Analysis of proteomic profile changes of Danio rerio embryos during exposure to doxorubicin,incorporated in the phospholipid transport nanosystem

The proteome profile of Danio rerio embryos grown in the medium containing doxorubicin, included in the phospholipid transport nanosystem (doxolip) has been investigated using combination of 1D-electrophoresis with subsequent MALDI-TOF-PMF mass spectrometry. Cultivation of growing of D. rerio embryos in the medium with doxolip caused a substantial increase in expression of the cytoskeletal proteins, a decrease in the number of nuclear proteins involved in DNA and RNA synthesis and disappearance of vitellogenin 2 in comparison with control (the cultivation medium containing the phospholipid transport nanosystem). Analysis of the proteomic profiles of doxolip-treated embryos suggests lower toxicity of doxorubicin incorporated in the phospholipid nanosystem.     

The rifampicin drug delivery system based on phospholipid nanoparticles

Low bioavailability of rifampicin, one of the main antituberculous agents, stimulates searches of its new optimized formulations. The present study has shown a possibility of rifampicin incorporation into nanoparticles from plant phosphatidylcholine (diameter of 20–30 nm). Addition of sodium oleate to the phospholipid system caused a 2-fold increase in the percent of rifampicin incorporation. The maximal concentration of rifampicin assayed in plasma samples by LC/MS was observed 1 h after oral administration to rats (6 mg/kg) and represented 0.5 and 4.2 μg/mL for free rifampicin and rifampicin incorporated in the phospholipids-oleate nanoparticles, respectively. These levels were maintained for more than 2 h of the experiment. High rifampicin bioavailability in the oleate containing phospholipid nanosystem suggests its prospects for practical use.     

Changes of doxorubicin distribution in blood and plasma after its inclusion into nanophospholipid formulations

Using original technology developed at the Institute of Biomedical Chemistry (Russian Academy of Medical Sciences) a drug composition based on plant phospholipids and the antitumor drug doxorubicin (particle size <30 nm) has been developed. In vitro experiments demonstrated that doxorubicin inclusion into the phospholipids nanoparticles decreased drug binding to blood cells and thus increase proportion of the potentially active drug in plasma. This was accompanied by changes in drug redistribution from the plasma protein fraction to high density lipoproteins. Importance of these changes for doxorubicin bioavailability and antitumor activity is discussed.     

Self-assembled virus-like particles from rotavirus structural protein VP6 for targeted drug delivery

Proteins of viral capsid may self-assemble into virus-like particles (VLPs) that can find many biomedical applications such as platform for drug delivery. In this paper, we describe preparation of VLPs by self-assembly of VP6, a rotavirus capsid protein that was chemically conjugated with doxorubicin (DOX), an anticancer drug. VP6 was first highly expressed in E. Coli, followed by purification and renaturation. DOX was then covalently attached to VP6 to form DOX-VP6 (DVP6) conjugates, which were subsequently self-assembled into VLPs under appropriate condition. Next, lactobionic acid (LA) was chemically linked to the surface of the VLPs. We demonstrated that the aforementioned nanosystem shows specific targeting to hepatoma cell line HepG2. The chemically functionalized VLPs, a kind of biological nanoparticles with excellent biocompatibility and biodegradability, can be prepared in large scale from E. Coli through our method, which may find practical applications in biomedicine.     

The effect of resveratrol and dihydroquercetin inclusion into phospholipid nanopatricles on their bioavalability and specific activity

Bioavailability and activity of natural polyphenols, resveratrol (RES) and dihydroquercetin (DHQ), included in phospholipid nanoparticles have been investigated. Specific features of RES and DHQ in these compositions have been analyzed in vivo and in vitro experiments in comparison with free substances. Preincubation of low density lipoproteins (LDL), isolated from plasma of healthy donors, with RES or DHQ included in phospholipid nanoparticles caused a more pronounced delay in Cu²⁺ induced lipid oxidation compared with the free substances, and reduced the formation of lipid peroxidation (LPO) products. In phospholipid formulations bioavailability of RES and DHQ orally administered to rats were 1.5–2-fold higher than that of free substances. Prophylactic administration of phospholipid formulations containing RES or DHQ for two weeks resulted in the 25% increase of animal survival in the acute hypoxia model and 1.5-fold increase of catalase activity assayed in brain homogenates as compared with free substances. Using the model of endothelial dysfunction in rats induced by L-NAME, nitric oxide synthase inhibitor, it was shown, that RES markedly attenuated the inhibitory effect of L-NAME on NO synthesis. RES administered in phospholipid nanoparticles demonstrated the same efficiency at a dose one order of magnitude lower compared than that of free RES. The load test with resistance (clamping of the ascending aorta for 30 s) showed that the phospholipid formulation of RES exhibited a more pronounced protective effect due to stimulation of endothelial NO-synthase.     

Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and intracellular distribution

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to alpha-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), alpha,omega-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or alpha-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02-1.5 microm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.     

Comparable interaction of doxorubicin with various acidic phospholipids results in changes of lipid order and dynamics

We have characterized the interaction of the antitumor drug doxorubicin with model membranes of the anionic phospholipids dioleoylphosphatidic acid (DOPA), dioleoylphosphatidylserine (DOPS), cardiolipin and dioleoylphosphatidylglycerol (DOPG) as compared to the zwitterionic dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE). The saturating binding levels were: 2.4 (DOPA), 1.3 (cardiolipin), 1.5 (DOPS, DOPG) and 0.02 (DOPC) doxorubicin per lipid phosphorus (mol/mol). The half-saturating free drug concentrations were comparable for DOPA, cardiolipin, DOPS and DOPG: 20, 16, 35 and 18 microM, respectively. Doxorubicin fluorescence revealed the simultaneous existence of at least two populations of bound drug in the various anionic phospholipids: (1) fluorescent molecules with chromophores that reside between the lipid molecules and (2) above 0.01-0.02 doxorubicin bound per lipid phosphorus: non-fluorescent drug-stacks that are closer to the aqueous phase than the fluorescent molecules. Small-angle X-ray scattering indicated that doxorubicin can reorganize anionic phospholipid dispersions into closely-packed multilamellar structures. Addition of the drug caused leakage of entrapped 6-carboxyfluorescein. Neither 2H-NMR on [2-2H]serine-labelled DOPS nor 31P-NMR revealed any significant effect of doxorubicin on headgroup conformation, but 2H-NMR on di[11,11-2H2]oleoyl-labelled phospholipids showed that the drug had a strong acyl chain-disordering effect on anionic phospholipids. 2H-NMR relaxation measurements indicated that the drug immobilized the headgroups and acyl chains of anionic phospholipids. The implications of these observations for the cellular activity of the drug are indicated.     

Efficacy,Safety and Anticancer Activity of Protein Nanoparticle-Based Delivery of Doxorubicin through Intravenous Administration in Rats

Background and Aims

Doxorubicin is a potent anticancer drug and a major limiting factor that hinders therapeutic use as its high levels of systemic circulation often associated with various off-target effects, particularly cardiotoxicity. The present study focuses on evaluation of the efficacy of doxorubicin when it is loaded into the protein nanoparticles and delivered intravenously in rats bearing Hepatocellular carcinoma (HCC). The proteins selected as carrier were Apotransferrin and Lactoferrin, since the receptors for these two proteins are known to be over expressed on cancer cells due to their iron transport capacity.

Methods

Doxorubicin loaded apotransferrin (Apodoxonano) and lactoferrin nanoparticles (Lactodoxonano) were prepared by sol-oil chemistry. HCC in the rats was induced by 100 mg/l of diethylnitrosamine (DENA) in drinking water for 8 weeks. Rats received 5 doses of 2 mg/kg drug equivalent nanoparticles through intravenous administration. Pharmacokinetics and toxicity of nanoformulations was evaluated in healthy rats and anticancer activity was studied in DENA treated rats. The anticancer activity was evaluated through counting of the liver nodules, H & E analysis and by estimating the expression levels of angiogenic and antitumor markers.

Results

In rats treated with nanoformulations, the numbers of liver nodules were found to be significantly reduced. They showed highest drug accumulation in liver (22.4 and 19.5 µg/g). Both nanoformulations showed higher localization compared to doxorubicin (Doxo) when delivered in the absence of a carrier. Higher amounts of Doxo (195 µg/g) were removed through kidney, while Apodoxonano and Lactodoxonano showed only a minimal amount of removal (<40 µg/g), suggesting the extended bioavailability of Doxo when delivered through nanoformulation. Safety analysis shows minimal cardiotoxicity due to lower drug accumulation in heart in the case of nanoformulation.

Conclusion

Drug delivery through nanoformulations not only minimizes the cardiotoxicity of doxorubicin but also enhances the efficacy and bioavailability of the drug in a target-specific manner.     

Temperature-Tunable Iron Oxide Nanoparticles for Remote-Controlled Drug Release

Herein, we report the successful development of a novel nanosystem capable of an efficient delivery and temperature-triggered drug release specifically aimed at cancer. The water-soluble 130.1 ± 0.2 nm iron oxide nanoparticles (IONPs) were obtained via synthesis of a monodispersed iron oxide core stabilized with tetramethylammonium hydroxide pentahydrate (TMAOH), followed by coating with the thermoresponsive copolymer poly-(NIPAM-stat-AAm)-block-PEI (PNAP). The PNAP layer on the surface of the IONP undergoes reversible temperature-dependent structural changes from a swollen to a collapsed state resulting in the controlled release of anticancer drugs loaded in the delivery vehicle. We demonstrated that the phase transition temperature of the prepared copolymer can be precisely tuned to the desired value in the range of 36°C–44°C by changing the monomers ratio during the preparation of the nanoparticles. Evidence of modification of the IONPs with the thermoresponsive copolymer is proven by ATR-FTIR and a quantitative analysis of the polymeric and iron oxide content obtained by thermogravimetric analysis. When loaded with doxorubicin (DOX), the IONPs-PNAP revealed a triggered drug release at a temperature that is a few degrees higher than the phase transition temperature of a copolymer. Furthermore, an in vitro study demonstrated an efficient internalization of the nanoparticles into the cancer cells and showed that the drug-free IONPs-PNAP were nontoxic toward the cells. In contrast, sufficient therapeutic effect was observed for the DOX-loaded nanosystem as a function of temperature. Thus, the developed temperature-tunable IONPs-based delivery system showed high potential for remotely triggered drug delivery and the eradication of cancer cells.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0131-x) contains supplementary material, which is available to authorized users.KEY WORDS: drug delivery, IONPs, remote-triggered drug release, thermoresponsive copolymer, tunable LCST     

In vitro cytotoxic study of immunoliposomal doxorubicin targeted to human CD34(+) leukemic cells

The expression of CD34 antigen in acute myelogenous leukemias is considered an unfavourable prognosis marker for response to anticancer drugs and duration of remission. This study investigated the applicability of long-circulating immunoliposomes loaded with doxorubicin targeted to CD34 antigen present on MDR(+) human myelogenous leukemia KG-1a cell line. Immunoliposomal doxorubicin showed a higher cytotoxicity against KG-1a cells than non-targeted liposomal doxorubicin, but it did not improve over that of free drug. Although no reversal of doxorubicin resistance was found to occur through its liposomal encapsulation, a therapeutic benefit can be obtained by the selective cytotoxicity observed. Endocytosis studies demonstrated that, after binding to CD34 antigen, the immunoliposomes are not internalized by the KG-1a cells and so the cytotoxic effect might be due to drug released into the space near the cell membrane. Thus, immunotargeting of liposomal doxorubicin to CD34(+) leukemic cells may only provide an ex vivo strategy for site-selective CD34(+) leukemia cell killing.     

Two-dimensional separation of human plasma proteins using iterative free-flow electrophoresis

Blood plasma is the most complex human-derived proteome, containing other tissue proteomes as subsets. This proteome has only been partially characterized due to the extremely wide dynamic range of the plasma proteins of more than ten orders of magnitude. Thus, the reduction in sample complexity prior to mass spectrometric analysis is particularly important and alternative separation methodologies are required to more effectively mine the lower abundant plasma proteins. Here, we demonstrated a novel separation approach using 2-D free-flow electrophoresis (FFE) separating proteins and peptides in solution according to their pI prior to LC-MS/MS. We used the combination of sequential protein and peptide separation by first separating the plasma proteins into specific FFE fractions. Tryptic digests of the separated proteins were generated and subsequently separated using FFE. The protein separation medium was optimized to segregate albumin into specific fractions containing only few other proteins. An optimization of throughput for the protein separation reduced the separation time of 1 mL of plasma to approximately 3 h providing sufficient material for digestion and the subsequent peptide separation. Our approach revealed low-abundant proteins (e.g., L-selectin at 17 ng/mL and vascular endothelial-cadherin precursor at 30 ng/mL) and several tissue leakage products, thus providing a powerful orthogonal separation step in the proteomics workflow.     

Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells

The aim of this study was to observe the cytotoxicity and apoptotic effects of nickel oxide nanoparticles on human cervix epithelioid carcinoma cell line (HeLa). Nickel oxide precursors were synthesized by an nickel sulphate-excess urea reaction in boiling aqueous solution. The synthesized NiO nanoparticles (<200 nm) were investigated by X-ray diffraction analysis and transmission electron microscopy techniques. For cytotoxicity experiments, HeLa cells were incubated in 50-500 μg/mL NiO for 2, 6, 12 and 16 hours. The viable cells were counted with a haemacytometer using light microscopy. The cytotoxicity was observed low in 50-200 μg/mL concentration for 16 h, but high in 400-500 μg/mL concentration for 2-6 h. HeLa cells' cytoplasm membrane was lysed and detached from the well surface in 400 μg/mL concentration NiO nanoparticles. Double staining and M30 immunostaining were performed to quantify the number of apoptotic cells in culture on the basis of apoptotic cell nuclei scores. The apoptotic effect was observed 20% for 16 h incubation.     

The synthesis of doxorubicin-conjugated magnetite nanoparticles and their magnetic resonance and cytotoxic properties

The possibility of increasing the effectiveness of antitumor drugs such as doxorubicin by preparing its complex with ultrafine magnetic iron oxide nanoparticles is considered. A method for binding doxorubicin molecules to magnetic nanoparticles via citric acid is proposed. The main magnetic properties of the obtained conjugates were studied by proton relaxometry and Mössbauer spectroscopy, while their cytotoxic activity was evaluated via spectrophotometric MTT assay in HeLa cells. It was shown that the conjugates of magnetite nanoparticles with doxorubicin are characterized by a high level of contrast in magnetic resonance imaging. The magnetic properties of doxorubicin-free and bound magnetite nanoparticles are mainly determined by the average size of nanoobjects and the phase composition and slightly depend on the composition of the stabilizing shell. The cytotoxic effect of the synthesized conjugates of magnetite nanoparticles with doxorubicin is higher than that of unbound doxorubicin. This makes it possible to increase the antitumor effect of doxorubicin and control the dynamics of its delivery in the form of a conjugate into the disease focus due to the magnetic contrast properties of nanoparticles.     

Proteomic analysis of doxorubicin‐induced changes in the proteome of HepG2cells combining 2‐D DIGE and LC‐MS/MS approaches

HepG‐2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2‐D gel‐based and gel‐free methods. The analysis of crude HepG2 cell extracts by 2‐D DIGE provided data on 1835 protein spots which was then complemented by MS‐centered analysis of stable isotope labeling by amino acids in cell culture‐labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin‐induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin‐associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.     

Reduction-Responsive Disassemblable Core-Cross-Linked Micelles Based on Poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)-Lipoic Acid Conjugates for Triggered Intracellular Anticancer Drug Release

Reduction-sensitive reversibly core-cross-linked micelles were developed based on poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)-lipoic acid (PEG-b-PHPMA-LA) conjugates and investigated for triggered doxorubicin (DOX) release. Water-soluble PEG-b-PHPMA block copolymers were obtained with M(n,PEG) of 5.0 kg/mol and M(n,HPMA) varying from 1.7 and 4.1 to 7.0 kg/mol by reversible addition-fragmentation chain transfer (RAFT) polymerization. The esterification of the hydroxyl groups in the PEG-b-PHPMA copolymers with lipoic acid (LA) gave amphiphilic PEG-b-PHPMA-LA conjugates with degrees of substitution (DS) of 71-86%, which formed monodispersed micelles with average sizes ranging from 85.3 to 142.5 nm, depending on PHPMA molecular weights, in phosphate buffer (PB, 10 mM, pH 7.4). These micelles were readily cross-linked with a catalytic amount of dithiothreitol (DTT). Notably, PEG-b-PHPMA(7.0k)-LA micelles displayed superior DOX loading content (21.3 wt %) and loading efficiency (90%). The in vitro release studies showed that only about 23.0% of DOX was released in 12 h from cross-linked micelles at 37 °C at a low micelle concentration of 40 μg/mL, whereas about 87.0% of DOX was released in the presence of 10 mM DTT under otherwise the same conditions. MTT assays showed that DOX-loaded core-cross-linked PEG-b-PHPMA-LA micelles exhibited high antitumor activity in HeLa and HepG2 cells with low IC(50) (half inhibitory concentration) of 6.7 and 12.8 μg DOX equiv/mL, respectively, following 48 h incubation, while blank micelles were practically nontoxic up to a tested concentration of 1.0 mg/mL. Confocal laser scanning microscope (CLSM) studies showed that DOX-loaded core-cross-linked micelles released DOX into the cell nuclei of HeLa cells in 12 h. These reduction-sensitive disassemblable core-cross-linked micelles with excellent biocompatibility, superior drug loading, high extracellular stability, and triggered intracellular drug release are promising for tumor-targeted anticancer drug delivery.     

Hybrid nanoreservoirs based on dextran-capped dendritic mesoporous silica nanoparticles for CD133-targeted drug delivery

In this study, the chemical features of dendritic mesoporous silica nanoparticles (DMSNs) provided the opportunity to design a nanostructure with the capability to intelligently transport the payload to the tumor cells. In this regard, doxorubicin (DOX)-encapsulated DMSNs was electrostatically surface-coated with polycarboxylic acid dextran (PCAD) to provide biocompatible dextran-capped DMSNs (PCAD-DMSN@DOX) with controlled pH-dependent drug release. Moreover, a RNA aptamer against a cancer stem cell (CSC) marker, CD133 was covalently attached to the carboxyl groups of DEX to produce a CD133-PCAD-DMSN@DOX. Then, the fabricated nanosystem was utilized to efficiently deliver DOX to CD133+ colorectal cancer cells (HT29). The in vitro evaluation in terms of cellular uptake and cytotoxicity demonstrated that the CD133-PCAD-DMSN@DOX specifically targets HT29 as a CD133 overexpressed cancer cells confirmed by flow cytometry and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. The potentially promising intelligent-targeted platform suggests that targeted dextran-capped DMSNs may find impressive application in cancer therapy.     

Cytotoxicity of the anticancer drug doxorubicin for human embryonic stem cells

The cytotoxic effect of the anticancer drug doxorubicin (DR) on human embryonic stem cells (ESCs) C910 and fibroblasts spontaneously differentiated from these cells has been examined. The fibroblasts retained a diploid karyotype. It was found that ESCs are more sensitive to DR than fibroblasts: the DR dose killing 20% of cells was 0.01 and 0.1 μg/mL, respectively. DR induced ESC apoptotic death and reduced both ESC and fibroblast proliferation. DR reversibly inhibited ESC, but not fibroblast, proliferation. Thus, we demonstrated that ESCs and differentiated derivatives thereof are distinguished by sensitivity and response to the genotoxic agent.     

双向电泳法在家蚕蛋白质分离中的应用

家蚕Bombyxmori(L.)既是重要的经济昆虫,又是鳞翅目昆虫研究的典型模式生物。开展家蚕蛋白质组研究,将有助于阐明家蚕绢丝蛋白的分泌机理,也是研究鳞翅目昆虫及其他生物生命本质的需要。双向电泳是蛋白质分离的关键技术。为探讨适宜家蚕蛋白质组研究的双向电泳条件,以家蚕丝腺、丝腺内容物、蚕卵和血液为材料,在不同条件下进行双向电泳,并对分离的蛋白点进行质谱分析。结果表明:通过改进的蛋白质裂解液辅以超声破碎制备的蛋白质,双向电泳后能够得到较好的2-DE图,也能满足进行MALDI-TOFMS分析的需要。因此本研究方法适用于家蚕不同组织中蛋白质的提取和双向电泳。     

Synthesis of biocompatible chitosan decorated silver nanoparticles biocomposites for enhanced antimicrobial and anticancer property

Numerous studies investigated the biosynthesis of silver nanoparticles (AgNPs); however, there is a large gap for the ideal time-consuming process and their cytotoxicity. Herein, for the first time, rapid AgNPs was synthesized in a short time span, using Piper betle leaf (PBL) extract by applying microwave exposure. PB-AgNPs antibacterial activity and cell compatibility were enhanced by capping with chitosan (CS@PB-AgNPs). The synthesized nanoparticles were characterized by bioanalytical techniques. PB-AgNPs expressed significant antibacterial activity against Gram-positive and Gram-negative bacterial pathogens, while hybrid CS@PB-AgNPs presented the enhanced bactericidal activity. In addition, PB-AgNPs exhibited IC₅₀ value of 140 μg/mL against RAW 264.7 macrophages and 100 μg/mL against lung cancer cells while, CS capping reduced its toxicity at IC₅₀ values of 400 μg/mL and 180 μg/mL respectively were affirmed by MTT, apoptosis and DNA damage detection. Overall it was demonstrated that CS capping could be a phenomenal finding to improve the biomedical potential of AgNPs.