The importance of radiochemical purity: Cellular binding and internalization of different radiometal chlorides in prostate cancer cells.

Radiometals play an important role in nuclear medicine, both for imaging and therapy. Binding studies represent an important step in the development of new radiolabeled ligands, as valuable insights into the binding properties can be gained. However, this technique requires high radiochemical purity, otherwise results may lead to wrong assumptions or misinterpretations of affinities or uptake rates. Therefore, this in vitro study aimed at investigating the cell binding and internalization characteristics of different radiometal chlorides ([111In]InCl3, [68Ga]GaCl3 and [177Lu]LuCl3) commonly applied in nuclear medicine, as well as the clinically applied [177Lu]Lu-PSMA-I&T in comparison, by using prostate cancer cells. PC-3 and LNCaP cells were incubated with 100 kBq of the respective radiometal chloride or [177Lu]Lu-PSMA-I&T for 1 h. For [177Lu]LuCl3, nuclei isolations and colloid determinations in saline and cell medium were also performed. Results showed that [111In]InCl3 and [68Ga]GaCl3 bind and are internalized up to 3 % to PC-3 and LNCaP cells, whereas [177Lu]LuCl3 showed cell binding of up to 25 %, internalization up to 2.5 % and a nuclear uptake below 0.3 %. In comparison, [177Lu]Lu-PSMA-I&T showed only 3 % total cell binding to LNCaP cells. Further analysis of [177Lu]LuCl3 stability in NaCl and cell medium showed only low amounts of colloids, which are not increasing over time, and negligible unspecific binding to the used cell culture plates. In conclusion, the results demonstrate the importance of high radiochemical purity, especially with regard to Lu-177 labeled compounds. Even if radiopharmaceuticals comply with common release-criteria, significant uptake can be derived from [177Lu]LuCl3 impurities and lead to wrong estimations of a compound's uptake behavior. Assuming an experimental result of 2 % membrane binding of the applied product, and 5 % residual [177Lu]LuCl3 in the final product (thereof 25 % membrane binding, as described above), would lead to 1.25 % membrane binding resulting from [177Lu]LuCl3 and only 0.75 % from the radiopharmaceutical.

Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

BACKGROUND: In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, a phenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. METHODS: The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). RESULTS: This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. CONCLUSIONS: We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

A New Water-Soluble Thermosensitive Star-Like Copolymer as a Promising Carrier of the Chemotherapeutic Drug Doxorubicin.

A new water-soluble thermosensitive star-like copolymer, dextran-graft-poly-N-iso-propilacrylamide (D-g-PNIPAM), was created and characterized by various techniques (size-exclusion chromatography, differential scanning calorimetry, Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering (DLS) spectroscopy). The viability of cancer cell lines (human transformed cervix epithelial cells, HeLa) as a model for cancer cells was studied using MTT and Live/Dead assays after incubation with a D-g-PNIPAM copolymer as a carrier for the drug doxorubicin (Dox) as well as a D-g-PNIPAM + Dox mixture as a function of the concentration. FTIR spectroscopy clearly indicated the complex formation of Dox with the D-g-PNIPAM copolymer. The size distribution of particles in Hank's solution was determined by the DLS technique at different temperatures. The in vitro uptake of the studied D-g-PNIPAM + Dox nanoparticles into cancer cells was demonstrated by confocal laser scanning microscopy. It was found that D-g-PNIPAM + Dox nanoparticles in contrast to Dox alone showed higher toxicity toward cancer cells. All of the aforementioned facts indicate a possibility of further preclinical studies of the water-soluble D-g-PNIPAM particles' behavior in animal tumor models in vivo as promising carriers of anticancer agents.

In Vitro and In Vivo Studies on HPMA-Based Polymeric Micelles Loaded with Curcumin.

Curcumin-loaded polymeric micelles composed of poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) were prepared to solubilize and improve the pharmacokinetics of curcumin. Curcumin-loaded micelles were prepared by a nanoprecipitation method using mPEG5kDa-b-p(HPMA-Bz) copolymers with varying molecular weight of the hydrophobic block (5.2, 10.0, and 17.1 kDa). At equal curcumin loading, micelles composed of mPEG5kDa-b-p(HPMA-Bz)17.1kDa showed better curcumin retention in both phosphate-buffered saline (PBS) and plasma at 37 °C than micelles based on block copolymers with smaller hydrophobic blocks. No change in micelle size was observed during 24 h incubation in plasma using asymmetrical flow field-flow fractionation (AF4), attesting to particle stability. However, 22-49% of the curcumin loading was released from the micelles during 24 h from formulations with the highest to the lowest molecular weight p(HPMA-Bz), respectively, in plasma. AF4 analysis further showed that the released curcumin was subsequently solubilized by albumin. In vitro analyses revealed that the curcumin-loaded mPEG5kDa-b-p(HPMA-Bz)17.1kDa micelles were internalized by different types of cancer cells, resulting in curcumin-induced cell death. Intravenously administered curcumin-loaded, Cy7-labeled mPEG5kDa-b-p(HPMA-Bz)17.1kDa micelles in mice at 50 mg curcumin/kg showed a long circulation half-life for the micelles (t1/2 = 42 h), in line with the AF4 results. In contrast, the circulation time of curcumin was considerably shorter than that of the micelles (t1/2α = 0.11, t1/2ß = 2.5 h) but ∼5 times longer than has been reported for free curcumin (t1/2α = 0.02 h). The faster clearance of curcumin in vivo compared to in vitro studies can be attributed to the interaction of curcumin with blood cells. Despite the excellent solubilizing effect of these micelles, no cytostatic effect was achieved in neuroblastoma-bearing mice, possibly because of the low sensitivity of the Neuro2A cells to curcumin.

Solid lipid nanoparticles as a novel formulation approach for tanespimycin (17-AAG) against leishmania infections: Preparation, characterization and macrophage uptake.

17-N-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) is an inhibitor of heat shock protein 90 (Hsp90), which has been studied in the treatment of cancer such as leukemia or solid tumors. Alternatively, 17-AAG may represent a promising therapeutic agent against leishmaniasis. However, the delivery of 17-AAG is difficult due to its poor aqueous solubility. For exploring the therapeutic value of 17-AAG, we developed solid lipid nanoparticles (SLN) by double emulsion method. SLN exhibited ~100 nm, PDI < 0.2 and zeta potential ~20 mV. In addition, SLN were morphologically spherical with negligible aggregation. The entrapment efficiency of 17-AAG into the lipid matrix reached at nearly 80%. In a separate set of experiments, fluorescent SLN (FITC-labeled) showed a remarkable macrophage uptake, peaking within 2 h of incubation by confocal microscopy. Regarding the drug internalization as critical step for elimination of intracellular Leishmania, this finding demonstrates an important feature of the developed SLN. Collectively, these data indicate the feasibility of developing SLN as potential delivery systems for 17-AAG in leishmaniasis chemotherapy.

Gd-GLU toward NMR imaging: synthesis, characterization and breast cell uptake assay

Breast cancer cell uptake of Gd-metal is investigated based on the formation of coordinate compounds of gadolinium and glucose (Glu) molecules in solution. The hypothesis is that glucose helps Gd-internalization by complex formations constituted of Gd3+ coordinate to m-glucose molecules, whose valence was complemented by Cl- anions. Such a proposal is an insight toward a metabolic-dependent contrast-agent for cancer and inflammation in magnetic resonance image. A solution was prepared based on anhydrous d-glucose and gadolinium chloride (Gd-Glu). Uptake assays for MDA-MB-231(c231) cells were elaborated collecting incubated c231-cells with Gd-Glu and measuring metal-uptake and their concentrations by Nuclear Activation Analysis (NAA). The ionic solution was studied using Direct-Infusion Electrospray Ionization Mass-Spectrometry (ESI-MS) to identify Gd-Glu interactions. Means and standard deviations of Gd-masses were 13.3±0.8 and 12.5±0.7µg, at 361.5 µg of Gd in 3mL Gd-Glu/PBS solution, in times of 30-50 min, equivalent to the concentrations of 13404±2104 and 11347±2742 µg.g-1 in dried cells. Such values were statistically higher than the control with metal presence. ESI-MS demonstrated the m/z-signals at 516, 552, 696, 923, attributed to positively loaded-species containing Glu, Gd+3 and Cl-. In conclusion, Gd-internalization was increased in aqueous solution due to the gadolinium-glucose coordination. Such findings drive the research to MRI with Gd-Glu complexes.

Preparation and evaluation of effect on Escherichia coli and Staphylococcus aureus of radiolabeled ampicillin-loaded graphene oxide nanoflakes.

Ampicillin is a one of effective antibiotics against Gram-positive and Gram-negative bacteria. This study aimed to label ampicillin-loaded graphene oxide nanoflake (AMP-GO) with 99m Tc and evaluate of its in vitro binding to Staphylococcus aureus and Escherichia coli. Firstly, ampicillin was loaded into graphene oxide nanoflake prepared. AMP-GO was characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) techniques, and the amount of loaded ampicillin onto GO was determined by UV-Vis absorption spectroscopy. AMP and AMP-GO were labeled with 99m Tc using stannous chloride reducing agent. Labeling efficiency of 99m Tc-AMP-GO was found to be 97.66 ± 2.06%. 99m Tc-AMP-GO has higher binding efficiencies to both S. aureus and E. coli than 99m Tc-AMP. 99m Tc-AMP-GO could be promising candidate as agent infection nuclear imaging. Furthermore, in vivo studies of 99m Tc-AMP-GO with infected rats are planned to be performed.

Phage-derived protein-mediated targeted chemotherapy of pancreatic cancer.

Pancreatic cancer has been a life-threatening illness associated with high incidence and mortality rates. Paclitaxel (PCT) that causes mitotic arrest in cancer cells disrupting microtubule function is used for pancreatic cancer treatment. Nausea, anorexia and abdominal pain are some of the typical dose-limiting toxicity associated gastrointestinal side effects of the drug. Here, we present the use of polymeric mixed micelles to enable a targeted delivery of PCT and to provide additional advantages such as enhanced drug solubility, bioavailability and minimal dose-limiting toxicity. Also, these micelles self-assemble with pancreatic cancer cells-specific phage proteins P38, L1 and with the hydrophobic drug PCT resolving the issue of complex chemistry efforts normally needed for any conjugation. Our cytotoxicity and binding experiment results in vitro in 2 D and 3 D models suggested that the phage protein-targeted drug-loaded micelles bind and exhibit higher cell killing over the non-targeted ones.

Synthesis and Functional Characterization of Novel Sialyl LewisX Mimic-Decorated Liposomes for E-selectin-Mediated Targeting to Inflamed Endothelial Cells.

Sialyl LewisX (sLeX) is a natural ligand of E-selectin that is overexpressed by inflamed and tumor endothelium. Although sLeX is a potential ligand for drug targeting, synthesis of the tetrasaccharide is complicated with many reaction steps. In this study, structurally simplified novel sLeX analogues were designed and linked with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG) for E-selectin-mediated liposomal delivery. The sLeX structural simplification strategies include (1) replacement of the Gal-GlcNAc disaccharide unit with lactose to reduce many initial steps and (2) substitution of neuraminic acid with a negatively charged group, i.e., 3'-sulfo, 3'-carboxymethyl (3'-CM), or 3'-(1-carboxy)ethyl (3'-CE). While all the liposomes developed were similar in particle size and charge, the 3'-CE sLeX mimic liposome demonstrated the highest uptake in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs), being even more potent than native sLeX-decorated liposomes. Inhibition studies using antiselectin antibodies revealed that their uptake was mediated primarily by overexpressed E-selectin on inflamed HUVECs. Molecular dynamics simulations were performed to gain mechanistic insight into the E-selectin binding differences among native and mimic sLeX. The terminally branched methyl group of the 3'-CE sLeX mimic oriented and faced the bulk hydrophilic solution during E-selectin binding. Since this state is entropically unfavorable, the 3'-CE sLeX mimic molecule might be pushed toward the binding pocket of E-selectin by a hydrophobic effect, leading to a higher probability of hydrogen-bond formation than native sLeX and the 3'-CM sLeX mimic. This corresponded with the fact that the 3'-CE sLeX mimic liposome exhibited much greater uptake than the 3'-CM sLeX mimic liposome.

Structural dependence of in vitro cytotoxicity, oxidative stress and uptake mechanisms of poly(propylene imine) dendritic nanoparticles.

The in vitro cytotoxic and intracellular oxidative stress responses to exposure to poly(propylene imine) (PPI) dendritic nanoparticles of increasing generation (number of repeated branching cycles) (G0-G4) were assessed in an immortal non-cancerous human keratinocyte cell line (HaCaT). Confocal fluorescence microscopy with organelle staining was used to explore the uptake and intracellular trafficking mechanisms. A generation- and dose-dependent cytotoxic response was observed, increasing according to generation and, therefore, number of surface amino groups. A comparison of the cytotoxic response of G4 PPI and the related G4 poly(amido amine) dendrimer indicates that the PPI with the same number of surface amino groups elicits a significantly higher cytotoxic response. The trend of cytotoxicity versus dendrimer generation and, therefore, size is discontinuous in the region of G2, however, indicating a difference in uptake mechanism for higher compared to lower generations. Whereas the higher generations elicit an oxidative stress response at short exposure times, the lower generations indicate an antioxidant response. Confocal microscopy indicates that, whereas they are prominent at early exposure times for the larger PPI dendrimers, no evidence of early stage endosomes was observed for lower generations of PPI. The results are consistent with an alternative uptake mechanism of physical diffusion across the semipermeable cell membrane for the lower generation dendrimers and are discussed in terms of their implications for predictive models for nanotoxicology and design strategies for nanomedical applications.