Curcumin-loaded albumin submicron particles with potential as a cancer therapy: an in vitro study.

Curcumin (CUR), a polyphenolic compound, shows promising biological properties, particularly antioxidant activity. However, its medical applications are limited due to its low water solubility, bioavailability, and pH-instability. CUR-loaded albumin microparticles (CUR-HSA-MPs) of submicron size in the range of 800 to 900 nm and a zeta potential of -15 mV were prepared. The CUR loading efficiency was up to 65%. A maximum release of 37% of the encapsulated CUR was observed within 6 h when the CUR-HSA-MPs were dispersed in 50% ethanol in PBS at pH 7, while in RPMI 1640 medium the release was 7%. This demonstrates a sustainable release. The in vitro cytotoxicity of CUR-HSA-MPs showed promising anticancer potential against human hepatocellular carcinoma (Huh-7) and human breast adenocarcinoma (MCF-7) cell lines, although this effect was less pronounced in human dermal fibroblasts (HDFB) and human cholangiocyte (MMN) cell lines. Confocal microscopy was used to confirm the uptake of CUR-HSA-MPs by cancer cells. Our studies revealed that HSA-MPs are potentially promising vehicles for increasing the solubility and bioavailability of CUR.

Tumor Cell Capture Using Platelet-Based and Platelet-Mimicking Modified Human Serum Albumin Submicron Particles.

The co-localization of platelets and tumor cells in hematogenous metastases has long been recognized. Interactions between platelets and circulating tumor cells (CTCs) contribute to tumor cell survival and migration via the vasculature into other tissues. Taking advantage of the interactions between platelets and tumor cells, two schemes, direct and indirect, were proposed to target the modified human serum albumin submicron particles (HSA-MPs) towards tumor cells. HSA-MPs were constructed by the Co-precipitation-Crosslinking-Dissolution (CCD) method. The anti-CD41 antibody or CD62P protein was linked to the HSA-MPs separately via 1-ethyl-3-(-3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) EDC/NHS chemistry. The size of modified HSA-MPs was measured at approximately 1 µm, and the zeta potential was around -24 mV. Anti-CD41-HSA-MPs adhered to platelets as shown by flowcytometry and confocal laser scanning microscopy. In vitro, we confirmed the adhesion of platelets to tumor lung carcinoma cells A549 under shearing conditions. Higher cellular uptake of anti-CD41-HSA-MPs in A549 cells was found in the presence of activated platelets, suggesting that activated platelets can mediate the uptake of these particles. RNA-seq data in the Cancer Cell Lineage Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) database showed the expression of CD62P ligands in different types of cancers. Compared to the non-targeted system, CD62P-HSA-MPs were found to have higher cellular uptake in A549 cells. Our results suggest that the platelet-based and platelet-mimicking modified HSA-MPs could be promising options for tracking metastatic cancer.

Fabrication and Characterization of Human Serum Albumin Particles Loaded with Non-Sericin Extract Obtained from Silk Cocoon as a Carrier System for Hydrophobic Substances.

Non-sericin (NS) extract was produced from the ethanolic extract of Bombyx mori silk cocoons. This extract is composed of both carotenoids and flavonoids. Many of these compounds are composed of substances of poor aqueous solubility. Thus, this study focused on the development of a carrier system created from biocompatible and biodegradable materials to improve the biological activity of NS extracts. Accordingly, NS was incorporated into human serum albumin template particles with MnCO3 (NS-HSA MPs) by loading NS into the preformed HAS-MnCO3 microparticles using the coprecipitation crosslinking dissolution technique (CCD-technique). After crosslinking and template dissolution steps, the NS loaded HSA particles are negatively charged, have a size ranging from 0.8 to 0.9 µm, and are peanut shaped. The degree of encapsulation efficiency ranged from 7% to 57% depending on the initial NS concentration and the steps of adsorption. In addition, NS-HSA MPs were taken up by human lung adenocarcinoma (A549 cell) for 24 h. The promotion of cellular uptake was evaluated by flow cytometry and the results produced 99% fluorescent stained cells. Moreover, the results from CLSM and 3D fluorescence imaging confirmed particle localization in the cells. Interestingly, NS-HSA MPs could not induce inflammation through nitric oxide production from macrophage RAW264.7 cells. This is the first study involving the loading of non-sericin extracts into HSA MPs by CCD technique to enhance the bioavailability and biological effects of NS. Therefore, HSA MPs could be utilized as a carrier system for hydrophobic substances targeting cells with albumin receptors.

Doxorubicin-Loaded Human Serum Albumin Submicron Particles: Preparation, Characterization and In Vitro Cellular Uptake.

Doxorubicin (DOX) is an effective anthracycline antibiotic drug which is commonly used in a broad range cancer therapy. However, due to dose depending side effects and toxicity to non-cancerous tissues, its clinical applications are restricted. To overcome these limitations, human serum albumin (HSA) has been investigated as a biocompatible drug delivery vehicle. In this study, human serum albumin submicron particles (HSA-MPs) were fabricated by using the Co-precipitation-Crosslinking-Dissolution technique (CCD technique) and DOX was loaded into the protein particles by absorption. DOX-HSA-MPs showed uniform peanut-like shape, submicron size and negative zeta-potential (-13 mV). The DOX entrapment efficiency was 25% of the initial amount. The in vitro release in phosphate buffered saline pH 7.4 was less than 1% within 5 h. In contrast, up to 40% of the entrapped DOX was released in presence of a protein digesting enzyme mixture (Pronase®) within the same time. In addition, in vitro cytotoxicity and cellular uptake of DOX-HSA-MPs were evaluated using the lung carcinoma cell line A549. The results demonstrated that DOX-HSA-MPs reduced the cell metabolic activities after 72 h. Interestingly, DOX-HSA-MPs were taken up by A549 cells up to 98% and localized in the cell lysosomal compartment. This study suggests that DOX-HSA-MPs which was fabricated by CCD technique is seen as a promising biopolymer particle as well as a viable alternative for drug delivery application to use for cancer therapy.

Riboflavin: The Health Benefits of a Forgotten Natural Vitamin.

Riboflavin (RF) is a water-soluble member of the B-vitamin family. Sufficient dietary and supplemental RF intake appears to have a protective effect on various medical conditions such as sepsis, ischemia etc., while it also contributes to the reduction in the risk of some forms of cancer in humans. These biological effects of RF have been widely studied for their anti-oxidant, anti-aging, anti-inflammatory, anti-nociceptive and anti-cancer properties. Moreover, the combination of RF and other compounds or drugs can have a wide variety of effects and protective properties, and diminish the toxic effect of drugs in several treatments. Research has been done in order to review the latest findings about the link between RF and different clinical aberrations. Since further studies have been published in this field, it is appropriate to consider a re-evaluation of the importance of RF in terms of its beneficial properties.

Detection of CD33 expression on monocyte surface is influenced by phagocytosis and temperature.

CD33 is a myeloid-associated marker and belongs to the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family. Such types of receptors are highly expressed in acute myeloid leukemia, which could be used in its treatment. CD33 shows high variability in its expression levels with still unknown reasons. Here, we investigated the CD33 expression of monocytes in human blood samples processed at different temperatures and in dependence on their phagocytic activity against opsonized Escherichia coli. The samples were stained by fluorescently labelled anti-human CD14 to specify the monocyte population, anti-human CD33 antibodies to evaluate CD33 expression and analyzed by flow cytometry and confocal laser scanning microscopy. In blood samples kept at 37°C or first pre-chilled at 0°C with subsequent warming up to 37°C, the percentage of CD33-positive monocytes as well as their relative fluorescence intensity was up-regulated compared to samples kept constantly at 0°C. After exposure to E. coli the CD33 relative fluorescence intensity of the monocytes activated at 37°C was 3 to 4 times higher than that of those cells kept inactive at 0°C. Microscopic analysis showed internalisation of CD33 due to its enhanced expression on the surface followed by engulfment of E. coli.

Inflammatory activation of human serum albumin- or ovalbumin-modified chitosan particles to macrophages and their immune response in human whole blood.

Nanomaterials have been extensively used in the biomedical field. These nanoscale objects may either promote or restrain immune responses depending on their surface characteristics and compositions. In this study, chitosan (CS) particles prepared using an emulsion-crosslinking method were modified with different amounts of human serum albumin (HSA) and ovalbumin (OVA), resulting in four types of modified CS particles, i.e. CS@HSA-10, CS@HSA-57, CS@OVA-13 and CS@OVA-65, respectively. They had a similar size of about 150 nm in a dry state, and were swollen 2-3 fold in PBS. No significant cytotoxicity was determined toward in vitro cultured RAW264.7 and THP-1 cells. However, all the modified CS particles, in particular the OVA-modified ones (CS@OVA-13 and CS@OVA-65), induced significantly higher secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) compared with the negative control. In human whole blood, CS@OVA-13 and CS@OVA-65 were phagocytosed with a significantly higher ratio by granulocytes and monocytes, leading to the higher secretion of TNF-α, IL-1ß and IL-8, and a larger extent of platelet activation than CS@HSA-10 and CS@HSA-57, respectively.

Effects of heat and freeze on isolated erythrocyte submembrane skeletons.

In this study we heated insoluble residues, obtained after Triton-X-100 (0.1 v/v%) extraction of erythrocyte ghost membranes (EGMs). Specific heat capacity, electric capacitance and resistance, and optical transmittance (280 nm) sustained sharp changes at 49°C (TA) and 66°C (TC), the known denaturation temperatures of spectrin and band 3, respectively. The change at TA was selectively inhibited by diamide (1 mM) and taurine mustard (1 mM) while its inducing temperature was selectively decreased by formamide in full concert with the assumed involvement of spectrin denaturation. In the residues of EGMs, pretreated with 4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid (DIDS), the change at TC was shifted from 66 to 78°C which indicated the involvement of band 3 denaturation. The freeze and rapid thaw of EGM residues resulted in a strong reduction of cooperativity of band 3 denaturation while the slow thaw completely eliminated the peak of this denaturation. These effects of freeze-thaw were prevented in residues obtained from DIDS-treated EGMs. The freeze-thaw of residues slightly affected spectrin denaturation at 49°C although an additional denaturation appeared at 55°C. The results indicate preserved molecular structure and dynamics of the membrane skeleton in Triton-X-100 extracts of EGMs. The freeze-thaw inflicted strong damage on band 3 and spectrin-actin skeleton of EGM extracts which is relevant to cryobiology, cryosurgery and cryopreservation of cells.

Kinetics and efficiency of a methyl-carboxylated 5-Fluorouracil-bovine serum albumin adduct for targeted delivery.

5-Fluorouracil (5-FU) is a clinically well-established anti-cancer drug effectively applied in chemotherapy, mainly for the treatment of breast and colorectal cancer. Substantial disadvantages are adverse effects, arising from serious damage of healthy tissues, and shortcoming pharmacokinetics due to its low molecular weight. A promising approach for improvement of such drugs is their coupling to suitable carriers. Here, a 5-FU adduct, 5-fluorouracil acetate (FUAc) is synthesized and covalently coupled to bovine serum albumin (BSA) as model carrier molecule. On average, 12 molecules FUAc are bound to one BSA. Circular dichriosm (CD)-spectra of BSA and FUAc-BSA are identical, suggesting no significant conformational differences. FUAc-BSA is tested on T-47D and MDA-MB-231 breast cancer cells. Proliferation inhibition of membrane albumin-binding protein (mABP)-expressing T-47D cells by FUAc-BSA is similar to that of 5-FU and only moderate for MDA-MB-231 cells that lack such expression. Therefore, a crucial role of mABP expression in effective cell growth inhibition by FUAc-BSA is assumed.

The effect of olive oil polyphenols on antibodies against oxidized LDL. A randomized clinical trial.

BACKGROUND & AIM: Oxidized LDL (oxLDL) is a highly immunogenic particle that plays a key role in the development of atherosclerosis. Some data suggest a protective role of OxLDL autoantibodies (OLAB) in atherosclerosis. Our aim was to assess the effect of olive oil polyphenols on the immunogenicity of oxLDL to autoantibody generation. METHODS: In a crossover, controlled trial, 200 healthy men were randomly assigned to 3-week sequences of 25 mL/day of 3 olive oils with high (366 mg/kg), medium (164 mg/kg), and low (2.7 mg/kg) phenolic content. RESULTS: Plasma OLAB concentration was inversely associated with oxLDL (p < 0.001). Olive oil phenolic content increased OLAB generation, with the effect being stronger at higher concentrations of oxLDL (p = 0.020 for interaction). A direct relationship was observed between OLAB and the total olive oil phenol content in LDL (r = 0.209; p = 0.014). OLAB concentrations, adjusted for oxLDL, increased directly in a dose-dependent manner with the polyphenol content of the olive oil administered (p = 0.023). CONCLUSION: Olive oil polyphenols promote OLAB generation. This effect is stronger at higher concentrations of lipid oxidative damage.

Biodegradable insulin-loaded PLGA microspheres fabricated by three different emulsification techniques: investigation for cartilage tissue engineering.

Growth, differentiation and migration factors facilitate the engineering of tissues but need to be administered with defined gradients over a prolonged period of time. In this study insulin as a growth factor for cartilage tissue engineering and a biodegradable PLGA delivery device were used. The aim was to investigate comparatively three different microencapsulation techniques, solid-in-oil-in-water (s/o/w), water-in-oil-in-water (w/o/w) and oil-in-oil-in-water (o/o/w), for the fabrication of insulin-loaded PLGA microspheres with regard to protein loading efficiency, release and degradation kinetics, biological activity of the released protein and phagocytosis of the microspheres. Insulin-loaded PLGA microspheres prepared by all three emulsification techniques had smooth and spherical surfaces with a negative zeta potential. The preparation technique did not affect particle degradation nor induce phagocytosis by human leukocytes. The delivery of structurally intact and biologically active insulin from the microspheres was shown using circular dichroism spectroscopy and a MCF7 cell-based proliferation assay. However, the insulin loading efficiency (w/o/w about 80%, s/o/w 60%, and o/o/w 25%) and the insulin release kinetics were influenced by the microencapsulation technique. The results demonstrate that the w/o/w microspheres are most appropriate, providing a high encapsulation efficiency and low initial burst release, and thus these were finally used for cartilage tissue engineering. Insulin released from w/o/w PLGA microspheres stimulated the formation of cartilage considerably in chondrocyte high density pellet cultures, as determined by increased secretion of proteoglycans and collagen type II. Our results should encourage further studies applying protein-loaded PLGA microspheres in combination with cell transplants or cell-free in situ tissue engineering implants to regenerate cartilage.

New 4-maleamic acid and 4-maleamide peptidyl chalcones as potential multitarget drugs for human prostate cancer.

PURPOSE: The objective of this study was to investigate the effect of new 4-maleamic acid and 4-maleamide peptidyl chalcone derivatives against human prostate cancer in vitro and in vivo. METHODS: From a series of 21 chalcones, the effects of the three best inhibitors of PC-3 and LNCaP cell viability on growth, including cell cycle changes, adhesion, migration, and cell invasion, as well as their ability to inhibit angiogenesis, clonogenic activity, and matrix metalloproteinases MMP-2 and MMP-9, were tested. The effects in vivo were studied in PC-3 and LNCaP xenografts. RESULTS: Three of the examined chalcones reduced cell viability in both cell lines in a strong dose- and time-dependent manner. An inhibition of the cell cycle progress was observed. These changes were accompanied with the inhibition of cell adhesion, migration, and invasion as well as with reduced neovascularization in chick embryos, tumor colony formation, and MMP-9 activity. The in vivo results demonstrated the strong activity of these structures as inhibitors of tumor development in nude mice compared to non-treated animals. CONCLUSION: The results suggest the multitarget efficacy of 4-maleamic acid and 4-maleamide peptidyl chalcones against human prostate cancer cells and emphasize the potential therapeutic relevance of these compounds.

Measurement conditions for flow cytometry analyses of cell lines from urological carcinomas.

Prerequisites for successful flow cytometry investigations are specific antibodies labeled with appropriate fluorochromes and negligible autofluorescence of the untreated cells at the wavelength of interest. The aim of this study was (a) to characterize frequently used urological carcinoma cell lines with regard to their autofluorescence properties, (b) to demonstrate the autofluorescence as a serious interfering factor on FACS analysis of urological carcinoma cell lines and (c) to suggest an alternative to avoid interfering autofluorescence. Twenty-one cell lines originating from prostate carcinoma, renal cell carcinoma and bladder cancer were included in this study. The various cell lines were read on a flow cytometer in comparison to human erythrocytes as cells with low fluorescence intensity. Urological cell lines show a high autofluorescence when flow cytometry analyses are performed at the frequently used excitation wavelengths at 405 and 488 nm. At excitation wavelength of 633 nm, this problem was reduced and most of the cell lines (14/21) were without autofluorescence at the emission wavelength of 785 nm. In addition, with a spectrofluorometer three exemplary cell lysates were investigated. The above observations were confirmed. The dye APC-Cy7 is one suitable fluorochrome for successful investigation under these measurement conditions.

Modification of aminosilanized superparamagnetic nanoparticles: feasibility of multimodal detection using 3T MRI, small animal PET, and fluorescence imaging.

PURPOSE: The aim of our study was to modify an aminosilane-coated superparamagnetic nanoparticle for cell labeling and subsequent multimodal imaging using magnetic resonance imaging (MRI), positron emission tomography (PET), and fluorescent imaging in vivo. PROCEDURES: We covalently bound the transfection agent HIV-1 tat, the fluorescent dye fluorescein isothiocyanate, and the positron-emitting radionuclide gallium-68 to the particle and injected them intravenously into Wistar rats, followed by animal PET and MRI at 3.0 T. As a proof of principle hepatogenic HuH7 cells were labeled with the particles and observed for cell toxicity as well as detectability by MRI and biodistribution in vivo. RESULTS: PET imaging and MRI revealed increasing hepatic and splenic accumulation of the particles over 24 h. Adjacent in vitro studies in hepatogenic HuH7 cells showed a rapid intracellular accumulation of the particles with high labeling efficiency and without any signs of toxicity. In vivo dissemination of the labeled cells could be followed by dynamic biodistribution studies. CONCLUSIONS: We conclude that our modified superparamagnetic nanoparticles are stable under in vitro and in vivo conditions and are therefore applicable for efficient cell labeling and subsequent multimodal molecular imaging. Moreover, their multiple free amino groups suggest the possibility for further modifications and might provide interesting opportunities for various research fields.

Changes in LDL fatty acid composition as a response to olive oil treatment are inversely related to lipid oxidative damage: The EUROLIVE study.

OBJECTIVE: The aim of our study was to assess the changes in the fatty acid composition of low density lipoproteins (LDL) after sustained consumption of olive oil at real-life doses (25 mL/day) and their relationship with lipid oxidative damage. METHODS: A multi-center randomized, cross-over, clinical trial with 3 similar types of olive oils, but with differences in the phenolic content, was conducted on 200 healthy European subjects. Intervention periods were of 3 weeks separated by 2-week washout periods. The LDL fatty acid content was measured in samples drawn at baseline and after the last intervention period. RESULTS: After olive oil ingestion oleic acid concentration in LDL increased (1.9%; p < 0.001) and those of linoleic (1.1%; p < 0.002) and arachidonic acid (0.5%; p < 0.001) decreased. Monounsaturated/polyunsaturated fatty acid and oleic/linoleic acid ratios in LDL increased after olive oil consumption. An inverse relationship between the oleic/linoleic acid ratio and biomarkers of oxidative stress was observed. One unit increase in the oleic/linoleic acid ratio was associated with a decrease of 4.2 microg/L in plasma isoprostanes. CONCLUSION: Consumption of olive oil at real-life doses improved the fatty acid profile in LDL, the changes being associated with a reduction of the oxidative damage to lipids.

Effect of olive oils on biomarkers of oxidative DNA stress in Northern and Southern Europeans.

High consumption of olive oil in the Mediterranean diet has been suggested to protect DNA against oxidative damage and to reduce cancer incidence. We investigated the impact of the phenolic compounds in olive oil, and the oil proper, on DNA and RNA oxidation in North, Central, and South European populations. In a multicenter, double-blind, randomized, controlled crossover intervention trial, the effect of olive oil phenolic content on urinary oxidation products of guanine (8-oxo-guanine, 8-oxo-guanosine and 8-oxo-deoxyguanosine) was investigated. Twenty-five milliliters of three olive oils with low, medium, and high phenolic content were administered to healthy males (n=182) daily for 3 wk. At study baseline the urinary excretion of 8-oxo-guanosine (RNA oxidation) and 8-oxo-deoxyguanosine (DNA oxidation) was higher in the Northern regions of Europe compared with Central and Southern European regions (P=0.035). Urinary excretion of the 8 hydroxylated forms of guanine, guanosine, deoxyguanosine and their nonoxidized forms were not different when comparing olive oils with low, medium, and high phenolic content given for 2 wk. Testing the effect of oil from urinary 8-oxo-deoxyguanosine changes from baseline to post-treatment showed a reduction of DNA oxidation by 13% (P=0.008). These findings support the idea that ingestion of olive oil is beneficial and can reduce the rate of oxidation of DNA. This effect is not due to the phenolic content in the olive oil. The higher DNA and RNA oxidation in Northern European regions compared with that in Central and Southern regions supports the contention that olive oil consumption may explain some of the North-South differences in cancer incidences in Europe.

The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial.

BACKGROUND: Virgin olive oils are richer in phenolic content than refined olive oil. Small, randomized, crossover, controlled trials on the antioxidant effect of phenolic compounds from real-life daily doses of olive oil in humans have yielded conflicting results. Little information is available on the effect of the phenolic compounds of olive oil on plasma lipid levels. No international study with a large sample size has been done. OBJECTIVE: To evaluate whether the phenolic content of olive oil further benefits plasma lipid levels and lipid oxidative damage compared with monounsaturated acid content. DESIGN: Randomized, crossover, controlled trial. SETTING: 6 research centers from 5 European countries. PARTICIPANTS: 200 healthy male volunteers. MEASUREMENTS: Glucose levels, plasma lipid levels, oxidative damage to lipid levels, and endogenous and exogenous antioxidants at baseline and before and after each intervention. INTERVENTION: In a crossover study, participants were randomly assigned to 3 sequences of daily administration of 25 mL of 3 olive oils. Olive oils had low (2.7 mg/kg of olive oil), medium (164 mg/kg), or high (366 mg/kg) phenolic content but were otherwise similar. Intervention periods were 3 weeks preceded by 2-week washout periods. RESULTS: A linear increase in high-density lipoprotein (HDL) cholesterol levels was observed for low-, medium-, and high-polyphenol olive oil: mean change, 0.025 mmol/L (95% CI, 0.003 to 0.05 mmol/L), 0.032 mmol/L (CI, 0.005 to 0.05 mmol/L), and 0.045 mmol/L (CI, 0.02 to 0.06 mmol/L), respectively. Total cholesterol-HDL cholesterol ratio decreased linearly with the phenolic content of the olive oil. Triglyceride levels decreased by an average of 0.05 mmol/L for all olive oils. Oxidative stress markers decreased linearly with increasing phenolic content. Mean changes for oxidized low-density lipoprotein levels were 1.21 U/L (CI, -0.8 to 3.6 U/L), -1.48 U/L (-3.6 to 0.6 U/L), and -3.21 U/L (-5.1 to -0.8 U/L) for the low-, medium-, and high-polyphenol olive oil, respectively. LIMITATIONS: The olive oil may have interacted with other dietary components, participants' dietary intake was self-reported, and the intervention periods were short. CONCLUSIONS: Olive oil is more than a monounsaturated fat. Its phenolic content can also provide benefits for plasma lipid levels and oxidative damage. International Standard Randomised Controlled Trial number: ISRCTN09220811.