Biomedical Applications of Synthetic Microgels

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1 Biomedical Applications of Synthetic Microgels Andrés J. García Woodruff School of Mechanical Engineering Petit Institute for Bioengineering & Bioscience Georgia Institute of Technology Atlanta, GA, U.S.A. Cellular & Biomaterials Engineering Laboratory

2 Disclosures AJG is an inventor on IP related to technologies presented here. This IP is owned in part by the Georgia Tech Research Corp. AJG is a co-founder and/or sits on the Board of Directors of the start-ups CellectCell and CorAmi.

3 Outline Intro: Why microgels for biomedical/regenerative medicine applications? Common fabrication approaches Application 1: Protein delivery for vascularization Application 2: Protein presentation for immunomodulation Application 3: Cell encapsulation

4 Microgels as Delivery Vehicles Advantages of microgels over bulk hydrogels microgels bulk hydrogel microgels bulk hydrogel microgels less trauma improved filling of defect/cavity ease of generating mixtures Liu & Garcia ABME (2016)

(trapping) proteasedegradable crosslinks (light) swelling of

5 Microgels as Delivery Vehicles Strategies to Control Protein Release change in ph, [ion], T microgel mesh ( nm) reduced mesh size (trapping) proteasedegradable crosslinks (light) swelling of stimuliresponsive network secondary containment tethering to microgel network Liu & Garcia ABME (2016)

6 Microgels as Delivery Vehicles Common Fabrication Methods Liu & Garcia ABME (2016)

7 Hydrogels for Controlled Delivery of Therapeutics PEG-4maleimide hydrogels flexible delivery platform well-defined structure mild rx minimal toxicity stoichiometric incorporation of biomolecules control over polymerization kinetics (macrogels, injectable, microgels) degradation products no toxicity/inflammation, renal excretion precise control over biophysical & biochemical properties elastic modulus, adhesive ligand type & density, degradation sensitivity Phelps+ Adv Mater (2012) Phelps+ Biomaterials (2013) Enemchukwu+ J Cell Biol (2016)

8 PEG-4MAL Microgels Microfluidics polymerization: control over size & chemical composition Headen+ Adv Mater (2014) US Patent 9,381,217

µm 400 300 200 100 Q Oil = 5 µl min -1 Q Oil = 25 µl min

Q PEG Droplet size / µm 300 200 100 Q PEG -1 Q xlink (µl

) PEG 50 30 20 20 Mean Diameter (µm) - 137 315 150 CV

6 22 Droplet size / µm 400 300 200 100 Q Oil = 50 µl min

= 20 µl min Q xlink = 30 µl min Droplet size / µm 300

min -1-1 -1-1 -1 Q xlink= 50 µl min Q xlink= 100 µl min

9 PEG-4MAL Microgels Microfluidics polymerization: control over size & chemical composition (a) (b) Droplet size / µm Q Oil = 5 µl min -1 Q Oil = 25 µl min / µl min -1 / µl min -1 Q PEG Droplet size / µm Q PEG -1 Q xlink (µl min ) Qoil (µl min ) Q (µl min ) PEG Mean Diameter (µm) CV (%) Droplet size / µm Q Oil = 50 µl min -1 Q Oil = 100 µl min Q xlink = 10 µl min Q xlink = 20 µl min Q xlink = 30 µl min Droplet size / µm Q PEG / µl min -1 / µl min Q xlink= 50 µl min Q xlink= 100 µl min -1 Q PEG Q xlink= 200 µl min Q xlink= 400 µl min -1-1 Headen+ Adv Mater (2014) US Patent 9,381,217

10 Outline Intro: Why microgels for biomedical/regenerative medicine applications? Common fabrication approaches Application 1: Protein delivery for vascularization Application 2: Protein presentation for immunomodulation Application 3: Cell encapsulation

of vasculogenic proteins (VEGF) critical

11 Microgels: Protein Delivery for Vascularization Motivation: Need for Therapeutic Vascularization Injectable biomaterial platform for controlled delivery of vasculogenic proteins (VEGF) critical limb ischemia transplant grafts (e.g., islets) wound healing healing post-mi

biomolecules control over polymerization kinetics degradation products no

12 Microgels: Protein Delivery for Vascularization PEG-4MAL hydrogels flexible delivery platform well-defined structure mild reaction minimal toxicity stoichiometric incorporation of biomolecules control over polymerization kinetics degradation products no toxicity/inflammation, renal excretion VEGF vascularization PBS protease Phelps+ Adv Mater (2012) Phelps+ Biomaterials (2013)

Microgels: Protein Delivery for Vascularization PEG-4MAL Microgels

defined chemistry DTT crosslinked Headen+ Adv Mater (2014); US Patent

considerations conformability mix-and-match /mosaic Re-engineer to generate

13 Microgels: Protein Delivery for Vascularization PEG-4MAL Microgels Microgels: microfluidics polymerization controlled size ( µm ± 2-5%) defined chemistry DTT crosslinked Headen+ Adv Mater (2014); US Patent 9,381,217 Advantages over bulk hydrogels injectability no in situ gelling considerations conformability mix-and-match /mosaic Re-engineer to generate peptide cross-linked microgels crosslinker oil 50 µm 200 µm PEG 400µm Foster + Biomaterials (2016)

14 Microgels: Protein Delivery for Vascularization Protease-Dependent Degradation of Microgels Microgels crosslinked with VPM degradable peptide protease-dependent degradation rate 100 U/mL collagenase Foster + Biomaterials (2016)

DTT/VPM cross-linker ratio DTT crosslinked microgels do not degrade in the

15 Microgels: Protein Delivery for Vascularization Tuning Protease-Dependent Protein Release Rate Release rate of IgG from microgels is dependent on DTT/VPM cross-linker ratio DTT crosslinked microgels do not degrade in the presence of enzyme Foster + Biomaterials (2016) mixed microgels DTT/VPM collagenase

16 Microgels: Protein Delivery for Vascularization Degradable Microgels with VEGF Release Controlled VEGF release in presence of protease AF488-VEGF Released VEGF maintains bioactivity Foster + Biomaterials (2016)

17 Microgels: Protein Delivery for Vascularization In Vivo Degradation of Microgels Dylight750-RGD microgels, s.c. implantation IVIS imaging D a y 1 Foster + Biomaterials (2016)

18 Microgels: Protein Delivery for Vascularization VEGF-releasing Microgels Promote Vascularization s.c. implantation 14 d DTT microgels + VEGF VPM microgels - VEGF VPM microgels + svegf VPM microgels + VEGF Lectin/Microgels Protease degradable microgels that release VEGF promote local vascularization Sustained release of VEGF is required for the formation of blood vessels Foster + Biomaterials (2016)

generate peptide-cross-linked PEG-4MAL microgels.

presence of proteases & release tethered molecules.

Microgels for facile, minimally invasive delivery

19 Microgels: Protein Delivery for Vascularization Summary Microfluidic flow focusing device to generate peptide-cross-linked PEG-4MAL microgels. Microgels cross-linked with VPM degrade in the presence of proteases & release tethered molecules. Release rate of tethered molecules can be controlled by modifying the ratio of degradable/non-degradable cross-linking peptides. Microgels for facile, minimally invasive delivery via injections. Degradable microgels containing VEGF and RGD promote vascularization in vivo.

20 Outline Intro: Why microgels for biomedical/regenerative medicine applications? Common fabrication approaches Application 1: Protein delivery for vascularization Application 2: Protein presentation for immunomodulation Application 3: Cell encapsulation

Biomaterials for Islet Vascularization & Function Juvenile type 1 diabetes 1:400

(2-4 donor/recipient) instant blood-mediated inflammatory reaction islet loss due

drugs Alternative Transplant Site to Liver highly vascularized avoid IBMIR

sizes Vasculogenic Hydrogels for Islet Delivery Transplant Sites sub-cutis (SUBQ)

21 Biomaterials for Islet Vascularization & Function Juvenile type 1 diabetes 1:400 children in US, $15B healthcare costs Islet transplantation inadequate islet supply (2-4 donor/recipient) instant blood-mediated inflammatory reaction islet loss due to inadequate vascularization immune rejection & complications of immunosuppressive drugs Alternative Transplant Site to Liver highly vascularized avoid IBMIR physiological glycoinsular responses easily accessible accommodate large graft sizes Vasculogenic Hydrogels for Islet Delivery Transplant Sites sub-cutis (SUBQ) small bowel mesentery (SBM) epididymal fat pad (EFP) Weaver + Sci Adv (2017) Robertson, Diabetes (2010)

Immunomodulatory Microgels for Islet Acceptance Fas/FasL: central regulator of peripheral tolerance Activated T cells express Fas & become sensitive to FasL-mediated apoptosis Fas/FasL deficiency:

22 Immunomodulatory Microgels for Islet Acceptance Fas/FasL: central regulator of peripheral tolerance Activated T cells express Fas & become sensitive to FasL-mediated apoptosis Fas/FasL deficiency: lymphoproliferation & autoimmune pathologies No compensatory mechanisms FasL-induced apoptosis to block Teff cell killing of islet grafts Gene therapy: technical & translational limitations FasL protein presentation is critical to function Membrane-bound: apoptotic Soluble form: anti-apoptotic, chemotactic SA-FasL: immunomodulatory regulator for islet modification localized tolerance to allogeneic islets via apoptosis of Teff cells via induction/expansion of Treg cells Yolcu + J Immunol (2011)

Immunomodulatory Microgels for Islet Acceptance Microgels

microfluidics polymerization X-linker oil phase PEG-4MAL

20-400 µm dia (± 2-5%) biotin-peg microgel bioactivity

23 Immunomodulatory Microgels for Islet Acceptance Microgels for controlled presentation of SA-FasL Microgels: microfluidics polymerization X-linker oil phase PEG-4MAL density Microgel size: nozzle dimensions, flow rates µm dia (± 2-5%) biotin-peg microgel bioactivity SA-FasL Headen+ Adv Mater (2014) US Patent 9,381,217 Headen + Nat Mater (2018)

24 Immunomodulatory SA-FasL Microgels Microgels increase in vivo retention & localization of SA-FasL SA-FasL-AF750 (1 µg/1000 microgels): KC, IVIS imaging SA-FasL retention time (t 50 ) microgel 3.2 ± 0.7 days free 0.7 ± 0.3 days p < Headen + Nat Mater (2018)

25 Immunomodulatory SA-FasL Microgels Co-transplantation of allogeneic islets & SA-FasL-microgels prolongs islet acceptance & function p < functioning graft rejected graft SA-FasL-microgel+ rapa SA-FasL: 1 ug/1000 PEG, rapa: 0.2mg/kg x15 Headen + Nat Mater (2018)

26 Immunomodulatory SA-FasL Microgels Immune profiling Increased Treg:Teff in graft & graft-draining LN for SA-FasL-microgel + rapa Headen + Nat Mater (2018)

Immunomodulatory SA-FasL Microgels Co-transplantation of allogeneic islets & SA-FasL-microgels prolongs islet acceptance & function via Treg cells Treg depletion: C57BL/6.

27 Immunomodulatory SA-FasL Microgels Co-transplantation of allogeneic islets & SA-FasL-microgels prolongs islet acceptance & function via Treg cells Treg depletion: C57BL/6.FoxP3 EGFP/DTR mice BALB/c islets SA-FasL biotin-peg microgel STZ C57BL/6.FoxP3 EGFP/DTR kidney capsule SA-FasL: 1 ug/1000 PEG, rapa: 0.2mg/kg x15 Headen + Nat Mater (2018)

28 Immunomodulatory Microgels for Islet Acceptance Summary & Next Steps Immunomodulatory materials for islet acceptance SA-FasL-microgels + islets achieves permanent allograft survival Biomaterial-dependent retention of SA-FasL at transplant site Localized immune acceptance Tregs involved in immune acceptance Off-the-shelf application Microgels prepared at time of transplantation Simple mixing: no islet manipulation Ongoing studies NOD mice Non-human primate FasL study

29 Intro: Outline Why microgels for biomedical/regenerative medicine applications? Common fabrication approaches Application 1: Protein delivery for vascularization Application 2: Protein presentation for immunomodulation Application 3: Cell encapsulation

30 Microgels: Islet Encapsulation & Transplantation PEG-4MAL microgels vs. alginate capsules synthetic microgels defined composition reduced size

31 Microgels: Islet Encapsulation & Transplantation PEG-4MAL microgels vs. alginate capsules Equivalent transport properties PEG microgel alginate capsules

32 Microgels: Islet Encapsulation & Transplantation PEG-4MAL microgels vs. alginate capsules Engineering microenvironment

33 Microgels: Islet Encapsulation & Transplantation PEG-4MAL microgels outperform alginate capsules Syngeneic islets into STZ-diabetic mice (EFP) 600 IEQ

34 Microgels: Islet Encapsulation & Transplantation PEG-4MAL microgels improved vascularization vs. alginate capsules Syngeneic islets into STZ-diabetic mice EFP, wks

35 Microgels: Islet Encapsulation & Transplantation PEG-4MAL microgels provide allogeneic islet protection Allogeneic islets into STZ-diabetic mice EFP, 1200 IEQ

36 Summary Intro: Why microgels for biomedical/regenerative medicine applications? Common fabrication approaches Application 1: Protein delivery for vascularization Application 2: Protein presentation for immunomodulation Application 3: Cell encapsulation