INTRODUCTION:

Over the years we have witnessed unprecedented growth in the area of nanoscience and nanotechnology. There is increasing hope that this technology will significantly improve the diagnosis and treatment of diseases. Nanotechnology mainly focuses on the design, synthesis, characterization, and application of materials at the nanoscale. Application of nanotechnology to medicine led to the emergence of a new area called “nanomedicine”. Nanoparticles are particles of size in the range of 1-100 µm that are used as carriers for drugs. They are basically polymers. The drug is either entrapped within the polymer matrix or covalently bound to it. Because of their small size, nanoparticles can easily interact with bio molecules on the cell surface or inside cells. This small size also enables them to penetrate tissues such as tumours in depth with a high level of specificity improving the targeted delivery of drug.

One of the major applications of nanotechnology is the use of nanosensors. For example, Nanoparticle (NP)-based assay helps in the early detection of diseases such as cancer and cardiovascular diseases, saving millions of lives by the prevention and early treatment of these diseases. The nanodrug and gene delivery system is another technique which has potential in various applications such as anti-tumour therapy by targeted delivery of therapeutic agents to the tumour cell mass. Cancer treatment is a great challenge for drug delivery. The unique properties of cancer make cancer treatment a great challenge for drug delivery. Hence the development of a multifunctional drug delivery system is required so that it can specifically target the tumour cells and leave the healthy cells from damage¹. In the case of chemotherapy, cancer drugs reach the tumour tissue with poor specificity. However, Nanoparticle mediated drug delivery provides a more specific mode of treatment where it is able to localize the drug directly to its target. Nanoscale carriers allow the continuous and controlled release of therapeutic drugs into the body and maintain the drug dosage level within a desired concentration. Hence the patients need not suffer possible toxicity of high dosage drugs.

TYPES OF NANO PARTICLES USED IN DRUG DELIVERY SYSTEM:

Polymer-Based: The drug is physically entrapped inside or covalently bound to a polymer. The compounds are in the form of capsules, micelles or dendrimers.

Polymer-drug conjugates:

Polymeric drug conjugates, due to their passive tumour targeting properties make effective delivery vehicles. Their prolonged exposure within the blood stream allows them to reach the tumour target without being eliminated by the body defence mechanism (which includes opsonisation by macrophages, excretion etc.) Polymers used as nanoparticles can be either natural or synthetic. Various natural polymers have been used as materials for delivering DNA, proteins, and even drugs.

The major synthetic polymers used are N-(2-hydroxypropyl)- methacrylamide copolymer (HPMA), polystyrene-maleic anhydride copolymer, polyethylene glycol (PEG), and poly-L-glutamic acid (PGA). These are basically agents which impart hydrophilicity to the drug. PGA was the first biodegradable polymer to be used in conjugation with the Nanoparticle. Several chemotherapeutics that are used widely in the clinic have been tested in conjugation with PGA and had the abilities which other drugs lacked. PK1, which is a conjugate of HPMA with doxorubicin, is the synthetic polymer-drug conjugate which is can be a possible anti cancer agent. It was also proved that subcutaneous injection with PLG nanoparticles loaded with RMP, INH, and PZA into mice injected with M. tuberculosis demonstrated a better chemotherapeutic efficacy².

Polymeric micelles:

Polymeric micelles assemble to form a nanosized core/shell structure in aqueous media. The hydrophobic core region is a reservoir for drugs and the hydrophilic shell region stabilizes the hydrophobic core and renders the polymer water-soluble, making the particle suitable for intravenous drug administration³. The drug can be loaded into a polymeric micelle in two ways: physical encapsulation or chemical covalent attachment. The polymeric micelles are formed by association of amphiphilic block copolymers into the nanoshell. Multifunctional polymeric micelles containing targeting ligands are being developed⁴.

Fig.1. Multifunctional nanoparticle drug delvery system³

Dendrimers:

Dendrimers are precisely defined, synthetic nanomaterials that are approximately 5-10 nanometres in diameter composed of multiple highly branched monomers that emerge radially from the central core. The dendrimer surface contains many different sites to which drugs may be attached and also attachment sites for materials such as polyethylene glycol (PEG) which can be used to modify the way the dendrimer interacts with the body. PEG can be attached to the dendrimer to 'disguise' it and prevent the body's defence mechanisms in detecting it, thus slowing the process of breakdown. This allows the drug to circulate in the body for a long time, incresing the chances of the drug to reach the target site. Dendrimers have specific size, multivalency, water solubility, and available internal cavity which make them suitable for drug delivery. Polyamidoamine dendrimer is a dendrimer which is used as a scaffold is conjugated with cisplatin. This is used as a drug delivery vehicle⁵. As the surface of the dendrimers can be modified easily, they can be conjugated with various molecules such as ligands, drugs, etc which gives a multifunctional drug delivery system. Research was conducted where folate (which targets the high affinity folate receptor found on malignant cells), along with an indicator (luorescein) and an anti cancer drug (such as paclitaxel) was attached to a single dendrimer⁶. Both in vitro and in vivo experiments showed that this system delivered the drug specifically to the folate receptor containing cells while simultaneously labelling these cells for detection.

LIPID-BASED DRUG CARRIERS:

Liposomes: Liposomes are microscopic spheres with a core surrounded by an outer shell of lipids arranged in the form of a bilayer. This fatty layer protects and confines the enclosed drug until the liposome binds to the outer membrane of target cells. Nanoparticles made with liposomes are the simplest form and have the advantage of a long circulation time, reduced toxicity and increased uptake into tumor cells. Several types of anti-cancer drugs have been included in this lipid-based system using different types of preparation methods. The next generation of liposomal drugs are immunoliposomes, which selectively deliver the drug to the desired site⁷.

Viral nanoparticles: Viral nanoparticles are emptied virus cells that can carry drugs directly to cancer cells to kill them. They have been engineered from plant viruses, insect viruses, and animal viruses including cowpea mosaic virus, cowpea mosaic virus, canine parvovirus have been developed for drug delivery⁸. Targeting molecules can be displayed on the viral capsid surface. Several ligands like transferrin and folic acid are conjugated with viruses for specific tumor targeting. Protein cages are used which serve as a nanocontainer for the drug. These have dual nature- specific targeting and encapsulation of doxorubicin drug⁹. One of the major benefits of using viral nanoparticles in a drug delivery system is that molecules can easily be attached to the nanoparticles’ surfaces to enable the virus cells to bond only to the cancer cells, instead of the surrounding cells thus protecting the surrounding healthy cells.

Carbon nanotubes: Carbon nanotubes are carbon cylinders composed of benzene rings that have been applied in biology as sensors for detecting DNA and protein, as diagnostic devices for the distinguishing between different proteins from serum samples, and as carriers to deliver vaccines. Carbon nanotubes can be chemically modified to make them functionalised where they can be attached to peptides, nucleic acids and other molecules. Carbon nanotubes are linked with anticancer drugs (methotrexate) along with a fluorescent agent (FITC). In an in vitro study, drugs attached to carbon nanotubes were shown to be more effectively enter cells when compared to the free drug alone and they were also found to have antifungal activity. Multiple covalent functionalizations on the tips of the carbon nanotubes allow them to carry several molecules at once which help in cancer treatment¹⁰.

Gold nanoparticles have also been utilized as nontoxic drug carriers for drug delivery. PEG-coated colloidal gold nanoparticles incorporated with a TNF (tumor necrosis factor) receptor can delay tumor growth in mice when they are given proper dosage¹¹.

Fig.2. Types of nanocarriers²⁴

TARGETED DELIVERY OF NANOPARTICLES:

Ideally, for anticancer drugs to be effective in cancer treatment, they should first, after administration, be able to reach the desired tumor tissues through the penetration of barriers (such as blood-brain barrier etc) in the body with minimal loss of their activity in the blood stream. After reaching the tumor tissue, drugs should be able to selectively kill the tumor cells without affecting the surrounding normal cells. Along with this, the toxicity must also be reduced. Hence nanoparticles have this potential for effective cancer treatment.

CHARACTERISTICS OF NANOPARTICLES:

To effectively deliver the drug to the targeted tumor tissue, nanoparticles must have the ability to remain in the bloodstream for a long time without being eliminated. Hence the size and surface characteristics have to be adjusted to prolong their time in the bloodstream.

Size:

Nanoparticles are large enough so that they do not leak into blood capillaries and also small enough to escape capture (opsonisation) by macrophages. The size of the sinusoid in the spleen is 150-200 nm and the size of the gap junction between endothelial cells of the leaky tumour vasculature is 100-600 nm. Hence, the size of nanoparticles should be up to 100 nm to reach tumour tissues by passing these structures¹².

Surface:

The surface of nanoparticles is also an important factor which determines their life span during circulation. Nanoparticles should have a hydrophilic surface to escape macrophage capture. This is done either by coating the surface of nanoparticles with a hydrophilic polymer (PEG- protects them from opsonisation by macrophages) or from block copolymers with hydrophilic and hydrophobic domains¹³.

PASSIVE TARGETING:

Passive targeting involves preparing the drug-carrier complex such that it avoids elimination by body defence mechanisms. As the fast growing cancer tissue requires rapid vascularisation. It has a leaky structure which gives rise to permeability. This is a major advantage in passive targeting as the drug can easily enter the cancer cells. Some drugs can be given in an inactive form, which when exposed to the tumour environment, can be switched on to become highly active. Nanoparticles that satisfy the size and surface characteristics requirements have the ability to stay in the blood circulation for a long time and hence have a greater chance of entering the tumour tissue.

Leaky vasculature:

Fast-growing cancer cells require the recruitment of new vessels (neovascularisation) to supply them with oxygen and nutrients. The imbalance in growth factors and MMPs (matrix metalloproteinase) makes tumour cells disorganized and dilated with pores showing enlarged gap junctions between endothelial cells and lymphatic drainage. This is called the enhanced permeability and retention effect (EPR), by which the nanoparticles can selectively accumulate in the tumour interstitial space¹⁴.

Tumor environment:

The microenvironment surrounding tumour cells is different from that of normal cells. Fast-growing cancer cells show a high metabolic rate. As the oxygen and nutrient supply is not enough to maintain this rate, tumour cells use glycolysis to obtain extra energy, which results in an acidic environment. Liposome is generally designed to be stable at a pH 7.4. However, on release of the drug into the target cell (with an acidic environment), they are degraded. Also, cancer cells express and release unique enzymes such as matrix metalloproteinase (MMPs), which influence their movement¹⁵.

ACTIVE TARGETING:

A non specific mode of entry is by active targeting. Passive targeting has specificity limitations. To overcome these, a targeting ligand or antibody is included in polymer-drug conjugates¹⁶. Direct conjugation of an antibody to a drug did not work due to the limited number of drug molecules that can be loaded on the antibody.

Carbohydrate-Directed Targeting: Carbohydrates expressed on the tumour cell surface affect interactions of the tumour cells with the extracellular matrix during metastasis. For example: Lectin-carbohydrate. These interactions can be regulated by tumour cell carbohydrates and their binding proteins (lectins)¹⁷

Antigen expression: Cell-surface antigens are expressed only on tumour cells and not on normal cells which make them suitable tumour targets. However, these antigens should not be released into the blood circulation¹⁸.

Local application:

This allows the drug to be given directly to the tumour tissue, avoiding circulation. Various methods have been carried out to improve the delivery of the anticancer agents, such as intraperitoneal administration etc. The only problem is that exposure to high concentrations of the anticancer agents is required, which is not possible. Intratumoral administration is another method in local drug application.

INTERNALIZATION OF POLYMER-DRUG CONJUGATES:

Internalization occurs by receptor-mediated endocytosis. For example, when a drug along with folate-targeted conjugate binds to folate receptor on the cell surface, by receptor mediated endocytosis, the plasma membrane invaginates and envelopes the complex of the receptor and ligand to form an endosome which is transferred to target organelles. At low pH, lysozymes within the endosome get activated leading to release of the drug into the cytoplasm which is then sent to the target organelle. The folate receptor that is then released returns to the cell membrane so it is available for new folate-targeted conjugates for binding.

Fig.3. Internalization of nanoparticles by receptor-mediated endocytosis. Tumour-specific ligands on the nanoparticles bind to cell-surface receptors, which triggers internalization of the complex into the cell through endosome. The drug is then released into the cytoplasm²⁴

Biodegradable nanoparticles:

Biocompatible nanoparticles have been researched for controlled-release and sustainable drug delivery. Researchers have found that PLGA (poly D, L-lactide co-glycolide) has a sustained drug release²⁰. PLGA nanoparticles are generally made by emulsion solvent evaporation or by solvent displacement techniques. Drugs encapsulated inside the nanoparticles can be released at a sustained rate through diffusion and by the degradation of the nanoparticles.

Nanoparticles for Gene Delivery:

Viral Vectors: Viral vectors with diameters of 100 nm or less, are the most efficient and stable transgene vectors into the cell and hence suitable for vaccine and gene therapy. Viruses are able to stably insert the cell genome into the host and provide a long-term transgene expression in cells. Use of viral vectors as gene delivery systems has been researched. Retrovirus, lentivirus, adenovirus and adeno-associated virus gene transfers are used in cancer therapy due to their ability to deliver the genome into the nucleus where the transgene can be transcribed²¹. Adenoviruses are the most widely used vector models for human gene therapy.

Nanoparticle self assembly: The positive charge of nanoparticles will be a problem in vivo as a high cationic charge density can lead to the aggregation of nanoparticles in organs such as the spleen and liver. To overcome these problems, scientists have developed a nucleic acid-lipid self assembly which can provide small stable nanoparticles for targeted gene delivery in tumours. An antibody such as the F5-cys-mal-PEG (2000)- DSPE can be conjugated to the nanoparticles for targeted gene delivery. Antibody-conjugated self-assembled nanoparticles can selectively deliver plasmid DNA.

siRNA Delivery using nanoparticles: Gene silencing is done by double-stranded small interfering RNA (siRNA) which has strong therapeutic potential for treatment of genetic defects. But siRNA therapy is hindered due to their poor stability in fluids. Research has shown that intraperitoneal administration of targeted nanoparticles can be used to specifically deliver siRNA and maintain a sufficient local concentration of siRNA²².

CONCLUSIONS:

Nanotechnology is a rapidly expanding area of science which could lead to the development of multifunctional applications that can recognize tumours and deliver the drugs to the target tissue with high efficacy. Photosensitizes used in photodynamic therapy, in which light is used to generate reactive oxygen within tumours causing their destruction, have also been entrapped in targeted nanoparticles. Further steps are being taken to entrap a light-generating system (such as luciferin-luciferase pair) to trigger production of light after internalization of nanoparticles by the targeted cell²³. Such approaches would be very useful in treating tumours deep within the body.

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Received on 25.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 295-299