| 1. |
- Bergström, Sara K., et al.
(författare)
-
Polyamine deactivation of integrated poly(dimethylsiloxane) structures investigated by radionuclide imaging and capillary electrophoresis experiments
- 2005
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 77:3, s. 938-942
-
Tidskriftsartikel (refereegranskat)abstract
- The poly(dimethylsiloxane) (PDMS) material provides a number of advantageous features, such as flexibility, elasticity, and transparency, making it useful in integrated analytical systems. Hard fused-silica capillary structures and soft PDMS channels can easily be combined by a tight fit, which offers many alternatives for structure combinations. PDMS and fused silica are in different ways prone to adsorption of low levels of organic compounds. The need for modification of the inner wall surface of PDMS channels may often be necessary, and in this paper, we describe an easy and effective method using the amine-containing polymer PolyE-323 to deactivate both fused-silica and PDMS surfaces. The adsorption of selected peptides to untreated surfaces was compared to PolyE-323-modified surfaces, using both radionuclide imaging and capillary electrophoresis experiments. The polyamine modification displayed a substantially reduced adsorption of three hydrophobic test peptides compared to the native PDMS surface. Filling and storage of aqueous solution were also possible in PolyE-323-modified PDMS channels. In addition, hybrid microstructures of fused silica and PDMS could simultaneously be deactivated in one simple coating procedure.
|
|
| 2. |
- Blom, Elisabeth, 1979-, et al.
(författare)
-
68Ga-Labeling of Biotin Analogues and their Characterization
- 2009
-
Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 20:6, s. 1146-1151
-
Tidskriftsartikel (refereegranskat)abstract
- Biotin- and Ga-68-based tracers have been suggested as tools that could be used to monitor the survival of avidin-coated islets of Langerhans isolated from pancreas and used in transplantation, i.e., to liver. Three biotin analogues with various alkyl and poly(ethylene glycol) (PEG) chains coupled to DOTA were synthesized and labeled with Ga-68. The Ga-68 labeling was studied at room temperature as well as elevated temperature using either conventional or microwave heating. Radioactivity incorporation reached 95% within 5 and 2 min using the, respectively, conventional and microwave heating modes. The specific activity of the tracers was improved by preconcentration and purification of the generator eluate. The binding of the labeled and nonlabeled conjugates to avidin in solution was compared to the binding of native biotim. All compounds maintained good affinity for avidin, though introducing the linkers and chelator, especially the PEG-groups, somewhat decreased the binding affinity. The extent of binding of the labeled compounds to avidin was 54-91% after 5 min. Blocking experiments were performed confirming the specificity of the binding of biotin analogues to avidin. The stability of the three labeled compounds in human serum was studied. The stability of the biotin analogue 8 (65% within 30 min) and avidin-biotin complex (80% within 120 min) might be sufficient for the monitoring of the islets of Langerhans. The tracers will be evaluated in in vitro experiments of avidin-coated islets of Langerhans and in transplantation models in vivo.
|
|
| 3. |
- Blom, Elisabeth, 1979-
(författare)
-
Development of 18F- and 68Ga-Labelled Tracers : Design Perspectives and the Search for Faster Synthesis
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis deals with the design of 18F- and 68Ga-labelled positron emission tomography (PET) tracers and the development of technologies that enable faster and simpler preparation with high specific radioactivity. Techniques like microwave heating and reducing the concentrations of the precursor were investigated with this perspective. A few applications were explored using molecular design perspectives. A nucleophilic 18F-labelling strategy using perfluoro-containing leaving groups was explored. We observed that [18F]fluoride was interacting with the perfluoro alkyl chains of the substrate, preventing the nucleophilic substitution from taking place. When a perfluoroaryl group was instead used in the leaving group, the substitution took place and purification by fluorous solid-phase extraction was possible. 18F-Labelled analogues of the monoamine oxidase-A inhibitor harmine were prepared by one-step nucleophilic fluorinations and evaluated by in vitro autoradiography, showing high specific binding. Biotin analogues labelled with 18F and 68Ga were prepared and their binding to avidin evaluated. All analogues retained their binding ability and will be further evaluated in transplantation models with avidin-coated islets of Langerhans. Peptide design perspectives were used in some examples where the Arg-Gly-Asp (RGD) sequence and a single-chain version of vascular endothelial growth factor (VEGF) protein functionalized with 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) or 2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (NOTA) as chelators were labelled with 68Ga. The RGD motif and VEGF have high affinity for, respectively, αvβ3 integrin and VEGFR-2 receptor that are overexpressed in angiogenesis process. The 68Ga-labelled scVEGF maintained its functional activity in vitro. A polypeptide conjugate containing phosphocholine, which has affinity for the C-reactive protein released during the inflammatory process, was labelled with 68Ga for the development of an imaging agent for inflammation in vivo. Finally [18F]/19F exchange in fluorine-containing compounds was studied in order to investigate whether the exchange reaction can be of practical use for labelling.
|
|
| 4. |
- Lavén, Martin, et al.
(författare)
-
Imaging of peptide adsorption to microfluidic channels in a plastic compact disc using a positron emitting radionuclide
- 2005
-
Ingår i: Lab Chip. ; :5, s. 756-763
-
Tidskriftsartikel (refereegranskat)abstract
- A method for studying peptide-surface interactions within microfluidic channels by radionuclide imaging is described. With the high surface area-to-volume ratio of channels in miniaturised devices, combined with low amunts of analyte, non-specific peptide adsorption is a critical issue. The objective of the study was therefore to develop a method capable of direct detection of adsorbed peptide within microfluidic channels. A micro-device consisting of channels moulded in a plastic compact disc was chosen for the study, together with two selected peptides of different lengths and isolelectric point (pI) values. A bifunctional chelator, DOTA, was attached to the peptide by conjugation and labelled with the short-lived positron emitting radionuclide 68Ga. Quantitative images of radiotracer distribution within the microfluidic channels were obtained using a PhosphorImager system. The power of the method was demonstrated by the ability to clearly measure changes in adsorption when varying a number of parameters that typically affect peptide adsorption. These included surface modifications, analyte concentration, pH, and ionic strength. Additionally, two quantification methods were developed and compared. Radionuclide imaging also permitted visualisation of adsorption and release processes in microchannel chromatographic columns. The results suggest that radionuclide imaging is a suitable tool not only for the study of peptide adsorption to the microchannels presented in this study but also as a versatile tool to measure peptide-surface interactions in a wide variety of miniaturised structures and devices.
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
- Lendvai, Gabor, et al.
(författare)
-
Biodistribution of Ga-68-labeled LNA-DNA mixmer antisense oligonucleotides for rat Chromogranin-A
- 2008
-
Ingår i: Oligonucleotides. - : Mary Ann Liebert Inc. - 1545-4576 .- 1557-8526. ; 18:1, s. 33-49
-
Tidskriftsartikel (refereegranskat)abstract
- In vivo monitoring of gene expression may be accomplished using a most advanced imaging technology such as positron emission tomography (PET). However, a range of methodological and biological hurdles needs exploration. In the present study, 20-mer DNA-LNA (locked nucleic acid) mixmer oligonucleotides specific for rat Chromogranin-A (Chg-A) mRNA were labeled with Ga-68 and their biodistribution were investigated in rats; namely, two Antisense (LNA1, LNA2-differing only in the positioning of LNA modification), Mismatched, and Sense sequences. In addition, in vivo and in vitro metabolite analysis of LNA1 and LNA2 was compared, and hybridization in solution was performed to verify the hybridization ability after labeling. Furthermore, semiquantitative polymerase chain reaction was carried out to find organs expressing Chg-A mRNA in the rat. The biodistribution patterns altered according to the sequence and the positioning of LNA modification. The pattern of Mismatched-differing only in two nucleotides from the two Antisenses-was similar to that of Sense, whereas the pattern of LNA1 and LNA2 showed differences. Uptake in the adrenal gland was twofold higher with LNA2 compared to the other three oligonucleotides. Intact LNA2 could be observed in the 60-minute sample in vivo, whereas in vitro, the intact compound of both Antisenses could also be detected after 2 hours. Hybridization in solution revealed that the two Antisenses retained their hybridization abilities after Ga-68-labeling. With decreasing magnitude, Chg-A mRNA was expressed in the adrenal gland, intestine, testis, and pancreas. This study further supported LNA-DNA mixmer to be a favorable modification for antisense targeting approach with respect to hybridization and longer plasma residence; however, the organ uptake was dominated by processes irrelevant to specific hybridization.
|
|
| 8. |
- Lendvai, Gabor, et al.
(författare)
-
Non-hybridisation saturable mechanisms play a role in the uptake of 68Ga-labelled LNA-DNA mixmer antisense oligonucleotides in rats
- 2009
-
Ingår i: Oligonucleotides. - : Mary Ann Liebert Inc. - 1545-4576 .- 1557-8526. ; 19:3, s. 223-231
-
Tidskriftsartikel (refereegranskat)abstract
- Oligonucleotides (ODN) are key molecules for the aim of preventing translation of a gene product or monitoring gene expression in tissues. However, multiple methodological and biological hurdles need to be solved before in vivo application in humans will be possible. For positron emission tomography (PET) investigations, a 20-mer DNA-locked nucleic acid (LNA) mixmer ODN specifi c for rat chromogranin-A mRNA was labeled with Ga-68 and its uptake was examined in vivo in rats with and without blocking of scavenger receptors by polyribo-nucleotides. In addition, uptake studies of Ga-68-LNA were performed with respect to time and concentration in human and rat cell lines. The human cell lines did not express the target mRNA. Both polyinosinic acid (poly-I) and polyadenylic acid (poly-A) reduced the uptake in rat tissues and in human cell lines. Poly-I was found to be more effective in the liver whereas poly-A was more effective in the kidney. In addition, the blockade by poly-I was statistically significant in the pancreas, adrenal gland, bone marrow, intestine, testis, urinary bladder, muscle, parotid gland, and heart, whereas poly-A also caused significant reduction in pancreas, adrenal gland, and bone marrow but not as much as in kidney. Cell culture study showed a 2-phase dose-dependent uptake characteristic with a saturable and a passive diffusion-like phase; however, these 2 phases were not so well expressed in the rat cell line. The results suggest that scavenger receptors or other saturable processes unrelated to hybridization may be involved in the tissue uptake of Ga-68-LNA and in the clearance of antisense ODN through the liver, kidney, spleen, and bone marrow. The fact that these processes may be sequence-dependent suggests that proof of in vivo hybridization through imaging may not be obtained by only comparing sense and antisense sequences and proving dose-dependency.
|
|
| 9. |
|
|
| 10. |
- Velikyan, Irina, et al.
(författare)
-
Preparation and evaluation of (68)Ga-DOTA-hEGF for visualisation of EGFR expression in malignant tumours
- 2005
-
Ingår i: Journal of Nuclear Medicine. - 0161-5505 .- 1535-5667. ; 46:11, s. 1881-1888
-
Tidskriftsartikel (refereegranskat)abstract
- Detection of epidermal growth factor receptor (EGFR) overexpression in many carcinomas provides important diagnostic information, which can influence patient management. The use of PET may enable such detection in vivo by a noninvasive procedure with high sensitivity. The aim of this study was to develop a method for preparation of a positron-emitting tracer based on a natural ligand to EGFR, the recombinant human epidermal growth factor (hEGF), and to perform a preclinical evaluation of the tracer.METHODS: DOTA-hEGF (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was prepared by coupling of a N-sulfosuccinimide ester of DOTA to hEGF. The conjugate was labeled with a generator-produced positron-emitting nuclide, (68)Ga (half-life = 68 min), using microwave heating. Binding specificity, affinity, internalization, and retention of (68)Ga-DOTA-hEGF was studied in 2 EGFR-expressing cell lines, U343 glioma cells and A431 cervical carcinoma cells. Biodistribution and microPET visualization studies were performed in BALB/c nu/nu mice bearing A431 carcinoma xenografts.RESULTS: A 1-min-long microwave-assisted labeling provided radioactivity incorporation of 77% +/- 4%. Both cell lines demonstrated receptor-specific uptake of the conjugate, rapid internalization of the tracer, and good retention of radioactivity. Binding to both cell lines occurred with high affinity, approximately 2 nmol/L. The biodistribution study demonstrated accumulation of radioactivity in xenografts and in EGFR-expressing organs. The microPET imaging study enabled visualization of tumors and demonstrated quick--within 5 min--localization of radioactivity in tumors.CONCLUSION: (68)Ga-DOTA-hEGF has potential for imaging EGFR overexpression in tumors.
|
|
| 11. |
- Velikyan, Irina, 1966-
(författare)
-
Synthesis, Characterisation and Application of 68Ga-labelled Macromolecules
- 2005
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The positron emitting radionuclide 68Ga (T1/2 = 68 min) might become of practical interest for clinical positron emission tomography (PET). The metallic cation, 68Ga(III), is suitable for complexation with chelators, either naked or conjugated with biological macromolecules. Such labelling procedures require pure and concentrated preparations of 68Ga(III), which cannot be sufficiently fulfilled by the presently available 68Ge/68Ga generator eluate. This thesis presents methods to increase the concentration and purity of 68Ga obtained from a commercial 68Ge/68Ga generator. The use of the preconcentrated and purified 68Ga eluate along with microwave heating allowed quantitative 68Ga-labelling of peptide conjugates within 15 min. The specific radioactivity of the radiolabelled peptides was improved considerably compared to previously applied techniques using non-treated generator eluate and conventional heating. A commercial 68Ge/68Ga generator in combination with the method for preconcentration/purification and microwave heated labelling might result in an automated device for 68Ga-based radiopharmaceutical kit production with quantitative incorporation of 68Ga(III).Macromolecules were labelled with 68Ga(III) either directly or via a chelator. The bifunctional chelator, DOTA, was conjugated in solution to peptides, an antibody and oligonucleotides. The peptides had varied pI values, constitution, and length ranging from 8 to 53 amino acid residues. The oligonucleotides were of various sequences and length with modifications in backbone, sugar moiety and both 3' and 5' ends with a molecular weight up to 9.8 kDa. The bioconjugates were labeled with 68Ga(III), and the resulting tracers were characterised chemically and biologically. The identity of the 68Ga-labelled bioconjugates was verified. The tracers were found to be stable and their biological activity maintained. Specific radioactivity was shown to be an important parameter influencing the feasibility of accurate imaging data quantification.Furthermore, 68Ga-labelled peptide imaging was shown to be a useful tool to study peptide adsorption to microstructures in a chemical analysis device.
|
|
| 12. |
- Velikyan, Irina, et al.
(författare)
-
The importance of high specific radioactivity in the performance of Ga-68-labeled peptide.
- 2008
-
Ingår i: Nuclear Medicine and Biology. - : Elsevier BV. - 0969-8051 .- 1872-9614. ; 35:5, s. 529-536
-
Tidskriftsartikel (refereegranskat)abstract
- The Use of Ga-68-labeled peptides in diagnosis, dosimetry, therapy planning and follow-up of response to chemo- and radiotherapy requires accurate quantification of tracer binding characteristics in vivo, which may be influenced by the specific radioactivity (SRA) of the tracer. Systematic study of the complexation reaction of DOTA-D-Phe(1)-Tyr(3)-Octreotide (DOTATOC, where DOTA is the chelator 1,4,7,10-tetrauzacyclododecane-1,4,7,10-tetraacetic acid) with Ga-67, Ga-68, Ga-69,Ga-71 and in the presence of competing metal cations [Al(III), Fe(III), In (III)] was performed using conventional and microwave heating techniques and assessed by mass spectrometry. Saturation binding of Ga-68-DOTATOC to Rhesus monkey brain slices was performed using frozen section autoradiography. High SRA was necessary in order to characterize the saturation binding of Ga-68-DOTATOC to somatostatin receptors in Rhesus monkey brain sections. The complexation of Ga(III) with DOTATOC suggested more favorable formation compared to Fe(III) and In(III). The microwave heating mode might influence the selectivity of the complexation reaction, especially when comparing the behavior of Ga(III) and In(III). Al(III)was less critical with contamination and could be tolerated up to a concentration equal to that of the peptide bioconjugate. The SRA of Ga-67-DOTATOC and Ga-67-NODAGA-TATE (NODAGA-Tyr(3)-Octreotate, where NODAGA is 1,4,7-triazacyclononaiie-1-glutaric acid-4,7-diacetic acid) exceeded literature data by a factor of 7 and 5-15, respectively. High SRA was critical for providing sufficient contrast and accurate quantification of PET images. Microwave heating mode apart from the acceleration of the labeling reaction also improved the selectivity of the complexation reaction towards gallium. Fe(III) was shown to be the most critical competitor deteriorating the Ga-68-labeling efficiency.
|
|
