A facile one-pot sonochemical synthesis of surface-coated mannosyl protein microspheres for detection and killing of bacteria

Article abstract of DOI:10.1039/c1cc13518j

We report a remarkably facile one-pot sonochemical approach to prepare protein microspheres whose shells are covalently decorated with a mannosyl derivative to target Escherichia coli (E. coli), while their cores are encapsulated with tetracycline. Conjugated microspheres induced the agglutination of E. coli and increased the anti-bacterial activity of the encapsulated tetracycline by five fold.

Full text of DOI:10.1039/c1cc13518j

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Chemical Communications
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Volume 47 | Number 45 | 7 December 2011 | Pages 12241–12404
ISSN 1359-7345
COMMUNICATION
Rahimipour et al.
A facile one-pot sonochemical
synthesis of surface-coated mannosyl
protein microspheres for detection
and killing of bacteria
1359-7345(2011)47:45;1-7

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Cite this: Chem. Commun., 2011, 47, 12277–12279
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A facile one-pot sonochemical synthesis of surface-coated mannosyl
protein microspheres for detection and killing of bacteriaw
Natalia Skirtenko,ab Michal Richman,a Yeshayahu Nitzan,c Aharon Gedankenab and
Shai Rahimipour*a
Received 14th June 2011, Accepted 2nd August 2011
DOI: 10.1039/c1cc13518j
We report a remarkably facile one-pot sonochemical approach
to prepare protein microspheres whose shells are covalently
decorated with a mannosyl derivative to target Escherichia coli
(E. coli), while their cores are encapsulated with tetracycline.
Conjugated microspheres induced the agglutination of E. coli
and increased the anti-bacterial activity of the encapsulated
tetracycline by five fold.
to accumulate.9 However, in order to be effective and selective as
potential drug delivery carriers, the surface of the microspheres
should be modified with vectors to target a specific cell type or a
cell component. Surface-modified BSA microspheres have been
previously prepared by a non-covalent electrostatic layer-by-
layer (LBL) method.10 The overall negative charge of the BSA
surface, at physiological pH, was used as a template for electro-
static deposition of the positively charged polyelectrolyte,
RGDKKKKKK (R = Arg, G = Gly, D = Asp, K = Lys),
to target colon tumors over-expressing integrin receptors.10
Protein–carbohydrate interactions regulate many vital biological
processes.11 These interactions are however characterized by
intrinsically low binding affinity that hampers the development
of new therapeutics. To compensate for the low binding affinity of
these interactions, nature usually uses a polyvalent presentation
of the ligands and receptors on the cell surface.12 Therefore, there
is a growing interest in neoglycoconjugates as multivalent
ligands for diverse biological applications.13 Multivalent carbo-
hydrate systems including polymers, dendrimers, nano-
particles and self-assembled supermolecular polymers have been
demonstrated to bind and detect pathogens such as Escher-
ichia coli (E. coli) that express the corresponding receptors.14
Targeting and detection of bacteria by surface-modified
protein microspheres has not been reported yet. Here, we report
on a versatile one-pot sonochemical method to covalently modify
the surface of protein microspheres with a thiol-containing
mannosyl derivative 3 via thiol/disulfide exchange reaction. The
conjugated microspheres (CM) are formed through intermolecular
disulfide exchange reaction between the mannosyl derivative 3
containing a thiol group and the BSA shell. These micro-
spheres have a shell composed of mannosyl conjugated BSA
that can bind and detect E. coli expressing mannose-binding
lectin, and a core that can encapsulate different compounds—
including tetracycline as a broad-spectrum antibiotic. The main
advantages of this method over the LBL method are that it does
not require the preparation of a charged targeting vector that may
exhibit reduced biological activity. This is especially true in the
case of a small molecule, such as mannose. Moreover, surface
modification in this method is unrelated to the net charge of
the protein and can be applied to any protein expressing either
Cys or disulfide bonds. Most importantly, in contrast to the
LBL method, the modification of the protein by the sonochemical
approach is carried out under ‘‘one-pot’’ conditions.
Protein microspheres have attracted increasing interest in recent
years due to their potential applications in different bio-medical
imaging modalities and drug delivery systems. Methods
commonly used to prepare protein microspheres include spray-
and freeze-drying, crystallization, calcium carbonate templating
and emulsion polymerization.1 Suslick and Grinstaff have devel-
oped a remarkably facile sonochemical technique to prepare gas-
or liquid-filled protein microspheres from Cys-containing pro-
teins, including bovine serum albumin (BSA), human serum
albumin and hemoglobin.2 Oxidation of the intermolecular
ꢀ
protein Cys residues to disulfide bonds by the HO2 generated
during sonochemical irradiation3 was suggested to be responsible
for holding the microsphere shells together.4 Similar procedures
have been used to prepare microspheres from Cys-less proteins5
or even from the polysaccharide, chitosan.6 Intermolecular inter-
actions between the protein molecules and intermolecular imine
formation between the b-D-glucosamine subunits in chitosan
were suggested to assist the microsphere formation.6
Protein microspheres are highly biocompatible and non-
immunogenic.7 Thus, liquid-filled serum albumin microspheres
have been used for various in vivo imaging applications8 whereas
air-filled microspheres have been approved for clinical use as
contrasting agents (e.g., Albunex) in echocardiography by FDA.
In addition, the capability of these microspheres to encapsulate
different therapeutics has been utilized for drug delivery to
organs, such as lung and liver, where the microspheres tend
a Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900,
Israel. E-mail: rahimis@biu.ac.il; Fax: +972 3 7384053
b Institute of Nanotechnology and Advanced Materials, Bar-Ilan
University, Ramat-Gan 52900, Israel
c Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900,
Israel
w Electronic supplementary information (ESI) available: Detailed
experimental details, Table S1 and Fig. S1. See DOI: 10.1039/
c1cc13518j
c
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Chem. Commun., 2011, 47, 12277–12279 12277

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spheres detected in Fig. 2b. The diameter size and spherical shape
of these microspheres may be advantageous over the linear or
nano-sized structures as they mimic more closely the surface of
the cells with which the bacteria interact.16 Moreover, the micro-
spheres exhibited extended stability in aqueous solutions over
several months at 4 1C as their size and content did not change
over the time.
Support for the core–shell structure of the microspheres and
the presence of the bioactive mannosyl ligands on their surface
was provided by fluorescent-microscopy studies. Conjugated
microspheres were incubated with fluorescein labeled mannose-
binding protein-concanavalin A (FITC-Con A) and visualized by
confocal microscopy. The data demonstrated that FITC-Con A
is evenly associated on the surface of the microspheres (Fig. 2d).
This suggests that the mannosyl moieties present on the surface
of the BSA microspheres retained their ability to interact with the
carbohydrate-binding protein, Con A. Moreover, the binding of
the FITC-Con A to the microspheres was found to increase with
increasing amount of the mannosyl precursor 3 used for the
microspheres preparation (Fig. 2e). The presence of mannosyl
derivative on the microspheres surface and their bioactivity were
further confirmed by fluorescence-activated cell sorting (FACS).
Fig. 2f suggests that although FITC-Con A can bind non-
selectively to the surface of the BSA-microspheres (tinted gray),
it accumulates to an approximately 10 fold greater extent on the
surface of mannosyl-CM (black line).
Fig. 1 Synthesis of thiol-containing mannosyl derivative.
The requisite O-linked mannosyl mercaptoethanol 3 was easily
synthesized by glycosylation of 2-bromoethanol with peracylated
mannose, using BF3ÁOEt2, followed by reaction with thioacetic
acid. The acetyl protecting groups of 2 were then removed by
NaOMe in MeOH (Fig. 1).15
To generate the protein microspheres coated with the mannose
derivative, an aqueous solution of 3 was mixed with different
amounts of BSA (mole ratio BSA : 3 1 : 1 to 1 : 1000) and over-
layered by a vegetable oil or dodecane. The mixture was then
subjected to a sonochemical irradiation for three minutes while
cooling to produce microspheres filled with the organic
co-solvent. The HPLC analyses of the aqueous phase before
and after sonication suggest that more than 95% of 3 was
incorporated into the microspheres in each preparation.
Having established that the mannosyl moieties on the BSA
microspheres are capable of binding Con A, we have micro-
scopically examined whether they can bind to the E. coli bacteria
and cause their agglutination. Microspheres were incubated with
either an E. coli strain expressing FimH mannose-binding protein
(K-12) or with the E. coli isolate that is deficient of FimH (1313).
Incubation of the mannosyl-CM with K-12 resulted in the
formation of large bacterial clusters and their agglutination that
could be easily detected by optical microscopy (Fig. 3 and Fig. S1,
ESIw). In contrast, only negligible bacterial binding or
aggregation was induced when the E. coli strain (1313) was
incubated with the conjugated microspheres (Fig. 3b). This
suggested that the mannosyl moiety 3 and its corresponding
lectin are responsible for the specific binding of the bacteria to
the microspheres and the eventual agglutination.
The optical microscopy and SEM images show that the
microspheres are spherical with a smooth outer surface (Fig. 2a
and b). The average diameter size of the conjugated microspheres
measured by optical microscopy was 1.80 Æ 0.5 mm (Fig. 2c).
However, the SEM analysis of the microspheres (Fig. 2b) clearly
indicated the presence of smaller particles that could not be
observed by optical microscopy. The size of these microspheres is
therefore somewhat smaller than those reported for the naked
BSA microspheres and is expected to be suitable for in vivo
applications.2a,8 This is especially true for the small 100–200 nm
The selective binding of the mannosyl-CM with the bacteria
encouraged us to evaluate the capability of these microspheres
for targeted delivery of tetracycline (TTCL) as a broad-spectrum
antibiotic. Among the promising new therapeutic approaches
developed in recent years, targeted drug carriers have received
increasing interest.17 These carriers aim to release their payloads
at the affected site, protecting the active substance from fast
degradation and elimination, which leads to dose reduction and
avoids side effects. In order to encapsulate TTCL within the
mannosyl-CM, a solution of BSA and 3 in molar ratio of 1 : 1000
was over-layered with a solution of dodecane containing different
amounts of TTCL (10 and 60 mg), and sonochemically irradiated
in a one-pot reaction.9a Spectroscopic examination of the loaded
TTCL suggested that about 60% of TTCL was encapsulated
inside the mannosyl-CM—similar to that of BSA microspheres
(Table S1, ESIw).9a Higher amounts of TTCL may be encapsulated
within the BSA microspheres by dissolving increasing amounts
of TTCL in the organic phase.9a
Fig. 2 Physical properties of mannosyl-CM. (a) Optical micrograph
of the mannosyl-CM and (b) the corresponding SEM. (c) Size
distribution analysis of the mannosyl-CM as measured with an optical
microscope. (d) Fluorescence-microscopy images of the mannosyl-CM
incubated with FITC-Con A. (e) Dose-dependent binding of FITC-
Con A to mannosyl-CM generated from increasing amount of 3.
(f) The extent of binding of FITC-Con A to mannosyl-CM (black) and
naked BSA microspheres (tinted gray) as studied by flow cytometry.
c
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Moreover, the mannosyl-CM selectively bind and cluster E. coli
strain containing the corresponding lectin. We have also shown
that encapsulation of TTCL by the targeted microspheres
significantly increased the toxicity of the drug to bacteria that
express mannose-binding protein. This method could be easily
generalized for the preparation of other microspheres containing
different targetable ligands on their surface that can be applicable
for selective drug delivery and imaging purposes. However,
further experiments are needed to probe the toxicity, immuno-
genicity and pharmacokinetics of the conjugated microspheres in
related animal models.
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corresponding antibacterial activity of the TTCL encapsulated microspheres.
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