Graphene Multilayers as Gates for Multi-Week Sequential Release of Proteins from Surfaces

Abstract

The ability to control the timing and order of release of different therapeutic drugs will play a pivotal role in improving patient care and simplifying treatment regimes in the clinic. The controlled sequential release of a broad range of small and macromolecules from thin film coatings offers a simple way to provide complex localized dosing in vivo. Here we show that it is possible to take advantage of the structure of certain nanomaterials to control release regimes from a scale of hours to months. Graphene oxide (GO) is a two-dimensional charged nanomaterial that can be used to create barrier layers in multilayer thin films, trapping molecules of interest for controlled release. Protein-loaded polyelectrolyte multilayer films were fabricated using layer-by-layer assembly incorporating a hydrolytically degradable cationic poly(β-amino ester) (Poly1) with a model protein antigen, ovalbumin (ova) in a bilayer architecture along with positively and negatively functionalized GO capping layers for the degradable protein films. Ova release without the GO layers takes place in less than 1 hour, but can be tuned to release from 30 to 90 days by varying the number of bilayers of functionalized GO in the multilayer architecture. We demonstrate that proteins can be released in sequence with multi-day gaps between the release of each species by incorporating GO layers between protein loaded layers. In vitro toxicity assays of the individual materials on proliferating hematopoietic stem cells (HSCs) indicated limited cytotoxic effects with HSCs able to survive for the full 10 days of normal culture in the presence of Poly1 and the GO sheets. This approach provides a new route for storage of therapeutics in a solid-state thin film for subsequent delivery in a time-controlled and sequential fashion.

Synthesis, toxicity, biocompatibility, and biomedical applications of graphene and graphene-related materials

“…215 Drug delivery systems aim to localize delivery of therapeutic agents, in which GO is predominantly used because it can create barrier layers in multilayer thin films, trapping molecules of interest for controlled release. 216 NGO-PEG has been used as a nanocarrier for delivery of water-insoluble aromatic anticancer drugs into cells. The NGO-PEG loaded with SN38 exhibited high cytotoxicity for HCT-116 cells, 1,000-fold more potent than CPT-11.…”

“…219 Functionalization of GO with the active targeting ligand TRC105 increased therapeutic efficacy in angiogenesis. 216 A multiple supramolecular assembly was fabricated with a folic acid-modified beta-cyclodextrin, adamantanyl porphyrin, and GO through non-covalent interactions. Owing to the cooperative contribution of these three units, the DOX showed better drug activity and much lower toxicity.…”

Delivery of bone morphogenetic protein-2 and substance P using graphene oxide for bone regeneration

“…29 Prepared GO was exfoliated onto each GO-COO − sheet by ultrasonification in an ice bath. The resultant brown dispersion was centrifuged at 3,470 g for 5 minutes to remove any unexfoliated powder.…”

“…29,34,35 Coating with multiple layers of GO resulted in a full coverage of the Ti substrate ( Figure 1A). Atomic force microscopy revealed that, unlike the bumpy surface of an uncoated Ti substrate, the GOcoated Ti surfaces became much smoother after multiple cycles of LbL deposition.…”

“…28 The ionized groups of GO enable protein delivery by binding through electrostatic interaction. [28][29][30] In addition, GOs' hydrophobic domains can interact with those of proteins' by hydrophobic π-π stacking. [31][32][33] Therefore, GO can allow for a sustained release of loaded protein, which is critical for clinical use of protein delivery.…”