Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition

Journal name:
Nature Materials
Volume:
13,
Pages:
653–661
Year published:
DOI:
doi:10.1038/nmat3922
Received
Accepted
Published online

Abstract

Inhibitors of matrix metalloproteinases (MMPs) have been extensively explored to treat pathologies where excessive MMP activity contributes to adverse tissue remodelling. Although MMP inhibition remains a relevant therapeutic target, MMP inhibitors have not translated to clinical application owing to the dose-limiting side effects following systemic administration of the drugs. Here, we describe the synthesis of a polysaccharide-based hydrogel that can be locally injected into tissues and releases a recombinant tissue inhibitor of MMPs (rTIMP-3) in response to MMP activity. Specifically, rTIMP-3 is sequestered in the hydrogels through electrostatic interactions and is released as crosslinks are degraded by active MMPs. Targeted delivery of the hydrogel/rTIMP-3 construct to regions of MMP overexpression following a myocardial infarction significantly reduced MMP activity and attenuated adverse left ventricular remodelling in a porcine model of myocardial infarction. Our findings demonstrate that local, on-demand MMP inhibition is achievable through the use of an injectable and bioresponsive hydrogel.

At a glance

Figures

  1. The fabrication of injectable and MMP-sensitive hydrogels for therapeutic delivery.
    Figure 1: The fabrication of injectable and MMP-sensitive hydrogels for therapeutic delivery.

    a, HA and DS polymers were modified with ALD groups through periodate oxidation of diols along the polysaccharide backbone. ALD modifications for HA and DS were ~15% and 5% of the repeat disaccharides, respectively. A separate HA batch was modified with complementary HYD groups through conjugation of the peptide GCNSGGRMSMPVSNGG–HYD where C is a thiol-containing cysteine to couple to a MAHA through a thiol–maleimide click reaction, HYD is the terminal HYD for hydrogel crosslinking, NS are hydrophilic spacers, and GGRMSMPV is the MMP-cleavable sequence. b, Hydrogel crosslinking was designed through hydrazone bond formation of complementary ALD and HYD groups to form hydrogels under physiologic conditions. c, On mixing ALD (2.4 wt% HA–ALD, 1.4 wt% DS–ALD) and HYD (3.2 wt% HA–MMP–HYD) polymers, robust hydrogels formed rapidly as evidenced by development of storage (G′) and loss (G′′) moduli within minutes. d,e 3.5 wt% hydrogels were incubated with or without active MMPs (collagenase type 4). MMPs were refreshed every 2 days to maintain enzyme activity throughout the study. d, Hydrogels were stable in the absence of enzyme activity (0 U ml−1) and degraded in response to increasing enzyme concentration (20 and 200 U ml−1 collagenase type 4). e, Encapsulated FITC–BSA released in proportion to hydrogel degradation with higher rates of molecule release corresponding with higher levels of MMP activity (mean ± s.d.,n = 3 hydrogels per condition).

  2. The non-covalent incorporation of rTIMP-3 into hydrogels and on-demand release in response to MMP activity.
    Figure 2: The non-covalent incorporation of rTIMP-3 into hydrogels and on-demand release in response to MMP activity.

    Release of encapsulated rTIMP-3 was controlled through incorporation of sulphate groups (in DS polymers) to bind rTIMP-3 through electrostatic interactions and MMP-cleavable crosslinks to release encapsulated rTIMP-3 as the hydrogel degrades in the presence of MMPs. a, rTIMP-3 bound to polymer-coated wells was detected with an ELISA. rTIMP-3 bound to DS–ALD with a greater capacity than HA–ALD, and approached rTIMP-3 binding to heparin (Hp) n = 3, mean ± s.d. b, rTIMP-3 activity was measured by its ability to inhibit a 4 nM rMMP-2 solution. rTIMP-3 binding to DS–ALD did not significantly reduce rMMP-2 inhibition. c, Passive release of rTIMP-3 (no MMP) encapsulated in 3.5 wt% ALD/HYD-crosslinked hydrogels was markedly reduced by incorporation of sulphated DS–ALD polymers into the hydrogels, n = 3 hydrogels per group, mean ± s.d. d, Hydrogels with (filled symbols) and without (open symbols) encapsulated rTIMP-3 (10 μg per 50 μl gel) were incubated with (squares) or without (triangles) 20 nM rMMP-2. rMMP-2 was refreshed every two days to maintain enzyme activity (indicated by green arrows). Encapsulated rTIMP-3 attenuated rMMP-2-mediated hydrogel degradation, confirming activity of rTIMP-3 across the 14-day study, n = 3 hydrogels per condition, mean ± s.d. e, In this hydrogel system, MMPs degrade the hydrogel crosslinks (1), liberating polysaccharide-bound rTIMP-3, which inhibits local MMP activity (2), and attenuates further hydrogel degradation (3).

  3. The visualization of injected hydrogel distribution and degradation within porcine myocardium.
    Figure 3: The visualization of injected hydrogel distribution and degradation within porcine myocardium.

    a, A pig model of MI was used to investigate the responsiveness of hydrogels post MI. Hydrogels (3.5 wt%) containing rTIMP-3 were injected into the myocardium at 9 equally spaced sites (100 μl per injection) within a 2 cm by 2 cm grid. Hydrogels were formed in situ by blending ALD- and HYD-modified polysaccharides immediately before entering the tissue using a dual-barrel syringe. Hydrogels were injected into tissue with normal MMP activity (sham) and pathological MMP activity (experimental MI) and hydrogels were analysed immediately after injection and after 14 days in vivo. Transmural myocardial explants of the injection grid were imaged with MRI, which allowed visualization of hydrogels within the myocardium. b, Three-dimensional reconstruction of the MRI images using ITKsnap software shows pockets of hydrogel throughout the myocardium (scale bars, 2 cm). c, Representative MRI images of injection grid tissue immediately following hydrogel injection and 14 days following injection in sham and MI animals (scale bars, 10 mm). Hydrogel pockets (white arrowheads) are still visible 14 days following injection in sham pigs but not in MI animals. d, Representative histological sections of injection grid tissue following haematoxylin and eosin staining. Pockets of hydrogels were visible in sham pigs 14 days following injection but not in MI animals (scale bars, 2 mm). Black arrowheads indicate regions of hydrogel identified by dark purple staining. Inset indicated by black square (scale bars, 200 μm). e, Quantification of hydrogels within tissue across groups showed significantly less hydrogel in the myocardium from MI pigs compared with sham pigs after 0 and 14 days following injection (mean ± s.e.m.,n = 3 pigs per group, pairwise t-test with Bonferroni correction).

  4. Hydrogel delivery of rTIMP-3 alters the MMP/TIMP imbalance post MI.
    Figure 4: Hydrogel delivery of rTIMP-3 alters the MMP/TIMP imbalance post MI.

    Hydrogels with and without encapsulated rTIMP-3 were injected immediately following experimental MI and myocardial MMP/TIMP levels were assessed 14 days post MI. a, Hydrogel delivery of rTIMP-3 restored TIMP-3 levels within the MI region to normal, non-MI levels. b, Blood samples revealed no significant increase in systemic TIMP-3 levels with hydrogel-mediated rTIMP-3 delivery to the MI region. c, Interstitial microdialysis of a fluorogenic MMP substrate into myocardium at 14 days post MI showed a significant increase in MMP activity within the MI region following MI induction. Injection of the MMP-cleavable hydrogels significantly inhibited MMP activity, and rTIMP-3 encapsulation further inhibited MMP activity. d, Myocardial tissue extracts were collected 14 days post MI and subjected to MMP activity assays using substrates for global MMP activity and membrane type-1 MMP (MT1-MMP) activity. A significant increase in both global MMP activity and MT1-MMP activity was observed following MI induction. This increase was abrogated with hydrogel/rTIMP-3 delivery. All values are mean ± s.e.m.; sham n = 5, MI n = 6, MI/hydrogel n = 7, MI/hydrogel/rTIMP-3 n = 7; pairwise t-test with Bonferroni correction; *P <0.05 versus sham, +P <0.05 versus MI, #P <0.05 versus MI/hydrogel.

  5. Hydrogel delivery of rTIMP-3 attenuates adverse LV remodelling and improves cardiac function post MI.
    Figure 5: Hydrogel delivery of rTIMP-3 attenuates adverse LV remodelling and improves cardiac function post MI.

    Hydrogels with and without encapsulated rTIMP-3 were injected immediately following experimental MI and LV geometry and function was assessed with echocardiography. a, MI induction caused a gradual decline in ejection fraction (EF) over 14 days that was significantly attenuated by hydrogel/rTIMP-3 delivery. b, MI induction caused a gradual dilation of the LV end diastolic volume (LVEDV). Hydrogel/rTIMP-3 delivery significantly attenuated LVEDV compared with hydrogel delivery alone. c, MI induction caused progressive thinning of the LV posterior wall thickness at diastole (LVPWThd) that was significantly attenuated by both hydrogel and hydrogel/rTIMP-3 injections at early time points, but only hydrogel/rTIMP-3 injections significantly attenuated wall thinning by day 14. d, MI induction caused a steady increase in pulmonary capillary wedge pressure (PCWP) that was significantly attenuated by hydrogel/rTIMP-3 delivery. e, Representative short-axis views (top) and m-mode targeted images (bottom) for each treatment group 14-days post MI (scale bars, 1 cm). The posterior wall (PW) at the site of the infarct induction is shown by the arrows. Significant chamber dilation and wall thinning occurred following MI, consistent with the adverse remodelling process, which was unaffected by hydrogel injection alone. However, the degree of LV dilation and wall thinning was attenuated in the hydrogel/rTIMP-3 group. f, Hydrogel/rTIMP-3 injections continued to show a therapeutic benefit 28 days following MI induction, a critical time in the progression of adverse LV remodelling. All values are mean ± s.e.m.; (ad) sham n = 5, MI n = 6, MI/hydrogel n = 7, MI/hydrogel/rTIMP-3 n = 7; f, n = 3 for all groups; pairwise t-test with Bonferroni correction; *P <0.05 versus sham, +P <0.05 versus MI, #P <0.05 versus MI/hydrogel.

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Author information

Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Brendan P. Purcell,
    • Manoj B. Charati,
    • Shauna M. Dorsey,
    • Ryan J. Wade &
    • Jason A. Burdick
  2. Cardiovascular Translational Research Center, University of South Carolina School of Medicine and the WJB Dorn Veteran Affairs Medical Center, Columbia, South Carolina 29208, USA

    • David Lobb,
    • Kia N. Zellars,
    • Heather Doviak,
    • Sara Pettaway,
    • Christina B. Logdon,
    • James A. Shuman,
    • Parker D. Freels &
    • Francis G. Spinale
  3. Gorman Cardiovascular Research Laboratory, Department of Surgery, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Joseph H. Gorman III &
    • Robert C. Gorman

Contributions

B.P.P., M.B.C., R.C.G., F.G.S. and J.A.B. conceived the ideas and designed the experiments. B.P.P., D.L., M.B.C., S.M.D., R.J.W., K.N.Z., H.D., S.P., C.B.L., J.A.S. and P.D.F. conducted the experiments and analysed the data. B.P.P., J.H.G., R.C.G, F.G.S. and J.A.B. interpreted the data and wrote the manuscript.

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The authors declare no competing financial interests.

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