Hydrophobically modified chitosan nanoliposomes for intestinal drug delivery.

BACKGROUND: Encapsulation of hydrophilic drugs within liposomes can be challenging. METHODS: A novel chitosan derivative, O-palmitoyl chitosan (OPC) was synthesized from chitosan and palmitoyl chloride using methane-sulfonic acid as a solvent. The success of synthesis was confirmed by Fourier transform infra-red (FT-IR) spectroscopy and proton NMR spectroscopy (H-NMR). Liposomes encapsulating ferrous sulphate as a model hydrophilic drug for intestinal delivery were prepared with or without OPC inclusion (Lipo-Fe and OPC-Lipo-Fe). RESULTS: Entrapment of iron was significantly higher in OPC containing liposomes compared to controls. Quantitative iron absorption from the OPC liposomes was significantly higher (1.5-fold P<0.05) than free ferrous sulphate controls. Qualitative uptake analysis by confocal imaging using coumarin-6 dye loaded liposomes also indicated higher cellular uptake and internalization of the OPC-containing liposomes. CONCLUSION: These findings suggest that addition of OPC during liposome preparation creates robust vesicles that have improved mucoadhesive and absorption enhancing properties. The chitosan derivative OPC therefore provides a novel alternative for formulation of delivery vehicles targeting intestinal absorption.

Design and development of novel mitochondrial targeted nanocarriers, DQAsomes for curcumin inhalation.

Curcumin has potent antioxidant and anti-inflammatory properties but poor absorption following oral administration owing to its low aqueous solubility. Development of novel formulations to improve its in vivo efficacy is therefore challenging. In this study, formulation of curcumin-loaded DQAsomes (vesicles formed from the amphiphile, dequalinium) for pulmonary delivery is presented for the first time. The vesicles demonstrated mean hydrodynamic diameters between 170 and 200 nm, with a ζ potential of approximately +50 mV, high drug loading (up to 61%) and encapsulation efficiency (90%), resulting in enhanced curcumin aqueous solubility. Curcumin encapsulation in DQAsomes in the amorphous state was confirmed by X-ray diffraction and differential scanning calorimetry analysis. The existence of hydrogen bonds and cation-π interaction between curcumin and vesicle building blocks, namely dequalinium molecules, were shown in lyophilized DQAsomes using FT-IR analysis. Encapsulation of curcumin in DQAsomes enhanced the antioxidant activity of curcumin compared to free curcumin. DQAsome dispersion was successfully nebulized with the majority of the delivered dose deposited in the second stage of the twin-stage impinger. The vesicles showed potential for mitochondrial targeting. Curcumin-loaded DQAsomes thus represent a promising inhalation formulation with improved stability characteristics and mitochondrial targeting ability, indicating a novel approach for efficient curcumin delivery for effective treatment of acute lung injury and the rationale for future in vivo studies.

Primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder of cilia structure and function, leading to chronic infections of the respiratory tract, fertility problems and disorders of organ laterality. Making a definitive diagnosis is challenging, utilizing characteristic phenotypes, ciliary functional and ultra-structural defects in addition to newer screening tools such as nasal nitric oxide and genetic testing. There are 21 known PCD causing genes and in the future, comprehensive genetic testing may help diagnosis young infants prior to developing symptoms thus improving survival. Therapy includes surveillance of pulmonary function and microbiology in addition to, airway clearance, antibiotics and early referral to bronchiectasis centers. Standardized care at specialized centers using a multidisciplinary approach is likely to improve outcomes. In conjunction with the PCD foundation and lead investigators and clinicians are developing a network of PCD clinical centers to coordinate the effort in North America and Europe. As the network grows, care and knowledge will undoubtedly improve.

Ascorbyl palmitate/DSPE-PEG nanocarriers for oral iron delivery: preparation, characterisation and in vitro evaluation.

The objective of this study was to encapsulate iron in nanocarriers formulated with ascorbyl palmitate and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine polyethylene glycol (DSPE-PEG) for oral delivery. Blank and iron (Fe) loaded nanocarriers were prepared by a modified thin film method using ascorbyl palmitate and DSPE-PEG. Surface charge of the nanocarriers was modified by the inclusion of chitosan (CHI) during the formulation process. Blank and iron loaded ascorbyl palmitate/DSPE nanocarriers were visualised by transmission electron microscopy (TEM) and physiochemical characterisations of the nanocarriers carried out to determine the mean particle size and zeta potential. Inclusion of chitosan imparted a net positive charge on the nanocarrier surface and also led to an increase in mean particle size. Iron entrapment in ascorbyl palmitate-Fe and ascorbyl palmitate-CHI-Fe nanocarriers was 67% and 76% respectively, suggesting a beneficial effect of chitosan on nanocarrier Fe entrapment. Iron absorption was estimated by measuring Caco-2 cell ferritin formation using ferrous sulphate as a reference standard. Iron absorption from ascorbyl palmitate-Fe (592.17±21.12 ng/mg cell protein) and ascorbyl palmitate-CHI-Fe (800.12±47.6 ng/mg, cell protein) nanocarriers was 1.35-fold and 1.5-fold higher than that from free ferrous sulphate, respectively (505.74±23.73 ng/mg cell protein) (n=6, p<0.05). This study demonstrates for the first time preparation and characterisation of iron loaded ascorbyl palmitate/DSPE PEG nanocarriers, and that engineering of the nanocarriers with chitosan leads to a significant augmentation of iron absorption.

A novel approach to oral iron delivery using ferrous sulphate loaded solid lipid nanoparticles.

Iron (Fe) loaded solid lipid nanoparticles (SLN's) were formulated using stearic acid and iron absorption was evaluated in vitro using the cell line Caco-2 with intracellular ferritin formation as a marker of iron absorption. Iron loading was optimised at 1% Fe (w/w) lipid since an inverse relation was observed between initial iron concentration and SLN iron incorporation efficiency. Chitosan (Chi) was included to prepare chitosan coated SLN's. Particle size analysis revealed a sub-micron size range (300.3±31.75 nm to 495.1±80.42 nm), with chitosan containing particles having the largest dimensions. As expected, chitosan (0.1%, 0.2% and 0.4% w/v) conferred a net positive charge on the particle surface in a concentration dependent manner. For iron absorption experiments equal doses of Fe (20 µM) from selected formulations (SLN-FeA and SLN-Fe-ChiB) were added to Caco-2 cells and intracellular ferritin protein concentrations determined. Caco-2 iron absorption from SLN-FeA (583.98±40.83 ng/mg cell protein) and chitosan containing SLN-Fe-ChiB (642.77±29.37 ng/mg cell protein) were 13.42% and 24.9% greater than that from ferrous sulphate (FeSO4) reference (514.66±20.43 ng/mg cell protein) (p≤0.05). We demonstrate for the first time preparation, characterisation and superior iron absorption in vitro from SLN's, suggesting the potential of these formulations as a novel system for oral iron delivery.

Attenuation of plasma annexin A1 in human obesity.

Obesity-related metabolic disorders are characterized by mild chronic inflammation, leukocyte infiltration, and tissue fibrosis as a result of adipocytokine production from the expanding white adipose tissue. Annexin A1 (AnxA1) is an endogenous glucocorticoid regulated protein, which modulates systemic anti-inflammatory processes and, therefore, may be altered with increasing adiposity in humans. Paradoxically, we found that plasma AnxA1 concentrations inversely correlated with BMI, total percentage body fat, and waist-to-hip ratio in human subjects. Plasma AnxA1 was also inversely correlated with plasma concentrations of the acute-phase protein, C-reactive protein (CRP), and the adipocytokine leptin, suggesting that as systemic inflammation increases, anti-inflammatory AnxA1 is reduced. In addition, AnxA1 gene expression and protein were significantly up-regulated during adipogenesis in a human adipocyte cell line compared to vehicle alone, demonstrating for the first time that AnxA1 is expressed and excreted from human adipocytes. These data demonstrate a failure in the endogenous anti-inflammatory system to respond to increasing systemic inflammation resulting from expanding adipose tissue, a condition strongly linked to the development of type 2 diabetes and cardiovascular disease. These data raise the possibility that a reduction in plasma AnxA1 may contribute to the chronic inflammatory phenotype observed in human obesity.

Annexin-A1 protein and its relationship to cortisol in human saliva.

Salivary cortisol is commonly used as a clinical biomarker of endocrine status and also as a marker of psychosocial stress. Annexin-A1 (AnxA1) is an anti-inflammatory protein whose expression is modulated by glucocorticoids. Our principal objectives were to (i) detect the presence of and (ii) measure AnxA1 protein in whole human saliva and to (iii) investigate whether salivary cortisol and AnxA1 are correlated in healthy humans. A total of 37 healthy participants (male and female) were used in the study. Saliva was collected using salivette tubes. Salivary cortisol and AnxA1 protein were sampled at between 3 and 6 time points over 24h and measured for cortisol and AnxA1 protein using specific ELISA's. The presence of salivary AnxA1 protein was confirmed by Western blotting. AnxA1 protein is detectable in whole human saliva, as detected by Western blot analysis and ELISA. A diurnal rhythm was evident in both salivary cortisol (P<0.01) and AnxA1 (P<0.01) and was defined as a significant difference in time 0 (waking) samples compared to 'bed' (2300 h) samples. AnxA1 protein did not exhibit an awakening response (P>0.05), whereas salivary cortisol was significantly elevated between time 0 and 30 min post waking (P<0.001). AnxA1 protein correlates positively with salivary cortisol, indicating that cortisol is most likely a regulator of AnxA1 in human saliva.

PPARs and the orchestration of metabolic fuel selection.

This contribution describes recent advances in our knowledge of the regulatory interactions between the two major oxidative fuels glucose and lipid. It also addresses how the metabolic abnormalities associated with insulin resistance and ischemic diseases impair the ability of skeletal muscle to switch between the use of alternative metabolic fuels and the ability of adipose tissue to function appropriately in relation to the body's requirements for triglyceride mobilisation or storage, as appropriate to nutritional status. We discuss how targeting PPARs might ameliorate metabolic inflexibility in muscle through altered expression of pyruvate dehydrogenase kinase (PDK) isoforms and impact the functions of the adipocyte in lipid buffering and energy homeostasis. Focus has been placed on the participation of the regulatory pyruvate dehydrogenase kinases, PPAR targets, both in the beneficial and the potentially adverse actions of the PPARs in metabolic control.

Diet, obesity and diabetes: a current update.

The prevalence of obesity has been increasing at a rapid rate over the last few decades. Although the primary defect can be attributed to an imbalance of energy intake over energy expenditure, the regulation of energy balance is now recognized to be complex. Adipose-tissue factors play a central role in the control of energy balance and whole-body fuel homoeostasis. The regulation of adipose-tissue function, in particular its secretion of adipokines, is impaired by increases in adipose mass associated with obesity, and with the development of insulin resistance and Type 2 diabetes. This review analyses adipose-regulated energy input and expenditure, together with the impact of dietary macronutrient composition on energy balance in relation to susceptibility to the development of obesity and Type 2 diabetes, and how these metabolic conditions may be exacerbated by the consequences of abnormal adipose function. By gaining a greater understanding of how energy balance is controlled in normal, and in obese and diabetic states, a more practical approach can be employed to prevent and better treat obesity and metabolic disorders.

Germline mutations in an intermediate chain dynein cause primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal recessive disorder caused by abnormal ciliary ultrastructure and function, characterized clinically by oto-sino-pulmonary disease. Mutations in an intermediate chain dynein (DNAI1; IC78) have recently been described in PCD patients, with outer dynein arm (ODA) defects. The aims of the current study were to test for novel DNAI1 mutations in 13 PCD patients with ODA defects (from 7 unrelated families) and to assess genotype/phenotype correlations in patients and family members. A previously reported mutation (219+3insT) was detected in three PCD patients from two families. The opposite allele had the novel missense mutation G1874C (W568S) in both affected individuals from one family, and a nonsense mutation G1875A (W568X) in an affected individual from another family. The tryptophan at position 568 is a highly conserved residue in the WD-repeat region, and a mutation is predicted to lead to abnormal folding of the protein and loss of function. None of these mutations were found in 32 other PCD patients with miscellaneous ciliary defects. Mutations in DNAI1 are causative for PCD with ODA defects, and are likely the genetic origin of clinical disease in some PCD patients with ultrastructural defects in the ODA.

Prevalence of human papilloma virus DNA in head and neck cancers carrying wild-type or mutant p53 tumor suppressor genes.

The normal function of the p53 tumor suppressor protein can be perturbed by non-mutational mechanisms. The E6 protein encoded by high risk strains of human papilloma virus (HPV) targets the p53 protein resulting in enhanced degradation via the ubiquitin pathway. We have used nested PCR for detecting the presence of HPV DNA in 58 primary head and neck tumors and 15 metastatic lymph nodes, which had been prescreened for p53 mutations in exons 5 to 8. HPV DNA sequences were detected in 12 tumors (20.6%) and 4 metastatic lymph nodes (21%). HPV type 16 DNA was predominantly found in tumors (n = 11) and lymph nodes (n = 4), one tumor was positive for HPV type 18 sequences. Five of 12 HPV-positive tumors (41%) carried a p53 mutation. Of 46 HPV-negative tumors, 16 (34.8%) carried a p53 mutation. Thus, HPV positivity and p53 mutations were not mutually exclusive in head-and-neck cancer. Three of 6 normal tissues adjacent to the tumor were positive for HPV type 16, while no viral DNA was found in the corresponding tumors. Thus, the presence of HPV type 16 DNA did not directly confer a growth advantage on the population of emerging tumor cells. Instead, these tumors lack normal p53 function due to mutation.

Cyclin E2, a novel human G1 cyclin and activating partner of CDK2 and CDK3, is induced by viral oncoproteins.

G1 cyclin E controls the initiation of DNA synthesis by activating CDK2, and abnormally high levels of cyclin E expression have frequently been observed in human cancers. We have isolated a novel human cyclin, cyclin E2, that contains significant homology to cyclin E. Cyclin E2 specifically interacts with CDK inhibitors of the CIP/KIP family and activates both CDK2 and CDK3. The expression of cyclin E2 mRNA oscillates periodically throughout the cell cycle, peaking at the G1/S transition, and exhibits a pattern of tissue specificity distinct from that of cyclin E1. Cyclin E2 encodes a short lived protein whose turnover is most likely governed by the proteasome pathway and is regulated by phosphorylation on a conserved Thr-392 residue. Expression of the viral E6 oncoprotein in normal human fibroblasts increases the steady state level of cyclin E2, but not cyclin E1, while expression of the E7 oncoprotein upregulates both. These data suggest that the expression of these two G1 E-type cyclins may be similarly regulated by the pRb function, but distinctly by the p53 activity.

Parathyroid tumor suppressor on 1p: analysis of the p18 cyclin-dependent kinase inhibitor gene as a candidate.

Loss of chromosome arm 1p DNA is the most common molecular defect thus far observed in human parathyroid adenomas, suggesting that 1p is the location of a putative tumor suppressor gene (or genes) whose inactivation contributes frequently to parathyroid tumorigenesis. To narrow the genomic location of this tumor suppressor gene, we analyzed 25 sporadic parathyroid adenomas for allelic loss of polymorphic DNA loci on chromosome 1 using 11 microsatellite markers not previously scored for this set of tumors. Allelic loss on chromosome arm 1p DNA was observed in 8 of 25 adenomas. Marker deletion patterns showed some complexity, with the regions most commonly deleted in these tumors being 1p36 and 1p35-p31. The 1p35-p31 region contains an excellent candidate tumor suppressor gene, p18, whose product is a cell cycle regulator that inhibits the cyclin D1-associated kinase CDK6. Given that cyclin D1 is a parathyroid oncogene, inactivation of an inhibitor of cyclin D1 function, like p18, might also cause excessive parathyroid growth. To examine the involvement of p18 in parathyroid tumorigenesis, we analyzed 25 parathyroid adenomas for mutations of the p18 coding exons by single strand conformational polymorphism analysis and sequencing. No point mutations were found in any of the 25 adenomas. These observations indicate that inactivating mutation of the p18 gene occurs uncommonly, if at all, in parathyroid adenomas. In addition, the data raise the important possibility that more than a single tumor suppressor gene on 1p could contribute to parathyroid neoplasia.

Mutation of the p53 tumour suppressor gene occurs preferentially in the chromophobe type of renal cell tumour.

Genomic DNA from 30 non-papillary and 20 chromophobe renal cell carcinomas (RCCs), 30 papillary renal cell tumours, and 20 renal oncocytomas was screened for the presence of mutations in exons 5-8 of the p53 tumour suppressor gene by polymerase chain reaction-single strand conformation polymorphism analysis and direct DNA sequencing. Mutations leading to an amino acid change were found only in 6 out 20 chromophobe RCCs. Microsatellite analysis of chromophobe RCCs revealed the loss of one allele at chromosome 17p in 14 out of 18 informative cases. No mutation of the p53 gene was found in five sarcomatous RCCs or in seven tumours of stage IV. This study shows that mutation of the p53 tumour suppressor gene does not correlate with the specific loss of DNA sequences at chromosome 17 in chromophobe RCCs, nor can it be used as a prognostic parameter for RCCs in general.

Molecular analysis of the cyclin-dependent kinase inhibitor genes p15INK4b/MTS2, p16INK4/MTS1, p18 and p19 in human cancer cell lines.

Cyclin-dependent kinase-4 inhibitor genes (INK4) regulate the cell cycle and are candidate tumor-suppressor genes. To determine if alterations in the coding regions of the p18 and p19 genes, which are novel members of the INK4 family and if they correlate with the development of human cancer, 100 human cancer cell lines were analyzed. Two other INK4 gene family members, p15INK4b/MTS2 and p16INK4/MTS1 genes were also analyzed. Homozygous deletions of the p15INK4b/MTS2 gene were detected in 29 cancer cell lines. Thirty-five homozygous deletions and 7 intragenic mutations of the pl6INK4/MTS1 gene were also detected in these cell lines. Neither homozygous deletions nor intragenic mutations of the p18 and p19 genes were found except in an ovarian cancer cell line, SKOV3, harboring a single base pair deletion in exon 1 of p19. In p16INK4/MTS1 expression analysis, 5 cell lines with both authentic and alternative spliced p16INK4/MTS1 mRNA had no detectable p16INK4/MTS1 protein. These results suggest the hypotheses that either post-translational modification or enhanced degradation may be responsible for the lack of detection of the p16INK4/MTS1 protein. Using Western blot analysis, subsets of 26 human cancer cell lines were examined for p18 expression and 39 cell lines for p19 expression. All of these cell lines expressed the p18 or p19 protein, with the exception of SKOV3, which did not express p19. Therefore, the INK4 gene family may be divided into 2 groups. One group includes p15INK4b/MTS2 and p16INK4/MTS1, in which genetic and epigenetic alterations might contribute to the development of human cancers. The other group includes p18 and p19, in which somatic mutations are uncommon in many types of human cancer, and their role in human carcinogenesis and cancer progression is uncertain.

Lack of mutation in the cyclin-dependent kinase inhibitor, p19INK4d, in tumor-derived cell lines and primary tumors.

Inhibitors of cyclin-dependent kinases provide a major mechanism of negative regulation on cell cycle progression. Defects in the function of the CDK inhibitors may lead to uncontrolled cell proliferation and potentially facilitate tumorigenesis. The p16INK4 family of CDK inhibitors specifically prevent the phosphorylation of the retinoblastoma susceptibility gene product, pRb, by inhibiting the kinase activity of CDK4 and CDK6, thereby keeping pRb in its active form as a growth suppressor. The loss of p16INK4 inhibitory activity would, therefore, have the same consequence as the loss of pRb growth suppressing activity. The p16INK4 family currently includes four members, p15INK4b, pl6INK4a, pl8INK4c and p19INK4d. Two members, p15INK4b and pl6INK4a have been found to be deleted and mutated in a variety of human tumor-derived cell lines and primary tumors. In the present study we have examined the genomic status of the newly isolated p19INK4d gene in 75 tumor-derived cell lines; 13 immortalized, transformed or normal cell lines; 19 ovarian tumors and 18 acute myelogenous leukemias. No deletions or point mutations were observed in the pl9INK4d gene. A genetic polymorphism at codon 30 (CGC-->CGG) in exon 1 of the pl9INK4d gene was observed in 10% of the samples under investigation. In the same set of samples, p16INK4a was found to be homozygously deleted in 32% of the tumor derived cell lines. These results together with our previous data that showed a 22% deletion frequency in p15INK4b and rare alterations in the pl8INK4c gene, indicating that the p16INK4a and pl5INK4b, but not the p18INK4c and pl9INK4d genes, are frequently mutated in human tumors. Hence, members of the p16INK4 CDK inhibitor family, while evolutionary related and biochemically indistinguishable, carry out distinct biological functions.

Mutational analysis of the p16 family cyclin-dependent kinase inhibitors p15INK4b and p18INK4c in tumor-derived cell lines and primary tumors.

The growth suppressing activity of the retinoblastoma suspectibility gene product, pRb, is down regulated by cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) whose kinase activity is negatively regulated by CDK inhibitors of the p16 family. We have examined the genomic status of two recently isolated p16-related CDK inhibitors, p15 and p18, in 15 normal and 73 tumor-derived cell lines established from 23 different tissues, as well as 26 invasive primary breast cancers and 20 acute myelogenous leukemias. p15 was found to be homozygously deleted in 22% of the tumor derived cell lines, but no point mutations were found in either the cultured cells or the two types of primary tumors. With the exception of one breast cancer cell line, no deletions or mutations were found in the p18 gene in either cultured cell lines or primary tumors. These results indicate that mutation of the p18 gene occurs rarely in human tumors. Thus, while they share a very similar biochemical mechanism of inhibiting the kinase activity of CDK4 and CDK6, members of the p16 gene family play different roles in controlling cell proliferation and suppressing tumor growth.

Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK6 and CDK4.

Cyclin-dependent kinases 4 and 6 are complexed with many small cellular proteins in vivo. We have isolated cDNA sequences, INK4d, encoding a 19-kDa protein that is associated with CDK6 in several hematopoietic cell lines. p19 shares equal similarity and a common ancestor with other identified inhibitors of the p16/INK4 family. p19 interacts with and inhibits the activity of both CDK4 and CDK6 and exhibits no detectable interaction with the other known CDKs. p19 protein is present in both cell nuclei and cytoplasm. The p19 gene has been mapped to chromosome 19p13.2, and the level of its mRNA expression varies widely between different tissues. In contrast to p21 and p27 whose interaction with CDK subunits is dependent on or stimulated by the cyclin subunit, the interaction of p19 and p18 with CDK6 is hindered by the cyclin protein. Binary cyclin D1-p18/p19 or cyclin D1-CDK6 complexes are highly stable and cannot be dissociated by excess amounts of cyclin D1 or p19/p18 proteins, suggesting that p16 inhibitors and D cyclins may interact with CDKs 4 and 6 in a competing or potentially mutually exclusive manner.

Deletion of cyclin-dependent kinase 4 inhibitor genes P15 and P16 in non-Hodgkin's lymphoma.

B-cell non-Hodgkin's lymphoma (NHL) is a heterogeneous lymphoid malignancy consisting of several histologic types. Alterations in proto-oncogenes caused by reciprocal chromosome translocations have been implicated in the etiology of specific histologic groups. In this study, we examined the contribution of the cell cycle inhibitor genes P15, P16, and P18 to pathogenesis in a large panel of 209 cytogenetically characterized B-cell NHL tumors representing varied histologic groups. We identified the homozygous deletion of P15 and P16 genes in 13 tumors from 12 patients, all belonging to diffuse large-cell histology; 10 had this diagnosis made on presentation, 1 had transformed from small lymphocytic lymphoma, and 1 had transformed from Hodgkin's disease. Tumor-specific point mutations were not identified in the coding regions of these genes. Cytogenetically, chromosome 9p was normal in all but one tumor. On the other hand, eight tumors hemizygous for 9p by cytogenetic analysis showed wild-type configuration of these genes. Our study, therefore, indicates that deletion of P15 and P16 occurs in about 15% of diffuse large-cell NHL and is not usually detected by cytogenetic analysis. P18 was wild-type in all tumors including the 13 tumors hemizygous for 1p.