-
Doxorubicin physical state in solution and inside liposomes
loadedvia a pH gradient
Xingong Li a, Donald J. Hirsh a, Donna Cabral-Lilly a, Achim
Zirkel b,Sol M. Gruner b, Andrew S. Jano a, Walter R. Perkins
a;*
a The Liposome Company, Inc., One Research Way, Princeton, NJ
08540, USAb Department of Physics, Cornell University, Ithaca, NY
14853, USA
Received 26 May 1998; accepted 9 September 1998
Abstract
We have examined doxorubicins (DOX) physical state in solution
and inside EPC/cholesterol liposomes that were loadedvia a
transmembrane pH gradient. Using cryogenic electron microscopy
(cryo-EM) we noted that DOX loaded to 200^300mM internal
concentrations in citrate containing liposomes formed linear,
curved, and circular bundles of fibers with nosignificant
interaction/perturbation of the vesicle membrane. The individual
DOX fibers are putatively comprised of stackedDOX molecules. From
end-on views of bundles of fibers it appeared that they are aligned
longitudinally in a hexagonal arraywith a separation between fibers
of approx. 3^3.5 nm. Two distinct small angle X-ray diffraction
patterns (oblique and simplehexagonal) were observed for
DOX-citrate fiber aggregates that had been concentrated from
solution at either pH 4 or 5. Thedoxorubicin fibers were also
present in citrate liposomes loaded with only one-tenth the amount
of doxorubicin used above(approx. 20 mM internal DOX concentration)
indicating that the threshold concentration at which these
structures form isrelatively low. In fact, from cryo-EM and
circular dichroism spectra, we estimate that the DOX-citrate fiber
bundles canaccount for the vast majority (s 99%) of DOX loaded via
a pH gradient into citrate buffered liposomes. DOX loaded
intoliposomes containing lactobionic acid (LBA), a monoanionic
buffer to control the internal pH, remained disaggregated
atinternal DOX concentrations of approx. 20 mM but formed
uncondensed fibers (no bundles) when the internal DOXconcentration
was approx. 200 mM. This finding suggests that in the citrate
containing liposomes the citrate multianionelectrostatically
bridged adjacent fibers to form the observed bundles. 13C-NMR
measurements of [1,5-13C]citrate insideliposomes suggested that
citrate bound to the DOX complex and free citrate rapidly exchange
indicating that the citrate-DOX interaction is quite dynamic. DOX
release into buffer was relatively slow (6 4% at 1 h) from
liposomes containingDOX fibers (in citrate loaded to a low or high
DOX concentration or in LBA liposomes loaded to a high internal
DOXconcentration). LBA containing liposomes loaded with
disaggregated DOX, where the internal DOX concentration was
onlyapprox. 20 mM, experienced an osmotic stress induced vesicle
rupture with as much as 18% DOX leakage in less than 10 min.The
possible implications for this in vivo are discussed. 1998 Elsevier
Science B.V. All rights reserved.
Keywords: Adriamycin; Remote loading; Cryo-electron microscopy;
TLC D-99; Evacet
0005-2736 / 98 / $ ^ see front matter 1998 Elsevier Science B.V.
All rights reserved.PII: S 0 0 0 5 - 2 7 3 6 ( 9 8 ) 0 0 1 7 5 -
8
Abbreviations: CD, circular dichroism; cryo-EM, cryogenic
electron microscopy; DOX, doxorubicin; EPC, egg
phosphatidylcholine;HBS, HEPES buered saline; LBA, lactobionic
acid; LUV, large unilamellar vesicle ; MLV, multilamellar vesicle ;
NMR, nuclearmagnetic resonance; SAXS, small angle X-ray
diraction
* Corresponding author. Fax: (609) 520-8250; E-mail :
[email protected]
BBAMEM 77484 1-12-98 Cyaan Magenta Geel Zwart
Biochimica et Biophysica Acta 1415 (1998) 23^40
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1. Introduction
The major limitation of doxorubicin (DOX), oneof the most widely
used anticancer agents, has beenits cumulative dose related
cardiotoxicity [1]. Lipo-some encapsulation has been shown to
reduce thecardiotoxicity of anthracyclines in animal models[2^5]
and one formulation of liposomal DOX (Eva-cet, formerly TLC D-99)
has been shown to be lesscardiotoxic in humans [6]. In this
formulation, en-capsulation has been made highly ecient (s 95%)by
taking advantage of DOXs accumulation to theliposome interior in
response to an inside-acidictransmembrane pH gradient. The
encapsulationprocedure has been referred to as remote
loading[7^9].
Transmembrane pH gradients can be created di-rectly, as in the
above case, by forming the liposomesin a well-buered solution of
low pH (e.g., 300 mMcitrate at pH 4) and then adding a more basic
sol-ution to raise the external solution pH [7]. pH gra-dients can
also be created indirectly by an electricalpotential which drives
protons to the vesicle interior[8] or from the electroneutral
outward ow of thecounterion to an entrapped acid (e.g.,
ammoniumsulfate [9]). With a pH gradient established,
DOXaccumulates in the vesicle interior and the ideal dis-tribution
of DOX in solution inside (Din) and outside(Dout) the liposomes is
expected to be related to theinner and outer H concentrations
by
DinDout
V inVout
K HinK Hout
1
where Vin and Vout are the internal and externalaqueous volumes
and K is the dissociation constantfor DOX (pK = 8.22 [10]).
However, we and others[11] have found that Din/Dout can exceed the
valuepredicted by Eq. 1. One explanation for this en-hanced
accumulation is that internalized DOX doesnot remain in solution.
Precipitation of DOX, forexample, from the internal solution would
facilitatemovement of additional DOX from the outside tosatisfy the
equilibrium relationship of Eq. 1. Infact, Lasic et al. [12,13],
employing an ammoniumsulfate gradient to load DOX into liposomes,
haveshown that in these systems internalized DOX formsaggregates.
Using cryo-electron microscopy, theywere able to visualize the
internalized DOX aggre-
gates and found structures similar to those caused
byprecipitation of DOX with sulfate anion in solution.
Aggregation could also explain the restriction ofDOX molecular
motions noted by Cullis and co-workers who studied DOX entrapped by
a trans-membrane pH gradient into citrate containing lipo-somes.
Although Cullis and co-workers have sug-gested that citrate could
cause precipitation of thisinternalized DOX [7,11], in more recent
work theyhave proposed that the attenuation of the DOX nu-clear
magnetic resonance (NMR) signal they ob-served was due entirely to
its binding to the lipo-somes inner surface [14]. Moreover, in
order toexplain the internalized structures they observed in-side
DOX liposomes by cryo-electron microscopy[15] they proposed that
DOX produces invaginationof the liposome bilayer membrane. Because
imagesof these liposomes were similar in appearance tothose
containing DOX-sulfate aggregates [13] andbecause we have noted
that DOX forms a precipitatein citrate solutions, we believed the
nature of DOXsphysical state in citrate bearing liposomes to
remainin question.
It was our desire here to study in detail the phys-ical state of
DOX inside citrate containing liposomes.We examined DOX in solution
and inside liposomesby various methods and found that DOX can
formhighly organized bundles of brous structures in thepresence of
citrate. Our results indicate that s 99%of DOX internalized in
liposomes at 20^300 mM ispredominantly self-associated and not
bound to theinner membrane surface.
To assess the extent to which counterion valencyinuences DOX
organization, we also examined lip-osomes in which the monoanionic
compound lacto-bionic acid (LBA) was used to buer the vesicle
in-terior. When a pH gradient was established and thesesystems were
loaded with DOX (approx. 200 mMDOX inside liposomes), brous
structures were ob-served, but unlike the structures we found in
citrateliposomes these bers were disorganized and notcondensed into
bundles. When these systems wereloaded with low internal DOX
concentrations no -bers were observed. Whether in this case DOX
wasbound to the liposome inner surface remains unclear,but membrane
invagination did not occur.
Because the physical state of DOX inside lipo-somes could be
manipulated, we ventured to examine
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23^4024
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its impact upon release. Leak proles were not dis-tinguishable
for DOX loaded at high internal con-centrations (approx. 200 mM
DOX) into either cit-rate or LBA containing liposomes. For DOX
loadedto low internal concentrations (approx. 20 mMDOX) where DOX
was either in ber bundles (cit-rate) or disaggregated (LBA),
dierent leak rateswere noted. However, we found this dierence tobe
due to an osmotic stress related rupture for theLBA liposomes. When
osmotic stress was avoidedthe leak proles were similar. Because in
the LBAliposomes DOX was disaggregated at the low DOXconcentration,
we speculate that ber formation mayplay an important role in
curtailing DOX loss invivo, an important concern considering the
long res-idence time that DOX liposomes have in the circu-lation
[16] and the fact that plasma constituents in-crease liposome leak
[17].
2. Materials and methods
2.1. Materials
Egg phosphatidylcholine (EPC) and cholesterolwere obtained from
Avanti Polar Lipids (Alasbaster,AL). DOX-HCl (Adriamycin RDF) was
purchasedfrom Farmitalia Carlo Erba (Milan, Italy). ForNMR, DOX
from Sigma (St. Louis, MO) wasused. Citric acid monohydrate,
ammonium sulfate,chromiumoxalate (trihydrate) (potassium
salt),4-oxo-TEMPO (TEMPONE) and 4-amino-TEMPOwere obtained from
Aldrich (Milwaukee, WI). [1,5-13C]Citrate was purchased from Isotec
(Miamisburg,OH). HEPES, cholesterol calibrator, and
cholesteroldiagnostic kits were purchased from Sigma. Octa-ethylene
glycol monododecyl ether (C12E8) was ob-tained from Fluka (Buchs,
Switzerland). Tetra(sulfo-natophenyl)porphine was purchased from
PorphyrinProducts (Logan, UT). Sodium carbonate anhydrouswas
purchased from J.T. Baker (Phillipsburg, NJ).All the chemicals used
were the highest purity avail-able.
2.2. Liposome preparation and lipid assay
Liposomes were made by extrusion of lipid suspen-sions rst made
by solvent evaporation. These struc-
tures were used instead of multilamellar vesicles(MLVs) because
we wanted to avoid localized soluteexclusion [18,19]. Freezing and
thawing the MLVswould not have attained an ideal solute
distributionbecause the high concentration of solutes used
hereaorded these systems cryoprotection. For the initialliposome
suspensions, chloroform stock solutions ofEPC and cholesterol
(approx. 20 mg/ml lipid) weremixed at 55:45 (mole) ratio,
accordingly. After add-ing 2.5 ml of either 300 mM citric acid (pH
4.0) or650 mM lactobionic acid (pH 3.6) into the chloro-form
mixture (approx. 25 ml), the chloroform wasremoved by rotary
evaporation at 40C. When essen-tially all of the chloroform was
judged to be removedan additional 2.5 ml of the appropriate buer
wasadded to the paste that had formed and the samplewas subjected
to further rotary evaporation at 40C.If necessary, the resulting
suspensions were then ad-justed with distilled water to a total
volume of 5 ml.These liposomes were then extruded through
twostacked lters of pore size 400 nm (one pass) andthen 200 nm (ten
passes) at 40C; lters were poly-carbonate Nuclepore membranes from
NucleporeCorporation (Pleasanton, CA). Liposome size wasdetermined
by light scattering using a Nicomp Model270/370 Submicron Particle
Sizer from Pacic Scien-tic (Menlo Park, CA).
The phospholipid concentration of all liposomesamples was
measured by a modied version of theprocedure of Chen et al. [20].
Cholesterol concentra-tions were determined using the cholesterol
diagnos-tic kit from Sigma. Absorbances were recorded on
anUV-2101PC UV-scanning spectrophotometer fromShimadzu Scientic
Instruments (Princeton, NJ).
2.3. Doxorubicin loading and leak assays
2.3.1. Loading of DOX into liposomesUnless stated otherwise
transmembrane pH gra-
dients were created by adding an aliquot of sodiumcarbonate
solution (17.6 mg/ml) into the liposomesamples. The pH of the
aqueous solution outsidethe liposomes ranged from 7.8 to 8.1. This
liposomesuspension was then mixed with a DOX saline sol-ution. The
nal overall DOX concentration was ei-ther 3.4 mM or 0.34 mM,
whereas the lipid concen-tration was approx. 12.8 mM. An aliquot of
10 mM4-amino-TEMPO was added if the pH gradient
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across the lipid bilayer was to be measured. To ac-celerate
loading, the sample was then heated to 55^60C for 10 min and cooled
to room temperature.
The loading eciency was determined using gelchromatography to
remove unencapsulated material.Aliquots of each sample were passed
down columnspre-equilibrated with 10 mM HEPES, 150 mM NaCl(HBS) at
pH 7.5. The liposome fraction was gatheredand this material and an
aliquot not passed down thecolumn were adjusted to an equivalent
volume withsaline and then assayed for lipid by phosphate
anal-ysis. Each sample was also assayed for DOX contentby
dissolution in methanol and measurement of theabsorbance at 490 nm.
The percent entrapment wascalculated as that percent of DOX
remaining withthe liposomes following elution; DOX concentra-tions
were normalized using the lipid concentrations.
For LBA, we found that 650 mM LBA maintainedpH gradients (3
units) for several hours without anymeasurable decrease thus
indicating that LBAs per-meability to these liposome membranes is
low; allsubsequent experiments were performed well withinthis time
frame. Additionally, 650 mM LBA allowedthe complete loading of DOX
at 3.4 mM DOX and12.8 mM lipid. At 500 mM LBA, the buer capacitywas
insucient to achieve complete DOX loading.
2.3.2. Doxorubicin leakage from liposomesTo compare DOX leak
from liposomes, an aliquot
of liposomal DOX was diluted 300-fold by injectioninto a cuvette
containing a solution of 10 mM HBS(pH 7.5). The uorescence
intensity of DOX wasmeasured continually with data collected every
10s. The excitation and emission wavelengths were480 nm and 590 nm,
respectively. At the end ofeach measurement, C12E8 was added to
dissolvethe liposomes and attain the 100% leakage value.The
percentage of leakage of DOX from liposomesat a given time was
calculated using Eq. 2:
% leakage I3I0I1003I0U100 2
where I was the uorescence intensity at a given timeand I0 and
I100 were intensities immediately after theinitial dilution into
HBS or after addition of C12E8,respectively. DOX uorescence
intensity (without lip-osomes) decreased linearly with time for
samplesmaintained above pH 7, due to DOX degradation
at higher pH values [10,21]. The magnitude of thisdecrease at pH
7 was small (6 2% per hour). There-fore, we did not adjust the leak
equation to reectthis loss. At pH 4 no decrease in DOX
uorescenceintensity was noted up to 7 h.
2.3.3. Measurement of vpH and captured volumeTo measure internal
pH, and thus vpH, we used
the distribution of an electron spin resonance (ESR)amine probe
[22]. The ESR probe 4-amino-TEMPOwas added to liposomes at the time
of DOX loading(nal concentration 200 WM). The sample was sepa-rated
into fractions and these fractions were thendiluted equally with
either a solution of buer (10mM HBS at pH 7.5) alone or with buer
containingthe broadening agent chromiumoxalate (trihydrate).The
solution containing broadening agent was rstadjusted to the same
osmotic strength as that of theliposome solution to avoid vesicle
shrinkage or swell-ing. All osmolarity measurements were
performedwith a 5500 Vapor Pressure Osmometer from Wescor(Logan,
UT). The ESR spectra of both fractionswere then recorded and the
vpH was calculated us-ing the following relationship:
vpH log10 AinAtot3AinUVoutV in
3
where Ain and Atot were the amplitudes of the ESRI = +1
resonance with and without the broadeningagent, respectively. Vin
and Vout were the aqueousvolume inside and outside the liposomes,
respec-tively. The values of Vin and Vout were obtained sep-arately
from the measurement of captured volume.
The captured volume of the liposomes was deter-mined using an
ESR procedure previously described[23] which is a slight modication
of the method ofAnasi et al. [24]. The ESR spin probe TEMPONE
(inthe appropriate buer) was added to the sample inquestion which
had rst been adjusted to pH 7^8 byaddition of sodium carbonate
(17.6 mg/ml) solution.Adjustment of the pH to above pH 5 was
necessaryto prevent leakage of the chromiumoxalate broaden-ing
agent that was to be added later. The liposomesample (38 mg/ml
lipid) containing TEMPONE (1mM) was separated into 100 Wl
fractions. To onefraction the same liposome buer solution (100
Wl)was added and to another faction the chromiumox-alate broadening
agent solution (100 Wl) was added.
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The ESR signal amplitudes with and without broad-ening agent
were then related to the inside and totalaqueous volumes,
respectively. Captured volume (Wl/Wmole lipid) was then derived as
previously describedusing a correction to account for the volume
occu-pied by the lipid [23].
2.4. Microscopy
For confocal microscopy, uorescence images wereacquired with a
Biorad 1000 Confocal Microscopefrom Olympus (Hercules, CA), using a
Kr/Ar laserwith Vex at 568 nm and Vem at 605 nm. For cryo-electron
microscopy, frozen-hydrated samples wereprepared by placing 5 Wl of
a liposomal suspensionon a copper grid with a holey carbon support.
Eachsample was blotted to a thin lm and immediatelyplunged into
liquid ethane. The grids were thenstored under liquid nitrogen
until used. The gridswere viewed on a Philips CM12 transmission
electronmicroscope (Mahwah, NJ) operating at an accelerat-ing
voltage of 120 kV. The microscope was equippedwith a Gatan 626
cryoholder (Warrendale, PA), andthe samples were maintained at
3177C duringimaging. Electron micrographs were recorded underlow
electron dose conditions at a magnication of60 000U and 1.4^1.7 Wm
defocus.
2.5. X-Ray diraction
Solution aggregates for X-ray diraction were pre-pared by mixing
equal parts of a solution of DOX(15 mg/ml) and 600 mM (nal conc.
300 mM) citrateat the appropriate pH. Aggregates immediatelyformed
and after 10 min the samples were concen-trated via centrifugation
(14 000Ug) and the super-natant removed. For the heated samples,
after aggre-gate formation the samples were heated to 55^60Cand
cooled back to room temperature prior to con-centration via
centrifugation. For liposomes contain-ing DOX, loaded liposomes and
empty liposomes (tocorrect for liposome scattering) were pelleted
via cen-trifugation at 210 000Ug for 2 h. Pelleted sampleswere
placed in 1.5 mm diameter glass X-ray capilla-ries which were then
ame sealed. X-Rays were pro-duced by a Rigaku RU-200 rotating anode
generatorat a typical loading of 40 kV and 60 mA. The Cu KKline at
1.54 A was selected and focused by Ni lter-
ing and double mirror Franks optics, which resultedin a typical
ux at the sampled of 7U107 X-rays/s.The sample temperature stage
was thermoelectrical-ly adjusted to the desired temperature to
within 0.1C. A two-dimensional X-ray detector basedon a 512U512
pixel Thomson CCD [25] was usedto acquire the data. The diraction
patterns wereconcentric powder pattern rings which were
azi-muthally integrated into one-dimensional plots of in-tensity
versus scattering angle. Typical integrated ex-posures ranged from
20 to 60 min in duration.
Diraction signals were analyzed over a q-rangeof 0.1^0.8 A 31,
where the wave vector q is denedby
q 4ZV
sina2
4
where a is the scattering angle and V is the wave-length of the
incident radiation. Based on the cryo-EM data, we assumed that the
X-rays basicallyprobed only two dimensions of the structure withthe
third dimension (along the bers axis) beingtoo large to be seen.
The diraction was analyzedin terms of a general oblique planar
lattice in whichthe two unit cell basis vector lengths, a and b,
are notconstrained to have the same length and in which thesmallest
angle between them, Q+Z/2, is constrainedonly in that 06 Q6 Z/2.
The general formula in twodimensions for the peak positions in
reciprocal spacewith Miller indices h, k is given by:
q2
2Z2 h2
a2 sin2 Q 2hk cos Q
ab sin2 Q k
2
b2 sin2 Q5
2.6. Fluorescence and resonance light scattering
DOX uorescence in solution was measured usingan Alpha Scan
Spectrouorometer from PTI (SouthBrunswick, NJ). Excitation and
emission wave-lengths were set at 480 nm and 590 nm,
respectively.For resonance light scattering measurements,
excita-tion and emission wavelengths were kept identical asthe
spectrum was scanned from 200 nm to 900 nm;this is essentially a
U90 light scattering experimentas a function of wavelength (see
Pasternack et al.[26,27] for details).
Tetra(sulfonatophenyl)porphineat pH 1 was used as a positive
control for RLS (R.Pasternack, personal communication).
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2.7. Spectrophotometric assays
2.7.1. Circular dichroism (CD) spectroscopyLiposomal DOX
suspensions, or DOX aqueous
solutions/suspensions containing saline, water, cit-rate, or
lactobionic acid were typically placed in a1 mm CD cell. The CD
spectra were taken atroom temperature (20^23C) from 700 nm to200 nm
on a J-710 spectropolarimeter (Jas-co, Easton, MD) and typically
the average of vescans was reported with removal of high
frequencynoise.
2.7.2. Turbidity and UV-Vis spectra measurementThe turbidity
(absorbance) and UV-visible spectra
of dierent DOX aqueous solutions/suspensions weremeasured using
a 1 cm pathlength quartz cell in aUV-2101 Spectrophotometer from
Shimadzu Scien-tic Instruments. For turbidity, absorbance
wasmeasured at 800 nm. This relatively high wavelengthwas chosen to
avoid any interference from DOX ab-sorption.
2.8. Dierential scanning calorimetry (DSC)
Calorimetry was performed using a MC2 Ultra-sensitive Scanning
Calorimeter (MicroCal, North-hampton, MA). Heating scans were
obtained from15C to 85C at a rate of 20C/h.
2.9. NMR spectroscopy
2.9.1. NMR sample preparationFor NMR samples, liposomes were
prepared as
described in Section 2.2 except that for citrate con-taining
liposomes, [1,5-13C]citrate was mixed withunlabeled citrate to give
either 17 or 47 mole %[1,5-13C]citrate. The nal concentrations of
citrateand LBA in which the liposomes were formed were270 mM and
650 mM, respectively. To establish apH gradient and to remove the
outer buer solu-tions, liposomes were passed down
polyacrylamidecolumns (Pierce, Rockford, IL) equilibrated with20 mM
HBS at pH 7.6. Only the rst 2/3 or so ofthe eluting liposomes were
used so as to guaranteeremoval of as much external buer as possible
andthe lipid concentration was then analyzed. DOX
(from Sigma) was then hydrated with saline and thenmixed with an
aliquot of liposomes and additionalHBS such that the nal lipid
concentration was 12.8mM lipid and the nal DOX concentrations
wereeither 3.4 or 0.34 mM DOX (2 mg/ml or 0.2 mg/ml). Upon mixing,
the liposomes were then heatedto 55^60C for 10 min. The samples
were then exam-ined by CD to conrm typical spectra and then usedfor
NMR measurements. Samples were stored at ap-prox. 5C. The NMR
signal of the citrate bueralone was used to gauge the amount of
citratetrapped inside the empty liposomes. This amountof citrate
was approx. 6.2 mM (total solution con-centration). At 3.4 mM
overall DOX and assumingcomplete loading, this corresponds to a
ratio of ap-prox. 1.8 citrate molecules per DOX molecule in
theliposome interior.
2.9.2. 13C-NMR spectroscopyData were acquired on a Bruker AC 300
NMR
spectrometer using a 10 mm broadband probe fromNalorac
Cryogenics Corporation. The resonantfrequency for 13C was 75.5 MHz.
Free inductiondecays (FIDs) were acquired with proton
decouplingusing a 20 kHz sweep width and 16K points. Nointernal eld
lock signal was used. Data for the T1measurements were acquired
using the saturation-re-covery method. Signal intensities (peak
areas) wereplotted versus time and t by the equation:I(t) =
I(0)[13exp(3t/T1)]. Data for the T2 measure-ments were collected
with the Carr-Purcell-Mei-boom-Gill pulse sequence. Where signal
intensitiesof dierent samples were compared, the FIDs werecollected
using a 14.8 Ws 90 pulse, a recycle delayv 5UT1, and proton
decoupling applied only duringthe data acquisition period. Other
FIDs were col-lected with the minimum recycle delay allowed bythe
spectrometer and the pulse width set to the Ernstangle for the
spins of interest. At the end of dataacquisition for a particular
sample, ethylene glycol,0.2% v/v, was added as an internal chemical
shiftreference (64.0 ppm) and a nal set of FIDs weresummed. The
Fourier transform of this data wasused to assign the chemical
shifts. Prior to Fouriertransformation, all FIDs were zero-lled to
32Kpoints and multiplied by an exponential decay corre-sponding to
3 Hz.
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3. Results
To load DOX into liposomes, Cullis and co-work-ers have used
transmembrane pH gradients(pHinW4, pHoutW7.5) created using either
citrate[7] or glutamate [28] to buer the vesicle interior.This work
was the basis for a liposomal DOX for-mulation (Evacet, formerly
TLC D-99) that is cur-rently being evaluated in clinical trials
[29^31]. Withthis strategy, DOX can be loaded to high
concentra-tions inside liposomes (200^300 mM internal
concen-trations). This is far in excess of DOXs solubilitylimit in
solution, which is 960 mM in water [12].The question then arises as
to what physical struc-ture does DOX adopt or does it bind to the
innermonolayer of the liposome. The purpose of ourstudy here was to
investigate the physical state ofDOX inside liposomes.
3.1. Doxorubicin in solution
As indicated in Table 1, the physical state of DOXin solution is
clearly inuenced by the valency of thecounterion. When combined
with the multianionscitrate or sulfate, DOX formed a viscous gel
whichupon shaking yielded brous structures (Fig. 1). Theresulting
structures were similar in appearance tothose previously reported
for DOX-sulfate aggre-gates [9]. No such structures were observed
forDOX in water or in solutions of glutamate or themonoanionic buer
lactobionic acid (LBA). Why
glutamate, which has two carboxylic acid groups,did not initiate
aggregate formation is unclear butit does also contain a cationic
amine group whichmight obviate assemblage of the DOX
structureselectrostatically. Because we were interested in
whathappens to DOX inside citrate containing liposomeswe focused
our attention upon citrate-doxorubicininteractions for the
remainder of the studies dis-cussed here.
The DOX-citrate structures we observed in solu-tion were
assembled into linear brous-like struc-tures. Upon close inspection
the larger strands werefound to be bundles of smaller bers, similar
in ap-pearance to micellar bers resulting from
porphyrinstacking/aggregation [32]. These brous
structuresdisappeared upon heating and reappeared with cool-ing.
Calorimetric scans of 4 mg/ml DOX in 300 mMcitrate revealed a broad
endotherm which was near43C at pH 4 but which was shifted upward to
57Cat pH 5; vH was 1.8 and 2.8 kcal/mole, respectively(data not
shown). The melting of DOX-citrate pre-cipitate may be similar to
the melt of hydrated mi-cellar crystals (Krat point) that has been
describedfor local anesthetics [33,34]. An exothermic peak wasalso
observed in the scans of both samples at approx.75 and 65C,
respectively, which was absent uponsubsequent heating cycles. A
visual check of heatedsamples conrmed that disaggregation was
associ-ated with the endothermic transition and not theexothermic
event. From TLC analysis, it appearedthat the exothermic transition
represented loss ofDOXs amino sugar moiety (not shown). In thework
we report here, samples in solution or invesicles were never heated
to beyond 60C.
3.2. Cryo-EM of DOX inside citrate liposomes
Shown in Fig. 2 are cryo-EM images of citratebearing liposomes
loaded with DOX. In all casesDOX formed brous aggregates inside the
liposomesand the liposome membranes (both inner and
outermonolayers) were well resolved with no observablemembrane
invagination. The arrow in Fig. 2A pointsto an end-on view of a
U-shaped bundle of DOXbers that are packed hexagonally. A similar
U-shaped bundle is seen in prole in Fig. 2B. FibrousDOX bundles
were apparent in nearly all liposomesand were either linear, curved
(some having a U-
Table 1Formation of DOX aggregates in solution
Counterion pH Aggregateformation
[DOX] at whichaggregationbeginsa (mM)
Citrate 4^7 yes 0.5^1.5Sulfate 4^7 yes 1.0LBA 4 no s 20b
Glutamate 4 no s 20b
aAggregation onset was measured by the increase in
turbidity(A800 nm) of DOX at 4 mM in the indicated
solutions.bDetermined visually not spectrophotometrically. DOX
predis-solved in water was mixed 1:1 with solutions. Final
counterionconcentrations were 300 mM for citrate, 650 mM for LBA,
110mM for sulfate, and 175 mM for glutamate. These
counterionconcentrations were selected as they are the
concentrations usedpreviously with DOX liposomes.
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shape), or circular where the bers apparently closedback upon
themselves. For many of the longer linearDOX structures, liposomes
were obviously elongatedto accommodate ber growth. Many of the
bundlesappeared to twist, as for example, the large
circularstructure in Fig. 2A. From the hexagonal pattern inFig. 2A,
the number of bers in that particular bun-dle appeared to be
approx. 50. This value wouldappear to vary since end-on views of
other U-shapedstructures (not shown) revealed a range of
12^60bers/bundle, all in a hexagonal arrangement. Fromthis end-on
perspective the average spacing betweenbers was estimated to be
3^3.5 nm. In some cases,striations could be observed along the
length of thebundles that alternated with non-striated regions.For
example, the arrows in Fig. 2C indicate repeat-ing striated regions
in the DOX brous bundles. Thedistance between the centers of these
striated regionsappeared to be approx. 50 nm. Interestingly,
meas-uring across the striations (perpendicular to bundleslong
axis) we estimated that the striated lines them-selves were approx.
3^3.5 nm apart suggesting that
these features are the side view of aligned rows ofbers. Such a
periodic alignment would occur if thehexagonally packed bundles
twisted about their cen-ter. In which case, as viewed from the side
the rowswould align with every 60 rotation and give theappearance
of striations. For some circular struc-tures, the striations
appeared to persist. Perhaps thepitch of the rotation and the
curvature weresynchronized or perhaps there was some
interactionwith the inner monolayer that allowed alignmentwith that
surface.
3.3. Small angle X-ray diraction of DOX aggregates
X-Ray diraction was used to better understandthe packing details
of the aggregates. Although wecould not suciently concentrate
liposomes for peakanalysis by small angle X-ray diraction (SAXS)
wewere able to examine the DOX-citrate aggregatespelleted via
centrifugation. Fig. 3 shows the dirac-tion from samples produced
at pH 4 and pH 5. ThepH 4 material at room temperature showed
similar
Fig. 1. Confocal light microscopy of DOX-citrate brous bundles.
DOX in water was mixed 1:1 with a citrate solution for a
nalconcentration of 4 mM DOX and 300 mM citrate at pH 4. Similar
DOX aggregates were observed in solutions of citrate at pH 5and
ammonium sulfate at pH 4 and 7 (not shown). Image was colored to
better visualize structures three-dimensionally. Bar length is5
Wm.
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diraction both before heating (Fig. 3A) and after aheating
protocol (see Section 2.5) meant to mimic thetemperature protocol
used to load liposomes, e.g.,heat briey to 55^60C and then cool
back downto 20C (data not shown). The arrows in Fig. 3Ashowed the
expected peak positions of an oblique
lattice of a = 33.6 A , b = 29.2 A , and Q= 40, as ob-tained by
a least squares t of Eq. 5 to the data.Because of the paucity of
diraction peaks, and theabsence of certain peaks (which may simply
be tooweak to be seen), the lattice assignment should betaken as
possible, but not unequivocal. The pH 5material, before heating,
yielded diraction very sim-ilar to the pH 4 material (data not
shown). However,upon heating to 55^60C and then cooling back to20C,
the diraction pattern changes to that seen inFig. 3B. An
unconstrained t to Eq. 5 yields a =33.7 A , b = 35.7 A , and Q=
61.48. This is withinthe measurement errors of the simple hexagonal
lat-tice (34.5 A basis length), indicated by the arrows inthe
gure.
A weak peak corresponding to a real space repeatof 10.1 A is
also seen in the data, as shown in Fig.3A (see the peak in between
the ones indexed with(2,0) and (3,3) in the gure). This does not t
theplanar lattice assignment and might be due to corre-lations
along the direction of the bers, that is to sayperpendicular to the
lattice plane.
From these data it would appear that doxorubicin-citrate ber
bundles can adopt two distinct packingarrangements. A simple
hexagonal packing arosewhen both a pH 5 environment and a
heating/cool-ing (recrystallization) step were employed for the
un-encapsulated solution bers. These conditions maybe met for
liposomal DOX since the interior pH ofthe loaded vesicles increases
to near pH 5 (see exam-ples in Table 3) and, as per our protocol,
liposomesare always heated during loading (see Section 2.3).
We tried to determine whether the oblique struc-ture is present
in the liposomes as well. As mentionedbefore, it was dicult to
directly index peak posi-
Fig. 2. Cryo-EM images (three elds) of DOX loaded into
lipo-somes buered by citrate. The nal DOX solution concentra-tion
was 3.4 mM which is estimated to be 200^300 mM DOXinternally. This
internal concentration was estimated basedupon DOX loading and the
liposome captured volume (see Ta-ble 2). The total lipid
concentration was 12.8 mM and the sam-ple was undiluted for
microscopy. The arrow in A indicates theend of a brous bundle in
which the bers are hexagonally ar-ranged with an approx. 3^3.5 nm
separation. In B, a circular,U-shaped, and straight DOX-citrate
complex are noted. The ar-rows in C indicate striated regions in
the DOX brous bundleswith a repeating distance of approx. 50 nm.
Bar represents 100nm.6
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tions for the DOX-citrate loaded liposomes ^ Fig. 3C(upper
pattern) shows an example of the diractionpattern of pelleted DOX
loaded liposomes. Any sig-nal arising from a DOX-citrate lattice in
the lipo-
somes would be superimposed on a signal arisingfrom the liposome
shells. Hence we adopted the fol-lowing procedure: an image was
taken of the DOXloaded liposomes as well as one of the empty
lipo-somes (not shown). Assuming that the total scatter-ing arises
from the product of the liposome formfactor and the structures
factor arising from theDOX-citrate complex, we divided the
diractionfrom the DOX loaded liposomes by that of theempty
liposomes (Fig. 3C, top pattern). Even withpoor statistics, one can
see that the resultant peakscoincide with those of the DOX-citrate
complexformed in solution at pH 4 (lower pattern of Fig.3C). This
suggests that the oblique lattice may alsobe present in the
liposomes. Note that the resolutionof the cryo-EM images does not
allow us to discrim-inate between a simple hexagonal lattice and
aslightly skewed hexagonal lattice.
3.4. Circular dichroism of doxorubicin in liposomes
As expected, unentrapped DOX that was mixedwith empty liposomes
exhibited a CD pattern consis-tent with disaggregated dimeric DOX
[35,36] (seeFig. 4). This spectrum was identical to that for
Fig. 3. Small angle X-ray diraction patterns of
DOX-citratecomplexes in solution at (A) pH 4 and (B) pH 5 or in
pelletedliposomes (C). For the pH 4 sample (A), the pattern is from
anunheated sample: heating and cooling the sample did not
ap-preciably change the diraction pattern. The arrows show
theexpected peak positions for the oblique lattice described in
thetext. Diraction from the samples at pH 5 before heatinglooked
very similar to samples at pH 4, e.g., as shown in A.The pattern
for pH 5 samples changed to that shown in Bupon heating and
cooling. The arrows show the expected peakpositions for the
hexagonal lattice described in the text. Theupper pattern of C is
that derived for the DOX-citrate complexinside loaded liposomes
(see text for details). Shown for refer-ence, the lower pattern of
C is that for DOX-citrate complexpelleted from solution at pH
4.
Fig. 4. CD spectra of DOX loaded into citrate containing
lipo-somes via a pH gradient where the nal DOX concentrationwas
either 3.4 mM (solid line, left y-axis) or 0.34 mM (dashedline,
right y-axis). Internal DOX concentrations were estimatedto be
200^300 mM and 20^30 mM, respectively. Also shownis 3.4 mM DOX
mixed with empty liposomes with no pHgradient (dotted-dashed line).
Liposomes (approx. 100 nmLUVs) contained 300 mM citrate and lipid
concentration was12.8 mM.
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DOX in water without liposomes (data not shown).Loading of DOX
into citrate containing liposomesdramatically changed its CD
spectrum (Fig. 4). TheCD spectrum of loaded DOX indicated that a
sig-nicant change in DOX interactions had occurred,consistent with
the observed structure formation.
When DOX was loaded to a ten times lower con-centration into
liposomes the CD signal was nearlythe same (see Fig. 4). As shown
in Fig. 5, cryo-EMimages of these loaded liposomes revealed
similarinternalized structures to those found in liposomesloaded to
the ten times higher internal DOX concen-tration. The dimensions of
the internal DOX struc-tures were smaller in both length and
breadth ascompared to those of Fig. 2 with fewer bers perbundle.
Because of these images and because thereseemed to be little
contribution to the CD patternfrom disaggregated DOX we believe the
complexedform is the predominant DOX species inside
citratecontaining liposomes. In fact, for liposomes loadedto the
higher internal concentration of DOX we es-timate from analysis of
CD spectra that the DOX-ber species accounts for greater than 99%
of thetotal DOX.
3.5. Inuence of citrate upon doxorubicin dimerization
To understand more about how citrate interactswith DOX we
examined whether the citrate anioninuences the association of DOX
monomers intodimers. This association occurs at very low
DOXconcentrations in water (from 5 to 20 WM) and onemight expect
citrate to inuence this conversion if thedimer is arranged such
that the two positive chargesfrom the sugar moieties can be bridged
by the citratemultianion. We compared the CD spectra of DOX
ineither water or a solution of 300 mM citrate over therange of
concentrations at which monomer to dimerconversion is expected
(data not shown). At 5 WMDOX, CD spectra were similar to each other
and tothat previously reported for monomeric DOX anddaunorubicin
[35,36]. While there were changesfrom 5 WM to 1 mM that were
consistent with mono-mer to dimer conversion [35^37], the spectra
forDOX in water and citrate were similar to one anoth-er up to 1 mM
DOX. However, at 2 mM DOX theCD patterns began to dier signicantly,
consistentwith our assessment of aggregation by turbidity (Ta-ble
1). From these data it does not appear that cit-rate inuences DOX
dimerization. This might sug-gest that dimerized DOX molecules are
orientedsuch that the cationic amines cannot be bridged bythe
citrate multianion. This nding, along with ourother data, also
suggests that citrate induces aggre-gation by coupling DOX
dimers/oligomers (dimersinto oligomers and/or oligomers into
side-by-sidepacked ber bundles).
3.6. 13C-NMR measurements of citrate
To better understand the interactions betweenDOX and the citrate
counterion inside liposomes,we used 13C-NMR to examine internalized
citrate.The terminal carboxylic acid carbons of citrate, car-bons 1
and 5, are chemically identical and, at anyparticular pH, give rise
to a single resonance in the13C-NMR spectrum. In our NMR studies of
citratebuered liposomes, the 13C-NMR signal arising fromthe
terminal carboxylic acid carbons was enhancedby the presence of
[1,5-13C]citrate. To simplify ourdescription of the NMR results, we
will refer to theresonance arising from these terminal carboxylic
acidcarbons as the citrate resonance.
Fig. 5. Cryo-EM image of DOX loaded into citrate
containingliposomes where the nal DOX concentration was 0.34 mM.The
internal DOX concentration was estimated to be 20^30mM. The lipid
concentration was 12.8 mM. Bar represents 100nm.
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In citrate buered liposomes, the citrate resonancefrom inside
the liposome was shifted and broadenedby the loading of DOX (Fig.
6). The chemical shiftsobserved are consistent with an increase in
the inter-nal pH of the DOX loaded liposomes (see Table 3).We
believe that the broadening of the citrate reso-nance is due to a
distribution of internal pH valueswithin the liposomes, produced by
DOX loading.The modest decrease in T2 of the citrate resonancewhich
occurs with DOX loading (Table 2) cannotaccount for the magnitude
of the line broadeningobserved. In LBA buered liposomes, loading
withDOX induced similar changes in the chemical shiftand line width
of the resonance arising from the
carboxylic acid carbon of internal LBA, (data notshown).
Because cryo-EM images show that DOX formedbrous structures
inside citrate buered liposomes,one might expect that some fraction
of the citratewould also be immobilized. If so, this citrate
mightnot be observed in the solution state spectrum due tolifetime
broadening or dipolar broadening of its res-onance. Assuming a
charge ratio of 3:1, citrate toDOX, and complete loading of the DOX
into theliposomes, one would expect that approx. 18% ofthe citrate
would be associated with DOX throughelectrostatic interactions
alone. However, the signalintensity from citrate is, to within the
uncertainty inour measurement, unchanged by the loading of
DOX(approx. 200 mM inside) (Table 2). What we do ob-serve is a
modest decrease in the T1 and T2 of thecitrate signal (Table 2),
suggesting that citratebound to DOX bers may be in rapid
exchangewith free citrate.
3.7. LBA liposomes
The DOX structures inside liposomes that formedwith citrate were
similar in appearance to thoseformed with the divalent sulfate
anion [13]. To de-termine whether these internalized DOX
structuresrequired a multivalent counterion in order to form,we
next examined liposomes loaded with DOX usingthe monoanionic buer
LBA to control pH; the ini-tial internal pH was approx. 4 and the
outside pHwas raised to approx. 7.4. Cryo-EM of LBA lipo-somes
containing DOX at internal concentrations
Fig. 6. Eect of DOX loading on the 13C chemical shift andline
width of the citrate resonance. 13C-NMR spectra of EPC/cholesterol
liposomes with an initial pH gradient created by270 mM citrate, pH
4.0, inside and 20 mM HBS at pH 7.5 out-side. After the exchange of
external citrate buer for 20 mMHBS, pH 7.0, some residual citrate
remained outside the lipo-somes. This resonance is labeled Co in
the gure above. Theresonance due to internal citrate is labeled Ci.
Subsequent tocreation of the pH gradient the liposomes were loaded
with:(A) 0 mM, (B) 0.34 mM (20^30 mM internal concentration)and (C)
3.4 mM DOX (200^300 mM internal concentration).The lipid
concentration was 12.8 mM. Internal pH values asdetermined from the
chemical shifts were (A) pH 4.0, (B) pH4.2^4.0, and (C) pH 5.5^4.1,
consistent with the values of Table3.
Table 2T1, T2, and normalized intensity of the 13C resonance
from[1,5-13C]citrate inside liposomes
[DOX](mM)
T1 (s) T2 (s) Normalized intensitya
0.0 6.1 0.5 0.16 0.03 ^0.34 4.9 0.2 ^ ^3.4 2.6 0.4b 0.09 0.03c
1.01 0.08c
4.5 1.0aIntensities were normalized to that of citrate with 0
mMDOX.bThe internal citrate gave rise to two broad resonances in
theseliposomes. The rst T1 corresponds to the more
downeldpeak.cThese numbers reect the integrated area of both
resonances.
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of 20^30 mM DOX did not reveal any noticeablestructures (Fig.
7A); an occasional ber was the ex-ception. Again no membrane
invagination wasnoted. The CD pattern for DOX loaded into
LBAliposomes at this low concentration was consistentwith
disaggregated dimeric DOX (Fig. 8). At internalDOX concentrations
of approx. 200^300 mM, far inexcess of DOXs solubility limit, what
appeared to benumerous uncondensed bers were observed in
theliposomes (Fig. 7B). The CD pattern for these un-condensed DOX
bers was similar to that for DOXber bundles in citrate bearing
liposomes except that
the shoulder at 520 nm was decreased relative to theother peaks
(Fig. 8); this shoulder could be sensitiveto side-by-side ber
interactions.
Apparently, DOX ber formation occurs in LBAliposomes only at DOX
concentrations in excess ofits aqueous solubility limit, which you
will recall isapprox. 60 mM in water [12]. LBA is incapable
ofbridging charges so the bers that formed above thesolubility
limit of DOX remained uncondensed. Thisimplies that the DOX ber
bundles observed insidecitrate containing liposomes are the result
of the cit-rate multianion electrostatic bridging between sepa-rate
bers. Although citrate may have simply elimi-nated charge
repulsion, thus allowing an otherwisefavorable association to
occur, this seems less likelyto be the reason for condensation
since LBA couldhave also served this purpose but did not.
3.8. Eect of DOX physical state upon liposomalrelease
Having established that the DOX aggregationstate inside the
liposome can be manipulated, wenext ventured to make comparable
liposomes con-taining either citrate or LBA to establish the eectof
DOXs physical state upon the release rate. Thephysical properties
of these liposomes are listed inTable 3. Liposomes contained either
650 mM LBA
Fig. 7. Cryo-EM images of DOX loaded into liposomes bu-ered by
LBA. (A) Liposomes contained LBA (650 mM) asbuer and the solution
concentration of DOX was 0.34 mM,which would give an internal [DOX]
of approx. 20^30 mM. (B)LBA liposomes loaded such that the nal
solution concentra-tion of DOX was 3.4 mM, which we estimate to be
200^300mM DOX internally. The lipid concentration was 12.8 mM
andsamples were examined undiluted for microscopy. Bar repre-sents
100 nm.
Fig. 8. CD spectra of DOX loaded into liposomes containingLBA as
buer. Final solution DOX concentrations were 3.4mM (solid lines,
left y-axis) or 0.34 mM (dashed lines, righty-axis). The nal lipid
concentration was 12.8 mM. For thelower concentration of DOX in LBA
liposomes, the CD signa-ture indicated that DOX was predominantly
disaggregateddimers.
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or 300 mM citrate, both at pH approx. 4 initially,and were
loaded to low (16^29 mM) or high (160^290 mM) internal
concentrations of DOX. (ForLBA, 650 mM was required to achieve
completeDOX loading; 500 mM LBA was insucient (notshown).) The
liposomes were diluted into hepes bu-ered saline and leak monitored
as described in Sec-tion 2.3.
In all cases where the internalized DOX formedbers, the leak
proles were indistinguishable withless than approx. 4% leak after
60 min (data notshown). For those LBA liposomes in which DOXwas
loaded to a low internal concentration (no -bers), the leak proles
were biphasic and variablewith, in some cases, a signicant loss of
material inthe rst few minutes (see leak values at 10 min ^Table
3).
When samples were diluted into a hyperosmotic(approx. 1060 mOsM)
solution of buer containing20% (w/v) sucrose the liposomes
displayed similarleak proles (6 4% leak at 60 min) for all four
sys-tems of Table 3 (bers and no bers) (data notshown). This
indicated that osmotic stress was in-volved for the LBA containing
liposomes with a
low internal DOX concentration (no bers). Thiswas not too
surprising since the expected osmoticdierential for the LBA
containing liposomes dilutedinto hepes buered saline is approx. 565
mOsMwhich is close to the rupture threshold dierentialof 600 mOsM
reported by Mui et al. [38] for spher-ical EPC/cholesterol
liposomes of similar size andcomposition. Considering this, the
variability inleak for DOX from LBA containing liposomes (nobers)
was not too surprising either since those lip-osomes may have been
teetering on the thresholdamount of stress required to cause
rupture.
The reason why DOX leak from the LBA lipo-somes loaded to a high
DOX internal concentration(bers) did not also display a rapid
component isunclear, but in those liposomes DOX did form bersthat
may have been restricted sterically from escap-ing the vesicle.
Alternatively, it may be that only theLBA liposomes containing the
lower amount of dis-aggregated-DOX were osmotically stressed. That
is,the cationic DOX bers that formed at higher DOXinternal
concentrations may have bound a signicantportion of the LBA
molecules, and thus reduced theosmotic dierence.
Table 3Properties of loaded liposomes
Buer Physical state ofDOX inside
[DOX](mM)
Internal [DOX](mM)
Cap. volume(Wl/Wmole)
Mean vesicle size(nm)
FinalvpH
InternalpH
% leak at10 min
Citrate bers (bundles) 3.4 290 0.9 124 2.5 4.9 0.83.4 290 0.9 99
1.8 5.6 1.1
bers (bundles) 0.34 29 0.9 124 2.9 4.7 0.70.34 29 0.9 124 ^ ^
0.10.34 29 0.9 99 2.6 4.6 0.10.34 29 0.9 99 2.6 4.8 0.1
LBA bers 3.4 260 1.0 120 1.0 6.4 0.83.4 250 1.1 120 0.6 6.8
0.13.4 160 1.6 94 2.4 5.0 6 0.13.4 200 1.3 115 2.4 5.0 1.3
no bers 0.34 24 1.1 120 2.4 5.0 1.30.34 16 1.6 94 3.2 4.2
1.70.34 16 1.6 94 3.2 4.2 0.50.34 20 1.3 115 3.1 4.3 13.10.34 20
1.3 115 ^ ^ 18.40.34 20 1.3 115 3.1 4.3 9.7
Liposomes were made in either 300 mM citrate or 650 mM LBA at pH
4. Vesicle sizes, captured volumes, and pH measurementswere
performed as outlined in Section 2. The listed DOX concentrations
were the overall solution values prior to dilution;
internalconcentrations were estimated based on captured volume,
lipid concentration, and loading eciency (for all cases s 95%) (see
text).vpH and internal pH values were determined for vesicles
following DOX loading. Leakage experiments were performed
immediatelyafter DOX loading (6 1 h) and the data represent the
percent leakage at the 10 min point along the leak curves (not
shown) for sam-ples diluted into HEPES buered saline.
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4. Discussion
In solution, citrate, like sulfate, caused DOX ag-gregation to
occur at concentrations 100 times lowerthan its aqueous solubility
limit. Inside citrate con-taining liposomes, DOX loaded via a pH
gradientwas found to exist in ber bundles by cryo-EMwith no
observable membrane invagination. Whilethe straight ber bundles
were somewhat similar inappearance to the DOX-sulfate aggregates
observedby Lasic and co-workers [12,39], we also notedcurved and
circular ber bundles. These appearedto result from the bending of
the linear structuresthat had grown in length beyond what the
liposomewas capable of accommodating. End-on views ofthese
DOX-citrate ber bundles indicated that thebers were packed
longitudinally in a hexagonal ar-rangement with a separation of
approx. 3^3.5 nmbetween bers. Two distinct diraction patternswere
also observed by SAXS for DOX-citrate berbundles in solution, an
oblique and a simple hexag-onal lattice. Which of the two, or if
both, are presentinside liposomes remains unclear since cryo-EM
does
not have the resolution to distinguish between a sim-ple
hexagonal and a slightly skewed hexagonal pack-ing. Even so the
basis lengths noted by SAXS likelycorrespond to the interber
distance estimated bycryo-EM, both approx. 30^35 A . The repeating
stria-tions observed for ber bundles suggests that theentire bundle
twists 60 approximately every 50 nm.This is consistent with a
twisting hexagonal latticesince, as viewed from the side, the
rotation of a hex-agonal array aords spatial alignment every 60
(seeFig. 9). The fact that the peaks of Fig. 3B do notcorrespond
exactly to a hexagonal pattern may beconsistent with our assumption
that we have aslightly tilted hexagonal structure that is
twisting.
Because for DOX-sulfate aggregates only straightbundles (rods)
were noted by cryo-EM [12,13], theappearance of curved and circular
bundles herewould seem to indicate that our DOX-citrate berbundles
are more exible. Since only individual bersformed in LBA liposomes
(Fig. 2B) we believe thatbundles formed in citrate containing
liposomes dueto interber crosslinking by the citrate
multianion.This would then suggest that the dierence between
Fig. 9. Schematic representation of twisting hexagonally
arranged bers. In this proposed model each ber is comprised of
stackedDOX molecules. The lower illustration depicts how the
stacked DOX molecules (circles) would appear as viewed end-on. As
viewedfrom the side (upper illustration), the bers are aligned
along the line of sight at rotation multiples of 60 thus giving the
appearanceof repeating striations. For an oblique lattice, the
interber distance across each striated zone would vary. This
variation might not beresolvable in the cryo-EM images if the
obliquity is only slightly dierent from hexagonal, as seems to be
the case here, based onSAXS results.
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DOX-sulfate and DOX-citrate ber bundles stemsdirectly from
dierences in the counterions them-selves. Although the interber
spacing for DOX-cit-rate aggregates was 30^35 A the separation
noted forDOX-sulfate bers was reported to be approx. 27 A
[12]. This tighter packing is consistent with the factthat
sulfate is a smaller ion. Additionally, ber ex-ibility may also
involve dierences in the multi-anions binding anity. Although we
have no senseof sulfate binding we did nd here from NMR
ex-periments with 13C-citrate that the citrate-DOX in-teraction was
quite dynamic with rapid exchange offree and bound citrate.
We have not detailed how DOX forms bers butgiven its planar
structure it seems likely that DOXmolecules stack. Because citrate
did not aect mono-mer to dimer conversion one might argue that
theamino sugar moieties are separated far enough thatcitrate could
not bridge/couple monomers. Interest-ingly, upon stacking/
aggregating porphyrins exhibita signicantly increased scattering
intensity at theabsorption wavelengths; this phenomenon has
beentermed resonance light scattering (RLS) [26,27,40]. Inour
hands, the DOX brous structures did not ex-hibit RLS (data not
shown). This result, though notconclusive, may indicate that RLS is
a specic phe-nomenon of the large porphyrin ring systems.
For DOX loaded into liposomes containing themonoanionic buer
LBA, its physical state was de-pendent upon the internal DOX
concentration.
When loaded to only approx. 20 mM DOX, therewere no discernible
structures inside LBA liposomesand the CD pattern was consistent
with disaggre-gated DOX. It is quite interesting that this CD
pat-tern was identical to that for dimeric and not mono-meric DOX
because Gallois et al. [36] using CD havereported that DOX
partitioned onto large unilamel-lar vesicles (LUVs) was monomeric
and not dimeric.While this might suggest that no binding of DOX
tothe membrane occurred we cannot rule out that forour liposomes
DOX binds to the membrane surfaceas the dimeric species. In any
case, no membraneinvagination was noted.
When loaded into LBA liposomes at high internalconcentrations
DOX formed bers. Again no mem-brane invagination was noted by
cryo-EM. Unlikethe DOX in citrate containing liposomes, these
berswere disorganized and not condensed into bundles
(Fig. 8B). Apparently DOX stacking to form bersoccurs above its
solubility limit regardless if whetherthere is a multivalent
counterion present or not.These results with LBA would also seem to
conrmthat the ber bundles in citrate containing liposomesarise
because the citrate multianion facilitates inter-ber
crosslinking.
Interestingly, this crosslinking of bers by citratedid not
dramatically slow the rate of DOX release asit was similar to that
for DOX out of LBA liposomeswhere the DOX was disaggregated. From
13C-NMRexperiments it was apparent that bound DOX wasin rapid
exchange with free DOX. Such a dynamicrelationship would be
consistent with the unhinderedleak of DOX from the citrate
containing liposomes.However, because liposome integrity/stability
is amajor concern in vivo, ber formation (and berbundles in
particular) may enhance the stability ofDOX loaded liposomes by
providing a possible sterichindrance to vesicle rupture. It has
been shown thatlipoprotein adsorption onto PC/cholesterol
LUVssignicantly decreased their ability to withstand os-motic
stress [41]. Consequently, liposomes with un-complexed DOX may leak
more signicantly in vivo(work now underway).
Cullis and co-workers proposed that DOX is pre-dominantly bound
to the inner monolayer [14] andthat this binding leads to an
invagination of themembrane [15]. From the work presented here it
isobvious that citrate produced organized aggregatedDOX-citrate
structures inside liposomes. Also ob-vious from our cryo-EM images
is that membranemorphology is unaected by DOX loading. The
con-clusion by Cullis and co-workers that membrane in-vagination
occurred was based upon less well re-solved cryo-EM images (DOX
liposomes lookedlike coee beans) where the DOX-citrate ber
ag-gregates appeared as blurred lines inside the lipo-somes which
could be mistaken for a membraneedge [15]. To discard any
possibility that DOXfrom dierent vendors might yield a dierent
inter-action, we examined three dierent DOX vendor ma-terials (one
which included methyl paraben and twothat did not) and found
identical structures insideliposomes (data not shown). Cullis and
co-workersfound that DOXs 13C-NMR signal was signicantlybroadened
when internalized in citrate bearing lip-osomes [14]. While they
interpreted this as due to
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inner monolayer binding, our data clearly indicatethat the
explanation for this molecular immobiliza-tion is caused by DOX
complexation with citrate. Aswe have demonstrated here, packing of
adjacentDOX bers into bundles was facilitated by citratewhich
lowered the solubility limit of DOX signi-cantly. Although we
cannot rule out that someDOX may be partitioned into the inner
monolayer,we believe that this DOX accounts for only a
smallpercentage of the total internalized DOX.
Acknowledgements
This work was supported by the Liposome Com-pany, Inc. We thank
Dr. Eric Mayhew for his assist-ance performing the confocal
microscopy, Dr. Rob-ert Pasternack for a fruitful discussion of
resonancelight scattering, and Rao Fu for help creating Fig. 9color
graphic. X-Ray instrumentation developmentin SMGs lab is supported
by the Dept. of Energy(DEFG02-97ER624434).
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