Advanced Colloids Experiment (ACE) Science Overview

The Advanced Colloids Experiment is being conducted on the International Space Station (ISS) using the Light Microscopy Module (LMM) in the Fluids Integrated Rack (FIR). Work to date will be discussed and future plans and opportunities will be highlighted. The LMM is a microscope facility designed to allow scientists to process, manipulate, and characterize colloidal samples in micro-gravity where the absence of gravitational settling and particle jamming enables scientists to study such things as:a.The role that disordered and ordered-packing of spheres play in the phase diagram and equation of state of hard sphere systems,b.crystal nucleation and growth, growth instabilities, and the glass transition, c.gelation and phase separation of colloid polymer mixtures,d.crystallization of colloidal binary alloys,e.competition between crystallization and phase separation,f.effects of anisotropy and specific interactions on packing, aggregation, frustration and crystallization,g.effects of specific reversible and irreversible interactions mediated in the first case by hybridization of complementary DNA strands attached to separate colloidal particles,h.Lock and key interactions between colloids with dimples and spheres which match the size and shape of the dimples,i.finding the phase diagrams of isotropic and interacting particles,j.new techniques for complex self-assembly including scenarios for self-replication, k.critical Casimir forces,l.biology (real and model systems) in microgravity,m.etc. By adding additional microscopy capabilities to the existing LMM, NASA will increase the tools available for scientists that fly experiments on the ISS enabling scientists to observe directly what is happening at the particle level. Presently, theories are needed to bridge the gap between what is being observed (at a macroscopic level when photographing samples) with what is happening at a particle (or microscopic) level. What is happening at a microscopic level will be directly accessible with the availability of the Light Microscopy Module (LMM) on ISS. To meet these goals, the ACE experiment is being built-up in stages, with the availability of confocal microscopy being the ultimate objective. Supported by NASAs Physical Sciences Research Program, ESAESTEC, and the authors respective governments

Synthesis of a new HYNIC-DAPI derivative for labelling with ⁹⁹ᵐTechnetium and its in vitro evaluation in an FRTL5 cell line

4′,6-Diamidine-2-phenylindole (DAPI) is a common fluorochrome that is able to bind to deoxyribonucleic acid (DNA) with distinct, sequence-dependent enhancement of fluorescence. This work presents the synthesis of a new multifunctional compound that includes the fluorescent dye as a ⁹⁹ᵐTechnetium (⁹⁹ᵐTc) carrier. A new technique for the bioconjugation of DAPI with 6-hydrazinonicotinic acid (HYNIC) through an amide linkage was developed. The radiolabelling was performed with HYNIC as a chelator and N-IJ2-hydroxy-1,1-bisIJhydroxymethyl)ethyl)glycine (tricine) as a coligand. Furthermore, experimental evidence showed that ⁹⁹ᵐTc complexes with DAPI as DNA-binding moieties are detectable in living Fischer rat thyroid follicular cell line 5 (FRTL5) and their nuclei. The investigations indicated further that the new HYNIC-DAPI derivative is able to interact with double-stranded DNA. This establishes the possibility of locating ⁹⁹ᵐTc in close proximity to biological structures of living cells, of which especially the genetic information-carrying cell compartments are at the centre of interest. In this context, further investigations are related to the radiotoxic effects of DNA-bound ⁹⁹ᵐTc-HYNIC-DAPI derivatives and dosimetric calculations

99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

(99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99m)Tc-labeled HYNIC-DAPI compound with that of (99m)Tc pertechnetate ((99m)TcO4(-)). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by (99m)TcO4(-) (0.51), and the number of DSBs increased fivefold in the (99m)Tc-HYNIC-DAPI-treated sample compared with the (99m)TcO4(-) treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99m)TcO4(-) treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99m)Tc-HYNIC-DAPI-treated samples. These results indicated that (99m)Tc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99m)Tc-labeled compound with DNA. In contrast to these results, (99m)TcO4(-) induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99m)Tc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs

Fluorescence intensity with respect to DNA-bound activity.

<p>Dependence of the relative fluorescent intensity of open circle and linear plasmid DNA on the DNA-bound activity. A good correlation was observed between the activity and the radiation damage. The higher fluorescent intensity after the 24 h incubation time was due to the increased number of radioactive decays.</p

Dose-Response of ^99mTc-pertechnetate and ^99mTc-HYNIC-DAPI.

<p>Dose-dependent decrease in supercoiled DNA exposed to ^99mTc-pertechnetate or ^99mTc-HYNIC-DAPI for 2 (A) or 24 hours (B). The dose-dependent increase in open circular DNA was similar for both radiotracers, but the fraction of linear DNA was greater in the presence of ^99mTc-HYNIC-DAPI compared with ^99mTc-pertechnetate.</p

Number of DSBs and SSBs per DNA molecule.

<p>Analysis of the mean number of DSBs (A) and SSBs (B) per plasmid molecule as a function of ^99mTc-HYNIC-DAPI decays during a 2 h irradiation. The numbers of DSBs and SSBs were calculated based on the fluorescence intensity of the linear and supercoiled DNA fractions in the presence (□) or absence (▪) of DMSO.</p

Example images of agarose gel indicating the effect of DMSO.

<p>Representative images of agarose gels in the absence (A) or presence (B) of DMSO. The plasmid DNA samples were irradiated for 24 h with 50–1250 MBq/mL ^99mTc-HYNIC-DAPI. The influence of DMSO on DNA damage (OC: open circular, L: linear, SC: super coiled plasmid DNA fraction) caused by ^99mTc-HYNIC-DAPI was compared with that elicited by ^99mTc-pertechnetate (^99mTcO₄⁻). Notably, the formation of open circular and linear DNA in response to ^99mTc-pertechnetate could be prevented by DMSO. The first lane is a non-irradiated plasmid sample, L is a non-irradiated linear plasmid, and M is the marker. The DNA bands at the bottom represent supercoiled DNA.</p

Measurement of the Plasmid DNA-bound radioactivity.

<p>The measurements were performed using a gamma counter, and the activity was decay-corrected based on the application time. The data were obtained from a single experiment. A good linear correlation was observed between the applied activity and the bound activity.</p

RP-HPLC Chromatogram of ^99mTc-HYNIC-DAPI.

<p>HPLC trace of the radioactivity 60(3.6%). The signals from 8.49 to 12.88 min represent different ^99mTc-HYNIC-DAPI derivatives (96.4%). After 12 h, the peak at 1.33 min increased to 100%.</p

Emission Spectrum of ^99mTc (Data from Howell [17]).

<p>Emission Spectrum of ^99mTc (Data from Howell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104653#pone.0104653-Howell1" target="_blank">[17]</a>).</p