Intestinal Epithelial Membrane Research Articles

Cryo-Milled β-Glucan Nanoparticles for Oral Drug Delivery.

Gemcitabine is a nucleoside analog effective against a number of cancers. However, it has an oral bioavailability of less than 10%, due to its high hydrophilicity and low permeability through the intestinal epithelium. Therefore, the aim of this project was to develop a novel nanoparticulate drug delivery system for the oral delivery of gemcitabine to improve its oral bioavailability. In this study, gemcitabine-loaded β-glucan NPs were fabricated using a film-casting method followed by a freezer-milling technique. As a result, the NPs showed a small particle size of 447.6 ± 14.2 nm, and a high drug entrapment efficiency of 64.3 ± 2.1%. By encapsulating gemcitabine into β-glucan NPs, a sustained drug release profile was obtained, and the anomalous diffusion release mechanism was analyzed, indicating that the drug release was governed by diffusion through the NP matrix as well as matrix erosion. The drug-loaded NPs had a greater ex vivo drug permeation through the porcine intestinal epithelial membrane compared to the plain drug solution. Cytotoxicity studies showed a safety profile of the β-glucan polymers, and the IC50s of drug solution and drug-loaded β-glucan NPs were calculated as 228.8 ± 31.2 ng·mL-1 and 306.1 ± 46.3 ng·mL-1, respectively. Additionally, the LD50 of BALB/c nude mice was determined as 204.17 mg/kg in the acute toxicity studies. Notably, pharmacokinetic studies showed that drug-loaded β-glucan NPs could achieve a 7.4-fold longer T1/2 and a 5.1-fold increase in oral bioavailability compared with plain drug solution. Finally, in vivo pharmacodynamic studies showed the promising capability of gemcitabine-loaded β-glucan NPs to inhibit the 4T1 breast tumor growth, with a 3.04- and 1.74-fold reduction compared to the untreated control and drug solution groups, respectively. In conclusion, the presented freezer-milled β-glucan NP system is a suitable drug delivery method for the oral delivery of gemcitabine and demonstrates a promising potential platform for oral chemotherapy.

A review on oral novel delivery systems of insulin through the novel delivery system formulations: A review.

Parenteral administration of insulin remains the most common route of administration, causing local hypertrophy at the injection sites because of multiple daily injections. Because of this, there is an interest and effort in oral insulin administration that is convenient and mimics the physiology of endogenous insulin secreted in the liver. However, oral insulin encountered different challenges due to abundant enzyme degradation, the presence of a mucus layer, and the underlying intestinal epithelial membrane barrier in the gastrointestinal tract. This narrative review reviewed the literature dealing with novel oral insulin delivery approaches. Various pieces of literature were searched, filtered, and reviewed from different sources, and the information obtained was organized, formulated, and finalized. Oral insulin has been formulated and extensively studied in various novel delivery approaches, such as nanoparticles, microspheres, mucoadhesive patches, encapsulations, hydrogels, ionic liquids, liposomes, and complexation. The efficiency of these formulations demonstrated improved efficiency and potency compared to free oral insulin delivery, but none of them have greater or equivalent potency to subcutaneous insulin. Future studies regarding dose-dependent therapeutic efficacy and the development of new novel formulations to produce comparable oral insulin to subcutaneous insulin are warranted to further support the suitability of the current platform for oral insulin delivery.

Immunomodulatory Properties of Vitamin D in the Intestinal and Respiratory Systems

Vitamin D plays a crucial role in modulating the innate immune response by interacting with its intracellular receptor, VDR. In this review, we address vitamin D/VDR signaling and how it contributes to the regulation of intestinal and respiratory microbiota. We additionally review some components of the innate immune system, such as the barrier function of the pulmonary and intestinal epithelial membranes and secretion of mucus, with their respective modulation by vitamin D. We also explore the mechanisms by which this vitamin D/VDR signaling mounts an antimicrobial response through the transduction of microbial signals and the production of antimicrobial peptides that constitute one of the body's first lines of defense against pathogens. Additionally, we highlight the role of vitamin D in clinical diseases, namely inflammatory bowel disease and acute respiratory distress syndrome, where excessive inflammatory responses and dysbiosis are hallmarks. Increasing evidence suggests that vitamin D supplementation may have potentially beneficial effects on those diseases.

Effects of Lombok Island's local moringa (Moringa oleifera) leaf powder on the decrease of liver fructose levels and GLUT5 expression in albino rats (Rattus novergicus) fed a high fructose diet.

Consumed fructose enters enterocytes of the intestinal epithelial apical membrane mediated by glucose transporter 5 (GLUT5). To determine the effects of Lombok Island's local Moringa leaf powder on reducing liver fructose levels and GLUT5 expression in the small intestine of albino rats (Rattus novergicus) fed a high-fructose diet. Moringa leaf (Moringa oleifera) was obtained from Lombok Island, Indonesia. Subsequently, 30 male albino rats (R. novergicus) were used, divided into the normal group (NG), treatment group 1 (T1G), treatment group 2 (T2G), Quercetin group (QG), Moringa group (MG). Quercetin and moringa leaf powder (M. oleifera) was administered at 50 and 500 mg/kgbw for 28 days. Enzyme-linked immunosorbent assay (Elisa) method was used to examine liver fructose levels. The observation of GLUT5 expression in the small intestine was performed by the Immunofluorescence method. The ANOVA test proved that there are significant differences (p < 0.005) in liver fructose levels in all groups. Further post hoc tests show no significant difference (p > 0.005) in liver fructose levels in rats fed a high fructose diet in T1G and T2G with QG and MG rats. However, Moringa leaf powder reduces liver fructose levels by 32.1% and 17.2% in T1G and T2G rats, respectively. The ANOVA test showed a significant difference (p < 0.005) in the expression of GLUT5 in all groups. Further post hoc tests showed a significant difference (p < 0.005) in the expression of GLUT5 in the duodenum, jejunum, and ileum between the NG and the T1G rats. Meanwhile, in T2G rats, significant differences were only found in the jejunum. Moringa leaf powder reduces GLUT5 expression in T1G rats by 44.5%, 59.5%, and 57.2% in the duodenum, jejunum, and ileum, whereas in T2G rats is by 33.5%, 50.2%, and 48.1%, respectively. The administration of local moringa (M. oleifera) leaf powder in Lombok Island had an effect on reducing GLUT5 expression in the small intestine, however, did not in liver fructose levels of albino rats (R. novergicus) fed a high-fructose diet.

Vitamin D receptor involves in the protection of intestinal epithelial barrier function via up-regulating SLC26A3

BackgroundVitamin D receptor (VDR) and SLC26A3 (DRA) have been identified as pivotal protective factors in maintaining gut homeostasis in IBD patients. However, the specific mechanism underlying the increased intestinal susceptibility to inflammation induced by the loss of VDR and whether DRA participates in the role of VDR regulating intestinal epithelial barrier function are undefined. AimThe current study is undertaken to elucidate the regulatory effects of VDR on DRA and VDR prevents intestinal epithelial barrier dysfunction via up-regulating the expression of DRA. MethodsWT and VDR-/- mice are used as models for intestinal epithelial response. Paracellular permeability is measured by TEER and FD-4 assays. Immunohistochemistry, immunofluorescence, qPCR and immunoblotting are performed to determine the effects of VDR and DRA on gut epithelial barrier function. ResultsVDR-/- mice exhibits significant hyperpermeability of intestine with greatly decreased levels of ZO-1 and Claudin1 proteins. DRA is located on the intestinal epithelial apical membrane and is tightly modulated by VDR in vivo and in vitro via activating ERK1/2 MAPK signaling pathway. Notably, the current study for the first time demonstrates that VDR maintains intestinal epithelial barrier integrity via up-regulating DRA expression and the lack of DRA induced by VDR knockdown leads to a more susceptive condition for intestine to DSS-induced colitis. ConclusionOur study provides evidence and deep comprehension regarding the role of VDR in modulating DRA expression in gut homeostasis and makes novel contributions to better generally understanding the links between VDR, DRA and intestinal epithelial barrier function.

Autoimmunity to extracellular matrix in inflammatory bowel disease: antibody to intestinal epithelial basement membrane and renal tubules in severe ulcerative colitis and Crohn disease

Autoimmunity to extracellular matrix in inflammatory bowel disease: antibody to intestinal epithelial basement membrane and renal tubules in severe ulcerative colitis and Crohn disease

Epithelial HVEM maintains intraepithelial T cell survival and contributes to host protection.

Intraepithelial T cells (IETs) are in close contact with intestinal epithelial cells and the underlying basement membrane, and they detect invasive pathogens. How intestinal epithelial cells and basement membrane influence IET survival and function, at steady state or after infection, is unclear. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily, is constitutively expressed by intestinal epithelial cells and is important for protection from pathogenic bacteria. Here, we showed that at steady-state LIGHT, an HVEM ligand, binding to epithelial HVEM promoted the survival of small intestine IETs. RNA-seq and addition of HVEM ligands to epithelial organoids indicated that HVEM increased epithelial synthesis of basement membrane proteins, including collagen IV, which bound to β1 integrins expressed by IETs. Therefore, we proposed that IET survival depended on β1 integrin binding to collagen IV and showed that β1 integrin-collagen IV interactions supported IET survival in vitro. Moreover, the absence of β1 integrin expression by T lymphocytes decreased TCR αβ+ IETs in vivo. Intravital microscopy showed that the patrolling movement of IETs was reduced without epithelial HVEM. As likely consequences of decreased number and movement, protective responses to Salmonella enterica were reduced in mice lacking either epithelial HVEM, HVEM ligands, or β1 integrins. Therefore, IETs, at steady state and after infection, depended on HVEM expressed by epithelial cells for the synthesis of collagen IV by epithelial cells. Collagen IV engaged β1 integrins on IETs that were important for their maintenance and for their protective function in mucosal immunity.

Ligands for oral delivery of peptides across the blood-brain-barrier

Peptides are short chains of amino acids linked by peptide bonds. Many peptides and proteins are limited by their poor enzymatic stability and permeability across the intestinal epithelial membranes and/or blood-brain barrier (BBB). Parenteral administration of these peptides is unfavorable because of procedural complications and low patient compliance with treatments. Instead, oral delivery is the preferred route of administration because it allows for self-administration and has a high degree of patient acceptability and compliance. Oral delivery of these peptides poses a major challenge, because the peptide drug must overcome both the physical and biochemical barriers of the gastrointestinal tract and BBB. An oral drug delivery system is beneficial because it can protect peptide drugs against degradation and deliver them to the brain, where they exert their pharmacological actions. The use of active-targeting ligands and/or cell-penetrating peptides increases penetration and uptake across the BBB. This review focuses on the diverse combinations of drug delivery systems, active-targeting ligands, and cell-penetrating peptides used to deliver peptides to the brain.

Characterizing nanoplastics‐induced stress and its SERS fingerprint in an intestinal membrane model

AbstractEpithelium membranes provide important barrier functions, and it is important to understand how nanoparticle (NP) exposure affects their barrier function. In this manuscript, we investigate NP‐induced stress in a Caco‐2 intestinal epithelial membrane model and its effect on the vibrational spectrum of the extracellular medium that can be sampled and investigated without perturbation of the cells. Monolayers of Caco‐2 cells were incubated with 50 nm diameter polystyrene (PS) NPs functionalized with amine or carboxylic acid groups and concentrations of 1 × 1012–1 × 1014PS NPs mL–1for 6 and 18 hours. Reactive oxygen species (ROS) generation, cell viability, and intestinal membrane integrity measurements were performed to detect and quantify PS NP‐induced membrane damage under the acute exposure conditions. After identifying conditions that result in NP‐induced stress, Surface Enhanced Raman Spectroscopy (SERS) was applied to monitor the composition of the medium in direct contact with the intestinal cells and to detect potential PS NP‐induced changes in the cellular metabolism in real time and in a minimally invasive fashion. The analysis of the SERS spectra through artificial intelligence algorithms and chemometric tools revealed concentration‐, exposure time‐, and surface chemistry‐dependent differences in the cellular metabolism in response to PS NPs. The SERS spectral analysis identifies the ring breathing mode of hypoxanthine (C4H4N4O), as a spectroscopic marker for the PS NP‐induced loss in membrane integrity.

A pharmacokinetic study of morphine-6-O-sulfate in rat plasma and brain.

Morphine-6-O-sulfate (M6S), a polar, zwitterionic sulfate ester of morphine, is a powerful and safe analgesic in several rat models of pain. A sensitive liquid chromatography-tandem mass spectrometry bioanalytical method was developed and validated for the simultaneous determination of M6S and morphine (MOR) in rat plasma and brain after M6S administration. Morphine-d6 was used as internal standard. Multiple reaction monitoring was used for detection and quantitation of M6S, MOR, and morphine-d6 in the turbo ion spray positive mode. The chromatographic separation was carried out on an Alltech Altima C18 column. The analytical method was validated for linearity, precision, accuracy, specificity, and stability over a concentration range of 3-8000 ng/ml in rat plasma and 10-10,000 ng/ml in brain samples for both M6S and MOR. The validated method was applied to determine the PK profile of M6S in plasma after i.v., i.p., and oral dosing in male Sprague-Dawley rats. Rats were administered M6S by i.p. administration (5.6 and 10.0mg/kg) or orally (10 and 30 mg/kg) and bioavailability compared to an i.v. injection (1mg/kg) of M6S. The in vivo results indicate that M6S is not a prodrug of morphine, since M6S is not biotransformed into MOR in plasma after either i.p. or oral administration, and MOR was not detected in brain. The bioavailability of M6S was >93% and about 5% after i.p. and oral dosing, respectively. The low oral bioavailability of M6S may be due to poor permeation of the intestinal epithelial membrane. After i.p.-administration, M6S appears to reach brain tissues in low, but significant, concentrations.

Slc26a3 (DRA) in the Gut: Expression, Function, Regulation, Role in Infectious Diarrhea and Inflammatory Bowel Disease.

The transport of transepithelial Cl- and HCO3- is crucial for the function of the intestinal epithelium and maintains the acid-based homeostasis. Slc26a3 (DRA), as a key chloride-bicarbonate exchanger protein in the intestinal epithelial luminal membrane, participates in the electroneutral NaCl absorption of intestine, together with Na+/H+ exchangers. Increasing recent evidence supports the essential role of decreased DRA function or expression in infectious diarrhea and inflammatory bowel disease (IBD). In this review, we give an overview of the current knowledge of Slc26a3, including its cloning and expression, function, roles in infectious diarrhea and IBD, and mechanisms of actions. A better understanding of the physiological and pathophysiological relevance of Slc26a3 in infectious diarrhea and IBD may reveal novel targets for future therapy. Understanding the physiological function, regulatory interactions, and the potential mechanisms of Slc26a3 in the pathophysiology of infectious diarrhea and IBD will define novel therapeutic approaches in future.

Evaluation of Cell-Penetrating Peptides as Versatile, Effective Absorption Enhancers: Relation to Molecular Weight and Inherent Epithelial Drug Permeability.

The poor permeability of new drug candidates across intestinal epithelial membranes complicates their development in oral form. This study investigated the potential of cell-penetrating peptides (CPPs) to improve the intestinal permeation and absorption of low-permeable low-molecular-weight (low-MW) drugs. The in vitro epithelial permeation of six different drugs (metformin, risedronate, zanamivir, methotrexate [MTX], tacrolimus, and vincristine [VCR]) across Caco-2 cell monolayers was examined in the presence and absence of L- or D-penetratin, and the correlation between permeation enhancement efficiency and the properties of tested drugs was analyzed. In addition, a rat closed ileal loop absorption study was conducted to determine the in vivo effects of penetratin. MTX and VCR efficiently permeated Caco-2 monolayers in the presence of L- and D-penetratin, suggesting that CPPs enhanced the epithelial permeation of drugs with relatively high molecular weight and resultant limited intrinsic permeability. The in vivo rat closed ileal loop absorption study revealed the stimulatory effect of L- and D-penetratin on the intestinal absorption of MTX and VCR. CPPs are useful as oral absorption enhancers for low-permeable drugs.

Effects of Chitosan-Gentamicin Conjugate Supplement on Non-Specific Immunity, Aquaculture Water, Intestinal Histology and Microbiota of Pacific White Shrimp (Litopenaeus vannamei).

When the aquaculture water environment deteriorates or the temperature rises, shrimp are susceptible to viral or bacterial infections, causing a large number of deaths. This study comprehensively evaluated the effects of the oral administration of a chitosan–gentamicin conjugate (CS-GT) after Litopenaeus vannamei were infected with Vibrio parahaemolyticus, through nonspecific immunity parameter detection, intestinal morphology observation, and the assessment of microbial flora diversification by 16S rRNA gene sequencing. The results showed that the oral administration of CS-GT significantly increased total hemocyte counts and reduced hemocyte apoptosis in shrimp (p < 0.05). The parameters (including superoxide dismutase, glutathione peroxidase, glutathione, lysozyme, acid phosphatase, alkaline phosphatase, and phenoloxidase) were significantly increased (p < 0.05). The integrity of the intestinal epithelial cells and basement membrane were enhanced, which correspondingly alleviated intestinal injury. In terms of the microbiome, the abundances of Vibrio (Gram-negative bacteria and food-borne pathogens) in the water and gut were significantly reduced. The canonical correspondence analysis (CCA) showed that the abundances of Vibrio both in the water and gut were negatively correlated with CS-GT dosage. In conclusion, the oral administration of CS-GT can improve the immunity of shrimp against pathogenic bacteria and significantly reduce the relative abundances of Vibrio in aquaculture water and the gut of Litopenaeus vannamei.

Development of a modified enrichment method for the rapid immunoassay of Escherichia coli O157 strains in fresh cut vegetables

Escherichia coli O157:H7 is a pathogen that colonizes human intestinal epithelial membrane and causes a characteristic lesion and severe syndromes, including diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. It is difficult to isolate and identify this pathogen. The purpose of this research was to establish a two-stage enrichment method for the detection of E. coli O157:H7 in fresh cut vegetables. Fresh cut vegetable samples were first enriched in modified EC broth with novo-biocin at 42°C for 6 hr, and then transferred to sorbitol MacConkey broth containing cefixime and tellurite at 35°C for 18 hr. Finally, the enriched broth was verified for the existence of E. coli O157 by using an automated enzyme-linked fluorescence immunoassay (mini-VIDAS) or an immunostrip test. This two-stage enrichment method would decrease the false-positive E. coli O157 tested by immunostrip or mini-VIDAS. The pathogen in food sample could be as low as 10 CFU/g and selectively enriched to 105 or 107 CFU/mL required for the detection by mini-VIDAS or immunostrip. The presence of E. coli O157:H7 in enriched broth was confirmed by multiplex polymerase chain reaction (PCR). Thus, the two-stage enrichment method can be used to rapidly screen food samples for E. coli O157.

Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation.

Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

ПАТОЛОГОАНАТОМИЧЕСКИЕ ИЗМЕНЕНИЯ ПРИ ЭНТЕРОПАТИЯХ ВЫЗВАННЫМИ ДИФИЛЛОБОТРИОЗОМ У СОБАК

Pathomorphological research of dogs intes-tine allowed us to describe in more detail all encountered cases of pathological manifesta-tion and effect of broad tapeworm on differ-ent tissue structures and their frag-ments.Enteropathies are polymorphous and are introduced with different etiological fac-tors. Intestine helminthiases have heavy share of ethiofactors.Microscopic signs of intestinal lesion of dogs during diphylloboth-riasis were characterized with obvious changes in mucosa and submucosa and their damage pathological changes in a smooth muscle layer were rarely noted.Serous coat and all its structural fragments complied with the norm and didn't have pathological. Extensive zones of damage from mehanical injury were not noted,there were areas of gut wall changes with damage and intestinal villi destruction and epithelial membrane destruc-tion possibly caused by morphological and physiological structural features of the para-site. Characteristic pathological signs were symptomatic desquamation of tessellated epithelium.Extensive damage of serous coat tracedin all the microslides prepared for his-tologic study.All these changes can be a pos-sible reason of development of reduction of nutrient intake.Such kind of research as pathological process encourages more de-tailed identifying of clinical status during diphyllobothriasis and its not characteristic or specific manifestations and will be the additional criteria of high-impact therapeu-tics or its evidence.

Stimulation of constitutive nitric oxide uniquely and compensatorily regulates intestinal epithelial cell brush border membrane Na absorption.

In the mammalian small intestine, sodium is primarily absorbed by Na+/H+ exchange (NHE3) and Na‐glucose cotransport (SGLT1) in the brush border membrane (BBM) of villus cells. However, how enhanced cellular constitutive nitric oxide (cNO) may affect NHE3 and SGLT1 remains unclear. Both in vivo in rabbit intestinal villus cells and in vitro IEC‐18 cells, administration of NO donor, GSNAP, modestly increased cNO. GSNAP stimulated SGLT1 in villus and IEC‐18 cells. The mechanism of stimulation was secondary to an increase in the affinity of SGLT1 for glucose. The change in SGLT1 was not secondary to altered Na‐extruding capacity of the cell since Na+/K+‐ATPase was decreased by GSNAP treatment. In contrast, GSNAP inhibited NHE3 activity in villus cell BBM. The mechanism of NHE3 inhibition was secondary to reduced BBM transporter numbers. These studies demonstrated that the physiological increase in cNO uniquely regulates mammalian small intestinal NHE3 and SGLT1 to maintain Na homeostasis.

Modulation of Intestinal Transport and Absorption of Topotecan, a BCRP Substrate, by Various Pharmaceutical Excipients and Their Inhibitory Mechanisms of BCRP Transporter

Breast cancer resistance protein transporter (ABCG2/BCRP) is highly expressed on the intestinal epithelial membrane and has a significant impact on the oral absorption of topotecan. In this study, we examined 6 pharmaceutical excipients including BL-9EX, Brij97, Cremophor EL, Labrasol, Pluronic F68, and Tween 20 for their BCRP inhibitory effects. A bidirectional transport study using Caco-2 cells demonstrated that Tween 20 and Cremophor EL significantly increased the absorptive transport of topotecan, while simultaneously decreasing secretory transport. Interestingly, Labrasol selectively increased absorptive transport, whereas Pluronic F68 selectively decreased the secretory transport, of topotecan. Further investigation using an in situ closed loop experiment showed that 0.05% (w/v) Tween 20 and Cremophor EL significantly increased the intestinal absorption of topotecan in rats. An LDH assay demonstrated that 0.05% (w/v) Tween 20 and Cremophor EL did not cause significant damage to intestinal epithelial membranes. Furthermore, we examined the absorption-enhancing mechanisms of these excipients and found that Cremophor EL, Tween 20, and Labrasol increased the membrane fluidity of the inner lipid bilayers of the intestine. Therefore, this might be one of the most important mechanisms for inhibition of BCRP function by these excipients in the intestine.

The use of low molecular weight protamine to enhance oral absorption of exenatide

Although oral delivery of exenatide has significant advantages, its poor permeability through intestinal epithelial membranes and rapid digestion by pepsin and ereptase in the gastrointestinal tract make effective oral delivery of exenatide a formidable challenge. In this study, we constructed a zinc ion (Zn2+) and exenatide complex functionalized nanoparticle (NP) oral delivery system to overcome the above-mentioned issue. Polyethylene glycol-poly(lactic-co-glycolic acid) (PEG-PLGA) was used as a drug carrier to escape enzymatic degradation in the gastrointestinal tract, and low molecular weight protamine (LMWP) was used as a functional group to increase penetration of NPs into the intestinal epithelium. The functionalized NPs exhibited significantly improved penetration across the intestinal epithelium, as shown by cell uptake and transmembrane transport experiments. Moreover, a significant hypoglycemic effect was observed in diabetic rats. The relative bioavailability of the orally administered functionalized NPs vs. subcutaneous injection was 7.44%, 29-fold that of the exenatide-Zn2+ solution group. These findings indicate that our modification could effectively improve exenatide treatment.

N-trimethyl chitosan nanoparticles and CSKSSDYQC peptide: N-trimethyl chitosan conjugates enhance the oral bioavailability of gemcitabine to treat breast cancer

Gemcitabine is a nucleoside analogue effective against a number of cancers. However, the full potential of this drug has not been realised, in part due to low oral bioavailability and frequent dosing requirements. This study reports the synthesis, in-vitro, ex-vivo and in-vivo evaluation of trimethyl chitosan (TMC) - CSKSSDYQC (CSK) peptide conjugates capable of enhancing the oral bioavailability of gemcitabine due to the ability to target intestinal goblet cells and promote intestinal cellular uptake. TMC was synthesized by a novel two-step methylation method to improve quanternization and yield. The CSK-TMC conjugates were prepared by ionic gelation to achieve particles sized at 173.6 ± 6.8 nm, zeta potential of +18.5 ± 0.2 mV and entrapment efficiency of 66.4 ± 0.1%, capable of sustained drug release. By encapsulating gemcitabine into CSK-TMC conjugates, an increased amount of drug permeated through porcine intestinal epithelial membranes compared with the unconjugated TMC nanoparticles (NPs). The rate of cellular uptake of drug loaded conjugates into HT29-MTX-E12 intestinal goblet cells, was time- and concentration-dependant. The conjugates underwent active transport associated with adsorptive mediated, clathrin and caveolae mediated endocytosis. In cellular transport studies, drug loaded conjugates had greater drug transport capability compared with drug solution and TMC NPs over the co-cultured Caco-2/HT29-MTX-E12 cell monolayer. The drug loaded conjugates exhibited electrostatic interaction with the intestinal epithelial cells. Both P-glycoprotein (P-gp) and multiple resistance protein-2 (MRP2) efflux affected the cellular transport of the conjugates. Importantly, during the pharmacokinetic studies, the orally administrated drug loaded into TMC NPs showed an improved oral bioavailability of 54.0%, compared with gemcitabine solution of 9.9%. Notable, the CSK-TMC conjugates further improved oral bioavailability to 60.1% and reduced the tumour growth rate in a BALB/c nude mouse model, with a 5.1-fold and 3.3-fold reduction compare with the non-treated group and gemcitabine solution group. Furthermore, no major evidence of toxicity was discernible on histologic studies of selected organs. In conclusion, the presented CSK-TMC conjugates and TMC nanoparticles both significantly improve the oral bioavailability of gemcitabine and have the potential for the treatment of breast cancer.