| Background:Chinese herb drugs have been widely applied to deal with different kinds of diseases for a long history. Due to the progress of technology, nowadays more elements have been abstracted and purified from Chinese herb drug. Among so many kinds of Chinese herb drugs, traditional Tibetan medicine has driven a lot of public attention because of its some unique therapeutic effects.’Ershiwuwei Shanhu’ Pill (ESP), a kind of traditional Tibetan medicine, is a classic and practical prescription using extensively in patients. It was developed by Dimar Tenzing, a famous Tibetan traditional physician in the 18th century AD, and was reprinted in’The Set Drug Tetra Library’ in the 19th century AD, recording the formulas and indications of ESP. ESP is comprised of 25 kinds of active ingredients, including Chebuloe fructus, Swertia, Carthamiflos, Red coral and so on. Clinically, ESP is applied to the treatment of cerebrovascular diseases and neurological disorders and proved to be eutherapeutic.Drug-loaded nanoparticles from’Ershiwuwei Shanhu’ Pill (ESP) inducing cellular swelling of the SH-SY5Y neuroblastoma cells were investigated. Electron microscope was used to observe nanoparticles existing in the freeze-dried supernatant of ’Ershiwuwei Shanhu’ Pill. Drug-free nanoparticles were obtained from the solution of drug-loaded nanoparticles via dialysis. The size and zeta potential of two kinds of nanoparticles were tested by granularmetric analysis and surface charge analysis. Results showed that nanoparticles could penetrate into cellular nucleus and caused cell swelling. CCK8 analysis implied that low concentration of drug-free nanoparticles from ’Ershiwuwei Shanhu’ Pill can induce cell proliferation of the SH-SY5Y neuroblastoma cells, while drug-loaded nanoparticles can reduce cell viability through NF-κB pathway. Drug-loaded nanoparticles existed in ’Ershiwuwei Shanhu’ pill might play a vital role during pharmacotherapy, which served as nanocarriers in delivering drugs into cells.1. INTRODUCTIONChinese herb drugs have been widely applied to deal with different kinds of diseases for a long history. Due to the progress of technology, nowadays more elements have been abstracted and purified from Chinese herb drug. Among so many kinds of Chinese herb drugs, traditional Tibetan medicine has driven a lot of public attention because of its some unique therapeutic effects.’Ershiwuwei Shanhu’ Pill (ESP), a kind of traditional Tibetan medicine, is a classic and practical prescription using extensively in patients. It was developed by Dimar Tenzing, a famous Tibetan traditional physician in the 18th century AD, and was reprinted in’The Set Drug Tetra Library’ in the 19th century AD, recording the formulas and indications of ESP. ESP is comprised of 25 kinds of active ingredients, including Chebuloe fructus, Swertia, Carthamiflos, Red coral and so on. Clinically, ESP is applied to the treatment of cerebrovascular diseases and neurological disorders and proved to be eutherapeutic. It has been verified that the pharmacological effects of ESP may be related to the absorbed components and metabolites from ESP tested in plasma and tissues. Nevertheless, clinical researches, concerning the specific composition of ESP taking effects on cells, have been blocked by its complicated components and extreme insolubility. Bioavailability of ESP being employed is also decreased by poor water solubility. However, when ESP was delivered by nanoparticles, the water dispersivity of ESP will be improved.Recently, nanotechnology-based traditional medicine has received increasing attention. Nanoparticles can work as carriers to deliver drugs for detection, prevention and treatment of diseases, which raises the use in biomedical application. Decreased particle size increases solubility of poorly water-soluble drugs, which improves the ratio of the drugs that get into the cells and the biological activity of them. Nanoparticles could offer a valid drug delivery system, allowing drugs to reach the intracellular site exactly and therefore, greatly improve therapeutic effect. Hence, we propose the hypothesis whether there has lots of drug-loaded nanoparticles existing in ESP and improving its clinic therapeutic effects.In this study, we extracted drug-loaded nanoparticles from ESP. Two different kinds of nanoparticles, drug-loaded nanoparticles and drug-free nanoparticles, were used to treat SH-SY5Y neuroblastoma cells respectively. The variations of cellular morphology, viability and molecule under different conditions were observed by electron microscope, confocal laser scanning microscopic observation, CCK-8 testing and Western blot analysis.Objective:1. nanotechnology-based and Dialysis-Technology based traditional medicine has received increasing attention. Nanoparticles can work as carriers to deliver drugs for detection, prevention and treatment of diseases, which raises the use in biomedical application.2. To explore effects of the different nanoparticles on SHSY-5Y cell behavior in vitro co-culture environment, in our experiment, human neuroblastoma cells SHSY-5Y and two different drug-loaded nanoparticles were in co-cultured conditions,we detected nanoparticles’intracellular localization, SHSY-5Y activity, apoptosis and proliferation, protein expression and so on. Contrasting different drug-loaded nanoparticles influence on SHSY-5Y cells on biological characteristics on whether there are differences in the comparative analysis.Methods:1. Preparation of drug-loaded nanoparticle and drug-free nanoparticle from ESPlg ’Ershiwuwei Shanhu’ pill was resuspended in 10 ml deionized water. The aqueous mixture was bathed at 37℃ for 1h, and the supernatant was obtained after being centrifuged at 1500g for 1h. The supernatant was dealt with ultrasound at 130 watts, 60% peak output frequency at 0℃ for 10 min, then centrifuged at 16000g for 30 min. Finally, the sample containing nanoparticle was divided into two different groups. In one group, the supernatant was directly freeze-dried to obtain drug-loaded nanoparticles. As the control, the supernatant in another group was dialyzed against deionized water using 8kd MWCO dialysis for four days (changing the water every 4-6 hours), and then freeze-dried to obtain the drug-free nanoparticles.2. Granularmetric analysis and zeta potential distribution Particle size distribution (mean diameter and polydispersity index), and zeta potential were captured utilizing a particle analyzer Malvern Zetasizer (Malvern Instruments, UK). Prior to measurement, samples were suspended in distilled water and then measured at a temperature of 25℃and a scattering angle of 90 degree. For each sample, the mean diameter±tandard deviation were calculated applying multimodal analysis. Values reported are the mean value±tandard deviation for two replicate samples.3. Cell cultureHuman neuroblastoma SH-SY5Y cell line, was grown in high glucose DMEM medium supplemented with 10% fetal bovine serum, 100U/ml penicillin, and 100μg/ml streptomycin. Cells were placed in an incubator at 37℃ with a humid atmosphere of 5% CO2 and the culture medium was refreshed every day.4. Immunofluorescence staining assaysCellular uptake and intracellular distribution of nanoparticle was tracked by confocal laser scanning microscopy by incubating the SH-SY5Y cells with drug-loaded nanoparticles or drug-free nanoparticles. SH-SY5Y cells were seeded in the culture dish with a cover slip at a density of 2×105 cells/dish for 24 h. Then the cells were exposed to drug-loaded nanoparticles and drug-free nanoparticles. After a predetermined incubation time, the cover slip was washed with cold PBS for three times. The cells were fixed by 4% paraformaldehyde at room temperature for 15 min, followed by incubation with DAPI for 30 min. Then the cover slip was set on a microscope slide and then examined by CLSM using a blue laser 405 nm, a green laser 488 nm and a red laser 568 nm for excitation.5. Transmission electron microscopy (TEM)Visualization of particle-cell interaction and intracellular translocation was observed using TEM. The SH-SY5Y cells cultured for 24h were exposed to drug-loaded nanoparticles and drug-free nanoparticles for 8h. After exposure, the particle suspensions were removed and cells were washed with PBS, digested with trypsin and collected into Eppendorf tube respectively. Then the samples were fixed in 2.5% glutaraldehyde, post-fixed in osmic acid and dehydrated in graded acetone. The samples were embedded in epoxy resin. Ultrathin sections were double stained with uranyl acetate and lead citrate, then observed under TEM.6. CCK-8 analysisThe cytotoxicity was determined using a Cell Counting Kit-8 assay (CCK-8). The SH-SY5Y cells were seeded into a 96-well culture plate at a density of 9000 cell per well and cultured at 37℃ in 5% CO2. The culture medium was replaced with fresh DMEM after 24 h. Then drug-loaded nanoparticles solution and drug-free nanoparticles solution were added into every well respectively (six well per sample). After being incubated for 6 h,10 μL CCK-8 solution and 90 μL medium was added to each well and incubated for another 2 h at 37℃. The absorbance at 450 nm was measured using a microplate reader.7. Western blottingThe SH-SY5Y cells were seeded into a 25 cm2 cell culture flask and maintained at 37℃in 5% CO2 for 24 h. Then, the culture medium was replaced with DMEM containing drug-loaded nanoparticles. After incubation for 6 h, cells were washed with cold PBS for three times, and lysed with moderate cell RIPA buffer (1% NP40, 1% DOC,0.1% SDS,150 mM NaCl,10 mM Tris/HCl, pH 7.4) containing 5μl of phosphatase inhibitors, 1μl of Protease inhibitor and 5μl of Phenylmethanesulfonyl fluoride (PMSF) in each 1 ml lysis buffer. Total protein concentrations were measured using BCA Protein Assay Kit. After boiling for 5min at 100℃, equal amounts of the samples were subjected to 10% SDS-PAGE for separation. After electrophoresis, proteins were transferred to PVDF membrain in transfer buffer at 4℃. Membranes were blocked with 5% fat-free milk for 1 h at room temperature, washed with 1 x TBST (8g Nacl,0.2g Kcl,3g Tris-base and 1ml Tween 20 in 1L ultrapure water) and incubated with primary antibody, including anti-NF-κB p65 (1:1000), anti-NF-KB p-p65 (1:1000) and anti-β-actin (1:1000) at 4℃ overnight. The HRP-Labeled goat anti-rabbit IgG (1:5000) and goat anti-mouse IgG (1:5000) were used as the secondary antibodies. Images were visualized using Western Blot Super ECL Plus Detection Reagent kit under Tanon-5500 automatic chemiluminescence system.8. Data are presented as means standard deviation (SD). Statistical analysis of differences between multiple groups was performed using the one-way analysis of variance (ANOVA). Statistical analyses were performed with SPSS (version 13.0), p values of less than 0.05 were considered significant.Results:1. Nanoparticles appeared in ’Ershiwuwei Shanhu’PillThe prepared powder of ESP was obtained by grinding ESP in a porcelain mortar. The powder of ESP exhibited different fluorescence in different excitation light. Most of large-scale insoluble components in ESP solution have difficulties in penetrating into cells considering the particle size. Here, we tried to extract drug-loaded nanoparticles from ESP. Due to the poor water solubility, the powder of ESP suspended in water was centrifuged twice at different G-force to collect the supernatant in turn. The supernatant, containing drug-loaded nanoparticles was freeze-dried. The various size of nanoparticles could be observed under transmission electron microscope.2. Most of the tibetan medicines, ESP included, are in poor water solubility, high toxicity and short valid period in vivo, which has always been the main limitation of biotechnology industries. Thus, nanotechnology-based drug delivery system has driven public attention. This delivery system is known for delivering drugs more targeted and easily controllable release. To explore the influence of nanoparticles from ESP on drugs’ bioactivity, two kinds of nanoparticles, drug-loaded nanoparticles and drug-free nanoparticles were separated in this study. The former was directly extracted from ESP, while the latter was obtained from processing the supernatant of ESP by dialysis (MWCO 7000Daldo). Then the granularmetric and surface charge were analyzed on drug-loaded nanoparticles and drug-free nanoparticles respectively. As for sizes, about 84.4% drug-loaded nanoparticles ranged from 68.6nm to 122.4 nm. By contrast, almost 90% drug-free nanoparticles ranged from 37.84 nm to 58.77 nm. Obviously, the drug-loaded nanoparticles were larger than the drug-free nanoparticles. The magnified diameter of nanoparticles implied that drugs were loaded on the nanocarriers. The sizes of drug-loaded nanoparticles whose intensity could be detected ranged from 78.82 nm to 1106 nm, a continuous interval. While the sizes of drug-free nanoparticles whose intensity could be detected ranged from 37.84 nm to 78.82 nm and from 220 nm to 615 nm, a segmented interval. Drug-free nanoparticles lacked nanoparticles in size ranging from 78.82 nm to 220 nm. This showed that drug-free nanoparticles were in different sizes and took on discrete values and indicated that different drug-loading rate was displayed in different nanocarriers. The further study showed that most of the drug-loaded nanoparticles were in-21.5mV zeta potential, while most of the drug-free nanoparticles were in -9.31mV zeta potential. Distinctively, after drug-loaded nanoparticles got rid of the carrying drugs, the number of negative charge ions had been decreased, indicating that the drugs were with negative zeta potential. Judging from the results, we supposed that nanocarriers can reduce the negative zeta potential of drugs and make them more easily penetrating into cells.3. Cell Swelling Induced by Drug-loaded NanoparticlesMost of the tibetan medicines, ESP included, are in poor water solubility, high toxicity and short valid period in vivo, which has always been the main limitation of biotechnology industries. Thus, nanotechnology-based drug delivery system has driven public attention. This delivery system is known for delivering drugs more targeted and easily controllable release.Because of the poor water solubility and complicated constituents of ESP, the active ingredients which played a part in cells was still not clear. In order to detect the effects of drug-loaded nanoparticles on cells, the SH-SY5Y cells challenged with drug-loaded nanoparticles and drug-free nanoparticles respectively, were observed under the view of confocal microscopy. Because nanoparticles from ESP had different fluorescence under different excitation light, their tracks could be detected by confocal microscopy. It had been shown that drug-loaded nanoparticles entered into cell nucleuses, while the drug-free nanoparticles could fill the whole cells. Cellular uptake of nanoparticles could be divided into two steps. First, nanoparticles bound on the cell membrane, which was affected by the surface charge of nanoparticles. Second, it was the internalization of nanoparticles. A passageway could be seen in the nuclear membrane of cell nucleuses. It might suggest that nanoparticles formed a passageway in nucleus membrane to realize the internalization. This result could be further demonstrated, which was the view of electron microscope.By comparing, the nucleuses of SH-SY5Y cells treated with 500μg/ml drug-loaded nanoparticles were larger than those of SH-SY5Y neuroblastoma cells treated with 41.5μg/ml drug-free nanoparticles(equivalent to the total nanoparticles dialyzed from 500μg/ml drug-loaded nanoparticles), which indicated that drug-loaded nanoparticles induced the swelling of cell nucleuses, that is, the swelling of cells, while drug-free nanoparticles would not. As shown, cell borders of the SH-SY5Y cells treated with drug-loaded nanoparicles almost couldn’t be seen. While the cell borders of the SH-SY5Y cells treated with drug-free nanoparicles were clear to recognize. Thus we supposed that the swelling of cells caused by drug-loaded nanoparticles might result in the hardness of recognition of cell borders. By comparing, the intensity of red fluorescence of the cells treated with drug-loaded nanoparticles and drug-free nanoparticles was almost the same, but via comparing, the intensity of green fluorescence of the cells treated with drug-free nanoparticles became obviously weaker than that treated with drug-loaded nanoparticles. It indicated that most of nanoparticles exposed to excitation light were in red fluorescence and most of drugs exposed to excitation light were in green fluorescence, so the rid of the drugs would weaken the intensity of green fluorescence.4. After the SH-SY5Y cells being challenged with drug-loaded nanoparticles or drug-free nanoparticles, CCK-8 analysis was used to detect the cellular bioactivity. The higher the OD values were, the stronger the cellular bioactivity was, which meant that the speed of cellular multiplication was faster.In this study, cells were treated with different concentrations of drug-loaded nanoparticles and drug-free nanoparticles for 6 hrs respectively. Each experimental group was repeated three times independently. As shown, after being challenged with 16.6ug/ml drug-free nanoparticles (NP200, corresponding to the total nanoparticles dialyzed from 200μg/ml drug-loaded nanoparticles) for 6 h, OD value was higher than the control group, indicating that drug-free nanoparticles in a low concentration could induce cell proliferation. However,200μg/ml drug-loaded nanoparticles treatment could not induce cell proliferation under the same conditions. After being treated with 2000μg/ml drug-loaded nanoparticles for 6 h, the cell proliferation was inhibited, while 166μg/ml drug-free nanoparticles (NP2000, corresponding to the total nanoparticles dialyzed from 2000μg/ml drug-loaded nanoparticles) had no effect on them. These results indicated that drugs loaded in nanoparticles are the main toxic sources for cells, while the drug-free nanoparticles were relatively non-toxic.5. Western blot analysis showed that 500μg/ml drug-loaded nanoparticles treatment for 8 h could reduce NF-κB activation through inhibiting the expression of NF-κB/p65 proteins and its phosphorylation. According to above data, drug-loaded nanoparticles had effects on the NF-Kappa B, a family of transcription factors which is related to cell apoptosis. P65 contains Rel homology domain (RHD) on N-terminal and transactivation domain (TD) on C-terminal. RHD is responsible for DNA binding, dimerization and nuclear translocation, while TD is related to transcription activation. So when drug-loaded nanoparticles decreased the contents of p65 subunits and its phosphorylation, cell transcription activation would be hindered. Then the process of protein translation would also be blocked, which influenced cell proliferation. It meant that drug-loaded nanoparticles inhibit cell proliferation and induce cell swelling mediated by NF-Kappa B pathway.Conclusions:1. According to this study, nanoparticles, which played a part in carrying drugs, widely existed in ESP.2. It was demonstrated that nanoparticles had no toxicity on cells, that is, could not induce the swelling of cells. Meanwhile, drug-loaded nanoparticles could increase the toxicity of drugs as carriers on the SHSY-5Y neuroblastoma cells. The nanoparticles can form passageway in nucleus membrane, which help to deliver drugs into nucleus more easily. This is the main reason that toxic drug-loaded nanoparticles from ESP penetrated into nucleus and triggered cellular swelling of the SH-SY5Y neuroblastoma cells. As shown in the results of western blotting, the toxicity of drug-loaded nanoparticles was related to NF-κB activation through decreasing the expression of NF-κB/p65 protein and its phosphorylation. The results in this study facilitate further studies on the therapeutic effect of particularly component from ESP for the treatment of cerebrovascular diseases and neurologic disorders. |
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