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)


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

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)

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 .

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

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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