01 September 2010

CONTROLLED DRUG DELIVERY SYSTEMS


CONTROLLED DRUG DELIVERY SYSTEMS

Deepu



Controlled drug delivery occurs when a polymer, whether natural or synthetic, is judiciously combined with a drug or other active agent in such a way that the active agent is released from the material in a predesigned manner. The release of the active agent may be constant over a long period, it may be cyclic over along period, or it may be triggered by the environment or other external events. In any case, the purpose behind controlling the drug delivery is to achieve more effective therapies while eliminating the potential for both under- and overdosing.

In recent years, controlled drug delivery formulations and the polymers used in these systems have become much more sophisticated, with the ability to do more than simply extend the effective release period for a particular drug. . In addition, materials have been developed that should lead to targeted delivery systems, in which a particular formulation can be directed to the specific cell, tissue, or site where the drug it contains is to be delivered.

Providing control over the drug delivery can be the most important factor at times when traditional oral or injectable drug formulations cannot be used. These include situations requiring the slow release of water-soluble drugs, the fast release of low-solubility drugs, drug delivery to specific sites, drug delivery using nanoparticulate systems, delivery of two or more agents with the same formulation, and systems based on carriers that can dissolve or degrade and be readily eliminated. The ideal drug delivery system should be inert, biocompatible, mechanically strong, comfortable for the patient, capable of achieving high drug loading, safe from accidental release, simple to administer and remove, and easy to fabricate and sterilize.


DRUG DELIVERY CARRIERS

Colloidal drug carrier systems such as micellar solutions, vesicle and liquid crystal dispersions, as well as nanoparticle dispersions consisting of small particles of 10-400 nm diameter show great promise as drug delivery systems. When developing these formulations, the goal is to obtain systems with optimized drug loading and release properties, long shelf-life and low toxicity. The incorporated drug participates in the microstructure of the system, and may even influence it due to molecular interactions, especially if the drug possesses amphiphilic and mesogenic properties.

Recently, as a result of as a result of rapid development of novel nanotechnology-derived materials, a new generation of polymer therapeutics has emerged, using materials and devices of nanoscale size for the delivery of drugs, genes and imaging molecules. These materials include polymer micelles, polymer-DNA complexes (polyplexes), liposomes, and other nanostructured materials for medical use that are collectively known as nanomedicines.



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Figure 1. Pharmaceutical carriers


MICELLES


Micelles formed by self-assembly of amphiphilic block copolymers (5-50 nm) in aqueous solutions are of great interest for drug delivery applications. The drugs can be physically entrapped in the core of block copolymer micelles and transported at concentrations that can exceed their intrinsic water- solubility. Moreover, the hydrophilic blocks can form hydrogen bonds with the aqueous surroundings and form a tight shell around the micellar core. As a result, the contents of the hydrophobic core are effectively protected against hydrolysis and enzymatic degradation. Total molecular weight and block length ratios can be easily changed, which allows control of the size and morphology of the micelles. Functionalization of block copolymers with crosslinkable groups can increase the stability of the corresponding micelles and improve their temporal control. Substitution of block copolymer micelles with specific ligands is a very promising strategy to a broader range of sites of activity with a much higher selectivity.



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Figure 2. Block copolymer micelles



LIPOSOMES

Liposomes are a form of vesicles that consist either of many, few or just one phospholipid bilayers. The polar character of the liposomal core enables polar drug molecules to be encapsulated. Amphiphilic and lipophilic molecules are solubilized within the phospholipid bilayer according to their affinity towards the phospholipids. Channel proteins can be incorporated without loss of their activity within the hydrophobic domain of vesicle membranes, acting as a size-selective filter, only allowing passive diffusion of small solutes such as ions, nutrients and antibiotics.



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Figure 3. Drug encapsulation in liposomes


DENDRIMERS

Dendrimers are nanometer-sized, highly branched and monodisperse macromolecules with symmetrical architecture. They consist of a central core, branching units and terminal functional groups. The core together with the internal units, determine the environment of the nanocavities and consequently their solubilizing properties, whereas the external groups the solubility and chemical behaviour of these polymers. Targeting effectiveness is affected by attaching targeting ligands at the external surface of dendrimers, while their stability and protection from the Mononuclear Phagocyte System (MPS) is being achieved by functionalization of the dendrimers with polyethylene glycol chains (PEG).

LIQUID CRYSTALS

Liquid Crystals combine the properties of both liquid and solid states. They can be made to form different geometries, with alternative polar and non-polar layers (i.e., a lamellar phase) where aqueous drug solutions can be included.

NANOPARTICLES (BOTH NANO SPHERES AND NANO CAPSULES)

Nanoparticles (including nanospheres and nanocapsules of size 10-200 nm) are in the solid state and are either amorphous or crystalline. They are able to adsorb and/or encapsulate a drug, thus protecting it against chemical and enzymatic degradation. Nanocapsules are vesicular systems in which the drug is confined to a cavity surrounded by a unique polymer membrane, while nanospheres are matrix systems in which the drug is physically and uniformly dispersed. Nanoparticles as drug carriers can be formed from both biodegradable polymers and non-biodegradable polymers. In recent years, biodegradable polymeric nanoparticles have attracted considerable attention as potential drug delivery devices in view of their applications in the controlled release of drugs, in targeting particular organs / tissues, as carriers of DNA in gene therapy, and in their ability to deliver proteins, peptides and genes through the peroral route.





HYDROGELS

Hydrogels are three-dimensional, hydrophilic, polymeric networks capable of imbibing large amounts of water or biological fluids. The networks are composed of homopolymers or copolymers, and are insoluble due to the presence of chemical crosslinks (tie-points, junctions), or physical crosslinks, such as entanglements or crystallites. Hydrogels exhibit a thermodynamic compatibility with water, which allows them to swell in aqueous media. They are used to regulate drug release in reservoir-based, controlled release systems or as carriers in swellable and swelling-controlled release devices. On the forefront of controlled drug delivery, hydrogels as enviro-intelligent and stimuli-sensitive gel systems modulate release in response to pH, temperature, ionic strength, electric field, or specific analyte concentration differences. In these systems, release can be designed to occur within specific areas of the body (e.g., within a certain pH of the digestive tract) or also via specific sites (adhesive or cell-receptor specific gels via tethered chains from the hydrogel surface). Hydrogels as drug delivery systems can be very promising materials if combined with the technique of molecular imprinting.

In recent years, there has been a rapid growth in the area of drug discovery, facilitated by novel technologies such as combinatorial chemistry and high-throughput screening. These novel approaches have led to drugs which are generally more potent and have poorer solubility than drugs developed from traditional approaches of medicinal chemistry (Lipinsky, 1998) [1]. The development of these complex drugs has resulted in a more urgent focus on developing novel techniques, to deliver these drugs more effectively and efficiently.


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Figure 4: Conventional and Ideal drug release profiles

As can be seen in Figure 4, the conventional oral and intravenous routes of drug administration do not provide ideal pharmacokinetic profiles especially for drugs, which display high toxicity and/or narrow therapeutic windows. For such drugs the ideal pharmacokinetic profile will be one wherein the drug concentration reached therapeutic levels without exceeding the maximum tolerable dose and maintains these concentrations for extended periods of time till the desired therapeutic effect is reached. One of the ways such a profile can be achieved in an ideal case scenario would be by encapsulating the drug in a polymer matrix. The technology of polymeric drug delivery has been studied in details over the past 30 years and numerous excellent reviews are available (Gombotz and Pettie [2], 1995; Sinha and Khosla [3], 1998; Langer[4], 1998).

The three key advantages that polymeric drug delivery systems can offer are:

1. Localized delivery of drug: The product can be implanted directly at the site where drug action is needed and hence systemic exposure of the drug can be reduced. This becomes especially important for toxic drugs which are related to various systemic side effects (such as the chemotherapeutic drugs).

2. Sustained delivery of drugs: The drug encapsulated is released over extended periods and hence eliminates the need for multiple injections. This feature can improve patient compliance especially for drugs for chronic indications, requiring frequent injections (such as for deficiency of certain proteins).

3. Stabilization of the drug: The polymer can protect the drug from the physiological environment and hence improve its stability in vivo. This particular feature makes this technology attractive for the delivery of labile drugs such as proteins.



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Figure 5. Possible drug release mechanisms for polymeric drug delivery

As shown in Figure 5, the drug will be released over time either by diffusion out of the polymer matrix or by degradation of the polymer backbone. This continuous release of the drug could potentially lead to a pharmacokinetic profile close to the ideal case scenario depicted in Figure 4.

The continuous release of drugs from the polymer matrix could occur either by diffusion of the drug from the polymer matrix, or by the erosion of the polymer (due to degradation) or by a combination of the two mechanisms. Several reviews have been presented on the mechanisms and the mathematical aspects of release of drugs from polymer matrices (Batycky et al., 1997) [5]; Brazel and Peppas, 2000 [6]; Comets et al., 2000). For a given drug, the release kinetics from the polymer matrix are governed predominantly by three factors, viz. the polymer type, polymer morphology and the excipients present in the system. The subsequent sections will focus on each of these three factors to describe their role on drug release characteristics of a polymeric system

The Polymer

An appropriate selection of the polymer matrix is necessary in order to develop a successful drug delivery system. The polymer could be non-degradable or degradable. A major disadvantage with non-degradable polymers is that a surgery is required to harvest these polymers out of the body once they are depleted of the drug. Hence, non-degradable polymers can be used only if removal of the implant is easy (such as an ocular implant). Degradable polymers on the other hand do not require surgical removal and hence are preferred for drug delivery applications. However, since they degrade to smaller absorbable molecules, it is important to make sure that the monomers are non-toxic in nature. The most commonly used polymers for this application are Polylactide (PLA) and Poly(Lactide-co-Glycolide) (PLGA). These polymers have been used in biomedical applications for more than 20 years and are known to be biodegradable, biocompatible and non-toxic. Degradation of lactide based polymers and in general all hydrolytically degradable polymers, depends on the following properties:


  1. Chemical composition: The rate of degradation of polymers depends the type of degradable bonds present on the polymer. In general, the rate of degradation of different chemical bonds follows as Anhydride > Esters > Amides.

  2. Crystallinity: Higher than crystallinity of a polymer, slower is its rate of degradation.

  3. Hydrophilicity: If the polymer has a lot of hydrophobic groups present on it, then it is likely to degrade slower than a polymer which is hydrophilic in nature.

Polylactides are known to be more hydrophobic as compared to PLGA and take a longer time to degrade. Among the polylactides, DL-PLA, which is a polymer of D and L-lactide, degrades faster than L-PLA, which is a homopolymer of L-lactide, presumably due to lesser crystallinity. Similarly, the more hydrophobic end-capped PLGA polymers degrade faster than the carboxyl-ended PLGA.

In spite of the several apparent advantages of PLA and PLGA based polymers, commercialization of products based on these polymers has certain limitations. One of the major concerns is that more than 500 patents have been issued for various applications of these polymers. Hence, patent infringement may become a concern in developing new products. In addition, PLA and PLGA polymers have certain inherent limitations in terms of flexibility for applications. Due to these concerns, several new polymers are presently being explored for applications in drug delivery. Some of the new polymers which are in clinical or preclinical development stage are:

  1. Polyorthoesters (Heller[7] et al., 2000)

  2. Polyphosphazenes
  3. Polyanhydrides (Shieh[8] et al., 1994)
  4. Polyphosphoesters.

The use of new natural polymers as drug carriers has received considerabl attention in the last few years. One of the goals of such systems is to prolong the residence time of a drug carrier in the gastrointestinal (GI) tract. The bioadhesive bond can be of a covalent, electrostatic, hydrophobic, or hydrogen bond nature. Ionic polymers are reported to be promising for biadhesive medical applications, and an increased charge density will also give better adhesion, suggesting that the electrostatic interactions are of great importantace.

Polymer Morphology

Morphology of the polymer matrix plays an important role in governing the release characteristics of the encapsulated drug. The polymer matrix could be formulated as either micro/nano spheres, gel, film or an extruded shape (such as cylinder, rod etc). The shape of the extruded polymer can be important to the drug release kinetics. For example, it has been shown that zero order drug release can be achieved using a hemispherical polymer form. Polymer microspheres are the most popular form due to manufacturing advantages as well as ease of administration (injectability by suspending in a vehicle). Polymer microspheres can be manufactured by using various techniques such as spray drying, solvent evaporation etc (O'Donnell and McGinity 1997 [9]; Hermann and Bodmeier 1998; Witschi and Doelker 1998). The type of techniques used affects, factors such as porosity, size distribution and surface morphology of the microspheres and may subsequently affect the performance of the drug delivery product.

Excipients

Polymeric drug delivery products can be formulated with excipients added to the polymer matrix. The main objective of having excipients in the polymer matrix could be either to modulate the drug release, or to stabilize the drug or to modulate the polymer degradation kinetics. Recent studies by Schwendeman and coworkers (Zhu and Schwendeman, 1999; Zhu et al., 2000)[10] have shown that by incorporating basic salts as excipients in polymeric microspheres, the stability of the incorporated protein can be improved. It has shown that these basic salts however also slow the degradation of the polymer. Similarly, hydrophilic excipients can accelerate the release of drugs, though they may also increase the initial burst effect.

Nanoparticles prepared from hydrophilic polymers

Other than commonly used synthetic hydrophobic polymers active research is now focused on the preparation of hydrophilic polymers like chitosan, sodium alginate, gelatin,etc. Different methods have been adopted to prepare naanoparticles from hydrophilic polymers. Several hydrophobic-hydrophilic carriers having limited protein-loading capacity have prepared by using organic solvents.

Hydrophilic nanoparticles based on Chitosan receive currently increasing interest as they could control the rate of drug release, prolonging the duration of the therapeutic effect, and deliver the drug to specific sites in the body. Chitosan can form nanoparticles using, amongst other methods, ionotropic gelation. This method is based on the gelation of Chitosan when it comes in contact with specific polyanions due to the formation of inter and intramolecular cross-linkages mediated by these polyanions.

















REVIEW OF LITERATURE



















Over the past few decades, there has been considerable interest in developing biodegradable efficiency nanoparticles (NPs) as effective drug delivery devices. Various polymers have been used in drug delivery research as they can effectively deliver to a target site and thus increase the efficiency, while minimizing side effects [11]. The controlled release (CR) of pharmacologically active agents to the specific site of action at the therapeutically optimal rate and dose regimen has been major goal in designing such devices. Liposomes have been used as potential drug carriers instead of conventional dosage forms because of their unique advantages which include ability to protect drugs from degradation, target the drug to the site of action and reduce the toxicity or other side effects [12].

However developmental works on liposomes, due to their inherent problems such as low enscapsulation efficiency, rapid leakage of water-soluble drug in the presence of blood components and poor storage stability. On the other hand, polymeric nanoparticless offer the some specific advantages over liposomes. Polymeric nanoparticles help to increase the stability of drugs and controlled release of drugs especially protein drugs.

Nanoparticles generally vary in size from 10- 1000 nm. The drug is dissolved, entrapped, or attached to a nanoparticle matrix. Depending upon the method of preparations, nanospheres or nanocapsules can be obtained. Nanocapsules are vesicular systems in which the drug is confined to a cavity surrounded by a unique polymer membrane, while nanospheres are matrix systems in which the drug is physically and uniformly dispersed. In recent years, biodegradable polymeric nanoparticles have attracted attention as potential drug delivery devices in view of their applications, the controlled release of drugs, their ability to target organs / tissues, as carriers of DNA in gene therapy, and in their ability to deliver proteins, peptides genes through a peroral route of administration [13, 14].

The present review details latest on the above mentioned polymers as well as nanoparticles based on chitosan [16], gelatin [15], sodium alginate[17] and other hydrophilic biodegradable polymers. The PLA, PGA and PLGA polymers become tissue compactible have been used earlier as controlled release formulations as in parental and drug delivery applications [18,19].


PREPARATION OF NANO PARTICLES


Conventionally nps have been prepared mainly by two methods:

(1) dispersion of the pre formed polymers,

(2) polymerization of polymers.


DISPERSION OF THE PRE FORMED POLYMERS


Several methods have to be suggested to prepare biodegradable nanoparticles from PLA, PGA and PLGA.

SOLVENT EVAPORATION METHOD


In this method polymer is dissolved in an organic solvent like dichloromethane, chloroform or ethyl acetate. The drug is dissolved or dispersed into the pre-formed solution, and this mixture is then emulsified into an aqueous solution to make an oil (O) in water (W) ie., O/W emulsion by using a surfactant or emulsifying agent like gelatin, polyvinyl alcohol, polysorbate-80 etc. After the formation of the stable emulsion, organic solvent is evaporated by increasing the temperature/under pressure or by continuous stirring. The W/O/W method has also been prepared the water soluble drug-loaded nanoparticles [20]. Both the above method use the high-speed homogenization or sonication, however these procedures are good for a laboratory scale operation, but for a large scale pilot production, alternative methods using low-emulsification are required.



SPONTANEOUS EMULSIFICATION OR SOLVENT DIFFUSION METHOD


This is the modified version of the solvent evaporation method [21, 22]. Here the water soluble solvents like acetone or methanol along with the water insoluble organic solvent like dichloromethane or chloroform were used as the oil phase. Due to the spontaneous diffusion of water soluble solvent, an interfacial turbulence is created between the two phases leading to the formation of smaller particles. As concentration of water-soluble solvent increases, a considerable decrease in particle size can be achieved.


SALTING OUT /EMULSIFICATION-DIFFUSION METHOD


The above methods require the use of the organic solvents which are hazardous to the environment as well as the physiological system, in order to meet the requirements. Allemann and co workers have developed two methods of preparing nanoparticles. The first one is the salting-out method and second one is the emulsification-solvent diffusion technique.



PREPARATION OF NANOPARTICLES BY SUPERCRITICAL FLUID TECHNOLOGY


Conventional methods like solvent evaporation, coacervation and in-situ polymerization often require the toxic solvents and or surfactants, therefore research efforts have been directed to develop the environmentally safer encapsulation methods to produce drug -loaded micron and submicron size particles. If solvent impurities remain in the drug-loaded nanoparticles, then these become toxic and may degrade the pharmaceuticals within the polymer matrix. Supercritical fluids have now became the attractive alternatives, because these are environmentally friendly solvents and the method can be profitably used to process particles in high purity and without any trace amount of the organic solvent.



NANOPARTICLES PREPARED FROM HYDROPHILIC POLYMERS


Other than commonly used synthetic hydrophobic polymers active research is now focused on the preparation of hydrophilic polymers like chitosan, sodium alginate, gelatin etc. Different methods have been adopted to prepare nanoparticles from hydrophilic polymers. Several hydrophobic-hydrophilic carriers having limited protein-loading capacity have prepared by using organic solvents. Calvo and co-workers have reported a method to prepare hydrophilic chitosan nanoparticles [23] [24] .

The preparation method involves ionic gelation, with a mixture of two aqueous phases, one of which contains chitosan and a di block copolymer of ethylene oxide (EO) and other contains a polyanion sodium tripolyphosphate (TPP). In this method, the positively charged amino group of chitosan interacts with the negatively charged TPP. The size (200-1000nm) and zeta potential (+20mV and +60mV) of the nanoparticles produced were modulated by varying the composition of chitosan with the PEO-PPO diblock polymer.

DRUG LOADING


A successful nanoparticle system may be the one which has a high loading capacity to reduce the quantity of the carrier required for administration. Drug loading into nanoparticles is achieved by two methods: one is by incorporating the drug at the time of the nanoparticle preparation or secondly by absorbing the drug after the formation of nanoparticles by incubating them in the drug solution. It is thus evident that a large amount of the drug can be entrapped by the incorporation method.

The capacity of absorption is thus related to the hydrophobicity of the polymer and specific areas of the nanoparticles. In case of the entrapment method, an increase in the concentration of the monomer, increases the association of the drug, but a reverse trend is observed with drug concentration in the dispersed solution. These results indicate that there is a need to optimize the amount of monomer available for the drug entrapment. In addition to the absorption and incorporation anew method of drug loading for water-soluble drugs.In this method, the drug was chemically conjugated to the nps.



DRUG RELEASE


Drug release from nanoparticles and the subsequent biodegradation are important for developing the successful formulations. The release rate of nanoparticles depends on

(1) desorption of the surface-bound /absorbed drug

(2) diffusion through the np matrix

(3) diffusion (in case of nanocapsules) through the polymer wall .

(4) nanoparticle matrix erosion

(5) Combined erosion/diffusion process.

Thus diffusion and the biodegradation govern the process of drug release.

Methods to study in-vitro release are

(1) side-by-side diffusion cells with artificial or biological membranes

(2) dialysis bag diffusion technique

(3) reverse dialysis sac technique

(4) ultracentrifugation

(5) ultrafiltration


SURFACE PROPERTIES OF NANO PARTICLES



SURFACE CHARECTERIZATION METHODS


Many techniques have been developed and used to study the surface modification of nanoparticles. The efficiency of surface modification can be measured either by estimating the surface charge, density of functional groups or an increase in surface hydrophilicity. One method used to determine the surface modification is to determine zeta potential of aqueous suspension containing nanoparticles. In this method the mobility of the charged particles are monitored by applying an electrical potential. The zeta potential values may be negative or positive depending upon the nature of the polymer or material used for the surface modification. The extent of surface hydrophilicity can be predicted from the values of zeta potential. This is the widely used technique to understand the surface charges of nanoparticles.

Another commonly used technique is Electron Spectroscopy for Chemical Analysis (ESCA) also called X-ray Photoelectron Spectroscopy (XPS). This technique is based on the emission of electrons from the materials in response to the irradiation of photons of sufficient energy to cause the ionization of core level electrons. These electrons emitted at energy characteristic of atoms from which they are emitted. Since photons have low penetration energy, only those electrons pertaining to atoms at or near the surface (up to 100Ao) escape and these can be counted. for each atom type ,the number of electrons emitted is related to the number of atoms of a particular type of atom.

In another technique, surface hydrophobicity of nanoparticles can be directly measured by hydrophobic interaction chromatography. This technique involves column chromatography which is able to separate materials based on interaction with a hydrophobic gel matrix. The nanoparticle and the gel interaction is a function of surface hydrophobicity of nanoparticles.































AIMS AND OBJECTIVES





















Nanoparticles and nanoformulations have already been applied as drug delivery systems with great success; and nanoparticulate drug delivery systems have still greater potential for many applications including anti-tumour therapy, gene therapy, and AIDS therapy, radiotherapy, in the delivery of proteins, antibiotics, virostatics, vaccines and as vesicles to pass the blood - brain barrier.

Nanoparticles provide massive advantages regarding drug targeting, delivery and release and, with their additional potential to combine diagnosis and therapy, emerge as one of the major tools in nanomedicine. The main goals are to improve their stability in the biological environment, to mediate the bio-distribution of active compounds, improve drug loading, targeting, transport, release, and interaction with biological barriers. The cytotoxicity of nanoparticles or their degradation products remains a major problem, and improvements in biocompatibility obviously are a main concern of future research.

The aims of present work are to further purify the purchased low molecular weight chitosan, to investigate the different compositions to obtain an optimized nanoparticulate formation of chitosan -TPP nanoparticles and to encapsulate chloramphenicol antimicrobial drug to chitosan nanoparticles. The focus of this work is to find out how systematically manipualating processing parameters in the TPP initiated chitosan gelation to obtain predictable and optimal chitosan nanoparticles properties for application of antibacterial drug delivery.



















MATERIALS AND METHODS

















MATERIALS


1. Chitosan polymers, derived from crab shell, in the form of fibrils flakes were obtained from Sigma-Aldrich.

2. Sodium Tripolyphosphate was purchased from Sigma-Aldrich.

3. Chloramphenicol from Sigma-Aldrich.

4. All other materials and reagents used were of analytical grade of purity.


METHODS

1. Purification of Chitosan polymer

2. Preparation of chitosan nanoparticles (ionic gelation of chitosan with sodium tripolyphosphate).

3. Drug (chloramphenicol) encapsulation.

4. Optmization and characterization of chitosan nanoparticles.






















CHITOSAN-A UNIQUE POLYMER



Chitosan is a natural polymer obtained by the hydrolysis of chitin, a native polymer present in shellfish. Together with chitin, chitosan is considered the second most abundant polysaccharide after cellulose. However, unlike cellulose, the use of chitosan as an excipient in pharmaceutical formulations is a relatively new development. Chitosan (poly[-(1,4)-2-amino-2-deoxy-D-glucopiranose]) differs from chitin in that a majority of the N-acetyl groups in chitosan are hydrolyzed. The degree of hydrolysis (deacetylation) has a significant effect on the solubility and rheological properties of the polymer. The amine group on the polymer has a pKa in the range of 5.5 to 6.5, depending on the source of the polymer. At low pH, the polymer is soluble, with the sol-gel transition occurring at approximate pH 7. The pH sensitivity, coupled with the reactivity of the primary amine groups, make chitosan a unique polymer.




CHITOSAN IN CONVENTIONAL DOSAGE FORM

Chitosan's film forming abilities lend itself well as a coating agent for conventional solid dosage forms such as tablets. Furthermore its gel- and matrix-forming abilities makes it useful for solid dosage forms, such as granules, microparticles, etc. Crystallinity, molecular weight, and degree of deacetylation were seen to be factors that affected the release rates from the chitosan-based granules. Combination of positively charged chitosan with negatively charged biomolecules, such as gelatin, alginic acid, and hyalouronic acid, has been tested to yield novel matrices with unique characteristics for controlled release of drugs

SODIUM TRI POLYPHOSPHATE

Industrially, sodium tripolyphosphate is prepared by heating a stoichiometric mixture of disodium phosphate,Na2HPO4 and monosodium phosphate, NaH2PO4 under carefully controlled conditions.

2Na2HPO4 + NaH2PO4 → Na5P3O10 + 2H2O

CHLORAMPHENICOL(DRUG)

Chloraphenicol (INN) is a bacteriostatic antimicrobial. It is considered prototypical broad-spectrum antibiotics alongside the tetracyclines.

The most serious adverse effect associated with chloramphenicol treatment is bone marrow toxicity, which may occur in two distinct forms: bone marrow suppression, which is a direct toxic effect of the drug and is usually reversible, and a plastic anemia which is idiosyncratic (rare, unpredictable, and unrelated to dose).

PURIFICATION OF CHITOSAN

Since medical application of animal derived biomaterials entails an inherent risk of protein conatamination which has in recent years aroused great awareness and anziety among the public, drug companies, and the industry regulators, it is of utmost importance to ensure that chitosan intended for medical applications is of the highest purity and free of protein contamination. The origin and purity of purchased chitosan material depends on its source, season, and conditions of the chemical deacetylation process, which may vary across different suppliers. Further purification process is crucial to ensure that the starting chitosan material for nanoparticle fabrication possesses the highest purity and integrity.

PREPERATION OF CHITOSAN NANO PARTICLE USNG IONIC GELATION METHOD

The preparation method involves ionic gelation, with a mixture of two aqueous phases, one which contains chitosan in acetic acid and other contains poly anion like sodium tri poly phosphate (TPP) in deionized water. TPP is nontoxic and has multivalent anions. It can form a gel by ionic interaction between positively charged amino groups of chitosan and negatively charged counterions of TPP. This interaction can be controlled by charge density of TPP and chitosan, which is dependent on the pH of the solution. The formation of particles was a result of the interaction between the negative groups of TPP and the positively charged amino groups of chitosan (ionic gelation). Formation of nanoparticles can be visualized by the formation of turbidity of the solution.


DRUG ENCAPSULTION

For the preparation of drug loaded nanoparticles, Chloramphenicol solution was added to Chitosan solution, prior to the formation of the nanoparticles. Three different drug concentrations were used, in order to study the effect of the drug loading on the morphological and physicochemical characteristics of nanoparticles.



EQUIPMENTS


1. High Performance Liquid Chromatography (HPLC)


2. Sonicator


3. UV spectrophotometer


4. Particle Size analyzer


HPLC (HIGH PERFOMANCE OR PRESSURE LIQUID CHROMATOGRAPHY)

An HPLC system is unique among laboratory instruments because it can be assembled using components from different manufacturers and suppliers. Although many systems are sold as complete packages, a far greater number are assembled by bench level scientists and customized for specific needs. Pumps, detectors, columns, column ovens, and data management systems can all be interchanged. For example, when a pump is removed for service or the result of a breakdown, a new one can be swapped in with little or no interruption of service. Most components have their own keypad as well as a computer connection. The operator thus has a choice of running them off a central computer or via the keypads. Compared with classical column chromatography, where the columns are gravity fed and a separation can take hours or even days, HPLC can offer analysis times of 5-30 minutes. Such times are comparable to that needed for GLC analyses.

HPLC is especially suited for the analysis of compounds not readily assayed by GLC. For example, thermally labile compounds can be analyzed by HPLC at ambient temperatures, and highly polar or nonvolatile compounds can be analyzed. Sample treatment is often minimal since aqueous solutions can be used in HPLC. Since its inception in the late 1960's, HPLC has made significant practical impact on the areas of pharmaceutical, clinical, forensic, environmental and industrial research and development analyses

COLOUMN


STEP-1

Preparing the System Column - If the column mounted on the system is not the correct the requisite assay, pump an appropriate storage liquid through the column and removing it. Cap the ends and return the column to the correct drawer. A column rack helps keep columns organized and makes them easy to find.

Check the column's direction of flow. This is identified by an arrow stamped on the side of the column or marked on the label. After installing the correct column, tighten the connections so they do not leak. Firm but gentle pressure should be applied to the connections. Excessive force is not required.

Mobile Phase - The mobile phase must be free of dissolved gasses so that no bubbles form inside the instrument during the run. Each system in the chromatography laboratory has a connection to a helium tank. Gently bubbling helium through the mobile phase for a few minutes will generally remove all dissolved gasses. Some laboratories use sonic baths to degas the mobile phases and many new HPLC systems are sold with degassing units built into the pump. Often the mobile phase is mixed in large quantities. When preparing a mobile phase with two or more components, be aware that large amounts of heat can be generated by the mixing process. Glass bottles may crack! The selection of mobile phases is based on the relative polarity of the analytes and the column packing. Often the addition of just a few milliliters of some ionic species will dramatically affect the analysis.


STEP-2

Prime the pump. Select a mobile phase by either placing the intake line in the bottle or using the keypad on the pump to select a specific bottle. Open the "prime" or the "purge" valve on the pump module. Place a beaker under the outlet. Activate the "prime" or "purge" function on the pump. If the pump does not have this function, turn up the flow and switch it on. Run the system for a few minutes. When the pump is properly primed, the system will deliver a smooth flow of mobile phase from the outlet, produce a steady sound with no burping or grinding, and the inlet line will be free of air bubbles. Turn off the pump and close the valve.


STEP-3

Set the detector wavelength. Turn on the detector power and allow the unit to warm up. Using the keypad or the control computer GUI, set the wavelength(s).


STEP-4

Start the mobile phase flow. Use the pump controller, to set the flow rate for the mobile phase. Restart the pump. Watch the system's pressure indicator or gauges to see that it does not exceed the maximum allowed for the various components. If pressures become too high, slow down the flow rate.

If pressure continues to rise, turn off the pump, and perform the following procedures:

· If the system has a guard column, replace it.

· The analytical column may need to be back flushed. Remove the column and reverse it so that mobile phase flows through it in the wrong direction. Catch the outflow in a beaker instead of allowing it to enter the detector.To condition the column, run mobile phase through it for a few minutes. Some operators recycle their mobile phase by running the waste line outlet back into their mobile phase reservoir. Care must be taken to avoid contaminating the reservoir with old samples, or impurities from the column. Monitor the detector output, when the signal is stable, begin running the samples.


STEP-5:

Inject the samples. Before injecting a sample, check the needle's tip. HPLC Needles have a smooth or blunt tip. Do not use a needle with a sharp tip or a tip with metal burrs. These will scratch the inner surfaces of the injector and cause it to leak. Remember this is a two-step process; syringe injection followed by turning the valve from "load" to "inject." Open the injection valves by turning them to "load." Insert needle into the plastic needle guide as far as it will go. Smoothly inject the appropriate amount of sample. After the syringe is completely empty, quickly and smoothly turn the valve to "inject." It is safe to leave the syringe in place. Start the data system recording. Behind each injector, there is a small coil of tubing. Known as the "sample loop," it holds the sample during the interval between the syringe injection and the start of the run. The sample loop can be identified by a small tag listing its volume. When the injector is set to the 'load" position this loop is isolated from the mobile phase flow. Turning the valve to "inject" diverts the mobile phase flow through the sample loop and sweeps the sample onto the column. For manual injections, as long as the sample volume is less than or equal to the loop volume, changing the loop is not necessary. More experienced with HPLC will teach how to determine a good injection volume. Column capacity, detector sensitivity, and column size, must all be balanced to obtain the best results.

SONICATOR


Sonication is the act of applying sound (usually ultrasound) energy to agitate particles in a sample, for various purposes. In the laboratory, it is usually applied using an ultrasonic bath or an ultrasonic probe, colloquially known as a sonicator. In a paper machine, an ultrasonic foil can distribute cellulose fibres more uniformly and strengthen the paper.

Sonication can be used to speed dissolution, by breaking intermolecular interactions. It is especially useful when it is not possible to stir the sample. It may also be used to provide the energy for certain chemical reactions to proceed. Sonication can be used to remove dissolved gases from liquids (degassing) by sonicating the liquid while it is under a vacuum. This is an alternative to the freeze-pump-thaw and sparging methods. In biological applications, sonication may be sufficient to disrupt or deactivate a biological material. For example, sonication is often used to disrupt cell membranes and release cellular contents. This process is called sonoporation.

Sonication is commonly used in nanotechnology for evenly dispersing nanoparticles in liquids. Sonication can also be used to initiate be crystallisation processes and even control polymorphic crystallisations. Sonication is used to intervene in anti-solvent precipitations (crystallisation) to aid mixing and isolate small crystals. Sonication is the mechanism used in ultrasonic cleaning loosening particles adhering to surfaces.

UV SPECTROSCOPY

Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) involves the spectroscopy of photons in the UV-visible region. This means it uses light in the visible and adjacent (near ultraviolet (UV) and near infrared (NIR)) ranges. The absorption in the visible ranges directly affects the color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions. This technique is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.

The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length. Thus, for a fixed path length, UV/VIS spectroscopy can be used to determine the concentration of the absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration cure.

A UV/Vis is spectrophotometer may be used as a detector for HPLC. The presence of an analyte gives a response which can be assumed to be proportional to the concentration. For accurate results, the instrument's response to the analyte in the unknown should be compared with the response to a standard; this is very similar to the use of calibration curves. The response (e.g., peak height) for a particular concentration is known as the response factor.

PARTICLE SIZE ANALYZER (BECKMANN COULTER DELSA NANO AT)

The delsa nano utilizes photon correlation spectroscopy and electrophoretic light scattering techniques to determine particle size and zeta potential of materials,

offering an excellent degree of accuracy, resolution and reproducibility.

FEATURES


  • Provides accurate size measurements in the range from 0.6nm to 7micro meter
  • Sample concentration ranging from 0.001% to 40%(sample dependent)
  • Analysis of aqueous and non-aqueous samples
  • It offers accurate and reliable particle size analysis.

The delsa nano AT is an optional accessory used with the delsa nano C to

titrate sample suspensions in a pH range from 1-13. It automatically controls the PH of solutions and performs titrations during Zeta potential or size analysis measurements.















EXPERIMENTAL METHODS




























EXPERIMENTAL PROCEDURES



PURIFICATION OF CHITOSAN

Purchased chitosan materials were subjected to a vigorous purification process, which involved mixing the chitosan flakes in 1M sodium hydroxide solution and continuously heated and stirred for 2h at 700C, and filtered. The recovered flakes were washed thoroughly and dried at 400C for 12h. These flakes were dissolved in 0.1M acetic acid and were filtered to remove residues of insoluble particles. The filtrate was made alkaline to a pH 8.0 to result purified chitosan in the form of white precipitates.

The precipitated chitosan was washed thoroughly with deionised water and the product was vacuum dried at room temperature for 24h. These dried samples were used for the preparation of chitosan-TPP nanoparticles.

PREPARATION OF CHITOSAN NANOPARTICLES (BLANK)

Preparation of Chitosan stock solution

1% chitosan solution was dissolved in glacial acetic acid with the help of sonicator.

General Method for the preparation of chitosan nanoparticles

Chitosan solution of different concentrations and molecular weight were prepared by dissolving purified chitosan with sonication in 1% (w/v) acetic acid solution until the wolution was transparent. Once dissolved, the chitosan solution was diluted with deionized water to produce different concentrations at 0.05%, 0.10%, 0.15%, 0.20%, and 0.25% (weight/volume). Sodiumtripolyphosphate (TPP) was dissolved in deionized water at concentration 0.7 mg/mL.( Table 1)


Preparation of 0.05% chitosan/TPP nanoparticle solution

1. 1.25 mL of chitosan stock solution was transferred to a beaker and made upto 25 mL using deionised water.

2. The chitosan solution was flush mixed with an equal volume of TPP solution (0.7%) using a magnetic stirrer for 2h.

3. The Chitosan nanoparticle solution was formed spontaneously via TPP initiated ionic gelation mechanism.

4. The particle size and polydispersity index of the chitosan nanoparticles were measured using particle size analyzer.

Preparation of 0.10% chitosan/TPP nanoparticle solution

1. 2.50 mL of chitosan stock solution was transferred to a beaker and made upto 25 mL using deionised water.

2. The chitosan solution was flush mixed with an equal volume of TPP solution (0.7%) using a magnetic stirrer for 2h.

3. The Chitosan nanoparticle solution was formed spontaneously via TPP initiated ionic gelation mechanism.

4. The particle size and polydispersity index of the chitosan nanoparticles were measured using particle size analyzer.

Preparation of 0.15% chitosan/TPP nanoparticle solution

1. 3.75 mL of chitosan stock solution was transferred to a beaker and made upto 25 mL using deionised water.

2. The chitosan solution was flush mixed with an equal volume of TPP solution (0.7%) using a magnetic stirrer for 2h.

3. The Chitosan nanoparticle solution was formed spontaneously via TPP initiated ionic gelation mechanism.

4. The particle size and polydispersity index of the chitosan nanoparticles were measured using particle size analyzer.

Preparation of 0.20% chitosan/TPP nanoparticle solution

1. 5.0 mL of chitosan stock solution was transferred to a beaker and made upto 25 mL using deionised water.

2. The chitosan solution was flush mixed with an equal volume of TPP solution (0.7%) using a magnetic stirrer for 2h.

3. The Chitosan nanoparticle solution was formed spontaneously via TPP initiated ionic gelation mechanism.

4. The particle size and polydispersity index of the chitosan nanoparticles were measured using particle size analyzer.

Preparation of 0.25% chitosan/TPP nanoparticle solution

1. 6.25 mL of chitosan stock solution was transferred to a beaker and made upto 25 mL using deionised water.

2. The chitosan solution was flush mixed with an equal volume of TPP solution (0.7%) using a magnetic stirrer for 2h.

3. The Chitosan nanoparticle solution was formed spontaneously via TPP initiated ionic gelation mechanism.

4. The particle size and polydispersity index of the chitosan nanoparticles were measured using particle size analyzer.


Table 1. Preparation of Chitosan nanoparticles



CS nanoparticles(%)



CS polymer from stock solution(mL)



CS polymer solution (mL)



Sodium tripolyphate solution (mL)



0.05


1.25


25


25



0.10


2.5


25


25



0.15


3.75


25


25



0.20


5.0


25


25



0.25


6.25


25


25



PREPARATION OF CHLORAMPHENICOL LOADED CHITOSAN NANOPARTICLES


Preparation of Chloramphenicol stock solution

200 mg of accurately weighed chlaramphenicol was made upto 1mL (0.2mg/µL) was prepared.


Preparation of drug loaded chitosan nanoparticles

For the preparation of drug loaded nanoparticles, chloramphenicol solution was added to the chitosan solution before the preparation of nanoparticles. Three different drug concentrations were used, i.e. (drug /polymer ratio) 1:2, 1:1 and 2:1 were used in order to study the morphology, particle size and Polydispersity index of drug loaded nanoparticles. (Table 2)



Table 2: Drug encapsulated Chitosan (CS) nanoparticles



Drug/Polymer ratio



Chloramphenicol from stock solution(µL)



CS polymer solution


(%)



2:1


1.25


0.02



1:1


2.5


0.02



1:2


3.75


0.02





































RESULTS AND DISCUSSIONS




























CHITOSAN PURIFICATION

Since medical applications of animal derived biomaterials entail an inherent risk of protein contamination which has recent years aroused great awareness and anxiety among the public, drug companies, and the industry regulators, it is of utmost importance to ensure that chitosan intended for medical applications is of the highest purity and free of protein contamination. It is therefore decided to further purify the purchased chitosan materials and examine whether there are changes in chemical as well as physical properties before and after purification. The origin and purity of purchased chitosan material depends on its source, season, and conditions of the chemical deacetylation process, which may vary across different suppliers. Further purification process is crucial to ensure that the starting chitosan material for nanoparticle fabrication possesses the highest purity and integrity.

In this work, purchased chitosan materials were subjected to vigorous purification process which involved mixing, heating and stirring the solid chitosan flakes in 1M NaOH solution. Chitosan was insoluble in caustic solution, and the recovered flakes were washed thoroughly and dried at 400C for 12 h. The NaOH treated chitosan flakes were dissolved in 0.1M acetic acid solution, and was filtered to remove insoluble residues. The purified chitosan flakes was reprecipited by adjusting pH to 8 with 1 M NaOH. The precipitated chitosan was washed thoroughly using deionized water, and product was vacuum-dried at room temperature for 24h. The dried samples were used for the preparation of chitosan-TPP nanoparticles.

PREPARATION OF CHITOSAN NANOPARTICLES (BLANK)

Preparation of chitosan nanoparticles

Chitosan's ability of quick gelling on contact with polyanions like sodiumtripolyphosphate (TPP) relies on the formation of inter-and intramolecular cross linkages mediated by these polyanions. Nanoparticles aare formed immediately upon mixing of TPP and chitosan solutions as molecular linkages were formed between TPP phosphates and chitosan amino groups. The ratio between chitosan and TPP is critical and controls the size and the size distribution of nanoparticles. The Size characteristics have been found to affect the biological performance of chitosan nanoparticles. For this reason, before drug encapsulation into chitosan nanoparticles, the effect of chitosan/TPP ratios is presented.

Chitosan solutions of different concentrations were prepared by dissolving purified chitosan with sonication in 1% acetic acid. Once didissolved, the chitosan solution was diluted with deionized water to produce chitosan solution of different concentrations at 0.05, 0.10, 0.15, 0.20, 0.25% (w/v). The particle size and polydispersity index of the synthesized nanoparticles were compared and found out optimum particle size and polydispersity index of the chitosan solution and this chitosan composition were used for chloramphenicol drug entrapment.

CHITOSAN NANOPARTICLE CHARACTERIZATION

Freshly formed nanoparticle suspensions with various chitosan percentages and TPP were analyzed by Beckman Zeta sizer and compared. Table 3. (Appemdix 1,2,3,4,5)



Table 3. Measurement of particle size and poly dispersity index of chitosan nanoparticles



Sl. No



Chitosan (%)



Particle size (nm)



PDI value



Time in hours


1


0.05


3151.5 nm


0.915


2h


2


0.10


2543.1 nm


0.759


2h


3


0.15


171.9 nm


0.229


2h


4


0.20


100.3 nm


0.308


2h


5


0.25


127.1 nm


0.266


2h



The chitosan solution (0.2%) was found to be optimum particle size of 100.3nm and polydispersity index (PDI value) of 0.308 were found to be suitable composition for drug entrapement. (Graph 1)


image011.gif


CHLORAMPHENICOL ENTRAPPED CHITOSAN NANOPARTICLES

For the preparation of drug loaded nanoparticles, chloramphenicol solution was added to chitosan solution with three different drug/polymer ratios. i.e. 1:2, 1:1, 2:1. Chloramphenicol loaded chitosan nanoparticles and their particle size and polydispersity index were shown. (Table 4)



Sl. No



Drug/polymer ratio



Particle size (nm)



PDI value



Time in h



1


1:2


106.6


0.296


2 h



2


1:1


110.5


0.234


2 h



3


2:1


133.9


0.295


2 h



Graph 2. Particle size and Polydispersity index of drug loaded nanoparticles (1:2)


Graph 2. Particle size and Polydispersity index of drug loaded nanoparticles (1:1)image013.gif



image015.gif

Graph 2. Particle size and Polydispersity index of drug loaded nanoparticles (2:1)


image017.gif







SUMMARY AND CONCLUSIONS


In this work, the potential of Chitosan/TPP nanoparticles as drug carriers for antimicrobial agents was investigated. Chloramphenicol, an antimicrobial agent was successfully formulated in the form of a chitosan nanoparticle system and the formulation was optimized by considering the concentration of chitosan, chitosan/TPP ratio and nanoparticle size distribution. Initially we chemically purified the commercially available chitosan polymer using NaOH and glacial acetic acid. Chitosan nanoparticles with a size of below 200 nm were obtained with chitosan concentrations of 0.15, 0.20, and 0.25 %. We found out optimum chitosan nanoparticles with 0.2% polymer with a polydispersity index of 0.308. This particular chitosan/TPP composition was successfully used for chloramphenicol drug entrapment.
We successfully entrapped chitosan nanoparticles with antibacterial drug chloramphenicol with different drug/polymer compositions, i.e. 1:2, 1:1, 2:1. All these compositions gave very good results with nanoparticles size in the range of 110-135 nm. Among these three compositions, the drug/polymer (1:1) ratio was found to be lowest size of 110.5nm and a polydispersity index of 0.234. This composition (drug/polymer; 1:1) was found to be successful candidate for the further studies for chloramphenicol drug delivery.

REFERENCES


[1] Lipinsky C. American Association of Pharmaceutical Sciences,

Meeting 1998

[2] Gombotz WR, Pettit DK. Biodegradable polymers for protein and peptide

drug delivery. Bioconjug Chem. 1995 Jul-Aug;6(4):332-51

[3] Sinha VR, Khosla L. Bioabsorbable polymers for implantable therapeutic

systems. Drug Dev Ind Pharm. 1998 Dec;24(12):1129-38

[4] Langer R. Drug delivery and targeting. Nature. 1998 Apr 30;392(6679

. Suppl):5-10

[5] Batycky RP, Hanes J, Langer R, Edwards DA. A theoretical model of erosion

. and macromolecular drug release from biodegrading microspheres. J Pharm

Sci. 1997 Dec;86(12):1464-77

[6] Peppas NA, Sahlin JJ. Hydrogels as mucoadhesive and bioadhesive materials:

a review. Biomaterials. 1996 Aug;17(16):1553-61.

[7] Heller J, Barr J, Ng SY, Shen HR, Schwach-Abdellaoui K, Emmahl S,

Rothen-Weinhold A, Gurny R. Poly(ortho esters) - their development and some

recent applications. Eur J Pharm Biopharm. 2000 Jul;50(1):121-8.

[8] Shieh L, Tamada J, Chen I, Pang J, Domb A, Langer R. Erosion of a new

family . of biodegradable polyanhydrides. J. Biomed. Mater. Res., 28, 1465.

[9] Patrick B. O'Donnell and James W. McGinity, Preparation of microspheres by

the solvent evaporation technique, Advanced Drug Delivery Reviews 28(1)

(1997) pp. 25

[10] Zhu G, Mallery SR, Schwendeman SP. Stabilization of proteins encapsulated in

injectable poly (lactide- co-glycolide). Nat Biotechnol 2000 Jan;18(1):52-7

[11] J.kreuter, nanoparticles,in: J.kreuter(Ed),colloidal Drug Drug delivery systems,

Marcel Dekker , NewYork ,1994 PP 219-342

[12] C.G .Knight (Ed), Liposomes from physical structure to Therapeutic

Applications ,Elsevier , Amsterdam,1981

[13] R.Langer ,biomaterials in drug delivery and tissue Engineering : one laboratory

experience ,Acc. Chem.Res .33(2000) 94-101


[14] R.P Lanza . R. Langer, W.L. Chick principles of tisseue Engineering in;

Academic press , Austin ,TX 1997 PP 405-427.


[15] C.A farrugia ,MJ. Grover ,Gelatin behavior in the Dilute aqueous solutions:

designing a nano particulate Formulations ,J .pharmacol . 51(1999) 643-649



[16] R. Fernandz -Urrusuno, P. calvo ,C. Remunan -Lopez , Of insulin using

chitosan nanoparticles , pharm .Res.16(1999) 1576-1581.


[17] I.C.Aynie,C.vauthier,E.Fattal,R.Muler(Eds),future Strategies of drug delivery

with particulate systems,Med .pharm scientific publisher ,Stuttgart,1998,pp 5-10


[18] D.F .Raney, biommertic transport,rotational drug Delivery. ,

Biochem.pharmacol.59(2000) 105-114


[19] L.C.Andersson .D.L. wise ,J.F .Howes, an injectable Sustained release fertility

control system.contraception 13 (1976) 375-384


[20] M.F .Zambaux ,F.Bonneaux,R.Gref ,P.Maincent ,E.Dellacherie ,MJ.Alonso

,P. Labrude ,C.Vigneron ,influence of experimental parameters on the

Characteristic Of poly(lactic acid )nanoparticles prepared by double

Emulsion.Rel.50(1998) 31-40


[21] T.Niwa ,H.Takeuchi , T.Hino ,Y.Kawashima ,Preparations of biodegradable

nanospheres of Water soluble and insoluble drugs with D,L- Lactide /glycolide

copolymer by a novel spontaneous Emulsification solvent diffusion method

and drug Release behaviour ,J.control . Rel. 25 (1993) 89-98


[22] H.murakami ,H.Yoshino ,M.Mizobe ,M.Kobayashi -lactide -co-glycolide) latex

for surface modifying Material by a double coacervation method,proced

Intern.symp.control .Rel.bioact .mater .23(1996) 361-362


[23] P.Calvo, C.Remunan Lopez, J.L Vila Jato M.J Alonso, novel hydrophilic chitosan

And chitosan /polyethylene oxide as nanoparticle carriers ,J.Appl. polym .sci.63

(1997) 125-132.


[24] P.Calvo, C.Remunan Lopez, J.L Vila Jato M.J Alonso, novel hydrophilic chitosan

And chitosan /ethylene Oxide-propylene Oxide block copolymer nano particles

As novel carriers for proteins and vaccines ,Pharm,Res 14 (1997) 1431-1436.





























































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