The aim of this work was to evaluate the mucus-permeating properties of nanocarriers using zein nanoparticles (NPZ) coated with a Gantrez (R) AN-thiamine conjugate (GT). NPZ were coated by incubation at different GT-to-zein ratios: 2.5% coating with GT (GT-NPZ1), 5% (GT-NPZ2) and 10% (GT-NPZ3). During the process, the GT conjugate formed a polymer layer around the surface of zein nanoparticles. For GT-NPZ2, the thickness of this corona was estimated between 15 and 20 nm. These nanocarriers displayed a more negative zeta potential than uncoated NPZ. The diffusivity of nanoparticles was evaluated in pig intestinal mucus by multiple particle tracking analysis. GT-NPZ2 displayed a 28-fold higher diffusion coefficient within the mucus layer than NPZ particles. These results align with in vivo biodistribution studies in which NPZ displayed a localisation restricted to the mucus layer, whereas GT-NPZ2 were capable of reaching the intestinal epithelium. The gastro-intestinal transit of mucoadhesive (NPZ) and mucus-permeating nanoparticles (GT-NPZ2) was also found to be different. Thus, mucoadhesive nanoparticles displayed a significant accumulation in the stomach of animals, whereas mucus-penetrating nanoparticles appeared to exit the stomach more rapidly to access the small intestine of animals.

Re-activation of the healing process is a major challenge in the field of chronic wound treatment. For that purpose, lipid-nanoparticles, especially nanostructured lipid carriers (NLC), possess extremely useful characteristics such as biodegradability, biocompatibility and long-term stability, besides being suitable for drug delivery. Moreover, they maintain wound moisture due to their occlusive properties, which have been associated with increased healing rates. In the light of above, NLC have been extensively used topically for wound healing; but to date, there are no safety-preclinical studies concerning such type of application. Thus, in this work, biodistribution studies were performed in rats with the NLC previously developed by our research group, using technetium-99 m (99mTc-NLC) as radiomarker, topically administered on a wound. 99mTc-NLC remained on the wound for 24 h and systemic absorption was not observed after administration. In addition, toxicological studies were performed to assess NLC safety after topical administration. The results obtained demonstrated that NLC were non-cytotoxic, non-sensitizing and non-irritant/corrosive. Overall, it might be concluded that developed NLC remained at the administration area, potentially exerting a local effect, and were safe after topical administration on wounds.

Thiamine-coated nanoparticles were prepared by two different preparative methods and evaluated to compare their mucus-penetrating properties and fate in vivo. The first method of preparation consisted of surface modification of freshly poly(anhydride) nanoparticles (NP) by simple incubation with thiamine (T-NPA). The second procedure focused on the preparation and characterization of a new polymeric conjugate between the poly (anhydride) backbone and thiamine prior the nanoparticle formation (T-NPB). The resulting nanoparticles displayed comparable sizes (about 200 nm) and slightly negative surface charges. For T-NPA, the amount of thiamine associated to the surface of the nanoparticles was 15 mu g/mg. For in vivo studies, nanoparticles were labelled with either Tc-99m or Lumogen (R) Red. T-NPA and T-NPB moved faster from the stomach to the small intestine than naked nanoparticles. Two hours post-administration, for T-NPA and T-NPB, > 30% of the given dose was found in close contact with the intestinal mucosa, compared with a 13.5% for NP. Interestingly, both types of thiamine-coated nanoparticles showed a greater ability to cross the mucus layer and interact with the surface of the intestinal epithelium than NP, which remained adhered in the mucus layer. Four hours post-administration, around 35% of T-NPA and T-NPB were localized in the ileum of animals. Overall, both preparative processes yielded thiamine decorated carriers with similar physico-chemical and biodistribution properties, increasing the versatility of these nanocarriers as oral delivery systems for a number of biologically active compounds.

Resveratrol is a naturally occurring polyphenol that provides several health benefits including cardioprotection and cancer prevention. However, its biological activity is limited by a poor bioavailability when taken orally. The aim of this work was to evaluate the capability of casein nanoparticles as oral carriers for resveratrol. Nanoparticles were prepared by a coacervation process, purified and dried by spray-drying. The mean size of nanoparticles was around 200 nm with a resveratrol payload close to 30 ¿g/mg nanoparticle. In vitro studies demonstrated that the resveratrol release from casein nanoparticles was not affected by the pH conditions and followed a zero-order kinetic. When nanoparticles were administered orally to rats, they remained within the gut, displaying an important capability to reach the intestinal epithelium. No evidence of nanoparticle "translocation" were observed. The resveratrol plasma levels were high and sustained for at least 8 h with a similar profile to that observed for the presence of the major metabolite in plasma. The oral bioavailability of resveratrol when loaded in casein nanoparticles was calculated to be 26.5%, 10 times higher than when the polyphenol was administered as oral solution. Finally, a good correlation between in vitro and in vivo data was observed.

Background: The feasibility of beta cell mass (BCM) imaging and quantification with positron emission tomography (PET) in the pancreas is controversial. In an effort to shed some light on this topic, we have used a xenograft model of rat insulinoma (RIN) in mice, mimicking an intramuscular islet transplantation situation. Methods: A total of 105 RIN cells were subcutaneously implanted in nude mice (N.=8). Tumor size and glycaemia levels were determined daily. Rat C-peptide was measured to demonstrate rat insulin production. PET imaging with 11C-(+)-¿-dihydrotetrabenazine (11C-DTBZ) was done at 3 and 4 weeks and compared with 18F-FDG and 18F-DOPA studies in the same mice. Ex-vivo autoradiography with 11C-DTBZ was carried out in frozen sections of tumors. VMAT2 expression was measured by Western-blot and immunohistochemistry in tumors and RIN cells. Results: Functional rat insulin production in mice was demonstrated by substantial decrease in glycaemia (<50 mg/dL by week 4) and rat C-peptide levels (7.2±2.6 ng/mL) similar to those measured in control rats. PET studies showed that tumor imaging with 11C-DTBZ at four (N.=8) and five (N.=5) weeks was negative; only bigger tumors could be seen with 18F-DOPA. In explanted tumors 11C-DTBZ autoradiography was negative, albeit VMAT2 expression measured by Western-blot and immunohistochemistry was lower than in cultured RIN cells. Conclusions: Although insulinomas are fully functional it does not seem feasible to use 11C-DTBZ for in-vivo measuring of BCM. This might either be due to inherent technical limitations of PET, decrease in VMAT2 expression in the tumors due to unknown reasons, or other biological limiting facts.

The aim of this work was to investigate the mucus-permeating properties of poly(ethyleneglycol)-coated nanoparticles prepared from the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN) after oral administration in rats. Nanoparticles were ¿decorated¿ with PEGs of different molecular masses (PEG2000, PEG6000 and PEG10000) at a PEG-to-polymer ratio of 0.125. All the PEG-coated nanoparticles displayed a mean size of ~150 nm, slightly negative ¿ values and a ¿brush¿ conformation as determined from the calculation of the PEG density. For in vivo studies, nanoparticles were labelled with either 99mTc or fluorescent tags. Naked nanoparticles displayed a higher ability to interact with the mucosa of the stomach than with the small intestine. However, these interactions were restricted to the mucus layer covering the epithelial surface, as visualised by fluorescence microscopy. On the contrary, PEG-coated nanoparticles moved rapidly to the intestine, as determined by imaging, and, then, were capable to develop important interactions with the mucosa, reaching the surface of the epithelium. These mucus permeating properties were more intense for nanoparticles coated with PEG2000 or PEG6000 than with PEG10000. However, the capability of nanocarriers to develop adhesive interactions within the mucosa decreased when prepared at excessive PEG densities.

Background: [F-18]-tetrafluoroborate is a PET radiotracer taken up by the sodium/iodide symporter (NIS). Albeit the in vivo behavior in rodents is similar to the (99)mTc-pertechnetate, no studies exist in primates or in humans. The aims of this study were to evaluate the biodistribution of [F-18]-tetrafluoroborate in non-human primates with PET and to estimate the absorbed dose in organs. Methods: Whole-body PET imaging was done in a Siemens ECAT HR+ scanner in two male Macaca fascicularis monkeys. After an i.v. injection of 24.93 +/- 0.05 MBq/kg of [F-18]-tetrafluoroborate, prepared by isotopic exchange of sodium tetrafluoroborate with [F-18]-fluoride under acidic conditions, eight sequential images from the head to the thigh (five beds) were collected for a total duration of 132 min. The whole-body emission scan was reconstructed applying attenuation and scatter corrections. After image reconstruction, three-dimensional volumes of interest (VOIs) were hand-drawn on the PET transaxial or coronal slices of the frame where the organ was most conspicuous. Time-activity curves for each VOI were obtained, and the organ residence times were calculated by integration of the time-activity curves. Human absorbed doses were estimated using the OLINDA/EXM software and the standard human model. Results: [F-18]-tetrafluoroborate was able to discriminate clearly the thyroid gland with an excellent signal-to-noise ratio. Most of the radiotracers (residence time) are localised in the orga

The aim of this work was to prepare and evaluate the capability of zein nanoparticles for oral drug delivery. More particularly, in this work, the ability of these nanoparticles to improve the oral bioavailability of folic acid is reported. The nanoparticles were prepared by a desolvation process, followed by purification via ultrafiltration and drying in a spray-drier apparatus. The resulting nanoparticles displayed a mean size close to 200 nm with negative zeta potential and a payload of 54 ¿g folic acid per mg nanoparticle. From the in vitro release studies, it was observed that folic acid was only released from nanoparticles in simulated intestinal conditions. In vivo biodistribution studies, with radiolabelled or fluorescently marked nanoparticles, revealed that nanoparticles remained within the gut and were capable of interacting with the protective mucus layer of the jejunum. For the pharmacokinetic study, folic acid was orally administered to rats as a single dose of 1 mg/kg. The relatively oral bioavailability of folic acid, when encapsulated in zein nanoparticles, was around 70%: two-times higher than the value obtained with an aqueous solution of the vitamin. This fact might be explained by the mucoadhesive properties of these nanoparticles.

Purpose: To optimize radiolabeling with (99m)Tc of mannosylated Gantrez(®) nanoparticles loaded with the Brucella Ovis antigen (Man-NP-HS) and to carry out biodistribution studies in mice after ocular administration of the nanoparticles. Material and methods: Man-NP-HS nanoparticles were prepared by the solvent displacement method. They were purified, lyophilized and characterized. Following this, they were radiolabeled with 74 MBq of (99m)TcO4(-) previously reduced with an acidic stannous chloride solution, working in absence of oxygen and at a final pH of 4. Radiolabeling yield was evaluated by TLC. Biodistribution studies were carried out in mice after ocular administration of the formulation and control of free (99m)TcO4(-). To do so, the animals were humanely killed at 2 and 24hours after the ocular administration and activity in organs was measured in a Gamma counter. Results: Radiolabeling yield obtained was greater than 90%. Biodistribution studies of (99m)Tc-Man-NP-HS showed radioactivity accumulated at 2 and 24hours in nasal and ocular mucosa and gastrointestinal tract, in contrast to biodistribution of free (99m)TcO4(-) that remained concentrated in the skin around the eye and gastrointestinal tract. Conclusion: Biodistribution studies of (99m)Tc-Man-NP-HS after ocular instillation have made it possible to demonstrate its biodistribution in nasal mucosa and gastrointestinal tract. This characteristic is essential as an antigenic delivery system throughout the ocular mucosa. This, together with its elevated immune response, effective protection and intrinsic avirulence make them a suitable anti-Brucella vaccine candidate.

Neuronal loss in Alzheimer's disease, a better correlate of cognitive impairment than amyloid deposition, is currently gauged by the degree of regional atrophy. However, functional markers, such as GABA(A) receptor density, a marker of neuronal integrity, could be more sensitive. In post-mortem hippocampus, GABA(A) messenger RNA expression is reduced even in mild cognitive impairment. We measured whole-brain GABA(A) binding potential in vivo using [(11)C]-flumazenil positron emission tomography and compared GABA(A) binding with metabolic and volumetric measurements. For this purpose, we studied 12 subjects, six patients with early Alzheimer's disease and six healthy controls, with [(11)C]-flumazenil and [(18)F]-fluorodeoxyglucose positron emission tomography, as well as with high-resolution magnetic resonance imaging. Data were evaluated with both voxel-based parametric methods and volume of interest methods. We found that in early Alzheimer's disease, with voxel-based analysis, [(11)C]-flumazenil binding was decreased in infero-medial temporal cortex, retrosplenial cortex and posterior perisylvian regions. Inter-group differences reached corrected significance when using an arterial input function. Metabolism measured with positron emission tomography and volumetric measurements obtained with magnetic resonance imaging showed changes in regions affected in early Alzheimer's disease, but, unlike with [(11)C]-flumazenil binding and probably due to sample size, the voxel-based findings failed to reach corrected significance in any region of the brain. With volume of interest analysis, hippocampi and posterior cingulate gyrus showed decreased [(11)C]-flumazenil binding. In addition, [(11)C]-flumazenil hippocampal binding correlated with memory performance. Remarkably, [(11)C]-flumazenil binding was decreased precisely in the regions showing the greatest degree of neuronal loss in post-mortem studies of early Alzheimer's disease. From these data, we conclude that [(11)C]-flumazenil binding could be a useful marker of neuronal loss in early Alzheimer's disease

PURPOSE: The aim of the study was to evaluate the volumetric integration patterns of standard MRI and (11)C-methionine positron emission tomography (PET) images in the surgery planning of gliomas and their relationship to the histological grade. METHODS: We studied 23 patients with suspected or previously treated glioma who underwent preoperative (11)C-methionine PET because MRI was imprecise in defining the surgical target contour. Images were transferred to the treatment planning system, coregistered and fused (BrainLAB). Tumour delineation was performed by (11)C-methionine PET thresholding (vPET) and manual segmentation over MRI (vMRI). A 3-D volumetric study was conducted to evaluate the contribution of each modality to tumour target volume. All cases were surgically treated and histological classification was performed according to WHO grades. Additionally, several biopsy samples were taken according to the results derived either from PET or from MRI and analysed separately. RESULTS: Fifteen patients had high-grade tumours [ten glioblastoma multiforme (GBM) and five anaplastic), whereas eight patients had low-grade tumours. Biopsies from areas with high (11)C-methionine uptake without correspondence in MRI showed tumour proliferation, including infiltrative zones, distinguishing them from dysplasia and radionecrosis. Two main PET/MRI integration patterns emerged after analysis of volumetric data: pattern vMRI-in-vPET (11/23) and pattern vPET-in-vMRI (9/23). Besides, a possible third pattern with differences in both directions (vMRI-diff-vPET) could also be observed (3/23). There was a statistically significant association between the tumour classification and integration patterns described above (p¿<¿0.001, ¿¿=¿0.72). GBM was associated with pattern vMRI-in-vPET (9/10), low-grade with pattern vPET-in-vMRI (7/8) and anaplastic with pattern vMRI-diff-vPET (3/5). CONCLUSION: The metabolically active tumour volume observed in (11)C-methionine PET differs from the volume of MRI by showing areas of infiltrative tumour and distinguishing from non-tumour lesions. Differences in (11)C-methionine PET/MRI integration patterns can be assigned to tumour grades according to the WHO classification. This finding may improve tumour delineation and therapy planning for gliomas.

Purpose: Study by molecular imaging the biodistribution of poly(anhydride) nanoparticles after oral administration. Procedures: Poly (anhydride) nanoparticles (NP) and cyclodextrin-tagged nanoparticles (CD-NP) were radiolabelled with Tc-99m. Radiochemical purity was measured with a double-solvent chromatography system and the absence of undesirable components was confirmed by size and polydispersion measurement of the technetium-labelled nanoparticles by photon correlation spectroscopy. Single photon emission computed tomography (SPECT) fused computed tomography (CT) in vivo molecular imaging was used for biodistribution studies in small animals. Results: SPECT-CT images revealed activity only in the gastrointestinal tract. Thirteen percent of the given dose of CD-NP and 3% of the given dose of conventional NP were found in the stomach at 8 h. Conclusion: No evidence of translocation or distribution out of gastrointestinal tract was found. CD-NP moved significantly more slowly inside the gut than conventional NP, probably due to their physico-chemical structure that allows stronger interactions with the gut mucosa.