About Authors:
L.Kalaiselvi1*, Mr.R.Ulaganathan2
1 B.Pharm, II M.Tech Nanoscience&Technology,
2 Assistant Professor, B.Sc, M.Sc., M.Tech
Department of Biotechnology, Udaya school of Engineering,
Nagercoil, Kanyakumari dt.
*klkselvi@gmail.com
ABSTRACT:
Nanoparticle based systems have significant prospective for diagnosis, treatment and prevention of tuberculosis (TB) Gelatin nanoparticle derived from marine sources (fish skin, bone and fins) has been looked upon as a possible alternative to bovine and porcine. Fish gelatin nanoparticle synthesis by two step desolvation method, it was stable nanoparticles and confirm through Scanning electron microscopy (SEM).These nanoparticle were used as carrier for rifampicin. Our aim to develop a Nano particulate carrier of rifampicin for controlled delivery as well as reduced toxicity. In this study, rifampicin loaded fish gelatin nanoparticle was fabricated by an absorption/adsorption method. The effect of several variables on the Nanoparticle’s characteristics was calculated.
Reference Id: PHARMATUTOR-ART-1351
1. INTRODUCTION
Nanotechnology can be defined as the science and engineering involved in the design, Synthesis, characterization, application materials and devices whose smallest functional organization in at least one dimension is on the nanometer scale (One billionth a meter). It can prove to be a boon for human healthcare. Because Nano science and nanotechnologies have a huge potential to bring benefits in areas as diverse as drug development, water decontamination, information and communication technologies and the production of stronger, lighter materials. Human health care nanotechnology research can definitely result in immense health benefits. The genesis it nanotechnology can be traced to the promise of revolutionary advances across medicine, communications, genomics, and robotics. A complete list the potential applications nanotechnology is too vast and diverse to discuss in detail, but without doubt, one the greatest values nanotech will be in the development of new and effective medical treatments.
1.1.1 APPLICATION OF NANOTECHNOLOGY:
Nanotechnology in drug delivery from nanotechnology there is only one step Nano medicine, which may be defined as the monitoring, repair, Construction, and control of human biological systems at the molecular level, using engineer’s Nano devices and nanostructures. If can also be regarded as another implementation if nanotechnology in the field if medical science and diagnostics. One of the most important issues is the proper distribution of drugs and other therapeutic agents with in the patient’s body. During the past two decades, however, researchers involved in the development of pharmaceuticals have understood that drug delivery is a fundamental part of drug development, and a wide range of drug delivery systems has thus been designed. Ideally all these systems would improve the stability, absorption, and therapeutic concentration if the drug with in the target issue, as well as permit reproducible and longtime release if the drug at the target site.
1.1.2 THE NEED FOR INNOVATIVE NANOTECHNOLOGY BELOW DRUG TARGETING
Research into the delivery and targeting if therapeutic and diagnostic agents with nanoparticles is at the forefront, if Nano medicine for several reasons. First traditional oral or inject able drugs are not necessarily the most efficient formulations for a given product. This is particularly true for new biologics such as proteins and nucleic acids that require novel delivery technologies to optimize efficacy, minimize side effects and lead to better patient compliance.
Since the efficiency related to particle size nanoparticle formulations can enhance bioavailability, improve times/ controlled release of drugs and enable none precise targeting to the level of direct intracellular delivery. Because of their small size, Nano particulate drug carriers by pass the blood brain barrier, the branching pathways of the pulmonary system, and the tight epithelial junctions if the skin that normally delivery if drugs to the designed target. These reformulation drugs have the potential advantages of drug side effects, increasing patient compliance, and reducing health care costs. The current strategy for enhancing the therapeutic activity if currently available drugs is to entrap drugs with in a delivery system from where they are slowly released over an external time period. A novel drug carrier system plays an important role in controlled delivery as a pharmaceutical agent to the target at therapeutically optimal rate and dose. Several reports are available regarding the use of carrier systems like liposomes, dendrimers, micro spheres, and solid, lipid Nano particles for delivery of bio actives. Amongst various drug delivery systems, Nano particles NP represent a very promising approach for deliver bio actives.
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1.1.3 NANO PARTICLES:
Nano particles are defined as particulate dispersions or solid particles with a size in the range of 10-1000nm. The drug is dissolved, entrapped, encapsulated or attached to a nanoparticle matrix. Depending upon the method of preparation, nanoparticles, Nano spheres or Nano capsules can be obtained. The major goals in designing, Nano particles as a delivery system and to control particle size, Surface properties and release of pharmacologically agents in order to achieve the site- specific action of the drug at the therapeutically optimal rate and dose regimen.
The advantages of using nanoparticles as a drug delivery system include the following
* Manipulated to achieve both passive and active drug targeting after parental administration.
* They control and sustain release of the drug during the transportation and at the site of localization, altering organ distribution of the drug so as to achieve increase in drug therapeutic efficacy and reduction in side effects.
* Controlled release and particle degradation characteristics can be readily modulated by the choice of matrix constituents.
* Site specific targeting can be achieved by attaching targeting ligands to surface of particles or use of magnetic guidance.
* The system can be used for various routes of administration including oral, nasal, parental, intraocular etc. Nanoparticles can be prepared from a variety of materials such as proteins, polysaccharides and synthetic polymers. The selection of matrix materials is dependent on many features including,
Size of nanoparticles required, Inherent properties of the drug, e.g. aqueous solubility and stability, Surface characteristics such as charge and permeability, Degree of biodegradability, bio compatibility and toxicity. Drug release profile desired and antigenicity of the final product. Among the available potential colloidal drug carrier systems covering the size range described, protein based nanoparticles play an important role. Most often serum albumin obtained from human, bovine, legumin, etc., as well as gelatin was used as the starting material for the preparations. The present review details the latest development of protein nanoparticles drug delivery systems its preparation methods, characterization and potential application of nanoparticles.
1.1.4 PROTEIN NANO PARTICLES
The most important advantage of colloidal drug carrier systems is the possibility of drug targeting by a possibility if drug distribution as well as the improvement of the cellular uptake if a number of substances. Among if colloidal systems those based on proteins may be very capable. Proteins and a class of natural molecules that have unique functionalities and potential application in both biological as well as material fields. Nano materials derived from proteins, especially protein nanoparticles are biodegradable, non antigenic, metabolizable and can also be easily amenable for surface modification and covalent attachment of drugs and ligands. Because of the defined primary structure of proteins the protein based nanoparticles may suggest various possibilities for surface alteration and covalent drug attachment. Now a day’s active research is focused on the preparation of nanoparticles using proteins of nanoparticles using proteins like albumin gelatin, gliadin and legumin.
1.1.5 GELATIN
Gelatin is one of the protein materials that can be used for the production of Nano particles. It’s derived by hydrolytic degradation of collagen, the principle component of animal connective tissue. Gelatin has found application in food, photographic, cosmetic and pharmaceutical industries over the years. Recently, its usage as colloid stabilizer, foaming agent and emulsifiers. The source and type of collagen will influence the properties of the resulting gelatin. The main raw material for gelatin production is skin and bones from bone and porcine source. With the outbreak of bovine spongy form encephalopathy (BSE) in bone animals, there has been an interest in the gelatin production from non-bovine source. Traditional source of gelatin have been primer pig skin and cow hide, however for a number of reasons such as religious prohibition and concerns over the spreading of bovine spongiform encephalopathy alternative for these mammalian based gelatins are increasing demand.
1.1.6 PROPERTIES OF GELATIN
Gelatin is biodegradable and bio compatibility in physiological environments. These characteristics have contributed to selections proven record of safety as a plasma expander, as an ingredient in drug formulations, and as a sealant for vascular prosthesis. Although to date up to 27 different types of collagen have been identified type I collagen is the most occurring collagen in connective tissue. Gelatins qualify for a particular application depends largely on its rheological properties. Apart from basic physio chemical properties such as composition parameters, solubility, transparency color, odor and taste, the main attributes that best define the overall commercial quality of gelatin one gel strength and thermal physical properties of gelatin influence its quality and potential application since they are related to gelatin structure.
1.1.7 SOURCE OF GELATIN
The most abundant sources of gelatin one pig skin (46%), bovine hide, (29.4%) pork and cattle bones (23.1%). Fish gelatin accounted for less than 1.5% of total gelatin production. Due to their availability, animal skins and bones have been used extensive as a source of raw material for the formulation of the shell of gelatin capsules. Their excellent film forming ability and mechanical stability properties of gelatin result in the desired physical properties. Furthermore they can be recycled and retain their good performance. The gelatin made from the traditional material like animal skin, bone, tendon and collagen have been noted to meet the necessary pharmaceutical requirements as being quickly hydrolyzed by gastrointestinal enzymes. In addition they contain a variety of nutritious amino acids. The purified products obtained therefore are easily swallowed and rapidly absorbed.
1.1.8 ALTERNATIVE SOURCE OF GELATIN
Raw materials from fish and poultry have received considerable attention in recent years. As for as fish gelatin is concerned the huge number of species having very different intrinsic charareristics.This range from natural resources like fish to polymers, the fish gelatin has been obtained from fish skin products and formulated as natural hollow capsules of fish oil, spirulina and cellulose. Natural herbal capsules have been reported as concentrated herbal capsules of gold lipo plant capsules. The polymer material hydroxyl propyl methyl cellulose (HPMC) has also been explored. The odorless, milky, fibrous or, granular powder, from lint or wood pulp, dissolves completely in cold water. But it is almost insoluble in ethanol and other organic solvents. The global demand for gelatin has been over the years. Recent reports indicate that the annual world output of gelatin is nearly 326,000 tons. However, althoughgelatin has such a wide range of useful applications, pessimism and strong concerns still pre list among consumers with regard its usage. This is mainly due to religious sentiments (both Judaism and Islam forbid the consumption of any pork related products, while Hindus do not consume cow related products) whether animal tissue derived collagens and gelatin are capable of transmitting pathogenic vectors such as prions.
Gelatin from marine sources (warm and cold water fish skins, bones, and fins) is a possible alternative to bovine gelatin. One major advantage of gelatin sources is that they are not associated with the risk of out breaks of bovine spongiform Encephalopathy Furthermore, Fish Skin, which is a major byproduct of the Fish processing industry, causing waste and pollution, could provide a valuable source of gelatin. Production of fish gelatin is actually not new as it has been produced since 1960 by acid extraction, although most of it has been used industrial applications. Gelatin has been extracted from skins and bones of various cold water (e.g., cod hate, Alaska Pollock, and salmon) and warm water (e.g. Tuna, Catfish, Tilapia, Nile perch and Shark) fish.
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1.1.9 APPLICATION OF FISH GELATIN
* Low melting temperature of gelatin from cold water fish also makes it useful as the base for light sensitive coatings.
* Coldwater micro encapsulation of vitamins and other pharmaceutical additives.
* Minor volumes of fish gelatin one used to make soft gel capsules in nutrition supplements.
* Fish gelatin is also a good medium for precipitating silver halide emulsions.
* Gelatin is low in calories and melts in the mouth to give excellent sensory properties resembling fat making it ideal for use in low fat protein. Fish gelatin has also been used in the preparation if pharmaceutical products
1.1.10 NANOTECHNOLOGY IN TREATMENT OF TUBERCULOSIS
Treatments with improved sustained release profiles and bioavailability can increase compliance through reduced drug requirements and there in minimize chemotherapy of TB is complex due to the requirement of multidrug regimens that need to be administered over long periods. The poor patient compliance is the single most common reason for chemotherapy failure in TB. The micro encapsulation of pharmaceutical substances in biodegradable polymers used in controlled drug delivery has seen as an emerging technology. carrier or delivery systems such as liposome’s and micro spheres have been developed for the sustained delivery of antiTB drugs and have found better chemotherapeutic efficacy when investigated in animal models.(e.g. mice)the following are among the important technological advantages of nanoparticles as drug carriers, high stability, high carrier capacity (i.e., many drug molecules can be incorporated in the particle matrix ), feasibility of incorporation of both hydrophilic and hydrophobic substances; and feasibility of variable routes of administration, including oral administration and inhalation. These carriers can also be designed to enable controlled (sustained) drug release from the matrix.
1.1.11 THE NEED FOR ANTITUBERCULAR DRUG DELIVRY SYSTEMS
Although effective drugs are available for treatment of TB, yet daily multiple drug therapy for several months, poor patient compliance, drug toxicity and emergence of multi drug resistance lead to failure of chemotherapy. The current strategy for enhancing the therapeutic activity of currently available drugs is to entrap drugs within the delivery system from where they are slowly release over and extended time period. Rifampicin is first line drug currently used for treatment of latent mycobacterium Tuberculosis infections in adults. But a number of side effect like lack of appetite, nausea, hepatotoxicity, fever, chill allergic rashes, itching and immunological disturbances. Modern drug carrier systems play an important role in controlled delivery of a pharmaceutical agent to the target at a therapeutically optimal rate and dose. Nano particles represent a class of drug delivery vehicles which can serve as promising perspective for ferrying large doses of the drug to the target site with interception of minimal side effects.
As a drug delivery carrier they offer several advantages, such as ease of purification and sterilization, possibility of drug targeting, and a sustained release action. Targeted drug delivery systems can optimize the therapeutic index of antitubercular drugs by increasing the drug concentration ratio of diseased tissue to normal tissue compared to other systems, nanoparticles are known to have better accumulation in macrophage rich organs, e.g. lungs, liver and spleen, because of their preferential phagocytosis it is well known that pulmonary TB is the commonest form of TB and alveolar macrophages of lungs being the abode of Mycobacterium tuberculosis. Therefore, nanoparticles represent an interesting carrier system for the delivery of anti-tubercular agents to the macrophages as an attempt to reduce the required dose. To minimize toxicity, to minimize dose dependent side effects, to sustain the drug release and selectively deliver the drug to infected cells. So the Rifampicin loaded gelatin Nano particles represent a class of drug delivery vehicle which can serve as promising perspective for ferrying large closes of the drug to the site with interception of minimal side effects. Gelatin nanoparticles containing RIF were prepared using two step desolvation methods.
Fish gelatin quality and nutritional properties like physiochemical characteristics, rheological properties and amino acid analysis are subject of many investigations. Fish gelatin to have similar physical and chemical properties compared to porcine gelatin and to be rated superior in a blind sensory test. In this study comparison of yield, physiochemical and rheological properties of acid and alkaline fitofague’s gelatin
2. Manufacturing Methods
2.1.1 EXTRACTION OF FISH GELATIN
The procedure was essentially based on a mild acid pretreatment for collagen swelling, followed by extraction in water at moderate Temperature (45ºC). The entire process takes about 24hrs. Because of the acid liability of the cross links found in fish skins collagen. Skins were washed under running tap water to remove superfluous materials. The wash skins were drip dried and soaked in the saturated lime solution [Ca (OH)2], at 20ºC for 14days for each kilogram of wet skins.Ca (OH)2 solution was used at the soaking medium. After soaking, the skins were then removed and washed with abundant tap water (1:10) to remove excessive Ca (OH)2while maintaining the skins at pH10. This was followed by the soaking in distilled water at 48ºC overnight to solubilize the gelatin. The solution was then filtered through whattman No 541 filter papers before passing through a strong acid cationic exchange resin, which reduced the pH of gelatin to approximately 5.The filtrates were then freezing, dried and further analyzed for physio chemical properties.
The better recovery of gelatin is based on the wet and dry conditions of the skin and the use of Ca (OH)2 prior to gelatin extraction. The yield of gelatin was calculated using the following expression
% of yield (wet weight basis) = Dry weight of gelatin x 100
------------------------
Wet weight of skin
2.1.2 FABRICATION METHODS OF GELATIN NANO PARTICLES
Two fundamental methods of cross linking have been described for gelatin Physical and chemical. Physical methods include UV irradiation and dehydrothermal treatment, although these are inefficient and make it difficult to control the cross linking density of the gelatin matrix. Chemical cross linking agents have been categorized into two types as non zero length and zero length. Non zero length cross linkers are functional or poly functional and operate by bridging free carboxylic acid resides or amine groups between adjacent protein molecules, e.g., glutaraldehyde, glyceraldehyde
2.1.2. TWO STEP DESOLVATION METHOD
Briefly 10g gelatin was dissolved in distilled water (100ml) under constant heating at 40±1ºC. Acetone (50ml) was added to the gelatin solution as a desolvation agent to precipitate the high molecular weight (HMW) gelatin. The supernatant was discarded, and the HMW gelatin was re dissolved by adding distilled water (10ml) with stirring at 600rpm under constant heating. The pHof the gelatin solution at the second desolvation step was adjusted (between 2 and 12) drop wise addition of acetone (30ml) to form GNPs. At the end of the process, glutraldehyde solution (25% V/V aqueous solution) was added 2 µl. It act as a cross linking agent, and the solution was stirred for 12 hours at 600rpm. Effect of parameters like pH, temperature, amount of glutaraldehyde was studies
2.1.3 COLOR DETERMINATION
Color of gelatin samples were measured by putting them on white background and compared with each other. Gelatin color must be pale yellow to amber.
2.2 CHARACTERIZATION THE NANOPARTICLES (FGNP)
2.2.1 SHAPE AND SIZE
Fig 1a. SEM Coater, 1b. Photograph Of Scanning Electron Microscopy
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The morphology of FGPs was determined by Scanning Electron Microscopy (ZEISS from CarlZeiss, India). A Scanning Electron Microscope (SEM) is a type of electron microscope that images a sample by scanning it with a beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition, and other properties such as electrical conductivity. The powdered sample was coated with gold and the accelerating voltage of 95KV.
2.2.2 DRUG LOADING
Drug loading can be done by two methods namely an Incorporation method and an Adsorption/ absorption technique. Ideally, a successful Nano particulate system has a high drug loading capacity. Adsorption /absorption technique followed to drug loading, absorbing the drug after formation of nanoparticles by incubating the carrier with a concentrated drug solution. Drug loading of nanoparticles was determined by the method proposed by the amount of RIF loaded was determined by incubating the nanoparticle suspension (1.0ml) in 5.0ml phosphate buffer saline (pbs.pH 7.4) for 2hrs at 800rpm at 25±1ºC. The amount of unloaded and loaded drug was determined UVspectrophotometrically in the supernatant obtained after separation of nanoparticles by centrifugation.
Fig2. Photograph of UV- Spectrometer
The instrument used here was P.C based UV 5704SS Spectrometer. It consists of a spectrophotometer unit and a computer unit. The amount of radiant energy absorbed by a sample at a certain wavelength depends on how much of that substance, which absorbs that wavelength of radiation, is present in the sample. In other words, the absorption of radiant energy is proportional to the absorbing materials. Transmittance (T) is defined as the ratio of intensity of transmitted light and intensity of the light that is failing upon the sample when light falls upon a homogenous medium a portion is transmitted. It is the transmitted light that is actually detected by the instrument.
2.2.3 STABILITY UPON STORAGE OF THE NANOPARTICLES
Nanoparticles are intended to be administered as pharmaceutical dosage forms in humans. Among other requirements, they must be free of impurities. The necessity for and degree of purification are dependent on the final purpose of the formulation developed, the most commonly reported procedures are gel filtration, ultracentrifugation, dialysis, and, recently, cross flow filtration. Another requirement relates to the sterilization of the formulation. The choice of the sterilizing treatment depends on the physical susceptibility of the system. Finally, nanoparticles should be easily stored and administered. Nanoparticles constitute a relatively stable physical system because of their colloidal nature. Many variables can affect the stability of nanoparticles generally a colloidal suspension is stable and does not tend to separate as a result of slow deposition due to the mixing tendencies of diffusion and convection. However, some agglomeration can occur. To prevent a complete precipitation, it is necessary to incorporate some additives.
Chemical stability of drug is also a fundamental aspect of the overall stability evaluation of the nanoparticles. Some parameters are crucial for the stability, such as the duration of contact with the aqueous environment when the drug is water soluble, the surrounding pH when drug degradation is pH dependent and light exposure when the drug is light sensitive. Stability studies are thus important and can be performed according to the drug and to the polymer properties. There are some methods to increase the stability of the nanoparticles lyophilization (freeze drying) seems to be a highly stabilizing process. It is generally applied to enhance the physiochemical stability of the nanoparticles to achieve a pharmaceutically acceptable product, especially in cases in which the storage conditions are unfavorable. This technique involves the freezing of the suspension and subsequent elimination of its water content by sublimation under reduced pressure. After complete desiccation, nanoparticles are obtained in the form of a dry powder that is easy to handle and store.
3. CONCLUSION
A novel approach was followed for the preparation of RIF loaded FGNPs. These nanoparticles possess various therapeutic and biomedical applications. Fish gelatin was extracted successfully from the skin and fin of cat fish by acidic methods. The fish gelatin nanoparticles were successfully extracted by a two-step desolvation method, encapsulation and characterization. Rifampicin is a bactericidal antibiotic with a wide spectrum of activity and is used to treat Mycobacterium including tuberculosis and leprosy. It is less active against gram-negative bacteria. RIF inhibits DNA dependent RNA polymerase in bacterial cells by binding to its B subunit. The results demonstrated that the RIF loaded NPs were more effective against gram positive bacteria. The advantage of using gelatin nanoparticles as drug vehicle is that they enable controlled release of drugs. The dosage of drug required is reduced with gelatin nanoparticles, when compared with that through the oral route; hence side effects are significantly reduced.
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