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65993 Peptide Conjugated Hollow, Degradable Nanoparticles Bind to Exposed Hyaluronic Acid for the Prevention and Treatment of Osteoarthritis
- Marcus Deloney, Parssa Garoosi, Blaine Christiansen, Alyssa Panitch
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- Journal:
- Journal of Clinical and Translational Science / Volume 5 / Issue s1 / March 2021
- Published online by Cambridge University Press:
- 30 March 2021, p. 142
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ABSTRACT IMPACT: Our research would be the first therapeutic to both prevent and treat osteoarthritis - helping 27 millions U.S. citizens alone immediately. OBJECTIVES/GOALS: Our objective is to conjugate hyaluronic acid binding peptides (HABP) to anionic hollow nanoparticle (hNP), and allowing the HABP-hNP complex to penetrate into osteoarthritic cartilage, bind to exposed HA, prevent further degradation, and restore the compressive strength of articular cartilage. METHODS/STUDY POPULATION: N-isopropyl acrylamide, 2-acrylamindo-2-methyl-1-propanesulfonic acid, N,N’-bis(acryoyl)cystamine, and Acrylic Acid, in fluorescent batches rhodamine b isothiocyanate (RBITC), were polymerized via precipitation reaction. HA binding peptide, GAHWQFNALTVRGSG-Hydrazide (GAH-Hyd), was covalently bonded to the hNP using DMTMM chemistry. The reaction was halted by diluting the solution 10:1 with milliQ water and purified using tangential flow filtration. The dynamic viscosity of the six treatments were analyzed in a 70 kDa HA. Using a rheometer (Discovery HR-3) with a 20 mm parallel plate geometry, TA Instruments, New Castle, DE), a frequency sweep (0.01 -1000 Hz, 2.512 Pa) was conducted to measure the storage modulus of each solution. RESULTS/ANTICIPATED RESULTS: GAH-Hyd was successfully conjugated to the surface of the hNP and zeta-potential shows a significant increase in surface charge from -21.41 mV for unconjugated hNP to -8.94 mV for 65 GAH conjugated hNP, confirming conjugation. The hNPs need 65 ±10 GAH per nanoparticle to significantly bind to HA, shown by increasing the dynamic viscosity of the solution. The minimum concentration of 65 GAH-hNP required to significantly bind to HA is 313 µM. These data from our study display the ability to functionalized the surface of polymeric hNPs with site specific peptides and their ability to bind to diseased tissue. We expect the GAH-hNP system will restore the compressive strength of OA cartilage and prevent further HA degradation in ex vivo aggrecan depleted cartilage plugs. DISCUSSION/SIGNIFICANCE OF FINDINGS: Binding to exposed HA within the ECM of cartilage protects the HA from further degradation, halting the progression of OA. 65 GAH-hNP binds to HA at a 313 µM. Our system can be translated and used to treat a multitude of conditions by conjugating tissue specific peptides to the surface of our hNPs and delivery site specific therapeutics to diseases tissue.
4260 Hollow, Degradable poly(N-Isopropylacrylamide) Derived Nanoparticles for the Delivery of Anti-Inflammatory Peptides for the Treatment and Prevention of Post-Traumatic Osteoarthritis
- Marcus A Deloney, Kyra Smart, Blaine Christiansen, Alyssa Panitch
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- Journal:
- Journal of Clinical and Translational Science / Volume 4 / Issue s1 / June 2020
- Published online by Cambridge University Press:
- 29 July 2020, p. 109
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OBJECTIVES/GOALS: Knocking down the inflammatory response following joint trauma may halt the cytokine cascade and prevent the resulting cyclic degradation of articular cartilage. MK2 inhibiting (MK2i) peptides are an emerging and promising class of pharmaceutical to treat post-traumatic osteoarthritis (PTOA); however, these peptides are susceptible to proteolytic degradation in the extracellular space. Our objective is to encapsulate MK2i in thermoresponsive hollow nanoparticles (hNPs) to knockdown the inflammatory cytokine IL-6 to prevent the cyclic degradation of articular cartilage. METHODS/STUDY POPULATION: NP Synthesis: N-isopropyl acrylamide (NIPAm) cores was initiated by potassium persulfate (KPS) in aqueous solution with sodium dodecylsulfate (SDS) at 70°C under a nitrogen for 2 hours. Then exposed to oxygen for 45 min, followed by a nitrogen purge. NIPAm, 2-acrylamindo-2-methyl-1-propanesulfonic acid (AMPS), N,N’-bis(acryoyl)cystamine (BAC), and Acrylic Acid (AAc), in fluorescent batches rhodamine b isothiocyanate (RBITC), were polymerized around the core to form the shell. NPs were purified using tangential flow filtration. The NPs were dialyzed at 4°C for 14 days to remove the core and form hNPs. Loading & Release: hNPs and MK2i were incubated at 1 mg/ml at 4°C for 24 h. MK2i released into 1x PBS and analyzed on HPLC. IL-6 Expression: Bovine chondrocytes seeded at 10,000 cell/cm2 were stimulated with 20 ng/ml IL-1b daily and treated once with 100 µg/ml MK2i loaded-NP or 100 µg/ml free MK2i treatment on day 2. Analyzed on bovine IL-6 ELISA. In Vivo Intra Articular Injections: 75 µl of 2 mg/ml hNPsRHB or a PBS control was injected into the right knee of 4-month old Fischer 344 (Envigo) rats. Rats were imaged daily for 7 days then euthanized, legs dissected, and imaged. RESULTS/ANTICIPATED RESULTS: Core removal facilitated increased MK2i release from hNPs, Fig 1A, allowing up to 63% after 5 days in PBS. The hNPs generated here offer a continual sustained release of MK2i and hNPs are non-cytotoxic (data not shown) up to 12 mg/ml. MK2i loaded-NPs significantly knocked down IL-6 production after a single treatment after 2 days, Figure 1B, and continued knockdown for up to 4 days. hNPsRBITC was successfully injected into rat joint space and was retained for at least 7-days compared to pre-injection and PBS control, Fig 1 B-C. DISCUSSION/SIGNIFICANCE OF IMPACT: hNPs protect MK2i from ECM degradation and offer continual sustained release into chondrocytes. Core removal allows for MK2i release in vitro with further sustained release compared to previous non-degradable model. The single MK2i treatment lead to a significant IL-6 knockdown bovine chondrocytes for up to 4 days in hNPs. We were able to successfully inject and retain fluorescently labeled hNPs within rat knees for 7 days. Our translational therapeutic shows the promise of delivering a degradable, non-cytotoxic hNP into the joint space to knockdown the inflammatory response to halt the cyclic progression of articular cartilage degradation and progression of PTOA. CONFLICT OF INTEREST DESCRIPTION: The authors declare the following competing financial interest(s): Moerae Matrix, Inc. has a worldwide exclusive license to the CPP (MK2 inhibitor peptide). A. Panitch owns greater than 5% of Moerae Matrix, Inc.
2229: Development of an angiogenic proteoglycan mimic to accelerate ischemic diabetic foot ulcer repair
- Jenny Lin, Alyssa Panitch
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- Journal:
- Journal of Clinical and Translational Science / Volume 1 / Issue S1 / September 2017
- Published online by Cambridge University Press:
- 10 May 2018, p. 4
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OBJECTIVES/SPECIFIC AIMS: This project aims to synthesize an angiogenic decorin mimic (VEGFp-DS-SILY) with varying densities of QK and characterize its angiogenic potential and synergism with VEGF by evaluating (1) endothelial cell (EC) migration and proliferation, (2) EC VEGF receptor activation, (3) EC tubule formation in collagen scaffolds, and (4) angiogenesis from a chick chorioallantoic membrane (CAM assay) growing into the scaffold, reflecting the ability of the collagen scaffold to integrate into existing vasculature. The next main goal is to develop and characterize an MMP-degradable nanoparticle system for controlled release of VEGF. Future work will evaluate in vivo effects of VEGFp-DS-SILY bound to a 3D collagen scaffold on ischemic wound repair in a combined excisional wound/bipedical dorsal skin flap rat model. METHODS/STUDY POPULATION: Peptide hydrazides are conjugated to the free carboxylic acid functional groups on dermatan sulfate using EDC chemistry. We added a 3 amino acid spacer (-Gly-Ser-Gly) to the C-terminus of the established QK sequence before the hydrazide functional group and refer to this modified QK as “VEGFp.” VEGFp, SILY, and N-terminal biotinylated versions were synthesized using standard Fmoc solid-phase peptide synthesis protocols and purified using reverse phase HPLC. Coupling efficiencies of peptides to dermatan sulfate were determined spectroscopically at 280 nm measuring the aromatic residues (Trp or Tyr) using a NanoDrop system. Dermatan sulfate with 1 or 4 VEGFp peptides coupled were termed DSV1 and DSV4, respectively. After further conjugation with SILY, we will blend this VEGFp-DS-SILY with unmodified DS-SILY to a total 10 μM to test increasing densities of VEGFp. To verify that the collagen-binding properties of VEGFp-DS-SILY are not compromised by the addition of VEGFp, we will use a streptavidin-HRP system to detect bound biotinylated VEGFp-DS-SILY on collagen-coated plates by established protocols. DSV1 and DSV4 were tested for their effects on endothelial VEGFR2 phosphorylation using an MSD ELISA-type assay and endothelial proliferation using an MTS assay. Cell migration was monitored using an ORIS assay where cells are grown to confluence around a silicone stopper that is then removed to allow cells to migrate inward. Tubulogenesis was evaluated by examining tubule formation on matrigel. Finally, in vivo angiogenesis will be evaluated using a chorioallantoic membrane assay. For extracellular VEGF release, hollow MMP-degradable thermoresponsive nanoparticles [NIPAM, 5 mol% 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 1% Acrylic Acid (AAc), 2 mol% MMP-degradable peptide diacrylate, and potassium persulfate initiator] will be synthesized around noncross-linked polymer cores. The cores will then be diffused out through the shell by dialysis prior to drug loading. SILY (and some biotinylated SILY for visualization) will be conjugated with EDC chemistry for targeting nanoparticles to collagen. NPs size and zeta-potential will be measured on a Malvern Zetasizer. VEGF will be loaded into NPs by co-incubating a loading solution of 1 µg/mL VEGF with 1 mg of NPs, incubating overnight at 4°C. VEGF loading and release will be measured by ELISA. Biological activity of the released VEGF from particles will be determined on ECs using assays similar to those outlined previously. RESULTS/ANTICIPATED RESULTS: Preliminary data have verified the synthesis and purification of SILY and VEGFp (QK-Gly-Ser-Gly-hydrazide), as well as an N-terminal biotinylated version, through mass spectrometry and reverse-phase HPLC, respectively. For proof-of-concept, we have verified binding of VEGFp to the VEGF receptor 2 using a ForteBio Blitz interferometry instrument. In addition to support based on published reports showing retained bioactivity of QK after conjugation using other spacers, our preliminary data suggests that VEGFp still binds to VEGF receptor 2, albeit with decreased affinity like QK as compared with VEGF. Circular dichroism also shows that VEGFp has retained its α-helical structure necessary for bioactivity; however it appears that it has some uncoiling when conjugated to dermatan sulfate. We hypothesize that varying densities of VEGFp conjugated to the decorin mimetic (DS-SILY) will modulate the degree of angiogenic activity and synergy with VEGF. We determined that we can achieve ~70% VEGFp conjugation completion to dermatan sulfate after 3.5 hours. We have quantified VEGFR2 phosphorylation after 5 minute treatments by using phospho-specific antibodies and an ELISA-type protocol in a mesoscale discovery system. Preliminary data with human umbilical vein endothelial cells shows that VEGFp exhibits synergism with VEGF at levels similar to QK. DSV1 and DSV4 data suggests synergy with VEGF, although free-peptides and engineered compounds alone did not show effects similar to VEGF in the conditions tested. Prelminary data with 30 minute treatments suggests that the peptides and compounds may require longer exposures to induce activation, as they may have slower binding rates. In contrast, prolonged stimulation with VEGF causes a sharp increase in receptor activation, peaking around 10 minutes and decreasing significantly by 30 minutes. Peptides QK and VEGFp both slightly increased proliferation of dermal microvascular endothelial cells (HMVECs) after 60 hours incubation. However, incubation with dermatan sulfate and DSV caused significant cell death after 24 hours in reduced growth factor media, likely due to sequestering of growth factors. It is possible that VEGFp-DS-SILY may better stimulate proliferation since it would be presented as a surface bound proteoglycan mimic, rather than as a soluble factor. HMVECs migrated farther for all treatment groups (10 µM QK, 10 µM VEGFp, 1 µM DSV4, and 10 µM DSV4) than the 10 ng/mL VEGF positive control, although more cells migrated in response to VEGF. This may be accounted at least in part by the more pronounced proliferation induced by VEGF. Migration will also be tested in 3D culture within a collagen gel. We are currently testing a 2D matrigel system for tubulogenesis. We have found that 10 µM DSV4 forms qualitatively more well-defined tubules than the untreated control on reduced growth factor matrigel. However, we were not able to quantify the improved tubule formation and are still troubleshooting the tubule analysis. After seeding ECs and culturing for 4, 8, and 12 hours, cells will be fluorescently stained with anti-CD31 and imaged for 3D tubule formation. CAM assay angiogenesis growing into a collagen scaffold. In brief, fertilized chicken embryos are incubated for 2 days before exposing the CAM. VEGFp-DS-SILY bound to a collagen gel will be placed onto the CAM. Some treatment groups will receive additional VEGF to investigate synergistic effects. Light microscope images of angiogenesis into the collagen gel coated with VEGFp-DS-SILY, taken every day from days 10 to 13, will reflect the ability of the collagen scaffold to integrate into existing vasculature and 3D angiogenic potential of VEGFp-DS-SILY with or without VEGF. We expect that VEGFp-DS-SILY treatment will increase the number of vessels formed on the CAM. Preliminary data using a Fluoraldehyde assay indicates that loading of ~300 ng VEGF per mg of nanoparticles can be achieved. We expect that using an MMP-degradable peptide diacrylate crosslinker will allow nanoparticles to degrade in protease-rich environments like the chronic wound bed and release VEGF. Adjustments to the formulation, such as crosslinker density, may need to be modified to control the rate of VEGF release. DISCUSSION/SIGNIFICANCE OF IMPACT: We expect that our angiogenic decorin mimetic will lead to a novel treatment to accelerate healing of ischemic diabetic foot ulcers, thereby reducing the need for limb amputation and mortality rate of diabetic patients. We anticipate that the diabetes research and regenerative medicine communities will (1) gain a platform for targeted delivery of growth factors, (2) understand the dependence of vascularization within 3D collagen constructs on VEGFp densities and VEGF receptor activation in controlling the degree of angiogenesis, and (3) gain the benefits of controlled angiogenesis in ischemic diabetic wound healing.
Biologically-Based Self-Assembling Hydrogels
- Brandon L. Seal, Alyssa Panitch
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- Journal:
- MRS Online Proceedings Library Archive / Volume 724 / 2002
- Published online by Cambridge University Press:
- 01 February 2011, N3.2
- Print publication:
- 2002
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We have developed polymers, which borrowing from biology, assemble into networks. The self-assembly regions of fibrinogen were cloned to form a scaffolding that either interacts with fibrin or assembles independently. Peptides consisting of a binding pocket (BP), ligand (L), and/or a Factor XIIIa substrate were synthesized and conjugated to methacroylated dextran or acrylated poly(ethylene glycol). Peptide-conjugated dextran was added to polymerizing fibrin, and the resulting hydrogels were evaluated rheologically. These conjugates significantly affected the mechanical properties of fibrin while the addition of unconjugated dextran did not. The BP and L peptides were conjugated to PEG star polymer. Mixtures of conjugated PEG-BP and PEG-L were found to assemble. This work shows that peptides directing assembly can be designed using motifs found in proteins. The peptides in this study not only alter the mechanical properties of fibrin, but also allow a mechanism for creating a self-assembling network.