Burst
Release of Lipophilic Drugs from Poly (Ethylene Oxide)-B-Polystyrene Micelles
is not Caused by Micelle Disassembly
Peng Zou, Hong-Wei
Chen, Hayley J Paholak, Du-Xin Sun
Peng
Zou, Hong-Wei Chen, Hayley J Paholak, Du-Xin Sun, Department of Pharmaceutical Sciences,
College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, the
United States
Correspondence
to: Du-Xin Sun, Department of Pharmaceutical Sciences,
College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, the
United States. duxins@umich.edu
Tel:
+01-734-615-8740
Fax:+01-734-615-6162
Received: May 4, 2013
Revised: June 21, 2013
Accepted:
June 23, 2013
Published
online: July 18, 2013
ABSTRACT
Burst release is one of the
challenges which limit clinical successes of block copolymer micelles for
targeted delivery of lipophilic anticancer drugs. It¡¯s generally assumed that
burst drug release in blood circulation is mainly due to blood dilution and
subsequent micelle disassembly after i.v. administration. Hence, investigating
the integrity of polymeric micelles is therefore essential to understanding the
mechanisms of burst release. In this study, lipophilic cargo was observed to be
rapidly released from poly(ethylene oxide)-b-polystyrene (PEG-PS) micelles into
PC-3 and MDA-MB-231 cancer cells before the micelles were internalized into
cells. Fluorescence resonance energy tranfer (FRET) imaging and fluorescence
quecnhing by iron oxide nanoparticles (IONPs) were used to assess in vitro
and in vivo integrity of PEG-PS micelles. The results showed that the
disassembly of PEG-PS micelles in cell culture media and fetal bovine serum was
very limited over 24 h. When PEG-PS micelles were internalized into cancer
cells, a majority of internalized PEG-PS micelles kept integrity. Rapid in
vivo disassembly of micelles upon blood dilution was not observed and the
micelles in mouse body gradually disassembled over 24 h. In summary, burst
release of lipophilic drugs from PEG-PS micelles was not caused by micelle
disassembly. This finding is useful for developing polymeric micelles with
controlled release of lipophilic drugs.
©
2013 ACT. All rights reserved.
Key words: Burst release; Fluorescence resonance energy transfer;
Polymeric micelle; Micelle disassembly; Fluorescence quenching; Anticancer drug delivery
Zou P, Chen HW, Paholak HJ, Sun DX. Burst Release
of Lipophilic Drugs from Poly (Ethylene Oxide)-B-Polystyrene Micelles is not
Caused by Micelle Disassembly. Journal of tumor 2013; 1(2): 7-15
Available from: URL: http://www.ghrnet.org/index.php/jt
INTRODUCTION
Premature release is one of the challenges which limit clinical
successes of block copolymer micelles for targeted anticancer drug delivery[1,2].
In polymeric micelles, polymer unimers always exist in a dynamic equilibrium
with the micelles at concentrations above the critical micelle concentration
(CMC). It¡¯s generally assumed that burst drug release in blood circulation is
mainly due to blood dilution and subsequent micelle disassembly after i.v.
administration[3,4]. However, rapid cargo release was detected even
when the blood concentrations of polymers were much higher than their aqueous
CMC[5,6], suggesting burst release may not be caused by blood
dilution. In addition, serum proteins alpha- and beta- globulins were found to
accelerate lipophilic cargo release from poly (ethylene glycol)-poly
(D,L-lactic acid) (PEG-PDLLA) micelles[5,7]. It is generally
believed that contacts with biological fluids, macromolecules, proteins, lipids
and cells might cause disassembly of polymeric micelles[8]. However,
no direct evidence was provided to support that serum protein could trigger
rapid disassembly of micelles. To better predict and reduce burst release, it
would be worthwhile to investigate if the burst release is caused by micelle
disassembly.
Although the
disassembly of polymeric micelles in simple aqueous solutions has been studied
using classical microscopy-, spectroscopy-, and chromatography-based methods,
intracellular and in vivo disassembly is still hard to be monitored and direct
evidence for rapid in vivo disassembly of micelles is very limited[8,9].
The only reported effort to detect intracellular and in vivo disassembly
of polymeric micelles was the development of a fluorogenic-based approach[8].
In this design, a fluorogenic dye fluorescein-5-carbonyl azide diacetate
(F-5-CADA) was covalently conjugated to the lipophilic block end of poly
(caprolactone)-b-poly (ethyleneoxide) (PEG-PCL). The fluorescence was not
detectable until the PEG-PCL micelle was disrupted and the ester group of the
dye was cleaved. In vitro and in vivo micelle disruption was
monitored by the increase of fluorescence. However, the limitation of this
fluorogenic-based approach is obvious since it requires an additional F-5-CADA
activation step (ester cleavage). The estimation of disassembly was based on
two assumptions: F-5-CADA in the core of intact micelles cannot be activated
and F-5-CADA is immediately and totally activated upon in vivo
disassembly. However, ester cleavage might occur even though micelles keep
integrity. Furthermore, disassembly of micelles does not guarantee ester
cleavage.
Fluorescence
resonance energy transfer (FRET) has been employed to investigate lipophilic
cargo release from micelles and liposomes[5-7,10-12]. The proximity
(less than 10 nm) between FRET donor dye and FRET acceptor dye gives rise to
FRET effect, which is utilized to measure the distance between the two dyes.
The release of donor dye and acceptor dye from nanocarriers causes decrease or
disappearance of FRET. Although the reported FRET approaches can detect cargo
release from micelles, they are unable to detect micelle disassembly.
Covalently conjugating donor and acceptor dyes to lipophilic end of polymers
may provide an alternative FRET approach to detect micelle disassembly.
Due to their
high optical absorption, gold nanoparticles and iron oxide nanoparticles
(IONPs) are able to quench fluorescent dyes[13,14] and quantum dots[15,16]
in close proximity by attenuating both the excitation beam and the fluorescence
signal (inner filter effect). In previous reports, cationic
tetramethylrhodamine (TAMRA)[17] and sulforhodamine 101[18]
were conjugated to lipophilic end of block copolymers and the fluorescent
dye-labeled polymers were used to encapsulate oleic acid-coated lipophilic
IONPs. However, fluorescent dyes could not be self-quenched or quenched by
IONPs since the positive charge of the dyes prevented their intermolecular
interaction and close interaction with lipophilic IONPs. Furthermore,
conjugation with cationic or anionic dyes might change cellular internalization
process and intracellular distribution of nonionic block copolymers[19,20].
To achieve efficient fluorescence quenching by core-loaded IONPs and monitor
the integrity of nonionic micelles, neutral and nonpolar dyes are required.
Bodipy dyes
are relatively nonpolar and the chromophore is electrically neutral[21-23].
These properties tend to minimize dye-induced perturbation of conjugate
functional properties. In current study, a pair of dyes Bodipy-FL (FRET donor)
and Bodipy-TMR (FRET acceptor) was conjugated to polystyrene end of PEG-PS. The
conjugation with neutral and nonpolar Bodipy dyes allowed the formation of a
compact micelle core, which resulted in FRET effect between the two dyes. The
FRET effect was utilized to monitor in vitro disassembly of PEG-PS micelles.
Furthermore, IONPs were encapsulated into the core of PEG-PS micelles to quench
the Bodipy dye conjugated to polystyrene end. Fluorescence recovery was
utilized to detect in vivo disassembly of polymeric micelles.
MATERIALS AND METHODS
Materials
Lipophilic IONPs (10 nm) and SuperMag Separator™ were supplied by Ocean
NanoTech (Springdale, AR). Carboxyl-PEG-PS (9.5 kD-b-18 kD) and PEG-b-PS-NH2
(5 kD-b-5 kD) were purchased from Polymer Source Inc. (Dorval, Quebec, Canada).
Fluorescein-5-carboxamide cadaverine (5-FAM cadaverine, ex/em 490/521 nm) was
purchased from AnaSpec (Fremont, CA). Bodipy-FL (ex/em 504/513 nm), Bodipy-TMR
(ex/em 535/574 nm), Bodipy-665 (ex/em 650/665 nm) succinimidyl ester and DiI
were purchased from Invitrogen (Carlsbad, CA). Sephadex LH-20 and PD-10
desalting columns were purchased from GE Healthcare (Piscataway, NJ). Centrifugal
filter units (MWCO 10 kD) were purchased from Millipore (Billerica, MA).
Dialysis tubing (MWCO 3.5-5 kD) was supplied by Spectrum Laboratories, Inc
(Rancho Dominguez, CA). Tetrahydrofuran (THF) and other chemical reagents were
purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO).
Synthesis of
fluorescence-labeled PEG-PS
Bodipy dyes were conjugated to polystyrene end of PEG-PS (5 kD-b-5 kD)
polymer. PEG-PS-FL polymer: A total of 30 mg of PEG-PS-NH2 (3 µmol) was
dissolved in 2 mL anhydrous THF. 10 mg of Bodipy-FL succinimidyl ester (24
µmol) was added under stirring. To the mixture, 10 ¦ÌL of triethylamine was
added and the reaction continued overnight under stirring. Bodipy-FL labeled
PEG-PS (PEG-PS-FL) was purified using Sephadex LH-20 columns in triplicate. The
purified PEG-PS-FL in THF was filtered through a 0.45 µM filter and dried under
vaccum. 30.6 mg of powder was obtained and stored at -20ºC. 1H-NMR
(400 MHz, CDCl3) ¦Ä ppm 7.39 (s, 0.3 H), 6.25-7.2 (brm, 250 H),
3.57(t, 450 H), 3.28 (t, 1 H) 3.21 (t, 1 H), 2.60 (t, 1 H), 2.51 (s, 1.3 H),
2.26 (s, 1.3 H), 2.16 (t, 1 H), 1.78 (brm, 50 H), 1.36 (brm, 100 H). PEG-PS
polymer was labeled with Bodipy-TMR (PEG-PS-TMR polymer) and Bodipy-665
(PEG-PS-665 polymer) following similar procedures. PEG-PS-TMR: 1H-NMR
(400 MHz, CDCl3) ¦Ä ppm 7.87 (d, 1 H), 7.27 (d, 1 H), 6.25-7.2 (brm,
250 H), 3.85 (s, 1.4 H), 3.58(t, 450 H), 3.20 (t, 1 H), 2.75(t, 1 H), 2.53 (s,
1.3 H), 2.41 (t, 1 H), 2.17 (t, 1 H), 2.13 (s, 1.3 H), 1.79 (brm, 50 H), 1.36(brm,
100 H). PEG-PS-665: 1H-NMR (400 MHz, CDCl3) ¦Ä ppm 7.56
(m, 1 H), 7.40 (s, 0.4 H), 6.25-7.2 (brm, 250 H), 4.57 (s, 1 H), 3.57 (t, 450
H), 3.29 (t, 1 H), 2.17 (t, 1 H), 1.79 (brm, 50 H), 1.36 (brm, 100 H).
5-FAM was
conjugated to PEG end of PEG-PS (9.5 kD-b-18 kD) polymer. A total of 110 mg of
carboxyl-PEG-b-PS (4 µmol) was dissolved in 2.5 mL anhydrous THF. 82.5 mg of
DCC (0.4 mmol) and 69 mg of NHS (0.6 mmol) were added to the polymer solution
and stirred for 20 min. Following the addition of 92.1 mg of 5-FAM cadaverine
(200 µmol), 10 ¦ÌL of triethylamine was added to the mixture and the reaction
continued overnight under stirring at room temperature. 5-FAM labeled PEG-b-PS
(5FAM-PSO-PS) was purified using Sephadex LH-20 columns. The purified
5FAM-PEG-PS was dried under vaccum and stored at -20ºC. 1H-NMR (400 MHz, CDCl3)
¦Ä ppm 10.44 (s, 0.44 H), 8.41 (d, 0.69 H), 8.02 (dd, 0.64 H), 7.64
(d, 0.72 H), 7.47 (d, 0.70 H), 6.25-7.20 (brm, 880 H), 3.58 (t, 906 H), 3.24
(t, 1.6 H), 2.98 (t, 1.5 H), 2.55 (t, 1.5 H), 1.67-1.84 (brm, 180 H), 1.20-1.36
(brm, 350 H).
Fabrication of
polymeric micelles
IONP-loaded PEG-PS-665 micelles were prepared by a precipitation
method. 10 mg of PEG-PS-665 was dissolved in 0.5 mL of THF and 1mg of IONPs was
added to the solution. 2 mL of deionized water was dropped at a speed of 6
mL/min using a syringe pump (Fisher Scientific, Pittsburgh, PA) under vigorous
stirring and followed by 20 min of stirring. The solution was then dialyzed
against 2 liters of deionized water for 2 days. Water was changed every day.
IONP-loaded nanoparticles were isolated and concentrated using a magnetic
separator (Ocean NanoTech, Springdale, AR). Finally, the IONP-loaded
nanoparticles were resuspended in PBS (equivalent to 0.5 mM
polymer) and filtered through a 0.45 µm filter, and stored at 4¡ãC. By following
this procedure, IONP-free PEG-PS-FL, PEG-PS-TMR, and PEG-PS-665 micelles were
prepared. 10 mg of PEG-PS-FL and 10 mg of PEG-PS-TMR were dissolved in 0.5 mL
of THF to prepare FRET micelles. The micelles were concentrated using a 10 kD
MWCO centrifugal filter unit and suspended in PBS (equivalent to 1 mM of
polymers). 5-FAM labeled and DiI-loaded PEG-PS micelles were prepared by
following similar procedures. Briefly, 10 mg of 5FAM-PEG-PS and 0.1 mg of DiI
were dissolved in 0.5 mL of THF. 2 mL of deionized water were added under
vigorous stirring. After dialysis, the micelle solution passed through a PD-10
desalting column (GE Healthcare, Piscataway, NJ) to remove unencapsulated DiI.
The micelles were suspended in PBS (equivalent to 0.5 mM polymer).
Characterization
of polymeric micelles
Average hydrodynamic sizes of micelles were measured using a Zetasizer
Nano ZS particle sizer (Malvern Instruments Ltd, Westborough, MA). The
morphology of IONP-loaded polymeric micelles was obtained on a Philips CM-100
transmission electron microscope (TEM). Fluorescence spectra of various
micelles were measured on an LS55 PerkinElmer luminescence spectrometer
(Waltham, MA) with an excitation at 488 nm for Bodipy-FL and FRET effect, 520
nm for Bodipy-TMR and 610 nm for Bodipy-665. To evaluate quenching effect of
IONPs on fluorescence of Bodipy-665, IONP-loaded micelles were suspended in PBS
or THF to record the fluorescent spectra. To measure FRET ratios, FL/TMR FRET
micelles and mixed FL micelles and TMR micelles were excited at 488 nm and the
emission scan was 500-620 nm. The FRET ratios were calculated as FRET
ratio=IFRET/ (IFRET+IFL)[6]. IFRET and IFL are the fluorescence
intensity at 570 nm and 513 nm respectively.
Integrity of
micelles in PBS, cell culture media and fetal bovine serum
Aliquots of FL-TMR FRET micelles were incubated in phosphate-buffered
saline, RPMI 1640 with 10% fetal bovine serum (FBS), or 100% FBS in triplicate
in a 96-well plate at 37ºC for 24 h. The final incubation concentrations of
polymers were 50 µM and 5 µM. Fluorescence of Bodipy-FL (ex/em 488/513 nm) and
FRET (ex/em 488/570 nm) were measured using a Synergy plate reader (BioTek,
Winooski, VT) at 0.5 h, 6 h and 24 h. The FRET ratios were calculated as FRET
ratio=IFRET/ (IFRET+IFL). Meanwhile, the FRET ratio of FRET micelles in
THF/water (50%/50%) was measured as a control. The FRET ratio of mixed
PEG-PS-FL micelles and PEG-PS-TMR micelles (molar ratio 1:1) in PBS was also
measured as a negative control.
Fluorescence
microscopy and FRET confocal microscopy
A prostate cancer cell line PC-3 and a breast cancer cell line
MDA-MB-231 obtained from American Type Culture Collection (ATCC, Rockville, MD)
were cultured on 8-well Lab-Tek glass chamber slides (Thermo Fisher Scientific,
Rochester, NY) for FRET and fluorescent imaging. 104 cells per well were
incubated for 2 days to allow cell adherence. To visualize DiI release and
micelle internalization, 5-FAM labeled and DiI-loaded PEG-PS micelles
(equivalent to 10 µM polymer) were incubated with cancer cells at 37¡ãC for the
desired lengths of time before imaging. Nuclei were stained with Hoechst.
Images were obtained with TRITC (DiI), FITC (5-FAM) and DAPI channels on a
Nikon TE2000S epifluorescence microscope coupled with a standard mercury bulb
illumination, a CCD camera (Roper Scientific, Tucson, AZ). Images were overlaid
using MetaMorph® software (Molecular Devices Corporation, Sunnyvale,
CA).
To track
intracellular disassembly of micelles, cancer cells were incubated with 50 µM
of FL/TMR FRET micelles for 8 h. In parallel, cells were incubated with mixed
PEG-PS-FL micelles (25 µM) and PEG-PS-TMR micelles (25 µM) for 8 h as a
control. The FRET confocal images were acquired by using a digital camera
(C9100, Hamamatsu Photonics, Japan) mounted on a Visitech VT Infinity 3
array-scanning confocal system (VisiTech International Ltd., United Kingdom)
attached to a Nikon TE-2000U microscope with a 60X Nikon Plan Apo
water-immersion objective at room temperature. Images were recorded under
Bodipy-FL channel (488 nm excitation, 535¡À20 nm emission), FRET channel (488 nm
excitation, 580¡À20 nm emission) and Bodipy-TMR channel (543 nm excitation,
580¡À20 nm emission). The exposure time was 200 ms. Images were obtained using
MetaMorph v6.5.3 (Universal Imaging, Malvern, PA). The images were
background-subtracted using the ¡°Background Correction¡± tool in MetaMorph.
Crosstalk correction and FRET ratios calculation were carried out using an
in-house FRETCalculator program and Matlab. To correct crosstalk, correction
coefficients ¦Á and ¦Â were determined from cells incubated with PEG-PS-TMR
micelles only (¦Á=IFRET/ITMR) and PEG-PS-FL micelles only (¦Â=IFRET/IFL)[24-26].
IFRET, ITMR, and IFL were intensities in each region of interest (ROI) under
FRET, Bodipy-TMR, and Bodipy-FL filter sets, respectively. FRET ratios were
calculated as: FRET ratio=IFRET / (IFRET+IFL).
Xenograft mice,
in vivo and ex vivo fluorescent imaging
The animal procedures were performed according to a protocol approved
by the University Committee for the Use and Care of Animals (UCUCA) at
University of Michigan. Female athymic nude mice (nu/nu), obtained from
National Cancer Institute (Bethesda, MD) at 8 weeks of age, were subcutaneously
inoculated in the back with 5¡Á106 PC-3 cells suspended in a mixture
of 50 µL of PBS and 50 µL of matrixgel basement membrane (BD Biosciences, San
Jose, CA). When the tumor implants reached 0.8 cm in diameter, the tumor-bearing
mice were subjected to the imaging studies.
In vivo
fluorescence imaging was performed with an IVIS Spectrum imaging system
(Xenogen, Alameda, CA). The exposure time was 1 s. Images were acquired and
analyzed using Living Image 2.5 software (Xenogen, Alameda, CA). Images were
recorded under an excitation of 640 nm and an emission of 680 nm at 2 h and 24
h after i.v. administration of PEG-PS-665 micelles (equivalent to 0.1 µmole of
polymer) or IONP-loaded PEG-PS-665 micelles (equivalent to 0.1 µmole of
polymer). Simultaneously, 0.2 mL of PEG-PS-665 micelles or IONP-loaded
PEG-PS-665 micelles in an eppendorf
tube (10 µM in THF, PBS or mouse plasma) were imaged with mice. At the
end of experiment, the mice were sacrificed using CO2. Blood and
tissues (tumor, heart, lung, liver, spleen, and kidneys) were collected and
tissues were rinsed with PBS. Blood and tissue samples were imaged using the
identical settings as in vivo imaging.
RESULTS
Rapid release of DiI from 5FAM-PEG-PS micelles
Rapid release of lipophilic cargos from PEG-PDLLA,
PEG-PCL, PEG-b-poly 4-(vinylpyridine) (PEG-PVPy) and
PEG-b-distearoylphosphatidylethanolamine (PEG-DSPE) micelles has been detected in
vitro[6,7,12,27] and in vivo[5]. To
investigate cargo release from PEG-PS micelles, PC-3 cells were incubated with
5-FAM and DiI dual-labeled PEG-PS micelles (Figure1). After incubation for 1 h,
encapsulated DiI was released into cells and accumulated in endosomes and
lysosomes. No significant uptake of 5-FAM labeled micelles was observed (Figure
1). In contrast, at 8 h, both 5-FAM labeled micelles and DiI were internalized
into cells and co-localized in endosomes and lysosomes. Similar results were
observed in MDA-MB-231 cells (Figure 2). The results suggested that lipophilic
cargo and PEG-PS micelles were not internalized into cells simultaneously. This
is consistent with previous reports which revealed that DiI dye was rapidly
released from PEG-PDLLA[6], PEG-PCL[6], PEG-PVPy[12]
and PEG-DSPE[7] micelles prior to cellular uptake of the micelles.
Characterization
of FRET micelles and PEG-PS-665 micelles
To elucidate the mechanisms of rapid release of DiI
from PEG-PS micelles, it is worthwhile to test in vitro and in vivo integrity
of PEG-PS micelles. Bodipy dyes (FL, TMR and 665) were conjugated to the amine
group at the end of polystyrene block. Lipophilic IONPs were entrapped in the
core of PEG-PS-665 micelles to quench Bodipy-665. The average hydrodynamic
sizes of Bodipy dye labeled micelles ranged from 35 nm to 46 nm (Table 1).
Incorporation of 10% IONPs increased the average size of PEG-PS-665 micelles
from 40 nm to 167 nm.
Figure 3a shows the diagrams and fluorescence spectra of FL micelles,
TMR micelles and FL/TMR FRET micelles. FL/TMR micelles in PBS were excited at
488 nm. The formation of a compact lipophilic core of FL/TMR FRET micelles gave
rise to the FRET effect between FL and TMR with 480 nm excitation (Figure 3a,
red line). FRET ratio was determined to be 0.78¡À0.02 (Figure 3b). When mixed FL
micelles and TMR micelles (molar ratio 1:1) in PBS was excited at 480 nm, no
FRET effect was observed (Figure 3a, blue line). The fluorescence at 570 nm was
due to spectral crosstalk. The FRET ratio was 0.24¡À0.02. The disassembly of
FL/TMR FRET micelles in 50% THF decreased FRET ratio to 0.31, indicating that
the decrease of FRET ratio can be utilized to detect the disassembly of
micelle.
Integrity of
PEG-PS micelles in PBS, cell culture media and FBS
FL/TMR FRET micelles were incubated in PBS, RPMI 1640 with 10% of FBS and
100% FBS at concentrations of 50 µM and 5 µM. Fluorescence of Bodipy-FL and
FRET were measured at 0.5 h, 6 h and 24 h. The decrease of FRET ratio was used
to detect the disassembly of FRET micelles. As shown in Figure 4a, FRET ratios
of FRET micelles were 0.75 at 5 µM and 0.77 at 50 µM. At concentrations of 50
µM and 5 µM, the FRET ratios of micelles in PBS were constant over 24 h,
indicating that PEG-PS micelles were not disrupted in PBS within 24 h.
In RPMI 1640
media with 10% of FBS (Figure 4b), the FRET ratio of FL/TMR micelles (5 µM)
decreased from 0.76 at 0.5 h to 0.73 at 24 h, indicating a slight increase of
the mean particle size of micelles or the disassembly of a small fraction of
micelles. Similarly, when FL/TMR micelles (5 µM) were incubated in 100% of FBS,
FRET ratios decreased from 0.74 at 0.5 h to 0.72 at 24 h (Figure 4c). The
results suggested that a majority of micelles kept their integrity in PBS, RPMI
1640 and FBS within 24 h.
FRET confocal
imaging
To detect intracellular disassembly of micelles, PC-3 cells were
incubated with FL/TMR FRET micelles (Figure 5 a1-a4) and mixed FL micelles and
TMR micelles (Figure 5 b1-b4) in parallel. FRET signals were detected on both
cell membranes and intracellular organelle membranes after incubation with
FL/TMR FRET micelles for 8 h (Figure 5 a4), indicating the integrity of
micelles during cellular internalization process. The FRET ratios detected from
endosomes/lysosomes were slightly lower than that from cell membranes,
suggesting a gradual disassembly of micelles in endosomes/lysosomes. In
contrast, no FRET was observed when the cells were incubated with mixed FL
micelles and TMR micelles (Figure 5 b4). Similar results were observed when MDA-MB-231
cells were incubated with FL/TMR FRET micelles (Figure 5 c1-c4) and mixed FL
micelles and TMR micelles (Figure 5 d1-d4).
Fluorescence
quenching by the formation of micelles and IONP-loaded micelles
As shown in figure 6a, the formation of core-shell micelles caused
quenching of Bodipy-665 fluorescence. The disassembly of PEG-PS-665 micelles (5
µM) in THF resulted in a fluorescent intensity (blue line) 5 fold higher than
that of intact micelles in PBS (green line). IONP-loaded PEG-PS-665 micelles (5
µM) showed a 21.4 fold higher fluorescence at 670 nm in THF (red line) than in
PBS (yellow line). TEM image of IONP-loaded PEG-PS micelles showed clusters of
10 nm IONPs in the cores of PEG-PS-665 micelles (Figure 6b).
In vivo micelle disassembly in xenograft
mice
Formation of compact PEG-PS-665 micelles and incorporation of IONPs
into PEG-PS-665 micelles dramatically quenched fluorescence of Bodipy-665.
Hence, the recovery of Bodipy-665 fluorescence was used to detect in vivo
disassembly of micelles. As shown in figure 7a, PEG-PS-665 micelles and
IONP-loaded PEG-PS-665 micelles in PBS and mouse plasma exhibited much lower
fluorescence than in THF, suggesting that both PEG-PS-665 micelles and
IONP-loaded PEG-PS-665 micelles kept their integrity in PBS and mouse plasma.
PEG-PS-665
micelles and IONP-loaded PEG-PS-665 micelles were i.v. injected to nude mice
with xenograft PC-3 tumors. At 2 h following the injection of PEG-PS-665
micelles, due to very limited disassembly of micelles, only very low
fluorescence was detectable (Figure 7a). At 2 h, fluorescence was not
detectable from the mouse treated with IONP-loaded PEG-PS-665 micelles.
However, at 24 h, both the mouse treated with PEG-PS-665 micelles and the mouse
treated with IONP-loaded PEG-PS-665 micelles showed dramatically increased
fluorescence. The large particle sizes of IONP-loaded PEG-PS-665 micelles
(167¡À43 nm) caused rapid elimination from the mouse body, which might explain
the lower fluorescence than that of the mouse treated with PEG-PS-665 micelles.
The data showed that micelle disassembly within 2 h was very limited and most
micelles gradually disassembled in mice.
The mice were
sacrificed at 2 h and 24 h following the injection of micelles to collect the
blood and tissues. Figure 7b showed the image of blood and tissue samples
collected from blank control mouse, the mice treated with PEG-PS-665 micelles,
and the mice treated with IONP-loaded PEG-PS-665 micelles. At 2 h after the
injection of IONP-loaded micelles, compared with blank control, slight
fluorescence increase was observed only in spleen and liver, which suggested
that micelles kept integrity in blood circulation, tumor, heart, lung and
kidneys. The disassembly in liver and spleen might be due to rapid uptake and
digestion by macrophages. At 24 h post the injection of IONP-loaded micelles,
increased fluorescence was detected in blood and tissues, indicating gradual
disassembly of micelles in mouse over 24 h. Consistently, blood and tissues
collected from the mice treated with PEG-PS-665 micelles showed dramatically
increased fluorescence from 2 h to 24 h, suggesting that a majority of PEG-PS
micelles disassembled between 2-24 h and there was no burst disassembly of
micelles.
DISCUSSION
To achieve targeted drug delivery and sustained drug release, it is
essential to understand the mechanisms of burst release and reduce the burst
release. In this study, rapid DiI release from 5-FAM labeled PEG-PS micelles to
cancer cells was observed prior to the cellular internalization of micelles
(Figure 1 and figure 2). Similar rapid lipophilic cargo release from PEG-PCL
micelles and PEG-PDLLA micelles was previously reported[6,27]. In
vivo burst release of lipophilic dyes from PEG-PDLLA micelles was observed
in mice[5]. A majority of dyes was released at 15 min post i.v.
injection. Our recent study showed that the in vivo release half-life of
lipophilic dyes from PEG-PS micelles was only 9.2 min (unpublished results).
The rapid release is unlikely caused by disassembly of micelles since polymer
concentrations in incubation and blood circulation were much higher than their
CMC. Furthermore, another study revealed that a majority (80%) of PEG-PCL
micelles kept their integrity even 20 h after their internalization into bladder
cancer cells[8], indicating disassembly is not the major cause of
rapid lipophilic cargo release.
One of the
reasons for limited understanding of in vivo integrity of micelles is that
currently there are no practical experimental methods available to detect the
disassembly of micelles without micelle modification[3]. Various
fluorescent dyes such as tetramethylrhodamine (TAMRA)[17,19] and
sulforhodamine 101[18] have been used to label the hydrophobic end
of block copolymers. Although the formation of micelles was observed, the
cationic dyes could not form a compact core and efficient fluorescence
quenching was not achieved. In our experiment, PEG-PS was labeled with 5-FAM or
TAMRA, IONPs entrapped in the core of micelles were not able to quench 5-FAM or
TAMRA (data not shown). In contrast, neutral and lipophilic Bodipy dye-labeled
PEG-PS formed micelles with a compact core, which allowed FRET effect between
Bodipy-FL and Bodipy-TMR. The encapsulation of IONPs could dramatically quench
the fluorescence of Bodipy-665.
The incubation
of FL/TMR FRET micelles with PBS showed that micelles kept integrity in PBS
within 24 h, which was consistent with previous report[8]. In RPMI
1640 media containing 10% of FBS and 100% of FBS, FRET ratios of micelles only
slightly decreased, suggesting that most micelles kept integrity and the
decrease of FRET ratio was likely due to particle size changes in different
media. In a previous report[8], incubation with RPMI 1640 media and
100% of FBS for 24 h caused 36% and 74% integrity loss of
poly(caprolactone)-b-poly(ethylene oxide) (PEG-PCL) micelles. However, the
micelles disassembly was probably overestimated since this method was based on
the activation of a dye F-5-CADA (to cleave an ester of the fluorescent dye to
generate fluorescence). Considering the dynamic equilibrium between PEG-PCL
unimers and micelles in the incubations, endogenous esterases in FBS could
rapidly activate F-5-CADA even without the disassembly of micelles. There
exists some evidence that enzymes possibly penetrate the hydrophilic shell of
the micelles. For example, it was observed that a micelle consisting of PEG-PCL
was slowly degraded in the presence of lipase K[28]. Additionally,
compared with PEG-PCL, PEG-PS polymer has a much lower CMC and higher glass
transition temperature of polystyrene (107ºC)[29], which might
explain the less disassembly of PEG-PS micelles in FBS.
FRET has been
utilized to investigated lipid exchange between micelles and between liposomes[30,31].
In current study, the FRET between Bodipy-FL and Bodipy-TMR was used to monitor
micelles disassembly. The FRET confocal imaging showed that PEG-PS micelles
kept integrity during cellular internalization and the micelles gradually lost
integrity in endosomes/lysosomes of cancer cells. This was consistent with a
previous study, in which 20% of internalized PEG-PCL micelles were disrupted
after incubation with bladder cancer cells for 20 h[8]. Ideally,
FRET effect between Bodipy dyes can be used to visualize the in vivo disassembly
of micelles. Unfortunately, a pair of near-infrared Bodipy dyes
(emission>700 nm) is still not commercially available. The available Bodipy
dye with the longest emission is Bodipy-665. Although IONP-loaded PEG-PS-655
micelles are able to detect micelle disassembly in dissected mouse tissues,
poor tissue penetration limits its application to noninvasive imaging of deep
tissues. Furthermore, the autofluorescence from tumors and liver detected at
emission 680 nm (Figure 7b, blank control) affects accurate detection of
micelle disassembly in these two tissues.
The exclusion
of micelle disassembly as a major cause of burst release from PEG-PS polymeric
micelles has its implications for reducing burst release. Various strategies
have been employed to reduce burst release by preventing micelle
disintegration, such as varying the lengths of hydrophilic and hydrophobic
blocks of the block copolymers, crosslinking of the core, and/or crosslinking
the shell. These approaches decrease CMC of micelles and prevent micelle
disintegration in the bloodstream but might not necessarily eliminate burst
release. On the other hand, a drug release kinetics study implied that the
release of lipophilic drugs from micelles could be triggered by ubiquitous
nanoscale acceptors in the body such as cell membranes and other lipids[32].
Hence, increasing lipophilicy of both the core of micelles and encapsulated
drugs might be a more efficient approach to reduce burst release. For example,
burst release of paclitaxel in mice was successfully reduced using
lipophilicity-enhanced PEG-PS micelles[33]. Specifically,
lipophilicity of paclitaxel was increased by conjugating lipid anchors to the
drug. The lipid-paclitaxel prodrugs and phopholipids were coencapsulated into
PEG-PS nanoparticles. The reduced burst release of paclitaxel by incorporating lipids into
micelles suggests that lipophilicity of micelle core plays a more inportant
role in burst release of lipophilic drugs than micelle disassembly.
Our in
vitro FRET imaging and in vivo fluorescence quenching study showed that no
burst disassembly of PEG-PS micelles was obsevred and internalized micelles
grdually disassembled in cells. Because micelle integrity depends on many
factors such as lipophilicity of core-forming block, the ratio of hydrophilic
and lipophilic blocks, and the degradation rate of polymer, extrapolations of the herein observed
integrity of PEG-PS micelles cannot be generalized. Rather, the integrities of
block copolymer micelles made of different polymers need to be investigated
separately.
ACKNOWLEDGMENTS
This work was partially supported by the National Institutes of Health
(RO1 CA120023 and R21 CA143474); University of Michigan Cancer Center Research
Grant (Munn); and University of Michigan Cancer Center Core Grant to DS. We
sincerely thank Dr. Andrew Wang (Ocean NanoTech, Springdale, Arkansas) for
generously providing iron oxide nanoparticles.
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Peer reviewers:
Myth T.S. Mok, PhD, Institute of Digestive Disease,
Li Ka Shing Institute of Health Sciences, Department of Medicine and
Therapeutics, The Chinese University of Hong Kong, Hong Kong, China; Sarah Van
Loo, Resident Vascular and Thoracic Surgery, Imelda Hospital, Imeldalaan,
2820 Bonheiden, Mechelen, Belgium.
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