INTRODUCTION:

Blood brain barrier structural and functional barrier, which impedes, and regulates the influx of most compounds from blood to brain formed by brain microvascular endothelial cells (BMEC), Astrocyte end feet, Pericytes, Regulates passage of molecules in and out of brain to maintain neutral environment. Responsible for metabolic activities such as the metabolism of L-dopa to regulate its concentration in the brain. The blood brain barrier is a highly sophisticated organ that acts as the biological equivalent of a computer firewall: it selectively allows nutrients into the brain, while keeping out harmful components [1].

The blood brain barrier function results from a combination of this:

A physical barrier:

Tight junctions between cell reducing flux via the intercellular cleft or paracellular pathway.

A transport barrier:

A specific transport mechanism mediating solute flux and the targeted transport mechanism provided that the endogenous function of the transporter and its endogenous ligand (s) is not influenced by the technology in a major way.

b. Blood brain barrier physiology:

The blood brain barrier blocks all molecules except those that cross cell membranes by means of lipid solubility (such as oxygen, carbon dioxide, and ethanol). Despite weighing only about 3 pounds, the brain consumes as much as 20% of the oxygen and glucose taken in by the body. Nervous tissue in the brain has a very high metabolic rate due to the sheer number of decisions and processes taking place within the brain at any given time. This organ is protected against various harmful substances due to the presence of two types of barriers: the blood-brain barrier and the blood cerebrospinal fluid barrier [2]. Fig.1 represents the blood brain barrier, CSF, and brain-CSF. Many drugs are unable to pass the barrier since 98% of them are heavier than 500 Dalton hormones generally do not penetrate the brain from the blood except at the “circum ventricular organs.’

MECHANISM TO OVERCOME BBB:

The BBB is the most restrictive membrane in the body. As far as small molecule drugs are concerned, more than 98 % cannot enter the brain [3]. In fact, only a few class of drugs and small lipophilic molecules with low molecular mass (<400–500 Da) actually cross the BBB [4]Hence, the structural compositions, the factors able to regulate BBB permeability, and their functions are important to understand – in order to overcome this barrier and achieve brain to treat diseases [4]. The various systems that mediate the transport across BBB can be divided into three categories – small molecule, large molecule, and efflux transporters (Fig. 1). Within the small molecule transporters, there are two possibilities, the diffusion

Fig.1.

Fig. 1 Different type of blood brain barrier (BBB) transporters. The scheme is divided into three large groups. The first group is about small molecule transporters. The second group is about macromolecule transporters. And the last group is about active efflux transporters (AET). Each group is divided in various small groups which have different biologic and physical characteristics – diffusion and carrier-mediated transport (CMT); receptor-mediated transcytosis (RMT), adsorptive-mediated transcytosis (AMT), and cell-mediated transcytosis (CMT); adenosine triphosphate-binding cassette (ABC) transporters transport – simply diffusion or facilitated transport through aqueous channels – and the active transport, mediated by a carrier like proteins (carrier-mediated transport, CMT). In the first, the passage of molecules across the EC of the BBB can occur between adjacent cells (the paracellular pathway) or through the cells (the transcellular pathway). Regarding passive diffusion, Lipinski and coresearchers developed five rules that determine if a compound is more likely to be membrane permeable and easily absorbed by the body. In order to achieve his goal, Lipinski analyzed the physicochemical properties of more than 2,000 drugs and candidate drugs in clinical trials. His work resulted in the establishment of five criteria that must be fulfilled by the compounds. These are: no more than five hydrogen bond donors (nitrogen or oxygen atoms with one or more hydrogen atoms); no more than ten hydrogen bond acceptors (nitrogen or oxygen atoms); a molecular mass lower than 500 Da; an compound’s lipophilicity, expressed as a quantity known as log P (the logarithm of the partition coefficient between water and 1-octanol) lower than 5, and compound classes that are substrates of biological transporters are exceptions to the rule [5,6,7]. It is important to state that the rule of five is applied only to absorption by passive diffusion of compounds through cell membranes; compounds that are actively transported through cell membranes by transporter proteins are exceptions to the rule. Therefore, it is of limited significance nowadays [6,8].

Lastly, macromolecule transporters include receptor-mediated transcytosis (RMT), adsorptive-mediated transcytosis (AMT), and cell-mediated transcytosis. RMT occurs in three steps: first the endocytosis of macromolecules which bind to a receptor on the endothelial surface of BBB, followed by diffusion across the endothelium and further exocytosis on the other site [9]. ECs comprise different receptors, such as transferring receptor [10], insulin receptor [11], lipoprotein receptors, and insulin-like growth factors [12]. AMT, also named pinocytosis, is mediated by electrostatic interactions between ligands (positively charged) and BBB membranes (negatively charged) [13]. The last one refers only to immune cells transport [14].

INFORMATION RELATED TO NANOPARTICLES:

The small size promotes the NPs penetration to the BBB and facilitates drug delivery across the barrier [14]. Usually NPs are also non-toxic, biodegradable, and biocompatible; stable in blood (no opsonization by proteins); they do not induce activation of neutrophils (non-inflammatory reaction) or platelet aggregation; they avoid reticulo endothelial systems and as a consequence increased their circulation time (clearance protection); regarding manufacturing process, they are cost effective; willing to small molecules, peptides, proteins, or nucleic acids [15]. Simultaneously, NPs should also retain the drug stability and ensure that early degradation of drugs does not occur. Many factors such as target-specific affinity and ability to facilitate controlled and slow drug release (or modulated release profiles) need to be considered during manufacture of the NPs (Fig. 2.).

Fig. 2. Nanoparticles’ properties and how they can be used to do brain targeting and delivery

PREPARATION OF NANOPARTICLES:

There are different type of nanoparticles used to target the brain such as magnetic, solid-lipid, polymeric nanoparticles. (Fig. 3) represents the various prepartion of these nanoparticles are as follows.

Fig.3.various methods of preparations of nanoparticles

Method of preparation of nanoparticle:

1. solvent emulsification method:

The solid lipid nanoparticles (SLNs) were prepared by a solvent emulsification method. Glyceryl monostearate (GMS) was dissolved in organic phase (ethanol) by heating at 70-80°C add in to the solution. The prepared solution was added to a hot poloxamer 407 (P407) solutions (5°C above melting point of the lipid) under stirring using a polytron homogenizer at 15,000 rpm for 15 minutes. The resultant dispersion was immediately sonicated using a probe sonicator at amplitude of 50% with the pulse of 4 sec intervals. After probe sonication the organic solvents present in the solution was evaporated using rotavapour for 2 hours at 80 rpm. The prepared SLN samples were freeze dried at -48 °C for 24 hours to yield dry powder.

2. High Shear Homogenization and/or Ultrasound:

High shear homogenization and ultrasonication are dispersing techniques. (16) described the use of high sheer homogenization followed by ultrasonication to prepare lipid nano pellets as an oral drug carrier. This method primarily involves heating of a solid lipid to approximately 5_10_C above its melting point Following this process, drug dispersed in melted lipid phase is homogenized by a rotor-stator homogenizer with a hot surfactant water solution to obtain a nanoemulsion that is then cooled to form SLN (17). The lipid nano pellets obtained had an average particle diameter of 80_800 nm and were suitable for per oral administration.

Homogenization and ultrasound technique (16). Process parameters that can affect particle size are: emulsification time, stirring rate, and cooling conditions .The simplicity of this production technique and the use of simple instruments make this method attractive; in this method the use of a high concentration of the surfactant is always disadvantageous .The main possible disadvantages of this method include high energy input, broad particle size distribution, and potential damage to sensitive biomolecules. When sonication is used, potential metal contamination and temperature increase have to be considered (16).

3. Lyophilization:

Aqueous dispersions of NP may not be stable physically for a long period of time; moreover, drug release properties may be altered on storage. To avoid these problems, it is necessary to convert such aqueous dispersions into dry product by lyophilization or spray drying Lyophilization (freeze drying) involves the process of removal of water through sublimation using reduced pressure (17). The protective effect of the surfactant can be compromised by Lyophilization. It has been discovered that the lipid content of the SLN dispersion should not exceed 5% to prevent an increase in particle size (18). The dried SLNs produced through the Lyophilization method remain stable in the range of 12_36 months. Transformation into a solid form will prevent Ostwald ripeningand avoid hydrolysis reactions (18). Lyophilization also offers real possibilities for SLN incorporation into pellets, tablets, or capsules (17).

4. Phase Inversion Temperature Technique:

Emulsification by the phase inversion temperature (PIT) method was first reported in 1968 by schinodha. The basic principle behind the method is the ability of poly ethoxylated surfactants to change their affinities toward oil and water as a function of the temperature. The transformation of an o/w type to a w/o type of emulsion is termed “phase inversion,” which can be induced by changing the temperature and the temperature at which the inversion occurs is referred to as the PIT. Heurtault et al. developed a novel solvent-free technique for the formulation of lipid nanocapsules that was based on the phase inversion of an emulsion. In this PIT technique the formulation ingredients (i.e., lipid, surfactant, drug, and water) are thoroughly mixed under constant magnetic agitation. The mixture is then subjected to three cycles of heating and cooling (from room temperature to 85_C [the PIT], to 60_C to 85_C to 60_C to 85_C to room temperature) applied at a constant rate of 4_C/min. In the final step, the emulsion is diluted under cooling conditions.

5. Dispersion of Preformed Polymer:

Dispersion of drug in preformed polymers is a common technique used to prepare biodegradable nanoparticles from poly (D, L-glycolide) (PLG), poly (lactic acid) (PLA), poly (cyanoacrylate) (PCA) and poly (D, L-lactide-co-glycolide) (PLGA).

6. Preparation by w/o/w Double-Emulsion Method:

The production of lipospheres by the double-emulsion method was first described [6]. The double-emulsion method was introduced to solubilize hydrophilic drugs in the internal water phase of a w/o/w emulsion. In a w/o/w double-emulsion procedure, an aqueous solution of drug is emulsified in molten lipid to give a primary w/o emulsion and stabilized by adding stabilizers such as gelatin or poloxamer in the aqueous phase. Subsequent dispersion of the primary emulsion in a second aqueous solution of stabilizer under constant stirring generates a w/o/w double emulsion. Constant stirring for longer periods leads to precipitation of SLNs (5). The instabilities associated with multiple emulsions have been their most striking drawbacks. Coalescence of the internal aqueous droplets within the oil phase, coalescence of the oil droplets, and splitting of the surface layer of the internal droplets are major instabilities that occur in the final products prepared using this technique. (19)

Characterization of Nanoparticles:

some of the characterization of Nanoparticles is as follows

1. Particle size and zeta potential measurements:

The particle size and surface charge of nanoparticles represent the two most important factors determining the capacity to cross the BBB. Functionalized and non-functionalized nanoparticles were characterized by DLS to verify whether the surface modification of SLNs affected the mean diameter of the particles. Therefore, the functionalized SLNs developed here exhibit an ideal size for brain Targeting and increased blood circulation time. In fact, most of the successfully used nanoparticles for the transport of drugs across the BBB present a size ranging from 150–300 nm [20].

2. Morphology determination:

NP are almost spherical and uniform in shape. It is possible to see particles, mainly in the range of 100–200 nm or smaller, and there is no visible aggregation or agglomeration of particles. Apparently, functionalized SLNs using palmitate have an average size greater than the functionalized SLNs using DSPE. Undoubtedly, for all formulations the most frequent population of particles has a diameter less than 200 Nm (21)

3. LDH ASSAY:

The LDH assay was performed to access cytotoxicity after SLN exposure. The medium resulting from the incubation of SLNs with cells was centrifuged (250 g, 10 min, at RT) the supernatant separated from the deposited cells in each well. This centrifugation process allowed us to remove any wastes and cellular debris and also SLNs. The LDH release into culture supernatants was detected by adding catalyst and dye solutions of an LDH cytotoxicity detection kit (Takara Bio Inc., Shiga, Japan). The absorbance values were read at 490 nm and 690 nm. Cytotoxicity was expressed as a percentage compared to the maximum LDH release in the presence of triton X-100 (positive control). EBM-2 medium was used in the LDH assay as the negative control, since no cytotoxicity was detected in such conditions.

4. Statistical analysis:

Statistical analyses were performed using SPSS software (v 20.0; IBM, Armonk, NY, USA). The measurements were repeated at least three times, and data were expressed as mean ± SD. Data were analyzed using one-way analysis of variance (one-way ANOVA), followed by Bonferroni, Tukey, and Dunnett post-hoc tests. A p value less than 0.05 was considered statistically significant for nanoparticles.

CONCLUSION:

Nanoparticles have the potential to deliver not only traditional small molecule drugs, but also nucleic acids [22], proteins [23], and diagnostic agents. These are easier to prepare and offer better control over other release agents. As this technology moves forward, some of the major challenges to clinical translation will be the ability to scale-up this system in a cost-effective manner. The nanometer size, smaller size causes protection of the drug against chemical and enzymatic degradation and allow them to cross BBB, and bypass the liver. These nanoparticles can be given through the oral, ocular, transdermal routes. These nanoparticles will open a new channel for effective delivery of a vast variety of drug molecules including antitubercular, anticancer, antiaging, analgesics, antianxiety,

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Received on 29.02.2020 Modified on 08.04.2020

Asian J. Res. Pharm. Sci. 2020; 10(3):199-203.

DOI: 10.5958/2231-5659.2020.00038.7