Affiliations of authors: Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, K.U. Leuven, Leuven, Belgium (AD, AK, PDW); Pre-Authorisation Unit of Medicines for Human Use, EMEA, London, U.K. (AG); Department of Morphology and Molecular Pathology, K.U. Leuven, Leuven, Belgium (TR); Centre for Surface Science and Catalysis, K.U. Leuven, Heverlee, Belgium (DDV); Department of Clinical Immunology, K.U. Leuven Campus Gasthuisberg O/N, Leuven, Belgium (AK); F. Hoffmann-La Roche, Ltd., CNS Research, PRPD, Basel, Switzerland (JH); Laboratory for Physiology, Faculty of Medicine, K.U. Leuven Campus Gasthuisberg O/N, Leuven, Belgium (LM)
Correspondence to: Peter A. M. de Witte, PhD, Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, K.U. Leuven, Van Evenstraat 4, B-3000 Leuven, Belgium (e-mail: peter.dewitte{at}pharm.kuleuven.ac.be)
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ABSTRACT |
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INTRODUCTION |
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Most bladder cancers (98%) occur within the transitional epithelium as superficial transitional-cell carcinomas. Bladder cancer is ideally suited to treatment with photodynamic therapy because the lumen of the bladder is easily accessible by endoscopy and bladder tissue is more translucent than other human tissues (3). Photodynamic therapy of urothelial malignancies initially involved focal light delivery to one area, but the frequent multifocal and occult character of the disease requires irradiation of the whole bladder (4). The safety of whole-bladder photodynamic therapy depends on the selective uptake and retention of the photosensitizer by the malignant tissue to avoid major side effects, such as fibrosis and contraction of the bladder with upper tract obstruction due to oxidative damage of the muscularis (5,6).
One way to target the photosensitizer to malignant tissue is to take advantage of iron transport systems. Iron is transported within the body between sites of absorption, storage, and use by transferrin. Cells that require iron express transferrin receptors on their cell surface that mediate the cellular uptake of iron from the plasma transferrin. Because iron is an essential element for cell proliferation and metabolism, and because the increased proliferation of malignant cells is accompanied by a higher demand for iron, many tumor cells overexpress transferrin receptors on the cell surface and have a higher turnover of these receptors compared with normal cells (7). The magnitude of transferrin receptor expression and turnover is proportional to the proliferative ability of the tumor tissue, because a higher proliferation rate requires more iron (8). For example, bladder transitional-cell carcinoma cells overexpress transferrin receptors compared with normal bladder mucosa, in whichexcept for the proliferating cells of the basal layertransferrin receptors are undetectable (9,11). Moreover, the degree of transferrin receptor expression is associated with the histologic grade and pathologic stage of a tumor (10). In addition, superficial bladder tumors that overexpressed transferrin receptors compared with their normal cell counterparts had a higher recurrence rate than tumors that did not (11,12).
These data raise the possibility that transferrin can be used to target therapeutic compounds to bladder transitional-cell carcinoma cells. Previous investigations using a transferrin conjugate to target an HT29 human colon cancer growing in nude mice resulted in a poor tumor accumulation after systemic administration of the compound (16), probably because of the large amount of competing transferrin present in blood (13). However, whole-bladder photodynamic therapy provides an ideal situation in which transferrin conjugates can be administered directly to the bladder through catheterization and intravesical instillation. Intravesical instillation, unlike systemic administration, allows direct exposure of bladder tumor cells to transferrin conjugates without interference by competing transferrin.
We examined whether transferrin-mediated targeting of the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS4) is an effective strategy to attain a tumor-selective behavior of this compound when applied intravesically. For that purpose, the photosensitizer was stably encapsulated in polyethylene glycol (PEG) liposomes, and transferrin was conjugated to the functionalized terminal ends of the PEG chains. An advantage of liposomal delivery is that it provides the opportunity to increase the ratio of the entrapped compound to the targeting molecule, resulting in enhanced cellular uptake and therapeutic efficacy of the liposome-associated drug. We chose AlPcS4 over other photosensitizers because its hydrophilic character permits stable encapsulation in the aqueous internal compartment of the liposome. Moreover, AlPcS4 exhibits high molar absorption at 672 nm, a wavelength that is not absorbed or dispersed by endogenous tissue components (15). We prepared sterically stabilized liposomes by incorporating a fraction of PEG-derivatized phospholipids into the liposomal membranes. These highly hydrophilic polymers form a water shell at the liposome surface, repelling the absorption of opsonins (proteins or peptides that label targets for phagocytosis) and resulting in a reduced clearance of the liposomes by the mononuclear phagocyte system (16). This steric barrier allows the liposomes to be applied intravenously and enables them to transit more easily across tissue when applied topically because of its lubricating properties (17). We analyzed the accumulation of free AlPcS4 and AlPcS4 in transferrin-conjugated and unconjugated liposomes in both human transitional-cell carcinoma cells in vitro and in a rat orthotopic model system in vivo.
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MATERIALS AND METHODS |
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PEG liposomes were prepared as described previously by Huwyler et al. (18) by dissolving 5.2 µmol of distearoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL), 0.3 µmol of distearoyl phosphatidylethanolaminePEG [2000 d] (Shearwater Polymers, Huntsville, AL), 0.17 µmol of distearoyl phosphatidylethanolaminePEG [3400 d]maleimide (custom synthesized by Shearwater Polymers), and 4.5 µmol of cholesterol (Sigma, St. Louis, MO) into 5 mL of chloroform. The chloroform was evaporated over 1 hour with the use of a rotavapor, forming a thin lipid film that was then hydrated for 15 minutes at 40 °C with 1 mL of a 10 mM solution of AlPcS4 (Porphyrin Products, Logan, UT) in phosphate-buffered saline (PBS [pH 7.4]; Gibco-BRL, Paisley, Scotland). The hydrated phospholipids were mixed with 20 3-mm glass beads and resuspended by rotating on a rotavapor (180 rpm) without applying vacuum for 15 minutes at 65 °C. The resulting PEG liposomes were subjected to five freezethaw cycles (at 80 °C and 60 °C, respectively) and homogenized by extrusion through a 100-nmpore-size polycarbonate membrane (Avestin, Ottawa, Ontario, Canada). The PEG liposome suspension was then divided into two aliquots: One was used to prepare transferrin-conjugated PEG liposomes, and the other was used without further modification as nonconjugated PEG liposomes (Lip-AlPcS4).
All manipulations involving the photosensitizer AlPcS4 (preparation, purification, and analysis of liposomes) were performed under low-light conditions (i.e., <1 µW/cm2).
Transferrin Conjugation
Holo-transferrin (Sigma) was thiolated by adding 10 nmol of the compound to a fresh solution of 400 nmol of Traut's reagent (2-iminothiolane; Pierce, Rockford, IL) in 2 mL of borateEDTA buffer, pH 8.5 (0.15 M sodium borate, 0.1 mM EDTA). The mixture was shaken in the dark for 1 hour. The thiolated transferrin was then concentrated by ultrafiltration (Centricon-30; Amicon, Beverly, MA) to a volume of 0.2 mL, washed with 2 mL of PBS, pH 8.0 (0.15 M sodium chloride, 0.1 M Na2HPO4·2H2O), and reconcentrated by ultrafiltration to a volume of 0.2 mL. The thiolated transferrin was immediately added to one aliquot of PEG liposomes, and the mixture was incubated for 24 hours at 4 °C to allow reaction with the maleimide linkers of the PEG liposomes to generate transferrin-conjugated AlPcS4-containing PEG liposomes (Tf-LipAlPcS4).
Purification of Liposome Constructs
Lip-AlPcS4 and Tf-LipAlPcS4 were purified on Sephacryl S-500 HR gel filtration columns (1.6 x 16 cm; Pharmacia, Uppsala, Sweden) at 4 °C. The columns were equilibrated and eluted with PBS (pH 7.4). This method allowed us to separate AlPcS4-containing PEG liposomes from free AlPcS4 and transferrin-conjugated PEG liposomes from nonconjugated transferrin.
Analysis of Liposome Constructs
Average liposome diameter was determined by dynamic light scattering ( = 632.8 nm [HeNe laser],
= 173°, T = 26 °C) with the use of a noninvasive back-scattering high-performance particle sizer (ALV Laser, Langen, Germany). After incubation of freshly prepared liposomes at 37 °C or 4 °C (i.e., the temperatures of the experimental and storage conditions) for 24 hours or 1 month, respectively, the average diameters of the liposomes were reanalyzed.
The number of liposomes per preparation was determined from the average number of phospholipids per liposome and the amount of initially added phospholipid that was effectively incorporated into liposomes. The average number of phospholipids per liposome was based on the area (per polar head group) of the phospholipids and cholesterol, taking into account their molar ratios, and the total surface area of a liposome (19). The amount of initially added phospholipid that was effectively incorporated into liposomes was determined by complexation of the phospholipids with ferrothiocyanate as previously described (20).
To determine the average number of AlPcS4 molecules per liposome, we diluted an aliquot of the liposome suspension 100-fold in methanol : water (80 : 20 [vol/vol]) and measured the fluorescence of the released AlPcS4 at 360 nm (excitation wavelength) and 645 nm (emission wavelength) with the use of a microplate fluorescence reader (FL600; Bio-Tek Instruments, Winooski, VT). The concentration of AlPcS4 was calculated by using a calibration curve and converted to the number of AlPcS4 molecules per liposome.
The average number of transferrin molecules conjugated to a PEG liposome was quantified by the bicinchoninic acid assay as described by Wessel et al. (21). To test for leakage of AlPcS4 from the liposomes, we incubated a 10 µM suspension of liposomes (liposome concentration is expressed as a function of the AlPcS4 concentration) in cell culture medium (see below) at 37 °C for 24 hours or 1 week. The mixtures were then subjected to Sephacryl S-500 HR gel filtration, and fractions containing the liposome-encapsulated AlPcS4 were pooled and diluted in methanol. The fluorescence of the released AlPcS4, measured at 360 nm (excitation wavelength) and 645 nm (emission wavelength) using a microplate fluorescence reader, was used to calculate the AlPcS4 concentrations, which were compared with the AlPcS4 concentration of a 10 µM suspension of liposomes that was purified immediately after suspension in cell culture medium.
Tumor Cells and Culture Conditions
We used AY-27 rat bladder carcinoma cells, which originated from an N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamideinduced bladder transitional-cell carcinoma of Fischer CDF rats (cells originally developed by Dr. S. Selman and Dr. J. Hampton, Medical College of Ohio, Toledo, OH and obtained from Dr. F. Guillemin, University of Nancy, Nancy, France). J-82 and T-24 cells (transitional-cell carcinoma, urinary bladder, human) and RT-4 cells (transitional-cell papillary carcinoma, urinary bladder, human) were obtained from the American Type Culture Collection (Manassas, VA). RT-112 cells (transitional-cell papillary carcinoma, urinary bladder, human) were obtained from the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [DSMZ], Braunschweig, Germany). Cells were grown as monolayers at 37 °C in a humidified atmosphere with 5% CO2 in minimum essential medium containing Earle's salts and 2 mM L-glutamine supplemented with penicillin (100 IU/mL), streptomycin (100 µg/mL), amphotericin B (0.25 µg/mL), tylocin (60 µg/mL), 1% (vol/vol) nonessential amino acids, and 10% (vol/vol) fetal calf serum. The medium and all cell culture ingredients were obtained from Gibco-BRL. All cell manipulations involving AlPcS4 were performed under low-light conditions (<1 µW/cm2).
Flow Cytometry
AY-27 cells (0.5 x 106 cells) were fixed for 5 minutes in 1 mL of 0.5% paraformaldehyde (Sigma) in PBS (pH 7.4) at 4 °C and then washed with 1.7 mL of PBS and labeled with 3 µM (100 µL) transferrinpolylysinefluorescein isothiocyanate conjugate (TfpolylysineFITC; conjugate concentration expressed as a function of the FITC concentration; Sigma) in PBS for 25 minutes at 4 °C. The cell suspension was washed with 1.7 mL of PBS, and the resulting cell pellet was resuspended in 300 µL of 1% paraformaldehyde in PBS and subjected to flow cytometry. As a control for nonspecific binding of the fluorescent dye, AY-27 cells were similarly fixed, labeled with 3 µM fluorescein (Sigma), and subjected to flow cytometry. We used a FACSort flow cytometer (Becton Dickinson, Mountain View, CA) utilizing an argon laser with an excitation wavelength of 488 nm. The fluorescence was measured after passage through a 530-nm band-pass filter. A total of 5 x103 events were analyzed per sample. Three replicate experiments were performed, and each sample was assayed in triplicate. The data were analyzed using CellQuest software (Becton Dickinson).
In Vitro Quantification of Intracellular AlPcS4 Accumulation
AY-27 cells were seeded onto transparent 24-well tissue culture plates (Costar, Cambridge, MA) at 3 x 105 cells per well and incubated for 24 hours at 37 °C. Under low-light conditions, the culture medium was replaced with fresh medium containing 10 µM free AlPcS4, 10 µM Lip-AlPcS4, or 10 µM Tf-LipAlPcS4 (photoactive compound concentration expressed as a function of the AlPcS4 concentration). The cells were incubated for 4 hours or 24 hours, after which the cellular accumulation of the photoactive compounds was determined as previously described (22). Three replicate experiments were performed.
We conducted two additional experiments for AY-27 cells incubated with Tf-LipAlPcS4. In the first experiment, AY-27 cells were incubated for 4 hours with Tf-LipAlPcS4 in the presence of 50 µM free transferrin. In the second experiment, AY-27 cells were first incubated for 2 hours in a 10 µM solution of colchicine or cytochalasin B (Sigma) in medium supplemented with 1% fetal calf serum, as described previously (2325), and then immediately incubated for an additional 2 hours in medium supplemented with 10% fetal calf serum and 10 µM Tf-LipAlPcS4 (concentration expressed as a function of AlPcS4 concentration). The cellular accumulation of the photoactive compounds was then determined as previously described (22). Three replicate experiments were performed.
In Vitro Fluorescence Microscopy
AY-27 cells were seeded onto Lab-Tek two-chamber glass slides for tissue culture (Nunc, Naperville, IL) at 6 x 104 cells per chamber and incubated for 24 hours at 37 °C. Under low-light conditions, the culture medium was then replaced with fresh medium containing 10 µM free AlPcS4, 10 µM Lip-AlPcS4, or 10 µM Tf-LipAlPcS4 (photoactive compound concentration expressed as a function of the AlPcS4 concentration). The cells were incubated for 2, 4, or 24 hours, and then the medium was removed and the cells were washed three times with PBS (pH 7.4). Fluorescence microscopy was used to localize the intracellular accumulation of AlPcS4. We used an AxioSkop 2+ fluorescence microscope (Carl Zeiss, Göttingen, Germany) equipped with a 100-W mercury lamp. The filter set used for fluorescence imaging included a 575625-nm band-pass excitation filter and a 660710-nm band-pass emission filter. Fluorescence images were acquired with a light-sensitive charge-coupled device digital camera (AxioCam HR; Carl Zeiss). Rapid observation and electronic image storage were used to prevent photobleaching of the AlPcS4 fluorescence. For uniformity, all parameters pertaining to fluorescence excitation and detection (e.g., exposure time and gain) were held constant throughout the study. Three replicate experiments were performed.
In Vitro Photodynamic Therapy
AY-27 cells were seeded onto transparent six-well tissue culture plates (Costar) at 9 x 105 cells per well and incubated for 24 hours at 37 °C. For each six-well plate, we used only a single well, the size of which corresponded to the diameter of the light beam used for photodynamic therapy (see below). Under low-light conditions, the culture medium was then replaced with fresh medium containing 1, 5, or 10 µM free AlPcS4, Lip-AlPcS4, or Tf-LipAlPcS4 (photoactive compound concentration expressed as a function of the AlPcS4 concentration). The cells were incubated for 2 or 4 hours, the medium was removed, and the cells were washed two times with PBS (pH 7.4) and then irradiated for 10 minutes in 2.5 mL of PBS. For irradiation, the six-well plate was mounted 10 cm above a water-cooled 1000-W halogen lamp equipped with a filter (center wavelength = 651 nm; band width = 85 nm at 50% transmission; Melles Griot, Irvine, CA). The output at the absorption maximum of AlPcS4 (672 nm) was 84% of the peak transmission of the filter. The uniform fluence rate at the surface of the irradiated well was 20 mW/cm2, as measured with an IL 1400 radiometer (International Light, Newburyport, MA). During irradiation, the temperature of the cell culture medium never exceeded 25 °C. Immediately after irradiation, the cells were harvested by trypsinization and pelleted by centrifugation (5 minutes, 500g), and the cell pellets were resuspended in fresh cell culture medium lacking photoactive compounds. The number of cells per well was quantified by using a Coulter Z1 particle counter (Coulter Electronic, Luton, U.K.). An appropriate number of cells sufficient to yield 50100 colonies was plated onto a 100-mm Petri dish in 15 mL of cell culture medium lacking photoactive compounds and incubated for 9 days. The cells were then fixed and stained for 2 minutes with 1% (wt/vol) methylene blue in methanol.
Colonies with more than 50 cells were counted during viewing with a dissection microscope. Because photodynamic therapy can lead to an immediate and complete fragmentation of cells, a fraction of the cells originally present in the irradiated wells are not quantified by the Coulter counter and therefore are not processed in the clonogenic assay. This fraction (R1) was quantified from the ratio of the number of cells harvested immediately after photodynamic therapy to the number of untreated, control cells (as assessed by using the Coulter counter). The final survival fraction (SF) was then calculated according to SF = R1 x R2, in which R2 is the ratio of the cloning efficiency of treated cells to the cloning efficiency of untreated, control cells (as assessed by the clonogenic assay).
Three replicate experiments were performed.
Rat Orthotopic Superficial Bladder Tumor Model
Ten- to fifteen-week-old female Fischer CDF (F-344)/CrlBR rats weighing 150175 g (Charles River Laboratories, Sulzfeld, Germany) were used to establish a superficial urothelial orthotopic bladder tumor model as previously described (26,27). The rats were anesthetized with intraperitoneal injections of sodium pentobarbital (45 mg/kg of body weight) and placed in a supine position on a homeothermic blanket to maintain their body temperature. We next inserted a catheter into the bladder via the urethra with the use of an 18-gauge plastic intravenous cannula and rinsed the bladder for 15 seconds with 0.4 mL of 0.1 N hydrochloric acid, followed by a 15-second rinse with 0.4 mL of 0.1 N sodium hydroxide. The bladder was then drained and washed five times with PBS (pH 7.4). A suspension of AY-27 cells (106 cells in 0.5 mL of medium) was then instilled and maintained in the bladder for 1 hour. The rats were turned 90° laterally every 15 minutes to ensure exposure of the entire bladder wall to the tumor cells. The catheters were removed, and the rats were allowed to void spontaneously. The rats were then used for intravesical administration of the photoactive compounds 6 days after tumor cell instillation. All animal procedures were carried out in compliance with national and European regulations and were approved by the Animal Care and Use Committee of the Katholieke Universiteit Leuven.
Intravesical Administration of Photoactive Compounds
We used intravesical instillation to introduce Lip-AlPcS4, Tf-LipAlPcS4, and free AlPcS4 into the bladders of rats that either had or had not been instilled with AY-27 cells. To allow the liposomes to penetrate the bladder wall, the glycocalyx of the bladder transitional epithelium was partially removed by pretreating the bladder with Proteus vulgaris chondroitinase ABC (Sigma), an enzyme that selectively digests chondroitin and dermatan sulfates (28). Rats were anesthetized and catheterized, and 3 U of chondroitinase ABC in 0.5 mL of PBS or 0.5 mL of PBS alone was instilled into the bladder through the catheter and maintained in the bladder for 1 hour. The rats were turned 90° laterally every 15 minutes to ensure homogeneous exposure of the bladder wall to the enzyme solution. The bladder was then drained, and 0.5 mL of AlPcS4, Lip-AlPcS4, or Tf-LipAlPcS4 in PBS (at 10, 100, or 400 µM; concentration expressed as a function of AlPcS4 concentration) was instilled into the bladder through the catheter for 2 hours before biodistribution evaluation. Rats were separated into 36 experimental groups (three rats per experiment group; i.e., three photoactive compounds at three different concentrations; tumoral versus nontumoral bladder; with versus without chondroitinase ABC treatment).
Biodistribution Studies
After exposure of the bladders to the compounds, rats were killed by pentobarbital overdose. Their bladders were immediately removed, snap-frozen, and stored in liquid nitrogen.
Immunohistochemical staining for chondroitin. We performed immunohistochemical staining for chondroitin on 5-µm frozen bladder sections from rats that were instilled with AY-27 cells, with and without chondroitinase ABC pretreatment. Bladder sections were dried onto glass slides and fixed in acetone. The sections were rehydrated in PBS and then incubated for 30 minutes with a murine monoclonal antichondroitin sulfate antibody (10-fold dilution in PBS; Sigma), which reacts specifically with chondroitin sulfate types A and C but not with dermatan sulfate type B. The sections were washed with PBS and then incubated for 30 minutes with a horseradish peroxidaseconjugated goat antimouse immunoglobulin G secondary antibody (100-fold dilution in PBS; Sigma). The slides were rinsed with PBS and incubated for 30 minutes with a horseradish peroxidaseconjugated rabbit antigoat immunoglobulin G (100-fold dilution; Sigma) that was previously absorbed with rat liver acetone powder (Sigma) to block nonspecific binding. The slides were rinsed with PBS and placed in a solution of 2.4 mM 3-amino-9-ethyl-carbazole (Janssen-Cilag, Geel, Belgium) in 50 mM sodium acetate buffer (pH 4.9) for 10 minutes. The slides were then washed with 50 mM acetate buffer, and the cell nuclei were counterstained for 1 minute with Mayer's hematoxylin solution (Sigma). The slides were mounted with coverslips by using glycerol jelly (British Drug House, Dorset, U.K.).
Localization and quantitation of AlPcS4 fluorescence in the bladder wall. Imaging of AlPcS4 fluorescence in sections of bladder tissues from rats that had and had not received AY-27 cells was carried out by fluorescence microscopy. Two hours after instillation of the liposome constructs or AlPcS4, the bladders were drained and rinsed twice with PBS (pH 7.4) through the catheter. The rats were killed, and their bladders were removed, cut open, and immediately embedded in Tissue Tek OCT embedding medium (Miles Laboratories, Elkhart, IN) and immersed in liquid nitrogen. Two consecutive 5-µm tissue sections were cut transversely from each frozen bladder with a cryostat. The first section was stained with hematoxylineosin, and the second section was examined by fluorescence microscopy as described above. We used KS imaging software (Carl Zeiss, Hallbergmoos, Germany) to measure the mean AlPcS4 fluorescence in specific areas of the bladder. We measured the fluorescence in arbitrary units of 20 sections (size = 3.8 x 150 µm) taken randomly from regions of interest in the bladders from three rats and corrected those measurements for the autofluorescence measured in the respective tissue layer of bladders from three control rats with tumoral bladders in which PBS (pH 7.4) (no photoactive compounds) was instilled.
Statistical Analysis
The statistical significance of differences was calculated using unpaired Student's t test (two-sided) that assumed unequal variance (Welch correction) (Instat; GraphPad Software, San Diego, CA).
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RESULTS |
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Dynamic light-scattering measurements demonstrated that the average liposome diameter was 146 nm (95% confidence interval [CI] = 117 to 174 nm; based on data obtained from five different liposomal preparations). The average liposomal diameter did not change appreciably after liposomes were incubated for 24 hours at 37 °C or 1 month at 4 °C (<1% change in diameter; based on data from three independent experiments performed on one batch of liposomes).
Given the area occupied by the polar head groups of the phospholipids and cholesterol, and taking into account their molar ratio versus the total surface of a liposome (19), we estimated that a 146-nm liposome contains approximately 144 000 molecules of phospholipid. Based on an initial phospholipid concentration of 5.67 µmol, the average incorporation efficiency of phospholipids into liposomes was 62.0% (95% CI = 59.8% to 64.1%; data obtained from three different liposomal preparations). We used these data to determine the exact number of liposomes per preparation and to calculate the numbers of AlPcS4 and transferrin molecules per liposome.
Fluorescence measurements revealed that the intraliposomal concentration of AlPcS4 was 29.0 mM (95% CI = 27.0 to 31.0 mM), which corresponded to 22 995 AlPcS4 molecules per liposome (95% CI = 21 532 to 24 458 AlPcS4 molecules per liposome). As quantified by the bicinchoninic acid assay, the coupling of transferrin to the liposomes yielded 112 transferrin molecules per liposome (95% CI = 106 to 118 transferrin molecules per liposome), corresponding to a conjugation efficiency of 13.9% (data obtained from seven different liposomal preparations).
We also examined the stability of AlPcS4-containing liposomes under the conditions used for the later experiments. Tf-LipAlPcS4 incubated at 37 °C in cell culture medium for 24 hours and for 1 week retained 100% (95% CI = 94.8% to 105.2%) and 96.7% (95% CI = 93.1% to 100.3%), respectively, of the original amount of AlPcS4. These results were based on data from three independent experiments performed on one batch of liposomes.
Transferrin Receptor Expression on AY-27 Cells
To examine whether rat AY-27 bladder cancer cells express transferrin receptors, cells were stained with TfpolylysineFITC, a transferrin conjugate that binds specifically to transferrin receptors, and analyzed by flow cytometry. As shown in Fig. 1, more than 99.7% of AY-27 cells stained with TfpolylysineFITC, showing a mean fluorescence intensity (MFI) of 1306 (95% CI = 691 to 1921). The specificity of TfpolylysineFITC binding was confirmed by comparing the Tf-polylysine-FITCstained AY-27 cells with AY-27 cells that were stained with fluorescein alone, which had an MFI of 4.4 (95% CI = 2.1 to 6.9). Moreover, similar experiments performed on several other bladder transitional-cell carcinoma cell lines of human origin (T24, RT4, J82, and RT112) indicated that these cell lines expressed similar levels of transferrin receptors (results not shown).
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A 4-hour incubation of AY-27 cells with free AlPcS4 and Tf-LipAlPcS4 resulted in intracellular AlPcS4 concentrations of 52.7 µM (95% CI = 36.8 to 68.6 µM) and 384.1 µM (95% CI = 223.2 to 545.0 µM) (calculated as AlPcS4 concentration), respectively. By contrast, very little intracellular accumulation of the photosensitizer was observed for cells incubated with Lip-AlPcS4 (i.e., 3.7 µM, 95% CI = 1.1 to 6.3 µM) (Fig. 2). The intracellular concentration of AlPcS4 was statistically significantly lower for cells incubated with Lip-AlPcS4 than for cells incubated with free AlPcS4 (difference = 49 µM, 95% CI = 32.9 to 65.1 µM; P = .0061) or Tf-LipAlPcS4 (difference = 380.4 µM, 95% CI = 219.4 to 541.3 µM; P = .0095). For AY-27 cells incubated with Tf-LipAlPcS4 in the presence of a competing concentration of transferrin (i.e., 50 µM), the intracellular concentration of AlPcS4 was 7.7 µM, a statistically significant decline from the intracellular AlPcS4 concentration for cells incubated with Tf-LipAlPcS4 in the absence of 50 µM transferrin (difference = 376.4 µM, 95% CI = 215.4 to 537.4 µM;; P = .0097). Extending the incubation period from 4 to 24 hours did not statistically significantly increase the intracellular AlPcS4 concentrations of cells exposed to the different photosensitizer preparations (Fig. 2). For cells incubated with Tf-LipAlPcS4, this result suggests that their transferrin receptors had reached saturation during the first 4 hours of incubation.
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Visualization of Intracellular AlPcS4 Accumulation
Consistent with our quantitative results for intracellular AlPcS4 accumulation, we found that cells incubated with Tf-LipAlPcS4 exhibited a stronger AlPcS4-induced fluorescence (Fig. 3, D) than cells incubated with free AlPcS4 (Fig. 3, B). By contrast, cells incubated with Lip-AlPcS4 displayed a very weak fluorescence signal, indicating that almost no AlPcS4 had accumulated (Fig. 3, C). Increasing the incubation time from 2 to 4 hours or 24 hours resulted in an increase in the fluorescence signal, except for cells incubated with Tf-LipAlPcS4. Control cells (i.e., cells incubated in the absence of any photoactive compound) displayed no fluorescence (Fig. 3, A).
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We evaluated AY-27 cell killing in response to photodynamic therapy for cells incubated with each of the photoactive compounds for two different times: a 2-hour interval, which corresponded to the in vivo incubation time, and a 4-hour interval, which corresponded to the exposure time used for in vitro quantification of the intracellular accumulation. The survival curves are shown in Fig. 4. Photodynamic therapy of AY-27 cells incubated with Lip-AlPcS4 resulted in cell viabilities greater than 90% for all concentrations and incubation times tested. By contrast, photodynamic therapy of cells incubated with 1 µM Tf-LipAlPcS4 for 2 and 4 hours resulted in cell viabilities of 1.34% (95% CI = 1.04% to 1.64%) and 0.19% (95% CI = 0.02% to 0.36%), respectively. For AlPcS4, these conditions led to cell viabilities of 25.49% (95% CI = 22.65% to 28.30%) and 1.32% (95% CI = 0.46% to 2.19%), respectively. Moreover, higher concentrations of either AlPcS4 or Tf-LipAlPcS4 further decreased the tumor cell viability, resulting in cell kills of more than 3 logs (Fig. 4). For both photoactive compounds, we observed a similar cell killing profile at these higher concentrations, regardless of the incubation period. Conversely, Lip-AlPcS4 at 5 µM and 10 µM induced a moderate photocytotoxic effect. For instance, 2-hour incubations with 5 µM AlPcS4 or Tf-LipAlPcS4 resulted in cell viabilities of 0.012% (95% CI = 0.010% to 0.014%) and 0.021% (95% CI = 0.017% to 0.025%), respectively, whereas a 2-hour incubation with 5 µM Lip-AlPcS4 resulted in a cell viability of 30.2% (95% CI = 26.3% to 34.1%). Therefore, the difference in cell viabilities recorded after photodynamic therapy with AlPcS4 and Lip-AlPcS4 on the one hand (0.012% versus 30.2%; difference = 30.2%, 95% CI = 26.3% to 34.0%; P<.001) and with Tf-Lip-AlPcS4 and Lip-AlPcS4 on the other (0.021% versus 30.2%, difference = 30.2%, 95% CI = 26.3% to 34.0%; P<.001), were very similar.
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We next investigated the in vivo relevance of transferrin-conjugated liposome targeting of AlPcS4 for photodynamic therapy of superficial bladder carcinoma by instilling each of the photoactive compounds into healthy and tumoral rat bladders and examining the accumulation of AlPcS4 in the different tissues of the bladder. Preliminary results of in vivo experiments showed that Tf-LipAlPcS4 failed to accumulate in AY-27 cellderived bladder tumor tissues of rats. We suspected that this lack of accumulation was due to the presence of the glycocalyx layer in the bladder, which is important for maintaining the impermeability of the bladder mucosa. We therefore examined accumulation of Tf-LipAlPcS4 in bladders that had been pretreated with chondroitinase ABC, an enzyme that selectively digests chondroitin and dermatan sulfates. In addition, we investigated whether treatment of the bladder wall with this enzyme results in specific digestion of chondroitin present in the glycocalyx of the bladder wall.
Immunohistochemical staining showed that sections of bladders from untreated rats had a layer on the epithelial surface that stained positive with a monoclonal antibody for chondroitin sulfate (Fig. 5, A), a ground substance of the bladder surface mucin. By contrast, bladder sections from rats pretreated with chondroitinase ABC showed no such staining (Fig. 5, B). As a negative control for nonspecific staining, we omitted the murine monoclonal antichondroitin sulfate antibody from the staining procedure and found that, apart from some staining of blood vessels, there was no staining of the luminal surface of the urothelium in either untreated or chondroitinase ABCpretreated rat bladders (Fig. 5, C and D). These results indicate that the chondroitinase ABC pretreatment of the bladder results at least in a partial digestion of the mucopolysaccharide layer.
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These results indicate that Tf-LipAlPcS4 accumulates selectively in bladder tumor tissues but not in the underlying tissue layers of the tumoral bladder or the normal urothelium. Moreover, the nontargeted liposomes (i.e., Lip-AlPcS4) do not show such a pattern of accumulation.
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We examined whether targeting the photosensitizer AlPcS4 by means of transferrin-conjugated liposomes is an effective strategy to attain the tumor-selective behavior of the compound when applied intravesically. The rationale for this approach is based on the finding that transitional-cell carcinoma cells overexpress transferrin receptors on their cell surface (912). Indeed, we found that all of the transitional carcinoma cell lines we examined, including some human cell lines, expressed substantial levels of the transferrin receptor. Results of our in vitro experiments showed that transferrin-targeted liposomes statistically significantly increased (by approximately 100-fold) the cellular uptake of AlPcS4 compared with that of cells exposed to AlPcS4 encapsulated in nontargeted (i.e., nontransferrin conjugated) liposomes. The transferrin dependence of the cellular uptake of the liposomes was further corroborated by the observation that the intracellular AlPcS4 accumulation underwent a statistically significant 50-fold drop, in the presence of a competing concentration of transferrin. From these findings, we deduced that the substantial amount of transferrin in human blood (as much as 50 µM) (14) would compete for transferrin receptors and render the transferrin-targeted liposomes unsuitable for intravenous application.
To investigate the practical applicability of photodynamic therapy using targeted liposomes, we measured the in vitro photocytotoxicity of Tf-LipAlPcS4 as well as its in vivo capacity to selectively accumulate in rat tumoral bladders. We found that transferrin targeting increased the in vitro photocytotoxicity of the encapsulated photosensitizer, resulting in a cell kill of more than 3 logs compared with the nontargeted liposomal photosensitizer, which showed only limited photocytotoxicity (<1 log cell kill). At a 1 µM concentration of the photoactive compound, Tf-LipAlPcS4 was more photocytotoxic than AlPcS4, consistent with the results obtained in our studies of in vitro intracellular accumulation. The photocytotoxicity of Tf-LipAlPcS4 and AlPcS4 in AY-27 cells further increased as a function of increasing AlPcS4 concentration, with the higher concentrations of both compounds resulting in a similar loss of cell viability. However, this result does not imply that Tf-LipAlPcS4 has no advantage over free AlPcS4 in photodynamic therapy because the therapeutic benefit that can be expected from the targeted liposomes resides mainly in an enhancement of the tumor selectivity, not just in their ability to increase the accumulation or photocytotoxicity of the photosensitizer.
Despite our in vitro results, preliminary results of in vivo experiments showed that Tf-LipAlPcS4 failed to accumulate in AY-27 cellderived bladder tumor tissues of rats. We suspected that this lack of accumulation was due to the presence of the glycocalyx layer in the bladder, which is important for maintaining the impermeability of the bladder mucosa. The glycocalyx is synthesized mainly by the umbrella cells and composed of a dense layer of glycosaminoglycans, most commonly present as constituents of proteoglycans, and glycoproteins or mucin (28,31). We therefore examined accumulation of Tf-LipAlPcS4 in bladders that had been pretreated with chondroitinase ABC, an enzyme that can be produced in large quantities by transconjugates of Flavobacterium heparinum (32) and that permeabilizes the glycocalyx layer without damaging the urothelium. Of importance, the glycocalyx can be resynthesized by the umbrella cells in less than 24 hours and completely replaced within 48 hours (33,34). This short recovery period guarantees that a mild (partial) enzymatic digestion of the glycocalyx could be employed in clinical practice.
We found that, in bladders that had been pretreated with chondroitinase ABC, Tf-LipAlPcS4 appeared to concentrate selectively in AY-27 cellderived tumor tissue. We concluded that this retention was tumor selective because Tf-LipAlPcS4 was not retained in normal urothelium of healthy rats whose bladders were pretreated with chondroitinase ABC. Furthermore, because the liposomal diameters of Lip-AlPcS4 and Tf-LipAlPcS4 were similar, we expected that both constructs would show similar penetration through the tumor tissue. However, Lip-AlPcS4 was virtually absent from the urothelial tumor tissue, indicating that the observed high and specific retention of Tf-LipAlPcS4 in the tumor was due to its specific interaction with transferrin receptors followed by intracellular accumulation. Although ligands such as transferrin typically bind to the first target they encounter, which in malignant bladder tissue will tend to be the cells at the tumor periphery, this "binding-site barrier" (35) obviously did not prevent the transferrin-conjugated liposomes from penetrating into the deeper layers of tumor tissue. Because of its much smaller size, free AlPcS4 showed a higher level of uptake in tumor tissue compared with AlPcS4 delivered by Tf-LipAlPcS4. However, as observed previously (36), free AlPcS4 does not accumulate selectively in tumors (fluorescence ratio of bladder tumor to normal bladder = 2 : 1), precluding its use for photodiagnosis and wholebladder-wall photodynamic therapy.
In summary, our results suggest that transferrin-targeted, sterically stabilized liposomes are promising vehicles to selectively deliver photosensitizers, such as AlPcS4, to tumor cells that overexpress transferrin receptors. These targeted liposomes could also be used to visualize and, as suggested by the results of our in vitro clonogenic assay, selectively eradicate urothelial carcinoma lesions. In addition, these liposomal constructs may be useful for the targeted delivery of other anticancer agents. Thus, the potential of the transferrin-liposomal constructs may be far beyond the application described in this study.
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REFERENCES |
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Manuscript received March 4, 2004; revised September 13, 2004; accepted September 14, 2004.
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