U.S. patent application number 12/313784 was filed with the patent office on 2014-02-06 for method for preparing hydro/organo gelators from disaccharide sugars by biocatalysis and their use in enzyme-triggered drug delivery.
The applicant listed for this patent is George John, Praveen Kumar Vemula. Invention is credited to George John, Praveen Kumar Vemula.
Application Number | 20140037731 12/313784 |
Document ID | / |
Family ID | 42196514 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140037731 |
Kind Code |
A9 |
John; George ; et
al. |
February 6, 2014 |
Method for preparing hydro/organo gelators from disaccharide sugars
by biocatalysis and their use in enzyme-triggered drug delivery
Abstract
A method for preparing hydro/organo gelators from disaccharide
sugars by biocatalysis and their use in enzyme-triggered drug
delivery. Controlled delivery of an anti-inflammatory,
chemopreventive drug is achieved by an enzyme-triggered drug
release mechanism via degradation of encapsulated hydrogels. The
hydro- and organo-gelators are synthesized in high yields from
renewable resources by using a regioselective enzyme catalysis and
a known chemopreventive and anti-inflammatory drug, curcumin, is
encapsulated in the gel matrix and released by enzyme triggered
delivery. The release of the drug occurs at the physiological
temperature and control of the drug release rate is achieved by
manipulating the enzyme concentration and temperature. The
by-products formed after the gel degradation clearly demonstrated
the site specificity of degradation of the gelator by enzyme
catalysis. The present invention has applications in developing
cost effective, controlled drug delivery vehicles from renewable
resources, with a potential impact on pharmaceutical research and
molecular design and delivery strategies.
Inventors: |
John; George; (Edison,
NJ) ; Vemula; Praveen Kumar; (Yonkers, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
John; George
Vemula; Praveen Kumar |
Edison
Yonkers |
NJ
NY |
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20100129451 A1 |
May 27, 2010 |
|
|
Family ID: |
42196514 |
Appl. No.: |
12/313784 |
Filed: |
November 24, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US2007/012333 |
May 22, 2007 |
|
|
|
12313784 |
|
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|
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60802412 |
May 22, 2006 |
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Current U.S.
Class: |
424/488 ;
514/733 |
Current CPC
Class: |
A61Q 19/00 20130101;
A61K 8/60 20130101; A61K 31/12 20130101; A61K 8/11 20130101; A61K
9/06 20130101; A61K 9/48 20130101; A61K 8/0216 20130101; A61K 31/05
20130101; A61K 2800/10 20130101; A61K 2800/56 20130101; A61K 47/26
20130101 |
Class at
Publication: |
424/488 ;
514/733 |
International
Class: |
A61K 9/10 20060101
A61K009/10; A61K 31/05 20060101 A61K031/05 |
Claims
1.-11. (canceled)
12. A drug-delivery composition, comprising: a) a hydro/organo gel
prepared from disaccharide sugars or derives thereof; and b) a
hydrophobic drug encapsulated in said gel; wherein said drug is
capable of release upon enzyme mediated degradation of said
gel.
13. The composition of claim 12, wherein the hydro/organo gel is
prepared by self-assembly of hydro/organo gelators prepared from a
disaccharide sugar or derivatives thereof via a biosynthesis
reaction.
14. The composition of claim 12, wherein the disaccharide sugar is
amygdalin.
15. The composition of claim 12, wherein the gelator is an
esterification reaction product of a fatty acid and amygdalin.
16. The composition of claim 15, wherein the gelator is selected
from the group consisting of amygdalin butyrate, amygdalin
tetradecanoate, and amygdalin octadecanoate.
17. The composition of claim 12, wherein the hydrophobic drug is
curcumin and the gelator is an amygdalin-derived gelator.
18. A method for preparing hydro/organo gelators, comprising the
step of attaching a fatty acid chain to disaccharide sugars via a
regiospecific transesterification reaction on a primary sugar
hydroxyl via enzyme catalysis.
19. The method as described in claim 18, wherein the said
disaccharide sugar is amygdalin.
20. The method as described in claim 18, wherein the glator is
selected from the group consisting of amygdalin butyrate, amygdalin
tetradecanoate, and amygdalin octadecanoate.
21. The method as described in claim 18, where the said catalyzing
enzyme is a lipase or a lipolase.
22. A method for using hydro/organo gelators for drug delivery or
cosmetic delivery, comprising the steps of: a) encapsulating and
solubilization of a hydrophobic drug molecule or a hydrophobic
molecule which is a component of a cosmetic formula in a hydrogel;
and b) releasing the drug or the hydrophobic cosmetic molecule by
using a gel-breaking enzyme.
23. The method as described in claim 22, where the gel-breaking
enzyme is a hydrolase.
24. The method as described in claim 22, where the activity of the
gel-breaking enzyme is controlled by physiological temperature and
concentration.
25. A method of using hydro-organo gelators prepared from
disaccharide sugars in generating inorganic nanomaterial template.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to hydro/organo gelators, and
more particularly, the present invention relates to a method for
preparing hydro/organo gelators from disaccharide sugars by
biocatalysis and their use in enzyme-triggered drug delivery,
cosmetic components delivery, and making of templated materials to
generate inorganic and soft nanomaterials.
[0003] 2. Description of the Prior Art
[0004] Use of renewable resources for production of valuable
chemical commodities is becoming a topic of great interest and an
objective of promoting the industrial bio-refinery concept in which
a wide array of valuable chemicals, fuel, food, nutraceuticals, and
animal feed products all result from the integrated processing of
grains, oil seeds, and other biomass materials..sup.1 .sup.1Lorenz,
P. & Zinke, H. White biotechnology: differences in US
approaches? Trends Biotechnol. 23, 570-574 (2005); Business and
regulatory news. OECD says industrial biotech not realizing
potential. Nat. Biotechnol. 19, 493-494 (2001).
[0005] An article by Stephan Harrera.sup.2 illustrates that
industrial or `white` biotechnology.sup.3 is making an increasingly
important contribution to the development of a sustainable,
biobased economy by an environmental benign approach..sup.4 It uses
enzymes and micro-organisms to make products in sectors, such as
chemistry, food and feed, paper, textile, and medicine. As opposed
to chemical synthesis, enzyme catalysis is highly selective and has
been used to generate various specialty chemicals,.sup.5 including
sugar-based esters..sup.6 .sup.2Herrera, S. Industrial
biotechnology-a chance at redemption. Nat Biotechnol. 22, 671-675
(2004)..sup.3Industrial biotechnology and sustainable chemistry.
Royal Belgian Academy of Applied Sciences, Brussels (January
2004)..sup.4Eissen, M., Metzger, J. 0., Schmidt, E. &
Schneidewind, U. 10 Years after rio-concepts on the contribution of
chemistry to a sustainable development. Angew. Chem. Int. Ed 41,
414-436 (2002); Biermann, U. et al. New synthesis with oils and
fats as renewable raw materials for the chemical industry. Angew.
Chem. Int. Ed. 39, 2206-2224 (2000); Gibson, J. M. et al.
Benzene-free synthesis of phenol. Angew. Chem. Int. Ed. 40,
1945-1948 (2001)..sup.5Wandrey, C., Liese, A. & Kihumbu, D.
Industrial biocatalysis: past, present and future. Org. Process
Res. Dev. 4, 286-290 (2000)..sup.6Yan, Y., Bornschener, U. T. &
Schmid, R. D. Lipase-catalyzed synthesis of vitamin C esters.
Biotechnol. Lett. 21, 1051-1054 (1999).
[0006] Thus, there exists a need for developing building blocks
from renewable resources to generate soft nanomaterials, such as
new surfactants, liquid crystals, organic gelling materials, and
hydrogels..sup.7 .sup.7John, G., Masuda, M. & Shimizu, T.
Nanotube formation from renewable resources via coiled nanofibers.
Adv. Mater. 13, 715-718 (2001); John, G., Masuda, M., Jung, J. H.,
Yoshida, K. & Shimizu, T. Unsaturation influenced gelation of
aryl glycolipids. Langmuir 20, 2060-2065 (2004); John, G., Mason,
M., Ajayan, P. M. & Dordick, J. S. Lipid-based nanotubes as
functional architectures with embedded fluorescence and recognition
capabilities. J. Am. Chem. Soc. 126, 15012-15013 (2004).
[0007] Hydrogels have a range of biomedical applications in areas
such as tissue engineering,.sup.8 controlled released drug delivery
systems,.sup.9 and medical implants..sup.10 Design and synthesis of
low-molecular-weight hydrogelators has received considerable
attention in soft materials research in terms of its potential
applications in cosmetics, toiletries, and pharmaceutical
formulations. Literature study reveals that there are only limited
reports on easily achievable and efficient low-molecular-weight
gelators that are able to gel water or even water mixtures with
other solvents,.sup.11 and which are often achieved by multi-step
chemical synthesis. Surprisingly, to the best of applicants'
knowledge, to date there are no examples in the literature where
low-molecular-weight hydrogelators were synthesized from renewable
resources by using regioselective enzyme catalysis. .sup.8Lee, K.
Y. & Mooney, D. J. Hydrogels for tissue engineering. Chem. Rev.
101, 1869-1879 (2001)..sup.9Friggeri, A., Feringa, B. L. & van
Esch, J. Entrapment and release of quinoline derivatives using a
hydrogel of low molecular weight gelator. J. Controlled Release 97,
241-248 (2004); Yang, Z., Liang, G., Wang, L. & Xu, B. Using a
kinase/phosphatase switch to regulate a supramolecular hydrogel and
forming the supramolecular hydrogel in vivo. J. Am. Chem. Soc.
DOI:10.1021/j057412y (2006); van Bommel, K. J. C., Stuart, M. C.
A., Feringa, B. L. & van Esch, J. Two-stage enzyme mediated
drug release from LMWG hydrogels. Org. Biomol. Chem. 3, 2917-2920
(2005)..sup.10Lee, K. Y. & Mooney, D. J. Hydrogels for tissue
engineering. Chem. Rev. 101, 1869-1879 (2001); Miyata, T., Uragami,
T. & Nakamae, K. Biomolecule-sensitive hydrogel. Adv. Drug
Delivery Rev. 54, 79-98 (2002)..sup.11Menger, F. M. & Caran, K.
L. Anatomy of a gel. Amino acid derivatives that rigidify water at
submillimolar concentrations. J. Am. Chem. Soc. 122, 11679-11691
(2000); Jokic, M., Makarevic, J., Zinic, M. & Makarevic, J. A
novel type of small organic gelators: bis(amido acid) oxalyl
amides. J. Chem. Soc., Chem. Commun. 1723-1724 (1995); Makarevie,
J. et al. Bis(amino acid) oxalyl amides as ambidextrous gelators of
water and organic solvents: supramolecular gels with temperature
dependent assembly/dissolution equilibrium. Chem. Eur. J. 7,
3328-3341 (2001); Oda, R., Huc, I. & Candau, S. J. Gemini
surfactants as new, low molecular weight gelators of organic
solvents and water. Angew. Chem. Int. Ed. 37, 2689-2691 (1998);
Estroff, L. A. & Hamilton, A. D. Effective gelation of water
using a series of bis-urea dicarboxylic acids. Angew. Chem. Int.
Ed. 39, 3447-3450 (2000); Kobayashi, H. et al. Molecular design of
"super" hydrogelators: understanding the gelation process of
azobenzene-based sugar derivatives in water. Org. Lett. 4,
1423-1426 (2002); Luboradzki, R., Gronwald, O., Ikeda, M., Shinkai,
S. & Reinhoudt, D. N. An attempt to predict the gelation
ability of hydrogen-bond-based gelators utilizing a glycoside
library. Tetrahedron 56, 9595-9599 (2000); Gronwald, 0. &
Shinkai, S. Sugar-integrated gelators of organic solvents. Chem.
Eur. J. 7, 4328-4334 (2001); Jung, J. H. et al. Self-assembly of a
sugar-based gelator in water: Its remarkable diversity in gelation
ability and aggregate structures. Langmuir 17, 7229-7232 (2001);
Wang, G. & Hamilton, A. D. Low molecular weight organogelators
for water. Chem. Commun. 310-311 (2003).
[0008] Thus, there exists a need to use biocatalysis as a tool to
make gelators from biomass and their assembly to form hierarchical
superstructures in water, i.e., formation of hydrogel and soft
nanomaterials, encapsulation of hydrophobic drug or hydrophobic
cosmetic components, as well as enzyme mediated hydrogel
degradation, which will give new insights into low-molecular-weight
hydrogelators-based drug delivery.
[0009] Controlled delivery of drugs or cosmetic material occurs
when a polymer, whether natural or synthetic, is judiciously
combined with a drug or other active agent in such a way that the
active agent is released in a pre-designed manner..sup.12 While
these advantages can be significant, the potential disadvantages
cannot be ignored, such as the possible toxicity or
non-biocompatibility of the materials used, the undesirable
by-products from gel degradation, and the higher cost of
controlled-release systems compared with traditional pharmaceutical
formulations. .sup.12Dorski, C. M., Doyle, F. J. & Peppas, N.
A. Preparation and characterization of glucose-sensitive
P(MMA-a-EG)hydrogels. Polym. Mater. Sci. Eng. Proceed. 76, 281-282
(1997); Vert, M., Li, S. & Garreau, H. More about the
degradation of LA/GA derived matrices in aqueous media. J.
Controlled Release 16, 15-26 (1991).
[0010] Thus, there exists a need for sugar amphiphiles by
regioselective synthesis of amygdalin esters as new hydrogelators,
which are low cost, efficient, safe, and with high gelation
efficiency.
[0011]
[O-.beta.-D-glucopyranosyl-(1-6)-.beta.-D-glucopyranosyloxy]benzene-
acetonitrile known as D-Amygdalin is a naturally occurring
glycoside found in many food plants, for example, the kernels of
apples, almonds, peaches, cherries, and apricots..sup.13 Amygdalin
(a by-product of apricot, almonds and peach industry, see FIG. 1,
which are pictures of: (a) an apricot pit that is a source of
amygdalin; (b) Curcuma longa; and, (c) powdered curcumin.sup.14
that is commonly known as turmeric and used in traditional Indian
culinary and medicine--has been used as a main ingredient in
commercial preparations of laetrile, a purported therapeutic
agent..sup.15 .sup.13Jones, D. A. Why are so many food plants are
cyanogenic? Phytochemistry 47, 155-162 (1998)..sup.14Curcumin is
just one example as a drug model..sup.15Turczan, J. W. &
Medwick, T. Qualitative and quantitative analysis of amygdalin
using NMR spectroscopy. Anal. Lett. 10, 581-590 (1977); Syrigos, K.
N., Rowlinson-Busza, G. & Epenetos, A. A. In vitro cytotoxicity
following specific activation of amygdalin by .beta.-glucosidase
conjugated to a bladder cancer-associated monoclonal antibody. Int.
J. Cancer 78, 712-719 (1998).
[0012] Thus, there exists a need to synthesize amygdalin
derivatives that can form nanoaggregates through self-assembly and
encapsulation of a hydrophobic drug followed by release of the
encapsulated drug upon enzyme mediated degradation, i.e.,
enzyme-triggered drug-delivery.
[0013] In amygdalin-fatty acid conjugates, sugar moiety can
facilitate the stacking of molecules through hydrogen bonding,
phenyl ring can facilitate intermolecular interactions through
.pi.-.pi. stacking, and hydrophobic hydrocarbon chain not only
decreases the solubility in water, it also helps the molecular
association through the van der Waals interactions.
[0014] In general, multi-step synthesis, arduous separation
procedures, and lower yields often keep low-molecular-weight
gelators away from commercial use due to high production cost.
Strikingly, the hydrogelators of the present invention were
synthesized from renewable resources in a single-step process in
high yields (>90%), and unpurified crude products showed
unprecedented gelation abilities like their counter pure products,
allowing the development of versatile gelators which can be made
from low cost starting materials and without purification.
[0015] Thus, there exists a need for gelator molecules with various
chain lengths. See FIG. 2, which is a synthetic scheme of
amygdalin-based amphiphiles.
SUMMARY OF THE INVENTION
[0016] Thus, an object of the present invention is to provide a
method for preparing hydro/organo gelators from disaccharide sugars
by biocatalysis and their use in enzyme-triggered drug delivery,
which avoids the disadvantages of the prior art.
[0017] Briefly stated, another object of the present invention is
to provide a method for preparing hydro/organo gelators from
disaccharide sugars by biocatalysis and their use in
enzyme-triggered drug delivery. Controlled delivery of an
anti-inflammatory, chemopreventive drug is achieved by an
enzyme-triggered drug release mechanism via degradation of
encapsulated hydrogels. The hydro- and organo-gelators are
synthesized in high yields from renewable resources by using a
regioselective enzyme catalysis and a known chemopreventive and
anti-inflammatory drug, curcumin, is encapsulated in the gel matrix
and released by enzyme triggered delivery. The release of the drug
occurs at the physiological temperature, and control of the drug
release rate is achieved by manipulating the enzyme concentration
and temperature. The by-products formed after the gel degradation
clearly demonstrate the site specificity of degradation of the
gelator by enzyme catalysis. The present invention has applications
in developing cost effective, controlled drug delivery vehicles
from renewable resources, with a potential impact on pharmaceutical
research and molecular design and delivery strategies.
[0018] The novel features considered characteristic of the present
invention are set forth in the appended claims. The invention
itself, however, both as to its construction and its method of
operation together with additional objects and advantages thereof
will be best understood from the following description of the
specific embodiments when read and understood in connection with
the accompanying drawing.
[0019] Another object of the present invention is to provide a
method for preparing hydro/organo gelators from disaccharide sugars
by biocatalysis and their use in enzyme-triggered or
thermo-triggered cosmetic delivery. Controlled delivery of
components of cosmetic formula is achieved by an enzyme-triggered
release mechanism via degradation of encapsulated hydrogels. The
hydro- and organo-gelators are synthesized in high yields from
renewable resources by using a regioselective enzyme catalysis. The
release of the cosmetic components occurs at the physiological
temperature and control of their release rate is achieved by
manipulating the enzyme concentration and temperature. The present
invention has applications in developing cost effective, controlled
cosmetic delivery vehicles from renewable resources.
[0020] Another object of the present invention is to provide a
method for preparing hydro/organo gelators from disaccharide sugars
by biocatalysis and their use in making templated materials to
develop inorganic nanomaterials.
BRIEF DESCRIPTION OF THE DRAWING
[0021] The figures of the drawing are briefly described as
follows:
[0022] FIG. 1 are pictures of: (a) an apricot pit that is a source
of amygdalin; (b) Curcuma longa; and, (c) powdered curcumin that is
commonly known as turmeric and used in traditional Indian culinary
and medicine. It is also a known chemopreventive and
anti-inflammatory drug;
[0023] FIG. 2 is a synthetic scheme of amygdalin-based
amphiphiles;
[0024] FIG. 3 is a table of gelation ability of amygdalin
derivatives 1-3 in various solvents;
[0025] FIG. 4 are SEM micrographs, wherein scale bar is equivalent
to 1 .mu.M, of: (a) the organogel of derivative 1 prepared from
acetonitrile; (b) the aqueous gel from derivative 2; (c) the
aqueous gel from derivative 3; and, (d) a higher magnification of
hydrogel derivative 2;
[0026] FIG. 5 are scanning electron micrographs of
curcumin-embedded hydrogels of derivative 3;
[0027] FIG. 6 are: (a) the crystal structure analysis of derivative
1 in water; and, (b) a top view showing the .pi.-.pi. stacking of
phenyl rings and hydrogen boding between two amygdalin molecules,
wherein hydrogen bonding acts as a bridge between the stacked
amygdalin molecules along the b-axis shown as blue arrows and
between these two stacks along the a-axis shown as black arrows,
and wherein: oxygen is shown as red; nitrogen is shown as blue;
carbon is shown as white circles; and, hydrogen-bonding is shown as
black dashed lines;
[0028] FIG. 7 are schematic representations of possible molecular
packing models for the: (a) hydrogels; and, (b) organogels of
derivative 2;
[0029] FIG. 8 are: (a) a schematic representation of drug
encapsulation in a supramolecular hydrogel and subsequent release
of the drug by enzyme mediated degradation of hydrogel at
physiological temperature; and, (b) real images of the hydrogels of
derivative 3 with (i-iv) and without (v-vi) curcumin, wherein after
complete gel degradation, the remained white fluffy powder that
settled at the bottom was characterized as a water insoluble fatty
acid that formed after gel degradation by the enzyme;
[0030] FIG. 9 are UV absorption spectra of curcumin in various
types of solution mixtures, including: (a) an curcumin-entrapped
hydrogel in the presence of an enzyme; (b) an enzyme added to the
hydrogel that does not contain curcumin; (c) an curcumin-entrapped
hydrogel in the absence of an enzyme; and, (d) a methanolic
solution of curcumin;
[0031] FIG. 10 is a table of the effect of enzyme concentration and
temperature on drug release time;
[0032] FIG. 11 are a comparison of curcumin-drug-release time at
different concentrations and different temperatures from hydrogels
of amygdalin derivatives by enzyme degradation, wherein: (a) is the
time required for 5% release; and, (b) is the time required for
100%;
[0033] FIG. 12 is a comparison of the .sup.1H-NMR spectra of
commercially available stearic acid and the white fluffy solid
obtained after gel degradation, which was filtered, freeze-dried,
and NMR recorded in Chloroform; and
[0034] FIG. 13 is a table of the crystallographic parameters of
derivative 1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Synthesis of Hydrogelators by Enzyme Catalysis
[0035] By taking advantage of the supreme control on
regioselectivity of enzyme catalysis, a series of amygdalin
derivatives were made where selectively introduced acyl moiety on
primary hydroxyl group gave excellent yields.
[0036] Amygdalin is a disaccharide containing one primary hydroxyl
group that forms ester bonds with fatty acids. Vinyl esters were
used as acyl donors. The detailed synthesis procedures are shown in
the METHODS section below and the synthetic route to the
amphiphilic amygdalin derivatives is shown FIG. 2.
[0037] In general, multi-step synthesis, arduous separation
procedures, and lower yields often keep low-molecular-weight
gelators away from commercial use due to high production
cost..sup.16 Strikingly, the hydrogelators of the present invention
were synthesized from renewable resources in a single-step process
in high yields (>90%), and unpurified crude products showed
unprecedented gelation abilities like their counter pure products,
allowing the development of versatile gelators which can be made
from low cost starting materials and without purification. In
particular, this property gives the opportunity to develop these
gelators in industrial scales for various applications in
cosmetics, toiletries, drug deliveries, nanomaterials, and
pharmaceutical formulations..sup.17 .sup.16Zhang, S. Hydrogels: Wet
or let die. Nat. Mater. 3, 7-8 (2004)..sup.17Id.
Gelation Abilities of Derivatives 1-3
[0038] Amygdalin derivatives 1-3 encompass all required functional
groups, such as hydrogen bond forming `sugar` headgroup, phenyl
ring for .pi.-.pi. stacking, and hydrocarbon chain for van der
Waals interactions. These groups together can synergistically act
to form strong intermolecular interactions leading to the gelation.
The gelation abilities of the derivatives 1-3 in water and in
organic--polar and nonpolar--solvents are compared in FIG. 3, which
is a table of gelation ability of amygdalin derivatives 1-3 in
various solvents.
[0039] Typically, a gelator (0.01-2 mg) in a required solvent
(0.1-1 mL) was heated until the solid was completely dissolved. The
resulting solution was slowly allowed to cool to room temperature
and gelation was visually observed. The gel sample obtained
exhibited no gravitational flow in an inverted tube. All gels
obtained were thermally reversible. Above their gelation
temperature, the gels dissolved in water but could be returned to
their original gel state upon cooling.
[0040] The amygdalin amphiphiles derivatives 1-3 showed
unprecedented gelation abilities in a broad range of solvents at
extremely low concentrations [0.05-0.2 wt % (MGC)], while
displaying excellent thermal and temporal stabilities.
Scanning Electron Microscopic Studies
[0041] Molecular self-aggregation features can be observed on an
electron microscope, since the initial stage of physical gelation
is the self-assembly of gelator monomers. FIG. 4, which are SEM
micrographs, wherein scale bar is equivalent to 1 .mu.M, of: (a)
the organogel of derivative 1 prepared from acetonitrile; (b) the
aqueous gel from derivative 2; (c) the aqueous gel from derivative
3; and, (d) a higher magnification of hydrogel derivative
2--presents the scanning electron microscope (SEM) images of the
organogels formed by derivative 1 shown in FIG. 4(a) and the
aqueous gels formed by derivatives 2 and 3 shown in FIGS. 4(b) and
(c), respectively.
[0042] The images of their xerogels reveal two different types of
morphologies. The organogel formed in acetonitrile by derivative 1
showed `grass` like morphology. Hydrogels of derivatives 2 and 3
showed helical ribbon morphology at microscopic level. Analysis of
these aggregates clearly showed that the individual fibers are
approximately 50 nm in width, about 100-125 nm in pitch, and up to
several micrometers in length. These helical nanofibers are
entangled and formed a dense fibrous network resulting in
immobilization of the solvent. Gels were also made in the presence
of the drug curcumin, and SEM images suggest that inclusion of
curcumin does not change the basic twisted fibrous morphology of
the hydrogel. See FIG. 5, which are scanning electron micrographs
of curcumin embedded hydrogels of derivative 3.
XRD Measurements and Crystal Structure Analysis
[0043] From X-ray diffraction patterns of the xerogels of
derivatives 1-3 prepared from xylene and water, the long spacings
(d) were calculated and discussed to postulate the possible mode of
aggregation in the gel state. Possibly lamellar structures were
formed by these amphiphiles in gels. The xerogels of derivatives
1-3 were prepared by a freezing-and-pumping method and were
sponge-like materials. Amygdalin butyrate (derivative 1) gave a
single crystal in water that was successfully analyzed by X-ray
crystallography. The crystal structure of derivative 1 is shown in
FIG. 6, which are: (a) the crystal structure analysis of derivative
1 in water; and, (b) a top view showing the .pi.-.pi. stacking of
phenyl rings and hydrogen boding between two amygdalin molecules,
wherein hydrogen bonding acts as a bridge between the stacked
amygdalin molecules along the b-axis shown as blue arrows and
between these two stacks along the a-axis shown as black arrows,
and wherein: oxygen is shown as red; nitrogen is shown as blue;
carbon is shown as white circles; and, hydrogen-bonding is shown as
black dashed lines. The information obtained from the single
crystal analysis was combined with the XRD data to postulate
possible molecular packing of amygdalin amphiphiles within the
hydro- and organogels.
Drug Encapsulation and Enzyme Triggered Controlled Release
[0044] Solubilization of hydrophobic drugs and developing suitable
drug delivery systems is a challenging task in drug discovery
research..sup.18 A conceptual approach of single-step
enzyme-triggered drug delivery at physiological conditions was
performed where a hydrophobic drug molecule was
encapsulated--solubilized without chemical modification--in a
hydrogel and subsequent release of the drug by breaking the gel by
using hydrolase enzyme (Lipolase 100L, Type EX). The preformed
hydrogel was degraded completely by the lipolase while the
encapsulated chemopreventive hydrophobic drug curcumin was
released. See FIGS. 1(b) and (c) for images of curcumin. Drug
release was monitored by absorbance spectra of the drug. Control of
the drug release rate was achieved by manipulating the enzyme
concentration and/or temperature. The by-products formed after the
gel degradation were characterized and the cleavage site of the
gelator by enzyme was determined. Gel degradation occurred due to
the cleavage of the ester bond in the gelator by the hydrolase
enzyme. .sup.18Miyata, T., Uragami, T. & Nakamae, K.
Biomolecule-sensitive hydrogel. Adv. Drug Delivery Rev. 54, 79-98
(2002).
Discussion
[0045] Gelation is the delicate balance between solubility and
precipitation. To obtain this, the structural features in the
gelator molecules need to be fined tuned.
[0046] The amphiphiles of derivatives 1-3 were generated by
attaching a fatty acid chain to amygdalin via regiospecific
transesterification reaction on a primary sugar hydroxyl.
Inspection of FIG. 3 reveals that the amygdalin derivatives are
versatile gelators for water and polar/nonpolar organic solvents,
derivative 1 formed gels in two solvents out of ten tested, whereas
derivative 3 gelled in all ten solvents. This explains the
importance of chain length on gelation ability. Noteworthy to
mention is that derivatives 2 and 3 did not require any co-solvent
to form the hydrogel despite their gelation ability in less polar
solvents like benzene, toluene, and xylene. These gelators showed
excellent gelation in a broad range of solvents.
[0047] Robustness of a gelator can be determined by considering
three parameters: i) gelation ability in a broad range of solvents;
ii) low minimum gelation concentration (MGC); and, iii) thermal
stability of the gels.
[0048] For example, these gelators--derivatives 2 and 3--formed
gels in highly polar solvents like water, methanol, and non-polar
solvents like nonane, benzene, and toluene. Minimum gelation
concentration (MGC) of these gels are very low, typically 0.05 and
0.2 wt % for derivative 3 in water and benzene, respectively. This
is one of the lowest gelation concentrations reported in the
literature for any class of gelators..sup.19 Similarly, the other
derivatives also exhibit lower MGC values for various solvents
typically between 0.07 to 0.5 wt %. In addition, they show good
thermal and temporal stabilities. .sup.19van Bommel, K. J. C.,
Stuart, M. C. A., Feringa, B. L. & van Esch, J. Two-stage
enzyme mediated drug release from LMWG hydrogels. Org. Biomol.
Chem. 3, 2917-2920 (2005); Kobayashi, H. et al. Molecular design of
"super" hydrogelators: understanding the gelation process of
azobenzene-based sugar derivatives in water. Org. Lett. 4,
1423-1426 (2002).
[0049] Gel to solution transition temperature (T.sub.gel) was
determined by typical `inversion tube method`.sup.20 and from
differential scanning calorimeter (DSC). The T.sub.gel values of
these gels were in the range of 40 to 85.degree. C. for 0.5 wt %
gels depending on the solvent used. All gels were stable for
months. Hence, together satisfying all three parameters, the
reported amygdalin based gelators could be considered as excellent
gelators. .sup.20Menger, F. M. & Caran, K. L. Anatomy of a gel.
Amino acid derivatives that rigidify water at submillimolar
concentrations. J. Am. Chem. Soc. 122, 11679-11691 (2000).
[0050] In X-ray diffraction experiments, p-xylene gel of derivative
2 showed long distance spacing of 4.3 nm, which is higher than the
molecular length--2.8 nm from the optimized geometry
calculations--and much lower than double that of the extended
molecular length of derivative 2. Thus, there could be two possible
ways to explain how these molecules could form self-assembly, which
is shown in FIGS. 7(a) and (b), which are schematic representations
of possible molecular packing models for the: (a) hydrogels; and,
(b) organogels of derivative 2. First, a highly interdigitated
bilayer structure with the alkyl chain tilting with respect to the
normal to the layer plane shown in FIG. 7(a), and second, the
hydrophilic parts face inside the assembly and hydrophobic chains
are exposed to the outer side of the assembly shown in FIG. 7(b).
On the other hand, the long distance spacing for the hydrogel of
derivative 2 is 4.0 nm strongly supports that interdigitated
molecular packing would be possible at the nanoscopic level. It is
unlikely that hydroxyls containing sugar headgroup will face inside
and lipophilic hydrocarbon chains face bull polar solvent, hence
model shown in FIG. 7(b) would be ruled out. Thus, molecular
packing in the hydrogels of derivative 2 would be similar to that
shown in FIG. 7(a). In this model, hydrophilic groups are exposed
to the outer solvent while hydrophobic chains are highly
interdigitated, which is consistent with previous reports..sup.21
On the basis of long distance spacing of the hydro-and organogels
of derivative 3, it is proposed that most likely the molecular
packing of the amphiphiles are similar to the gels of 2 and it
might be possible that in the case of the gels of derivative 3, the
allyl chain tilt would be more than that of derivative 2.
Therefore, layered structures for self-assembly of these gels are
also supported by their solid-state crystal structure. .sup.21John,
G., Masuda, M. & Shimizu, T. Nanotube formation from renewable
resources via coiled nanofibers. Adv. Mater. 13, 715-718 (2001);
Gronwald, 0. & Shinkai, S. Sugar-integrated gelators of organic
solvents. Chem. Eur. J. 7, 4328-4334 (2001).
[0051] Amygdalin butyrate--derivative 1--gives single crystals in
water. The isolated single crystal was successfully analyzed by
X-ray crystallography. Interestingly, these molecules were well
packed in the crystal lattice due to the extensive hydrogen
bonding. Strong well-arranged intra- and inter-molecular hydrogen
bonding was observed. Intramolecular hydrogen bonding between N
(nitrogen) of the nitrile group and of sugar hydroxyl (O--H)
hydrogen helps to form a locked conformation that apparently
participated in forming the stacked structures.
[0052] Stacked layered structure was stabilized by .pi.-.pi.
stacking and van der Waals interactions between the alkyl chains.
These two stacks were arranged in `head-to-tail` fashion to give
the extended porous structure shown in FIG. 6(b).
[0053] Water molecules were involved in two types of hydrogen
bonding. In one type, water molecules formed hydrogen bonding with
sugar hydroxyls while acting as bridged molecules between stacked
amygdalin amphiphiles and stabilized the stacked layers as shown in
open arrows in FIG. 6(b). In the second mode, water molecules were
involved in hydrogen bonding with sugar hydroxyls while acting as
bridged molecules between two different stacks of amygdalin
amphiphiles to stabilize the two adjacent layers as shown in filled
arrow in FIG. 6(b). In addition to that, the intermolecular
hydrogen bonding between sugar hydroxyls of two amygdalin molecules
from opposite stacks, which also indicates the greater ability to
form self-assembled structures by amygdalin derivatives, was
observed.
[0054] By collecting the information from the crystal structure of
derivative 1, most likely in the gel state similar self-assembly
would be possible. Previously in literature two reports explained
the aggregation modes of the gelators based on single crystal
analysis..sup.22 As evidenced in the crystal structure, there are
several interactions, such as extensive hydrogen bonding, .pi.-.pi.
stacking, and van der Waals interactions existing. Such cooperative
interactions play an important role in stabilizing the fiber
structures in the gel state. .sup.22Kiyonaka, S. et al. Semi-wet
peptide protein array using supramolecular hydrogel. Nat. Mater. 3,
58-64 (2004); Kumar, D. K., Jose, D. A., Das, A. & Dastidar, P.
First snapshot of a nonpolymeric hydrogelator interacting with its
gelling solvents. Chem. Commun. 32, 4059-4062 (2005).
[0055] The possible applications of these robust gels to utilize
the hydrophobic pockets within the gel to encapsulate hydrophobic
drugs were investigated. Hence, these hydrogels were tested as a
drug delivery vehicle model. In the process of developing drug
delivery systems, chemical modification of the drug and cleavage
induced by external stimuli, such as increasing temperature
followed by enzyme-mediated cleavage, has been shown
recently..sup.23 Such an approach has limitations when applying to
different drugs. Covalently connecting the drugs to the
hydrogelators may not be achievable trivially in all types of
drugs. In the process of chemical modification, there is a
potential chance of loosing its original drug activity. It would be
an ideal system to have encapsulated drug models, where drug
release can be triggered by enzymes without the need of altering pH
or temperature. An enzyme triggered drug delivery at physiological
condition was demonstrated where a hydrophobic drug molecule was
encapsulated--solubilized without chemical modification--in an
hydrogel. Subsequent release of the drug was by breaking the gel
with an hydrolase enzyme (Lipolase 100L, Type EX). .sup.23van
Bommel, K. J. C., Stuart, M. C. A., Feringa, B. L. & van Esch,
J. Two-stage enzyme mediated drug release from LMWG hydrogels. Org.
Biomol. Chem. 3, 2917-2920 (2005).
[0056] The success of this approach in drug delivery model systems
for possible in vivo applications rely on a few factors, such as:
(a) selected hydrogels should be able to provide the hydrophobic
pockets to solubilize the hydrophobic drugs; (b) gel
degradation--to release the drug--should take place at mild
conditions like physiological pH and temperature; and, (c) the
products formed after degradation should be biocompatible.
[0057] Selected as a model drug was one of the best-characterized
chemopreventive agents, curcumin--or
diferuloylmethane.sup.24--extracted from the root of Curcuma longa,
which presents strong anti-oxidative, anti-inflammatory, and
antiseptic properties..sup.25 In addition, curcumin also inhibits
purified human immunodeficiency virus type 1 (HIV-1)
integrace,.sup.26 HIV-1 and HIV-2 proteases,.sup.27 and HIV-1 long
terminal repeat-directed gene expression of acutely or chronically
infected HIV-1 cells..sup.28 Despite such astounding drug activity,
unfortunately curcumin has an extremely low aqueous solubility and
poor bioavailability limiting its pharmaceutical use..sup.29 One
possible way to increase its aqueous solubility is to form
inclusion complexes, i.e. to encapsulate curcumin as a guest within
the internal cavities of a water-soluble host or encapsulate within
the nanoaggregates--formed by self-assembly--that have hydrophobic
pockets within. .sup.24Duvoix, A. et al. Chemopreventive and
therapeutic effects of curcumin. Cancer Lett. 223, 181-190 (2005).
L6 .sup.25Hergenhahn, M. et al. The chemopreventive compound
curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1
transcription in Raji DR-LUC cells. Mol. Carcinog. 33, 137-145
(2002)..sup.26Hergenhahn, M. et al. The chemopreventive compound
curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1
transcription in Raji DR-LUC cells. Mol. Carcinog. 33, 137-145
(2002); Mazumder, A., Raghavan, K., Weinstein, J., Kohn, K. W.
& Pommier, Y. Inhibition of human immunodeficiency virus type-1
integrase by curcumin. Biochem. Pharm. 49, 1165-1170
(1995)..sup.27Burke, T. R. Jr. et al. Hydroxylated aromatic
inhibitor of HIV-1 integrase. J. Med. Chem. 38, 4171-4178
(1995)..sup.28Sui, Z., Salto, R., Li. J., Craik, C. & Ortiz de
Montellano, P. R. Inhibition of the HIV-1 and HIV-2 proteases by
curcumin and curcumin boron complexes. Bioorg. Med. Chem. 1,
415-422 (1993)..sup.29Khodpe, S. M., Priyadarsini, K. I., Palit, D.
K. & Mukherjee, T. Effect of solvent on the excited-state
photophysical properties of curcumin. Photochem. Photohiol. 72,
625-631 (2000).
[0058] Schematic representation of curcumin encapsulation and
enzyme-mediated release is depicted in FIG. 8(a) wherein FIG. 8 are
(a) a schematic representation of drug encapsulation in a
supramolecular hydrogel and subsequent release of the drug by
enzyme-mediated degradation of hydrogel at physiological
temperature; and, (b) real images of the hydrogels of derivative 3
with (I-iv) and without (v-vi) curcumin, wherein after complete gel
degradation, the remained white fluffy powder that settled at the
bottom was characterized as a water insoluble fatty acid that
formed after gel degradation by the enzyme. The release of curcumin
into the solution in the presence of enzyme was monitored by
measuring the curcumin UV-absorption spectrum. The absorption
spectrum recorded in aqueous gel solution was compared with the
curcumin spectra recorded in methanol. The effect of solvent
polarity on the absorbance spectrum of curcumin has previously been
reported as minimal..sup.30 High concentration of curcumin
(1.times.10.sup.-3 M) was solubilized in 0.5 wt % hydrogel of
derivative 3--reported.sup.31 solubility of curcumin in water is
3.times.10.sup.-8 M, i.e., .about.33,000 times more than
solubilized in the hydrogel. The resulted gel was yellow in color
as shown in FIG. 8(b), and due to the hydrophobic nature, curcumin
might be located at hydrophobic pockets of the gel. To test this
hypothesis, water was added to the preformed gel and left for 12
hrs. The UV-absorption of the supernatant was recorded. Absence of
any absorbance peak concluded the unavailability of curcumin on the
gel surface by adsorption. .sup.30Id..sup.31Tonnesen, H. H.,
Masson, M. & Loftsson, T. Studies of curcumin and curcuminoids.
XXVII. Cyclodextrin complexation: solubility, chemical and
photochemical stability. Int. J. Pharm. 244, 127-135 (2002).
[0059] First, 0.5 mL of lipase--Lipolase 100L, Type EX, lipase
units 100 KLU/g--was added to the preformed gel and kept at
37.degree. C.--far lower than gel melting temperature. Initially
the added solution was colorless as shown in FIG. 8(b)(iii). After
12 hrs visual changes occurred as shown in FIG. 8(b)(iv), i.e.,
100% of the gel has been degraded and the top solution has became
yellow in color, which indicates that upon enzyme mediated gel
degradation, encapsulated curcumin has been released into the
solution. This was confirmed by spectroscopic experiments. Aliquots
were collected after addition of an enzyme to the hydrogel after 10
min and 12 hrs and absorbance spectrum were recorded.
Interestingly, initial aliquots after 10 min did not show any
absorbance peak, but aliquots collected after 12 hrs showed
absorption maxima at 425 nm, which corresponds to the absorption
peak of curcumin. See FIG. 9, which are UV absorption spectra of
curcumin in various types of solution mixtures, including: (a) an
curcumin-entrapped hydrogel in the presence of an enzyme; (b) an
enzyme added to the hydrogel that does not contain curcumin; (c) an
curcumin-entrapped hydrogel in the absence of an enzyme; and, (d) a
methanolic solution of curcumin.
[0060] To find out the role of the enzyme on hydrogel degradation,
similar experiments were carried out by adding only water without
an enzyme. As expected, the curcumin-encapsulated gel was still
intact after incubating for a few days at 37.degree. C. There was
no visual change in the gel volume and in the added solution. See
FIG. 8(b). And, the absorbance peak corresponding to the curcumin
was not shown. See FIG. 9(a). In addition, control experiments with
the same hydrogel of derivative 3 without curcumin, which is opaque
and white in color as shown in FIG. 8(b), were performed. To this,
0.5 mL of lipolase was added and incubated at 37.degree. C. After
12 hrs, the gel degraded completely. The absorption spectrum of the
solution shown in FIG. 9(b) was then recorded. Absence of the
absorption peak at 425 nm suggested that the previously observed
peak, FIG. 9(c), corresponded to the curcumin released into the
solution.
[0061] To obtain control on the rate of release, the role of enzyme
concentration and temperature on gel degradation or controlled drug
release was investigated. In a first set of experiments, the
temperature was changed while keeping enzyme concentration
constant. After addition of the enzyme to the preformed gel, the
vial was kept at room temperature for two days, and as explained
previously, curcumin release was monitored by absorption spectra.
Interestingly, even after two days at room temperature in the
presence of the enzyme, there was no gel degradation observed, and
thus, there was no release of encapsulated curcumin. When the vial
was placed at 37.degree. C. in an incubator, after 120 min slow
release of curcumin was observed, and within 720 min encapsulated
curcumin was released completely. Similarly when incubated at
45.degree. C., release was initiated within 30 min and complete
release was observed in 270 min.
[0062] In a second set of experiments, the enzyme concentration was
changed while keeping the temperature constant. 10 times lower
concentrated lipolase--units 10 KLU/g--added to the preformed
curcumin encapsulated gel. At room temperature, even after several
days, there was no release. Then the vial was placed at 37.degree.
C. in incubation and took 300 min to start the drug release, which
eventually took 4,320 min to release completely. In addition to
this, a similar low enzyme concentrated vial was directly incubated
at 45.degree. C. In this case, drug release was started after 180
min and in 2,880 min complete release was observed. Hence at
constant temperature, drug release can be controlled by lowering
the enzyme concentration. These results are summarized in FIG. 10,
which is a table of the effect of enzyme concentration and
temperature on drug release time, and FIG. 11, which are a
comparison of curcumin-drug-release time at different
concentrations and different temperatures from hydrogels of
amygdalin derivatives by enzyme degradation, wherein: (a) is the
time required for 5% release; and, (b) is the time required for
100%--which clearly demonstrate the achieved control over the
release of an encapsulated drug from a hydrogel.
[0063] It is important to characterize the products/compounds
formed after gel degradation. To find out the other components
formed after gel degradation, thin layer chromatography (TLC) of
the solution was performed and it was found that this solution
contains amygdalin, curcumin, and enzyme--confirmed by comparing
R.sub.f values. This indicates that the enzyme is degrading the gel
by cleaving the ester bond of derivative 3. Upon gel degradation, a
white fluffy solid was produced, which is not soluble in water, and
thus settled down in the vial. See FIG. 8(b)(iv). The solid was
isolated and characterized by .sup.1H-NMR, and it matched with the
NMR of pure stearic acid. See FIG. 12, which is a comparison of the
.sup.1H-NMR spectra of commercially available stearic acid and the
white fluffy solid obtained after gel degradation, which was
filtered, freeze-dried, and NMR recorded in Chloroform. Hence, it
is undoubtedly suggesting that gel degradation is occurring through
the cleavage of the ester bond of amygdalin derivatives by lipolase
enzyme.
[0064] These results unambiguously explain the drug encapsulation
abilities of hydrogels formed by amygdalin derivatives and
enzyme-triggered drug release. Noteworthy, these gelators were
generated via enzyme catalysis, and gels were degraded--converting
from gelators to starting materials--by yet again using enzyme
catalysis in environmentally benign conditions.
Methods
General Information
[0065] Amygdalin and curcumin was purchased from Acros Chemicals
(Fisher Scientific Company, Suwane, Ga.). The Novozyme 435 [lipase
B from Candida Antarctica, (CALB)] and Lipolase 100L were obtained
from Novozymes through Brenntag North America. Other reagents were
obtained from TCI America (Portland, Oreg.).
General Synthesis Procedure of Amygdalin Esters by Enzyme
Catalysis
[0066] Typically, 40 ml of acetone containing 0.1 mol/L amygdalin
and 0.3 mol/L vinyl esters--vinyl butyrate, vinyl myristate, and
vinyl stearate--was added to 1 g of Novozyme 435. The reaction
mixtures were placed in an incubator and shook at 200 rpm at
45.degree. C. for 48 hr. The reactions were terminated by the
filtration of reaction mixtures. After evaporating the solvent, the
obtained crude products were purified by silica gel flash
chromatography using ethyl acetate--methanol (4:1) as eluent,
afforded pure products as white solids. The yields were above 90%
for all reactions.
Amygdalin Butyrate--Derivative 1
[0067] .sup.1H-NMR, (Acetone-d.sub.6, 300 MHz) .delta. 7.58 (m,
2H), 7.48 (m, 2H), 7.46 (m, 1H), 5.98 (s, 1H), 5.1-5.33 (br m, 6H),
4.45 (d, 1H), 4.29 (d, 1H), 4.22 (dd, 1H), 4.04 (dd, 1H), 3.99 (dd,
1H), 3.59 (dd, 1H), 3.38 (m, 1H), 3.35 (m, 1H), 3.25 (m, 2H), 3.18
(m, 2H), 3.13 (m, 1H), 3.1 (m, 1H), 2.2 (t, 2H), 1.24 (m, 2H), 0.83
(t, 3H). Anal. Calcd. for C.sub.24H.sub.33NO.sub.12: C, 54.64; H,
6.31; N, 2.66. Found: C, 54.68; H, 6.30; N. 2.70.
Amygdalin Tetradecanoate--Derivative 2
[0068] .sup.1H-NMR, (CDCl.sub.3, 300 MHz) .delta. 7.58 (m, 2H),
7.48 (m, 2H), 7.46 (m, 1H), 5.98 (s, 1H), 5.1-5.33 (br m, 6H), 4.45
(d, 1H), 4.29 (d, 1H), 4.22 (dd, 1H), 4.04 (dd, 1H), 3.99 (dd, 1H),
3.59 (dd, 1H), 3.38 (m, 1H), 3.35 (m, 1H), 3.25 (m, 2H), 3.18 (m,
2H), 3.13 (m, 1H), 3.1 (m, 1H), 2.2 (t, 2H), 1.24 (m, 22H), 0.83
(t, 3H). Anal. Calcd. for C.sub.34H.sub.53NO.sub.12: C, 61.15; H,
8.00; N, 2.10. Found: C, 61.20; H, 8.02; N. 2.14.
Amygdalin Octadecanoate--Derivative 3
[0069] .sup.1H-NMR, (CDCl.sub.3, 300 MHz) .delta. 7.58 (m, 2H),
7.48 (m, 2H), 7.46 (m, 1H), 5.98 (s, 1H), 5.1-5.33 (br m, 6H), 4.45
(d, 1H), 4.29 (d, 1H), 4.22 (dd, 1H), 4.04 (dd, 1H), 3.99 (dd, 1H),
3.59 (dd, 1H), 3.38 (m, 1H), 3.35 (m, 1H), 3.25 (m, 2H), 3.18 (m,
2H), 3.13 (m, 1H), 3.1 (m, 1H), 2.2 (t, 2H), 1.24 (m, 30H), 0.83
(t, 3H). Anal. Calcd. for C.sub.38H.sub.61NO.sub.12: C, 63.05; H,
8.49; N, 1.93. Found: C, 63.11; H, 8.51; N. 1.95.
Preparation of Supramolecular Gels: Self-Assembly
[0070] Typically, the gelator (0.01-2 mg) and required solvent
(0.1-1 mL) were placed into a 2 mL scintillation vial, which was
then sealed with a screw cap. The vial was heated and shook until
the solid was completely dissolved. The solution was set aside and
allowed to cool to room temperature. Gelation was considered to
have occurred when no gravitational flow in the inverted tube was
observed.
Gel Melting Temperatures
[0071] The Gel to Sol transition temperature (T.sub.gel) was
determined by two methods. One was the typical `inversion tube
method`,.sup.32 where the gel was prepared in a 2 mL glass vial by
dissolving a 0.5 wt % gelator in a required amount of solvent and
closed with a tight screw cap. The vial was immersed in water `up
side down` and slowly heated. The temperature where the viscous gel
melted and dropped down was considered as the T.sub.gel. The second
method, T.sub.gel was determined by using a Mettler DSC-822
Differential Scanning Calorimeter equipped with a nitrogen-gas
intra cooling system. The gel was hermitically sealed in a silver
pan and measured against a pan containing alumina as reference.
Thermograms were recorded at a heating rate of 5.degree. C./min.
The T.sub.gel values determined by these two methods were
identical. .sup.32Menger, F. M. & Caran, K. L. Anatomy of a
gel. Amino acid derivatives that rigidify water at submillimolar
concentrations. J. Am. Chem. Soc. 122, 11679-11691 (2000).
Observation of Gel Structure by Microscopy
[0072] The xerogel samples were prepared by the
freezing-and-pumping method from their gel phases below the sol-gel
transition temperature. It is important to note that the SEM images
of xerogels and the following drying under ambient condition show
similar morphologies. Therefore, morphology with the gels dried
under ambient conditions, which was called xerogels, was
studied.
X-Ray Powder Diffraction (XRD)
[0073] XRD measurements were conducted using a Bruker AXS D-8
Discover with GADDS diffractometer using graded d-space elliptical
side-by-side multilayer optics, monochromated Cu--K.alpha.
radiation (40 kV, 40 mA), and an imaging plate. The gels were used
as prepared in the wet condition for the analysis. A small portion
of the gel sample was transferred to the sample holder and sealed
off immediately using capstone tape to avoid any drying off of the
solvent. The typical exposure time was 1 min for self-assembled
structures with a 100 mm camera length.
X-Ray Single Crystal Analysis
[0074] X-ray quality colorless single crystals of derivative 1 were
obtained from water in a flat rod shape. X-ray diffraction data
were collected using Mo--K.alpha. (.lamda.=0.7 107 .ANG.) radiation
on a graphite monochromatized Bruker X8 APEX II diffractometer at
173(2) K. Data collection, data reduction, and structure solution
refinement were carried out using the software package of
SHELX97..sup.33 All structures were solved by direct methods
(S1R92) and refined in a routine manner. Non-hydrogen atoms were
treated anisotropically. Whenever possible, the hydrogen atoms were
located on a different Fourier map and refined. The
crystallographic parameters are listed in FIG. 13, which is a table
of the crystallographic parameters of derivative 1.
.sup.33Sheldrick, G. M. SHELEXL-97, A program for crystal structure
solution and refinement; University of Gottingen: Gottingen,
Germany, 1993.
Conclusions
[0075] In conclusion, hydro/organogelators from renewable resources
were successfully developed. Low-molecular-weight hydrogelators
were synthesized by regioselective enzyme catalysis for the first
time. Yields were quantitative and crude reaction mixtures
exhibited equally unprecedented gelation properties like their
counter pure products. This capability may allow development of
hydrogelators in industrial scale for future applications..sup.34
The hierarchical structural characteristic of supramolecular gels
were clearly demonstrated and the self-assembly based on XRD and
single crystal analysis were explained. .sup.34Zhang, S. Hydrogels:
Wet or let die. Nat. Mater. 3, 7-8 (2004).
[0076] The gel fibers were self-assembled and stabilized by various
interactions, such as intra- and inter-molecular hydrogen bonding,
.pi.-.pi. stacking, and van der Waals interactions. The
encapsulation of chemopreventive curcumin in the hydrogel was
shown, and enzyme-triggered gel degradation was performed to
release the encapsulated drug into the solution at physiological
temperature. Controlled drug release rate was achieved by
manipulating the concentration of enzyme or temperature.
[0077] The by-products formed after gel degradation were
characterized and clearly demonstrated the site specificity of
degradation of the gelator by enzyme catalysis. Supramolecular
chemistry is now a powerful strategy for developing new molecularly
defined materials in material and medicinal science. This would be
a possible model system for drug encapsulation and enzyme mediated
delivery for in vivo formulations and may have potential
applications in pharmaceutical research and molecular design of
value added products from biobased materials, otherwise under
utilized.
[0078] It will be understood that each of the elements described
above or two or more together may also find a useful application in
other types of constructions differing from the types described
above.
[0079] While the invention has been illustrated and described as
embodied in a method for preparing hydro/organo gelators from
disaccharide sugars by biocatalysis and their use in
enzyme-triggered drug delivery, however, it is not limited to the
details shown, since it will be understood that various omissions,
modifications, substitutions, and changes in the forms and details
of the device illustrated and its operation can be made by those
skilled in the art without departing in any way from the spirit of
the present invention.
[0080] Without further analysis, the foregoing will so fully reveal
the gist of the present invention that others can by applying
current knowledge readily adapt it for various applications without
omitting features that from the standpoint of prior art fairly
constitute characteristics of the generic or specific aspects of
the invention.
* * * * *