Declaration 2 Publications
2.2 CLASSIFICATION OF AMPHIPHILIC DENDRIMERS
2.3.1 Non-stimuli-responsive (NSR) self-assembling dendrimers
2.3.1.1 Amphiphilic layered dendrimers based NSR delivery systems
The structure of amphiphilic layered dendrimers utilizes layer-wise segregation of hydrophobic core and hydrophilic corona, thus creating specialized self-assembling structures known as unimolecular dendritic micelles (Hawker, Wooley and Frechet, 1993). These unimolecular micelles are considered to be effective micellar delivery vehicles due to their single molecular self- assembly pattern (Fan, Li and Loh, 2016).
Using the Fréchet and company design of a layer-wise segregation of hydrophobic and hydrophilic hydrophobic regions, Morgan and coworkers constructed an amphiphilic layered dendrimer from biocompatible polyester amphiphilic layered G3 and G4 poly(glycerol-succinic acid) (PGLSA) dendrimers from natural components, such as glycerol and succinic acid (Fig. 3). PGLSA-OH and
PGLSA-COONa (G3 & G4) dendrimers were synthesized divergently by propagating through esterification with 2-(cis-1,3-O-benzylideneglycerol) succinic acid and deprotecting with H2/Pd/C (hydrogenolysis). To obtain PGLSA-COONa dendrimers, in the final synthesis step, hydroxyl terminals of PGLSA-OH were converted to carboxylate terminals by reaction with succinic anhydride in pyrene and the molecular weights for G4-PGLSA-OH and G4-PGLSA-COONa were found to be 10715 and 18500 Daltons respectively.
An aggregation study performed on G4-PGLSA-OH and G4-PGLSA-COONa by using quasi- elastic light scattering method showed the formation of unimolecular micelles. The hydrodynamic diameter of the PGLSA-OH (G4) dendrimer was found to be 7 nm, which was further reduced to 4 nm after encapsulation of Richard’s dye, this decrease of the unimolecular micelles being attributed to the collapse of aliphatic the amphiphilic dendrimer around the hydrophobic dye. The G3 PGLSA amphiphilic dendrimers encapsulated approximately one dye molecule, while the G4 dendrimer showed encapsulation of two molecules of the dye, increasing the solubility of Richard’s dye 2000-fold compared to water. These observations showed that an increase in glomerular structure of the amphiphilic dendrimer enhances the encapsulation efficiency.
Drug encapsulation studies were performed using 10-hydroxycamptothecin (10HCPT) as a model hydrophobic guest molecule. Encapsulation of 10HCPT was performed using G4-PGLSA- COONa, as solubilization with G4-PGLSA-OH amphiphilic dendrimers resulted in the formation of precipitate on storage. Results of the in-vitro anticancer activity against human breast cancer cell (MCF-7) demonstrated that dendrimers alone were inactive, whereas anticancer activity of 10HCPT was retained after encapsulation within the dendrimers (Cytotoxicity results as indicated in Fig. 4). The study concluded that the unimolecular micelles formed by these amphiphilic dendrimers were suitable as delivery vehicle for encapsulating hydrophobic anticancer drugs (Morgan et al., 2003).
Fig. 3. Structure of G4-PGLSA dendrimers and hydrophobic guest molecules (Morgan et al., 2003). Reproduced with license from American Chemical Society.
Fig. 4. Cytotoxicity assay with human breast cancer, MCF-7, cells (5000 cells/well; n ) 8) (Morgan et al., 2003). Reproduced with license from American Chemical Society.
In another study, the same research group expanded the potential use of these PGLSA dendrimers to deliver poorly water soluble 7-butyl-10-aminocamptothecin (BACPT) along with 10HCPT (Morgan et al., 2006). Encapsulation studies were carried out using a solvent evaporation method, where G4-PGLSA-COONa amphiphilic dendrimers and camptothecins were used at a 1:1 ratio.
The results of the encapsulation study indicated a 10 -fold solubility enhancement for 10HCPT and BACPT in amphiphilic dendrimer solution compared to water. The drug release profile of the encapsulated 10HCPT/G4-PGLSA-COONa vehicle was monitored by dialysis method using phosphate buffer solution (pH 7.4), with the drug release being complete within 6 h with linearity over the period of 2 h (release results are shown in Fig. 5. Encapsulation into the dendrimers enhanced the cytotoxic potency of both 10HCPT and BACPT towards human cancer cell lines [human breast adenocarcinoma (MCF-7), colorectal adenocarcinoma (HT-29), non–small cell lung carcinoma (NCI-H460), and glioblastoma (SF-268) (Morgan et al., 2006). The two studies reported by Morgan and co-workers provided information on the solubility and activity enhancement of the hydrophobic anticancer drug molecules by their encapsulation into self- assembled amphiphilic dendrimers, with the morphology of the formed self-assemblies not being investigated. Morphological investigations using scanning electron microscopy (SEM) and/or transmission electron microscopy (TEM) would have been helpful to understand the effect of size
and shape on encapsulation efficiency, as there were differences in the number of molecules encapsulated in the G3 and G4 PGLSA.
Fig. 5. Release profile of [G4.5]-PGLSA-COONa encapsulated 10HCPT. Points, mean; bars, range (n = 2) (Morgan et al., 2006). Reproduced with permission from American Association for Cancer Research.
Alternatively, amphiphilic layered dendrimers can also be constructed by coupling hydrophobic moieties to hydrophilic polyamidoamine (PAMAM) dendrimers to form different self-assembling nano structures. This approach was employed by Hung et al., who coupled a hydrophilic PAMAM dendron shell to the poly(L-lactide) (PLLA) core, which self-assembled to unimolecular micelles (Hung et al., 2013). Cao and Zhu applied a very similar strategy, where a hydrophilic segment of the amphiphilic PAMAM, G2 to G5 core, was attached to a hydrophobic shell of the aniline pentamer, whose self-assembled spherical aggregates formed bilayer vesicular structures (Cao and Zhu, 2011b).
Linking the block linear polymers to dendrimers has also resulted in amphiphilic layered dendrimers. Such a method was employed by Wang et al who designed a dendritic micellar system with a G2 PAMAM core, and a linear block copolymer from a poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) shell. These PAMAM-PCL-PEG hybrids self-assembled to micelles that were able to encapsulate etoposide with 22% loading capacity. Furthermore, the cytotoxicity
assay of the hybrid against porcine kidney epithelial cells (LLC-PK) demonstrated that the PAMAM-PCL-PEG amphiphilic molecule was nontoxic, and that the entrapped drug produced significantly better anticancer effect than the free drug (Wang et al., 2005). These types of core shell assemblies could be employed to non-covalently entrap the drug within the dendrimer structure. The advantage of these systems is that the release process is not chemical dependent, but purely dependent on ‘soft-bonds’, such as the ionic pairing, hydrogen and halogen bonds, which requires lower energy interactions and more subtle conditions, such as shifts in local equilibria, to release the drug (Jain and Asthana, 2007).