卟啉-脂质体化合物P-GlyL的紫外可见及荧光光谱性质研究

以溴代肽脂质BrC5Gly2C16与5,10,15-三苯基-20-对羟基苯基卟啉(HPTPP)为原料,合成一种可用作新型分子器件的卟啉-脂质体化合物P-GlyL。采用元素分析、紫外可见光谱、红外光谱以及核磁共振等进行表征。并对该化合物在不同浓度的金属离子Zn2+溶液中的紫外可见光谱和荧光光谱进行了研究。结果表明,当加入Zn2+之后,形成的金属卟啉化合物荧光强度在650.0 nm处下降,而在437.0 nm和595.8 nm处产生新的荧光发射峰,且荧光强度随着Zn2+浓度增加而增强,而同时349.0 nm和698.5 nm处的发射峰发生了0.5 nm的红移且荧光强度增强。

人降钙素基因相关肽脂质体质量标准的研究

建立完整的人降钙素基因相关肽脂质体 (LipohCGRP)药物的质量标准。用家兔球睫膜血管扩张法测定hCGRP的生物学活性 ;采用RP HPLC、等电聚焦、薄层层析等方法分别测定样品的纯度、等电点和迁移率及脂质体嵌入率的测定 ,并按照中国生物制品规程的要求完成了甲醇和氯仿等残留物质的分析和动物安全试验。建立了活性测定方法 ,能准确测定样品中不到 1ng的样品活性单位 ,连续三批样品的各项指标均符合质量标准的要求。建立了脂质体多肽药物的质量标准 ,并用于质量检定。

Effect of Liposome Double-Coated with Chitosan and Chitosan-EDTA Conjugates on Oral Absorption of Insulin

Aim To evaluate the gastrointestinal uptake of the insulin liposomes double-coated with chitosan （Ch） and chitosan-EDTA conjugates （CEC）, and verify their efficiencies. Methods Insulin-liposomes were prepared by reversed-phase evaporation. The hypoglycemic effects of the insulin liposomes coated with Ch or/and CEC were investigated using the glucose oxidase method after oral administration in diabetic rats, normal rats, and beagle dogs. Serum insulin concentrations in beagle dogs were determined by radioimmunoassay and were assessed by Pkanalyst computer program. Results The animals fed the insulin liposomes coated with Ch or/and CEC were able to regulate better the glucose load than the animals receiving PBS or uncoated insulin liposome, and the regulative effects of the insulin liposomes double-coated with Ch and CEC were better than those of the insulin liposomes coated with Ch or CEC alone. After oral administration of the insulin-liposomes double-coated with Ch and CEC to animals, a significant （P 〈 0. 05 ） blood glucose reduction was observed. Their relative pharmacological bioavailability was higher than 9 % in comparison with subcutaneous injection of insulin. In addition, in comparison with subcutaneous injection of insulin, the relative bioavailability was 12. 67 % calculated by area under the curve of serum insulin concentration versus time profile after oral administration of the insulin-liposomes double-coated with Ch and CEC to beagle dogs. Conclusion The insulin-liposomes double-coated with Ch and CEC were conducive to improving oral bioavailability of insulin.

SOD-like Activities of SOD and Cu(Ⅱ)Complexes inLiposomes

The SOD-like activities of SOD, four Cu(Ⅱ)Complexes, SOD-and Cu(Ⅱ)com-plex-liposomes were determined respectively by using cytochrome C method. The results showed that all of these systems had SOD-like activities to some extent of which the Cu(His)_2-liposome displayed the highest activity and the Cu(Ⅱ)complexes and liposomes had some positive cooperative action.

Systemic delivery of full-length C/EBPβ / liposome complex suppresses growth of human colon cancer in nude mice

C/EBPβ (CCAAT/enhancer-binding protein β) is an important transcription factor involved in cellular proliferation and differentiation. Overexpression of the full-length C/EBPβ protein results in cellular growth arrest and apoptosis. Using a nonviral liposome as carrier, we delivered the full-length C/EBPβ expression plasmid, pCN, into nude mice bearing CW-2 human colon cancer tumors via tail vein. Southern blots revealed that the major organs and tumors were transfected. Experimental gene therapy showed that a strong suppression of tumor growth was observed in the pCN- treated mice, and such suppression was due to the overexpression of C/EBPβ, leading to the increased apoptosis in tumors of pCN-treated mice. No apparent toxic effects of pCN/liposome complex were observed in the animals. Thus, C/EBPβ has tumor suppression effect in vivo and may be used in gene therapy for cancers.

Theoretical Study on Electronic Gain-and-loss Properties of TEMPO and Its Derivates in Charge/Discharge Processes

Theoretical study on the electronic structures and related properties of 2,2,6,6-tetramethyl- l-piperidinyloxy （TEMPO） and its cationic lipid derivates in the charge/discharge processes has been carried out using the density functional theory （DFT） at the （U）B3LYP/6-31G（d,p） or 6-31＋G（d,p） level. The changes and regularities of geometric and electronic properties of these compounds in the charge/discharge processes were revealed in detail. The compu- tational results show that the substitute group plays a very important role in the electronic structures and related properties of TEMPOs during the charge/discharge processes. It is very interesting to find that after getting an electron, TEMPO is more stable in singlet state but the lipid is more stable in triplet state. For TEMPO, both the charge and the discharge processes greatly influence the electronic properties of N and O atoms of the radical part. For the cationic lipid, the discharge process mainly influences the pyridinium head and the charge process mainly influences the free radical head. Moreover, the solvent effect plays an important role in some bond lengths and the charge population of the free radical head. In addition, the UV-Vis absorption spectra simulated using TDDFT at the 6-31G（d,p） with the experimental ones. of TEMPO and the lipid were calculated and or 6-31＋G（d,p） level, in satisfying agreement

Moro orange juice prevents fatty liver in mice

AIM:To establish if the juice of Moro,an anthocyaninrich orange,may improve liver damage in mice with diet-induced obesity.METHODS:Eight-week-old mice were fed a high-fat diet(HFD) and were administrated water or Moro juice for 12 wk.Liver morphology,gene expression of lipid transcription factors,and metabolic enzymes were assessed.RESULTS:Mice fed HFD displayed increased body weight,insulin resistance and dyslipidemia.Moro juice administration limited body weight gain,enhanced insulin sensitivity,and decreased serum triglycerides and total cholesterol.Mice fed HFD showed liver steatosis associated with ballooning.Dietary Moro juice markedly improved liver steatosis by inducing the expression of peroxisome proliferator-activated receptor-α and its target gene acylCoA-oxidase,a key enzyme of lipid oxidation.Consistently,Moro juice consumption suppressed the expression of liver X receptor-α and its target gene fatty acid synthase,and restored liver glycerol-3-phosphate acyltransferase 1 activity.CONCLUSION:Moro juice counteracts liver steatogenesis in mice with diet-induced obesity and thus may represent a promising dietary option for the prevention of fatty liver.

Targeting of AUF1 to vascular endothelial cells as a novel anti-aging therapy

Background Inhibition of aging of vascular endothelial cells （VECs） may delay aging and prolong life. The goal of this study was to prepare anti-CD31 monoclonal antibody conjugated PEG-modified liposomes containing the AU-rich region connecting factor 1 （AUF1） gene （CD31-PILs-AUF1） and to explore the effects of targeting CD31-PILs-AUF1 to aging VECs. Methods The mean particle sizes of various PEGylated immunoliposomes （PILs） were measured using a Zetasizer Nano ZS. Gel retardation assay was used to confirm whether PILs had encapsulated the AUF1 plasmid successfully. Fluorescence microscopy and flow cytometry were used to quantify binding of CD31-PILs-AUF1 to target cells. Flow cytometry was also used to analyze the cell cycles of aging bEnd3 cells treated with CD31-PILs-AUF1. We also developed an aging mouse model by treating mice with D-galactose. Enzyme-linked immunosorbent assay （ELISA） was used to evaluate the levels of interleuldn-6 （IL-6） and tumor necrosis factor-or （TNF-ct）. The malondialdehyde （MDA） and the superoxide dismutase （SOD） levels were detected by commercial kits. Hematoxylin-eosin （HE） staining was used to determine whether treatment with CD31-PILs-AUF 1 was toxic to the mice. Results CD31-PILs-AUF 1 specifically could targeted bEnd3 VECs and increased the percentage of cells in the S and G2/M phases of aging bEnd3 cells. ELISA showed that content of the IL-6 and TNF-ct decreased in CD31-PILs-AUF1 group. The level of SOD increased, whereas MDA decreased in the CD31-PILs-AUF1 group. Additionally, CD31-PILs-AUF 1 was not toxic to the mice. Conclusion CD31-PILs-AUF 1 targets VECs and may delay their senescence.

Antitumor activity and biodistribution of DHA-NLC formulation in sarcoma 180-bearing mice

Lipid nanoparticles have become attractive for its prominent properties recent years. In this paper, in vivo anti-tumor efficacy of nanostructured lipid carrier of dihydroartemisinin （DHA-NLC） were evaluated in sarcoma 180-bearing mice model through intraperitoneal （i.p.） administration. In vivo biodistribution was also investigated in Kunming mice bearing S180. Results demonstrated that the intraperitoneally injected DHA-NLC could significantly inhibit tumor growth at the dose levels of 20, 40 and 80 mg/kg, and their inhibition rates were 71.24%, 79.20% and 85.74%, respectively. The biodistribution of DHA after intraperitoneal injection of DHA-NLC in S180-bearing mice is remarkably different from the DHA solution. Therefore, DHA encapsulated in NLC does demonstrate superior anticancer effect to DHA suspension on S 180-bearing mice at the same dose and displayed a dose-dependent antitumor efficacy.

Detection of bacterial endotoxin in paclitaxel liposome

Based on current research, there are three technologies during the test of bacterial endotoxin of liposomes：（1） extraction of bacterial endotoxin from liposomes;（2） addition of bacterial endotoxin in the process of recovery test; and（3） elimination of the interference factors from drugs and excipients. In the present study, we pointed out that the key technologies to test bacterial endotoxin from paclitaxel liposome included following steps： extraction of bacterial endotoxins from ethanol-dissolved liposomes; preparation of positive control of recovery solution by adding 0.01 m L standard endotoxins in 1 m L liposome ethanol solution; and the use of 0.5% human albumin to eliminate the interference from detection, and accurate detection of the bacterial endotoxin of liposomes.

Age-related changes of yolk precursor formation in the liver of laying hens

A rapid decline in egg production of laying hens begins after 480 d of age. Such a rapid decrease results predominantly from the ovarian aging, accompanied by endocrine changes, decreased yolk synthesis and accumulation, and the reduction in follicles selected into the preovulatory hierarchy. In this study, hens at 90, 150, 280, and 580 d old （D90, D150, D280, and D580, respectively） were compared for yolk precursor formation in the liver to elucidate effects of aging on laying performance. The results showed that liver lipid synthesis increased remarkably in hens from D90 to D150, but decreased sharply at D580 as indicated by the changes in triglyceride （TG） levels. This result was consistent with the age-related changes of the laying performance. The levels of liver antioxidants and total antioxidant capacity decreased significantly in D580 hens and the methane dicarboxylic aldehyde in D580 hens was much higher than that at other stages. The serum 17β-estradiol level increased from D90 to D280, but decreased at D580 （P〈0.05）. The expression of estrogen receptor a and 13 mRNAs in the liver displayed similar changes to the serum 17β-estradiol in D580 hens. Expressions of the genes related to yolk precursor formation and enzymes responsible for fat acid synthesis were all decreased in D580 hens. These results indicated that decreased yolk precursor formation in the liver of the aged hens resulted from concomitant decreases of serum 17β-estradiol level, transcription levels of estrogen receptors and critical genes involved in yolk precursor synthesis, and liver antioxidant status.

A mechanism study on the tamoxifen-mediated cellular internalization of liposomes

It was reported previously that tamoxifen （TAM） could increase the intracellular accumulation of drug-loaded liposomes, but the exact mechanism is unknown although it was supposed that TAM might enhance the cell uptake by inhibiting the drug efflux caused by P-glycoprotein （P-gp）. To identify the mechanism of increased cellular uptake of liposomes induced by tamoxifen, PEGgylated liposomes （SSL） ofP-gp-substrate doxorubicin （DOX） or non-P-gp-substrate coumarin （Cou） were prepared with or without TAM. The cell uptake of these liposome systems was investigated in cell lines with different P-gp-expressing levels and the interaction of TAM with lipid membrane was also studied. As the results, the co-encapsulation of TAM with DOX-SSL increased the intracellular uptake in all three tumor cell lines. In P-gp-highly-expressing MCF-7/Adr cells, the effect of TAM was the strongest and in negative control Hela cells, the impact weakened but still significant. The improvement was also observed in the cellular uptake of Cou-SSL. Surface plasmon resonance （SPR） studies demonstrated that TAM-SSL exhibited a much stronger atYmity with model biomembrane compared with empty SSL, and ft^her test with isothermal titration calorimetry （ITC） showed that free TAM had an obvious interaction with lipid membrane. In conclusion, TAM could increase the affinity of liposomes with biomembrane and enhance the intracellular accumulation of liposomes via both TAM-mediated P-gp inhibition and the increased interaction between hydrophobic TAM molecules and lipid membrane.

A novel type of functional epirubicin liposomes modified with DSPE-PEG_(2000)-cyclopamine conjugate for eliminating heterogeneous breast cancer cells

Common chemotherapy is unable to eliminate the heterogeneous side population of cancer cells （such as cancer stem-like cells）, resulting in poor prognosis. The heterogeneity of cancer cells causes an extensive multidrug resistance through the aberrantly active Hedgehog （Hh） signaling pathway. Cyclopamine is a chemical compound that can block Hh signaling pathway, and a combination use of cyclopamine with anticancer drug would be beneficial for killing heterogeneous cancer cells. In the present study, we aimed to develop a kind type of fimctional drug liposomes for eliminating heterogeneous cancer, The study was performed on human breast cancer cells. A distearoylphosphoethanolamine polyethylene glycol （DSPE-PEG2000）-cyclopamine conjugate was newly synthesized by a nucleophilic substitution reaction, and confirmed by MALDI-TOF mass. An HPLC method was established and validated for qualification of epirubicin. Functional epimbicin liposomes were successful constructed by modifying with DSPE-PEG2o00-cyclopamine, displaying a particle size in nano-scale （approximately 98 nm） and a high epirubicin encapsulation （〉97%）. The CD44＋/CD24-side population was characterized in defining heterogeneous breast cancer cells. As compared with regular epirubicin liposomes, fimctional epirubicin liposomes exhibited an evidently enhanced cellular drug uptake and a significant killing effect in overall breast cancer cells. In conclusion, the functional epirubicin liposomes could be a useful drug delivery carrier for eliminating heterogeneous breast cancer cells.

Enantiomers separation using avidin-liposome complex as a chiral selector in capillary electrophoresis

Avidin-liposome complex is a specific chiral selector used to separate the enantiomers of D,L-tryptophan,D,L-phenylthiohydantoin -serine（D,L-PTH-Ser）,D,L-phenylthiohydantoin-threonine（D,L-PTH-Thr） and R,S-pioglitazone hydrochloride in capillary electrochromatography（CEC）.The avidin is immobilized on the phospholipids coated in the capillary.The liposome used for the phospholipid coating contains l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-（Cap biotinyl）（sodium salt） （Biotinyl-cap-DPPE）（16：0） and different proportions of L-α-phosphatidylserine（PS） in Tris（hydroxymethyl） aminomethane （Tris） buffer at pH 7.4.A good resolution and reproducibility was obtained by coating the capillary with Biotinyl-cap-DPPE/PS （80：20,mol%） followed by immobilization of 1 mg/mL of avidin solution in N-（2-hydroxyethyl） piperazine-N＇-（2-ethanesulfonic acid） （HEPES） buffer at pH 7.4.A comparative study of chiral separation efficiency with different capillary coating methods and preconditioning conditions was conducted.Finally,the electrochromatographic method was successfully used to separate enantiomers of pioglitazone hydrochloride.Therefore,coated CEC will be a promising tool for pharmaceutical enantiomers separation in new drug development.

Preparation and evaluation of liposomal clarithromycin with antimicrobial effect

In this study, we developed a novel liposomal delivery system modified by Tat peptide and wheat germ agglutinin（WGA） with antimicrobial effect. Physicochemical parameters, in vitro antimicrobial, time-kill study, cellular uptake, biofilm formation inhibition and in vivo antibacterial efficacy against Methicillin-resistant Staphylococcus aureus（MRSA） were investigated. Minimum inhibitory concentrations（MICs） and colony-forming units（CFUs） in the time-kill study for Tat-WGA-modified liposomal clarithromycin（CLA-Tat WGALip） were lower than those of free and other modified liposomal CLA. Flow cytometry analysis disclosed that Tat WGALip delivered more coumarin 6 into bacteria. Furthermore, Tat-WGA-modified liposomal CLA efficiently inhibited the formation of MRSA biofiom. CFU of MRSA in the abscess of mice treated with CLA-Tat WGALip was significantly lower than that of any others（P〈0.01）. Collectively, liposomal delivery system modified by Tat and WGA could be a promising anti-resistant infection strategy.

Interferon-liposomes prepared to make macroglia maintain M1 phenotype

Macroglia is a crucial macrophage only existing in the central nervous system.During the development of glioma,it can be activated as M2 anti-inflammatory type to promote glioma growth.Interferon-γ(IFN-γ)is an important immunomodulator in glioma microenvironment,which can also activate macroglia as M1 pro-inflammatory type to enhance anti-tumor immune response and lead to inhibition of glioma growth.Therefore,we utilized IFN-γto make macroglia maintain M1 phenotype,so that prospectively achieving anti-tumor immunity for glioma therapy.We prepared interferon-γ-liposomes(IFN-Lp)to protect IFN-γfrom elimination.IFN-Lp was proved to have strong capability to be phagocytosed and accumulate in macroglia(BV2 cells)to achieve long-term effect.In addition,IFN-Lp could allow BV2 cells to maintain M1 phenotype,showing no impact on its cell viability.These findings will offer new opportunities to achieve enhanced immunotherapy of glioma.