Construction and application of biotin-poloxamer conjugate micelles for chemotherapeutics.

Biotin was conjugated on poloxamer to prepare biotin-poloxamer (BP) conjugate micelles for chemotherapeutics. Epirubicin (EPI) was encapsulated in BP micelles. The EPI-loaded BP micelles were characterized in terms of size, ζ-potential, morphology, drug loading, drug encapsulation and drug release. Marrow leukemic HL-60 cells were used for evaluating the in vitro cytotoxicity of EPI-loaded BP micelles. Nude mice were axillainoculated subcutaneously HL-60 cells to establish tumour model for investigating the inhibition effects of EPI-loaded BP micelles. From the results, the sizes of these nanoparticles were about 100 nm. Fluorescence microscope observation supported the enhanced cellular uptake of the micelles. The order of the inhibition on tumour volume growth was: EPI-loaded BP micelles >EPI-loaded MATP micelles >EPI-loaded poloxamer micelles >EPI. BP micelles showed significant antitumour activity and low toxicity, compared with the non-targeted micelles. With the advantage of EPR effect and tumour-targeting potential, BP conjugate micelles might be developed as a new system for chemotherapeutics.

Chlorin e6 (Ce6)-loaded plaque-specific liposome with enhanced photodynamic therapy effect for atherosclerosis treatment.

Recently, photodynamic therapy (PDT) has been considered as a new strategy for atherosclerosis treatment. Targeted delivery of photosensitizer could significantly reduce its toxicity and enhance its phototherapeutic efficiency. CD68 is an antibody that can be conjugated to nano-drug delivery systems to actively target plaque sites, owing to its specific binding to CD68 receptors that are highly expressed on the surfaces of macrophage-derived foam cells. Liposomes are very popular nanocarriers due to their ability to encapsulate a wide range of therapeutic compounds including drugs, microRNAs and photosensitizers, and their ability to be surface-modified with targeting moieties leading to the development of nanocarriers with an improved targeted ability. Hence, we designed a Ce6-loaded liposomes using the film dispersion method, followed by the conjugation of CD68 antibody on the liposomal surface through a covalent crosslinking reaction, forming CD68-modified Ce6-loaded liposomes (CD68-Ce6-mediated liposomes). Flow cytometry results indicated that Ce6-containing liposomes were more effective in promoting intracellular uptake after laser irradiation. Furthermore, CD68-modified liposomes significantly strengthened the cellular recognization and thus internalization. Different cell lines have been incubated with the liposomes, and the results showed that CD68-Ce6-mediated liposomes had no significant cytotoxicity to coronary artery endothelial cells (HCAEC) under selected conditions. Interestingly, they promoted autophagy in foam cells through the increase in LC3-Ⅰ, LC3-Ⅱ expression and the decrease in p62 expression, and restrained the migration of mouse aortic vascular smooth muscle cells (MOVAS) in vitro. Moreover, the enhancement of atherosclerotic plaque stability and the reduction in the cholesterol content by CD68-Ce6-mediated liposomes were dependent on transient reactive oxygen species (ROS) generated under laser irradiation. In summary, we demonstrated that CD68-Ce6-mediated liposomes, as a photosensitizer nano-drug delivery system, have an inhibitory effect on MOVAS migration and a promotion of cholesterol efflux in foam cells, and thereby, represent promising nanocarriers for atherosclerosis photodynamic therapy.

Using acoustic cavitation to enhance chemotherapy of DOX liposomes: experiment in vitro and in vivo.

Experiments in vitro and in vivo were designed to investigate tumor growth inhibition of chemotherapeutics-loaded liposomes enhanced by acoustic cavitation. Doxorubicin-loaded liposomes (DOX liposomes) were used in experiments to investigate acoustic cavitation mediated effects on cell viability and chemotherapeutic function. The influence of lingering sensitive period after acoustic cavitation on tumor inhibition was also investigated. Animal experiment was carried out to verify the practicability of this technique in vivo. From experiment results, blank phospholipid-based microbubbles (PBM) combined with ultrasound (US) at intensity below 0.3 W/cm² could produce acoustic cavitation which maintained cell viability at high level. Compared with DOX solution, DOX liposomes combined with acoustic cavitation exerted effective tumor inhibition in vitro and in vivo. The lingering sensitive period after acoustic cavitation could also enhance the susceptibility of tumor to chemotherapeutic drugs. DOX liposomes could also exert certain tumor inhibition under preliminary acoustic cavitation. Acoustic cavitation could enhance the absorption efficiency of DOX liposomes, which could be used to reduce DOX adverse effect on normal organs in clinical chemotherapy.

The ethanolic extract of Artemisia anomala exerts anti-inflammatory effects via inhibition of NLRP3 inflammasome.

BACKGROUND: Artemisia anomala S. Moore (Compositae), known as "Nan-Liu-Ji-Nu" in traditional Chinese medicine (TCM), has been used to treat many inflammatory diseases, including enteritis, acute icteric hepatitis, rheumatism, toothache, tonsillitis, and chronic bronchitis, for centuries. Our preliminary studies have demonstrated that the ethanolic extract of A. anomala (EAA) might be with the potential of inhibiting the activation of the NLRP3 inflammasome. However, the anti-inflammatory activity of EAA based on NLRP3 inflammasome inhibition is still unclear. PURPOSE: This work aimed to elucidate the anti-inflammatory mechanism of EAA by inhibiting NLRP3 inflammasome activation. METHODS: Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs) were used to evaluate the inhibitory effects on NLRP3 inflammasome activation. The level of IL-1ß was determined by ELISA. The expression levels of IL-1ß, caspase-1, NLRP3, and ASC were assayed using western blot analysis. ASC oligomerization and speck formation were detected by immunofluorescence microscopy. The measurements of intracellular chloride and potassium were conducted using N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) probe assay and inductively coupled plasma-optical emission spectrometry (ICP-OES), respectively. Mitochondrial reactive oxygen species (mtROS) were examined using the MitoSOX method. Acridine orange (AO) staining was used to detect the permeability of the lysosomal membrane. A DSS-induced ulcerative colitis model was established to evaluate the anti-inflammatory effects of EAA in vivo. Finally, high-performance liquid chromatography (HPLC) was employed to identify and quantify the major constituents of EAA. RESULTS: In BMDMs, EAA significantly inhibited the release of IL-1ß induced by LPS. The mechanistic study revealed that EAA inhibited NLRP3 inflammasome activation by blocking the oligomerization of ASC and suppressed the LPS-induced priming step. Furthermore, EAA protected lysosomes by inhibiting the TAK1-JNK pathway, thereby inhibiting the assembly of downstream NLRP3 inflammasome and the production of IL-1ß. In addition, EAA exerted potent protective effects in an ulcerative colitis model by decreasing the content of colonic IL-1ß and alleviating the process of ulcerative colitis. HPLC analysis identified eight main components of EAA, including isofraxidin (1), quercetin-7-O-ß-D-glucopyranoside (2), apigenin-7-O-ß-D-glucopyranoside (3), 7-methoxycoumarin (4), quercetin (5), luteolin (6), kaempferol (7), and eupatorin (8), Of these compounds, quercetin and kaempferol were found to be the most potent ingredients. CONCLUSION: These findings collectively reveal that EAA exerts anti-inflammatory effects by both suppressing the NLRP3 priming step and protecting lysosomes to inhibit NLRP3 inflammasome activation, suggesting that this traditional herbal medicine might be used to treat NLRP3-driven inflammatory diseases.

RNA interference-mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice.

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.

[Antitumor effects of matrix protein of vesicular stomatic virus on EL4 lymphoma mice].

OBJECTIVE: To explore antitumor effects of plasmid pcDNA3. 1-MP encoding matrix protein of vesicular stomatitis virus (VSV) complexed with cationic liposome (DOTAP:CHOL) in mice with EL4 lymphoma. METHODS: C57BL/6 mouse model with EL4 lymphoma was established. Sixty mice bearing EL4 lymphoma were divided randomly into five groups including Lip-MP, Lip-pVAX, Lip, ADM and NS groups, which were intravenously injected with liposome-pcDNA 3. 1-MP complex, liposome-pVAX complex, empty liposome, Adriamycin and normal saline respectively every three days. Tumor volumes and survival time were monitored. Microvessel density and tumor proliferative index in tumor tissues were determined by CD31, Ki-67 immunohistochemistry staining, meanwhile the tumor apoptosis index was measured by TUNEL method. RESULTS: From 6 days after treatments on, the tumor volume in Lip-MP group was much smaller than that in Lip-pVAX, Lip and NS group (P < 0.05). The median survival time of mice in Lip-MP group, 44 days after inoculation of tumor cells, was significantly higher than that in other groups (P < 0.05), which was 39 days, 38.5 days and 34 days in Lip-pVAX, Lip and NS groups respectively. The MVD value in tumor tissues in Lip-MP group was less than that in Lip-pVAX, Lip and NS groups (P < 0.05). Ki67 staining revealed that Lip-MP complex apparently suppressed the proliferation of EL4 tumor cells in vivo (P < 0.05). TUNEL assays showed that apoptosis index of tumor cells in Lip-MP group, 10.60 +/- 1.71, was much higher than that in other three groups (P < 0.05). CONCLUSIONS: Lip-MP complex, the plasmid encoding matrix protein of VSV (VSV-MP) encapsulated in cationic liposome, significantly inhibited the growth of tumor and prolonged the survival of mice bearing EL4 lymphoma, which may be related to the induction of tumor cell apoptosis, inhibition of tumor angiogenesis, and suppression of tumor cell proliferation.

A novel drug and gene co-delivery system based on Poly(epsilon-caprolactone)-Poly(ethylene glycol)-Poly(epsilon-caprolactone) grafted polyethyleneimine micelle.

In this paper, we prepared a novel cationic self-assembled micelle from poly(epsilon-caprolactone)-poly(ethyl glycol)-poly(epsilon-caprolactone) grafted polyethyleneimine (PCEC-g-PEI). The PCEC-g-PEI micelles, formed by self-assembly method, had mean particle size of ca. 82 nm and zeta potential of +22.5 mV at 37 degrees C, and could efficiently transfer pGFP into HEK293 cells in vitro. Meanwhile, as a model hydrophobic chemotherapeutic drug, honokiol was loaded into PCEC-g-PEI micelles by direct dissolution method assisted by ultrasonication. The honokiol loaded cationic PCEC-g-PEI micelles could effectively adsorb DNA onto its surface, while it could release honokiol in an extended period in vitro. This study demonstrated a novel DNA and hydrophobic chemotherapeutic drug co-delivery system.

Liposomal honokiol inhibits VEGF-D-induced lymphangiogenesis and metastasis in xenograft tumor model.

Lymph nodes metastasis of tumor could be a crucial early step in the metastatic process. Induction of tumor lymphangiogenesis by vascular endothelial growth factor-D may play an important role in promoting tumor metastasis to regional lymph nodes and these processes can be inhibited by inactivation of the VEGFR-3 signaling pathway. Honokiol has been reported to possess potent antiangiogenesis and antitumor properties in several cell lines and xenograft tumor models. However, its role in tumor-associated lymphangiogenesis and lymphatic metastasis remains unclear. Here, we established lymph node metastasis models by injecting overexpressing VEGF-D Lewis lung carcinoma cells into C57BL/6 mice to explore the effect of honokiol on tumor-associated lymphangiogenesis and related lymph node metastasis. The underlying mechanisms were systematically investigated in vitro and in vivo. In in vivo study, liposomal honokiol significantly inhibited the tumor-associated lymphangiogenesis and metastasis in Lewis lung carcinoma model. A remarkable delay of tumor growth and prolonged life span were also observed. In in vitro study, honokiol inhibited VEGF-D-induced survival, proliferation and tube-formation of both human umbilical vein endothelial cells (HUVECs) and lymphatic vascular endothelial cells (HLECs). Western blotting analysis showed that liposomal honokiol-inhibited Akt and MAPK phosphorylation in 2 endothelial cells, and downregulated expressions of VEGFR-2 of human vascular endothelial cells and VEGFR-3 of lymphatic endothelial cells. Thus, we identified for the first time that honokiol provided therapeutic benefit not only by direct effects on tumor cells and antiangiogenesis but also by inhibiting lymphangiogenesis and metastasis via the VEGFR-3 pathway. The present findings may be of importance to investigate the molecular mechanisms underlying the spread of cancer via the lymphatics and explore the therapeutical strategy of honokiol on antilymphangiogenesis and antimetastasis.

Induction of tumor inhibition and apoptosis by a candidate tumor suppressor gene DRR1 on 3p21.1.

Down-regulated in renal cell carcinoma gene (DRR1) is one of the candidate tumor suppressor genes (TSGs) on human 3p21.1. This study was performed to validate the expression status of DRR1 gene in cancer cells and the expression pattern of the protein in clinical specimens of human lung cancer and to examine its potential as a molecular target for treatment of lung cancer in vivo. DRR1 expression was analyzed in 7 human lung cancer cell lines. DRR1 protein expression was also examined in clinical non-small cell lung cancer (NSCLC) specimens. Furthermore, effects of DRR1 re-expression on A549 cells in vitro and A549 xenograft tumors in nude mice were evaluated. Loss of DRR1 mRNA expression was detected in 6 of the 7 human cancer cell lines, the exception was the renal cancer cell line OS-RC-2. DRR1 protein expression was absent in 15 of 20 (75%) human NSCLC specimens by immunostaining. Transfection of DRR1 gene into DRR1-negative-expressing A549 cells resulted in significant cell growth suppression and apoptosis. Plasmids containing DRR1 cDNA complexed with DOTAP:Chol liposomes were administered intravenously via tail vein to nude mice bearing A549 xenograft tumors resulting in tumor growth inhibition and elevation of apoptosis compared with the controls. DRR1 is a potent growth suppressor of NSCLC, acting through apoptosis pathway in vivo and it may be a potential therapeutic gene for human lung cancer.

Fracture resistance after simulated crown lengthening and forced tooth eruption of endodontically-treated teeth restored with a fiber post-and-core system.

PURPOSE: To evaluate the effect of ferrule preparation length on the fracture resistance after simulated surgical crown lengthening and after forced tooth eruption of endodontically-treated teeth restored with a carbon fiber-reinforced post-and-core system. METHODS: 40 extracted endodontically-treated mandibular first premolars were decoronated 1.0 mm coronal to the buccal cemento-enamel junction. The teeth were divided randomly into five equal groups. The control group had no ferrule preparation (Group A). Simulated crown lengthening provided ferrule preparations of 1.0 mm (Group B) and 2.0 mm (Group C). Simulated forced tooth eruption provided ferrule preparations of 1.0 mm (Group D) and 2.0 mm (Group E). After restoration with a carbon fiber post-and-core system, each root was embedded in an acrylic resin block from 2.0 mm apical to the margins of a cast Ni-Cr alloy crown, and loaded at 150 degrees from the long axis in a universal testing machine at a crosshead speed of 1.0 mm/minute until fracture. Data were analyzed using ANOVA with Tukey HSD tests, and Fisher's exact test, with alpha = 0.05. RESULTS: Mean failure loads (kN) for Groups A, B, C, D and E were: 1.13 (SD = 0.15), 1.27 (0.18), 1.02 (0.11), 1.63 (0.14) and 1.92 (0.19), respectively. Significant differences were shown for the effects of treatment method and ferrule length, with significant interaction between these two sources of variation (P < 0.0001). Increased apical ferrule preparation lengths resulted in significantly increased fracture resistance for simulated forced tooth eruption (P < 0.0001), but not for simulated crown lengthening (P > or = 0.24).

Methotrexate-loaded biodegradable polymeric micelles for lymphoma therapy.

Drug resistance and recurrence are the main clinical challenges in chemotherapy of lymphoma. Methotrexate (MTX), especially high dose MTX (HD MTX), is extensively used to treat some aggressive subtypes of lymphoma, such as Burkitt's lymphoma, in order to overcome drug resistance. But poor solubility of the free drug and severe side effects of HD MTX limit its clinical application. Polymeric micelle, as an ideal nano delivery system, provides effective solutions to these problems. In this work, monomethyl poly (ethylene glycol)-poly (ε-caprolactone) (MPEG-PCL) was employed to load MTX through a one-step solid dispersion method. MTX loaded micelles had a small particle size of 25.64 ±â€¯0.99 nm and polydisperse index (PDI) of 0.176 ±â€¯0.05. Drug loading and encapsulation efficiency of MTX loaded micelles were 5.57 ±â€¯0.14% and 92.46 ±â€¯2.38%. Compared with free MTX, MTX loaded micelles demonstrated a much slower and sustained release behavior in vitro. MTT assay and cell apoptosis study suggested that MTX loaded micelles were more effective in inhibiting proliferation and inducing apoptosis on Raji lymphoma cells than MTX injection, which was especially distinct in high dose groups. Cellular uptake study indicated that MPEG-PCL micelle had a 1.5 times higher uptake rate in Raji cells. As for in vivo studies, MTX loaded micelles were more competent to suppress tumor growth and prolong survival time than MTX injection in the subcutaneous Raji lymphoma model. Notably, the high dose group of micelle formulation exhibited the strongest anti-tumor effect without additional toxicity. Furthermore, immunofluorescent and immunohistochemical studies showed that tumors of MPEG-PCL-MTX treated mice had more apoptotic cells and fewer proliferative cells. In conclusion, MPEG-PCL-MTX micelle is an excellent intravenously injectable formulation of MTX with both good solubility and enhanced anti-tumor activity, which perfectly meets clinical demands, especially for administration of HD MTX.

Liposomal honokiol, a potent anti-angiogenesis agent, in combination with radiotherapy produces a synergistic antitumor efficacy without increasing toxicity.

Honokiol is an active compound purified from magnolia that has been shown to induce cell differentiation, apoptosis, and anti-angiogenesis effects, as well as an enhancement in tumor growth delay in combination with chemotherapeutic agents in several mouse xenograft models. Our goal was to investigate the radiosensitization effect of honokiol on lung carcinoma. The radiosensitization effect of liposomal honokiol in Lewis lung carcinoma cells (LL/2) was analyzed using an in vitro clonogenic survival assay. For an in vivo study, Lewis lung carcinoma-bearing C57BL/6 mice were treated with either liposomal honokiol at 25 mg/kg or 5 Gy of single tumor radiation, or a combination of both over 12 days of treatment. The tumor growth delay and the survival time were evaluated. In addition, histological analysis of tumor sections was performed to examine changes by detecting the microvessel density and apoptosis in tumor tissues. In the clonogenic survival assay, LL/2 cells treated with IC(50) Lipo-HNK for 24 h showed a radiation enhancement ratio of 1.9. After 12 days of combination treatment, the tumor volume decreased 78% and produced an anti-tumor activity 1.3-fold greater than a predicted additive effect of honokiol and radiation alone. This combination treatment also caused an 8.7 day delay in tumor growth. The cell cycle distribution and histological analysis demonstrated that liposomal honokiol has an anti-tumor effect via inducing apoptosis and inhibiting angiogenesis. Liposomal honokiol can enhance tumor cell radiosensitivity in vitro and in vivo, indicating that radiotherapy combined with liposomal honokiol can lead to greater anti-tumor efficacy.

Liposomal honokiol, a promising agent for treatment of cisplatin-resistant human ovarian cancer.

PURPOSE: Honokiol has been receiving attention as an anticancer agent because of its anti-tumor effect. In the current study, we encapsulated honokiol with liposome and tested it on cisplatin-sensitive (A2780s) and -resistant (A2780cp) human ovarian cancer models. METHODS: The anti-tumor activity of liposomal honokiol (Lipo-HNK) was evaluated in nude mice bearing A2780s and A2780cp s.c. tumors. Mice were treated twice weekly with i.v. administration of Lipo-HNK (10 mg/kg), control liposome (10 mg/kg), 0.9% NaCl solution or weekly with intraperitoneally administered cisplatin (5 mg/kg) for 3 weeks. Tumor volume and survival time were observed. Assessment of apoptotic cells by TUNEL assay was conducted in tumor tissue. Microvessel density within tumor tissue was determined by CD34 immunohistochemistry. For in vitro study, induction of apoptosis by Lipo-HNK was examined by PI staining fluorescence microscopy, DNA fragmentation assay and flow cytometric analysis. RESULTS: Administration of Lipo-HNK resulted in significant inhibition (84-88% maximum inhibition relative to controls) in the growth of A2780s and A2780cp tumor xenografts and prolonged the survival of the treated mice. These anti-tumor responses were associated with marked increases in tumor apoptosis, and reductions in intratumoral microvessel density. CONCLUSIONS: The present findings suggest that Lipo-HNK may provide an effective approach to inhibit tumor growth in both cisplatin sensitive and -resistant human ovarian cancer with minimal side effects.

Improved therapeutic effectiveness by combining liposomal honokiol with cisplatin in lung cancer model.

BACKGROUND: Honokiol is a major bioactive compound extracted from Magnolia. The present study was designed to determine whether liposomal honokiol has the antitumor activity against human lung cancer as well as potentiates the antitumor activity of cisplatin in A549 lung cancer xenograft model, if so, to examine the possible mechanism in the phenomenon. METHODS: human A549 lung cancer-bearing nude mice were treated with liposomal honokiol, liposomal honokiol plus DDP or with control groups. Apoptotic cells and vessels were evaluated by fluorescent in situ TUNEL assay and by immunohistochemistry with an antibody reactive to CD31 respectively. RESULTS: The present study showed that liposomal honokiol alone resulted in effective suppression of the tumor growth, and that the combined treatment with honokiol plus DDP had the enhanced inhibition of the tumor growth and resulted in a significant increase in life span. The more apparent apoptotic cells in the tumors treated with honokiol plus DDP was found in fluorescent in situ TUNEL assay, compared with the treatment with control groups. In addition, the combination of honokiol and DDP apparently reduced the number of vessels by immunolabeling of CD31 in the tissue sections, compared with control groups. CONCLUSION: In summary, our data suggest that honokiol alone had the antitumor activity against human lung cancer in A549 lung cancer xenograft model, and that the combination of honokiol with DDP can enhance the antitumor activity, and that the enhanced antitumor efficacy in vivo may in part result from the increased induction of the apoptosis and the enhanced inhibition of angiogenesis in the combined treatment. The present findings may be of importance to the further exploration of the potential application of the honokiol alone or the combined approach in the treatment of lung carcinoma.

Biocompatibility of tetramethylpyrazine-eluting stents in normal porcine coronary arteries.

OBJECTIVE: Drug-eluting stents have been used to markedly decrease in-stent restenosis in 6 months, but they are noticed due to the late thrombogenicity. The purpose of the present study was to evaluate the biocompatibility of Tetramethylpyrazine-eluting stents by investigating the intimal response and thrombogenicity in normal porcine coronary arteries by quantitative coronary angiography (QCA), intravascular ultrasound (IVUS) and histomorphometry. METHODS: Bare metal stents (BMS) were uniformly spray-coated with Tetramethylpyrazine (TMP 200 microg) and prepared for TMP-eluting stents (TES). Fourteen coronary arteries in 14 pigs underwent stent implantation. Seven TES were implanted in 7 pigs and 7 BMS in other 7 pigs. The stents were deployed with a stent-to-artery ratio of 1.1-1.2/1.0 in order to induce vascular wall injury. QCA and IVUS were performed before and immediately after the implantations and at 28 days (end time point). The analysis on blood cell count, biochemical parameters, status of behavior of pigs were evaluated before the implantation and at the time of 1 and 28 days. Stented-coronary arteries, stented-coronary arteries related ventricular wall, lung, liver and kidney were harvested after euthanasia of animals at the endpoint. Histopathology and histomorphometry had been done to assess the local toxicity of TES to these organs. RESULTS: All the stents were successfully implanted, however, 4 pigs died of cardiac tamponade or anesthesia. No bone marrow depression and hemolysis was seen. No damage to the function and metabolism of liver and kidney was discovered. No thrombosis was found in control and test groups. Few inflammatory cells were found in the stented-coronary artery walls at each endpoint in both groups. No damage to stented-coronary arteries related ventricular wall, lung, liver and kidney was detected due to TES implantation. Compared with the control group, the neointimal area was significantly reduced in the TES group (60.2+/-23.5% vs 10.0+/-2.1%, P=0.01) by IVUS analysis, but the lumen area in the TES group was increased (4.34+/-0. 93 mm(2) vs 1.29+/-1.02 mm(2), P=0.011), the neointimal area was reduced markedly (1.51+/-0.45 mm(2) vs 4.60+/-1.39 mm(2), P=0.004). CONCLUSIONS: The biocompatibility of TES in porcine model at 28 days seems to be good and acceptable. Biocompatibility can be evaluated by IVUS and histopathology in a porcine restenosis model.

[BFGF entraped in cationic liposomes as adjuvant: optimization of technical parameters].

OBJECTIVE: To test the immune efficiency of bFGF entraped in cationic liposomes as adjuvant in vivo. METHODS: The technical parameters on encapsulation were tested in each step to gain high encapsulation efficiencies, which included lipid composition, weight ratio of protein and lipids, liposome extrusion, and different conditions of freeze-thawing. The bFGF in cationic liposome, Freund's adjuvant, or PBS were injected (four times) to the four-week old Balb/c mice to test the immune responses. The serum antibody was measured by ELISA 13 days after each injection. RESULTS: Maximal encapsulation efficiency (about 50%) was achieved through optimized technical parameters. Cationic liposome demonstrated satisfied immune efficiency as adjuvant. CONCLUSION: Cationic liposome is a safe and effective immunological adjuvant.

Liposomal quercetin efficiently suppresses growth of solid tumors in murine models.

PURPOSE: Quercetin is a potent chemotherapeutic drug. Clinical trials exploring different schedules of administration of quercetin have been hampered by its extreme water insolubility. To overcome this limitation, this study is aimed to develop liposomal quercetin and investigate its distribution in vivo and antitumor efficacy in vivo and in vitro. EXPERIMENTAL DESIGN: Quercetin was encapsulated in polyethylene glycol 4000 liposomes. Biodistribution of liposomal quercetin i.v. at 50 mg/kg in tumor-bearing mice was detected by high-performance liquid chromatography. Induction of apoptosis by liposomal quercetin in vitro was tested. The antitumor activity of liposomal quercetin was evaluated in the immunocompetent C57BL/6N mice bearing LL/2 Lewis lung cancer and in BALB/c mice bearing CT26 colon adenocarcinoma and H22 hepatoma. Tumor volume and survival time were observed. The mechanisms underlying the antitumor effect of quercetin in vivo was investigated by detecting the microvessel density, apoptosis, and heat shock protein 70 expression in tumor tissues. RESULTS: Liposomal quercetin could be dissolved in i.v. injection and effectively accumulate in tumor tissues. The half-time of liposomal quercetin was 2 hours in plasma. The liposomal quercetin induced apoptosis in vitro and significantly inhibited tumor growth in vivo in a dose-dependent manner. The optimal dose of liposomal quercetin resulted in a 40-day survival rate of 40%. Quantitative real-time PCR showed that liposomal quercetin down-regulated the expression of heat shock protein 70 in tumor tissues. Immunohistochemistry analysis showed that liposomal quercetin inhibited tumor angiogenesis as assessed by CD31 and induced tumor cell apoptosis. CONCLUSIONS: Our data indicated that pegylated liposomal quercetin can significantly improve the solubility and bioavailability of quercetin and can be a potential application in the treatment of tumor.

Epirubicin loaded with propylene glycol liposomes significantly overcomes multidrug resistance in breast cancer.

Multidrug resistance (MDR) is one of the major reasons for the failure of cancer chemotherapy. A newly reported liposome carrier, propylene glycol liposomes (EPI-PG-liposomes) were made to load epirubicin (EPI) which enhanced EPI absorption in MDR tumor cells to overcome the drug resistance. MDA-MB 435 and their mutant resistant (MDA-MB 435/ADR) cells were used to examine the cellular uptake and P-gp function in vitro for EPI-PG-liposomes by fluorescence microscopy and FCM, respectively. Mammary tumor model was also established to investigate the tumor growth inhibition and pharmacodynamics of EPI-PG-liposomes in vivo. Morphology evaluation showed that EPI-PG-liposomes had a homogeneous spherical shape with an average diameter of 182 nm. Based on cell viability assay, fluorescent microscopy examination, and EPI uptake assay, EPI-PG-liposomes exhibited an effective growth inhibition not only in MDA-MB-435 cells, but also in MDA-MB 435/ADR cells. EPI-PG-liposomes have high permeability not only on tumor cell membrane, but also on cell nucleus membrane. P-gp function assay showed that the anticancer action of EPI-PG-liposomes was not related to P-gp efflux pump, suggesting that PG-liposomes would not affect the normal physiological functions of membrane proteins. EPI-PG-liposomes also showed a better antitumor efficacy compared to EPI solution alone. With high entrapment efficiency, spherical morphology and effective inhibition on MDR cancer cells, EPI-PG-liposomes may represent a better chemotherapeutic vectors for cancer targeted therapy.

Evaluation of a novel thermosensitive heparin-poloxamer hydrogel for improving vascular anastomosis quality and safety in a rabbit model.

Despite progress in the design of advanced surgical techniques, stenosis recurs in a large percentage of vascular anastomosis. In this study, a novel heparin-poloxamer (HP) hydrogel was designed and its effects for improving the quality and safety of vascular anastomosis were studied. HP copolymer was synthesized and its structure was confirmed by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy ((1)H-NMR). Hydrogels containing HP were prepared and their important characteristics related to the application in vascular anastomosis including gelation temperature, rheological behaviour and micromorphology were measured. Vascular anastomosis were performed on the right common carotid arteries of rabbits, and the in vivo efficiency and safety of HP hydrogel to achieve vascular anastomosis was verified and compared with Poloxamer 407 hydrogel and the conventional hand-sewn method using Doppler ultrasound, CT angiograms, scanning electron microscopy (SEM) and histological technique. Our results showed that HP copolymer displayed special gel-sol-gel phase transition behavior with increasing temperature from 5 to 60 °C. HP hydrogel prepared from 18 wt% HP solution had a porous sponge-like structure, with gelation temperature at approximately 38 °C and maximum elastic modulus at 10,000 Pa. In animal studies, imaging and histological examination of rabbit common jugular artery confirmed that HP hydrogel group had similar equivalent patency, flow and burst strength as Poloxamer 407 group. Moreover, HP hydrogel was superior to poloxamer 407 hydrogel and hand-sewn method for restoring the functions and epithelial structure of the broken vessel junctions after operation. By combining the advantages of heparin and poloxamer 407, HP hydrogel holds high promise for improving vascular anastomosis quality and safety.

A systemic administration of liposomal curcumin inhibits radiation pneumonitis and sensitizes lung carcinoma to radiation.

Radiation pneumonitis (RP) is an important dose-limiting toxicity during thoracic radiotherapy. Previous investigations have shown that curcumin is used for the treatment of inflammatory conditions and cancer, suggesting that curcumin may prevent RP and sensitize cancer cells to irradiation. However, the clinical advancement of curcumin is limited by its poor water solubility and low bioavailability after oral administration. Here, a water-soluble liposomal curcumin system was developed to investigate its prevention and sensitizing effects by an intravenous administration manner in mice models. The results showed that liposomal curcumin inhibited nuclear factor-κB pathway and downregulated inflammatory factors including tumor necrosis factor-α, interleukin (IL)-6, IL-8, and transforming growth factor-ß induced by thoracic irradiation. Furthermore, the combined treatment with liposomal curcumin and radiotherapy increased intratumoral apoptosis and microvessel responses to irradiation in vivo. The significantly enhanced inhibition of tumor growth also was observed in a murine lung carcinoma (LL/2) model. There were no obvious toxicities observed in mice. The current results indicate that liposomal curcumin can effectively mitigate RP, reduce the fibrosis of lung, and sensitize LL/2 cells to irradiation. This study also suggests that the systemic administration of liposomal curcumin is safe and deserves to be investigated for further clinical application.