Carrier-Based Drug Delivery - ACS Publications - American Chemical

Chapter 9

Downloaded by MICHIGAN STATE UNIV on November 29, 2013 | http://pubs.acs.org Publication Date: May 5, 2004 | doi: 10.1021/bk-2004-0879.ch009

Polymeric Micelles as Targetable Pharmaceutical Carriers Vladimir P. Torchilin*, Anatoly N. Lukyanov, Zhonggao Gao, Junping Wang, and Tatyana S. Levchenko Department of Pharmaceutical Sciences, Bouve College of Health Sciences, Northeastern University, Boston, MA 02115 *Corresponding author: email: [email protected]

Micelles formed by Poly(ethylene glycol)/phospatidylethanol -amine (PEG-PE) conjugates are stable under physiological conditions, accumulate spontaneously in tumors via the enhanced permeability and retention (EPR) effect, and can be loaded with various anticancer agents. These micelles were modified with anticancer monoclonal 2C5 antibody to prepare targeted PEG-PE micelles able to recognize a broad variety of tumors. Modified micelles carry between 10 and 50 antibody molecules each. The micelle-bound antibody retains its specific activity. 2C5-targeted immunomicelles recognize tumors of different types in vitro, and show an enhanced efficiency of their tumor accumulation in vivo.

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© 2004 American Chemical Society

In Carrier-Based Drug Delivery; Svenson, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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Introduction Poly(ethylene glycol)/phospatidylethanolamine (PEG-PE) conjugates form very stable micelles in aqueous media. The protection provided by the PEGbased corona makes these micelles long-circulating, while the hydrophobic PEcore may be used as a cargo space for poorly soluble compounds, including anti-cancer drugs. (1-4) Certain features of PEG-PE micelles such as their characteristic size, stability, and the longevity in circulation make them a very promising drug carrier for tumor delivery utilizing the enhanced permeability and retention (EPR) effect. The EPR effect is based on a spontaneous penetration of the particles into the tumor interstitium through enlarged fenestrations typical, in particular, for tumor vasculature. (5-7) Because of their small diameter, micelles may be a valuable alternative to other particulate delivery systems, such as liposomes and microparticles, for tumor delivery. It has been shown that in some cases particles with a diameter of 100 nm or larger do not penetrate tumor interstitium, apparently because of a small tumor vasculature cutoff size. (8, 9) The small size of 5-50 nm, therefore, makes the use of micelles advantageous in the case of such tumors. (10, 11) Similar to other particulate delivery systems, the drug delivery potential of PEG-PE micelles can be greatly enhanced by attaching targeting ligands, such as antibodies, to their surface. (12, 13) In an attempt to develop a drug delivery system that would recognize a broad variety of tumors, we have used a nucleosome-specific antibody, 2C5, which can recognize tumors via tumor cell surface-bound nucleosomes, to prepare targeted PEG-PE immunomicelles. (14, 15) We report here the results of the preparation and characterization of 2C5 targeted immunomicelles, and their ability to recognize tumors of different types in vitro and in vivo.

Materials and Methods PEG-PE with a PEG residue of 2000 Da, and phosphatidylethanolamine lissamine rhodamine B (Rh-PE) were purchased from Avanti Polar Lipids, Inc., (Alabaster, AL) and were used without further purification. I n with specific radioactivity of 395 Ci/mg of In equivalent was purchased from Perkin Elmer Life Sciences, Inc. (Boston, MA) and was used within 5 days upon arrival. Distilled and deionized water was used in all experiments. Diethylene triamine pentaacetic acid-phosphatidylethanolamine conjugate (DTPA-PE) and p-nitrophenylcarbonyl-PEG-dipalmitoyl phosphatidylethanolamine (pNP-PEG-PE) were synthesized according to published procedures. (4,16) m


122 Monoclonal 2C5 antibody was produced using a 2C5E3 hybridoma cell line (American Type Culture Collection, Manasas, VA). Cells were cultivated in RPMI 1640 cell culture medium supplemented with 10% fetal calf serum, streptomycin/penicillin, Na-pyruvate, and p-mercaptoethanol. After a sub stantial level of antibody had been detected in the cell-cultivating medium using ELISA (5-6 days after inoculation), cells were removed by centrifugation at 2000xg for 15 minutes. The supernatant was concentrated 10-fold by ultra filtration using Amicon filters with a cutoff size of 100 kDa (Millipore Corp., Bedford, MA). The protein was precipitated by addition of (NH4) S0 to the final concentration of 47% and centrifiiged at 14,000xg for 60 min. Precipi tated protein was dissolved in 0.01 M Tris, pH 8.6, and dialyzed against a 1000-fold excess of the same buffer. 2C5 antibody was purified using ionexchange chromatography on DEAE-Toyopearl 650M and subjected to affinity chromato-graphy on Protein-G Sepharose. The micelles were formed by extensive 5-15 min vortexing of PEG-PE at a polymer concentration of 5 mM in 10 mM HEPES buffered saline at pH 7.4 (HBS). To prepare 2C5-modified micelles, PEG-PE was supplemented with 2 mol% of pNP-PEG-PE, and the micelles were formed in 5 mM Na-citrate buffered saline at pH 5.0. To 0.5 ml of the micellar solution obtained, an equal volume of 12 mM solution of 2C5 antibody in 40 mM Tris-buffered saline, pH 9.0, was added. In some cases, the protein was labeled with fluorescein (1-3 residues per protein molecule) using carboxyfluorescein succinimidyl (Molecular Probes, Inc., Eugene, OR). The mixture was incubated for 3 hours at room temperature and dialyzed against a 5000-fold excess of HBS, using cellulose ester membranes with a cutoff size of 300,000 Da (Spectrum Medical Industries, Inc., Rancho Dominguez, CA). The amount of micelle-bound 2C5 was quantified following the fluorescence of fluorescein probe at the exitation wavelength of 490 nm and emission wavelength of 520 nm (F /52o)Size and size distributions of micelles were determined by dynamic light scattering using a N4 plus M D Coulter Submicron Particle Size Analyzer (Coulter Corporation, Miami, FL). The immunological activity of micelle-attached 2C5 was estimated by standard ELISA. ELISA plates were coated with 50 ^1 of 1 jig/ml nucleosomes (water soluble fraction of calf thymus nucleohistone, Worthington Biochemical Corp., Lakewood, NJ) and incubated overnight at 4°C. To the plates coated with nucleosomes, 50 |xl of 2C5 immunomicelles at 20 ng/ml of PEG-PE were added and incubated for 4 hours at room temperature. The plates were washed 3 times with HBS and coated with horseradish peroxidase-anti mouse IgG conjugate (ICN biomedical, Inc., Aurora, OH), diluted according to the manu facturer recommendation. The conjugate was removed after 3 hours incubation


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123 at room temperature, and the plates were washed 3 times with HBS. Bound peroxidase was quantified by degradation of its substrate, diaminobenzidine supplied as a ready for use solution (Neogen, Lexington, KY). The intensity of color developed was analyzed by an ELISA reader, Labsystems Multiscan MCC/340 (Labsystems and Life Sciences International, LTD, UK). Murine Lewis lung carcinoma (LLC) and EL4 T lymphoma cells as well as human mammary adenocarcinoma BT20 cells were purchased from American Type Culture Collection. The cells were propagated under ATCC recommen ded conditions. The immunomicelles-cell interaction was studied using micelles containing 0.5 mol % of Rh-PE. EL4 cells were grown in suspension to the density of about 2xl0 cells/ml and transferred to Hank's solution by centrifugation at 700xg for 10 minutes. The cells were washed twice by centrifugation under the same conditions and resuspended in Hank's buffer at about lxlO cells/ml density. Rh-PE-labeled antibody-free micelles or immunomicelles were added to the suspension of EL4 cell at the PEG ooo-PE concentration of 4.2X10" M . Following, the cells were incubated for 1 hour at 37°C under 5% C 0 , washed trice with Hank's solution, concentrated to the density of lxlO cells/ml by centrifugation, and mounted individually on fresh glass slides using a fluores cence-free Trevigen™ mounting medium (Trevigen, Gaithersburg, MD). Mounted slides were studied with a Nikon Eclipse E400 microscope under bright light, or under epi-fluorescence using a Rh/TRITC filter. BT-20 cells were grown on cover slips placed into 6-well tissue culture plates. After the cells reached a confluence of 60 to 70%, the plates were washed twice with Hank's solution. Rhodamine-labeled 2C5 immunomicelles containing micelles dispersed in Hank's buffer at a PEG ooo-PE concentration of 4.2xl0" M were added to the washed cells, and the cells were incubated for 1 hour at 37°C under 5% C 0 . After incubation, the cover slips were washed twice with Hank's buffer, mounted, and studied for the presence of Rh fluores cence as described above for EL4 cells. For animal experiments, the micelles were radiolabeled with I n via an amphiphilic chelating agent, DTPA-PE, added to their composition. The study was performed in female C57B1/6J mice. The mice were inoculated subcutaneously into the left rear flanks with 20,000 LLC cells dispersed in 100 \xl of HBS. After the tumor diameters reached ca. 0.5-1 cm (1-2 weeks post inoculation), the mice were injected with 100 ml of 0.5 mM In-labeled micellar formulations via the tail vein. At 0.5-2 hours post injection, the mice were sacrificed by cervical dislocation. Blood, tumors, and samples of muscle were extracted and analyzed for the presence of micelle-associated I n radioactivity. 4

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Results and Discussion PEG-PE conjugates form very stable, long-circulating micelles that selec tively accumulate in tumors via the EPR effect. (1,2,4) Table I summarizes the data on PEG-PE micelles loaded with anticancer agents from earlier publi cations as well as some new formulations. As Table I indicates, PEG-PE micelles can be loaded with various anticancer agents. In most cases, drugloaded micelles have a size very close to that of the blank ("empty") particles, indicating that drug-loaded micelles, similar to blank particles, should be able to penetrate leaky vasculature typical for tumors and deliver their load via passive targeting (EPR effect). Table 1. PEG-PE micelles loaded with anticancer agents Size (nm)

Publication

N/A Chlorine e6 trimethyl ester to 32 wt % Vitamin K3 to 50 wt %

7-15 nm 5-20 nm

(4) N/A

2-5 nm

N/A

N/A Chlorine e6 trimethyl ester to 30 wt % Tamoxifen to 25 wt % Taxol to 1.5 wt% Vitamin K3 to 30 wt %

7-20 nm 7-20 nm

(3,4) (3)

7-20 nm 7-20 nm 10-20 nm

N/A (3) N/A

ePC/PEG ooPE, 4/1 mol

N/A Taxol to wt 4%

20-60 nm 20-60 nm

N/A N/A

PEGsooo'PE

N/A Dequalinium to 3 wt % Tamoxifen to 20 wt % Taxol to wt 1% Vitamin K3 to 30 wt %

10-35 nm 10-40 nm 10-40 nm 13-40 nm 7-40 nm

(3,4) (2) (3) (3) N/A

Micelle Composition* PEG rPE 75(

PEG2ooo"PE

20

Drug load (drug/carrier)

* - ePC - egg phosphatidylcholine; the number associated with PEG shows its molecular weight.


125 PEG-PE micelles were modified with monoclonal 2C5 antibodies, known to recognize the surface of various tumor cells but not normal cells, to further improve their drug targeting potential. (14) The use of this antibody as a drug carrier targeting ligand would allow preparation of drug delivery systems able to recognize tumors of various types. 2C5-targeted PEG-PE immunomicelles were prepared utilizing a new coupling reagent, pNP-PEG-PE, earlier developed by us for attaching specific amino group-containing ligands to normal and longcirculating liposomes. (16,17) The amphiphilic reagent pNP-PEG-PE is capable of inserting into the hydrophobic phase of liposomes and micelles with its lipid part, and of covalent attaching to primary NH -groups of protein and peptides with its p-nitrophenol-activated PEG terminus. A typical result of bound 2C5 quantification obtained using a fluorescein labeled protein is shown in Figure 1. (18) Under the experimental conditions used, 30% of added 2C5 was attached to the micelles. This amount corresponds to between 10 and 50 protein molecules bound to a single micelle. Protein binding to control micelles lacking pNP-PEG-PE was negligibly small.


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Figure 1. Binding of 2C5 antibodies to PEG-PE micelles. One of the advantages of micelles over alternative particulate drug carriers is their relatively small size. This small size allows micelles to penetrate the interstitium of tumor cells with small vasculature cutoff size, which are nonaccessible for other drug carriers with larger particle diameters. (8, 9) The data shown in Figure 2 demonstrate that modification of micelles with 2C5 mono clonal antibodies has a very small effect on the micelle size, indicating that 2C5



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Figure 3. Binding of PEG-PE micelles to a monolayer of nucleosomes.



127 targeted PEG-PE immunomicelles will be capable of delivering drugs to tumors with a low vasculature cutoff size. The preservation of specific activity of micelle-bound antibody is crucial for drug targeting. The data shown in Figure 3 indicate that the 2C5 antibody attached to PEG-PE micelles via pNP-PEG-PE retains its affinity. (18) 2C5immunomicelles bind to a 2C5-specific nucleosomes, whereas control micelles, prepared by the same method but lacking pNP-PEG-PE, show only very minor binding, comparable to the background signal typical for this method. The use of 2C5 antibody as a targeting ligand allows for the preparation of drug delivery systems with specificity against tumors of various types (Figure 4). Binding of 2C5-immunomicelles to cancer cells was observed for two totally different cell lines, murine EL4 T lymphoma and human mammary adenocarcinoma BT20. Incubation of the same cell lines with micelles lacking antibodies resulted in virtually no micelle-to-cell binding.

Figure 4. Binding of Rh-tabeled PEG-PE micelles to EL4 cells (top panel) and to BT20 cells (bottom panel). Left half: bright field andfluorescent microsc of 2C5-micelles; right half: bright field and fluorescent microscopy of pla micelles. (See page 6 of color insert.) The accumulation of plain and 2C5-targeted PEG-PE micelles in sub cutaneous LLC tumors in mice is shown in Figure 5. At all time points studied, both plain and 2C5-targeted formulations demonstrated a much higher accu mulation in the tumor cells compared to non-targeted muscle tissue. The enhanced accumulation of plain micelles in the tumor can be explained by the EPR effect, whereas improved accumulation of 2C5-targeted micelles clearly indicates that immunomicelles are capable of specific recognition and binding to tumor cells. This observation allows to assume that micelles targeted with 2C5 will be capable of delivering their load not only to tumors with a mature vasculature but also to tumors at earlier stages of their growth, and to meta stases. This ability is not expected for plain micelles.


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Figure 5. Accumulation of PEG-PE micelles in subcutaneous LLC tumor in mice.

Conclusions Antibodies, such as the 2C5 monoclonal antibody, can be covalently attached to PEG-PE micelles utilizing the amphiphilic modifying agent, pNPPEG-PE, with preservation of the antibody's specific activity. 2C5-targeted PEG-PE micelles specifically bind to tumor cells of various types in vitro, and demonstrate an enhanced accumulation in tumors in vivo.

Acknowledgements This work was supported by NIH grant 5R01 GM60200-03 to Vladimir P. Torchilin.

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Gao, Z.; Lukyanov, A.N.; Singhal, A.; Torchilin, V.P. Nano Letters 2002, 2, 979-982. Lukyanov, A.N.; Gao, Z.; Mazzola, L.; Torchilin, V.P. Pharm. Res. 2002, 19, 1424-1429. Gabizon, A.A. Adv. Drug Deliv. Rev. 1995, 16, 285-294. Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Control. Release 2000, 65, 271-284. Maeda, H. Adv. EnzymeRegul.2001, 41, 189-207. Parr, M.J.; Masin, D.; Cullis, P.R.; Bally, M.B. J. Pharmacol. Exp. Ther. 1997, 280, 1319-1327. Weissig, V.; Whiteman, K.R.; Torchilin, V.P. Pharm. Res. 1998, 15, 15521556. Jain, R.K. Cancer Metastasis Rev. 1987, 6, 559-593. Jain, R.K. Cancer Metastasis Rev. 1990, 9, 253-66. Torchilin, V.P. Eur. J. Pharm. Sci. 2000, 11 (Suppl. 2), S81-91. Torchilin, V.P. J. Control. Release 2001, 73, 137-172. Iakoubov, L.Z.; Torchilin, V.P. Oncol. Res. 1997, 9, 439-446. Iakoubov, L.Z.; Torchilin, V.P. Cancer Detect. Prev. 1998, 22, 470-475. Torchilin, V.P.; Levchenko, T.S.; Lukyanov, A.N.; Khaw, B.A.; Klibanov, A.L.; Rammohan, R.; Samokhin, G.P.; Whiteman, K.R. Biochim. Biophys. Acta2001, 1511, 397-411. Torchilin, V.P.; Rammohan, R.; Weissig, V.; Levchenko, T.S. Proc. Natl. Acad. Sci. USA 2001, 98, 8786-8791. Torchilin, V.P.; Lukyanov, A.N.; Gao, Z.; Mazzola, L. Polym. Prepr. 2002, 43, 677-678.


Carrier-Based Drug Delivery - ACS Publications - American Chemical

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