Koss Lab: Research

Building Better Biomolecules

Biophysics of Self-Assembling Cell Scaffolds
Discovery of Neuroinflammatory Modulating Peptides
Hyaluronic Acid (HA)-Binding Peptides
3D Cell Scaffold Models for Intraspinal Microstimulation (ISMS), Traumatic Brain Injury (TBI) and Glia in Limb Transplantation and Injury

Biophysics of Self-Assembling Cell Scaffolds

Dr. Koss’ initial research at the University of Alberta and NINT focused on the biostability of (RADA)4, a novel self-assembling peptide scaffold. These scaffolds exhibit remarkable self-assembly properties under physiological conditions, making them promising candidates for injectable cell and drug delivery platforms. However, their stability under extreme pH and temperature conditions remained largely unexplored.

In a pioneering study, he demonstrated the robust stability of (RADA)4 scaffolds across a spectrum of pH and temperature extremes typically encountered during disease states, suggesting their potential for in vivo applications.

Building upon this, he conducted the first comprehensive biocompatibility studies of peptide-based self-assembling scaffolds with human immune cells. Specifically, we investigated the interaction of (RADA)4 scaffolds with human platelets and assessed their impact on platelet aggregation. Furthermore, he evaluated the biocompatibility of these scaffolds with primary rat microglia, key immune cells of the nervous system. His findings demonstrated excellent biocompatibility with these immune cells, indicating minimal inflammatory response.

Leveraging these findings, he developed an on-demand drug delivery system triggered by inflammatory proteases. This system was successfully employed to induce neural differentiation in PC-12 cells, showcasing its potential for controlled drug release and targeted cellular responses.

Illustration showing microglia actively degrading material and phagocytosing debris, alongside astrocytes forming dense networks of astrogliosis.
A potential host response is portrayed by active amoeboid microglia degrading and phagocytosing the material and astrocytes forming the dense interlinked networks of astrogliosis. Proposed biocompatibiliy of (RADA)4 scaffold with inclusion laminin receptors and IKVAV peptides, where microglia are ramified and astrocytes are circular and non-fibrous. (PMID: 26850147).
Neural cells seeded in a (RADA)4 nanoscaffold showing spherical morphology without MMP-2 treatment and differentiated with neurites after MMP-2 treatment. Cells adhere to IKVAV motifs in both conditions.
Neural cells, seeded into a (RADA)4 nanoscaffold, responding to bound and cleaved neurotrophic peptides without and with MMP-2 protease treatment, respectively. Cells are spherical when untreated and differentiate with outgrown neurites when treated with enzyme. Cells are adhering to IKVAV motifs. Full sequences for cleavable peptides and drug peptides are shown with (RADA)4. (PMID: 27720765).

Discovery of Neuroinflammatory Modulating Peptides

A significant aspect of the Koss lab’s research involves a collaborative project aimed at identifying novel peptides capable of modulating microglial activity and neuroinflammatory processes. This project utilizes bacteriophage display libraries, which present a vast repertoire of over 2 million unique peptide sequences.

The primary objective of this research is to develop novel coatings for implantable devices that minimize rejection by the host immune system. These peptides hold immense potential for broader applications, including the development of therapeutics and image contrast agents for a wide range of neuroinflammatory diseases and injuries.

Through this comprehensive screening effort, we have successfully identified 58 unique peptide sequences. We are currently conducting in-depth analyses to elucidate their origin, function, and potential therapeutic applications.

SX7 phage panning of activated microglia and astrocytes, treated with LPS, minocycline, dexamethasone, and conditioned medium. Peptide sequences sorted and sequenced for future biosensor applications.
SX7 phage panning of ramified, activated, and primed neuroinflammatory phenotypes of microglia and astrocytes (PMID: 37171455). Cells are treated with the phage, sorted, and sequenced for displayed peptide sequences. LPS: lipopolysaccharide, Mino: minocycline, Dex: dexamethasone, CM: conditioned medium. Fifty-eight unique sequences were identified for future work with biosensors.

Hyaluronic Acid-Binding Peptides

In an effort to modulate neuroinflammation mediated by CD44, we designed a novel family of peptides utilizing our custom biophysical codex. We engineered these peptides to mimic the active sites of RHAMM (Receptor for Hyaluronan-Mediated Motility) and CD44, key receptors involved in hyaluronan (HA) signaling.

Our findings demonstrated that these peptides exhibit significantly enhanced binding affinity to HA compared to previously reported peptides. Furthermore, we observed that these peptides possess unique self-assembling properties, forming potent complexes with HA to create novel gel-like structures. Notably, these peptides maintain a distinct helical structure, reminiscent of molecular inductions, which lead to inspiration in recent electron transfer designs.

These findings led us to develop of a novel pro-HA scaffold wound healing system, leveraging the unique properties of these peptide-HA complexes to promote tissue regeneration.

Illustration of the B(X7)B hyaluronic acid binding site in mPEP35, showing N-C termini ribbon model, spatial locations of K4, V8, K12, and helical net. Includes secondary structure shift to nanofiber with peptide concentration.
An example illustration of mapping single B(X7)B hyaluronic acid binding site in mPEP35, showing N to C termini (blue to red) ribbon model, side and overview stick model with lysine 4 (K4), valine 8 (V8), and lysine 12 (K12) spatial locations, and helical net with drawn line (B(X7)B domain) between these residues. The domain is drawn by connecting the two positive charges, i.e., B1 (residue #4) and B2 (residue #12) of a B(X7)B domain, where I5 to L11 are the residues in the C7 region of the B(X7)B domain. (B) Secondary structure shift from 310 helical, to beta sheet (intra/intermolecular), and eventually self-assembled nanofiber with increase peptide concentration. (PMID: 37178990).
Helical wheel model of HA target peptide 17x-316 showing the fourth face 17x-3-iv with seven helical faces and five B1(X7)B2 HA binding domains. Residues and pair designations are color-coded as per Figure 4.
Multifaceted analysis of HA target peptide 17x-3 with a representative model of the fourth face 17x-3-iv and a helical wheel. The helical nets depict the seven helical faces composed of three 7-residue diagonals. The five B1(X7)B2 HA binding domains are contained in five of the seven helical faces, i.e., 17x-3-i, 17x-ii, 17x-3-iv, 17x-3-v and 17x-3-vii. B1(X7)B2 positive residues in the helical face are colored in blue, hydrophobic in yellow, hydroxy-amino acids in purple and amide amino acids in pink. (PMID: 39655381).

3D Cell Scaffolds Models for Intraspinal Microstimulation (ISMS), Traumatic Brain Injury and Glia in Limb Transplantation and Injury

Our lab focuses on optimizing the synthesis and design of methacrylated hyaluronic acid (HAMA) scaffolds for 3D cell culture. By incorporating various basal lamina components, we have successfully enhanced the integration of mixed glial populations within these scaffolds.

Further, we have developed a multi-layered scaffold system that enables the inclusion of multiple cell types and facilitates the reproducible insertion of functional electrical stimulation (ISMS) electrodes. We are currently investigating the effects of various electrical stimulation paradigms on rat brain-derived glia and exploring surface modifications to the electrodes to minimize glial adhesion.

These 3D scaffold models have also been adapted to study traumatic brain injury (TBI) using a torsion impact system. This system allows for the recreation of varying degrees of TBI and can also be utilized to model peripheral nerve injury in a hind limb transplant setting.

We are currently applying this system in glial biology and neuroinflammation to the complex field of limb transplantation. Specifically, we are investigating strategies to mitigate rejection by engineering specific microglial phenotypes within peptide-based self-assembling scaffolds. These tailored microglial populations are designed to promote a reparative response within the transplanted nerve.

graphic and cross-sectional overview of femur surgery with 18-gauge intramedullary rod. Includes micrographs of femoral nerve, artery, vein anastomoses, and sciatic nerve co-aption before bone fusion.
(A) Operative design is depicted in cartoon format with (B) cross-sectional overview. Representative images were taken through the operating microscope of the 18-gauge needle intramedullary rod before fully co-apting the femur bone, femoral artery, vein, and nerve anastomoses, sciatic nerve anastomosis. Cross-section cartoon and respective micrographs are presented as (i) fermoral nerve, artery, and vein anastomoses, (ii) sciatic nerve anastomoses, and (iii) femoral bone co-aption. (PMID: 32925888).
Schematic of a 3D cell culture using methacrylated hyaluronic acid (HAMA). Glial cells are mixed with HAMA, pipetted into a mold, photopolymerized, and incubated. Microglia (red), astrocytes (yellow), and oligodendrocytes (green) are shown.
Schematic of methacrylated hyaluronic acid (HAMA)-based 3D cell culture and photopolymerization protocol. Glial cells (2 week primary rat brain culture) are mixed with HAMA and pipetted into a polydimethylsiloxane (PDMS) mold on a glass coverslip. This mixture is exposed to a high intensity green LED light to polymerize the hydrogel for 5 minutes. The mold is removed and the gel is incubated in media. Representative cells include microglia (red), astrocytes (yellow), and oligodendrocytes (green). (PMID: 29286415).
Schematic of neural glia transplantation strategies, including microglia, astrocytes, Schwann cells, olfactory ensheathing glia, oligodendrocytes, and radial glia, for CNS repair and neurogenesis.
Overall schematic for major strategies employing neural glia for transplantation. Microglia (MG) and astrocytes (AC) are modeled and engineered (peptides, scaffolds, coatings) to mediate the inflammatory microenvironment and provide anti-gliotic function. Schwann cells (SC), olfactory ensheathing glia (OEG), and oligodendrocytes (OG) are used in various transplant applications to remyelinate CNS injury. Radial glia and ACs are employed to generate artificial rostral migratory streams to guide and differentiate neural progenitors (NPC) into neurons. (PMID: 39396630).