Publications by authors named "Molly S Shoichet"

183 Publications

Designing Hydrogels for 3D Cell Culture Using Dynamic Covalent Crosslinking.

Adv Healthc Mater 2021 May 14:e2100234. Epub 2021 May 14.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada.

Designing simple biomaterials to replicate the biochemical and mechanical properties of tissues is an ongoing challenge in tissue engineering. For several decades, new biomaterials have been engineered using cytocompatible chemical reactions and spontaneous ligations via click chemistries to generate scaffolds and water swollen polymer networks, known as hydrogels, with tunable properties. However, most of these materials are static in nature, providing only macroscopic tunability of the scaffold mechanics, and do not reflect the dynamic environment of natural extracellular microenvironment. For more complex applications such as organoids or co-culture systems, there remain opportunities to investigate cells that locally remodel and change the physicochemical properties within the matrices. In this review, advanced biomaterials where dynamic covalent chemistry is used to produce stable 3D cell culture models and high-resolution constructs for both in vitro and in vivo applications, are discussed. The implications of dynamic covalent chemistry on viscoelastic properties of in vitro models are summarized, case studies in 3D cell culture are critically analyzed, and opportunities to further improve the performance of biomaterials for 3D tissue engineering are discussed.
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http://dx.doi.org/10.1002/adhm.202100234DOI Listing
May 2021

Stable oxime-crosslinked hyaluronan-based hydrogel as a biomimetic vitreous substitute.

Biomaterials 2021 04 4;271:120750. Epub 2021 Mar 4.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College St, Toronto, ON, M5S 3E5, Canada; Institute of Biomedical Engineering, University of Toronto, 160 College St, Toronto, ON, M5S 3E1, Canada; Institute of Medical Sciences, University of Toronto, 1 King's College Circle, ON, M5S 1A8, Canada. Electronic address:

Vitreous substitutes are clinically used to maintain retinal apposition and preserve retinal function; yet the most used substitutes are gases and oils which have disadvantages including strict face-down positioning post-surgery and the need for subsequent surgical removal, respectively. We have engineered a vitreous substitute comprised of a novel hyaluronan-oxime crosslinked hydrogel. Hyaluronan, which is naturally abundant in the vitreous of the eye, is chemically modified to crosslink with poly(ethylene glycol)-tetraoxyamine via oxime chemistry to produce a vitreous substitute that has similar physical properties to the native vitreous including refractive index, density and transparency. The oxime hydrogel is cytocompatible in vitro with photoreceptors from mouse retinal explants and biocompatible in rabbit eyes as determined by histology of the inner nuclear layer and photoreceptors in the outer nuclear layer. The ocular pressure in the rabbit eyes was consistent over 56 d, demonstrating limited to no swelling. Our vitreous substitute was stable in vivo over 28 d after which it began to degrade, with approximately 50% loss by day 56. We confirmed that the implanted hydrogel did not impact retina function using electroretinography over 90 days versus eyes injected with balanced saline solution. This new oxime hydrogel provides a significant improvement over the status quo as a vitreous substitute.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120750DOI Listing
April 2021

Single-cell chromatin accessibility profiling of glioblastoma identifies an invasive cancer stem cell population associated with lower survival.

Elife 2021 Jan 11;10. Epub 2021 Jan 11.

Princess Margaret Cancer Centre, University Health Network, Toronto, Canada.

Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In-depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival, in line with the higher invasive properties of Invasive state GSCs compared to Reactive and Constructive GSCs as shown by in vitro and in vivo assays. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.
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http://dx.doi.org/10.7554/eLife.64090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847307PMC
January 2021

An Injectable Hyaluronan-Methylcellulose (HAMC) Hydrogel Combined with Wharton's Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair.

Int J Mol Sci 2020 Oct 7;21(19). Epub 2020 Oct 7.

Department of Neurosurgery, CHA University School of medicine, CHA Bundang Medical Center, Seongnam-si, 13496, Korea.

Intervertebral disc (IVD) degeneration is one of the predominant causes of chronic low back pain (LBP), which is a leading cause of disability worldwide. Despite substantial progress in cell therapy for the treatment of IVD degeneration, significant challenges remain for clinical application. Here, we investigated the effectiveness of hyaluronan-methylcellulose (HAMC) hydrogels loaded with Wharton's Jelly-derived mesenchymal stromal cell (WJ-MSCs) in vitro and in a rat coccygeal IVD degeneration model. Following induction of injury-induced IVD degeneration, female Sprague-Dawley rats were randomized into four groups to undergo a single intradiscal injection of the following: (1) phosphate buffered saline (PBS) vehicle, (2) HAMC, (3) WJ-MSCs (2 × 10 cells), and (4) WJ-MSCs-loaded HAMC (WJ-MSCs/HAMC) ( = 10/each group). Coccygeal discs were removed following sacrifice 6 weeks after implantation for radiologic and histologic analysis. We confirmed previous findings that encapsulation in HAMC increases the viability of WJ-MSCs for disc repair. The HAMC gel maintained significant cell viability in vitro. In addition, combined implantation of WJ-MSCs and HAMC significantly promoted degenerative disc repair compared to WJ-MSCs alone, presumably by improving nucleus pulposus cells viability and decreasing extracellular matrix degradation. Our results suggest that WJ-MSCs-loaded HAMC promotes IVD repair more effectively than cell injection alone and supports the potential clinical use of HAMC for cell delivery to arrest IVD degeneration or to promote IVD regeneration.
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http://dx.doi.org/10.3390/ijms21197391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582266PMC
October 2020

Attenuated diphtheria toxin mediates siRNA delivery.

Sci Adv 2020 May 1;6(18). Epub 2020 May 1.

Department of Chemistry, University of Toronto, Toronto, ON, Canada.

Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-β1 ()-a gene that promotes invasion and metastasis-and siRNA against eukaryotic translation initiation factor 3 subunit b ()-a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.
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http://dx.doi.org/10.1126/sciadv.aaz4848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195190PMC
May 2020

Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability.

Sci Adv 2020 Aug 19;6(34):eabc6378. Epub 2020 Aug 19.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON M5S 3E5, Canada.

Maintaining biocatalyst stability and activity is a critical challenge. Chondroitinase ABC (ChABC) has shown promise in central nervous system (CNS) regeneration, yet its therapeutic utility is severely limited by instability. We computationally reengineered ChABC by introducing 37, 55, and 92 amino acid changes using consensus design and forcefield-based optimization. All mutants were more stable than wild-type ChABC with increased aggregation temperatures between 4° and 8°C. Only ChABC with 37 mutations (ChABC-37) was more active and had a 6.5 times greater half-life than wild-type ChABC, increasing to 106 hours (4.4 days) from only 16.8 hours. ChABC-37, expressed as a fusion protein with Src homology 3 (ChABC-37-SH3), was active for 7 days when released from a hydrogel modified with SH3-binding peptides. This study demonstrates the broad opportunity to improve biocatalysts through computational engineering and sets the stage for future testing of this substantially improved protein in the treatment of debilitating CNS injuries.
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http://dx.doi.org/10.1126/sciadv.abc6378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438101PMC
August 2020

Hydrogel-mediated co-transplantation of retinal pigmented epithelium and photoreceptors restores vision in an animal model of advanced retinal degeneration.

Biomaterials 2020 10 30;257:120233. Epub 2020 Jul 30.

Institute of Biomedical Engineering, University of Toronto, Toronto, ON, M5S 3G9, Canada; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, M5S 3E1, Canada; Institute of Medical Science, University of Toronto, Toronto, ON, M5S 1A8, Canada; Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada; Department of Chemistry, University of Toronto, Toronto, ON, M5S 3H6, Canada. Electronic address:

We demonstrate a novel approach to reverse advanced stages of blindness using hydrogel-mediated delivery of retinal pigmented epithelium (RPE) and photoreceptors directly to the degenerated retina of blind mice. With sodium iodate (NaIO) injections in mice, both RPE and photoreceptors degenerate, resulting in complete blindness and recapitulating the advanced retinal degeneration that is often observed in humans. We observed vision restoration only with co-transplantation of RPE and photoreceptors in a hyaluronic acid-based hydrogel, and not with transplantation of each cell type alone as determined with optokinetic head tracking and light avoidance assays. Both RPE and photoreceptors survived significantly better when co-transplanted than in their respective single cell type controls. While others have pursued transplantation of one of either RPE or photoreceptors, we demonstrate the importance of transplanting both cell types with a minimally-invasive hydrogel for vision repair in a degenerative disease model of the retina.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120233DOI Listing
October 2020

Evaluation of ASCs and HUVECs Co-cultures in 3D Biodegradable Hydrogels on Neurite Outgrowth and Vascular Organization.

Front Cell Dev Biol 2020 16;8:489. Epub 2020 Jun 16.

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal.

Vascular disruption following spinal cord injury (SCI) decisively contributes to the poor functional recovery prognosis facing patients with the condition. Using a previously developed gellan gum hydrogel to which the adhesion motif GRGDS was grafted (GG-GRGDS), this work aimed to understand the ability of adipose-derived stem cells (ASCs) to impact vascular organization of human umbilical vein endothelial cells (HUVECs), and how this in turn affects neurite outgrowth of dorsal root ganglia (DRG) explants. Our data shows that culturing these cells together lead to a synergistic effect as showed by increased stimulation of neuritogenesis on DRG. Importantly, HUVECs were only able to assemble into vascular-like structures when cultured in the presence of ASCs, which shows the capacity of these cells in reorganizing the vascular milieu. Analysis of selected neuroregulatory molecules showed that the co-culture upregulated the secretion of several neurotrophic factors. On the other hand, ASCs, and ASCs + HUVECs presented a similar profile regarding the presence of angiotrophic molecules herein analyzed. Finally, the implantation of GG-GRGDS hydrogels encapsulating ASCs in the chick chorioallantoic membrane (CAM) lead to increases in vascular recruitment toward the hydrogels in comparison to GG-GRGDS alone. This indicates that the combination of ASCs with GG-GRGDS hydrogels could promote re-vascularization in trauma-related injuries in the central nervous system and thus control disease progression and induce functional recovery.
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http://dx.doi.org/10.3389/fcell.2020.00489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308435PMC
June 2020

Inverse Electron-Demand Diels-Alder Methylcellulose Hydrogels Enable the Co-delivery of Chondroitinase ABC and Neural Progenitor Cells.

Biomacromolecules 2020 06 22;21(6):2421-2431. Epub 2020 Apr 22.

Department of Chemical Engineering & Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON M5S 3E5, Canada.

A hydrogel that can deliver both proteins and cells enables the local microenvironment of transplanted cells to be manipulated with a single injection. Toward this goal, we designed a hydrogel suitable for the co-delivery of neural stem cells and chondroitinase ABC (ChABC), a potent enzyme that degrades the glial scar that forms after central nervous system (CNS) injury. We leveraged the inverse electron-demand Diels-Alder reaction between norbornene and methylphenyltetrazine to form rapidly gelling (<15 min) crosslinked methylcellulose (MC) hydrogels at physiological temperature and pH, with Young's modulus similar to that of brain tissue (1-3 kPa), and degradable, disulfide-containing crosslinkers. To achieve tunable, affinity-controlled release of a ChABC-Src homology 3 (SH3) fusion protein, we immobilized norbornene-functionalized SH3-binding peptides onto MC-methylphenyltetrazine and observed release of bioactive ChABC-SH3 over 4 days. We confirmed cytocompatibility by evaluating neural progenitor cell survival and proliferation. The combined encapsulation of neural stem cells and chondroitinase ABC from one hydrogel lays the framework for future in vivo studies to treat CNS injuries.
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http://dx.doi.org/10.1021/acs.biomac.0c00357DOI Listing
June 2020

Injectable hydrogel enables local and sustained co-delivery to the brain: Two clinically approved biomolecules, cyclosporine and erythropoietin, accelerate functional recovery in rat model of stroke.

Biomaterials 2020 03 16;235:119794. Epub 2020 Jan 16.

Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON, M5S 3G9, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada; Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, M5S 3H6, Canada; Department of Surgery, University of Toronto, 149 College Street, Toronto, ON, M5S 3E1, Canada. Electronic address:

Therapeutic delivery to the brain is limited by the blood-brain barrier and is exacerbated by off-target effects associated with systemic delivery, thereby precluding many potential therapies from even being tested. Given the systemic side effects of cyclosporine and erythropoietin, systemic administration would be precluded in the context of stroke, leaving only the possibility of local delivery. We wondered if direct delivery to the brain would allow new reparative therapeutics, such as these, to be identified for stroke. Using a rodent model of stroke, we employed an injectable drug delivery hydrogel strategy to circumvent the blood-brain barrier and thereby achieved, for the first time, local and sustained co-release to the brain of cyclosporine and erythropoietin. Both drugs diffused to the sub-cortical neural stem and progenitor cell (NSPC) niche and were present in the brain for at least 32 days post-stroke. Each drug had a different outcome on brain tissue: cyclosporine increased plasticity in the striatum while erythropoietin stimulated endogenous NSPCs. Only their co-delivery, but not either drug alone, accelerated functional recovery and improved tissue repair. This platform opens avenues for hitherto untested therapeutic combinations to promote regeneration and repair after stroke.
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http://dx.doi.org/10.1016/j.biomaterials.2020.119794DOI Listing
March 2020

Induction of Rod and Cone Photoreceptor-Specific Progenitors from Stem Cells.

Adv Exp Med Biol 2019 ;1185:551-555

Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.

Retinal degeneration includes a variety of diseases for which there is no regenerative therapy. Cellular transplantation is one potential approach for future therapy for retinal degeneration, and stem cells have emerged as a promising source for future cell therapeutics. One major barrier to therapy is the ability to specify individual photoreceptor lineages from a variety of stem cell sources. In this review, we focus on photoreceptor genesis from progenitor populations in the developing embryo and how this understanding has given us the tools to manipulate cultures to specific unique rod and cone lineages from adult stem cell populations. We discuss experiments and evidence uncovering the lineage mechanisms at play in the establishment of fate-specific rod and cone photoreceptor progenitors. This may lead to an improved understanding of retinal development in vivo, as well as new cell sources for transplantation.
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http://dx.doi.org/10.1007/978-3-030-27378-1_90DOI Listing
February 2020

Benchmarking to the Gold Standard: Hyaluronan-Oxime Hydrogels Recapitulate Xenograft Models with In Vitro Breast Cancer Spheroid Culture.

Adv Mater 2019 Sep 19;31(36):e1901166. Epub 2019 Jul 19.

The Donnelly Centre, University of Toronto, Toronto, 160 College St, Ontario, M5S 3E1, Canada.

Many 3D in vitro models induce breast cancer spheroid formation; however, this alone does not recapitulate the complex in vivo phenotype. To effectively screen therapeutics, it is urgently needed to validate in vitro cancer spheroid models against the gold standard of xenografts. A new oxime-crosslinked hyaluronan (HA) hydrogel is designed, manipulating gelation rate and mechanical properties to grow breast cancer spheroids in 3D. This HA-oxime breast cancer model maintains the gene expression profile most similar to that of tumor xenografts based on a pan-cancer gene expression profile (comprising 730 genes) of three different human breast cancer subtypes compared to Matrigel or conventional 2D culture. Differences in gene expression between breast cancer cultures in HA-oxime versus Matrigel or 2D are confirmed for 12 canonical pathways by gene set variation analysis. Importantly, drug response is dependent on the culture method. Breast cancer cells respond better to the Rac inhibitor (EHT-1864) and the PI3K inhibitor (AZD6482) when cultured in HA-oxime versus Matrigel. This study demonstrates the superiority of an HA-based hydrogel as a platform for in vitro breast cancer culture of both primary, patient-derived cells and cell lines, and provides a hydrogel culture model that closely matches that in vivo.
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http://dx.doi.org/10.1002/adma.201901166DOI Listing
September 2019

Triggered Release Enhances the Cytotoxicity of Stable Colloidal Drug Aggregates.

ACS Chem Biol 2019 07 25;14(7):1507-1514. Epub 2019 Jun 25.

Department of Chemical Engineering & Applied Chemistry , University of Toronto , 200 College Street , Toronto , Ontario M5S 3E5 , Canada.

Chemotherapeutics that self-assemble into colloids have limited efficacy above their critical aggregation concentration due to their inability to penetrate intact plasma membranes. Even when colloid uptake is promoted, issues with colloid escape from the endolysosomal pathway persist. By stabilizing acid-responsive lapatinib colloids through coaggregation with fulvestrant, and inclusion of transferrin, we demonstrate colloid internalization by cancer cells, where subsequent lapatinib ionization leads to endosomal leakage and increased cytotoxicity. These results demonstrate a strategy for triggered drug release from stable colloidal aggregates.
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http://dx.doi.org/10.1021/acschembio.9b00247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136197PMC
July 2019

Local delivery of FK506 to injured peripheral nerve enhances axon regeneration after surgical nerve repair in rats.

Acta Biomater 2019 09 26;96:211-221. Epub 2019 May 26.

Division of Plastic and Reconstructive Surgery, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario M5G1X8, Canada; Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Toronto, Toronto, Ontario, Canada; Program in Neuroscience, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.

Administration of FK506, an FDA approved immunosuppressant, has been shown to enhance nerve regeneration following peripheral nerve injuries. However, the severe side effects of the systemically delivered FK506 has prevented clinicians from the routine use of the drug. In this study, we analyzed the effectiveness of our fibrin gel-based FK506 delivery system to promote axon regeneration in a rat peripheral nerve transection and immediate surgical repair model. In addition, biodistribution of FK506 from the local delivery system to the surrounding tissues was analyzed in vivo. Rats in the negative control groups either did not receive any delivery system treatment or received fibrin gel with empty microspheres. The experimental groups included rats treated with fibrin gel loaded with solubilized, particulate, and poly(lactic-co-glycolic) acid microspheres-encapsulated FK506. Rats in experimental groups receiving FK506 microspheres and the particulate FK506 regenerated the highest number of motor and sensory neurons. Histomorphometric analysis also demonstrated greater numbers of myelinated axons following particulate FK506 and FK506 microspheres treatment compared to the negative control groups. In biodistribution studies, FK506 was found at the nerve repair site, the sciatic nerve, and spinal cord, with little to no drug detection in other vital organs. Hence, the local application of FK506 via our delivery systems enhanced axon regeneration whilst avoiding the toxicity of systemic FK506. This local delivery strategy represents a new opportunity for clinicians to use for cases of peripheral nerve injuries. STATEMENT OF SIGNIFICANCE: This work for the first time investigated the influence of locally administered FK506 to the site of nerve injury and immediate repair directly on the number of motor and sensory neurons that regenerated their axons. Furthermore, using the immediate nerve repair model, we obtained valuable information about the biodistribution of FK506 within the nervous system following its release from the delivery system implanted at the site of nerve injury and repair. The strategy of local FK506 delivery holds a great promise in the clinical translation, as the localized delivery circumvents the main limitation of the systemic delivery of FK506, that of immunosuppression and toxicity.
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http://dx.doi.org/10.1016/j.actbio.2019.05.058DOI Listing
September 2019

Effect of Sugar 2',4'-Modifications on Gene Silencing Activity of siRNA Duplexes.

Nucleic Acid Ther 2019 08 14;29(4):187-194. Epub 2019 May 14.

1Department of Chemistry, McGill University, Montreal, Canada.

In this study, we explore the effect of a library of 2'-, 4'-, and 2',4'-modified uridine nucleosides and their impact on silencing firefly luciferase and on down-regulated in renal cell carcinoma (DRR) gene targets. The modifications studied were 2'-F-ribose, 2'-F-arabinose, 2'-OMe-ribose, 2'-F,4'-OMe-ribose, 2'-F,4'-OMe-arabinose, and 2'-OMe,4'-F-ribose. We found that 2',4'-modifications are well tolerated within A-form RNA duplexes, leading to virtually no change in melting temperature as assessed by UV thermal melting. The impact of the dual (2',4') modification was assessed by comparing gene silencing ability to 2'- or 4'- (singly) modified siRNA counterparts. siRNAs with (2',4')-modified overhangs generally outperformed the native siRNA as well as siRNAs with a 2'- or 4'-modified overhang, suggesting that 2',4'-modified nucleotides interact favorably with Argonaute protein's PAZ domain. Among the most active siRNAs were those with 2'-F,4'-OMe-ribose or 2'-F,4'-OMe-arabinose at the overhangs. When modifications were placed at overhangs and internal positions, a duplex with the 2'-F (internal) and 2'-F,4'-OMe (overhang) combination was found to be the most potent, followed by the duplex with 2'-OMe (internal) and 2',4'-diOMe (overhang) modifications. Given the nuclease resistance exhibited by 2',4'-modified siRNAs, particularly when the modification is placed at or near the overhangs, these findings may allow the creation of superior siRNAs for therapy.
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http://dx.doi.org/10.1089/nat.2019.0792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686699PMC
August 2019

Cationic block amphiphiles show anti-mitochondrial activity in multi-drug resistant breast cancer cells.

J Control Release 2019 07 6;305:210-219. Epub 2019 May 6.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON M5S 3E5, Canada; Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON M5S 3G9, Canada; Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada. Electronic address:

Currently, there are limited treatment options for multi-drug resistant breast cancer. Lipid-modified cationic peptides have the potential to reach the mitochondria, which are attractive targets for the treatment of multi-drug resistant (MDR) breast cancer; yet, little is known about their mitochondrial targeting and anti-cancer activity. Interestingly, lipid-modified cationic peptides, typically used as gene transfection agents, exhibit similar structural features to mitochondrial targeted peptides. Using octahistidine-octaarginine (HR) as a model cationic peptide for cell penetration and endosomal escape, we explored the anti-cancer potential of lipid-modified cationic peptides as a function of amphiphilicity, biodegradability and lipid structure. We found that cationic peptides modified with a lipid that is at least 12 carbons in length exhibit potent anti-cancer activity in the low micromolar range in both EMT6/P and EMT6/AR-1 breast cancer cells. Comparing degradable and non-degradable linkers, as well as L- and D-amino acid sequences, we found that the anti-cancer activity is mostly independent of the biodegradation of the lipid-modified cationic peptides. Two candidates, stearyl-HR (Str-HR) and vitamin E succinate-HR (VES-HR) were cytotoxic to cancer cells by mitochondria depolarization. We observed increased reactive oxygen species (ROS) production, reduced cell bioenergetics and drug efflux, triggering apoptosis and G1 cell cycle arrest. Compared to Str-HR, VES-HR showed enhanced cancer cell selectivity and drug efflux inhibition, thereby serving as a potential novel therapeutic agent. This study deepens our understanding of lipid-modified cationic peptides and uncovers their potential in multi-drug resistant breast cancer.
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http://dx.doi.org/10.1016/j.jconrel.2019.04.045DOI Listing
July 2019

A hyaluronan/methylcellulose-based hydrogel for local cell and biomolecule delivery to the central nervous system.

Brain Res Bull 2019 05 18;148:46-54. Epub 2019 Mar 18.

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada; Donnelly Centre for Cellular & Biomolecular Research, University of Toronto, Toronto, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada; Department of Chemistry, University of Toronto, Toronto, Canada. Electronic address:

Regenerative medicine strategies rely on exogenous cell transplantation and/or endogenous cell stimulation. Biomaterials can help to increase the regenerative potential of cells and biomolecules by controlling transplanted cell fate and provide a local, sustained release of biomolecules. In this review, we describe the use of a hyaluronan/methylcellulose (HAMC)-based hydrogel as a delivery vehicle to the brain, spinal cord, and retina to promote cellular survival and tissue repair. We discuss various controlled release strategies to prolong the delivery of factors for neuroprotection. The versatility of this hydrogel for a diversity of applications highlights its potential to enhance cell- and biomolecule-based treatment strategies.
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http://dx.doi.org/10.1016/j.brainresbull.2019.03.005DOI Listing
May 2019

Modulated Protein Delivery to Engineer Tissue Repair.

Tissue Eng Part A 2019 07 14;25(13-14):925-930. Epub 2019 Jun 14.

1Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada.

Impact Statement: Achieving targeted protein delivery to injured tissues is a core focus of the field of tissue engineering and has enormous clinical potential. This article highlights significant advances made in biomaterial-based protein delivery strategies over the last 25 years and how they will influence research in the next 25 years. These advances will enable protein release rates to be tuned with increased flexibility to deliberately address the challenges of the dynamic injury environment and ultimately lead to better solutions for patients.
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http://dx.doi.org/10.1089/ten.TEA.2019.0066DOI Listing
July 2019

Colloidal Drug Aggregate Stability in High Serum Conditions and Pharmacokinetic Consequence.

ACS Chem Biol 2019 04 12;14(4):751-757. Epub 2019 Mar 12.

Department of Chemical Engineering and Applied Chemistry , University of Toronto , 200 College Street , Toronto , Ontario M5S 3E5 , Canada.

Colloidal drug aggregates have been a nuisance in drug screening, yet, because they inherently comprise drug-rich particles, they may be useful in vivo if issues of stability can be addressed. As the first step toward answering this question, we optimized colloidal drug aggregate formulations using a fluorescence-based assay to study fulvestrant colloidal formation and stability in high (90%) serum conditions in vitro. We show, for the first time, that the critical aggregation concentration of fulvestrant depends on media composition and increases with serum concentration. Excipients, such as polysorbate 80, stabilize fulvestrant colloids in 90% serum in vitro for over 48 h. Using fulvestrant and an investigational pro-drug, pentyloxycarbonyl-( p-aminobenzyl) doxazolidinylcarbamate (PPD), as proof-of-concept colloidal formulations, we demonstrate that the in vivo plasma half-life for stabilized colloids is greater than their respective monomeric forms. These studies demonstrate the potential of turning the nuisance of colloidal drug aggregation into an opportunity for drug-rich formulations.
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http://dx.doi.org/10.1021/acschembio.9b00032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474797PMC
April 2019

Transplantation of Directly Reprogrammed Human Neural Precursor Cells Following Stroke Promotes Synaptogenesis and Functional Recovery.

Transl Stroke Res 2020 02;11(1):93-107

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.

Stroke is one of the leading causes of long-term disability. Cell transplantation is a promising strategy to treat stroke. We explored the efficacy of directly reprogrammed human neural precursor cell (drNPC) transplants to promote functional recovery in a model of focal ischemic stroke in the mouse sensorimotor cortex. We show that drNPCs express neural precursor cell markers and are neurally committed at the time of transplantation. Mice that received drNPC transplants recovered motor function, irrespective of transplant vehicle or recipient sex, and with no correlation to lesion volume or glial scarring. The majority of drNPCs found in vivo, at the time of functional recovery, remained undifferentiated. Notably, no correlation between functional recovery and long-term xenograft survival was observed, indicating that drNPCs provide therapeutic benefits beyond their survival. Furthermore, increased synaptophysin expression in transplanted brains suggests that drNPCs promote neuroplasticity through enhanced synaptogenesis. Our findings provide insight into the mechanistic underpinnings of drNPC-mediated recovery for stroke and support the notion that drNPCs may have clinical applications for stroke therapy.
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http://dx.doi.org/10.1007/s12975-019-0691-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957566PMC
February 2020

Modeling of Photoreceptor Donor-Host Interaction Following Transplantation Reveals a Role for Crx, Müller Glia, and Rho/ROCK Signaling in Neurite Outgrowth.

Stem Cells 2019 04 14;37(4):529-541. Epub 2019 Feb 14.

Donald K. Johnson Eye Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada.

The goal of photoreceptor transplantation is to establish functional synaptic connectivity between donor cells and second-order neurons in the host retina. There is, however, limited evidence of donor-host photoreceptor connectivity post-transplant. In this report, we investigated the effect of the host retinal environment on donor photoreceptor neurite outgrowth in vivo and identified a neurite outgrowth-promoting effect of host Crx retinas following transplantation of purified photoreceptors expressing green fluorescent protein (GFP). To investigate the noncell autonomous factors that influence donor cell neurite outgrowth in vitro, we established a donor-host coculture system using postnatal retinal aggregates. Retinal cell aggregation is sensitive to several factors, including plate coating substrate, cell density, and the presence of Müller glia. Donor photoreceptors exhibit motility in aggregate cultures and can engraft into established aggregate structures. The neurite outgrowth-promoting phenotype observed in Crx recipients in vivo is recapitulated in donor-host aggregate cocultures, demonstrating the utility of this surrogate in vitro approach. The removal of Müller glia from host aggregates reduced donor cell neurite outgrowth, identifying a role for this cell type in donor-host signaling. Although disruption of chondroitin sulfate proteoglycans in aggregates had no effect on the neurite outgrowth of donor photoreceptors, disruption of Rho/ROCK signaling enhanced outgrowth. Collectively, these data show a novel role of Crx, Müller glia, and Rho/ROCK signaling in controlling neurite outgrowth and provide an accessible in vitro model that can be used to screen for factors that regulate donor-host connectivity. Stem Cells 2019;37:529-541.
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http://dx.doi.org/10.1002/stem.2985DOI Listing
April 2019

Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury.

Nat Commun 2019 01 31;10(1):518. Epub 2019 Jan 31.

Axe neurosciences du Centre de recherche du Centre hospitalier universitaire (CHU) de Québec-Université Laval et Département de médecine moléculaire de l'Université Laval, Québec, QC, G1V 4G2, Canada.

The role of microglia in spinal cord injury (SCI) remains poorly understood and is often confused with the response of macrophages. Here, we use specific transgenic mouse lines and depleting agents to understand the response of microglia after SCI. We find that microglia are highly dynamic and proliferate extensively during the first two weeks, accumulating around the lesion. There, activated microglia position themselves at the interface between infiltrating leukocytes and astrocytes, which proliferate and form a scar in response to microglia-derived factors, such as IGF-1. Depletion of microglia after SCI causes disruption of glial scar formation, enhances parenchymal immune infiltrates, reduces neuronal and oligodendrocyte survival, and impairs locomotor recovery. Conversely, increased microglial proliferation, induced by local M-CSF delivery, reduces lesion size and enhances functional recovery. Altogether, our results identify microglia as a key cellular component of the scar that develops after SCI to protect neural tissue.
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http://dx.doi.org/10.1038/s41467-019-08446-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355913PMC
January 2019

Local delivery of stabilized chondroitinase ABC degrades chondroitin sulfate proteoglycans in stroke-injured rat brains.

J Control Release 2019 03 25;297:14-25. Epub 2019 Jan 25.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada; Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada; Institute of Biomaterials and Biomedical Engineering, 164 College Street, Toronto, Ontario M5S 3G9, Canada; Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada. Electronic address:

Central nervous system (CNS) injuries, such as stroke and spinal cord injuries, result in the formation of a proteoglycan-rich glial scar, which acts as a barrier to axonal regrowth and limits the regenerative capacity of the CNS. Chondroitinase ABC (ChABC) is a potent bacterial enzyme that degrades the chondroitin sulfate proteoglycan (CSPG) component of the glial scar and promotes tissue recovery; however, its use is significantly limited by its inherent instability at physiological temperatures. Here, we demonstrate that ChABC can be stabilized using site-directed mutagenesis and covalent modification with poly(ethylene glycol) chains (i.e. PEGylation). Rosetta protein structure modeling was used to screen >20,000 single point mutations, and four potentially stabilizing mutations were tested in vitro. One of the mutations, N1000G (asparagine ➔ glycine at residue 1000), significantly improved the long-term activity of the protein, doubling its functional half-life. PEGylation of this ChABC mutant inhibited unfolding and aggregation and resulted in prolonged bioactivity with a 10-fold increase in activity compared to the unmodified protein after two days. Local, affinity-controlled release of the modified protein (PEG-N1000G-ChABC) was achieved by expressing it as a fusion protein with Src homology 3 (SH3) and delivering the protein from a methylcellulose hydrogel modified with SH3 binding peptides. This affinity-based release strategy provided sustained PEG-N1000G-ChABC-SH3 release over several days in vitro. Direct implantation of the hydrogel delivery vehicle containing stabilized PEG-N1000G-ChABC-SH3 onto the rat brain cortex in a sub-acute model of stroke resulted in significantly reduced CSPG levels in the penumbra of 49% at 14 and 40% at 28 days post-injury compared to animals treated with the vehicle alone.
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http://dx.doi.org/10.1016/j.jconrel.2019.01.033DOI Listing
March 2019

Local Delivery of Brain-Derived Neurotrophic Factor Enables Behavioral Recovery and Tissue Repair in Stroke-Injured Rats.

Tissue Eng Part A 2019 08 27;25(15-16):1175-1187. Epub 2019 Feb 27.

1Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada.

Impact Statement: We developed a biocomposite that can be mixed with brain-derived neurotrophic factor (BDNF) and dispensed onto the surface of the brain to provide sustained, local release of the protein using a procedure that avoids additional damage to neural tissue. The composite is simple to fabricate, and provides sustained release without nanoparticle encapsulation of BDNF, preserving material and protein bioactivity. We demonstrate that when delivered epicortically to a rat model of stroke, this composite allows BDNF to diffuse into the brain, resulting in enhanced behavioral recovery and synaptic plasticity in the contralesional hemisphere.
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http://dx.doi.org/10.1089/ten.TEA.2018.0215DOI Listing
August 2019

Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.

Adv Mater 2019 Feb 27;31(7):e1806214. Epub 2018 Dec 27.

Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, M5S 3G9, Canada.

Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.
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http://dx.doi.org/10.1002/adma.201806214DOI Listing
February 2019

Human Oligodendrogenic Neural Progenitor Cells Delivered with Chondroitinase ABC Facilitate Functional Repair of Chronic Spinal Cord Injury.

Stem Cell Reports 2018 12 21;11(6):1433-1448. Epub 2018 Nov 21.

Division of Genetics and Development, Krembil Research Institute, University Health Network, 60 Leonard Avenue, Toronto, ON M5T 2S8, Canada; Institute of Medical Sciences, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Surgery and Spinal Program, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Surgery, Division of Anatomy, Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada. Electronic address:

Treatment of chronic spinal cord injury (SCI) is challenging due to cell loss, cyst formation, and the glial scar. Previously, we reported on the therapeutic potential of a neural progenitor cell (NPC) and chondroitinase ABC (ChABC) combinatorial therapy for chronic SCI. However, the source of NPCs and delivery system required for ChABC remained barriers to clinical application. Here, we investigated directly reprogrammed human NPCs biased toward an oligodendrogenic fate (oNPCs) in combination with sustained delivery of ChABC using an innovative affinity release strategy in a crosslinked methylcellulose biomaterial for the treatment of chronic SCI in an immunodeficient rat model. This combinatorial therapy increased long-term survival of oNPCs around the lesion epicenter, facilitated greater oligodendrocyte differentiation, remyelination of the spared axons by engrafted oNPCs, enhanced synaptic connectivity with anterior horn cells and neurobehavioral recovery. This combinatorial therapy is a promising strategy to regenerate the chronically injured spinal cord.
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http://dx.doi.org/10.1016/j.stemcr.2018.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294173PMC
December 2018

Initial cell maturity changes following transplantation in a hyaluronan-based hydrogel and impacts therapeutic success in the stroke-injured rodent brain.

Biomaterials 2019 02 15;192:309-322. Epub 2018 Nov 15.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S3E1, Canada; Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, M5S3E1, Canada; Department of Chemistry, University of Toronto, Toronto, Ontario, M5S3E1, Canada. Electronic address:

Ischemic stroke results in a loss of neurons for which there are no available clinical strategies to stimulate regeneration. While preclinical studies have demonstrated that functional recovery can be obtained by transplanting an exogenous source of neural progenitors into the brain, it remains unknown at which stage of neuronal maturity cells will provide the most benefit. We investigated the role of neuronal maturity on cell survival, differentiation, and long-term sensorimotor recovery in stroke-injured rats using a population of human cortically-specified neuroepithelial progenitor cells (cNEPs) delivered in a biocompatible, bioresorbable hyaluronan/methylcellulose hydrogel. We demonstrate that transplantation of immature cNEPs result in the greatest tissue and functional repair, relative to transplantation of more mature neurons. The transplantation process itself resulted in the least cell death and phenotypic changes in the immature cNEPs, and the greatest acute cell death in the mature cells. The latter negatively impacted host tissue and negated any potential positive effects associated with cell maturity and the hydrogel vehicle, which itself showed some functional and tissue benefit. Moreover, we show that more mature cell populations are drastically altered during the transplantation process itself. The phenotype of the cells before and after transplantation had an enormous impact on their survival and the consequent tissue and behavioral response, emphasizing the importance of characterizing injected cells in transplantation studies more broadly.
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http://dx.doi.org/10.1016/j.biomaterials.2018.11.020DOI Listing
February 2019

Induction of rod versus cone photoreceptor-specific progenitors from retinal precursor cells.

Stem Cell Res 2018 12 13;33:215-227. Epub 2018 Nov 13.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address:

During development, multipotent progenitors undergo temporally-restricted differentiation into post-mitotic retinal cells; however, the mechanisms of progenitor division that occurs during retinogenesis remain controversial. Using clonal analyses (lineage tracing and single cell cultures), we identify rod versus cone lineage-specific progenitors derived from both adult retinal stem cells and embryonic neural retinal precursors. Taurine and retinoic acid are shown to act in an instructive and lineage-restricted manner early in the progenitor lineage hierarchy to produce rod-restricted progenitors from stem cell progeny. We also identify an instructive, but lineage-independent, mechanism for the specification of cone-restricted progenitors through the suppression of multiple differentiation signaling pathways. These data indicate that exogenous signals play critical roles in directing lineage decisions and resulting in fate-restricted rod or cone photoreceptor progenitors in culture. Additional factors may be involved in governing photoreceptor fates in vivo.
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http://dx.doi.org/10.1016/j.scr.2018.11.005DOI Listing
December 2018

Controlled release strategy designed for intravitreal protein delivery to the retina.

J Control Release 2019 01 10;293:10-20. Epub 2018 Nov 10.

Chemical Engineering & Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON M5S 3E5, Canada; Institute of Biomaterials & Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON M5S 3G9, Canada. Electronic address:

Therapeutic protein delivery directly to the eye is a promising strategy to treat retinal degeneration; yet, the high risks of local drug overdose and cataracts associated with bolus injection have limited progress, requiring the development of sustained protein delivery strategies. Since the vitreous humor itself is a gel, hydrogel-based release systems are a sensible solution for sustained intravitreal protein delivery. Using ciliary neurotrophic factor (CNTF) as a model protein for ocular treatment, we investigated the use of an intravitreal, affinity-based release system for protein delivery. To sustain CNTF release, we took advantage of the affinity between Src homology 3 (SH3) and its peptide binding partners: CNTF was expressed as a fusion protein with SH3, and a thermogel of hyaluronan and methylcellulose (HAMC) was modified with SH3 binding peptides. Using a mathematical model, the hydrogel composition was successfully designed to release CNTF-SH3 over 7 days. The stability and bioactivity of the released protein were similar to those of commercial CNTF. Intravitreal injections of the bioengineered thermogel showed successful delivery of CNTF-SH3 to the mouse retina, with expected transient downregulation of phototransduction genes (e.g., rhodopsin, S-opsin, M-opsin, Gnat 1 and 2), upregulation of STAT1 and STAT3 expression, and upregulation of STAT3 phosphorylation. This constitutes the first demonstration of intravitreal protein release from a hydrogel. Immunohistochemical analysis of the retinal tissues of injected eyes confirmed the biocompatibility of the delivery vehicle, paving the way towards new intravitreal protein delivery strategies.
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http://dx.doi.org/10.1016/j.jconrel.2018.11.012DOI Listing
January 2019

Colloidal aggregation: from screening nuisance to formulation nuance.

Nano Today 2018 Apr 10;19:188-200. Epub 2018 Mar 10.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, ON,Canada.

It is well known that small molecule colloidal aggregation is a leading cause of false positives in early drug discovery. Colloid-formers are diverse and well represented among corporate and academic screening decks, and even among approved drugs. Less appreciated is how colloid formation by drug-like compounds fits into the wider understanding of colloid physical chemistry. Here we introduce the impact that colloidal aggregation has had on early drug discovery, and then turn to the physical and thermodynamic driving forces for small molecule colloidal aggregation, including the particulate nature of the colloids, their critical aggregation concentration-governed formation, their mechanism of protein adsorption and subsequent inhibition, and their sensitivity to detergent. We describe methods that have been used extensively to both identify aggregate-formers and to study and control their physical chemistry. While colloidal aggregation is widely recognized as a problem in early drug discovery, we highlight the opportunities for exploiting this phenomenon in biological milieus and for drug formulation.
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http://dx.doi.org/10.1016/j.nantod.2018.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150470PMC
April 2018