Albumin-micelles via a one-pot technology platform for the delivery of drugs

Article abstract of DOI:10.1039/c4cc00616j

A new micelle delivery platform based on albumin coated nanoparticles is able to selectively deliver the payload to cancerous cells while healthy cells remain less affected. The technology is simple and can be used in a one-pot procedure. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00616j

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Albumin-micelles via a one-pot technology
platform for the delivery of drugs†
Cite this:DOI: 10.1039/c4cc00616j
Yanyan Jiang, Mingtao Liang, Domenic Svejkar, Gene Hart-Smith, Hongxu Lu,
Wei Scarano and Martina H. Stenzel*
Received 24th January 2014,
Accepted 24th April 2014
DOI: 10.1039/c4cc00616j
www.rsc.org/chemcomm
A new micelle delivery platform based on albumin coated nano- molecules which act like glue holding the albumin molecules
particles is able to selectively deliver the payload to cancerous cells in place. More hydrophilic drug molecules and charged drugs
while healthy cells remain less affected. The technology is simple like nucleic acids are therefore not suitable for this technique.
and can be used in a one-pot procedure.
In some cases, albumin has been modified to conjugate to
DNA,6,7 but these extensive modifications of albumin may have
Abraxaner, is an injectable formulation of paclitaxel where the altered the protein structure, which in turn loses its specificity
drug is bound to albumin as a delivery vehicle (nab technology). towards gp60.
This is one of the few nano-formulations that have made it on
In order to modify albumin without affecting its bioactivity,
the market today. The secret of this technology developed by one method is to only modify one amino acid with a water-
Desai and co-workers1 lies within the simplicity of the approach soluble polymer as it has been done in the past 40 years.8
while being highly effective during treatment of cancer. The Conjugation of hydrophobic polymer chain is a less explored
capacity for albumin to adsorb hydrophobic molecules as well pathway resulting in biohybrid amphiphiles with a hydrophilic
as the advantage of discharging the drug via desorption without protein head group conjugated to a hydrophobic polymer tail
significant burst effects makes it a unique carrier for a con- which self-assembles to form micelles in water.9–13 The synth-
trolled release system.2 Due to pathophysiological conditions in esis of a well-defined biohybrid amphiphile requires precise
neoplastic tissue, high amounts of albumin accumulate in control over both the protein/polymer ratio and the site at
tumours, and metabolization in malignant cells is enhanced which the protein is modified.9,14
by specific receptor glycoproteins gp60 on the endothelial cell
In this Communication, we have demonstrated the success-
surface. These provide a mediated albumin transport pathway ful synthesis of albumin polymer conjugated nanocarriers and
to the subendothelial space and more importantly, even dis- also its ability to deliver drugs without losing the specificity of
tribution of the drug in tumour site.3 Many formulations so far albumin (Fig. 1).15 Maleimide-terminated poly(methyl metha-
use a single albumin molecule as carrier with the drug either crylate) (PMMA) has been prepared via the reversible addition
physically absorbed or chemically bound.3 Processing many fragmentation chain transfer (RAFT) polymerization. After drug
albumin molecules into nanoparticles represents a quantum loading the formation of micelles took place in an aqueous
leap in performance since additional accumulation of the drug
in the tumour site was caused by the enhanced permeation
retention (EPR) effect.3,4
The nab technology produces particles of sizes of well above
100 nm. This may limit its use to certain application since some
cancers, such as pancreatic cancers, seem to have an appetite
for particles below 50 nm.5 Moreover, the driving force of
nanoparticle formation is the presence of hydrophobic drug
Centre for Advanced Macromolecular Design (CAMD), School of Chemistry,
University of New South Wales, Sydney, NSW 2052, Australia.
E-mail: M.Stenzel@unsw.edu.au
† Electronic supplementary information (ESI) available: All experimental proce-
Fig. 1 Synthesis approach to albumin micelles tailored to hydrophobic
drugs.
dures as well as all raw data and the investigations in the optimization of the
process. See DOI: 10.1039/c4cc00616j
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Table 2 IC50 values of curcumin deliver with or without micelle, evaluated
after 48 hours (1: curcumin, 2: POEGMEMA-PMMA micelle, 3: albumin-PMMA
micelle)
environment. These biohybrid micelles possess a built-in bio-
functionality through the presence of the protein based head
group compared to PEG based hydrophilic head groups, which
is typically used for polymeric micelles.
For the attachment of hydrophobic polymer chains to albu-
min, one of the prerequisite is that the polymer chains must
have high reactive end group fidelity for the conjugation.
PMMA was primarily chosen as the suitable hydrophobic block
to be attached to albumin, but the process should be suitable
IC50 (mM) A 2870
(cancerous)
IC50 (mM) AsPC-1
(cancerous)
IC50 (mM) CHO
(healthy)
1
2
3
15.4
8.38
2.09
11.5
12.89
4.33
10.07
6.6
11
for any hydrophobic block including degradable polymers. For polymers attached. It is commonly assumed that dimerization
proof of concept, PMMA was prepared with a RAFT agent that is the result of coupling of Cys-34, which is possibly the main
contains the functionality of furan protected maleimide, but a attachment point for polymer conjugation, but other reports
myriad of other reactive groups for protein conjugation are highlight that dimerization is not necessarily prevented when
possible.16 The main challenge is the slow addition of the Cys-34 has been reacted.17 Further investigations are therefore
albumin solution in PBS buffer to the polymer solution in necessary to understand the co-existence of dimers and
DMSO. Increasing amounts of water in the DMSO solution polymer-conjugates. In addition, the conjugation efficiency
can lead to the precipitation of polymer while the presence of could be measured using GPC (ESI,† Fig. S5 and S6). Resuspen-
DMSO causes the protein to precipitate. The rate of polymer– sion of the freeze dried final product in THF led to the
protein conjugation needs to be faster than the change in extraction of the unreacted PMMA into the THF. In contrast,
solvent quality to obtain a stable system. The system was water will only separate free BSA while the conjugate remains
optimised by the slow addition of albumin containing aqueous insoluble. Based on the results from the GPC measurements,
solution to the DMSO solution over several hours with the help equimolar amount of albumin and PMMA led to a conjugation
of a syringe pump, followed by dialysis to remove DMSO. The efficiency of about 58%. (ESI,† Fig. S6) Results from the THF
size of albumin coated PMMA particles obtained were found to GPC, which suggests a conjugation efficiency of 87% (ESI,† Fig.
be dependent on the molecular weight of the hydrophobic S5), were neglected since the incomplete removal of unreacted
polymers and larger PMMA chains lead to larger nanoparticles PMMA from the particle is very likely. A control experiment was
(ESI† and Table 1). Therefore by simply changing the polymer carried out that enlisted PMMA without any reactive endgroup
length from Mn = 5000 g molÀ1 to 42 500 g molÀ1 the particle and BSA. Mixing of both compounds using similar conditions
size can be adjusted between 68 to 210 nm.
led to the formation of a cloudy solution with a broad particle
The formation of polymer–protein conjugates and the simul- size distribution. Analysis of the reaction mixture by washing it
taneous self-assembly into nanoparticles was not only con- with THF to extract unreacted PMMA revealed the easy separa-
firmed using DLS and TEM, but also with MALDI-TOF and tion of PMMA and BSA (ESI,† Fig. S7).
GPC using THF and water as the mobile phase. MALDI-TOF
It should be noted here that the resulting core–shell nano-
analysis gives direct evidence for the presence of the protein particles are not micelles in the traditional sense, but rather
drug conjugate at around 71 000 g molÀ1 (ESI,† Fig. S3 and S4), nano-precipitated aggregates with an albumin surface layer.
although it needs to be considered that ionization of the The detailed internal structure will be subject to further
polymer–protein conjugate compared to the free protein is investigations.
difficult and the height of the signals is not representative of
Subsequently, curcumin was employed as the model-drug,
the actual amounts. The spectrum shown in ESI,† Fig. S4 also not only because of its bright yellow colour which facilitates the
shows the presence of BSA dimers, which have potentially analysis, but also because of its anti-cancer properties.18 Cur-
cumin has recently been delivered using the nab-technology,
but the particles were well above 200 nm.19 Incorporating
curcumin in the synthesis of albumin–polymer conjugates
Table 1 Effect of the amount of curcumin and the ratio between DMSO
and water on the hydrodynamic diameter
results in three processes that take place simultaneously: poly-
mer–protein conjugation, self-assembly into micelles and drug
encapsulation into the PMMA compartments of the nano-
particle. For the following experiment, PMMA with a molecular
weight of Mn = 5400 g molÀ1 was employed. Initial investiga-
tions were devoted to the effect of the amount of curcumin used
during micelle formation process. Molar ratios between cur-
cumin and PMMA of 1 : 5 to 1 : 15 were employed for the
investigation (Table 1). The DLS analysis of the only slightly
opaque (ESI,† Fig. S8) curcumin loaded nanoparticles reveals a
desirable aggregation sizes below 100 nm with a clearly-
defined, narrow peak (ESI,† Fig. S9). A clear relationship
between the amount of curcumin used and the final particle
Curcumin :
polymer
Volume
Molecular
Volume of of PBS
weight/g molÀ1 molar ratio DMSO/mL buffer/mL Dh/nma
1
2
3
4
5
6
7
8
9
10
42 500
20 000
12 000
8500
5400
5400
5400
5400
5400
5400
—
—
—
—
2
2
2
2
2
3
3
3
2
4
8
8
8
8
8
7
7
7
8
6
213 Æ 37
164 Æ 22
122 Æ 15
99 Æ 12
68 Æ 11
104 Æ 25
59 Æ 18
50 Æ 12
112 Æ 20
119 Æ 23
—
1 : 5
1 : 10
1 : 15
1 : 15
1 : 15
a
After dialysis against water.
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size is shown in Table 1 and Fig. S4 (ESI†). Higher amounts of
curcumin lead to larger particles. Included in this study was the
optimum ratio between DMSO and water. The drug-loaded
nanoparticle formation is a sensitive interplay between differ-
ent parameters. Drug encapsulation, polymer–protein conjuga-
tion and self-assembly need to occur prior to precipitation in
the DMSO–water mixture that can neither dissolve albumin nor
PMMA. A volume ratio of 30/70% (v/v) was deemed the most
suitable resulting in narrow particle size distributions (ESI,†
Fig. S10) while the overall hydrodynamic diameter was small
compared to the other mixtures (Table 1). Higher DMSO con-
tent led to premature precipitation of albumin while PMMA
cannot be dissolved in high water-contents.
Prior to further investigations in regards to the drug loading
efficiency, drug release and in vitro investigations a control
experiment was set-up to be able to compare the performance
of the albumin micelle with a tradition polymer micelle with
a poly[oligo(ethylene glycol) methylether methacrylate] (POEG-
MEMA) surface layer. To simulate the negative charge of the
albumin micelle (x = À30.1 mV), methacrylic acid (MAA) was
co-polymerized with OEGMEMA using RAFT polymerization.
The macroRAFT agent was then chain extended with MMA to
yield P(OEGMEMA104-co-MAA5)-b-PMMA76, yielding micelles
Fig. 2 (a) Size distribution and zetapotential measured by DLS of the
curcumin-carrying albumin-PMMA micelle (concentration of micelle
2 mg mLÀ1 in water); (b) the corresponding TEM image of the albumin
curcumin-carrying micelle (scale bar is 200 nm); (c) release of curcumin
from both micelles; (d) size distribution and zetapotential measured by DLS
of the curcumin POEGMEMA-PMMA micelle (concentration of micelle
2 mg mLÀ1 in water). (e) The corresponding TEM image of the curcumin
loaded POEGMEMA-PMMA micelle (scale bar is 200 nm); (f) cellular uptake
of albumin micelle by A2870 showing the localization of the green
fluorescent nanoparticles with the stained (red) lysosomes.
with the same hydrophobic compartment as the albumin- (CHO) cells (Table 2, entry 1). It needs to be noted here that the
micelles, but a PEG-based shell with a similar negative charge empty albumin-PMMA micelle is non-toxic. Drugs loaded into
(x = À28.9 mV). Two systems – one with albumin shell, the PEGMEMA based micelles increases the toxicity simply due to
other with a PEG based shell – were compared to identify the its improved drug uptake. This improved effect is due to the
effect of albumin. The drug encapsulation efficiency of albumin- change in uptake pathway where the drugs are now entering the
loaded micelle was depicted in Fig. 1 was determined using UV/Vis cell via an endocytic pathway instead of diffusion. Therefore
analysis and calculated to be 72% (ESI,† Fig. S11). The curcumin in compared to free drug, the PEG based micelles shows a higher
the POEGMEMA based micelle was loaded using similar concen- toxicity towards cancer cells, but also shows high toxicity
trations, but with a more traditional solvent exchange technique towards healthy CHO cells (Table 2, entry 2). Remarkably, the
(ESI†). The drug encapsulation efficiency was determined to be delivery of the drug into the two cancerous cell lines using
79%. The physical parameter of both albumin and POEGMEMA albumin-coated micelles seems to be enhanced, which leads to
based micelles are summarized in Fig. 2 highlighting their simila- higher toxicities. In contrast, the healthy CHO cells seemed to
rities. In addition, the rate of drug release was found to be similar be less affected when incubated with the albumin micelle
as well (Fig. 2(c)). This is not unexpected considering that the (Table 2, entry 3). This clearly highlights the selectivity of the
retention of the drug is influenced partly by the compatibility with albumin micelles towards cancerous cells. It has been high-
the polymer matrix, in both cases here PMMA.20
lighted earlier that the accumulation of albumin and albumin-
It is hypothesised that the replacement of the more tradition bound drugs in the tumour is facilitated by SPARC (secreted
PEG shell of the micelle by albumin has more advantages, not protein, acidic and rich in cysteine). It was reported that SPARC
just the biocompatibility factor as albumin is already abundant is an albumin bound protein which may play an important role
in the body. The uptake efficiency of both micelles was tested in the increased tumour accumulation of albumin-bound
against different cell lines and was determined visually by drugs.21 Interestingly, SPARC is overexpressed in several aggres-
confocal fluorescent microscopy (Fig. 2(f) and ESI,† Fig. S12). sive cancers but is absent in the corresponding normal tissues.
The fluorescent labelled micelles were found to be localized
In conclusion, initial results show that the use of albumin-
inside the endosomes within a time window of a few minutes. micelles is a simple and effective approach to deliver anti-
The efficient delivery of the anti-cancer drugs into the cell can cancer drugs. As depicted in Fig. 1, the nanoparticles can often
often be correlated to amount of cell death, which is expressed be prepared in one pot while the size can be adjusted by the
by the IC50 value. To investigate the cytotoxicity of these drug molecular weight of the polymer. The advantage of this system
loaded micelles the ovarian cancer cell line (A2870) and the is not only the simplicity in synthesis, but also the specificity of
pancreatic cancer cell line (AsPC-1) were used. A non-cancerous albumin towards cancerous cells. This technique can therefore
cell line, Chinese hamster ovary (CHO) was also tested to evaluate be described as inexpensive while being efficient to select
the selectivity of the albumin-carrier towards tumour cells.
cancer cells and considered to be able to deliver different types
In vitro studies (ESI,† Fig. S13–S15) show that free curcumin of drugs which will be further investigated in future. One aspect
is toxic to all cell lines including healthy Chinese hamster ovary that has not yet been touch upon is the activity of the albumin
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