Lipoic acid based core cross-linked micelles for multivalent platforms: Design, synthesis and application in bio-imaging and drug delivery

Article abstract of DOI:10.1039/c7ob00927e

Natural lipoic acid derived small-molecule amphiphiles self-assemble into micelles in water. The presence of numerous disulfides accumulated in the core makes the micelles readily cross-linked to achieve the establishment of core cross-linked micelles (CCMs). Thanks to the inherent biocompatibility, the resulting lipoic acid based CCMs (LA-CCMs) are good multivalent platforms for biomedical applications.

Full text of DOI:10.1039/c7ob00927e

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Lipoic Acid Based Core Cross-linked Micelles for Multivalent
Platform: Design, Synthesis and Application in Bio-imaging and
Drug Delivery
Jingsheng Huang,a Fang Wu,a Yunlong Yu,*b Haolong Huang,b Shiyong Zhang*ab and Jingsong You*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Natural lipoic acid derived small-molecule amphiphiles self-
Lately, the cross-linked small-molecule self-assemblies
assemble into micelles in water. The presence of numerous have been received substantial attentions for fabrication of
disulfides accumulated in the core makes the micelles readily be multivalent platform, which shows great potentials in
cross-linked to achieve the establishment of core cross-linked controlled release,8 catalysis,9 sensors,10 and template
micelles (CCMs). Thanks to the inherent biocompatibility, the synthesis.11 However, the use of non-biocompatible synthetic
resulting lipoic acid based CCMs (LA-CCMs) are good multivalent small-molecule and hazard solvents severely limit their bio-
platforms for biomedical application.
functional applications. Lipoic acid is a totally biocompatible
and natural product, which normally exists in mitochondrion,
and acts as a kind of coenzyme for the anti-oxidation. Besides,
some reports give evidence that the natural product lipoic acid
might have potential as a drug for some diseases.12 Quite
recently, our group presented the first clean and clinically
feasible core cross-linked micelles (CCMs) based multivalent
drug delivery systems (MDDSs) formed by a conjugated
amphiphile with lipoic acid as the hydrophobic tail and
anticancer drug gemcitabine (Gem) as the hydrophilic head.13
The strategy of lipoic acid conjugated MDDSs conceptually
overcomes the essential challenges faced by the other MDDSs
such as polymeric micelles. However, with the up-to-down
method, only hydrophilic molecules containing functional
groups, which can react with the carboxylic group of lipoic acid,
can be employed to construct the MDDSs. This seriously limits
the application prospect of the strategy.
In this communication, we report a general lipoic acid based
core cross-linked micelles (LA-CCMs) for multivalent platform
through bottom-to-up fabrication. First, modifiable micelles are
constructed by using readily available lipoic acid based functional
amphiphiles, as shown in Scheme 1a. Overcoming the defects faced
by original CCMs, the mono-functional LA-CCMs can be easily post-
functionalized by either hydrophobic or hydrophilic drug or
fluorophore for drug delivery or bio-imaging. In particular, multi-
functional LA-CCMs can afford the multivalent platforms to carry
two or more drugs or other functional molecules relying on the
specific requirements (Scheme 1b). Therein, the LA-CCMs can be
employed as general multivalent platforms for biomedical
application for numerous advantages, such as the readily available
surfactant, the simple preparation of cross-linked nanoparticles, the
convenient surface functionalization, the adjustable surfactant
combination, and the excellent biocompatibility etc.
The phenomena of multivalency exist ubiquitously in nature.1
With the help of multivalent interaction, a strong and
reversible attachment or adhesion can be gained between
various surfaces bearing multiple ligands and receptors
compared to the single pair. Especially in biological systems,
the multivalent interactions play an essential and decisive role
in conducting numerous vital activities/biological functions in
living organism, such as cell self-organization, immune
recognition, pathogen-cell adhesion and signal transduction.2
In recent years, lots of efforts have been devoted to
understand and synthesize the optimal multivalent ligands for
biological and pharmaceutical application.3 Especially,
numerous multivalent platforms are manufactured in drug
delivery systems, such as polymeric micelles (PMs),
dendrimers, and nano-inorganic particles etc.4 However, some
of the intrinsic flaws of these materials inhibit their further
clinical applications.5 For example, PMs suffer from low drug
loading contents (˂ 5%) and meta-stability in vascular system.5c
Dendrimers seem to be able to overcome some disadvantages
of PMs, but the tedious synthetic route and toxicity in high
generation limit their prospect as practical drug delivery
systems.6 Nano-inorganic particles have unique therapeutic
effect on cancerous cells, while, the in vivo metabolism
difficulty is
a vital problem for their pharmaceutical
applications.7
a.College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu, 610064,
China
b.National Engineering Research Center for Biomaterials, Sichuan University, 29
Wangjiang Road, Chengdu, 610064, China
* Email: yuyunlong666@gmail.com; jsyou@scu.edu.cn; szhang@scu.edu.cn
†
Electronic Supplementary Information (ESI) available. See DOI:
10.1039/x0xx00000x
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2 months (Fig. 5S). The excellent stability of the cross-linked
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DOI: 10.1039/C7OB00927E
micelles also gives evidence to the successful cross-linking of
disulfide bonds. When DTT is added, the disulfide bonds are broken
and the micelles are degraded as shown in Fig. 1c. As to evaluate
the cytotoxicity of the platform, the cell viability of LA-CCM/L2 is
given as an example to be incubated with A549 and HeLa cells for
o
24 h at 37 C with series of concentrations. Fig. 1d shows the
platform is biocompatible, which has almost no toxicity even at a
high concentration of 400 μg/mL. Therein, the LA-CCM platform can
be potentially employed as nano-carriers for biomedical
application.
Scheme 1. a) Synthesis of small molecules L1-L5 with different end-
functional groups, and the details are shown in SI; b) Schematic
illustration of the formation of multivalent platforms and
conjugation with drugs and dyes.
Typically, five functional small-molecule amphiphiles L1-L5 are
synthesized simply by one or two-step modification of lipoic acid
(See Supporting Information for details, SI), and various functional
groups can be easily achieved for further modification, such as
alkyne, carboxylic acid, amine, hydroxyl and azido groups. With a
hydrophilic head and hydrophobic tail, all five compounds are
readily soluble in water and gave rise to the formation of micelles.
The critical micelle concentration (CMC) of the amphiphiles is
determined by using fluorescence method, as shown in SI. The DLS
results show that the hydrodynamic diameter of all the small-
molecule micelles is around 50 nm (Fig. 2S). After ruptured by DTT
and exposed in air, the cores of the micelles are polymerized and
chemically and physically cross-linked due to formation of new
disulfide bonds. The successful cross-linking was confirmed by the
1H NMR spectra (see Fig. 3S). The proton peaks after cross-linking
become more broad and flat compared to the uncross-linked one.
While, only polymerization can happen between the disulfide below
the CMC, which is evidenced in Fig. 5S. Fig. 1a shows the DLS results
of L3 micelles before and after cross-linking. After cross-linking, the
hydrodynamic diameter of the micelles increases from 51 nm to
122 nm, which is probably due to the rearrangement of the core
structure of the micelles during cross-linking.13 The transmission
electron microscope (TEM) image of LA-CCM/L3 in Fig. 1b further
shows the micelles shape like a sphere, and with a relatively smaller
diameter (~43 nm in average) compared to the DLS results, as TEM
characterizes the dimension of micelles at collapsed state. Cross-
linking is of crucial importance to endow enhanced thermal stability
and the prolonged blood circulation of our LA-CCMs. The stability of
the LA-CCM/L3 in liquid media is evaluated by transferring the
micelles from water to methanol, and the DLS results show no
distinct changes in diameter (see Fig. 5S). Fig. 1c shows the stability
of L3 micelles before and after cross-linking in 10% FBS at 37 oC. The
uncross-linked micelles disassemble in just 2 h as the ions in FBS can
disturb the dynamic balance of the micelles. While, the cross-linked
micelles can maintain stable in the experimental circumstance, as
its size has no obvious change, that can be kept in water for at least
Fig. 1 a) Particle size distribution (PSD) of uncross-linked
micelles (LA-UCM/L3) and core cross-linked micelles (LA-
CCM/L3) in aqueous solution; b) TEM micrograph of the LA-
CCM/L3; (c) Stability test of LA-UCM and LA-CCM/L3 ([L3] = 8
μg/mL); (d) Cell viability of LA-CCM/L2 against A549 and HeLa
cells.
First, the LA-CCMs platform is studied as use of mono-
component carrier system. Around the core of the platform, various
functional groups are suspended. Depending on applications,
certain kind of functional molecules can be covalently bonded on
the LA-CCMs, such as fluorophore or drug, for bio-imaging and drug
delivery. Rhodamine B (RhB) is a well-known bright fluorescence
dye with excellent photo-stability, and has many applications in bio-
imaging.14 Here, with one step esterification, RhB can be covalently
linked on the LA-CCM/L5. The resulting functional RhB@LA-CCM/L5,
compared with free RhB, demonstrates a blue shift in the
fluorescence spectrum (see Fig. 7S), presumably as a result of the
perturbing of molecular resonance after the chemical modification
of RhB.15 The HeLa cells are employed to test the imaging efficiency
of RhB@LA-CCM/L5 by confocal laser scanning microscopy (CLSM).
As shown in Fig. 2, after 0.5 h incubation, partial fluorescence
micelles are accumulated in cell membrane with less fluorescence
intensity. Furthermore, after 2 h, the fluorescence of RhB is more
visible and stronger within cytoplasm, which indicates that the
cellular intake of fluorescence micelles is time-dependent. The
results show that the RhB-bearing LA-CCMs system has potential
application in bio-imaging.
Besides fluorophore, mono-drug can also be attached on the LA-
CCMs by easily one step modification. For example, as a hydrophilic
drug, gemcitabine (Gem) behaves good curative effect on lung
2 | J. Name., 2012, 00, 1-3
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cancer. By one step amidation with LA-CCM/L2, hydrophilic drug
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Besides hydrophilic drug, the hydrophobic drug can also be
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nano-carriers Gem@LA-CCM/L2 are obtained with a drug loading tethered on the LA-CCMs platforms. ForDOeIx:a1m0.1p0le3,9/Cd7oOxoBr0u0b9i2c7inE
efficiency of 26.8%, much higher than most of the PM systems.16 hydrochloride (DOX·HCl) is one of the most common anti-cancer
1
The successful reaction is confirmed by both UV-Vis spectra and H drugs. After neutralizing, it becomes a hydrophobic drug. Through
NMR, as shown in Fig. 8S and 9S. After that, the in vitro cell viability one step reaction, DOX can be covalently connected on the
of the system is measured by culturing A549 cells with the micelles, which can be confirmed by the red shift of the maximum
Gem@LA-CCM/L2 in various concentrations for 24 h. Free Gem is absorption of DOX@LA-CCM/L2 compared to the platform LA-
also cultured with cells at the same condition as a reference. Fig. 3a CCM/L2 in UV-Vis spectra. Also a relatively high drug loading
shows both free and Gem@LA-CCM/L2 can effectively kill the efficiency (13.6%) is obtained. The in vitro cytotoxicity of DOX@LA-
cancerous cell with a half-maximal inhibitory concentration (IC50) of CCM/L2 is characterized by measuring the IC50 of cancer cell
15.59 and 11.09, respectively. Though the incorporation of proliferation. After cultured with human lung carcinoma HeLa cells
biocompatible LA-CCM/L2 lowers down the IC50, the system for 24 h at 37 °C, the cell viability of free DOX and DOX@LA-CCM/L2
behaves relatively good curative effect. Especially at a relatively are shown in Fig. 3b. Free DOX gives significant anticancer activity
high dose of 20 μg/mL, Gem@LA-CCM/L2 has similar cytotoxicity (IC50 = 0.65 μg/mL). Compared to that, the DOX@LA-CCM/L2 shows
with free Gem, which can effectively kill cancer cells.
less anticancer efficiency (IC50 = 11.2 μg/mL), probably due to the
lower drug loading efficiency compared to Gem@LA-CCM/L2. Then
the CLSM is used to observe the cellular uptake behaviour of
DOX@LA-CCM/L2 using HeLa cells. As exhibited in Fig. 3c, after
incubation for 1 h, only weak fluorescence can be found around the
cell, which displays a low fluorescence density due to low uptake
efficiency in a short time. After 3 h, the intracellular fluorescence is
enhanced, which reveals a better cell-uptake of DOX@LA-CCM/L2.
Above all, the mono-platform can be afforded with potential
applications in both bio-imaging and drug delivery.
In biology, tandem repeats formed by protein domains can be
used for the multivalent cell-adhesion, in which more than two
receptors are needed on the multivalent platform.17 In clinical
studies, the curative effect of solid tumour is not apparent by using
mono-drug due to the inhomogeneity inside the tumour and drug
resistance.18 Multiple drugs incorporation becomes a popular and
effective way to overcome the drug resistance. However, most of
the reported nano-carriers can only load mono-drug for a specific
cancer, which significantly limited the therapeutic effect and field.
Block copolymers could be used to carry two different drugs at the
same nanoparticle.19 However, the complex and tedious synthesis
and low drug-loading efficiency inhibit their broad application. In
contrast, by using the multivalent LA-CCMs multi-functional
platform, two or more kinds of drugs can be easily loaded on one
micelle and delivered simultaneously for the cancer therapy. Here,
two dyes are selected to replace two drugs loading on the platform
to imitate multi-drugs delivery. 1,5-EDANS (donor) and CF-N3
(acceptor) are chosen (See Scheme 1 for details), as there is an
overlap between their fluorescence and absorption spectra (Fig.
4b), which can cause Förster resonance energy transfer (FRET)
phenomena when located in a distance less than 10 nm.20 Firstly, L2
and L3 are mixed and core cross-linked to form multi-functional LA-
CCM/L23 platform, and the spherical morphology is characterized
by TEM, as shown in Fig. 4a. After that, the donor-acceptor pairs are
connected on the platform by one step reaction, and the details are
shown in SI. The free donor molecule D has a maximum
fluorescence emission at 475 nm, and the acceptor A at 520 nm.
However, after forming the LA-CCM/L23, the donor’s emitting
signal is abruptly reduced in aqueous solution. While, the acceptor’s
emission is greatly enhanced that is caused by the FRET effect.
Meanwhile, the FRET phenomena also indicate that the two dyes
are successfully grafted on the micelles with a closed distance
between each other.
Fig. 2 CLSM images of HeLa cells treated with RhB@LA-CCM/L5 with
concentration of RhB about 2.0 μg/mL after 0.5 h and 2 h. Blue
represents the fluorescence of Hoechst 33342 for cell nuclei
staining and red indicates the fluorescence of RhB. The scale bars
are 25 μm in all images.
Fig. 3 (a) Cell viability of free Gem and Gem@LA-CCM/L2 against
A549 cells after incubation for 24 h at 37 oC with series of
concentrations; (b) Cell viability of free DOX and DOX@LA-CCM/L2
o
against HeLa cells after incubation for 24 h at 37 C with series of
concentrations. Data are presented as the average ± SD (mean ± SD,
n = 5); (c) CLSM images of HeLa cells treated with DOX@LA-CCM/L2
([c] = 2.5 μg/mL). Blue represents the fluorescence of Hoechst
33342 for cell nuclei staining and red indicates the fluorescence of
DOX. The scale bar is 25 μm in all images.
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As known, the glutathione (GSH) concentration is higher in specific bio-imaging, drug-delivery, catalysis and anti-bacterial
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DOI: 10.1039/C7OB00927E
tumour cells than the normal cells. Such that disulfide can be used fields.
to design nano-carriers, which can be degraded in the reductive
In summary, an easy and feasible LA-CCMs multivalent platform
condition to release drugs or degradation of nanocarriers in the is synthesized based on natural lipoic acid. The platform has various
pathological location.13,21 In order to evaluate the degradation functional groups surrounded, which can be further modified with
capability of our dual-functional carrier system, the reductive DTT fluorophore, hydrophilic and hydrophobic drugs to form mono-
with various concentrations are added and vibrated for 5 min. Fig. functional system. Due to the excellent biocompatibility of the
4c shows that the FRET effect is weakened by adding the DTT. platform, applications on bio-imaging and drug delivery system can
Especially in 30 mM DTT, the fluorescence intensity ratio of be achieved. Moreover, by choosing numerous platform
acceptor to donor I1/I2 is significantly increased from 8.8% to 27.3% combinations, multi-functional groups can be carried on the
(Fig. 10S), which indicates the cross-linked micelles are degraded. complex platform for the multi-functional applications, such as
As the coupling pairs are separated after degradation, no exited- dual-drug delivery for improving drug resistance. Furthermore,
state energy can be transferred between the FRET molecules as a other functional groups can also be attached on the LA-CCMs
long separation distance. Though the degradation weakens the platform for specific multi-functional application, such as drug
FRET effect, the fluorescence emission of donor is not totally delivery, anti-fouling, catalysis and so on.
recovered to the default, which might be due to the uncompleted
We thank the National Natural Science Foundation of China
degradation of the cross-linker. As explained in Fig. 4d, after adding (21372170 and 51673130) for supporting the research.
DTT, the cross-linked core is broken to several branches. In some
branches, there is only one donor or acceptor, which is not
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Fig. 4 (a) DLS and TEM of the multivalent platform LA-CCM/L23; (b)
Fluorescence spectra of D@LA-CCM/L23 and adsorption spectra of
A@LA-CCM/L23; (c) Fluorescence spectra of D and A@LA-CCM/L23
broken with different DTT concentration in 5 mins. (d) Illustration of
platform with FRET phenomenon by conjugating two dyes and
breaking with DTT. Donor dye: 1,5-EDANS (D), acceptor dye: CF-N3
(A).
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