Protein Based Biomaterials for Therapeutic and Diagnostic Applications

Abstract

Proteins are some of the most versatile and studied macromolecules with extensive biomedical applications. The natural and biological origin of proteins offer such materials several advantages over their synthetic counterparts, such as innate bioactivity, recognition by cells and reduced immunogenic potential. Furthermore, proteins can be easily functionalized by altering their primary amino acid sequence and can often be further self-assembled into higher order structures either spontaneously or under specific environmental conditions. This review will feature the recent advances in protein-based biomaterials in the delivery of therapeutic cargo such as small molecules, genetic material, proteins, and cells. First, we will discuss the ways in which secondary structural motifs, the building blocks of more complex proteins, have unique properties that enable them to be useful for therapeutic delivery. Next, supramolecular assemblies, such as fibers, nanoparticles, and hydrogels, made from these building blocks that are engineered to behave in a cohesive manner, are discussed. Finally, we will cover additional modifications to protein materials that impart environmental responsiveness to materials. This includes the emerging field of protein molecular robots, and relatedly, protein-based theranostic materials that combine therapeutic potential with modern imaging modalities, including near-infrared fluorescence spectroscopy (NIRF), single-photo emission computed tomography/computed tomography (SPECT/CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound/photoacoustic imaging (US/PAI).

Keywords: Biomaterials; drug delivery; protein; self-assembly.

Figure 1.

Schematic depicting general approaches to self-assembled drug delivery vehicles. NIRF = near-infrared fluorescence spectroscopy. SPECT = single-photon emission computer tomography. PET = positron emission tomography. CT = computed tomography. MRI = magnetic resonance imaging. US/PAI = ultrasound/photoacoustic imaging.

Figure 2.

A standard helical wheel diagram showing interactions between helical domains in a coiled-coil. Reprinted with permissions from Utterström et al. under Creative Commons license.

Figure 3.

(A) Schematic and (B) sequence of the collagen triple helix. X and Y most commonly represent proline and hydroxyproline. under the Creative Common License. (C) Axial (left) and lateral (right) interactions between amino acid side chains. (A) and (B) Adapted from [65]. CC BY 4.0. (C) Adapted with permission from [67]. Copyright 2020 American Chemical Society.

Figure 4.

β-sheet assembly and edge interactions of two representative peptides FEFKFEFK (F8) and KFEFKFEFKK. (A) β-strands assemble into more complex β-motifs such as sheets, turns, or hairpins (an antiparallel β-sheet is shown here). These motifs may then form fibers or hydrogels as shown. C = concentration, CGC = critical gelation concentration. (B) β-sheet assembly into fibers with fiber axis orthogonal to strand axis (‘cross β’ conformation). (C) Chemical structure of hydrophobic-driven assembly into β-sheets. Mutating the end residues to lysine caused the edges of the sheet to be more hydrophilic, decreasing aggregation with other hydrophobic β-sheets. Reproduced from [42]. CC BY 3.0.

Figure 5.

Schematic showing suckerin containing nanoparticles loaded with drug in a protein matrix. Reprinted with permission from Ping et al. Copyright (2017) American Chemical Society.

Figure 6.

Elastin-like polypeptide properties from amino acid sequence. A variety of factors determine the chain’s disordered state, including the proline/glycine content, the identity of the guest residues, and the number of tandem repeats, allowing for rich engineering of thermally responsive materials. Reprinted with permission from Roberts et al. Copyright (2015) John Wiley and Sons.

Figure 7.

Ferritin nanocages surface modified with A) 36-unit long XTEN and B) 288-unit long XTEN. Reprinted with permission from Lee et. al. Copyright (2017) Elsevier. C) Recombinant genetic design of fusion protein for self-assembling micelles developed from NF-H IDP domain. D) The IDPs provide mechanical and structural integrity to the neurofilament core and were further engineered with a hydrophobic extension to self-assemble into micelles. Reprinted with permission from Klass et al. Copyright (2019) American Chemical Society.

Figure 8.

Self-assembly of β-sheets into fibers. Adapted with permission from Das et al. Copyright (2018) American Chemical Society.

Figure 9.

A) Amino acid sequences of wildtype COMPCC (wt) and Q, along with L, a nonhelical variant. B) Surface charge distribution in various views of Q. Red is positive charge, Blue is negative. Reprinted with permission from Hume et al. Copyright (2014) American Chemical Society.

Figure 10.

Cell specificity of antimicrobial SAANs developed by Chen et al. (A) Schematic showing SAANs and their differential effects on mammalian vs microbial cells. (B) Confocal microscopy and Live/dead assays of E. coli treated with Free Mel and SAANs compared to 3T3, a mouse fibroblast line. Reprinted with permission from [117]. Copyright 2019 American Chemical Society.

Figure 11.

ELP and globular proteins fused to leucine zippers form coacervates upon an increase in temperature and further assemble into micelles at room temperature. The globular protein (green circle) is fused to one of two leucine zippers (Z_E, red), and the other leucine zipper (Z_R, blue) is fused to ELP (gray). Adapted with permission from Dautel et al. Copyright 2021 American Chemical Society.

Figure 12.

Multidomain peptide (MDPs) self-assemble into β-fibers and even further into hydrogels upon a salt or pH switch. A) General structure of MDPs. MDPs are flanked by polar residues (C) with an interior sequence composed of alternating hydrophilic (X) and hydrophobic (Y) residues. B) The peptide forms β-sheets with a hydrophobic face and a hydrophilic face. C) The sheets self-assemble into nanofibers via packing of the hydrophobic face. D) While the exterior of the fiber binds to hydrophilic small molecule compounds, the interior peptide sequence can be mutated with alanines to form pockets suitable for hydrophobic drug encapsulation. Panels A, B, C, and D adapted with permissions from Moore and Hartgerink found at https://pubs.acs.org/doi/10.1021/acs.accounts.6b00553. Further permissions related to the material excerpted should be directed to the American Chemical Society. E) MDPs have been shown to support encapsulation of NIH-3T3 fibroblasts. Red = ethidium homodimer (nonviable cells). Green = Calcein AM (viable cells). Scale bar = 100 μm. F) Viability of fibroblasts remained above 75% over a period of 14 days. G) MDPs implanted into a full thickness dorsal wound of diabetic mice facilitated wound closure over 28 days. Scale bar = 5 mm. H) Wound contraction (dashed line) and wound closure (solid line) of full thickness wounds with MDP implants. *p < 0.05; ** p < 0.01. Panels E, F, G, and H adapted with permissions from Carrejo et al. Copyright (2018) American Chemical Society.

Figure 13.

A) The RADA16–1 peptide, with alternating residues of hydrophilic and hydrophobic residues, forms b-sheet secondary structure with a hydrophilic face and a hydrophobic face. The hydrophobic face of multiple units self-assembles into elongated fibers. Reproduced from Wang et al. under Creative Commons license. B) Circular dichroism of RADA16 showing typical b-sheet signature. Reproduced with permissions from Yang et al. Copyright 2018 American Chemical Society.

Figure 14.

A). Chemical structure of A1 at pH 7. B). Matrix-like structure of A1 hydrogel. C). Release of various drugs from A1 hydrogel over 72 hrs. Adapted from Roy et al. Copyright (2020) American Chemical Society.

Figure 15.

Image displaying thermoresponsive supramolecular self-assembly of α/β₆. Reprinted with permissions from Nagarkar et al. Copyright (2018) John Wiley and Sons.

Figure 16.

Illustration of pH-dependent VLP nanoparticle assembly and disassembly. To the right, ELP-dependent VLP assembly is shown. Reprinted with permissions from van Eldijk et al. Copyright (2012) American Chemical Society.

Figure 17.

Illustration of self-assembled Nap-GFYE nanofibers forming a hydrogel upon crosslinking with iron oxide magnetic nanoparticles. Reprinted with permissions from Nowak et al. Copyright (2021) Royal Society of Chemistry.

Figure 18.

Self-assembled contractile active network. A) The various protein components of the network. B) Contractile unit formed by the assembly of these protein components. C) Exposure to UV light increases concentration of Ca²⁺ ions, inducing the binding of K465m13 to CaMLMM self-assembled scaffolds and organizing around microtubules. D) Schematic of using localized light to direct contraction in macroscopic systems. Reprinted from Nitta et al. Copyright (2021) Springer Nature.

Figure 19.

Suckerin-derived self-propelled protein motors. a, b) Fabrication of motors at various length scales to determine morphology effects on locomotion. Smaller motors (l=<1 mm) demonstrate greater forward propulsion while larger motors (l=10 mm) exhibit greater lateral propulsion. c) Mechanism of HFIP replacement by H₂O and subsequent crystallization of suckerin with release of HFIP. Reprinted with permission from Pena-Francesh et al. (2019) under Open Access guidelines. Copyright Nature Communications.

Figure 20.

Protein microtubules developed by the Komatsu group with urease motors situated on the interior of layers of protein. A) LBL synthesis process. A porous polycarbonate (PC) template is used where the diameter of the pores corresponds to the diameter of the nanotubes and the depth corresponds to the length Alternating layers of protein and a positively charged compound used as a “glue” e.g. poly-L-arginine (PLA) are deposited sequentially in the pore, then air dried. The PC template is then removed by solubilizing in dimethylfuran (DMF). Reprinted with permission from Qu et al. Copyright (2010) American Chemical Society B) Completed microtubules with layers of PLA and human serum albumin (HSA) comprising the bulk of the nanotube along with an inner layer of poly-L-glutamic acid (PLG) bonded to avidin (Avi) and biotinylated urease (bUre) Reprinted with permission from Sugai et al. Copyright (2019) Wiley and Sons.

Figure 21.

Image illustrating the process of synthesis of small ultra-red fluorescent protein (smURFP) nanoparticles via emulsification. Reprinted with permission from An et al. Copyright (2020) Elsevier.

Figure 22

(A) Structure of NIR dye ZW800–3a and its incorporation within gelatin forming NIR-gelatin (B) Scheme showing fabrication of NIR-gelatin hydrogel by crosslinking HA-tyramine conjugate and NIR-gelatin using tyrosinase for later intracranial animal injection. Reprinted with permission from Park et al. (2019) under Creative Commons Attribution (CC BY-NC) license.

Figure 23.

A) Flow chart illustrating the fabrication of ^99mTc labeled Cy@Silk SPECT theranostic agent. B) Figure illustrating multimodal functions of these nanoagents: dynamic photoacoustic imaging, photothermal imaging and SPECT of osteosarcoma cells. Reprinted with permission from Wang et al. Copyright (2019) American Chemical Society.

Figure 24.

Image illustrating Au-BSA-DOX-FA nanocomposite and its application in multimodal CT imaging, targeted chemotherapy via FA, and pH-sensitive chemotherapy in a mouse model. Reprinted with permission from Huang et al. . Copyright (2017) Dove Medical Press.

Figure 25.

A) Engineering of Fmoc-L-L/Mn²⁺ nanoparticles (FMCNP’s) from Fmoc-L-L and Ce6 via Mn²⁺ coordination complexing based self-assembly. B) MRI-guided PDT causes their smart disassembly (bottom). Reprinted with permission from Zhang et al. Copyright (2018) American Chemical Society.

Figure 26.

PemFE peptide monomers self-assembling into nanofilaments that in turn form a fibrous 3D network within a hydrogel under appropriate environmental conditions. Reprinted with permission from Lock et al. Copyright (2017) American Chemical Society.

Figure 27.

Figure illustrating self-assembled porphyrin-MB nanoparticles and its components. Adapted from Moon et al. Copyright (2015) Elsevier.