Nanoparticulate drug delivery systems for cancer chemotherapy

Abstract

Nanoparticles (NPs) are, in general, colloidal particles, less than 1000 nm, that can be used for better drug delivery and prepared either by encapsulating the drug within a vesicle and or by dispersing the drug molecules within a matrix. Nanoparticulate drug delivery systems have been extensively studied in recent years for spatial and temporal delivery, especially in tumour and brain targeting. NPs have great promise for better drug delivery as found in both pharmaceutical and clinical research. As a drug carrier, NPs have significant advantages like better bioavailability, systemic stability, high drug loading, long blood circulation time and selective distribution in the organs/tissues with longer half life. The selective targeting of NPs can be achieved by the enhanced permeability and retention effect (EPR-effect), attaching specific ligands, or by making selective distribution due to change of the physiological conditions of specific systems like nature, pH, temperature, etc. It has been observed that drug-loaded NPs can have selective distribution to organs/tissues using different types of and proportions of polymers. The current aim of researchers is to prepare NPs that are long-lived with and that demonstrate the appropriate selective distribution for better therapy and thus improved clinical outcomes. Nanoparticulate drug delivery systems have the potential to deliver a drug to the target site with specificity and to maintain the desired concentration at the site for the intended time without untoward effects. In this review article, the methods for the preparation of NPs, their characterization, biodistribution, and pharmacokinetic characteristics are discussed.

Keywords::

• Polymeric/lipid nanoparticles
• opsonization
• intracellular trafficking
• cytotoxicity
• brain targeting

Introduction

Development in nanotechnology and nanoscience has opened up potential applications in medical science. Interest and investment in research and application of nanotechnology have been increased extensively from both private and government sector for various applications in life sciences areas. Nanoscience and nanotechnology have invaded pharmacy and opened up new opportunities in drug delivery for improved therapy. Although nanotechnology has plenty of promise, it brings new challenges in safety and ethical considerations (Aston et al. 2005, Couvreur and Vauthier 2006). Formulation pharmacists are continuously working on novel drug delivery systems, as conventional dosage forms have various shortcomings. Though new generation drugs have entered the market with potent action, most of them have found to have drawbacks, such as poor water solubility, low gastro-intestinal permeability, high first pass metabolism, poor stability, non-selective distribution and others (Grau et al. 2000, Gill et al. 2007). Thus, in recent years, novel particulate drug delivery systems (PDDS), either polymer or lipid based, have been evaluated as agents to overcome one or more of these drawbacks. Nanoparticulate systems (NS) have been found to show promising results in overcoming these problems with the potential to improve therapeutic outcomes. Biological membranes are a major obstacle for conventional drug delivery systems, especially in cancer chemotherapy. However, drug NPs or NS have shown better membrane permeability leading to improved therapy, not only in cancer but in the treatment of other diseases (Osborne et al. 1991, Kemken et al. 1992, Trotta 1999).

NPs are well known for improving the dissolution rate and bioavailability of drugs, but they can also be used to target drugs, including peptide based drugs, to specific sites or organs and to control drug release (Amidi et al. 2006, Dillen et al. 2006). Delivery of nanoparticulate insulin by the nasal route was found to produce enhanced absorption of insulin (Yang et al. 2008). It is expected that within 10 years, 50% of all drug delivery and design approaches may be shifted to Nanoparticulate Drug Delivery Systems (NPDDS) technology (Gill et al. 2007). The ultimate goal of NPDDS is to provide clinically useful formulations for better therapy and patient quality of life. Chemotherapy is one area where NS has shown great promise, as conventional dosage forms produced severe toxic effects due to nonselective distribution of cytotoxic drugs. NS have shown selective targeting to tumour sites. The available literature shows that the NPDDS are not only useful in chemotherapy, but can be employed to deliver other class of drugs to their site of action through different routes of administration (Osborne et al. 1991, Gao et al. 2007, Gill et al. 2007). NPs can also provide a better alternative for ophthalmic administration, because they can be retained at the application site and prolong drug release to the eye (Osborne et al. 1991). The NPs based tuberculosis treatment reduces the dosing frequency and increases the patient compliances (Gill et al. 2007). It is also possible to deliver central nervous system active drugs selectively to brain by NPDDS (Gao et al. 2007).

Challenges in cancer treatment

Cancer is a major cause of death globally. Although localized primary solid tumours can be removed successfully by surgical process, the treatment of spreading tumours and tumour metastases require extensive chemotherapy (Stewart and Kleihues 2003). Anticancer drugs are mostly associated with side-effects, in particular nephrotoxicity, neurotoxicity, ototoxicity etc. Strident efforts have been made to reduce side effects by means of biological and pharmacological strategies. Cancer chemotherapy is a delicate balance between response and toxicity, while under-dosing undermines effective therapy, over-dosing results in excessive toxicity. As the cytotoxic action is not selective for malignant cells, but also affects normal cells, non selective distribution of drug in the body will inevitably cause significant side or toxic effect. It is a great challenge to formulation pharmacists to balance the two by designing novel delivery systems for selective distribution.

NPDDS promise to overcome the drawbacks of chemotherapeutic treatment by modifying the biodistribution and pharmacokinetics of the drug. Particulate and colloidal systems like NPs and liposomes have been studied as a physical approach to alter and improve the biodistribution, pharmacokinetic and pharmacodynamic properties of various types of drug molecules. They have been used to protect the drug entity in the systemic circulation, to restrict access of the drug to chosen sites and to provide drug release at a controlled or sustained rate to the site of action. NPDDS can be selective and effective in localization of pharmacologically active ingredient at pre-selected targets at therapeutic concentration, restricting its access to non-target areas, thus maximizing the effectiveness of the drug (Cummings et al. 1994).

Tumour-selective targeted drug delivery systems have become one of the most important advances of the 21st century leading to improvements in the cure rate of advanced stage cancers (Couvreur et al. 1979, Miklos 2005). Numerous investigations have shown that both the tissue and cell distribution profile of anticancer drugs can be controlled or modified by their entrapment in NS (Snehalatha et al. 2008a). Maeda et al. (2001, 2009) reported styrene-maleic acid copolymer-conjugated neocarzinostatin (SMANCS) as a tumour-targeted drug delivery system, producing an enhanced permeability and retention (EPR) effect on solid tumours. The EPR-effect appears to be a universal phenomenon in solid tumours which warrants the development of new polymeric drug delivery systems or nanomedicines. The polymer-based new drug delivery systems, also termed as polymer therapeutics, which target tumours are a novel strategy for improving drug performance exploiting the pathophysiological uniqueness of tumours (Langer 1998, Duncan 2006).

NPDDS in chemotherapy

In general, NPs are polymeric or lipidic sub micron-sized colloidal pharmaceuticals, in which, drugs may be embedded in a polymeric/lipidic nanomatrix, or dissolved, encapsulated, entrapped in the polymeric/lipidic matrix or adsorbed on their surface. Specifically for anticancer drugs, NPs modify the biodistribution and pharmacokinetic character of drugs and hence reduces the systemic side-effects accrued due to non-specific delivery of anticancer drugs to normal cells. The NPDDS can increase the half-life (t_1/2) of drugs up to 10 times in tissue and blood, leading to less frequent administrations of dose with increased patient compliance and quality of life (Maeda et al. 2009). The half life of L-asparaginase, when used for the treatment of lymphocytic leukemia, is short (t_1/2 between 8 and 30 h) and requires daily administration for four weeks. By contrast, treatment with Poly ethylene glycol (PEG)-L-asparaginase conjugate (Oncaspar^®, t_1/2 approx. 14 days) requires only one infusion every two weeks (Graham 2003, Harris and Chess 2003). Different NPs, like lipid nanoparticles, micelles, nanospheres, nanocapsules, niosomes, nanoemulsion, and nanosuspension have been formulated and their applications have also been evaluated (Letchford and Burt 2007, Snehalatha et al. 2008b, Xiong et al. 2008).

The cancer chemotherapy began in the early 1940s and efforts are continuing to provide better drugs. Several new drugs are at different phases of clinical trials. Presently, efforts are being made to enhance the therapeutic effectiveness by modified delivery systems or formulations. NPDDS has received tremendous attention from researchers for its potential to target cancer cells. NPDDS can overcome the drawbacks of the conventional cancer chemotherapy by providing a continuous and controlled supply of drug selectively to the cancer cells without the development of drug resistance. Treatment with NPDDS allows the oncologist to administer the cytotoxic drugs in lower doses because of its selective delivery, with the opportunity to further enhance the dose if required (Skipper et al. 1964).

NPDDS can be designed and developed selectively depending on the intended route of administration as nanoemulsion or nanosuspension. The oncologists may prefer the simple drug NPs to target organs like liver, lungs, kidney and spleen, because they have the inherent ability to target reticulocyte endothelial system organs (Owens and Peppas 2006). In some cases, ligand molecules may be coated on the surface of NPs for the active docking to particular macrophage receptors in cancer cells, triggering phagocytosis. If the target tissue is not part of the reticulocyte endothelial system, then the initial opsonization process has to be inhibited. This can be done by coating the NPs with substances like PEG, or polysorbates-80, that prevent capture by opsonin proteins. This results in an increase in the circulation time of the particles in the blood stream and gives time to the NPs to identify the target area and increase the therapeutic efficacy and decrease systemic toxicity (Owens and Peppas 2006). It has been found that Poly(lactic-co-glycolic acid) (PLGA) NPs coated with biodegradable Polylactic acid (PLA)-PEG copolymer, are less susceptible to hepatic uptake than uncoated PLGA NPs (Stolnik et al. 1994). The di-block and tri-block copolymer (e.g., PEG-PLGA, methoxy-PEG-PLA, PLA-PEG-PLA NPs) NPs evade the reticulocyte endothelial system and have extended circulation times. There is a significant decrease in drug distribution to liver and spleen for the same copolymer NPs. Thus, NPDDS avoiding the reticulocyte endothelial system result in increased circulation half-life and enhanced therapeutic efficacy of anticancer drugs (Gref et al. 1997, Otsuka et al. 2003).

The desired pharmacological action of anticancer drugs is achieved not only by targeting the cell surface, but further by reaching specific cell organelles (endosomes, lysosomes, mitochondria, endoplasmic reticulum and nucleus). Hence, the NPDDS provide an improved option for chemotherapy. The NPDDS can provide better cancer treatment through dual action of providing site-specific drug delivery at cancer cells with control release, overcoming the major impediment of multidrug resistances (MDR). A wide variety of structurally unrelated compounds such as doxorubicin, daunourubicin, vincristine, vinblasine, EP, teniposide, paclitaxel, docetaxel, have shown acquired resistance during therapy (Links and Brown 1999, Shabbits et al. 2001). The NPDDS delivers drugs directly to tumour cells, in vivo, thereby avoiding the resistance problem at vascular, interstitial and cellular levels (Links and Brown 1999, Shabbits et al. 2001).

Clinical trials of neoplastic gene therapy have showed a selective targeting of cationic liposomes to tumour vascular endothelial cells (Dass 2002). Liposomes target very specifically to lung and liver when given by the intravenous (i.v) route. Addition of helper lipid like dioleoylphosphatidylethanolamine to liposomes facilitates the rate of cellular uptake by increasing the fusion of liposomes with cell membranes, and this property is extensively used to target anticancer drugs to neoplastic cells (Dass 2002).

Types of nanoparticles for drug delivery

NS for drug delivery can be classified into solid lipid nanoparticles (SLN), nanospheres, nanocapsules, liposomes and polymersomes and micelles, based on the methods of preparation. If solid nanoparticles are prepared by incorporating drug in lipid, they are called SLN, whereas nanospheres are matrix systems in which the drug is embedded throughout the solid polymers. Nanocapsules are vesicular systems in which the drug alone or drug, confined to an aqueous or oily drops, is surrounded by a single polymeric membrane. Nanocapsules are usually used to encapsulate lipophilic drugs. If the polymeric membrane is of multiple layers then it is called a polymersome. Nanosized liposomal systems have also been studied extensively with encouraging results, where various phospholipids (saturated and unsaturated) have been used. Polymeric NPs are typically prepared from biodegradable polymers to avoid accumulation of the polymer matrix following repeat dosing (Discher and Eisenberg 2002, Hassan et al. 2009).

Method of preparation of nanoparticles

In general, NPs are prepared by processes such as, solvent evaporation, solvent diffusion/displacement, reverse salting-out and droplet gelation, emulsification and polymerization, dispersion polymerization, interfacial condensation polymerization and interfacial complexation. The SLN is prepared by emulsification and solvent evaporation techniques. The simplest method of NP preparation is nanoprecipitation. PLA, PLGA and poly-ϵ-caprolactone (PCL) NPs can be prepared by emulsification and solvent evaporation process, with average particle size of 250 nm and above (Mu and Feng 2003, Mainardes and Evangelista 2005, Letchford and Burt 2007). Amphiphilic block copolymer NPs can be prepared by using this method with polymers such as PEG -PCL, PEG-PLGA, PEG-PLA and PEG-PACA. Though, this method is used for preparing lipophilic drug loaded NPs extensively; even hydrophilic drugs can also be encapsulated by this process. Solvents used for this method are methylene chloride, chloroform, ethyl acetate etc. Another method used for preparation of NPs is emulsification and solvent diffusion/displacement process. As the name implies diffusion of organic solvent in to the aqueous phase is the key step in emulsion solvent displacement method. The organic solvent should be partially soluble in water and is selected from a wide range of solvents such as benzyl alcohol, 2-butanone, methyl acetate, propylene carbonate, ethyl acetate, isopropyl acetate, methyl acetate, methyl ethyl ketone and isovaleric acid. This method can be used to prepare NPs of size around 150 nm for poorly water soluble drugs. Nanocapsules can be prepared by the same method just by adding a small amount of oil into the organic phase (Desgouilles et al. 2003, Sinjan et al. 2005, Vauthier and Bouchemal 2008).

In emulsification-reverse salting-out method, water miscible acetone is emulsified with aqueous phase containing high concentration of salts or sucrose. Magnesium chloride, calcium chloride and magnesium acetate salts are preferably used, because of their high salting out effect in aqueous phase (Allenmann et al. 1993). When, acetone is added to aqueous phase, the miscibility of water to acetone decreases, due to the presence of the large quantity of electrolyte which holds water molecules, resulting in emulsion droplet formation. The precipitation of polymer from the emulsion is induced by adding excess water, which results in a sudden drop of the salt or sucrose concentration in the continuous phase of the emulsion and hence inducing the organic solvent to migrate out of the emulsion droplets; this process is called reverse salting out. The gelling property of the polymers is used to prepare the NPs from the emulsion, the polymers used being agarose, alginate and pectin. This method of preparation is called emulsion droplet gelation. NPs prepared by in situ polymerization use monomer (alkylcyanoacrylates) to produce polymerization of alkylcyanoacrylate while forming NPs. The NPs are synthesized by an in situ spontaneous polymerization reaction. This polymer can encapsulate both lipophilic and hydrophilic drugs and is used to prepare nanospheres and nanocapsules containing an aqueous or oily core. The NPs prepared by in situ method follow anionic polymerization reaction mechanism and the reaction is spontaneously initiated by hydroxyl groups of water or any nucleophilic groups (Nguyen et al. 2006, Vauthier et al. 2007).

Nanoprecipitation is the simplest, fastest and most reproducible and economical method of preparing NPs especially for lipophilic drugs. In this method the polymer, drug and lipophilic surfactant are dissolved in a semi-polar water miscible solvent such as acetone, ethanol, dimethylformamide, dimethylsulfoxide. The basic requirement of the selection of solvent is miscibility with aqueous phase and the aqueous phase has to be a non solvent of the polymer. Once the organic solvent is added to aqueous phase, NPs form instantaneously because of the rapid diffusion of water miscible solvent in to the aqueous phase. Because of the instantaneous process, the nanoprecipitation method provides very fine particles (about 200 nm) with a narrow size distribution. This method can also be used to encapsulate hydrophilic drugs (Bilati et al. 2005, Zili et al. 2005, Derakhshandeh et al. 2007).

The SLNs are composed of physiological lipid, dispersed in water or a slotion of aqueous surfactant. The lipid matrices used in the preparation of SLNs are Acidan N 12, B-CD21C6, Cetyl palmitate, Dynasan 114 (Trimyristin), Dynasan 116 (Tripalmitin), Glyceral behanate, Glycerol monostearate, Monostearin, Stearic acid, Tristearin, Tricaprin, Witepsol E 85 and Precirol ATO 5. The proposed advantages of these polymeric NPs is increased drug stability, high drug payload (both hydrophilic and lipophilic drugs), no biotoxicity of the carrier, ease of scale up and sterilization. The nanoemulsion is prepared mainly by the spontaneous emulsification or titration method. In this method the nanoemulsion is prepared by blending oil, water, surfactant and cosurfactant in the appropriate proportions with mild agitation. Nanoemulsions are thermodynamically stable preparations with a particle size of less than 100 nm. Nanoemulsions are mainly used to improve the transdermal and dermal delivery of drugs (Bilati et al. 2005, Snehalatha et al. 2008a, 2008b).

Critical steps in nanoparticles preparation

Prepared NPs require concentrating to reduce the volume of administration; this reduces the systemic over exposure of excipients. The concentration process plays an important role in final particle size and its aggregation in the final formulations. There are several methods for concentrating NPs, such as centrifugation, lyophilization, evaporation and dialysis. Concentrating to the desired volume by evaporation is usually performed by rotory evaporation. Based on the solvent and polymer, the temperature and vacuum are optimized. During this process the polymer layer of the NPs is also solidified. Using lyophilization, the NPs are transformed into a free flowing dry powder, and this approach also helps to avoid microbiological degradation, premature polymer degradation, physico-chemical instability and loss of drug activity. To avoid damage to NPs during the freezing and lyophilization process, special excipients, for cryoprotectant (to overcome freezing stress) and lyoprotectant (to overcome drying stress) actions, are added to the nanosuspension before freezing (Saez et al. 2000, Tsinontides et al. 2004, Mainardes and Evangelista 2005). Some of the very frequently used cryo or lyoprotectant are glucose, sucrose, lactose, mannitol, sorbitol, Poly(vinyl pyrrolidone), glycerol, Poly(vinyl alcohol) and dextran. Other methods of concentrating NPs are centrifugation and ultracentrifugation. Normal centrifugation, performed at low g forces, can remove aggregates and large particles from the polymeric nanoparticle suspension, but this method will not guarantee removal of all particles above the nanometer size in the formulation. Ultracentrifugation can sediment particles with slightly higher density than water. Ultracentrifugation is performed at 100,000–110,000 g for 30–45 min to form pellet of NPs. These pellets can be reconstituted to the desired volume of dispersion medium. NPs concentration by dialysis can be performed using different cellulose membranes with various molecular weight cut-offs. In the simple dialysis method, the concentration of the suspension is performed against a polymer solution. This causes an osmotic stress producing a displacement of water from the nanosuspension towards the counter-dialysis solution. The dialysis method results showed that the amount of water removed can be controlled and the process is reproducible (Vauthier et al. 2008).

Characterization of nanoparticles

Even though the Federal Drug Administration has not released any specific guidelines for NPDDS, the following characterizations are usual for nanomedicine formulations; particle size and its distribution, surface morphology (surface charge/zeta potential) and surface properties (poly-dispersity index), drug loading/drug entrapment efficiency and in vitro drug release evaluation. The mean size, distribution and polydispersity index of the NPs are identified using laser light scattering or photon correlation spectroscopy. Particles morphology is examined by microscopy techniques, such as, scanning electron microscopy, transmission electron microscopy and atomic force microscopy. Photon correlation spectroscopy, X-ray photon correlation spectroscopy, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy and electrophoretic mobility can be used to examine surface properties of NPs, such as, zeta potential and surface hydrophobicity. The drug release from NPs is mainly governed by diffusion and biodegradation processes. The methods normally used to study the in vitro release of NPs include the use of side-by-side diffusion cells with an artificial or biological membrane, the dialysis bag diffusion technique, the reverse dialysis sac technique, ultra-centrifugation, ultra-filtration and/or centrifugal ultra-filtration.

Particle size

Particle size plays a major role in determining the in vivo fates of NPs. Researchers have demonstrated that opsonization and subsequent recognition and phagocytosis by macrophages of mononuclear phagocytic system (MPS) are strongly correlated with NP size. Particles less than 200 nm in diameter have slow rate of clearance and thus have extended circulation times compared to those with larger diameters (Owens and Peppas 2006, Champion et al. 2007). Opsonin cannot bind to the smaller particles which have a high radius of curvature and hence, the circulation time of smaller NPs is very high and its clearance rate is low. These smaller NPs are referred to as long circulating NPs. Gaur and Sahoo (2000) observed that the hydrophilic poly (vinyl pyrrolidone) NPs of 35 nm diameter show less than 1% uptake by the spleen and liver, and even after 8 h of injection 5–10% of NPs remain circulating in the blood stream. So, particles with smaller diameters can circulate for longer and have improved ability to target their site of action. If NPs are administered through the i.v route, smaller particles, less than 20–30 nm, are eliminated by renal excretion and larger particles, greater than 200 nm, will be removed by opsoninization leading to localization in the liver, kidney, lung, spleen and to a lesser extent in bone marrow. Hence the ideal size for targeted drug delivery is between 70 and 200 nm (Moghimi et al. 2001). An improved pharmacokinetic, profile, area under the curve (AUC) of etoposide (EP) loaded PLGA NPs (105.1 ± 2.38 nm) was observed over PCL NPs, (257.2 ± 3.96 nm) (Snehalatha et al. 2008a, 2008b).

Surface properties

Surface properties of NPDDS are critical in determining their drug delivery potential as these properties govern the overall in vivo performance of the drug delivery system. These properties also modulate the in vitro performance such as stability, drug entrapment efficiency and drug release kinetics. The specific surface area, surface charge and surface hydrophobicity are very important as these govern the physical-chemical and electrostatic interactions with biological membranes and the overall biodistribution of drug-loaded NPs (Lamprecht et al. 2001, Hasani et al. 2009).

Entrapment/encapsulation efficiency (EE) and drug loading

Ideally nanomedicines should have high drug loading and entrapment efficiency to reduce the volume of nanosuspension required to be administered. Numerous different terms have been used to represent the drug content and the efficiency of the preparation method. These include entrapment or encapsulation efficiency, nanoparticles recovery, drug loading, process efficiency, loading capacity, association efficiency and drug incorporation efficiency.

The entrapment of drug into the NPs can be determined directly or indirectly. In the indirect method the prepared NPs are recovered or separated by ultracentrifugation (100,000 g, 25 min) and the supernatant is analyzed for the free drug content. In the direct method the incorporated drug in NPs is determined by dissolving the NPs in a suitable solvent, followed by analysis of drug content (Snehalatha et al. 2008b).

The entrapment or encapsulation efficiency can be calculated by:

To determine the amount of drug in the sediment, specified volume of the prepared nanoparticle suspension was taken and centrifuged at 45,000 g for three cycles for 10 min/cycle. The sediment NPs was taken and processed to determine the drug content in NPs. It can also be determined by indirect method:

The drug loading in NPs can be determined by taking required amount NPs and dissolving with suitable solvent, filtered and the drug content was determined by:

Fate of NPDDS in biosystems

On the basis of the rate of removal of NPs from the circulation they can be classified as conventional NPs and long circulating NPs. NPs are considered as non-self by the immune system and are removed by opsonization. NPs thus identified by the immune system undergo rapid phagocytosis by the macrophages of the mononuclear phagocytic system. Mononuclear phagocytic system is one of the most important biological barriers to targeted drug delivery systems, because the delivery system is removed from the body before reaching the target and performing their expected therapeutic action (Brigger et al. 2002, Owens and Peppas 2006). NPs are also targeted by the RES macrophages for phagocytosis, which decreases the circulation time of the particles and hence reduces the duration of the therapeutic effect. There are no methods available to completely and effectively block the opsonization of particles, hence researchers have tried to develop a nanoparticle formulation which is invisible to MPS and/or which can slow down the opsonization process. This led to development of Stealth^TM nanoparticles, which are characterized by a prolonged half-life in the blood compartment (Harris and Chess 2003). The surface characteristics of NPs, as well as size, play a vital role in the fate of NPs in biological systems. It has been observed that smaller size NPs (< 200 nm) and or hydrophilic surface, obtained by adsorption of surfactant/hydrophilic molecules (PEG) or by using block/branched copolymer, which are amphiphilic block copolymers nature, can reduce the opsonization reaction and subsequent clearance by macrophages (Owens and Peppas 2006). The amphiphilic block copolymer is of interest to researchers to formulate NPs because of its hydrophilic surface nature. It has also been observed that this nanoparticle modulates the activity of the efflux pump, P-glycoprotein (Letchford and Burt 2007). Polymers which are normally used to shield the NPs are polysaccharides, polyacrylamide, poly (vinyl alcohol), poly (N-vinyl-2-pyrrolidone), PEG and PEG-containing copolymers such as poloxamers, poloxamines, and polysorbates. A review of the literature indicates that PEG and PEG-containing copolymers is most effective and very commonly used polymer to prepare long-lived circulating NPs (Brigger et al. 2002, Otsuka et al. 2003).

Targeting of nanoparticles: Mechanisms

Intracellular trafficking

NPs enter the body through the skin, lungs or intestinal tract, and distributed to target organs and tissues. Targeting of the therapeutic agents to organelles within the cell is referred to as intracellular trafficking or targeting. The therapeutic trafficking is initiated by different modes of phagocytosis including, fluid-phase pinocytosis and receptor mediated endocytosis, clathrin-coated pits, caveoli and non-endocytic pathways may be involved. The mechanism of intracellular trafficking is tissue dependent. Panyam et al. (2002) showed that the NP internalization in vascular smooth muscle cells is through fluid phase pinocytosis and in part through clathrin-coated pits. The therapeutic efficacy of prepared NPs depends on size, surface charge and hydrophobicity. The concentration of drug in the intracellular environment also depends on the release characteristic, which can be altered by varying the composition of the formulation and preparation method (Panyam et al. 2002, Panyam and Labhasetwar 2003a, 2003b).

Once the particulate drug delivery systems reach the target cell, it is transported to primary endosomes or early endosomes by different mechanism of endocytosis (phagocytosis for uptake of large particles and pinocytosis for uptake of fluid and solutes), and then to sorting endosomes. A portion of the NPs are then recycled back to the cell exterior through recycling endosomes and the remaining NPs are transported to the secondary endosomes where they fuse with lysosomes. During this transport, NPs change their surfaces charge from anionic to cationic, a process referred to as surface charge reversal (Breunig et al. 2008). This mechanism is responsible for the escape of NPs from endo-lysosomes to the cytoplasm, where the particles become anionic. The transmission electron microscopy study of Panyam and co-workers showed that, cells exposed to NPs interact with vesicular membranes inside the cells, due to cationization in the vesicles. This leads to localized destabilization of the membrane and the escape of NPs into the cytoplasmic compartment. Thus by modifying the surface charge one can target the NPs to different intracellular targets, such as, endo-lysosomes, cytoplasm, nucleus and mitochondria (Panyam et al. 2002).

Enhanced permeability and retention effect – passive targeting

Enhanced permeability and retention is a phenomenon of enhanced extravasations of macromolecules from tumour blood vessels, and their retention in tumour tissue, which is not observed in normal vasculature. In cancer tissues, in addition to leaky vascular blood vessels and a discontinuous endothelial cell lining, the matrix metalloproteinases (collagenases) effect disintegration of the matrix tissue surrounding blood vessels, which in addition, permits the entry of macromolecules and particles that have limited access to normal tissue. Even lymphatic drainage is impaired in cancer tissues which helps macromolecules larger than 40 kDa and particles between 250 and 300 nm to be retained in tumours (Maeda et al. 2001, 2009). The EPR-effect is applicable to any biocompatible macromolecular compound above 40 kDa, or even the size of bacteria. When SMANCS/lipiodol is administered via a tumour feeding artery, the drug concentration in the tumour compared to blood (T/B ratio) can be as high as 2000 which is due to EPR-effect. In a clinical setting SMANCS/lipiodial given via the hepatic artery accumulates selectively in hepatocellular carcinomas (Maeda et al. 2001). A similar result was observed for doxorubicin liposomal drug delivery (Daemen et al. 1995).

There are many endogenous substances which facilitates the extravasations (EPR-effect) including, vascular endothelial growth factor (VEGF/VPF), bradykinin, 3-hydroxypropyl bradykininin, nitric oxide, peroxynitrite, prostaglandins, matrix metalloproteinases, kinin, kallikrein system and other cytokines. Bradykinin is known to induce vascular permeability and play an important role in the EPR-effect. Thus, the site of infection/inflammation where an excess of bradykinin is generated also exhibits the EPR-effect. The difference between the infection-induced EPR-effect and that of cancer cells effect is the duration of the retention period. In cancer cells the enhanced extravasation of macromolecules is long lived. In support of the EPR-effect, Hashizume et al. (2000) reported structural abnormalities in the endothelium of tumour blood vessels in mouse mammary carcinomas, whose intercellular openings were found to be as large as 4.7 μm (mean size 1.7 μm) in diameter. In addition, Yuan et al. (1995) examined the pore size of tumour vessels in LS-147 human colon adenocarcinoma implanted in SCID mouse and found that they could be as large as 0.4 μm in diameter. These findings gave way to a new concept for drug delivery. This abnormal vascular architecture and overproduction of permeability enhancers, with impairment of lymphatic drainage result in the preferential extravasations and retention of high molecular weight macromolecules in tumours. By using this pathophysiological uniqueness of tumours (EPR-effect) tumour-targeted drug delivery systems were developed and successfully used to reduce the morbidity and mortality rate due to cancers in animal models or human trials (Matsumoto et al. 1984). By EPR-effect, the drug concentration in tumour can be increased typically to 10–30 times of the blood concentration, i.e., the T/B ratio can be made very high for NPDDS which results in effective chemotherapy. This results in selective delivering of drug to tumour tissue which reduces the systemic side-effects and improves patient quality of life. The EPR-effect has forced researchers to consider tumour-selective targeted drug delivery system to treat different cancers at different stages (Maeda et al. 2009).

Oral absorption

Absorption of NPs in oral administration has been found to be enhanced, particularly in inflamed tissue or ulcerated tissue where there is excess production of mucus. Residence time of NPs is enhanced in the gastrointestinal tract due to better adhesion to mucus, leading to enhanced absorption. Another mechanism for the enhanced deposition of NPs around ulcerated tissue is through surface charge interactions. It has been reported that the ulcerated tissues contain high concentrations of positively charged proteins. Hence targeting to ulcerated tissue may be achieved with negatively charged NPs. Many researchers showed that the bioadhesion and deposition of particulate drug delivery to gastrointestinal tract depends on the particle size and the surface properties. Hasani et al. (2009) studied the effect of particle size on the gastrointestinal deposition of NPs after oral administration to mice. The fluorescence spectrophotoscopy study revealed that the gastrointestinal depositition were decreased with increase in average particle size of NPs (50 nm > 200 nm > 750 nm). In addition, in intestinal inflammatory disease, epithelium permeability is modified and hence the NPs can easily be transported. It has also been shown that reduction in particle size from 10–0.1 μm increases the uptake of particles in experimentally-induced colitis in rats when compared to control animals. It has been shown that NPs deposition is very high in ulcerated area of stomach due to Helicobacter pylori (Hasani et al. 2009).

The paracellular and the transcellular route are the two pathways used by PDDS to cross the intestinal epithelium. The paracellular pathway is the tight junction of the intestinal epithelium which is made up of proteins (including, occludins and claudins) and imparts a negative charge overall at the junction (Miller and Johnston 2005). In addition, it has been reported that there are some aqueous pores in the junction for diffusion of hydrophilic substance (Florence and Hussain 2001). The multiple layers of tight junctional strands present a rate-limiting barrier for the free diffusion of hydrophilic molecules. It has been reported that paracellular permeability enhancers, such as surfactants, fatty acids, salicylates, chelating agents, selectively and reversibly open the tight junctions of the intestinal epithelial layer for the diffusion of hydrophilic molecules for enhanced absorption. It has been shown that the anionic surfactant sodium dodecyl sulfate increases the permeability of tight junctions by shortening microvilli, actin disbandment and structural separation of tight junctions with reversible damage to the apical cell membrane. The space for paracellular transport is very small, (200–2000 cm²), in comparison to the total surface area of intestinal epithelium (2 × 10⁶ cm²). Even this space is sufficient for absorption of certain therapeutic substances whose pharmacological action can be in pico mole/picogram or nanomole/nanogram (Breunig et al. 2008).

In oral drug delivery, transcytosis is the main mechanism for transcellular transport of NPs. The intestinal epithelium is composed of a number of cell types. In particular, the enterocytes and follicle associated epithelium-M cells of Peyer's patches have been investigated for their role in transcellular transport. The M cells play a role in immuno surveillance transferring material from the lumen of the gut to the lymphoid tissue. The transport of this material involves a number of mechanisms including, endocytosis, clathrin coated pits, fluid phase endocytosis and phagocytosis. This action is exploited to deliver particulate drug delivery systems through the oral route. Hence, to target intestinal cells one can enhance the delivery to M cells or the enterocytes by surface physico-chemical modification or coupling targeting molecules to NPs. A number of studies have shown that the surface properties of NPs are significant for the uptake of carrier by the intestinal epithelial cells. The surface modification of NPs by the addition of hydrophilic substances such as, polyethylene glycol, chitosan, or polyoxyethylen-sorbitan-monooleate, enhances their transport across the intestinal mucosa by interaction with intestinal epithelium (Miller and Johnston 2005). The coupling of ligand to NPs is another way of targeting carrier to intestinal cells; lectins and peptidic ligands (arginine-glycine-aspartate) have been evaluated (Thanou et al. 2001). The transport of NPs by transcytosis can be altered by physico-chemical properties of particles (particle size, zeta potential, surface hydrophobicity/polymer properties and presence of ligand), physiology of the GI tract, and mucoadhesion. The main mechanisms for the absorption of SLN after oral administration are enhancement of dissolution or solubilization by biliary and pancreatic secretions, gallbladder contraction, prolongation of gastric residence time, stimulation of lymphatic transport affecting intestinal permeability and reduced metabolism and efflux activity (Miller and Johnston 2005).

Mechanism of drug transport to brain

The blood-brain barrier is a continuous layer of brain capillary endothelial cells, connected by highly developed tight junctions. These cells express numerous efflux transporters and metabolizing enzymes. Drug-loaded NPs cross the blood brain barrier into the central nervous system by an endocytotic process. The brain capillary endothelial cells are the major place for endocytotic mechanism. An antinociceptive effect was observed when apolipoprotein E was adsorbed to dalargin-loaded poly-(butyl cyanoacrylate) and to loperamide-loaded albumin NPs (Kreuter 2001). Tosi et al. (2007) showed that the biocompatible polyester poly (D, L-lactide-co-glycolide) derivatized with the peptide g7 would enhance permeation through the blood brain barrier. Gao et al. (2007) reported that the PEG-PLA NPs coupled with wheat germ agglutinin were effective in transporting vasoactive intestinal peptide into the brain following intranasal administration. Kytorphin (dipeptide), tubocurarine, NMDA-receptor antagonists (MRZ 2/576 and MRZ 2/596) and doxorubicin are some drugs transported across the blood brain barrier using the polysorbates 80-coated Poly(butyl cyanoacrylate) NPs. These studies confirm that when the NPs, coated or attached to the apolipoprotein E or B, can interact with the LDL receptors on the surface of cells of the blood brain barrier resulting in endocytotic uptake (Vauthier et al. 2007). It has been shown that only polysorbates 20, 40, 60 or 80 adsorb apolipoprotein E to facilitate endocytosis and release of drug to brain (Kreuter 2001). Other potential mechanisms for the delivery of drug as NPs to the brain are, by increasing the concentration gradient of drug in brain capillaries, membrane fluidization by exploiting surfactant coated NPs, opening of tight junctions due to presence of surfactants, endocytotic uptake of NPs, transcytosis and inhibition of efflux system (P-gp) by surfactants (Kreuter 2001).

Nanoparticles for topical application

The stratum corneum is the main barrier for percutaneous absorption of topically applied drugs. Particulate drug delivery systems can be used for dermatological application to improve the therapeutic action and reduce systemic side-effects. The horny layer of the skin functions as barrier to prevent the entry of hydrophilic compounds and allows the passage of lipophilic substance selectively. Complete stripping of the horny layer increases the uptake of acyclovir (logP = – 1.76) absorption 440-fold, indicating that this barrier is clearly most effective against hydrophilic compounds (Kreuter 2001). Highly lipophilic compound can penetrate via the hair follicle and through the follicular pathway (Borgia et al. 2005).

SLN and nanostructured lipid carrier have been widely investigated for use in dermatological applications. SLNs enhance the penetration through the stratum corneum membrane by increasing the thickness of the occlusive film and with improved skin hydration. Small, 200 nm or less, SLNs permit site-specific delivery to the skin. SLN has high affinity for the stratum corneum and therefore enhanced bioavailability of the encapsulated material to the skin is achieved. Lipid particles of nanostructured lipid carrier form an adhesive occluding layer over the skin, which increases the hydration of the stratum corneum and reduces corneocyte packing. In addition, inter-corneocytes widening takes place which facilitates drug penetration in to deeper skin strata. All the above actions are mainly dependent on the particle size of the carrier. NPs, less than 400 nm and with a minimum of 35% lipid, provide more therapeutic effect than the microparticles, as observed with 2 times increased concentration of SLN vitamin A, when compared with conventional gel (Pople and Singh 2006).

Miyazaki et al. (2003) prepared indomethacin loaded poly n-butylcyanoacrylate nanocapsules to promote its systemic action through topical application. The in vitro permeation study in rat skin showed that the poly n-butylcyanoacrylate nanocapsules were able to permeate rat skin. The fluorescence photomicrography study in rat skin showed that the NPs can penetrate through the stratum corneum and reach the epidermis. They showed that the permeation of the drug through rat skin was mostly due to permeation of the intact nanocapsules. The study reported higher plasma drug concentration (2.24 μg/ml) in 6 h in the case of poly n-butylcyanoacrylate nanocapsule formulation compared with gel formulation (0.88 μg/ml) which is in agreement with in vitro results.

The reason for the penetration of NPs through the stratum corneum and epidermis into the systemic circulation is attributed to the small size, lipophilic nature of the polymers, hydrophilic and hydrophobic surface characteristics of the material. Sometimes penetration enhancers (e.g., benzyl benzoate/oleic acid) are used in the nano-formulation to enhance the penetration of the drug. Drugs, loaded in lipid carriers, appear to enhance dermal absorption. The nanosize carries can make very close contact and adhere with corneocyte clusters and furrows between corneocyte islands, which provides lager surface to the drug molecules to penetrate the barrier (Muller et al. 2002, Korting et al. 2007).

Applications for nanoparticles

Targeted drug delivery

Newly developed anticancer drugs may not be therapeutically useful due to poor water-solubility and poor or non-selective distribution of the drug to site of action. A significant amount of work has been done in developing polymeric tumour-targeted NPDDS for anticancer drugs and promising results were reported with improved solubility and better therapeutic efficacy with selective distribution. Recent attempts to improve the bioavailability and targeting of anticancer drugs, EP, imatinib mesylate (IM) and paclitaxel have provided encouraging results (Girish et al. 2007, Snehalatha et al. 2008a, 2008b, Sekar et al. 2009, Kollipara et al. 2010). It was observed that after i.v and oral administration of radio-labeled EP loaded PCL and PLGA NPs, the serum residence time of EP was increased. Also a low distribution of EP to the heart was observed: NPs are likely to produce no or low cardiac toxicity. The EP-loaded NPs were found to reach the lungs at relatively higher levels suggesting that such formulations may be useful in the treatment of lung cancer (Snehalatha et al. 2008a). The results also showed that free EP disappeared from the liver faster than NPs, indicating that nanoparticulate EP may distribute selectively and stay for a longer time in liver than the free drug. Therefore, these NP formulations can be used potentially in the treatment of liver carcinomas. After i.v. and oral administration of free EP and NPs, NS produced a higher AUC_0-inf, MRT and t_1/2 when compared with free EP. Furthermore, it was observed that a PLGA 85/15 formulation has enhanced AUC_0-inf, MRT and t_1/2 when compared to PCL NPs suggesting that the polymer plays an important role in distribution profile (Snehalatha et al. 2008a).

Selective delivery to brain

The pharmacokinetic and biodistribution study for the Poly (ethyl cyanoacrylate) (PECA) and Poly(lactide-co-glycolic acid) (PLGA) NPs of IM in healthy rats by single dose i.v. (Figure 1) and oral administration (Figure 2) indicated that, both PECA and PLGA NPs were effectively produced increased serum drug concentration (AUC_inf 262.28 mg h/l and 166.85 mg h/l, respectively) for longer serum circulation time (Half-life 21.59 h and 12.47 h) with approximate two- and three-fold increase in drug residence time in the serum (MRT 31.15 h and 17.99 h) as compared to pure drug (AUC_inf 102.72 mg h/l, t _½ 6.81 h and MRT 9.83 h) (unpublished work; PhD thesis, Girish B, 2008).

Figure 1. Rats were injected with imatinib mesylate pure drug (IM-PD) and nanoparticles formulations [(Poly (ethyl cyanoacrylate) (PECA-NP) and Poly (lactide-co-glycolic acid) (PLGA-NP) nanoparticle] via intravenous route. Serum samples were collected at respective time points and the drug concentrations in serum (mg/l) were analyzed by high-performance liquid chromatography method.

Figure 2. Rats were injected with imatinib mesylate pure drug (IM-PD) and nanoparticles formulations [(Poly (ethyl cyanoacrylate) (PECA-NP) and Poly (lactide-co-glycolic acid) (PLGA-NP) nanoparticle] via intravenous route. Brain tissue samples were collected after sacrificing the animal at respective time points and the drug concentrations in brain (μg/g) were analyzed by high-performance liquid chromatography method.

In the above biodistribution studies (unpublished work; PhD thesis, Girish B, 2008) of IM after i.v. administration of the pure drug indicated a slow and poor permeation of free drug to the brain. The maximum drug concentration (C_max) observed in brain was 0.6 μg/g at 3 h (T_max) post dosing. Administration of PECA and PLGA NPs by i.v, indicated increased distribution and retention of the drug with increased AUC_inf to 26.24 and 23.54 μg h/g. MRT was found to increased (29.06 h and 31.91 h) with increased elimination half-life (20.14 h and 22.11 h) in brain (Figure 2). In pharmacokinetic studies on oral administration revealed similar results and that the PLGA nanoparticulate preparation has relatively more bioavailability when compared to free drug (AUC free drug 200.4 μg·h·l^-1, NPs 242.2 μg·h·l^-1).

Although there was no significant increase in C_max mean residence time was increased significantly with the nanoparticulate preparations indicating a controlled release of drug from NPs (Figure 3). The plasma half life of the drug was increased approximately 1.5-fold. Availability in brain was considerably increased (more than twice) with nanoparticulate preparations when compared to free drug (AUC_inf free drug 14.7 μg·h·l^-1, NPs 31.3 μg·h·l^-1). The same was observed with the mean residence time in brain (unpublished work; PhD thesis, Girish B, 2008). Nanoparticulate preparations have shown a bi-phasic profile in serum and brain with a two fold increase in tissue half life of drug (Figure 4).

Figure 3. Rats were administered with imatinib mesylate pure drug (IM-PD) and nanoparticles formulations [(Poly (ethyl cyanoacrylate) (PECA-NP) and Poly (lactide-co-glycolic acid) (PLGA-NP) nanoparticle] via oral route. Serum samples were collected at respective time points and the drug concentrations in serum (mg/l) were analyzed by high-performance liquid chromatography method.

Figure 4. Rats were administered with imatinib mesylate pure drug (IM-PD) and nanoparticles formulations [(Poly (ethyl cyanoacrylate) (PECA-NP) and Poly (lactide-co-glycolic acid) (PLGA-NP) nanoparticle] via oral route. Brain tissue samples were collected after sacrificing the animal at respective time points and the drug concentrations in brain (μg/g) were analyzed by high-performance liquid chromatography method.

Wilson et al. (2008) prepared and studied polysorbate 80-coated poly (n-butylcyanoacrylate) NPs of anti-Alzheimer's drug tacrine to target brain. The study reported that tacrine concentration in the brain increased by 4.07-fold when compared to the free drug tacrine. The i.v pharmacokinetic and biodistribution study of EP-loaded tripalmitin positively charged NPs showed that NPs produced a higher brain concentration (0.07% of injected dose/organ) when compared to negatively charged SLN and EP solution. The prepared nitrendipine-loaded SLN for brain targeting, has found to be taken up to a greater extent by brain and hence the concentration of nitrendipine was higher and maintained for 6 h as compared to suspension (3 h). Xiong et al. (2008) prepared a nanosuspensions, average particle size of 300 and 650 nm, containing nimodipine (a calcium-channel blocker for the treatment of senile dementia and subarachnoid haemorrhage related vasospasm) to target the brain and the results were compared with nimodipine ethanol formulation and Tween 80-coated nanoparticle. The i.v. biodistribution study demonstrated suggested that the 300 nm nanoparticle effectively increased brain drug concentration and reduced drug concentration in the liver, spleen and lungs, indicating that the 300 nm particles are not taken up by Kupffer cells as are the 650 nm NPs.

Selective delivery to lungs

Administration of IM PECA NPs by i.v revealed that, maximum drug concentration (C_max) was 31.94 μg/g in lungs at 3 h (T _max) (unpublished work; PhD thesis, Girish B, 2008). The AUC _inf was observed to increase to 608.01 μg·h·l^-1 against the AUC _inf 316.9 μg·h·l^-1 for drug alone. MRT was also found to increase to 24.73 h (drug alone MRT 14.6 h) with increased lungs elimination half-life 17.14 h (drug alone half-life 14.6 h). Similarly, i.v. administration of PLGA NPs also showed increased distribution of the drug to lungs with increased AUC _inf to 611.36 μg·h·l^-1 (Figure 5). The maximum drug concentration of 30.99 μg/g was observed in the lungs at 1.5 h post dosing. The MRT was found to increase to 27.1 h with increased elimination half life in the lungs (18.78 h).

Figure 5. Rats were injected with imatinib mesylate pure drug (IM-PD) and nanoparticles formulations [(Poly (ethyl cyanoacrylate) (PECA-NP) and Poly (lactide-co-glycolic acid) (PLGA-NP) nanoparticle] via intravenous route. Lungs tissue samples were collected after sacrificing the animal at respective time points and the drug concentrations in brain (μg/g) were analyzed by high-performance liquid chromatography method.

The oral biodistribution study of PECA NPs also revealed similar findings (Figure 6) as observed in the i.v. biodistribution study, with an increased concentration in lungs (41.96 μg/g) and increased MRT (27.33 h) and elimination half life (27.33 h). The pharmacokinetic and biodistribution studies confirmed that the biodistribution of the IM can be modulated efficiently by forming NPDDS of IM. Though PECA and PLGA NPs showed enhanced but different level of distribution in brain, whereas distribution in the lungs did not vary, indicating different profile of distribution in brain and in lungs presumably due to the different polymers employed (unpublished work; PhD thesis Girish B, 2008).

Figure 6. Rats were administered with imatinib mesylate pure drug (IM-PD) and nanoparticles formulations [(Poly (ethyl cyanoacrylate) (PECA-NP) and Poly (lactide-co-glycolic acid) (PLGA-NP) nanoparticle] via oral route. Lungs tissue samples were collected after sacrificing the animal at respective time points and the drug concentrations in brain (μg/g) were analyzed by high-performance liquid chromatography method.

Selective delivery to liver and bone

EP-loaded NPs with narrow size range (105.1 ± 2.38 and 257.2 ± 3.96) and homogenous drug distribution (Polydispersity index 0.11 ± 0.01 and 0.1 ± 0.01) were prepared successfully in our laboratory using the nanoprecipitation method (PLGA-NP) and solvent evaporation method (PCL) in the presence of PF 68 as stabilizer. The pharmacokinetic and biodistribution studies were performed with radio-labeled free drug EP, empty NPs (NP/F68/17), drug-loaded PLGA NPs (ENTP/F68/17) and drug-loaded PCL NPs (ETNP/PCL/F68/03) (Figure 7 and 8). All the nanoparticle formulations showed higher distribution to liver and bone and longer circulating time than the drug alone. The study showed that, after 24 h post injection PLGA (1.005%) and PCL (0.332%) NPs amount of EP in bone were found to be 15.02 and 4.96 times more than free EP (0.066%), respectively (Snehalatha et al. 2008a).

Figure 7. Rabbits were injected with etoposide pure drug and nanoparticles formulations [Poly (lactide-co-glycolic acid) (ETNP/F68/17), Poly-ϵ-caprolactone (ETNP/PCL/F68/03) and empty (without drug) (NP/F68/17) nanoparticle] via intravenous route. The drug concentration in blood (%A/g) was analyzed by gamma scintillation counter method.

Figure 8. Rabbits were administered with etoposide pure drug and nanoparticles formulations [Poly (lactide-co-glycolic acid) (ETNP/F68/17) and empty (without drug) (NP/F68/17) nanoparticle] via oral route. The drug concentration in blood (%A/g) was analyzed by gamma scintillation counter method.

The biodistribution studies also showed that the radioactivity levels of the nanoparticle formulations in tissues/organs were significantly higher than those of EP alone except in the heart. Therefore using EP-loaded PLGA and PCL NPs can reduce the accumulation of EP in the heart, which may lead to reduced cardiac toxicity. In tumour-induced mice, it was observed that tumour uptake of nanoparticulate EP was much higher than free drug. The free EP disappeared faster from the liver than the formulations indicating that the nanoparticulate EP may distribute more widely and stay for a longer time than drug alone. Relatively high radioactivity was found in bone for NPs than free EP. This is one more useful indicator that such preparations may be of value in treating bone related malignancies (Snehalatha et al. 2008a).

Selective delivery to eye

Targeting drug-loaded NPs to the eye has advantages over conventional dosage forms. It can prolong drug residence time and can hence reduce the required dose and related side-effects of the drug. By using the polyalkylcyanoacrylate, poly-ϵ-caprolactone, polyester and albumin NPs, the short elimination half life of drugs in aqueous eye drops can be extended from the usual 1–3 min to 15–20 min. The polyalkylcyanoacrylate-loaded pilocarpin and betaxolol NPs prolong and maintain the normal intraocular pressure more than 9 h in rabbits. Kim et al. (2009) prepared PLGA-loaded doxorubicin NPs, the targeted drug delivery system to treat intraocular tumours (retinoblastoma). The diffusion study demonstrated that the PLGA NPs deliver drug at a slower rate across the sclera. The in vitro release study of PLGA NPs using the dialysis bag technique shows that 100% of drug is released in 10 h in the doxorubicin solution preparation; however, in PLGA NPs, after 24h only 3.39% was released indicating sustained release of drug from the NPs. The cumulative release of doxorubicin after seven days, was only 12.72 ± 1.89% from the NPs. De Campos et al. (2001) prepared chitosan-loaded cyclosporine A NPs (293 nm) by ionic gelation with an association efficiency of 73% and a loading efficiency of 9%. The in vivo experiment result showed that the topical application of NPs to rabbit eye, provided a therapeutic concentration in external ocular tissues (cornea and conjunctiva) for 48 h and negligible or undetectable drug level in inner ocular structures (iris/ciliary body and aqueous humour), blood and plasma. This drug delivery system provided potential selective application for extraocular application.

Liposomes in cancer therapy

Liposomes are useful drug delivery vehicles. Liposomes, made up of amphiphilic phospholipids and cholesterol, generally are in the size range of 50–1000 nm and can encapsulate both hydrophobic and hydrophilic drugs. The aqueous vesicle of liposome encapsulates hydrophilic drugs and the lipidic bilayer encapsulates lipophilic drugs. The most significant advantage of liposomes is their biologically inertness and biocompatibility (Vyas and Khar 2001). The long circulating liposomes have gained much attention as like the polymeric NPs they can be engineered for selectivity and targeting. A commercial PEG-coated liposome (Caelyx) designed to extend the circulation time of doxorubicin is available for the selective treatment of Kaposi's sarcoma and advanced breast and ovarian cancer (Torchilin 2007). Nanosized liposomal drug carriers have shown higher bioavailability, selective distribution, controlled release and better therapeutic efficacy. PEGylated liposomes have been used as long circulating nanocarrier for better therapeutic action (Fahmy et al. 2007). Such liposomes are valuable for the treatment of cancer because they are long-lived, without triggering an immune response, and they reach the tumour interstitium where the liposomes can release the drug leading to higher drug availability for action in the required area (EPR-effect). Stimuli sensitivity liposomes (SSL) are the ‘holy grail’ of the drug delivery scientist. The stimuli-sensitive drug delivery in cancer treatment is based on the nature of the cell. In general, cancer cells have a lower pH value and high temperature than normal cells and these conditions can be exploited in SSL delivery for drug release and better cancer treatment (Torchilin 2007). Liposomes, as drug carriers, are the most successful cytosolic drug delivery systems. Cellular drug delivery by endocytosis is mediated by pH. Hence pH-sensitive liposomal drug delivery is a potential way to deliver drug molecules to the intracellular compartment (Kisel et al. 2001).

Cytotoxicity of nanoparticles

Despite the enormous potential value of nanotechnology for drug delivery, much work still needs to be done to improve our appreciation of the cytotoxicity of NPs. The in vivo administration of NPs will require a thorough understanding of the toxicology of polymeric NPs. Attention has been given to the in vitro cytotoxicity of polymeric NPs using cell lines (Medina et al. 2007). There are reports of toxic effects of NPs in a variety of organs (Serpe et al. 2004, Medina et al. 2007, Stephanie et al. 2007). Hence it is necessary to find the risk/benefit ratio of the prepared NPs by performing cytotoxicity studies.

It has been demonstrated that the gelatin NPs are physiologically very well tolerated. Cationized gelatin NPs show hardly any significant cytotoxic effect when compared to PEI polyplexes in transfected B16 F10 cells. The major benefit of gelatin NPs is not only the very low cell toxicity, but also their simple production combined with low cost and multiple modification opportunities offered by the matrix molecule (Klaus et al. 2004). Researchers showed that cells treated in vitro with NPs showed relatively high viability, when compared to negative controls, supporting the non-toxic nature of the NPs; even at high concentration (100 μg/ml, which is equivalent to 800,000 particles/cell) (Stephanie et al. 2007).

Recently an in vitro cytotoxicity study was performed in IOBA-NHC cell lines for chitosan NPs (CSNP) to determine whether this new CSNP system was noxious for the conjunctival epithelium. Based on cell survival and viability values, a 0.5 mg/ml concentration was found to be optimal, leading to localization of CSNPs within the cells, but still resulting in maximal cell survival. Cell survival in cultures exposed to CSNPs for 30 min was very high. The significantly higher cell recovery levels at 30 min compared 60 min may be related to the NPs aggregation observed after 1 h of incubation at 37°C. Cell survival at 24 h after CSNP exposure was also high at all tested concentrations and exposure times, except the 2 h exposure to 1 mg/ml CSNPs. Although survival was high, levels were significantly lower than that of control cells exposed to culture medium alone. The difference was probably because of the effect of PBS on cells, since similar survival rates were recorded immediately and after 24 h in culture medium for cells exposed to PBS and all CSNP concentration. Thus, no inherent toxicity can be attributed to CSNPs, per se. This was further confirmed by the viability of recovered cells, which was approximately 90% when measured immediately after CSNP exposure (> 92%) and after the 24 h recovery period (> 86%) (Salamanca et al. 2006).

The in vitro cytotoxicity study of doxorubicin, paclitaxel, doxorubicin-loaded SLN, paclitaxel-loaded SLN and unloaded SLN in human colorectal adenocarcinomal cell line, HT-29 was performed and percentage survival of cell lines was observed after 24, 48 and 72 h exposure of cells to the SLN formulation and free drugs. The amount of doxorubicin and paclitaxel required to achieve 50% inhibitory concentration (IC₅₀ value) was lower with loaded SLN than with free drug. The IC₅₀ of free doxorubicin and paclitaxel, SLN-loaded doxorubicin and paclitaxel were found to be 126.75 ± 0.72 nM and 33.43 ± 1.67 nM, 81.87 ± 4.11nM, 37.36 ± 6.41 nM, respectively. In the case of unloaded SLN, 100% no cytotoxicity was observed indicating the safety of SLN drug delivery to cancer treatment (Serpe et al. 2004).

Scientists working on particulate drug delivery systems should consider not only the beneficial effect, they should focus on the potential areas of hazard and risk assessment for PDDS used in medical profession.

Conclusion

Nanotechnology has opened up opportunities for newer drug delivery systems with better therapy and better patient compliance. NPs can increase drug targeting to specific sites, and hence can increase drug availability both at the cell surface and intracellularly. It has been observed that selective distribution is made possible by altering the properties of the nanoparticulate delivery system. For the same drug, the distribution can be modified and can be targeted to different organs and tissues by using different polymer(s) or different proportions of drug and polymer or by using different combinations of polymers. It is not only anticancer drugs but central nervous system active drugs, bronchodilators, antibacterial, antiviral and hormones that can benefit in terms of targeting or selective distribution using NPDDS. This can lead to decreased dosage, more economic therapy, reduced side-effects and better patient compliance with overall improvement of quality of life. However, toxicity of NPDDS will need to be studied before their full potential for human therapy can be explored.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.