ABSTRACT
Trace elements are found in most drugs as a result of the drug formulation and drug production methods. An inductively coupled plasma-mass spectrometry method for the determination of 24 trace elements (Mg, Ti, V, Cr, Mn, Cu, Fe, Co, Ni, Zn, As, Se, Mo, Ru, Rh, Pd, Ag, Cd, Sb, Ba, Ir, Pt, Au, and Pb) in solid ibuprofen tablets was established in relation to the ICH Q3D(R1) guideline, to evaluate the possibility of linking trace elemental profiles to drug formulation strategies, and to differentiate between drug products based on the trace elemental profiles. Ten European ibuprofen drug products were evaluated (n=3). The sample preparation was performed by microwave-assisted acid digestion using only 10 mg of homogenized sample and 900 µL of a mix of 65% HNO3, 37% HCl, and 30% H2O2. Solid residuals primarily composed of insoluble SiO2 excipients were removed by centrifugation. Only concentrations of Mg, Fe, Ti, Mn, Cr, and Ni were detected above the limits of detection and did not exceed the ICH Q3D(R1) guideline permitted daily exposure limits. The trace elemental profiles were evaluated through principal component analysis. Three principal components describing 96% of the variance were useful in grouping the ibuprofen drug products, and the detected trace elemental remnants could be related to drug formulation and drug production strategies. An in-house quality control material was used in lack of certified reference materials and was in combination with spike recoveries used for method validation. Good spike recoveries (94-119%) were obtained for all measured trace elements except Mg. Mg showed acceptable spike recoveries (75-155%) for mid and high-spike concentrations, but poor recoveries (30-223%) were detected with low spike concentrations in spike matrices containing high amounts of Mg. Overall, the method is suggested applicable for solid drugs containing insoluble SiO2 excipients and drugs comparable to ibuprofen.
ABSTRACT
Binuclear molybdenum sulfur complexes are effective for the catalytic conversion of cyanide into thiocyanate. The complexes themselves exhibit low toxicity and high aqueous solubility, which render them suitable as antidotes for cyanide poisoning. The binuclear molybdenum sulfur complex [(thr)Mo2O2(µ-S)2(S2)]- (thr - threonine) was subjected to biological studies to evaluate its cellular accumulation and mechanism of action. The cellular uptake and intracellular distribution in human alveolar (A549) cells, quantified by inductively coupled plasma mass spectrometry (ICP-MS) and cell fractionation methods, revealed the presence of the compound in cytosol, nucleus, and mitochondria. The complex exhibited limited binding to DNA, and using the expression of specific protein markers for cell fate indicated no effect on the expression of stress-sensitive channel components involved in cell volume regulation, weak inhibition of cell proliferation, no increase in apoptosis, and even a reduction in autophagy. The complex is anionic, and the sodium complex had higher solubility compared to the potassium. As the molybdenum complex possibly enters the mitochondria, it is considered as a promising remedy to limit mitochondrial cyanide poisoning following, e.g., smoke inhalation injuries.
ABSTRACT
The histone deacetylase inhibitor trichostatin A (TSA) reduces cell viability in cisplatin-sensitive (A2780WT) and cisplatin-resistant (A2780RES) human ovarian cancer cells due to progression of apoptosis (increased caspase-9 activity), autophagy (increased LC3-II expression), and cell cycle arrest (increased p21 expression). The TSA-mediated effect on p21 and caspase-9 is mainly p53 independent. Cisplatin increases DNA-damage (histone H2AX phosphorylation) in A2780WT cells, whereas cisplatin, due to reduced uptake [inductively coupled-plasma-mass spectrometry (Pt) analysis], has no DNA-damaging effect in A2780RES cells. TSA has no effect on cisplatin accumulation or cisplatin-induced DNA-damage in A2780WT/A2780RES cells. Tracer technique indicates that TSA inhibits the volume-sensitive organic anion channel (VSOAC) in A2780WT/A2780RES cells and that the activity is restored by exogenous H2O2. As TSA reduces NOX4 mRNA accumulation and concomitantly increases catalase mRNA/protein accumulation, we suggest that TSA increases the antioxidative defense in A2780 cells. Inhibition of the kinase mTOR (rapamycin, palomid, siRNA), which is normally associated with cell growth, reduces VSOAC activity synergistically to TSA. However, as TSA increases mTOR activity (phosphorylation of 4EBP1, S6 kinase, S6, ULK1, SGK1), the effect of TSA on VSOAC activity does not reflect the shift in mTOR signaling. Upregulation of the protein expression and activity of the taurine transporter (TauT) is a phenotypic characteristic of A2780RES cells. However, TSA reduces TauT protein expression in A2780RES cells and activity to values seen in A2780WT cells. It is suggested that therapeutic benefits of TSA in A2780 do not imply facilitation of cisplatin uptake but more likely a synergistic activation of apoptosis/autophagy and reduced TauT activity.
Subject(s)
Cell Size/drug effects , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Ovarian Neoplasms/metabolism , Taurine/metabolism , A549 Cells , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Female , Humans , Ovarian Neoplasms/pathologyABSTRACT
The feasibility of quantitatively tracking platinum, derived from platinum-based compounds, during subcellular fractionation was studied. Cisplatin-exposed murine Ehrlich Lettré Ascites cells were fractionated into cytosolic and crude nuclear fractions. The latter was subsequently purified. Every residue and fraction produced during the fractionation procedure were collected and the platinum content determined by inductively coupled plasma mass spectrometry. Western blotting verified that the nuclear and cytosolic fractions were pure. It was found that 18% of platinum taken up by the cells was located in the nuclear fraction while 66% was located in the cytosolic fraction. Accumulated uncertainty originating from invariable sample characteristics and giving fraction purity priority had a negative effect on platinum recovery. Thus, overall 81% (n = 3, RSD = 3.4%) of the platinum taken up by the cells was recovered in the residues and final fractions. In conclusion, a reliable intracellular localization and quantitation of platinum following administration of Cisplatin can be determined by application of the method.
Subject(s)
Antineoplastic Agents/metabolism , Carcinoma, Ehrlich Tumor/metabolism , Cisplatin/metabolism , Mass Spectrometry/methods , Animals , Blotting, Western , Cell Nucleus/metabolism , Cytosol/metabolism , Mice , Subcellular Fractions/metabolism , Tumor Cells, CulturedABSTRACT
Gold-based compounds are of great interest in the field of medicinal chemistry as novel therapeutic (anticancer) agents due to their peculiar reactivity and mechanisms of action with respect to organic drugs. Despite their promising pharmacological properties, the possible toxic effects of gold compounds need to be carefully evaluated in order to optimize their design and applicability. This study reports on the potential toxicity of three experimental gold-based anticancer compounds featuring lansoprazole ligands (1-3) studied in an ex vivo model, using rat precision cut kidney and liver slices (PCKS and PCLS, respectively). The results showed a different toxicity profile for the tested compounds, with the neutral complex 2 being the least toxic, even less toxic than cisplatin, followed by the cationic complex 1. The dinuclear cationic gold complex 3 was the most toxic in both liver and kidney slices. This result correlated with the metal uptake of the different compounds assessed by ICP-MS, where complex 3 showed the highest accumulation of gold in liver and kidney slices. Interestingly compound 1 showed the highest selectivity towards cancer cells compared to the healthy tissues. Histomorphology evaluation showed a similar pattern for all three Au(i) complexes, where the distal tubular cells suffered the most extensive damage, in contrast to the damage in the proximal tubules induced by cisplatin. The binding of representative gold compounds with the model ubiquitin was also studied by ESI-MS, showing that after 24 h incubation only 'naked' Au ions were bound to the protein following ligands' loss. The mRNA expression of stress response genes appeared to be similar for both evaluated organs, suggesting oxidative stress as the possible mechanism of toxicity. The obtained results open new perspectives towards the design and testing of bifunctional gold complexes with chemotherapeutic applications.
ABSTRACT
Cisplatin occupies a crucial role in the treatment of various malignant tumors. However, its efficacy and applicability are heavily restricted by severe systemic toxicities and drug resistance. Our study exploits the active targeting of supramolecular metallacages to enhance the activity of cisplatin in cancer cells while reducing its toxicity. Thus, Pd2L4 cages (L = ligand) have been conjugated to four integrin ligands with different binding affinity and selectivity. Cage formation and encapsulation of cisplatin was proven by NMR spectroscopy. Upon encapsulation, cisplatin showed increased cytotoxicity in vitro, in melanoma A375 cells overexpressing αvß3 integrins. Moreover, ex vivo studies in tissue slices indicated reduced toxicity toward healthy liver and kidney tissues for cage-encapsulated cisplatin. Analysis of metal content by ICP-MS demonstrated that the encapsulated drug is less accumulated in these organs compared to the "free" cisplatin.
Subject(s)
Antineoplastic Agents/administration & dosage , Cisplatin/administration & dosage , Drug Carriers/metabolism , Integrin alphaVbeta3/metabolism , Melanoma/drug therapy , Metal-Organic Frameworks/metabolism , Palladium/metabolism , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cisplatin/pharmacology , Drug Carriers/chemistry , Drug Delivery Systems , Humans , Ligands , Male , Melanoma/metabolism , Metal-Organic Frameworks/chemistry , Palladium/chemistry , Rats, WistarABSTRACT
Systemic chemotherapy with the anticancer agent cisplatin is approved for advanced non-melanoma skin cancer (NMSC), but topical treatment is limited by insufficient cutaneous penetration. We studied the impact of ablative fractional laser (AFL) exposure on topical cisplatin's pharmacokinetics and biodistribution in skin, using microscopic ablation zones reaching the mid- (MAZ-MD; 620 µm depth) and deep dermis (MAZ-DD; 912 µm depth) (λ = 10,600 nm, 196 MAZ/cm2). Assessed in an in vitro Franz cell model after 0.5-, 4-, 24 h topical exposure (n = 8), cisplatin delivery was greatly accelerated by AFL, shown by quantitative- and imaging-based inductively coupled plasma-mass spectrometry (ICP-MS). After 30 minutes, cisplatin concentrations were 91.5, 90.8 and 37.8 µg/cm3 in specific 100-, 500, and 1500 µm skin layers respectively, contrasting to 8.08, 3.12, 0.64 µg/cm3 in non-laser-exposed control skin (p < .001; control vs MAZ-MD). Supported by element bioimaging, the greatest relative increases occurred in the deep skin compartment and at later time points. After 24 h, cisplatin concentrations thus rose to 1829, 1732 and 773 µg/cm3, representing a 25-, 103- and 447-fold enhancement in the 100, 500, and 1500 µm deep skin layers versus corresponding controls (p < .001; MAZ-MD). A significant difference in cutaneous uptake using MAZ-MD and MAZ-DD was not shown at any time point, though deeper laser channels resulted in increased transdermal cisplatin permeation (p ≤ .015). In conclusion, AFL is a rapid, practical and existing skin treatment that may provide greatly enhanced uptake of topical cisplatin for treatment of superficial and deep skin cancer.
Subject(s)
Antineoplastic Agents/administration & dosage , Cisplatin/administration & dosage , Drug Delivery Systems/methods , Laser Therapy/methods , Skin Absorption/radiation effects , Skin/radiation effects , Animals , Antineoplastic Agents/pharmacokinetics , Cisplatin/pharmacokinetics , Female , In Vitro Techniques , Skin/metabolism , Swine , Time Factors , Tissue DistributionABSTRACT
The molecular mechanisms of toxicity and cellular transport of anticancer metallodrugs, including platinum-based agents, have not yet been fully elucidated. The aim of our study was to investigate the relevance of copper transporters (CTR1 and ATP7A/B), organic cation transporters (OCT2) and the multidrug and toxin extrusion proteins (MATE) in the intracellular accumulation of a novel organometallic cytotoxic Au(III) compound in cancer cells in comparison to cisplatin. Specifically, the synthesis and characterization of the gold complex [Au(pyb-H)(PPh2Ar)Cl]PF6 (PPh2Ar = 3-[4-(diphenylphosphino)phenyl]-7-methoxy-2H-chromen-2-one] (1), featuring a coumarin ligand endowed with "smart" fluorescence properties, have been achieved. Initially, the cytotoxic effects of both cisplatin and 1 were studied in a small panel of human cancer cells, and against a non-tumorigenic cell line in vitro. Thus, the human ovarian cancer cell line A2780 and its cisplatin resistant variant A2780cisR, were selected, being most sensitive to the treatment of the gold complex. Co-incubation of the metallodrugs with CuCl2 (a CTR1 substrate) increased the cytotoxic effects of both the Au(III) complex and cisplatin; while co-incubation with cimetidine (inhibitor of OCT2 and MATE) showed some effect only after 72 h incubation. ICP-MS (Inductively Coupled Plasma Mass Spectrometry) analysis of the cell extracts showed that co-incubation with CuCl2 increases Au and Cu accumulation in both cancer cell lines, in accordance with the enhanced antiproliferative effects. Conversely, for cisplatin, no increase in Pt content could be observed in both cell lines after co-incubation with either CuCl2 or cimetidine, excluding the involvement of CTR1, OCT2, and MATE in drug accumulation and overall anticancer effects. This result, together with the evidence for increased Cu content in A2780 cells after cisplatin co-treatment with CuCl2, suggests that copper accumulation is the reason for the observed enhanced anticancer effects in this cell line. Moreover, metal uptake studies in the same cell lines indicate that both 1 and cisplatin are not transported intracellularly by CTR1 and OCT2. Finally, preliminary fluorescence microscopy studies enabled the visualization of the sub-cellular distribution of the gold compound in A2780 cells, suggesting accumulation in specific cytosolic components/organelles.
ABSTRACT
Cisplatin is a widely used chemotherapeutic drug. Due to severe side effects and intrinsic or acquired resistance, there is a great interest in developing new platinum-based anticancer agents and a need for robust and validated analytical methods for determination of platinum accumulation in biological samples. A validated ICP-MS method for quantification of total carbon and platinum in cell samples is presented, applicable for cellular drug accumulation studies of platinum-based drugs, enabling estimation of drug accumulation while simultaneously determining carbon to monitor the sample digestion efficiency. Adequate precision (RSD <6%), accuracy and sensitivity were achieved for carbon and platinum determinations. Limits of detection were 0.9-3.0â¯mg/L for carbon and 0.11-0.50⯵g/L for platinum. Determination of platinum by ICP-MS in cell samples digested applying either open-vessel or microwave-assisted acid digestion produced similar concentrations, although the residual carbon content in the sample solutions were significantly higher following open-vessel acid digestion compared to microwave-assisted acid digestion. Experiments showed that the residual carbon content after acid digestion did not have an influence on determination of total platinum by ICP-MS.
Subject(s)
Carbon/analysis , Mass Spectrometry/methods , Platinum/analysis , A549 Cells , Acids/chemistry , Calibration , Humans , Mass Spectrometry/instrumentation , Microwaves , Sensitivity and SpecificityABSTRACT
Cisplatin ( cis-diamminedichloroplatinum(II)) is among the most potent cytotoxic agents used in cancer chemotherapy. The encapsulation of cisplatin in lipid-based drug carriers has been challenging owing to its low solubility in both aqueous and lipid phases. Here, we investigated cisplatin encapsulation in nonlamellar liquid-crystalline (LC) nanodispersions formed from a ternary mixture of phytantriol (PHYT), vitamin E (Vit E), and an anionic phospholipid [either phosphatidylglycerol (DSPG) or phosphatidylserine (DPPS)]. We show an increase in cisplatin encapsulation efficiency (EE) in nanodispersions containing 1.5-4 wt % phospholipid. The EE was highest in DPPS-containing nanodispersions (53-98%) compared to DSPG-containing counterparts (25-40%) under similar experimental conditions. Through structural and morphological characterizations involving synchrotron small-angle X-ray scattering and cryogenic transmission electron microscopy, we further show that varying the phospholipid content of cisplatin-free nanodispersions triggers an internal phase transition from a neat hexagonal (H2) phase to a biphasic phase (internal H2 phase coexisting with the lamellar (Lα) phase). However, cisplatin encapsulation in both DPPS- and DSPG-containing nanodispersions generates the coexistence of morphologically different multicompartments in the internal nanostructures comprising vesicles as a core, enveloped by an inverted-type surface bicontinuous cubic Im3 m (primitive, QIIP) phase or H2 phase. We discuss the biophysical basis of these drug-induced morphological alterations and provide insights into the potential development of inverted-type LC nanodispersions for cisplatin delivery.
Subject(s)
Cisplatin/chemistry , Drug Carriers/chemistry , Liquid Crystals/chemistry , Nanostructures/chemistry , Phase Transition , Phospholipids/chemistry , X-Ray DiffractionABSTRACT
Mechanisms of toxicity and cellular transport of anticancer metallodrugs, including platinum-based agents, have not yet been fully elucidated. Here, we studied the toxic effects and accumulation mechanisms of cisplatin in healthy rat kidneys ex vivo, using the Precision Cut Tissue Slices (PCTS) method. In addition, for the first time, we investigated the nephrotoxic effects of an experimental anticancer cyclometallated complex [Au(pyb-H)(PTA)Cl]PF6 (PTA = 1,3,5-triazaphosphaadamantane). The viability of the kidney slices after metallodrug treatment was evaluated by ATP content determination and histomorphology analysis. A concentration dependent decrease in viability of PCKS was observed after exposure to cisplatin or the Au(iii) complex, which correlated with the increase in slice content of Pt and Au, respectively. Metal accumulation in kidney slices was analysed by ICP-MS. The involvement of OCTs and MATE transporters in the accumulation of both metal compounds in kidneys was evaluated co-incubating the tissues with cimitedine, inhibitor of OCT and MATE. Studies of mRNA expression of the markers KIM-1, villin, p53 and Bax showed that cisplatin damages proximal tubules, whereas the Au(iii) complex preferentially affects the distal tubules. However, no effect of cimetidine on the toxicity or accumulation of cisplatin and the Au(iii) complex was observed. The effect of temperature on metallodrug accumulation in kidneys suggests the involvement of a carrier-mediated uptake process, other than OCT2, for cisplatin; while carrier-mediated excretion was suggested in the cases of the Au(iii) complex.
Subject(s)
Antineoplastic Agents/toxicity , Cisplatin/toxicity , Cytotoxins/toxicity , Gold/toxicity , Kidney/metabolism , Adenosine Triphosphate/metabolism , Animals , Antiporters/metabolism , Kidney/drug effects , Kidney/pathology , Male , Organic Cation Transport Proteins/metabolism , Organic Cation Transporter 2/metabolism , Rats , Rats, WistarABSTRACT
Acquired resistance to chemotherapeutic drugs in cancer cells can reflect an ability to limit cellular drug availability, to repair drug induced DNA damage, and to limit initiation/progression of cell death (apoptosis). The leucine-rich-repeat-containing 8A (LRRC8A) protein is an essential component of volume sensitive channels for organic osmolytes (VSOAC) and volume regulated anion channels (VRAC), which are activated during the apoptotic process. Here we illustrate that cisplatin resistance in human ovarian cancer cells (A2780) correlates with a reduced expression of LRRC8A and copper transporter receptor 1 (CTR1), as well as a concomitant increased expression of copper-transporting P-type ATPases (ATP7A/ATP7B). We also find that cisplatin (Pt) accumulation correlates with LRRC8A protein expression and channel activity, i.e., the cellular Pt content is high when VSOAC is activated by depolarization of the plasma membrane or hypoosmotic cell swelling, and reduced when channel activity/LRRC8A expression is reduced by genetically silencing/pharmacological inhibition, or the cells have acquired a resistant phenotype with low LRRC8A protein expression. It is suggested that reduced LRRC8A expression in cisplatin-resistant A2780 cells ensures cell survival through limitation in cisplatin accumulation and a concomitant reduction in osmolytes loss via VSOAC/VRAC and hence instigation of the apoptotic process.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Drug Resistance, Neoplasm/genetics , Epithelial Cells/drug effects , Gene Expression Regulation, Neoplastic , Membrane Proteins/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cell Line, Tumor , Cell Membrane/drug effects , Cell Survival/genetics , Copper Transporter 1 , Copper-Transporting ATPases , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Ovary/drug effects , Ovary/metabolism , Ovary/pathology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal TransductionABSTRACT
The paper presents an analytical method for quantification of low molecular weight (LMW) selenium compounds in human plasma based on liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) and post column isotope dilution-based quantification. Prior to analysis, samples were ultrafiltrated using a cut-off value of 3000 Da. The method was validated in aqueous solution as well as plasma using standards of selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenite, and the selenosugar Se-methylseleno-N-acetylgalactosamine (SeGal) for linearity, precision, recoveries, and limits of detection and quantitation with satisfactory results. The method was applied for analysis of a set of plasma samples from cancer patients receiving selenite treatment in a clinical trial. Three LMW selenium compounds were observed. The main compounds, SeGal and selenite were tentatively identified by retention time matching with standards in different chromatographic systems, while the third minor compound was not identified. The identity of the selenosugar was verified by ESI-MS-MS product ion scanning, while selenite was identified indirectly as the glutathione (GSH) reaction product, GS-Se-SG.
Subject(s)
Antineoplastic Agents/administration & dosage , Selenious Acid/administration & dosage , Selenium/blood , Antineoplastic Agents/therapeutic use , Chromatography, Liquid , Humans , Limit of Detection , Mass Spectrometry , Molecular Weight , Neoplasms/drug therapy , Reference Standards , Selenious Acid/therapeutic useABSTRACT
Cellular uptake of vitamin B12-cisplatin conjugates was estimated via detection of their metal constituents (Co, Pt, and Re) by inductively coupled plasma mass spectrometry (ICP-MS). Vitamin B12 (cyano-cob(iii)alamin) and aquo-cob(iii)alamin [Cbl-OH2](+), which differ in the ß-axial ligands (CN(-) and H2O, respectively), were included as control samples. The results indicated that B12 derivatives delivered cisplatin to both cellular cytosol and nuclei with an efficiency of one third compared to the uptake of free cisplatin cis-[Pt(II)Cl2(NH3)2]. In addition, uptake of charged B12 derivatives including [Cbl-OH2](+), [{Co}-CN-{cis-PtCl(NH3)2}](+), [{Re}-{Co}-CN-{cis-PtCl(NH3)2}](+), and [{Co}-CN-{trans-Pt(Cyt)(NH3)2}](2+) (Cyt = cytarabin) was high compared to neutral B12, which implied the existence of an additional internalization pathway for charged B12 vitamin analogs. The affinities of the charged B12 derivatives to the B12 transporters HC, IF and TC were similar to that of native vitamin B12.
Subject(s)
Endocytosis , Metals/metabolism , Vitamin B 12/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cytosol/metabolism , Endocytosis/drug effects , Humans , K562 Cells , Protein Transport/drug effects , Proton Magnetic Resonance Spectroscopy , Vitamin B 12/chemical synthesis , Vitamin B 12/chemistry , Vitamin B 12/pharmacologyABSTRACT
The inverted-type liquid-crystalline dispersions comprising cubosomes and hexosomes hold much potential for drug solubilization and site-specific targeting on intravenous administration. Limited information, however, is available on the influence of plasma components on nanostructural and morphological features of cubosome and hexosome dispersions, which may modulate their stability in the blood and their overall biological performance. Through an integrated approach involving SAXS, cryo-TEM, and nanoparticle tracking analysis (NTA) we have studied the time-dependent effect of human plasma (and the plasma complement system) on the integrity of the internal nanostructure, morphology, and fluctuation in size distribution of phytantriol (PHYT)-based nonlamellar crystalline dispersions. The results indicate that in the presence of plasma the internal nanostructure undergoes a transition from the biphasic phase (a bicontinuous cubic phase with symmetry Pn3m coexisting with an inverted-type hexagonal (H2) phase) to a neat hexagonal (H2) phase, which decreases the median particle size. These observations were independent of a direct effect by serum albumin and dispersion-mediated complement activation. The implication of these observations in relation to soft nanocarrier design for intravenous drug delivery is discussed.
Subject(s)
Liquid Crystals/chemistry , Nanostructures/chemistry , Cryoelectron Microscopy , Drug Carriers/chemistry , Fatty Alcohols/chemistry , Humans , Liquid Crystals/ultrastructure , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Nanostructures/ultrastructureABSTRACT
In the present work a novel C,N-cyclometalated benzimidazole Ru(ii) arene complex (GY34) was characterized by applying an alternative, diverse approach considering both chemical and biological aspects. RP-HPLC-ICP-MS and RP-HPLC-ESI-MS analysis proved that GY34 in both RPMI-1640 cell medium and ammonium acetate buffer was transformed into several subspecies and the importance of evaluating and controlling analyte stability throughout experiments was demonstrated. Applying a novel cell fractionation protocol GY34 was found to target cell nuclei and mitochondria in Ehrlich Lettré Ascites (ELA) cells, with the intracellular distribution depending on GY34 concentration in the cell medium during incubation. In ELA cells 96 ± 0.2% of cytosolic GY34 was bound to high-molecular species. Furthermore, using the tracer technique GY34 was found to reduce uptake and increase release of the organic osmolyte taurine in ELA cells, with innate resistance to Cisplatin and in A2780 human ovarian cancer cells, with acquired resistance to Cisplatin. Importantly, FACS analysis revealed that GY34 induced apoptosis in ELA cells. The present data suggest the potential of GY34 in overcoming Cisplatin resistance. The methodology applied can be used as a general protocol and an additional tool in the initial evaluation of novel metal-based drugs.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Ruthenium/chemistry , Ruthenium/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Benzimidazoles/pharmacokinetics , Carcinoma, Ehrlich Tumor/drug therapy , Carcinoma, Ehrlich Tumor/metabolism , Carcinoma, Ehrlich Tumor/pathology , Cell Line, Tumor , Female , Homeostasis/drug effects , Humans , Mice , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Ovary/drug effects , Ovary/metabolism , Ovary/pathology , Ruthenium/pharmacokinetics , Taurine/metabolismABSTRACT
Human serum albumin (HSA) is the most abundant protein in the human plasma. HSA has several physiological roles in the human body, including storage and transport. Owing to the predominance of albumin in plasma, HSA is often involved in the protein binding of drugs. The aim of this work was to develop a selective, quantitative method for determining albumin in plasma with the purpose of clarifying the fate of metal-based drugs in biological systems. The method can also be applied for determination of urine albumin, which is of relevance in diagnostics of kidney disease. A selective method for quantification of HSA based on labelling the protein with iophenoxic acid (IPA) was developed. Samples were subjected to size exclusion chromatography (SEC) and detection by inductively coupled plasma mass spectrometry (ICP-MS) monitoring iodine and platinum. The iodine signal for the HSA-IPA complex showed linearity in the range 1 to 250 mg L(-1). The precision was 3.7% and the accuracy 100.7% determined by analysis of a certified HSA reference material. The limit of detection (LOD) and limit of quantification (LOQ) were 0.23 and 9.79 mg L(-1), respectively. The method was applied for analysis of HSA in human plasma and urine samples and for studying the binding of cisplatin to proteins in the human plasma.
Subject(s)
Chromatography, Gel/methods , Iopanoic Acid/chemistry , Mass Spectrometry/methods , Serum Albumin/analysis , Albuminuria/urine , Cisplatin/metabolism , Humans , Limit of Detection , Reproducibility of Results , Sensitivity and Specificity , Serum Albumin/chemistry , Serum Albumin/metabolismABSTRACT
In the present study, the impact on peptide properties of labelling peptides with the fluorophore TAMRA or the selenium (Se) containing amino acid SeMet was evaluated. Three differently labelled variants of the cell-penetrating peptide (CPP) penetratin (Pen) were synthesized, PenM(Se), TAMRA-PenM(Se) and TAMRA-Pen. The labelled peptides were characterized in terms of hydrodynamic radius, secondary structure during peptide-membrane interaction, effect on membrane leakage induction, uptake efficiency in HeLa cells. Furthermore, stability of peptides and identities of degradation products in cell media and cell lysate were evaluated. TAMRA-labelling increased the hydrodynamic radius of Pen and PenM(Se) significantly. Labelling with Se caused no or minimal changes in size, secondary structure and membrane leakage induction in concentration levels relevant for cellular uptake studies. Similar degradation patterns of all labelled peptides were observed in HBSS media; degradation was mainly due to oxidation. Cellular uptake was significantly higher for the TAMRA labelled peptides as compared to Se-labelled Pen. Extensive degradation was observed in media during cellular uptake studies, however, in all cell lysates, primarily the intact peptide (PenM(Se), TAMRA-PenM(Se) or TAMRA-Pen) was observed. Selenium labelling caused minimal alteration of the physicochemical properties of the peptide and allowed for absolute quantitative determination of cellular uptake by inductively coupled plasma mass spectrometry. Selenium is thus proposed as a promising alternative label for quantification of peptides in general, altering the properties of the peptide to a minor extent as compared to commonly used peptide labels.
Subject(s)
Carrier Proteins/chemistry , Cell-Penetrating Peptides/chemistry , Fluorescent Dyes/chemistry , Rhodamines/chemistry , Selenium/chemistry , Biological Transport , Carrier Proteins/pharmacology , Cell-Penetrating Peptides/pharmacology , Cholesterol/chemistry , Circular Dichroism , Drug Stability , Fluorescent Dyes/pharmacology , HeLa Cells , Humans , Liposomes , Molecular Weight , Phosphatidylcholines/chemistry , Phosphatidylglycerols/chemistry , Protein Structure, Secondary , Rhodamines/pharmacology , Selenium/pharmacologyABSTRACT
The aim of the present work was to demonstrate how selenium labelling of a synthetic cell-penetrating peptide may be employed in evaluation of stability and quantitative estimation of cellular uptake by inductively coupled plasma mass spectrometry (ICP-MS). Two analogues of the cell-penetrating peptide, penetratin, were synthesized, one with selenomethionine (SeMet) added at the N-terminal of the peptide (N-PenM(Se)) and the other with the internal methionine (Met) replaced with SeMet (i-PenM(Se)). The purity of the synthesized peptides was 92% for N-PenM(Se) and 89% for i-PenM(Se) as determined by liquid chromatography (LC)-ICP-MS. The selenium-labelled peptides were investigated by cell uptake studies in HeLa WT cells. The stability of the peptides was monitored in water, cell medium and during cell uptake studies. Total uptake of selenium was quantified by flow injection (FI)-ICP-MS. Speciation analysis of cell samples by LC-ICP-MS showed mainly uptake of the intact peptides, while the amount of intact peptides in cell lysates was semi-quantitatively determined. The selenium-containing penetratin analogues were to some extent degraded in pure cell medium, while an extensive degradation was observed during cell uptake studies. The major degradation products were determined by LC-electrospray ionization mass spectrometry (ESI-MS). The labelling method in combination with FI-ICP-MS, LC-ICP-MS and LC-ESI-MS techniques provided detailed information on the fate of penetratin in cellular uptake studies. Most pharmaceutical peptides, including penetratin, are synthetic analogues of endogenous peptides, and incorporation of selenium may improve the critical assessment of the native drug or drug delivery candidate early in the drug development process.
Subject(s)
Peptides/analysis , Pharmaceutical Preparations/analysis , Selenium/analysis , Staining and Labeling , Amino Acid Sequence , Cell Extracts , Endocytosis , HeLa Cells , Humans , Mass Spectrometry , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistryABSTRACT
Exposure to arsenic in drinking water is associated with increased respiratory disease. Alpha-1 antitrypsin (AAT) protects the lung against tissue destruction. The objective of this study was to determine whether arsenic exposure is associated with changes in airway AAT concentration and whether this relationship is modified by selenium. A total of 55 subjects were evaluated in Ajo and Tucson, Arizona. Tap water and first morning void urine were analyzed for arsenic species, induced sputum for AAT and toenails for selenium and arsenic. Household tap-water arsenic, toenail arsenic and urinary inorganic arsenic and metabolites were significantly higher in Ajo (20.6±3.5 µg/l, 0.54±0.77 µg/g and 27.7±21.2 µg/l, respectively) than in Tucson (3.9±2.5 µg/l, 0.16±0.20 µg/g and 13.0±13.8 µg/l, respectively). In multivariable models, urinary monomethylarsonic acid (MMA) was negatively, and toenail selenium positively associated with sputum AAT (P=0.004 and P=0.002, respectively). In analyses stratified by town, these relationships remained significant only in Ajo, with the higher arsenic exposure. Reduction in AAT may be a means by which arsenic induces respiratory disease, and selenium may protect against this adverse effect.