Review Article

WATER SOLUBLE CHITOSAN NANOPARTICLE FOR THE EFFECTIVE DELIVERY OF LIPOPHILIC DRUGS: A REVIEW

KUSHAGRI SINGH*, ABHA MISHRA

School of Biochemical Engineering, Indian Institute of Technology, Banaras Hindu University, Uttar Pradesh, India. Email: kushagri.singh@gmail.com

Received: 24 Nov 2011, Revised and Accepted: 05 Jan 2013

ABSTRACT

Several polymeric nanoparticulate systems have been prepared and characterized in recent years, based on both natural and synthetic polymers, each with its own advantages and drawbacks. Among the natural polymers, chitosan has been studied extensively for preparation of nanoparticles. Chitosan nanoparticles have been reported with different characteristics with respect to drug delivery. Chitosan nanoparticle have gained more attention as drug delivery carriers because of their better stability, low toxicity, simple and mild preparation method, and providing versatile routes of administration. This review here presents the importance of chitosan nanoparticle as water soluble matter for the delivery of some lipophilic drugs mainly with the propofol delivery, the application of explored chitosan nanoparticles and its preparation methods.

Keywords: Chitosan Nanoparticles, Drug delivery carriers, Polymers, Lipophilic drugs, Toxicity.

INTRODUCTION

Nanoparticle

In nanotechnology, a particle is defined as a small object that behaves as a whole unit with respect to its transport and properties. Particles are further classified according to diameter.[1] Coarse particles cover a range between 10,000 and 2,500 nanometers. Fine particles are sized between 2,500 and 100 nanometers. Ultrafine particles or nanoparticles are sized between 1 and 100 nanometers. The reason for this double name of the same object is that, during the 1970-80's, when the first thorough fundamental studies with "nanoparticles" were underway in the USA (by Granqvist and Buhrman) [2] and Japan, (within an ERATO Project)[3] they were called "ultrafine particles" (UFP). However, during the 1990s before the National Nanotechnology Initiative was launched in the USA, the new name, "nanoparticle" had become fashionable (see, for example the same senior author's paper 20 years later addressing the same issue, lognormal distribution of sizes [4]). Nanoparticles may or may not exhibit size-related properties that differ significantly from those observed in fine particles or bulk materials [5][6]. Although the size of most molecules would fit into the above outline, individual molecules are usually not referred to as nanoparticles.

Nanoparticles are of great scientific interest as they are effectively a bridge between bulk materials and atomic or molecular structures. A bulk material should have constant physical properties regardless of its size, but at the nano-scale size-dependent properties are often observed. Thus, the properties of materials change as their size approaches the nanoscale and as the percentage of atoms at the surface of a material becomes significant. For bulk materials larger than one micrometer (or micron), the percentage of atoms at the surface is insignificant in relation to the number of atoms in the bulk of the material. The interesting and sometimes unexpected properties of nanoparticles are therefore largely due to the large surface area of the material, which dominates the contributions made by the small bulk of the material.

Nanoparticles often possess unexpected optical properties as they are small enough to confine their electrons and produce quantum effects. For example gold nanoparticles appear deep red to black in solution.

Nanoparticles of usually yellow gold and grey silicon are red in color. Gold nanoparticles melt at much lower temperatures (~300 °C for 2.5 nm size) than the gold slabs (1064 °C);[7]And absorption of solar radiation in photovoltaic cells is much higher in materials composed of nanoparticles than it is in thin films of continuous sheets of material.

History of Nanoparticles

The history of nanoparticles is older than nanotechnology. The first known use of nanoparticles, made by Artisans in 9th century in Mesopotamia. The purpose of using them was to create a shiny effect on the pots surfaces. From middle Ages and Renaissance, pots usually had been glittered with distinct gold or copper colored metalics.

The method to create this glitter was, to make a metallic film which was applied to a transparent surface of a covering. As long as the oxidation and other weathering conditions continued on the film surface, the glitter stayed visible. The glitter which created the film contained silver and copper nanoparticles, dispersed homogeneously in the glassy matrix of the ceramic cover. [8]

Artisans created these nanoparticles by adding salts of copper and silver and oxides them withclay, vinegar and ochre, on the surface of the cover which has been made before. After creation, the object was put into a special oven and heated until it reaches about 600 °C in a decreasing atmosphere. As temperature increases in the oven, silver and copper ions slide to the outer layers of glitter. However ions were going back to metals when the atmosphere reduced, later the ions came together to form the nanoparticles which give the optical effects and color. It has been also discussed from different sources that, the first usage of nanoparticles found in early dynasty Chinese porcelain. [9]

However, Michael Faraday was the one who made the first scientific description about nanoparticles. He pointed out the optical proper ties of nanometascale metals in his classic1857 paper.

Usage of Nanoparticle in Medicine and Biology

In medicine and biology, nanoparticles are one of the basic things for applications.The main reason is their size and high ratio of surface area to mass. The most known applications are;

Fluorescent biological labels,

Drug and gene delivery

Bio detection of pathogens

Detection of proteins

Probing of DNA structure

Tissue engineering

Tumour destruction via heating (hyperthermia)

Separation and purification of biological molecules and cells

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MRI contrast enhancement

Phagokinetic studies

Toothpastes

Some Biodegradable Nanoparticles

Biodegradable nanoparticles have been used for site-specific delivery of drugs, vaccines and various other biomolecules. A few of the most extensively used biodegradable polymer matrices for preparation of nanoparticles are:

Poly-D-L- lactide-co-glycolide (PLGA)

Poly-D-L- lactide-co-glycolide (PLGA) is one of the most successfully used biodegradable polymers. It undergoes hydrolysis in the body to produce biodegradable metabolite monomers such as lactic acid and glycolic acid. Since lactic acid and glycolic acids are normally found in the body and participate in a number of physiological and biochemical pathways, there is very minimal systemic toxicity associated with the use of PLGA for the drug delivery or biomaterial applications. PLGA NPs have been mostly prepared by the emulsification-diffusion, the solvent evaporation and the nanoprecipitation methods [10]. PLGA nanoparticles have been used to develop protein and peptide based nanomedicines, nano-vaccines, and genes containing nanoparticles for in-vivodelivery systems [11,12].

Polylactic acid (PLA)

PLA is a biocompatible and biodegradable polymer which is broken down to monomeric units of lactic acid in the body. Lactic acid is a natural intermediate/by product of anaerobic respiration, which is converted into glucose by the liver during the Cori cycle. Glucose then is used as an energy source in the body. The use of PLA nanoparticles is therefore safe and devoid of any major toxicity. PLA nanoparticles have been mostly prepared by the solvent evaporation, solvent displacement, salting out and solvent diffusion methods [13]. The salting out procedure is based on the separation of a water- miscible solvent from aqueous solution by adding a salting out agent like magnesium chloride or calcium chloride. The main advantage of the salting out procedure is that it minimizes stress to protein encapsulants [10].

Poly-ε-caprolactone (PCL)

Poly-ε-caprolactone is degraded by hydrolysis of its ester linkages under the normal physiological conditions in the human body and has minimal or no toxicity. Therefore, PCL has grabbed the attention of researchers as a candidate of choice for use in drug delivery and long-term implantable devices. PCL's slower rate of degradation compared to polylactides has made it better candidate for making long-term implantable devices. PCL nanoparticles have been prepared mostly by nanoprecipitation, solvent displacement and solvent evaporation [10, 14, 15].

Chitosan

Chitosan is a modified natural carbohydrate polymer prepared by the partial N-deacetylation of the crustacean-derived natural biopolymer chitin. There are at least four methods reported for the preparation of chitosan nanoparticles. The four methods are ionotropic gelation, microemulsion, emulsification solvent diffusion and polyelectrolyte complex formation [10, 16, and 17].

Gelatin

Gelatin is extensively used in food and medical products and is a nontoxic alternative. Gelatin NPs are very efficient in delivery and controlled release of the drugs. They are nontoxic, biodegradable, bioactive and inexpensive. Gelatin is a poly-ampholyte consisting of both cationic and anionic groups along with a hydrophilic group.

It is known that the mechanical properties such as swelling behavior and thermal properties of gelatin NPs depend significantly on the degree of cross-linking between cationic and anionic groups. These properties of gelatin can be manipulated to prepare desired type of NPs from gelatin. Gelatin nanoparticles can be prepared by the desolvation/coacervation or emulsion methods [10, 18, and 19].

Poly-alkyl-cyano-acrylates (PAC)

The biodegradable as well as biocompatible poly-alkylcyanoacrylates are degraded by enzyme esterases found in the body. On degradation they produce some toxic products that may stimulate or damage the central nervous system. Thus this polymer is not authorized for application in humans. PAC nanoparticles are prepared mostly by emulsion polymerization, interfacial polymerization and nanoprecipitation [10].

Fig. 1: Schematic presentation of Chitosan production

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Chitosan nanoparticle: water soluble

Chitosan is a natural polymer obtained by deacetylation of chitin from crustacean shells such as crabs, shrimps and lobsters. . After cellulose chitin is the second most abundant polysaccharide in nature. It is biologically safe, non-toxic, biocompatible and biodegradable polysaccharide.

Chitosan nanoparticles have gained more attention as drug delivery carriers because of their better stability, low toxicity, simple and mild preparation method and providing versatile routes of administration. Their sub-micron size is also suitable for mucosal routes of administration i.e. oral, nasal and ocular mucosa which is non-invasive route. Chitosan nanoparticles showed to be a good adjuvant for vaccine.

Chitosan is also found in some microorganisms, yeast and fungi (Illum, 1998). The primary unit in the chitin polymer is 2-deoxy-2-(acetylamino) glucose. These units combined by ↓-(1, 4) glycosidic linkages, forming a long chain linear polymer. Although chitin is insoluble in most solvents, chitosan is soluble in most organic acidic solutions at pH less than 6.5 including formic, acetic, tartaric it is insoluble in phosphoric and sulfuric acid [20, 21].

Chitin is similar to cellulose both in chemical structure and in biological function as a structural polymer. The crystalline structure of chitin has been shown to be similar to cellulose in the arrangements of inter- and intrachain hydrogen bonding. Chitosan is made by alkaline N-deacetylation of chitin (as shown in fig.1.). The term chitosan does not refer to a uniquely defined compound; it merely refers to a family of copolymers with various fractions of acetylated units.

It consists of two types of monomers; chitin-monomers and chitosan-monomers. Chitin is a linear polysaccharide consisting of (1-4)-linked 2-acetamido-2-deoxy-b-D-glucopyranose. Chitosanis a linear polysaccharideconsisting of(1-4)-linked2-amino-2-deoxy-b-D glucopyranose. Commercial chitin and chitosan consists of both types of monomers. Chitosan is found in nature, to a lesser extent than chitin, in the cell walls of fungi. Chitin is believed to be the second most abundant biomaterial after cellulose.

The annual biosynthesis of chitin has been estimated to 109 to 1011 tons. Chitin is widely distributed in nature. Among several sources, the exoskeleton of crustaceans consists of 15% to 20 % chitin of dry weight. Chitin found in nature is a renewable bioresource [22, 23].

Fig. 2: Chemical structure of Chitin, Chitosan, Cellulose and Starch

Properties of Chitosan

For the past few decades, there has been a growing interest in the modification and application of chitosan in medical and health fields. Chitosan has been the material of choice for the preparation of nanoparticles in various applications due to its biodegradable and nontoxic properties. Chitosan is soluble in acidic condition and the free amino groups on its polymeric chains protonates and contributes to its positive charge [24]. Chitosan nanoparticles are formed spontaneously on the incorporation of polyanion such as tripolyphosphate (TPP) in chitosan solution under continuous stirring condition. These nanoparticles are then harvested and used for gene therapy and drug delivery applications [25,26]. However, due to its poor solubility at pH above 6.5, various chitosan derivatives with enhanced water solubility are introduced through chemical modification process, for example, N-trimethyl chitosan (TMC).

Chitosan in its free polymer form has been proved to have antifungal activity against Aspergillus niger, Alternaria alternata, Rhizopus oryzae, Phomopsis asparagi, and Rhizopus stolonifer [27,28]. From these findings, it could be concluded that antifungal activity of chitosan was influenced by its molecular weight, degree of substitution, concentration, types of fungus, and types of functional groups in chitosan derivatives chains [29-33]. Basically, the antifungal activity is contributed by the polycationic nature of chitosan. Therefore, chitosan exhibits natural antifungal activity without the need of any chemical modification [29].

There are three mechanisms proposed as the inhibition mode of chitosan. In the first mechanism, plasma membrane of fungi is the main target of chitosan. The positive charge of chitosan enables it to interact with negatively charged phospholipid components of fungi membrane. This will increase the permeability of membrane and causes the leakage of cellular contents, which subsequently leads to cell death [34,35]. For the second mechanism, chitosan acts as a chelating agent by binding to trace elements, causing the essential

nutrients unavailable for normal growth of fungi [36]. Lastly, the third mechanism proposed that chitosan could penetrate cell wall of fungi and bind to its DNA. This will inhibit the synthesis of mRNA and, thus, affect the production of essential proteins and enzymes. Currently, most of the research has focused on the antifungal activity of chitosan solution.

Despite of its superiority as a biomaterial, chitosan is not fully soluble in water and then soluble in acidic solution. Aqueous solubility of chitosan only in acidic solution is because of its rigid crystalline structure and the deacetylation which limits its application to bioactive agents such as gene delivery carriers, peptide carriers, and drug carriers. Water-soluble chitosan is easily soluble in neutral aqueous solution. Its advantage is ease of modification, useful as gene, peptide and lipophilic drug carrier. Therefore, water-soluble chitosan and functional property have been developing for pharmaceutical and new drug candidate.

Chitosan nanoparticles are good drug carriers because of their good biocompatibility and biodegradability, and can be readily modified. As a new drug delivery system, they have attracted increasing attention for their wide applications in, for example, loading protein drugs, gene drugs, and anticancer chemical drugs, and via various routes of administration including oral, nasal, intravenous, and ocular. This paper reviews published research on chitosan nanoparticles, including its preparation methods, characteristics, modification, in vivo metabolic processes, and applications.

Criteria for ideal polymeric carriers for nanoparticles & nanoparticle delivery systems:

Polymeric carriers

Easy to synthesize and characterize Inexpensive Biocompatible Biodegradable

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Non-immunogenic Non-toxic Water soluble

Nanoparticle delivery systems

Simple and inexpensive to manufacture and scale-up No heat, high shear forces or organic solvents involved in their

preparation process Reproducible and stable Applicable to a broad category of drugs; small molecules,

proteins and polynucleotides Ability to lyophilize Stable after administration Non-toxic

Preparation method for chitosan nanoparticle

Ionotropic gelation

Chitosan NP prepared by ionotropic gelation technique was first reported by Calvo et al.,(1997b) and has been widely examined and developed (Janes et al., 2001; Pan et al., 2002). The mechanism of chitosan NP formation is based on electrostatic interaction between amine group of chitosan and negatively charge group of polyanion such as tripolyphosphate (Bodmeier et al., 1989; Xu and Du, 2003). This technique offers a simple and mild preparation method in the aqueous environment. First, chitosan can be dissolved in acetic acid in the absence or presence of stabilizing agent, such as poloxamer, which can be added in the chitosan solution before or after the addition of polyanion. Polyanion or anionic polymers was then added and nanoparticles were spontaneously formed under mechanical stirring at room temperature. The size and surface charge of particles can be modified by varying the ratio of chitosan and stabilizer (Calvo et al., 1997a).

Microemulsion method

Chitosan NP prepared by microemulsion technique was first developed by Maitra et al. This technique is based on formation of chitosan NP in the aqueous core of reverse micellar droplets and subsequently cross-linked through glutaraldehyde. In this method, a surfactant was dissolved in N-hexane. Then, chitosan in acetic solution and glutaraldehyde were added to surfactant/hexane mixture under continuous stirring at room temperature. Nanoparticles were formed in the presence of surfactant. The system was stirred overnight to complete the cross- linking process,

which the free amine group of chitosan conjugates with glutaraldehyde. The organic solvent is then removed by evaporation under low pressure. The yields obtained were the cross-linked chitosan NP and excess surfactant. The excess surfactant was then removed by precipitate with CaCl2 and then the precipitant was removed by centrifugation. The final nanoparticles suspension was dialyzed before lyophilyzation. This technique offers a narrow size distribution of less than 100 nm and the particle size can be controlled by varying the amount of glutaraldehyde that alters the degree of cross-linking. Nevertheless, some disadvantages exist such as the use of organic solvent, time-consuming preparation process, and complexity in the washing step.

Emulsification solvent diffusion method

El-Shabouri reported chitosan NP prepared by emulsion solvent diffusion method, (El-Shabouri, 2002) which originally developed by Niwa et al. employing PLGA (Niwa et al., 1993). This method is based on the partial miscibility of an organic solvent with water. An o/w emulsion is obtained upon injection an organic phase into chitosan solution containing a stabilizing agent (i.e. poloxamer) under mechanical stirring, follow by high pressure homogenization. The emulsion is then diluted with a large amount of water to vercome organic solvent miscibility in water. Polymer precipitation occurs as a result of the diffusion of organic solvent into water, leading to the formation of nanoparticles. This method is suitable for hydrophobic drug and showed high percentage of drug entrapment. The major drawbacks of this method include harsh processing conditions (e.g., the use of organic solvents) and the high shear forces used during nanoparticle preparation.

Polyelectrolyte complex (PEC)

Polyelectrolyte complex or self assemble polyelectrolyte is a term to describe complexes formed by self-assembly of the cationic charged polymer and plasmid DNA. Mechanism of PEC formation involves charge neutralization between cationic polymer and DNA leading to a fall in hydrophilicity as the polyelectrolyte component self assembly. Several cationic polymers (i.e. gelatin, polyethylenimine) also possess this property. Generally, this technique offers simple and mild preparation method without harsh conditions involved. The nanoparticles spontaneously formed after addition of DNA solution into chitosan dissolved in acetic acid solution, under mechanical stirring at or under room temperature (Erbacher et al., 1998). The complexes size can be varied from 50 nm to 700nm.

Fig. 3: Different preparation methods for Chitosan nanoparticles

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Applications of Chitosan Nanoparticles

As antibacterial agents, gene delivery vectors and carriers for protein release and drugs

Used as a potential adjuvant for vaccines such as influenza, hepatitis B and piglet paratyphoid vaccine

Used as a novel nasal delivery system for vaccines. These nanoparticles improve antigen uptake by mucosal lymphoid tissues and induce strong immune responses against antigens.

Chitosan has also been proved to prevent infection in wounds and quicken the wound-healing process by enhancing the growth of skin cells.

Chitosan nanoparticles can be used for preservative purposes while packaging foods and in dentistry to eliminate caries.

It can also be used as an additive in antimicrobial textiles for producing clothes for healthcare and other professionals.

Chitosan nanoparticles show effective antimicrobial activity against Staphylococcus saprophyticus and Escherichia coli.

These materials can also be used as a wound-healing material for the prevention of opportunistic infection and for enabling wound healing.

The nanoparticles have also been proven to show skin regenerative properties when materials were tested on skin cell fibroblasts and keratinocytes in the laboratory, paving the way to anti-aging skin care products.( Chitosan for biomedical applications – University of Iowa)

Use of Chitosan nanoparticle for the delivery of lipophilic drugs

Chitosan nanoparticles can be used as a carrier molecule for the delivery of some Lipophilic drugs such as propofol (lipophilic in nature) and some antifungal drugs like Amphotericin B (a highly lipophilic, polyene antifungal substance).

With some modifications in the chitosan molecule i.e linkage of this with functional derivatives leads to its effective and stable binding with the lipophilic drugs. For example, use of propofol often results in pain on injection, which is sometimes very distressing to patients. In analysis of 6,264 patients in 56 reports, 70% of all control patients reported some degree of pain or discomfort on injection with propofol [37]. In 1998, Tan and Onsiong [38] suggested that a wise choice would be to use a combination of techniques, such as alfentanil pretreatment, mixing lidocaine with the propofol and injecting into a large vein with no carrier fluid to decrease the incidence and severity of propofol injection pain.

McCrirrick and Hunter [39] found that administering propofol at 4℃, significantly reduces the incidence of injection pain. Prior injection of cold saline reduces the incidence of pain and discomfort significantly compared with unmodified propofol and is similar to that after cold propofol and propofol with lidocaine [40]. However, the incidence of pain with cold propofol and lidocaine is not statistically different from room temperature propofol and lidocaine in children [41]. Cho et al. [42] assessed the effectiveness of cold propofol and pretreatment with remifentanil in minimizing pain associated with the injection of propofol and investigated whether a combination of cold propofol and remifentanil produced additional analgesic efficacy. They found that a combination treatment with 0.5 µg/kg remifentanil pretreatment and cold propofol significantly reduces the incidence and severity of propofol injection pain compared with each treatment used alone.

The aqueous phase, which contains free propofol, is known to be associated with the intensity of pain at the injection site. Yamakage et al. [43] recommended the use of propofol medium-chain triglyceride (MCT)/long-chain triglyceride (LCT) for reducing pain on injection, because the concentrations of free propofol are significantly smaller by 30-45% than those in propofol LCT. Mallick et al. [44] reported that premixing 40 mg of lidocaine to Lipuro® propofol (a propofol formulated as an emulsion of 50% MCT and 50% LCT) almost abolishes the pain on injection, making induction of anesthesia less painful compared to Lipuro® propofol alone or Diprivan® propofol (a propofol formulated as an emulsion in a solution of 10% soybean oil containing only LCT) with lidocaine.

A lipid-free microemulsion propofol (Aquafol®) was developed to avoid the risk of lipid solvent-related adverse drug reactions, and the efficacy and safety are not different from those of Diprivan® [45]. Microemulsion propofol produces more frequent and severe pain on injection than lipid emulsion propofol, a difference that may be attributable to the seven-fold higher concentration of aqueous free propofol [46]. Kim et al. [47] investigated the effect of a lidocaine mixture on microemulsion propofol injection pain and sought to determine the optimal dose of lidocaine that reduced pain on injection of propofolidocaine mixture. They used 20, 30, and 40 mg lidocaine, and the incidence of pain was 80%, 65%, and 50%, respectively, compared with 97.5% in a control group.

Within this lidocaine dose range, increasing the lidocaine dose significantly lessened the pain during injection of microemulsion propofol. However, it is difficult to conclude that 40 mg of lidocaine is an optimal dose because a 50% incidence of injection pain is too high.

Although several single or combination methods with lipid emulsion propofol nearly abolish pain on injection, further studies to search for an effective and safe method to reduce pain on injection, especially with microemulsion propofol, are needed until a better and safer propofol is available for clinical use in the near future.

Therefore there is a need to search for another tool or delivery system that is more efficient and leads to minimum pain on intravenous injection of drugs as well as targeted delivery. In addition, chitosan NP less than 100 nm in size have been developed which showed to be RES evading and circulate in the blood for considerable amount of time. Delivery of anti-infectives such as antibacterial, antiviral, antifungal and antiparasitic drugs, is another common use of nanoparticles [48]. The low therapeutic index of antifungal drugs, short half-life of antivirals and the limited ability of antibiotics to penetrate infected cells in intracellular compartments make them ideal candidates for nanoparticle delivery. Thus, it has been suggested that nanoparticles should improve the therapeutic efficacy while decreasing the toxic side effects of these drugs. In theory, chitosan NP are very attractive carrier system for these drugs as they offer many advantages such as hydrophilic surface particles, nano-size of less than 100 nm [49,50].

Chitosan nanoparticles are now being modified for sustained/ controlled release and targeting. . These systems have great utility in controlled release and targeting studies of almost all class of bioactive molecules as discussed in this review. While great progress has been achieved in the application of chitosan nanoparticles as drug carriers, some problems remain to be resolved urgently. Majority of studies carried out so far are only in in vitro conditions. More in vivo studies need to be carried out. Chemical modifications of chitosan are important to get the desired physicochemical properties such as solubility, hydrophilicity, etc. For example, chitosan has poor solubility and unmodified chitosan nanoparticles can encapsulate only some hydrophilic drugs. However, studies toward optimization of process parameters and scale up from the laboratory to pilot plant and then, to production level are yet to be undertaken Although chitosan can be modified easily to encapsulate hydrophobic drugs.

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