Impact of regulatory T cell therapy on immune cell composition and fetal survival rate in abortion prone mice.

CONTEXT: Implantation of fertilised eggs and survival of a semi-allogenic embryo rely on the interactions between the cells and molecules preparing the uterus. We investigated the effect of regulatory T cell (Treg) therapy on the mechanism of local immune tolerance of mice prone to spontaneous abortion. METHODS: Naive T cells were stimulated in vitro with 17ß-oestradiol (E2), progesterone (P4) and TGF-ß1 for 96h to generate induced Tregs (iTreg). The iTregs were injected into DBA/2-mated pregnant CBA/J female mice (abortion prone model). On day 14 of pregnancy, mice were killed and decidual and placental tissues were collected for cellular composition analysis. RESULTS: Abortion prone mice (PBS treated) showed significantly lower survival rates (P <0.0001), increased CD3+ CD8+ (P <0.05), lower IDO+ (P <0.05) and increased natural killer cells (uNK) cell numbers (P <0.001) in the uterus, as well increased NK cells in the placenta (P <0.05) than in normal pregnant mice (CBA/J×BALB/c). Adoptive transfer of iTregs increased fetal survival in abortion-prone mice (P <0.01) and histopathological evaluation revealed a significantly decreased number of uNK cells in the uterus of TGF-ß1-, E2- and P4-iTregs (P<0.05, P<0.0001 and P<0.05, respectively) than in the PBS treated group. In the placenta, we found significantly lower numbers of uNK cells from TGF-ß1-, E2- and P4-iTregs than in the PBS treated group (P <0.05, P <0.05 and P <0.01, respectively). CONCLUSIONS: We propose that modulation of uterine NK cell activity through immunotherapy using Treg cells should be given more attention as an immunological strategy in the treatment of recurrent miscarriage.

Adoptive cell therapy with induced regulatory T cells normalises the abortion rate in abortion-prone mice.

Ovarian hormones drive invivo generation of regulatory T cells (Tregs) during pregnancy. Little is known about the therapeutic potential of invitro hormone-derived Tregs in pregnancy loss. We investigated the effects of hormone-induced Tregs in a murine model of abortion. CD4+CD25- T cells were isolated from the spleens of CBA/J mice and stimulated with either 17ß-oestradiol (E2), progesterone (P4) or transforming growth factor-ß1 (TGFB1) plus retinoic acid (RA) for 4 days to generate induced Tregs (iTregs). On Days 1-4 of gestation, DBA/2-mated pregnant CBA/J female mice (abortion prone) were injected intravenously with iTregs or Tregs isolated from normal BALB/c-mated pregnant CBA/J mice (np-Tregs). On Day 14, the number of resorbed fetuses was assessed. Serum interferon (IFN)-γ and uterine forkhead box p3 (Foxp3) expression was analysed by ELISA and immunohistochemistry respectively. Using a 3H-thymidine incorporation assay, isolated CD4+CD25+ Tregs induced by the different treatments suppressed the proliferation of CD4+CD25- T cells. Adoptive transfer of iTregs (from all induction groups) significantly decreased fetal resorption in abortion-prone mice. There were no significant changes in serum IFN-γ concentrations after the adoptive transfer of iTregs or np-Tregs. Immunohistochemistry revealed significantly higher Foxp3 expression in gravid uteri from mice injected with np-Tregs and P4-induced iTregs than in the phosphate-buffered saline-treated group. The findings of this study indicate a potential therapeutic benefit of invitro-induced Tregs in patients with recurrent abortion.

Spectrofluorimetric method for determination of bupropion in pharmaceutical dosage forms.

OBJECTIVE: Bupropion is a noradrenaline and dopamine reuptake inhibitor which is used as an antidepressant drug. Few HPLC and spectrophotometric methods have been reported before for the determination of bupropion. Most of the previous methods reported for determination of bupropion in pharmaceutical dosage forms are somehow dangerous to health and environment because of using organic solvents. METHOD: In the present method bupropion was determined in pharmaceutical dosage forms by spectrofluorimetry after ion-pair complex formation with eosin Y. The ion-pair complex formation was optimized for reagent amount, buffer pH and time. RESULT: The developed method was linear over the range of 3-120µgmL-1 with an acceptable precision (CV<1.5%) and accuracy (Error<1%). CONCLUSION: The present method is applicable for determination of bupropion in pharmaceutical dosage forms for routine quality control analysis.

Evaluation of the immune response in BALB/c mice induced by a novel DNA vaccine expressing GRA14 against Toxoplasma gondii.

Toxoplasma gondii can cause severe and even fatal disease in human beings and animals. Effective vaccines may contribute to control toxoplasmosis. GRA14, a novel secreted dense granule protein of T. gondii, has been proposed as a vaccine candidate due to its intervacuolar transport and unique topology in the parasitophorous vacuole membrane. In this study, we constructed a DNA vaccine encoding GRA14 of T. gondii. BALB/c mice were immunized intramuscularly three times at 2 week intervals and challenged with T. gondii RH strain 5 weeks later. The immune responses were evaluated using lymphocyte proliferation assay, cytokine and antibody measurements. In addition, the survival times and parasite load of mice challenged with the virulent T. gondii RH strain were evaluated. The results showed that the mice immunized with pcGRA14 induced both enhanced specific humoral and Th1 cellular immune responses, and also mice immunized with the pcGRA14 showed an increased survival time and decreased parasite load compared with control groups (P<.05). The results indicated, for the first time, that the GRA14 is a potential DNA vaccine against toxoplasmosis.

The metabolomics and lipidomics window into thyroid cancer research.

CONTEXT: Thyroid carcinoma is the most common endocrine system malignancy with a fast rising incidence in the last decade for unknown reasons. Fine needle aspiration (FNA) biopsy, the gold standard in thyroid cancer (TC) screening has still its own challenges and in some cases needs a proceeding surgery. OBJECTIVE: This review highlights the role of the two most recent "omics" approaches, "metabolomics" and "lipidomics", in the field of TC research. METHODS: All the previous studies have been extracted from the literature and major concepts were detailed in the field of TC metabolomics and lipidomics. RESULTS: Metabolomics and lipidomics, have potential in finding biomarkers related to thyroid carcinoma. Among the previous studies, the most important introduced altered tissue metabolites and lipids included glucose and galactose, lactate, Scyllo- and Myo inositol, hypoxanthine, citrate, cholesterol and choline. CONCLUSION: Metabolomics methods have been widely used in the field of biomarker discovery in TC and attempts are still in progress to use these methods to find a reliable biomarker panel besides current diagnostic tools.

Formulation, in vitro evaluation and kinetic analysis of chitosan-gelatin bilayer muco-adhesive buccal patches of insulin nanoparticles.

The present study was performed to optimise the formulation of a muco-adhesive buccal patch for insulin nanoparticles (NPs) delivery. Insulin NPs were synthesised by an ionic gelation technique using N-di methyl ethyl chitosan cysteine (DMEC-Cys) as permeation enhancer biopolymer, tripolyphosphate (TPP) and insulin. Buccal patches were developed by solvent-casting technique using chitosan and gelatine as muco-adhesive polymers. Optimised patches were embedded with 3 mg of insulin-loaded NPs with a homogeneous distribution of NPs in the muco-adhesive matrix, which displayed adequate physico-mechanical properties. The drug release characteristics, release mechanism and kinetics were investigated. Data fitting to Peppas equation with a correlation coefficient indicated that the mechanism of drug release followed an anomalous transport that means drug release was afforded through drug diffusion along with polymer erosion. In vitro drug release, release kinetics, physical and mechanical studies for all patch formulations reflected the ideal characteristics of this buccal patch for the delivery of insulin NPs.

Nonlinear damping and quasi-linear modelling.

The mechanism of energy dissipation in mechanical systems is often nonlinear. Even though there may be other forms of nonlinearity in the dynamics, nonlinear damping is the dominant source of nonlinearity in a number of practical systems. The analysis of such systems is simplified by the fact that they show no jump or bifurcation behaviour, and indeed can often be well represented by an equivalent linear system, whose damping parameters depend on the form and amplitude of the excitation, in a 'quasi-linear' model. The diverse sources of nonlinear damping are first reviewed in this paper, before some example systems are analysed, initially for sinusoidal and then for random excitation. For simplicity, it is assumed that the system is stable and that the nonlinear damping force depends on the nth power of the velocity. For sinusoidal excitation, it is shown that the response is often also almost sinusoidal, and methods for calculating the amplitude are described based on the harmonic balance method, which is closely related to the describing function method used in control engineering. For random excitation, several methods of analysis are shown to be equivalent. In general, iterative methods need to be used to calculate the equivalent linear damper, since its value depends on the system's response, which itself depends on the value of the equivalent linear damper. The power dissipation of the equivalent linear damper, for both sinusoidal and random cases, matches that dissipated by the nonlinear damper, providing both a firm theoretical basis for this modelling approach and clear physical insight. Finally, practical examples of nonlinear damping are discussed: in microspeakers, vibration isolation, energy harvesting and the mechanical response of the cochlea.

Polyphyly, gene-duplication and extensive allopolyploidy framed the evolution of the ephemeral Vulpia grasses and other fine-leaved Loliinae (Poaceae).

The fine-leaved Loliinae is one of the temperate grass lineages that is richest in number of evolutionary switches from perennial to annual life-cycle, and also shows one of the most complex reticulate patterns involving distinct diploid and allopolyploid lineages. Eight distinct annual lineages, that have traditionally been placed in the genus Vulpia and in other fine-leaved ephemeral genera, have apparently emerged from different perennial Festuca ancestors. The phenotypically similar Vulpia taxa have been reconstructed as polyphyletic, with polyploid lineages showing unclear relationships to their purported diploid relatives. Interspecific and intergeneric hybridization is, however, rampant across different lineages. An evolutionary analysis based on cloned nuclear low-copy GBSSI (Granule-Bound Starch Synthase I) and multicopy ITS (Internal Transcribed Spacer) sequences has been conducted on representatives of most Vulpia species and other fine-leaved lineages, using Bayesian consensus and agreement trees, networking split graphs and species tree-based approaches, to disentangle their phylogenetic relationships and to identify the parental genome donors of the allopolyploids. Both data sets were able to reconstruct a congruent phylogeny in which Vulpia was resolved as polyphyletic from at least three main ancestral diploid lineages. These, in turn, participated in the origin of the derived allopolyploid Vulpia lineages together with other Festuca-like, Psilurus-like and some unknown genome donors. Long-distance dispersal events were inferred to explain the polytopic origin of the Mediterranean and American Vulpia lineages.

Prevalence and associated factors of T-score discordance between different sites in Iranian patients with spinal cord injury.

OBJECTIVES: The present study was conducted to determine the prevalence of T-score discordance and its risk factors in a group of patients with spinal cord injury in a university teaching hospital in the Iranian capital of Tehran. METHODS: This cross-sectional study was conducted on paraplegic men undergoing bone density testing in an outpatient clinic at a hospital in the Iranian capital, Tehran, between March 2011 and 2012. A questionnaire on demographic and anthropometric characteristics, including age, height, weight, engagement in physical activity and personal smoking habits, was filled out for each subject. All the subjects underwent bone mineral density measurement and blood samples were sent for laboratory testing. RESULTS: Major T-score discordance between two sites was noted in 54 (41.22%) patients. Multivariate logistic regression revealed that every unit increase in serum calcium levels, as the only factor influencing T-score discordance, was associated with a 2.49-fold increased risk in T-score discordance in the area. As for the spine and radius, BMI was the only influencing factor as every unit increase in BMI was associated with a 14% lower risk for T-score discordance in these regions. Body mass index was the only factor, based on the multivariate model, affecting the risk of developing T-score discordance between two sites. CONCLUSION: Our study revealed the high prevalence of T-score discordance in patients with spinal cord injury. Physicians should therefore be encouraged to perform BMD at three sites when visiting patients with such injuries.

Frequency and expression of inhibitory markers of CD4(+) CD25(+) FOXP3(+) regulatory T cells in patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is the most symptomatic primary antibody deficiency associated with recurrent infections and chronic inflammatory diseases as well as autoimmunity. CD4(+) CD25(+) FOXP3(+) regulatory T cells (Tregs) are critical T cell subsets for maintaining self-tolerance and regulation of immune response to antigens thus play a pivotal role in preventing autoimmunity. Thirty-seven CVID patients and 18 age-/sex-matched controls were enrolled. Peripheral blood mononuclear cells (PBMCs) were obtained from both groups, and the percentage of Tregs was calculated using flow cytometry method. The mRNA expression of Tregs' surface markers cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoid-induced tumour necrosis factor receptor (GITR), which are associated with Tregs' inhibitory function, was compared between patients and controls by quantitative real-time PCR TaqMan method. The results revealed that the frequency of Tregs was significantly lower in CVID patients than normal individuals (P < 0.001). In addition, CVID patients with autoimmunity were found to have markedly reduced proportion of Tregs compared to those cases without autoimmune diseases (P = 0.023). A significant difference was seen in factor forkhead box P3 (FOXP3) expression between CVID patients and controls (P < 0.001). The mRNAs of CTLA-4 and GITR genes were expressed at lower levels in CVID patients compared to control group (P = 0.005 and <0.001, respectively). Our findings showed reduced proportion of Tregs in CVID patients together with downregulation of FOXP3 protein and diminished expression of inhibitory Tregs' markers. It could be concluded that all of these changes may be responsible for cellular immune dysregulation observed in these patients especially those with autoimmune manifestation.

Hybrid carbon fiber/carbon nanotube composites for structural damping applications.

Carbon nanotubes (CNTs) were grown on the surface of carbon fibers utilizing a relatively low temperature synthesis technique; graphitic structures by design (GSD). To probe the effects of the synthesis protocols on the mechanical properties, other samples with surface grown CNTs were prepared using catalytic chemical vapor deposition (CCVD). The woven graphite fabrics were thermally shielded with a thin film of SiO2 and CNTs were grown on top of this film. Raman spectroscopy and electron microscopy revealed the grown species to be multi-walled carbon nanotubes (MWCNTs). The damping performance of the hybrid CNT-carbon fiber-reinforced epoxy composite was examined using dynamic mechanical analysis (DMA). Mechanical testing confirmed that the degradations in the strength and stiffness as a result of the GSD process are far less than those encountered through using the CCVD technique and yet are negligible compared to the reference samples. The DMA results indicated that, despite the minimal degradation in the storage modulus, the loss tangent (damping) for the hybrid composites utilizing GSD-grown MWCNTs improved by 56% compared to the reference samples (based on raw carbon fibers with no surface treatment or surface grown carbon nanotubes) over the frequency range 1-60 Hz. These results indicated that the energy dissipation in the GSD-grown MWCNTs composite can be primarily attributed to the frictional sliding at the nanotube/epoxy interface and to a lesser extent to the stiff thermal shielding SiO2 film on the fiber/matrix interface.

Microbial Profile and Antibiotic Susceptibility Pattern in Diabetic Patients with Mild, Moderate, and Severe Foot Infections in Tehran.

It is estimated that 10-25% of diabetic patients will encounter diabetic foot ulcers (DFU) during their lifetime. This study evaluated the microbiology of DFUs and determined the antibiotic resistance pattern of bacterial isolates based on the severity of wounds and infections in different grades of ulcer. The specimens were collected from115 diabetic foot infections (DFI) deep tissue by needle aspiration and biopsy. The aerobic and anaerobic cultures and antimicrobial susceptibility testing were carried out. The presence of resistance genes including metallo-beta-lactamases (MBL), extended-spectrum ß-lactamase (ESBL), ermA, ermC, and mecA was also determined. A total of 222 microorganisms were isolated. The prevalence of poly-microbial infections was 69.6%. Bacterial isolates comprised 64.2% Gram-positive bacteria (GPB), 33.5% Gram-negative bacteria (GNB), and five isolates of anaerobic bacteria were also detected. The most prevalent GPB and GNB were Staphylococcus spp. (52.2%) and Escherichia coli (33.3%), respectively. The prevalence of poly-microbial infections and GNB was positively associated with increased grades of Wagner and IDSA classifications. Among Staphylococcus aureus isolates, resistance to clindamycin (73.5%), ciprofloxacin (70.6%), and erythromycin (70.6%) were noticeable. GNB was also highly resistant to cephalosporins and ciprofloxacin. ESBL genes were detected in approximately 40% of isolates of Enterobacteriaceae. The prevalence of ermA, ermC, and mecA genes in S. aureus isolates were 8.8%, 32.3%, and 14.7%, respectively. In conclusion, our data suggest that GPBs are the most common isolates from DFIs. Furthermore, with the development of wounds and infection, the prevalence of GNB in DFIs are increased.

Synthesis of WS2 nanostructures from the reaction of WO3 with CS2 and mechanical characterization of WS2 nanotube composites.

Tungsten disulfide (WS(2)) nanometer sheets, spheres, fibers and tubes were generated by a synthetic pathway that avoids the use of H(2)S as the source of sulfur and employs instead CS(2) vapor, carried by an Ar or N(2)/H(2) stream in a heated tubular furnace, for the reaction with WO(3) precursor powders. The experiments were conducted at temperatures between 700 and 1000 °C, while the reaction times expanded between 30 min and 24 h. Characterization methods used to analyze the products of the synthesis include TEM, SEM, XRD and EDX. We found a strong correlation between precursor and product microstructure, although the temperature and reaction times play a critical role in the products' microstructural features as well. WS(2) inorganic fullerene (IF) nanospheres are generated in a wide window of conditions, while nanotubes and nanofibers are only produced at high temperatures or long reaction times. A proposed growth mechanism based on the CS(2) synthetic approach is presented. Nanoindentation and nano-impulse techniques were used to characterize the mechanical properties of polymer matrix-WS(2) nanotube composites, finding them superior to equivalent SWCNT composites. The improvements in toughness of nanocomposites based on WS(2) can be attributed to geometrical and morphological effects that assisted several toughening mechanisms such as crack pinning and the formation of an immobilized polymeric interphase around the nanotubes.

Detectability of Her2 positive tumors using monoclonal antibody conjugated iron oxide nanoparticles in MRI.

Detecting an imaging signal from a small number of cells is vital when a disease needs to be diagnosed in an early stage of development. Molecular and genetic information from cancer cell types provide a guide for specific imaging based on gene expression and their activities on the cell membrane. Various physical and biological parameters affect the capability of an imaging system to provide an efficient procedure for biomarker imaging. Iron oxide based magnetic nanoparticles conjugated to breast cancer monoclonal antibody (Her2) were used as a specific contrast agent for detection of the tumor cells in nude mice models. All processes for the nanoparticle synthesis, antibody development, and conjugation strategies were designed and evaluated in the current work. The final engineered product was found to be without precipitate containing 20 microg antibody/mg magnetic nanoparticles at 10 mg Fe/ml solution. This contrast agent has a high affinity for the BT474 breast cancer cells. MRI images of nude mice bearing tumor cells confirm this specific biomarker based imaging protocol.

Anisotropic damage of soft tissues in supra-physiological deformations.

Soft tissues may undergo mechanical damage under supra-physiological deformations caused by medical treatments such as balloon-angioplasty and stent deployment. This damage is exhibited as a softening in the mechanical behavior of tissues. In this work, alteration of the collagen network is treated as the origin of damage and loss of stiffness. Inspired by the hierarchical structure of the collagen network, the mechanical properties of collagenous tissues are connected to model parameters. Softening of esophageal and arterial tissues under directional cyclic loading is investigated to determine evolution of the associated material parameters through damage progress. An evolution law is proposed which predicts the mechanical behavior of tissues after excessive deformations. Various deformation measures are examined as the damage parameter to determine the most appropriate one for general 3D loading. It is observed that, if the Green-Lagrange strain in the direction of the fibers is used as the damage parameter, the proposed law well describes the evolution of the collagen network's stiffness. The results not only facilitate prediction of the deformation-induced damage under supra-physiological deformations but also are useful for surgeons in better planning of surgical procedures and stents design.

Comparison home care service versus hospital-based care in patients with diabetic foot ulcer: an economic evaluation study.

INTRODUCTION: Providing health care to patients at home could be causing the mortality and readmission rates reduction in addition to satisfaction of both patients and health care providers increase. The aim of this study was to assess the cost-effectiveness of home care service compared to hospital based care in patients with diabetic foot ulcer. METHODS: An economic evaluation study and a trial study were simultaneously conducted in Iran. In trial phase, patients with diabetic foot ulcer were randomly assigned to the home care or hospital care. The Cost and Quality of life data were determined as measures of the study. Incremental cost-effectiveness ratio was calculated for comparative purposes. The model consisted of five stages of the disease. The Tree Age Pro 2009 and R software's were used for data analysis. RESULTS: 120 patients were enrolled in our trial; among which 30 patients were in home care service group and 90 patients in hospital based care group. The rate of ulcer size reduction in hospital based care was significant (P value = 0.003) in comparison with home care service. The total cost of the home care and hospital strategies were 1720.4 US$, 3940.3 US$ and the total effectiveness were 0.31 and 0.29, respectively. The incremental cost-effectiveness ratio (ICER) was 117,300 US$ per quality-adjusted life year for home care intervention compared to hospital based care. Based on ICER plane home care treatment will be placed on the southeastern quadrant of the Cost-Effectiveness Plane, and is suggested as a more dominant treatment alternative. CONCLUSIONS: Regarding current evidence, home care strategy for patients suffering diabetic foot ulcer enjoys more cost effectiveness compared to hospital care. It is suggested that healthcare policy makers determine the tariff for health care services for disease groups according to the activity based costing approach.

Expression profiling of plac1 in murine cancer cell lines

AIM: Placenta-specific 1 (PLAC1) is among recently-discovered placental antigens which exerts fundamental role in placental function and development. Increasing body of literature shows that PLAC1 is frequently activated and expressed in a wide variety of human cancers and promote cancer progression. However, no data is available regarding the expression of mouse orthologue, plac1, in murine cancer cell lines. Materials and Methods: We investigated the expression of plac1 in a series of murine cell lines from different histological origins, mammary carcinoma (4T1), melanoma (B16F10), colorectal carcinoma (CT26), renal carcinoma (Renca), glioma (GL26), B-cell lymphoma (A20 and BCL1) and also two fibroblast cell lines (NIH3T3 and L929), using RT-PCR, Western blotting and flow cytometry. Results: Our data demonstrated that plac1 transcript and plac1 protein were expressed in all examined cell lines, as judged by RT-PCR and Western blot, respectively. The molecular weight of mouse plac1 was experimentally observed to be approximately 24 kD. Flow cytometric analysis showed surface expression of plac1 in aforesaid cell lines ranging from 2% to 42.5%. Conclusion: Based on the ubiquitous expression of plac1, the investigated cancer cell lines or immortalized cell lines can be used to examine the role of plac1 in the process of immortalization.

Permeation enhancer effect of chitosan and chitosan derivatives: comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells.

In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50+/-5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC>DMEC>DEMC=TEC>chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC>DMEC>DEMC>TEC>chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.

Preparation, characterization and antibacterial activities of chitosan, N-trimethyl chitosan (TMC) and N-diethylmethyl chitosan (DEMC) nanoparticles loaded with insulin using both the ionotropic gelation and polyelectrolyte complexation methods.

TMC and DEMC, quaternized derivatives of chitosan, have been shown to have penetration enhancement properties and able to open the tight junctions of the intestinal epithelia at neutral and alkaline pH environments. The use of the nanoparticulate systems has the advantage of protecting the peptidic drugs from the harsh environment of the gastrointestinal tract. Hence, the aim of this study was to synthesize and characterize TMC and DEMC, both with quaternization degrees of 50+/-5%, which were then used to prepare insulin nanoparticles with two different methods: ionotropic gelation and the polyelectrolyte complexation (PEC) techniques. The obtained nanoparticles were then characterized for size, zeta potential, insulin loading and release as well as antibacterial activities. The results showed that nanoparticles prepared by the PEC method had higher insulin loading efficiency and zeta potential than those made by the ionotropic gelation method and may subsequently be used for further in vitro, ex vivo and in vivo studies. Moreover, the antibacterial studies suggest that the polymers in free form have higher antibacterial activity against Gram-positive bacteria than in the nanoparticulate form.