) at 37°C under an atmosphere containing 5% CO2. Next, the cells were collected after 24 h in 0.6 ml of Qiazol as a lysis reagent (without pooling of the material) and frozen at –20°C.

Gene expression analysis of lipid metabolism in HepG2 cells after incubation with HTCC and Pull-GTMAC

The procedure of RNA isolation from HepG2 cells by the Chomczynski method with modifications was applied (samples of cell lysate in Qiazol at a final volume of 600 µl in the Eppendorf type of tubes).

The sample was defrosted and then 100 µl of chloroform was added. Next, each sample was shaken twice with chloroform for 30 seconds. The samples were incubated on ice for 20 minutes. The tube was centrifuged for 15 min at 12,000 rpm (centrifuge Biofuge 28RS, Heraeus Sepatech, rotor: HFA 22.2 No # 3042, Heraeus Instruments) at 4°C. After separation the upper phase was transferred to a new chilled Eppendorf tube at a volume of about 200 µl per sample). Then, an equal volume of isopropanol (about 200 µl per sample) was added, the samples were mixed by inversion and precipitated at –20°C for 1 hour. After precipitation each sample was centrifuged for 15 min at 12,000 rpm at 4°C. After decantation of the supernatant, 1 ml of chilled 70% ethanol (EtOH) was added for a sample, the samples were mixed by inversion to detach the pellet and centrifuged for 10 min at 12,000 rpm at 4°C (this step of procedure was performed twice).

RNA pellets were dried airing to remove ethanol remains. The pellets were dissolved in 15 µl of RNase-free water and shaken. The samples were incubated for 10 min at 65°C and gently shaken. Then, the samples were centrifuged with a speed of 0 to 5000 rpm for 5 s (Mini Spin centrifuge, rotor no. F-45-12-11, Eppendorf).

Next, spectrophotometric measurement of RNA quality was performed. RNA level [ng/µl] (based on optical density at the wavelenght (λ) of 260 nm, purity of RNA (measurement of the ratio of the absorbance at λ = 260 nm and 280 nm – carried out on Epoch spectrophotometer, Bio-Tek), and the amount of RNA for reverse transcription reaction were measured (1000 ng per sample).

The reaction of reverse transcription was performed according the same procedure described in the section ‘Evaluation of the influence of HTCC on gene expression of lipid metabolism in apoE(–/–) mice tissues’.

Analysis of HMG-CoA reductase expression in HepG2 cells incubated with modified polysaccharides for 24 hours

The TE buffer at pH = 8.0 was added to the main solutions of primers for human 3-hydroxy-3-methylglutaryl-CoA reductase (human HMG-CoAR) and for human glyceraldehyde 3-phosphate dehydrogenase (human GAPDH) as a reference gene (to obtain solutions with a concentration of 100 µM of both sense and antisense primers). The primers were diluted 10 times in RNase-free water (resulting concentration of 10 µM for each primer to the real-time PCR reaction). Primers for HMG-CoAR were used in the following sequences for sense primer: 5’-CTTGTGTGTCCTTGGTATTAGAGCTT-3’ and antisense primer: 5’-TTATCATCTTGACCCTCTGAGTTACAG-3’ (13). Primers for GAPDH were used in the sequences: for the sense primer: 5’-GCTGGCGCTGAGTACGTCGT-3’, for the antisense primer: 5’-ATGACCTTGGCCAGGGGTGCT-3’.

The primers for HMG-CoA reductase and GAPDH as reference gene were checked using standard polymerase chain reaction (PCR) and the real-time PCR reaction (dilution curves were made for HMG-CoAR and GAPDH primers). The cDNA was diluted 8 times in RNase free water in a separate, sterile Eppendorf tube for each sample. Next, the 13 µl of reaction mixture was added to the wells of a 96-well plate. The mixture contained: 7.5 µl of a mixture containing the SYBR Green dye, 0.75 µl of solution of sense starter, 0.75 µl of solution of antisense starter, and 4 µl of RNase-free water. The mixture was shaken using vortex mixer. Then, two microliters of diluted cDNA was added to the wells in 3 replications for each of the genes (GAPDH and HMG-CoAR). The plate was sealed with foil and briefly centrifuged (centrifuge Labofuge 400R, Heraeus, rotor no: # 8177). Next, the reaction plate was transferred to the thermocycler for real-time PCR (7900 HT Fast Real-Time PCR System with high-speed, 96-well block (96-Well Fast Block) for experiments with HTCC or ViiA™ 7 Real-Time PCR System, Applied Biosystems, for experiments with Pull-GTMAC). The following temperature profile was used in the reaction of real-time PCR: Step 1 – 95.0°C for 10 min, Step 2 – (40 cycles) 95.0°C for 30 s, 60.0°C for 1 min (annealing), 72.0°C for 45 s, Step 3 – (step of dissociation): 95.0°C for 15 s, 60.0°C for 15 s, 95.0°C for 15 s for experiments with HTCC or 95.0°C for 15 s, 60.0°C for 1 min, 95.0°C for 15 s for experiments with Pull-GTMAC. The results from the real-time PCR reaction was initially analyzed using the sequence detection software (Sequence Detection Systems Version 2.4 or ViiA 7 RUO Software v. 1.2.2., Applied Biosystems). The gene expression analysis was performed using ΔΔCt method (relative quantitative analysis of results). Optimized results of the reaction were shown as a mean value of relative expression change for the material from 3 – 4 wells with cell culture medium ± SEM.

Analysis of the effect of Pull-GTMAC on genes expression of lipid metabolism in apoE(–/–) mice brown fat tissue

The analysis of insulin induced gene 1 (INSIG1) and LDL receptor gene expression in dorsal brown fat tissue was performed using real-time PCR method addapted to array of primers for lipoprotein metabolism pathway on cDNA material from female apoE-knockout mice fed with a feed containing Pull-GTMAC at a dose of 300 mg/kg b.w./day for 18 weeks. Isolation of RNA was performed by column-based method using RNA isolation kit for lipid tissue. After isolation, one thousand nanograms of RNA was dissolved in RNase free water in a final volume of 8 µl used for a sample prepared to the reaction of a reverse transcription using RT² First Strand Kit. Next, 2 µl of buffer GE was added to each sample to prepare genomic DNA elimination mix in final volume 10 µl that was incubated for 5 min at 42°C in T3 Thermocycler (Biometra). Then, the sample was placed on ice for 1 min. Afterwords, 10 µl of a reverse-transcription mix was added, which contained 4 µl of 5 × Buffer BC3, 1 µl of Control P2, 2 µl of RE3 Reverse Transcriptase Mix, and 3 µl of RNase-free water. The prepared samples were incubated in the thermocycler with the following profile of temperature: 42°C for 15 min, 95°C for 5 min. Then, 91 µl of RNase-free water was added to each reaction. To carry out the real-time PCR reaction, commercial array of sense and antisense primers called Mouse Lipoprotein Signaling and Cholesterol Metabolism RT² Profiler™ PCR Array was used with mouse glyceraldehyde-3-phosphate (mouse GAPDH) as a reference gene. Obtained cDNA from one mouse per one plate of array of primers as a one replicate was used in each performed real-time PCR reaction. For preparation of real-time PCR reaction mixture for one 96-well plate 1350 µl of 2 × RT2 SYBR Green Mastermix was added to 102 µl of cDNA synthesis reaction, and than the 1248 µl of RNase-free water was added to obtain final volume of 2700 µl. Next, 25 µl of the reaction mixture was added to each well of the 96-well plate. Then, the reaction plate was transferred to the thermocycler for real-time PCR (ViiATM 7 Real-Time PCR System with 96-Well Fast Block, Applied Biosystems). The following temperature profile was used in real-time PCR method: Step 1 – (1 cycle) 95.0°C for 10 min, Step 2 – (40 cycles) 95.0°C for 15 s, 60.0°C for 1 min (annealing), Step 3 – (dissociation stage): 95.0°C for 15 s, 60.0°C for 15 s, 95.0°C for 15 s. The results from the real-time PCR reaction was initially analyzed using the sequence detection software (ViiA 7 RUO Software v. 1.2.2., Applied Biosystems). The gene expression analysis was performed using ΔΔCt method (relative quantitative analysis of results). Volcano plot analysis and statistical analysis of the results was carried out using web-based PCR Array Data Analysis Software from web site: www.SABiosciences.com/ pcrarraydataanalysis.php. The number of the animals in groups was 3 individuals (n = 3) in both the tested and control group of mice (3 reactions was performed using 3 separate plates of primer array for the tested group and 3 reactions on 3 96-well plates for the control group). The results after optimisation for performed real-time PCR reaction were shown as the mean value of the expression change (in relative units) for 3 mice.

Statistical analysis

The T-test was used for statistical analysis of the results. The P value < 0.05 was considered as statistically significant. Continuous variables (for example area of the atherosclerotic plaque) were expressed as the arithmetic mean ± SEM (where SEM is a standard error of the mean).

RESULTS

Characterization of the structure and chemical properties of the HTCC

In the FT-IR spectrum of HTCC a band at wavenumber of 1499 cm–1 was visible (indicated with an arrow in Fig. 1). This band is characteristic for methyl groups of the quaternary amine and clearly confirmed the cationic modification (1-3). In the FT-IR spectrum of unmodified chitosan this band did not exist.

The value of the zeta potential of HTCC in PBS buffer at pH = 7.4 was 13.3 ± 1.2 mV at 25°C.

Fig. 1. FT-IR spectrum of native chitosan (dotted line) and HTCC (solid line). The arrow indicates the band characteristic of the methyl groups of the quaternary amine).

Influence of the HTCC on a lipid plasma profile

A statistically significant 15% decrease of the total serum cholesterol level of apoE-knockout mice (males) fed with a feed containing 200 mg of HTCC per 1 kg of body weight/day was observed after 16 weeks of feeding in relation to the control group of apoE(–/–) mice whose diet was not supplemented with cationically modified chitosan (T-test, P = 0.0003; Fig. 2).

Fig. 2. Total cholesterol level in plasma of apoE(–/–) mice in the absence and presence of HTCC administered orally at a dose
of 200 mg/kg b.w./day for 16 weeks. **P < 0.001.

A statistically significant decrease in LDL cholesterol level by 32% was observed under the influence of HTCC at a dose of 200 mg/kg b.w./day after 16 weeks of the experiment compared to the level of LDL cholesterol in the control group of mice fed with a feed without modified chitosan (T-test, P = 0.0097; Fig. 3). However, HTCC did not cause a statistically significant change in the HDL and triglycerides levels. These results indicate that chitosan HTCC at a dose of 200 mg/kg b.w./day has beneficial effect on cholesterol level in blood and exhibits hypolipidemic effect.

Fig. 3. Analysis of LDL cholesterol level in plasma of apoE(–/–) mice in the absence and presence of HTCC administered orally at a dose of 200 mg/kg b.w./day for 16 weeks. *P < 0.05.

Influence of HTCC on the area of an atherosclerotic plaque

In the experiment carried out on female apoE-knockout mice fed with a feed mixed with HTCC at a dose of 300 mg/kg b.w./day for 18 weeks (using ‘cross-section’ method of imaging of the aortic root), statistically significant reduction by 33% of the area of an atherosclerotic plaque under the influence of cationically modified chitosan was observed compared to the control group of the apoE-knockout mice whose diet was not supplemented with HTCC (T-test, P = 0.003; Fig. 4 and 5).

Fig. 4. Effect of HTCC on the area of atherosclerotic plaque in the aortic root of apoE-knockout mice. *P < 0.05 versus control.

Fig. 5. Microscope imaging of aortic root stained with ORO using a ‘cross-section method’ of histological analysis in a group treated with HTCC at a dose of 300 mg/kg b.w./day (A) and in a control group of apoE(–/–) mice (B).

Impact of Pull-GTMAC on the development of atherosclerotic lesions

In the second experiment carried out in apoE-knockout female mice fed with a feed containing Pull-GTMAC at a dose of 500 mg/kg b.w./day for 18 weeks a statistically significant decrease of an atherosclerotic plaque area under the influence of Pull-GTMAC by 22% was noticed, compared to the control group of mice whose diet did not contain the tested compound (T-test, P = 0.0003; Fig. 6). Measured area of an atherosclerotic plaque was 3.73 × 105 ± 0.11 × 105 µm2 in the control group versus 2.89 × 105 ± 0.05 × 105 µm2 in the group of mice fed with a feed containing Pull-GTMAC. These results clearly confirmed that Pull-GTMAC attenuates atherosclerotic plaque development.

Fig. 6. Area of the atherosclerotic lesion presented as a mean ± S.E.M. in a control group of apoE-knockout mice and in a group of apoE(–/–) mice treated with Pull-GTMAC at a dose of 500 mg/kg b.w./day for 18 weeks (cross-section method with ORO staining). **P < 0.001 versus control.

Influence of HTCC on low density lipoprotein receptor gene expression in the apoE(–/–) mice liver

HTCC at a dose of 300 mg/kg b.w./day administered orally for 18 weeks caused a statistically significant increase in LDL receptor expression in female apoE-knockout mice compared to the control group of apoE-knockout mice (T-test, P = 0.02; Fig. 7).

Fig. 7. Influence of HTCC on LDLR gene expression in the liver of apoE-knockout mice after feeding with feed containing the polymer at a dose of 300 mg/kg b.w./day for 18 weeks. Results from real-time PCR (mean value ± SEM). *P < 0.05 versus control.

Expression of the 3-hydroxy-3-methylglutaryl-CoA reductase in HepG2 cells incubated with HTCC

A significant decrease in the level of mRNA for HMG-CoAR after 24-hour in vitro incubation of HTCC with HepG2 cell line (hepatocellular cancer cells) was shown (Fig. 8). Statistically significant differences in comparison to the gene expression level in the control sample (without incubation with HTCC) were noticed under the influence of HTCC at a concentration of 30 µg/ml of cell culture medium (T-test, P = 0.04). These results indicate that HTCC affects the expression of a gene involved in a lipid metabolism (HMG-CoAR) causing its down-regulation.

Fig. 8. HTCC impact on the expression of HMG-CoAR after 24 hours of incubation with HepG2 cells. Results from real-time PCR (mean ± SEM), *P < 0.05 versus control.

Analysis of the expression of the 3-hydroxy-3-methylglutaryl-CoA reductase in HepG2 cells after an incubation with Pull-GTMAC

No statistically significant changes in the level of mRNA for HMG-CoAR (hepatocellular cancer cells) were observed after 24-hour in vitro incubation of Pull-GTMAC with HepG2 cell line (Fig. 9). These results indicated that in contrast to HTCC, pullulan-GTMAC didn’t affect the expression of a gene involved in a lipid metabolism (HMG-CoAR).

Fig. 9. Pull-GTMAC treatment and influence on the expression of HMG-CoAR after 24 hours of incubation with HepG2 cells. Results from real-time PCR reaction (mean ± SEM).

Impact of Pull-GTMAC on the insulin induced gene 1 and low density lipoprotein receptor gene expression in brown fat tissue of apoE(–/–) mice

It was demonstrated that Pull-GTMAC affected the expression of lipid metabolism genes by statistically significant up-regulation of both INSIG1 and LDL receptor genes in brown fat tissue of apoE-knockout mice after oral administration with feed at a dose of 300 mg/kg b.w./day for 18 weeks (Fig. 10). The results of volcano plot analysis have shown that Pull-GTMAC caused a statistically significant increase in an INSIG1 expression and an increase in mRNA level of LDL receptor in female apoE-knockout mice in comparison to the control group of apoE(–/–) mice.

Fig. 10. Results of volcano plot analysis of data from the real-time PCR method in brown fat tissue of apoE(–/–) mice after treatment with Pull-GTMAC; (A): volcano plot graph; (B): table with upregulated genes of lipid metabolism, (C): table with downregulated genes of lipid metabolism; P < 0.05 versus control. Abbreviations: Insig1, insulin induced gene 1; Ldlr, low density lipoprotein receptor; Mvd, mevalonate (diphospho) decarboxylase; Npc1l1, NPC1-like 1; Akr1d1, aldo-keto reductase family 1 member D1; Apoa1, apolipoprotein A-I; Apoa2, apolipoprotein A-II; Apob, apolipoprotein B; Olr1, oxidized low density lipoprotein (lectin-like) receptor 1.

DISCUSSION

In the present work, it was clearly demonstrated that both cationically modified polysaccharides (HTCC and pullulan-GTMAC) inhibit an atherosclerotic plaque development and control the expression of genes involved in lipid metabolism.

It has also been shown that HTCC acts as hypolipidemic agent and can favorably affects cholesterol level in the blood. In turn, Pull-GTMAC does not show lipid lowering effect in blood (2), but, like HTCC, shows antiatherogenic activity. It is assumed that the mechanism of antiatherosclerotic action of cationically modified polysaccharides is independent on their effects on serum lipid profile. On the basis of the molecular simulation it is also supposed that between the chains of HTCC and cholesterol molecules there are electrostatic interactions (betwixt the -NH3+ groups of HTCC and OH groups of cholesterol) (1, 14). It seems to that the same interactions may be present between Pull-GTMAC and cholesterol (2). Furthermore, HTCC and Pull-GTMAC chains may form pseudomicelles with cholesterol molecules. It is believed that a similar effect occurs in the case of fatty acids and their salts (1). Moreover, it was shown that in normal conditions the expression of HMG-CoAR in the muscles is very low but increases during regeneration after damage and as a result of statin therapy (statins cause up-regulation of HMG-CoAR) (15). It is believed that oxLDLs cause damage of the smooth muscle cells of the rat (SMCs), which leads to increased expression of HMG-CoAR in these cells. Therefore, expression of HMG-CoAR should be high in the aorta of apoE-knockout mice. Probably, in the cells of HepG2 cell line, that are tumour cells, the level of the expression of HMG-CoA is also high. In the current study it was shown that HTCC acted therapeutically by reduction of the level of the expression of HMG-CoA reductase. What is interesting, Pull-GTMAC did not show this effect in HepG2 cells. This difference in in vitro activity of these cationically modified polysaccharides suggests a difference in the mechanisms of their antiatherogenic activity.

It has been also noticed that with increasing substitution degree, the zeta potential also increases, but this relationship does not translate into an increase in the antiatherosclerotic potential of cationically modified polysaccharides (degree of substitution of Pull-GTMAC was equal 71.25%; the zeta potential of Pull-GTMAC was measured at pH 7.4 and was equal +15.2 mV; HTCC was received with substitution degree equal to 63.6%; the value of the zeta potential of HTCC was 13.3 ± 1.2 mV).

It is supposed that HTCC influences the expression of lipid metabolism genes, resulting in restoring the balance of the lipids in the body, especially in pathological inflammatory disorders (hyperlipidemia, tumours, atherosclerosis). In patients with paraneoplastic hypercholesterolemia in the course of hepatocellular carcinoma, there was an overproduction of cholesterol and upregulation of HMG-CoAR (16). Moreover, it was also shown that proinflammatory cytokines such as tumor necrosis factor-a (proatherogenic factor) and IL-6 led to upregulation of the expression of both the mRNA and protein levels of HMG-CoAR and LDLR in THP-1 macrophages (17). The results of the test performed on macrophages suggest that inflammation (inevitably associated with atherosclerosis as one of the factors causing this disease) impairs feedback control of HMG-CoAR and LDLR and increases accumulation of cholesterol by activating the sterol regulatory element-binding protein (SREBP) pathway. Stress associated with inflammation stimulates the formation of foam cells through disruption of LDLR feedback regulation in macrophages (17).

Although it was believed that the mechanism of action of Pull-GTMAC could be slightly different from the mechanism of HTCC, there are some similarities in the activity of these two polymers. Current in vivo studies showed a statistically significant reduction of the area of an atherosclerotic plaque in apoE(–/–) mice fed with a diet containing Pull-GTMAC at a dose of 500 mg/kg b.w./day for 18 weeks in comparison to the control group of the mice. This study clearly confirmed that Pull-GTMAC inhibits atherogenesis in a mouse model of atherosclerosis. What is more, the reduction of atherosclerotic lesion area was greater in the experiment with higher dose of Pull-GMAC: 500 mg/kg b.w./day (by 22% in comparison to the control group) than in the previous experiment with dose of 300 mg/kg b.w./day administered for 18 weeks to apoE(–/–) mice (reduction by 13% relative to the control mice) (2). These results suggest dose-dependent action of Pull-GTMAC. The previous results of the real-time PCR reaction have also shown that Pull-GTMAC caused a statistically significant increase in the mRNA level of LDLR in the liver of apoE(–/–) mice fed with a diet containing Pull-GTMAC at a dose of 300 mg/kg b.w./day for 18 weeks (2) as well as HTCC at the same dose in the present study. Similarly to Pull-GTMAC administered in higher dose, HTCC also caused significant reduction of an atherosclerotic lesion area (about 33% in relation to the control group of mice whose diet did not contain the polysaccharide).

To fully explain the mechanism of action of Pull-GTMAC, its chemical structure should be considered. Pullulan is a polysaccharide composed of maltotriose units. Receptor for this compound, an asialoglycoprotein receptor (ASGPR), is present on the surface of the hepatocytes and specifically recognizes carbohydrates, and binding these molecules by the ASGP receptor is followed by their internalization into the cell in a process of endocytosis. It is thought that Pull-GTMAC, internalized by hepatocytes via asialoglycoprotein receptors, can influence gene expression of lipid metabolism and show antiatherogenic activity (2).

Although it has been shown that the cationically modified polysaccharides are biologically active compounds (1) and exhibit antiatherogenic potential (2), the particular mechanism of action of these polymers on lipid metabolism and circulatory system is still not fully elucidated. In the GI tract HTCC may be degraded to oligochitosan, which undergoes intestinal absorption more easily (1, 18). It seems to that chitooligosaccharides as the oligomeric form of the polysaccharide are the active substance released from a polymeric form of the HTCC drug. More importantly, it was proved that chitooligosaccharides have the angiotensin-I-converting enzyme inhibitory activity (1, 19, 20). So it is considered that the cationically modified chitosan degraded to oligomeric form has similar effects on the cardiovascular system as angiotensin converting enzyme inhibitors. The particular mechanism of activity of HTCC on gastric system is unknown yet and seems to be very important to check the influence of this polymer on the renin-angiotensin system (RAS) to fully characterize the biological properties of cationically modified chitosan. It is established, that both the blocking of angiotensin (Ang) AT-1 receptors and the inhibition of angiotensin-converting enzyme, cause protection against an acute gastric mucosal injury (21). The relationship between the gastric vasculature and RAS has been newly considered by the proof that a peptide metabolites of RAS can counteract and oppose the proinflammatory, vasoconstrictor, pro-oxidant and hypertrophic effects of Ang II in the gastric mucosa (21-23). In order to fully compare the strength and mechanism of action of cationically modified chitosan and pullulan, it is necessary to do further research of a gene expression of individual pathways involved in a regulation of lipid metabolism. It seems to be the key aspect of further studies likewise to determine whether the oligo- or monomeric forms of pullulan shaped in the gastrointestinal tract during digestion are the active substance released from the polymeric form of the Pull-GTMAC drug (as analogous to chitosan) and what role do they play in the regulation of the lipid metabolism of the body.

It has been suggested that INSIG1 protein plays a central role in the feedback control of lipid synthesis regulating the synthesis of cholesterol due to binding in sterol-dependent way to HMG-CoA reductase and causing proteosomal degradation of this enzyme. In current article we have shown the influence of Pull-GTMAC on the expression of INSIG1 in the brown fat tissue. What is more, the Pull-GTMAC also caused upregulation of LDLR gene in the brown fat tissue, so the mechanism of action of this polymer on lipid metabolism is complex and similar to mechanism of HTCC acting.

Abbreviations: Ang, angiotensin; ASGPR, asialoglycoprotein receptor; DS, degree of substitution; FBS, fetal bovine serum; FT-IR, Fourier Transform Infrared; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GTMAC, glycidyltrimethylammonium chloride; HDL, high density lipoprotein; HMG-CoAR, 3-hydroxy-3-methylglutaryl-CoA reductase; HPDLC, human periodontal ligament cells; HTCC, cationically modified chitosan, INSIG1, insulin induced gene 1; LDL, low-density lipoprotein; LDLR, low density lipoprotein receptor gene; LMWC, low molecular weight chitosan; LMWH, low-molecular-weight heparin; ORO, oil red O; UFH, unfractionated heparin;

Acknowledgements: Authors JS and RK acknowledge the financial support from the grant from National Science Centre, Poland (NCN) No. 2014/13/N/NZ7/00255 for years 2015-2017.

Conflict of interests: None declared.

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