Optimization of radioprotective dose of Juglans nigra leaf extract using response surface methodology.

J. nigra leaf contains mixture of various pharmacologically active compounds and it is assumed that they may have the potential radioprotective effect. The purpose of this work was to predict radioprotective doses by correlating changes in organ distribution of radiopharmaceuticals with extract dose levels and rat body weight using response surface methodology (RSM) based on a second-order polynomial equation. The extract was administered daily by oral gavage to rats at dose of 6.9, 10.3, or 13.7 mg kg-1 body weight (bw) day-1 for 10 days. On the eleventh day, 0.1 ml of the one radiopharmaceutical (Na99mTcO4, 99mTc-dimercaptosuccinic acid and 99mTc-tin colloid) was injected into the tail vein. The statistical parameters: the coefficient of determination (0.81-0.95), the coefficient of variation (3.05-11.1), the adequate precision (8.82-19.12) and the mean relative percentage deviation (± 2.3-8.2) were indicated the precision and reliability of RSM. Using RSM, the predicted daily dose of leaf extract ranging from 11.19 to 11.99 mg kg-1 bw may be considered as an optimal daily radiopotective dose for protection of organs from radiation in cases of planned radiation exposures.

Synthesis, Characterization, and Therapeutic Efficacy of 177Lu-DMSA@SPIONs in Nanobrachytherapy of Solid Tumors.

As an alternative to classical brachytherapy, intratumoral injection of radionuclide-labeled nanoparticles (nanobrachytherapy, NBT) has been investigated as a superior delivery method over an intravenous route for radionuclide therapy of solid tumors. We created superparamagnetic iron oxide nanoparticles (SPIONs) coated with meso-1,2-dimercaptosuccinic acid (DMSA) and radiolabeled with Lutetium-177 (177Lu), generating 177Lu-DMSA@SPIONs as a potential antitumor agent for nanobrachytherapy. Efficient radiolabeling of DMSA@SPIONS by 177Lu resulted in a stable bond with minimal leakage in vitro. After an intratumoral injection to mouse colorectal CT-26 or breast 4T1 subcutaneous tumors, the nanoparticles remained well localized at the injection site for weeks, with limited leakage. The dose of 3.70 MBq/100 µg/50 µL of 177Lu-DMSA@SPIONs applied intratumorally resulted in a high therapeutic efficacy, without signs of general toxicity. A decreased dose of 1.85 MBq/100 µg/50 µL still retained therapeutic efficacy, while an increased dose of 9.25 MBq/100 µg/50 µL did not significantly benefit the therapy. Histopathology analysis revealed that the 177Lu-DMSA@SPIONs act within a limited range around the injection site, which explains the good therapeutic efficacy achieved by a single administration of a relatively low dose without the need for increased or repeated dosing. Overall, 177Lu-DMSA@SPIONs are safe and potent agents suitable for intra-tumoral administration for localized tumor radionuclide therapy.

Design and preparation of proline, tryptophan and poly-l-lysine functionalized magnetic nanoparticles and their radiolabeling with 131I and 177Lu for potential theranostic use.

Surface modification of magnetic nanoparticles with poly-l-lysine, proline, and tryptophan was used to design potential theranostic agents for the application in cancer diagnosis and radionuclide-hyperthermia therapy. Characterization of bare and functionalized magnetic nanoparticles was performed in detail. The transparency of the examined magnetic nanoparticles was measured in the non-alternating magnetic field for a complete and better understanding of hyperthermia. For the first time amino acid-functionalized magnetic nanoparticles were labeled with theranostic radionuclides 131I and 177Lu. The specific absorption rate (SAR) procured for poly-l-lysine functionalized magnetic nanoparticles (SAR values of 99.7 W/g at H0 = 15.9 kA/m and resonant frequency of 252 kHz) demonstrated their possible application in magnetic hyperthermia. Poly-l-lysine functionalized magnetic nanoparticles labeled with 177Lu showed the highest radiochemical purity (>99.00 %) and in vitro stability in saline and serum (>98.00 % up to 96 h). The in vivo analysis performed after their intravenous administration in healthy Wistar rats presented good in vivo stability for several days. Encouraging results as well as magnetic and radiochemical properties of 177Lu-PLL-MNPs (80 °C) justify their further testing toward the potential use as theranostic agents for diagnostic and combined radionuclide-hyperthermia therapeutic applications.

90Y-CA/SPIONs for dual magnetic hyperthermia-radionuclide nanobrachytherapy of solid tumours.

Radiolabelled superparamagnetic iron oxide nanoparticles (SPIONs) are a promising nanomaterial for the development of dual radiation/hyperthermia cancer therapy. To that purpose, flower-shaped SPIONs with an exceptional heating capability were synthesised and coated with citrate, dextran or (3-aminopropyl)triethoxysilane. Both non-coated and coated SPIONs were nontoxic to CT-26 mouse colon cancer cells up to 1.0 mg ml-1in vitro. In an oscillating magnetic field, citrate-coated SPIONs (CA/SPIONs) displayed the highest heating rate (SAR âˆ¼ 253 W g-1) and the strongest hyperthermia effects against CT-26 cells. Labelling of the CA/SPIONs by the90Y radionuclide, emitting ß-radiation with an average/maximum energy of 0.94/2.23 MeV, and deep tissue penetration generated90Y-CA/SPIONs intended for the therapy of solid tumours. However, intravenous injection of90Y-CA/SPIONs in CT-26 xenograft-bearing mice resulted in low tumour accumulation. On the contrary, intratumoural injection resulted in long-term retention at the injection site. A single intratumoural injection of 0.25 mg CA/SPIONs followed by 30-min courses of magnetic hyperthermia for four consecutive days caused a moderate antitumour effect against CT-26 and 4T1 mouse tumour xenografts. Intratumoural application of 1.85 MBq/0.25 mg90Y-CA/SPIONs, alone or combined with hyperthermia, caused a significant (P ≤ 0.01) antitumour effect without signs of systemic toxicity. The results confirm the suitability of90Y-CA/SPIONs for monotherapy or dual magnetic hyperthermia-radionuclide nanobrachytherapy (NBT) of solid tumours.

177Lu-doxycycline as potential radiopharmaceutical: electrochemical characterization, radiolabeling, and biodistribution in tumor-bearing mice.

PURPOSE: Recent studies with doxycycline as adjuvant therapy to conventional chemotherapy have shown promising results in cancer therapy. The current study aimed to examine the capability of 177Lu-labeled tetracycline ligand, doxycycline hyclate, to use as an anticancer agent. MATERIALS AND METHODS: Doxycycline was radiolabeled with beta-emitting radioisotope 177Lu. Complex formation and its interaction with DNA were investigated electrochemically. Binding of 177Lu-doxycycline to CT 26 cell line was done. Biodistribution of 177Lu-doxycycline was examined in healthy Wistar rats and CT26 colon carcinoma tumor-bearing mice by i.v. and i.p. administration, respectively. RESULTS: Doxycycline hyclate was successfully radiolabeled with 177Lu in high radiolabeling yield (>99%). The radiolabeled complex was stable in vitro in saline and human serum over 72 h. Non-radioactive Lu-doxycycline complex formation was demonstrated electrochemically as well. Intercalative interactions of the doxycycline and Lu-doxycycline with DNA were proved using simultaneously spectrophotometric and electrochemical methods. The binding of the radiolabeled complex with plasma proteins was 4.0 ± 0.4%. The partition coefficient showed the lipophilic nature of the complex similar to the free ligand. The binding curve demonstrates binding from 0.1 nM concentrations of 177Lu-doxycycline, with half-binding estimated ∼100 nM. Biodistribution studies of 177Lu-doxycycline in CT26 colon tumor-bearing mice showed a satisfactory accumulation rate in the tumor (2.88 ± 0.85% ID/g) 3 h after intraperitoneal injection. Both the hepatobiliary system and the urinary system were prominent as excretory routes of the radiolabeled complex. CONCLUSION: Considering obtained results, 177Lu-doxycycline complex, due to its excellent electrochemical and biological characteristics, with emphasis on the binding ability to DNA via intercalative interaction as well as significant accumulation in the tumor, is suitable for further in vivo studies to investigate its potential use in cancer treatment.

177Lu-labeled micro liposomes as a potential radiosynoviorthesis therapeutic agent.

Micro-sized multivesicular liposomes were prepared, radiolabeled with 177Lu, and tested in vitro and in vivo to evaluate the potential of 177Lu-labeled micro liposomes in radiosynoviorthesis (RSO) therapy. A standard reverse-phase procedure of liposome preparation with a lipid mixture of DPPC: CHOL (80:20%) was used for the synthesis. TEM and fluorescence microscopy imaging were performed to determine the size, shape, and structure of the prepared liposomes. Both measurements are in good agreement while TEM micrographs additionally indicate to a large multivesicular inner structure of prepared liposomes. A simple and straightforward procedure was used for liposome radiolabeling with 177Lu, a well-known and commonly used radionuclide in radiotherapy with favorable properties, that can be exploited in RSO therapy. Radiolabeled 177Lu-liposomes were tested in vitro for stability and then injected into the knee joints of Wistar rats where liposome in vivo behavior was followed up to 30 days post injection. Results from both ex vivo biodistribution and in vivo imaging studies presented a high stability and retention (>94 %ID) of 177Lu-micro liposomes in the synovial liquid for the entire observation period. Leakage of free 177Lu or 177Lu-liposomes from the synovial fluid has not been detected, indicating to a possible application of 177Lu-liposomes in radiosynoviorthesis (RSO) therapy.

Bioevaluation of glucose-modified liposomes as a potential drug delivery system for cancer treatment using 177-Lu radiotracking.

Liposomes are promising drug's delivery systems due to decreased toxicity of the liposome-encapsulated drug, but wider clinical application requires their more efficient tumor targeting with uptake, controlled drug release and higher shelf life. The unique metabolic characteristics of cancer cells based on higher demand for energy and therefore increased glucose utilization were exploited in the design of glucose modified liposomes (GML) with the aim to provide increased tumor targeting via glucose transporters and increased ability of drug delivery into tumor cells. Tumor accumulating potential of GML and non-glucose liposomes (NGL) were investigated on CT26 and LS174T tumor-bearing mice by simple and reliable radiotracer method using 177Lu as radioactive marker. Both liposomes, GML and NGL were radiolabeled in high radiolabeling yield, showing high in vitro stability in biological media, as the main prerequisite for the biodistribution studies. Tumors displayed significantly better accumulation of 177Lu-GML with the maximum uptake 6 h post-injection (5.8 ± 0.2%/g in LS174T tumor and 5.1 ± 0.5%/g in CT26 tumor), compared to negligible uptake of 177Lu-NGL (0.6 ± 0.1%/g in LS174T tumor and 0.9 ± 0.2%/g in CT26 tumor). Results of comparative biodistribution studies of 177Lu-NGL and 177Lu-GML indicate that increased accumulation of GML is enabled by glucose transporters and subsequent endocytosis, resulting in their prolonged retention in tumor tissues (up to 72 h). Direct radiolabeling of liposomes with 177Lu may be used not only for biodistribution studies using radiotracking, but also for cancer treatment.

Aminosilanized flower-structured superparamagnetic iron oxide nanoparticles coupled to 131I-labeled CC49 antibody for combined radionuclide and hyperthermia therapy of cancer.

Combined radionuclide therapy with magnetic nanoparticles-mediated hyperthermia has been under research focus as a promising tumor therapy approach. The objective of this study was to investigate the potential of 131I-radiolabeled superparamagnetic iron oxide nanoparticles (SPIONs) prepared as the ~40 nm flower-shaped structures with excellent heating efficiency (specific absorption rate at H0 = 15.9 kA∙m-1 and resonant frequency of 252 kHz was 123.1 W∙g-1) for nano-brachytherapy of tumors. 131I-radiolabeled CC49 antibody attached to SPIONs via reactive groups of 3-aminopropyltriethoxysilane (APTES) provided specificity and long-lasting localized retention after their intratumoral application into LS174T human colon adenocarcinoma xenografts in NOD-SCID mice. The results demonstrate feasibility and effectiveness of magnetic hyperthermia (HT), radionuclide therapy (RT) and their combination (HT + RT) in treating cancer in xenograft models. Combined therapy approach induced a significant (p < 0.01) tumor growth suppression in comparison to untreated groups presented by the tumor volume inhibitory rate (TVIR): 54.38%, 68.77%, 73.00% for HT, RT and HT + RT, respectively in comparison to untreated group and 48.31%, 64,62% and 69,41%, respectively, for the SPIONs-only injected group. Histopathology analysis proved the necrosis and apoptosis in treated tumors without general toxicity. Obtained data support the idea that nano-brachytherapy combined with hyperthermia is a promising approach for effective cancer treatment.

Evidence of acute phase reaction in asymptomatic dogs naturally infected with Babesia canis.

Asymptomatic outdoor dogs can be carriers of Babesia canis, but data describing the development of an acute phase response (APR) are not available. We hypothesised that these dogs have a moderate APR that could be detected by hematological and biochemical changes. Two groups of Babesia-exposed dogs were represented by nine B. canis PCR-positive and twenty B. canis PCR-negative, seroreactive dogs. The control group consisted of ten Babesia-naïve dogs. Serum amyloid A (SAA), paraoxonase-1 (PON-1), complete blood count, and biochemistry parameters were analysed by standard methodologies. Protein and lipoprotein fractions were separated using agarose gel electrophoresis (GE), and the dominant diameters of lipoproteins were assessed on gradient GE. Results were evaluated using non-parametric tests and the Receiver Operating Characteristic curve. SAA (median 39.0 µg/mL, range 2.2-48.8 µg/mL), total protein (median 74.7 g/L, range 57.1-98.3 g/L) and the dominant diameter of α-lipoproteins (median 13.31 nm, range 12.09-14.17 nm) in B. canis PCR-positive dogs were higher relative to dogs in the control group or dogs that were PCR-negative but seroreactive (p < 0.001 for both groups). Mild to moderate anemia (4/29), thrombocytopenia (7/29), and leukocyte counts that were close to the upper limit of the reference range were encountered in both Babesia-exposed groups. When compared to controls, Babesia-exposed dogs displayed decreased a PON-1 activity and protein GE pattern consistent with low-grade chronic inflammation (p < 0.001 for both groups). Dogs with detectable amounts of B. canis DNA in blood contain increased levels of SAA and total protein along with α-lipoproteins that display an increased diameter relative to those dogs with positive Babesia serology but undetectable levels of B. canis DNA in blood.

Association of acute Babesia canis infection and serum lipid, lipoprotein, and apoprotein concentrations in dogs.

BACKGROUND: Babesia canis infection induces a marked acute phase response (APR) that might be associated with alteration in lipid and lipoprotein metabolism and disease prognosis. HYPOTHESIS: Dogs with B. canis-induced APR develop dyslipidemia with altered lipoprotein concentration and morphology. ANIMALS: Twenty-nine client-owned dogs with acute B. canis infection and 10 clinically healthy control dogs. METHODS: Observational cross-sectional study. Serum amyloid A (SAA) was measured using ELISA. Cholesterol, phospholipids, and triglycerides were determined biochemically. Lipoproteins were separated using agarose gel electrophoresis. Lipoprotein diameter was assessed by polyacrylamide gradient gel electrophoresis; correlation with ApoA-1 (radioimmunoassay) and SAA was determined. RESULTS: Dogs with B. canis infection had a marked APR (median SAA, 168.3 µg/mL; range, 98.1-716.2 µg/mL) compared with controls (3.2 µg/mL, 2.0-4.2 µg/mL) (P < .001). Dogs with B. canis infection had significantly lower median cholesterol (4.79 mmol/L, 1.89-7.64 mmol/L versus 6.15 mmol/L, 4.2-7.4 mmol/L) (P = .02), phospholipid (4.64 mmol/L, 2.6-6.6 mmol/L versus 5.72 mmol/L, 4.68-7.0 mmol/L) (P = .02), and α-lipoproteins (77.5%, 27.7%-93.5% versus 89.2%, 75.1%-93.5%) (P = .04), and higher ApoA-1 (1.36 U, 0.8-2.56 U versus 0.95 U, 0.73-1.54 U) concentrations (P = .02). Serum amyloid A correlated with high-density lipoproteins (HDLs) diameter (rho = .43; P = .03) and ApoA-1 (rho = .63, P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Major changes associated with B. canis-induced APR in dogs are related to concentration, composition, and morphology of HDL particles pointing to an altered reverse cholesterol transport. Parallel ApoA-1 and SAA concentration increase is a unique still unexplained pathophysiological finding.

99mTc-bisphosphonate-coated magnetic nanoparticles as potential theranostic nanoagent.

Novel theranostic nanoplatform is expected to integrate imaging for guiding and monitoring of the tumor therapy with great therapeutic efficacy and fewer side effects. Here we describe the preparation of a multifunctional 99mTc-bisphosphonate-coated magnetic nanoparticles (MNPs) based on Fe3O4 and coated with two hydrophilic bisphosphonate ligands, i.e., methylene diphosphonate (MDP) and 1-hydroxyethane-1,1- diphosphonate (HEDP). The presence of the bisphosphonates on the MNPs surface, enabled their biocompatibility, colloidal stability and successful binding of the radionuclide. The morphology, size, structure, surface charge and magnetic properties of obtained bisphosphonate-coated Fe3O4 MNPs were characterized by transmission electron microscopy, X-ray powder diffraction, dynamic light scattering, laser Doppler electrophoresis, Fourier transform infrared spectroscopy and vibrating sample magnetometer. The specific power absorption values for Fe3O4-MDP and Fe3O4-HEDP were 113 W/g and 141 W/g, respectively, indicated their heating ability under applied magnetic field. Coated MNPs were radiolabeled with 99mTc using stannous chloride as the reducing agent in a reproducible high yield (95% for Fe3O4-MDP and 97% for Fe3O4-HEDP MNPs) and were remained stable in saline and human serum for 24 h. Ex vivo biodistribution studies presented significant liver and spleen uptake in healthy Wistar rats after intravenous administration at all examined time points due to the colloidal nature of both 99mTc-MNPs. Results of scintigraphy studies are in accordance with ex vivo biodistribution studies, demonstrating high in vivo stability of radiolabeled MNPs and therefore results of both methods were proved as accurate information on the biodistribution profile of investigated MNPs. Overall, in vitro and in vivo stability as well as heating ability, indicate that biocompatible radiolabeled bisphosphonate magnetic nanoparticles exhibit promising potential as a theranostic nanoagent.

Molecular and Serological Prevalence of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeenses, E. ewingii, Borrelia burgdorferi, Babesia canis, B. gibsoni and B. vogeli among Clinically Healthy Outdoor Dogs in Serbia.

Data concerning combined molecular and serological prevalence of emerging canine tick-borne pathogens in Serbia are lacking. A large population of outdoor living dogs in Belgrade, Serbia's' capital, present an excellent population for epidemiology study. Blood samples were collected from 111 dogs, including 46 shelter, 31 free roaming, and 34 hunting dogs. Species-specific real-time polymerase chain reaction (PCR) (IDEXX Laboratories, Inc., Westbrook Maine, USA) was applied for the molecular detection of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, Babesia canis, B. gibsoni and B. vogeli. A research based SNAP assay (SNAP® M-A, IDEXX Laboratories, Inc., Westbrook Maine, USA) that uses genus and species-specific peptides was used to asses Anaplasma spp., A. phagocytophilum, A. platys, Ehrlichia spp., E. canis, E. chaffeensis, E. ewingii and Borrelia burgdorferi antibody status. B. canis, B. gibsoni and B. vogeli antibody status was assessed with an indirect immunofluorescence test (MegaCor Diagnostic, Horbranz, Austria). Anaplasma spp. and Ehrlichia spp. DNA was not amplified. One quarter of the dogs were A. phagocytophilum, one dog was A. platys, one was E. ewingii and two dogs were B. burgdorferi seroreactive with the SNAP® M-A. Babesia canis or B. gibsoni DNA was amplified by PCR from 16.2% of dogs, whereas 67.6% were seroreactive to one or more Babesia spp. Babesia vogeli was not PCR amplified. We conclude that outdoor dogs in this territory are reservoirs for B. canis and B. gibsoni and are frequently co-exposed to combinations of Anaplasma and Babesia spp.

Acute-phase response in Babesia canis and Dirofilaria immitis co-infections in dogs.

Babesia canis and Dirofilaria immitis are emerging and geographically overlapping vector-borne pathogens in dogs. Infection with B. canis leads to acute-phase response (APR) that can be mild to severe and results in either non-complicated or complicated forms of the disease. The aim of this study was to determine whether acute B. canis infection is more severe in dogs with underlying asymptomatic D. immitis infection. Dogs of both sexes, different ages and breeds, with naturally occurring mono-infections with B. canis (n=13) and D. immitis (n=18) and co-infected dogs (n=7) were enrolled as well as healthy controls (n=15). Routine haematology and biochemistry, agarose gel electrophoresis (agEF) protein fraction separation and enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA) were performed. Based on clinical and laboratory findings, sepsis was diagnosed in the majority of dogs with acute B. canis infection with or without underlying asymptomatic D. immitis infection. Overall, haematology, biochemistry and agEF pattern changes were induced and dictated by acute B. canis infection whether or not the dogs had an asymptomatic D. immitis infection. D. immitis infection slightly influenced the level of anaemia, slightly aggravated the level of dehydration and increased the concentration of γ-globulins in acute-phase B. canis infection. D. immitis infection prevented B. canis-induced leukopenia. SAA equally increased in dogs with acute B. canis infection with or without underlying D. immitis infection. The level of SAA was not changed in dogs with asymptomatic D. immitis when compared to the controls. In conclusion, asymptomatic D. immitis infection does not influence overall APR after acute B. canis infection.

Assessment of using recombinant Ixodes ricinus AV422 saliva protein for confirmation of tick bites in hunting dogs as naturally infested hosts.

Due to the recorded spreading of ticks in past years, a higher incidence of tick-borne diseases (TBDs) can be expected in the future in endemic areas, but can also pose an emerging public health concern in areas where they have not yet been recognized. Assessment of the exposure of vulnerable hosts to ticks would be a very helpful tool for TBD epidemiological studies, as well as for their proper managing. To confirm previous tick bites, the method of choice is detection of antibodies in host serum as markers developed against injected tick saliva proteins during feeding. We recently showed that the recombinant form of Ixodes ricinus AV422 saliva protein (rIrAV422) can serve for detection of markers in experimentally infested rats. Here we examine whether it can be used in the same manner in naturally exposed hosts. We chose hunting dogs as good sentinel animals. The study group consisted of 15 dogs that varied in breed, age, sex, previous tick infestation history and repellent treatment. Western blot analysis with rIrAV422 as an antigen confirmed the presence of tick bite markers in all analysed dogs. For some of the dogs, their previous tick infestation history was unclear, which emphasizes the usefulness of rIrAV422 for revealing it. Since hunting dogs are naturally infested with different ticks, the potential of rIrAV422 in assessment of general exposure to ticks is highlighted. Use of rIrAV422 can also be helpful in veterinary practice and research as a tool for validation of the efficiency of tick repellent products.