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Experimental Therapeutics |
Departments of Biomedical Engineering [G. K.] and Mechanical Engineering and Material Science [G. A., D. N.], Duke University, Durham, North Carolina 27708, and Departments of Medicine [W. P. P., O. M. C.] and Radiation Oncology [R. D. B., M. W. D.], Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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42–45°C and releases drug over 30 min, and LTSL is a
new low temperature sensitive liposome that triggers in the range of
39–40°C and releases drug in a matter of seconds. Because
of the different attributes of the liposomes, it was possible to
delineate the relative importance of liposome drug encapsulation, HT
cytotoxicity, HT-drug interaction, HT-induced liposomal delivery, and
HT-triggered liposomal drug release in achieving antitumor activity.
Athymic nude mice bearing the FaDu human tumor xenograft were given a
single i.v. dose of 5 mg/kg of DOX (free drug or liposome
encapsulated), and the tumors were then heated to either 34°C or
42°C for 1 h at 34°C. All treatment groups were similar,
achieving low concentrations of DOX (0–4.5 ng/mg). At 42°C, the LTSL
(25.6 ng/mg) achieved the highest DOX concentration
(P < 0.04), but all three liposomal
formulations (7.3–25.6 ng/mg) were higher than saline or DOX (0–0.7
ng/mg; P < 0.02). LTSL + HT
was also the only group that resulted in significant amounts of
DNA-bound DOX (silver nitrate-extractable fraction;
P < 0.02). Tumor tissue sections were
visualized for DOX fluorescence to investigate the local distribution
of the drug in the tumor and confirm the relative drug concentrations
based on fluorescence intensity. There was relatively little
fluorescence seen with treatment groups at 34°C. At 42°C, the LTSL
showed the most DOX fluorescence (P < 0.01), and the fluorescence, although not homogeneous, was pervasive
throughout the tumor sections. Therapeutic efficacy of treatments was
determined from tumor growth time. At 34°C, the only treatment group
significantly better than the saline group (9.8 days) was the NTSL
group, with a growth time of 20.9 days (P < 0.02). At 42°C, all three liposomal formulations were more
efficacious than DOX. LTSL + HT had the longest growth time
(51.4 days) and the most number of local controls at 60 days (six of
nine tumors). With HT, the DOX concentrations and fluorescence were
tightly correlated with tumor growth delay, indicating that adequate
(increased) drug delivery can be predictive of therapeutic effect.
Overall, the LTSL + HT group showed the largest DOX
concentration, the highest and most pervasive DOX fluorescence, and the
most antitumor effect. Thus, HT-triggered liposomal drug release may
account for the largest differential therapeutic effect and
demonstrates the importance of rapid drug release from the drug
carriers at the tumor site. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the search to overcome these delivery issues, liposomes have been identified as promising carriers for therapeutic agents in the treatment of cancer (4 , 5) . By encapsulating cancer chemotherapeutic agents within liposomes, preferential delivery to the tumor can be attained (6) . Initially, liposomes were plagued with rapid opsonization and uptake by the reticuloendothelial system, resulting in a short circulation time on the order of minutes. This has largely been resolved by incorporation of lipid-grafted PEG3 into the liposome membrane, which reduces opsonization and increases the circulation time to hours or days (7) . PEG also increases vascular permeability to liposomes facilitating increased accumulation of drug containing liposomes in tumor tissue (8) . Despite these advances, current clinically approved liposomal formulations (6) have still resulted in only modest increased efficacy for the treatment of cancer (9 , 10) . The actual advantage of current liposomal formulations is reduced toxicity and not increased therapeutic effect. Increasing the circulation time of liposomes has not been sufficient, and other modifications to liposomal therapy are needed to improve the treatment efficacy. Efforts to design liposomes that are pH sensitive, temperature sensitive, antibody targeted, or fusogenic have all been pursued with various degrees of success (11) .
HT has been used to modify the local tumor environment to increase liposomal drug delivery to tumors (12) . Although classically viewed as a form of adjuvant therapy to increase the efficacy of radiation and chemotherapy, HT can be applied to augment liposomal drug delivery by increasing tumor blood flow and microvascular permeability. At temperatures of 41–43°C, HT has been shown to increase blood flow (13) and oxygenation (14) . HT has also been shown to increase permeability of tumor vessels to antibodies (15, 16, 17, 18) , ferritin (19) , and Evans blue dye (20) . More specifically, HT has been shown to increase permeability of tumor vessels to liposomes (21) .
Additionally, HT can be used as a modality for increasing liposomal drug delivery to tumor by triggering release of drug from the liposome. Temperatures of 40–43°C have been shown to trigger drug release from specially designed thermosensitive liposomes (22, 23, 24) , making it possible to release liposome contents at the heated site. Besides targeting and triggering release in liposomes, preclinical data have indicated that several cancer chemotherapeutic agents in combination with HT have supra-additive cytotoxic effects (25, 26, 27, 28, 29) . Furthermore, HT itself has been shown to be directly cytotoxic (30) . The therapeutic benefits from liposomes and HT individually, coupled with the potential advantages seen by their combination, make the use of the two modalities together an attractive method for drug delivery to tumors.
There have been many studies showing increased drug delivery to the tumor when the drug is encapsulated in a liposome and administered with HT (31, 32, 33) . The therapeutic efficacy of combination HT and liposomal drug delivery has also been shown with several drugs, ranges of thermal doses, different tumor models, and various liposomal formulations (34, 35, 36) . These positive findings are difficult to evaluate across studies because they have usually been limited to one liposome formulation. Thus, the different treatment regimens have not been directly compared in the same system, and the characteristics of an optimal liposome formulation remain poorly defined (12) .
This report directly compares three long-circulating liposomal
formulations with and without HT by tumor drug concentrations, tissue
fluorescence, and growth delay using a human tumor xenograft model. The
three different liposomes represent fundamentally different classes of
liposomal carriers that can be used in combination with HT. The NTSL
(21)
is not thermosensitive; the TTSL (37)
is
thermosensitive, but only in the range 42–45°C and releases drug
over 30 min; and the LTSL (38)
is thermosensitive at
the range of 39–40°C and releases drug in a matter of seconds.
Some of the results in this report have been published in a preliminary
form (38)
, where the focus was to introduce the LTSL
formulation, its physical properties, and effects on tumor growth delay
when combined with HT. This report will provide detailed analysis of
tumor drug concentration, tissue drug fluorescence, and growth delay
results that have not been presented so that the key factors for
optimal combination HT and liposomal therapy can be better understood.
Using direct comparison of saline, DOX, NTSL, TTSL, and LTSL with and
without HT, it was possible to determine the relative importance of
liposomal drug encapsulation, HT cytotoxicity, HT-drug interaction,
HT-induced liposomal delivery, and HT-triggered liposomal drug release
to therapeutic efficacy.
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MATERIALS AND METHODS |
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140 nm by dynamic light scattering) were prepared by the lipid film
hydration and extrusion method (39)
. Encapsulation of DOX
into the liposomes was carried out using the pH gradient-driven loading
protocol (40)
.
Mice and Tumors.
Homozygous NCr athymic nude mice (20 ± 3g) were
purchased from Taconic (Germantown, NY). Animals were housed in
appropriate isolated caging with sterile rodent food and acidified
water ad libitum and a 12-h light/dark cycle. A human
squamous cell carcinoma xenograft line, FaDu, was used in this study.
The right lower leg of each mouse was inoculated s.c. with 1 x 106 single-cell suspension in a volume of
50 µl. Tumors were allowed to grow to 4–6 mm in diameter before
starting treatment. Mice were carefully monitored for general
well-being, weight, and tumor volume. Any mice exhibiting weight loss
15% of the initial weight were scheduled to be euthanized, but no
animals fit this criterion. All protocols were approved by the Duke
Institutional Animal Care and Use Committee.
Treatment Protocol.
Mice were stratified by tumor volume and randomized to 1 of 10
treatment groups: saline, DOX, NTSL, TTSL, and LTSL; at 34°C or
42°C for 1 h. Except for the saline group, all treatment groups
were given an equivalent single dose of 5 mg/kg of DOX. Mice were
anesthetized with an i.p. injection of pentobarbital (80 mg/kg). This
dose of anesthesia provided adequate immobilization for the 1-h heat
treatment period, and no redosing was necessary. The mice were then
given an injection of 100 µl of treatment solution via the tail vein.
Immediately after injection, the mice were positioned in specially
designed holders that allowed the isolated leg tumor to be placed in a
water bath for 1 h. The isolated leg tumor was covered in a thin
plastic sleeve to prevent excessive water absorption and subsequent
limb edema (18)
. The water bath temperature was set to
35°C or 43°C, temperatures that had been previously calibrated to
give tumor temperatures of either 34°C or 42°C, respectively,
depending on the treatment group (18)
.
Tumor Drug Concentrations.
Tumors were surgically excised (six tumors/group) immediately after the
1-h water bath treatment and frozen at -70°C. Samples were then
evaluated via a liquid extraction of homogenates with chloroform and
the addition of silver nitrate to ascertain total DOX concentrations
(41)
. This method has been shown to extract >95% of DOX
present in tissue. The presence of silver nitrate allows for the
extraction of DOX bound to DNA and RNA that cannot be extracted with
only chloroform. Samples were also extracted with only chloroform for
comparison to determine the amount of DOX bound to DNA and RNA.
Subsequently, concentrations of DOX in the tumor samples were
quantified using a modification of a previously published gradient,
reverse phase-high performance liquid chromatography assay with
florescence detection (42)
. High and low concentrations of
DOX in quality control samples were required to be within 15% of
spiked concentrations for an assay run to be acceptable. Unless
specified, data are reported as the amount of total DOX (extracted with
chloroform and silver nitrate) in each sample per weight of extracted
tumor tissue. The average value of six treated tumors is given for each
of the 10 treatment groups.
Tumor Fluorescence Sections.
Tumors were surgically excised (six tumors/group) immediately after the
1-h water bath treatment, and two 4-µm-thick frozen sections from
each sample taken at different levels of the tumor were prepared. The
tumor sections were examined under fluorescence microscopy (H546;
Zeiss) for DOX. Using an objective with x20 and epi-illumination with
light from a 100-W mercury lamp passing through a dichroic filter set
suitable for DOX, images of the tumor sections were captured and
analyzed off-line for fluorescence intensity. All tumor sections were
analyzed with image processing software (NIH Scion Image) under the
same settings and reported in arbitrary fluorescence intensity units.
Tumor Growth Delay.
Mice were treated by 1 of the 10 treatments (8–12 mice/group): saline,
DOX, NTSL, TTSL, and LTSL; at 34°C or 42°C for 1 h. Animals
were weighed, and tumors were measured 3 times/week. Tumor volume was
determined using the equation: volume =
(width)2
x length x 
/6. Tumor
measurements were taken by one individual and performed in duplicate to
confirm measurements. The individual measuring the tumors was blinded
to the treatment groups. Animals were followed until five times the
initial tumor volume was reached or 60 days after treatment, at which
point they were euthanized. Growth times are reported as the average of
all animals in a treatment group.
Statistics.
The Mann Whitney U test was used to determine statistical
significance for all comparisons except for evaluating 60-day local
control, where the Fisher exact probability test was used.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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80% bound
in vivo across a wide range of concentrations
(43)
, most DOX that is released from a liposome becomes
bound. The total DOX tumor concentrations (concentration extracted with
chloroform and silver nitrate) are reported unless otherwise stated.
At 34°C, the DOX concentrations achieved by all of the treatments
were similar. The mean values ranged from 0 to 4.5 ng/mg, with saline
being the lowest and the TTSL being the highest (Fig. 1)
. DOX concentrations at 42°C for
three liposomes (NTSL, 8 ng/mg; TTSL, 7.2 ng/mg; and LTSL, 25.6 ng/mg)
were higher than saline (0 ng/mg) and free DOX (1 ng/mg;
P < 0.04). In addition, the DOX
concentration achieved by the LTSL at 42°C was significantly more
than the other two liposomes (P < 0.04).
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DOX extraction from tumor tissue was done with and without silver
nitrate (chloroform only; Fig. 1
). In all groups except for LTSL + HT, there was no significant difference in DOX concentration
with or without silver nitrate, although the presence of silver nitrate
tended to increase the amount of DOX extracted in most of those groups.
Tumors treated with LTSL + HT showed a significant difference
in DOX concentration when extracted with silver nitrate (25.6 ng/mg)
compared to without silver nitrate (13.1 ng/mg; P < 0.02), indicating that there was a substantial amount of DOX
bound to DNA and RNA in these tumors.
Tumor Fluorescence Sections.
The goal of examining tumor tissue sections was to directly evaluate
the relative levels of DOX and the local distribution of DOX achieved
in the tumor immediately after treatment. All values are denoted in
arbitrary DOX fluorescence intensity units, and the control group was
used to determine background fluorescence. At 34°C, DOX tumor
sections (0.89 units; Fig. 2B
)
showed slightly more fluorescence than the control group (Fig. 2A
; P < 0.02). All three
liposomal formulations (Fig. 2, C–E)
were essentially
equivalent, ranging from 2.23 to 2.43 units, but were all more
fluorescent than DOX (P < 0.01). Overall,
the treatment groups at 34°C were low in DOX fluorescence intensity.
HT did not result in any difference in fluorescence for the control
group. At 42°C, DOX tumor sections (0.99 units; Fig. 2G
)
were again slightly more fluorescent than the saline group
(P < 0.03). All three liposomal formulations
(Fig. 2, H–J)
were more fluorescent than DOX
(P < 0.001). LTSL + HT was the
most fluorescent (27.38 units; P < 0.005),
followed by TTSL + HT (10.76 units) and NTSL + HT
(4.93 units). The distribution of DOX fluorescence in the tumor
sections was fairly faint and sparse for all treatment groups except
for the TTSL and LTSL at 42°C. TTSL + HT showed
intermittent clusters of fluorescence throughout the tumor sections
(Fig. 2I)
. The LTSL + HT showed dense clusters of
high fluorescence throughout the tumor sections, although not
completely homogeneous (Fig. 2J)
.
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Within the normothermic group (34°C), the growth time for tumors
treated with saline was 9.8 days (Fig. 3A)
. DOX and two of three of
the liposomal formulations (TTSL and LTSL) had growth times that were
not statistically different from the saline group (9.8–13.5 days; Fig. 3
, B, D, and E). An exception was the NTSL group,
which had a growth time of 20.9 days (P < 0.02, compared with saline). The NTSL group also had the largest
variation in growth times as seen in Fig. 3C
.
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) were not statistically different. All three liposomal
formulations in combination with HT were significantly more effective
than HT + DOX or HT alone (P < 0.05). NTSL + HT and TTSL + HT had growth times of
31.9 and 35.1 days, respectively (Fig. 4, C and D)
. TTSL + HT had local control at 60 days in 1 of
12 tumors treated. LTSL + HT showed the most growth delay
with a growth time of 51.4 days (P < 0.002;
Fig. 4E
). This group had local controls at 60 days in six of
nine tumors treated. In this study, within all formulations, HT was
statistically more efficacious than no HT (P < 0.05). The HER, defined as the ratio of the growth time at
42°C to that at 34°C, ranged from 1.75 to 4.51, with the LTSL
having the largest HER (Table 1)
. None of
the mice in any of the treatment groups showed any obvious signs of
morbidity or weight loss, indicating that the single-dose drug + HT was well tolerated.
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. Comparison of results with a second,
previously published tumor growth delay experiment (38)
,
are shown (Fig. 5A)
. Both correlations were similar with
slopes near unity, indicating a very direct relationship between DOX
concentration, fluorescence, and growth delay. Thus, when substantial
tumor DOX concentrations or tumor DOX fluorescence was achieved
(e.g., at 42°C), they were predictive of tumor growth
delay.
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DISCUSSION |
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Contribution of Liposomal Encapsulation.
Results from all three liposome types (NTSL, TTSL, and LTSL) at 34°C
demonstrate the effect of liposomal drug encapsulation on tumor drug
delivery, DOX tissue fluorescence, and therapeutic efficacy. Under
normothermic conditions (34°C), there was little difference among
liposomal treatment groups (tumor DOX concentrations, DOX fluorescence,
or growth time) except for the NTSL group growth time (Table 1)
.
Presumably all three liposome types, which are all long circulating and
of similar size, would be equally likely to extravasate into the tumor
interstitium because of the permeable tumor microvasculature. The DOX
concentrations and fluorescence attained immediately after treatment by
all three liposomes were similar, supporting this interpretation (Figs. 1
and 2)
. This degree of extravasation, however, for the TTSLs and
LTSLs did not result in any therapeutic gains over saline (Fig. 3A)
or DOX (Fig. 3B)
at 34°C. This may be
because of the slow drug release rate of the TTSLs and LTSLs at 34°C
(37)
. Thus, the tumors in the TTSL and LTSL groups were
ineffectively exposed to drug over time after liposomes accumulated in
the tumor. The increased efficacy of the NTSLs compared with controls
(11.1 days growth delay; Fig. 3C
) might be explained by a
slightly higher amount of drug released at some time later than the
measured time point. Although not designed for triggered release, the
NTSL has the highest cholesterol content, and this has been observed
previously to result in some drug loss (more than the TTSL) over time
in vivo (37)
.
Contribution of HT Cytotoxicity.
By testing the saline groups at 34°C (Fig. 3A)
and 42°C
(Fig. 4A)
, the effect of HT cytotoxicity could be isolated.
HT alone (42°C for 1 h) showed significant growth delay (10
days), and every treatment group in combination with HT was more
efficacious than without HT (P < 0.05).
These findings are consistent with the literature regarding HT
cytotoxicity (30)
and HT interactions with liposomes
(12)
.
Contribution of HT-DOX Interaction for Free Drug Administration.
There have been several studies examining the interaction of HT and
chemotherapeutic agents (26)
. With some agents,
supra-additive effects have been seen with HT (26
, 27)
.
For DOX, the current literature is not definitive. There have been
studies showing HT + DOX synergy (25
, 47
, 48)
and others showing only additive effects (49, 50, 51)
. Still
others describe a threshold temperature of 42°C for HT + DOX; only above this temperature is synergy seen (52)
. The
tumor DOX concentrations and the DOX fluorescence for the DOX group
were statistically similar at both 34°C and 42°C (Fig. 1)
; thus
there was no increased DOX delivery attributable to HT. DOX is known to
have a short initial half-life in circulation because of its rapid
binding to cells and tissues (53)
, and this characteristic
may limit local ability of HT to increase DOX delivery to the tumor.
For the growth delay study, the HT growth time (defined as difference
in growth time at 42°C and 34°C) for DOX was 10.2 days, which was
not statistically different from the saline HT growth time (10 days;
Table 1
). Thus, no evidence for HT and DOX interaction was observed in
this study. Because DOX showed no activity at 34°C or 42°C compared
with saline, it can be concluded that free DOX, given at this dose,
showed no activity in this tumor model. Higher doses of DOX (7.5 and 10
mg/kg) were also tested in a pilot study but resulted in significant
toxicity to the animals (data not shown). On the basis of these
results, 5 mg/kg was determined to be the maximally tolerated dose.
Contribution of HT-induced Liposomal Delivery.
To evaluate the contribution of HT-induced liposomal delivery, the
NTSL + HT group was examined. HT might cause additional
delivery of NTSL to the tumor because of increased tumor blood flow
(13
, 54) , vascular permeability (17, 18, 19, 20)
, or
increased vascular pore size (55)
. Because the NTSL cannot
be triggered to release drug, its therapeutic effect with HT is limited
to HT cytotoxicity, HT-induced liposomal delivery, and any interaction
between DOX that is released and HT. The NTSL does not release drug
because of HT; therefore, it is unlikely that tumor cells were exposed
to HT and significant levels of drug simultaneously. The NTSL tumor
drug concentrations show approximately double the DOX tumor
concentrations at 42°C (8 ng/mg) compared with that seen at 34°C
(4.4 ng/mg; P < 0.05). This ratio is also
seen for the tumor DOX fluorescence (42°C = 4.93 and
34°C = 2.38; P < 0.01). The
results show a tumor growth delay of 11 days for NTSL + HT
(Fig. 4C)
, but this is not significantly different from the
effect attributable to HT alone (10 days). Therefore, although there
may have been increased total liposome delivery with HT, this effect
was not significant enough to translate into increased therapeutic
efficacy for the NTSL because of the lack of significant release within
a relevant time frame (i.e., DOX remained in the liposome).
There have been several studies using NTSL showing some increased
efficacy in combination with HT (36
, 56)
. Despite these
results, the effects of HT on tumor blood flow and vascular
permeability, as discussed in the "Introduction," are not always
consistent. In different models, HT has not led to increased tumor
blood flow (57)
or vascular permeability (58
, 59)
.
Contribution of HT-triggered Liposomal Drug Release.
By using two temperature-sensitive liposomes (TTSLs and LTSLs) that
have different triggering temperature ranges and release rates, we were
able to explore the role of HT-triggered liposomal drug release in drug
delivery and how differences in release characteristics affected
efficacy. Triggered release occurs because of a lipid membrane phase
transition that results in increased permeability across the liposome
membrane (60)
. A peak in the membrane permeability occurs
coincident with the midpoint of the gel-liquid crystalline transition
(61)
. It is important to note that although the peak in
permeability occurs at this midpoint, drug permeability can be
appreciable 1°C or 2°C below this point. The TTSL starts drug
release in the range of 42–45°C, and 40% content release is
achieved within 30 min at the appropriate temperature
(38)
. This release temperature is not clinically optimized
because most clinical HT treatments yield nonuniform temperatures with
averages of 40–41°C and ranges from 40 to 43°C
(62)
. As discussed above, the LTSL is a novel liposome
composition that incorporates lysolecithin into the gel-phase lipid
membrane, which acts to slightly lower the phase transition temperature
of the lipid mixture to 39–40°C (24)
. This lowered
transition temperature then brings down the temperature at which drug
starts to be released into a temperature range that is clinically
attainable in the tumor. The LTSL also has a higher total percentage of
drug release and a faster rate of drug release than the TTSL
(38)
. Forty-five % of content release occurs within
20 s, which may be expected to enable higher local concentrations
of drug to accumulate in the tumor and even be released in the blood
stream as the liposomes are circulating in the tumor. The results from
this study confirm this expectation.
The highest DOX concentrations and fluorescence achieved were seen with the LTSL + HT group. It was the only group to show significant DOX binding to DNA and RNA (determined by the difference in the amount of DOX extracted with chloroform compared with that extracted with chloroform and silver nitrate; P < 0.02). DNA binding is one of the main mechanisms by which DOX exerts cytotoxicity (63) . These differences between the LTSL and other liposomes reflect the large amount of DOX released by the LTSL at 42°C and may explain the significant therapeutic effect seen with this group. The large amount of DOX released by the LTSL most likely occurs by two mechanisms: tumor interstitial release and vascular release.
If LTSL extravasation and subsequent drug release were the only
mechanisms for LTSL drug delivery, then the DOX concentration achieved
should be similar to the TTSL and NTSL because all three of these
liposomes have PEG and are similar in size. However, the DOX
concentration achieved by the LTSL at 42°C was much higher than other
groups, and an additional mechanism for LTSL drug delivery is likely
present. This other mechanism is most likely LTSL release of drug in
the tumor vasculature. A simple estimation of LTSL tumor circulation
time can be useful for elucidating this mechanism of intravascular drug
release. Erythrocyte velocity in tumor vessels has been shown to be
approximately 0.1–0.2 mm/s (64)
. Assuming that LTSL
velocity is similar to erythrocyte velocity in tumor vessels, in a 1-cm
diameter tumor, LTSL will travel through the entire diameter of the
tumor in 50–100 s. This time frame is long enough for LTSL to release
its contents. Because most tumor vasculature is chaotic and tortuous,
LTSL circulation would most likely be even longer than estimated in
this calculation. This occurrence of drug release in the local tumor
blood stream would result in high intravascular drug concentration and
the resultant concentration gradient that would drive drug into the
tumor interstitium. The combination of interstitial and vascular drug
release by the LTSL explains the high DOX concentration achieved. The
TTSL is unable to release drug in the tumor vessels for two main
reasons: time frame and vessel temperature. The estimated tumor
circulation time for the TTSL would be similar to the LTSL but not long
enough for appreciable TTSL drug release (1800 s). Additionally, the
TTSL would need to be continuously exposed to intravascular
temperatures of 42°C. This condition is unlikely when the tumor
interstitium is heated to 42°C (i.e., the vessel
temperature would most likely be <42°C due to blood flow).
For both temperature-sensitive liposomes, augmented release of drug
translated into an increased therapeutic effect (Table 1)
. The HT
growth time for the TTSL was 24 days (P < 0.02). In the situation of the TTSL + HT group, heating to
42°C for 1 h, although suboptimal because of the higher
transition temperature of the liposome, was sufficient to trigger some
drug release (Figs. 2I
and 4D)
. This drug release
then translated into efficacy by increasing the growth time in addition
to 60-day local control in 1 of 12 tumors. HT also triggered the LTSL,
but to a larger degree. LTSL + HT led to a HT growth time of
40 days (P < 0.01) and 60-day local control
in six of nine tumors (statistically different from NTSL and TTSL at
42°C, P < 0.01; Table 1
). The higher
efficacy for the LTSL compared with the TTSL is likely attributable to
the significant tumor drug levels achieved in a short time frame, which
is a function of the lower triggering temperature, the higher total
percentage of drug release, and the faster rate of drug release. These
characteristics allow for more elevated tissue drug concentrations
(Fig. 1)
in the tumor and help overcome any potential threshold dose
for efficacy. Therefore, although DOX levels are increased with the
NTSL and TTSL + HT, it is the difference of triggered and
rapid drug release of the LTSL that appears to be instrumental in
achieving sufficient concentrations of drug to allow for a larger
therapeutic effect.
One of the many drawbacks to cancer chemotherapeutic regimens is the
associated toxicity for normal tissues. In this situation, single-dose
treatment of free or liposomal DOX (5 mg/kg) in combination with local
HT (42°C for 1 h) was well tolerated. There were no cases of
weight loss 15% of the initial weight and, in most cases, the
animals gained weight during the study. In the case of the LTSL at
42°C, this further strengthens the case for a very high therapeutic
index.
Correlation between Tumor DOX Concentrations, DOX Fluorescence, and
Growth Delay.
Because the groups with the highest and lowest tumor DOX concentrations
and tumor DOX fluorescence yielded the largest and smallest growth
delay, the possibility of a correlative relationship between tumor drug
concentration, tumor DOX fluorescence, and growth delay became
apparent. When correlating the relationships between these three
results for the groups that received HT, the correlation coefficients
were >0.98, and the slopes were all similar to unity (Fig. 5)
. These
tight correlations show that the actual tumor drug concentration and/or
tumor DOX fluorescence can be good predictors of each other and,
ultimately, of therapeutic effect. These observations could become
clinically useful as prognostic factors (e.g., determine
tumor drug concentration or tissue fluorescence from a biopsy). In
addition to these relationships, perhaps a threshold response needs to
be overcome to obtain more long-lasting therapeutic effects. Only the
LTSL at 42°C, which was triggered for significant drug release,
resulted in consistent local controls (i.e., complete
regressions at 60 days after treatment). No correlations could be made
for the groups that did not receive HT because little tumor growth
delay was seen.
In conclusion, this study aimed to explore whether liposome drug
encapsulation, HT cytotoxicity, HT-drug interaction, HT-induced
liposomal delivery, and HT-triggered liposomal drug release can
contribute to more effective therapy. A schematic of these critical
factors is presented in Fig. 6
. Although
much of the current literature has established that these factors are
important, this study highlights the need for HT-triggered liposomal
drug release at a high rate and at clinically attainable temperatures.
It is also important to note that DOX alone was unable to delay tumor
growth, but the same systemic dose of drug delivered in a liposome and
triggered for release was able to produce a therapeutic effect.
Although current liposomal formulations may emphasize one or two of
these factors, it is necessary to optimize all of these factors to
obtain the highest therapeutic intervention.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH Grants CA40355
and CA42745 and a grant from the Celsion Corporation. 
2 To whom requests for reprints should be
addressed, at Duke University Medical Center, Box 3455, Durham, NC
27710. E-mail: dewhirst{at}radonc.duke.edu
3 The abbreviations used are: PEG, polyethylene
glycol; HT, hyperthermia; DOX, doxorubicin; NTSL, nonthermosensitive
liposome; TTSL, traditional thermosensitive liposome; LTSL, low
temperature-sensitive liposome; MPPC,
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine; DPPC,
1,2-dipalmitoyl-sn-glycero-3-phosphocholine; HSPC,
hydrogenated soy sn-glycero-3-phosphocholine;
DSPE-PEG-2000,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG
2000; HER, HT enhancement ratio.
Received 5/10/00. Accepted 10/17/00.
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,
extracted with chloroform only; 
, extracted with chloroform and
silver nitrate. Bars, SE.
, TTSL; 
, LTSL. Open symbols, groups
repeated in another study 