Biological characterization of cetuximab-conjugated gold nanoparticles in a tumor animal model.

Gold nanoparticles (AuNPs) are widely applied to the diagnosis and treatment of cancer and can be modified to contain target-specific ligands via gold-thiolate bonding. This study investigated the pharmacokinetics and microdistribution of antibody-mediated active targeting gold nanoparticles in mice with subcutaneous lung carcinoma. We conjugated AuNPs with cetuximab (C225), an antibody-targeting epidermal growth factor receptor (EGFR), and then labeled with In-111, which created EGFR-targeted AuNPs. In vitro studies showed that after a 2 h incubation, the uptake of C225-conjugated AuNPs in high EGFR-expression A549 cells was 14.9-fold higher than that of PEGylated AuNPs; furthermore, uptake was also higher at 3.8-fold when MCF7 cells with lower EGFR-expression were used. MicroSPECT/CT imaging and a biodistribution study conducted by using a A549 tumor xenograft mouse model provided evidence of elevated uptake of the C225-conjugated AuNPs into the tumor cells as a result of active targeting. Moreover, the microdistribution of PEGylated AuNPs revealed that a large portion of AuNPs remained in the tumor interstitium, whereas the C225-conjugated AuNPs displayed enhanced internalization via antibody-mediated endocytosis. Our findings suggest that the anti-EGFR antibody-conjugated AuNPs are likely to be a plausible nano-sized vehicle for drug delivery to EGFR-expressing tumors.

Evaluation of EGFR-targeted radioimmuno-gold-nanoparticles as a theranostic agent in a tumor animal model.

This study evaluated the tumor targeting and therapeutic efficacy of a novel theranostic agent (131)I-labeled immuno-gold-nanoparticle ((131)I-C225-AuNPs-PEG) for high epidermal growth factor receptor (EGFR)-expressed A549 human lung cancer. Confocal microscopy demonstrated the specific uptake of C225-AuNPs-PEG in A549 cells. (131)I-C225-AuNPs-PEG induced a significant reduction in cell viability, which was not observed when incubated with AuNPs-PEG and C225-AuNPs-PEG. MicroSPECT/CT imaging of tumor-bearing mice after intravenous injection of (123)I-C225-AuNPs-PEG revealed significant radioactivity retention in tumor suggested that (131)I-labeled C225-conjugated radioimmuno-gold-nanoparticles may provide a new approach of targeted imaging and therapy towards high EGFR-expressed cancers.

Tc-99m-HL91 imaging in the early detection of neuronal injury in a neonatal rat model of hypoxic ischemia.

OBJECTIVE: Hypoxic-ischemic insult in newborns results in progressive neuronal loss. For neuroprotective therapy to be effective, it is important to identify high-risk neonates soon after birth. 99mTc-labeled imaging agent, Tc-99m-HL91, developed as a putative hypoxic reagent, has been reported to demonstrate increased uptake in ischemic myocardium. We hypothesized that Tc-99m-HL91 is sensitive for the early identification of hypoxic-ischemic injury in neonatal rat brains. DESIGN: Laboratory investigation. SETTING: University research laboratory. SUBJECTS: Sprague-Dawley rat pups. INTERVENTIONS: Postnatal day-7 pups were divided into four groups: hypoxic-ischemia, hypoxia-only, ischemia-only, and controls. In the early (2 hrs), intermediate (20 hrs), and late (44 hrs) reoxygenation phases, Tc-99m-HL91 in vivo and ex vivo imaging and quantitative autoradiography were performed. Regions of interest were drawn to calculate the contrast ratio of Tc-99m-HL91 uptake between the ipsilateral and contralateral hemispheres. Pathology, cerebral blood flow, and blood-brain barrier damage were determined. MEASUREMENTS AND MAIN RESULTS: After hypoxic-ischemia, there were very few pyknotic neurons in the early phase, many pyknotic neurons in the intermediate phase, and extensive neuronal loss in the late phase postreoxygenation. Blood-brain barrier damage occurred in the early phase, progressed in the intermediate phase, and became extensive in the late phase. The hypoxia-only and ischemia-only pups showed no neuronal or blood-brain barrier damage and had higher cerebral blood flow postreoxygenation compared with the hypoxia-ischemia pups. Regions of interest analysis of in vivo and ex vivo images and autoradiography revealed significantly higher Tc-99m-HL91 contrast ratio at early and intermediate phases, not late phase of hypoxic-ischemic group. Hypoxic-ischemia group had significantly higher contrast ratio values in the early and intermediate phases than the hypoxia-only and ischemia-only groups. A contrast ratio value of 0.15 in the early phase on postnatal day 7 had a sensitivity of 0.95 and specificity of 0.89 in detecting significant hypoxic-ischemic lesions on postnatal day 21. CONCLUSION: Tc-99m-HL91 uptake is sensitive for the early detection of hypoxic-ischemic injury in neonatal brains.

Bromelain Ameliorates Atherosclerosis by Activating the TFEB-Mediated Autophagy and Antioxidant Pathways.

Bromelain, a cysteine protease found in pineapple, has beneficial effects in the treatment of inflammatory diseases; however, its effects in cardiovascular pathophysiology are not fully understood. We investigated the effect of bromelain on atherosclerosis and its regulatory mechanisms in hyperlipidemia and atheroprone apolipoprotein E-null (apoe-/-) mice. Bromelain was orally administered to 16-week-old male apoe-/- mice for four weeks. Daily bromelain administration decreased hyperlipidemia and aortic inflammation, leading to atherosclerosis retardation in apoe-/- mice. Moreover, hepatic lipid accumulation was decreased by the promotion of cholesteryl ester hydrolysis and autophagy through the AMP-activated protein kinase (AMPK)/transcription factor EB (TFEB)-mediated upregulation of autophagy- and antioxidant-related proteins. Moreover, bromelain decreased oxidative stress by increasing the antioxidant capacity and protein expression of antioxidant proteins while downregulating the protein expression of NADPH oxidases and decreasing the production of reactive oxygen species. Therefore, AMPK/TFEB signaling may be crucial in bromelain-mediated anti-hyperlipidemia, antioxidant, and anti-inflammatory effects, effecting the amelioration of atherosclerosis.

Characteristics of Thermosensitive and Targeted F127-Triethoxysilane/Gold Nanorod Combined with Photothermal Therapy in Tumor Model Mice.

Even with all the recent technological improvements, cancer remains to be the disease with the highest impact on global health. Due to obviously disadvantages or limitations on traditional therapy, researchers are engaged to search for safely and effective methods in cancers' therapy. Photothermal therapy (PTT) has been employed in treating cancers and several of other diseases. In this study, novel thermosensitive and targeting nanoparticle, C225-silane-F127/gold nanorod (C-SFGR) combined with PTT was investigated in EGFR-overexpressing xenografts mice model. For better light to heat transformation exposed with 808 nm near-infrared (NIR) laser, the diameter of thermosensitive C-SFGR was designed at about 120 nm. To address the biocompatibility, the viability of A549 cell line was greater than 80% under high concentrations of C-SFGR (1,000 µg/mL), indicating its low cytotoxicity. After intravenous injection of C-SFGR and combined with NIR treatment for 2 min in A549 bearing mice, tumors were almost completely shriveled after 2 weeks. For developing as theranostic agent, C-SFGR was then labeled with 67Ga, with radiochemical purity over 98%. These present results suggest that C-SFGR could be also applied as a SPECT-imaging agent and as an effective antitumor agent.

Accumulation of Tc-99m HL91 in tumor hypoxia: in vitro cell culture and in vivo tumor model.

Hypoxic cells within a tumor can account, in part, for resistance to radiotherapy and chemotherapy. Indeed, the oxygenation status has been shown to be a prognostic marker for the outcome of therapy. The purpose of this study was to determine whether Tc-99m HL91 (HL91), a noninvasive imaging tracer, detects tumor hypoxia in vitro in cell culture and in vivo in a tumor model. Uptake of HL91 in vitro into human lung cancer cells (A549) and murine Lewis lung cancer cells (LL2) was investigated at oxygen concentrations of 20% O2 (normoxia), and 1% O2 (hypoxia). HL91 biodistribution was studied in four groups: severe combined immune deficiency (SCID) mice bearing A549 tumors, C57BL/6NCrj (B6) mice bearing LL2 tumors, SCID controls, and B6 controls. Accumulation of the tracer was compared between tumors treated with hydralazine or phosphate-buffered saline (PBS). Scintigraphic images were obtained for hydralazine-treated mice and PBS-treated mice in each of the four study groups. Autoradiography of tumor slices was also acquired. In vitro studies identified hypoxia-selective uptake of HL91, with significantly increased uptake in the hypoxic state than in the normoxic state. Biodistribution and scintigraphy showed increased HL91 uptake during tumor hypoxia at 0.5 hours, and there was progressively increased activity for up to 4 hours after tracer administration. HL91 accumulation in tumor hypoxia was markedly increased in mice treated with hydralazine compared with those treated with PBS. Autoradiography revealed high HL91 uptake in the peripheral areas around the necrotic regions of the tumor, which were identified by histologic examination. HL91 exhibits selectivity for tumor hypoxia both in vitro and in vivo and provides a successful imaging modality for the detection of tumor hypoxia in vivo.

Reducing renal uptake of 111In-DOTATOC: a comparison among various basic amino acids.

PURPOSE: Several studies have reported significant renal toxicity after the use of a high dose of 90Y-DOTATOC. Thus, renal protection is necessary in treatments with 90Y-DOTA Tyr3-octreotide (DOTATOC). The infusion of certain positively charged amino acids has been shown to effectively reduce renal uptake of DOTATOC. In this study, we compared the effectiveness of three kinds of amino acids, D-lysine (lysine), L-arginine (arginine) and histidine, on renal protection in healthy rats and tried to determine which one was the most effective. METHODS: Twenty SD healthy male rats were divided into 4 groups: lysine, histidine, arginine, and control. The rats were injected with a dose of 400 mg/kg of amino acid or 2 ml of phosphate-buffered saline (PBS) (as control) intraperitoneally. All rats were sacrificed at 4 hrs after the injection of 1 MBq 111In-DOTATOC. Samples of the kidney were taken and weighed carefully. The counts of radioactivity were measured by a gamma counter and renal concentrations were calculated and expressed as percent injected dose per gram (% ID/g). RESULTS: The renal uptake of 111In-DOTATOC was significantly lower for all three kinds of amino acids when compared to the control group. The renal uptake of 111In-DOTATOC in the lysine group was significantly lower than those in the histidine and arginine groups. The renal uptake of 111In-DOTATOC in the histidine group was lower than that in the arginine group, but no statistical difference was noted. CONCLUSION: Among these three amino acids, lysine had the best reduction rate of renal uptake of DOTATOC. Histidine was more effective than arginine but no statistical difference was noted.

A comparison of biodistribution between 111In-DTPA octreotide and 111In-DOTATOC in rats bearing pancreatic tumors.

111In-DTPA octreotide (DTPAOC) has been used for detecting somatostatin receptor positive tumor for years. In-111 DOTA-Tyr3-octreotide (DOTATOC) is newly developed for diagnostic and therapeutic purposes. In this study, we compared the biodistribution and tumor uptake ratio after injection of In-111 DTPAOC and In-111 DOTATOC in rats. Twelve rats bearing pancreatic tumors were divided into two groups: six rats were sacrificed at 4 hr after injection of 3.7 MBq of In-111 DTPAOC and another 6 rats were sacrificed at the same time after injection of 3.7 MBq of In-111 DOTATOC. Samples of various organs were obtained and counted to calculate the tissue concentration. In addition, 12 rats bearing pancreatic tumors were scanned at 4, 24, and 48 hr after injection of 37 MBq of In-111 DTPAOC or In-111 DOTATOC. The tumor uptake ratios (T/N ratio) were calculated. The biodistribution data showed that the activity in the tumor as well as in the kidney was significantly higher in the In-111 DOTATOC group than in the In-111 DTPAOC group, although both radiopharmaceuticals had the expected high affinity to the tumor. The T/N ratios in the In-111 DOTATOC group were also significantly higher than those in the In-111 DTPAOC group at 24 hr after injection. We conclude that In-111 DOTATOC showed lower clearance than In-111 DTPAOC in the rats bearing pancreatic tumors, although both of these radiopharmaceuticals showed expected high tumor uptake.

In vitro comparison of conventional hyperthermia and modulated electro-hyperthermia.

Radiofrequency-induced hyperthermia (HT) treatments for cancer include conventional capacitive coupling hyperthermia (cCHT) and modulated electro-hyperthermia (mEHT). In this study, we directly compared these methods with regard to in vitro cytotoxicity and mechanisms of action under isothermal conditions. Hepatoma (HepG2) cells were exposed to HT treatment (42°C for 30 min) using mEHT, cCHT or a water bath. mEHT produced a much higher apoptosis rate (43.1% ± 5.8%) than cCHT (10.0% ± 0.6%), the water bath (8.4% ± 1.7%) or a 37°C control (6.6% ± 1.1%). The apoptosis-inducing effect of mEHT at 42°C was similar to that achieved with a water bath at 46°C. mEHT also increased expression of caspase-3, 8 and 9. All three hyperthermia methods increased intracellular heat shock protein 70 (Hsp70) levels, but only mEHT greatly increased the release of Hsp70 from cells. Calreticulin and E-cadherin levels in the cell membrane also increased after mEHT treatment, but not after cCHT or water bath. These results suggest that mEHT selectively deposits energy on the cell membrane and may be a useful treatment modality that targets cancer cell membranes.

The biological characterization of (99m)Tc-BnAO-NI as a SPECT probe for imaging hypoxia in a sarcoma-bearing mouse model.

OBJECTIVES: Tumor growth beyond the region where vascular oxygen can reach creates a hypoxic domain. In this study, BnAO, a ligand that had been labeled with (99m)Tc-pertechnetate for hypoxia imaging, was conjugated with 2-nitroimidazole to give 3,3,10,10-tetramethyl-1-(2-nitro-1H-imidazo-1-y1)-4,9-diazadodecane-2,11- dionedioxime (BnAO-NI) as a potential ligand for hypoxia detection. Pentoxifylline is a peripheral vasodilator and has been used as a radiosensitizer in tumor radiotherapy. (99m)Tc-BnAO-NI/SPECT was applied to noninvasively assess the pharmacological effect of pentoxifylline in reducing tumor hypoxia in vivo. METHODS: BnAO-NI was synthesized and formulated with methylene diphosphonate (MDP), stannous chloride and carbonate buffer to afford kits. After mixing with (99m)Tc-pertechnetate, (99m)Tc-BnAO-NI injection can be readily prepared. The partition coefficient, radiochemical purity and in vitro stability were determined. Cellular uptake of radiotracers in KHT cells under hypoxia was conducted in a CO(2) incubator at 37°C under hypoxia or normoxia. A biodistribution study after intravenous injection of (99m)Tc-BnAO-NI in KHT sarcoma-implanted C3H mice was performed. The effect of pentoxifylline (100 mg/kg) on reducing tumor hypoxia was also studied. RESULTS: The radiochemical purity (RCP) of the (99m)Tc-BnAO-NI preparation was greater than 96% and stable at ambient temperature for 24h (RCP>90%). The accumulation of (99m)Tc-BnAO-NI and (99m)Tc-BnAO in KHT cells under hypoxia were 3.57 and 4.13-fold higher than those under normoxic environment, indicating unambiguous oxygen-dependent uptakes of these two probes. The distribution of (99m)Tc-BnAO-NI in KHT sarcoma-bearing mice revealed rapid clearance from the blood circulation. The tumor uptake peaked at 2h post-injection (0.32 ± 0.05%ID/g) with tumor-to-blood and tumor-to-muscle ratios of 10.32 and 3.96, respectively. The effect of pentoxifylline on the tumor blood perfusion was obvious. The tumor-to-muscle ratios at 2h post-injection of (99m)Tc-BnAO-NI with and without pentoxifylline pretreatment were 1.67 ± 0.38 and 2.59 ± 0.25, respectively (p = 0.025, n = 3). CONCLUSION: This study demonstrates that (99m)Tc-BnAO-NI is a hypoxia-sensitive radio probe for monitoring hypoxic regions in a malignant neoplasm. However, (99m)Tc-BnAO-NI, though with higher lipophilicity than (99m)Tc-BnAO, did not achieve better specific accumulation in hypoxic tissues. (99m)Tc-BnAO-NI/SPECT could be applied in clinics to noninvasively evaluate the feasibility of using pentoxifylline as a radiosensitizer by reducing tumor hypoxia in vivo.

The preparation and biological characterization of a new HL91-derivative for hypoxic imaging on stroke mice.

AIM: (99m)Tc-HL91 (Prognox, GE-Healthcare) was the first nonnitro-aryl-based radiotracer for evaluating hypoxic fraction in neoplasm, stroke and myocardium infarction regions. However, the high hydrophilicity of (99m)Tc-HL91 might hamper its penetration into cells. In this study, we prepared a new ligand 4,4,11,11-tetramethyl- 5,10-diazatetradecane- 3,12-dionedioxime (HL91-ET) with higher lipophilicity but structurally similar compared with that of HL91. The chemical and biological characterizations of (99m)Tc-HL91-ET as a scintigraphic probe for hypoxia were performed with a stroke-bearing mouse model. MATERIALS AND METHODS: HL91-ET was synthesized and formulated with stannous chloride and buffer to afford kits. After mixing with (99m)Tc-pertechnetate, (99m)Tc-HL91-ET can be prepared in high yield and high radiochemical purity (both >96%). The partition coefficient of (99m)Tc-HL91-ET was determined in n-octanol/PBS system. Cellular uptake assays under normoxic and hypoxic conditions were performed in an oxygen-controlled CO(2) incubator. Brain stroke in the mouse model was induced by the electrocautery of the middle cerebral artery. After intravenous injection of (99m)Tc-HL91-ET into the Balb/c mouse suffering brain stroke, small-animal SPECT images were acquired at designated time points and autoradiography of the brain slides was conducted. Parallel studies of (99m)Tc-HL91 were also conducted at the same conditions for comparison. RESULTS: The higher partition coefficient of (99m)Tc-HL91-ET (0.294+/-0.007) indicated higher lipophlicity compared with that of (99m)Tc-HL91 (0.089+/-0.005). The (99m)Tc-HL91-ET preparation was stable at ambient temperature for 24h. Cellular uptake assay showed that (99m)Tc-HL91-ET was less selectively retained in hypoxic cells than (99m)Tc-HL91. The target-to-normal brain ratios derived from the autoradiograms of the brains of stroke mice were 1.31+/-0.02 and 17.47+/-0.10 (n=3), respectively, at 2h post injection of (99m)Tc-HL91-ET and (99m)Tc-HL91. CONCLUSIONS: This study revealed that (99m)Tc-HL91-ET, though with higher lipophilicity than (99m)Tc-HL91, did not suggest better specific accumulation in hypoxic cells or tissues than (99m)Tc-HL91. The uptake mechanism of (99m)Tc-HL91 was at least not solely by passive diffusion. Lipophilicity should not be the major consideration in designing HL91-derivatives for hypoxia imaging.