Locoregional cancer treatment with magnetic drug targeting.

The specific delivery of chemotherapeutic agents to their desired targets with a minimum of systemic side effects is an important, ongoing challenge of chemotherapy. One approach, developed in the past to address this problem, is the i.v. injection of magnetic particles [ferrofluids (FFs)] bound to anticancer agents that are then concentrated in the desired area (e.g., the tumor) by an external magnetic field. In the present study, we treated squamous cell carcinoma in rabbits with FFs bound to mitoxantrone (FF-MTX) that was concentrated with a magnetic field. Experimental VX-2 squamous cell carcinoma was implanted in the median portion of the hind limb of New Zealand White rabbits (n = 26). When the tumor had reached a volume of approximately 3500 mm3, FF-MTX was injected intraarterially (i.a.; femoral artery) or i.v. (ear vein), whereas an external magnetic field was focused on the tumor. FF-MTX i.a. application with the external magnetic field resulted in a significant (P < 0.05), complete, and permanent remission of the squamous cell carcinoma compared with the control group (no treatment) and the i.v. FF-MTX group, with no signs of toxicity. The intratumoral accumulation of FFs was visualized both histologically and by magnetic resonance imaging. Thus, our data show that i.a. application of FF-MTX is successful in treating experimental squamous cell carcinoma. This "magnetic drug targeting" offers a unique opportunity to treat malignant tumors locoregionally without systemic toxicity. Furthermore, it may be possible to use these magnetic particles as a "carrier system" for a variety of anticancer agents, e.g., radionuclides, cancer-specific antibodies, and genes.

Coordination sphere and structure of the Mn cluster of the oxygen-evolving complex in photosynthetic organisms.

The great similarity between the binding of Fe(II) and the high-affinity Mn-binding site in the Mn-depleted PSII membranes (Semin et al. (1996) FEBS Lett. 375, 223-226) suggests that the coordination sphere of Mn in PSII is also suitable for iron. A comparison is performed between the primary amino acid sequences of D1 and D2 and diiron-oxo enzymes with the function of oxygen activation. All conservative motifs (EXXH) and residues binding and stabilizing the diiron cluster in diiron-oxo enzymes have been found in the C-terminal domains of D1 and D2 polypeptides. On the basis of these sequence similarities we suggest a structural model for the manganese cluster in the oxygen-evolving complex.

Highly purified D1/D2/cyt b559 preparations from spinach do not contain the non-heme iron center.

57Fe enriched D1/D2/cyt b559 preparations were isolated from spinach grown hydroponically on a 57Fe containing medium. In terms of polypeptide and pigment composition these samples are of high purity and functional integrity of P680. Mössbauer spectra measured in D1/D2/cyt b559 complexes revealed that these preparations are completely deprived of the non-heme iron center. Possible implications of this finding are discussed for the electron transfer from Pheo-. to exogenous electron acceptors.

Comparative stability studies on the iron and manganese forms of the cambialistic superoxide dismutase from Propionibacterium shermanii.

The superoxide dismutase of Propionibacterium shermanii shows similar activity with iron and manganese bound at the active site of the protein. On the other hand, the iron form, in comparison to the manganese form, exhibits higher stability towards thermal- and pH-dependent inactivation. Upon inactivation the metal ions are released from the active site. Thus, in comparison to the manganese form, a higher stability of the iron-protein complex might be the triggering reason for this behavior.

High-specific binding of Fe(II) at the Mn-binding site in Mn-depleted PSII membranes from spinach.

The interaction of Fe(II) and Fe(III) with the 'high-affinity Mn-binding site' in Mn-depleted photosystem II (PSII) was investigated using diphenilcarbazide (DPC)/2,6-dichlorophenol-indophenol (DCIP) inhibition assay. Fe(III) was ineffective in the inhibition of DPC-DCIP reaction while Fe(II) decreased the rate of DCIP photoreduction supported by DPC in the same concentration range as Mn(II). The effectivity of the interaction of Fe(II) with the high affinity Mn-binding site depends on different anions in the same manner as for Mn(II) and coincides with hierarchy observed for the stimulation of O2 evolution. The Fe(II) binding is accompanied by its oxidation. By using reductants it was shown that the high affinity site contains a redox-active component and the reduction of this component totally prevents the binding of Fe(II).

Isolation and properties of PS II membrane fragments depleted of the non heme iron center.

The functional properties and the content of non heme iron and cytochrome b559 were investigated by measuring flash induced transient changes of the relative fluorescence quantum yield and applying Mössbauer spectroscopy. It was found that untreated PS II membrane fragments contain a heterogeneous population of two types of non heme iron centers and about 2 cytochrome b559 per PS II. Twofold treatment of these samples with a recently described 'iron depletion' procedure (MacMillan, F., Lendzian, F., Renger, G. and Lubitz, W. (1995) Biochemistry 34, 3144-3156) leads to a complete loss (below the detection limit of Mössbauer spectroscopy) of the non heme iron center while more than 50% of the PS II complexes retain the functional integrity for light induced formation of the 'stable' radical pair Y(OX)(Z) P680Pheo Q(-.)(A). This sample type deprived of virtually all non heme iron in PS II provides a most suitable material for magnetic resonance studies that require an elimination of the interaction between Fe2+ and nearby radicals.

Vibrational dynamics of myoglobin determined by the phonon-assisted Mössbauer effect.

The phonon-assisted Mössbauer effect is used to determine the partial phonon density of states of the iron within the active center of deoxymyoglobin, carboxymyoglobin, and dry and wet metmyoglobin between 40 and 300 K. Between 0 and 1 meV the iron density of states increases quadratically with the energy, as in a Debye solid. Mean sound velocities are extracted from this slope. Between 1 and 3 meV a nearly quadratic "Debye-like" increase follows due to the similar strength of intermolecular and intramolecular forces. Above 3 meV, optical vibrations are characteristic for the iron-ligand conformation. The overall mean square displacements of the heme iron atom obtained from the density of states agree well with the values of Mössbauer absorption experiments below 180 K. In the physiological temperature regime the data confirm the existence of harmonic vibrations in addition to the protein specific dynamics measured by Mössbauer absorption. In the Debye energy regime the mean square displacement of the iron is in agreement with that of the hydrogens measured by incoherent neutron scattering demonstrating the global character of these modes. At higher energies the vibration of the heavy iron atom at 33 meV in metmyoglobin is as large as that of the lightweight hydrogens at that energy. A freeze dried, rehydrated (h=0.38 g H2O/g protein) metmyoglobin sample shows an excess of states above the Debye law between 1 and 3 meV, similar to neutron scattering experiments. The room temperature density of states below 3 meV exhibit an increase of the density compared to the low temperature data, which can be interpreted as mode softening.

[Polychromatic kinetics of conformational and spin relaxation of reduced intermediates of myoglobin]. / Polikhromaticheskaia kinetika konformastionno'i i spinovo'i relaksastiivosstanovlennykh intermediatov mioglobina.

Moessbauer spectroscopy was used to study the relaxation of a non-equilibrium myoglobin state produced at 77 K by reduction of metmyoglobin Fe(III) with thermalized electrons. The intermediate is characterized with the metMb (r) conformation of the protein globule and a low spin heme Fe(II) with a water molecule on the sixth coordination site. The intermediate is stable at 77 K but relaxes with increasing temperature into equilibrium deoxymyoglobin with dissociation of the bond Fe(II)-H2O and the transition of the Iron into the high spin state. In the temperature range 147-195 K the relaxation kinetics is nonexponential in time and can be described in terms of the polychromatic kinetics. A fitting of the kinetics data supposing a gamma-distribution of activation enthalpies of the relaxation shows a shift and a narrowing of the distribution at T > 180 K. The effect of structural rearrangements and equilibrium conformational fluctuations in the protein on the shape of the observed barrier distribution is discussed.

[Magnetic Drug Targeting--a new approach in locoregional tumor therapy with chemotherapeutic agents. Experimental animal studies]. / Magnetisches Drug Targeting--ein neuer Ansatz in der lokoregionären Tumortherapie mit Chemotherapeutika. Tierexperimentelle Untersuchungen.

BACKGROUND: Advanced squamous cell carcinomas of the head and neck region were often treated with combined radio-chemotherapy. Radiotherapy allows a focused treatment of the tumor, and healthy tissue can be protected from radiation. Chemotherapy, however, is mostly given systemically and the unwanted negative side effects also develop in many other organs. AIM OF THE STUDY: Locoregional application of chemotherapeutic agents with Magnetic Drug Targeting on an animal experimental study. METHODS AND RESULTS: Magnetic Drug Targeting is a new approach to the locoregional treatment of tumors. Ferrofluids (colloidal dispersion of magnetic nanoparticles) were reversibly bound to chemotherapeutic agents and injected intra-arterially, while focused with an external magnetic field to a certain body compartment (i.e. the tumor). With only 20% or 50% percent of the regular systemic chemotherapeutic dose, we achieved an up to 26 times higher concentration in the tumor region with this application compared to the usual systemic administration. CONCLUSION: Magnetic Drug Targeting offers an unique opportunity to treat tumors locoregionally with chemotherapeutic agents.

Mössbauer spectroscopy on oxygenated sperm whale myoglobin: evidence for an Fe3+-O2- coupling at the active center.

57Fe Mössbauer spectra of oxygenated sperm whale myoglobin (MbO2) show a well resolved quadrupole doublet with a temperature dependent splitting. The temperature dependence of the corresponding electric field gradient tensor (EFG) can be calculated from a Fe3+ term scheme for the iron at the active center. The Mössbauer spectra as well as the diamagnetc character of the MbO2-complex are then understood by an exchange coupling of the Fe3+-ion with O2- oxygen molecule ion. The resulting groundstate is a diamagnetic singlet. In order to keep the whole complex diamagnetic at room temperature, an exchange coupling with [J] greater than or equal to 300cm-1 is necessary. As the whole model is in fair agreement with many other spectroscopic data, it is believed to be a good starting point for further detailed calculations.

Investigation of the electronic term scheme of deoxygenated human haemoglobin by a least squares fit procedure using simultaneously magnetic susceptibility and Mössbauer data.

The calculation of the magnetic susceptibility from a published term scheme for the ferrous iron in deoxygenated human haemoglobin is discussed and a procedure for the simultaneous least squares fit of susceptibility and Mössbauer data is presented. The application of this procedure to the appropriate measurements on human haemoglobin leads to a rearrangement of the low lying electronic levels of the iron. The term schemes received as results of two different sets of susceptibility data used in combination with one set of Mössbauer data overlap with their error bars. The obtained level scheme of the Fe is correlated with the distance of the iron atom from the haem plane and the distance Fe-HIS F8, and some biological implications of these correlations are discussed.

A consistent picture of protein dynamics.

Information about the protein dynamics of myoglobin obtained by x-ray and Mössbauer investigations is analyzed and compared with computer simulations. Computer simulations give correct amplitudes of mean-square displacements but fail in the description of the time dependence of motions. Our model describes protein dynamics at physiological temperatures as an overdamped diffusion-like motion in a restricted space. The fluctuations occur around the average conformation determined by x-ray structure analysis. The gain in entropy drives the molecule into the transition state and, in this way, accounts for its flexibility.