Concentration Of FITC-dextran Research Articles

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran.

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

Gegen Qinlian decoction activates AhR/IL-22 to repair intestinal barrier by modulating gut microbiota-related tryptophan metabolism in ulcerative colitis mice

Ethnopharmacological relevanceGegen Qinlian decoction (GQD) is a traditional Chinese medicine derived from Treatise on febrile diseases and is clinically used for the treatment of acute ulcerative colitis (UC). However, the potential mechanism of GQD treatment for UC remains elusive. Aim of studyIn this study, we aimed to explore the involvement of gut microbiota-related tryptophan metabolism in mediating protective effects of GQD against intestinal barrier damage. Materials and methodsMice with colitis were treated with 3% dextran sulfate sodium (DSS) for 6 days. The therapeutic effects of GQD in UC mice were examined based on body weight, disease activity index (DAI), organ index, length and pathological changes in the colon. The distribution of fluorescein isothiocyanate dextran (FITC-dextran) in the intestinal tract was observed using small animal imaging, while concentration of FITC-dextran in serum was detected using a fluorescein microplate analyser. Bacterial infiltration in colon tissues was observed by fluorescence in situ hybridisation (FISH), and the bacterial load in mesenteric lymph nodes (MLNs) was further examined through bacterial culture. Subsequently, colonic goblet cells were detected using Alcian blue staining. The tight junctions of the colonic epithelium were observed using transmission electron microscopy, and the expression of tight junction proteins was detected by immunofluorescence (IF) and western blot. In addition, flow cytometry was used to analyse the proportion of interleukin-22-positive (IL-22+) ILC3 cells in lamina propria lymphocytes, and the content of IL-22 in colon homogenates was determined using an ELISA kit. In addition, targeted tryptophan metabolomics was used to detect the concentration of indole derivatives produced by tryptophan metabolism in faeces, and 16S rDNA was used to investigate the composition and abundance of gut microbiota-related tryptophan metabolism. ResultsAdministration of GQD significantly alleviated the pathological symptoms, including weight loss, increased DAI score, changes in organ index, colon shortening, and colon pathological injury in UC mice. In addition, GQD reduced the diffusion of FITC-dextran in the intestinal tract, the content of FITC-dextran in serum, and bacterial infiltration in MLNs and colon tissues. Additionally, GQD significantly increased the number of colonic goblet cells, repaired the structure of epithelial tight junctions and increased the expression of tight junction proteins. Furthermore, GQD significantly increased the proportion of IL-22+ ILC3 in the lamina propria, the expression of CYP1A1 protein in colon tissue, and the level of IL-22 in colon homogenates. However, the above protective effects of GQD were inhibited by co-administration of GQD and aryl hydrocarbon receptor (AhR) antagonist. Additionally, GQD restored the content of indole derivatives generated by tryptophan metabolism, regulated the diversity of the gut microbiota, and significantly increased the abundance of genes related to tryptophan metabolism. ConclusionOur results confirmed that GQD repaired the damaged intestinal barrier in UC mice by regulating gut microbiota-related tryptophan metabolism and restoring the generation of indole derivatives to activate AhR-mediated IL-22 production.

Glycyrrhiza Polysaccharide Alleviates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice.

Background Licorice is one of the most ubiquitous herbs in traditional Chinese medicine, with notable anti-inflammatory and antiulcerative effects as well as potent digestive disease therapeutic impacts; yet, its active components and mechanisms remain unclear. There is a lot of evidence that Glycyrrhiza polysaccharide (GPS) has antioxidants, improving intestinal flora, anti-inflammatory effects, etc. Hypothesis/Purpose. Here, we investigated the effects of GPS on dextran sulfate sodium (DSS)-induced acute ulcerative colitis (UC) mice and its possible mechanisms. Methods GPS (100, 200, and 400 mg/kg) or the positive control drug sulfasalazine (SASP) (200 mg/kg) were orally administered to mice for 8 days. Body weight was recorded daily. Symptoms associated with UC, such as disease activity index (DAI), colon length, spleen weight, and mucosal damage were detected. The possible mechanism of GPS ameliorating enteritis symptoms was explored by detecting intestinal permeability and serum levels of inflammatory factors, and changes in intestinal permeability were expressed by serum concentration of FITC-dextran and D-lactic acid. Results The results demonstrated that GPS administration alleviated UC symptoms in colitis mice, including weight loss, DAI index, shorting colon length, and mucosal damage. Mechanistic evaluation revealed that GPS treatment reduced intestinal permeability and serum levels of inflammatory factors: IL-1, IL-6, and TNF-α, while increasing serum levels of the anti-inflammatory factor IL-10, suggesting that GPS's mechanism in UC is related to reducing intestinal permeability and inhibiting the inflammatory response, with intestinal permeability implicated as the initiating mechanism. Conclusion This study highlights GPS as a promising therapeutic agent, with high therapeutic efficacy and a good safety profile, for enteritis and beyond.

Dealcoholized Muscadine Wine Alleviates Inflammation and Preserves Intestinal Barrier in Il-10–/– Mice With Spontaneous Colitis

ObjectivesMuscadine wine has a unique polyphenol profile consisting of anthocyanin 3, 5-diglucosides, ellagitannins, ellagic acids, and flavonols. This study aimed to determine whether dealcoholized muscadine wine (DMW) alleviated inflammatory bowel diseases (IBD) in IL-10–/– mice. MethodsDMW was prepared by concentrating muscadine wine to 10% of its original volume. Male IL-10–/– and wild-type C57BL/6 mice (6∼7-week-old) were fed an AIN-93M diet containing DMW (5.5%, v/w) or wine-free diet for 154 days. Body weight, food intake, stool consistency, rectum bleeding, and disease activity index of mice were recorded twice a week. Colon, spleen, and serum were collected after euthanasia for histological and biochemical analysis. Fluorescence of serum were measured 4 hours after oral gavage of fluorescein isothiocyanate (FITC)-dextran to determine the intestinal permeability. ResultsIL-10–/– mice developed ulcerative colitis, inflammation, and impaired gut barrier spontaneously whereas wild type mice remained healthy. DMW in diet preserved colon length and decreased colon histology score by 21.2% in IL-10–/– mice (P < 0.05). IL-10–/– mice on DMW diet had a significantly lower disease activity index than those on wine-free diet. Serum level of keratinocyte chemokine, IL-1β, and TNF-α were decreased by 31.8%, 62.8%, and 29.3% (P < 0.05), respectively, by DMW in IL-10–/– mice, whereas IL-6 remained unchanged. Compared to the wine-free diet, concentration of FITC-dextran in the serum of IL-10–/– mice were decreased by 18% by DMW (P < 0.05), indicating improved intestinal barrier function. DMW had no effect on wild type mice. IL-10–/– mice had comparable body weight to wild type ones although their overall food intake was lower. ConclusionsDMW alleviated colon inflammation via inhibiting the secretion of inflammatory cytokines and preserving intestinal barrier. Muscadine wine has a potential to manage IBD in patients. Funding SourcesThis research is supported by NIFA 2018–07,941.

Intestinal HuR deficiency exacerbates diet‐induced fatty liver disease

Background Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. However, no effective treatment is available due to the limited knowledge regarding the complex disease pathology. We and others have previously reported that impaired intestinal barrier function promotes the progression of various liver diseases, including NAFLD. Human antigen R (HuR) is a member of the Hu family of RNA-binding proteins. It is reported that HuR is essential for maintaining gut epithelial integrity, and targeted deletion of HuR in the intestinal epithelium disrupts mucosal regeneration in mice. However, the role of intestinal HuR in NAFLD remains unclear and is the focus of this study. Methods: Intestinal epithelial tissue-specific HuR deletion (IE-HuR–/–) mice were generated by crossing the HuRflox/flox (HuRfl/fl) and villin-Cre mice. Age and gender-matched IE-HuR–/–and control mice were fed ad libitum a high-fat diet (HFD, Harlan TD.88137) for 4 weeks. At the end of the treatment, FITC-Dextran was administered to mice by oral gavage. The serum concentration of FITC-dextran was measured. The serum lipids and liver function enzymes were measured using the Alfa Wassermann Vet ACE Axcel® System. Small intestine and liver tissues were processed for H&E staining and isolation of total RNA. Hepatic lipid was stained by Oil Red O. RNAseq transcriptome analysis was used to profile hepatic gene expression. The mRNA levels of the key genes were confirmed by qRT-PCR. Results Compared to control mice, the serum levels of FITC-Dextran were significantly higher in IE-HuR–/– mice fed an HFD. Consistently, the H&E staining of small intestine tissues revealed that the intestinal barrier was disrupted in IE-HuR–/– mice fed an HFD. Besides, the serum levels of ALP, ALT and AST significantly increased in HFD-fed IE-HuR–/– mice. The H&E and Oil Red O staining of liver tissues showed a significant increase in hepatic lipid accumulation in IE-HuR–/– mice. Bioinformatics analysis of RNAseq data showed that intestinal-specific deficiency of HuR significantly affected hepatic metabolic pathways, activated inflammatory and ER stress response. The qRT-PCR further confirmed that in IE-HuR–/– mice, the key genes involved in bile acid metabolisms (Cyp7a1, Cyp27a1, Cyp7b, and Cyp8b1) were significantly reduced, but the expression of genes involved in inflammation (Mcp-1, Il1α, Cd63, and Cxcl1) and endoplasmic reticulum stress (Chop, Atf4, Xbp-1s, and Grp78) were increased. Furthermore, the key genes involved in sphingosine lipid pathways, such as Smpd3 and Spt1c1, were significantly increased while the expression of Sphk2 was decreased. Conclusions Intestinal HuR deficiency exacerbates HFD-induced fatty liver disease via inducing intestinal barrier dysfunction, activating ER stress, and inflammatory response in the liver.

Altered integrity of hepatocyte tight junctions in rats with triptolide-induced cholestasis.

Triptolide (TP), an active component of Tripterygium wilfordiiHook. f. (TWHF), has been widely used for centuries as a traditional Chinese medicine. However, the clinical application of TP has been restricted due to multitarget toxicity, such as hepatotoxicity. In this study, 28 days of oral TP administration (100, 200, or 400 μg·kg-1·d-1) induced the occurrence of cholestasis in female Wistar rats, as evidenced by increased serum levels of γ-glutamyl transpeptidase (γ-GGT), alkaline phosphatase (ALP) and hepatic total bile acids (TBAs). In addition, the heptocyte polarity associated with the strcture of tight junctions (TJs) was disrupted in both rats and sandwich-cultured primary hepatocytes. Immunoblotting revealed decreased expression of the TJ-associated proteins occludin, claudin-1, and zonula occludens protein (ZO-1), and downregulated mRNA levels of these TJs was also detected by real-time PCR. An immunofluorescence analysis showed abnormal subcellular localization of occludin, claudin-1 and ZO-1, which was also confirmed by transmission electron microscopy. Moreover, the concentration of FITC-dextran, a marker of paracellular penetration, was found to increase rapidly in bile increased rapidly (within 6 minutes) after treatment with TP, which indicated the functional impairment of TJs. Taken together, these results suggest that the administration of TP for 28 consecutive days to rats could induce cholestatic injury in the liver, and the increased paracellular permeability might play an important role in these pathological changes.

Hyaluronic Acid and Poly-l-Lysine Layers on Calcium Alginate Microspheres to Modulate the Release of Encapsulated FITC-Dextran.

Alginate solutions crosslink into microspheres in calcium alginate, enabling the encapsulation and subsequent release of biological macromolecules and drugs. However, release from calcium alginate into PBS is relatively fast because it will decrosslink the gel relatively quickly. In this research, FITC-dextran (MW 10kDa) was encapsulated in 2% (w/v) calcium alginate microspheres by electrospraying. The resulting microspheres (diameter=247±13μm) were then layered with thin polyelectrolyte films of hyaluronic acid (HA) and poly-l-lysine (PLL) to attempt to slow the diffusion of FITC-dextran out of the microspheres and the coating parameters were modified to modulate diffusion and release. Increasing the concentration of FITC-dextran encapsulated in the microspheres resulted in an increase in its release over time into PBS. Crosslinking PLL/HA layers on the microspheres did not decrease the invitro release rates of encapsulated FITC-dextran into PBS. Increasing the number of layers on the microspheres from 3 to 5 layers significantly decreased the amount of encapsulated FITC-dextran released. However, increasing the number of layers to 7 did not further sustain the release of FITC-dextran, likely because these microspheres collapsed to a smaller size during the coating procedure, resulting in release controlled by both diffusion and swelling. Multiple layers of PLL and HA provided a robust mechanism to sustain and control release of large molecules from calcium alginate.

Compound sophorae decoction enhances intestinal barrier function of dextran sodium sulfate induced colitis via regulating notch signaling pathway in mice

BackgroundCompound sophorae decoction (CSD), a Chinese Herbal decoction, is frequently clinically prescribed for patients suffered from ulcerative colitis (UC) characterized by bloody diarrhea. Yet, the underlying mechanism about how this formulae works is remain elusive. MethodsIn the present study, the experimental colitis in C57BL/6 J mice was induced by oral administration of standard diets containing 3% dextran sodium sulfate (DSS), and CSD was given orally for treatment at the same time. The clinical symptoms including stool and body weight were recorded each day, and colon length and its histopathological changes were observed. Apoptosis of colonic epithelium was studied by detecting protein expression of cleaved caspase-3, and cell proliferation by Ki-67 immunohistochemistry. Tight junction complex like ZO-1 and occludin were also determined by transmission electron microscope and immunofluorescence. The concentration of FITC-dextran 4000 was measured to evaluate intestinal barrier permeability and possible signaling pathway was investigated. Mucin2 (MUC2) and notch pathway were tested through western blot. The M1/M2 ratio in spleen and mesenteric lymph nodes were detected by flow cytometry. And the mRNA levels of iNOS and Arg1 were examined by qRT-PCR. ResultsCSD could significantly alleviate the clinical manifestations and pathological damage. Body weight loss and DAI score of mice with colitis were improved and shortening of colon was inhibited. The administration of CSD was able to reduce apoptotic epithelial cells and facilitate epithelial cell regeneration. Increased intestinal permeability was reduced in DSS-induced colitis mice. In addition, CSD treatment obviously up-regulated the expression of ZO-1 and occludin and the secretion of MUC2, regulated notch signaling, and decreased the ratio of M1/M2. ConclusionsThese data together suggest that CSD can effectively mitigate intestinal inflammation, promote phenotypic change in macrophage phenotype and enhance colonic mucosal barrier function by, at least in part, regulating notch signaling in mice affected by DSS-induced colitis.

Decapsulation of Dextran by Destruction of Polyelectrolyte Microcapsule Nanoscale Shell by Bacillus subtilis Bacteria.

One of the prerequisites of successful address delivery is controlling the release of encapsulated drugs. The new method of bacterial spore encapsulation in polyelectrolyte microcapsules allows for degrading the nanoscale membrane shell of microcapsules. The possibility of encapsulating spore forms of Bacillus subtilis in polystyrenesulfonate sodium/ polyallylamine hydrochloride (PSS/PAH) polyelectrolyte microcapsules was demonstrated. The activation and growth on a nutrient medium of encapsulated bacterial spores led to 60% degradation of the microcapsules nanoscale membrane shell. As a result, 18.5% of Fluorescein isothiocyanatedextran was encapsulated into polyelectrolyte microcapsules, and 28.6% of the encapsulated concentration of FITC-dextran was released into the solution.

Pulmonary permeability assessed by fluorescent-labeled dextran instilled intranasally into mice with LPS-induced acute lung injury.

BackgroundSeveral different methods have been used to assess pulmonary permeability in response to acute lung injury (ALI). However, these methods often involve complicated procedures and algorithms that are difficult to precisely control. The purpose of the current study is to establish a feasible method to evaluate alterations in lung permeability by instilling fluorescently labeled dextran (FITC-Dextran) intranasally.Methods/Principal FindingsFor the mouse model of direct ALI, lipopolysaccharide (LPS) was administered intranasally. FITC-Dextran was instilled intranasally one hour before the mice were euthanized. Plasma fluorescence intensities from the LPS group were significantly higher than in the control group. To determine the reliability and reproducibility of the procedure, we also measured the lung wet-to-dry weight ratio, the protein concentration of the bronchoalveolar lavage fluid, tight and adherens junction markers and pathological changes. Consistent results were observed when the LPS group was compared with the control group. Simultaneously, we found that the concentration of plasma FITC-Dextran was LPS dose-dependent. The concentration of plasma FITC-Dextran also increased with initial intranasal FITC-Dextran doses. Furthermore, increased fluorescence intensity of plasma FITC-Dextran was found in the intraperitoneally LPS-induced ALI model.Conclusion/SignificanceIn conclusion, the measurement of FITC-Dextran in plasma after intranasal instillation is a simple, reliable, and reproducible method to evaluate lung permeability alterations in vivo. The concentration of FITC-Dextran in the plasma may be useful as a potential peripheral biomarker of ALI in experimental clinical studies.

Mo1805 Ablation of DCLK1 in Intestinal Epithelium Exacerbates Colonic Epithelial Barrier Damage in Response to DSS Treatment

Background: During normal homeostasis the intestinal epithelium maintains a dynamic architecture with rapid renewal every 3-5 days. Cytotoxic and genotoxic insults cause excessive epithelial damage and crypt injury. Intestinal stem cells (ISCs) are responsible for homeostasis and crypt regeneration. It is believed that the quiescent ISCs may represent a population that can be called upon to rescue the crypt following severe injury. Doublecortinlike kinase 1 (Dclk1) is a putative quiescent ISC and tuft cell marker. It is predominantly expressed in relatively quiescent cells near the traditional +4 location within the intestinal crypt. Dclk1+ cells sorted from murine small intestine form spheroids in suspension culture, and when transplanted into nude mice develop into nodules demonstrating early intestinal epithelial secretory lineage commitment. These data suggest that Dclk1 is a putative ISC marker. The role of Dclk1 in epithelium regeneration in response to injury is not clear. In this study, we investigate the role of Dclk1 in colonic epithelial restitution following injury. Aim: To determine whether Dclk1 regulates colonic epithelial barrier function following DSS-induced colitis. Methods: Age and gender matched control (Dclk1flox/flox) and Dclk1 KO (Villin-Cre;Dclk1flox/flox) mice were fed 3% Dextran Sulfate Sodium (DSS) in drinking water for 7 days, followed by water for 2 days. Body weight and occult blood were monitored daily. To assess epithelial barrier permeability, both Dclk1 KO and control mice were gavaged with FITC-dextran. Blood was collected 4 hours later and the serum concentration of FITC-dextran was measured. Corresponding colon tissues were subjected to histological examination, gene expression, and protein expression analysis. Results: Dclk1 KO mice lost significantly more weight than control mice following DSS treatment. The serum concentration of FITC-dextran in Dclk1 KO mice was approximately 2 fold higher than in control mice. The gene expression levels of ISC markers, Dclk1, Lgr5, Bmi1, mTert, and Msi-1, were significantly downregulated in the colon of Dclk1 KO mice compared to control mice following DSS treatment. Protein levels of Ecadherin, Claudin-1, and Claudin-7 were similar in untreated control and Dclk1 KO mice, but decreased dramatically in Dclk1 KO mice following DSS treatment. H&E staining of colon from DSS-treated mice indicated increased inflammation in Dclk1 KO mice relative to controls. Conclusions: In summary, these results show that loss of Dclk1 in colonic epithelial cells results in exacerbated damage to barrier function following DSS treatment. These data also demonstrate a role for Dclk1 in the regulation of other stem cell markers following DSS-induced injury. Taken together, this study suggests that Dclk1 plays an important role in colonic epithelial restitution in response to severe injury.

Therapeutic hypothermia reduces intestinal ischemia/reperfusion injury after cardiac arrest in rats

To investigate the effects of therapeutic hypothermia (TH) on the morphology and function of intestine after cardiac arrest and resuscitation, 45 male rats were randomly assigned into three groups: (1) normothermia group, animals underwent ventricular fibrillation (VF) and cardiopulmonary resuscitation (CPR) with the rectal temperature maintained at 36.8 ± 0.2°C until 4 h after return of spontaneous circulation (ROSC); (2) hypothermia group, TH was induced with the aid of ice packs and an electrical fan because VF occurred and was maintained at 33.5 ± 0.5°C for 4 h after ROSC; (3) sham-operated group, animals underwent identical anesthetic and surgical procedures without VF, CPR or defibrillation. Five animals in each group were sacrificed at 4, 24 and 72 h post resuscitation. Serum diamine oxidase (DAO) and apoptosis rate of intestinal epithelial cells were tested by ELISA and flow cytometry, respectively. The concentration of FITC-Dextran that leaked out of enteric cavity was used to analyze the permeability of intestine. Histological changes were graded and compared among the three groups. Serum DAO concentrations in normothermia group reached the peak at 4 h post resuscitation, and then decreased at 24 and 72 h. In comparison with normothermia group, serum DAO concentrations were lower at 4 h in hypothermia group (0.97 ± 0.16 vs. 1.24 ± 0.29, P < 0.05). The amount of FITC-Dextran that passed the wall of small intestine in hypothermia group was signii¬cantly lower than that in normothermia group at 24 h after ROSC (7.81 ± 1.11 vs. 13.07 ± 3.07, P < 0.05). The amount of FITC-Dextran had no difference between normothermia and hypothermia groups at 4 and 72 h post resuscitation. The detached intestinal epithelial cells in hypothermia group showed significant lower frequency of apoptosis than those in normothermia group at 4 h (17.30 ± 2.56 vs. 25.63 ± 4.09, P < 0.05) and 24 h (9.38 ± 1.29 vs. 11.98 ± 1.78, P < 0.05). No obvious injury was observed in both normothermia and hypothermia groups at 4 h with grade of 0 to 1. The histopathological injury in normothermia group reached the peak at 24 h with grade of 2 to 3, which was significantly severe than that in hypothermia group with grade of 1 to 2. At 72 h post resuscitation, an almost complete restitution of the intestinal mucous could be observed both in hypothermia and normothermia groups. This study demonstrates that short term ischemia induced by cardiac arrest and resuscitation resulted in intestinal ischemia/reperfusion (IR) injury, which could be attenuated by therapeutic hypothermia. Key words: Rat, intestine, cardiac arrest, cardiopulmonary resuscitation, therapeutic hypothermia.

Pharmacokinetics of 5-fluorouracil in the hamster following inhalation delivery of lipid-coated nanoparticles

The inhalation delivery of 5-fluorouracil (5-FU) in lipid-coated nanoparticles (LNPs) to hamsters was evaluated to determine the feasibility for use in lung cancer chemotherapy. The inhaled dose, 30 mg LNPs/kg body weight (1.5 mg/kg 5-FU), was delivered over an 8-min interval. Fluorescein isothiocyanate dextran (FITC-dextran) was included within the LNPs to provide an estimate of the particle concentration. The concentration of FITC-dextran and total 5-FU (released and LNP-associated) was determined as a function of time in the lung, trachea, larynx, esophagus, and serum. Concentrations of 5-FU and FITC-dextran were initially high in the trachea, larynx, and esophagus, and lower in the lung. Within 24 h, greater than 99% of the LNPs were cleared from the respiratory tract and total 5-FU concentrations mirrored the LNP concentration. An eight-compartment pharmacokinetic model was used to describe the observed trends in concentrations of LNPs and total 5-FU and to estimate the released 5-FU concentration in the above tissues. From this analysis, effective local targeting as well as sustained efficacious concentrations of 5-FU in the expected tumor sites were demonstrated.

Thermosensitive poly(organophosphazene) hydrogels for a controlled drug delivery

Thermosensitive poly(organophosphazenes) were synthesized for a controlled release of hydrophilic polymeric model drugs such as dextran and albumin in this study. The solutions of the present polymers bearing both hydrophobic side groups of l-isoleucine ethyl ester (IleOEt) and hydrophilic groups of α-amino-ω-methoxy-PEG ( M w 550) (AMPEG550) exhibited reversible sol–gel transition behaviors with changes of temperature. Viscometric measurement indicated that the thermosensitive hydrogels with good strength could be formed from the solutions in the range of the concentrations of 7–15 wt% around body temperature. For increasing their biodegradabililites, depsipeptides of ethyl-2-( O-glycyl)lactate (GlyLacOEt) were also introduced to the polymer, showing enhanced degradation of hydrogels. In vitro release behaviors of hydrophilic FITC-dextran ( M w 71,600) and human serum albumin from these polymer hydrogels were sustained for about 2 weeks while those from poloxamer (Pluronic F-127) hydrogel showed a distinct initial burst. The release of FITC-dextran exhibited concentration-dependent behavior ranging from 7 to 15 wt% of the polymer solution while it was almost independent of the concentration of FITC-dextran.

Characterization of ocular pharmacokinetics of beta-blockers using a diffusion model after instillation.

To characterize the ocular pharmacokinetics of beta-blockers (timolol and tilisolol) after instillation in the albino rabbit using a mathematical model that includes a diffusion process. The disposition of fluorescein isothiocyanate-dextran (FITC-dextran, molecular weight 4400), timolol, and tilisolol was determined in tear fluid and aqueous humor after instillation or ocular injection in rabbits. The in vivo penetration parameters were estimated by fitting the concentration-time profiles to the Laplace equations based on a diffusion model using MULTI(FILT) program. The in vitro permeability of drugs was measured across cornea using a two-chamber diffusion cell. Concentration-time profiles of drugs in the tear fluid after instillation showed a monoexponential curve. Although a monoexponential curve was observed in the aqueous humor concentration of FITC-dextran after injection into the aqueous chamber, timolol and tilisolol showed a biexponential curve. On the basis of these results, an in vivo pharmacokinetic model was developed for estimation of penetration parameters. The in vitro partition parameters were higher than those of the in vivo parameters. The ocular absorption of timolol and tilisolol was characterized using an in vivo pharmacokinetic model and in vivo penetration parameters.

Interrelation of intracellular proteases with total parenteral nutrition-induced gut mucosal atrophy and increase of mucosal macromolecular transmission in rats.

Total parenteral nutrition (TPN) is known to induce mucosal atrophy and to increase macromolecular transmission of the small intestine. The potential participation of various proteases in that process was investigated. Male Wistar rats were randomly divided into two groups: the TPN group (n = 11) received a standard TPN (250 kcal/kg per day, 1.78 g nitrogen/kg per day) and the FED group (n = 10) received a standard rat food for 1 week. This was followed by an examination of gut macromolecular transmission of fluorescein isothiocyanate dextran 70,000 (FITC-dextran) after intragastric injection and of the activities of gut mucosal cathepsins B, H, and L and of proteasome. Mucosal wet weight and protein content decreased significantly by TPN for 1 week. In both groups, the activities of all proteases in the ileum were significantly greater than in the jejunum. In the TPN group, cathepsin L and H activities in the ileum, and cathepsin B activity in both the jejunum and the ileum, were greater than those in the FED group. The portal concentration of FITC-dextran was higher than arterial and venous concentrations in the both groups. In the TPN group, the portal FITC-dextran concentration increased significantly compared with the FED group. In conclusion, active proteolysis is not associated with TPN-induced mucosal atrophy. Cathepsins activities in the ileum increase as a result of TPN. Interrelationship is implicated between increase of lysosomal protease activity and the deterioration of the intestinal barrier function, which permits macromolecular transmission.