Biochemical and Biophysical Research Communications 378 (2009) 503–506
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Mannosylated self-assembled structures for molecular confinement
and gene delivery applications
Nidhi Gour a, Chandra Shekhar Purohit a, Sandeep Verma a,*, Rajat Puri b, Subramaniam Ganesh b
a
b
Department of Chemistry, Indian Institute of Technology-Kanpur, Kanpur 208016 (UP), India
Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, Kanpur 208016 (UP), India
a r t i c l e
i n f o
Article history:
Received 12 November 2008
Available online 24 November 2008
Keywords:
Self-assembly
Nanocontainers
Hollow spheres
Nanoreactor
Cell transfection
COS-7 cells
a b s t r a c t
This paper reports self-assembly of a lysine conjugated with a biantennary mannose to form spherical
structures. These supramolecular structures are found to be hollow in nature and they afford effective
encapsulation of alkaline phosphatase enzyme, plasmid DNA and a GFP reporter gene, which was transfected in COS-7 cells. Loaded hollow structures also get disrupted upon mild sonication, releasing encapsulated molecules thereby illustrating their potential for confinement and delivery applications.
Ó 2008 Elsevier Inc. All rights reserved.
Self-assembled hollow structures have attracted considerable
attention due to their potential applications as supramolecular
containers [1,2] and as delivery vehicles [3,4]. Such applications
are feasible as the empty core of hollow spheres can encapsulate
guest molecules and also act as reaction chambers by bringing
two or more reactants into close proximity [5,6]. In addition, several reports also describe covalent decoration of such structures
by multiple carbohydrate residues to elicit interactions with specific cell surface receptors [7–11].
We have been interested in studying peptide-based soft structures from the viewpoint of generating diverse morphologies and
to discover enabling mechanisms that can trigger disruption of
such soft structures. As a result of our investigations, we have discovered peptide-based supramolecular ensembles where some of
them respond to colchicine, physiologically relevant cations, and
covalently attached structure modifiers [12–15].
In this report, we demonstrate that bis-mannosylated lysine (3)
(Fig. 1A) self assembles in aqueous medium to reveal formation of
hollow spheres which can complex/or encapsulate guest molecules. These soft structures disrupt when ultrasonicated and release guest molecules. Remarkably, these structures complex
pEGFP-N1 expression construct, encoding the green florescent protein, and upon transfection with this complex, a number of mammalian cells exhibit nuclear fluorescence.
* Corresponding author.
E-mail address: [email protected] (S. Verma).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.11.070
Materials and methods
Bis-mannosylated lysine (3) was prepared by a slightly modified previously reported procedure [16,17] via Scheme 1 [18]. Sample Preparation for different microscopy analysis, i.e., AFM, TEM,
SEM, fluorescence microscopy, and optical microscopy, was done
by dissolving 1 mM of 3 in deionized water for 16 h at 37 °C. It
was necessary to incubate samples for 16 h because 3 did not show
instantaneous self assembly and required 16 h for formation of stable self assemblies. Enzyme assays were done by adding 5 U of
CIAP to 1 mL of 1 mM solution. AFM and gel electrophoresis was
done by adding 20 lL of DNA to 180 lL of 1 mM of 3 dissolved
in autoclaved water for 16 h at 37 °C. Compound 3 (1 mM) dissolved in autoclaved water was filter sterilized and then incubated
with DNA (1 lg) at 37 °C for 16 h for cell transfection experiments.
Details of all procedures are given in supporting information [18].
Results and discussion
Bis-mannosylated lysine (3) thus synthesized was fully characterized by various spectroscopic methods which showed it to be in
full agreement with molecular structure of 3. Several glycosylated
lysine and lysine containing peptides are reported in the literature
[19–21] and more specifically, the interaction of 3 and its galactose
analog with macrophages and HepG2 cells has been described
[22,23]. This background provided us an impetus to investigate
self-assembly properties of mannosylated 3.
Scanning electron microscopy (SEM) and atomic force microscopy (AFM) confirmed formation of spherical structures (Fig. 1B
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N. Gour et al. / Biochemical and Biophysical Research Communications 378 (2009) 503–506
Fig. 1. (A) Molecular structure of 1. (B) SEM of 1 (1 mM, aqueous solution) after 16 h incubation, (C) TEM image showing a contrast between periphery and core of spherical
structures (marked by two arrows), (D) AFM, (E) fluorescence micrograph, (F) DLS analysis.
and D). Further evidence was obtained from transmission electron
(TEM) (Fig. 1C) which interestingly revealed a contrast between
the periphery and core of spherical structures, a typical characteristic of the projection images of hollow spheres [24].
It was possible to stain these structures with rhodamine B and
fluorescence microscopy image once again afforded brightly
stained spherical aggregates (Fig. 1E). Size distribution of these
spherical aggregates was determined by dynamic light scattering
(DLS) measurements which revealed a broad peak (Fig. 1F) with
an average size of 700 nm, which was in accordance with the size
measured by other microscopy methods. Notably, gross spherical
structure was observed in all the microscopy studies confirming
an inherent preference of this morphology.
Looking at the molecular structure of 3, it can be proposed that
more hydrophilic mannose groups will prefer being displayed at
the outer surface of the spherical structures for a favourable interaction with water. We decided to confirm whether mannose
appendages are indeed projected outwards through an assay
exploiting carbohydrate–lectin interactions. It is known that mannose–Concanavalin A (Con A) interaction leads to aggregation, thus
enhancing turbidity of the solution [25]. Therefore, a turbidimetric
assay was employed to study the interaction of 3 with Con A. Increase in the absorbance with respect to time was observed at
400 nm which indicated aggregation of Con A by mannose-coated
spherical structures, thus providing an indirect proof of mannose
display at the surface of these soft structures [18].
We decided to probe whether these structures could be disrupted by ultrasonication to expand application of these spherical
structures. Interestingly, we observed that these structures could
be completely disrupted by ultrasonication over a 5 min exposure
in an ultrasonic bath (Fig. 2). Taken together with the possibility of
guest molecule interaction, a non-invasive release method appeared promising enough for us to further investigate these soft
structures for complexation, confinement and delivery of biological
molecules of interest.
Calf intestine alkaline phosphatase (CIAP) and plasmid DNA
pBR322 was used for confinement studies. CIAP (5 U, 1 lM) was
incubated with 3 (1 mM) in (10 mM Tris buffer, pH 7.9) for 16 h
in the aqueous medium. To confirm complete encapsulation (or
complexation) of the enzyme, p-nitrophenol phosphate (pNPP)
was added to this solution. Enzymatic activity was not observed
as judged by the lack of detection of the yellow color corresponding to hydrolysis product p-nitrophenolate (pNP) anion, even after
4 h of addition of substrate (Trace 3, Fig. 3).
This suggests that either the enzyme has simply complexed
with 3 or it got encapsulated inside hollow spherical particles, thus
rendering the enzyme ineffective towards pNPP hydrolysis. However, the enzymatic action of alkaline phosphatase was restored
when the solution of enzyme complexed or loaded spherical structures were ultrasonicated for 5 min, as evidenced by the appearance of the yellow color (Trace 5, Fig. 3). In control experiments,
alkaline phosphatase was added to preformed spherical structures
Fig. 2. SEM images of self-assembled structures of 3 before and after 5 min sonication.
N. Gour et al. / Biochemical and Biophysical Research Communications 378 (2009) 503–506
Fig. 3. Enzymatic activity (pNPP hydrolysis) of alkaline phosphatase when encapsulated inside self-assembled spherical structures of 3. Trace 1 (black), CIAP alone;
Trace 2 (blue), CIAP added to preformed spheres of 3, Trace 3 (bluish green),
encapsulated CIAP with added pNPP; Trace 4 (red), pNPP alone, Trace 5 (green),
after 5 min of sonication of encapsulated CIAP. Inset shows release of CIAP from
spherical structures, aided by ultrasonication. A solution colorless even after 4 h of
addition of pNPP substrate turns yellow after 5 min of sonication. (For interpretation of color mentioned in this figure the reader is referred to the web version of
the article.)
and incubated for 2 h prior to the addition of the substrate pNPP.
Under these conditions, alkaline phosphatase exhibited enzymatic
activity to an extent similar to that of free enzyme thus ruling out
the possibility of simple complexation of spherical structures. This
strongly suggests for the encapsulation of enzyme within the confines of these hollow spheres.
Being aware of several reports concerning the use of glycoclusters for delivery applications, we decided to probe mannosylated
hollow spherical structures as potential carriers for gene delivery
[26]. AFM micrographs of 3, coincubated with pBR322 plasmid
DNA, revealed formation of irregular shaped structures suggesting
interaction of DNA with carrier molecules as previously reported
for DNA–micelle complexation [27] (Fig. 4A). Interestingly, agarose
505
gel electrophoresis did not show any band corresponding to 3pBR322 (Fig. 4B, lanes 2 and 3) plasmid complex suggesting effective complexation or encapsulation of plasmid DNA which prevents visualization when stained with ethidium bromide.
Notably, plasmid DNA was released when the samples were ultrasonicated for 5 min making the DNA band visible in gel (Fig. 4B,
lane 5) confirming effective complexation with DNA. Moreover
DNA band was visible when preformed spheres of 3 were incubated with DNA, ruling out possibility of simple complexation
(Fig. 4B, lane 4).
As a further proof of plasmid DNA encapsulation within the
mannosylated hollow spheres, we decided to transfect African
green monkey derived COS-7 mammalian cells with a pEGFP-N1
reporter plasmid which encodes for an enhanced green fluorescent
protein (GFP) When the self-assembled structures of mannosylated
spheres encapsulating the expression construct pEGFP-N1 were
added to the medium, and incubated for a duration of 36 h, a number of cells showed nuclear green fluorescence as expected for the
GFP protein (Fig. 4C). Transfection efficiency of pEGFP-3 complex
was about 8–10% which is less, but definitely proves that 3 may
be used for plasmid delivery applications, if this efficiency might
be improved by inducing some structural changes. A comparison
of transfection efficiency is being carried out vis-à-vis standard
transfection reagents, by modifying structure of 3 and these results
will be reported in due course.
Nuclear localization of GFP was further confirmed by staining
the cells with a nucleus specific dye, 40 ,6-diamidino-2-phenylindole (DAPI). DAPI is a fluorescent stain that avidly binds to the
stranded DNA and gives a blue emission restricted around cell nuclei (Fig. 4D). When DAPI stained image was merged with GFP image nuclear localization was confirmed (Fig. 4E).
In the control experiments, cells were incubated with either a
solution of 3 without plasmid DNA or vice-versa. Toxicity of 3
was minimal and unaided transfection of cells was not detected
in the second case. Cell were also incubated with ultrasound treated, after 5 min of sonication, pEGFP-3 complex did not produce
any transfection, probably because DNA was released from these
spherical structures. Moreover, pEGFP-N1 reporter plasmid incubated with preformed spheres for a 2 h duration exhibited extremely low transfection efficiency [28]. Thus, preliminary studies
Fig. 4. (A) AFM images of 3 loaded with DNA; (B) Gel of DNA with 3: lane 1, DNA alone; lanes 2 and 3, hollow spheres of 3 encapsulating DNA after 16 h of incubation; lane 4,
DNA added to preformed spheres of 3; lane 5, DNA released from spheres after 5 min of sonication. (C–E) Nuclear GFP expression in a cell from the COS-7 cell line when
transfected with pEGFP-N1 plasmid-loaded self-assembled structures of 3. Nucleus specific expression of GFP was confirmed by DAPI staining and merging GFP expressed
and DAPI stained images (scale bar = 10 lm).
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suggest that plasmid DNA most likely gets inside the hollow
spheres during the self-assembly process.
We surmise that cellular delivery of pEGFP-N1 reporter plasmid
is probably occurring through endocytosis, whereby the plasmidencapsulated structures of 3 are engulfed and released inside the
cell and express its gene product. Taken together, these observations suggest that self-assembling property of 3 can be used for
DNA encapsulation and for designing novel carriers for gene
delivery.
Conclusions
We demonstrated that bis-mannosylated lysine exhibits selfassociation to reveal formation of hollow soft spherical structures.
Morphological features of these structures ware studied by microscopy analysis and dynamic light scattering. Notably, these structures afford facile encapsulation of alkaline phosphatase, plasmid
DNA and facilitate transfection of mammalian cells. These experiments are significant as direct application of mannosylated selfassembled hollow spheres is demonstrated. The display of mannose units raises the possibility of invoking specific recognition
events at cell surfaces. Future endeavours will include further
refinement in the design of hollow capsules in order to permit targeted delivery of drugs and biomolecules and their use as reaction
vessels.
Acknowledgments
N.G. thanks IIT-Kanpur for a pre-doctoral research fellowship.
We thank Profs. D.C. Agarwal and A. Sharma for DLS and AFM measurements. ACMS, IIT Kanpur is thanked for the access to SEM.
Alexander von Humboldt Stiftung, Bonn, Germany, is thanked for
an equipment donation to S.V. This work is partially supported
by an Indo-US Frontier of Science Award to SV from the Indo-US
Science and Technology Forum, New Delhi. SV is a Swarnajayanti
Fellow (DST) in Chemical Sciences.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2008.11.070.
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