Published OnlineFirst March 9, 2010; DOI: 10.1158/0008-5472.CAN-09-2821
Integrated Systems and Technologies
Combined Delivery and Magnetic Resonance Imaging of Neural Cell Adhesion Molecule–Targeted Doxorubicin- Containing Liposomes in Experimentally Induced Kaposi’s Sarcoma
Cristina Grange1, Simonetta Geninatti-Crich2, Giovanna Esposito2, Diego Alberti2, Lorenzo Tei3, Benedetta Bussolati1, Silvio Aime2, and Giovanni Camussi1
Abstract
Specific targeting of tumors by combined delivery of drugs and of imaging agents represents an attractive strategy for treatment of cancer. The aim of the present study was to investigate whether neural cell adhesion molecule (NCAM)–targeted liposomes may enhance drug delivery and allow magnetic resonance imaging (MRI) in a severe combined immunodeficient mouse model of NCAM-positive Kaposi’s sarcoma. NCAM-binding peptide–coated liposomes loaded with both doxorubicin and a lipophilic gadolinium (Gd) derivative were generated. NCAM-targeted liposomes induced an enhanced in vitro doxorubicin internaliza- tion within Kaposi’s cells as detected by MRI with respect to untargeted polyethylene glycol liposomes. Internalization resulted in enhanced apoptosis. In vivo weekly administration of NCAM-targeted liposomes containing 5 mg/kg doxorubicin for 4 consecutive weeks induced a significant reduction of tumor mass and vascularization and enhanced cell necrosis and apoptosis with respect to untargeted liposomes. These ef- fects were associated with an enhanced concentration of doxorubicin within the tumor and a reduced sys- temic toxicity of doxorubicin. By electron microscopy, NCAM-targeted liposomes were detected mainly within tumor cells whereas the untargeted liposomes were mainly accumulated in the extracellular space. Gd-labeled liposomes allowed the MRI visualization of drug delivery in the tumor region. The intensity of MRI signal was partially hampered by the “quenching” of the attainable relaxation enhancement on endosomal entrapment of the Gd-labeled liposomes. In conclusion, targeting NCAM may be a suitable strategy for specific drug delivery and imaging by liposomes in NCAM-expressing tumors. Moreover, treatment with NCAM-targeted liposomes showed enhanced therapeutic effect and reduced toxicity with respect to untargeted liposomes. Cancer Res; 70(6); 2180–90. ©2010 AACR.
Introduction
Targeting tumors or tumor endothelial cells (TEC) with systems that combine delivery of drugs and imaging agents represents one of the most important challenges for the treatment of cancer. Lipid-based nanosized particles have been proved to be suitable as carrier for the delivery of several therapeutic agents (1–3). Due to its superb spatial
Authors’ Affiliations: 1Department of Internal Medicine and 2Center for Molecular Imaging, Department of Chemistry IFM, University of Turin, Turin, Italy and 3Department of Environmental and Life Science, University of Piemonte Orientale “A. Avogadro,” Alessandria, Italy
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
C. Grange and S. Geninatti-Crich contributed equally to this work.
Corresponding Author: Giovanni Camussi, Dipartimento di Medicina Interna, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14, 10126 Turin, Italy. Phone: 39-11-6336708; Fax: 39-11-6631184; E-mail: giovanni.camussi@unito.it.
doi: 10.1158/0008-5472.CAN-09-2821
©2010 American Association for Cancer Research.
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resolution, magnetic resonance imaging (MRI) seems to be the technique of choice for monitoring drug delivery process and therapeutic output (4). Liposomes seem to be good can- didates for carrying both therapeutic and contrast agents, as the payload can be encapsulated in their internal aqueous cavity and/or intercalated in the phospholipid bilayer, thus allowing the transport of both hydrophilic and hydrophobic compounds (5, 6). Clinical applications of liposomes in the delivery of anticancer agents for the treatment of different solid tumors are well established. A specific class of lipo- somes, named “long time circulating” polyethylene glycol (PEG)–coated liposomes, are able to accumulate in solid tu- mors as a consequence of increased microvascular perme- ability and defective lymphatic drainage (7, 8). The extent of passive extravasations is directly dependent on the pro- longed residence time of liposomes in the blood stream (9, 10). The most common and widely used chemotherapeutic agent encapsulated in liposomes is doxorubicin (11, 12). The efficacy of doxorubicin incorporated in PEG-coated lipo- some is under investigation in several tumor models and human clinical trials, such as ovarian, breast, brain, and
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hepatocellular tumors (13–16). Moreover, doxorubicin- containing liposomes are currently used for treatment of some solid tumors, such as Kaposi’s sarcoma (17, 18).
The targeting of liposomes to tumor cells is a promising strategy to reduce side effects and improve drug delivery to tumors. To this purpose, liposomes have been functionalized with specific ligands (peptides, monoclonal antibodies, and small organic molecules) able to bind and to internalize re- ceptors expressed on tumor vasculature or on tumor cells (19–24).
We recently reported the expression of the embryonic form of neural cell adhesion molecule (NCAM) by Kaposi’s cells and TECs (25). NCAM is an adhesion molecule structur- ally belonging to the immunoglobulin superfamily that med- iates cell-to-cell interactions (26, 27). Moreover, NCAM is expressed by several solid tumors and some leukemias (28). By exploiting a high-affinity NCAM-binding peptide (C3d; refs. 29, 30), we showed the possibility to target NCAM in vivo in neo-formed vessels (31).
The aim of this work was to investigate the possibility to use C3d-coated liposomes to enhance drug delivery and allow MRI visualization in an in vivo model of Kaposi’s sarcoma expres- sing NCAM. For this purpose, we generated C3d-coated PEG liposomes (C3d lipo) loaded with both doxorubicin and a lipo- philic gadolinium (Gd)–DOTA-monamide (DOTAMA) deriva- tive. In vitro, we tested the incorporation of C3d lipo within Kaposi’s cells and TECs by MRI, the delivery of doxorubicin, and its apoptotic effect. In vivo, we evaluated the therapeutic effect of C3d lipo in a model of human Kaposi’s sarcoma in severe combined immunodeficient (SCID) mice and the concomitant drug delivery and MRI. The effect of C3d lipo was compared with that of C3d-uncoated PEG liposomes (PEG lipo) containing the same dose of doxorubicin and Gd-DOTAMA derivative.
Materials and Methods
Preparation of doxorubicin-loaded liposomes. POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DSPE-PEG-PDP {1,2-distearoyl-sn-glycero-3-phosphoethano- lamine-N-poly(ethyleneglycol)-2000-N-[3-(2-(pyridyldithio) propionate] ammonium salt}, and DSPE-PEG {1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt} were purchased from Avanti Polar Lipids. Cholesterol and all other chemicals were pur- chased from Sigma-Aldrich. The lipophilic Gd-DOTAMA (C18)2 was synthesized according to a previously reported procedure (32). Liposomes were prepared by following the thin lipid film hydration method (33). Doxorubicin (hydro- chloric salt) incorporation in the internal liposome aqueous phase was done by incubating 0.03 mg of drug per 1 mg of phospholipids for 1 h at 55°C as described (34). The NCAM mimetic C3d peptide (C3 peptide: ASKKPKRNIKA) and the control peptide C3d-Ala, in which Lys6 and Arg7 residues are substituted with alanines, were synthesized as previously described (25). For conjugation to thiol-containing mole- cules, a cysteine residue was added at the COOH terminus
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of the tetrameric peptides before the lysine backbone (C3d- Cys and C3d-Ala-Cys). A scramble tetrameric peptide (S3d) containing the COOH-terminal cysteine (S3d-Cys-S3d pep- tide: KAKSRINKAKP) was also synthesized as described in Supplementary Data. The conjugation of the peptides to the liposome was done as described (ref. 35; Supplementary Data). The amount of Gd-DOTAMA(C18)2 incorporated in the liposome was determined by 1H nuclear magnetic reso- nance T1 measurement at 20 MHz, 25°C (Stelar Spinmaster) of the mineralized complex solution (in 6 mol/L HCl at 120°C for 16 h). Doxorubicin encapsulation yield was ∼80% and was measured after the peptide conjugation. The hydrated mean diameter of liposomes and the ζ potential were deter- mined using a Malvern dynamic light scattering spectropho- tometer (Malvern Zetasizer 3000HS). All samples were analyzed at 25°C in filtered (cutoff, 100 nm) HBS buffer (pH 7). The polydispersity index for all the liposomes prepared in this work was smaller than 0.2. The liposomes were stored at 4°C under argon and used within 2 d from the preparation. FITC-labeled liposomes have been prepared by adding to the lipid mixture 0.1% 1,2-dioleoyl-sn-glycero-3-phosphoethano- lamine-N-(carboxyfluorescein) (ammonium salt).
Cell lines. A primary culture of Kaposi’s cells was obtained from a cutaneous lesion of a patient bearing renal allograft under immunosuppressive therapy and previously character- ized (36). Kaposi’s cells were maintained in culture in RPMI with 10% FCS. TECs were obtained from renal clear cell car- cinomas as previously described (37); TECs were maintained in culture in EBM complete medium (Cambrex Bioscience) supplemented with 10% FCS.
Uptake experiments and MRI analysis. Cells were incu- bated with the liposomes at different Gd concentrations for 3 h at 37°C in the complete medium. Cells were then washed, detached with EDTA (1 mmol/L), washed, and transferred in- to glass capillaries placed in an agar phantom for MRI anal- ysis. MRI was acquired on a Bruker Avance 300 spectrometer (7 T) equipped with a Micro 2.5 microimaging using a stan- dard T1-weighted multislice multiecho sequence (TR/TE/ NEX, 200:3.3:16; FOV, 1.2 cm; one slice = 1 mm; in-plane resolution, 94 × 94 μm). T1 measurements of cells were done by using a standard saturation recovery sequence.
Labeling of cell lines and liposomes for in vitro binding assay. We labeled the cells with carboxyfluorescein diacetate succinimidyl ester (Vybrant CFDA SE Cell Tracer Kit, Molec- ular Probe) and the liposomes with the PKH26 Red Fluores- cent Cell Linker Kit (Sigma) following the manufacturer’s instruction. Cells were incubated for 30 min at 37°C with liposomes (C3d, C3d-Ala, or PEG) and, after washing, were fixed in 3.5% paraformaldehyde containing 2% sucrose. Confocal microscopy analysis was done using a Zeiss LSM 5 Pascal model confocal microscope (Carl Zeiss Internation- al). Hoechst 33258 dye (Sigma) was added for nuclear stain- ing. Four different liposome preparations were tested.
Apoptosis assay. For in vitro experiments, we incubated cells (Kaposi and TEC), cultured in a 24-well plate, for 1 h with three different liposomes (C3d, C3d-Ala, or PEG) and with doxorubicin alone as positive control. For each stimulus, we used increasing doses of doxorubicin (100, 250, and
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Figure 1. A, schematic representation of liposomes loaded with both doxorubicin and the lipophilic Gd-DOTAMA(C18)2 MRI contrast agent. B, representative T1-weighted spin echo MRI (measured at 7 T) of an agar phantom containing unlabeled cells (1) or cells incubated with 0.1 mmol/L Gd of C3d lipo (2), C3d-Ala lipo (3), or PEG lipo (4) for 3 h at 37°C. C, doxorubicin intracellular concentration measured in Kaposi’s cells and TECs after incubation (for 3 h) with C3d lipo, PEG lipo, and doxorubicin alone, all at 50 μg/mL doxorubicin. Columns, mean of six different experiments; bars, SD. *, P < 0.01, C3d lipo versus PEG lipo and doxorubicin alone (ANOVA with Newman-Keuls multicomparison test). D, correlation between observed relaxation rates (R1obs s−1) measured at
7 T and doxorubicin delivered to target cells after 3-h incubation with C3d lipo at increasing concentrations of Gd (0.01–0.1 mmol/L).
500 ng/mL). After 1 h, each sample was washed and medium was replaced with fresh medium. Apoptosis was evaluated af- ter 48 h by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (ApopTag Oncor, Milli- pore). Results are expressed as the mean (± SD) percentage of apoptotic cells per field of four different experiments done in triplicate.
Moreover, we assessed apoptosis by TUNEL assay on slices of tumors recovered at the end of the in vivo experiment in SCID mice; paraffin-embedded slices of tumors were depar- affinized, and TUNEL assays were done following the manu- facturer’s instruction. We counted the number of apoptotic cells per field in 20 randomly chosen sections under ×200 magnification. We expressed the number of apoptotic cells as mean ± SD.
In vivo therapeutic studies. All animal experiments were done according to the guidelines for the care and use of re- search animals and were approved by the local ethics com- mittee. Male mice were 6 to 8 wk of age and their weights were ∼22 to 24 g. Kaposi’s cells were s.c. implanted in the flank of SCID mice (Charles River Laboratories, Inc.) within growth factor Matrigel (Sigma; day 0); cells (2 × 106) were
2182 Cancer Res; 70(6) March 15, 2010
resuspended in 150 μL of DMEM and added to 150 μL of Matrigel. Ten days after the inoculation of tumor cells, mice were randomly divided into three groups. The first group (n = 12) received the functionalized liposomes (C3d lipo), the second group (n = 12) was treated with PEG lipo, and the third (n = 12), as control, was treated with equal volume of PBS. Each mouse received in the tail vein a dose of lipo- somes corresponding to 5 mg/kg doxorubicin once per week for 4 consecutive weeks. Mice were monitored two times weekly for body weight and for evidence of tumor or doxoru- bicin treatment–associated morbidity. Four animals per each group received FITC-labeled liposome in the last i.v. injection for fluorescence staining of liposomes in tumors. Twenty-four hours after the last liposome injection (36 d after the injection of tumor cells) was considered the end of the experiment. Mice were weighed and sacrificed, and the organs (kidney, liver, tumor, brain, and spleen) were recovered for histology. Doxorubicin alone (5 mg/kg) given with the same regimen in six mice or liposomes coated with scramble peptide (see Supplementary Data) were used as controls.
Histologic analysis. For histology, 5-μm-thick paraffin sections of tumor, kidney, liver, brain, and spleen were
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routinely stained with H&E (Merck). For the determination of vessel number, sections were stained with Masson tri- chrome (Masson trichrome Golden, Bio-Optica) and the number of vessels with blood cells inside was counted in 20 randomly chosen sections (×400).
Immunofluorescence. For confocal microscopy analysis, sections from paraffin-embedded tumors treated with FITC liposomes were stained with HLA class I polyclonal antibody (BioLegend). Texas Red goat anti-rabbit IgG (Molecular Probes) was used as secondary antibody. Hoechst 33258 dye (Sigma) was added for nuclear staining.
Electron microscopy. Transmission electron microscopy was done on Karnovsky’s fixed, osmium tetraoxide–postfixed tissues and embedded in epoxy resin according to standard procedures (38). Ultrathin sections were stained with uranyl acetate and lead citrate and were examined with a Jeol JEM 1010 electron microscope.
Quantification of doxorubicin in tumor. In eight animals per group, the doxorubicin concentration was measured by spectrofluorimetric analysis as described (ref. 39; see Supple- mentary Data). Tumors of untreated mice were used as neg- ative control. The results were expressed as mean (± SD) micrograms of doxorubicin per gram of tissue. Fluorescence intensity was measured with a Fluoromax-4 spectrofluorim- eter (Horiba Jobin Yvon). Excitation wavelength was posi- tioned at 480 nm and emission wavelength was set at 595 nm. The release of doxorubicin in plasma samples of three healthy individuals and in PBS was evaluated at 5 and 24 h.
Biodistribution and MRI. MRI was acquired at 7 T on a Bruker Advance spectrometer as described above, using a T1- weighted, fat-suppressed, multislice multiecho protocol (TR/ TE/NEX, 250:3.2:6; FOV, 3 cm; one slice = 1 mm). Fat suppres- sion was done by applying a presaturation pulse (90° BW = 1,400 Hz) at the absorption frequency of fat (−1,100 Hz from water). MRI images were acquired 1 d before every adminis- tration and 5 and 24 h after treatments. Tumor volumes were estimated from MRI images two times per week. Before MRI examination, animals were anesthetized by injecting tileta- mine/zolazepam (Zoletil 100, Virbac, 20 mg/kg) and xylazine (Rompun, Bayer SpA). The mean signal intensity values were calculated in regions of interest drawn on the whole tumors, liver, muscle, spleen, cortical regions of kidneys, and an ab- dominal vein. Treatment outcome was assessed by calculat- ing the percentage of tumor growth inhibition in the group treated with C3d lipo compared with the group that received PEG lipo or vehicle alone (PBS).
Statistical methods. Data are presented as means ± SD. Differences were determined by Student’s t test or ANOVA with the Newman-Keuls multicomparison tests when appro- priate. A P value of <0.05 was considered significant.
Results
Preparation and characterization of liposomes. To perform MRI-guided drug delivery, liposomes were loaded with doxorubicin and the Gd-based MRI contrast agent Gd-DOTAMA(C18)2 (Fig. 1A). The millimolar relaxivity (20 MHz, 25°C) of Gd-DOTAMA(C18)2 when incorporated
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in the liposome formulation was 15.8 (mmol/L)−1 s−1. This value is particularly high as a consequence of both the de- crease of the molecular mobility after the insertion in the li- posome membrane and the relatively fast water exchange rate through the phospholipid bilayer (40). The NCAM target- ing liposomes (C3d lipo) were generated using a C3d peptide having a cysteine residue at the COOH terminus conjugated with the PDP functions on the liposome surface. A control liposome (C3d-Ala lipo) functionalized with a nonspecific peptide (C3d-Ala), in which a lysine and an arginine involved in the binding to the receptor of the C3d peptide were re- placed by two alanines, was prepared with the same protocol. A liposome functionalized with the C3d scrambled peptide S3d was also prepared as control. The amount of doxorubicin encapsulated in the liposomes remained almost unchanged on conjugation with the peptides. The average liposome hy- drodynamic diameters were unaffected on peptide conjuga- tion, and they were 120 ± 20 nm for all investigated formulations as detected by dynamic light scattering mea- surements. Liposome ζ potentials were −12.5 ± 0.6, +21 ± 2, +9 ± 2, and +18 ± 2 mV for PEG lipo, C3d lipo, C3d-Ala lipo, and S3d lipo, respectively.
As shown in Supplementary Fig. S1, C3d lipo and PEG lipo became stable on incubation at 37°C in PBS and in plasma for 5 and 24 hours. The amount of Gd remained unchanged for PEG and C3d lipo in both media (not shown).
In vitro liposome cell uptake and MRI. We previously showed that Kaposi’s cells and TECs express on their surface the polysialylated embryonic form of NCAM (25). Liposomes were tested on Kaposi’s cells and TECs in vitro to evaluate the amount of Gd and doxorubicin taken up by cells and the cyto- toxic effect. The results obtained using C3d and C3d-Ala tar- geted liposomes were compared with those obtained using nontargeted PEG lipo. The MRI acquired at 7 T on Kaposi’s cell and TEC pellets (Fig. 1B) showed the specific accumulation of C3d lipo in tumor cells with respect to C3d-Ala lipo and PEG lipo. Figure 1C shows that the intracellular concentration of doxorubicin in Kaposi’s cells and TECs incubated with C3d lipo was significantly higher than that found in cells treated with PEG lipo. A good correlation (r = 0.89 and 0.94 for Kaposi’s cells and TECs, respectively) between Gd relaxation enhance- ment and doxorubicin delivered to target cells after the incu- bation with C3d lipo was found (Fig. 1D). The Gd millimolar relaxivity of internalized C3d lipo was 1.8 (mmol/L)−1 s−1 at 7 T, a value significantly lower than that measured at the same magnetic field in a 1 mmol/L aqueous solution of C3d lipo [r1p = 4.2 (mmol/L)−1 s−1]. This observation supports the view that the liposomes are internalized through receptor-mediated endocytosis. As previously shown (41, 42), the occurrence of a low r1p value is a good indication that the Gd complexes are confined in endosomal vesicles, thus resulting in a “quenching” of the observed relaxivity.
In vitro apoptotic effect of C3d lipo internalization. By confocal microscopy, C3d lipo labeled with the red fluores- cent dye PKH26 were detected inside Kaposi’s cells and TECs after 1 hour of incubation, whereas only minimal internaliza- tion was seen using PEG lipo (Fig. 2A). When cells were incubated with liposomes coated with the control C3d-Ala
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Figure 2. Internalization of liposomes and apoptosis in Kaposi’s cells and TECs.
A, representative confocal micrographs showing the internalization of C3d lipo
after 1 h of incubation at 37°C. Minimal internalization was observed with PEG lipo. Liposomes were labeled with the red dye PKH26, cells with green CSFE, and nuclei with Hoechst 33258 (magnification, ×630). B, apoptosis detected by TUNEL assay. Kaposi’s cells and TECs were incubated for 1 h at
37°C with C3d lipo, C3d-Ala lipo, PEG lipo, or doxorubicin alone as described in Materials and Methods. Apoptosis was evaluated
after 48 h. Columns, mean of four different experiments performed in triplicate; bars,
SD. *, P < 0.01, Student’s t test.
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Figure 3. Effect of liposome treatment on SCID mice bearing Kaposi’s sarcomas. Ten days after s.c. injection of 2 × 106 tumor cells, mice were
treated weekly with 5 mg/kg doxorubicin encapsulated in C3d lipo or in PEG lipo or with vehicle alone (PBS; n = 12 per group). A, C3d lipo induced a significant reduction of tumor volume compared with PEG lipo or PBS alone, measured weekly by MRI. *, P < 0.05, C3d lipo versus PEG lipo or PBS (ANOVA). B, mortality of mice treated up to day 40 with 5 mg/kg doxorubicin containing C3d lipo, PEG lipo, PBS, or 5 mg/kg doxorubicin alone. *, P > 0.01, C3d lipo versus PEG lipo and doxorubicin; #, P < 0.01, PEG lipo versus doxorubicin (ANOVA with Newman-Keuls multicomparison test). C, representative micrographs of kidney histology of mice treated with doxorubicin alone showing glomerular injury, tubular casts, necrosis, and atrophy. D, representative micrographs of kidney histology of mice treated with C3d lipo; no significant renal alteration was observed. C and D, left, ×150; right, ×400.
peptide, no internalization was observed (not shown). Apo- ptosis was evaluated by incubating the cells with different doses of doxorubicin (100, 250, and 500 ng/mL) incorporated within liposomes or used alone as control. A dose-dependent increase in apoptosis was observed. C3d lipo induced a significant higher degree of apoptosis in both Kaposi’s cells
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and TECs compared with apoptosis induced by PEG lipo or C3d-Ala lipo (Fig. 2B).
In vivo effect of C3d lipo in an experimental model of Kaposi’s sarcoma. SCID mice injected s.c. with 2 × 106 human Kaposi’s cells developed a tumor that reached a di- ameter ranging from 4 to 7 mm after 1 week. Starting from
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day 10, mice were treated weekly with 5 mg/kg doxorubicin loaded in C3d lipo or PEG lipo or with vehicle (PBS). As shown in Fig. 3A, the size of tumors detected by MRI of mice treated with C3d lipo was significantly lower than that of mice treated with PEG lipo or PBS. The Kaposi’s sarcomas in SCID mice treated with PBS grew subcutaneously without a significant mortality up to day 40 when mice were sacri- ficed. When treated with doxorubicin alone, all mice died be-
fore day 32 as a consequence of doxorubicin toxicity. In these mice, a severe renal injury characterized by tubular necrosis and atrophy, glomerulosclerosis (Fig. 3C), and proteinuria (3,000 ± 500 mg/dL after 15 days) was observed. In contrast, none of the mice treated with C3d lipo and only 30% of those treated with PEG lipo died before the end of the observation period. No significant proteinuria (0–50 mg/dL) or renal his- topathologic lesions were observed in mice treated with C3d
Figure 4. C3d lipo treatment induced apoptosis and necrosis in Kaposi’s sarcomas and inhibited vascularization. A, representative micrographs of tumors treated with C3d lipo, PEG lipo, or PBS showing increased cell necrosis and reduced vascularization in C3d lipo with respect to PEG lipo–treated or PBS-treated mice (magnification, ×250). The number of vessels was calculated on trichromic sections as described in Materials and Methods. §, P < 0.01, C3d lipo versus PEG lipo and PBS; *, P < 0.01, PEG lipo versus PBS (ANOVA with Newman-Keuls multicomparison test). B, representative micrographs
and quantitative evaluation of apoptosis detected by TUNEL assay on tumors treated with C3d lipo, PEG lipo, and PBS (magnification, ×200). §, P < 0.01, C3d lipo versus PEG lipo and PBS (ANOVA with Newman-Keuls multicomparison test). C, representative micrographs showing transmission electron microscopy of Kaposi’s sarcomas developed in SCID mice treated with C3d lipo, PEG lipo, or PBS. Tumors of mice treated with C3d lipo show diffuse necrosis and numerous apoptotic cells (arrows) with characteristic nuclear fragmentation, which are absent or scarce in tumors treated with PEG lipo or PBS (magnification, ×6,000).
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Figure 5. Intratumor accumulation of liposomes, concentration of doxorubicin, and MRI detection. A, representative confocal microscopy of Kaposi’s sarcomas treated with C3d lipo, PEG lipo, or PBS analyzed 24 h after the i.v. treatments. Liposomes were labeled with FITC-conjugated lipids (green). The human Kaposi’s cells were stained with anti–HLA class I antibodies (red; magnification, ×630). B, extraction and quantification of doxorubicin from tumor tissue (n = 8 per group) 24 h after treatment. *, P < 0.01 (Student’s t test). C, representative T1-weighted MRI of Kaposi’s sarcomas acquired 24 h after the administration of C3d lipo and PEG lipo at a doxorubicin dose of 5 mg/kg corresponding to a Gd dose of 0.023 ± 0.02 mmol/kg. D, percentage of signal intensity enhancement measured in the regions of interest normalized using a standard Gd solution. #, P < 0.05 (Student’s t test).
lipo. No histologic alteration was observed in the liver, spleen, and brain in all the studied groups.
On histopathologic examinations, Kaposi’s sarcomas in mice treated with C3d lipo showed marked areas of necrosis, reduced vascularization (Fig. 4A), and enhanced apoptosis as seen by TUNEL assay and electron microscopy (Fig. 4B and C). The reduction of vessels and the enhanced apoptosis were significantly higher than in mice treated with PEG lipo or PBS. By electron microscopy, tissue dissociation by edema and characteristic fragmentation of apoptotic nuclei were observed in tumors of mice treated with C3d lipo, but not in tumors of mice treated with PEG lipo or PBS (Fig. 4C).
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By confocal microscopy, FITC-labeled C3d lipo injected in SCID mice were localized after 24 hours within the human tumor cells, detected as HLA class I–positive cells. In con- trast, only very limited incorporation was observed in mice treated with PEG lipo (Fig. 5A). This observation is in agree- ment with the significantly higher amount of doxorubicin extracted from tumors of mice treated with C3d lipo with respect to those treated with PEG lipo (Fig. 5B).
The accumulation of C3d lipo in tumors was also assessed by MRI. As seen in Fig. 5C, the localization of C3d lipo was clearly detectable within the tumors that showed a reduced size with respect to those treated with PEG lipo. However,
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the signal enhancement was lower than that elicited on PEG lipo administration (Fig. 5D). The electron microscopy of tu- mors showed that PEG lipo were mainly detectable outside of the cells in the interstitial and perivascular areas, whereas the C3d lipo were almost exclusively seen within the cyto- plasm of tumor cells (Fig. 6).
Liposome biodistribution, measured by MRI, showed that 5 and 24 hours after injection, the C3d lipo blood content was much lower than that of PEG lipo, with a concomitant increase in liver and spleen uptake (see Supplementary Data). To understand if the different biodistribution and ef- ficacy of PEG and C3d liposomes were due to their different surface charges, a control liposome functionalized with 0.5% of a scrambled peptide of C3d was injected at the same doxo- rubicin dose (5 mg/kg) in a group of mice. The percentage enhancement measured by MRI (see Supplementary Fig. S2) shows that the amount of scrambled peptide lipo- some (S3d lipo) accumulated in the tumor is significantly lower than that measured after the injection of C3d lipo.
Discussion
The results of the present study showed that the herein developed liposome functionalized with an anti-NCAM pep- tide may be used for a combined delivery of a cytotoxic drug
and MRI in a model of human Kaposi’s sarcoma in SCID mice. Moreover, the targeted liposomes were found to be more efficient for the delivery of drug for the regression of the tumor and less toxic with respect to nontargeted liposomes.
The Kaposi’s sarcoma is an angiogenic tumor often asso- ciated with conditions of immune system impairment, such as HIV1 infections or posttransplantation immunosuppres- sive therapy (43–45). We have recently found that Kaposi’s cells and tumor-derived endothelial cells express NCAM on their surface. NCAM is a widely expressed molecule during embryogenesis, which is downregulated in the course of dif- ferentiation to be reexpressed in some tumors and TECs (25). By exploiting NCAM expression, we recently developed a strategy to detect in vivo by MRI the neo-formed vessels us- ing a NCAM peptide (C3d; ref. 31). In the present study, the model of Kaposi’s sarcoma implanted in SCID mice was cho- sen because treatment with liposome containing doxorubicin is currently used as a therapeutic strategy for this tumor (46, 47). Doxorubicin incorporation into liposomes seems to reduce the systemic toxicity of the drug (48).
In the present study, we found that C3d-coated liposomes loaded with both doxorubicin and Gd-DOTAMA(C18)2 were internalized and induced apoptosis of Kaposi’s cells and TECs significantly more efficiently than did the uncoated
Figure 6. Representative transmission electron microscopy of Kaposi’s sarcomas treated with PEG lipo or C3d lipo. A, Gd-labeled PEG lipo are predominantly observed in perivascular interstitial space. Arrows, liposomes;
*, extracellular matrix. B, PEG lipo internalization is absent in tumor cells. C and D, representative electron microscopy showing the internalization of C3d lipo within tumor cells, which display alteration in the cytoplasmic organic
swelling and disorganization of mitochondria (M). E, liposome internalization in perivascular cells. F, representative micrograph
of tumor cells of mice injected with PBS. Original magnifications, ×15,000 (A, B, E, and F) and ×20,000 (C and D).
Cancer Research
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PEG liposomes. This indicated that the NCAM-C3d interac- tion allowed a specific internalization of C3d lipo whereas PEG lipo mainly remained extracellular.
The incorporation of doxorubicin in PEG-coated lipo- somes alters the pharmacokinetics of the drug, resulting in a smaller volume of distribution and slower plasma clearance than free doxorubicin, thus allowing an extravasa- tion at the site of enhanced permeability. In contrast, C3d lipo were rapidly internalized within the Kaposi’s sarcoma cells bearing NCAM, with a reduced persistence in plasma. This resulted in a more efficient reduction of tumor mass and vascularization. These effects were related to an enhanced in vivo intracellular accumulation of C3d lipo within the tumors associated with an enhanced intratumor concentration of doxorubicin with respect to PEG lipo. Moreover, C3d lipo were found to be less toxic than free doxorubicin and PEG lipo.
The specific delivery of C3d lipo to targeted Kaposi’s cells was confirmed by MRI both in vitro and in vivo. MRI showed the reduced size of tumors in C3d lipo–treated mice and al- lowed detection of drug delivery within the tumors. However, the intensity of MRI signal enhancement within the tumors was lower than that obtained with PEG liposomes. This is likely due to the internalization of paramagnetic liposomes into cells that can limit the attainable relaxation enhance- ment as a consequence of the reduced water exchange across the barriers among the different compartments. The efficien- cy of PEG lipo is more than two times higher than that of C3d lipo because of its prevalent extracellular distribution. In fact, by electron microscopy, PEG lipo were mainly de- tected outside of the cells in the interstitial and perivascular areas whereas the C3d lipo were almost exclusively seen
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    Disclosure of Potential Conflicts of Interest
    No potential conflicts of interest were disclosed.
    Acknowledgments
    We thank Dr. Massimo Visigalli (Bracco Imaging, Colleretto Giacosa, Italy) for the synthesis of Gd-DOTAMA(C18)2.
    Grant Support
    Regione Piemonte Nano-IGT project (Converging Technologies) and Ricer- ca Scientifica Applicata, Istituto Superiore di Sanità (40F.19), Associazione Italiana per la Ricerca sul Cancro, ENCITE project (FP7-HEALTH-2007-A), DiMI (CT-2005-512146) EU NoEs, and Meditrans (NMP4-CT-2006-026668).
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Combined Delivery and Magnetic Resonance Imaging of Neural Cell Adhesion Molecule−Targeted Doxorubicin-Containing Liposomes in Experimentally Induced Kaposi’s Sarcoma
Cristina Grange, Simonetta Geninatti-Crich, Giovanna Esposito, et al. Cancer Res 2010;70:2180-2190. Published OnlineFirst March 9, 2010.
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