MUCOADHESIVE MICROSPHERES: AN EMINENT ROLE IN CONTROLLED DRUG DELIVERY

ABSTRACT Mucoadhesion is simply known as interfacial force interactions between polymeric materials and mucosal tissues. In the last two decades mucoadhesive microspheres have received considerable attention for design of novel drug delivery systems due to their ability to prolong the residence time of dosage forms and to enhance drug bioavailability. Mucoadhesive microspheres have advantages like efficient absorption and enhanced bioavailability of the drugs due to a high surface to volume ratio, a much more intimate contact with the mucus layer, controlled and sustained release of drug from dosage form and specific targeting of drugs to the absorption site. Microspheres are the carrier linked drug delivery system in which particle size is ranges from 1-1000 μm range in diameter having a core of drug and entirely outer layers of polymer as coating material. Keywords: mucoadhesion, microspheres, controlled release, residence time. INTRODUCTION Since many years several kinds of diseases that may be acute or chronic diseases can be treated by using pharmaceutical dosage form like solutions, tablets, capsules, syrups, suspension, emulsion, ointments, creams, gels which can be used as orally, topically, or intravascular route. To get the proper therapeutic effect of these pharmaceutical dosage forms they should be administered several times a day, this results consequently undesirable toxicity, fluctuation in drug level and poor efficiency or therapeutic effect. Controlled release dosage form plays eminent role to overcome the problems which are discussed above. The most important example of controlled drug delivery system is mucoadhesive microspheres which can improve the therapeutic effect of administered drug. Also bioavailability of drug is also better than other conventional system because mucoadhesive microspheres remain close to the mucous membrane and absorption tissue. Drug delivery systems (DDS) that can precisely control the release rates or target drugs to a specific body site have had an enormous impact on the healthcare system. The last two and developing novel delivery systems referred to as "mucoadhesive microspheres". [1] Physiology of mucin Mucus is produced in the eye, ear, nose and mouth. It also lines the respiratory, gastrointestinal and reproductive tracts. Its primary functions are the protection and lubrication of the underlying epithelium. Human cervical mucus, for instance, plays an integral role in both conception and contraception. It is essential to understand the structure and physical chemistry of mucus if the latter is to be exploited as a site for bioadhesive controlled drug release. Since the gastrointestinal tract is the primary site for drug absorption, the physiology of this site will be the focus of this discussion. The gelling properties which are essential to the function of mucus are the direct result of the glycoprotein present in the mucosal secretion. This glycoprotein is generally the same for various secretion sites within the body; however, specific and subtle biochemical differences have been identified. Mucus may be either constantly or intermittently secreted. The amount of mucus secreted also varies. The glycoproteinic component of mucus is a high molecular weight, highly glycosylated macromolecular system. This polydisperse natural polymer makes up between 0.5 and 5% of the fully hydrated mucus secretion. [10] The size of the intact molecule is approximately 1.8 x 10 6 , but the molecular weight of undegraded gastric mucin is as high as 4.5 x 10 7 . These macromolecules are highly expanded random coils made up of monomeric glycoproteins which for humans range from 5.5 x 1o 5 in the stomach to 2.4 x lo 5 in the small intestine. Oligosaccharide branches are attached to 63% of the protein core while the remainder of There are 34 disulphide bridges per molecule of rat goblet cell mucin, which has a molecular weight of 2 x 10 6 , while porcine intestinal mucin has 28 bridges per molecule. Human mucin has a similar density of disulphide bonds. The protein spine of the macromolecule has about 800 amino acid residues. Sugar chains are attached at about every three residues along the glycosylated regions; this results in approximately 200 side chains per molecule. This molecule is resistant to proteolytic attack in the glycosylated regions only. Thus, charge interactions may have a significant effect on the behaviour of mucus glycoproteins. The mucous gel covering the epithelium varies in thickness. In the human stomach, the mean thickness is 192 pm, while in the duodenum the thickness ranges from 10 to 400 pm In the gastrointestinal tract, mucus facilitates the passage of food and boluses through the alimentary canal. It also helps shield the epithelium from shear forces induced by peristaltic waves, and resists auto digestion. These functions are promoted by the constant secretion of mucus to replenish losses from turbulence and degradation. In response to an irritant, the amount of acidic side chains in the glycoprotein increases from 50 to 80%, making the macromolecule more negatively charged. The submucosal gland layer increases in depth and the number of goblet cells increases. The total content of non dialysable solids and pH also increase. In the GI tract, DNA and albumin thicken mucus in the diseased state. Mucosal irritation, such as exposure to alcohol or bile salts, elicits accelerated mucin release. Disease can significantly alter the nature and thickness of the mucus. This may lead to a change in the behaviour of the delivery system. Any drug delivery system which is intended to adhere to the mucus epithelium will need to adapt to a substrate which varies in depth and consistency, and may also change biochemically. Hypersecretion, which is more common than hyposecretion during disease, increases the transit rate through the GI tract, and thus reduces the residence time of a mucoadhesive device. Thus, it is essential to consider the physiology of the system when optimizing the formulation of an adhesive controlled release device. CLASSIFICATION OF MUCOADHESIVE POLYMERS Mucoadhesion is defined as interfacial force interactions between polymeric materials and mucosal tissues. In the last two decades mucoadhesive polymers have received considerable attention for design of novel drug delivery systems due to their ability to prolong the residence time of dosage forms and to enhance drug bioavailability. Various administration routes, such as ocular, nasal, gastrointestinal, vaginal and rectal, make mucoadhesive drug delivery systems attractive and flexible in dosage forms development. Mucoadhesive polymers can be classified as,- I. Traditional non-specific first-generation mucoadhesive polymers First-generation mucoadhesive polymers may be divided into three main subsets, namely: (1) Anionic polymers:-Anionic polymers are widely employed for its greatest mucoadhesive strength and low toxicity. These polymers are characterised by the presence of sulphate and carboxyl group that gives rise to net negative charge at PH values exceeding the pka of polymer. Example:-polyacrylic acid (PAA) & its weakly cross linked derivatives, Sodium carboxymethyl cellulose (NACMC) [30] (2) Cationic polymers: -The most conveniently and widely used cationic polymer is chitosan which is produced by deacetylation of chitin. Chitin is a natural polysaccharide found predominantly in the shells of crustaceans such as crabs and shrimp, the cuticles of insects, and the cell walls of fungi. It is one of the most abundant biopolymers next to cellulose Most of the naturally occurring polysaccharides, e.g. cellulose, dextran, pectin, alginic acid, agar, agarose and carrageenans, are neutral or acidic in nature, whereas chitin and chitosan are examples of highly basic polysaccharides. The unique properties include II.Novel second-generation mucoadhesive polymers: The major disadvantage in using traditional nonspecific mucoadhesive systems (first generation) is that adhesion may occur at sites other than those intended. Unlike first-generation non-specific platforms, certain second-generation polymer platforms are less susceptible to mucus turnover rates, with some species binding directly to mucosal surfaces; more accurately termed ''cytoadhesives". Furthermore as surface carbohydrate and protein composition at potential target sites vary regionally, more accurate drug delivery may be achievable. MUCOADHESION Due its relative complexity, it is likely that the process of mucoadhesion cannot be described by just one of these theories. In considering the mechanism of mucoadhesion, a whole range 'scenarios' for in-vivo mucoadhesive bond formation are possible. These include: A). Dry or partially hydrated dosage forms contacting surfaces with substantial mucus layers (typically particulates administered into the nasal cavity). B). fully hydrated dosage forms contacting surfaces with substantial mucus layers (typically particulates of many 'First Generation'mucoadhesives that have hydrated in the luminal contents on delivery to the lower gastrointestinal tract). C). Dry or partially hydrated dosage forms contacting surfaces with thin/discontinuous mucus layers (typically tablets or patches in the oral cavity or vagina). D). fully hydrated dosage forms contacting surfaces with thin/discontinuous mucus layers (typically aqueous semisolids or liquids administered into the oesophagus or eye). It is unlikely that the mucoadhesive process will be the same in each case. In the study of adhesion generally, two steps in the adhesive process have been identified Step 2 -Consolidation stage: Various physicochemical interactions occur to consolidate and strengthen the adhesive joint, leading to prolonged adhesion. THEORIES ON MUCOADHESION [4, 5] Various kinds of theories are there which can explain the mechanism of mucoadhesion they are discussed below, TYPES OF MICROSPHERES Mucoadhesive microspheres:-Adhesion can be defined as sticking of drug to the membrane by using the sticking property of the water soluble polymers. Adhesion of drug delivery device to the mucosal membrane such as buccal, ocular, rectal, nasal etc can be termed as bio -adhesion. These kinds of microspheres exhibit a prolonged residence time at the site of application and causes intimate contact with the absorption site and produces better therapeutic action. [26] Magnetic microspheres:-This kind of delivery system is very much important which localises the drug to the disease site. In this larger amount of freely circulating drug can be replaced by smaller amount of magnetically targeted drug. Magnetic carriers receive magnetic responses to a magnetic field from incorporated materials that are used for magnetic microspheres are chitosan, dextran etc. The different type are, Therapeutic magnetic microspheres: Are used to deliver chemotherapeutic agent to liver tumour. Drugs like proteins and peptides can also be targeted through this system.6 Diagnostic microspheres: Can be used for imaging liver metastases and also can be used to distinguish bowel loops from other abdominal structures by forming nano size particles supramagnetic iron oxides. Floating microspheres:-In this type of microspheres the bulk density is less than the gastric fluid and so remains buoyant in stomach without affecting gastric emptying rate. The release rate of drug is slow at the desired rate, if the system is floating on gasteric content and increases gastric residence and increases fluctuation in plasma concentration

Natural Terpenes Prevent Mitochondrial Dysfunction, Oxidative Stress and Release of Apoptotic Proteins during Nimesulide-Hepatotoxicity in Rats

Nimesulide, an anti-inflammatory and analgesic drug, is reported to cause severe hepatotoxicity. In this study, molecular mechanisms involved in deranged oxidant-antioxidant homeostasis and mitochondrial dysfunction during nimesulide-induced hepatotoxicity and its attenuation by plant derived terpenes, camphene and geraniol has been explored in male Sprague-Dawley rats. Hepatotoxicity due to nimesulide (80 mg/kg BW) was evident from elevated SGPT, SGOT, bilirubin and histo-pathological changes. Antioxidants and key redox enzymes (iNOS, mtNOS, Cu/Zn-SOD, Mn-SOD, GPx and GR) were altered significantly as assessed by their mRNA expression, Immunoblot analysis and enzyme activities. Redox imbalance along with oxidative stress was evident from decreased NAD(P)H and GSH (56% and 74% respectively; P<0.001), increased superoxide and secondary ROS/RNS generation along with oxidative damage to cellular macromolecules. Nimesulide reduced mitochondrial activity, depolarized mitochondria and caused membrane permeability transition (MPT) followed by release of apoptotic proteins (AIF; apoptosis inducing factor, EndoG; endonuclease G, and Cyto c; cytochrome c). It also significantly activated caspase-9 and caspase-3 and increased oxidative DNA damage (level of 8-Oxoguanine glycosylase; P<0.05). A combination of camphene and geraniol (CG; 1∶1), when pre-administered in rats (10 mg/kg BW), accorded protection against nimesulide hepatotoxicity in vivo, as evident from normalized serum biomarkers and histopathology. mRNA expression and activity of key antioxidant and redox enzymes along with oxidative stress were also normalized due to CG pre-treatment. Downstream effects like decreased mitochondrial swelling, inhibition in release of apoptotic proteins, prevention of mitochondrial depolarization along with reduction in oxidized NAD(P)H and increased mitochondrial electron flow further supported protective action of selected terpenes against nimesulide toxicity. Therefore CG, a combination of natural terpenes prevented nimesulide induced cellular damage and ensuing hepatotoxicity

Analysis of tail-anchored protein translocation pathway in plants

Tail-anchored (TA) proteins are a special class of membrane proteins that carry out vital functions in all living cells. Targeting mechanisms of TA proteins are investigated as the best example for post-translational protein targeting in yeast. Of the several mechanisms, Guided Entry of Tail-anchored protein (GET) pathway plays a major role in TA protein targeting. Many in silico and in vivo analyses are geared to identify TA proteins and their targeting mechanisms in different systems including Arabidopsis thaliana. Yet, crop plants that grow in specific and/or different conditions are not investigated for the presence of TA proteins and GET pathway. This study majorly investigates GET pathway in two crop plants, Oryza sativa subsp. Indica and Solanum tuberosum, through detailed in silico analysis. 508 and 912 TA proteins are identified in Oryza sativa subsp. Indica and Solanum tuberosum respectively and their localization with respect to endoplasmic reticulum (ER), mitochondria, and chloroplast has been delineated. Similarly, the associated GET proteins are identified (Get1, Get3 and Get4) and their structural inferences are elucidated using homology modelling. Get3 models are based on yeast Get3. The cytoplasmic Get3 from O. sativa is identified to be very similar to yeast Get3 with conserved P-loop and TA binding groove. Three cytoplasmic Get3s are identified for S. tuberosum. Taken together, this is the first study to identify TA proteins and GET components in Oryza sativa subsp. Indica and Solanum tuberosum, forming the basis for any further experimental characterization of TA targeting and GET pathway mechanisms in crop plants

Evaluation of Three-Dimensional Chitosan-Agarose-Gelatin Cryogel Scaffold for the Repair of Subchondral Cartilage Defects: An In Vivo Study in a Rabbit Model

In this study, the potential of a chitosan-agarose-gelatin (CAG) cryogel scaffold for the repair of subchondral cartilage defects was explored in female New Zealand white rabbits. Custom-made CAG cryogel scaffold was implanted in a surgically created subchondral defect (diameter of 4 mm, depth of 4 mm) in knee joint of rabbit. The repair of the subchondral defect was evaluated at regular time interval by both macroscopic as well as microscopic examinations. The gross evaluation of the scaffold-implanted site showed integration of the scaffold with the surrounding tissue. Scanning electron microscopy and histological staining of the remnants of implanted cryogel scaffold showed infiltration of the host cells. The repair of the subchondral defect along with well-integrated regenerated cartilage was confirmed by the histology analysis of the joint. Results showed significant cartilage regeneration by the fourth week until eighth week after implantation. Immunohistochemical analysis confirmed that regenerated tissue is hyaline cartilage and absence of hypertrophy marker was reported. In addition, the CAG scaffolds did not elicit any adverse immunological rejection as shown by hematological analysis. Enzyme-linked immunosorbent assay did not show any statistically significant change in the concentration of tumor necrosis factor-alpha in the serum, and remained in a nontoxic range. Rabbits with a surgically created defect but no scaffold did not show any cartilage regeneration throughout the experiment of 8 weeks. These results demonstrate that CAG cryogel scaffolds promote repair of an osteochondral defect at a load-bearing site in rabbits

Correction: Topical Application of Ochratoxin A Causes DNA Damage and Tumor Initiation in Mouse Skin.

[This corrects the article DOI: 10.1371/journal.pone.0047280.]

Dietary administration of Nexrutine inhibits rat liver tumorigenesis and induces apoptotic cell death in human hepatocellular carcinoma cells

Epidemiological studies suggested that plant-based dietary supplements can reduce the risk of liver cancer. Nexrutine (NX), an herbal extract from Phellodendronamurense, has been shown to have anti-inflammatory, anti-microbial and anti-tumor activities. In the present study, we have shown the anti-tumor potential of NX against Solt-Farber model with elimination of PH, rat liver tumor induced by diethylnitrosoamine (DEN) as carcinogen and 2-acetylaminofluorene (2-AAF) as co-carcinogen. The elucidation of mechanistic pathways was explored in human liver cancer cells. Dietary intake of NX significantly decreased the cell proliferation and inflammation, as well as increased apoptosis in the liver sections of DEN/2-AAF-treated rats. Moreover, NX (2.5–10 μg/ml) exposure significantly decreased the viability of liver cancer cells and modulated the levels of Bax and Bcl-2 proteins levels. NX treatment resulted in increased cytochrome-c release and cleavage of caspases 3 and 9. In addition, NX decreased the expression of CDK2, CDK4 and associated cyclins E1 and D1, while up-regulated the expression of p21, p27 and p53 expression. NX also enhanced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, p38 and JNK1/2. Collectively, these findings suggested that NX-mediated protection against DEN/2-AAF-induced liver tumorigenesis involves decrease in cell proliferation and enhancement in apoptotic cell death of liver cancer cells

Topical application of ochratoxin A causes DNA damage and tumor initiation in mouse skin.

Skin cancer is one of the most common forms of cancer and 2-3 million new cases are being diagnosed globally each year. Along with UV rays, environmental pollutants/chemicals including mycotoxins, contaminants of various foods and feed stuffs, could be one of the aetiological factors of skin cancer. In the present study, we evaluated the DNA damaging potential and dermal carcinogenicity of a mycotoxin, ochratoxin A (OTA), with the rationale that dermal exposure to OTA in workers may occur during their involvement in pre and post harvest stages of agriculture. A single topical application of OTA (20-80 µg/mouse) resulted in significant DNA damage along with elevated γ-H2AX level in skin. Alteration in oxidative stress markers such as lipid peroxidation, protein carbonyl, glutathione content and antioxidant enzymes was observed in a dose (20-80 µg/mouse) and time-dependent (12-72 h) manner. The oxidative stress was further emphasized by the suppression of Nrf2 translocation to nucleus following a single topical application of OTA (80 µg/mouse) after 24 h. OTA (80 µg/mouse) application for 12-72 h caused significant enhancement in- (a) reactive oxygen species generation, (b) activation of ERK1/2, p38 and JNK MAPKs, (c) cell cycle arrest at G0/G1 phase (37-67%), (d) induction of apoptosis (2.0-11.0 fold), (e) expression of p53, p21/waf1, (f) Bax/Bcl-2 ratio, (g) cytochrome c level, (h) activities of caspase 9 (1.2-1.8 fold) and 3 (1.7-2.2 fold) as well as poly ADP ribose polymerase cleavage. In a two-stage mouse skin tumorigenesis protocol, it was observed that a single topical application of OTA (80 µg/mouse) followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate for 24 week leads to tumor formation. These results suggest that OTA has skin tumor initiating property which may be related to oxidative stress, MAPKs signaling and DNA damage

Cell factory-derived bioactive molecules with polymeric cryogel scaffold enhance the repair of subchondral cartilage defect in rabbits.

We have explored the potential of cell factory-derived bioactive molecules, isolated from conditioned media of primary goat chondrocytes, for the repair of subchondral cartilage defects. Enzyme-linked immunosorbent assay (ELISA) confirms the presence of transforming growth factor-β1 in an isolated protein fraction (12.56 ± 1.15 ng/mg protein fraction). These bioactive molecules were used alone or with chitosan-agarose-gelatin cryogel scaffolds, with and without chondrocytes, to check whether combined approaches further enhance cartilage repair. To evaluate this, an in vivo study was conducted on New Zealand rabbits in which a subchondral defect (4.5 mm wide × 4.5 mm deep) was surgically created. Starting after the operation, bioactive molecules were injected at the defect site at regular intervals of 14 days. Histopathological analysis showed that rabbits treated with bioactive molecules alone had cartilage regeneration after 4 weeks. However, rabbits treated with bioactive molecules along with scaffolds, with or without cells, showed cartilage formation after 3 weeks; 6 weeks after surgery, the cartilage regenerated in rabbits treated with either bioactive molecules alone or in combinations showed morphological similarities to native cartilage. No systemic cytotoxicity or inflammatory response was induced by any of the treatments. Further, ELISA was done to determine systemic toxicity, which showed no difference in concentration of tumour necrosis factor-α in blood serum, before or after surgery. In conclusion, intra-articular injection with bioactive molecules alone may be used for the repair of subchondral cartilage defects, and bioactive molecules along with chondrocyte-seeded scaffolds further enhance the repair. Copyright © 2015 John Wiley & Sons, Ltd

Treatment schedule and division of groups.

<p>Treatment schedule and division of groups.</p

Structure of the drug - nimesulide, and camphene and geraniol.

<p>Nimesulide is a non-steroidal anti-inflammatory drug (NSAID). Camphene is a bicyclic mono-terpenoid whereas geraniol is acyclic monoterpene-alcohol.</p