Although nonspecific contrast agents have appreciably improved computed tomography (CT) and magnetic resonance imaging (MRI), specific tissue enhancement represents the next logical progression of improved patient care. Specific targeting of cell-surface proteins has been exploited for the delivery of chemotherapeutic agents and for many types of nuclear medicine studies, but cellular targeting of contrast agents has met with only limited success. This article reports the development of CT and MRI contrast agents specific for cell-surface proteins, accomplished through adaptation of an existing immunoliposome system used for the delivery of chemotherapy to breast tumors. We chose to develop contrast agents specific toward three cell-surface proteins, namely Fas ligand, Fc receptor, and CD5. Fas ligand, which is expressed on immune-privileged cells, binds to the Fas receptor present on T cells and induces apoptosis. The Fc receptor is specific for the terminal constant region of the immunoglobulin molecule, which is utilized by the reticuloendothelial system to bind immunoglobulins. Finally, CD5 is a signal transduction molecule that modulates signaling through the T cell or B cell receptors. We demonstrate specific in vitro enhancement of testicular tissue through Fas ligand targeting, and enhancement of the reticulo-endothelial system (RES) through Fc receptor and CD5 targeting.
is a fourth-year Resident in the Department of Radiology,
University of California, Los Angeles. He received his PhD from
Mayo Graduate School in 1997 and his MD from Mayo Medical School
in 1998. He completed a Postdoctoral Fellowship in Surgery in
1999. He has accepted a Fellowship in Vascular and Interventional
Radiology at UCLA, starting July 2005.
is a Graduate Student in the laboratory of Dr. Hawthorne, and
plans to attend medical school after receiving his PhD.
is a University Professor in the Department of Chemistry,
University of California, Los Angeles.
Although nonspecific contrast agents have appreciably
improved computed tomography (CT) and magnetic resonance imaging
(MRI), specific tissue enhancement represents the next logical
progression of improved patient care. Specific targeting of
cell-surface proteins has been exploited for the delivery of
chemotherapeutic agents and for many types of nuclear medicine
studies, but cellular targeting of contrast agents has met with
only limited success. This article reports the development of CT
and MRI contrast agents specific for cell-surface proteins,
accomplished through adaptation of an existing immunoliposome
system used for the delivery of chemotherapy to breast tumors. We
chose to develop contrast agents specific toward three
cell-surface proteins, namely Fas ligand, Fc receptor, and CD5.
Fas ligand, which is expressed on immune-privileged cells, binds
to the Fas receptor present on T cells and induces apoptosis. The
Fc receptor is specific for the terminal constant region of the
immunoglobulin molecule, which is utilized by the
reticuloendothelial system to bind immunoglobulins. Finally, CD5
is a signal transduction molecule that modulates signaling
through the T cell or B cell receptors. We demonstrate specific
in vitro enhancement of testicular tissue through Fas ligand
targeting, and enhancement of the reticulo-endothelial system
(RES) through Fc receptor and CD5 targeting.
Currently, contrast-enhanced imaging by computed tomography (CT)
and magnetic resonance imaging (MRI) is limited to nonspecific
delivery of macromolecules containing specific properties, which
cause either increased attenuation of X-rays by iodinated materials
or T1 signal shortening by the paramagnetic effect on hydrogen
Although this technique has obvious diagnostic advantages and is
commonplace, specific tissue enhancement has the potential to
dramatically improve sensitivity and specificity. The application
of tissue-specific imaging in nuclear medicine has received
considerable research attention. Progress has been substantial and,
recently, diagnostic tests for colorectal and prostate cancer have
entered clinical practice.
Tissue-specific imaging by other modalities has met with only
however, and is confined mainly to the examination of gene
expression and protein synthesis.
We approached the problem of extending tissue-specific imaging
to CT and MRI by modifying and expanding a system widely used in
drug-delivery research, namely immunoliposomes.
Liposomes are multipurpose biological and immunological agents
whose use in drug delivery, nuclear imaging, and radiotherapy has
been studied extensively over the last quarter-century.
More recently, liposomes have been used in the development of
ultrasound and MRI contrast agents. The targeting of liposomes to
specific cell types through conjugation to antibodies has made this
system ideal for research into cellular-specific contrast agents.
Initial research into the use of liposomes for drug delivery was
hindered by several factors. First, their design had to be
optimized so that liposomes could carry the desired chemical
"payload." Otherwise, electrostatic forces of the passenger
chemical or charged surface lipid membranes could cause rapid
instability of the liposome.
Small, neutral liposomes with bilayers composed of "rigid"
phospholipids of high-phase-transition temperature were required
for prolonged in vitro stability.
Subsequent in vivo experimentation demonstrated rapid clearance of
liposomes by the reticuloendothelial system (RES) in a
This problem was overcome by the development of sterically
stabilized liposomes (SSL), which were covered by a polymeric
coating of polyethylene glycol (PEG). These "stealth" liposomes
avoided clearance, presumably by steric inhibition of liposomal
Using SSLs, Huang et al
demonstrated preferential extravasation of chemical payloads in
solid tumors, presumably because of vascular abnormalities
associated with tumor angiogenesis. However, Horowitz et al
also showed that SSLs do not interact directly with tumor cells in
vitro or in vivo, instead accomplishing targeted delivery by
eventual degradation and local payload diffusion into target
Conventional nuclear imaging agents, such as gallium 67 and
indium 111, have been coupled to antibodies, chemotactic peptides,
and cytokines in an attempt to enhance imaging specificity.
Liposomes have also been evaluated for directed delivery of
radionuclides for either therapy or imaging, in both the central
nervous system and the rest of the body.
One form of radionuclide therapy that has potential efficacy
against malignant gliomas is boron neutron capture therapy (BNCT).
This two-step radiotherapy involves first targeting the tumor with
B, then exposing the boron to low-energy neutrons. The reaction
yields intensively ionizing particles that travel a maximum of
approximately 1 cell width, thereby minimizing collateral tissue
Delivery of large quantities of boron atoms through liposome
encapsulation is an active area of research.
Ultrasound contrast agents consisting of coated gas microbubbles
are used in conjunction with ultrasound imaging for diagnostic
studies. Two commercially available systems use human serum
albumin-encapsulated microbubbles, while a third uses a
galactose-based microbubble stabilized with fatty acids.
Air-filled monolayers of phospholipids have also been used
experimentally and demonstrate increased in vivo stability in
comparison with commercially available products.
Initial research into the use of liposomes in MRI incorporated
paramagnetic molecules into the liposome membrane in an attempt to
increase the blood-pool circulation time of contrast agents. In the
formation of the liposome membrane, paramagnetic polymerized
liposomes (PPLs) incorporated a derivative of gadopentetate
dimeglumine as the hydrophilic head group and diacetylene groups in
the hydrophobic acyl chains.
Following in vivo administration, 80% of the PPLs remained in the
blood pool after 2 hours; the half-life of elimination from the
blood pool was 19 hours.
Nonspecific enhancement of the kidneys and liver was also observed.
Additional research in this field was accomplished through the
mannan-coated liposomes containing gadolinium diethylenetriamine
penta-acetic acid (Gd-DTPA),
which demonstrated nonspecific enhancement of the liver and lung at
lower doses than intravenously injected gadolinium. Recently,
vascular endothelial-cellspecific enhancement was accomplished.
Vascular structures were targeted by antibody LM609 (Chemicon
International, Inc., Temecula, CA), specific to endothelial cell
receptors, which were conjugated to multivalent polymerized
vesicles (PVs) chelated to gadolinium to allow imaging through MR.
This protocol demonstrated tumoral enhancement, a result of
increased tumor vasculature.
Antibodies as targeting agents
Tremendous research effort has been focused on tumor-specific
antibodies as a potential therapy for various forms of cancer. A
prime example is the development of monoclonal antibodies (mAb)
directed against the p185HER2 receptor tyrosine kinase.
Human epidermal growth factor receptor 2 (HER2) is a protein found
on the surface of cells that, when functioning normally, has been
found to be a key component in regulating cell growth.
This protein is highly overexpressed in a significant portion of
breast and other cancers.
The anti-HER2 mAb trastuzumab induces an antitumor response as a
single agent but is most efficacious when coupled with
Recently, immunoliposomes constructed using the anti-HER2 mAb have
demonstrated improved antitumor effect over chemotherapy or
monoclonal antibody therapy alone, further highlighting the
importance of immunoliposomes for cell-specific targeting and drug
In vivo refinements
Pioneering studies in tumor targeting by chemotherapeutic
liposomes relied on the leaky capillaries of tumors for increased
The initial use of monoclonal antibodies as targeting agents
revealed increased uptake by the RES and subsequent decreased
In addition, comparatively poor tissue penetrance of the
immunoliposomes was observed.
The immunoglobulin Fc portion, which is the crystallizable fragment
of the immunoglobulin molecule formed after specific proteolysis,
may have directly caused these effects, as Fc is efficiently
recognized by the RES for normal opsonized pathogen removal.
Experiments with immunoliposomes that were created through use of
the immunoglobulin Fab' fragment demonstrated increased tissue
penetrance and prolonged circulatory time.
In our model system, we adapted for CT and MRI refinements
originally made for immunoliposome-based drug delivery. We
accomplished this by using Omnipaque (Amersham Health, Princeton,
NJ), an iodinated macromolecule widely used for CT contrast
enhancement, and Omniscan (Amersham Health), a
gadolinium-containing macromolecule widely used for MRI contrast
enhancement, as the chemical payloads, instead of chemotherapeutic
Our initial experiments sought to determine the theoretical
detection limit of our system with both CT and MR. We attempted in
vitro solid-organ tissue targeting using liposomes conjugated to
one of three antibodies. The first antibody was specific to
whole-rat anti-mouse Fc receptor (FcR). The Fc receptor is a
cell-surface protein utilized by the RES to bind immunoglobulins,
as they are specific for the terminal constant region of the
These liposomes were targeted toward the RES and lymphoid
structures, specifically liver and spleen. We also used lipopsomes
conjugated to anti-CD5 Fab' (a smaller immunoglobulin fragment
generated through proteolysis), which recognizes CD5, a signal
transduction molecule that modulates signaling through the T cell
or B cell receptors, and is present on the cell surface of T-helper
and B-cell subpopulations.
This construct would also target the RES and lymphoid structures.
Finally, we utilized Fas ligand (FasL), expressed on the surface of
immune-privileged cells, which binds to the Fas receptor present on
T cells to induce apoptosis.
The testicles, which highly express Fas ligand in order to confer
were specifically targeted through liposomes conjugated to
anti-FasL. Untargeted contrast-laden and targeted immunoliposomes
were incubated with a variety of freshly obtained murine tissues,
and the relative tissue enhancement was calculated.
Materials and methods
Rat anti-mouse FcR IgG antibodies were kindly provided by Sherie
Morrison, MD, Professor of Immunology, University of California,
Los Angeles. Humanized Fab' fragments of mouse anti-CD5 were kindly
provided by XOMA (Berkeley, CA). Commercially available anti-FasL
IgG antibodies were purchased through Research Diagnostics, Inc.
Preparation of liposomes and immunoliposomes
The lipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DSPC),
and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG),
as well as cholesterol were obtained from Avanti Polar Lipids
(Alabaster, AL). The PD-10 and Sephadex G-50 (Amersham Biosciences,
Piscataway, NJ) columns were purchased from Pharmacia Corporation
(Peapack, NJ). All other reagents were American Chemical Society
(ACS) reagent grade.
A 1000-mg mixture of DSPC, cholesterol, and DSPG was prepared in a
molar ratio of 2:1:0.1. The mixture was dissolved in
dichloromethane, placed in a rotary evaporator to remove the
solvent, and dried under vacuum overnight. Each batch of liposomes
was formed using 200 mg of this mixture.
To form liposomes with osmolarities below 800 mOsM, and because
the Omnipaque solution is quite viscous, the solutions were diluted
using phosphate-buffered saline (PBS), pH 7.4. Two mL of Omnipaque
was diluted 1:1, and 3.0 mL of Omniscan was diluted 3:1. These
specific dilutions were initially chosen in part for future in vivo
murine model studies, in which a clinical practice-equivalent
amount of contrast would be delivered.
For each batch of liposomes, 200 mg of the lipid mixture was
placed in a 16- * 100-mm culture tube under a nitrogen stream.
Next, 3.0 mL of the contrast agent/PBS solution was placed into the
tube. The tube was mounted into a VibraCell sonicator equipped with
a 1Ž8-inch probe and a water bath set at 65° C. The sample was
sonicated continuously for 1 minute at a setting of 4.5. After 1
minute, the remaining 1.0 mL of contrast agent/PBS solution was
added, and the sample sonicated for an additional 10 minutes.
During sonication, the liposome suspension became milky-white and
Using an Avestin Liposofast extrusion device (Avestin, Ottawa, CA),
2.0 mL of each liposome suspension was extruded through a 0.1 µm
polycarbonate membrane. The material was forced through the
membrane 11 times. The extruded liposomes were cleaned using PD-10
columns equilibrated with PBS pH 7.4 and diluted to 10 mg/mL total
lipid. The remaining liposome suspensions were stored at 4° C.
A 0.6 M solution of sodium periodate was prepared by dissolving 512
mg in 4.0 mL of water. Next, 2.0 mL of this solution was added to
each extruded liposome suspension. The reactions were carried out
in amber vials at room temperature for 30 minutes. After the
glycerol was oxidized to the reactive aldehyde, the suspensions
were cleaned using 15- * 100-mm Sephadex G-50 columns equilibrated
with 20 mM sodium borate buffer and 150 mM sodium chloride at pH
Antibodies and immunoliposomes
Each of the 6 conjugation reactions were carried out in 1.5-mL
plastic vials. The antibodies were added to the oxidized liposomes
and allowed to form Schiff-base linkages for 2 hours at room
temperature. These were then reduced to secondary amines by
reacting overnight with sodium cyanoborohydride at 4° C.
A 2-M solution of sodium cyanoborohydride was prepared with
caution by dissolving 125 mg in 1.0 mL of water. The antibodies
were dissolved in sodium borate buffer pH 8.4 as follows: anti-FcR,
100 µg in 15.6 µL; anti-CD5 Fab', 385 µg in 80 µL; and anti FasL
100 µg in 100 µL.
Anti-FcR immunoliposomes were prepared as follows: 150 µL of
oxidized Omnipaque liposomes were combined with 7.8 µL of anti-FcR
and 15.8 µL cyanoborohydride; 150 µL of oxidized Omniscan liposomes
were combined with 7.8 µL of anti-FcR and 15.8 µL cyanoborohydride.
Anti-CD5 Fab' immunoliposomes were prepared as follows: 400 µL of
oxidized Omnipaque liposomes were combined with 40 µL anti-CD5 and
40.0 µL cyanoborohydride; 400 µL of oxidized Omniscan liposomes
were combined with 40 µL anti-CD5 and 40.0 µL cyanoborohydride.
Anti FasL immunoliposomes were prepared as follows: 5.0 mL of
oxidized Omnipaque liposomes were combined with 100 µL anti-FasL
and 50.0 µL cyanoborohydride; 5.0 mL of oxidized Omniscan liposomes
were combined with 100 µL anti-FasL and 50.0 µL
Following reduction, the immunoliposomes were cleaned using 15-
* 100-mm Sephadex G-50 columns equilibrated with PBS pH 7.4. The
remaining oxidized liposomes were reduced under the same conditions
in order to serve as a control for the tissue experiments. The
blank, anti-FcR and anti-CD5 immunoliposomes were placed in vials
at a concentration of approximately 3 mg/mL total lipid. The
anti-FasL immunoliposomes were prepared at a concentration of
approximately 9 mg/mL total lipid. Each of the liposome suspensions
was measured for particle size using a Microtrac ultrafine particle
analyzer (UPA) (Leeds-Northrup, Phoenix, AZ) validated against
National Institute of Standards and Technology (NIST) standards
measuring 87 nm ± 10 nm. In each case, the liposomes measured
approximately 100 nm in diameter.
Murine tissue preparation and in vitro tissue
As protocols for in vivo studies including intravenous injection
were pending University of California Los Angeles Animal Research
Committee approval, an in vitro model system was initially
developed. Adult male C57BL/6 breeder mice euthanized for advanced
age were obtained immediately posteuthanization, having been
graciously donated by Genhong Cheng, MD, Associate Professor of
Immunology and Molecular Biology, University of California, Los
Angeles. All animals were maintained and euthanized following
protocols approved by the University of California Los Angeles
Animal Research Committee. Liver, spleen, peritoneal fat,
quadriceps muscle, and testes were harvested. The tissues were
gently teased apart in PBS buffer, in order to more approximate the
cell surface area accessible by an in vivo model system, and placed
in standard 24-well tissue-culture plates for incubation. Next, 100
µM targeted and nontargeted immunoliposomes were added to the
tissue medium, and the samples were incubated for 1 hour at 37° C,
followed by an overnight incubation at 4° C.
The tissue samples were washed 3 times with PBS buffer and then
transferred to standard 96-well tissue-culture plates in order to
concentrate tissue density with corresponding increase in
signal-to-noise ratios. These samples were then scanned on
CT and MRI experimental sample imaging
Following purification of contrast-laden liposomes, serial
dilutions were performed on these unconjugated immunoliposomes to
determine the lowest amount of contrast detectible in our model
system. Initial wells contained 100 µL of a 3 mg/mL suspension of
Omnipaque samples were imaged on a GE HiSpeed CT scanner (GE
Medical Systems, Waukesha, WI) using a 1-mm contiguous axial
volume-data acquisition, display FOV = 10 cm. The 96-well plates
containing dilutional or tissue samples were secured to the CT
gantry on sufficient padding to place the sample plate in the
center of the imaging bore.
Omniscan samples were imaged in the coronal plane on a Siemens
1.5T MRI scanner (Siemens Medical Systems, Iselin, NJ) with the
following parameters: TR = 15, TE = 6, FOV = 300 * 300, matrix =
128 * 256, slice thickness 1 mm. Samples containing labeled testes
tissues were imaged with an FOV of 400 * 400; imaging was not
repeated with a smaller FOV due to limited research magnet time.
Samples were secured on top of a saline bag to improve signal.
The sample-saline bag construct was placed in the center of an
extremity coil for further improved signal-to-noise ratio.
Imaging was performed on serial dilutions of unconjugated
immunoliposomes to determine the lowest amount of contrast
detectible in our model system (Figure 1). MR imaging demonstrated
progressive T1-weighted signal loss until a background level was
reached at 1:32 dilution (Figure 1A), with an air bubble partially
obscuring the signal in dilution well #4. The 1:32 dilution
corresponds to 9.4 µg unconjugated immunoliposome as our practical
limit of detection by MR.
Relative enhancement values were also calculated (Figure 1C).
From the digital image, the gray-scale pixel value of T1-weighted
enhancement was obtained utilizing ImageJ 1.29x, National
Institutes of Health public-domain software package. The absolute
values of the serial dilutions were plotted. A relative value of 1
was arbitrarily assigned to the gray scale value of dilution well
1:256, which demonstrated no increased signal in comparison with
downstream dilutional well samples. As the plot follows a near
linear progression, it is reasonable to conclude that at this
concentration range, increasing numbers of immunoliposomes will
produce proportional T1 signal increases.
CT examination of unconjugated Omnipaque immunoliposomes did not
demonstrate visible differences between well signal density. In
order to determine if this modality could be used to image
immunoliposomes at this concentration of payload contrast, the HU
of the various dilutional samples were measured. A maximal HU value
of 60 was seen in the first well, with progressive dilutions
resulting in background equilibration at a dilution of 1:8 (Figure
1B). This corresponds to 37.5 µg as the unconjugated immunoliposome
practical limit of detection by CT. Under our model conditions,
enhancement detection was approximately 4 times more sensitive by
MRI than by CT.
As we determined that it was possible to detect relatively small
amounts of immunoliposomes by both MR and CT imaging, tissue
enhancement patterns by cell-surface protein targeted
immunoliposomes were then explored. Freshly obtained C56BL/6 murine
liver, spleen, peritoneal fat, quadriceps muscle, and testes were
divided into 5 equal parts, incubated with uncongujated
immunoliposomes, anti-CD5 immunoliposomes, anti-FcR
immunoliposomes, anti-FasL immunoliposomes, and PBS, and processed
according to protocol. The tissues were then imaged by either CT or
MR T1-weighted imaging of murine tissue samples is shown in
Figure 2, which is a representative coronal slice. PBS-incubated
and unconjugated immunoliposome incubated tissues served as
negative and nonspecific controls, respectively. Relative
enhancement of tissues was calculated based on pixel values as
calculated in the Figure 1 studies, with PBS-incubated wells as the
reference value. Table 1 shows the relative enhancement values
compared with the native T1-weighted tissue signal. As testes and
splenic tissue comprised less mass than liver or muscle, pixel
values used in relative enhancement calculations were obtained from
coronal slices that contained the maximal amount of these
In comparison with the negative control, anti-CD5
immunoliposomes demonstrated liver enhancement, splenic
enhancement, and muscle enhancement on MRI, but did not result in
fat enhancement (Figure 2, Table 1). Anti-FcR immunoliposomes also
demonstrated selective 2-fold liver enhancement and slight splenic
enhancement, with less pronounced relative fat enhancement
(1.7-fold increase) (Figure 2). Anti-FasL immunoliposomes
demonstrated pronounced exclusive testes signal enhancement
(41-fold increase), with no appreciable increase compared with
background in the remaining tissues (Figure 2, Table 1).
Nonspecific binding of unconjugated immunoliposomes did not
result in any significant enhancement, as there was no significant
increase in signal between the unconjugated immunoliposome samples
and tissues incubated with PBS alone (Table 1).
Similar results were seen with CT imaging of cell-surface
protein targeted Omnipaque immunoliposomes incubated with murine
tissues (Table 2). As was demonstrated in the dilutional studies,
no visible difference in well density was obtained, so HU values
were again calculated for each sample well. No significant increase
in HU values was seen in peritoneal fat in any conjugated or
control well. As was seen in the MR studies, anti-CD5
immunoliposomes again enhanced liver (+27 HU) and splenic tissues
(+48 HU), and were also associated with an increase in muscle
attenuation (+27 HU). Anti-FcR immunoliposomes enhanced both liver
and spleen samples (+26 HU and +67 HU, respectively), while the
testes were enhanced only by anti-FasL conjugated immunoliposomes
Again, nonspecific binding of unconjugated immunoliposomes did
not result in any significant enhancement, as there was no
significant increase in HUs between the unconjugated immunoliposome
samples and tissues incubated with PBS alone (Table 2).
We have demonstrated tissue-specific enhancement by
contrast-laden immunoliposomes that are conjugated to monoclonal
antibodies directed against a variety of cell-surface proteins. The
highest tissue-specific enhancement was seen with anti-FasL
immunoliposomes, which is consistent with the tissue distribution
of this cell-surface protein and the known high surface
concentration of Fas Ligand on testes. Anti-FcR immunoliposomes
also successfully enhanced tissues of the RES known to express the
surface Fc receptor. Anti-CD5 immunoliposomes enhanced tissues that
would be expected to house B- and T-cell subtypes, but also
enhanced muscle tissue. One possible explanation is that inguinal
lymph nodes were included in the sample, but cross-reactivity with
this murine anti-human immunoglobulin against other murine tissues
is not excluded.
Our pilot in vitro studies also demonstrated the need to improve
the discriminatory capabilities of our CT immunoliposomes, as a
maximum enhancement of 67 HU was seen in this study. Conventional
contrast agents use macromolecules not only to improve the
solubility of constituent elements but also to provide a method to
control for osmolarity. As liposomes would naturally buffer iodine,
experiments in which more elemental forms of iodine are
encapsulated in the immunoliposome will be performed prior to in
vivo testing. As we are interested in increasing the ability of MRI
to discriminate target tissues, experiments using superparamagnetic
compounds in place of chelated gadolinium in the immunoliposomes
will also be performed.
Advances in both CT and MRI technology have been realized
through improvements in hardware, software, and contrast materials.
The use of nonspecific contrast and its differential uptake by
various tissues has widely expanded the diagnostic capabilities of
CT and MRI. Under certain circumstances, the specificity of these
imaging modalities now approaches that of histology. Cellular
targeting of contrast agents represents the next logical
progression. By merging the fields of molecular biology and
radiology, we have demonstrated one avenue toward this goal.
The synthesis of contrast-laden immunoliposomes, conjugated to
any of a wide arsenal of antibodies, can be accomplished in
relatively short order. As monoclonal antibody generation is an
established science, novel tissue-specific antibodies can be
developed to further increase the utility of this technique. While
multiple uses of this technique can be devised, we believe certain
applications of MRI and CT will initially be explored.
With MRI, the utility of contrast immunoliposomes will likely be
greatest in the characterization of the central nervous system.
Further research is needed to enable diagnostic reagents to
successfully cross the blood-brain barrier, however. Recently,
immunoliposomes conjugated to the transferrin receptor have
demonstrated successful translocation and delivery of
brain-specific promoter-region DNA to the parenchyma.
One possible experimental approach for enhanced delivery of
contrast would be double conjugation of the immunoliposomes with
transferrin and antibodies specific for tissues of the central
nervous system. Using this method, it may be possible to further
explore demyelinating diseases, as selective myelin enhancement may
lead to improved sensitivity for the detection of demyelinated
In both the central nervous system and in the remainder of the
body, contrast immunoliposomes may improve tumor characterization,
as tumor-specific agents could differentiate cancer from a
nonspecifically enhancing mass. Also, radiation necrosis could
reliably be differentiated from recurrent tumor through selective
enhancement. In the same way, the detection of small foci of
metastastic cells could also be improved through tissue-specific
With CT, differential cellular enhancement could improve
diagnosis in many clinical conditions. Lung nodules that are too
small to be detected by PET, for example, must now undergo a
complicated clinical and imaging protocol. Evaluation with a panel
of cell-specific contrast agents could instead allay patient
concerns, if granulomatous disease were confirmed, or increase
patient survival, if resectable lung cancer were detected.
In vivo histological characterization of masses could also be
accomplished. For example, specific tissue enhancement could
determine whether an adrenal mass originated from medullary or
cortical tissue, or a pelvic mass from germ or stromal cells.
Therapeutic response could be determined more accurately, as the
progress of tumoral enhancement in primary or metastatic cell foci
could be more accurately assessed. Micrometastatic disease might be
detected more readily, especially in lymph nodes containing tumor
foci that would otherwise be discounted due to CT size criteria for
malignant lymph nodes. Also, the extent of lymph node involvement
in breast and other cancers could be determined without surgical
dissection, thereby reducing morbidity and mortality.
We have shown that immunoliposomes targeted to cell-surface
proteins can successfully be used for tissue-specific contrast
enhancement in CT and MRI. This approach has the potential to
increase diagnostic specificity and sensitivity, and has broad
clinical applications. The study reported here will serve as the
foundation for our future research, which will first focus on
improving signal enhancement in both CT and MRI and, ultimately, on
developing tumor-specific immunoliposomes for in vivo detection of
both primary and metastatic lesions. Further research is needed
into techniques that enable crossing the blood-brain barrier for
central nervous system imaging before the benefits of
tissue-specific contrast are fully realized.
This work was supported in part by a grant from the Department
of Energy DE-FG03-95ER61975. The authors thank Brian Zarnagar for
his work in animal husbandry.