PURPOSE:Spherical reconstituted high density lipoprotein (rHDL) can target atherosclerotic lesions by the very low density lipoprotein (VLDL) receptor, which is seldom expressed in liver. By promoting this pathway, the targeting efficiency was hyphothesized to be improved due to avoiding undesired uptake in liver mediated by the scavenger receptor class B type I (SR-BI). In this study, how fatty acid modification in spherical rHDL influenced the VLDL receptor-mediated endocytosis pathway was investigated.METHODS:Stearic acid (SA) and arachidonic acid (AA) with different saturation levels were utilized to modify the lovastatin-loaded rHDL (LS-rHDL). Phagocytosis test on foam cells with or without cholesteryl ester transfer protein (CETP) expression was conducted to observe the cellular uptake of the SA or AA modified rHDL and the non-modified one. Raman spectroscopy, guanidine hydrochloride (Gdn-HCl) denaturation experiment and in vitro evaluation of drug release were used to analyze the related mechanism.RESULTS:In comparison with the non-modified rHDL, AA modification could reduce the packing order of the rHDL phospholipid acyl chains, leading to the decreased apoA-I binding extent with lipid and the increased drug release, while the opposite was true for SA modification. The AA-modified rHDL exhibited a higher uptake of foam cells expressing CETP than the non-modified one, while the SA-modified one showed the lowest cellular uptake among the three rHDLs.CONCLUSIONS:Increased unsaturation level can facilitate lipid-interchange process where the cargo in rHDL core may transfer to VLDL more easily, and then promote the endocytosis mediated by the VLDL receptor.

Hepatic macrophages have been considered as a therapeutic target for liver fibrosis treatment, and phosphatidylserine (PS)-containing nanoparticles are commonly used to mimic apoptotic cells that can specifically regulate macrophage functions, resulting in anti-inflammatory effects. This study was designed to test the efficacy of PS-modified nanostructured lipid carriers (mNLCs) containing curcumin (Cur) (Cur-mNLCs) in the treatment of liver fibrosis in a rat model. Carbon tetrachloride-induced liver fibrosis in rats was used as an experimental model, and the severity of the disease was examined by both biochemical and histological methods. Here, we showed that mNLCs were spherical nanoparticles with decreased negative zeta potentials due to PS decoration, and significantly increased both mean residence time and area under the curve of Cur. In the rats with liver fibrosis, PS-modification of NLCs enhanced the nanoparticles targeting to the diseased liver, which was evidenced by their highest accumulation in the liver. As compared to all the controls, Cur-mNLCs were significantly more effective at reducing the liver damage and fibrosis, which were indicated by in Cur-mNLCs-treated rats the least increase in liver enzymes and pro-inflammatory cytokines in the circulation, along with the least increase in collagen fibers and alpha smooth muscle actin and the most increased hepatocyte growth factors (HGF) and matrix metalloprotease (MMP) two in the livers. In conclusion, PS-modified NLCs nanoparticles prolonged the retention time of Cur, and enhanced its bioavailability and delivery efficiency to the livers, resulting in reduced liver fibrosis and up-regulating hepatic expression of HGF and MMP-2.

磷脂酰丝氨酸(phosphatidylserine,PS)大量存在于凋亡的细胞膜,是一种重要的生物膜磷脂.研究发现,伴随细胞凋亡的发生,PS由细胞膜内侧翻转到细胞膜外侧,表现出特异的“食我”信号,这种信号能被巨噬细胞上的特异性受体识别,表现出巨噬细胞靶向性.PS被巨噬细胞吞噬进而产生一系列的抗炎反应,也有研究者借助PS的这种巨噬细胞靶向性,以PS为膜材构建生物靶向递药系统.本文调研了近年来国内外研究者利用PS这一特性实现巨噬细胞靶向性应用的文献报道,并对其进行了总结和概括,以期为PS的后续研究提供参考.

Purpose: To develop a simple method to extract the whole apolipoproteins (apo) including apoA-I in native high density lipoproteins (HDLs) and prepare discoidal Tanshinone IIA-loaded reconstituted HDL (TA-rHDLs) as a dual functional drug delivery system with plaque-site target and therapeutic promises in atherosclerotic lesions. Methods: A method based on isoelectric precipitation coupled with organic solvent precipitation was developed to isolate the whole apolipoproteins (apos). TA-rHDLs were prepared by incubating the resultant apos with liposomes and the incubation conditions were optimized using fluorescence quenching experiment. TA-rHDLs were characterized in terms of size, zeta potential, morphology, interaction between lipid and apos, safety, and bionic function. Results: The extraction results showed that the yield of the HDL apos was 82.4%, with 59% being apoA-I type, similar ratio of apoA-I in the native apos. TA-rHDL prepared were disc-like with an average diameter of 157.6 ± 4.8 nm, zeta potential of -20.90 ± 0.15 mV, and entrapment efficiency of (90.13 ± 1.4) %. The interaction between the lipids and apos was electrostatic and hydrophobic force and was associated with amino acid sequence. Haemolysis and cytotoxicity assays showed good biocompatibility of TA-rHDL. Sterol efflux assay from macrophages mediated by TA-rHDLs and structure remodeling behavior from discs to spheres proved that TA-rHDL could resemble the biological activity of native nascent HDL irrespective of the size. Conclusions: The simple approach to isolate apos may provide a convenient and economical resource to support the development of rHDL as a potential targeting nanocarrier for lipophilic cardiovascular drugs. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Ardipusilloside I (ADS-I) is a natural compound that can be isolated from the Chinese medicinal herb Ardisiapusilla A.DC, and has been reported to inhibit the growth of glioblastoma cells in cultures. This study was designed to test its efficacy by the delivery using biodegradable implants against glioblastoma in vivo. ADS-I was incorporated into polymer microspheres, which were prepared by a mixture of poly (D, L-lactic acid) and poly (D, L-lactic-co-glycolic acid) polymers and then fabricated into wafers. The anti-glioma activities of ADS-I-loaded wafers were examined by methylthiazol tetrazolium (MTT) assay in cultured rat C6 glioma cells, and by magnetic resonance imaging (MRI) and survival monitoring in C6 glioma-bearing rats. Here, we showed that ADS-I-loaded wafers sustained ADS-I release in vitro for 36 days in Higuchi model of kinetics, and had the same cytotoxic activity as ADS-I in the solution against the growth of C6 glioma cells in cultures. In C6 glioma-bearing rats, ADS-I wafer implants inhibited tumor growth in a dose-dependent matter, and were more effective than the same dosage of ADS-I in the solution. The tumor suppression efficacies of ADS-I wafer implants were positively correlated with an increase in tumor cell apoptosis and prolonged animal survival, and were associated with a decrease in vascular endothelial growth factor, C-reactive protein, tumor necrosis factor-α and interleukin-6, and an increase in interleukin-2 expression. In conclusion, this study demonstrates significant efficacy of local delivery of ADS-I using polymer implants against glioma tumor growth in vivo, suggesting the potential of ADS-I-loaded wafers for glioma treatment.

Two Ru(II) polypyridyl complexes [Ru(phen)2(idpq)](ClO4)2 (1) and [Ru(dmp)2(idpq)](ClO4)2 (2) were synthesized and characterized. Cytotoxicity, apoptosis, cell cycle arrest, reactive oxygen species and mitochondrial membrane potential were assayed. The IC50 values of complexes 1 and 2 toward HepG-2, A549, MG-63 and HeLa cell lines range from 15.1±2.1 to 24.8±2.4μM. The complexes can effectively induce apoptosis and induce cell cycle arrest at G0/G1 phase by an increase of 4.88% for 1 and 6.64% for 2 at G0/G1 phase in HeLa cells. Complexes 1 and 2 can enter the cytoplasm and accumulate in the nuclei. The complexes can enhance the level of reactive oxygen species. The ratio of the red/green is 0.74 and 0.52 for complexes 1 and 2, which suggests that the complexes induce a decrease of mitochondrial membrane potential. These complexes induce apoptosis in HeLa through ROS-mediated mitochondrial dysfunction pathway.

Two new dibenzoxanthenes compounds 1 and 2 have been synthesized and characterized by analytical and spectral methods. The crystal structure of compound 2 informs that the five rings of compound are almost planar. The DNA binding properties of two compounds were studied by absorption titration, viscosity measurement and luminescence. These results indicate that two compounds interact with calf thymus DNA through intercalative mode. Agarose gel electrophoresis experiment shows that PBR 322 DNA can be induced to cleave by two compounds under photoactivated condition. Compound 1 exhibits higher cytotoxicity than compound 2 toward MG-63, BEL-7402 and A549 cells. The apoptosis and cellular uptake of MG-63 cells were studied by fluorescence microscopy. Two compounds can also enhance the level of reactive oxygen species (ROS) and decrease the mitochondrial membrane potential. Compound 1 induces cell cycle arrest in G2/M phase and compound 2 induces cell cycle arrest in G0/G1 phase in MG-63.

Two Ru(II) polypyridyl complexes [Ru(dmb)2(idpq)](ClO4)2 (1) (idpq = indeno[1,2-b]dipyrido[3,2-f:2′,3′-h]-quinoxaline-6-one, dmb = 4,4′-dimethyl-2,2′-bipyridine) and [Ru(bpy)2(idpq)](ClO4)2 (2) (bpy = 2,2′-bipyridine) have been synthesized and characterized. Their in vitro cytotoxicities, apoptosis, cellular uptake, production of reactive oxygen species (ROS), mitochondrial membrane potential assays and effects on cell cycle distribution were studied. The IC50 values range from 13.1 ± 1.1 to 30.9 ± 3.1 μM. Complex 1 is toxic to HeLa cells with an IC50 value of 13.1 ± 1.1 μM, while complex 2 shows relatively high cytotoxicity against HepG-2 cells. However, both complexes exhibit lower cytotoxicity than cisplatin toward selected cell lines under identical conditions. Both complexes can induce apoptosis and cell cycle arrest at the S and G0/G1 phases in HeLa cells, respectively. In addition, both Ru(II) complexes, which can be effectively taken up by HeLa cells, induced generation of intracellular ROS in a concentration-dependent manner and produced a decrease in mitochondrial membrane potential. The results show that these complexes induce apoptosis in HeLa cells through a ROS-mediated mitochondrial dysfunction pathway.

Two new ruthenium(II) polypyridyl complexes [Ru(dmb)(2)(dcdppz)](ClO4)(2) (I) and [Ru(bpy)(2) (dcdppz)](ClO4)(2) (2) were prepared and characterized. The crystal structure of the complex 2 was solved by single crystal X-ray diffraction. The complex crystallizes in the monoclinic system, space group P2(1)/n with a = 12.9622(14) angstrom, b = 17.1619(19) angstrom, c = 22.7210(3) angstrom, beta = 100.930(2)degrees R = 0.0536, R-omega = 0.1111. The DNA-binding constants for complexes 1 and 2 were determined to be 1.92 x 10(5) (s = 1.72) and 2.24 x 10(5) (s = 1.86) M-1, respectively. The DNA-binding behaviors showed that complexes 1 and 2 interact with DNA by intercalative mode. The antioxidant activities of the ligand and the complexes were performed. Ligand, dcdppz, has no cytotoxicity against the selected cell lines. Complex 1 shows higher cytotoxicity than complex 2, but lower than cisplatin toward selected cell lines. The apoptosis and cell cycle arrest were investigated, and the apoptotic mechanism of BEL-7402 cells was studied by reactive oxygen species (ROS), mitochondrial membrane potential and western blot analysis. Complex 1 induces apoptosis in BEL-7402 cells through ROS-mediated mitochondrial dysfunction pathway and by regulating the expression of Bcl-2 family proteins. (C) 2014 Elsevier B.V. All rights reserved.

Three Ru(II) polypyridyl complexes [Ru(dmb)2(HMSPIP)](ClO4)2 (1), [Ru(phen)2(HMSPIP)](ClO4)2 (2) and [Ru(dmp)2(HMSPIP)](ClO4)2 (3) were synthesized and characterized. The cytotoxicity in vitro, apoptosis, cell cycle arrest, reactive oxygen species and mitochondrial membrane potential were assayed. The IC50 values of complexes 1, 2 and 3 toward BEL-7402, A549, MG-63 and SK-BR-3 cell lines ranged from 10.9 ± 1.6 to 42.0 ± 3.4 μM. Complexes 1, 2 and 3 can effectively induce apoptosis and inhibit the growth of BEL-7402 cells at the G2/M phase. These complexes can enhance the level of reactive oxygen species and induce decrease in the mitochondrial membrane potential. Additionally, complex 2 can down-regulate the expression of antiapoptotic protein of Bcl-2 protein and up-regulate the levels of proapoptotic protein Bim in BEL-7402 cells.

A new ligand dmdppz and its four ruthenium(II) polypyridyl complexes [Ru(dmb)2(dmdppz)](ClO4)2 (1), [Ru(bpy)2(dmdppz)](ClO4)2 (2), [Ru(phen)2(dmdppz)](ClO4)2 (3) and [Ru(dmp)2(dmdppz)](ClO4)2 (4) (where dmb, bpy, phen, dmp and dmdppz stand for 4,4′-dimethyl-2,2′-bipyridine, 2,2′-bipyridine, 1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline and 5,8-dimethoxylpyrido[3,2-a:2′,3′-c]phenazine, respectively) have been synthesized and characterized. Their DNA binding behaviors show that the complexes bind to calf thymus DNA by intercalation. The complexes exhibit efficient photocleavage of pBR322 DNA on irradiation. The cytotoxicity of the ligand and the complexes toward HepG-2, HeLa, MG-63, A549 and BEL-7402 were assayed by MTT ((3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide)) method. The IC50 values of the complexes 1, 2, 3 and 4 toward BEL-7402 cells are 14.6, 16.8, 18.0 and 16.7μM, respectively. Dmdppz shows no cytotoxic activity against selected cell lines. The cellular uptake, apoptosis, comet assay, reactive oxygen species (ROS), mitochondrial membrane potential and western blot analysis were investigated. These results indicate that complexes 1–4 exert their toxicity through the intrinsic ROS-mediated mitochondrial pathway, which is accompanied by the regulation of Bcl-2 family proteins.

Our previous studies indicated that drug leaked from discoidal reconstituted high density lipoprotein (d-rHDL) during the remodeling behaviors induced by lecithin cholesterol acyl transferase (LCAT) abundant in circulation, thus decreasing the drug amount delivered into the target. In this study, arachidonic acid (AA)-modified d-rHDL loaded with lovastatin (LT) were engineered as AA-LT-d-rHDL to explore whether AA modification could reduce the drug leakage during the remodeling behaviors induced by LCAT and further deliver more drug into target cells to improve efficacy.

The aim of our study was to investigate DNA-binding and cytotoxic activity of the four new Ru(II) polypyridyl complexes [Ru(dmb)(2)(HMHPIP)](ClO4)(2) (1), [Ru(bpy)(2)(HMHPIP)I(ClO4)(2) (2), [Ru(Phen)(2) (HMHPIP)](ClO4)(2) (3) and [Ru(dmp)(2)(HMHPIP)](ClO4)(2) (4). The complexes interact with DNA through intercalative mode and show relatively high cytotoxic activity against A549 cells, no cytotoxicity toward MG-63 cells. Complexes 1-4 can enhance the levels of ROS in A549 cells and induce the decrease of the mitochondrial membrane potential. These complexes inhibit the cell growth in A549 cells at G0/G1 or S phase. Complex 3 activated caspase 7, and down-regulated the expression of the anti-apoptotic protein Bcl-2. Complexes 1-4 induce apoptosis in A549 cells through ROS-mediated mitochondrial dysfunction pathway. Crown Copyright (C) 2014 Published by Elsevier B.V. All rights reserved.

Three complexes were synthesized and characterized. The cytotoxicity, apoptosis, cellular uptake, reactive oxygen species, mitochondrial membrane potential, cell cycle arrest and western blot analysis were investigated.

To investigate the possibility of liquid proliposomes being carriers for oral delivery, nimodipine liquid proliposomes-based soft capsules (NPSC) were prepared. Nimodipine proliposomes were characterized by transmission electron microscopy (TEM), conversion rate from proliposomes to liposomes, entrapment efficiency, particle size, and zeta potential. Accelerated stability testing of NPSC was carried out for 3 months at 40±2°C, 75±5% RH. The concentration of nimodipine in plasma of New Zealand rabbits of NPSC, nimodipine soft capsules, and hydrated liposomes was studied. Results showed that nimodipine proliposomes were automatically converted into liposomes when exposed to a water phase in 30 s. The average diameter was 378.6±26.5 nm in distilled water with entrapment efficiency (EE%) of 84.7±5.9%, while the average diameter was 316.9±34.6 nm in 0.1 M hydrochloric acid solution with EE% of 72.8±4.7%. Accelerated stability test showed that there was no change in drug content, particle size, and EE% except for a decrease in dissolution of nimodipine. In vivo experiments, areas under the plasma level-time curve of NPSC and nimodipine-hydrated liposomes increased 2.41 and 2.34 times more than that of nimodipine soft capsules, peak concentration increased 2.87 and 2.92 times, time of peak concentration from 0.75 to 2 and 1 h, respectively. Nimodipine-hydrated liposomes presented similar pharmacokinetic parameters compared with NPSC. Results suggested that NPSC offered a potential way to improve oral delivery of nimodipine.

OBJECTIVE:Monocholesterylsuccinate (CHS)-modified paclitaxel-loaded discoidal reconstituted high density lipoproteins (cP-d-rHDL) as novel biomimetic nanocarriers that were developed for tumor targeting delivery to avoid unexpected drug leakage from discoidal reconstituted high density lipoproteins (d-rHDL) during remodeling process associated with lecithin-cholesterol acyltransferase (LCAT).METHODS:Their in vitro characterizations and biomimetic properties, simultaneously tumor distribution and pharmacodynamics in tumor bearing mice were elaborately investigated.RESULTS:In vitro characterization results showed that cP-d-rHDL had nano-size diameter, high negative zeta potential and high entrapment efficiency (EE). Furthermore, morphology study indicated that cP-d-rHDL did not remodel in the presence of LCAT, compared with that of paclitaxel-loaded d-rHDL (P-d-rHDL, not modified). And cellular uptake, together with cytotoxicity toward tumor cells of cP-d-rHDL was not affected after interaction with LCAT. Tumor distribution and pharmacodynamics tests revealed that cP-d-rHDL possessed specific targeting property and anti-tumor efficacy.CONCLUSION:cP-d-rHDL served to restrain remodeling process and drug leakage, at the same time reinforce the targeting effect, and could act as a potential drug delivery system for cancer therapy.

Spherical and discoidal tanshinone IIA-loaded reconstituted high density lipoproteins (TA-rHDL) with different formulations and techniques were prepared and characterized. The targeting mechanism was investigated using a foam cell model. Pharmacokinetics of four TA-rHDL formulations with or without apolipoproteins (apos) after a single dose intravenous injection to rabbits has been studied. The results showed that the sizes of spherical and discoidal TA-rHDL increased after coupling with apos from 55.38 nm to 157.26 nm, 61.03 nm to 166.19 nm, and zeta potential decreased from -29.2 mV to -35.4 mV, -5.2 mV to -11.82 mV, respectively. The results of circular dichroic spectroscopy indicated variations of apos in protein secondary structure after binding with lipids. Phagocytosis tests demonstrated that the spherical TA-rHDL had a targeting effect for foam cells through the scavenger receptor-BI and CE-TG interchange with TG-rich lipoproteins pathway under cholesteryl ester transfer protein. Discoidal TA-rHDL could reconstruct to spheres and target via a similar route as TA-rHDL spheres, showing a higher targeting efficiency. In vivo experiments showed that areas under the plasma level-time curve (AUC) of TA increased as a function of spherical and discoidal rHDL, which were 4 and 13 times more than that of TA suspensions, respectively. Spherical and discoidal TA-rHDL had long circulating times in blood with mean residence time (MRT) of 15.874 and 18.956h, respectively, compared to 1.802h of TA suspensions, 14.190h of spherical TA-rHDL without apos and 15.071 of discoidal TA-rHDL without apos. The distribution volume of spherical TA-rHDL was 2.143 and 1.552 times as that of discoidal TA-rHDL and TA suspensions, respectively. In conclusion, TA-rHDL may be a long-circulating, healthy and potentially targeted carrier for delivering lipophilic cardiovascular drugs.

High density lipoproteins (HDL) have been successfully reconstructed to deliver a large number of lipophilic drugs. Here, discoidal and spherical recombinant HDL loaded with cardiovascular drug tanshinone IIA (TA) were constructed (TA-d-rHDL and TA-s-rHDL), respectively. And next their in vitro physiochemical and biomimetic properties were characterized. Furthermore, pharmacokinetics, atherosclerotic lesions targeting effects and antiatherogenic efficacies were elaborately performed and compared in atherosclerotic New Zealand White (NZW) rabbits. In vitro characterizations results showed that both TA-d-rHDL and TA-s-rHDL had nano-size diameter, high entrapment efficiency (EE) and drug-loading capacity (DL). Additionally, similar to their native counterparts, TA-d-rHDL maintained remodeling behaviors induced by lecithin cholesterol acyltransferase (LCAT), and TA leaked during remodeling behaviors. Pharmacokinetic studies manifested that both TA-d-rHDL and TA-s-rHDL markedly improved pharmacokinetic behaviors of TA in vivo. Ex vivo imaging demonstrated that both d-rHDL and s-rHDL bound more avidly to atherosclerotic lesions than to normal vessel walls, and s-rHDL had better targeting effect than d-rHDL. Pharmacodynamic tests illustrated that both TA-d-rHDL and TA-s-rHDL had much stronger antiatherogenic efficacies than conventional TA nanostructured lipid carriers (TA-NLC), TA liposomes (TA-L) and commercially available preparation Sulfotanshinone Sodium Injection (SSI). Moreover, TA-s-rHDL had more potent antiatherogenic efficacies than TA-d-rHDL. Collectively our studies indicated that rHDL could be exploited as potential delivery vehicles of TA targeting atherosclerotic lesions as well as synergistically improving efficacies, especially for s-rHDL.