Mangiferin functionalized radioactive gold nanoparticles (MGF-198AuNPs) in prostate tumor therapy: green nanotechnology for production, in vivo tumor retention and evaluation of therapeutic efficacy.

We report here an innovative feature of green nanotechnology-focused work showing that mangiferin-a glucose functionalized xanthonoid, found in abundance in mango peels-serves dual roles of chemical reduction and in situ encapsulation, to produce gold nanoparticles with optimum in vivo stability and tumor specific characteristics. The interaction of mangiferin with a Au-198 gold precursor affords MGF-198AuNPs as the beta emissions of Au-198 provide unique advantages for tumor therapy while gamma rays are used for the quantitative estimation of gold within the tumors and various organs. The laminin receptor specificity of mangiferin affords specific accumulation of therapeutic payloads of this new therapeutic agent within prostate tumors (PC-3) of human prostate tumor origin induced in mice which overexpress this receptor subtype. Detailed in vivo therapeutic efficacy studies, through the intratumoral delivery of MGF-198AuNPs, show the retention of over 80% of the injected dose (ID) in prostate tumors up to 24 h. By three weeks post treatment, tumor volumes of the treated group of animals showed an over 5 fold reduction as compared to the control saline group. New opportunities for green nanotechnology and a new paradigm of using mangiferin as a tumor targeting agent in oncology for the application of MGF-198AuNPs in the treatment of cancer are discussed.

Long-term follow-up after non-myeloablative transplant of bone and marrow in BXSB mice.

We present long-term outcomes of BXSB mice after non-myeloablative bone marrow transplants using major histocompatability complex (MHC)-matched cells. Groups differed in sources of donor lymphocytes or mesenchymal stromal cells (MSC). Unfractionated marrow cells from green fluorescent protein (GFP) transgenic (Tg) mice (BMT group) or from RAG1-/- B6 mice (RAG group) were injected intravenously (i.v.) into irradiated (550 cGy) hosts. As a source of mesenchymal cells, bone chips from GFP-Tg were injected intraperitoneally alone (MSC group) or along with i.v. bone marrow cells (BMT + MSC group). Controls were untreated mice (UnTx) or mice exposed to radiation only (Rad Cont). At 62 weeks post-transplant, surviving mice were harvested for histopathology, flow cytometry and real time polymerase chain reaction (RT-PCR). The mice from BMT + MSC group had the best outcomes for survival rates (71.4% vs. 43.8%), renal scores (2.9% vs. 28.8% glomerular sclerosis) and percent splenic monocytes (4.2 vs. 11.3%) compared with mice from Rad Cont. Improvement in RAG and BMT groups was less prominent but were comparable with one another. Although MSC alone were not sufficient to control the renal pathology, it limited the expansion of CD4(-)CD8(-) T cell populations without a change in Foxp3 expression. The results suggest the importance of the innate immune system in disease pathogenesis and a role for MSC in immunomodulation.

Nuclear fractal dimension as a prognostic factor in oral squamous cell carcinoma.

Strong theoretical reasons exist for using fractal geometry in measurements of natural objects, including most objects studied in pathology. Indeed, fractal dimension provides a more precise and theoretically more appropriate approximation of their structure properties and especially their shape complexity. The aim of our study was to evaluate the nuclear fractal dimension (FD) in tissue specimens from patients with oral cavity carcinomas in order to assess its potential value as prognostic factor. Relationships between FD and other factors including clinicopathologic characteristics were also investigated. Histological sections from 48 oral squamous cell carcinomas as well as from 17 non-malignant mucosa specimens were stained with Hematoxylin-Eosin for pathological examination and with Feulgen for nuclear complexity evaluation. The sections were evaluated by image analysis using fractal analysis software to quantify nuclear FD by the box-counting method. Carcinomas presented higher mean values of FD compared to normal mucosa. Well differentiated neoplasms had lower FD values than poorly differentiated ones. FD was significantly correlated with the nuclear size. Patients with FD lower than the median value of the sample had statistically significant higher survival rates. Within the sample of patients studied, FD was proved to be an independent prognostic factor of survival in oral cancer patients. In addition this study provides evidence that there are several statistically significant correlations between FD and other morphometric characteristics or clinicopathologic factors in oral squamous cell carcinomas.

Syntheses, in vitro and in vivo characterization of a 99mTc-(I)-tricarbonyl-benzylamino-dihydroxymethyl phosphine (NP(2)) chelate.

Studies were performed to study the complexation chemistry of 99mTc(CO)(+)(3) with a new tridentate amino-dihydroxymethyl phosphine (NP(2)) ligand with the 99mTc(CO)(3)(OH(2))(+)(3) synthon at tracer levels. A single, well-defined 99mTc(CO)(3)NP(2) complex is formed at pH 7.5 within 10 min at 60 degrees C that exhibits high in vitro and in vivo stability.

Synthesis and characterization of (99m)Tc- and (188)Re-complexes with a diamido-dihydroxymethylenephosphine-based bifunctional chelating agent (N(2)P(2)-BFCA).

A diamido-dihydroxymethylenephosphine (N(2)P(2)) bifunction chelating agent (BFCA) was shown to form well-defined (99m)Tc- and (188)Re-chelate structures. The 4, 4-bis [bis-hydroxymethyl-phosphonyl-propylcarbonmoyl]-butyric acid bifunctional chelating agent (N(2)P(2)-BFCA) formed stable complexes with (99m)Tc and (188)Re in >95% yield with high radiochemical purity (RCP). The biodistribution of the (99m)Tc- and (188)Re-N(2)P(2)-BFCAs after intravenous injection studied in normal mice showed the activity was excreted primarily via renal-urinary pathway indicating their use for labeling peptides with (99m)Tc and (188)Re.

Development of novel water-soluble, organometallic compounds for potential use in nuclear medicine: synthesis, characterization, and (1)H and (31)P NMR investigations of the complexes fac-[ReBr(CO)3L] (L=bis(bis(hydroxymethyl)phosphino)ethane, bis(bis(hydroxymethyl)phosphino)benzene).

The bidentate, water-soluble phosphine ligands, bis(bis(hydroxymethyl)phosphino)benzene (HMPB, 1) and bis(bis(hydroxymethyl)phosphino)ethane (HMPE, 2) were reacted with the organometallic precursor fac-[ReBr(3)(CO)(3)](2-), 3, to produce the complexes fac-[Re(OH(2))(CO)(3)L](+) and fac-[ReBr(CO)(3)L] (L = HMPE, HMPB), respectively, in good yields. The rhenium complexes fac-[ReBr(CO)(3)HMPB], 5, and fac-[ ReBr(CO)(3)HMPE], 8, were characterized using (1)H and (31)P NMR spectroscopy. The structure of fac-[ReBr(CO)(3)HMPB] was confirmed by single-crystal X-ray spectroscopy. The substitution reactions of HMPE/HMPB with the rhenium precursor 3 in aqueous solution were monitored using time-dependent (31)P NMR techniques. A significant discrepancy in the reaction kinetics and the substitution mechanism between the two bidentate ligands could be observed presumably due to the different chemical backbones.

Synthesis, characterization, and labeling with 99mTc/188Re of peptide conjugates containing a dithia-bisphosphine chelating agent.

Radiolabeling of small receptor-avid peptides at specific predetermined chelation sites with radioactive metals has been an effective approach for production of target-specific radiopharmaceuticals for diagnosis and therapy of diseases. Among various electron-donating groups found on chelator frameworks, phosphines are unique because they display versatile coordination chemistry with a wide range of transition metals. We have recently reported the utility of a dithia-bis(hydroxymethyl)phosphine-based (P2S2) bifunctional chelating agent (BFCA) containing air-stable primary phosphine groups to form 99mTc-labeled receptor-avid peptides by the preconjugation approach. Here we report a novel strategy for labeling small peptides with both 99mTc and 188Re using the P2S2-COOH (6,8-bis[3-(bis(hydroxymethyl)phosphanyl)propylsulfanyl]octanoic acid) BFCA by a postconjugation radiolabeling approach. The first step in this approach involves the coupling of the corresponding (PH2)2S2-COOH intermediate to the N-terminus of the peptide(s). Formylation of P-H bonds with aqueous formaldehyde in the presence of HCl in ethanol affords the corresponding (hydroxymethyl)phosphine-P2S2-peptide conjugates in the form of an oxidatively stable phosphonium salt. The P2S2-peptide conjugates are generated (where the PH2 groups are converted to P(CH2OH)2 groups) by treatment of the P2S2-peptide phosphonium salt(s) with 1 M sodium bicarbonate solution at pH 8.5. Complexation of BFCA conjugates with 99mTc is achieved by direct reduction with Sn(II) tartarate to yield the 99mTc-P2S2-peptide conjugate in near quantitative yields. Complexation of the BFCA conjugates with 188Re is achieved by transchelation with 188Re citrate in yields of >/=90%. In this study, (PH2)2S2-COOH BFCA was conjugated to model peptides. The glycineglycine ethyl ester (GlyGlyOEt)-(PH2)2S2-COOH BFCA conjugate was converted to the hydroxymethylene phosphine form and complexed with 99mTc to produce the 99mTcO2-P2S2-GlyGlyOEt conjugate 8 in RCPs of >/=95%. This singular 99mTc product is stable over 24 h in aqueous solution as confirmed by HPLC. Identical retention times of the 99mTcO2-P2S2-GlyGlyOEt complex and its cold rhenium analogue (ReO2-P2S2-GlyGlyOEt) on HPLC indicates similarity in structures at the macroscopic and the tracer levels. The utility of this postconjugation strategy was further demonstrated by synthesizing a P2S2-D-Lys6-LHRH conjugate and producing its corresponding 99mTc complex in RCPs of >/=88%. Finally, the P2S2-5-Ava-BBN[7-14]NH2 bombesin (BBN) analogue was synthesized, the PH2 groups converted to P(CH2OH)2 groups and subsequently labeled with 188Re to yield a 188Re-labeled bombesin analogue with a RCP of >/=90%. The biological integrity of this conjugate was demonstrated in both in vitro and in vivo. The results of this investigation demonstrate that the (PH2)2S2-COOH BFCA can be conveniently used as a precursor for labeling small receptor-avid peptides with diagnostic (99mTc) and therapeutic (188Re) radionuclides via the postconjugation approach in high yields.

In vitro and in vivo evaluation of bidentate, water-soluble phosphine ligands as anchor groups for the organometallic fac-[99mTc(CO)3]+-core.

The organometallic precursor fac-[99mTc(OH2)3(CO)3]+, 1a, was reacted with the bidentate, water-soluble phosphine ligands bis(bis(hydroxymethyl)phosphino)ethane (HMPE) and bis(bis(hydroxymethyl)phosphino)benzene (HMPB) in 0.9% saline to produce complexes in >95% yields. High performance liquid chromatography analyses indicate the initial formation of the complexes fac-[99mTcCl(CO)3L] (L = HMPE 2a, HMPB 3a). The neutral complexes ultimately lose the coordinated chloride to produce the cationic species fac-[99mTc(OH2)(CO)3L]+ 2b/3b. In vitro studies showed a high stability of 2b/3b over a wide pH range for >24 h. No decomposition or alteration of the complexes was observed even in the presence of excess histidine, cysteine, or human serum albumin. Experiments performed in normal mice demonstrated a fast clearance of the cationic compounds 2b/3b from the blood pool and clearance through the hepatobiliary and the urinary pathways.

99mTc-labeling and in vivo studies of a bombesin analogue with a novel water-soluble dithiadiphosphine-based bifunctional chelating agent.

Recent progress in the synthesis of water-soluble phosphine ligand systems and their corresponding 99mTc complexes prompted the development of a new bifunctional chelating agent (BFCA) based on a tetradentate dithiadiphosphine framework (P2S2-COOH). The detailed synthesis of this new BFCA is described here. The corresponding 99mTc complex, 99mTc-P2S2-COOH, can be formed in >95% yield. To demonstrate the potential of this chelate to efficiently label peptides, 99mTc-P2S2-COOH was coupled to the N-terminal region of the truncated nine-amino acid bombesin analogue, 5-Ava-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 [BBN(7-14)], to form 99mTc-P2S2-BBN(7-14). Conjugation to the peptide was performed in borate buffer (pH 8.5) by applying the prelabeling approach in yields of >60%. In competitive binding assays, using Swiss 3T3 mouse fibroblast cells against [125I-Tyr4]bombesin, Re-P2S2-BBN(7-14) exhibited an IC50 value of 0.8 +/- 0.4 nM. The pharmacokinetic studies of 99mTc-P2S2-BBN(7-14) and its ability to target tissue expressing gastrin-releasing peptide (GRP) receptors were performed in normal mice. The 99mTc-P2S2-BBN(7-14) exhibited fast and efficient clearance from the blood pool (0.6 +/- 0.1% ID, 4 h postinjection) and excretion through the renal and hepatobiliary pathways (56.4 +/- 8.2 and 28.1 +/- 7.9% ID, 4 h postinjection, respectively). Significant uptake in the pancreas was observed (pancreatic acini cells express bombesin/GRP receptors), producing pancreas:blood and pancreas:muscle ratios of ca. 22 and 80, respectively, at 4 h postinjection.

198Au-labeled hydroxymethyl phosphines as models for potential therapeutic pharmaceuticals.

The development of novel gold-198 complexes with water-soluble phosphines is reported. A series of cationic and hydrophilic 198Au complexes containing the ligands tris(hydroxymethyl)phosphine (THP, 1) 1,2-bis[bis(hydroxymethyl)phosphino]benzene (HMPB, 2), and 1,2-bis[bis(hydroxymethyl)phosphino]ethane (HMPE, 3) were prepared and evaluated as models for potential gold-199 radiopharmaceuticals. The 198Au complexes were formed in high radiochemical purity by simply mixing H198AuCl4 with the respective ligand. The complexes were shown to exhibit high in vitro stability over wide pH ranges and temperatures. However, only the 198Au(HMPB)2+ complex was found to exhibit good in vivo stability. HPLC analyses indicated that the 198Au complexes with these three phosphine ligands produced singular species with similar retention times as compared to their known macroscopic complexes.

In vitro and in vivo characterization of novel water-soluble dithio-bisphosphine 99mTc complexes.

The water-soluble dithio-bis(hydroxymethyl)phosphine ligands (HOH2C)2P(CH2)2 S(CH2)3S(CH2)2P(CH2OH)2 and (HOH2C)2P(CH2)3S(CH2)3S(CH2)3P(CH2OH)2 were complexed with 99mTc. The 99mTc-P2S2 complexes were formed in high radiochemical purity by simple mixing of 99mTcO-4 with the ligands or by transchelation from 99mTc-citrate. The 99mTc-P2S2 complexes were stable over a wide range of pHs and did not undergo in vitro decomposition for < or = 24 h. High performance liquid chromatographic analysis indicated the formation of singular chemical species. Retention times for each of the new 99mTc-P2S2 complexes are identical to those of corresponding Re(V) complexes, suggesting similar chemical species at the tracer level. Results of this study suggest that the combination of thioether and (hydroxymethyl)phosphine donor centers in new, multidentate ligand frameworks might aid in the development of new bifunctional chelating agents for the use of radiolabeling specific biomolecules.

In vitro and in vivo characterization of a 99mTc complex with tris(hydroxymethyl)phosphine (THP).

The water-soluble, monophosphine ligand tris(hydroxymethyl)phosphine (THMP) was complexed with 99mTc. The 99mTc-THMP was formed in high radiochemical purity by simply mixing 99mTcO4- with THMP over a wide pH range. In vitro and in vivo studies indicated the complex to be highly stable under the respective conditions. HPLC analysis indicated a singular species with a retention time identical to the known [ReO2(THMP)4]1 complex. Results show that the hydroxymethylphosphine functionality is attractive for use in designing new 99mTc radiopharmaceuticals.

Transition metal chemistry of main group hydrazides, Part 14: Evaluation of new Tc-99m chelates of thiol functionalized phosphorus hydrazides.

Ligands containing a combination of amine or amide nitrogens and thiol functionalities have been found to form stable chelates with Tc-99m, presumably in oxidation state +5. Two new thio-phosphorus monohydrazides [(MeO)2P(S)NMeNHCH2C6H4SH], SL1 and [(MeO)2P(S)NMeNHC(O)C6H4SH], SL2 were synthesized and their complexation properties with Re(V) and Tc-99m have been studied. Neutral-lipophilic Tc-99m chelates with both SL1 and SL2 were formed in high yields (95-97%) as a single species ascertained by electrophoresis and reversed-phase HPLC. Biodistribution studies show good in vivo stability and primary clearance of both 99mTc chelates is via the hepatobiliary pathway. Re(V) complexes with SL1 and SL2 were also synthesized using the ReOCl3(PPh3)2 precursor to obtain the product ReOCl(L)(PPh3), where L = SL1 or SL2. H+ was lost from the N-atom and the thiol group in these Re chelates. Even though the Tc-99m chelates of SL1 and SL2 formed at tracer levels are not identical to the Re-chelates (different synthons were used), the Re data suggests complexation of Tc-99m by these hydrazido-thiol ligands will be similar to N,S ligand systems previously used. The good in vitro and in vivo stability and high yields of the Tc-99m complexes of SL1 and SL2 indicate the potential hydrazido-thiols hold for use as a basis in formulating new Tc-99m radiopharmaceuticals, particularly when thiol moieties are used in conjunction with multi-functional phosphorous hydrazide compounds.

A new 99mTc-complex with a germanium-hydrazide (GeTH) ligand.

A new tetrahydrazido-germanium (GeTH) ligand was synthesized, characterized and complexed with 99mTc. The negatively charged 99mTc-chelate was shown to form in high yields at neutral pH in the absence of other reducing agents and exhibits high in vitro and in vivo stability.

Formation of a stable 99mTc-tri-hydrazidophosphine oxide (99mTc-THP) chelate.

Atri-hydrazidophosphine oxide (THP) ligand was synthesized and used to complex 99mTc in high yield (> 95%) by Sn(II) reduction of 99mTcO4-. 99mTc-THP has excellent stability in both 0.9% acqueous NaCl at neutral pH and in human serum at 37 degrees C. Biodistribution studies indicate minimal organ specificity and no significant in vivo release of 99mTcO4-. The ease of 99mTc complexation with THP and the high stability of 99mTc-THP suggests that this ligand may be used as a basis for the development of new imaging agents.