Sorafenib and triptolide loaded cancer cell-platelet hybrid membrane-camouflaged liquid crystalline lipid nanoparticles for the treatment of hepatocellular carcinoma.

In addition to early detection, early diagnosis, and early surgery, it is of great significance to use new strategies for the treatment of hepatocellular carcinoma (HCC). Studies showed that the combination of sorafenib (SFN) and triptolide (TPL) could reduce the clinical dose of SFN and maintain good anti-HCC effect. But the solubility of SFN and TPL in water is low and both drugs have certain toxicity. Therefore, we constructed a biomimetic nanosystem based on cancer cell-platelet (PLT) hybrid membrane camouflage to co-deliver SFN and TPL taking advantage of PLT membrane with long circulation functions and tumor cell membrane with homologous targeting. The biomimetic nanosystem, SFN and TPL loaded cancer cell-PLT hybrid membrane-camouflaged liquid crystalline lipid nanoparticles ((SFN + TPL)@CPLCNPs), could simultaneously load SFN and TPL at the molar ratio of SFN to TPL close to 10:1. (SFN + TPL)@CPLCNPs achieved long circulation function and tumor targeting at the same time, promoting tumor cell apoptosis, inhibiting tumor growth, and achieving a better "synergy and attenuation effect", which provided new ideas for the treatment of HCC.

N-Acetyl-l-cysteine-Loaded Nanosystems as a Promising Therapeutic Approach Toward the Eradication of Pseudomonas aeruginosa Biofilms.

Bacterial biofilms are a major health concern, mainly due to their contribution to increased bacterial resistance to well-known antibiotics. The conventional treatment of biofilms represents a challenge, and frequently, eradication is not achieved with long-lasting administration of antibiotics. In this context, the present work proposes an innovative therapeutic approach that is focused on the encapsulation of N-acetyl-l-cysteine (NAC) into lipid nanoparticles (LNPs) functionalized with d-amino acids to target and disrupt bacterial biofilms. The optimized formulations presented a mean hydrodynamic diameter around 200 nm, a low polydispersity index, and a high loading capacity. These formulations were stable under storage conditions up to 6 months. In vitro biocompatibility studies showed a low cytotoxicity effect in fibroblasts and a low hemolytic activity in human red blood cells. Nevertheless, unloaded LNPs showed a higher hemolytic potential than NAC-loaded LNPs, which suggests a safer profile of the latter. The in vitro antibiofilm efficacy of the developed formulations was tested against Staphylococcus epidermidis (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) mature biofilms. The results showed that the NAC-loaded LNPs were ineffective against S. epidermidis biofilms, while a significant reduction of biofilm biomass and bacterial viability in P. aeruginosa biofilms were observed. In a more complex therapeutic approach, the LNPs were further combined with moxifloxacin, revealing a beneficial effect between the LNPs and the antibiotic against P. aeruginosa biofilms. Both alone and in combination with moxifloxacin, unloaded and NAC-loaded LNPs functionalized with d-amino acids showed a great potential to reduce bacterial viability, with no significant differences in the presence or absence of NAC. However, the presence of NAC in NAC-loaded functionalized LNPs shows a safer profile than the unloaded LNPs, which is beneficial for an in vivo application. Overall, the developed formulations present a potential therapeutic approach against P. aeruginosa biofilms, alone or in combination with antibiotics.

Hybrid of niosomes and bio-synthesized selenium nanoparticles as a novel approach in drug delivery for cancer treatment.

The current study intends to investigate a novel drug delivery system (DDS) based on niosomes structure (NISM) and bovine serum albumin (BSA) which was formulated to BSA coated NISM (NISM-B). Also, selenium nanoparticles (SeNPs) have been prepared by BSA mediated biosynthesis. Finally, the NISM-B was hybridized with SeNPs and was formulated as NISM-B@SeNPs for drug delivery applications. Physicochemical properties of all samples were characterized by UV-Vis spectroscopy, FT-IR, DLS, FESEM, and EDX techniques. The cytotoxicity of all samples against A549 cell line was assessed by cell viability analysis and flow cytometry for apoptotic cells as well as RT-PCR for the expression of MDR-1, Bax, and Bcl-2 genes. Besides, in vivo biocompatibility was performed by LD50 assay to evaluate the acute toxicity. The proposed formulation has a regular spherical shape and approximately narrow size distribution with proper zeta-potential values; the proposed DDS revealed a good biocompatibility. The compound showed a significant cytotoxic effect against A549 cell line. Although the Bax/Bcl-2 expression ratio was significantly in NISM-B@SeNPs- treated cancer cells, the expression of MDR-1 was non-significantly lower in NISM-B@SeNPs-treated cancer cells. The obtained results suggest that the proposed DDS presents a promising approach for drug delivery, co-delivery and multifunctional biomedicine applications.

Chemical characterization of liposomes containing nutraceutical compounds: Tyrosol, hydroxytyrosol and oleuropein.

Tyrosol, hydroxytyrosol and oleuropein are among the major phenolic compounds in fruits, leaves and oils from Olea europaea L. These natural antioxidants molecules revealed several beneficial effects on human health, but a low bioavailability and accessibility to targeted site. Liposomes are drug/nutraceutical delivery carriers, used for driving bioactive molecules to desired target tissues, decreasing potential side effects and protecting the encapsulated molecule from enzymatic metabolic processes. In this study, zwitterionic liposomes containing tyrosol, hydroxytyrosol and oleuropein were synthesized and characterized for their size and surface charge. Particular attention was devoted to the determination of encapsulation efficiency (EE%), quantifying the loaded Tyr, HTyr and Ole amount, by using three different techniques: direct UV spectrophotometry, High Performance Liquid Chromatography and Trolox Equivalent Antioxidant Capacity assay. The results revealed higher EE% for oleuropein. Cyto-toxicity and cyto-compatibility of liposomes were also tested on human chondrocyte cells.

Synthesis of lipid-black phosphorus quantum dot bilayer vesicles for near-infrared-controlled drug release.

Black phosphorus quantum dots are incorporated into liposomal bilayers to produce a drug delivery system with excellent near-infrared (NIR) photothermal properties and drug release capability controlled by light. In vitro experiments demonstrate its good biocompatibility and NIR-light-induced chemo-photothermal antitumor efficiency.

Biocompatibility and therapeutic evaluation of magnetic liposomes designed for self-controlled cancer hyperthermia and chemotherapy.

Magnetic liposome-mediated combined chemotherapy and hyperthermia is gaining importance as an effective therapeutic modality for cancer. However, control and maintenance of optimum hyperthermia are major challenges in clinical settings due to the overheating of tissues. To overcome this problem, we developed a novel magnetic liposomes formulation co-entrapping a dextran coated biphasic suspension of La0.75Sr0.25MnO3 (LSMO) and iron oxide (Fe3O4) nanoparticles for self-controlled hyperthermia and chemotherapy. However, the general apprehension about biocompatibility and safety of the newly developed formulation needs to be addressed. In this work, in vitro and in vivo biocompatibility and therapeutic evaluation studies of the novel magnetic liposomes are reported. Biocompatibility study of the magnetic liposomes formulation was carried out to evaluate the signs of preliminary systemic toxicity, if any, following intravenous administration of the magnetic liposomes in Swiss mice. Therapeutic efficacy of the magnetic liposomes formulation was evaluated in the fibrosarcoma tumour bearing mouse model. Fibrosarcoma tumour-bearing mice were subjected to hyperthermia following intratumoral injection of single or double doses of the magnetic liposomes with or without chemotherapeutic drug paclitaxel. Hyperthermia (three spurts, each at 3 days interval) with drug loaded magnetic liposomes following single dose administration reduced the growth of tumours by 2.5 fold (mean tumour volume 2356 ± 550 mm3) whereas the double dose treatment reduced the tumour growth by 3.6 fold (mean tumour volume 1045 ± 440 mm3) compared to their corresponding control (mean tumour volume 3782 ± 515 mm3). At the end of the tumour efficacy studies, the presence of MNPs was studied in the remnant tumour tissues and vital organs of the mice. No significant leaching or drainage of the magnetic liposomes during the study was observed from the tumour site to the other vital organs of the body, suggesting again the potential of the novel magnetic liposomes formulation for possibility of developing as an effective modality for treatment of drug resistant or physiologically vulnerable cancer.

Liposomal butamben gel formulations: toxicity assays and topical anesthesia in an animal model.

The aim of this study was to evaluate the in vitro cytotoxicity and the in vivo analgesic effect and local toxicity of the local anesthetic butamben (BTB) encapsulated in conventional or elastic liposomes incorporated in gel formulations. The results showed that both gel formulations of liposomal BTB reduced the cytotoxicity (p < 0.001; one-way ANOVA/Tukey's test) and increased the topical analgesic effect (p < 0.05; one-way ANOVA/Tukey's test) of butamben, compared to plain BTB gel. The gel formulations presented good rheological properties, and stability assays detected no differences in physicochemical stability up to 30 d after preparation. Moreover, histological assessment revealed no morphological changes in rat skin after application of any of the gel formulations tested.

Preclinical evaluation of recombinant human IFNα2b-containing magnetoliposomes for treating hepatocellular carcinoma.

Magnetoliposomes are phospholipid vesicles encapsulating magnetic nanoparticles that can be used to encapsulate therapeutic drugs for delivery into specific organs. Herein, we developed magnetoliposomes containing recombinant human IFNα2b, designated as MIL, and evaluated this combination's biological safety and therapeutic effect on both cellular and animal hepatocellular carcinoma models. Our data showed that MIL neither hemolyzed erythrocytes nor affected platelet-aggregation rates in blood. Nitroblue tetrazolium-reducing testing showed that MIL did not change the absolute numbers or phagocytic activities of leukocytes. Acute-toxicity testing also showed that MIL had no devastating effect on mice behaviors. All the results indicated that the nanoparticles could be a safe biomaterial. Pharmacokinetic analysis and tissue-distribution studies showed that MIL maintained stable and sustained drug concentrations in target organs under a magnetic field, helped to increase bioavailability, and reduced administration time. MIL also dramatically inhibited the growth of hepatoma cells. Targeting of MIL in the livers of nude mice bearing human hepatocellular carcinoma showed that MIL significantly reduced the tumor size to 38% of that of the control group. Further studies proved that growth inhibition of cells or tumors was due to apoptosis-signaling pathway activation by human IFNα2b.

Modulation of drug-resistant membrane and apoptosis proteins of breast cancer stem cells by targeting berberine liposomes.

The recurrence of breast cancer is associated with drug-resistance of cancer stem cells (CSCs), while overexpression of cell membrane ATP-binding cassette (ABC) transporters and resistance of mitochondrial apoptosis-related proteins are responsible for the drug-resistance of CSCs. The targeting berberine liposomes were developed to modulate the resistant membrane and mitochondrial proteins of breast CSCs for the treatment and prevention of breast cancer relapse. Evaluations were performed on human breast CSCs and CSC xenografts in nude mice. The targeting berberine liposomes were shown to cross the CSC membrane, inhibit ABC transporters (ABCC1, ABCC2, ABCC3, ABCG2) and selectively accumulate in the mitochondria. Furthermore, the pro-apoptotic protein Bax was activated while the anti-apoptotic protein Bcl-2 was inhibited resulting in opening of the mitochondrial permeability transition pores, release of cytochrome c, and activation of caspase-9/caspase-3 enzymes. Significant efficacy of the administrations in mice was observed, indicating that the targeting berberine liposomes are a potential therapy for the treatment and prevention of breast cancer relapse arising from CSCs.

Therapeutic efficacy of 188Re-liposome in a C26 murine colon carcinoma solid tumor model.

Nanoliposomes are good drug delivery systems that allow the encapsulation of drugs into vesicles for their delivery. The objective of this study is to investigate the therapeutic efficacy of a new radio-therapeutics of (188)Re-labeled pegylated liposome in a C26 murine colon carcinoma solid tumor model. The safety of (188)Re-liposome was evaluated before radiotherapy treatment. The anti-tumor effect of (188)Re-liposome was assessed by tumor growth inhibition, survival ratio and ultrasound imaging. Apoptotic marker in tumor was also evaluated by the TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling) method after injection of (188)Re-liposome. The group treated with (188)Re-liposome displayed slight loss in body weight and decrease in white blood cell (WBC) count 7 to 14 days post-injection. With respect to therapeutic efficacy, the tumor-bearing mice treated with (188)Re-liposome showed better mean tumor growth inhibition rate (MGI) and longer median survival time (MGI = 0.140; 80 day) than those treated with anti-cancer drug 5-FU (MGI = 0.195; 69 day) and untreated control mice (MGI = 0.413; 48 day). The ultrasound imaging showed a decrease in both tumor volume and number of blood vessels. There were significantly more apoptotic nuclei (TUNEL-positive) in (188)Re-liposome-treated mice at 8 h after treatment than in control mice. These results evidenced the potential benefits achieved by oncological application of the radio-therapeutics (188)Re-liposome for adjuvant cancer treatment.

Cationic amphiphiles with fatty acyl chain asymmetry of coconut oil deliver genes selectively to mouse lung.

Recent structure-activity studies have revealed a dramatic influence of hydrophobic chain asymmetry in enhancing gene delivery efficacies of synthetic cationic amphiphiles (Nantz, M. H. et al. Mol. Pharmaceutics2010, 7, 786-794; Koynova, R. et al. Mol. Pharmaceutics2009, 6, 951-958). The present findings demonstrate for the first time that such a transfection enhancing influence of asymmetric hydrocarbon chains observed in pure synthetic cationic amphiphiles also works for cationic amphiphiles designed with natural, asymmetric fatty acyl chains of a food-grade oil. Herein, we demonstrate that cationic amphiphiles designed with the natural fatty acyl chain asymmetry of food-grade coconut oil are less cytotoxic and deliver genes selectively to mouse lung. Despite lauroyl chains being the major fatty acyl chains of coconut oil, both the in vitro and In vivo gene transfer efficiencies of such cationic amphiphiles were found to be remarkably superior (>4-fold) to those of their pure dilauroyl analogue. Mechanistic studies involving the technique of fluorescence resonance energy transfer (FRET) revealed higher biomembrane fusibility of the cationic liposomes of the coconut amphiphiles than that of the symmetric dilauroyl analogue. AFM study revealed pronounced fusogenic nonlamellar structures of the liposomes of coconut amphiphiles. Findings in the FRET and cellular uptake study, taken together, support the notion that the higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 are likely to play a dominant role in making the coconut amphiphiles transfection competent.

Biological safety of liposome-fullerene consisting of hydrogenated lecithin, glycine soja sterols, and fullerene-C60 upon photocytotoxicity and bacterial reverse mutagenicity.

Various water-soluble derivatives of fullerene-C60 (C60) have been developed as detoxifiers for reactive oxygen species (ROS), whereas C60 incorporated in liposome (Lpsm) has not been reported yet. We prepared the liposome-fullerene (0.2% aqueous phase, Lpsm-Flln) which was composed of hydrogenated lecithin, glycine soja (soybean) sterols, and C60 in the weight ratio of 89.7:10:0.3, then examined the photocytotoxicity and bacterial reverse mutagenicity, as comparing with the Lpsm containing no C60. Photocytoxicity of Lpsm-Flln or Lpsm was examined using Balb/3T3 fibroblastic cells at graded doses of 0.49-1000 microg/mL under the condition of UVA- or sham-irradiation. The cells were irradiated with UVA (5 J/cm2, 320-400 nm, lambda max = 360 nm) at room temperature for 50 min. The resultant cell viability (% of control) did not decrease dose-dependently to 50% or less regardless of the UVA-irradiation. These results show that Lpsm-Flln or Lpsm does not possess photocytotoxicity to Balb/3T3 fibroblasts, and Lpsm-Flln may not exert a UVA-catalytic ROS-increasing action. A possibility for the reverse mutation by Lpsm-Flln or Lpsm was examined on four histidine-demanding strains of Salmonella typhimurium and a tryptophan-demanding strain of Escherichia coli. As for the dosages of Lpsm-Flln or Lpsm (313-5000 microg/plate), the dose-dependency of the number of reverse mutation colonies of each strain did not show a twice or more difference versus the negative control regardless of the metabolic activation, and, in contrast, marked differences for five positive controls (sodium azide, N-ethyl-N'-nitro-N-nitrosoguanidine, 2-nitrofluorene, 9-aminoacridine, and 2-aminoanthracene). The growth inhibition of bacterial strains and the deposition of Lpsm-Flln or Lpsm were not found. As a result, the bacterial reverse mutagenicity of Lpsm-Flln or Lpsm was judged to be negative under the conditions of this test. Thus, Lpsm-Flln and Lpsm may not give any significant biological toxic effects, such as photocytotoxicity and bacterial reverse mutagenicity.

Influence of steric stabilization of liposome-encapsulated hemoglobin on Listeria monocytogenes host defense.

Liposome-encapsulated hemoglobin (LEH) products are being investigated as potential blood substitutes. To determine if changes in LEH composition can modify the immune response, red blood cell substitutes based on conventional lipids containing phosphatidylinositol (LEH1) and sterically stabilized lipid vesicles containing polyethylene glycol phosphatidylethanolamine (LEH2) were tested for effects on host resistance. On Day 0, groups of 18 to 20 female CD-1 mice were given an intravenous (i.v.) infectious challenge with a 20% lethal dose of Listeria monocytogenes. Mice received a single i.v. dose of LEH1, LEH2, or albumin vehicle on Day +1 or Day -3 relative to infectious challenge. Mice dosed with LEH1 and LEH2 on Day +1 died rapidly from Listeria infection; but mice dosed with LEH2 lived significantly longer than did mice receiving LEH1. By contrast, when administered on Day -3, LEH1 had no significant effect on host immunity, while LEH2 increased susceptibility to Listeria infection. In addition, LEH1 and LEH2 both caused significant reduction of phagocytic activity as measured by rat alveolar macrophage (AM) ingestion of latex microspheres. AM incubated 4 hr with either LEH1 or LEH2 prior to addition of microspheres ingested fewer beads in a dose-dependent manner. No difference in in vitro phagocytic activity was observed between LEH1 or LEH2. The inability to differentiate LEH formulations based on in vitro phagocytic activity suggests that the in vivo Listeria infection model may be more relevant in discerning the immunotoxicity of the LEH formulations tested.

Acute toxicity and depression of phagocytosis in vivo by liposomes: influence of lysophosphatidylcholine.

Small unilamellar phospholipid vesicles (liposomes), intended as drug carriers, have recently been demonstrated to reversibly depress phagocytic activity in rats when injected in a single high dose (2g of lipid per kg body weight) as revealed by the carbon clearance test. Depression of the phagocytic function was found to vary widely depending on the lipid used [M. Brandl et al., Pharm. Pharmacol. Lett., 4 (1) 1-4, 1994]. This study has now been extended in two directions: Firstly, liposomes made of the same type of lipid but different batches of raw material were compared in terms of their influence on phagocytosis as well as for their contents of impurities. The test revealed great variability of RES suppression between different batches of hydrogenated soy PC, whereas the reproducibility of the carbon clearance test was satisfactory with liposomes made of a single batch of raw material. Thin layer chromatographic analyses of the used phosphatidylcholines (PCs) and limulus tests on lipopolysaccharides revealed lysophosphatidylcholine (lysoPC) as the only impurity which showed parallels with the observed differences in phagocytosis. Secondly by "spiking" phosphatidylcholine with increasing amounts of lysoPC the latter could be proven to enhance RES depression by liposomes in a dose-dependent manner. At the same time a strong and dose-limiting increase in acute toxicity of PC vesicles was observed with increasing contents of lysoPC. However, in cholesterol-containing vesicles lysoPC-spiking did not significantly alter their behaviour, for lysoPC contents of up to 10%. Only PC/cholesterol-vesicles containing lysoPC contents as high as 15% provoked enhanced RES depression and toxicity compared to lysoPC-free vesicles. LysoPC and cholesterol in liposomes are known to play a destabilizing and stabilizing role respectively within liposomal bilayers which might influence recognition and uptake of vesicles by macrophages and thus modulation of phagocytosis.

Toxicity screening of liposomes.

Phospholipids are the major components of most liposomes. Extensive testing of these naturally occurring compounds has revealed them to be remarkably safe for pharmaceutical use. Addition of other constituents to liposomes in order to alter stability or kinetics can result in an increase in toxic potential, particularly on parenteral administration of liposomes. This paper describes some simple in vitro cellular tests for direct toxicity of liposomes, particularly following intravenous (i.v.) or topical administration, including tests for haemolysis, thrombosis and cytotoxicity. In addition, an in vivo test for the effects on phagocytosis and for pyrogenicity are described, together with a brief outline of the requirements for the further toxicity testing of liposomal drugs at a later stage of development.