Intracellular self-assembly of nanoparticles for enhancing cell uptake

Article abstract of DOI:10.1039/c2cc34899c

A radioactive probe (1) has been developed and applied to a condensation reaction and self-assembly of radioactive nanoparticles (i.e., ¹²⁵I-NPs) intracellularly. Upon 160 min cellular efflux, the radioactivity retained in cells incubated with 1 was 4-fold more higher than that of those cells treated with a scrambled control probe (1-Scr). The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc34899c

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COMMUNICATION
Intracellular self-assembly of nanoparticles for enhancing cell uptakew
Qingqing Miao,a Xiaoyu Bai,a Yingying Shen,b Bin Mei,a Jinhao Gao,c Li Lib and
Gaolin Liang*ac
Received 9th July 2012, Accepted 17th August 2012
DOI: 10.1039/c2cc34899c
A radioactive probe (1) has been developed and applied to a
condensation reaction and self-assembly of radioactive nano-
particles (i.e., 125I-NPs) intracellularly. Upon 160 min cellular
efflux, the radioactivity retained in cells incubated with 1 was 4-fold
more higher than that of those cells treated with a scrambled
control probe (1-Scr).
Recently, Rao and co-workers developed a biocompatible
condensation reaction between the 1,2-aminothiol group of
cysteine and the cyano group of 2-cyanobenzothiazole (CBT)
which could be controlled by pH, reduction and protease at
concentrations as low as micromolar for self-assembly of
nanostructures in living cells. This condensation reaction has
shown promising applications for imaging protease activities
in living cells, designing smart MRI probes, protein labeling,
and so on.14
For cell imaging, most of the effort has been put on designing
effective probes which can either easily overcome the cell membrane
barriers or amplify their signals at the targeting site, or both.1 For
example, arginine-derived peptide for translocation through the
negative charged phospholipid bilayer cell membrane,2 ligand–
receptor (folate–folate receptor, RGD–integrin avb3) interactions
for effective cellular internalization.3 Avidin–biotin conjugations, or
enzymatic reactions were applied on the probes to amplify their
signals at the targeting sites.4 Some water-compatible ‘‘click’’
reactions also have been developed by chemists and applied for
effective cellular labeling and imaging.5 In recent years, nanoprobes
have emerged as an excellent tool for molecular imaging because of
their ability of loading as many small molecular probes as
needed to achieve the desired signal.6 Nevertheless, nanoprobes
face the problems of cell membrane translocation and targeting,
besides the difficulty and reproducibility of their fabrication.7
Self-assembly, a prevalent and important process in nature,8
provides an easy solution to the above problems. Up to date, a
lot of self-assembly systems have been developed to make
biocompatible nanomaterials for controlled drug release and
delivery,9 biosensing,10 tissue engineering,11 wound healing,12
and even controlling the fate of cells.13 Nevertheless, using a
lower concentration of small molecular probes to smartly self-
assemble nanoprobes at the desired location in cells has still
remained challenging and is less explored for molecular imagers.
The trans-Golgi protease furin is a protein convertase that
plays crucial roles in homeostasis, and in diseases ranging from
anthrax and Ebola fever to Alzheimer’s disease and cancer.15
Overexpression of furin offers a useful hint of early development
of certain cancers. One big advantage for chemists to study furin
is that it preferentially cleaves Arg–X–Lys/Arg–ArgkX motifs,
where Arg is arginine, Lys is lysine, X can be any amino acid
residue and k indicates the cleavage site.16
Inspired by these, as shown in Scheme 1, we designed
Acetyl–Arg–Val–Arg–Arg–Cys(StBu)–Tyr(I-125)–CBT (1) for
self-assembling radioactive nanoparticles (125I-NPs) under the
action of furin in living tumor cells. After entering cells, the
disulfide bond of the Cys motif of 1 is reduced by the intra-
cellular glutathione (GSH) and subsequently its RVRR motif
is cleaved by furin on site, resulting in the active intermediate
1-Core. Two 1-Cores condense quickly to yield the amphiphilic
dimer (i.e., 1-Dimer) which has a hydrophobic macrocyclic core
a CAS Key Laboratory of Soft Matter Chemistry, Department of
Chemistry, University of Science and Technology of China,
96 Jinzhai Road, Hefei, Anhui, 230026, P. R. China.
E-mail: gliang@ustc.edu.cn; Fax: +86-551-3600730;
Tel: +86-551-3607935
b State Key Laboratory of Oncology in Southern China,
Imaging Diagnostic and Interventional Center, Cancer Center,
Sun Yet-sen University, 651 Dongfeng Road East, Guangzhou,
510060, P. R. China
c The Key Laboratory for Chemical Biology of Fujian Province,
Xiamen University, Xiamen, 361005, P. R. China
w Electronic supplementary information (ESI) available: Experimental
details and methods, synthesis of 1, 1-Cold, 1-Scr, 1-Scr-Cold, HPLC
conditions, supplementary tables and figures (Scheme S1–S4, Table
S1–S2, Fig. S1–S13). See DOI: 10.1039/c2cc34899c
Scheme 1 Furin-controlled condensation of 1 to assemble radioactive
nanoparticles in tumor cells.
c
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for self-assembling 125I-NPs via p–p stacking among each
other. As-formed 125I-NPs could concentrate the radioactivity
inside cells on one hand. On the other hand, the big size and
the hydrophobic property of the 125I-NPs would prevent
themselves from being pumped out by the cells. Compared
with the control probes (e.g., scrambled control 1-Scr) which
could not be cleaved by furin or self-assemble in cells, 1 should
have an enhanced cellular uptake by tumor cells, suggesting its
promising application in tumor imaging in vivo.
We began the study with the synthesis and preparation of four
compounds (two pairs): 1 and 1-Cold, 1-Scr and 1-Scr-Cold
(Fig. 1 and ESIw). To validate furin-controlled condensation
and self-assembly, we used 1-Cold for in vitro study. As shown in
Fig. 2a, after 16 h incubation of 1-Cold at 100 mM and 30 1C
with 1 nmol UÀ1 of furin, we directly injected the incubation
mixture into a HPLC system and collected the peaks for
matrix-assisted laser desorption/ionization (MALDI) mass
spectroscopic analysis. Peaks on HPLC traces at retention
times of 44.2 min (41.8%), 47.3 min (3.0%) and 48.3 min
(4.6%) were identified as the condensation products of 1-Cold
(i.e., 1-Cold-Dimer, Fig. S1a and b, ESIw). The peak at
retention time of 49.7 min (6.2%) was identified as the reduced
product of 1-Cold (i.e., 1-Cold-Red, Fig. S1c, ESIw). Increasing
the concentration of furin affords more condensation product
1-Cold-Dimer within a shorter time. At 8 h and a furin
concentration of 0.5 nmol UÀ1, more of the 1-Cold was
cleaved and condensed to form 1-Cold-Dimer which self-
assembled into nanoparticles and changed the solution into
a monodispersion. UV-Vis spectrum at 500–700 nm of the
monodispersion showed an obvious increase of absorption
compared with that of starting solution, suggesting the aggrega-
tion of nanoparticles (Fig. S2, ESIw). Dynamic light scattering
(DLS) measurements indicated that the sizes of the nanoparticles
have a narrow distribution and a mean diameter of 534 nm
(Fig. 2b). Directly taking the above dispersion for transmission
electron microscopy (TEM) observations, we found the nano-
particles have an average diameter of 388 nm (Fig. 2c). High
magnification of the TEM images indicated that each nano-
particle is formed via the aggregation of smaller nanoparticles
with a mean diameter around 13 nm (Fig. S3, ESIw). HPLC
and MALDI mass spectroscopic analysis indicated that at
least 77.3% of 1-Cold was converted into its dimeric products
Fig. 2 (a) HPLC trace of the incubation mixture of 1-Cold at 100 mM
after 16 h incubation with 1 nmol UÀ1 of furin at 30 1C (upper), and
HPLC trace of 1-Cold in water (lower). (b) Dynamic light scattering
(DLS) analysis of particles-size distribution of the self-assembled
products of 1-Cold after 8 h incubation with furin at 0.5 nmol UÀ1
and 30 1C. (c) TEM images of the nanoparticles in b.
(i.e., 1-Cold-Dimer) and only 6.3% of 1-Cold was unacted
(Fig. S4, ESIw).
After the validation of furin-controlled condensation and
self-assembly, we started to use MDA-MB-468 cells to assemble
125I-NPs intracellularly because we have demonstrated that
MDA-MB-468 cells overexpress furin using furin immuno-
fluorescence staining (Fig. S5, ESIw). Firstly, we studied the
cell permeability of 1 and 1-Scr by incubating the probes with
MDA-MB-468 cells and quantitating the cellular uptake by
counting the radioactivity with a g-counter. Cellular uptake of
both radiotracers rapidly reached their plateaus at 30 min
(21.3% and 15.8% of total radioactivity for 1 and 1-Scr
respectively) during the 1 h experiments, with that of 1 being
significantly higher than that of 1-Scr at all time points
(Fig. S6, ESIw). This result suggested that both 1 and 1-Scr
have excellent properties for cell permeability, and 1 is better
than 1-Scr, proven by the fluorescence microscopic images of the
cells incubated with 1-Cold or 1-Scr-Cold respectively (Fig. S7,
ESIw). After 8 h incubation with 100 mM of 1-Cold, electron
microscope (EM) image of the cells undoubtedly indicated that
the locations of the intracellular nanoparticles were near/at Golgi
bodies, the sites where furin is activated (Fig. S8, ESIw).
Although 1 might be directly applied for intracellular self-
assembly of 125I-NPs, there are still two potential problems
which might hinder it from intracellular self-assembly. Firstly,
even the radioactivity of 1 entering cells is high enough for
detection, its chemical concentration might not high enough
for initiating the condensation reaction for self-assembly.
Secondly, intracellular free cysteine might react with the cyano
group of 1 before it condenses with each other, resulting in the
formation of 1-Dimer and rendering its subsequent self-assembly
infeasible.14c To overcome these difficulties, we proceeded the
studies with the experiments of concentration-dependent intra-
cellular condensation. This was done by incubating 1 million
Fig. 1 Chemical structures of C, 1 and 1-Cold for furin-controlled
condensation and self-assembly, and their scrambled controls H, 1-Scr
and 1-Scr-Cold respectively.
c
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group 2 was 4-fold more higher than those of other three groups
(4.4%, 17.9%, 3.7% and 3.7% for group 1–4 respectively, Fig. 3d),
suggesting that intracellular 125I-NPs actually help the retaining of
radionuclide in living cells. Subsequent 3-(4,5-dimethylthiazol-2-yl)
2,5 diphenyl tetrazolium bromide (MTT) assay indicated that
neither 1-Cold nor 1-Scr-Cold shows acute cytotoxicity to the
cells at 100 mM (Fig. S9, ESIw).
In summary, using a strategy of co-incubation, we have
successfully demonstrated that intracellular condensation and
subsequent self-assembly of nanoparticles could help the
retaining of radionuclide in living tumor cells. This strategy
starts with small molecules outside tumor cells but ends up
with nanostructures inside them, offering a new method of
designing ‘‘smart’’ probes for molecular imaging. Using this
strategy for specifically imaging tumors in vivo, this research
work is underway.
This work was supported by the National Natural Science
Foundation of China (21175122, 91127036), the Fundamental
Research Funds for Central Universities (WK2060190018),
and Anhui Provincial Natural Science Foundation (1108085J17).
Fig. 3 (a, b) Concentration-dependent intracellular self-assembly:
Time course of cellular efflux of 1 after 30 min incubation with
1 million MDA-MB-468 cells at 1 mCi and co-incubated with 1-Cold
at different concentrations (a). Radioactivity retained in cells after
160 minutes’ efflux (b). (c, d) Effect of co-incubation on cellular efflux
and retaining of radioactivity in MDA-MB-468 cells: Time course of
cellular efflux of 1 and 1-Scr after 30 min incubation with 1 million
MDA-MB-468 cells with/without co-incubation of 1-Cold or 1-Scr-Cold
at 100 mM (c). Radioactivity retained in cells after 160 minutes’ efflux
(d). The dosage was normalized with that of 0 min (i.e., after 30 minutes’
incubation and before the efflux starts).
Notes and references
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