Spherical Nucleic Acids For Advanced Wound Healing Applications Chad A. Mirkin

Size: px

Start display at page:

Download "Spherical Nucleic Acids For Advanced Wound Healing Applications Chad A. Mirkin"

Ada Cummings
5 years ago
Views:

1 Spherical Nucleic Acids For Advanced Wound Healing Applications Chad A. Mirkin Departments of Chemistry, Infectious Disease, Materials Science & Engineering, Chemical & Biological Engineering, and Biomedical Engineering International Institute for Nanotechnology

Spherical Nucleic Acids (SNAs) 1 nm 20 nm

Programmable Recognition Multivalency and

2 Spherical Nucleic Acids (SNAs) 1 nm 20 nm Fluorescein 37 atoms 40-mer Oligo- Nucleotide 1400 atoms 13 nm Au NP ~67,500 atoms Mirkin et al, Nature 1996 (382) Synthetically Programmable Recognition Multivalency and Multi-functionality New Properties: Cooperative binding, Catalysis

3 Synthesis of Spherical Nucleic Acids (~200 strands/ 30nm particle) Citrate 40-mer Oligonucleotide Sequence-Spacer-Attachment (e.g. SH, N 3)

4 SNAs Have Unique Properties Distinct From Their Linear Counterparts Property Spherical Nucleic Acids Linear Nucleic Acids Melting Transition Cooperative and Narrow (~2-8ºC) Broad (~20ºC) Binding Strength K eq = 1.8 x K eq = 1.8x10 12 Inorganic Core s INTERNATIONAL INSTITUTE FOR NANOTECHNOLOGY Plasmonic, Catalytic, Magnetic, Luminescent Cellular Uptake Transfection agents NOT required Lipofectamine TM, Dharmafect TM, etc Immune Response Minimal Elevated Interferon-β Stability Resistance to Nucleases Rapid Degradation N/A

5 SNAs Enter Cells Rapidly And Efficiently C166 Cell Free ssdna (Cy5 labeled) 50 μm 50 μm Cy5-labeled SNA 50 μm 50 μm + DharmaFECT 0 h 0.5 h 1 h 2 h

6 Commercial Forms and Uses of Spherical Nucleic Acids NanoFlares for intracellular measurements of genetic content > 1,700 commercial forms Verigene System TM Potent Single Entity Gene Regulation Agents 10 FDA-cleared panel assays

7 SNAs Naturally Enter over 60 Different Cell Types Rapid and Efficient Cellular Uptake of SNAs in Multiple Cell Lines Cy5-siRNA-SNA

8 Different Paths To Gene Regulation Linear Nucleic Acids: Positively Charged Carriers Required SNA Conjugates Negatively Charged, Single-Entity Agents Delivery agent and nucleic acids needed for transfection Specialty nucleic acids required Significant immune response and toxicity Short construct lifetime Single-entity agent Compatible with unmodified nucleic acids No significant immune response or toxicity Long construct lifetime

9 SNA Technical Advances In Dermatology Scavenger receptors are overexpressed in keratinocytes Straightforward topical application Penetration of mouse, pig, and human skin No toxicity measured after 3 weeks of continuous use Efficacy against targets in mouse skin (psoriasis and wound healing) SNA Penetration Through Human Skin

Ulcers >25% of diabetics develop chronic ulcers significantly increases rate of bacterial infections In 2010, over

10 Diabetes Impairs Wound Healing > 20 million in the US have Type 2 diabetes Number of Persons with Diagnosed Diabetes (US ) Increases risk of heart disease, retinopathy, neuropathy, and wound healing complications Chronic Ulcers >25% of diabetics develop chronic ulcers significantly increases rate of bacterial infections In 2010, over 70,000 amputations needed to be performed in diabetic patients diabetes.webmd.com/foot-ulcers

Current Therapies for Diabetic Wounds Are

Factors Degradation by proteases Lack of

Very expensive Requires surgery www.cdc.

11 Current Therapies for Diabetic Wounds Are Limited Surgical debridement and moist dressings Requires a clinician Manages symptoms but not curative Topical Growth Factors Degradation by proteases Lack of clinical success Bioengineered Skin Grafts Very expensive Requires surgery

12 GM3 Has Been Shown to be a Critical Mediator of Insulin Resistance Normal Keratinocytes State of Insulin Resistance IR localizes to Lipid Rafts in normal keratinocytes Excessive accumulation of GM3 in Lipid Rafts Elimination of IR from Lipid Rafts Appropriate signaling necessary for skin keratinocyte proliferation and migration in wound healing Adapted from Kabayama et al. PNAS. 2007, 104,

Paller Has Shown GM3S Depletion Increases Rate of Wound Healing Diabetic GM3 synthase deficient mice show a dramatic reversal of the wound healing impairment seen in their wild type diabetic

13 Paller Has Shown GM3S Depletion Increases Rate of Wound Healing Diabetic GM3 synthase deficient mice show a dramatic reversal of the wound healing impairment seen in their wild type diabetic littermates. GM3S is a viable target for gene regulation. sirna technology is a potent tool to downregulate GM3S in vivo Challenges to overcome Delivery into cells and skin RNA Stability WT Diabetic GM3S -/- Diabetic WT Normal Day 0 Day 3 Day 5 Day 7 Mice were fed a high fat diet for 10 weeks to induce type 2 diabetes Wang et al. J Inv Dermatology. 2014, 532, 1038.

14 sirna Spherical Nucleic Acids (SNAs) Synthesized by functionalizing thiol-modified duplex RNA onto AuNP and passivating AuNP surface with diluents (e.g. PEG) 13nm AuNP Thiolated PEG This architecture confers some very unique biological properties to these particles Thiolated Duplex sirna

sirna SNAs Are a Powerful Platform to Suppress Gene

Mammalian Cells No Apparent Cytotoxicity PBS Control

DAPI-stained nuclei 20 μm % Cell Viability (Normalized to

PBS Alone Significantly Reduced Off- Target Effects in

expression High Gene Suppression Untreated DharmaFECT sirna

15 sirna SNAs Are a Powerful Platform to Suppress Gene Expression High Penetration Through Skin and Uptake into Mammalian Cells No Apparent Cytotoxicity PBS Control Cy5-labeled SNA 100 μm 100 μm Red = Cy5-labeled SNA Blue = DAPI-stained nuclei 20 μm % Cell Viability (Normalized to untreated) Lipofectamine2000 SNA Total DNA (ng) : PBS Alone Significantly Reduced Off- Target Effects in Cells 2: sirna-sna 3: DharmaFECT-siRNA Relative EGFR mrna expression High Gene Suppression Untreated DharmaFECT sirna sirna SNA Zheng et al. PNAS. 2012, 110, Cutler et al. JACS. 2011, 133, 9254.

16 Hypothesis By topically delivering gene-suppressing SNAs to induce GM3 synthase (GM3S) depletion at the wound site, we will be able to accelerate wound healing in diabetic mouse models. GM3 synthase suppressing sirna GM3 synthase suppressing SNA

knockdown GM3S mrna and protein levels by over 80%

17 Concentration Dependent Down-Regulation of GM3S in Immortalized Mouse Keratinocytes GM3S SNA knockdown GM3S mrna and protein levels by over 80% SNA show sirna-sequence specific knockdown of target gene

GM3S SNAs Increase the Migration Rate of Immortalized Mouse Keratinocytes Scratch Assay Untreated Nonsense SNA (2 nm) GM3S SNA (2 nm) Hyperglycemic growth medium used

18 GM3S SNAs Increase the Migration Rate of Immortalized Mouse Keratinocytes Scratch Assay Untreated Nonsense SNA (2 nm) GM3S SNA (2 nm) Hyperglycemic growth medium used to simulate insulin resistance 1 millimeter wide scratch GM3S SNA treatment significantly increases keratinocyte migration from scratch edge 0 h 48 h 60 h scale = 120 μm

19 GM3S SNAs Increase the Proliferation Rate of Immortalized Mouse Keratinocytes Normalized 450 nm (Cell count) Untreated Nonsense SNA GM3S SNA (2 nm) (2 nm) 1 Proliferation Assay Hyperglycemic growth medium used to simulate insulin resistance > 2-fold increase in rate of keratinocyte proliferation with GM3S SNA treatment Days

(arrows) Silicone splints placed around each wound to

20 Mouse Model for Animal Study Two 6 mm wounds punch-biopsied on the backs of diet-induced diabetic mice (arrows) Silicone splints placed around each wound to minimize wound contracture more closely simulates wound healing in humans

21 Topical Application of GM3S SNAs Increases the Rate of Wound Healing in Diabetic Mice * * * SNA mixed in OTC moisturizer, Aquaphor 50 nm SNA applied every 48h around wound edge * = p < 0.05

GM3S mrna and protein at harvested wound sites (N = 6/treatment) Gel

22 SNAs Accelerate Wound Healing by Downregulating GM3S in Mice via RNA Interference Topical GM3S SNA application resulted in over 80% depletion of the GM3S mrna and protein at harvested wound sites (N = 6/treatment) Gel electrophoresis on amplified mrna confirms that GM3S downregulation is mediated by RNA interference

Conclusions GM3S sirna-snas penetrate through skin, downregulate a critical mediator of insulin resistance via RNA interference, and accelerate wound healing in diabetic mice

23 Conclusions GM3S sirna-snas penetrate through skin, downregulate a critical mediator of insulin resistance via RNA interference, and accelerate wound healing in diabetic mice SNA Penetration Through Human Skin SNAs have the potential to be a potent platform to deliver nucleic acid based therapeutics to treat a wide variety of skinrelated disorders

24 Acknowledgements Mirkin Lab Pratik Randeria Dr. Tim Merkel Mirkin Research Group Amy Paller Lab Dr. Mark Seeger Dr. Xiaoqi Wang Dr. Desmond Shipp Heather Wilson Funding NSF, NIH

25 Questions?

Fish Skin Grafts Promote Superior Cell Ingrowth Compared to Amnion Allografts, Human Cadaver Skin and Mammalian Extracellular Matrix (ECM)

Fish Skin Grafts Promote Superior Cell Ingrowth Compared to Amnion Allografts, Human Cadaver Skin and Mammalian Extracellular Matrix (ECM) Christopher L. Winters, DPM American Health Network Indianapolis,