Chapter V: Overall Summary and Conclusions
2.1 Introduction
2.1.1 Chemical Changes Experienced During Transcytosis - Reduction
To prepare nanoparticles with targeting ligands capable of detaching during transcytosis, potential stimuli occurring during this process first needed to be identified.
As discussed in section 1.6, in most cells, holo-Tf is endocytosed, undergoes a conformational change to release bound iron, and then recycles back to the extracellular space. In polarized cells, such as the BBB endothlium, this process is slightly modified to allow Tf to enter the CNS. After endocytosis, intracellular machinery sorts the Tf- containing vesicle to undergo transcytosis, sending it across the cell and to the basal membrane (1,2).
Several chemical differences exist within the endosome compared to the extracellular space. The two most useful for a drug delivery device are (i) slight drop in pH and (ii) exposure to redox agents. Changes in pH will be discussed in section 3.1.
Glutathoine (GSH) is the major non-protein redox agent in biological systems (3). In the extracellular space, GSH concentration is low, about 10 μM (4), producing a generally oxidizing environment outside the cell. Intracellularly, however, GSH concentration is in low mM concentrations with the ratio of GSH to its oxidized form (GSSG) at ~100:1.
These two factors create a strongly reducing environment within mammalian cells. In the endocytosis pathway several membrane associated enzymes help reduce the vesicle contents. Protein disulfide isomerase (PDI) is a plasma membrane enzyme that reduces macromolecules within the early endosome through disulfide-thiol exchange. GSH is also present within the endosome to maintain the catalytic activity of this and other reducing enzymes (5). Though the exact mechanism by which molecules are reduced in the
endosome occurs is unclear, reduction of a disulfide-containing conjugate within endosomes has been observed. This finding was also important because it occurred in Tf- containing endosomes (6), suggesting it can be taken advantage of during Tf transcytosis.
2.1.2 Stimuli-Responsive Nanodevices
Stimuli-responsive nanomedicines are commonplace (7-9) including those in clinical trials (10). In general, therapeutics are designed to take advantage of physical and/or chemical changes the nanomedicine experiences at the site of action to trigger release of loaded drug. This is typically done by incorporating the appropriate stimuli- responsive chemistry into the backbone of the nanomedicine, causing it to destabilize or degrade once it reaches its desired destination (e.g. a malignant tumor) (11). With polymeric nanoparticles, the chemistry can be implemented either within the polymer chain itself (12-14) or at the point of attachment for polymers that stabilize the nanoparticle core under biological conditions (15-18).
Implementing stimuli-responsive chemistry to facilitate intact nanoparticle transit across cellular barriers has not been performed prior to this work. Because of the reducing potential within the endosome, the first cleavable chemistry investigated was disulfide bonds. Disulfides are easily synthesized, reversible, and commonplace in biological systems (5). These bonds have been implemented in many different nanoparticles, with the vast majority designed to destabilize the device once in the desired intracellular compartment (19-21).
2.1.3 Drug Delivery Using PLGA Nanoparticles
PLGA is an FDA-approved biodegradable co-polymer composed of lactic acid and glycolic acid (Fig 2.1). It is used in a wide variety of medical devices, including sutures, prosthetic devices, and implants (22). The polymer has received much attention from the drug delivery community due to its flexibility, versatility, and excellent biocompatibility.
Depending on the synthesis method, PLGA can be formulated into nearly any shape (23) and loaded with either hydrophobic or hydrophilic small (22) and macromolecules (24),
including proteins and nucleic acids (25). PLGA nanoparticles can easily be prepared through simple methods at the desired diameter for BBB transcytosis (26). Targeting molecules can also be added to the surface of the PLGA nanoparticles after synthesis, allowing for controlled quantities of targeting ligand on the nanoparticle surface (27).
PLGA nanoparticles are also resistant to degradation under mildly acidic conditions, suggesting the nanoparticles will remain intact within the acidic endosome (28).
The drug payload is encapsulated within the core of PLGA nanoparticles during synthesis. Under biological conditions, the esters comprising the PLGA polymer hydrolyze, slowly breaking apart the polymer (22). The rate of breakdown can be controlled by the ratio of lactic acid:glycolic acid in the co-polymer. The methyl group of lactic acid makes it more hydrophobic compared to glycolic acid, so co-polymers enriched in lactic acid break down more slowly (29). As the nanoparticle breaks down, water-filled pores form, allowing encapsulated molecules to exit the particle core through diffusion (30).
Fig 2.1 Chemical structure of carboxylic acid-terminated PLGA. X and Y refer to the molar ratio of glycolic to lactic acid, respectively.
2.1.4 PLGA-PEG Nanoparticles
Nanoparticles are cleared from circulation primarily through the reticuloendothelial system (RES). Opsonin proteins in the bloodstream bind foreign objects triggering their phagocytosis by macrophages, especially within the liver and spleen (31). To reduce this opsonization process and improve circulation half-life, nanoparticles are routinely decorated with polyethylene glycol (PEG). PEG is a hydrophilic polymer of repeated ether
units that is highly biocompatible and FDA-approved for use in humans. The polymer is typically added to the surface of the nanoparticle to form a corona that inhibits serum protein binding. This, in effect, reduces nanoparticle clearance from the bloodstream by the RES (32).
PEG also provides steric stability to the nanoparticles in serum. Due to their high surface area to volume ratio and surface potentials, spherical nanoparticles are at risk of aggregating in solution (33). Surface PEG reduces nanoparticle aggregation by reducing van der Waals attractive forces and increasing the distance between adjacent particles.
Addition of PEG also increases the solubility of the nanoparticle in aqueous solution (34).
A final benefit of PEG addition relevant to this work is that nanoparticles under 100nm with a dense surface PEG coating are capable of diffusing through the extracellular space of brain tissue (35).
PEG can be conjugated to PLGA through simple chemistry to form a block co- polymer (36). Nearly all PLGA nanoparticles formulated for drug delivery are prepared in this manner (25).