Imaging cells and tissues with refractive index radiology.

Can individual cells, including live cells, be imaged using hard x rays? Common wisdom until now required sophisticated staining techniques for this task. We show instead that individual cells and cell details can be detected in culture solution and tissues with no staining and no other contrast-enhancing preparation. The sample examined can be much thicker than for many other microscopy techniques without sacrificing the capability to resolve cells. The key factor in our approach is the use of a coherent synchrotron source and of contrast mechanisms based on the refractive index. The first successful tests were conducted on a variety of cell systems including skin and internal leaf cells, mouse neurons, rabbit fibroblast cells, and human tumor cells.

NODAGATOC, a new chelator-coupled somatostatin analogue labeled with [67/68Ga] and [111In] for SPECT, PET, and targeted therapeutic applications of somatostatin receptor (hsst2) expressing tumors.

A monoreactive NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) derived prochelator (1-(1-carboxy-3-carbo-tert-butoxypropyl)-4,7-(carbo-tert-butoxymethyl)-1,4,7-triazacyclononane (NODAGA(tBu)(3))) was synthesized in five steps with an overall yield of 21%. It is useful for the coupling to the N-terminus of peptides on solid phase and in solution; it was coupled to [Tyr3]-octreotide (TOC) on solid phase, and the resulting peptide, NODAGA-Tyr3-octreotide (NODAGATOC), was labeled with the radiometals 111In and 67Ga in high yields and good specific activities. [67Ga]- and [111In]-NODAGA-Tyr3-octreotide appear to be useful to visualize primary tumors and metastases which express somatostatin receptors subtype 2 (sstr2), such as neuroendocrine tumors, because of their high affinity to this receptor subtype with IC(50) = 3.5 +/- 1.6 nM and 1.7 +/- 0.2 nM, respectively. NODAGATOC could be used as a SPECT and PET tracer, when labeled with 111In, 67Ga, or 68Ga, and even for therapeutic applications. Surprisingly, [111In]-NODAGATOC shows 2 times higher binding affinity to sstr2, but also a factor of 4 higher affinity to sstr5 compared to [67Ga]-NODAGATOC. [67Ga]-NODAGATOC is very stable in serum and rat liver homogenate. There is no difference in the rate of internalization into AR4-2J rat pancreatic tumor cells; both radioligands are highly internalized, at 4 h a 3 times higher uptake compared to [111In]-DOTA-Tyr3-octreotide ([111In]-DOTATOC) was found. The biodistribution of [67Ga]-NODAGATOC in AR4-2J tumor bearing nude mice is very favorable at short times after injection; there is fast excretion from all nontarget organs except the kidneys and high uptake in sst receptor rich organs and in the AR4-2J tumor. Again it is superior to [111In]-DOTATOC in this respect. The results indicate an improved biological behavior which is likely due to the fact that an additional spacer group separates the chelate from the pharmacophoric part of the somatostatin analogue.

Sonoporation of cultured cells in the rotating tube exposure system.

Suspensions of Chinese hamster ovary cells were exposed to ultrasound in the presence of fluorescent dextran to determine the conditions needed for sonoporation with uptake of the large molecules. Albunex, a gas-body- based ultrasound contrast agent, was added to enhance cavitation. Ultrasound was continuous wave at frequencies of 1.0, 1.68, 2.25, 3.3, 5.3, and 7.15 MHz. Sterile 4.5-mL exposure chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. After exposure, cells were tested for sonoporation by counting fluorescent cells and for cell lysis by counting cells stained by trypan blue. Sonoporation was a sensitive bioeffects indicator that was detected at pressure amplitudes lower than were needed for transient cavitation or cavitation-induced cell lysis. For 10% Albunex, apparent thresholds for sonoporation, which were comparable to the levels required to perturb the gas bodies, were 0.084 MPa (spatial peak negative pressure amplitude) from 1.0-3.3 MHz and 0.27 MPa at 5.3 and 7.15 MHz. Sonoporation decreased slightly if the tube was not rotated. The effects increased for increasing Albunex concentration (with rotation). The plating efficiency of cells exposed to 0.2 MPa at 2.25 MHz and sorted by a flow cytometer was 19% (3.6% standard deviation [SD]) for fluorescent cells, compared to 67% (1% SD) for nonfluorescent exposed cells and 62% (6% SD) for sham-exposed cells. The reduced viability represents an important consideration for possible applications of sonoporation.

Transfection of a reporter plasmid into cultured cells by sonoporation in vitro.

Cultured Chinese hamster ovary cells were exposed to 2.25-MHz ultrasound in sterile 4.5-mL polyethylene chambers and tested for cell lysis, sonoporation and DNA transfection. Ten percent of Albunex, a gas-body-based ultrasound contrast agent, was added to ensure cavitation nucleation, and the chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. Uptake of large fluorescent dextran molecules by some cells was observed for spatial peak pressure amplitudes as low as 0.1 MPa, which indicates transient permeabilization and resealing, i.e., sonoporation, of these cells during exposure. Significant lysis occurred for 0.2 MPa, and increased rapidly for exposures above the apparent cavitation threshold (using the H2O2 production test) of about 0.4 MPa spatial peak pressure amplitude. In the DNA transfection tests, 20 micrograms/mL luciferase reporter plasmid was added to the suspension during exposure, and cells were assayed for proliferation ability and luciferase gene expression 2 days after exposure. Cell proliferation was greatly reduced above the cavitation threshold. Luciferase production was significant for 0.20-MPa exposure, and reached 0.33 ng per 10(6) cells at 0.8-MPa exposure. The luciferase production was great for cells exposed in medium supplemented with serum than for cells exposed in serum-free medium. Cells harvested for exposure either in the log phase or in the stationary phase of culture gave similar proliferation and transfection results. The effects essentially disappeared when the Albunex was omitted from the suspension and the tube was not rotated. Thus, sonoporation by ultrasonic cavitation in the rotating tube system yields plasmid transfection with subsequent transient gene expression.

Oxido-reductive state: the major determinant for cellular retention of technetium-99m-HMPAO.

UNLABELLED: Several clinical observations have suggested that HMPAO cerebral uptake might be related not only to regional cerebral perfusion but also to the nature of the lesion. Our aim was to investigate at the cellular level the nature of the process(es) involved in HMPAO accumulation in vitro. METHODS: Time-course incorporation of HM-PAO was studied in a fast-growing human premonocytic line, U937, in a human astrocytic-derived cell line, U373 and a human hybridized endothelial cell line, EaHy926. Minimal differences of HMPAO retention between these cell lines were observed and plateau of %U(HMPAO) (cpm cells/cpm standard of injected) were achieved within 2 hr. Because HMPAO cell retention was related to the intracellular content in glutathione, experiments studying effects of redox were conducted by preexposing U937 cells to D, L dithiothreitol or 2-Mercaptoethanol. RESULTS: Overnight incubation with NAC or BSO did not significantly modified the kinetic of 99mTc-HMPAO incorporation while overnight incubation with NAC resulted in a 2-fold increase in intracellular glutathione content and overnight incubation with BSO nearly abolished the intracellular glutathione content. At the opposite, presence of these reducing agents in the medium during the experiments completely abolished 99mTc-HMPAO retention. CONCLUSION: Our data thus provide in vitro evidence to support that overall intracellular retention of HMPAO is more dependent upon the redox activity of the tissue than the intracellular glutathione content. SPECT-HMPAO may accurately reflect regional cerebral blood flow in a normal state but possibly not in all pathological situations in which cell metabolism disturbances are characterized by alterations in the redox status.