A simplified and miniaturized glucometer-based assay for the detection of ß-glucosidase activity.

ß-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of ß-glucosidase activity. Single-factor experiments showed that optimum ß-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of ß-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from ß-dextranase, snailase, ß-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for ß-glucosidase activity. The easy-to-operate method was successfully used to detect a series of ß-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of ß-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Drug-loadable Mesoporous Bioactive Glass Nanospheres: Biodistribution, Clearance, BRL Cellular Location and Systemic Risk Assessment via (45)Ca Labelling and Histological Analysis.

Mesoporous bioactive glass (MBG) nanospheres with excellent drug loading property have attracted significant attention in the field of nano-medicine. However, systemic metabolism and biosafety of MBG nanospheres which are crucial issues for clinical application are yet to be fully understood. Isotope quantitative tracing combined with biochemical parameters and histopatological changes were used to analyze biodistribution, excretion path and the effect on metabolism and major organs, and then we focused on the hepatocellular location and damaging effect of MBG. The results indicated MBG possessed a longer residence time in blood. After being cleared from circulation, nanospheres were mainly distributed in the liver and were slightly internalized in the form of exogenous phagosome by hepatocyte, whereby more than 96% of nanospheres were located in the cytoplasm (nearly no nuclear involvement). A little MBG was transferred into the mitochondria, but did not cause ROS reaction. Furthermore, no abnormal metabolism and histopathological changes was observed. The accumulation of MBG nanospheres in various organs were excreted mainly through feces. This study revealed comprehensively the systemic metabolism of drug-loadable MBG nanospheres and showed nanospheres have no obvious biological risk, which provides a scientific basis for developing MBG nanospheres as a new drug delivery in clinical application.

Evolution of a mesoporous bioactive glass scaffold implanted in rat femur evaluated by (45)Ca labeling, tracing, and histological analysis.

Mesoporous bioactive glass (MBG) as a biodegradable scaffold with a nanostructure has attracted significant attention. However, the in vivo evolution of MBG, which includes in situ degradation, the local effect induced by degradation, and the disposition of degradation products, remains unclear. In this study, we performed in situ labeling and synthesis of an MBG scaffold for the first time using (45)CaCl2. The obtained (45)Ca-MBG scaffolds possessed a mesoporous-macroporous cross-linked structure. These (45)Ca-MBG scaffolds were implanted in critical-sized rat femur defects (3 × 3 mm) for 1 day and for 1, 4, 8, and 12 weeks and analyzed by isotopic quantitative tracing. The results illustrated that the MBG scaffolds gradually degraded over time and persisted at a local level of approximately 9.63% at week 12. This finding suggests that only a very small amount of MBG-released calcium ions may have been transformed into calcium components of the new bone matrix. The research also confirmed that the active ingredients derived from the degradation of MBG scaffolds could actively regulate the mRNA expression levels of osteoblast-related genes in rat bone marrow-derived mesenchymal stem cells (rBMSCs) and promote bone regeneration in vivo. Moreover, through isotopic tracing of the entire body, (45)Ca, which disappeared in situ after implantation, could be detected in the heart, lungs, spleen, kidneys, intestines, and brain via the blood and was mainly accumulated in distal bone tissue, including the radius and cranium. However, (45)Ca radioactivity in the body tissues significantly decreased or disappeared after 12 weeks. Systemic toxicological studies on MBG scaffolds demonstrated the degradation products that spread to major organs did not cause abnormal histopathological changes. The above discoveries comprehensively address crucial issues regarding the application of MBG in vivo, and these findings provide a scientific basis for introducing a material with mesoporous structure into clinical applications.

Tissue distribution and excretion of intravenously administered titanium dioxide nanoparticles.

As the biosafety of nanotechnology becomes a growing concern, the in vivo nanotoxicity of nanoparticles (NPs) has been drawn an increasing attention. Titanium dioxide nanoparticles (TiO(2)-NPs) have been developed for versatile use, but the pharmacokinetics of intravenously administered TiO(2)-NPs have not been investigated extensively. In the present study, the rutile-type TiO(2)-NPs with a size about 20nm were labeled with CF680 and (125)I. The labeled TiO(2)-NPs were injected in mice or rats with the concentration of 1mg/ml and the dose of 10mg/kg body weight and their tissue distribution and excretion were investigated by using ex vivo fluorescent imaging, γ-counter and TEM. The results indicated that the TiO(2)-NPs mainly accumulated in liver and spleen and could be retained for over 30days in these tissues due to the phagocytosis by macrophages. The excretion assay found that the excretory rate of TiO(2)-NPs through urine was higher than that of feces, indicating that renal excretion was the main excretion pathway of TiO(2)-NPs. Overall results of the present study provided important information on distribution and excretion of TiO(2)-NPs in vivo, which would greatly promote the pharmacokinetics and in vivo nanotoxicity research of TiO(2)-NPs.

Pharmacokinetics, tissue distribution and excretion of porcine fibrinogen after intraperitoneal injection of a porcine-derived fibrin glue to rats.

The aim of the present study was to characterize the preclinical pharmacokinetics, tissue distribution and excretion profiles of porcine fibrinogen in rats after intraperitoneal injection of a porcine-derived fibrin glue. A sensitive and rapid isotope-labeled assay method was developed and validated for quantitative analysis in biological analysis. Porcine fibrinogen, the major composition of the fibrin glue, was radioiodinated with Na(125)I using the Iodo-Gen method. Following the purification and identification of (125)I-porcine fibrinogen, the fibrin glue containing (125)I-porcine fibrinogen was intraperitoneally administered to rats at three single dosages (100, 200, 400mg/kg of porcine fibrinogen). The results showed that the (125)I-labeled assay method was suitable for the quantification of porcine fibrinogen in plasma samples, tissue samples and excreta samples with satisfactory linear (r(2)>0.998), precision (<13%), accuracy (95.9-104.2%) and recovery (>85%). After three single administrations, plasma concentration profiles showed a slow absorption phase with the mean t(max) of 1.83-5.67 h and a slow elimination proceeding with the terminal elimination half-life (T(1/2)) of 84.5-96.3h. Porcine fibrinogen was widely distributed to most of the tissues examined after a single intraperitoneal administration at 200mg/kg to rats. The radioactive porcine fibrinogen showed substantial disposition in liver, kidneys, stomach and intestine. Approximately 79.3% and 17.2% of administered radioactivity were recovered in urine and feces within 528 h post-dosing, which indicated the major elimination route was urinary excretion.

Cyclic RGD-poly(ethylene glycol)-polyethyleneimine is more suitable for glioblastoma targeting gene transfer in vivo.

Arginine-glycine-aspartic acid (RGD) is a widely chosen ligand to improve the specific gene targeting transfection efficiency of polyethyleneimine (PEI) in vivo. However, the optimal RGD conjugating mode, RGD-poly(ethylene glycol)-PEI (RGD-PEG-PEI) or RGD-PEI-methoxyl poly(ethylene glycol) (RGD-PEI-mPEG) still remains controversial. In this study, RGD-PEG-PEI and RGD-PEI-mPEG were synthesized and compared with respects to their glioblastoma cell-binding capability and tumor-targeting ability of their complexes with plasmid DNA. These results demonstrated that RGD-PEG-PEI/plasmid enhanced green fluorescent protein (pEGFP)-N2 complexes had higher binding affinities with U87 cells than RGD-PEI-mPEG/pEGFP-N2 complexes. The gene transfection was also performed on U87 cells in vitro and in vivo. In vitro, both of the RGD-modified PEI derivatives enhanced the gene transfection efficiency to some extent. However, all of the complexes (with or without RGD modification) had high transfection efficiency. The biodistribution of RGD-PEG-PEI/pEGFP-N2 complexes in mice bearing subcutaneous glioblastomas were significantly greater than that of RGD-PEI-mPEG/pEGFP-N2 complexes, suggesting a more efficient gene transfection in vivo. In the RGD-PEG-PEI, the use of a PEG spacer was particularly important. These results indicated that RGD-PEG-PEI was more suitable for targeted gene transfer in vivo.

Effects of uranium depletion on 1α-hydroxylase in kidney of rats.

This study was designed to evaluate the effects of depleted uranium (DU) on 1α-hydroxylase in the kidney of rats and to delinerate the mechanism of damage to kidneys and bones by DU. Male Sprague-Dawley rats were surgically implanted with DU fragments at three dose levels (0.1 g, 0.2 g and 0.3 g). After 3, 6 or 12 months, the concentration of 1α,25(OH)(2)D(3) in the kidney was measured by radioimmunoassay. The activity of 1α-hydroxylase was shown by the production of 1α,25(OH)(2)D(3) after incubation. The results showed that the 1α-hydroxylase activity in the kidney was decreased after 3 months (27.2% at the medium dose DU group, p < 0.05; 33.4% at the high dose DU group, p < 0.01). In contrast, at 6 months and 12 months after implantation of DU, the activity of renal 1α-hydroxylase in DU-treated animals was not decreased significantly in comparison with the controls (p > 0.05). On the other hand, the activity of renal 1α-hydroxylase was decreased by 33.1% (p < 0.05) and 34.4% (p < 0.01) in blank control groups at 6 and 12 months, respectively, when compared with the blank control group at 3 months. In conclusion, this study showed that chronic DU exposure could induce renal damages and inhibit the synthesis of biologically active form of vitamin D, which may be the underlying mechanism of bone metabolic disorder caused by renal injury after DU exposure.

[Transthyretin-binding activity of hexabromocyclododecanes (HBCDs) and its thyroid hormone disrupting effects after developmental exposure].

In vivo and in vitro research approaches were carried out to survey the potential health risk of environmental exposure by hexabromocyclododecanes (HBCDs). Transthyretin-binding assay was designed to test for the potency of HBCDs to compete with thyroxine (T4) for binding to the transport protein. The results showed that the binding of 25I-T4 and T4 was only slightly inhabited even at the highest competitive concentration of HBCDs (75.08%, 80 micromol x L(-1)), indicating the marginally interfere potency of HBCDs in the transportation of T4. Sprague-Dawley rats of 3-days old were exposed to 0.2 mg/kg and 1 mg/kg HBCDs for 21 d to examine the thyroid hormones (THs) disrupting effects of HBCDs after developmental exposure. Compared with the controls, levels of total 3,3',5-triiodothyronine (TT3), free 3,3',5-triiodothyronine (FT3), increased significantly (p < 0.05, p < 0.05) in low- and high-dose exposures, thyroid stimulating hormone (TSH) also increased slightly while the total thyroxine (TT4), free thyroxine (FT4) had a decline about two-fold inversely. Combined both the in vivo and in vitro results, the possible mode of action of HBCDs on THs disruption may through the synergy or substitution effect of T3. The findings support further investigation of the potential THs disrupting effects of HBCDs on public health, especially on children during brain development.

Hydroxyapatite nanoparticles as a controlled-release carrier of BMP-2: absorption and release kinetics in vitro.

Recently, nanoparticles have been extensively developed as controlled-release carriers; however, there has been little research on hydroxyapatite nanoparticles (HANPs) and their potential applications. In this study, HANPs were investigated as a controlled-release carrier of bone morphogenetic protein-2 (BMP-2), the absorption and release kinetics of which were analyzed in vitro. Different concentrations of BMP-2 solution were used to evaluate the adsorptive properties of HANPs. It was observed that the amount of BMP-2 adsorbed onto HANPs could be as high as 70 mug/mg and that adsorption rate was highly correlated with the concentration of BMP-2 solution used. After absorption, the suspension of HANPs absorbed BMP-2 (HANPs/BMP-2) was incubated at 37 degrees C for 15 days and the release kinetics of BMP-2 from HANPs/BMP-2 was determined daily. The release profile showed sustained release of BMP-2 over the period of the investigation. Collectively, these results suggest that HANPs has the potential to function as a carrier for drug delivery systems and as a scaffold material in bone tissue engineering.

Roles of dextrans on improving lymphatic drainage for liposomal drug delivery system.

Our aim was to develop a novel liposomal drug delivery system containing dextrans to reduce undesirable retention of antineoplastic agents and thus alleviate local tissue damage. At the cell level, diethylaminoethyl-dextran (DEAE-Dx) showed the strongest inhibiting effect on liposome uptake by macrophages among tested dextrans. The distribution of radiolabeled liposomes mixed with dextrans in injection site and draining lymph node was investigated in rats after subcutaneous injection. DEAE-Dx substantially reduced the undesired local retention and promoted the draining of liposome into lymphatics, which was further confirmed by confocal microscopy images revealing the substantial prevention of rhodamine B-labelled liposome sequestration by macrophages in normal lymph node in rats. Pharmacokinetic data indicated the accelerated drainage of liposome through lymphatics back to systemic circulation by mixing with DEAE-Dx. In the toxicological study in rabbits, DEAE-Dx alleviated the local tissue damage caused by liposomal doxorubicin. In conclusion, dextrans, particularly DEAE-Dx, could efficiently enhanced liposomes drainage into lymphatics, which proves themselves as promising adjuvants for lymphatic-targeted liposomal drug delivery system.

Evaluation of inherent toxicology and biocompatibility of magnesium phosphate bone cement.

Magnesium phosphate cement (MPC) is a kind of novel biodegradable bone adhesive for its distinct performance. However, there is few research work concerning on the systemic biocompatibility and genetic toxicological evaluation of MPC. In this study, the investigation on the inherited toxicology of MPC including gene mutation assay (Ames test), chromosome aberration assay (micronucleus test), and DNA damage assay (unscheduled DNA synthesis test) were carried out. Fracture healing and degradation behavior were explored for the evaluation of the biocompatibility of MPC, using macroscopical histological, histomorphometrical, and scanning electron microscopical methods. The results of mutagenicity and potential carcinogenicity of MPC extracts were negative, and the animal implantation illustrated no toxicity and good resorption. The study suggested that bioresorbable MPC was safe for application and might have potential applications for physiological fracture fixation.

Biodistribution and toxicity of intravenously administered silica nanoparticles in mice.

As the biosafety of nanotechnology becomes a growing concern, the in vivo nanotoxicity of NPs has drawn a lot of attention. Silica nanoparticles (SiNPs) have been widely developed for biomedical use, but their biodistribution and toxicology have not been investigated extensively in vivo. Although investigations of in vivo qualitative distribution of SiNPs have been reported, the time-dependent and quantitative informations about the distribution of SiNPs are still lacking. Here we investigated the long-term (30 days) quantitative tissue distribution, and subcellular distribution, as well as potential toxicity of two sizes of intravenously administered SiNPs in mice using radiolabeling, radioactive counting, transmission electron microscopy and histological analysis. The results indicated that SiNPs accumulate mainly in lungs, liver and spleen and are retained for over 30 days in the tissues because of the endocytosis by macrophages, and could potentially cause liver injury when intravenously injected.

[Vascular endothelial cells targeted Tyr-RGD-PEG-PEI nano-drug synthesis and its biological activity].

The study is designed to synthesize nano-carrier Tyr-RGD (cyclo-[Arg-Gly-Asp-d-Tyr-Lys]) and poly(ethylene glycol) modified polyethylenimine (Tyr-RGD-PEG-PEI) targeting vascular endothelial cells, then analyze its nanoparticle properties and the characteristics of drug carrying and targeting properties in vivo / in vitro tumor. The nano-carrier Tyr-RGD-PEG-PEI was synthesized with the method of chemical synthesis and the properties of this nanoparticle and drug carrying characteristics were identified. Its effect of targeting vascular endothelial cells in vitro was studied with the method of competitive binding assay. The fluorescent labeled nano-drug was injected into tumor-bearing nude mice to observe its tumor-targeting. The mean size of nano-carrier Tyr-RGD-PEG-PE was about 145 nm, good in encapsulation efficiency of siRNA. After incubation in plasma for half an hour, only about 3 percent of siRNA out. It was confirmed that it was a single spot with TLC analysis, the R(f) value was 0.65. Receptor competition experiments showed that the nano could effectively compete with RGD in binding the receptors on endothelial cells. Tumor-bearing nude mice experiments showed that when containing a fluorescent-labeled siRNA of Tyr-RGD-PEG-PEI nano-drug was injected into mice, after 24 hours this nano-drug mainly distributed within the tumor tissue. However, nano-drug without Tyr-RGD appeared in tumor tissue as well as other organs such as livers, lungs, etc. The Tyr-RGD-targeted gene vector Tyr-RGD-PEG-PEI synthesized in this study has good nanoparticle properties and high efficiency of gene-drug encapsulation. Study of nude mice shows that the ability of its tumor-targeting is significantly better than nano-drug without Tyr-RGD.

[Folate-poly-L-lysine-Gd-DTPA as MR contrast agent for tumor imaging via folate receptor-targeted delivery].

OBJECTIVE: To study folate-conjugated Gd-DTPA-Poly-L-Lysine (folate-PL-Gd-DTPA) as MR targeting agent to tumor cells via folate receptor, to evaluate feasibility and effectiveness by observing MR signal variations and imaging feature of pulmonary tumor xenografts in nude mice using this contrast material. METHODS: (1) Using Poly-L-Lysine (PL) as linker, after PL was tethered with caDTPA, GdCl(3) was added to label DTPA-PL, then PL-Gd-DTPA was conjugated to folate, a specific MR contrast agent, was thus prepared. (2) Using high performance liquid chromatography (HPLC) to evaluate the conjugate purity, and the ICP-AES to test Gd(3+) concentration, while the activity evaluated by competitive folate receptor binding with folic acid. (3) Folate-PL-Gd-DTPA as specific contrast agents (study group, n = 6) and Gd-DTPA as non-specific contrast agents (control group, n = 4) was injected respectively into caudal vein of the nude mice which was pulmonary tumor xenografts as experimental model in the study. MRI was performed with plain scans, enhanced scans at 30 minutes, 3 hours, 6 hours, 14 hours, 24 hours, 38 hours, 48 hours, 62 hours and 72 hours after the success of injection. Signal intensities of tumors and muscles were measured. RESULTS: (1) folate-PL-Gd-DTPA was successfully synthesized with high affinity to folate receptor and high concentration of Gd(3+) (56 Gd(3+)/folate). (2) folate-PL-Gd-DTPA had an excellent tumor selectivity in pulmonary tumor xenografts in the animal model. After injection, the tumor signal intensity in the study group was significantly higher than that observed before injection; An average intensity increase of 125.4% was observed from pre-contrast to post-contrast images of the tumor, which was observed at 24 - 48 hours after injection; The muscle signal intensity at any time-point after injection showed no statistically difference with that observed before injection. In control group, the tumor signal intensity showed statistically difference with that observed before injection at 0.5 hour and 3 hours, the biggest difference appeared at 0.5 hour; The muscle signal intensity at 0.5 hour time-point showed statistically difference with that observed before injection. CONCLUSION: Folate-PL-Gd-DTPA could be combined to tumor cells appetencially via folate receptor and significantly targeted to tumor cells with rich folate receptors for MR imaging.

Synthesis and biological evaluation of diethylenetriamine pentaacetic acid-polyethylene glycol-folate: a new folate-derived, (99m)Tc-based radiopharmaceutical.

(99m)Technetium-labeled diethylenetriamine pentaacetic acid-polyethylene glycol-folate (DTPA-PEG-folate) was synthesized and tested as a radiopharmaceutical agent, which targeted the lymphatic system with metastatic tumor. Folic acid was reacted with H2N-PEG-NH2 to yield H2N-PEG-folate. After purification by anion-exchange chromatography, the product was reacted with cyclic DTPA. By removal of unreacted DTPA by size-exclusion chromatography, DTPA-PEG-Folate was obtained. Fluorescein-5-isothiocyanate (FITC)-labeled DTPA-PEG-folate and DTPA-PEG-OCH3 were prepared via a dicyclohexylcarbodiimide-mediated coupling. In vitro competitive binding test showed that the uptake of [125I] folic acid was inhibited by DTPA-PEG-folate and the 50% inhibitory concentration was 4.37 pmol/L (R2 = 0.9922). The relative affinity of DTPA-PEG-FITC was 0.18 for human folate receptor comparing with folic acid. In cultured tumor cells, uptake of fluorescence-labeled DTPA-PEG-folate was found to increase significantly in folate-deficient medium compared with that of untargeted DTPA-PEG-OCH3 and FITC-ethylenediamine. The competition with free folic acid blocked the cell uptake of DTPA-PEG-folate. These results confirmed the DTPA-PEG-folate entered into KB cells through the folate receptor endocytosis pathway in vitro. The radiolabeled yield of [(99m)Tc] DTPA-PEG-folate was in excess of 98%, and specific activities of 7.4 kBq (0.2 microCi/microg) were achieved. After subcutaneous injection, [(99m)Tc] DTPA-PEG-folate exhibited an initial increase and successive decline of accumulation in popliteal nodes in normal Wistar rats. Expect for the kidney, uptake by other tissues was rather low. In a normal rabbit imagine study, the lymphatic vessels were readily visualized by single-photon-emission computed tomography following subcutaneous injection of [(99m)Tc] DTPA-PEG-folate. In conclusion, the [(99m)Tc] DTPA-PEG-folate conjugate may have a potential as a lymphatic tumor-targeted radiopharmaceutical.

[Research on radioimmunotherapy of lung cancer in nude mice using lung cancer monoclonal antibody LC-1 combined with 9°Y].

BACKGROUND: To evaluate potential clinical roles of monoclonal antibody (MoAb)-based radioimmunotherapy in the treatment of lung cancer. METHODS: Anti-lung cancer monoclonal antibody LC-l IgM was combined with 9°Y to produce radioimmunological targeting drug. Human lung cancer tissue was inoculated subcutaneously in 35 nude mice. They were randomized into seven groups while the tumor was 5 mm in diameter. The groups were divided by the dose of the drug injected to the tail vein of the mice: blank group, LC-1 IgM alone, 9°Y 50 µCi alone, 50 µCi, 150 µCi, 300 µCi, and 400 µCi combined therapy groups. The size of the tumor was measured weekly and the mice were killed in four weeks after treatment. The tumors were resected and weighed. RESULTS: As compared to blank group, therapy groups' tumor growth was inhibited and the inhibition was dose- and time-dependent. The inhibition rates of 300 µCi and 400 µCi groups were significantly different (P < 0.05). The nuclei of tumor cells showed karyopycnosis, structure disorder and rupture after treatment by pathological exam. In some regions, the tumor cells were necrotic or disappeared. CONCLUSIONS: The results indicate that the radioimmunological drug made from lung cancer monoclonal antibody LC-1 IgM and 9°Y can specifically localise in tumor tissue and ensure radioimmunological targeting therapy, so has underlying clinical value.

[Study on the permeability of 125I-sinomenine via the transdermal delivery way in rats].

OBJECTIVE: To study the permeance and diffusion trend of 125I-sinomenine via the transdermal delivery way in rats. METHODS: Assay the permeance depth and diffusion area of sinomenine labeled by isotopic 125I in subcutaneous of rats certain time after sinomenine was administrated on the cervical skin of rats. RESULTS: The diffuse of sionmenine can reach cervical spine in longitudinal direction and form an area of 2 cm2 in transverse direction. The concentration gradient of both directions are significant. CONCLUSION: 125I-sinomenine can not only be absorbed into blood via the transdermal delivery way to have whole effect, but also accumulate large amounts in the skin to have a local effect.