| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Departments of Mechanical Engineering and Material Science [D. N., G. A.] and Biomedical Engineering [G. K.], Duke University, Durham, North Carolina 27708, and Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710 [M. W. D.]
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
|---|
Our efforts have focused on developing a new thermal-sensitive drug delivery system containing DOX4 that has been optimized for both mild hyperthermic temperatures (39°C to 40°C) that are readily achievable in the clinic and rapid release times of drug (tens of seconds). We report here the design, development, in vitro characterization, and in vivo testing of a new thermal-sensitive lipid formulation that can be triggered to release drug rapidly and at clinically attainable hyperthermic temperatures. Our in vitro studies show the advantages that the new lipid composition has compared with existing liposome formulations. The studies also provide insight into the mechanism of action, which involves only a few mol% of MPPC contained in the gel-phase DPPC bilayer of the LTSLs to increase the overall phase transition-induced permeability of the bilayer to the encapsulated drug.
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
|---|
140
nm) were prepared by the lipid film hydration and extrusion method
(10)
. Encapsulation of DOX into the liposomes was carried
out using the pH gradient-driven loading protocol (11)
.
Temperature-induced Release of DOX from Liposomes in
Vitro.
The release of entrapped DOX from liposomes at various temperatures
between 30°C and 45°C and time points between 0 and 3600 s was
determined by measuring the amount of entrapped DOX that was released
from a given sample of liposomes as a function of time at a given
temperature. Each test temperature was attained by rapidly heating each
sample from room temperature. In this way, the experiment represents a
temperature jump and release of contents at each test temperature. A
20-µl aliquot of incubated sample was withdrawn, and after suitable
dilution to 1 ml, the fluorescence intensity was measured on a
fluorescence spectrophotometer (Shimadzu, RF-1501) as described by
Maruyama et al. (12)
with minor modifications.
The relative percentage of fluorescence intensity after incubation at
different temperatures was calculated by comparison with the total
fluorescence intensity obtained after disrupting the liposomes by
adding 0.3 M HCl-50% ethanol to the samples. To
provide an environment somewhat closer to the preclinical situation of
the mouse tumor than simple buffers, all of the in vitro
drug release experiments and differential scanning calorimetry (DSC7;
Perkin-Elmer, 2°C/min heating rate) were carried out in the presence
of 50% bovine serum.
Mice and Tumors.
Homozygous NCr athymic nude mice (20 ± 3 g) 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 implanted s.c. with 1 x 106 cells in 50 µl of PBS. 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. Mice with weight loss 
15% of the initial weight or tumor
volume 1500 mm3 were scheduled to be
euthanized, but no mice in this study met that requirement for early
euthanasia. The Duke Institutional Animal Care and Use Committee
approved all protocols.
Treatment.
In study 1, mice were stratified by tumor volume and randomized to 1 of
10 treatment groups (8–12 mice/group): saline, free DOX, NTSLs, TTSLs,
and the LTSLs, at 34°C or 42°C. In study 2, mice were
stratified by tumor volume and randomized to one of four treatment
groups (10–11 mice/group): saline, NTSLs, TTSLs, and LTSLs, all at
42°C. In both studies, the stratification by volume assured that
there was an equal volume distribution within groups, as confirmed by
statistical analysis (data not shown). Except for the saline group, all
treatment groups were given an equivalent single dose of 5 mg/kg of
DOX. Mice in all treatment groups were anesthetized with an i.p.
injection of pentobarbital (80 mg/kg); treatment was administered in a
volume of 100 µl via tail vein injection. This dose of anesthesia
provided adequate immobilization for the 1-h treatment period. No
redosing was needed. We did not observe any attempts to withdraw the
foot from the water bath (as one would expect from a pain response) or
movement of the animals during the treatment period. 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. Depending on the treatment group, the water bath temperature was set
at 35°C or 43°C. These water bath temperatures have been calibrated
previously to give tumor temperatures of either 34°C or 42°C,
respectively (13)
.
Evaluation.
Animals were weighed, and tumors were measured three times/week. Tumor
volume was determined with 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 posttreatment, at which point they were euthanized. The
Mann-Whitney U test and Fisher’s exact tests were used to
determine statistical significance.
|
Results and Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
|---|
, tested in bovine serum, the release of encapsulated DOX
from the new LTSL was extremely fast upon heating the liposomes to
42°C compared with the pure DPPC liposome.
|
45% of its contents in the first 20 s of
being exposed to the elevated temperature of 42°C, compared with only
20% over 1 h for pure DPPC. Also shown for comparison are the
more traditional thermal-sensitive liposome (TTSL; Ref.
9
), and a NTSL (8)
. As reported previously by
Gaber et al. (8)
and checked here
independently, the TTSL took 30 min to release 40% of its contents,
and as expected, the NTSL did not release any drug upon heating to
42°C.
Moreover, as shown in Fig. 1b
, the onset temperature for
LTSL-triggered release was relatively narrow and occurred mainly
between 39°C and 40°C. This release profile fits within the limits
of even the worst cases for average temperatures usually encountered
using regional heating for deep-seated tumors. For example, when
treated with externally applied radiofrequency-phased arrays,
temperatures for prostate and ovarian cancers are nonhomogeneous and
range from a minimum of 39.3°C to a median of 40.4°C and
39.7–41.6°C, respectively (14
, 15)
. Compared with DPPC
alone, the presence of MPPC in the liposome bilayer lowered the drug
release temperature by almost 2°C and significantly increased the
amount released, thus maximizing the release parameters for clinical
use. As shown in the inset to Fig. 1b
, for the
DPPC:MPPC mixture, the contents release started to become significant
at a temperature 1.5 degrees below the main peak of the gel to
liquid crystalline bilayer phase transition. This indicates that
although not at a maximum (see below), the enhanced permeability was
sufficient to allow drug to be rapidly released at these relatively
lower temperatures. For the TTSL, the triggered release temperature was
broader, in the range 41°C to 43°C, slightly lower than that
reported by Gaber et al. (9)
, and nevertheless
slightly higher than what is easily attainable clinically. Once again,
the NTSL showed little release of drug.
The concept that underlies the enhanced release of encapsulated drug
from the liposomes relies on two properties of the lipid bilayer:
(a) an increased bilayer permeability at the gel-to-liquid
crystalline phase transition temperature
(Tm) compared with either the solid or
liquid phases; and (b) the ability of a water-soluble
lysolipid component to desorb from the bilayer as the first lipid
begins to melt. Previous experiments (16)
and theory
(17)
have shown that passive bilayer permeability has a
sharp maximum, coincident with the maximum in the transition enthalpy
attributable to mismatches in molecular packing, especially at the
interfacial boundary regions of gel and liquid domains. How might this
permeability be enhanced? We hypothesized that if a molecule could be
included in the gel-phase bilayer that was also fairly soluble in the
aqueous phase, then as the lipid melted, the molecule would desorb and
possibly enhance the boundary defect formation, and with it the passive
permeability to entrapped drug and other material (18)
. We
have shown previously that lysolipids readily desorb from liquid-phase
lipid bilayers once the vesicle is washed with lysolipid-free buffer
(19
, 20) . We therefore made a mixed bilayer of DPPC
doped with the head group and acyl chain-matched lysolipid MPPC.
The new concept then is that MPPC is kinetically trapped in the ideally
mixed solid phase and, at the gel-liquid crystalline phase transition,
leaves the bilayer upon melting and enhances the permeability compared
with the pure DPPC bilayer transition. As shown in Fig. 1
, the presence
of only a few mol% of MPPC contained in the gel-phase DPPC bilayer of
the LTSLs increased both the rate and amount of drug released and
showed minimal release at body temperatures of 37°C, where the
membrane permeability barrier was essentially maintained. These
characteristics of attainable and narrow triggering temperature, rapid
"burst" release of drug, and high cumulative drug release would
appear to offer potential advantages that could lead to an increased
therapeutic effect over and above traditional chemotherapy and current
liposomal and other drug carrier systems.
In Vivo Testing in a Human Tumor Xenograft Model.
To test this idea, we carried out two tumor growth delay studies in a
human squamous cell carcinoma tumor xenograft model (FaDu) and compared
saline controls to free DOX and each of three liposome DOX formulations
(NTSL, TTSL, and LTSL) ± hyperthermia (Table 1)
.
|
In the first study, saline control and free DOX were compared at 34°C and 42°C. The 34°C control group had a growth time (time to reach five times the initial tumor volume) of 9.8 days. The growth time of free DOX was 13.5 days, which was not significantly different from the control. At 42°C, the growth delay for control was 10 days longer than at 34°C, demonstrating some hyperthermic cytotoxicity. However, free drug at 42°C was equivalent to heat alone. This lack of DOX activity could be attributed to either insensitivity of this tumor line to DOX or lack of delivery of sufficient drug. In a pilot toxicity study, we observed tumor regressions at doses of 7.5 and 10 mg/kg, but the drug was too toxic to use at those doses. Five mg/kg was chosen as the maximum tolerated dose for free drug. Therefore, the free drug is in fact toxic to the FaDu tumor, if high enough doses are reached, and we conclude that the lack of free drug activity at 5 mg/kg was attributable to lack of delivery and not lack of activity.
The TTSL and LTSL groups also showed essentially no activity against
this tumor at 34°C, with growth times of 9.8 and 13.5 days. The
growth time for NTSL, however, was 20.9 days, a 10-day growth delay
compared with the control group (P < 0.02).
This result might be expected because NTSL has the highest cholesterol
content, which has been reported previously to result in some drug loss
over time in serum (9)
.
Of the heated groups in both studies, the growth delay results for the
LTSL were the most striking. As shown in Fig. 2d
, the LTSL formulation resulted in the most impressive
antitumor effect.
|
produced one disease-free animal at 60 days and one extended growth
delay, recapitulating the results of the first study (1 of 12 local
controls at 60 days). The NTSL and TTSL groups had extended growth
times compared with the heat alone (Fig. 2, b and c
; and Table 1
; P < 0.05). Of
note, none of the mice in any of the treatment groups showed any
obvious signs of morbidity. Most animals gained weight during the
posttreatment period. None of the animals treated at 42°C lost weight
in either experiment. In the first experiment, one or two animals in
each group treated at 34°C lost some weight, while the rest gained
weight. The maximum weight loss for those individuals that lost weight
after treatment at 34°C ranged from 6% for free drug to 12% for the
NTSL group. Therefore, the single dose of drug ± hyperthermia was well tolerated, perhaps even better than when these
drugs were given under normothermic conditions.
The increased tumor growth times seen with the NTSL and TTSL
formulations and hyperthermia demonstrate the importance of using heat
to enhance liposome accumulation in tumor tissue for carriers that do
not necessarily release drug rapidly and confirms previous studies
(7
, 8)
. Nevertheless, the importance of increased
liposomal delivery and the rapid release of drug in achieving the best
antitumor effect became apparent. As is shown in Fig. 1
, the rates and
amounts of drug released from LTSLs at 42°C are superior to TTSLs.
These properties translate into the large difference in growth times
in vivo between LTSLs (Fig. 2d)
and TTSLs (Fig. 2c)
at 42°C, thus showing the importance of enhanced drug release in
achieving the best antitumor effect. Additional studies testing how
this enhanced release in vivo is responsible for the
improved antitumor effects of this formulation are in progress. If
enhanced release is the primary mechanism underlying the improvement in
effect, one could argue that it might be analogous to drug applications
involving intra-arterial administration. The problem with this approach
is that not all of the vascular supply to tumors is arteriolar
(23)
. Therefore, one might not expect to achieve
complete vascular coverage of the tumor with an intra-arteriolar
approach. The liposomal approach is more likely to reach all vessels of
a tumor, whether arteriolar or venular in nature, because the liposomes
continue to circulate for several hours after administration.
An important issue with relation to this type of application is to keep the drug in the tumor tissue and not allow it to be reabsorbed back into the microcirculation. The rapid tissue binding properties of DOX (24) make it an attractive drug in this respect. Other drugs, with less avid tissue binding characteristics, may be less effective. However, the overall effectiveness of this approach may also be dependent upon the mechanism of the antitumor effect. Given the characteristics of drug transport from microvasculature, the highest tissue concentration is likely to be perivascular. Thus, it is possible that direct vascular damage might be part of the antitumor mechanism. This being the case, then one would expect that this approach might be more effective than simply administering drug intratumorally, for example. Additional studies are under way to investigate this issue further.
We did not test empty LTSLs, either alone or in combination with free drug, in these experiments. These are controls that should be considered for future studies. It is possible that the lysolipid in the LTSLs could cause some cytotoxicity, which could add to the overall antitumor effect. This will be determined from ongoing studies evaluating the antitumor effect as a function of drug release in vivo. Because no weight loss was seen in the LTSL group treated at 42°C and the degree of weight loss is less than a nonlysolipid-containing liposome at 34°C, it is not likely that any lysolipid released is causing any systemic toxicity.
The presence of lysolipid in the DPPC bilayers then leads to three key
features of facilitated drug release by LTSLs as compared with the
other temperature-sensitive lipid formulations, TTSLs, or pure DPPC
liposomes: a lowered bilayer phase transition temperature (Fig. 1b)
, an increase in the rate, and an increase in the amount
of drug release (Fig. 1a)
. All of these features contribute
positively to the overall therapeutic success of this new drug
formulation.
The high tumor cure rate achieved in this model makes this system very attractive to apply to other water-soluble and remote-loadable drugs and a variety of oncological as well as nonmalignant situations. Because of the very rapid release kinetics associated with this new triggered-release system, one potential use could be to match drug-release properties to the type of drug delivered. For example, the action of anthracyclines may be best suited to a single protracted release because such drugs bind avidly to cells and act in a non-cell cycle-specific manner. However, other cell cycle-specific agents, such as camptothecins and Vinca alkaloids, may benefit from multiple short pulses over extended times as the long-circulating, temperature-sensitive liposomes continue to accumulate in the tumor tissue. Another potential application could be for drugs that show enhanced efficacy when they act synergistically. By formulating and delivering the two drugs in the LTSL system and using mild hyperthermia, both drugs could be made to accumulate and release in the tumor at the same time. Nonmalignant diseases, such as psoriasis and rheumatoid arthritis, and the deployment of other agents, such as anesthetics, might also benefit from a temperature-triggered, local, and rapid release of drug.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by NIH Grants GM40162 (to D. N.) and
CA42745 (to M. W. D.) and a grant from the Celsion Corporation. 
2 To whom requests for reprints should be
addressed, at Department of Mechanical Engineering and Material
Science, Duke University, NC 27708-0300. E-mail: david.needham{at}duke.edu
3 These authors contributed equally to this
work.
4 The abbreviations used are: DOX, doxorubicin;
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-polyethylene
glycol 2000; NTSL, nonthermosensitive liposome DOX; TTSL, traditional
thermosensitive liposome DOX; LTSL, lysolecithin-containing
thermosensitive liposome DOX.
Received 11/19/99. Accepted 1/18/00.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  G. Galiana, R. T. Branca, E. R. Jenista, and W. S. Warren Accurate Temperature Imaging Based on Intermolecular Coherences in Magnetic Resonance Science, October 17, 2008; 322(5900): 421 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Dreher and A. Chilkoti Toward a Systems Engineering Approach to Cancer Drug Delivery J Natl Cancer Inst, July 4, 2007; 99(13): 983 - 985. [Full Text] [PDF]
|
|
|
|
|
|
A. M. Ponce, B. L. Viglianti, D. Yu, P. S. Yarmolenko, C. R. Michelich, J. Woo, M. B. Bally, and M. W. Dewhirst Magnetic Resonance Imaging of Temperature-Sensitive Liposome Release: Drug Dose Painting and Antitumor Effects J Natl Cancer Inst, January 3, 2007; 99(1): 53 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Hauck, S. M. LaRue, W. P. Petros, J. M. Poulson, D. Yu, I. Spasojevic, A. F. Pruitt, A. Klein, B. Case, D. E. Thrall, et al. Phase I Trial of Doxorubicin-Containing Low Temperature Sensitive Liposomes in Spontaneous Canine Tumors. Clin. Cancer Res., July 1, 2006; 12(13): 4004 - 4010. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ahmed, Z. Liu, A. N. Lukyanov, S. Signoretti, C. Horkan, W. L. Monsky, V. P. Torchilin, and S. N. Goldberg Combination Radiofrequency Ablation with Intratumoral Liposomal Doxorubicin: Effect on Drug Accumulation and Coagulation in Multiple Tissues and Tumor Types in Animals Radiology, May 1, 2005; 235(2): 469 - 477. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Yuh, S. G. Shulman, S. A. Mehta, J. Xie, L. Chen, V. Frenkel, M. D. Bednarski, and K. C. P. Li Delivery of Systemic Chemotherapeutic Agent to Tumors by Using Focused Ultrasound: Study in a Murine Model Radiology, February 1, 2005; 234(2): 431 - 437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Jensen, T. L. Andresen, J. Davidsen, P. Hoyrup, S. D. Shnyder, M. C. Bibby, J. H. Gill, and K. Jorgensen Secretory phospholipase A2 as a tumor-specific trigger for targeted delivery of a novel class of liposomal prodrug anticancer etherlipids Mol. Cancer Ther., November 1, 2004; 3(11): 1451 - 1458. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Chen, S. Tong, M. W. Dewhirst, and F. Yuan Targeting tumor microvessels using doxorubicin encapsulated in a novel thermosensitive liposome Mol. Cancer Ther., October 1, 2004; 3(10): 1311 - 1317. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. H. Lindner, M. E. Eichhorn, H. Eibl, N. Teichert, M. Schmitt-Sody, R. D. Issels, and M. Dellian Novel Temperature-Sensitive Liposomes with Prolonged Circulation Time Clin. Cancer Res., March 15, 2004; 10(6): 2168 - 2178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ahmed, W. E. Monsky, G. Girnun, A. Lukyanov, G. D'Ippolito, J. B. Kruskal, K. E. Stuart, V. P. Torchilin, and S. N. Goldberg Radiofrequency Thermal Ablation Sharply Increases Intratumoral Liposomal Doxorubicin Accumulation and Tumor Coagulation Cancer Res., October 1, 2003; 63(19): 6327 - 6333. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. J. R. Charrois and T. M. Allen Multiple Injections of Pegylated Liposomal Doxorubicin: Pharmacokinetics and Therapeutic Activity J. Pharmacol. Exp. Ther., September 1, 2003; 306(3): 1058 - 1067. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. L. Monsky, J. B. Kruskal, A. N. Lukyanov, G. D. Girnun, M. Ahmed, G. S. Gazelle, J. C. Huertas, K. E. Stuart, V. P. Torchilin, and S. N. Goldberg Radio-frequency Ablation Increases Intratumoral Liposomal Doxorubicin Accumulation in a Rat Breast Tumor Model Radiology, September 1, 2002; 224(3): 823 - 829. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Chtryt and K. Ulbrich Conjugate of Doxorubicin with a Thermosensitive Polymer Drug Carrier Journal of Bioactive and Compatible Polymers, November 1, 2001; 16(6): 427 - 440. [Abstract] [PDF]
|
|
|
|
|
|
G. Kong, G. Anyarambhatla, W. P. Petros, R. D. Braun, O. M. Colvin, D. Needham, and M. W. Dewhirst Efficacy of Liposomes and Hyperthermia in a Human Tumor Xenograft Model: Importance of Triggered Drug Release Cancer Res., December 1, 2000; 60(24): 6950 - 6957. [Abstract] [Full Text]
|
|
|
|
|
|
M. L. Matteucci, G. Anyarambhatla, G. Rosner, C. Azuma, P. E. Fisher, M. W. Dewhirst, D. Needham, and D. E. Thrall Hyperthermia Increases Accumulation of Technetium-99m-labeled Liposomes in Feline Sarcomas Clin. Cancer Res., September 1, 2000; 6(9): 3748 - 3755. [Abstract] [Full Text]
|
|
|
|
|
|
G. Kong, R. D. Braun, and M. W. Dewhirst Hyperthermia Enables Tumor-specific Nanoparticle Delivery: Effect of Particle Size Cancer Res., August 1, 2000; 60(16): 4440 - 4445. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, and NTSL 
, and for pure DPPC 
. Liposome
compositions are given in "Materials and Methods," and in all
cases, in vitro tests were carried out with PEGylated
liposomes in 50% bovine serum to more closely match the in
vivo situation than with normal buffers. a,
examples of the rates of DOX release in bovine serum from each liposome
at 42°C determined by fluorimetry. b, examples of the
amount of DOX released in bovine serum from each liposome formulation
as a function of temperature after 5 min (LTSL) or 30 min (DPPC, TTSL,
and NTSL). Inset, comparison between the heat flow (mW)
measured by differential scanning calorimetry for the LTSL and its
release profile from b.