Porous Protein Crystals with Controlled Loading and Delivery of Guest Molecules

At a Glance

Researchers at Colorado State University have developed a patented class of protein crystals comprising unusually large nanopores (diameter ~13 nm). As a result, these crystals can be loaded with different kinds of guest molecules such as proteins, nucleic acids, nanoparticles, and small molecules, or mixtures of such guests. The crystals can be easily grown at different and controlled sizes. Various guest molecules have been loaded into the protein crystals with controlled and replicable placement. There is also potential for controlled release in response to triggers, such as changes in pH.

Background

Scaffolds created from biological building blocks such as nucleic acids, viruses and protein cages, or other supramolecular assemblies can position macromolecules with nanometer scale precision and have many applications in imaging, sensing, and drug delivery. Porous protein crystals could be superior scaffolds because they have the same precise nanostructure regardless of the size of the crystal. However, due to the technical challenges of crystallizing proteins and understanding their structure, they have not previously been utilized for these applications.

Overview

This novel class of porous protein crystals typically have a hexagonal prism shape and can be grown to a wide range of sizes: from 100 nm diameter to 1 mm in hours or several days. They have unusually large (~13 nm) and small (~3nm) nanopores which are good at adsorbing guest molecules, with a particularly high affinity for adsorbing nucleic acids.

 

The nanocrystals have been successfully demonstrated to hold DNA, RNA, active enzymes, nanoparticles, conductive polymers, metal-organic frameworks, fluorescent proteins, dye molecules, polymers, polysaccharides, lipids, vitamins, and other guest materials. Data has been collected regarding the stability and recyclability of the porous crystals to store and release guest molecules in predictable and tunable methods. Tests regarding the interaction of these loaded nanocrystals with living tissue and organisms are in process. Research has shown that mosquitos or mice who consume the protein nanocrystals loaded with DNA do not experience adverse effects, and the crystals can be used for viral vector tracking by using guest DNA as a synthetic barcode. Further experiments regarding delivery of guest molecules into living biologic tissue is in process.

Figure 1. (A) The porous protein crystal loaded with fluorescently tagged enzyme. (B) Four unit cells of the protein crystal with a single fluorescent guest molecule modelled in the large nanopore. (C) A schematic of crystal pores and axes.

Figure 2. Three samples of protein crystals were loaded with horseradish peroxidase (hHRP) and the concentration of adsorbed hHRP into the crystal is calculated over time.

Benefits

  • Highly stable
  • Controlled loading and unloading
  • Engineered for non-covalent or covalent capture of guest macromolecules
  • Allow for programmed placement within materials
  • Integrated crystals have spatially segregated loading patterns
  • Biodegradable and biocompatible

Applications

  • Immobilized enzymes or enzyme pathways (protein zeolites)
  • Advanced delivery sensing or theragnostic materials
  • Transport of therapeutic macromolecules for advanced drug delivery applications
  • Viral vector tracking
Last Updated: July 2023
Fluorescent protein crystals loaded with a macromolecule
Opportunity

Available for Licensing
TRL: 5

Inventors

Christopher Snow
Thaddaus R Huber

Reference Number
16-002 and 2023-084
Licensing Manager

Aly Hoeher
Aly.Hoeher@colostate.edu
970-491-7100