Targeted delivery of 5-fluorouracil to HT-29 cells using high efficient folic acid-conjugated nanoparticles.

Abstract The incorporation of a high percentage of targeting molecules into drug delivery system is one of the important methods for improving efficacy of targeting therapeutic drugs to cancer cells. PLGA-based drug delivery carriers with folic acid (FA) as targeting molecule have a low targeting efficiency due to a low FA conjugation ratio. In this work, we fabricated a FA-conjugated PLGA system using a crosslinker 1, 3-diaminopropane and have achieved a high conjugation ratio of 46.7% (mol/mol). The as-prepared PLGA-based biomaterial was used to encapsulate therapeutic drug 5-fluorouracil (5-FU) into nanoparticles. In the in vitro experiments, an IC50 of 5.69 µg/mL has been achieved for 5-FU loaded PLGA-1, 3-diaminopropane-folic acid nanoparticles on HT-29 cancer cells and is significantly lower than that of 5-FU and 5-FU loaded PLGA nanoparticles which only have an IC50 of 22.9 and 14.17 µg/mL, respectively. The fluorescent microscopy images showed that nanoparticles with FA are largely taken up by HT-29 cancer cells and the targeting nanoparticles have more affinity to cancer cells than the pure drugs and untreated nanoparticles. Therefore, the 1, 3-diaminopropane can facilitate the conjugation of FA to PLGA to form a novel polymer and 5-FU loaded PLGA-1, 3-diaminopropane-folic acid nanoparticles can be a highly efficient system for specific delivery of drugs to cancer cells.

Self-assembled multimicellar vesicles via complexation of a rigid conjugated polymer with an amphiphilic block copolymer

A viable method of encapsulating block copolymer micelles inside vesicles using a conjugated polymer is reported in this study. Self-assembly and complexation between an amphiphilic block copolymer poly(methyl methacrylate)-b-poly(acrylic acid) (PMMA-b-PAA) and a rod-like conjugated polymer polyaniline (PANI) in aqueous solution were studied using transmission electron microscopy, atomic force microscopy and dynamic light scattering. The complexation and morphology transformation were driven by electrostatic interaction between PANI and the PAA block of the block copolymer. Addition of PANI to PMMA-b-PAA induced the morphology transformation from micelles to irregular vesicles through vesicles, thick-walled vesicles (TWVs) and multimicellar vesicles (MMVs). Among the observed morphologies, MMVs were observed for the first time. Morphology transformation was studied as a function of aniline/acrylic acid molar ratio ([ANI]/[AA]). Micelles were observed for the pure block copolymer, while vesicles and TWVs were observed at [ANI]/[AA] = 0.1 and 0.3, respectively. MMVs were observed at [ANI]/[AA] = 0.5 and irregular vesicles were observed for molar ratios at 0.7 and above. Clearly, a conjugated polymer like polyaniline can induce a morphology transformation even at its lower concentrations and produce complex morphologies.

Large compound vesicles from amphiphilic block copolymer/rigid-rod conjugated polymer complexes

Morphology evolution in complexes of amphiphilic block copolymers poly(styrene)-b-poly(acrylic acid) (PS-b-PAA) and poly(styrene)-b-poly(ethylene oxide) (PS-b-PEO) in the presence of polyaniline (PANI) in aqueous solution is reported. Transmission electron microscopy, atomic force microscopy, and dynamic light scattering techniques were used to study the morphologies at various PANI contents [aniline]/[acrylic acid] ([ANI]/[AA]) ranging from 0.1 to 0.7. The interpolyelectrolyte complex formed between PAA and PANI plays a key role in the morphology transformation. Spherical micelles formed from pure block copolymers were transformed into large compound vesicles upon increasing PANI concentration due to internal block copolymer segregation. In addition to varying PANI content, the kinetic pathway of nanoparticle formation was controlled through different water addition methods and was critical in the formation of multigeometry nanoparticles.

Synthesis and self-assembly of block copolymer/conjugated polymer complexes

 This thesis describes the procedure for preparing polymer nanoparticles of various morphologies via simple complexation technique. The nanoparticles observed in this study may find potential application in drug delivery, diagnostic imaging, nano reactors, catalysis and preparation of stimuli responsive materials.

Doxorubicin conjugated to immunomodulatory anticancer lactoferrin displays improved cytotoxicity overcoming prostate cancer chemo resistance and Inhibits tumour development in TRAMP mice

Advanced, metastatic, castration resistant and chemo-resistant prostate cancer has triggered change in the drug development landscape against prostate cancer. Bovine lactoferrin (bLf) is currently attracting attention in clinics for its anti-cancer properties and proven safety profile. bLf internalises into cancer cells via receptor mediated endocytosis, boosts immunity and complements chemotherapy. We employed bLf as an excellent functional carrier protein for delivering doxorubicin (Dox) into DU145 cells, CD44+/EpCAM+ double positive enriched DU145 3D prostaspheres and drug resistant ADR1000-DU145 cells, thus circumventing Dox efflux, to overcome chemo-resistance. Successful bLf-Dox conjugation with iron free or iron saturated bLf forms did not affect the integrity and functionality of bLf and Dox. bLf-Dox internalised into DU145 cells within 6 h, enhanced nuclear Dox retention up to 24 h, and proved significantly effective (p < 0.001) in reducing LC50 value of Dox from 5.3 μM to 1.3 μM (4 fold). Orally fed iron saturated bLf-Dox inhibited tumour development, prolonged survival, reduced Dox induced general toxicity, cardiotoxicity, neurotoxicity in TRAMP mice and upregulated serum levels of anti-cancer molecules TNF-α, IFN-γ, CCL4 and CCL17. The study identifies promising potential of a novel and safer bLf-Dox conjugate containing a conventional cytotoxic drug along with bLf protein to target drug resistance.

Aging studies on conducting polypyrrole

Change of tensile properties, electrical conductivity and microwave shielding of electrochemically synthesized polypyrrole films with time are presented. Highly doped films had good electrical stability, retaining high microwave reflectivity throughout the aging period. Lightly doped films were less stable and partially reflective and absorptive of microwaves. FT-IR spectral observations revealed a progressive increase in intensity of an unsaturated conjugated carbonyl peak, which was not observed in the highly doped films, suggesting that the concentration of the dopant had an influence on the mechanism of degradation of conductivity.

In situ localization associates biologically active plant natriuretic peptide immuno-analogues with conductive tissue and stomata

Plant natriuretic peptide immuno-analogues (irPNP) have previously been shown to affect a number of biological processes including stomatal guard cell movements, ion fluxes and osmoticum-dependent water transport. Tissue printing and immunofluorescent labelling techniques have been used here to study the tissue and cellular localization of irPNP in ivy (Hedera helix L.) and potato (Solanum tuberosum L.). Polyclonal antibodies active against human atrial natriuretic peptide (anti-hANP) and antibodies against irPNP from potato (anti-StPNP) were used for immunolabelling. Tissue prints revealed that immunoreactants are concentrated in vascular tissues of leaves, petioles and stems. Phloem-associated cells, xylem cells and parenchymatic xylem cells showed the strongest immunoreaction. Immunofluorescent microscopy with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG supported this finding and, furthermore, revealed strong labelling to stomatal guard cells and the adjacent apoplastic space as well. Biologically active immunoreactants were also detected in xylem exudates of a soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.) thus strengthening the evidence for a systemic role of the protein. In summary, in situ cellular localization is consistent with physiological responses elicited by irPNPs reported previously and is indicative of a systemic role in plant homeostasis.

Incorporation of a molecular hinge into molecular tweezers by using tandem gycloadditions onto 5,6-Dimenthylenenorbornene

Site-selective 1,3-dipolar coupling at the norbornene p-bond of 5,6-dimethylenenorbornene 1 yields cycloadducts with an end-fused 1,3-diene system which have been reacted with N=N (or C=C) dienophiles to produce ribbon molecules, in which the internal diazacyclohexene (or cyclohexene) subunits are capable of acting as conformational hinges. Direct coupling of 5,6-dimethylenenorbornene with 1,3,4-oxadiazoles or dual coupling with bis(cyclobutene epoxides) afforded bis(1,3-dienes) that diastereoselectively react with dienophiles to produce new, conformationally mobile, molecular tweezers.

Milkfat characteristics and functionality: opportunities for improvement

Manipulation of the composition of milkfat has the potential to improve the nutritional properties and physical functionality of milkfat and its acceptability in the market. The modifications that have been targeted from a nutritional perspective have included:
(a) reducing the ratio of saturated to unsaturated fatty acids;
(b) increasing the level of omega-3 polyunsaturated fatty acids; and
(c) increasing the content of conjugated linoleic acid.
From a physical functionality viewpoint, the outcome targeted has been an improvement in the spreadability of butter by altering milkfat composition to reduce the hardness of milkfat. Both on-farm strategies and the application of appropriate post-farm processing technologies may be used to alter the milkfat composition to enhance its nutritional image and its physical functionality for a range of product applications. However, changes in milkfat composition that are desirable for a specific nutritional purpose or for one type of milk-based product may not meet all the desirable requirements of another milkfat or dairy product. Furthermore, modification of the milkfat composition can also have an influence on the processing characteristics of milk and the quality of finished dairy products. It is essential to substantiate the benefits of specific target nutritional or physical functionality outcomes before the introduction of breeding goals, altered milk production systems or post-farm processing operations to manipulate milkfat composition. This paper reviews the variation in milkfat characteristics and the strategies that have been used to modify milkfat composition to achieve milkfat with altered nutritional and physical functional properties.

Macronutrient innovations: the role of fats and sterols in human health

Dietary intake of fats and sterols has long been known to play a critical role in human health. High proportions of saturated fat, which increase blood cholesterol levels, are mainly found in animal fat and some plant oil (e.g. cocoa butter, palm oil etc.). The predominant polyunsaturated fatty acid (PUFA) in the Western diet is linoleic acid (LA; 18:2n-6), an essential fatty acid, which is commonly found in vegetable seed oils. This is the parent fatty acid of n-6 series PUFA, which can be converted in vivo to C20 and C22 n-6 long chain (LC) PUFA. α‐linolenic acid (ALA; 18:3n-3) is less abundant than LA and is another essential fatty acid; ALA is also present in some vegetable oils such as perilla, flaxseed, canola, soybean and walnut oils, and is the precursor of C20 and C22 n-3 LC PUFA. Sterols are widely distributed in animal tissue and plants, with cholesterol being the major sterol in animal tissue and β-sitosterol, campesterol and stigmasterol being the main sterols in plants. It has long been recognized that an increased dietary intake of saturated fat and (to a lesser extent) cholesterol, raises plasma/serum total and low-density lipoprotein (LDL)-cholesterol, and PUFA decreases these levels. Results from recent studies have shown that plasma/serum levels of lipids and lipoprotein lipids can also be decreased by plant sterols (phytosterols) and diacylglycerol (DAG). Conjugated linoleic acid (CLA, cis-9,trans-11−18:2) has been reported to have anticancer and antidiabetic activities. Fat as the DAG form has also been reported to have anti-obesity effects. Omega-3 PUFA have a beneficial effect on increased heart rate variability, decreased risk of stroke, reduction of both systolic and diastolic blood pressure and may be effective in managing depression in adults. Gamma-linolenic acid (GLA) and phytosterols have an anti-inflammatory activity. The GLA, when combined with docosahexaenoic acid (DHA), have been reported to have a beneficial effect in hyperactive children. These data show that various lipids are powerful bioactive compounds.

Omega-3 polyunsaturated fatty acid contents of canned meats avaialbe in Australia

Total lipid content of 20 species of canned meats available in Australia ranged from 2% in chicken (Hormel, USA) to 41% in stewed pork (Ma Ling, China). Total n-3 polyunsaturated fatty acids (PUFA) ranged from 30 in canned chicken (Hormel) to 659 mg/100 g in chicken hot dog (Tulip, Denmark). The 18:2n-6 was the predominant PUFA, ranging from 187 in corned beef (Hamper, Australia) to 2832 mg/100 g in chicken luncheon meat (Tulip). Other main PUFA, in order of concentration, were 18:3n-3, ranging from 14 in canned chicken (Hormel) to 590 mg/100 g in chicken hot dog (Tulip); conjugated 18:2n-6 (CLA) from 1 in chicken (Hormel) to 135 mg/100 g in corned mutton (Colonial, Australia); 20:4n- 6 from 11 in camp pie (Tom Piper, Australia) to 73 mg/100 g in spiced ham (Hormel); and 22:5n-3 from 5 in chicken (Hormel) and chicken luncheon (Almaraai, Jordan) to 45 mg/100 g in stewed pork (Ma Ling). Total saturated fatty acids (SFA) ranged from 598 to 14 660 mg/100 g, with 16:0 predominant followed by 18:0. Total monounsaturated fatty acid concentration ranged between 813 to 20 218 mg/100 g with 18:1 the major fatty acid. Trans 18:1 ranged from 10 to 698 mg/100 g. The canned meats contained 20 and 22-carbon long chain n-3 PUFA at levels comparable with or greater than those in fresh lean meat.

Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development

Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. Drug targeting of these transporters to overcome MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy. Most MRP/ABCC transporters are subject to inhibition by a variety of compounds. Based on currently available preclinical and limited clinical data, it can be expected that modulation of MRP members may represent a useful approach in the management of anticancer and antimicrobial drug resistance and possibly of inflammatory diseases and other diseases. A better understanding of their substrates and inhibitors has important implications in development of drugs for treatment of cancer and inflammation.

Fibre coating composition

An abrasion-resistant, electrically conductive material comprising a natural fibre-containing substrate and an electrically conductive conjugated polymer coating thereon is disclosed. A process for preparing an abrasion-resistant, electrically conductive material is also disclosed. The process comprises providing at least one monomer capable of forming an electrically conductive conjugated polymer, and a suitable substrate having a substrate surface, subjecting the substrate surface to a surface treatment step to improve abrasion resistance, and exposing the substrate surface to a vapour of the monomer to form an electrically conductive conjugated polymer coating thereon.

Cellular adaption to repeated eccentric exercise-induced muscle damage

Eccentrically biased exercise results in skeletal muscle damage and stimulates adaptations in muscle, whereby indexes of damage are attenuated when the exercise is repeated. We hypothesized that changes in ultrastructural damage, inflammatory cell infiltration, and markers of proteolysis in skeletal muscle would come about as a result of repeated eccentric exercise and that gender may affect this adaptive response. Untrained male (n = 8) and female (n = 8) subjects performed two bouts (bout 1 and bout 2), separated by 5.5 wk, of 36 repetitions of unilateral, eccentric leg press and 100 repetitions of unilateral, eccentric knee extension exercises (at 120% of their concentric single repetition maximum), the subjects' contralateral nonexercised leg served as a control (rest). Biopsies were taken from the vastus lateralis from each leg 24 h postexercise. After bout 2, the postexercise force deficit and the rise in serum creatine kinase (CK) activity were attenuated. Women had lower serum CK activity compared with men at all times (P < 0.05), but there were no gender differences in the relative magnitude of the force deficit. Muscle Z-disk streaming, quantified by using light microscopy, was elevated vs. rest only after bout 1 (P < 0.05), with no gender difference. Muscle neutrophil counts were significantly greater in women 24 h after bout 2 vs. rest and bout 1 (P < 0.05) but were unchanged in men. Muscle macrophages were elevated in men and women after bout 1 andbout 2 (P < 0.05). Muscle protein content of the regulatory calpain subunit remained unchanged whereas ubiquitin-conjugated protein content was increased after both bouts (P < 0.05), with a greater increase after bout 2. We conclude that adaptations to eccentric exercise are associated with attenuated serum CK activity and, potentially, an increase in the activity of the ubiquitin proteosome proteolytic pathway.

Androgen metabolism in the Australian lizard Tiliqua rugosa

Nonmammalian vertebrates possess some unusual features in their hormonal systems/ when compared to mammals. As a consequence, they can make an important contribution in investigations concerning the fundamental mechanisms operating in endocrinology. Such studies concerning androgens include inter alia their effects on developmental aspects in the brain of birds and related singing behaviour; the role of neural enzymes in reproductive processes in fish; and the relation between androgens and the stages of spermatogenesis in amphibia, The present thesis examines the biochemistry of androgens in the Australian lizard Tiliqua rugosa. The major compounds studied were testosterone and epitestosterone, which are known to be present in high concentrations in the plasma of the male animal. Previous investigations are expanded, particularly in the areas of steroid identification and testicular biosynthesis. In addition, preliminary studies on the metabolism in the brain (and other tissues) and plasma protein binding are reported. The presence of epitestosterone as a major free androgen in the plasma of the male lizard was confirmed. Other steroids were found in the sulphate fraction. Testosterone sulphate was the most rigorously identified compound, while some evidence was also found for the presence of conjugated 5-androstene-3β,17-diols, etiocholanolone and dehydroepiandrosterone (DHA). Epitestosterone does not appear to be extensively conjugated in this animal. Steroids were not found to be conjugated as glucuronides. The identification studies employed a novel method of electrochemical detection of steroids. This technique was investigated and extended in the current thesis. Biosynthetic studies were carried out on androgen interconversions in the testis, in vitro. The major enzyme activities detected were 17α-arid 17β-oxidoreductases (17α-OR and l7β-OR) and 3β-hydroxysteroid dehydrogenase (3β-HSD)/isonerase. No evidence was found for the presence of a steroid-17-epimerase that would directly interconvert testosterone and epitestosterone. The 17-oxidoreductases were found to be dependent on the cofactor NBDFH. Testosterone appears to be formed mainly via the 4-ene pathway, whereas epitestosterone is formed from both the 4- and 5-ene routes. The compound 5-androstene-3β, 17α-diol was found to be an intermediate in the synthesis of epitestosterone from DHA. Temperature was found to significantly affect 17α-OR activity (maximum at 32°C). In contrast,17β-OR activity was independent of this factor in the testis. Androgen metabolism in the testis was found to be regulated by cofactors, temperature and season. The major enzyme activities found in the male brain were 17α- and 17β-OR. 3βHSD/isomerase was not found; however a low activity of 5α-reductase was identified. Aromatase activity was not positively identified, but preliminary results suggest that it may be present at low levels. The 17-oxidoreductases were widespread throughout the brain. The 17α-OR was significantly lower in the forebrain than other brain sections. The 170-OR activity did not vary significantly throughout the organ, although there was a trend for its activity to be higher in the midbrain region (containing the hypothalamus in these sections). The concentration of endogenous steroids in brain tissue was estimated by radioimmunoassay. Epitestosterone was found throughout the organ structure, whereas testosterone was found mainly in the midbrain (containing hypothalamic regions in these sections). Correlations between enzyme activities and steroid concentrations in brain regions suggested that the main function of 17α-OR is to produce epitestosterone, whereas the 17β-OR may catalyse a more reversible reaction in vivo. Temperature was found to significantly affect both 17α- and 17β-OR activities in the brain. In contrast to the testis, the maximum activity of the brain enzymes occurred at 37°C. The level of 17α-OR activity in the male lizard (100 nmol/g tissue/h) is the highest reported for this enzyme in vertebrates. Both activities were found to be quantitatively similar in the whole brain homogenates of male and female animals, and did not vary seasonally when examined in the male. The 17-oxidoreductases were also found in most other tissues in T.rugosa, including epididymis, adrenal, kidney and liver (but not blood). This suggests that the high activities of both 17α-OR and 17β-OR are dominant features of the steroid system in this animal. The formation of 11-oxygenated compounds was found in the adrenal, in addition to the formation of polar metabolites in the kidney and liver (possibly polyhydroxylated and conjugated steroids). A preliminary investigation into the plasma binding of androgens was carried out. The insults suggest that there are several binding sites for testosterone; one with high affinity and low capacity; the other with low affinity and high capacity. Binding experiments were carried out at 32°C. At this temperature, specific binding was greater than at 25 or 37°C. From the results of competition studies it was suggested that epitestosterone (with a K(i)= 3 X 10 (-6)M for testosterone binding) regulates the binding of testosterone (K(i)=10(-7)M) and hence the concentrations of the latter steroid as a free compound in plasma. In general, the study has shown that the biochemistry of androgens in the reptile T.rugosa is largely similar to that found in other vertebrates. The major difference is a greatly increased activity of 17α-OR, which causes a higher concentration of 17α-compounds to be present in the tissues of this lizard. The physiological roles for epitestosterone are not yet clear. However it appears from this study that this steroid regulates testosterone concentrations in several tissues by either steroidogenic or binding mechanisms. Several major influences on this regulation include temperature, availability of cofactors and seasonal effects.