Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

Journal name:
Nature Biotechnology
Volume:
34,
Pages:
345–352
Year published:
DOI:
doi:10.1038/nbt.3462
Received
Accepted
Published online
Corrected online

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient1, 2, 3, 4, 5, 6. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.

At a glance

Figures

  1. Combinatorially modified hydrogels with reduced subcutaneous inflammation and fibrosis.
    Figure 1: Combinatorially modified hydrogels with reduced subcutaneous inflammation and fibrosis.

    (a) Scheme for the synthesis of 774 alginate analogs. (b) Schematic and representative whole-animal image for the rapid evaluation of multiple analogs as bulk hydrogels implanted subcutaneously in each mouse. The injected Prosense 680 probe is activated by cathepsin activity at implant sites, showing increased fluorescence as a marker of early inflammation. Fluorescence is measured 7 d post-implantation. (c) Heat map summarizing gelation and cathepsin evaluation for the entire alginate analog library (mean values from n = 3 replicates for each material). Black (poor gelation) and white (low yield/not created) indicate untested combinations. 200 alginate analogs displayed lower levels of cathepsin activity than the control alginate UPVLVG, the starting material for synthesis. (d) Microspheres of alginate analogs formulated using electrojetting. Different alginate analogs were blended with 20–50% SLG100 alginate to produce microcapsules with good spherical morphology. Scale bar, 1,000 μm. (e) Secondary cathepsin evaluation of 69 top analogs from the initial screen formulated as 300-μm microcapsules. Data normalized to the fluorescence of V/S microcapsules (V/S = UPVLVG/SLG100 blend; mean values shown). The ten analog microcapsules with the lowest cathepsin levels are highlighted in yellow, n = 10 (controls) and n = 3 (experimental). (f) Masson's trichrome (MT) 28-day subcutaneous histology of the top ten alginate analog microcapsules and the ultrapure control alginate microcapsules (SLG20, V/S = UPVLVG/SLG100 blend) that were implanted in e; n = 10 (controls) and n = 3 (experimental). Abnormal microcapsule morphology is caused by histological processing (dehydration) of the tissue. Scale bars, 400 μm. (g) Quantification of collagen density (blue pixel density) in the MT-stained histology images of the three lead materials shown in f; n = 3. The collagen density is plotted as a function of the distance from the implant surface to tissue interface (mean values ± s.e.m.). One-way ANOVA with Bonferroni correction was used to allow for statistical comparison of multiple means. #P < 0.05.

  2. Three lead hydrogels show reduced fibrosis intraperitoneally in C57BL/6J mice.
    Figure 2: Three lead hydrogels show reduced fibrosis intraperitoneally in C57BL/6J mice.

    (a) Representative phase contrast images of 300-μm microcapsules of the top ten alginate analog microcapsules and control alginate microcapsules (SLG20, V/S) retrieved from the intraperitoneal space of C57BL/6J mice after 14 d. For each mouse cohort n = 5; scale bars, 2,000 μm. (b) Representative z-stacked confocal microscopy images of the retrieved microcapsules in a, n = 5. The microcapsules were stained for macrophage markers (CD68), myofibroblast markers (α-smooth muscle actin, SMA) and general cellular deposition (DAPI). Scale bars, 100 μm. (c) Western blot analysis of protein extracted from the top three alginate analog microcapsules and control microcapsules in a. Blots were stained for SMA and loading was normalized to β-actin. SMA protein levels determined by quantification of band intensities from the blots shown in Supplementary Figure 2d (mean values ± s.e.m., n = 5). One-way ANOVA with Bonferroni correction was used to allow for statistical comparison of multiple means. #P < 0.05, *P < 0.01; ns, not significant. (d) Collagen content using a hydroxyproline quantification assay of protein extracted from the top three alginate analog microcapsules and control microcapsules in a, (mean values ± s.e.m., n = 5). One-way ANOVA with Bonferroni correction was used to allow for statistical comparison of multiple means. #P < 0.05, *P < 0.01; ns, not significant. (e) Chemical structures of the three lead materials.

  3. Lead hydrogels show low immune cell recruitment in vivo with covalently modified surfaces.
    Figure 3: Lead hydrogels show low immune cell recruitment in vivo with covalently modified surfaces.

    (a) FACS analysis of macrophages (CD11b+, CD68+) and neutrophils (CD11b+, Ly6g+) isolated from Z2-Y12, Z1-Y15, Z1-Y19, SLG20 and V/S microcapsules retrieved after 14 d in the intraperitoneal space of C57BL/6J mice, n = 5. One-way ANOVA with Bonferroni correction was used to allow for statistical comparison of multiple means. #P < 0.05; ***P < 0.0001. (b) Intravital imaging and single z-sections of fluorescent 300-μm Z2-Y12 and SLG20 microcapsules in MAFIA mice 7 d after implantation (n = 3). Green, GFP-expressing macrophages; red, fluorescent hydrogel microcapsules. For full confocal reconstructions, see Supplementary Videos 1 and 2. (c) Confocal Raman cross-section mapping of 300-μm Z2-Y12 microcapsules. The Raman peak at 830 cm−1 (shown in red) is indicative of the tetrahydropyranal modification of Z2-Y12, and the intensity of this peak is two times higher at the surface of the microcapsules than at the core. The peak at 1,000 cm−1 is mapped in green as a reference to the alginate backbone structure. The Raman spectrum of V/S microcapsules is also shown for reference. (d) Freeze-fracture cryo-SEM imaging of 300-μm Z2-Y12, Z1-Y15, Z1-Y19, V/S and SLG20 microcapsules. Representative images of the microcapsule surface topography is shown. Scale bars, 3 μm. (e) Table reporting percent polymer modification (n = 3, mean values ± s.d.), Young's modulus (n = 5, mean values ± s.d.), surface roughness (n = 3, mean values ± s.d.) and protein adsorption (n = 8, mean values ± s.d.) for the three lead materials and controls.

  4. Lead hydrogels mitigate the foreign body response in non-human primates.
    Figure 4: Lead hydrogels mitigate the foreign body response in non-human primates.

    Z2-Y12, Z1-Y15, and Z1-Y19 spheres significantly reduce fibrosis in cynomolgus macaques, while conventional SLG20 spheres become fibrotic. (a) Phase contrast imaging of spheres retrieved after 4 weeks in the intraperitoneal space show less fibrosis on Z2-Y12, Z1-Y15 and Z1-Y19 spheres than on SLG20. Scale bars, 2,000 μm; n = 3. (b) Confocal imaging of retrieved spheres from a after 4 weeks in the intraperitoneal space show significantly less macrophage (CD68, CD11b), myofibroblast (SMA) and general cellular deposition (DAPI) on Z2-Y12 spheres. Scale bars, 200 μm; n = 3. Brightfield images of the stained spheres are inset; scale bars, 100 μm. (c) Western-blot analysis of protein extracted from the top three alginate analog spheres and control spheres in a; n = 3. Blots were stained for SMA and loading was normalized to β-actin. SMA protein levels determined by quantification of band intensities from the blots shown in Supplementary Figure 7b. Dots represent measurements from individual biological replicates, and lines show the average of the three replicates. One-way ANOVA with Bonferroni correction was used to allow for statistical comparison of multiple means. #P < 0.05; **P < 0.001; ns, not significant. (d) Collagen content using a hydroxyproline quantification assay of protein extracted from the top three alginate analog spheres and control spheres in a; n = 3. Dots represent measurements from individual biological replicates and lines show the average of the three replicates. One-way ANOVA with Bonferroni correction was used to allow for statistical comparison of multiple means. #P < 0.05, **P < 0.001, ns = not significant. (e) Representative phase contrast imaging (n = 3) of Z2-Y12 after 6 months in the intraperitoneal space. Scale bar, 2,000 μm. (f) Representative z-stacked confocal imaging (n = 3) of Z2-Y12 spheres retrieved after 6 months. Few macrophages and myofibroblasts are observed on Z2-Y12 spheres. Scale bars, 200 μm.

  5. Reagents used for polymer modification.
    Supplementary Fig. 1: Reagents used for polymer modification.

    Amines, alcohols, azides, and alkynes used for the chemical modification of alginate.

  6. Additional data for lead materials tested in C57BL/6J mice.
    Supplementary Fig. 2: Additional data for lead materials tested in C57BL/6J mice.

    (a) Evaluation of enzyme activity of cathespins B and L in the absence and presence of 20 mM barium chloride. No reduction in enzymatic activity is observed (n = 6, mean values ± SD). (b) Representative hematoxylin and eosin (HE) stained subcutaneous 28 day histology of the top ten alginate analogue microcapsules and control alginate microcapsules (SLG20, V/S) that were implanted in Figure 2, n = 3 mice per group. Abnormal microcapsule morphology is caused by histological processing (dehydration) of the tissue. Scale bar = 400 µm. (c) PCA functional group analysis of the entire modified alginate library, with top performing materials indicated. Triazole-containing modifications are enriched for favorable in vivo performance. (d) Western-blot images used for SMA quantification in Figure 2c. Protein was extracted from retrieved microcapsules (n = 5 mice per group) of the top three alginate analogues and controls after 14 days IP in C57BL/6J mice. (e) Cellular viability of RAW 264.7 cells exposed to formulated lead microcapsules (n = 12, mean values ± SD). (f) Cytokine panel analysis of protein extracted from retrieved microcapsules (n = 5 mice per group) in Figure 2a. Scale shows average fold signal above background.

  7. Nanostring gene expression analysis.
    Supplementary Fig. 3: Nanostring gene expression analysis.

    Gene expression analysis of seventy-nine known inflammatory factors and immune cell markers from retrieved IP fluid, fat pad, and microcapsules 14 days post-implantation in C57BL/6J mice. Gene expression from IP and fat pad was normalized to mock implant controls, while gene expression from microcapsule-associated tissue was normalized to the SLG20 sample.

  8. Determination of potential contaminants in controls and lead materials.
    Supplementary Fig. 4: Determination of potential contaminants in controls and lead materials.

    (a) Determination of potential contaminant levels of alginate microcapsules prior to implantation. Endotoxin and glucan levels were measured and reported by Charles River Laboratories. The standard curves used for quantification of (b) flagellin and (c) LTA levels are also shown.

  9. Additional FACS data.
    Supplementary Fig. 5: Additional FACS data.

    FACS analysis of (a) macrophages and (b) neutrophils isolated from retrieved 300 µm microcapsules after 14 days IP in C57BL/6J mice, n = 5 mice per group. One-way ANOVA with Bonferroni correction was utilized to allow for statistical comparison of multiple means, # = p < 0.05, ** = p < 0.001, *** = p < 0.0001, ns = not signficant. The data express the number of cells as a percentage of total cells measured. (c) Representative dot plots of FACS analysis for macrophages and neutrophils presented in (a) and (b).

  10. Additional confocal raman and cryo-SEM imaging of lead materials.
    Supplementary Fig. 6: Additional confocal raman and cryo-SEM imaging of lead materials.

    (a) Confocal raman cross-section mapping of 300 µm Z1-Y15 microcapsules. The raman peak at 857 cm-1 (shown in red) is indicative of the thiomorpholine dioxide end group of Z1-Y15, and peak intensity is enriched at the surface of the microcapsules than at the core. The peak at 884 cm-1 is mapped in green as a reference to the alginate backbone structure. (b) Confocal raman cross-section mapping of 300 µm Z1-Y19 microcapsules. The raman peak at 1563 cm-1 (shown in red) is indicative of the aniline end group of Z1-Y19, and peak intensity appears more uniform at both the surface of the microcapsules and at the core. The peak at 884 cm-1 is mapped in green as a reference to the alginate backbone structure. (c) Representative freeze-fracture cryo-SEM images of the core (scale bar = 10 µm) and fractured surface (scale bar = 10 µm) of 300 µm SLG20, V/S, Z2-Y12, Z1-Y15, and Z1-Y19 microcapsules.

  11. NHP omental histology and western blot images of lead material implants.
    Supplementary Fig. 7: NHP omental histology and western blot images of lead material implants.

    (a) Representative MT and HE stained histology of biopsied omental tissue 4 weeks post-implantation in cynomolgus macaque, n = 3. (b) Western-blot images used for SMA quantification of protein extracted from the top three alginate analogue spheres and control spheres (n = 3).

Videos

  1. Intravital imaging of 300 m SLG20 microcapsules.
    Video 1: Intravital imaging of 300 μm SLG20 microcapsules.
  2. Intravital imaging of 300 m Z2-Y12 microcapsules.
    Video 2: Intravital imaging of 300 μm Z2-Y12 microcapsules.
  3. NHP Laparoscopic procedure for the retrieval of Z2-Y12 spheres.
    Video 3: NHP Laparoscopic procedure for the retrieval of Z2-Y12 spheres.

Change history

Corrected online 18 April 2016
In the version of this article initially published, one author, Adam C. Graham, his affiliation, and his contribution were omitted. In addition, two acknowledgments, to W. Salmon and J. Wyckoff, were omitted. The errors have been corrected in the HTML and PDF versions of the article.

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Author information

  1. Present addresses: Department of Chemistry, Boston University, Boston, Massachusetts, USA (A.J.V.); Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York, USA (M.M.); Department of Materials Science and Engineering, Iowa State University, Ames, Iowa, USA, and Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, USA (K.B.); Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA (A.R.B.).

    • Arturo J Vegas,
    • Minglin Ma,
    • Kaitlin Bratlie &
    • Andrew R Bader

Affiliations

  1. David H Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Arturo J Vegas,
    • Omid Veiseh,
    • Joshua C Doloff,
    • Minglin Ma,
    • Hok Hei Tam,
    • Kaitlin Bratlie,
    • Jie Li,
    • Andrew R Bader,
    • Erin Langan,
    • Karsten Olejnik,
    • Patrick Fenton,
    • Alan Chiu,
    • Sean Siebert,
    • Katherine Tang,
    • Siddharth Jhunjhunwala,
    • Stephanie Aresta-Dasilva,
    • Nimit Dholakia,
    • Raj Thakrar,
    • Thema Vietti,
    • Michael Chen,
    • Robert Langer &
    • Daniel G Anderson
  2. Department of Anesthesiology, Boston Children's Hospital, Boston, Massachusetts, USA.

    • Arturo J Vegas,
    • Joshua C Doloff,
    • Minglin Ma,
    • Jie Li,
    • Andrew R Bader,
    • Erin Langan,
    • Karsten Olejnik,
    • Patrick Fenton,
    • Alan Chiu,
    • Sean Siebert,
    • Katherine Tang,
    • Siddharth Jhunjhunwala,
    • Stephanie Aresta-Dasilva,
    • Nimit Dholakia,
    • Raj Thakrar,
    • Thema Vietti,
    • Michael Chen,
    • Robert Langer &
    • Daniel G Anderson
  3. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Omid Veiseh,
    • Hok Hei Tam,
    • Kaitlin Bratlie,
    • Robert Langer &
    • Daniel G Anderson
  4. MIT Spectroscopy Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Jeon Woong Kang
  5. Section on Islet Cell and Regenerative Biology, Research Division, Joslin Diabetes Center, Boston, Massachusetts, USA.

    • Jennifer Hollister-Locke,
    • Josh Cohen,
    • Karolina Siniakowicz &
    • Gordon C Weir
  6. Department of Surgery, Division of Transplantation, University of Illinois at Chicago, Chicago, Illinois, USA.

    • Matthew A Bochenek,
    • Meirigeng Qi,
    • James McGarrigle &
    • Jose Oberholzer
  7. Center for Nanoscale Systems, Harvard University, Cambridge, Massachusetts, USA.

    • Adam C Graham
  8. Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Stephen Lyle
  9. Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • David M Harlan,
    • Dale L Greiner &
    • Daniel G Anderson
  10. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer &
    • Daniel G Anderson
  11. Division of Health Science Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer

Contributions

A.J.V., O.V., J.C.D., M.M., K.B., J.L., A.R.B., E.L., K.O., P.F., J.W.K., J.H.-L., M.A.B., A.C., S.S., K.T., S.J., S.A.-D., N.D., R.T., T.V., M.C., J.C., K.S., M.Q. and J.M. designed and performed experiments, and analyzed data. H.H.T. assisted with data processing and data presentation. A.C.G. assisted with SEM imaging. S.L. assisted with histology. D.M.H., D.L.G., J.O. and G.C.W. provided conceptual advice and technical support. A.J.V. and D.G.A. wrote the paper. R.L. and D.G.A. supervised the study. All authors discussed the results and commented on the manuscript.

Competing financial interests

A.J.V., O.V., J.C.D., M.M., K.B., R.L. and D.G.A. declare financial interest in patents filed by MIT on the material and hydrogel capsule technology.

Corresponding author

Correspondence to:

Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Reagents used for polymer modification. (159 KB)

    Amines, alcohols, azides, and alkynes used for the chemical modification of alginate.

  2. Supplementary Figure 2: Additional data for lead materials tested in C57BL/6J mice. (206 KB)

    (a) Evaluation of enzyme activity of cathespins B and L in the absence and presence of 20 mM barium chloride. No reduction in enzymatic activity is observed (n = 6, mean values ± SD). (b) Representative hematoxylin and eosin (HE) stained subcutaneous 28 day histology of the top ten alginate analogue microcapsules and control alginate microcapsules (SLG20, V/S) that were implanted in Figure 2, n = 3 mice per group. Abnormal microcapsule morphology is caused by histological processing (dehydration) of the tissue. Scale bar = 400 µm. (c) PCA functional group analysis of the entire modified alginate library, with top performing materials indicated. Triazole-containing modifications are enriched for favorable in vivo performance. (d) Western-blot images used for SMA quantification in Figure 2c. Protein was extracted from retrieved microcapsules (n = 5 mice per group) of the top three alginate analogues and controls after 14 days IP in C57BL/6J mice. (e) Cellular viability of RAW 264.7 cells exposed to formulated lead microcapsules (n = 12, mean values ± SD). (f) Cytokine panel analysis of protein extracted from retrieved microcapsules (n = 5 mice per group) in Figure 2a. Scale shows average fold signal above background.

  3. Supplementary Figure 3: Nanostring gene expression analysis. (406 KB)

    Gene expression analysis of seventy-nine known inflammatory factors and immune cell markers from retrieved IP fluid, fat pad, and microcapsules 14 days post-implantation in C57BL/6J mice. Gene expression from IP and fat pad was normalized to mock implant controls, while gene expression from microcapsule-associated tissue was normalized to the SLG20 sample.

  4. Supplementary Figure 4: Determination of potential contaminants in controls and lead materials. (96 KB)

    (a) Determination of potential contaminant levels of alginate microcapsules prior to implantation. Endotoxin and glucan levels were measured and reported by Charles River Laboratories. The standard curves used for quantification of (b) flagellin and (c) LTA levels are also shown.

  5. Supplementary Figure 5: Additional FACS data. (132 KB)

    FACS analysis of (a) macrophages and (b) neutrophils isolated from retrieved 300 µm microcapsules after 14 days IP in C57BL/6J mice, n = 5 mice per group. One-way ANOVA with Bonferroni correction was utilized to allow for statistical comparison of multiple means, # = p < 0.05, ** = p < 0.001, *** = p < 0.0001, ns = not signficant. The data express the number of cells as a percentage of total cells measured. (c) Representative dot plots of FACS analysis for macrophages and neutrophils presented in (a) and (b).

  6. Supplementary Figure 6: Additional confocal raman and cryo-SEM imaging of lead materials. (214 KB)

    (a) Confocal raman cross-section mapping of 300 µm Z1-Y15 microcapsules. The raman peak at 857 cm-1 (shown in red) is indicative of the thiomorpholine dioxide end group of Z1-Y15, and peak intensity is enriched at the surface of the microcapsules than at the core. The peak at 884 cm-1 is mapped in green as a reference to the alginate backbone structure. (b) Confocal raman cross-section mapping of 300 µm Z1-Y19 microcapsules. The raman peak at 1563 cm-1 (shown in red) is indicative of the aniline end group of Z1-Y19, and peak intensity appears more uniform at both the surface of the microcapsules and at the core. The peak at 884 cm-1 is mapped in green as a reference to the alginate backbone structure. (c) Representative freeze-fracture cryo-SEM images of the core (scale bar = 10 µm) and fractured surface (scale bar = 10 µm) of 300 µm SLG20, V/S, Z2-Y12, Z1-Y15, and Z1-Y19 microcapsules.

  7. Supplementary Figure 7: NHP omental histology and western blot images of lead material implants. (136 KB)

    (a) Representative MT and HE stained histology of biopsied omental tissue 4 weeks post-implantation in cynomolgus macaque, n = 3. (b) Western-blot images used for SMA quantification of protein extracted from the top three alginate analogue spheres and control spheres (n = 3).

Video

  1. Video 1: Intravital imaging of 300 μm SLG20 microcapsules. (2.58 MB, Download)
  2. Video 2: Intravital imaging of 300 μm Z2-Y12 microcapsules. (2.25 MB, Download)
  3. Video 3: NHP Laparoscopic procedure for the retrieval of Z2-Y12 spheres. (61.5 MB, Download)

PDF files

  1. Supplementary Text and Figures (2,132 KB)

    Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Note

Additional data