Cannabidiol: Nanoparticles

Consistent with the notion that cannabidiol represents a Class II pharmaceutical, the development of nanotechnological drug delivery systems, which by definition comprise a molecular composition such that at least one dimension is nanoscale, or less than 100 nm, have been sought as a method to enhance bioavailability via intrinsically increased surface area to volume ratio; however, the tunable properties of amphiphilic biomolecules (i.e. phospholipids) as well as natural and synthetic polymers has been the subject of a growing field of theranostics, or the targeted delivery of therapeutics (Bhatia 2016). Ideally, nanoparticle formation may result in either a nanocapsule, whereby the pharmacologically active compound is protected within a core milieu by a shell of pharmacologically benign chemical species (i.e. liposome, polymerosome) or in a nanosphere, whereby both pharmacologically active and inactive compounds homogenize into a solid matrix. Classically, the assembly of nanoparticles is heavily determined by non-covalent interactions and the resultant size, stiffness, and exposed surface moieties provide tunable chemical characteristics that effect cellular uptake. Nanoparticles are designed to enter cells via endocytosis, which may involve phagocytosis, clathrin-dependent pinocytosis, caveolin-dependent pinocytosis, or a variety of other clathrin-independent pinocytotic pathways specific to cellular phenotype (Zhao and Stenzel 2018). The greater endocytic network involves circuits that either recycle cell surface materials or internalize components for either utilization or degradation and endosomal maturation, whereby the fusion of primary endosomes form weakly acidic (pH 6.8-5.9) early endosomes to begin sorting intralumenal vesicles, which are in constant communication with the trans-Golgi network, that mature into the more acidic (pH 6.0-4.9) late endosomes that fuse via Ras-mediated and microtubule-facilitated mechanisms with highly acidic (pH 4.5 ) and morphologically distinct lysosomes along an ingression towards the peri-nuclear area (Huotari and Helenius 2011; see Figure 9A). While the decrease in endosomal pH, per the activity of V-ATPase proton pumps, serves many functions from ligand dissociation to eventual degradation, the overall acidification process is of immense concern for nanoparticle delivery as release of the packaged pharmaceutical, or a process termed as ‘endosomal escape’, is a major determinant of efficacy. Four primary mechanisms affecting endosomal escape have been found as owing to membrane fusion, which may be facilitated by deshielding polymers at specific pH, osmotic pressure, which may be facilitated by buffering polymers that increase proton influx with chloride and water molecules beyond endosomal capacity, polymer swelling, which via electrostatic repulsions in response to decreased pH mechanically ruptures the endosome, and direct membrane destabilization via pH induced disassembly (Smith et al. 2019). A common strategy in polymer chemistry has been to incorporate functional groups that ionize at specific pH, such as carboxylic acids (pKa ̴ 4.5) or tertiary amines (pKa ̴ 10) depending on substituents as well as acid-labile linkages, to offer controlled release (Colson and Grinstaff 2012). While the benefit of functionalized polymers may offer a mechanism for targeted delivery, especially towards anionic phospholipids that have increased binding interactions with cationic groups, charged as well as neutral particle surfaces are subject to opsonization processes, or adsorption and accumulation of circulating opsonin proteins (i.e. complement system proteins C3 and C4, immunoglobulins, fibrinogen, apolipoproteins, albumin, etc.), that recruit the mononuclear phagocytic system or reticuloendothelial system for rapid clearance (Wani et al. 2019; see Figure 9B). Researchers have developed methods of coating nanoparticles with hydrophilic polyethylene glycol (PEG; aka polyethylene oxide, PEO) or polypropylene glycol (PPG; polypropylene oxide, PPO) to offer a so-called ‘stealth effect’ quality that increases circulation half-life by evading opsonization (Wani et al. 2019; see Figure 8). The surface coating of either covalently linked or non-covalently adsorbed PEG, termed PEGylation, utilizes a collective repulsive force based on its concentration and may either exhibit a ‘mushroom’ configuration at low densities, or a ‘brush’ configuration at higher densities (Owens and Peppas 2006).

Figure 8: Chemical Structures for PEG, PPG, PLGA, and Poloxamers

Figure 9: A) Endosomal Maturation and Endosomal Escape B) Opsonization Process

Poly (lactic-co-glycolic acid) (PLGA) is a di-block copolymer of varying units of D/L-lactic acid and glycolic acid that has gained FDA approval for medical formulations due to its biocompatibility and minimal toxicity derived from its hydrolytic products, lactic acid and glycolic acid (Danhier et al. 2012). In fact, the rate of PLGA hydrolysis, as well as its amphiphilic character, is highly determined by the monomer ratio as the lactic acid monomer donates more hydrophobic character than the glycolic acid monomer, which donates a relative hydrophilic character; thus, more rapid hydrolysis due to hydrophilicity is observed in PLGA of higher glycolic acid content (Ding and Zhu 2018). Additionally, a decrease in PLGA crystallinity is observed in polymers of lower lactic acid content and molecular weight, or polymer length, is also a factor of degradation, such that larger molecules degrade slower showing less rapid burst-release than smaller molecules (Ding and Zhu 2018). Research into the functionalization of PLGA nanoparticles has altered solubility properties and shifted degradation from bulk-erosion to surface-erosion pattern (Mundargi et al. 2008). Three generations of PVA-based branched graft polyesters have incorporated PLGA as a hydrophobic substituent whereby the ‘first-generation’ exhibited greater hydrophilicity provided by the PVA linkers, the ‘second-generation’ exhibited a negative charge due to a covalently linked sulfobutyl group, and the ‘third-generation’ exhibited a positive charge via the incorporation of amine groups such as dimethylaminopropyl amine (DMAPA), diethylamino-ethyl amine (DEAEA), and diethyl-aminopropyl amine (DEAPA) (Mundargi et al. 2008; see Figure 10).

Figure 10: Three Generations of PVA-based PLGA graft polyesters

PEGylation is also a prominent form of surface functionalization and may occur via the non-covalent adsorption of poloxamers, and poloxamines, or via the covalent assemblage of PLGA-PEG block copolymers. Poloxamers and poloxamines are amphiphilic tri-block copolymers such that a middle hydrophobic polypropylene oxide unit anchors the polymer by adhering to the surface of the nanoparticle, allowing the hydrophilic ethylene glycol chains to project from the surface; this simplistic formulation has incurred stability issues provided by the desorption of adsorbed attachments, but has been shown to alter surface properties, especially in co-formulations whereby the poloxamer is entrapped (Owens and Peppas 2006, Betancourt et al. 2009). Betancourt et al. explored carbodiimide/ N-hydroxysuccimide (NHS)-mediated peptide conjugation of PLGA to aminated-PEG functionalized with terminal carboxyl group for further conjugation reaction, whereby a carbodiimide (i.e. N,N’-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)) dehydrates a carboxyl group to form an activated NHS-ester that undergoes substitution with a more stable amine to generate the amide bond (Betancourt et al. 2009; see Figure 11A). While conjugation of functionalized PEG on pre-formed PLGA nanoparticles did not demonstrate significant PEGyltaion, nanoparticle formation from pre-conjugated PLGA-PEG (with functional carboxyl) has proven a robust method in attaching ligands for targeted delivery (Betancourt et al. 2009, Perinelli et al. 2019). Preparation of functionalized PLGA-PEG involving the ring-opening polymerization with metal catalyst, such as stannous octoate, which facilitates the nucleophilic attack of an alkoxy group to a lactic or glycolic acid carbonyl, is perhaps the preferred method, yet carbodiimide/NHS peptide crosslinking has been used to attach targeting ligands such peptides, small molecules (i.e. folate), oligonucleotide aptamers, and antibodies (Perinelli et al. 2019, Dechy-Cabaret 2004; see Figure 11B). Interestingly, Wang et al. distinguished a greater surface charge conversion from anionic functionalized PLGA-PEG with lesser amounts of cationic chitosan when allowed to non-synthetically adsorb; however, peptide-linked chitosan exhibited higher burst effect and sustained release of anionic pharmaceutical 5-fluorouracil, which the authors claim aids in the complete erosion of the nanoparticle (Wang et al. 2013). Other researchers have explored free radical polymerization chemistry to alter surface functionality of PLGA and Zhang et al. had successfully shown large intestine absorption of orally administered surface immunogenic protein (SIP) to vaccinate against group B Streptococcus (GBS) in tilapia model via encapsulation in poly(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid) coated PLGA (PMMMA-PLGA), which aided in the solubility (i.e. surface carboxyl groups), protection from enzymatic degradation, and controlled release in alkaline conditions (Zhang et al. 2016).

Figure 11: A) PLGA Functionalization with PEG-Carboxylate B) PEG-Carboxylate Functionalization with Target Ligand (both using carbodiimide/NHS chemistry)