Nanoparticle-Based Brain Targeted Delivery Systems

Presenting a very dismal prognosis, glioblastomas present as one of the most malignant brain tumors [1]. In addition to its low prognosis, glioblastomas account for ~70% of all astrocytic and oligodendroglial tumors, making this one of the most common types of brain tumors [1]. Through the mutations of multiple pro-oncogenic genes, primary glioblastomas develop as other neoplasms but present as “full-blown tumors” upon diagnosis, designating their development as de novo [1]. In contrast, because secondary glioblastomas develop through the progression of multiple low-grade and less malignant astrocytomas, they tend to differ from primary glioblastomas in development, the expression of various proteins, and responses to therapy [1]. Attempts at therapeutic intervention fall prey to blockages by the blood-brain barrier (BBB).


Glioblastomas
Presenting a very dismal prognosis, glioblastomas present as one of the most malignant brain tumors [1]. In addition to its low prognosis, glioblastomas account for ~70% of all astrocytic and oligodendroglial tumors, making this one of the most common types of brain tumors [1]. Through the mutations of multiple pro-oncogenic genes, primary glioblastomas develop as other neoplasms but present as "full-blown tumors" upon diagnosis, designating their development as de novo [1]. In contrast, because secondary glioblastomas develop through the progression of multiple low-grade and less malignant astrocytomas, they tend to differ from primary glioblastomas in development, the expression of various proteins, and responses to therapy [1]. Attempts at therapeutic intervention fall prey to blockages by the blood-brain barrier (BBB).

The Blood-Brain Barrier
The BBB is an anatomic barrier to the brain developed by the coordinated function of multiple cell types aiming to limit the penetration of harmful substances to the brain. Microvascular endothelium, basement membrane, and glial cells such as astrocytes and pericytes work together to form the BBB [2]. The tightness of the endothelial cells lining the brain capillaries forms tight junctions, limiting substances crossing this barrier into the brain [2]. However, the cytoplasm of the microvascular endothelial cells contain pinocytic vesicles and high number of mitochondria for the active transport of certain molecules between the blood and brain [2]. However, the BBB holds a number of transport proteins that are essential for the permeation of molecules essential to proper brain health. An example of this is the glucose transporter (GLUT) [2]. It is possible that therapeutic compounds can be modified to be recognized by the GLUT protein so that they can be transported into the brain endothelium. However, the compound attempting to be transported across the BBB needs to be able to bypass the multidrug resistance protein, MDR1, an efflux transporter that limits the accumulation of certain compounds in the brain [2]. Given the presence of this highly selective barrier, the development of certain therapies that can bypass the BBB and efficiently deliver drugs to affected areas would be a huge gain towards the development of effective therapies against glioblastomas and other brain disorders ( Figure 1) represents a model of the BBB.

Manipulating Nanoparticles for BBB Permeability
The relatively-recent development of nanomedicines, specifically nanoparticles,provides a new platform upon which to develop potential therapies to pass through the BBB. Nanoparticles are engineered to have at least one functional dimension in the nanometer scale range (10 -9 meter). At this size range, a number of distinct properties in the nanoparticles emerge; these properties are markedly different than those that would be apparent at a larger scale, possibly due to quantum effects at the nanoscale. The development of nanomedicine technologies opens the door to the potential delivery of a number of therapeutic compounds across the BBB by manipulating the engineered conditions of nanoparticles. However, for nanoparticle technologies to be effective in targeting brain tissue, three main criteria have to be met: If the nanoparticle therapy is systemically administered, it would need to effectively find BBB without adversely effecting other cell types; It would have to be able to cross the BBB; and It would have to be able to effectively target the appropriate cells after BBB translocation to release the therapeutic compound [ 3 ] .

PEG-coated nanoparticles
Poly(ethylene glycol) (PEG) ( Figure 1A) acts as a potential hydrophilic cover for hydrophobic nanoparticles to increase particles' aqueous solubility and systemic retention in a nontoxic way [4]. By being complexed with nanoparticle carriers, PEG allows the nanoparticles to cross the BBB through interacting with microvascular transport proteins such as apolipoproteinE and utilizing the lowdensity lipoprotein (LDL) receptor-mediated pathway [5,6]. PEG may be complexed with PLGA nanoparticles to act as a cover to the hydrophobic PLGA core [6]. The hydrophilic surface of the PEG-PLGA nanoparticles reduces its clearance from the blood and significantly enhances its circulation rate by eluding opsonization and phagocytosis [6]. PEGylatingliposome vectors that target the brain's insulin receptor shows promising results for gene therapies [7], as studies in human and rat glioma cells have shown that targeting the insulin receptor yields 100-200 fold higher levels of gene expression as compared to targeting the human epidermal growth factor receptor (EGFR) or the rat transferrin receptor [8]. In addition, hexadecylcyanoacrylate nanoparticles coated with PEG were shown to target rat gliosarcoma cells and accumulate within the cells [9]. PEGylated micelles were shown to accumulate into rat brain glioma models [10].Furthermore, PEG-complexed nanoparticles were shown to permeate the BBB to therapeutically effect models of multiple sclerosis [3,11] and Parkinson's disease [12] among other diseases. (Figure 2) presents an example of a PEGylated PLGA nanoparticle ready to be used for therapeutic studies.

Albumin-coated nanoparticles
Human serum albumin is one of the most common proteins found in the blood. Nanoparticles, when entering systemic circulation, are commonly surrounded by a protein "coat, " known as a corona, most commonly composed of albumin [13]. Studies have shown that albumin-and albumin-coated nanoparticles ( Figure 1B) are able to cross the BBB [14,15] and are able to release doxorubicin in in vitro neuroblastoma cell models [15].

Transferrin-coated nanoparticles
The cells lining the BBB express transferrin-receptors, making these types of nanoparticles very appealing in delivering drugs across the BBB [16]. PLGA nanoparticles coated with transferring ( Figure  1C) and loaded with doxorubicin and paclitaxel were evaluated in in vitro-and in vivo-glimoa models to show enhanced inhibition of tumor growth [16].

Glutathione-coated nanoparticles
Glutathione is an antioxidant that protects cells from toxins created through oxidative stresses [17]. PLGA nanoparticles, coated with glutathione ( Figure 1D) and loaded with paclitaxel, were shown to inhibit the growth of an in vitro rat glioma cell model (RG-2) as well as translocate the BBB in in vivo models to inhibit glioma cell growth [17]. (Figure 3) shows a TEM image of the cellular uptake of glutathionecoated PLGA nanoparticles in a RG-2 cell model. The nanoparticles release the drug once they are incorporated into the cell, inducing cytotoxicity in the glioma model.

Thiamine-coated nanoparticles
Thiamine is a compound required by cells for proper function, growth, and development [18]. Because all eukaryotic cells have receptors for the uptake of thiamine, the association of thiamine ligands on nanoparticles make for a suitable candidate for the development of therapies that cross the BBB [18]. Studies show that thiamine-coated nanoparticles ( Figure 1E) show specificity for endothelial cells and accumulate at the BBB, diffusing across in a time-dependent manner [18]. The encapsulation of drugs within the nanoparticle could enhance the delivery of drugs across the BBB. However, because the uptake of the thiamine-coated nanoparticles is dependent on the thiamine receptor, the presence of thiamine in systemic circulation presents as a possible source of competitive inhibition in the uptake of thaminecoated nanoparticles [18].

Polysorbate-80-coated nanoparticles
Coating nanoparticles with Polysorbate-80 (PS80) ( Figure 1F) is a highly popular method to induce its transport across the BBB. Mechanistic studies with PS80 show that its transport across the BBB is due to its interactions with apolipoproteins B, E, and possibly A-1 to be transported into endothelial cells by receptor-mediated endocytosis [2,3]. Studies with temozolomide-loaded, PS80-coated, PLGA nanoparticles in an in vitro rat glioma (C6) cell model showed a decreased cell viability in the presence of the nanoparticles [19]. In addition, in vivo studies have also shown the effectiveness of PS80 coatings on the BBB transport of drug-loaded nanoparticles. Gulyaev   showed that carotid intravenous injections of doxorubicin-loaded, PS80-coated nanoparticles were found to be present in the brain two hours after injection, showing their ability to translocate the BBB into the brain in an in vivo rat model [20]. Many such studies have led to the popularity of PS80 as a nanoparticle-modifying agent, making it a "gold standard" in brain drug delivery [6].

Conclusion and Future Considerations
The development of biodegradable, nanoparticle drug-vectors is essential for brain therapies to prevent the toxic effects of nonbiodegradable nanoparticles such as quantum dots and carbon nanotubes [6]. As previously mentioned, there are a number of different, modified-nanoparticle vectors that are able to translocate into the BBB to deliver their drugs in a localized way to the effected tissues. Of the mentioned brain delivery systems, the glutathione-and thiaminecoated methods seem the most promising. PS80 tends to produce cytotoxic effects in treated cells, and albumin-and transferrin-coated nanoparticles show less efficient BBB translocation as compared to glutathione-and thiamine-coated nanoparticles. The unique properties of each nanoparticle carrier, along with the drug it encapsulate and its surface modification with PEG to induce BBB permeability, has the potential to expand current brain therapeutics. Further work using in vitro and in vivo models will help advance the science of brain therapy and help the scientific community in reducing the staggering number of deaths brought about each year by gliomas and other degenerative brain disorders.