8588 J. Am. Chem. Soc., Vol. 120, No. 34, 1998
Gill and Ballesteros
immunodiagnostics, biooptical devices,3,7 bioimplants and
3
,6
attributes: (i) a high water solubility; (ii) autohydrolysis in
aqueous media; (iii) hydrolysis that would liberate a nonvolatile,
bioprotective alcohol, which would also function as a drying
artificial organs,3 adsorbents for the removal of enzymes,
b,8
9
and the biosynthetic pathway stabilization.10
1
Despite the potential of this technology in the bioencapsu-
lation arena, several complications derived largely from the alkyl
silicate and alkoxyalkylsilane precursors employed for generat-
control additive (DCA). Polyol silicates were perceived as ideal
1
1
candidates, and after trials with various polyhydroxylated
12
compounds, glycerol was selected for its high biocompatibility
and functionality. In addition, it was decided to utilize
polysilicate esters because of their higher solids content, the
reduced tendency of the produced sol-gels toward aging
phenomena, and the greater robustness of the resultant xero-
1c,3
ing sol-gel matrices have restricted its wider utilization. The
low water solubility and reactivity of these compounds typically
necessitates cosolvents and catalysts, respectively, both of which
can adversely affect the biological material of interest. Also,
hydrolysis liberates alcohols, which are deleterious to bioactivity,
and their resulting evaporation generates large-scale shrinkages
and pore collapse during xerogel formation. Such complications
together with difficulties encountered in controlling aging effects
have meant that the reproducible production of stable, high-
activity bioencapsulates has so far proved elusive.1 Here we
describe an approach based around a novel class of biocom-
patible precursors, namely polyol esters of silicates and silox-
anes, which addresses many of the above difficulties and permits
the effective and reproducible fabrication of a diverse range of
bio-doped sol-gel polymers.
1
,13
gels.
Thus, the poly(glyceryl silicate) “SiO1.2Glc0.8” (PGS)
was prepared as a stable, water-soluble solid by the partial
hydrolysis and condensation of tetramethyl orthosilicate (TMOS)
to poly(methyl silicate) (PMS), followed by its transesterification
with glycerol, in a one-pot reaction catalyzed by hydrochloric
acid or poly(antimony(III) ethylene glycoxide). PGS rapidly
hydrolyzed and gelled in aqueous, buffered milieu in a matter
of minutes without the need for any catalyst, to form silica
hydrogels, which after aging, washing to remove glycerol, and
drying, produced transparent, mesoporous, and physically stable
c,3
1
4
silica xerogels (Table 1). Importantly, polymerization in the
presence of proteins and cells allowed their efficient entrapment
with surprisingly little loss of activity, to yield bioactive silica
glasses. The results obtained with this approach in the
encapsulation of representative proteins and cells are compared
in Table 2 with those of a protocol employing PMS as precursor.
It should be stressed that the entrapment of trypsin (Table 1)
and R-chymotrypsin in sol-gels synthesized from alcohol-free
Results and Discussion
We reasoned that the drawbacks of current sol-gel bioen-
capsulation protocols could be surmounted if one could prepare
biocompatible sol-gel substrates displaying the following
(
5) (a) Braun, S.; Shtelzer, S.; Rappoport, S.; Avnir, D.; Ottolenghi, M.
J. Non-Cryst. Solids 1992, 147/148, 739. (b) Yamanaka, S. A.; Nishida,
F.; Ellerby, L. M.; Nishida, C. R.; Dunn, B.; Valentine, J. S.; Zink, J. I.
Chem. Mater. 1992, 4, 495. (c) Pankratov, I.; Lev, O. J. Electroanal. Chem.
4
l
poly(silicic acid) (PSA) solutions gave results similar to those
obtained with PMS, despite the presence of 20-34% v/v of
1
995, 393, 35. (d) Yamanaka, S. A.; Dunn, B.; Valentine, J. S.; Zink, J. I.
methanol in the latter hydrogels. In view of the denaturing
J. Am. Chem. Soc. 1995, 117, 9095. (e) Wang, J.; Pamidi, P. V. A.; Park,
D. S. Anal. Chem. 1996, 68, 2705. (f) Wang, R.; Narang, U.; Prasad, P.;
Bright, F. V. Anal. Chem. 1993, 65, 2671. (g) Sampath, S.; Pankratov, I.;
Gun, J.; Lev, O. J. Sol-Gel Sci. Technol. 1996, 7, 123. (h) Akbarian, F.;
Lin, A.; Dunn, B. S.; Valentine, J. S.; Zink, J. I. J. Sol-Gel Sci. Technol.
997, 8, 1067. (i) Navas Dias, A.; Ramos Peinado, M. C. Sens. Actuators
997, B39, 426. (j) Coche-Guerente, L.; Cosnier, S.; Labbe, P. Chem. Mater.
997, 9, 1348. (k) Wang, J.; Pamidi, P. V. A.; Park, D. S. Electroanalysis
997, 9, 52. (l) Blyth, D. J.; Oytner, S. J.; Russell, D. A. Analyst 1996,
21, 1975. (m) Aylott, J. W.; Richardson, D. J.; Russell, D. A. Analyst
997, 122, 77. (n) Yamanaka, S. A.; Nguyen, N. P.; Dunn, B.; Valentine,
J. S.; Zink, J. I. J. Sol-Gel Sci. Technol. 1996, 7, 117. (o) Glezer, V.; Lev,
O. J. Am. Chem. Soc. 1993, 115, 2533. (p) Blyth, D. J.; Aylott, J. W.;
Richardson, D. J.; Russell, D. A. Analyst 1995, 120, 2725. (q) Narang, U.;
Prasad, P. N.; Bright, F. V.; Kumar, A.; Kumar, N. D.; Malhotra, B. D.;
Kamalasanan, M. N.; Chandra, S. Chem. Mater. 1994, 6, 1596. (r) Shtelzer,
S.; Braun, S. Biotechnol. Appl. Biochem. 1994, 19, 293. (s) Dosoretz, C.;
Armon, R.; Starosvetsky, J.; Rothschild, N. J. Sol-Gel Sci. Technol. 1996,
3-5
effects of alkoxysilanes and alcohols on proteins,
we at-
tempted to extend the PSA method to other biologicals, but in
several cases (e.g., thermolysin, lipoxygenase, sialic acid
aldolase, tyrosinase, and S. salmonicolor cells), poor results were
obtained due to protein precipitation, premature/partial gelation,
and/or hydrogel synerisis, especially at the higher PSA con-
centrations. Because of this, and the greater ease of preparation
and handling and higher stability of PMS over PSA, the former
was utilized as the precursor for the standard sol-gels.
However, it is entirely possible that better activities could be
obtained with the biologicals used in this study by using a
suitably optimized PSA method, especially in the cases of whole
cells which can be rather sensitive to alkoxysilanes and lower
alcohols.
1
1
1
1
1
1
7
, 7.
6) (a) Barreau, J. Y.; Da Costa, J. M.; Desportes, I.; Livage, J.; Monjour,
M.; Gentilini, M. Compt. Rend. Acad. Sci., Ser. III 1994, 317, 653. (b)
Livage, J.; Barreau, J. Y.; Da costa, J. M.; Desport, I. SPIE Proc. 1994,
288, 493. (c) Livage, J.; Roux, C.; Da Costa, J. M.; Desportes, I.; Quinson,
J. F. J. Sol-Gel Sci. Technol. 1996, 7, 45. (d) Wang, R.; Narang, U.; Prasad,
P. N.; Bright, F. V. Anal. Chem. 1993, 65, 2671.
(
Properties of PGS-Derived Silica Matrices. Several notable
features of the PGS approach can be appreciated. First, the
mild encapsulation chemistry and high precursor biocompat-
ibility significantly reduce toxicity effects, with the biogels
retaining 83-98% of the activity of the native biological,15 as
compared with 11-76% for the PMS procedure (Table 2). In
this context, one should note the moderate, but notable,
2
(
7) (a) Weetall, H. H. Biosens. Bioelectr. 1996, 11, 327. (b) Wu, S.;
Ellerby, L. M.; Cohan, J. S.; Dunn, B.; El-Sayed, M. A.; Valentine J. S.;
Zink, J. I. Chem. Mater. 1993, 5, 115. (c) Chen, Z.; Samuelson, L. A.;
Akkara, J.; Kaplan, D. L.; Gao, H.; Kumar, J.; Marx, K. A.; Tripathy, S.
K. Chem. Mater. 1995, 7, 1779. (d) Chen, Z.; Kaplan, D. L.; Yang, K.;
Kumar, J.; Marx, K. A.; Tripathy, S. K. J. Sol-Gel Sci. Technol. 1996, 7,
(13) Ellsworth, M.; Novak, B. M. Chem. Mater. 1993, 5, 839.
(14) FTIR analysis indicated complete hydrolysis, with no trace of
glycerol being observed in the xerogels. Also, the monoliths were found to
be mechanically and optically stable to repeated cycles of wetting with
aqueous or organic media, followed by drying.
(15) Direct evidence for the diffusion of substrates into xerogels and
their reaction with the internalized enzymes was obtained by incubating
monoliths with chromogenic substrates. Thus, when silicas containing C.
rugosa lipase, â-glucosidase, and horseradish peroxidase were respectively
contacted with aqueous solutions of 4-nitrophenyl palmitate, 4-nitrophenyl
â-D-glucopyranoside or hydrogen peroxide, and 1,4-hydroquinone, the
generation of colored products and accompanying color development was
observed throughout the sol-gel matrix. Control reactions where xerogels
were placed in solutions of the products were used to distinguish the simple
diffusion of these compounds into the gel interiors.
9
9.
(
8) (a) Pope, E. J. A.; Braun, K.; Peterson, C. M. J. Sol-Gel Sci. Technol.
997, 8, 635. (b) Pope, E. J. A. Ceram. Trans. 1996, 63, 17.
9) Narang, U.; Rahman, M. H.; Wang, J. H.; Prasad, P. N.; Bright, F.
V. Anal. Chem. 1995, 67, 1935.
1
(
(
10) Bressler, E.; Braun, S. J. Sol-Gel Sci. Technol. 1996, 7, 129.
(
11) Although additives such as PEG and PVA have been included in
1
a-c
sol-gel procedures,
to our knowledge, the polyol ester precursors
presented here have not been previously reported, nor have similar
compounds been utilized for bioencapsulation purposes.
12) Silicate and polysilicate esters of ethylene glycol, propylene glycol,
diglycerol, triethylene glycol, etc., were readily accessed via transesterifi-
cation.
(