A REVIEW ARTICLE ON NANOPARTICLE

ABOUT AUTHOR:
Shalu Shukla
M.Pharmacy
Bahra University
shukla5828@gmail.com

ABSTRACT
The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. The kind of hazards that are introduced by using nanoparticles for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices. For nanoparticles the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanoparticles. The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs. This may vary from a rather high local exposure in the lungs and a low or neglectable exposure for other organ systems after inhalation. However, absorbed species may also influence the potential toxicity of the inhaled particles. For nanoparticles the situation is different as their size opens the potential for crossing the various biological barriers within the body. From a positive viewpoint, especially the potential to cross the blood brain barrier may open new ways for drug delivery into the brain. In addition, the nanosize also allows for access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently under investigation for the preparation of nanoparticles for drug delivery, varying from biological substances like albumin, gelatin and phospholipids for liposomes, and more substances of a chemical nature like various polymers and solid metal containing nanoparticles. It is obvious that the potential interaction with tissues and cells, and the potential toxicity, greatly depends on the actual composition of the nanoparticle formulation. This paper provides an overview on some of the currently used systems for drug delivery.

REFERENCE ID: PHARMATUTOR-ART-1770

INTRODUCTION
Recent years have witnessed unprecedented growth of research and applications in the area of nanoscience and nanotechnology. There is increasing optimism that nanotechnology, as applied to medicine, will bring significant advances in the diagnosis and treatment of disease. Anticipated applications in medicine include drug delivery, both in vitro and in vivo diagnostics, nutraceuticals and production of improved biocompatible materials (Duncan 2003; De Jong et al 2005; ESF 2005; European Technology Platform on Nanomedicine 2005; Ferrari 2005). Engineered nanoparticles are an important tool to realize a number of these applications. It has to be recognized that not all particles used for medical purposes comply to the recently proposed and now generally accepted definition of a size ≤100 nm (The Royal Society and Royal Academy of Engineering 2004). However, this does not necessarily has an impact on their functionality in medical applications. The reason why these nanoparticles (NPs) are attractive for medical purposes is based on their important and unique features, such as their surface to mass ratio that is much larger than that of other particles, their quantum properties and their ability to adsorb and carry other compounds. NPs have a relatively large (functional) surface which is able to bind, adsorb and carry other compounds such as drugs, probes and proteins. However, many challenges must be overcome if the application of nanotechnology is to realize the anticipated improved understanding of the patho-physiological basis of disease, bring more sophisticated diagnostic opportunities, and yield improved therapies. Although the definition identifies nanoparticles as having dimensions below 0.1 μm or 100 nm, especially in the area of drug delivery relatively large (size >100 nm) nanoparticles may be needed for loading a sufficient amount of drug onto the particles. The composition of the engineered nanoparticles may vary. Source materials may be of biological origin like phospholipids, lipids, lactic acid, dextran, chitosan, or have more “chemical” characteristics like various polymers, carbon, silica, and metals. The interaction with cells for some of the biological components like phospholipids will be quite different compared to the non biological components such as metals like iron or cadmium. Especially in the area of engineered nanoparticles of polymer origin (1)

DEFINITION
A nanoparticle is a microscopic particle whose size is measured in nanometres (nm). It is defined as a particle with at least one dimension <200nm.or nanoparticles are solid colloidal particles ranging in size from 10nm to 1000nm. They consist of macromolecular materials in which the active principle is dissolved, entrapped or encapsulated, and/or to which the active principle is absorbed or attached.

Nanoparticle can be formulated, as injections consisting of spherical amorphous particles which do not aggregate, hence they can be safely administered by the intravenous route. Since no cosolvent is used to solubilize the drug, the overall toxicity of the formulation is decreased.

Nanoparticles represent very promising carrier system for the targeting of anti-cancer agents to tumors. Nanoparticles exhibit a significant tendency to accumulate in a number of tumors after iv injection. Nanoparticles can also be used in Brain Drug Targeting.Poly (butyl cyanoacrylate) nanoparticles represent the only nanoparticles that were so far successfully used for in vivo delivery of drugs to brain. This polymer has the advantage that it is very rapidly biodegradable.The first drug that was delivered to brain using nanoparticles was the Hexapeptide Dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg), a Leu-enkephalin analouge with opioid activity.Other drugs that have successfully been transported into the brain are loperamide, tubocurarine, and doxorubicin. Nanoparticles mediated drug transport to the brain depends on the overcoating of the particles with polysorbates, especially polysorbate 80. (2)

Nanoclusters have at least one dimension between 1 and 10 nanometers and a narrow size distribution. Nanopowders are agglomerates of ultrafine particles, nanoparticles, or nanoclusters. Nanometer-sized Single crystals or Single domain ultrafine particles, are often referred to as nanocrystals. Nanoparticle research is currently an area of intense scientific interest due to a wide variety of potential applications in biomedical, optical and electronic fields.

(a, b, and c) images of prepared mesoporous silica nanoparticles with mean outer diameter: (a) 20nm, (b) 45nm, and (c) 80nm. (d) image corresponding to (b). The insets are a hig magnification of mesoporous silica particle.(3)

TYPES OF NANOPARTICLES

1. Quantum Dot
A quantum dot is a semiconductor nanostructure that confines the motion of conduction band electrons, valence band holes, or excitons (pairs of conduction band electrons and valence band holes) in all three spatial directions. The confinement can be due to electrostatic potentials (generated by external electrodes, doping, strain, impurities), due to the presence of an interface between different semiconductor materials (e.g. in the case of self-assembled quantum dots), due to the presence of the semiconductor surface (e.g. in the case of a semiconductor nanocrystal), or to a combination of these. A quantum dot has a discrete quantized energy spectrum. A quantum dot contains a small integer number (of the order of 1-100) of conduction band electrons, valence band holes, or excitons, i.e., an integer number of elementary electric charges.

Small quantum dots, such as colloidal semiconductor nanocrystals, can be as small as 2 to 10 nanometers, corresponding to 10 to 50 atoms in diameter and a total of 100 to 100'000 atoms within the quantum dot volume. Self-assembled quantum dots are typically between 10 and 50 nanometers in size. Quantum dots defined by lithographically patterned gate electrodes or by etching on two-dimensional electron gases in semiconductor heterostructures can have lateral dimensions exceeding 100 nanometers. At 10 nanometers in diameter, nearly 3 million quantum dots could be lined up end to end and fit within the width of a human thumb.

Quantum dots can be contrasted to other semiconductor nanostructures:
1) Quantum wires, which confine the motion of electrons or holes in two spatial directions and allow free propagation in the third.
2) Quantum wells, which confine the motion of electrons or hles in one direction and allow free propagation in two directions.

Quantum dots with a nearly spherical symmetry or flat quantum dots with nearly cylindrical symmetry can show shell filling according to the equivalent of Hund's rules for atoms. Such dots are sometimes called "artificial atoms". In contrast to atoms, the energy spectrum of a quantum dot can be engineered by controlling the geometrical size, shape, and the strength of the confinement potential.

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2. Nanocrystalline silicon
Nanocrystalline silicon(nc-Si) - an allotropic form of silicon - is similar to amorphous silicon (a-Si), in that it has an amorphous phase. Where they differ, however, is that nc-Si has small grains of crystalline silicon within the amorphous phase. This is in contrast to polycrystalline silicon (poly-Si) which consists solely of crystalline silicon grains, separated by grain boundaries. nc-Si is sometimes also known as microcrystalline silicon (µc-Si) The difference comes solely from the grain size of the crystalline grains. Most materials with grains in the micrometre range are actually fine-grained polysilicon, so nanocrystalline silicon is a better term.

3. Photonic crystal
Photonic crystals are periodic dielectric or metallo-dielectric (nano) structures that are designed to affect the propagation of electromagnetic waves (EM) in the same way as the periodic potential in a semiconductor crystal affects the electron motion by defining allowed and forbidden electronic energy bands.

Since the basic physical phenomenon is based on diffraction, the periodicity of the photonic crystal structure has to be in the same length-scale as half the wavelength of the EM waves i.e. ~300 nm for photonic crystals operating in the visible part of the spectrum. This makes the synthesis cumbersome and complex. To circumvent nanotechnological methods with their big and complex machinery, different approaches have been followed to grow photonic crystals as self-assembled structures from colloidal crystals.

Photonic crystals are attractive optical materials for controlling and manipulating the flow of light. They are of great interest for both fundamental and applied research, and are expected to find commercial applications soon.

4. Liposomes
A liposome is a spherical vesicle with a membrane composed of a phospholipid bilayer used to deliver drugs or genetic material into a cell. Liposomes can be composed of naturally-derived phospholipids with mixed lipid chains (like egg, phosphatidylethanolamine), or of pure components like DOPE (dioleolylphosphatidylethanolamine).

The lipid bilayer can fuse with other bilayers (e.g., the cell membrane), thus delivering the liposome contents. By making liposomes in a solution of DNA or drugs,(which would normally be unable to diffuse through the membrane), they can be (indiscriminately) delivered past the lipid bilayer.

5. Gliadin nanoparticles
In an effort to improve bioavailability anti-H.pylori effects of antibiotics, mucoadhesive gliadin nanoparticles (GNP) which have the ability to deliver the antibiotics at the sites of infection were prepared. GNP bearing clarithromycin(CGNP) and omeprazole(OGNP) were prepared by desolvation method.

In vivo gastric mucoadhesive studies confirmed the strong mucoadhesive propensity and specificity and specificity of gliadin nanoparticles towards stomach. Gliadin nanoparticles show a higher tropism for the gastrointestinal regions and their presence in other intestinal regions is very low. This high capacity to interact with the mucosa may be explained by gliadin composition.

In fact, this protein is rich in neutral and lipophilic residues. Neutral amino acid can promote hydrogen bonding interaction with the mucosa whereas the lipophilic components can interact within biological tissue by hydrophilic interaction. The related protein gliadin possessing an amino and disulphide groups on the side chain has a good probability of developing bonds with mucin gel.

6. Polymeric Nanoparticles
Polymeric nanoparticles have been invented by Speiser et al. They represent interesting alternative as drug delivery systems to liposomes. They usually exhibit a long shelf life and a good stability on storage.

These are superior to liposomes in targeting them to specific organs or tissues by adsorbing and coating their surface with different substances.

Nanoparticles can be prepared either from preformed polymers, such as polyesters (i.e. polylactic acid), or from a monomer during its polymerization, as in the case of alkyl-cyanoacrylates.

Most of the methods based on the polymerization of monomers consists in adding a monomer into the dispersed phase of an emulsion, an inverse microemulsion, or dissolved in a non-solvent of the polymer.

7. Solid Lipid Nanoparticles (SLN)
Solid lipid nanoparticles have been developed as alternative delivery system to conventional polymeric nanoparticles.SLNs are sub-micron colloidal carriers (50-1000nm) which are composed of physiological lipid, dispersed in water or in an aqueous surfactant solution.

SLNs combine advantages of polymeric nanoparticles, fat emulsions and liposomes, but avoid some of their disadvantages.They are biodegradable, biocompatible and non-toxic.

8. Others:
Gold nanoparticles stabilized by thiol functionality are extraordinarily stable and therefore are a great system for studying nanostructure formation. They have many applications. Because gold nanoparticles are so easy to synthesize they have been studied intensely in recent years.

A common synthesis involves the reduction of a gold salt in the presence of capping agent molecules such as thiols, citrates or phosphines. The functionalities of these capping agents can be altered to yield various chemical properties. (5)

Mechanisms of cellular targeting

1. Nanoparticle uptake by tissues
A succession of several membrane layers provides an obstacle for therapeutic agents attempting to target intracellular structures. During this process, compound is lost due to ineffective partitioning across biological membranes. The extent of partition across a membrane is related directly to the polarity of a molecule; nonpolar or lipophilic molecules easily bypass this obstacle with greater membrane penetration, generally via diffusion. However, the situation is much more complicated, as a myriad of other cellular processes directly affect the intracellular concentrations and effectiveness of the therapeutic agent. Variable efficiencies of endocytosis mechanisms, intracellular trafficking, release of the therapeutic agent into the cytoplasm, diffusion and translocation of the therapeutic agent to its susceptible target, and partition into the nucleus or other organelles alter the actual activity of the therapeutic agent. Nanoparticles present an interesting opportunity for eliminating much of this ‘waste’ due to masking of the therapeutic agent from its biological environment; this effectively limits the influence of a compound’s physical properties on intracellular drug concentrations. Instead, the properties and surface characteristics of the nanoparticle play a greater role in compound delivery and resulting intracellular drug concentrations. Nanoparticles may be ingested and ‘sampled’ by curious cells.

Endocytosis encompasses the process of membrane manipulation to envelope and absorb materials and includes three subtypes: phagocytosis, pinocytosis, and receptor-mediated endocytosis. Phagocytosis involves the ingestion of materials up to 10 lm in diameter,and can be accomplished by fairly few cell types of the reticuloendothelial system, such as macrophages, neutrophils, and dendritic cells. Pinocytosis is an uptake mechanism that can be conducted by virtually all cell types, and normally involves ingestion of sub-micron material and substances in solution. Larger microparticles provide selective access to phagocytic cells, while smaller nanoparticles provide access to virtually all cell types.

2. Cellular phagocytosis/endocytosis
Receptor-mediated endocytosis affords the potential for even greater selectivity in cellular targeting. The cellular membrane is dotted with a myriad of receptors, which upon extracellular binding to their respective ligands (or to nanoparticles whose surface is functionalized with ligands), transduce a signal to the intracellular space. This signal can trigger a multitude of biochemical pathways; however it may also cause internalization of the ligand and its appended nanoparticle via endocytosis. Caveolin- and clathrincoated pits provide an illustration of receptor-mediated endocytosis. Typically, clathrin coats generate a membrane indentation with a radius of curvature as small as approximately 50 nm,and invaginate further upon binding of the ligand. Cross-linking of receptors via ligands attached to nanoparticles results in a more pronounced membrane crater with subsequent enfolding and reunification of the cellular membrane to form an endosome. It has been shown that nanoparticle sizes between 25 and 50 nm are a requisite for optimal endocytosis and intracellular localization.

Steps detailing the cytosolic delivery of therapeutic agents via nanoparticle carriers. (1) Cellular association of nanoparticles, (2) internalization of nanoparticles via endocytosis, (3) endosomal escape of nanoparticles or (4) lysosomal degradation of nanoparticle, (5) therapeutic agent freely diffuses into cytoplasm, (6) cytoplasmic transport of therapeutic agent to target organelle, (7) exocytosis of nanoparticles.(6)

Nanoparticle drug delivery for human therapeutics
Nanoparticles have found widespread use in drug delivery, counting more than a dozen FDA-approved variants with indications ranging from cancer to infection.

1. Neurological cancers (glioblastoma multiforme)
The central nervous system represents a formidable challenge for the delivery of therapeutic agents due to the blood-brain barrier (BBB). This physical barrier limits the brain uptake of the vast majority of neurotherapeutics and neuroimaging contrast agents. The anatomical and cellular morphology of neurovascular capillary endothelial cells, including limited pinocytosis and tight junctions, produces this unique central nervous system manifestation.The brain microvasculature involves four types of cells: endothelial cells, pericytes, astrocyte foot processes, and nerve endings. Endothelial cells share the capillary basement membrane with pericytes that participate in immune surveillance. The other side of this basement membrane is almost entirely surrounded by astrocyte foot processes. Brain capillary endothelial cells are cemented together by tight junctions; this is associated with a 100-fold reduction of pinocytosis across the endothelium .Therefore, substances may gain access to the central nervous system by lipid- mediated free diffusion or potentially by receptor-mediated endocytosis of nanoparticles. Nanotechnology may provide an effective means for circumventing this delivery issue past the BBB. Glioblastoma multiforme (GBM) is among the most devastating and lethal of neoplasms, often claiming the lives of patients within a median of one year following diagnosis. The treatment is multidisciplinary, including radiotherapy, chemotherapy, and surgery.Transport of many chemotherapeutic agents past the BBB has proven difficult. Recently, however, a series of discoveries have been made. In particular, low-density lipoprotein receptors (LDLR) are upregulated on GBM cellular surfaces to between 128,000 and 950,000 receptors per tumor cell.In contrast, average neurons have comparatively lower LDLR numbers, as evidenced by examining normal rat and monkey brain tissue.Thus, targeting LDLR may offer the opportunity for potential therapeutic selectivity in chemotherapeutic drug delivery. Previous studies have investigated the use of lowdensity lipoproteins (LDL). Natural LDL particles are roughly 22– 27 nm in diameter with a core of lipids primarily composed of cholesteryl esters with small amounts of triglyceride. Initial studies used plasma-derived LDL as delivery agent to GBM tumors.

However, due to the difficulty of isolating natural LDL, reconstituted and synthetic versions have become desirable.Synthetic LDL nanoparticles have been shown to effectively deliver a toxic payload of paclitaxel to GBM tumor cells, and this cytotoxic effect is overturned upon treatment with the LDL receptor inhibitor suramin.

Anatomy of BBB

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2. Neurovascular diseases (vascular targeting and stroke)
Vascular diseases, such as atherosclerosis and hypertension, are a primary cause of neurological ischemia (strokes), aneurysm formation, and intracranial hemorrhage. Nanoparticles have been used diagnostically for the detection of atherosclerotic plaques; similar targeting strategies may be used to deliver therapeutic agents to these plaques. Early recognition and intervention may prevent dire neurological and systemic outcomes occurring subsequent to plaque rupture and subsequent thrombosis or embolism, as an example.

The natural course of atherosclerotic plaques is detailed in Atherosclerosis is primarily an inflammatory disease, with accumulating oxidized LDL particles triggering an inflammatory cascade with monocyte recruitment. Macrophages ingest these particles and are transformed into foam cells. The lipid core of the atherosclerotic plaque forms while ‘Natural course of atherosclerotic plaque’ smooth muscle cells in the blood vessel lining become activated and migrate inward. Simultaneously, collagen deposition occurs, strengthening the plaque and containing the subendothelial inflammation. Once this occurs, the plaque ruptures and prothrombotic factors, such as tissue factor, are accessible by circulating fibrinogen and platelets. Moreover, fibrinolysis is inhibited by increased production of plasminogen activator inhibitor-1 (PAI-1). Thus, a pseudo-stable clot forms with resumption of the inflammatory cycle. Rupture of an atherosclerotic plaque is a dangerous event—such as those occurring within the internal carotid arteries—with a high chance of embolus formation—this is a primary cause of neurological stroke and functional deficit in patients. As an extension, smooth muscle cells were harvested from porcine aorta and incubated with tissue factor-targeted nanoparticles loaded with paclitaxel. Specific binding of the nanoparticles elicited a substantial reduction in smooth muscle cell proliferation; non-targeted paclitaxel loaded nanoparticle administration resulted in normal proliferation. More recent reports showed that intravenous administration of nanoparticles loaded with the antiangiogenic agent, fumagillin, targeted to aVb3-integrin epitopes on the vasa vasorum of growing plaques resulted in a clear inhibition of angiogenesis in cholesterol-fed rabbits. Kolodgie et al. utilized taxol-containing albumin nanoparticles to limit the restenotic response subsequent to angioplasty and stent placement in experimental animals.

3. Neurodegenerative diseases (Alzheimer’s disease andchelation)
Alzheimer’s disease (AD) is marked by a progressive and irreversible damage to memory, thought, and language. It is extremely prevalent and represents the most common form of dementia in geriatric populations over 65 years of age. Current therapies include acetylcholinesterase inhibitors,cholinesterase inhibitors, antioxidants, amyloid-b-targeted drugs, nerve growth factors, c-secretase inhibitors and vaccines against amyloid-b.Mounting evidence suggests that oxidative stress triggered by various mechanisms may be a primary factor in neurodegeneration in AD.Compared with other tissues, the central nervous system may be particularly susceptible to oxidative stress, especially those catalyzed by transition metals such as iron and copper via Fenton chemistry.In fact, iron metabolism has been shown to be involved in AD, as iron concentrations are elevated in patients with the disease.Moreover, aluminum has also been found in high concentrations in senile plaques and intraneuronal neurofibrillary tangles within the brain of AD patients. Unlike transition metals, aluminum is unable to participate in electron transfer reactions via redox cycling, as it assumes a fixed +3 oxidation state in biological systems. However, aluminum can act in synergy with iron to increase free radical damage.The promotion of oxidative damage by various metals in neurodegenerative diseases—such as AD—may represent a new target for drug design. In particular, chelation of these metals may reduce the pathophysiological development of AD. Metal chelators, such as desferrioxamine (DFO), have been used clinically. DFO has strong affinities for iron, aluminum, copper, and zinc; the affinity constants for Fe(III), Al(III), Cu(II), and Zn(II) are 30.6, 22.0, 14.1, and 11.1 (logK) respectively. Unfortunately, DFO exhibits serious toxicity, including neurotoxicity and neurological changes; it is poorly absorbed by the gastrointestinal tract and rapidly degrades following drug administration. Penetration through the BBB may also be an issue due to DFO’s hydrophilic nature, rendering it futile in neurodegenerative disease therapies. Polymeric nanoparticles may represent a potential means to transport drugs across the BBB. Nanoparticles may be designed to mimic LDL and interact with the LDL receptor, consequently triggering uptake by brain endothelial cells. Nanoparticles may effectively mask covalently bound chelators, thus facilitating their delivery past the BBB and minimize toxicity while improving the pharmacokinetics of the chelator itself. Seminal work by Liu et al. demonstrated the bidirectional transport of chelators into and from the brain.These chelators were synthetically optimized and examined in brain tissue sections from AD patients. The synthetic chelators removed iron from ferritin more efficiently than DFO and were capable of removing iron from the brain tissue sections. Conjugation of these synthetic chelators to nanoparticles was achieved via covalent bonding to amino and carboxyl groups on the nanoparticle surface. (7)

DRUG DELIVERY THERAPY OF NANOPARTICLES

1. Liposome-based Drug Delivery Therapy

Liposomes: Definition, Classification & Methods of Preparation
Liposomes are self-closed vesicular structures composed of phospholipids that entrap water in their interior. These structures result from self-assembly of the amphiphiles in an aqueous medium forming single or multiple concentric bilayers, where the polar headgroups are in contact with the aqueous media and the fatty acids form the hydrophobic core of the bilayers that are shielded from the water.

Liposomes are commonly prepared by hydration of a dry phospholipid film above the main phase transition temperature of the lipid. The diameter of liposomes ranges from 20 nm to several hundreds of nanometers, whereas the thickness of the phospholipid bilayer membrane is approximately 4–7 nm.The classification of liposomes is generally based upon their size and number of lipidic bilayers. Liposomes resultant from the thin film hydration method are multilamellar vesicles (MLVs), which consist of many concentric bilayers in a single particle with diameters that vary between a few hundred to thousands of nanometers. MLVs can be processed by sonication or by extrusion, through a filter, to form unilamellar vesicles (ULVs), which are liposomes with a single membrane bilayer. ULVs can be further classified, regarding their size, into small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs). Accordingly, SUVs show a diameter inferior to 100 nm while LUVs present a diameter superior to 100 nm. (8)

2. Targeted Drug Delivery for Cancer
Cancer drug delivery is no longer simply wrapping the drug in new formulations for different routes of delivery. Knowledge and experience from other technologies such as nanotechnology, advanced polymer chemistry, and electronic engineering, are being brought together in developing novel methods of drug delivery. Advances in our knowledge of molecular biology of cancer and pathways involved in malignant transformation of cells are revolutionizing the approach to cancer treatment with a focus is on targeted cancer therapy. There is a vast range of strategies available for drug delivery in cancer. It would be impossible to cover all of these in one issue of the Journal. TCRT has published several articles in this area over the past few years and this issue is devoted to these articles.

Targeted Drug Delivery
The current focus in development of cancer therapies is on targeted drug delivery to provide therapeutic concentrations of anticancer agents at the site of action and spare the normal tissues. Vasir and Labhasetwar present an overview of the problems related to targeted drug delivery

in cancer, and to provide an insight into the issues related to the development of targeted drug delivery systems for cancer . The authors have described several technologies for targeted drug delivery in cancer and suggest that combination of some of these approaches may provide solutions to some of the problems encountered.

Drug Delivery Using Monoclonal Antibodies
Monoclonal antibodies (MAbs) are used both for diagnosis and therapy in cancer. Several MAbs are in the market for cancer therapy. MAbs are being paired with powerful toxins and radiopharmaceuticals to create specific agents that seek out cancer cells and kill them. Govindhan et al. describe targeted cancer therapy with radiolabeled and drug/toxin-conjugated

MAbs and methods of producing these conjugates (6). The clinical potential of these therapies in hematological malignancies is promising. For the treatment of solid tumors, the authors suggest application of combination therapies and use in residual disease rather than in bulky tumors. Bethge and Sandmaier have shown how radioimmunotherapy combines the advantages of targeted radiation therapy and specific immunotherapy using MAbs to target tumor cells (7). Radiolabeled MAbs enable the reduction of toxicity of conventional strategies of radiation therapy and enhance the efficacy of MAbs. The authors provide an overview of available radionuclides and radioimmunoconjugates and discusses clinical results in hematological malignancies.(9)

3. The Potential Advantages of Nanoparticle Drug Delivery Systems in Chemotherapy of Tuberculosis

ORAL ADMINISTRATION OF NANOPARTICLE-BASED TB DRUGS
Stability of nanoparticles offers the possibility of oral administration. The fate of nanoparticles in the gastrointestinal tract has been investigated in a number of studies). In general, the uptake of nanoparticles occurs as follows:
(1) by transcytosis via M cells, (2) by intracellular uptake and transport via the epithelial cells lining the intestinal mucosa, (3) by uptake via Peyer's patches.

Pandey and colleagues demonstrated that the nanoparticles provided sustained release of the anti-TB drugs and considerably enhanced their efficacy after oral administration. Three frontline drugs, rifampin (RMP), isoniazid (INH), and pyrazinamide (PZA) were coencapsulated in poly(lactide-co-glycolide) (PLG) nanoparticles. After a single oral administration of this formulation to mice, the drugs could be detected in the circulation for 4 d (RMP) and 9 d (INH and PZA); therapeutic concentrations in the tissues were maintained for 9 to 11 d. In contrast, free (unbound) drugs were cleared from the plasma within 12 to 24 h after administration. Treatment of M. tuberculosis–infected mice with the nanoparticle-bound drugs (five oral doses every 10th day) resulted in complete bacterial clearance from the organs. Free drugs were able to produce bacterial clearance only after daily administration of 46 doses. Similar efficacy of the nanoparticle-bound drugs was also observed in guinea pigs

At the same time, incorporation in microparticles was less effective: their drug-loading capacity was lower as well as the plasma half-life of the bound drugs

Accordingly, the efficacy of PLG-based formulations of anti-TB drugs were further improved by covalent attachment of wheat germ agglutinin .Oral administration of wheat germ agglutinin– coated PLG nanoparticles loaded with RIF, INH, and PZA in mice produced considerably extended serum half-life: detectable RIF serum levels were observed for 6 to 7 d and INH and PZA for 13 to 14 d (vs. 4–6 d and 8–9 d for nonmodified nanoparticles). All three drugs were present in lungs, liver, and spleen for 15 d. The lectin-modified formulations produced bacterial clearance in these organs after three oral doses administered every 14 d (vs. 45 daily doses of free drugs). As suggested by the authors, the prolonged circulation of drugs encapsulated in wheat germ agglutinin–grafted nanoparticles might be attributed to the fact that lectins enhance prolonged adhesion of the particles to the intestinal surface to allow (1) an increase in the time interval available for absorption and (2) a localized increase in the concentration gradient between luminal and serosal sides of the membrane. (38-40)

POTENTIAL FOR THE INHALATION FORM APPLICATION
The potential advantages of direct delivery of the TB drug to the lungs include the possibility of reduced systemic toxicity, as well as achieving higher drug concentration at the main site of infection. Moreover, in contrast to the oral route of administration, inhaled drugs are not subjected to first-pass metabolism. A possible obstacle to using nanocarriers for pulmonary delivery is that their mass median aerodynamic diameter, an essential parameter for the particle deposition in the lungs, is often too small.

Administration to infected guinea pigs of nebulized RMP, INH, and PZA coencapsulated in wheat germ agglutinin–functionalized PLG nanoparticles was even more effective: three doses administered fortnightly for 45 d were sufficient to produce a sterilizing effect in lungs and spleen .

INTRAVENOUS ADMINISTRATION
In contrast to microparticles with a diameter of more than 1 μm that cannot be administered via intravascular routes, nanoparticles are small enough to allow intracapillary passage followed by an efficient cellular uptake. When administered intravenously, the nanoparticles follow the route of other foreign particulates, including intracellular pathogens. They are endocytosed by resident macrophages of the mononuclear phagocyte system and by circulating monocytes. On the other hand, in the case of infections caused by intracellularly persisting microbes (e.g., Brucella, Salmonella, Listeria, Mycobacterium), macrophages become reservoirs for pathogens, thus representing one of the targets for delivery of antimicrobial agents.

Preferential uptake of nanoparticles by macrophages (mainly by Kupffer cells in the liver) is achieved by the physicochemical properties of the carrier and by physiologic opportunity, thus representing an example of passive delivery. This technology improves drug delivery to macrophages, increasing the amount of the drug reaching this target site, which allows reduction of the overall therapeutic dose and decrease of the adverse effects. Accordingly, the enhanced efficacy of the nanoparticle-bound antibiotics was demonstrated in a number of experimental infections .

Clofazimine, a riminophenazine compound, is an agent considered for treating patients with M. avium infection. However, use of this drug was restricted because of its poor solubility. A relatively new approach was applied to solve the problem: clofazimine was formulated as a nanosuspension consisting only of the drug and a minimum amount of surfactants (particle size, 385 nm). Intravenous injection of the nanocrystalline formulation of clofazimine resulted in a considerable reduction of bacterial loads in the liver, spleen, and lungs of mice infected with M. avium.This result correlated with the pharmacokinetic data: drug concentrations in these organs reached high concentrations, well in excess of the minimal inhibitory concentration for most M. avium strains. Interestingly, the effects of the nanocrystalline formulation of clofazimine were similar to those of the liposomal formulation used as a control in this study. This study is a vivid example of application of nanotechnology for overcoming the solubility problems of poorly soluble drugs. (10)

4. Nanotechnology approaches to crossing the blood-brain barrier and drug delivery to the CNS

Introduction
An ability to cross the blood-brain barrier (BBB) to deliver drugs or other molecules (for example, oligonucleotides, genes, or contrast agents) while potentially targeting a specific group of cells (for instance, a tumor) requires a number of things to happen together. Ideally, a nanodelivery-drug complex would be administered systemically (for example, intravenously) but would find the CNS while producing minimal systemic effects, be able to cross the BBB and correctly target cells in the CNS, and then carry out its primary active function, such as releasing a drug. These technically demanding obstacles and challenges will require multidisciplinary solutions between different fields, including engineering, chemistry, cell biology, physiology, pharmacology, and medicine. Successfully doing so will greatly benefit the patient.

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Applications to drugs and other molecules
A significant amount of work using nanotechnological approaches to crossing the BBB has focused on the delivery of antineoplastic drugs to CNS tumors. For example, radiolabeled polyethylene glycol (PEG)-coated hexadecylcyanoarcylate nanospheres have been tested for their ability to target and accumulate in a rat model of gliosarcoma [1]. Another group has encapsulated the antineoplasitc drug paclitaxel in polylactic co-glycolic acid nanoparticles, with impressive results. In vitro experiments with 29 different cancer cell lines (including both neural and non-neural cell lines) demonstrated targeted cytotoxicity 13 times greater than with drug alone [2]. Using a variety of physical and chemical characterization methods, including different forms of spectroscopy and atomic force microscopy, the investigators showed that the drug was taken up by the nanoparticles with very high encapsulation efficiencies and that the release kinetics could be carefully controlled. Research focusing on the delivery of many of the commonly used antineoplastic drugs is important because most of these drugs have poor solubility under physiologic conditions and require less than optimal vehicles, which can produce significant side effects.

The delivery of other drugs is also being investigated. Dalargin is a hexapeptide analog of leucine-enkephalin containing D-alanine, which produces CNS analgesia when it is delivered intracerebroventricularly, but it has no analgesic effects if it is administered systemically, specifically because it cannot cross the BBB on its own .[3H]Dalargin was conjugated to the same poly(butylcyanoacrylate) nanoparticles described above, injected systemically into mice, and demonstrated by radiolabeling to cross the BBB and accumulate in brain .Other, similar studies have also demonstrated delivery of dalargin using polysorbate 80-coated nanoparticles .Other polysorbate 80 nanoparticles have been chemically conjugated to the hydrophilic drug diminazenediaceturate (diminazene) and proposed as a novel treatment approach for second stage African trypanosomiasis .In other work, PEG-treated polyalkylcyanoacrylate nanoparticles were shown to cross the BBB and accumulate at high densities in the brain in experimental autoimmune encephalomyelitis, a model of multiple sclerosis. (11)

5. Gold nanoparticles help earlier diagnosis of liver cancer

Cancer spotters A new diagnostic technique can spot tumor-like masses as small as 5 millimeters in the liver. Gold nanoparticles with a polyelectrolyte coating can make smaller tumors more visible through X-ray scatter imaging, enabling earlier diagnosis.(12)

Applications and potential benefits
With nanotechnology, a large set of materials with distinct properties (optical, electrical, or magnetic) can be fabricated. Nanotechnologically improved products rely on a change in the physical properties when the feature sizes are shrunk. Nanoparticles for example take advantage of their dramatically increased surface area to volume ratio. Their optical properties, e.g. fluorescence, become a function of the particle diameter. When brought into a bulk material, nanoparticles can strongly influence the mechanical properties, such as the stiffness or elasticity. Example, traditional polymers can be reinforced by nanoparticles resulting in novel materials e.g. as lightweight replacements for metals. Therefore, an increasing societal benefit of such nanoparticles can be expected.

1. Medicine
The biological and medical research communities have exploited the unique properties of nanomaterials for various applications (e.g., contrast agents for cell imaging and therapeutics for treating cancer). Terms such as biomedical nanotechnology, bionanotechnology, and nanomedicine are used to describe this hybrid field.Functionalities can be added to nanomaterials by interfacing them with biological molecules or structures. The size of nanomaterials is similar to that of most biological molecules and structures; therefore, nanomaterials can be useful for both in vivo and in vitro biomedical research and applications.Thus far, the integration of nanomaterials with biology has led to the development of diagnostic devices, contrast agents, analytical tools, physical therapy applications, and drug-delivery vehicles.

2. Diagnostics
Nanotechnology-on-a-chip is one more dimension of lab-on-a-chip technology. Biological tests 1measuring the presence or activity of selected substances become quicker, more sensitive and more flexible when certain nanoscale particles are put to work as tags or labels. Magnetic nanoparticles, bound to a suitable antibody, are used to label specific molecules, structures or microorganisms. Gold nanoparticles, tagged with short segments of DNA can be used for detection of genetic sequence in a sample. Multicolor optical coding for biological assays has been achieved by embedding different-sized quantum dots, into polymeric microbeads. Nanopore technology foranalysis of nucleic acids converts strings of nucleotides directly into electronic signatures.

3. Drug delivery
The overall drug consumption and side-effects can be lowered significantly by depositing the active agent in the morbid region only and in no higher dose than needed. This highly selective approach reduces costs and human suffering.A targeted or personalized medicine reduces the drug consumption and treatment expenses resulting in an overall societal benefit by reducing the costs to the public health system.

4. Tissue engineering
Nanotechnology can help to reproduce or to repair damaged tissue. This so called “tissue engineering” makes use of artificially stimulated cell proliferation by using suitable nanomaterial-based scaffolds and growth factors. Tissue engineering might replace today’s conventional treatments, e.g. transplantation of organs or artificial implants. On the other hand, tissue engineering is closely related to the ethical debate on human stem cells and its ethical implications.

5. Chemistry and environment
Chemical catalysis and filtration techniques are two prominent examples where nanotechnology already plays a role. The synthesis provides novel materials with tailored features and chemical properties e.g. nanoparticles with a distinct chemical surrounding (ligands) or specific optical properties. In this sense, chemistry is indeed a basic nanoscience. In a short-term perspective, chemistry will provide novel “nanomaterials” and in the long run, superior processes such as “self-assembly” will enable energy and time preserving strategies.In a sense, all chemical synthesis can be understood in terms of nanotechnology, because of its ability to manufacture certain molecules. Thus, chemistry forms a base for nanotechnology providing tailor-made molecules, polymers etc. and furthermore clusters and nanoparticles.

6. Filtration
A strong influence of nanochemistry on waste-water treatment, air purification and energy storage devices is to be expected. Mechanical or chemical methods can be used for effective filtration techniques. One class of filtration techniques is based on the use of membranes with suitable hole sizes, whereby the liquid is pressed through the membrane. Nanoporous membranes are suitable for a mechanical filtration with extremely small pores smaller than 10 nm (“nanofiltration”). Nanofiltration is mainly used for the removal of ions or the separation of different fluids. On a larger scale, the membrane filtration technique is named ultrafiltration, which works down to between 10 and 100 nm. One important field of application for ultrafiltration is medical purposes as can be found in renal dialysis.

Magnetic nanoparticles offer an effective and reliable method to remove heavy metal contaminants from waste water by making use of magnetic separation techniques. Using nanoscale particles increases the efficiency to absorb the contaminants and is comparatively inexpensive compared to traditional precipitation and filtration methods.

7. Energy
The most advanced nanotechnology projects related to energy are: storage, conversion, manufacturing improvements by reducing materials and process rates, energy saving e.g. by better thermal insulation, and enhanced renewable energy sources.

8. Reduction of energy consumption
A reduction of energy consumption can be reached by better insulation systems, by the use of more efficient lighting or combustion systems, and by use of lighter and stronger materials in the transportation sector. Currently used light bulbs only convert approximately 5% of the electrical energy into light. Nanotechnological approaches like light-emitting diodes (LEDs) or quantum caged atoms (QCAs) could lead to a strong reduction of energy consumption for illumination.

9. Recycling of batteries
Because of the relatively low energy density of batteries the operating time is limited and a replacement or recharging is needed. The huge number of spent batteries and accumulators represent a disposal problem. The use of batteries with higher energy content or the use of rechargeable batteries or supercapacitors with higher rate of recharging using nanomaterials could be helpful for the battery disposal problem.

10. Information and communication
Current high-technology production processes are based on traditional top down strategies, where nanotechnology has already been introduced silently. The critical length scale of integrated circuits is already at the nanoscale (50 nm and below) regarding the gate length of transistors in CPUs or DRAM devices.

11. Novel semiconductor devices
An example of such novel devices is based on spintronics. The dependence of the resistance of a material (due to the spin of the electrons) on an external field is called magnetoresistance. This effect can be significantly amplified (GMR - Giant Magneto-Resistance) for nanosized objects, for example when two ferromagnetic layers are separated by a nonmagnetic layer, which is several nanometers thick (e.g. Co-Cu-Co).

The GMR effect has led to a strong increase in the data storage density of hard disks and made the gigabyte range possible. The so called tunneling magnetoresistance (TMR) is very similar to GMR and based on the spin dependent tunneling of electrons through adjacent ferromagnetic layers.

Both GMR and TMR effects can be used to create a non-volatile main memory for computers, such as the so called magnetic random access memory or MRAM.

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12. Novel optoelectronic devices
In the modern communication technology traditional analog electrical devices are increasingly replaced by optical or optoelectronic devices due to their enormous bandwidth and capacity, respectively. Two promising examples are photonic crystals and quantum dots.

Photonic crystals are materials with a periodic variation in the refractive index with a lattice constant that is half the wavelength of the light used. They offer a selectable band gap for the propagation of a certain wavelength, thus they resemble a semiconductor, but for light or photons instead of electrons.

13. Displays
The production of displays with low energy consumption could be accomplished using carbon nanotubes (CNT). Carbon nanotubes can be electrically conductive and due to their small diameter of several nanometers, they can be used as field emitters with extremely high efficiency for field emission displays (FED). The principle of operation resembles that of the cathode ray tube, but on a much smaller length scale. (13)

Toxicological hazards of nanoparticles

General concepts
To use the potential of Nanotechnology in Nanomedicine, full attention is needed to safety and toxicological issues. For pharmaceuticals specific drug delivery formulations may be used to increase the so called therapeutic ratio or index being the margin between the dose needed for clinical efficacy and the dose inducing adverse side effects (toxicity). However, also for these specific formulations a toxicological evaluation is needed. This is particularly true for the applications of nanoparticles for drug delivery. In these applications particles are brought intentionally into the human body and environment, and some of these new applications are envisaged an important improvement of health care .Opinions started to divert when toxicologists claimed that new science, methods and protocols are needed .However, the need for this is now underlined by several and more importantly by the following concepts:

Nanomaterials are developed for their unique (surface) properties in comparison to bulk materials. Since surface is the contact layer with the body tissue, and a crucial determinant of particle response, these unique properties need to be investigated from a toxicological standpoint. When nanoparticles are used for their unique reactive characteristics it may be expected that these same characteristics also have an impact on the toxicity of such particles. Although current tests and procedures in drug and device evaluation may be appropriate to detect many risks associated with the use of these nanoparticles, it cannot be assumed that these assays will detect all potential risks. So, additional assays may be needed. This may differ depending on the type of particles used, ie, biological versus non-biological origin.
Nanoparticles are attributed qualitatively different physico-chemical characteristics from micron-sized particles, which may result in changed body distribution, passage of the blood brain barrier, and triggering of blood coagulation pathways. In view of these characteristics specific emphasis should be on investigations in (pharmaco)kinetics and distribution studies of nanoparticles. What is currently lacking is a basic understanding of the biological behavior of nanoparticles in terms of distribution in vivo both at the organ and cellular level.
Effects of combustion derived nanoparticles in environmentally exposed populations mainly occur in diseased individuals. Typical pre-clinical screening is almost always done in healthy animals and volunteers and risks of particles may therefore be detected at a very late stage.

The use of nanoparticles as drug carrier may reduce the toxicity of the incorporated drug. In general the toxicity of the whole formulation is investigated while results of the nanoparticles itself are not described. So, discrimination between drug and nanoparticle toxicity cannot be made. So, there should be a specific emphasis on the toxicity of the “empty” non-drug loaded particles. This is especially important when slowly or non degradable particles are used for drug delivery which may show persistence and accumulation on the site of the drug delivery, eventually resulting in chronic inflammatory reactions.(14)

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