Effects of Platycodonis Radix-Curcumae Rhizoma on oral nanoparticle uptake and in vitro inhibition against breast cancer metastasis / 中国中药杂志

This study combined the herbal pair Platycodonis Radix-Curcumae Rhizoma(PR-CR) possessing an inhibitory effect on tumor cell proliferation and metastasis with the active component of traditional Chinese medicine(TCM) silibinin-loaded nanoparticles(NPs) with a regulatory effect on tumor microenvironment based on the joint effect on tumor cells and tumor microenvironment to inhi-bit cell metastasis. The effects of PR-CR on the cellular uptake of NPs and in vitro inhibition against breast cancer proliferation and metastasis were investigated to provide an experimental basis for improving nanoparticle absorption and enhancing therapeutic effects. Silibinin-loaded lipid-polymer nanoparticles(LPNs) were prepared by the nanoprecipitation method and characterized by transmission electron microscopy. The NPs were spherical or quasi-spherical in shape with obvious core-shell structure. The mean particle size was 107.4 nm, Zeta potential was-27.53 mV. The cellular uptake assay was performed by in vitro Caco-2/E12 coculture cell model and confocal laser scanning microscopy(CLSM), and the results indicated that PR-CR could promote the uptake of NPs. Further, in situ intestinal absorption assay by the CLSM vertical scanning approach showed that PR-CR could promote the absorption of NPs in the enterocytes of mice. The inhibitory effect of NPs on the proliferation and migration of 4T1 cells was analyzed using 4T1 breast cancer cells and co-cultured 4T1/WML2 cells, respectively. The results of the CCK8 assay showed that PR-CR-containing NPs could enhance the inhibition against the proliferation of 4T1 breast cancer cells. The wound healing assay indicated that PR-CR-containing NPs enhanced the inhibition against the migration of 4T1 breast cancer cells. This study enriches the research on oral absorption of TCM NPs and also provides a new idea for utilizing the advantages of TCM to inhibit breast cancer metastasis.

Preparation and cellular uptake study of anemoside B4 and PD-L1 siRNA co-delivered cRGD-modified targeting liposomes / 中国药房

OBJECTIVE To prepare anemoside B4 （AB4） and programmed cell death ligand 1 （PDL1） siRNA （siP） co- delivered cRGD-modified targeting liposomes （AB4/siP-c-L）， and to study the cellular uptake in vitro. METHODS The cRGD- modified AB4-loaded targeted liposomes （AB4-c-L） were prepared by ethanol injection. AB4-c-L was mixed with 20 nmol/L siP in the same volume and AB4/siP-c-L was obtained through electrostatic adsorption. The particle size， Zeta potential， morphology， encapsulation efficiency and drug content， in vitro release behavior and serum stability of AB4/siP-c-L were investigated by laser scattering particle size tester， transmission electron microscopy， ultrafiltration centrifugation， dialysis and agar-gel electrophoresis block test. Cellular uptake of AB4/siP-c-L by Lewis lung cancer cells LLC and its intracellular localization were evaluated by flow cytometry and confocal laser scan technique. RESULTS The average particle size of AB4/siP-c-L was （187.4±3.1） nm， and the Zeta potential was （33.5±1.4） mV. AB4/siP-c-L was spheroidal in shape. The encapsulation efficiency and content of AB4 were （95.2±0.4） % and （1.0±0.2） mg/mL， respectively. AB4/siP-c-L could better package siP， and exhibited good serum stability， obvious pH sensitivity and sustained release property. The uptake rate of AB4/siP-c-L by LLC cells was significantly higher than that of free drug， and was able to accumulate in cytoplasm. CONCLUSIONS AB4/siP-c-L can effectively realize the co-loading of AB4 and gene drug siP， which has certain in vitro targeting to LLC cells.

Cellular Uptake of Catharanthus roseus-Silver Nanoparticles in Human Hepatocellular Carcinoma HepG2 Cells

@#Introduction: Nanoparticles exhibit unique features and currently at the forefront of cutting-edge research. Silver nanoparticles (AgNPs) are among the most promising and widely commercialised nanoproducts in various fields. The interaction of these AgNPs with cells remain unclear to connect with its toxicological endpoints. The aim of this study was to investigate the cellular uptake of C. roseus-AgNPs in hepatocellular carcinoma cells HepG2. Methods: The HepG2 cells were treated with the mean IC50 value of C. roseus-AgNPs which was 4.95±0.26 µg/mL for 24, 48 and 72 hours. The effects were compared with the untreated cells and other treatments which include camptothecin, C. roseus-aqueous extract, and AgNO3 . Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to quantify the intracellular Ag+ and Ca2+, while transmission electron microscopy (TEM) imaging was used to visualise the nanoparticle distribution. Results: The HepG2 cells have significantly taken up Ag+ from C. roseus-AgNPs with at least six times higher compared to Ag+ from AgNO3 . The intracellular Ca2+ detected in HepG2 cells for all treatments were significantly higher than the untreated cells, in time-dependent manner. TEM images indicated the endocytosis of C. roseus-AgNPs with the presence of endosomes and exocytic vesicles. Conclusion: The significant accumulation of intracellular Ag+ demonstrated the efficiency of the C. roseus-AgNPs uptake while the increased Ca2+ indicated the early sign of cell injury. The cellular uptake was mainly through endocytosis. These findings are crucial to correlate the physicochemical properties of C. roseus-AgNPs with the anticancer mechanisms towards the development of liver cancer therapy.

Pharmacokinetic analysis of mouse short interspersed nuclear element B1 antisense RNA / 中国生物制品学杂志

@#Objective To investigate the biological distribution of B1 antisense RNA(Blas RNA) of mouse short interspersed nuclear element in blood and tissues of normal mice after vein injection and detect the cell uptake efficiency of B1 as RNA using cultured normal mouse embryo cells after transfection.Methods Six 8～12-week-old BALB/c mice,three males and three females,were injected with 20 μg Blas RNA via tail vein,and blood samples were collected at different times after injection.54 BALB/c mice of 8～12-weeks were injected with 20 μg Blas RNA via tail vein,of which six mice,three males and three females,were euthanized at different times after injection,and various tissues,including the heart,liver,spleen,lung,kidney and thymus were harvested.Blas RNA was transfected into cultured mouse embryonic cells,and a certain amount of cells were taken at different time after transfection.The biological distribution of B1as RNA in mouse blood and different tissues and the persistence of Blas RNA in cultured embryo cells were detected by RT-qPCR.30naturally senescent BALB/c mice(≥ 14 months old) were divided into three groups:treatment group(20 μg Blas RNA injected via tail vein,once a week),irrelevant RNA control group(20 μg LacZ3F3R RNA injected via tail vein,once a week) and saline control group(injected with the same volume of saline),with 10 mice in each group,and a young control group(normal young 8～12-week-old BALB/c mice,five males and five females) was set.Four weeks after administration,mice in each group were euthanized,the liver tissues were taken,and the expression levels of aging-related genes(Sirtl,p21,p16~(Ink4a),p15~(Ink4b),p19~(Arf)) were detected by RT-qPCR.Results After tail vein injection,Blas RNA was available in the blood of mice for approximately 30 min,persisted for approximately 2～4 h in most detected tissues and persisted for approximately 48 h in lungs.The efficiency of cellular uptake of Blas RNA was approximately 400 molecules per mouse embryo culture cell 45 min after transfection with B1as RNA.Compared with the saline control group,Blas RNA treatment significantly down-regulated the mRN A expression of p21,p16~(In4a),p15~(In4b) and p19~(Arf) genes(t=10.01,4.461,4.420 and 5.309 respectively,each P <0.05),and significantly up-regulated the mRNA expression of Sirt1 gene(t=4.579,P <0.05).Conclusion Blas RNA was efficiently taken up by cells after transfection.After intravenous injection,Blas RNA stayed in the blood and tissues for a certain period of time and regulated the expression of aging-related genes in aged mice so as to make them approach to the expression level of young normal mice.

Cellular behavior of carbon dots derived from Poria cocos polysaccharide / 药学学报

The biological behavior of carbon dots, especially the mechanism of cellular uptake and intracellular distribution, is the basis of its biomedical applications. In this paper, blue fluorescent carbon quantum dots were synthesized by hydrothermal method with Poria cocos polysaccharide as raw material, and the specific biological behavior of carbon dots entering cells was explored to evaluate its biological activity. It was characterized by transmission electron microscopy, UV-vis absorption spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction and X-ray photoelectron spectroscopy. Two different cell lines, immunocytes-RAW264.7 cells (mouse mononuclear macrophages cells) and cancer cells-4T1 cells (mouse breast cancer cells), were used as the research objects to study the uptake kinetics, uptake pathway, distribution and efflux of polysaccharide carbon dots in cells. The results showed that the carbon dots have a size distribution of 2 to 10 nm, and the average size was 6.85 nm. The carbon dots were mainly composed of C, O and N elements, with abundant surface functional groups such as -OH, C=O, C-N and C=C, and the fluorescence quantum yield was 4.72%. Carbon dots enter cells in a certain concentration and time dependence. Different cell lines have different uptake pathways. RAW264.7 cells enter the cells mainly by macrophage-specific phagocytosis, and a small part of the endocytosis is mediated by caveolin, while 4T1 cells are mainly mediated by grid protein endocytosis and giant cell drinking process. In summary, the synthesized carbon dots have good fluorescence properties, low cytotoxicity and excellent biocompatibility, which can be used for cell imaging applications.

Synthesis of two folate conjugates and their targeting effect in vitro / 中国药科大学学报

@#The aim of this study was to study the synthesis of two folate conjugates and their application in the preparation of folate targeted liposome, and to investigate their targeting effect in hepatocellular carcinoma HepG2 cell line in vitro. In this study, Folate-PEG-Cholesteryl hemisuccinate(Folate-PEG2000-CHEMS and Folate-PEG4000-CHEMS)were synthesized by linking folate and cholesterol succinate with two kinds of PEG materials. Structures of Folate-PEG2000-CHEMS and Folate-PEG4000-CHEMS were characterized by 1H NMR and ultra-high resolution mass spectrometry. Calcein was selected as the model drug, and calcein liposomes FA-PEG2000-L and FA-PEG4000-L were prepared by film dispersion method using Folate-PEG2000-CHEMS and Folate-PEG4000-CHEMS, respectively. The particle size and Zeta potential of FA-PEG2000-L and FA-PEG4000-L were measured by laser particle size analyzer. The drug delivery effect of FA-PEG2000-L and FA-PEG4000-L was evaluated by cellular uptake experiment in HepG2 cell line in vitro. Flow cytometry and laser confocal scanning microscope were used to determine fluorescence in HepG2 cells in vitro. The results showed that the average particle size of calcein liposome was (205.8 ± 10.2) nm, and the Zeta potential of calcein liposome was -(1.19 ± 0.31) mV.There was no significant difference in particle size and Zeta potential between FA-PEG2000-L and FA-PEG4000-L. The fluorescence intensity of FA-PEG4000-L liposome group was about 3.6 and 3.1 times higher than that of non-targeted liposome group and FA-PEG2000-L liposome group, with statistically significant difference (P < 0.01). The drug delivery efficiency of FA-PEG4000-L group in HepG2 cells was higher than that in FA-PEG2000-L and non-targeted groups, and the results indicated that Folate-PEG4000-CHEMS can promote the uptake of liposome by HepG2 cells in vitro. All in all, Folate-PEG4000-CHEMS could be applied in the preparation of folate targeted liposome, which could promote the uptake of liposome by HepG2 cells.

Effect of physicochemical properties on

Current advances of immunotherapy have greatly changed the way of cancer treatment. At the same time, a great number of nanoparticle-based cancer immunotherapies (NBCIs) have also been explored to elicit potent immune responses against tumors. However, few NBCIs are nearly in the clinical trial which is mainly ascribed to a lack understanding of

Preparation of paclitaxel-loaded and folic acid-modified poly (lactic-co-glycolic acid) nano-micelles and in vitro anticancer effect on cervical cancer HeLa cells / 中国中药杂志

The paclitaxel-loaded and folic acid-modified poly(lactic-co-glycolic acid) nano-micelles(PTX@FA-PLGA-NMs) were prepared by the emulsion solvent evaporation method, and the parameters of paclitaxel-loaded nano-micelles were optimized with the particle size and PDI as evaluation indexes. The morphology of the nano-micelles was observed by transmission electron microscopy(TEM), and the stability, drug loading and encapsulation efficiency were systematically investigated. In vitro experiments were performed to study the cytotoxic effects of nano-micelles, apoptosis, and cellular uptake. Under the optimal parameters, the nano-micelles showed the particle size of(125.3±1.2) nm, the PDI of 0.086±0.026, the zeta potential of(-20.0±3.8) mV, the drug loading of 7.2%±0.75%, and the encapsulation efficiency of 50.7%±1.0%. The nano-micelles were in regular spherical shape as observed by TEM. The blank FA-PLGA-NMs exhibited almost no inhibitory effect on the proliferation and growth of tumor cells, while the drug-loaded nano-micelles and free PTX exhibited significant inhibitory effects. The IC_(50) of PTX@FA-PLGA-NMs and PTX was 0.56 μg·mL~(-1) and 0.66 μg·mL~(-1), respectively. The paclitaxel-loaded nano-micelles were potent in inhibiting cell migration as assessed by the scratch assay. PTX@FA-PLGA-NMs had good pro-apoptotic effect on cervical cancer HeLa cells and significantly promoted the uptake of HeLa cells. The results of in vitro experiments suggested that PTX@FA-PLGA-NMs could target and treat cervical cancer HeLa cells. Therefore, as nanodrug carriers, PTX@FA-PLGA-NMs with anti-cancer activity are a promising nano-system for improving the-rapeutic effects on tumors.

Preparation of citronellol self-emulsifying delivery system and evaluation of its in vitro antitumor activity / 中草药

Objective: To investigate the effect of citronellol (citronellol, CT) on the proliferation of HEp-2 and MCF-7 cells, and prepare CT self-emulsifying drug delivery system (CT-SMs). Its antitumor activity and cell uptake ability of HEp-2 cells in vitro was evaluated. Methods: The effect of CT on the cell proliferation of HEp-2 and MCF-7 were investigated by MTT assay. The pseudo- ternary phase diagram method was used to optimize the formulation of CT-SMs, and the appearance morphology, mean particle size, and Zeta potential were characterized. The effect of CT-SMs on the proliferation of HEp-2 cells was detected by MTT assay and cellular uptake was determined by fluorescence inversion microscopy and flow cytometry. Results: After a certain concentration of CT treatment, MCF-7 cells proliferation was not affected, and the difference was not statistically significant (P > 0.05 compared with the control group), while the proliferative capacity of HEp-2 cells was significantly inhibited (P < 0.05 compared with the control group) in a dose-time dependent manner. The best prescription for CT-SMs was as following: Km (emulsifier:co-emulsifier) was Kolliphor® HS 15:absolute ethanol = 7:3, CT:Km = 3:7, the mean particle size was (354.0 ± 9.5) nm, the appearance was round and spherical with uniform distribution, and the Zeta potential was (-13.4 ± 0.3) mV. The results of cellular uptake experiments showed that the intake of CT-SMs (545.70 ± 11.56) was higher than that of CT (230.00 ± 17.76) in HEp-2 cells treating the same concentration of CT-SMs and CT. Conclusion: CT-SMs could significantly inhibit the proliferation of HEp-2 cells. In this study, CT-SMs were successfully prepared by dropping water method and the quality of CT-SMs was stable and controllable.

Preparation of co-loading photosensitizer/UCNPs/dihydroartemisinin liposomes and evaluation of its in vitro antitumor activity / 中草药

Objective: To develop the photosensitizer rose-bengal (RB)/upconverting nanoparticles (UCNPs)/dihydroartemisinin (DHA) co-encapsulated liposomes (LIP-RUD) and preliminarily study the in vitro inhibition effects on human colon cancer. Methods: The hydrophilic UCNPs were synthesized by solvothermal and ligand conversion and RB/UCNPs/DHA were encapsulated by thin-film dispersion method to obtain LIP-RUD. HPLC was performed to determine the loading ratio (LR) of RB and DHA. Zetasizer was used to evaluate the physiochemical properties of liposomes. The production of ROS was investigated by SOSG probe. In vitro cellular uptake of LIP-RUD was observed by confocal laser scanning microscopy (CLSM) and the cytotoxicity on HCT-116 cells was estimated by MTT assay. Results: LIP-RUD showed an average particle diameter of 150 nm with zeta potential of -12 mV. The LR of RB and DHA were 54.5% and 86.5%, respectively. The energy conversion efficiency of UCNPs and RB reached 49.8%. After irradiation, the singlet oxygen (1O2) was generated and 74.9% of encapsulated DHA was released from LIP-RUD at 12 h, which showed an improvement of up to 25.6% compared to the absence of laser irradiation group. In cellular experiments, LIP-RUD exerted improved cytotoxicity on HCT-116 cells. IC50 was 15.33 μmol/L under laser irradiation. Conclusion: LIP-RUD provides a new thought in the treatment of human colon cancer by the combination of photodynamic therapy (PDT) and chemotherapy, which is expected to enhance the penetration depth of PDT and the therapeutic effect of combination therapy.

Preparation and preliminary evaluation of quercetin and microRNA-150 co-loaded cationic solid lipid nanoparticles for ocular administration / 中草药

Objective: To prepare the cationic solid lipid nanoparticles (Que/mR150 SLNs) co-loaded with quercetin (Que) and microRNA-150 (mR150) and investigate the preparation process, then assess its in vitro release, cell uptake capacity and safety of ocular administration. Method: First, thin-film dispersion method was used to prepare quercetin-encapsulated cationic solid lipid nanoparticles (Que-SLNs), and the preparation process was optimized based on the particle size, PDI and encapsulation rate; Using electrostatic adsorption method to co-load mR150 in nanoparticles (Que/mR150 SLNs), and the adsorption efficiency of the miRNA by the nanoparticles was examined by agarose gel electrophoresis experiment; The in vitro release performance of quercetin in Que/mR150 SLNs was investigated; The effect of Que/mR150 SLNs on the proliferation of HUVEC of human umbilical vein endothelial cells was measured by MTT method, and fluorescence labeling was used to observe their uptake in HUVEC; And the irritancy of Que/mR150 SLNs to rabbit eyes was examined by pathological tissue sections of rabbit eyes. Result: After process optimization, the cationic nano Que-SLNs had good drug-loading, particle size distribution and stability. The appearance of the cationic nano-Que-SLNs was spherical, and it could be kept stable for two months. The quercetin encapsulation rate was (85.25 ± 1.29)%, the drug load was (1.67 ± 0.02)%, the average particle size was (110.00 ± 2.10) nm, and the Zeta potential is (53.2 ± 5.12) mV; The in vitro drug release results showed that the release of quercetin in the nanoparticles was slow, and the cumulative release amount within 48 h was about (80.69 ± 1.29)%; When the mass ratio of dioctadecyl dimethyl ammonium bromide to mR150 (DDAB/RNA) of different cationic materials was 6:1, the cationic solid lipid nanoparticles basically encapsulated mR150 completely with little effect on its particle size and potential. MTT experiments showed that blank nanometer mass concentration of 50-150 mg/L had no significant proliferation toxicity on HUVEC cells; Cell uptake experiments showed that Cy5 and coumarin-6 dual fluorescently labeled and co-loaded nanometers could effectively enter HUVEC cells; Pathological tissues of rabbit eyes showed that Que/mR150 SLNs had no obvious damage to the eyes. Conclusion: The preparation process of Que/mR150 SLNs solid lipid nanoparticles is stable and reliable, with good reproducibility, storage stability and good biological safety, which is conducive to the efficient delivery of quercetin and mR150 into HUVEC cells, which provides the ideas for the treatment of diseases related to angiogenesis

Preparation of sialic acid-modified chlorogenic acid liposomes and its anti-tumor activity in vitro / 中草药

Objective: Sialic acid (SA)-modified chlorogenic acid (CA) liposomes (CA-SAL) was prepared by response surface design to investigate its in vitro cytotoxicity and uptake. Methods: CA-SAL was prepared by a modified reverse-phase ethanol injection method. Sephadex G50 column was used to separate the CA-loaded liposomes and the free CA. The drug concentration was determined by HPLC method and the encapsulation efficiency was calculated. With encapsulation efficiency and drug loading as indicators, Box-Behnken response surface design experiments were used to optimize the prescription process of CA-SAL. The MTT method was used to evaluate the cytotoxicity of CA-SAL on human lung cancer cells A549. Inverted fluorescence microscope was used to investigate the uptake of CA-SAL by A549 cells. Results: The optimized preparation conditions: hydrogenated soybean lecithin-CA ratio at 15:1, hydration temperature 60 ℃, ultrasonic power 400 W. The average particle size of CA-SAL was (90.13 ± 0.51) nm, the polydispersity index (PDI) was 0.16 ± 0.01, the zeta potential was (-25.3 ± 0.5) mV, the encapsulation efficiency was 57.8%, RSD was 0.1%. MTT results showed that the inhibitory effect of CA-SAL on A549 cells was significantly greater than CA-CL. Greater cellular uptake of CA-SAL was observed compared with CA-CL. Conclusion: CA-SAL prepared by response surface optimization has a uniform particle size and good stability. SA-modified CA-loaded liposomes could enhance cellular uptake and cytotoxicity of human lung cancer cell A549 in vitro.

Nusinersen as a Therapeutic Agent for Spinal Muscular Atrophy

The reduction of survival motor neuron (SMN) protein causes spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease. Nusinersen is an antisense oligonucleotide, approved by the FDA, which specifically binds to the repressor within SMN2 exon 7 to enhance exon 7 inclusion and augment production of functional SMN protein. Nusinersen is the first new oligonucleotide-based drug targeting the central nervous system for the treatment of SMA. This review of nusinersen will discuss its action mechanism, cellular uptake, trafficking mechanisms, and administration approaches to cross the blood-brain barrier. Furthermore, nusinersen clinical trials will be assessed in terms of pharmacokinetics, tolerability and safety, the clinical outcomes of multiple intrathecal doses, and a discussion on the primary and secondary endpoints.

Cytotoxicity and cellular uptake of paclitaxel-loaded carboxymethyl chitosan-rhein polymeric micelles in MCF-7 cells / 中国药科大学学报

@#In this study, in vitro cytotoxicity of carboxymethyl chitosan-rhein conjugate(CR conjugate)and paclitaxel-loaded carboxymethyl chitosan-rhein polymeric micelles(PTX/CR PMs)was evaluated by MTT method in MCF-7 cells. The results showed that CR conjugate displayed good security; PTX/CR PMs in 24 h showed better antitumor activity than Taxol® . Environment-responsive fluorescent probe P4 was used to determine the cellular uptake of PTX/CR PMs in MCF-7 cells. The results also showed that P4 and PTX co-loaded carboxymethyl chitosan-rhein polymeric micelles ［(P4+PTX)/CR PMs］ could be taken up by MCF-7 cells. There was no difference between(P4+PTX)/CR PMs group and(P4+PTX)/CR PMs with verapamil group, suggesting that CR PMs could protect fluorescent probe and/or drugs in their cores avoiding efflux by P-glycoprotein. These results will contribute to in vivo study of CR conjugate and PTX/CR PMs in the future.

Advances in the research and application of cell penetrating peptides / 生物工程学报

Cell-penetrating peptides (CPPs) are short peptides that can penetrate the cell membrane or tissue barrier. CPPs can deliver a variety of biomacromolecules, such as proteins, RNA and DNA, into cells to produce intracellular functional effects. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms for CPPs-mediated cargo delivery. Compared with other non-natural chemical molecules-based delivery reagents, the CPPs have better biocompatibility, lower cytotoxicity, are easily degraded after cargo delivery, and can be fused and recombined expressed with bioactive proteins. Because of these advantages, the CPPs have become an important potential tool for delivery of developing drugs which targets intracellular factors. As a novel delivery tool, the CPPs also show promising application prospects in biomedical researches. This review summarized recent advances regarding the classification characteristics, the cellular uptake mechanisms and therapeutic application potentials of CPPs.

In vitro evaluation, cellular uptake and anti-acute myocardial ischemia effect of puerarin PEG-PLGA micelles / 中国中药杂志

PUE@PEG-PLGA micelles has excellent characteristics such as small particle size, high drug loading and slow drug release. The results of TEM electron microscopy showed that PUE@PEG-PLGA micelles had obvious core-shell structure. The critical micelle concentration(CMC) of PEG-PLGA micelles determined by pyrene assay was about 4.8 mg·L~(-1). Laser confocal experiments showed that PEG-PLGA micelles can enhance the cellular uptake of coumarin-6 and aggregate around the mitochondria; quantitative results of extracellular drug residues also indirectly confirmed that PEG-PLGA micelles can promote cellular uptake of the drug. Acute ischemic myocardial model rats were prepared by coronary artery ligation, and then the model rats were randomly divided into six groups: Sham operation group, model group, puerarin(PUE) group, as well as low-, mid-, and high-dose PUE@PEG-PLGA micelles groups. Drugs were given by iv administration 5 min after the ligation. The ST segment changes in the electrocardiogram were monitored; serum creatine kinase(CK), lactate dehydrogenase(LDH), aspartate aminotransferase(AST), and malondialdehyde(MDA) levels were detected and myocardial infarct size was also measured. Both PUE and PUE@PEG-PLGA micelles can reduce the elevated ST segment, reduce serum CK, LDH, AST and MDA levels, and reduce myocardial infarct size. The efficacy of PUE@PEG-PLGA medium and high dose groups was significantly better than that in the PUE group, and the efficacy in PUE@PEG-PLGA low dose group was basically equivalent to that in the PUE group. PUE@PEG-PLGA micelles can greatly improve the cardiomyocytes uptake of PUE, enhance the anti-acute myocardial ischemia effect of drugs, and reduce its dosage.

Transmembrane mechanism and intracellular trafficking of folic acid-polyethyleneimine carbon dots / 吉林大学学报(医学版)

Objective:To investigate the transport pathway and intracellular distribution of the of fluorescent carbon dots(CDs)synthesized by folic acid and polyethyleneimine(PEI)through the membrane of MC3T3-E1 cells and its effect on the cells,and to clarify the mechanism.Methods:The fluorescent CDs with the function of cell imaging were synthesized by hydrothermal method using folic acid and PEI as the raw materials;MTT assay was applied to screen the best concentration of CDs.The MC3T3-E1 cells were divided into blank control group,folic acid group and CDs group.The biocompatibility of CDs was evaluated by the detection of cell cycle,apoptosis and cellular reactive oxygen species(ROS)level.Nystatin as a kind of caveolae inhibitor and nocodazole as a kind of macropinocytosis inhibitor were used to find out the pathway through which the cells took in the CDs.Using the charcteristic of CDs with blue fluorescence stimulated by ultraviolet ray,the organelle probes were used to observe the distribution of CDs.Results:Compared with blank control group,the cells in different concentrations(100-450 mg·L-1)of CDs groups showed no cytotoxicity at 24 h(P>0.05);at 48 h,the cell proliferation rate was reduced to 68.4% of blank control group when the concentration of CDs reached 350 mg · L -1(P<0.05). Compared with blank control group,the percentages of cells in G0phase and G1phase in CDs group were decreased (P<0.05),and the percentage of cells in S phase was increased(P<0.05);the percentages of cells in G2phase and M phase were increased,but there no was significant differences(P>0.05).Compared with blank control group,the apoptotic rates of the cells in folic acid group and CDs group had no significant differences(P>0.05). Compared with blank control group,the intracellular ROS levels in folic acid group and CDs group were significantly decreased(P<0.05).Compared with blank control group,the uptake amount of CDs in the cells was decreased in nystatin group(P<0.05).The blue fluorescence of CDs overlapped with the red fluorescence of mitochondria under an inverted fluorescence microscope,the blue fluorescence of CDs overlapped with the red fluorescence of lysosomes;they didn't overlap completely with the red fluorescence of the endoplasmic reticulum;the blue fluorescence of CDs overlapped poorly with the red fluorescence of Golgi apparatus.Conclusion:CDs perform well in biocompatibility and they can be distributed to different organelles after taken in by the cells.They can be used as a kind of gene carrier in transgenic therapy.

Study on mechanism of enhanced HaCaT cellular uptake of tetrahydropalmatine by asarum essential oil and sinapine in Sanfu Patch / 中草药

Objective To study the mechanism of enhanced HaCaT cellular uptake of tetrahydropalmatine (THP) by asarum essential oil (AEO) and sinapine thiocyanate (SPT) in Sanfu Patch. Methods Effect of SPT, AEO, and THP on cell viability of HaCaT were determined by MTT. HaCaT cellular uptake of THP and the enhancing effects of AEO and SPT on THP uptake were visualized with confocal laser scanning microscope (CLSM) based on the green autofluorescence of THP, and the THP uptake content by HaCaT was further determined with HPLC. Moreover, HaCaT cells were labeled with diphenylhexatriene (DPH). After the labeled cells were treated with AEO, SPT, and THP, respectively, the cellular membrane fluidity was determined with fluorescence polarization technology. Results THP fluorescence intensity within HaCaT cells was significantly increased when THP was co-delivered with AEO or SPT respectively, and the THP content with each group within the cells was also significantly higher than that of THP delivered alone. In addition, AEO was superior to SPT in enhancing THP uptake by HaCaT cells. The fluorescence polarization and membrane micro-viscosity of HaCaT cells were significantly decreased after AEO treatment, which indicated that membrane fluidity was increased by the treatment with AEO. However, SPT or THP did not present the character of increasing the membrane fluidity.Conclusion HaCaT cellular uptake of THP can be enhanced by AEO and SPT of Sanfu Patch, in which AEO enhances the cellular uptake of THP through increasing the cellular membrane fluidity, while the mechanism of SPT in enhancing THP cellular uptake remains further clarification.

Research on monoclonal antibody OX26 modified salvianolic acid B/baicalin nanostructured lipid carriers / 中草药

Objective To prepare, characterize, and study cellular uptake of transferrin receptor monoclonal antibody OX26 modified nanostructured lipid carrier loaded with salvianolic acid B and baicalin (Sal B/BA-NLC). Methods Sal B/BA-NLC was prepared by emulsification-solvent evaporation method. OX26 was thiolated with 2-iminothiolane hydrochloride and then conjugated to the surface of Sal B/BA-NLC. The morphology, particle size, Zeta potential, and encapsulation efficiency (EE) were evaluated for the physicochemical properties, and OX26 modified Sal B/BA-NLC was verified by differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). Coumarin-6 (C6) was used as the fluorescent probe instead of baicalin and salvianolic acid B to prepare the formulations in cellular uptake study. The cellular uptake study was conducted by brain microvascular endothelial cells bEnd.3 using high content cell imaging analysis system. Results The prepared OX26 modified Sal B/BA-NLC had particle size of (27.50 ± 3.37) nm, PDI of 0.39 ± 0.04, and Zeta potential of (-7.06 ± 1.85) mV. The DSC and NMR results indicated that the drug was encapsulated in the nanostructured lipid carrier in an amorphous form. The results of cell uptake showed that the fluorescence intensities of the three solutions in bEnd.3 cells were: OX26-C6-NLC > C6-NLC > C6-SL. Conclusion The prepared OX26 modified Sal B/BA-NLC has smaller particle size, uniform distribution, and high EE. The OX26-modified NLC group had a higher intake than the solution group and the unmodified NLC group.

Preparation and characterization of nanostructured lipid carrier modified by hyaluronic acid loaded with three components in Ginseng Radix / 中草药

Objective To prepare and characterize tri-components nanostructure lipid carrier of Ginseng Radix modified with hyaluronic acid (HA-OUR-NLC). Methods Nanostructured lipid carriers (NLC) was used to wrap mount three difficult soluble active ingredients in Ginseng Radix including oleanolic acid (OA), ursolic acid (UA), and ginsenoside Rg3. Then using hyaluronic acid (HA) as the target factor, NLC was modified by charge-adsorption. The dynamic dialysis method was used to test the release. The cellular uptake and cytotoxicity of HA-OUR-NLC on SMMC-7721 cells were investigated by flow cytometry instrument and MTT assay respectively. Results OUR-NLC was prepared by ultrasonic dispersion of solvent using NLC as carrier material and CTAB as emulsifier, and its appearance was light blue opalescence. Then HA-OUR-NLC was successfully prepared by charge-sorption method with round shape and uniform distribution. In vitro release showed that it had a sustained release effect. Cell uptake experiments showed that HA-OUR-NLC can be taken up by SMMC-7721 cells. MTT assay results showed that HA-OUR-NLC had inhibitory effect on SMMC-7721 cell proliferation. Conclusion HA-OUR-NLC prepared by solvent ultrasonic dispersion not only has good physical and chemical properties, but also has a certain sustained release effect.