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@#In order to improve the poor solubility and low bioavailability of paeonol (Pae), paeonol-nanoemulsion (Pae-NE) was prepared, and its effect on uptake of human umbilical vein endothelial cells (HUVECs) was investigated.Pae-NE was prepared by phase inversion composition (PIC), the formulation of Pae-NE was optimized by single factor method and central composite design-response surface method (CCD), and the pharmaceutical properties were further characterized.Moreover, MTT was applied to evaluate the toxicity of Pae-NE on HUVECs, and the cellular uptake efficiency of Pae-NE was detected by fluorescence microscopy and flow cytometry.The results showed that the optimal formulation of Pae-NE was 20 mg of Pae, 55.1 mg of LCT, 144.9 mg of MCT, 600 mg of HS15, and 200 mg of 1,2 propylene glycol.The Pae-NE appearance was a light blue emulsion, and the average particle size is (25.69 ± 0.03) nm, with PDI of 0.182 ± 0.09, Zeta potential of -(4.01 ± 0.30) mV and good stability.The drug loading of Pae-NE was (1.967 ± 0.28) mg/mL and encapsulation rate of (99.36 ± 0.1)%.Pae-NE performed no significant effect on HUVECs growth in the Pae concentration range of 10-1-10-3 μg/mL.Moreover, NE as a drug delivery carrier significantly enhanced the uptake efficiency of Pae on HUVECs.In conclusion, Pae-NE preparation method was simple and stable, and promotes HUVECs uptake efficiency of Pae, suggesting that NE was a better dosage form reference for the lipid-soluble drug of Pae.
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@#Polyethylene glycol (PEG) of different lengths were prepared to investigate their effects on oral absorption of nanostructured lipid carrier (NLCs).Three kinds of PEG-modified NLCs with different chain lengths, including polyethylene glycol (100) monostearate (S100), polyethylene glycol (55) monostearate (S55), polyethylene glycol (40) monostearate (S40), were prepared by film dispersion method.Coumarin 6 was chosen as a fluorescent probe to characterize the physicochemical properties of NLCs with different lengths.Meanwhile, the stability of NLCs in simulate buffer, the release behavior, cytotoxicity of NLCs, the uptake kinetics and cellular uptake mechanisms were evaluated. This work demonstrated that the thickness of the hydrated layer increased with the increase of PEG length. Of note, S100-modified NLCs (pNLC-EG100) exhibited higher cellular uptake efficiency compared with other formulations. Thus, S100 was optimized as the best molecular weight for PEG-modified NLCs on oral drug delivery system.
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When nanoparticles were introduced into the biological media, the protein corona would be formed, which endowed the nanoparticles with new bio-identities. Thus, controlling protein corona formation is critical to
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Objective: To optimize the formulation of 1,8-cineole self-microemulsifying drug delivery system (1,8-Cin-SMEDDS), characterize it and investigate its cell uptake. Methods: By drawing pseudo-ternary phase diagram, the effective self-emulsifying region of 1,8-Cin-SMEDDS was determined, and the preliminary prescription was screened. Taking the particle size and drug loading as the index, the central composite design-response surface method was used to optimize and verify the prescription. Fluorescence microscope was used to observe the uptake of human umbilical vein endothelial cells (HUVEC) injured by high glucose. Results: The results showed that the best prescription of 1,8-Cin-SMEDDS was a mixture of soybean oil (7.5%) and 1,8-Cin (22.5%), HS15 (56%) as emulsifier, ethanol (14%) as co-emulsifier, and dripping pure water to 8 mL to obtain a translucent slightly bluish emulsion. The appearance of spherical droplets was observed by transmission electron microscope, and the average particle size and Zeta potential measured by laser particle size Zeta tester was (131.68 ± 1.44) nm and (-10.03 ± 1.63) mV, respectively; The entrapment efficiency estimated by HPLC was (99.890 ± 0.012)%, and the drug loading was (224.750 ± 0.028) mg/g. The results of HUVEC cell uptake assay showed that the uptake of 1,8-Cin-SMEDDS by cells was higher than that of free 1,8-Cin. Conclusion: The preparation method of 1,8-Cin-SMEDDS is simple and reproducible. The obtained method has good appearance, high entrapment efficiency, stable physical, and chemical properties, which can also promote cell uptake.
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Objective: To prepare norcantharidin TPP-PEG-PCL nanomicelles and study its release in vitro, intracellular transport and promoting effect on hepatoma cell apoptosis. Methods: Thin film hydration method was used to prepare norcantharidin TPP-PEG-PCL nanomicelles, and the particle size, electric potential and microscopic electron microscopy morphological analysis were measured. At the same time, the nanomicelles were evaluated for stability, in vitro release, pharmacokinetics and critical micelle concentration. Coumarin-6 was used as a fluorescent probe to evaluate the uptake of TPP-PEG-PCL nanomicelles in liver tumor cells, lysosomal escape and mitochondrial targeting function; Under the same dosage conditions, the effect of norcantharidin TPP-PEG-PCL nanomicelles on promoting apoptosis of liver tumor cells was evaluated. Results: The cantharidin TPP-PEG-PCL nanomicelles had a particle size of (16.8 ± 0.2) nm, a Zeta potential of (14.3 ± 0.2) mV, and transmission electron microscopy images showed that nanomicelles had a regular spherical shape. The fluorescence test results showed that TPP-PEG-PCL nanomicelles can promote the cellular uptake of drugs, escape lysosomal capture, and finally target aggregation at the mitochondrial site; Cell survival rate and Hoechst staining results showed that cantharidin TPP-PEG-PCL nanomicelles had a good effect on promoting apoptosis of liver tumor cells. Norcantharidin TPP-PEG-PCL nanomicelles can significantly reduce mitochondrial membrane potential, increase intracellular ROS levels, increase pro-apoptotic protein Bcl-2, and reduce resistance. The expression of apoptotic proteins Bax and these pro-apoptotic related experimental results are significantly better than those of norcantharidin PEG-PCL nanomicelles and norcantharidin, which have statistical significance. Conclusion: Norcantharidin TPP-PEG-PCL nanomicelles have good liver tumor cell mitochondrial targeting and promote tumor cell apoptosis, and it is a potentially effective drug delivery system for targeting tumor cell mitochondria.
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Fluorescent polystyrene nanospheres (PS) were used to explore the impact of substrate stiffness on cell uptake of nanoparticles in the breast cancer cells. Polyacrylamide (PAA) gels with varying stiffness were prepared by photopolymerization, and type I rat tail collagen was covalently conjugated on the surface of PAA gels to facilitate cell adhesion. Type I rat tail collagen was also used to fabricate collagen gels for 3D cell culture. Cells of human breast cancer cell line MCF-7 were incubated in the 2D culture on PAA gels and 3D culture within collagen gels. Next, nanospheres of 20 nm and 50 nm polystyrene were applied to MCF-7 cells in the 2D or 3D cultures. Cell morphology and uptake efficiency were observed with confocal microscopy. Our study demonstrates that substrate stiffness differentially regulated the cell morphology as well as the cell uptake behavior of polystyrene nanospheres in MCF-7 cells under 2D or 3D culture conditions.
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Objective To investigate several related problems possibly affecting the measurement results in the experiment of 125I-FSH in vitro cells uptake by domestic GC-1200 gamma RIA counter.Methods Before entering the measuring room,the sample was performed the background measurement.CPM measured in the different locations of same measurement frame and the different locations in unmeasured area were performed the statistical comparison.Results In high count,the influence of single sample reaching 1.9 ×106CPM on the adjacent low counting tube count was 7%;its influence on low counting tube count in adjacent detector was 7.33%;all samples were arranged from high to low order and the high count sample holder was placed on the measured location close to the detector,its influence on low counting tube count was 5.33%.Conclusion The domestic GC-1200 γ RIA counter is suitable for the measurement of the in vitro cell uptake experiment of 125I nuclear labeling.
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OBJECTIVE:To study the in vitro uptake of Resibufogenin(RBG)lactic acid glycolic acid copolymer-water solu-ble vitamin E (PLGA-TPGS) in human liver cancer HepG2 cells,mouse ascites-type lymphatic metastasis of tumor HCa-F cells, and the toxicity on HepG2 cells. METHODS:RCPTN loading RBG and coumarin-6(C6)were prepared. Fluorescent inverted mi-croscope was used to observe the in vitro uptake by RCPTN HepG2,HCa-F cells. It was divided into negative control group,blank PLGA-TPGS nanoparticles(EPTN)group,5-fluorouracil solution(FS)group,RBG solution(RS)group,RBG/PLGA nanoparti-cles(RPN)group and RPTN group. WST-1 was conducted to investigate the optical density at 450 nm wavelength of HepG2 cells after 24,48,72 h incubated by FS,RS,RPN and RPTN with different final concentrations (1.25,2.5,5,10,20 μg/mL);the cell viability (CV) and half inhibitory concentration (IC50) were calculated. RESULTS:RCPTN distributed around the nucleus of HepG2,HCa-F cells. CV was decreased by RBG concentration increased in RPN group and RPTN group,and decreased by time prolonged;compared with FS group,CV in RPTN group was decreased(PFS>RPN>RPTN;IC50 incubated by RPN and RPTN for 48,72 h was obviously less than that of FS and RS(P<0.05 or P<0.01). CONCLUSIONS:RPTN can deliver RBG in-to HepG2,HCa-F cells,showing inhibition effect on HepG2 cells which is stronger than RPN,RS and FS.
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Objective: To prepare nanocomplex comprising tea polyphenol and melittin (EMN) with synergistic effect for antitumor therapy. Methods: Poly-epigallocatechin gallate (polyEGCG) was synthesized by Bayer reaction and characterized by 1H-NMR and MS spectra. The preparation process of EMN was optimized by orthogonal test, and the in vitro release of EMN was conducted at different medium with different pH. The cell uptake of FITC-labeled Mel and EMN was measured by flow cytometry. The synergistic antitumor effect of polyEGCG and Mel was investigated using B16F10 and A549 cells by MTT assay. Results: The structure of polyEGCG was confirmed by 1H-NMR and MS. The optimized preparation process of EMN was as follws: 0.5 mg/mL polyEGCG solution was added dropwise to 1 mg/mL equal volume of Mel solution, and then incubated at room temperature for 30 min. In vitro release studies showed that acidic environment could accelerate the release of Mel. Cell uptake experiments showed that the cell uptake of Mel and EMN was comparable. MTT assay results showed the combination index (CI) of polyEGCG and Mel was less than 1, which intimated the synergistic effect when combined therapy with polyEGCG and Mel. Conclusion: EMN was prepared by a self-assembly method, which has a simple preparation process, a suitable particle size, and synergistic antitumor effect, and it is worthy of further studies.
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OBJECTIVE: To prepare amphiphilic polycaprolactone-poly (arginine polymer) (PCL-R15)/siRNA Nanopexes, and two kinds of nanoparticles with different particle size were prepared by different process. After encapsulated siRNA with electrostatic interaction, both of two nanoplexes (NR60/siRNA and NR160/siRNA) were used to compare the effects in vitro cell levels. METHODS: The particle size and Zeta potential, siRNA loading and protection ability, cytotoxicity, cellular uptake mechanism and gene silencing efficiency of the two nanoplexes were investigated. RESULTS: The results show that the two nanoplexes have similar siRNA protection ability and cytotoxicity, but the difference between the two sizes is about 100 nm and the potential difference is about 20 mV. Moreover, NR160/siRNA complexes have higher cell uptake efficiency, more complex uptake pathways, and show greater gene silencing efficiency. CONCLUSION: These nanoplexes with different particle sizes can cause different transfection efficiency for siRNA delivery in cells.
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Objective: To investigate the effect of astragaloside IV on tumor cellular uptake and antitumor efficacy by ginsenoside compound K (GCK). Methods: The human lung adenocarcinoma cell line A549 was prepared as an antitumor model, the cytotoxicity of the mixtures of GCK and astragaloside IV to A549 cells was determined by MTT assay, the cellular uptake of GCK was detected by fluorescence microscopy, and the intracellular GCK was determined by HPLC. The enhancement of astragaloside IV in vivo efficacy by GCK was evaluated by nude mice bearing tumor model. Results: The effect of GCK on the inhibition of A549 cell proliferation was enhanced in the presence of astragaloside IV and astragaloside IV could increase the uptake rate of GCK in A549 cells, with the proportion of astragaloside IV increasing, the cytotoxicity was significantly stronger than GCK and the uptake rate was improved. In vivo antitumor assay of mice bearing tumor indicated that astragaloside IV could enhance the antitumor efficacy of GCK. Conclusion: Preliminary results indicate that the mixture of GCK and astragaloside IV have the effect of enhancing antitumor efficacy.
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Objective: To investigate the effect of D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) on the inhibition of proliferation of breast cancer cells MCF-7 by baohuoside I. Methods: The cytotoxicity of baohuoside I to MCF-7 cells was determined by MTT assay, the cellular uptake of baohuoside I was detected by fluorescence microscopy, and the intracellular baohuoside I was determined by HPLC. Results: The effect of baohuoside I on the inhibition of MCF-7 cell proliferation was enhanced in the presence of TPGS, especially on lower concentration. The uptake rates of MCF-7 within 2 h were 29.51%, 38.12%, and 40.37%, when the proportions of baohuosaide I and TPGS were 1:1, 1:2, and 1:4, respectively. The ratios were increased by 27.92%, 65.24%, and 74.99% compared with those using baohuoside I only. Conclusion: TPGS can increase the uptake rate of baohuoside I in MCF-7 cells and enhance the inhibition of MCF-7 cell proliferation.
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Objective: The study aims at preparing the didodecyldimethylammonium bromide (DMAB)-modified PLGA nanoparticles (NPs) loading tetrandrine (Tet) (DMAB-Tet-PLGA-NPs) and investigating the preparation process, physicochemical characterization, in vitro cytotoxicity, and particle cellular uptake. Methods: DMAB-Tet-PLGA-NPs were prepared by the emulsion solvent diffusion method and the preparation process was optimized with the uniform design experiment. The drug loading, entrapment efficiency (EE), and in vitro drug release were studied to evaluate the drug-loading property. The in vitro cytotoxicity against human lung cancer cell A549 was measured by the standard MTT assay. The particles cellular uptake in A549 was evaluated by qualitative and quantitative methods. Results: DMAB-Tet-PLGA-NPs in the mean size of (205.40±2.66) nm with spherical shape and showed positive surface charge. Drug loading and EE were (2.130±0.035)% and (50.780±3.253)%, respectively. DMAB-Tet-PLGA-NPs could retard drug release in pH 7.4 release media and the cumulative release was up to 64.56% over 48 h. And DMAB-Tet-PLGA-NPs showed the significant dose-and time-dependent cytotoxicity of Tet in vitro and well cellular uptake by A549. Conclusion: DMAB-Tet-PLGA-NPs shows the good EE, uniform particle size, and could retard drug release in vitro. And DMAB-Tet-PLGA-NPs shows the significant cytotoxicity in vitro and well cellular uptake by A549.
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Objective: To synthesize butyryl galactose ester (But-Gal) and prepare butyryl galactose ester-modified coix component microemulsions (But-Gal-CMEs) and to evaluate its physicochemical properties and anticancer activity in vitro. Methods: But-Gal was synthesized by enzyme-catalyzed reaction and the structure of the product was confirmed by 1H-NMR and FT-IR. The CMEs and But-Gal-CMEs were prepared by aqueous titration method using coix seed oil, Cremophor RH40, PEG400, But-Gal, and coixan solution as oil phase, surfactant, cosurfactant, target ligand, and aqueous phase, respectively. The average particle size, polydispersity index (PDI), and Zeta potential were detected by dynamic light scattering (DLS). The cytotoxicity of CMEs and But-Gal-CMEs aganist HepG2 cells was determined by MTT assay. The cellular uptake of CMEs and But-Gal-CMEs was detected by fluorescence microscopy. Results: The structure of But-Gal was confirmed by 1H-NMR and FT-IR. The But-Gal-CMEs displayed the spherical surface with mean droplet size of (57.68 ± 6.65) nm, PDI of 0.070 ± 0.006, and Zeta potential of (-2.95 ± 0.23) mV, respectively. MTT experiments showed that the half of HepG2 cell proliferation inhibition concentration (IC50) of But-Gal-CMEs and CMEs was 62.55 and 71.23 μg/mL. The HepG2 cell uptake results suggested that the fluorescence intensity of But-Gal-CMEs group was higher than that of CMEs group. Conclusion: The But-Gal-CMEs presents small particle size, good roundness, and good stability. In addition, But-Gal could increase the uptake rate of CMEs in HepG2 cells and enhance the inhibition of HepG2 cell proliferation.
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OBJECTIVE: To prepare baohuoside I phytosomes and study its anti-tumor activities in vitro.
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To investigate a new kind of tumor tracer 99mTc-YIGSR developed from a five amino structure (YIGSR) of the Laminin -chain,which can bind to the laminin receptors of tumor specifically, and radiolabeled with MAG3. (1) Preparation of the 99m Tc-YIGSR probe: with S-Acetly-NH3-MAG3as the chelator and with proper reductants YIGSR was labeled with 99mTc; (2) Cell culture and viability measurement: EAC was maintained in RPMI 1640 supplemented with calf serum; the trypan blue exclusion was applied to calculate the cell viability; (3) Study of the cell dynamic: The EACs uptake of 99mTc-YIGSR and99mTc-MIBI was observed at 37 ℃ and 22 ℃, respectively. (1)The labeling efficiencies of 99mTc-YIGSR and99mTc-MIBI were (62±3) % and (96±2) %, respectively; (2) The cell viability was declined with time of incubation; (3) At 37 ℃, the EACS uptake of 99mTc-YIGSR and99mTc-MIBI reached the peak of (43. 16±2.4) % and (24.4±1.8) % at 60min, respectively; and at 22 ℃, the highest uptake was (26.5±2.1) % and (9. 47±1.9) % at 60min, respectively. The in vitro study suggests that 99mTc-YIGSR is superior to 99mTc-MIBI in cell uptake and has potential value in tumor imaging.