pH-responsive poly(lactide-co-glycolide) nanoparticles containing near-infrared dye for visualization and hyaluronic acid for treatment of osteoarthritis.

This study focuses on intra-articular (IA) drug delivery system for the treatment of knee osteoarthritis (OA). In osteoarthritic condition the synovial fluid presents pockets with lower pH environment. To take advantage of these pH differences, poly(lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and pH- responsive PLGA NPs encapsulated with ammonium bicarbonate (NH4HCO3) were generated. The nanoparticles were loaded with hyaluronic acid (HA) as a possible model drug for OA and with near-infrared dye (NIR) that was used to visualize the NPs with molecular imaging techniques. These NPs were characterized by dynamic light scattering, transmission electron microscopy and compared in in vitro, in vivo and ex vivo experiments in the treatment of OA. The results indicate that the NPs were sufficiently small, displayed a uniform size distribution and were non-toxic both in vitro and in vivo. Both NPs treatment seem to induced a reduction in OA progression, with pH- responsive NPs showing the more pronounced effect. This is probably because the pockets of low pH environment in the synovial fluid trigger a burst release of the pH-responsive NPs. This result is corroborated by in vitro experiments since the pH- responsive NPs showed an extracellular burst release behavior and higher chondrocyte vitality than non-responsive NPs. This study demonstrates that PLGA NPs containing HA and NH4HCO3 are candidates for the treatment of knee OA.

Dissociation of binding and antiresorptive properties of hydroxybisphosphonates by substitution of the hydroxyl with an amino group.

The purpose of this study was to examine the role of the R1 moiety of bisphosphonates in binding to bone mineral and for antiresorptive action. For this, the R1 chain of three clinically useful hydroxybisphosphonates (etidronate, pamidronate, and olpadronate) was substituted with an amino group. The effects of the aminosubstituted bisphosphonates were compared with those of their hydroxy counterparts in a crystal growth assay and in fetal mouse long bone cultures which are representative of bisphosphonate actions in vivo. It was found that all three aminosubstituted compounds and their hydroxy analogs bound with similar affinity to bone mineral and inhibited the growth of calcium oxalate crystals to the same extent. Surprisingly, the antiresorptive effect of olpadronate was totally abolished by the amino substitution of the hydroxyl group while that of pamidronate was reduced by about six-fold and that of etidronate did not change. These studies demonstrate the involvement of the entire bisphosphonate molecule in the cellular mechanism of antiresorptive action. In addition, the aminosubstituted analog of olpadronate, which lacks any antiresorptive action but retains all other properties of olpadronate, provides an excellent tool for the study of specific cellular effects involved in bisphosphonate action.

Plasma nitrate+nitrite levels are regulated by ovarian steroids but do not correlate with trabecular bone mineral density in rats.

Nitric oxide (NO) is a mediator of bone metabolism and its production is under the control of gender hormones in several cell types or tissues. Changes in endogenous NO production, measured as plasma nitrate+nitrite levels, may therefore contribute to ovariectomy (OVX)-induced bone loss. We studied plasma nitrate+nitrite levels and trabecular bone mineral density (TBMD) 4 weeks after sham-operation or OVX in rats receiving various hormonal treatments. OVX decreased plasma nitrate+nitrite levels significantly and this was accompanied by a significant decrease in TBMD. Treatment with oral ethinyl oestradiol (EE) and subcutaneous 17beta-oestradiol dose-dependently prevented the decrease in plasma nitrate+nitrite levels after OVX, but treatment with oral 17beta-oestradiol did not. Oestrogen treatment, 17beta-oestradiol (s. c. or orally) or EE (orally), prevented the OVX-induced decrease in TBMD. Treatment of sham-operated rats with the anti-oestrogen ICI164, 384 induced a significant decrease in TBMD that corresponded to 54% of the decrease observed after OVX, but did not affect plasma nitrate+nitrite levels. Treatment of ovariectomized rats with Org 2058, a pure progestagen, did not prevent bone loss, but prevented the decrease in plasma nitrate+nitrite levels dose-dependently. Treatment with tibolone, a synthetic steroid with combined weak oestrogenic, progestagenic, and androgenic properties, or with progestagen in combination with EE completely prevented bone loss after OVX. These treatments, however, only partly prevented the OVX-induced decrease in plasma nitrate+nitrite levels. In conclusion, OVX decreased both TBMD and plasma nitrate+nitrite levels. Although plasma nitrate+nitrite levels were under the control of both oestrogen and progesterone, TBMD was affected by oestrogen only. Decreased systemic production of NO is, therefore, not involved in OVX-induced bone loss in rats.

Modulation of calcium oxalate monohydrate crystallization kinetics by urine of preterm neonates.

Preterm neonates frequently develop nephrocalcinosis (NC). However, the cause has not yet been elucidated. This study focuses on the effects of urine from preterm neonates on crystallization kinetics. Urine samples were collected and renal ultrasound examinations of preterm neonates (gestational age < 32 weeks) were performed during the first weeks of life, at term, and ages 6, 12, and 24 months. The effect of urine on crystallization was determined using a seeded crystal growth system, which measures the square root of solubility product ( radicalLc), percentage of growth inhibition (GI), and agglomeration inhibition ([tm]) of calcium oxalate crystals. Data for preterm neonates in the first weeks of life (n = 19) were compared with those for full-term neonates (n = 17) and healthy adults. Moreover, the correlation between [tm] and urinary (U)citrate level was studied. Mean radicalLc (0.27 +/- 0.1 versus 0.36 +/- 0.08 mmol/L) and mean [tm] (81 +/- 32 versus 143 +/- 97 minutes) were lower and mean Ucalcium-creatinine (2.20 +/- 1.74 versus 0.46 +/- 0.73 mol/mol) and Uoxalate-creatinine ratios (0.39 +/- 0.21 versus 0.16 +/- 0.09 mol/mol) were greater in preterm neonates in the first weeks of life compared with full-term neonates (p < 0.05). Furthermore, [tm] was less than the lower limit for healthy adults for all but one preterm neonate; [tm] increased and Ucalcium-creatinine and Uoxalate-creatinine ratios decreased with age (p < 0.005). There was a correlation between [tm] and citrate excretion (coefficient of 38; P < 0.001). Patients with and without NC at term did not differ statistically in mean radicalLc, percentage of GI, or [tm]. In conclusion, urine from preterm neonates in the first weeks of life is highly supersaturated and has a defective ability to inhibit calcium oxalate crystal agglomeration. This ability improves with age and is citrate mediated. We suggest that both the high level of supersaturation and defective ability to inhibit calcium oxalate crystal agglomeration contribute to the high incidence of NC.

Reestablishment of in vitro and in vivo iodide uptake by transfection of the human sodium iodide symporter (hNIS) in a hNIS defective human thyroid carcinoma cell line.

Uptake of iodide is a prerequisite for radioiodine therapy in thyroid cancer. However, loss of iodide uptake is frequently observed in metastasized thyroid cancer, which may be explained by diminished expression of the human sodium iodide symporter (hNIS). Strategies to restore iodide uptake in thyroid cancer include the exploration of hNIS gene transfer into hNIS defective thyroid cancer. In this study, we report the stable transfection of a hNIS expression vector into the hNIS defective follicular thyroid carcinoma cell line FTC133. Stablely transfected colonies exhibited high uptake of Na125I, which could be blocked completely with sodiumperchlorate. hNIS mRNA expression corresponded with iodide uptake in semiquantitative polymerase chain reaction. Iodide uptake was maximal after 60 minutes, whereas iodide efflux was complete after 120 minutes. hNIS transfected FTC133 and control cell lines injected subcutaneously in nude mice formed tumors after 6 weeks. Iodide uptake in the hNIS transfected tumor was much higher than in the nontransfected tumor, which corresponded with hNIS mRNA expression in tumors.

Expression of the human sodium/iodide symporter (hNIS) in xenotransplanted human thyroid carcinoma.

The uptake of iodide in thyroid epithelial cells is mediated by the sodium/iodide symporter (NIS). The uptake of iodide is of vital importance for thyroid physiology and is a prerequisite for radioiodine therapy in thyroid cancer. Loss of iodide uptake due to diminished expression of the human NIS (hNIS) is frequently observed in metastasized thyroid cancer. So far, no animal model for the study of radioiodine therapy in thyroid cancer has been available. Strategies to restore iodide uptake in thyroid cancer include the exploration of hNIS gene transfer into hNIS defective thyroid cancer. We have performed a stable transfection of hNIS into the hNIS defective follicular thyroid carcinoma cell line FTC133. Stably transfected colonies exhibited high uptake of Na125I, which could be blocked completely with sodium perchlorate. hNIS transfected FTC133 and non-transfected cell lines injected subcutaneously in nude mice formed tumors after 6 weeks. Iodide uptake in the hNIS transfected tumor was much higher than in non-transfected tumor, but a rapid release of radioactivity from the hNIS transfected tumor was observed. Further studies are necessary to investigate the role of hNIS in relation to other thyroid specific proteins in iodide metabolism in thyroid cancer.

Optical imaging of cell death in traumatic brain injury using a heat shock protein-90 alkylator.

Traumatic brain injury is a major public health concern and is characterised by both apoptotic and necrotic cell death in the lesion. Anatomical imaging is usually used to assess traumatic brain injuries and there is a need for imaging modalities that provide complementary cellular information. We sought to non-invasively image cell death in a mouse model of traumatic brain injury using a near-infrared fluorescent conjugate of a synthetic heat shock protein-90 alkylator, 4-(N-(S-glutathionylacetyl) amino) phenylarsonous acid (GSAO). GSAO labels both apoptotic and necrotic cells coincident with loss of plasma membrane integrity. The optical GSAO specifically labelled apoptotic and necrotic cells in culture and did not accumulate in healthy organs or tissues in the living mouse body. The conjugate is a very effective imager of cell death in brain lesions. The optical GSAO was detected by fluorescence intensity and GSAO bound to dying/dead cells was detected from prolongation of the fluorescence lifetime. An optimal signal-to-background ratio was achieved as early as 3 h after injection of the probe and the signal intensity positively correlated with both lesion size and probe concentration. This optical GSAO offers a convenient and robust means to non-invasively image apoptotic and necrotic cell death in brain and other lesions.

Normalized volume of interest selection and measurement of bone volume in microCT scans.

Quantification of osteolytic lesions in bone is pivotal in the research of metastatic bone disease in small animal models. Osteolytic lesions are quantified using 2D X-ray photographs, which often neglects to take into account any changes in 3D structure. Furthermore, measurement errors are inadvertently introduced when a region of interest with predefined dimensions is used during MicroCT analysis. To study osteolytic processes, a normalized method of selecting a region of interest is required. Here we describe a new method to select volumes of interest in a normalized way regardless of curvature, fractures or dislocations within the bone. In addition, this method enables the user to visualize normalized cross sections in an exact 90° angle or along the longitudinal axis of bone, at any given point. As a result, the user can compare measurements of diameter, volume and structure between different bones in a normalized manner.

Intraoperative near-infrared fluorescence imaging of colorectal metastases targeting integrin α(v)ß(3) expression in a syngeneic rat model.

AIM: Near-infrared (NIR) fluorescence optical imaging is a promising technique to assess the extent of colorectal metastases during curative-intended surgery. However, NIR fluorescence imaging of liver metastases is highly challenging due to hepatic uptake and clearance of many fluorescent dyes. In the current study, the biodistribution and the ability to demarcate liver and peritoneal metastases were assessed during surgery in a syngeneic rat model of colorectal cancer using an integrin α(v)ß(3)-directed NIR fluorescence probe. METHODS: Liver tumors and peritoneal metastases were induced in 7 male WAG/Rij rats by subcapsular inoculation of 0.5 × 10(6) CC531 colorectal cancer rat cells into three distinct liver lobes. Intraoperative and ex vivo fluorescence measurements were performed 24 (N = 3 rats, 7 tumors) and 48 h (N = 4 rats, 9 tumors) after intravenous administration of the integrin α(v)ß(3)-directed NIR fluorescence probe. RESULTS: Colorectal metastases had a minimal two-fold higher NIR fluorescence signal than healthy liver tissue and other abdominal organs (p < 0.001). The tumor-to-background ratio was independent of time of imaging (24 h vs. 48 h post-injection; p = 0.31), which facilitates flexible operation planning in future clinical applications. Total fluorescence intensity was significantly correlated with the size of metastases (R(2) = 0.92 for the 24 h group, R(2) = 0.96 for the 48 h group). CONCLUSION: These results demonstrate that colorectal intra-abdominal metastases can be clearly demarcated during surgery using an integrin α(v)ß(3) targeting NIR fluorescence probe. Translating these findings to the clinic will have an excellent potential to substantially improve the quality of cancer surgery.

Whole body optical imaging in small animals and its translation to the clinic: intra-operative optical imaging guided surgery.

Whole body optical imaging using bioluminescence or fluorescence is one of the most rapidly emerging technologies to non-invasively follow all kinds of molecular and cellular processes in small animals. Using tomographic approaches it is now also possible to get better quantitative data. Due to its sensitivity and simplicity it is now also widely used in drug development and drug screening. Finally, using near infrared fluorescent probes that have much deeper penetration also opens up new exciting applications such as intra-operative image guided surgery for sentinel lymph node mapping and radical resection of tumours. Recent advances in imaging strategies that reveal cellular and molecular biological events in real-time facilitate our understanding of biological processes occurring in living animals. The development of molecular tags, such as green fluorescent protein (GFP) from the jellyfish Aequorea victoria, red fluorescent proteins (RFP) from the Discosoma species (dsRed2) and luciferase (Luc) from the firefly Photinus pyralis (fLuc) and the sea pansy Renilla (rLuc), has revolutionised research over the past decade, allowing complex biochemical processes to be associated with the functioning of proteins in living cells. Optical technologies, both microscopic and macroscopic, are developing fast. Recent technical advances for imaging weak visible light sources using cooled charged coupled device (CCCD) cameras, peltier cooled detectors and micro-plate channel intensifiers allow detection of photon emission from inside the tissues of small animals. Whole body fluorescent imaging (FLI) and bioluminescent imaging (BLI) are now applied to study cell- and tissue-specific gene promoter activity and also to follow trafficking, differentiation and fate of i.e. GFP or RFP and/or luciferase expressing cells, or biological processes like apoptosis, protein-protein interaction, angiogenesis, proteolysis and gene-transfer. Optical imaging (OI) and optical reporter systems are also very cost-effective and time-efficient and they are particularly well suited for small animal imaging and for in vitro assays to validate different reporter systems.

Hyperlipidaemia is associated with increased insulin-mediated glucose metabolism, reduced fatty acid metabolism and normal blood pressure in transgenic mice overexpressing human apolipoprotein C1.

AIMS/HYPOTHESIS: Insulin resistance for glucose metabolism is associated with hyperlipidaemia and high blood pressure. In this study we investigated the effect of primary hyperlipidaemia on basal and insulin-mediated glucose and on non-esterified fatty acid (NEFA) metabolism and mean arterial pressure in hyperlipidaemic transgenic mice overexpressing apolipoprotein C1 (APOC1). Previous studies have shown that APOC1 transgenic mice develop hyperlipidaemia primarily because of an impaired hepatic uptake of very low density lipoprotein (VLDL). METHODS: Basal and hyperinsulinaemic (6 mU.kg-1.min-1), euglycaemic (7 mmol/l) clamps with 3(-)3H-glucose or 9,10(-)3H-palmitic acid infusions and in situ freeze clamped tissue collection were carried out. RESULTS: The APOC1 mice showed increased basal plasma cholesterol, triglyceride, NEFA and decreased glucose concentrations compared with wild-type mice (7.0 +/- 1.2 vs 1.6 +/- 0.1, 9.1 +/- 2.3 vs 0.6 +/- 0.1, 1.9 +/- 0.2 vs 0.9 +/- 0.1 and 7.0 +/- 1.0 vs 10.0 +/- 1.1 mmol/l, respectively, p < 0.05). Basal whole body glucose clearance was increased twofold in APOC1 mice compared with wild-type mice (18 +/- 2 vs 10 +/- 1 ml.kg-1.min-1, p < 0.05). Insulin-mediated whole body glucose uptake, glycolysis (generation of 3H2O) and glucose storage increased in APOC1 mice compared with wild-type mice (339 +/- 28 vs 200 +/- 11; 183 +/- 39 vs 128 +/- 17 and 156 +/- 44 vs 72 +/- 17 mumol.kg-1.min-1, p < 0.05, respectively), corresponding with a twofold to threefold increase in skeletal muscle glycogenesis and de novo lipogenesis from 3-(3)H-glucose in skeletal muscle and adipose tissue (p < 0.05). Basal whole body NEFA clearance was decreased threefold in APOC1 mice compared with wild-type mice (98 +/- 21 vs 314 +/- 88 ml.kg-1.min-1, p < 0.05). Insulin-mediated whole body NEFA uptake, NEFA oxidation (generation of 3H2O) and NEFA storage were lower in APOC1 mice than in wild-type mice (15 +/- 3 vs 33 +/- 6; 3 +/- 2 vs 11 +/- 4 and 12 +/- 2 vs 22 +/- 4 mumol.kg-1.min-1, p < 0.05) in the face of higher plasma NEFA concentrations (1.3 +/- 0.3 vs 0.5 +/- 0.1 mmol/l, p < 0.05), respectively. Mean arterial pressure and heart rate were similar in APOC1 vs wild-type mice (82 +/- 4 vs 85 +/- 3 mm Hg and 459 +/- 14 vs 484 +/- 11 beats.min-1). CONCLUSIONS/INTERPRETATION: 1) Hyperlipidaemic APOC1 mice show reduced NEFA and increased glucose metabolism under both basal and insulin-mediated conditions, suggesting an intrinsic defect in NEFA metabolism. Primary hyperlipidaemia alone in APOC1 mice does not lead to insulin resistance for glucose metabolism and high blood pressure.

Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding.

Endostatin, produced as recombinant protein in human 293-EBNA cells, inhibits the migration of human umbilical vein endothelial cells (HUVECs) in response to vascular endothelial growth factor (VEGF) in a dose-dependent manner and prevents the subcutaneous growth of human renal cell carcinomas in nude mice at concentrations and in doses that are from 1000- to 100 000-fold lower than those previously reported. The inhibition of migration is not affected by mutations which eliminate Zn or heparin binding and inhibition of tumor growth does not depend on Zn binding. The results of the migration assays suggest that endostatin causes a block at one or more steps in VEGF-induced migration, while VEGF in turn can cause a block of the inhibition by endostatin of VEGF-induced migration of HUVECs.