Selective in vitro labeling of white blood cells using 99mTc-labeled liposomes

Nuclear Medicine and Biology 29 (2002) 185–190

Selective in vitro labeling of white blood cells using 99m Tc-labeled liposomes Dimitrios Andreopoulosa, Leela P. Kasia, Panayiotis J. Asimacopoulosb, Satish G. Jhingranb, William Coleb, David Yanga, E. Edmund Kima,* a

The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA b The Methodist Hospital, Baylor College of Medicine, Houston, Texas 77030, USA

Received 21 April 2001; received in revised form 14 July 2001; accepted 28 July 2001

Abstract We describe a method by which endocytosis-based radiolabeling of WBC is achieved using 99mTc-liposomes of optimal size and charge, and of a composition that assures both in vitro (whole blood) and intracellular stability of the radiopharmaceutical. In our study, excellent in vitro stability of 99mTc-liposomes with 95% labeling efficiency was observed with ⬎90% stability up to 6 h and a minimum of 85% after 24 h of incubation either in normal saline or serum. Total WBC labeling efficiency using 99mTc-liposomes determined by radio-thin layer chromatographic analysis was 30.6 ⫾ 2.21%, 20.89 ⫾ 1.31% for monocytes and 9.7 ⫾ 1.74% for polymorphonuclears. Negligible activity was bound to red blood cells. The procedure did not affect the cell viability and the separation of the free 99mTc-liposomes from the cells was done by centrifugation. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Liposomes; Phagocytosis; Technetium-99m; Radiolabeled leukocytes; Radiolabeled liposomes

1. Introduction Nuclear imaging contributes significantly to the detection of inflammation and infection sites, and radiolabeled white blood cells (WBC) is an accepted modality for this purpose. The current methods commonly used for WBC radiolabeling in clinical practice are based on two approaches: The first approach is to separate the desired cell fraction and then label the cells with 111In-oxine or 99mTc-hexamethylenepropyleneamineoxime (99mTc-HMPAO). The second approach is based on phagocytic in vitro labeling of the WBCs with 99mTc-labeled colloids. Although the above-mentioned methods are used routinely, they have certain disadvantages. The 111In-oxine technique is time consuming, requiring skilled personnel as well as adequate equipment [2,4,21]. 111In is an expensive and not always available radioisotope with more unfavorable radiation characteristics than 99mTc [4]. 99m Tc-HMPAO can label WBC with high efficiency, but it has been observed that a significant amount of 99mTc * Corresponding author. Tel.: ⫹1-713-794-1052; fax: ⫹1-713-7945456. E-mail address: [email protected] (E.E. Kim).

elutes off the cells, resulting in undesirable early gastrointestinal and high reticuloendothelial activities [5,26]. Another serious drawback is that WBC labeling is accomplished in other media than native plasma or whole blood [30]. Recently, attention has been focused on labeling WBC by phagocytosis of 99mTc-labeled particles. But early attempts have resulted in poor cell labeling and/or relatively weak in vitro and in vivo stability [13,18,19,30,31]. The above methods require many manipulations of WBC, thus raising questions about the viability and in vitro activation of cells [26]. There is also the possible risk of laboratory personnel being exposed to contaminated blood products while handling the blood samples [4]. To overcome these shortcomings we have investigated and developed a new method for selectively labeling WBC in vitro with 99mTc-labeled anionic liposomes with mean size 1 ␮m. In the present work we describe: i) the preparation and radiolabeling of liposomes used immediately for WBC labeling, ii) the methodology to determine the distribution of the radioactivity in the blood sample after liposome-cell interaction, iii) and assays to determine the toxicity of the radiolabeled liposomes to WBC.

0969-8051/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 1 ) - 0 0 2 9 9 - 2

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2. Materials and methods 2.1. Materials Chromatographically pure lipids in chloroform solutions and a mini-extruder were purchased from Avanti Polar Lipids Inc. (Birmingham, AL). Cholesterol, stannous chloride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Histopaque-1077/Histopaque-1119, HEPES and acid citrate dextrose (ACD) were purchased from Sigma Chemical Company (St. Louis, MO). RPMI1640 culture medium and Hank’s Balanced Salt Solution (HBSS) were purchased from GIBCO, Life Technologies, Inc. (Grand Island, NY). All other reagents used were of analytical grade. Liposome size distribution and cell numbering were determined with a Coulter Counter and Channelizer (Coulter Electronics, Hialeah, FL). All procedures were carried out in a laminar airflow hood using aseptic techniques. Radiolabeling and extrusion of liposomes were performed at 50 – 600C. Activities were measured in a Capintec Dose Calibrator using the 99mTc energy window. Values are expressed as mean ⫾ SD. Statistical analyses were done by the Student’s t -test. Calculations involving radioactivity were corrected for decay. 2.2. Liposome preparation Liposomes were prepared from dipalmitoylphosphatidylcholine (DPPC), and dipalmitoyl-phosphatidylglycerol (DPPG) in a molar ratios of 5:2 containing 40% mole cholesterol (CH). The inclusion of the negatively charged DPPG determined the liposome charge. A dry lipid film was formed by passing nitrogen gas into flask containing the required amount of lipids to quickly evaporate the chloroform solvent. The dry lipid film was then flushed with argon, the flask was well sealed and stored at 4°C until further use. 2.3. Radiolabeling of liposomes The method used to radiolabel the phospholipid vesicles has been described elsewhere [22]. The required amount of 99m Tc-pertechnetate was reduced in the presence of stannous chloride, under conditions at which maximum labeling efficiency is achieved and the presence of excessive technetium-tin colloids is virtually eliminated [7]. The dry lipid film was hydrated with the required volume of reduced 99m Tc followed by vigorous vortexing of the preparation. The resulting multilamellar liposomes were allowed to stand for 15 min, and then extruded through a 1-␮m polycarbonate membrane mounted in the mini-extruder, resulting in multilamellar liposomes of 1070 ⫾ 110 nm. Labeling efficiency (LE) was estimated by instant thin-layer chromatography developed in normal saline. In this technique, the radiolabeled liposomes remain at the origin while free

99m

Tc moves with the solvent front. The labeling efficiency was 96 ⫾ 1.2% (n ⫽ 40) determined by thin layer chromatography. The concentration of the preparation was 3.5 mg/mL with a specific activity of 4 –5 mCi/mg. 2.4. Assays of in vitro stability of

99m

Tc-labeled liposomes

Aliquots of radiolabeled liposomes were incubated at 37°C in a shaking water bath, in normal saline and in 50% and 100% human serum for 24 h. After 6 and 24 h of incubation, samples were diluted in 0.9% saline and centrifuged at 30,000 ⫻ g for 30 min at 20°C. The pellet and the supernatant in each sample were then counted for radioactivity. 2.5.

99m

Tc-liposome-WBC interaction

For each experiment (n ⫽ 30), 20 mL blood was withdrawn from healthy volunteers using ACD as anticoagulant [19]. The blood was divided equally into two silicon-coated vacutainer tubes, one labeled WBC-t for liposomally treated cells and the other WBC-c for control cells. In the WBC-t tube, 1 mL 99mTc-liposomes was added and incubated at 370C in a shaking water bath for 45 min. In a series of experiments in whole blood we observed that the maximum WBC-liposome association occurred after 45 minutes of incubation. 2.6. Determination of WBC labeling efficiency The distribution of the radiolabeled liposomes in the WBC-t sample was determined by the double-gradient density separation procedure [1], following the manufacturer’s instructions. In brief, an equal volume of Histopaque-1077 was layered over Histopaque-1119. WBC-t sample was carefully layered onto the upper Histopaque-1077 medium. After centrifugation at 700 ⫻ g for 30 minutes, the red blood cells (RBC), polymorphonuclear cells (PMN), and monocytes (MNC) were separated obtaining, as an opaque layer, the noncell-associated 99mTc-liposomes floating on the top of plasma. RBC, PMN, MNC, and plasma activities were individually counted in order to determine the labeling efficiency in each cell fraction. Also PMN and MNC activities were counted after the two washings with HBSS required by the separation procedure. In both cell fractions, 95.3 ⫾ 1.2% of the (cell-associated) activity was found to remain cell associated. These results are indirectly suggesting that endocytosis had occurred, rather than liposome adherence on the cell surface, our findings are compatible with other’s results [16]. In the case of adherence, a considerable fraction of the radiolabeled liposomes would recovered in the supernatant after each washing. Finally, measured WBC aliquots were further assayed for cell viability and activation. In the WBC-c tube, blood cells were separated as above. The resulting PMN and MNC

D. Andreopoulos et al. / Nuclear Medicine and Biology 29 (2002) 185–190

Fig. 1. In vitro stability of

99m

187

Tc-labeled liposomes at 37°C, n ⫽ 30.

were properly incubated for use as controls for WBC viability and activation assays. 2.7. WBC viability and metabolic activation status assays The effect of the radiolabeled liposomes on WBC viability was evaluated by (a) determining the ability of cells to exclude trypan blue, (b) determining the ability of viable cells to convert tetrazolium salt MTT to purple formazan, thus measuring colorimetrically the cytotoxicity and cell activation [10,25,28]. Cell activation was also visually controlled by phase-contrast microscopy [27]. Trypan blue exclusion tests were performed immediately after WBC fractionation. The MTT assay was performed immediately after liposomal treatment and at 6 and 24 h posttreatment, (simultaneously with phase-contrast microscopy studies) on both WBC-t and control cells (WBC-c) as described by Hansen et al., [11]. In brief, 2 ⫻ 105 cells were plated in 96-well plates (Becton Dickinson, Lincoln Park, NJ) in 100 ␮L RPMI complete medium (10% fetal bovine serum, 1% HEPES, and 1% penicillin-streptomycin solution). Cells were then incubated at 37°C in 5% CO2. At each time point 10 ␮L MTT (5 mg/mL) was added to each well, and cells were reincubated for 60 min. Finally, 100 ␮L MTT lysing buffer was added per well and cells were incubated for a further 30 min. Absorbance was read at 570 nm in a microplate reader (Molecular Devices Corporation, Palo Alto, CA) against WBC-c.

2.8. In vitro stability of the

99m

Tc-labeled WBC

Radiolabeled WBC suspended in RPMI complete medium (10% human serum, 1% HEPES, and 1% penicillinstreptomycin solution) were incubated at 37°C in 5% CO2; after 2 and 6 h, aliquots were resuspended in 0.9% saline and centrifuged at 400 ⫻ g for 10 min. The radioactivity from the pelleted 99mTc-WBCs and supernatants were counted in order to estimate the 99mTc elution from the labeled WBC (n ⫽ 30).

3. Results 3.1. In vitro stability of radiolabeled liposomes The in vitro stability of the radiopharmaceutical for methods based in the endocytotic capacity of WBC, is of crucial importance. Excellent in vitro stability was observed with ⬎90% stability up to 6 h and a minimum of 85% after 24 h of incubation either in normal saline or serum (Fig. 1). 3.2. WBC labeling efficiency After cell fractionation (by the double-gradient density separation procedure), 69.38 ⫾ 2.2% of the activity was found in the plasma. Negligible activity was found in the

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Fig. 2. Labeling efficiency of blood cell fractions, n ⫽ 30.

RBC fraction (0.06 ⫾ 0.04%) and no activity was found in the Histopaque gradients-cell interfaces. The WBC-bound activity was distributed in an approximately 2:1 ratio of MNC (20.89 ⫾ 1.31%) to PMN (9.7 ⫾ 1.74%) (Fig. 2). 3.3. WBC viability and metabolic activation status Viability for both 99mTc-liposome-treated cells and control cells was estimated to be ⬎99% by both the MTT and trypan blue methods. Comparison of the MTT results from liposome-treated and control cells showed insignificant statistical differences (p ⬎ 0.05) in the absorbance values. The MTT assay also demonstrated that radiolabeled liposomes did not activate WBC. This correlates well with the phasecontrast microscopy results, in which ⬎98% of the liposomally treated WBC were found spherical (functionally intact) [27]. 3.4. In vitro stability of the

99m

Tc-WBC

After the WBC-99mTc-liposomes interaction, the percentage of 99mTc eluted from the labeled WBCs after washings at 2 and 6 h was found to be ⬍1.5 ⫾ 0.7%. These results are a strong indication of an endocytosed radiopharmaceutical that remained stable in the intracellular millieu. The intracellular fate of the radiopharmaceutical determined the overall efficacy of the method being an index for good in vivo stability.

4. Discussion Many attempts have been made to label WBC by phagocytosis of radiocolloids. The radiolabeled-liposome model was previously tested [19], but not further investigated. The effectiveness of WBC radiolabeling based on phagocytosis depends on preparation of particles of definite size and charge and on the in vitro and intracellular stability of the radiopharmaceutical. In the case of different colloids, such parameters are not effectively controlled, resulting in nonreproducible results [6,26,30]. On the other hand, the low labeling yields that have been achieved seem to be due to the fact that a considerable fraction of these particles adhere nonspecifically to the cell surface rather than become endocytosed [19,30]. Furthermore, the separation of the noncell-associated radiopharmaceutical is usually difficult or laborious. Our method is based on liposome technology and the liposome-WBC interactions. The physicochemical properties of the radiolabeled liposomes determine the type of vesicle-cell interaction as well as parameters such as the in vitro and intracellular stability of the radio-pharmaceutical. Liposomes are made of phospholipids, the same molecules that form the membranes of all animal cells. Vesicles prepared from these biocompatible and biodegradable materials are nontoxic vectors and offer an excellent route for introducing (foreign) molecules into cells and they are cur-

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rently used for diagnostic and therapeutical applications [5,15,20,23]. Some liposomal compositions including certain radiolabeled preparations are very stable in vivo and in vitro [7,16,17]. Phospholipid vesicles can be effectively labeled with 99m Tc when exposed to reduced sodium pertechnetate, which is very reactive and bonds avidly to phosphate groups of the phospholipid molecule. Under optimal conditions the binding is 100% efficient making 99mTc a reliable lipid marker [17,22]. We tested several 99mTc-labeled liposomal compositions and selected the anionic DPPC:DPPG:CH liposomes, because they fulfilled the radiopharmaceutical standards of labeling efficiency and in vitro and intracellular stability. Liposomes of 1 ␮m diameter, an optimal size for endocytosis by both PMN and MNC, were consequently prepared [21]. Liposome uptake by WBC (total 30.6 ⫾ 2.21%) is known to be a saturable process, and PMNs (9.7 ⫾ 1.74%) uptake is less than MNCs (20.89 ⫾ 1.31%) [6,8,9]. In our experiments, 3.5 mg 99mTc-liposomes (1 ␮m size), with a specific activity of 4.5 ⫾ 0.5mCi/mg were added in 10 mL blood (50 – 60 ⫻106 mixed WBC). After 45 minutes of incubation and 2 washings, approximately 1.05 mg of 99m Tc-liposomes was found WBC-associated, suggesting strongly that endocytosis had occurred, rather than liposome adherence on the WBC surface, without affecting cell viability and functionality. The in vitro, endocytoplasmatic stability of the 99mTcliposomes is related to the observation that if cholesterol exceeds a critical concentration in liposome composition, then the endocytosed liposomes cannot be easily degraded by lysosomes [20]. On the other hand, negligible radioactivity was observed in RBC, leading us to conclude that no phospholipid exchange took place between liposomes and RBC. Although other laboratories have reported significant activity in the RBC fraction when using colloids or liposomes, we could not evaluate their results because reports of these studies provided insufficient details regarding materials and methods. For an in vivo (clinical) application we suggest the use of 20 –30 mL whole blood to be incubated with the required amount of radiolabeled liposomes. After 45 min incubation the sample is centrifuged (400 ⫻ g for 10 min) and the upper layer of plasma that contains the free radiolabeled liposomes is aspirated and counted for cell LE estimation. The WBC layer can be further reinjected to the patient [24].

5. Conclusions We have developed an endocytotic method for selective WBC labeling accomplished directly in whole blood. For this purpose we prepared and used anionic and cholesterolrich 1 ␮m liposomes, labeled with the most useful, inexpensive, readily available, and routinely used radionuclide in nuclear imaging: 99mTc pertechetate. Liposomes can be

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stored for long periods in lyophilized kit form as preformed liposomes or proliposomes to be labeled when required. After reconstitution, their properties remain intact making them available for immediate applications [3]. The preparation of liposomes in a kit form will further develop this approach in a simple, fast and safe method that requires a minimum of cell and blood manipulations. The relative low WBC labeling efficiency is comparable to other similar phagocytic methods and can be improved by means of increasing the specific activity of the radiopharmaceutical and using active targeting techniques. Results from our data indicate that under controlled conditions, 99mTc-liposomes can label WBC without affecting their viability and function. We have not evaluated liposome interaction with lymphocytes and platelets as yet. Nonendocytosed 99mTc-liposomes were easily separated from WBC by centrifugation. 99m Tc-liposomal preparations could accomplish higher cell-labeling yield by taking advantage of the liposome technology’s potential for fulfilling the criteria of the ideal blood cell-labeling method [12]. Active targeting to PMN, MNC, lymphocytes, and platelets can be achieved as well by using liposomes of the appropriate size and lipid composition for targeting to each cell and also by modifying the liposome membrane with determinants that recognize sugar-specific lectins on blood cell plasma membranes or immunoliposomes [14,29], and taking into consideration the criteria for an ideal blood cell-labeling method [12]. Acknowledgments Authors wish to express their gratitude to Dr. Reeta T. Mehta, Jo Lauppe and April Durett for their valuable consultations and also Miguel Diaz and Richardo GomezFlores for their technical assistance. References [1] A. Boyum, Isolation of mononuclear cells and granulocytes from human blood, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97) (1968) 77– 89. [2] F.H.M. Corstens, W.J.G. Oyen, W.J. Becker, Radioimmuno-conjugates in the detection of infection and inflammation, Semin. Nucl. Med. 23 (1993) 148 –164. [3] J.H. Crowe, L.M. Crowe, Preservation of liposomes by freeze-drying, In: G. Gregoriadis (Ed.), Liposome Technology (Vol. I), CRC Press, Boca Raton, FL, 1993, pp. 229 –252. [4] F.L. Datz, Letter from the guest editor, Semin. Nucl. Med. 24 (1994) 89 –91. [5] F.L. Datz, K.A. Morton, New radiopharmaceuticals for detecting infection, Invest. Radiol. 28 (1993) 356 –365. [6] M.K, Dewanjee, The chemistry of 99mTc-labeled radiopharmaceuticals, Semin. Nucl. Med. 20 (1990) 5–27. [7] S.J. Farr, I.W. Kellaway, D.R. Parry-Jones, S.G. Wllfrey, 99mTechnetium as a marker of liposomal deposition and clearance in the human lung, Int. J. Pharm. 26 (1985) 303–316. [8] M.N. Finkelstein, S.H. Kuhn, G. Schieren, G. Weissmann, S. Hoffstein, Liposome uptake by human leukocytes, Biochim. Biophys. Acta 673 (1981) 286 –302.

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