Gerard C. Krijger

Targeted Multifunctional Lipid-Based Nanocarriers for Image-Guided Drug Delivery

Lipid-based nanocarriers have proven successful in the delivery of mainly chemotherapeutic agents, and currently they are being applied clinically in the treatment of various types of cancer. These drug delivery systems achieve increased therapeutic efficacy by altering the pharmacokinetics and biodistribution of encapsulated drugs, resulting in decreased drug toxicity and enhanced accumulation in tumor tissue. This increased accumulation is due to the relatively leaky immature vasculature of a tumor. After the clinical relevance of such drug delivery systems was demonstrated, research in this area focused on optimization, both by cell specific targeting and including controlled and triggered release concepts within the carrier. These more advanced targeted nanocarriers in general have clearly shown their potential in various animal tumor models and await clinical application. The development of targeted nanocarriers in which therapeutic and imaging agents are merged into a single carrier will certainly be of importance in the near future. Indeed, scientists active in the field of imaging (e.g. nuclear and magnetic resonance imaging) have already started to exploit nanocarriers for molecular imaging. Image-guided drug delivery using these multifunctional nanocarriers, containing therapeutic and imaging agents, will ultimately allow for online monitoring of tumor location, tumor targeting levels, intratumoral localization and drug release kinetics prior and during radio- and/or chemotherapeutic treatment. This review describes the current status and challenges in the field of nanocarrier-aided drug delivery and drug targeting and discusses the opportunities of combining imaging probes with these drug carriers and the potential of these multifunctional lipid-based nanocarriers within image-guided drug delivery.

Tungsten Transport Protein A (WtpA) in Pyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.

Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope (99)Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten- and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs.

Development of a generic approach to native metalloproteomics: application to the quantitative identification of soluble copper proteins in Escherichia coli

A combination of techniques to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system has been developed. The procedure involves four steps: (1) labeling of the target proteins with a suitable short-lived radioisotope (suitable isotopes are 64Cu, 67Cu, 187W, 99Mo, 69Zn, 56Mn, 65Ni); (2) separation of intact soluble holoproteins using native isoelectric focusing combined with blue native polyacrylamide gel electrophoresis into native–native 2D gel electrophoresis; (3) spot visualization and quantification using autoradiography; and (4) protein identification with tandem mass spectrometry. The method was applied to the identification of copper proteins from a soluble protein extract of wild-type Escherichia coli K12 using the radioisotope 64Cu. The E. coli protein CueO, which has previously been only identified as a multicopper oxidase following homologous overexpression, was now directly detected as a copper protein against a wild-type background at an expression level of 0.007% of total soluble protein. The retention of the radioisotope by the copper proteins throughout the separation process corroborates the method to be genuinely native. The procedure developed here can be applied to cells of any origin, and to any metal having suitable radioisotopes. The finding that the periplasmic protein CueO is the only major form of soluble protein bound copper in E. coli strengthens the view that the bacterial periplasm contains only a few periplasmic copper proteins, and that the cytosol is devoid of copper proteins.

Neutron activation of holmium poly(L-lactic acid) microspheres for hepatic arterial radioembolization: a validation study

Poly(L-lactic acid) microspheres loaded with holmium-166 acetylacetonate (166Ho-PLLA-MS) are a novel microdevice for intra-arterial radioembolization in patients with unresectable liver malignancies. The neutron activation in a nuclear reactor, in particular the gamma heating, damages the 166Ho-PLLA-MS. The degree of damage is dependent on the irradiation characteristics and irradiation time in a particular reactor facility. The aim of this study was to standardize and objectively validate the activation procedure in a particular reactor. The methods included light- and scanning electron microscopy (SEM), particle size analysis, differential scanning calorimetry, viscometry, thermal neutron flux measurements and energy deposition calculations. Seven hours-neutron irradiation results in sufficient specific activity of the 166Ho-PLLA-MS while structural integrity is preserved. Neutron flux measurements and energy deposition calculations are required in the screening of other nuclear reactors. For the evaluation of microsphere quality, light microscopy, SEM and particle size analysis are appropriate techniques.

Microspheres with ultrahigh holmium content for radioablation of malignancies

PurposeThe aim of this study was to develop microspheres with an ultra high holmium content which can be neutron activated for radioablation of malignancies. These microspheres are proposed to be delivered selectively through either intratumoral injections into solid tumors or administered via an intravascularly placed catheter.MethodsMicrospheres were prepared by solvent evaporation, using holmium acetylacetonate (HoAcAc) crystals as the sole ingredient. Microspheres were characterized using light and scanning electron microscopy, coulter counter, titrimetry, infrared and Raman spectroscopy, differential scanning calorimetry, X-ray powder diffraction, magnetic resonance imaging (MRI), and X-ray computed tomography (CT).ResultsMicrospheres, thus prepared displayed a smooth surface. The holmium content of the HoAcAc microspheres (44% (w/w)) was higher than the holmium content of the starting material, HoAcAc crystals (33% (w/w)). This was attributed to the loss of acetylacetonate from the HoAcAc complex, during rearrangement of acetylacetonate around the holmium ion. The increase of the holmium content allows for the detection of (sub)microgram amounts of microspheres using MRI and CT.ConclusionsHoAcAc microspheres with an ultra-high holmium content were prepared. These microspheres are suitable for radioablation of tumors by intratumoral injections or treatment of liver tumors through transcatheter administration.

Targeted radiotherapy of bone malignancies.

The severe pain associated with many disorders affecting bone account for a large proportion of cases of patient morbidity, due to the encumbrance of mobility and therefore, compromised quality of life. Skeletal metastasis is one such condition, which generally complicates the treatment of the primary cancers such as that of the breast, prostate and lung - causing intense pain and eventually even mortality. This paper presents examples of various approaches explored and proposed in the ongoing search to identify better radiopharmaceuticals for the treatment of bone disorders such as metastases. The primary objective of these developments is to alleviate the debilitating pain commonly associated with bone lesions. The efficacy of a radiotherapeutic agent intended for the treatment of diseased bone is particularly dependent on the radiation dose to the tumor cells and on the extent to which suppression of bone marrow or other critical organs can be avoided. Therefore, the design rationale requires careful consideration of the choice radionuclide and especially ensuring that the drug selectively targets the lesion or tumor site. The options pursued include the use of radioisotopes with an intrinsic affinity for bone, such as (89)Sr or (223)Ra, or the design of bone-seeking ligands, such as phosphonates, to selectively deliver the radionuclide to the target, e.g. [(153)Sm]Sm-EDTMP. A combination of the above may too be possible, where the bone seeking ligand facilitates the selective accumulation of a radionuclide, which by itself is also bone homing. In terms of therapeutic application radionuclides with various decay modes are proposed, including beta (-) emitters: (153)Sm, (89)Sr, (186)Re, (188)Re, (32)P, (177)Lu and (170)Tm; alpha (α) emitters: (223)Ra and (225)Ra; and Auger or conversion electron emitter: (117)mSn. From a purely diagnostic perspective, the radioisotopes used for imaging include the well known photon emitting (99)mTc, and positron emitters (18)F and (68)Ga. The current status in the development and application of internal radiotherapy for the palliative treatment of bone pain will be discussed, summarizing the progress made and challenges encountered in the process to realizing an effective drug candidate.

The tungsten metallome of Pyrococcus furiosus.

The tungsten metallome of the hyperthermophilic archaeon Pyrococcus furiosus has been investigated using electroanalytical metal analysis and native-native 2D-PAGE with the radioactive tungsten isotope (187)W (t(1/2) = 23.9 h). P. furiosus cells have an intracellular tungsten concentration of 29 μM, of which ca. 30% appears to be free tungsten, probably in the form of tungstate or polytungstates. The remaining 70% is bound by five different tungsten enzymes: formaldehyde ferredoxin oxidoreductase, aldehyde ferredoxin oxidoreductase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase and the tungsten-containing oxidoreductases WOR4 and WOR5. The membrane proteome of P. furiosus is devoid of tungsten. The differential expression, as measured by the tungsten level, of the five soluble tungsten enzymes when the cells are subjected to a cold-shock shows a strong correlation with previously published DNA microarray analyses.

Selectively Induced Death of Macrophages in the Synovial Lining of Murine Knee Joints Using 10B-liposomes and Boron Neutron Capture Synovectomy

Objective: To investigate whether macrophages in the synovial lining can be selectively eliminated by local administration of an improved boron-10 (10B) containing liposome formulation combined with neutron irradiation (boron neutron capture synovectomy [BNCS]). Methods: Disodium dodecahydrododecaborate (Na210B12H12) was encapsulated into unilamellar liposomes (10B-liposomes). 10B-liposomes were injected into the mouse knee joint. Amounts of 10B in synovial tissue were measured over time using inductively coupled plasma-optical emission spectrometry (ICP-OES). Arthritis was induced in knee joints of mice. Joint inflammation and cartilage destruction was measured using histology. Results: When a 10 μl 10B-liposome solution (containing 40 μg 10B) was injected into the murine knee joint, high concentrations of 10B were measured in macrophages in the synovial lining (At 24 h 306±226 μg. g−1 macrophages). Completing the BNCS by neutron irradiation of the legs 24 h after 10B-liposome injection showed a clear selective depletion of macrophages in synovial lining of the knee joints. An estimated total physical dose of 13±9 Gy was given to the macrophages. When arthritis was induced in the macrophage-depleted joints, swelling of the knee was significantly lower as compared to the controls (53% and 79% lower at days 1 and 3, respectively). Histology confirmed the influx of inflammatory cells was strongly decreased and severe cartilage destruction was almost completely prevented. Conclusion: BNCS using an improved 10B-containing liposome formulation can cause selective depletion of macrophages in the synovial lining of murine knee joints. As a result of this proof-of principle, future applications are recommended.

Microbrachytherapy using holmium-166 acetylacetonate microspheres: a pilot study in a spontaneous cancer animal model

PURPOSE Holmium-166 acetylacetonate microspheres ((166)Ho-AcAc-MS) are proposed as an intratumoral radioablation device. This article presents a pilot study in housecats with unresectable liver cancer. Feasibility and tolerability of intratumoral administrations of (166)Ho-AcAc-MS was investigated. METHODS AND MATERIALS Three cats with unresectable liver tumors of different histotype were included. One cat had hepatocellular carcinoma (HCC), one had cholangiocarcinoma (CC), and one had a malignant epithelial liver tumor (MELT) of unspecified histotype. (166)Ho-AcAc-MS were injected percutaneously under ultrasound guidance into the tumors. Followup consisted of physical examinations and hematologic and biochemical analyses. RESULTS (166)Ho-AcAc-MS were administered to three liver tumor-bearing cats. The treatment was well tolerated and the clinical condition, that is body weight, alertness, mobility, and coat condition of the animals improved markedly. Most biochemical and hematologic parameters normalized shortly after treatment. Life of all cats was extended and associated with a good quality of life. The HCC cat that received 33-Gy tumor-absorbed dose was euthanized 6 months after the first administration owing to disease progression. The MELT cat received 99-Gy tumor dose and was euthanized 3 months posttreatment owing to bacterial meningitis. The CC cat received 333Gy and succumbed 4 months after the first treatment owing to the formation of a pulmonary embolism. CONCLUSIONS Percutaneous intratumoral injection of radioactive (166)Ho-AcAc-MS is feasible in liver tumor-bearing cats. The findings of this pilot study indicate that (166)Ho-AcAc-MS may constitute safe brachytherapeutic microspheres and warrant studies to confirm the clinical utility of this novel brachytherapy device.