Dissipation of self-assemblies by fusion of complementary gels: An elegant strategy for programmed enzymatic reactions

Article abstract of DOI:10.1039/c7cc04269h

Cholesterol based phenylboronic acid and glucose tailored complementary gels were developed which underwent mutual self-destruction upon mixing due to the formation of a boronate-diol adduct. This dissipation of the complementary gels was employed in a programmed enzymatic reaction and in pro-drug activation.

Full text of DOI:10.1039/c7cc04269h

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
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DOI: 10.1039/C7CC04269H
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Dissipation of Self-assemblies by Fusion of Complementary Gels:
An Elegant Strategy for Programmed Enzymatic Reaction
Received 00th January 20xx,
Accepted 00th January 20xx
Deep Mandal, Pritam Choudhury, Deblina Sarkar and Prasanta Kumar Das*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Cholesterol based phenylboronic acid and glucose tailored close proximity to facilitate the reaction if those matrix are
complementary gels were developed which underwent mutual mutually self-destructing upon mixing. Till date report on
self-destruction on mixing due to the formation of boronate-diol degradation of molecular gels in response to another self-
adduct. This dissipation of complementary gels was employed in assembled gels is really scarce. Thus, designing gelators with
programmed enzymatic reaction and pro-drug activation.
complementary binding functionality to each other that would
undergo physicochemical change in response to one another
would be of great importance. The present study delineates
the development of complementary gels that underwent
mutual self-destruction upon mixing. This dissolution of
complementary gels was employed in programmed enzyme
catalysis including pro-drug activation.
To develop such interactive gelators, complementary
functional moieties need to be included in the molecular
design so that one gelator will interact with other one either
by covalently or non-covalently. In this regard, phenylboronic
acid (PBA), a recognized saccharide receptor and glucose are
well known for complementary binding functionalities that
form boronate-diol adduct through reversible covalent
bonding. Accordingly, we synthesized two gelators; (i) gelator-
Molecular gels originating from self-assembled amphiphiles
showed unique dynamic properties. In supramolecular gels,
low molecular mass amphiphiles are held together by means
of weak non-covalent interactions such as hydrogen bonding,
- stacking, hydrophobic interactions and thus it can be easily
1-3
disassembled in response to external stimuli. Development
of stimuli responsive functional gels like photo-responsive, pH-
responsive, heat-sensitive, enzyme-responsive are finding
notable importance in drug release, disease diagnosis, tissue
4-7
engineering and so on. Hamachi et al. developed phosphate-
based hydrogelator that exhibited macroscopic gel to sol
transition in response to multiple stimuli: temperature, pH,
2+
8
Ca , and light. In another work, Lutolf and co-workers had
reported rational control in the degradation of polymeric gels
via selection of suitable amino acid and consequent release of
1
was composed of hydrophobic cholesterol unit coupled with
9
drug. In this context, functionally sensitive molecules as well
as enzyme/protein are known to be immobilized within gel
10
matrix for better stabilization. Design of stimuli responsive
matrix that will entrap chemically active substances, variety of
catalyst/biocatalyst without deactivation and will release them
as when required through degradation of the matrix is in great
1
1,12
demand.
With this aim, we intend to develop
complementary gels that can serve as encapsulating agent of
chemical/biochemical species and will degrade upon mixing.
The gel dissolution upon mixing will be useful to program Fig.1 (a) Structures of gelator-
1
and
DMF-water (2:1 v/v) and sol state after mixing of two gels; (c) dye entrapped gel-
(red) and gel- (blue) and its mixed state (purple) after 6h incubation.
2; photographic images of (b) gel-1 and 2 in
chemical or biochemical processes. Separately immobilized
reactants in different gels will have the propensity to come in
1
2
terminal PBA by C-12 long chain spacer and (ii) gelator-2 was
made of same cholesterol unit attached with D(+)-gluconic
acid through C-6 long chain linker (Fig. 1a and Scheme S1,
ESI). The synthesized amphiphiles were characterized by H-
Department of Biological Chemistry, Indian Association for the Cultivation of
Science, Jadavpur, Kolkata – 700 032, India.
E-mail: bcpkd@iacs.res.in; Fax:+91-33-24732805
13
1
†
Electronic Supplementary Information (ESI) available: synthetic procedure, Mass NMR, high resolution mass spectrometry (HRMS) (Fig. S1-S4,
1
1
and H-NMR
1 and 2 and their adduct, TEM images, temperature dependent H-
ESI). Gelation ability of these amphiphiles was tested in water,
different organic, binary solvent mixtures and the minimum
NMR and FTIR spectra, UV-vis and fluorescence spectra of ANS in gelator solutions,
complex viscosity vs. angular frequency plot. See DOI: 10.1039/x0xx00000x.
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gelation concentration (MGC) was depicted in Table 1. Both
and
soluble
dimethylformamide
Amphiphile-
nitrobenzene and DMSO-water (4:1 v/v) with MGC of 5.0-7.9 for comprehensible macroscopic observation. Upon incubation
mg/mL. Interestingly, both amphiphiles ( and ) showed for 6h, the individual blue and red coloured gels became
gelation ability in DMF-water mixture (2:1 v/v) and in organic purple coloured viscous sol (Fig. 1c). Notably, in contrast to the
1
ability resulting in gel to sol transition. After 6h incubation, the
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DOI: 10.1039/C7CC04269H
2
were unable to form gel in pure water while they remain mixture (gel-1 + gel-2) completely lost the characteristic
in
dimethyl
sulphoxide
(DMSO),
N,N- semisolid behaviour of gels and became free flowing viscous
(DMF),
tetrahydrofuran
(THF). sol (Fig. 1b). We repeated the experiment by including two
2
formed gel in chloroform, chlorobenzene, different dyes in each of the gel and placed one over another
1
2
solvents like toluene, benzene, xylene.
gelation behaviour of individual amphiphile, the solid mixture
of amphiphile- and could not exhibit any gelation ability in
DMF-water (2:1 v/v) binary solvent mixture.
1
2
Table.1 Minimum gelation concentration (MGC) of gelator-
solvents (S=soluble, Ins=Insoluble).
1
and
2 in different
Solvents
MGC(mg/mL)
Solvents
MGC(mg/mL)
1
2
1
2
Water
Ins
S
S
n-Hexane
Chloroform
Benzene
Toluene
S
S
DMSO
S
S
7.9
6.1
5.0
4.8
DMF
S
S
9.2
10.3
7.8
THF
S
S
DMSO-water(4:1, v/v)
S
6.5
o-Xylene
DMF-water(2:1, v/v)
THF-water
10.1
S
8.2
S
Chlorobenzene
Nitrobenzene
S
S
5.4
5.0
Various intermolecular non-covalent interactions operating
between the amphiphilic molecules during self-aggregation
1
were investigated by FTIR and temperature dependent H-
NMR spectroscopy. FTIR spectra of
1 in non-self-assembled
state (KBr pellet) showed sharp transmittance at 1511, 1652
-1
and ~3329-3346 cm due to amide δN-H (amide II, bending),
νC=O (amide I, stretching) and coexistence of νN-H (amide A) with
νO-H peaks, respectively (Fig. S5a, ESI). Interestingly, in DMF-
D O (2:1 v/v), the transmittance signals were shifted to 1549,
2
-1
Fig. 2 FESEM images of (a) gel-
1
, (b) gel-
2
and (c) fused gels (
and and mixed gels; (h) rheology of
gels 1 and 2 and its mixtures after varying incubation period.
1+2) after 1h, (d) 3h,
1
634 and ~3319-3516 cm (broad band), respectively. Similar (e) 5h, (f) 6h incubation; (g) XRD of gel-
1
2
shifts were noticed for gelator-2 upon transition form non-self-
assembled to self-assembled state in DMF-D O (2:1 v/v, Fig.
2
This macroscopic physical change (gel-to-sol) caused by
S5b, ESI). These shifts in stretching and bending frequencies
indicate the participation of intermolecular hydrogen bonding
between carbonyl (C=O) and amide N-H moieties during self-
amalgamation of the two gels intrigued us to investigate the
morphological transformation of self-aggregates upon mixing
through microscopic techniques. According to field emission
scanning electron microscopic (FESEM) and transmission
electron microscopic (TEM) studies both gels showed the
existence of entangled fibrillar network (Fig. 2a,b and Fig. S8,
1
3
1
assembled gelation. Temperature dependent H-NMR study
was carried out for both gels in benzene-d6. With steady
increase in temperature up to 80-85 °C, the suppressed broad
peaks owing to cholesteryl, alkyl chain spacer, phenyl ring (for
ESI). Gel-
several micrometer while in gel-
1
has 40-50 nm thick fibre diameter with length of
, comparatively thinner fibre
2
) and amide protons became more prominent with increased
2
peak intensity (Fig. S6, ESI). Non-covalent forces involved in
gelation got dissociated at higher temperature leading to gel
diameter of 20-30 nm was observed. Next, we have taken
FESEM images of the incubated mixed gels at different time
interval. After 1h incubation, the fused gel showed different
characteristics. The mixed gel fibre diameters were larger
compared to native gels and loosening of the fibrillar network
was noticed (Fig. 2c). After 3h incubation the mixed gel lost its
well-defined fibril structure and formed small irregular bulks
1
to sol transition where the characteristic molecular H-NMR
signals were observed. Moreover, gradual increase in the
emission intensity of 8-anilino-1-naphthalenesulfonic acid, ANS
(ex = 360 nm) upon successive increase in the concentration of
both gelators ensured the involvement of hydrophobic
14
interaction during gelation (Fig. S7, ESI).
With the primary objective of the present study, we were
curious to investigate whether the mixing of gel- and gel-
(
Fig. 2d). These irregular bulks were further divided into tiny
fragments with increase in incubation period up to 5 h (Fig.
1
2
2
6
e). Finally, no well-defined morphologies were observed after
h incubation (Fig. 2f), indicating the complete transition of
the fused gel to sol. Mixing of the two complementary gels led
to the gradual swelling of gel fibres presumably owing to the
formation of boronate-diol adduct between two gelators and
lost its compact entangled fibrillar network. X-ray diffraction
leads to any kind of macroscopic and microscopic changes
through interaction between complementary functional
moieties. DMF-water (2:1 v/v) was used for the experiment
because this is the only water containing solvent system where
both amphiphiles exhibited gelation ability. Upon placing the
gel-
1 over the gel-2, the physical state of the mixed gel slowly
(
XRD) study further supported the destruction of self-
assembled fibrillar network after mixing of gels (Fig. 2g). Dried
got changed. With time it gradually lost the solvent imbibing
2
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sample of gel-
14.13° and 18.66° corresponding to the spacing between assembled solutions of individual DgOelI:a1t0o.r1039w/Ca7sCCa0l4m26o9sHt
intermolecular steroidal backbones and phenyl rings, comparable. ANS encapsulated within the hydrophobic
1 in DMF-water (4:1 v/v) showed an XRD peak at vicinity of ANS within the dissolute gel mixture andVieiwnAnrtoiclne-Osnelilnfe-
2Ɵ 
15
respectively. Gel-2 showed peaks at 2Ɵ = 17.02°, that may be domain of native gels got freed in the bulk domain when
correlated to the spacing between intermolecular steroidal ordered molecular arrangement was self-destructed due to
moieties. Interestingly, the incubated (6h) mixed gel did not the mixing of gels. Thus, the spectroscopic observation further
show any distinguishable XRD peak, which indicates the loss of confirmed the dissipation of self-assemblies owing to the
ordered molecular arrangement upon mixing of two gels. The fusion of complementary gels.
dissolution of complementary gels due to fusion was further
confirmed from rheological experiment. Herein, the storage
modulus (G’) represents the ability of a deformed material to
restore its native form while the loss modulus (G’’) refers to
the flow behaviour of the material under applied stress. For
viscoelastic materials like gels, G’>G’’ and in the sol state
2
G’’>G’ (G’ and G’’=angular frequency). In a typical
oscillatory frequency sweep experiment of the native gel-
gel- at a fixed strain (0.01%), G’ and G’’ were recorded to be
2760, 2690 and 320, 360 Pa, respectively (Fig. 2h). When
the gels were mixed and aged, the G’ value of the fused gel
dropped to 1630 Pa after 3h incubation. After 6h, it was
further decreased to 640 Pa. This gradual drop in G’ value of
1 and
2




the fused gels with aging clearly indicates the decrease in
mechanical stiffness of the resulting material owing to
dissolution of mixed gels to viscous sol. In a different mixing.
experiment, a strain controlled (strain fixed at 1%) frequency
Fig. 3 Schematic representation of the dissolution of complementary gels upon
sweep was carried out for gel-
1
, gel-
2
and their mixture to
The question tickles at this point that what could be the
measure complex viscosity (η*) as a function of angular reason behind dissolution of gels upon mixing. An optimum
−
3
−1
frequency (10 to 10 rads , Fig. S9, ESI). At low frequency hydrophilic lipophilic balance (HLB) in a small molecule leads
−
3
range (angular frequency
very high ( 16200 and

10 ), η* value was found to be to successful gelation. Amphiphile
1 and 2 possibly maintained


13500 Pa for gel- and gel-
1
2,
the suitable HLB for gelation in DMF-water (2:1 v/v). Mixing of
respectively) probably due to well ordered molecular packing gels yielded the formation of cyclic boronate-diol adduct
1
in the gel structure. However, after 3h and 6h incubation of (characterized by H-NMR and mass spectra, Fig. S11, S12, ESI)
both gels, η* value dropped to
respectively. This gradual drop in complex viscosity of the of
fused gels with aging suggested the decrease in compactness have the required HLB for gelation resulting in gel dissolution.
and stiffness of the resulting mixtures. Hence, the microscopic
Herein, we intend to use this mixing induced dissolution of
investigation, XRD and rheology analysis clearly delineate that complementary gels in programmed enzymatic reactions,
the compact entangled fibrillar network of gel- and gel-
specially for pro-drug activation. Enzyme catalyzed ester
gradually destroyed possibly through formation of boronate- hydrolysis within the mixed gels ( ) was monitored as a
diol adduct upon mixing of the two complementary gels.
representative bio-catalytic reaction (Fig. 4a). We entrapped
At this instant, we investigated the change in molecular hydrolytic enzyme Chromobacterium Viscosum (CV) lipase

6900 and

2000 Pa, via covalent linkage between complementary functional group
(PBA) and (gluconic acid) (Fig. 3). The adduct might not
1
2
1
2
1+2
packing arrangement of complementary gels before and after within gel-
mixing. Since the gelators are devoid of any chromophoric within gel-
1
2
and its substrate (p-nitrophenyl-n-octanoate)
without disconcerting the stability of the native
moiety, absorbance was recorded for ANS that was doped gels. Lipase entrapped gel-
within the different solutions of both gelators and its mixture. included gel- and the consequent change was monitored with
ANS doped solution of and
in the non-self-assembled state time. A yellow colour was appeared at the junction of the two
in DMF) showed absorbance maxima at 371 nm (Fig. S10, ESI). gels and slowly spread over the whole system. After 6h aging,
1 was placed over substrate
2
1
2
(
Upon transition to the self-assembled state of the gelators in complete gel dissolution took place with the formation of
DMF-water (2:1 v/v), ANS showed red shifted UV-maxima at yellow colour free flowing viscous sol (Fig. 4b). Lipase
3
75 and 378 nm for
1
and
2
, respectively. The bathochromic entrapped in gel-
at the junction point and yellow coloured p-
and nitrophenol was liberated at the junction. With gradual
). ANS doped within the two gelator's solutions exhibited dissolution of the complementary gels upon mixing, availability
1 came in contact with the substrate
shift during the self-assembly of gelators indicates the J-type entrapped in gel-
2
(head-to-tail) aggregation pattern of the amphiphiles (1
1
6
2
absorbance maxima at 376 nm and 373 nm within 1h and 3h of substrate to enzyme steadily increased. Consequently more
of mixing, respectively (Fig. S10, ESI). After 6h aging, it showed amount of p-nitrophenol was liberated resulting in the
UV maxima at 372 nm similar to that in native gelator's formation of yellow coloured viscous sol. The formation of p-
solutions (in DMF). It indicates that the microenvironment in nitrophenol was also monitored by UV-vis spectroscopy. Lipase
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catalyzed hydrolysis was carried out within the mixture of self- complementary binding gels can not only be VuiesweAdrticilne Ontlhinee
assembled solution of amphiphiles at five times lower than programmed pro-drug activation but wiDllObI:e10a.l1s0o39u/Cse7CfuCl0i4n26t9hHe
MGCs to avoid turbidity related error. The substrate showed sustained release of drugs. Considering biological relevance of
absorbance maxima at 303 nm while the hydrolysed product enzymatic reaction and pro-drug activation, further change in
exhibited absorbance maxima at 316 nm, which confirmed the the amphiphile's structure is needed (by inclusion of more
formation of p-nitrophenol in the mixed solutions (Fig. S13a, hydrophilic spacer) to make them pure hydrogelators and
17
ESI). A gradual increase in the absorbance of liberated p- carry out the biocatalysis within pure aqueous domain.
nitrophenol with time was noted within the mixed solutions. In conclusion, we have developed two cholesterol based
Higher amount of product formation was observed owing to gelators bearing phenylboronic acid and glucose units as
greater enzyme-substrate interaction within dissipated gel complementary binding sites in their motifs. Mixing of these
mixture.
two gels resulted in the gel to sol transition through gradual
destruction of their fibrillar network. Dissipation in the self-
assemblies took place due to the formation of cyclic boronate-
diol adduct, which lacks the required HLB for gelation. The
dissolution of complementary gels was judiciously employed in
the programmed enzymatic reaction and pro-drug activation.
The present report endows
a new understanding of
disassembly generated from mixed self-assembled systems as
well as its prospect in controlled enzymatic reaction.
P.K.D. is thankful to CSIR, India (ADD, CSC0302) for financial
assistance. D.M., P.C., D.S. acknowledge CSIR, India for
research fellowships.
Notes and references
1
2
3
M. D. S. Maset, V. J. Nebot, J. F. Miravet, B. Escuder, Chem.
Soc. Rev., 2013, 42, 7086.
Fig. 4 (a) Lipase catalyzed hydrolysis of p-nitrophenyl-n-octanoate (substrate); (b)
photographs of enzyme and substrate included gels and its dissolution upon
mixing with liberated yellow coloured p-nitrophenol; (c) Lipase catalysed
hydrolysis of a pro-drug (CPS) to form activated drug (CP).
D. Weiss, K. Kreger, H. W. Schmidt, Macromol. Mater. Eng.,
2
017, 302, 1600390.
F. Trausel, F. Versluis, C. Maity, J. M. Poolman, M. Lovrak, J.
H. van Esch, R. Eelkema, Acc. Chem. Res., 2016, 49, 1440.
S. Datta, S. Bhattacharya, Chem. Soc. Rev., 2015, 44, 5596.
Y. Qiu, K. Park, Adv. Drug Deliv. Rev., 2001, 53, 321.
B. O. Okesola, D. K. Smith, Chem. Soc. Rev., 2016, 45, 4226.
C. Maity, W. E. Hendriksen, J. H. van Esch, R. Eelkema,
Angew. Chem. Int. Ed., 2015, 54, 998.
The preceding observation encouraged us to employ this
fusion triggered dissolution of complementary gels in pro-drug
activation. A pro-drug is an inactive precursor of drug that is
converted into a pharmacologically active drug by enzymatic
or non-enzymatic reaction. Controlled degradation of the drug
encapsulating agent assists in the sustained release of drug,
thereby stirring up biological response over a prolonged time
period. Herein, we tried to tune pro-drug activation through
gradual dissolution of enzyme and pro-drug encapsulating gels.
We used chloramphenicolsuccinate sodium salt (CPS) as a
water soluble pro-drug, which upon enzymatic hydrolysis will
4
5
6
7
8
9
1
1
H. Shigemitsu, T. Fujisaku, S. Onogi, T. Yoshii, M. Ikeda, I.
Hamachi, Nat. Protoc. 2016, 11, 1744.
Y. S. Jo, J. Gantz, J. A. Hubbella, M. P. Lutolf, Soft Matter,
2009, 5, 440.
0 M. P. Conte, K. H. A. Lau, R. V. Ulijn, ACS Appl. Mater.
Interfaces, 2017, , 3266.
1 M. Araújo, I. M. Capdevila, S. D. Oltra, B. Escuder, Molecules,
016, 21, 744.
Accordingly, CV lipase and CPS were separately encapsulated 12 H. Sun, D. J. Dobbins, Y. Dai, C. P. Kabb, S. Wu, J. A.
in gel- and gel- , respectively. Enzyme triggered hydrolysis of
Alfurhood, C. Rinaldi, B. S. Sumerlin, ACS Macro Lett., 2016,
, 688.
3 D. Mandal, S. Dinda, P. Choudhury, P. K. Das, Langmuir,
016, 32, 9780.
9
18
produce antibiotic chloramphenicol (CP) (Fig. 4c).
2
1
2
5
CPS was followed by measuring the absorbance of liberated
CP. CPS showed UV absorbance maxima at 272 (Fig. S13b, ESI).
1
1
2
Upon addition of lipase included gel-
1 to the CPS entrapped
4 P. Choudhury, D. Mandal, S. Brahmachari, P. K. Das, Chem. -
Eur. J., 2016, 22, 5160.
gel- , initially the absorbance maxima red shifted to 278 nm
2
possibly due to enzymatic hydrolysis of CPS to CP. The 15 D. Ke, C. Zhan, A. D. Q. Li, J. Yao, Angew. Chem. Int. Ed.,
2
011, 50, 3715.
absorbance maxima of pure CP (as procured) was also found to
be at 278 nm. Hence, the enzymatic hydrolysis of CPS resulted
in the red shift in UV maxima due to the formation of CP.
Interestingly, the absorbance value of liberated CP gradually
1
1
6 A. Ajayaghosh, C. Vijayakumar, R. Varghese, S. J. George,
Angew. Chem. Int. Ed., 2006, 45, 456.
7 D. Mandal, M. Ghosh, S. Maiti, K. Das, P. K. Das, Colloids and
Surfaces B: Biointerfaces, 2014, 113, 442.
enhanced with time possibly due to higher accessibility of the 18 V. J. Stella, K. W. N. Addae, Adv. Drug Deliv. Rev., 2007, 59
,
6
77.
enzyme and CPS within dissipated gels resulting in the
formation of higher amount of CP. In absence of lipase, neither
p-nitrophenol nor CP formation was observed upon mixing of
gels. Thus, dissipation of self-assemblies owing to the fusion of
4
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