Layer-by-Layers of Polymeric Micelles as a Biomimetic Drug-Releasing Network

Abstract

Mucin networks are lubricous biofunctional coats formed through the continuous deposition of mucin glycoproteins. Previously, we demonstrated the synthesis of a mucin mimic using biotinylated-filomicelles crosslinked via streptavidin using a layer-by-layer approach. These networks recreate the fibrous nature of mucin and can serve as a drug-releasing network. In this work, the ability to vary the network properties by blending filomicelles with spherical micelles is demonstrated. In addition, the deposition of a dense polymer coating on the mucin network was shown to act as a barrier to control diffusion and improved the structural stability under simulated oral chemical conditions. These biomimetic coatings can be utilized as a delivery system, providing a tunable drug release for oral applications.

Keywords: biomimetic; biotin-streptavidin; filomicelles; mucin; oral drug delivery.

Figure 1

Size distribution of filamentous and spherical micelles. Length distribution of filomicelles (FM) are estimated from ImageJ post-processing on FM fluorescence micrographs. Size distribution of spherical micelles (SM) measured using dynamic light scattering (DLS), which showed an average particle diameter of ~245 nm.

Figure 2

a) SEM micrographs of in vitro natural mucin network depositions recapitulated under similar conditions as synthetic mucin networks formation over the polystryene substrate. b) Synthetic mucin networks formations were visualized under different levels of magnification in demonstrating porous network formation.

Figure 3

FM networks are visualized under tapping mode atomic force microscopy (AFM) at different levels of magnification. a) Topographic images of 7-layered network projecting surface height features b) AFM phase imaging showing finer surface features of the 7-layered FM network via measurement of subtle differences in material property.

Figure 4

Scanning electron micrographs of 7-layered micelle networks made from different spherical-filomicelle compositional blend ratios (% filomicelles = ~0, 20, 40, 60, 80 and 100). Inset shows those micelle-LBL networks at an higher magnification level (Scale bar = 5 µm).

Figure 5

Percent substrate coverage from 7-layered micelle networks formed from different micelle mixture (% filomicelle or sphere:filomicelle ratio). With increasing % filomicelle in the micellar blends better network-substrate coverage was achieved. (Refer to Figure 4 and Supplementary figure S3)

Figure 6

Bacterial growth on curcumin-loaded synthetic networks measured using crystal violet stain, after 24 h bacterial exposure. 7-layered networks were deposited using varying micellar compositional blends (filomicelle: sphere ratio). As FM content increased, there was an observable decrease in bacterial development. A 2-sample t test was performed comparing bacterial growth in substrate control with different micelle-commixture networks. The * represents significant difference from the substrate (control) with p <0.05, while ** represents p <0.1.

Figure 7

Generation of a LBL capping barrier on FM mucin construct. a) Simplified scheme of filomicelle networks with different numbers of polymeric capping layers (1, 3, 5, and 7). b) Polymeric capping layer growth over synthetic networks. Biotinylated polymer (Biotin-40/PAA225k) was able to exhibit capping layer growth ontop of the FM networks (7-layers). The non-biotinylated polymer (Biotin-0/PAA225k) due to lack of biotin-streptavidin affinity linkages was unable form capping layers with increasing polymeric-LBL addition cycles. c) Evaluating stability of capping layers. The capping layer loss was tracked to evaluate its stability under destabilizing harsh protease. The capping barrier coatings from biotinylated polymer (Biotin-40/PAA225) demonstrated better barrier stability, whereas weak charge based non-biotinylated polymers due to lack of affinity interactions demonstrated a relatively poor capping barrier intactness. The barrier tendency offered by biotinylated capping layers was expected to shield the underlying protein-based synthetic network via its polymeric properties (e.g. chain length or molecular conformation) and was expected to improve its bulk structural stability.

Figure 8

In vitro chemical stability of 7-layered filomicelle networks with polymeric capping barriers under effects of simulated oral chemical environment. a) Network destabilization was tracked independently from base streptavidin layer under in vitro salivary and protease environment. b) Network destabilization was tracked independently from streptavidin loss from FM network interlayers under in vitro salivary and protease environment. The effect of different number of polymeric capping layers on network destabilization under chemical effects was illustrated.

Figure 9

Drug release studies from filomicelle networks and effect of polymeric capping barrier in tuning its drug release under simulated oral environment. FM networks were developed over the polystyrene substrate with increasing number of layer additions (or network thickness) (No. of micelle layer additions = 1, 3, 5, and 7). The curcumin drug release from those networks without incorporating capping barriers was studied under a) simulated salivary environment b) protease environment. Also, drug release from 7-layered FM network was studied after incorporating polymeric capping barriers (No. of polymeric capping layer additions = 1, 3, 5, 7, 10, and 15) under c) simulated salivary environment and d) protease environment.

Figure 10

Scanning electron micrographs of 7-layered FM networks after subjecting to destabilizing chemical effects of protease environment a) at T= 0 hr b) at T= 6 hr and c) at T= 24 hr.

Scheme 1

Scheme showing hierarchical self-assembly approach adopted to develop a synthetic biomimetic network. By utilizing highly specific biotin-streptavidin interactions, the drug loaded biotinylated-micelles are cross-linked to form networks that mimic natural oral mucin coatings. This synthetic mucin is proposed to function as a drug releasing network in oral delivery. By incorporating polymeric capping barriers, the synthetic networks are expected to offer a tunable drug release.