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Preparation, characterization and controlled-release property of CS crosslinked MWCNT based on Hericium erinaceus polysaccharides
Journal Pre-proofs Preparation, characterization and controlled-release property of CS crosslinked MWCNT based on Hericium erinaceus polysaccharides Zhe Ren, Yang Luo, Xiaopan Liu, Junwen Zhang, Shixiong Chen, Ruihong Yu, Yongde Xu, Zhen Meng, Jian Li, Yufang Ma, Yifan Huang, Tao Qin PII: DOI: Reference:
S0141-8130(19)35854-4 https://doi.org/10.1016/j.ijbiomac.2019.10.266 BIOMAC 13767
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
29 July 2019 1 October 2019 28 October 2019
Please cite this article as: Z. Ren, Y. Luo, X. Liu, J. Zhang, S. Chen, R. Yu, Y. Xu, Z. Meng, J. Li, Y. Ma, Y. Huang, T. Qin, Preparation, characterization and controlled-release property of CS crosslinked MWCNT based on Hericium erinaceus polysaccharides, International Journal of Biological Macromolecules (2019), doi: https:// doi.org/10.1016/j.ijbiomac.2019.10.266
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Preparation, characterization and controlled-release property of CS crosslinked MWCNT based on Hericium erinaceus polysaccharides Zhe Rena,1, Yang Luoa,1, Xiaopan Liua, Junwen Zhanga, Shixiong Chena, Ruihong Yua, Yongde Xua, Zhen Menga, Jian Lia, Yufang Maa, Yifan Huanga,*, Tao Qina,**
a
Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health in
Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
b
Fujian Key Laboratory of Chinese Traditional and Western Veterinary Medicine and
Animal Health, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
*
Corresponding author at: Key Laboratory of Traditional Chinese Veterinary
Medicine and Animal Health in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China. *
Co-corresponding author: Yifan Huang, [email protected].
**
1
Corresponding author: Tao Qin, [email protected]; [email protected].
These authors contributed equally to this work.
Abstract: In present study, the optimal condition of prepared drug was determined by response surface methodology. In addition, their physicochemical properties, drug
release and uptake ability of CS-MWCNT-HEP were studied, and the distribution of the drug in ICR mice and the sites of action were further evaluated. Under the optimal condition, the mean experimental loaded efficiency 68.55±1.47% was corresponded well with the predicted value of 68.28%. The results of in vitro experiments proved that a release of the drug in a pH-dependent behavior. Flow cytometry and inverted microscope showed that the uptake of CS-MWCNT-HEP in Raw264.7 cells increased significantly as the time increased. In vivo experiment proved that the HEP and CS-MWCNT-HEP were mainly accumulated in the kidney, shown the characteristics of
kidney metabolism.
CS-MWCNT-HEP
in
On the
the
other
mice
could
hand,
the
enhance
extended the
retention
immune
of
function.
CS-MWCNT-HEP has high loaded efficiency and pH-responsive drug released, which could significantly improved the body's immunity and enhance the body's ability to absorbed
drugs.
These
findings
proposed
a
well
characterized
novel
CS-MWCNT-HEP formulation as drug delivery system, and its mechanism and application will be further investigated in our undergoing studies. Keywords:
Hericium
erinaceus
polysaccharide;
Controlled-release
property;
Multi-walled carbon nanotube (MWCNT) 1. Introduction Hericium erinaceus is a traditional medicinal food for the prevention and treatment of chronic atrophic gastritis (CAG), duodenal ulcers and several other diseases. At present, many researchers have demonstrated that Hericium erinaceus contains some important pharmacological constituents, such as polysaccharides,
alkaloids and steroids [1-4]. Polysaccharides isolated from Hericium erinaceus have been shown to be the bioactive constituents responsible for its health benefits, such as antibacterial [5], anti-oxidation [6], hypolipidemic [7], immunomodulatory [8] and so on. However, there are some negative characteristics of Hericium erinaceus polysaccharide (HEP), such as short half-life, tendency to degrade and denature in physiological environment, which hinders its clinical application. Thus, further studies are required to improve its bioavailability. Nano drug-loaded systems have been studied widely as a carrier for drugs due to their advantages over conventional drug delivery vehicles. A variety of nano drug carriers have been developed, such as liposomes [9], microemulsions [10], magnetic nanoparticles [11], gold nanoparticles [12], carbon nanotubes (CNTs) [13] and so on. Carbon nanotube have high aspect ratio, which enhances their cell penetration capability [14]. Drugs can be encapsulated inside CNTs and hence be protected from deactivation before reach the target sites. In addition, the properties of encapsulated drugs can be indirectly altered by functionalization of the external walls [15-18]. In our previous study, HEP was more efficiently absorbed when encapsulated HEP in PLGA [19]. However, the loaded efficiency is low, we found that CNTs have a large specific surface area, can entrap molecules via adsorption to the external and internal walls of the tubes or loading the drugs into the lumen and intercalation of substances between layers, and have excellent cell permeability and other properties, which can solve the problem of low drug load [20, 21]. However, MWCNT have limited their use in the field of pharmaceutical carriers due to their poor water
solubility and toxicity. The functional MWCNT has improved physiological solubility and biocompatibility, which lays a foundation for its application in the field of drug carriers [22, 23]. Chitosan (CS) is a natural polysaccharide exhibiting good biocompatibility and biodegradability, which have become an ideal excipient as carrier. In addition, since CS contains one amino group and two hydroxyl groups, which is hydrophilic and positive, it is suitable for modified the carboxylated MWCNT and improved the stability of nanocarriers in water [24, 25]. Based on the characteristics of CS, it is desirable to achieve a pH-responsive drug release after modifying MWCNT with CS and further increase the effective drug concentration in cells. This advantage expands the application of MWCNT in biomedical applications. In this experiment, we prepared a suitable vector by loaded CS into MWCNT by non-covalent modification. The HEP is loaded onto the CS-MWCNT by π-π stackedinteraction. Studied the physicochemical properties and drug released of CS-MWCNT-HEP, and the ability of uptake and transport of CS-MWCNT-HEP by RAW264.7 cells. In the end, the distribution of the drug in ICR mice and the site of action were further evaluated. 2. Materials and methods 2.1. Chemicals and reagents The purified HEP was prepared as previously reported [26]. Carboxylated carbon nanomaterials (MWCNT) (Outer diameter < 8 nm, length 0.5-2 μm) was purchased from Chinese Academy of Sciences Chengdu Organic Chemistry Co., Ltd. Chitosan
(CS) was obtained from Shanghai Ika Biotechnology Co., Ltd. sodium tripolyphosphate (TPP) was purchased from Sinopharm Chemical Reagent Co., Ltd. FITC-dextran was purchased from BD Pharmingen (San Diego, CA, USA). DMEM was
purchased
from
diphenyltetrazolium
Gibco
bromide
Co., (MTT),
Ltd.
3-(4,5-Dimethylthiazol-2-yl)-2,5-
Dimethyl
sulfoxide
(DMSO)
and
Lipopolysaccharide (LPS) were purchased from Sigma Co., Ltd. All other reagents were analytical pure. 2.2. Preparation of CS-MWCNT-HEP 2.2.1. Preprocessing of CS CS (1 g) was dissolved in 100 mL of 1% acetic acid solution, stirred until fully dissolved, and the insoluble matter was removed by filtration, and the filtrate was freeze-dried for used. 2.2.2. Preparation of CS-MWCNT The pretreated CS was weighed 0.2 g, dissolved in 1% acetic acid and made up to 100 mL, and then sonicated for 10 min using a sonicator until fully dissolved. 50 mg of MWCNT was added to the solution, and after ultrasonication for another 10 min, it was added dropwise. The aqueous solution (40 mL of 1 mg·mL-1 TPP solution) was stirred at room temperature for 12 h with a magnetic stirrer. Finally, the mixture was centrifuged (13000 rpm, 30 min), and then washed 3 times with ultrapure water. The obtained CS-MWCNT were stabilized using a freeze dryer at -50 ℃ for 3 days. 2.2.3. loaded of HEP
5 mg of CS-MWCNT was dissolved in 8 mL of deionized and sonicated for 5 min. HEP (10 mg) was dissolved in appropriate amount of deionized water and added to CS-MWCNTs solution. After sonication for 20 min, the mixture was centrifuged (13000 rpm 30 min), and the precipitate was washed with ultrapure water for 3 times, and the last supernatant was measured the sugar concentration with phenol-sulfuric acid method. Finally, the CS-MWCNT-HEP mixture was lyophilized. 2.3.4. Preparation of FITC-CS-MWCNT-HEP Accurately weighed fluorescein isothiocyanate (FITC) (10 mg) and were suspended in 10 mL of DMSO solution. FITC (0.2 mL) dissolved in DMSO was slowly added to the CS-MWCNT-HEP solution (20 mL). After reaction in the dark at room temperature for 6 h, the resulting product was centrifuged in 13000 rpm for 20 min, washed with distilled water 3-5 times to remove the excess amount of FITC, and then lyophilized in a freeze dryer. 2.2.5. RSM experimental design According to the box-benhnken experimental design principle, the effects of single factor on loaded efficiency were comprehensively compared. Three main factors affecting loaded efficiency were selected: ultrasonic time, ultrasonic power, and the proportion (HEP: CS-MWCNT). The table of three factors and three levels was designed on the basis of single factor, as shown in Table 1. Table.1. Table of the factors and levels for Box-Behnken design.
2.3. Characterization
2.3.1. Transmission electron microscope (TEM) The MWCNT, CS-MWCNT and CS-MWCNT-HEP solution was diluted to the appropriate concentration with deionized water, 10 μL of which was dropped on copper grid and dried at room temperature. 2.3.2. Zeta potential and particle size distribution The average particle size, particle size distribution of the nanoparticles and the zeta potential were measured using a NanoPlus 3 Zeta potential and a nanoparticle size analyzer. 2.3.3. Raman spectroscopy Raman spectroscopy was performed used MWCNT, CS-MWCNT and CS-MWCNT-HEP samples. The Raman scattering was excited by a frequency Nd/YAG laser at a wavelength of 633 nm. The Raman spectra acquisitions were managed by the LabSpec software. 2.4. In vitro release As for the in vitro controlled release performance, the HEP-loaded CS-MWCNT in 10 mL phosphate-buffered saline (PBS at pH=7.4 or 4.5) were transferred to dialysis tubes (molecular weight cut-off of 12000), and immersed in 120 mL of PBS at pH=7.4 or 4.5, respectively. Aliquots (5 mL) of solution were removed at certain intervals, and the drug concentration in the dialysate was analyzed using a UV spectrophotometer to assess the cumulative release of the HEP-loaded CS-MWCNT. 5 mL of the fresh PBS was added after each sampling to keep the total volume of the solution constant. The cumulative release was expressed as the total percentage of
drug that was released from the HEP-loaded CS-MWCNT and was transported through the dialysis membrane over time. 2.5. Analysis of GPSL on Raw264.7 cells 2.5.1. Cell culture Macrophage cell line (Raw264.7) was obtained from Saiqi (Shanghai) Biological Engineering Co., Ltd. The Raw264.7 cells were cultured in DMEM (Hyclone) supplemented with 10% FBS (Gibco) and 100 IU·mL-1 streptomycin/penicillin (Gibco) at 37 °C, 5% CO2. 2.5.2. Cell viability The effect of CS-MWCNT-HEP on the viability of Raw264.7 cells were determined by MTT assay. Briefly, Raw264.7 cells were seeded at 5 × 105 cells·mL-1 in a 96-well plated for 24 h and then were cultured with different concentrations of CS-MWCNT-HEP for 24 h. After incubation, MTT was added to each well 30 μL to a final concentration of 0.5 mg·mL-1. The plates were continued to incubate for 4 h, and then the supernatants were removed before adding DMSO (150 μL·well-1). Finally, the absorbance at 490 nm was measured by microplate ELISA reader. 2.5.3. Cell uptake 2.4.5.1. Flow cytometry to investigate the uptake of cells The Raw264.7 cells in logarithmic growth phase were seeded in 6-well plates at a density of 2 × 105 cells per well (each well added with 2 mL) and incubated for 24 h. After removing the medium, the cells were treated with 2 mL of FITC-labeled CS-MWCNT-HEP (1.56 μg·mL-1) or blank medium (negative control) and then
incubated for 1, 2, and 4 h. The treated cells were washed with PBS thrice and trypsinized by 0.25% trypsin, and then centrifuged at 1000 rpm for 5 min. The collected cells were washed twice with PBS again, and the cellular uptake of functionalized nanocarriers was analyzed with a flow cytometer. 2.4.5.2. Fluorescence microscopy to investigate the uptake of cells The Raw264.7 cells in logarithmic growth phase were seeded in 12-well plates at a density of 2 × 105 cells per well and incubated for 24 h. After removing the medium, the cells were treated with 1 mL of FITC-labeled CS-MWCNT-HEP (1.56 μg·mL-1) or blank medium, and then incubated for 1, 2, and 4 h. The treated cells were washed with PBS thrice and stained by 100 µL of DAPI staining solution (nuclear staining) for 15 min. The resulting samples were washed with PBS thrice and then observed under the fluorescence microscope. 2.6. The distribution of drugs in the body 2.6.1. Labeling tyramine onto polysaccharides HEP (200 mg) was added to PBS (8 mL). Tyramine (200 mg) and Sodium borohydride (75 mg) were added to the HEP solution. The mixture was stirred for 96 h at 200 rpm at 37 ℃. The product was collected by centrifugation at 4000 rpm for 10 min. The final product HEP-Tyr was lyophilized using a freeze dryer at -50 ℃ under reduced pressure. 2.6.2. Marking of Cy7-NHS HEP-Tyr was dissolved in 10 mL distilled water and the pH was adjusted to 8.5 with 0.5 mol·L-1 sodium carbonate. Then, FITC was added for 12 h in the dark.
Subsequently, 3 volumes of ethanol were added and the mixture was centrifuged at 5000 rpm for 10 min to obtain precipitation. After a sufficient volume of distilled water was added to dissolve the precipitate, 3 volumes of ethanol were added. Collected products by centrifugation, and the procedure was repeated three times. Finally, precipitation were dissolved in distilled water and the final product FITC-HEP was lyophilized using a freeze dryer at -50 ℃ under reduced pressure. 2.6.3. Preparation of Cy7-CS-MWCNT-HEP 5 mg of CS-MWCNT were dissolved in 8 mL of deionized and sonicated for 5 min. HEP-Cy7 (10 mg) was dissolved in appropriate amount of deionized water and added to CS-MWCNTs solution. After sonication for 20 min, the mixture was centrifuged (13000 rpm 30 min), and the precipitate was washed with ultrapure water for 3 times. Finally, the product CS-MWCNT-HEP-Cy7 was lyophilized used a freeze dryer at -50 ℃ under reduced pressure. 2.6.4. Preparation of Cy7-MWCNT and Cy7-CS-MWCNT 5 mg of Cy7-MWCNT or Cy7-CS-MWCNT were dissolved in 8 mL of deionized water and sonicated for 5 min. Cy7 (10 mg) was dissolved in appropriate amount of deionized water and added to CS-MWCNTs solution. After sonication for 20 min, the mixture was centrifuged (13000 rpm 30 min), and the precipitate was washed with anhydrous ethanol for 3 times. Finally, the product Cy7-MWCNT and Cy7-CS-MWCNT were lyophilized used a freeze dryer at -50 ℃ under reduced pressure. 2.6.5. loaded efficiency
The loaded efficiency was calculated accorded to the formula: loaded efficiency (%)= Weight of loaded HEP/Weight of CS-MWCNT-HEP×100% 2.6.6. Animal Animal studied were conducted at the Fujian Medical University Laboratory Animal Center under the Guidance on Animal Use and Nursing in Fujian Medical University. A total of 16 3-week-old male ICR mice (21 ± 3 g) were kept in specific pathogen-free facility. All of the mice were housed in well-ventilated rooms that were adjusted for temperature (22-25 ℃), humidity (45-65%) and 12-h day/night cycles. Mice were supplied with unlimited water and food. After 1 week of acclimatization, 4 mice were randomly selected as the control group, the other 12 mice were assigned to the CS-MWCNT, HEP, CS-MWCNT-HEP group. 2.6.7. Distribution of drugs in the body Twelve ICR mice were randomly divided into four groups and intravenously injected with 0.4 mL of Cy7-NHS-saline, HEP, CS-MWCNT and CS-MWCNT-HEP by intraperitoneal injection. Three mice in each group were anesthetized with isoflurane at different time intervals and a real-time in vivo fluorescence imaging was observed using a small animal imager at an excitation wavelength of 740 nm. On the other hand, another three mice in each group were sacrificed after 48 h, and the heart, liver, spleen, lung and kidney were obtained to evaluate the tissue distribution of MWCNT. The various organs were washed with physiological saline and imaged through fluorescence scanning. 2.7. Statistics and analysis
All data obtained in this study were presented as mean ± SD. The experimental data of the optimization of CS-MWCNT-HEP preparation were analyzed by using SPSS 22.0 and P < 0.05 indicated significance differences. 3. Result and discussion 3.1. Optimization of the procedure by RSM 3.1.1. Statistical analysis and the model fitting On single factor analysis, the design matrix and corresponding response values of RSM experiments were shown in Table 2. By applying multiple regression analysis, a final second-order polynomial equation according to the response variable and the test variables was obtained as follows: Y=+69.63-1.46*A+3.30*B+1.91*C+0.064*A*B-0.17*A*C+0.16*B*C-5.95*A2-3.81 *B2-1.06*C2 Where X1 is ultrasonic time, X2 is ultrasonic power, X3 is ratio of HEP to CS-MWCNT. Table. 2. The experimental design and results of RSM.
The analysis of variance (ANOVA) results of the response surface quadratic model were summarized in Table 3. The low P-values (P < 0.0001) and the high F value of 43.43 indicate that the regression model was highly significant [27]. The F-value of 2.11 and P value of 0.2418 indicated that the “lack-of-fit” was not significant relative to the pure error. The high value of R2 (0.9824) indicated that the general availability and accuracy of the polynomial model were adequate and could explain most of the changes in the dependent variables [28].
Table. 3. Statistic analysis of variance for the experimental results of the BBD.
3.1.2. Analysis of response surfaces Fig. 1 A and B described the combined effect of ultrasonic time and ultrasonic power while the ratio of HEP to CS-MWCNT was fixed at 4 : 1. The results demonstrated that with increasing of the two factors, the loaded efficiency experienced an earlier raised and later decreased. Fig. 1 C and D described the combined effect of ratio of HEP to CS-MWCNT and ultrasonic time to the response while the ultrasonic power was fixed at 60%. The results shown that the loaded efficiency increased in first and decreased at last with increasing of the two factors. Fig. 1 E and F described the effect of ratio of HEP to CS-MWCNT and ultrasound power on loaded efficiency when the ultrasound time is fixed at 30 min. The results showed that with the increase of ultrasonic power and the ratio of HEP to CS-MWCNT, the loaded efficiency increased first and then decreased. According to these results, the optimum loaded efficiency for CS-MWCNT-HEP were as follows: the ultrasonic time was 35.88 min, the ultrasonic power was 67.18%, and the ratio of HEP to CS-MWCNT was 4.89 : 1. Under these optimal conditions, the loaded efficiency rate of CS-MWCNT was 68.71%. Considering the operability and convenience in the production, the experimental parameters can be modified as follows: the ultrasonic time is 35 min, the ultrasonic power is 60%, and the ratio of HEP to CS-MWCNT is 5. The average experimental loaded efficiency of 68.55±1.47% was comparable to the predicted value of 68.42%. This similarity indicates that the
extraction regression model is highly significant and can be used to predict the loaded efficiency of CS-MWCNT-HEP.
Fig. 1. Response surface plots (A, C, and E) and contour plots (B, D, and F) of loaded efficiency affected by ultrasonic time (X1), ultrasonic power (X2), and the concentration of HEP: CS-MWCNT (X3). 3.2. Characterization 3.2.1. TEM The morphology of MWCNT, CS-MWCNT, and CS-MWCNT-HEP is shown in Fig. 2. CS-MWCNT-HEP is tubular, and black chitosan carbon nanoparticles are attached to the wall. The polysaccharide adheres to the surface of the tube wall. These results indicate that HEP is loaded on CNTs to constitute CS-MWCNT-HEP.
Fig. 2. Transmission electron microscope of (A, B, and C) MWCNT (A), CS-MWCNT (B), CS-MWCNT-HEP (C). 3.2.2. Zeta potential and particle size The sizes, zeta and poly dispersity index (PDI) of the CS-MWCNT-HEP were tested. The value of the size of the CS-MWCNT-HEP was 179.8 nm. The zeta of CS-MWCNT-HEP was -26.47±0.38 mv. The value of the PDI was 0.282±0.078 which indicated a homogeneous size distribution. 3.2.3 Raman spectroscopy The G-band peak of CS-MWCNT-HEP displayed shift-up, compared with that of MWCNT. It could be explained by the functionalization of CS and HEP on the
MWCNT. Due to hydrophobic and van der Waals attractions between CS, HEP and MWCNTs increase the energy necessary for vibrations to occur, Raman peaks were shifted to higher frequency [29-31]. The ID/IG ratios for MWCNT, CS-MWCNT and CS-MWCNT-HEP are 1.5852, 1.5297 and 1.2637, respectively. Those data indicated that the exist interactions among MWCNT, CS and HEP, and reduce the degree of defects on the surface of MWCNT. Fig. 3. Raman spectrum of MWCNT, CS-MWCNT, CS-MWCNT-HEP. 3.3 In vitro drug release The release curves of HEP from the CS-MWCNT-HEP at pH=5.0 and pH=7.4 was shown in Fig. 4. The result showed that 63.99% of neridronate was released after 72 h at pH 4.5. In comparison, at a pH of 7.4, only 63.99% of the HEP is released in the 72 h. which was shown that CS-MWCNT-HEP can released more readily in acidic environment [32-33]. It might be the charge-charge repulsion between the drug and CS partially responsible for decreasing the binding free energy, or the higher protonation of CS could trigger the release of drug molecules. Fig. 4. Release profiles of HEP at different pH condition. 3.4. Effects of CS-MWCNT-HEP on the viability of Raw264.7 cells After treated with CS-MWCNT-HEP (0.39-100 μg·mL-1) for 24 h, Raw264.7 cells were detected for the viability using MTT assay. Compared control group, the results of the toxic effect of CS-MWCNT-HEP decreased with time. To avoid the cytotoxicity of test sample, CS-MWCNT-HEP at the indicated concentrations of 1.56
μg·mL-1 were selected for following assays. 3.5. Cellular uptake The uptake of CS-MWCNT-HEP in Raw264.7 cells were observed by a fluorescence microscopy and flow cytometry are shown in Fig. 5 A and B. The blue color represents the nucleus of cells, the green represents the CS-MWCNT-HEP tagged by FITC, and the merged view is the overlapping of the previous two pictures. Based on the cytotoxicity and preliminary test of cellular uptake, the concentration of CS-MWCNT-HEP used was 1.56 µg·mL-1. The results of the fluorescence microscope are shown in Fig. 5A, it indicated that the functionalized MWCNT presents excellent transport ability and the CS-MWCNT-HEP was uptaken by Raw264.7 cells in a time-dependent way. The ability of the cells to take up the drug was examined by flow cytometry, and the results are shown in Fig. 5B. The uptake of functionalized MWCNT by cells increases with increasing incubation time. The uptake rates of Raw264.7 cells were 25.80%, 45.76% and 71.40%, this is consistent with the results of fluorescence microscopy.
Fig. 5. Fluorescence
microscopy of CS-MWCNT-HEP uptake by
Raw264.7cells (A) and Flow diagram of Raw264.7 cell uptake CS-MWCNT-HEP (B). 3.6. Animal living imaging The biodistribution of drugs in mouse model was studied. To this end, free Cy7, Cy7-HEP,
Cy7-CS-MWCNT and
Cy7-CS-MWCNT-HEP were
intragastrical
administration mice via the respectively, and the distribution of Cy7 fluorescence was
monitored via an in-vivo imaging system at different time intervals (1, 6, 12, 24 and 48 h) (Fig. 6A). 1 h after administration, the groups of Cy7, Cy7-HEP, Cy7-CS-MWCNT and Cy7-CS-MWCNT-HEP revealed that remarkable fluorescence imagesat, and the fluorescence intensity of drug gradually decreases with time (Fig. 6B). After 48 h, the mice were sacrificed for the tissue distribution analysis. The HEP and CS-MWCNT-HEP were mainly located in the liver and spleen. These results of Fig. 6C demonstrated that CS-MWCNT nanocarriers could accumulate effectively in liver with an excellent biocompatibility in vivo [34]. At the same dose, the ratio of organ fluorescence intensity to drug fluorescence intensity of CS-MWCNT-HEP in spleen is higher than HEP, which indicated that the body could better absorbed CS-MWCNT-HEP and could enhance the immunity of the body. Moreover, CS-MWCNT-HEP has more residual drugs in the intestine, indicated that it can act on the body for a long-term effect to enhance the therapeutic effect of the drug. According to reported that CNTs could improve the accumulation of a drug and its slow release inside cells, our research was consistent with the above findings [35].
Fig. 6. Distribution of drugs (a, b, c, d) Saline (a), CS-MWCNT (b), HEP (c), CS-MWCNT-HEP (d) in mice (A), fluorescence intensity of drugs over time (B) and ratio of organ fluorescence intensity to drug fluorescence intensity (C). 4. Conclusion In this study, the optimal preparation condition for CS-MWCNT-HEP was studied by RSM and listed as follows: the ratio of ratio of HEP to CS-MWCNT was
4.89 : 1; ultrasonic power was 69.13%; ultrasonic time was 16 min. Under this condition, the experimen-tal encapsulation efficiency of CS-MWCNT-HEP was 68.55±1.47%, and Raman spectroscopy, TEM, Zate potential and particle size confirmed the successful synthesis of functionalized MWCNT vectors. The release behavior of CS-MWCNT-HEP at pH=7.4 and 4.5 was significantly different. The cumulative drug release at pH=4.5 was higher than that at pH=7.4, indicating a pH-dependent release of the drug. Flow cytometry and inverted microscopy showed that the absorption rate of Raw264.7 cells increased with time, and the absorption rate was 71.40% at 4 h. The small animal in vivo imaging experiment is more intuitive to see that the drug mainly acts on the spleen, liver and kidney, and enhances the body's immunity and metabolism. And nano-sized polysaccharides can increase their time in the body. It can be seen that CS-MWCNT-HEP has a high loaded efficiency and pH-responsive drug release, which can significantly improve the body's immunity, enhance the body's ability to absorb drugs, and have good safety to animals. These findings proposed a well characterized novel CS-MWCNT-HEP formulation as drug delivery system, and its mechanism and application will be further investigated in our undergoing studies. Acknowledgements The project was supported by National Natural Science Foundation of China (31802236), Scientific Research Program of Outstanding Youth of Fujian Agriculture and Forestry University (XJQ201816), Natural Science Foundation of Fujian Province of China (2019J01378), The Education Department Foundation of Fujian Province
(JK2016010). We are grateful to all other staff in the Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health in Fujian province for their assistance in the experiments. References [1] Z. Ren, T. Qin, F.A. Qiu, Y.L. Song, D.D. Lin, Y.F. Ma, J. Li, Y.F. Huang, Immunomodulatory
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Table 1 Table of the factors and levels for Box-Behnken design. X1
X2
X3
(ultrasonic time)
(ultrasonic power)
(HEP: CS-MWCNT)
-1
20
40 %
3
0
30
60 %
4
+1
40
80 %
5
factor
Table 2 The experimental design and results of RSM. Levels of independent factors
Response loaded efficiency (%)
RUN A
B
C
Experimental
Predicted
1
40
80
4
61.97
61.76
2
40
60
3
58.84
59.41
3
30
40
3
60.63
59.71
4
30
80
3
66.34
65.98
5
40
40
4
54.69
55.05
6
20
40
4
57.89
58.10
7
30
60
4
70.47
69.63
8
30
60
4
69.48
69.63
9
20
80
4
64.91
64.56
11
40
60
5
63.62
62.90
11
20
60
5
66.73
66.16
12
30
80
5
69.20
70.13
13
30
60
4
69.79
69.63
14
30
40
5
62.85
63.21
15
20
60
3
61.28
62.00
16
30
60
4
70.05
69.63
17
30
60
4
68.36
69.63
Table 3 Statistic analysis of variance for the experimental results of the BBD. Sum of Source
Mean df
squares
F value
P-Value
Prob >F significant
square
Model
365.54
9
40.62
43.43
<0.0001
X1
17.12
1
17.12
18.30
0.0037
X2
86.91
1
86.91
92.92
<0.0001
X3
29.27
1
29.27
31.30
0.0008
X1 X2
0.017
1
0.017
0.018
0.8980
X1 X3
0.11
1
0.11
0.12
0.7395
X2 X3
0.10
1
0.10
0.11
0.7477
X12
149.15
1
149.15
159.47
<0.0001
X22
61.27
1
61.27
65.50
<0.0001
X32
4.76
1
4.76
5.09
0.0587
Residual
6.55
7
0.94
Lack of fit
4.01
3
1.34
2.11
0.2418
Pure error
2.54
4
0.63
Cor total
372.09
6
R2 = 0.9824; R2adj = 0.9598; R2Pred = 0.8169. Fig 1
not significant
Fig. 1 Response surface plots (A, C, and E) and contour plots (B, D, and F) of loaded efficiency affected by ultrasonic time (X1), ultrasonic power (X2), and the concentration of HEP: CS-MWCNT (X3).
Fig 2
Fig. 2 Transmission electron microscope of (A, B, and C) MWCNT (A), CS-MWCNT (B), CS-MWCNT-HEP (C).
Fig 3
Fig. 3. Raman spectrum of MWCNT, CS-MWCNT, CS-MWCNT-HEP.
Fig 4
Fig. 4 Release profiles of HEP at different pH condition.
Fig 5
Fig. 5 Fluorescence microscopy of CS-MWCNT-HEP uptake by Raw264.7 cells (A) and Flow diagram of Raw264.7 cell uptake CS-MWCNT-HEP (B).
Fig 6
Fig. 6. Distribution of drugs (a, b, c, d) Saline (a), CS-MWCNT (b), HEP (c), CS-MWCNT-HEP (d) in mice (A), fluorescence intensity of drugs over time (B) and ratio of organ fluorescence intensity to drug fluorescence intensity (C). Bars without the same superscripts (a-d) differ significantly (P<0.05).
S0141-8130(19)35854-4 https://doi.org/10.1016/j.ijbiomac.2019.10.266 BIOMAC 13767
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
29 July 2019 1 October 2019 28 October 2019
Please cite this article as: Z. Ren, Y. Luo, X. Liu, J. Zhang, S. Chen, R. Yu, Y. Xu, Z. Meng, J. Li, Y. Ma, Y. Huang, T. Qin, Preparation, characterization and controlled-release property of CS crosslinked MWCNT based on Hericium erinaceus polysaccharides, International Journal of Biological Macromolecules (2019), doi: https:// doi.org/10.1016/j.ijbiomac.2019.10.266
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© 2019 Published by Elsevier B.V.
Preparation, characterization and controlled-release property of CS crosslinked MWCNT based on Hericium erinaceus polysaccharides Zhe Rena,1, Yang Luoa,1, Xiaopan Liua, Junwen Zhanga, Shixiong Chena, Ruihong Yua, Yongde Xua, Zhen Menga, Jian Lia, Yufang Maa, Yifan Huanga,*, Tao Qina,**
a
Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health in
Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
b
Fujian Key Laboratory of Chinese Traditional and Western Veterinary Medicine and
Animal Health, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
*
Corresponding author at: Key Laboratory of Traditional Chinese Veterinary
Medicine and Animal Health in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China. *
Co-corresponding author: Yifan Huang, [email protected].
**
1
Corresponding author: Tao Qin, [email protected]; [email protected].
These authors contributed equally to this work.
Abstract: In present study, the optimal condition of prepared drug was determined by response surface methodology. In addition, their physicochemical properties, drug
release and uptake ability of CS-MWCNT-HEP were studied, and the distribution of the drug in ICR mice and the sites of action were further evaluated. Under the optimal condition, the mean experimental loaded efficiency 68.55±1.47% was corresponded well with the predicted value of 68.28%. The results of in vitro experiments proved that a release of the drug in a pH-dependent behavior. Flow cytometry and inverted microscope showed that the uptake of CS-MWCNT-HEP in Raw264.7 cells increased significantly as the time increased. In vivo experiment proved that the HEP and CS-MWCNT-HEP were mainly accumulated in the kidney, shown the characteristics of
kidney metabolism.
CS-MWCNT-HEP
in
On the
the
other
mice
could
hand,
the
enhance
extended the
retention
immune
of
function.
CS-MWCNT-HEP has high loaded efficiency and pH-responsive drug released, which could significantly improved the body's immunity and enhance the body's ability to absorbed
drugs.
These
findings
proposed
a
well
characterized
novel
CS-MWCNT-HEP formulation as drug delivery system, and its mechanism and application will be further investigated in our undergoing studies. Keywords:
Hericium
erinaceus
polysaccharide;
Controlled-release
property;
Multi-walled carbon nanotube (MWCNT) 1. Introduction Hericium erinaceus is a traditional medicinal food for the prevention and treatment of chronic atrophic gastritis (CAG), duodenal ulcers and several other diseases. At present, many researchers have demonstrated that Hericium erinaceus contains some important pharmacological constituents, such as polysaccharides,
alkaloids and steroids [1-4]. Polysaccharides isolated from Hericium erinaceus have been shown to be the bioactive constituents responsible for its health benefits, such as antibacterial [5], anti-oxidation [6], hypolipidemic [7], immunomodulatory [8] and so on. However, there are some negative characteristics of Hericium erinaceus polysaccharide (HEP), such as short half-life, tendency to degrade and denature in physiological environment, which hinders its clinical application. Thus, further studies are required to improve its bioavailability. Nano drug-loaded systems have been studied widely as a carrier for drugs due to their advantages over conventional drug delivery vehicles. A variety of nano drug carriers have been developed, such as liposomes [9], microemulsions [10], magnetic nanoparticles [11], gold nanoparticles [12], carbon nanotubes (CNTs) [13] and so on. Carbon nanotube have high aspect ratio, which enhances their cell penetration capability [14]. Drugs can be encapsulated inside CNTs and hence be protected from deactivation before reach the target sites. In addition, the properties of encapsulated drugs can be indirectly altered by functionalization of the external walls [15-18]. In our previous study, HEP was more efficiently absorbed when encapsulated HEP in PLGA [19]. However, the loaded efficiency is low, we found that CNTs have a large specific surface area, can entrap molecules via adsorption to the external and internal walls of the tubes or loading the drugs into the lumen and intercalation of substances between layers, and have excellent cell permeability and other properties, which can solve the problem of low drug load [20, 21]. However, MWCNT have limited their use in the field of pharmaceutical carriers due to their poor water
solubility and toxicity. The functional MWCNT has improved physiological solubility and biocompatibility, which lays a foundation for its application in the field of drug carriers [22, 23]. Chitosan (CS) is a natural polysaccharide exhibiting good biocompatibility and biodegradability, which have become an ideal excipient as carrier. In addition, since CS contains one amino group and two hydroxyl groups, which is hydrophilic and positive, it is suitable for modified the carboxylated MWCNT and improved the stability of nanocarriers in water [24, 25]. Based on the characteristics of CS, it is desirable to achieve a pH-responsive drug release after modifying MWCNT with CS and further increase the effective drug concentration in cells. This advantage expands the application of MWCNT in biomedical applications. In this experiment, we prepared a suitable vector by loaded CS into MWCNT by non-covalent modification. The HEP is loaded onto the CS-MWCNT by π-π stackedinteraction. Studied the physicochemical properties and drug released of CS-MWCNT-HEP, and the ability of uptake and transport of CS-MWCNT-HEP by RAW264.7 cells. In the end, the distribution of the drug in ICR mice and the site of action were further evaluated. 2. Materials and methods 2.1. Chemicals and reagents The purified HEP was prepared as previously reported [26]. Carboxylated carbon nanomaterials (MWCNT) (Outer diameter < 8 nm, length 0.5-2 μm) was purchased from Chinese Academy of Sciences Chengdu Organic Chemistry Co., Ltd. Chitosan
(CS) was obtained from Shanghai Ika Biotechnology Co., Ltd. sodium tripolyphosphate (TPP) was purchased from Sinopharm Chemical Reagent Co., Ltd. FITC-dextran was purchased from BD Pharmingen (San Diego, CA, USA). DMEM was
purchased
from
diphenyltetrazolium
Gibco
bromide
Co., (MTT),
Ltd.
3-(4,5-Dimethylthiazol-2-yl)-2,5-
Dimethyl
sulfoxide
(DMSO)
and
Lipopolysaccharide (LPS) were purchased from Sigma Co., Ltd. All other reagents were analytical pure. 2.2. Preparation of CS-MWCNT-HEP 2.2.1. Preprocessing of CS CS (1 g) was dissolved in 100 mL of 1% acetic acid solution, stirred until fully dissolved, and the insoluble matter was removed by filtration, and the filtrate was freeze-dried for used. 2.2.2. Preparation of CS-MWCNT The pretreated CS was weighed 0.2 g, dissolved in 1% acetic acid and made up to 100 mL, and then sonicated for 10 min using a sonicator until fully dissolved. 50 mg of MWCNT was added to the solution, and after ultrasonication for another 10 min, it was added dropwise. The aqueous solution (40 mL of 1 mg·mL-1 TPP solution) was stirred at room temperature for 12 h with a magnetic stirrer. Finally, the mixture was centrifuged (13000 rpm, 30 min), and then washed 3 times with ultrapure water. The obtained CS-MWCNT were stabilized using a freeze dryer at -50 ℃ for 3 days. 2.2.3. loaded of HEP
5 mg of CS-MWCNT was dissolved in 8 mL of deionized and sonicated for 5 min. HEP (10 mg) was dissolved in appropriate amount of deionized water and added to CS-MWCNTs solution. After sonication for 20 min, the mixture was centrifuged (13000 rpm 30 min), and the precipitate was washed with ultrapure water for 3 times, and the last supernatant was measured the sugar concentration with phenol-sulfuric acid method. Finally, the CS-MWCNT-HEP mixture was lyophilized. 2.3.4. Preparation of FITC-CS-MWCNT-HEP Accurately weighed fluorescein isothiocyanate (FITC) (10 mg) and were suspended in 10 mL of DMSO solution. FITC (0.2 mL) dissolved in DMSO was slowly added to the CS-MWCNT-HEP solution (20 mL). After reaction in the dark at room temperature for 6 h, the resulting product was centrifuged in 13000 rpm for 20 min, washed with distilled water 3-5 times to remove the excess amount of FITC, and then lyophilized in a freeze dryer. 2.2.5. RSM experimental design According to the box-benhnken experimental design principle, the effects of single factor on loaded efficiency were comprehensively compared. Three main factors affecting loaded efficiency were selected: ultrasonic time, ultrasonic power, and the proportion (HEP: CS-MWCNT). The table of three factors and three levels was designed on the basis of single factor, as shown in Table 1. Table.1. Table of the factors and levels for Box-Behnken design.
2.3. Characterization
2.3.1. Transmission electron microscope (TEM) The MWCNT, CS-MWCNT and CS-MWCNT-HEP solution was diluted to the appropriate concentration with deionized water, 10 μL of which was dropped on copper grid and dried at room temperature. 2.3.2. Zeta potential and particle size distribution The average particle size, particle size distribution of the nanoparticles and the zeta potential were measured using a NanoPlus 3 Zeta potential and a nanoparticle size analyzer. 2.3.3. Raman spectroscopy Raman spectroscopy was performed used MWCNT, CS-MWCNT and CS-MWCNT-HEP samples. The Raman scattering was excited by a frequency Nd/YAG laser at a wavelength of 633 nm. The Raman spectra acquisitions were managed by the LabSpec software. 2.4. In vitro release As for the in vitro controlled release performance, the HEP-loaded CS-MWCNT in 10 mL phosphate-buffered saline (PBS at pH=7.4 or 4.5) were transferred to dialysis tubes (molecular weight cut-off of 12000), and immersed in 120 mL of PBS at pH=7.4 or 4.5, respectively. Aliquots (5 mL) of solution were removed at certain intervals, and the drug concentration in the dialysate was analyzed using a UV spectrophotometer to assess the cumulative release of the HEP-loaded CS-MWCNT. 5 mL of the fresh PBS was added after each sampling to keep the total volume of the solution constant. The cumulative release was expressed as the total percentage of
drug that was released from the HEP-loaded CS-MWCNT and was transported through the dialysis membrane over time. 2.5. Analysis of GPSL on Raw264.7 cells 2.5.1. Cell culture Macrophage cell line (Raw264.7) was obtained from Saiqi (Shanghai) Biological Engineering Co., Ltd. The Raw264.7 cells were cultured in DMEM (Hyclone) supplemented with 10% FBS (Gibco) and 100 IU·mL-1 streptomycin/penicillin (Gibco) at 37 °C, 5% CO2. 2.5.2. Cell viability The effect of CS-MWCNT-HEP on the viability of Raw264.7 cells were determined by MTT assay. Briefly, Raw264.7 cells were seeded at 5 × 105 cells·mL-1 in a 96-well plated for 24 h and then were cultured with different concentrations of CS-MWCNT-HEP for 24 h. After incubation, MTT was added to each well 30 μL to a final concentration of 0.5 mg·mL-1. The plates were continued to incubate for 4 h, and then the supernatants were removed before adding DMSO (150 μL·well-1). Finally, the absorbance at 490 nm was measured by microplate ELISA reader. 2.5.3. Cell uptake 2.4.5.1. Flow cytometry to investigate the uptake of cells The Raw264.7 cells in logarithmic growth phase were seeded in 6-well plates at a density of 2 × 105 cells per well (each well added with 2 mL) and incubated for 24 h. After removing the medium, the cells were treated with 2 mL of FITC-labeled CS-MWCNT-HEP (1.56 μg·mL-1) or blank medium (negative control) and then
incubated for 1, 2, and 4 h. The treated cells were washed with PBS thrice and trypsinized by 0.25% trypsin, and then centrifuged at 1000 rpm for 5 min. The collected cells were washed twice with PBS again, and the cellular uptake of functionalized nanocarriers was analyzed with a flow cytometer. 2.4.5.2. Fluorescence microscopy to investigate the uptake of cells The Raw264.7 cells in logarithmic growth phase were seeded in 12-well plates at a density of 2 × 105 cells per well and incubated for 24 h. After removing the medium, the cells were treated with 1 mL of FITC-labeled CS-MWCNT-HEP (1.56 μg·mL-1) or blank medium, and then incubated for 1, 2, and 4 h. The treated cells were washed with PBS thrice and stained by 100 µL of DAPI staining solution (nuclear staining) for 15 min. The resulting samples were washed with PBS thrice and then observed under the fluorescence microscope. 2.6. The distribution of drugs in the body 2.6.1. Labeling tyramine onto polysaccharides HEP (200 mg) was added to PBS (8 mL). Tyramine (200 mg) and Sodium borohydride (75 mg) were added to the HEP solution. The mixture was stirred for 96 h at 200 rpm at 37 ℃. The product was collected by centrifugation at 4000 rpm for 10 min. The final product HEP-Tyr was lyophilized using a freeze dryer at -50 ℃ under reduced pressure. 2.6.2. Marking of Cy7-NHS HEP-Tyr was dissolved in 10 mL distilled water and the pH was adjusted to 8.5 with 0.5 mol·L-1 sodium carbonate. Then, FITC was added for 12 h in the dark.
Subsequently, 3 volumes of ethanol were added and the mixture was centrifuged at 5000 rpm for 10 min to obtain precipitation. After a sufficient volume of distilled water was added to dissolve the precipitate, 3 volumes of ethanol were added. Collected products by centrifugation, and the procedure was repeated three times. Finally, precipitation were dissolved in distilled water and the final product FITC-HEP was lyophilized using a freeze dryer at -50 ℃ under reduced pressure. 2.6.3. Preparation of Cy7-CS-MWCNT-HEP 5 mg of CS-MWCNT were dissolved in 8 mL of deionized and sonicated for 5 min. HEP-Cy7 (10 mg) was dissolved in appropriate amount of deionized water and added to CS-MWCNTs solution. After sonication for 20 min, the mixture was centrifuged (13000 rpm 30 min), and the precipitate was washed with ultrapure water for 3 times. Finally, the product CS-MWCNT-HEP-Cy7 was lyophilized used a freeze dryer at -50 ℃ under reduced pressure. 2.6.4. Preparation of Cy7-MWCNT and Cy7-CS-MWCNT 5 mg of Cy7-MWCNT or Cy7-CS-MWCNT were dissolved in 8 mL of deionized water and sonicated for 5 min. Cy7 (10 mg) was dissolved in appropriate amount of deionized water and added to CS-MWCNTs solution. After sonication for 20 min, the mixture was centrifuged (13000 rpm 30 min), and the precipitate was washed with anhydrous ethanol for 3 times. Finally, the product Cy7-MWCNT and Cy7-CS-MWCNT were lyophilized used a freeze dryer at -50 ℃ under reduced pressure. 2.6.5. loaded efficiency
The loaded efficiency was calculated accorded to the formula: loaded efficiency (%)= Weight of loaded HEP/Weight of CS-MWCNT-HEP×100% 2.6.6. Animal Animal studied were conducted at the Fujian Medical University Laboratory Animal Center under the Guidance on Animal Use and Nursing in Fujian Medical University. A total of 16 3-week-old male ICR mice (21 ± 3 g) were kept in specific pathogen-free facility. All of the mice were housed in well-ventilated rooms that were adjusted for temperature (22-25 ℃), humidity (45-65%) and 12-h day/night cycles. Mice were supplied with unlimited water and food. After 1 week of acclimatization, 4 mice were randomly selected as the control group, the other 12 mice were assigned to the CS-MWCNT, HEP, CS-MWCNT-HEP group. 2.6.7. Distribution of drugs in the body Twelve ICR mice were randomly divided into four groups and intravenously injected with 0.4 mL of Cy7-NHS-saline, HEP, CS-MWCNT and CS-MWCNT-HEP by intraperitoneal injection. Three mice in each group were anesthetized with isoflurane at different time intervals and a real-time in vivo fluorescence imaging was observed using a small animal imager at an excitation wavelength of 740 nm. On the other hand, another three mice in each group were sacrificed after 48 h, and the heart, liver, spleen, lung and kidney were obtained to evaluate the tissue distribution of MWCNT. The various organs were washed with physiological saline and imaged through fluorescence scanning. 2.7. Statistics and analysis
All data obtained in this study were presented as mean ± SD. The experimental data of the optimization of CS-MWCNT-HEP preparation were analyzed by using SPSS 22.0 and P < 0.05 indicated significance differences. 3. Result and discussion 3.1. Optimization of the procedure by RSM 3.1.1. Statistical analysis and the model fitting On single factor analysis, the design matrix and corresponding response values of RSM experiments were shown in Table 2. By applying multiple regression analysis, a final second-order polynomial equation according to the response variable and the test variables was obtained as follows: Y=+69.63-1.46*A+3.30*B+1.91*C+0.064*A*B-0.17*A*C+0.16*B*C-5.95*A2-3.81 *B2-1.06*C2 Where X1 is ultrasonic time, X2 is ultrasonic power, X3 is ratio of HEP to CS-MWCNT. Table. 2. The experimental design and results of RSM.
The analysis of variance (ANOVA) results of the response surface quadratic model were summarized in Table 3. The low P-values (P < 0.0001) and the high F value of 43.43 indicate that the regression model was highly significant [27]. The F-value of 2.11 and P value of 0.2418 indicated that the “lack-of-fit” was not significant relative to the pure error. The high value of R2 (0.9824) indicated that the general availability and accuracy of the polynomial model were adequate and could explain most of the changes in the dependent variables [28].
Table. 3. Statistic analysis of variance for the experimental results of the BBD.
3.1.2. Analysis of response surfaces Fig. 1 A and B described the combined effect of ultrasonic time and ultrasonic power while the ratio of HEP to CS-MWCNT was fixed at 4 : 1. The results demonstrated that with increasing of the two factors, the loaded efficiency experienced an earlier raised and later decreased. Fig. 1 C and D described the combined effect of ratio of HEP to CS-MWCNT and ultrasonic time to the response while the ultrasonic power was fixed at 60%. The results shown that the loaded efficiency increased in first and decreased at last with increasing of the two factors. Fig. 1 E and F described the effect of ratio of HEP to CS-MWCNT and ultrasound power on loaded efficiency when the ultrasound time is fixed at 30 min. The results showed that with the increase of ultrasonic power and the ratio of HEP to CS-MWCNT, the loaded efficiency increased first and then decreased. According to these results, the optimum loaded efficiency for CS-MWCNT-HEP were as follows: the ultrasonic time was 35.88 min, the ultrasonic power was 67.18%, and the ratio of HEP to CS-MWCNT was 4.89 : 1. Under these optimal conditions, the loaded efficiency rate of CS-MWCNT was 68.71%. Considering the operability and convenience in the production, the experimental parameters can be modified as follows: the ultrasonic time is 35 min, the ultrasonic power is 60%, and the ratio of HEP to CS-MWCNT is 5. The average experimental loaded efficiency of 68.55±1.47% was comparable to the predicted value of 68.42%. This similarity indicates that the
extraction regression model is highly significant and can be used to predict the loaded efficiency of CS-MWCNT-HEP.
Fig. 1. Response surface plots (A, C, and E) and contour plots (B, D, and F) of loaded efficiency affected by ultrasonic time (X1), ultrasonic power (X2), and the concentration of HEP: CS-MWCNT (X3). 3.2. Characterization 3.2.1. TEM The morphology of MWCNT, CS-MWCNT, and CS-MWCNT-HEP is shown in Fig. 2. CS-MWCNT-HEP is tubular, and black chitosan carbon nanoparticles are attached to the wall. The polysaccharide adheres to the surface of the tube wall. These results indicate that HEP is loaded on CNTs to constitute CS-MWCNT-HEP.
Fig. 2. Transmission electron microscope of (A, B, and C) MWCNT (A), CS-MWCNT (B), CS-MWCNT-HEP (C). 3.2.2. Zeta potential and particle size The sizes, zeta and poly dispersity index (PDI) of the CS-MWCNT-HEP were tested. The value of the size of the CS-MWCNT-HEP was 179.8 nm. The zeta of CS-MWCNT-HEP was -26.47±0.38 mv. The value of the PDI was 0.282±0.078 which indicated a homogeneous size distribution. 3.2.3 Raman spectroscopy The G-band peak of CS-MWCNT-HEP displayed shift-up, compared with that of MWCNT. It could be explained by the functionalization of CS and HEP on the
MWCNT. Due to hydrophobic and van der Waals attractions between CS, HEP and MWCNTs increase the energy necessary for vibrations to occur, Raman peaks were shifted to higher frequency [29-31]. The ID/IG ratios for MWCNT, CS-MWCNT and CS-MWCNT-HEP are 1.5852, 1.5297 and 1.2637, respectively. Those data indicated that the exist interactions among MWCNT, CS and HEP, and reduce the degree of defects on the surface of MWCNT. Fig. 3. Raman spectrum of MWCNT, CS-MWCNT, CS-MWCNT-HEP. 3.3 In vitro drug release The release curves of HEP from the CS-MWCNT-HEP at pH=5.0 and pH=7.4 was shown in Fig. 4. The result showed that 63.99% of neridronate was released after 72 h at pH 4.5. In comparison, at a pH of 7.4, only 63.99% of the HEP is released in the 72 h. which was shown that CS-MWCNT-HEP can released more readily in acidic environment [32-33]. It might be the charge-charge repulsion between the drug and CS partially responsible for decreasing the binding free energy, or the higher protonation of CS could trigger the release of drug molecules. Fig. 4. Release profiles of HEP at different pH condition. 3.4. Effects of CS-MWCNT-HEP on the viability of Raw264.7 cells After treated with CS-MWCNT-HEP (0.39-100 μg·mL-1) for 24 h, Raw264.7 cells were detected for the viability using MTT assay. Compared control group, the results of the toxic effect of CS-MWCNT-HEP decreased with time. To avoid the cytotoxicity of test sample, CS-MWCNT-HEP at the indicated concentrations of 1.56
μg·mL-1 were selected for following assays. 3.5. Cellular uptake The uptake of CS-MWCNT-HEP in Raw264.7 cells were observed by a fluorescence microscopy and flow cytometry are shown in Fig. 5 A and B. The blue color represents the nucleus of cells, the green represents the CS-MWCNT-HEP tagged by FITC, and the merged view is the overlapping of the previous two pictures. Based on the cytotoxicity and preliminary test of cellular uptake, the concentration of CS-MWCNT-HEP used was 1.56 µg·mL-1. The results of the fluorescence microscope are shown in Fig. 5A, it indicated that the functionalized MWCNT presents excellent transport ability and the CS-MWCNT-HEP was uptaken by Raw264.7 cells in a time-dependent way. The ability of the cells to take up the drug was examined by flow cytometry, and the results are shown in Fig. 5B. The uptake of functionalized MWCNT by cells increases with increasing incubation time. The uptake rates of Raw264.7 cells were 25.80%, 45.76% and 71.40%, this is consistent with the results of fluorescence microscopy.
Fig. 5. Fluorescence
microscopy of CS-MWCNT-HEP uptake by
Raw264.7cells (A) and Flow diagram of Raw264.7 cell uptake CS-MWCNT-HEP (B). 3.6. Animal living imaging The biodistribution of drugs in mouse model was studied. To this end, free Cy7, Cy7-HEP,
Cy7-CS-MWCNT and
Cy7-CS-MWCNT-HEP were
intragastrical
administration mice via the respectively, and the distribution of Cy7 fluorescence was
monitored via an in-vivo imaging system at different time intervals (1, 6, 12, 24 and 48 h) (Fig. 6A). 1 h after administration, the groups of Cy7, Cy7-HEP, Cy7-CS-MWCNT and Cy7-CS-MWCNT-HEP revealed that remarkable fluorescence imagesat, and the fluorescence intensity of drug gradually decreases with time (Fig. 6B). After 48 h, the mice were sacrificed for the tissue distribution analysis. The HEP and CS-MWCNT-HEP were mainly located in the liver and spleen. These results of Fig. 6C demonstrated that CS-MWCNT nanocarriers could accumulate effectively in liver with an excellent biocompatibility in vivo [34]. At the same dose, the ratio of organ fluorescence intensity to drug fluorescence intensity of CS-MWCNT-HEP in spleen is higher than HEP, which indicated that the body could better absorbed CS-MWCNT-HEP and could enhance the immunity of the body. Moreover, CS-MWCNT-HEP has more residual drugs in the intestine, indicated that it can act on the body for a long-term effect to enhance the therapeutic effect of the drug. According to reported that CNTs could improve the accumulation of a drug and its slow release inside cells, our research was consistent with the above findings [35].
Fig. 6. Distribution of drugs (a, b, c, d) Saline (a), CS-MWCNT (b), HEP (c), CS-MWCNT-HEP (d) in mice (A), fluorescence intensity of drugs over time (B) and ratio of organ fluorescence intensity to drug fluorescence intensity (C). 4. Conclusion In this study, the optimal preparation condition for CS-MWCNT-HEP was studied by RSM and listed as follows: the ratio of ratio of HEP to CS-MWCNT was
4.89 : 1; ultrasonic power was 69.13%; ultrasonic time was 16 min. Under this condition, the experimen-tal encapsulation efficiency of CS-MWCNT-HEP was 68.55±1.47%, and Raman spectroscopy, TEM, Zate potential and particle size confirmed the successful synthesis of functionalized MWCNT vectors. The release behavior of CS-MWCNT-HEP at pH=7.4 and 4.5 was significantly different. The cumulative drug release at pH=4.5 was higher than that at pH=7.4, indicating a pH-dependent release of the drug. Flow cytometry and inverted microscopy showed that the absorption rate of Raw264.7 cells increased with time, and the absorption rate was 71.40% at 4 h. The small animal in vivo imaging experiment is more intuitive to see that the drug mainly acts on the spleen, liver and kidney, and enhances the body's immunity and metabolism. And nano-sized polysaccharides can increase their time in the body. It can be seen that CS-MWCNT-HEP has a high loaded efficiency and pH-responsive drug release, which can significantly improve the body's immunity, enhance the body's ability to absorb drugs, and have good safety to animals. These findings proposed a well characterized novel CS-MWCNT-HEP formulation as drug delivery system, and its mechanism and application will be further investigated in our undergoing studies. Acknowledgements The project was supported by National Natural Science Foundation of China (31802236), Scientific Research Program of Outstanding Youth of Fujian Agriculture and Forestry University (XJQ201816), Natural Science Foundation of Fujian Province of China (2019J01378), The Education Department Foundation of Fujian Province
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Table 1 Table of the factors and levels for Box-Behnken design. X1
X2
X3
(ultrasonic time)
(ultrasonic power)
(HEP: CS-MWCNT)
-1
20
40 %
3
0
30
60 %
4
+1
40
80 %
5
factor
Table 2 The experimental design and results of RSM. Levels of independent factors
Response loaded efficiency (%)
RUN A
B
C
Experimental
Predicted
1
40
80
4
61.97
61.76
2
40
60
3
58.84
59.41
3
30
40
3
60.63
59.71
4
30
80
3
66.34
65.98
5
40
40
4
54.69
55.05
6
20
40
4
57.89
58.10
7
30
60
4
70.47
69.63
8
30
60
4
69.48
69.63
9
20
80
4
64.91
64.56
11
40
60
5
63.62
62.90
11
20
60
5
66.73
66.16
12
30
80
5
69.20
70.13
13
30
60
4
69.79
69.63
14
30
40
5
62.85
63.21
15
20
60
3
61.28
62.00
16
30
60
4
70.05
69.63
17
30
60
4
68.36
69.63
Table 3 Statistic analysis of variance for the experimental results of the BBD. Sum of Source
Mean df
squares
F value
P-Value
Prob >F significant
square
Model
365.54
9
40.62
43.43
<0.0001
X1
17.12
1
17.12
18.30
0.0037
X2
86.91
1
86.91
92.92
<0.0001
X3
29.27
1
29.27
31.30
0.0008
X1 X2
0.017
1
0.017
0.018
0.8980
X1 X3
0.11
1
0.11
0.12
0.7395
X2 X3
0.10
1
0.10
0.11
0.7477
X12
149.15
1
149.15
159.47
<0.0001
X22
61.27
1
61.27
65.50
<0.0001
X32
4.76
1
4.76
5.09
0.0587
Residual
6.55
7
0.94
Lack of fit
4.01
3
1.34
2.11
0.2418
Pure error
2.54
4
0.63
Cor total
372.09
6
R2 = 0.9824; R2adj = 0.9598; R2Pred = 0.8169. Fig 1
not significant
Fig. 1 Response surface plots (A, C, and E) and contour plots (B, D, and F) of loaded efficiency affected by ultrasonic time (X1), ultrasonic power (X2), and the concentration of HEP: CS-MWCNT (X3).
Fig 2
Fig. 2 Transmission electron microscope of (A, B, and C) MWCNT (A), CS-MWCNT (B), CS-MWCNT-HEP (C).
Fig 3
Fig. 3. Raman spectrum of MWCNT, CS-MWCNT, CS-MWCNT-HEP.
Fig 4
Fig. 4 Release profiles of HEP at different pH condition.
Fig 5
Fig. 5 Fluorescence microscopy of CS-MWCNT-HEP uptake by Raw264.7 cells (A) and Flow diagram of Raw264.7 cell uptake CS-MWCNT-HEP (B).
Fig 6
Fig. 6. Distribution of drugs (a, b, c, d) Saline (a), CS-MWCNT (b), HEP (c), CS-MWCNT-HEP (d) in mice (A), fluorescence intensity of drugs over time (B) and ratio of organ fluorescence intensity to drug fluorescence intensity (C). Bars without the same superscripts (a-d) differ significantly (P<0.05).