nature biotechnology: doi:10.1038/nbt · supplementary figure 6 additional confocal raman and...

Supplementary Figure 1

Reagents used for polymer modification.

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

Nature Biotechnology: doi:10.1038/nbt.3462


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.



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.



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.



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).



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.



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).


Supplementary Table 1. Chemical modifications of the 69 microcapsule formulations and controls.


Combinatorial hydrogel library enables identification of materials that mitigate the foreign

body response in primates

Arturo J Vegas1,2,11, Omid Veiseh1,3, Joshua C Doloff1,2, Minglin Ma1,2,11, Hok Hei Tam1,3, Kaitlin Bratlie1,3,11,

Jie Li1,2, Andrew R Bader1,2,11, Erin Langan1,2, Karsten Olejnik1,2, Patrick Fenton1,2, Jeon Woong Kang4,

Jennifer Hollister-Locke5, Matthew A Bochenek6, Alan Chiu1,2, Sean Siebert1,2, Katherine Tang1,2,

Siddharth Jhunjhunwala1,2, Stephanie Aresta-Dasilva1,2, Nimit Dholakia1,2, Raj Thakrar1,2, Thema Vietti1,2,

Michael Chen1,2, Josh Cohen5, Karolina Siniakowicz5, Meirigeng Qi6, James McGarrigle6, Stephen Lyle7,

David M Harlan8, Dale L Greiner8, Jose Oberholzer6, Gordon C Weir5, Robert Langer1–3,9,10 & Daniel G

Anderson1–3,9,10*

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

Cambridge, MA, USA

2. Department of Anesthesiology, Boston Children’s Hospital, Boston, MA USA

3. Department of Chemical Engineering, Massachusetts Institute of Technology,

Cambridge, MA, USA

4. MIT Spectroscopy Lab, Massachusetts Institute of Technology, Cambridge, MA,

USA

5. Section on Islet Cell and Regenerative Biology, Research Division, Joslin Diabetes Center, Boston, MA

USA

6. Department of Surgery, Division of Transplantation, University of Illinois at Chicago, Chicago, IL USA

7. Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts,

USA

8. Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA USA

9. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA,

USA

10. Division of Health Science Technology, Massachusetts Institute of Technology, Cambridge, MA, USA

11. Present Address: Department of Chemistry, Boston University, Boston, MA, USA (A.J.V.); Biological and

Environmental Engineering, Cornell University, Ithaca, NY (M.M.); Department of Materials Science and

Engineering, Iowa State University, Ames, IA, USA (K.B.); Department of Chemical and Biological

Engineering, Iowa State University, Ames, IA, USA (K.B.).

*Correspondence to Daniel G. Anderson, email: [email protected]; Tel.: +1 617 258 6843; fax: +1 617

258-8827.


Supplementary Note – Polymer and Compound Characterization

All characterization data was collected after thorough purification outlined in the methods. Optimized syntheses

for lead materials Z2-Y12, Z1-Y19, and Z1-Y15 require small molecule synthesis and the characterization for

those intermediates is also presented below. All NMR spectra were collected from a Bruker 400 MHz or a Varian

500 MHz magnet at the DCIF facility at MIT. All IR data was collected using a Bruker ALPHA FTIR. Mass

Spectrometry was performed on a Waters Xevo QToF system with positive electrospray ionization (ESI).

Elemental analysis was performed by Midwest Microlabs (Indianapolis, IN).

N7: 1H (400 MHz; D2O): 3.10–4.10 (m, alginate protons), 4.20 (2H, s, H2N-CH2-Ph), 4.40–5.20 (m, alginate protons),

7.41 (2H, m, Phenyl), 7.49 (3H, m, Phenyl)

IR (ATR): 3234, 1579, 1465, 1407, 1368, 1078, 810, 692, 517

N8: 1H (400 MHz; D2O): 3.00–3.20 (m, alginate protons), 3.60 (8H, m, ethoxy), 3.60–5.10 (m, alginate protons)

IR (ATR): 3233, 2927, 2358, 1591, 1405, 1318, 1022, 945, 810

O3: 1H (400 MHz; D2O): 1.90-2.10 (m, 4H, Furfuryl), 3.23 (m, 2H, Furfuryl) 3.26–4.00 (m, alginate protons), 4.03

(3H, m, O-CH2-C[furfuryl]), 4.10–5.20 (m, alginate protons)

IR (ATR): 3202, 3070, 2344, 1711, 1594, 1398, 1021, 715, 549

O6: 1H (400 MHz; D2O): 3.60–4.52 (m, alginate protons), 4.59 (2H, m, O-CH2-C[furfuryl]), 4.6–5.2 (m, alginate

protons), 6.45 (2H, m, CH-CH=CH-O Furfuryl), 7.53 (1H, m, CH-CH=CH-O Furfuryl)

IR (ATR): 3232, 2360, 1614, 1410, 1028, 538

O9: 1H (400 MHz; D2O): 0.20 (s, 9H, Furfuryl),) 3.10–5.20 (m, alginate protons)

IR (ATR): 3310, 2939, 2360, 1592, 1406, 1316, 1081, 1020, 902, 770

O11: 1H (400 MHz; D2O): 3.05–4.50 (m, alginate protons), 4.52 (2H, s, O-CH2-Ph), 4.52–5.2 (m, alginate protons),

6.88 (2H, m, Phenyl), 7.26 (2H, m, Phenyl)

IR (ATR): 3370, 3089, 1597, 1517, 1454, 1235, 1207, 989, 835, 801, 561

Z1-Y2: 1H (400 MHz; D2O): 3.05-3.40 (m, alginate protons), 3.40-3.66 (16H, m, ethoxy), 3.75 (3H, s, methoxy) 3.8–5.1

(m, alginate protons), 7.19 (1H, m, Phenyl), 7.50 (1H, m, Phenyl), 7.94 (1H, m, Phenyl), 8.00 (1H, m, Phenyl),

8.49 (1H, s, triazole)

IR (ATR): 3144, 2922, 1592, 1400, 1329 1019, 943

Z1-Y15: 1H (400 MHz; D2O): 3.07 (4H, s, N-CH2-CH2-S), 3.17-3.40 (m, alginate protons), 3.46 (4H, s, N-CH2-CH2-S),

3.50-3.70 (16H, m, ethoxy), 3.7–5.2 (m, alginate protons), 8.08 (1H, s, triazole)

IR (ATR):3268, 2933, 2250, 1602, 1409, 1292, 1119, 1023, 946

Elemental: C: 39.65%, H: 5.41%, N: 7.64%, O: 33.50%

By elemental analysis data there is 21.0% modification of the starting alginate.


Z1-Y19: 1H (400 MHz; D2O): 3.05-3.40 (m, alginate protons), 3.40-3.66 (16H, m, ethoxy), 4.4–5.1 (m, alginate protons),

6.96 (2H, m, Phenyl), 7.63 (3H, m, Phenyl), 8.23 (1H, s, triazole)

IR (ATR): 3234, 2929, 2361, 1593, 1406, 1317 1024, 947, 810

Elemental: C: 44.01%, H: 5.49%, N: 8.29%, O: 36.73%


Z2-Y12: 1H (400 MHz; D2O): 1.57-1.78 (m, 6H, pyran), 3.10-4.40 (m, alginate protons), 4.48 (4H, m, pyran), 4.50–5.10

(m, alginate protons), 7.56 (2H, m, Phenyl), 7.76 (3H, m, Phenyl), 8.51 (1H, s, triazole)

IR (ATR): 3235, 2933, 2111, 1592, 1405, 1290, 1023, 946

Elemental: C: 37.72%, H: 4.77%, N: 4.05%, O: 43.80%.


N4-N2: 1H (400 MHz; D2O): 2.72 (s, 3H, N-CH3 Dioxolane) 2.77 (s, 3H, N-CH3 Benzyl), 3.36 (2H, d, N-CH2-

Dioxolane), 3.55-4.20 (m, alginate protons), 4.22 (2H, m, N-CH2-Ph), 4.50–5.10 (m, alginate protons), 5.19 (1H,

m, CH2-CH-O Dioxolane), 7.51 (5H, m, Phenyl).

IR (ATR): 3250, 2894, 1601, 1409, 1127, 1088, 1029, 946

O3-O10: 1H (400 MHz; D2O): 1.60-2.20 (m, 4H, Tetrahydrofurfuryl), 3.55-5.10 (m, alginate protons), 3.78 (2H, m, CH2-

CH2-O Tetrahydrofurfuryl ), 3.85 (3H, s, COO-CH3), 4.13 -4.30 (3H, m, N-CH2- Tetrahydrofurfuryl).

IR (ATR): 3448, 2926, 2111, 1618, 1420, 1290, 1096, 948, 904

N9-O8: 1H (400 MHz; D2O): 1.28 (m, 3H, N-CH2-CH3), 1.32 (m, 3H, O-CH2-CH3), 1.63 (m, 3H, N-CH2-CH2-CH2-

CH2-OH) 1.74 (m, 3H, N-CH2-CH2-CH2-CH2-OH), 3.09-3.40 (m, 6H, CH3-CH2- N-CH2-CH2-CH2-CH2-OH),

3.55-5.10 (m, alginate protons), 4.06 (m, 3H, O-CH2-CH3)

IR (ATR): 3422, 1709, 1655, 1611, 1474, 1395, 1042, 798

Small molecule preparations:

Z2-Y12 amine:

1H (400 MHz; MeOD): 1.57 (m, 4H, pyran), 1.72 (m, 1H, pyran), 1.82 (m, 1H,pyran) 3.58 (m, 1H, pyran), 3.87

(s, 1H, NH2-CH2-Ph), 3.92 (m, 1H, pyran), 4.68 (d, 1H, J= 12Hz, O-CH2-triazole), 4.79 (m, 1H, O-CH-O pyran),

4.97 (d, 1H, J=12Hz, O-CH2-triazole), 7.54 (m, 2H, aromatic), 7.80 (m, 2H, aromatic), 8.49 (s, 1H, triazole) 13C (400 MHz; MeOD): 20.3 (CH2 pyran), 26.5 (CH2 pyran), 31.5 (CH2 pyran), 46.1 (NH2CH2), 61.0 (O-CH2-

C), 63.3 (CH2–O pyran), 99.5 (O-CH-O pyran), 121.6 (CH aromatic), 123.17 (CH triazole), 129.9 (CH aromatic),

137.1 (Cq-N aromatic), 144.9 (Cq-C aromatic), 146.9 (C triazole)

High resolution MS: M+1 = 289.1665 +3.1ppm


Z1-Y15 amine:

1H (400 MHz; D2O): 2.86 (2H, s, NH2), 3.01 (4H, m, N-CH2-CH2-S), 3.10 (4H, m, N-CH2-CH2-S), 3.55 (2H, t,

J=5.2Hz , NH2-CH2),3.61 (8H, m, PEG) 3.85 (2H, s, Thiomorpholine-CH2-Triazole), 3.90 (2H, t, J=5.2Hz, N-

CH2-CH2-O), 4.59 (t, 2H, J=5.2, N-CH2-CH2-O), 7.99 (1H, s, triazole) 13C (400 MHz; MeOH): 41.7 (NH2-CH2), 51.42 (N-CH2), 51.48 (N-CH2 Thiomorpholine) 52.1 (S-CH2

Thiomorpholine) 52.4 (Thiomorpholine-CH2-Triazole), 70.4-72.1 (m, PEG), 126.0 (CH triazole), 144.5 (C

triazole)

High resolution MS: M+1 = 392.1968 -6.1ppm

Z1-Y19 amine:

1H (400 MHz; MeOD): 2.79 (t, 2H, J=5.2Hz, NH2-CH2), 3.46 (t, 2H, J=5.2, NH2-CH2-CH2), 3.53 (m, 4H, PEG),

3.61 (m, 4H, PEG), 3.91 (t, 2H, J=5.2, N-CH2-CH2-O), 4.58 (t, 2H, J=5.2, N-CH2-CH2-O) 6.76 (m, 2H, aromatic),

7.54 (m, 2H, aromatic), 8.14 (s, 1H, triazole) 13C (400 MHz; MeOD): 41.6 (NH2-CH2), 51.4 (N-CH2), 70.3-71.9 (m, PEG), 116.4 (CH aromatic), 121.1 (Cq-

C), 121.5 (CH triazole), 127.7 (CH aromatic), 149.4 (C-NH2 aromatic), 149.5 (C triazole)

High resolution MS: M+1 = 336.2036 -8.3ppm


nature biotechnology: doi:10.1038/nbt · supplementary figure 6 additional confocal raman and...

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