Transferrin-Containing, Cyclodextrin Polymer-Based Particles for

1122

Bioconjugate Chem. 2003, 14, 1122−1132

Transferrin-Containing, Cyclodextrin Polymer-Based Particles for Tumor-Targeted Gene Delivery Nathalie C. Bellocq,† Suzie H. Pun,†,§ Gregory S. Jensen,† and Mark E. Davis*,‡ Insert Therapeutics, Inc., Pasadena, California and California Institute of Technology, Pasadena, California. Received July 17, 2003

Transferrin is a well-studied ligand for tumor targeting due to upregulation of transferrin receptors in numerous cancer cell types. Here, we report the development of a transferrin-modified, cyclodextrin polymer-based gene delivery system. The delivery system is comprised of a nanoparticle (formed by condensation of a cyclodextrin polycation with nucleic acid) that is surface-modified to display poly(ethylene glycol) (PEG) for increasing stability in biological fluids and transferrin for targeting of cancer cells that express transferrin receptor. A transferrin-PEG-adamantane conjugate is synthesized for nanoparticle modification. The transferrin conjugate retains high receptor binding and self-assembles with the nanoparticles by adamantane (host) and particle surface cyclodextrin (guest) inclusion complex formation. At low transferrin modification, the particles remain stable in physiologic salt concentrations and transfect K562 leukemia cells with increased efficiency over untargeted particles. The increase in transfection is eliminated when transfections are conducted in the presence of excess free transferrin. The transferrin-modified nanoparticles are appropriate for use in the systemic delivery of nucleic acid therapeutics for metastatic cancer applications.

INTRODUCTION

One of the most challenging applications of gene therapy is its use against metastatic cancer. The delivery of nucleic acid therapeutics to disseminated tumor sites demands a vehicle capable of systemic administration that will be able to localize in tumor areas. While viral vectors containing therapeutic genes have shown antitumor responses via direct tumoral injections, these vectors generally suffer from immunogenicity issues that make repeat systemic administrations problematic (1, 2). Nonviral vectors, whose assets include low T-cell and humoral immunogenicity, offer a viable alternative approach. In addition, nonviral vectors can be readily modified with ligands for cell targeting. Transferrin (Tf), an iron-binding glycoprotein, is a wellstudied ligand for tumor targeting (3, 4). Iron-loaded (holo-) transferrin is recognized by and binds to transferrin receptors on cell surfaces. Transferrin is then endocytosed into acidic compartments. The drop in pH triggers the iron dissociation, and the iron-poor (apo-) transferrin is recycled to the cell surface and released (4). Expression of transferrin receptor (Tf-R) is elevated in rapidly dividing cells due to a need for iron; therefore, Tf-R is often upregulated on surfaces of malignant cells and has been used as a tumor-targeting ligand for several drug delivery systems (5). Anticancer agents such as doxorubicin have been conjugated to transferrin for tumor targeting (6-8). Transferrin has also been used for targeting nonviral gene delivery vehicles to cancerous cells (9-19). However, the synthesis and characterization of these multicomponent materials is often not completely defined or con* To whom correspondence should be addressed. E-mail: [email protected]. † Insert Therapeutics, Inc. ‡ California Institute of Technology. § Current address: Department of Bioengineering, University of Washington, Seattle, WA 98195.

trolled. For example, doxorubicin-transferrin conjugates were initially prepared by glutaraldehyde cross-linking between the amines on the transferrin (lysine) and the doxorubicin. These conjugates exhibited cytotoxic activity to tumor cell lines (20, 21). However, polymeric products are likely to be formed using this cross-linking method, and the resulting conjugates are poorly defined from a chemical point of view (4). In addition, the binding affinities of the conjugates are significantly reduced from the affinity of the natural protein (21). The “second generation” doxorubicin-transferrin synthesis was improved by Kratz and colleagues by thiolating transferrin with Traut’s reagent and then reacting it with maleimide derivatives of doxorubicin (7). Advantages of this approach include less random cross-linking and also the incorporation of a potentially reversible hydrazone linkage. However, the resulting conjugates still contained ∼10% transferrin dimers that were not separated from the monomeric conjugates (7). Numerous other transferrin-doxorubicin conjugates have been prepared and one has even been explored for human use in a phase I clinical trial (22). For targeted gene delivery, approaches include direct transferrin conjugation onto activated surfaces of polycation/nucleic acid complexes (17) or linkage via biotin/ streptavidin interactions to plasmid DNA (19). One method of transferrin conjugation to poly(ethylenimine) that has yielded promising in vivo targeting of tumors involves oxidization of carbohydrate groups associated with the transferrin followed by reductive amination with the primary amines of poly(ethylenimine) (23). These literature examples show the potential of tumor-targeting via transferrin-Tf-R interactions. We have developed a cyclodextrin-based polymer delivery system that has the potential of being used for systemic nucleic acid delivery (24). The delivery system consists of two components. The first component is a cyclodextrin-containing polycation that is used for nucleic acid condensation into nanoparticles (25, 26), and the

10.1021/bc034125f CCC: $25.00 © 2003 American Chemical Society Published on Web 11/04/2003

Transferrin-Containing Particles for Gene Delivery

second component is an adamantane-terminated modifier for stabilizing the particles in order to (i) minimize interactions with plasma and (ii) target cell surface receptors (27). The specific ligands contained in the second component mediate cell targeting specificity, e.g., Tf ligands for cells with Tf-R. The two components selfassemble with nucleic acids to form stable and uniform sub-100 nm particles. The modular design of this delivery system allows each component to be synthesized and characterized separately. Here, we investigate the synthesis of transferrin-poly(ethylene glycol)-adamantane (Tf-PEG-AD) conjugates (second component of the delivery system) and their use in formulating a complete delivery system for systemic application. In subsequent reports, we will show that these delivery vehicles can successfully target and deliver either oligonucleotides (28, 29) or plasmids (30, 31) to subcutaneous tumors in nude mice. These constructs can be used as targeting ligands as described above. The process of attaching PEG to a protein is typically achieved by reacting an activated PEG reagent through either the carbohydrate moiety or through the lysine residues (32). Since a typical protein possesses a number of such groups, each having different reactivities and degrees of accessibility, the resulting product contains a family of species that is characterized by a distribution in both the number and position of attachment of the PEG group. Below, we describe an example of a fairly well-defined and characterized preparation of transferrin-PEG conjugates. We report on (i) the effect of the site of modification (lysine residue versus carbohydrate moiety), and (ii) the effect of the degree of modification (number of PEG chains per protein) on the binding affinity toward transferrin receptors. These transferrin-PEG conjugates are used to formulate transferrin-modified particles and some of their properties are presented. The particles are demonstrated to mediate transferrin-mediated delivery of nucleic acids to cultured cells.

Bioconjugate Chem., Vol. 14, No. 6, 2003 1123

bodiimide (EDC) (0.128 g, 0.666 mmol). Then, N-hydroxysuccinimide (NHS) (3.83 mg, 33.3 µmol) dissolved in 200 µL of 25 mM MES (pH 6.5) buffer was added immediately to the polymer solution. The resulting solution was stirred for 24 h at room temperature and then dialyzed against water using a Spectra Por membrane 1000 MWCO. The solution was lyophilized to dryness. The imidazole content was determined by the TNBS assay (34), followed by UV measurements to quantify the amount of unreacted polymer end groups. The imidazole conjugation was 73%. Adamantane-PEG5000 (AD-PEG). AD-PEG was synthesized according to published procedures (27). In brief, PEG5000-SPA (Nektar AL, Huntsville, AL) was reacted with 5 equiv of 1-adamantane-methylamine (Aldrich) dissolved in dichloromethane. The resulting solution was stirred at room temperature for 4 h. The solvent was removed in vacuo, and water was added to the remaining solution. The solution was centrifuged at 15K rcf for 15 min in order to remove unreacted 1-adamantane-methylamine. The aqueous portion was collected and dialyzed (Slide-A-Lyzer 3500 MWCO, Pierce, Rockford, IL) for 24 h against water. The dialyzed solution was then lyophilized to yield a white, fluffy powder. The product was analyzed on a Beckman Coulter Gold HPLC system equipped with a Richards Scientific Sedere Evaporative Light Scattering detector using a C18 reversed phase column. The product was found to be pure (retention time of PEG5000-SPA: 10.7 min; retention time of product: 12.1 min). The product was also confirmed by mass spectroscopy Maldi-ToF analysis.

EXPERIMENTAL PROCEDURES

Material Synthesis. β-Cyclodextrin Polymers (CDPImid). βCDP was synthesized according to previously described procedures (25, 26). The number of methylene groups separating the charges in the polycation backbone was chosen to be six to maximize transfection efficiency (26). Imidazole was conjugated to the βCDP polymer by amidation of the primary amines at the end of the polymer with 4-imidazoleacetic acid (Aldrich, St. Louis, MO) (33). In a typical experiment, 200 mg (33.3 µmol) of βCDP was dissolved in 800 µL of 25 mM MES (pH 6.5) buffer to which was added 4-imidazoleacetic acid, sodium salt hydrate (49.3 mg, 0.333 mmol). This solution was used to dissolve 1-ethyl-3-(3-dimethylaminopropyl)car-

Transferrin Conjugation via Carbohydrate Groups (Figure 1). The synthesis involves three steps. First, FMOC-NH-PEG5000-NHS (1) (Nektar AL, 0.2 mmol, 1 g) was added to a round-bottom flask equipped with a stir bar. To this was added tert-butyl carbazate (Aldrich, 1.6 mmol, 0.2112 g) dissolved in 7 mL of dichloromethane/ ethyl acetate (1:1). The resulting solution was stirred overnight at room temperature. The next day, the solvents were removed in vacuo. The FMOC group was removed by dissolving the resulting solid in 10 mL of 20% piperidine in dimethylformamide and stirring at room

1124 Bioconjugate Chem., Vol. 14, No. 6, 2003

Bellocq et al.

Figure 1. Synthesis of Tf-PEG-AD by carbohydrate conjugation.

temperature for 5 h. The solvent was removed in vacuo, and the residue was redissolved in water. The solution was centrifuged to remove the undissolved FMOC group and then dialyzed overnight in Pierce’s Slide-A-Lyser, 3500 MWCO. The solution was then lyophilized to afford 790 mg of H2N-PEG5000-NHNHCOOtBu (2). In the second step, N-hydroxysuccinimide (Aldrich, 0.24 mmol, 27.3 mg) and adamantanecarboxylic acid (Aldrich, 0.39 mmol, 71.2 mg) were dissolved in 7 mL of dichloromethane and added to 2 (0.16 mmol, 790 mg). To this resulting solution was added 1,3-dicyclohexylcarbodiimide (Aldrich, 1.6 mmol, 0.326 g) dissolved in 3 mL of dichloromethane. The resulting solution was stirred overnight at room temperature. The next day, the solid formed was filtered on a fine glass frit, and the filtrate was concentrated on a rotary evaporator under vacuum. The residue was dissolved in 10 mL of water and centrifuged to remove the unreacted adamantanecarboxylic acid. The solvent was removed in vacuo, and the residue was redissolved in 6 mL of 4 M HCl in dioxane in order to deprotect the tert-butoxycarbonyl group and stirred at room temperature for 4 h. The solvent was then removed in vacuo, and the residue was redissolved in water. The resulting solution was dialyzed overnight in Pierce’s Slide-A-Lyser (Rockford, IL), 3500 MWCO, and lyophilized to afford 635 mg of AD-PEG5000NHNH2 (3). This product was analyzed on a Beckman Coulter Gold HPLC system equipped with a Richards Scientific Sedere Evaporative Light Scattering detector using a C18 reversed phase column. The product was also confirmed by mass spectroscopy Maldi-ToF analysis. In the third step, 3 was conjugated to transferrin according to the protocol described previously by Wagner et al. (23). A solution of 100 mg (1.28 µmol) of human transferrin (iron poor) (Sigma-Aldrich) in 1 mL of a 30 mM sodium acetate buffer (pH 5) was subjected to gel filtration on a Sephadex G-25 (Supelco) column. The resulting 4 mL of solution containing transferrin (monitoring: UV absorption at 280 nm) was cooled to 0 °C and 80 µL of a 30 mM sodium acetate buffer (pH 5) containing

4 mg (19 µmol) of sodium periodate was added. The mixture was kept in an ice bath and in the dark for 2 h. For removal of the low molecular weight products, an additional gel filtration (Sephadex G-25, 30 mM sodium acetate buffer (pH 5)) was performed. This yielded a solution containing about 85 mg (1.09 µmol) of oxidized transferrin. The modified transferrin solution was promptly added to a solution containing 10.9 mg (2.2 µmol) of AD-PEG5000-NHNH2 (3) in 1 mL of 100 mM sodium acetate (pH 5). The resulting solution was stirred overnight at room temperature. The pH was then brought to 7.5 by addition of 1 M sodium bicarbonate, and four portions of 9.5 mg (150 µmol) of sodium cyanoborohydride each were added at 1 h intervals. After 4 h, the PEGylated transferrin was purified and concentrated using a Centricon YM-50,000 NMWI device (Millipore). This concentrated solution was subjected to hydrophobic interaction chromatography media (Tosoh Bioscience Butyl-650S) packed into a 10 cm bed height × 1 cm I.D. column. The products were eluted using a gradient mobile phase from 1.7 M to no ammonium sulfate in 0.1 M potassium phosphate buffered at pH 7.0. The column was run at room temperature, and the elution profile was monitored by UV absorbance at 280 nm. The UV-active fractions were then analyzed on a Beckman Coulter HPLC system equipped with a system gold 168 detector and a Butyl-NPR (Tosoh Bioscience) hydrophobic interaction column using a linear gradient from 1.7 to 0 M ammonium sulfate in 0.1 M potassium phosphate buffered at pH 7.0 at a 1 mL/min flow rate. Transferrin Conjugation via Lysine Groups (Figure 2). Vinyl sulfone-PEG5000-NHS (4) (Nektar AL, 0.147 mmol, 0.5 g) was added to a round-bottom flask equipped with a stir bar and dissolved in 5 mL of DMSO. To this was added adamantanemethylamine (Aldrich, 0.147 mmol, 0.0243 g). The resulting solution was stirred 1 h at room temperature. The solvent was removed in vacuo, and the residue was redissolved in water. The resulting mixture was dialyzed overnight against a 3500 MWCO membrane



Figure 2. Synthesis of Tf-PEG-AD by lysine conjugation.

(Spectra Por). The solution was then lyophilized to afford 0.49 g of vinyl sulfone-PEG5000-AD (5). In the second step,0.49 g (0.1 mmol) of vinyl sulfonePEG5000-AD (5) were added to a solution of 1 g (12.5 µmol) of human transferrin (iron poor) (Sigma-Aldrich) in 30 mL of a 0.1 M sodium tetraborate buffer (pH 9.4) and stirred at room temperature for 2 h. The PEGylated transferrin was purified from the unreacted vinyl sulfonePEG5000-AD (5) and concentrated using a Centricon plus YM-50,000 NMWI device (Millipore). This concentrated solution was subjected to hydrophobic interaction chromatography media (Tosoh Bioscience Butyl-650S) packed into a 30 cm bed height × 4 cm I.D. column. The products were eluted using a gradient mobile phase from 1.7 M to no ammonium sulfate in 0.1 M potassium phosphate buffered at pH 7.0. The column was run at room temperature, and the elution profile was monitored by UV absorbance at 280 nm. The UV active fractions were then analyzed on a Beckman Coulter HPLC system equipped with a system gold 168 detector and a Butyl-NPR (Tosoh Bioscience) hydrophobic interaction column using a linear gradient from 1.7 M to no ammonium sulfate in 0.1 M potassium phosphate buffered at pH 7.0 at a 1 mL/min flow rate (Figure 3A). The product was also confirmed by mass spectroscopy Maldi-ToF analysis (Figure 3B). Synthesis of Transferrin-Fluorescein. Fluorescein-5thiosemicarbazide, the hydrazide derivative of fluorescein (35) was conjugated to the transferrin via carbohydrate groups (23). A solution of 50 mg (0.64 µmol) of human transferrin (iron poor) (Sigma-Aldrich) in 1 mL of ice cold 30 mM sodium acetate buffer (pH 5) was subjected to gel filtration on a Sephadex G-25 (Supelco) column. The resulting 2 mL of solution containing transferrin (monitoring: UV absorption at 280 nm) was cooled to 0 °C, and 40 µL of a 30 mM sodium acetate buffer (pH 5) containing 2 mg (9.5 µmol) of sodium periodate was added. The mixture was kept in an ice bath and in the dark for 2 h. For removal of the low molecular weight products an additional gel filtration (Sephadex G-25, 30 mM sodium acetate buffer, pH 5) was performed. This yielded a solution containing about 43 mg (0.55 µmol) of oxidized transferrin. The modified transferrin solution was promptly added to 0.5 mg/mL fluorescein-5-thi-

osemicarbazide. The resulting solution was stirred 30 min in the dark at room temperature. The resulting transferrin-fluorescein was then subjected to gel filtration on a Sephadex G-25 (Supelco) column. Iron-Loading of Transferrin Conjugates. A 40 mg amount of apo-transferrin-based compound (apo-transferrin or apo-transferrin-PEG-AD) was dissolved in 700 µL of dH2O. To this solution was added 200 µL of 5 mM iron citrate and 100 µL of 84 mg/mL NaHCO3. This solution was allowed to stand for 2-3 h and then dialyzed against dH2O overnight. The iron-loading efficiency was calculated by determining the ratio of adsorbance at 465 nm (from the oxidized iron) to adsorbance at 280 nm (from the tryptophan residues in the protein) and normalizing to the A465/A280 ratio of commercially available holo-transferrin (ratio is denoted as iron-loading in the figures). Cell Culture and Cellular Studies. Cells and Plasmids. PC-3 (human prostate carcinoma) and K562 (chronic myelogenous leukemia) cells were purchased from the ATCC. Cells were cultured according to recommended procedures. Media and supplements were purchased from Gibco BRL (Gaithersburg, MD). Plasmid pGL3-CV (Promega, Madison, WI) containing the luciferase gene under the control of the SV40 promoter was amplified and purified by Elim Biopharmaceuticals (Hayward, CA). Competitive Binding Studies. PC-3 cells or K562 cells were plated at 300000 cells/well in six-well plates. After 24 h, cells were exposed to 250 nM of Tf-fluor (fluoresceinlabeled transferrin) with various amounts of unlabeled transferrin or Tf-PEG-AD in MEM media containing 1% BSA. 15 min after exposure, media was removed and cells washed with PBS and prepared for analysis by flow cytometry. For competitive binding studies using particle formulations, the same procedure was followed except Tf or Tf-PEG-AD was first formulated with particles (as described in the following paragraph) and then added to the media at a final concentration of 75 nM. Tf-Particle Formulations. Transferrin-modified particles were prepared at a final DNA concentration of 1 mg/mL. Equal volumes of the four components (plasmid pGL3-CV, CDP-Imid, AD-PEG, and Tf-(PEG-AD)x)


Bellocq et al.

Figure 3. (A) Purification and isolation of transferrin conjugates by hydrophobic interaction chromatography and (B) confirmation of desired products Maldi-ToF mass spectroscopy.

were mixed by adding the three polymer solutions (premixed) to the DNA solution and pipeting gently to mix. CDP-Imid was added at a 3 +/- to DNA. AD-PEG and Tf-(PEG-AD)x were included such that the total moles of AD-PEG and Tf-(PEG-AD)x is equal to the total moles of cyclodextrin. For particle size and zeta potential determination, particles were prepared, diluted to 5 µg/mL in dH2O, and analyzed using a ZetaPals dynamic light scattering detector (DLS, Brookhaven Instruments Corporation, Holtsville, NY). Tf-Particle Transfection Studies. K562 cells were plated in 24-well plates at 40,000 cells/well in 0.2 mL Opti-MEM (Invitrogen). Particle-containing solutions (with or without holo-transferrin as a competitive inhibitor) were then added to each well. Five hours after transfection, 0.8 mL of complete media was added to each well. Cells were collected by centrifugation 48 h post-transfection, washed with PBS, lysed and analyzed for luciferase activity by addition of Promega’s luciferase assay reagent and light

measurements integrated over 10 s with a Monolight 3010C luminometer (Pharmingen, San Diego, CA). RESULTS

Tf-PEG-AD Synthesized by Carbohydrate Conjugation. Transferrin-poly(ethyleneglycol)-adamantane (Tf-PEG-AD) was synthesized by carbohydrate conjugation according to literature procedures (Figure 1) (12, 23). In brief, the vicinal diols in the apo-transferrin glycan chains were oxidized to aldehydes with sodium periodate and reacted by reductive amination with adamantane-PEG-hydrazide. The Tf-PEG-AD conjugates were purified by hydrophobic interaction chromatography and iron-loaded with iron citrate in sodium bicarbonate. The iron-loading efficiency of the conjugates was measured by the ratio of the UV absorption at 465 nm (ironloaded transferrin) to the absorption at 280 nm (total protein)


Figure 4. (A). Comparison of iron-loading efficiency of transferrin, oxidized transferrin, and Tf-PEG-AD (synthesized by carbohydrate conjugation). Proteins were purified at each step of the reaction by gel filtration and iron-loaded with iron citrate in sodium bicarbonate. Iron-loading efficiency was measured by the ratio of UV absorption at 465 nm to 280 nm and normalized by the A465/A280 ratio for holo-transferrin. (B) Relative binding of Tf-PEG-AD conjugates (synthesized by carbohydrate conjugation) to Tf-R. PC-3 cells were exposed to 250 nM of Tffluor in the presence of transferrin or various transferrin-based conjugates for 15 min. Cells were collected, washed, and analyzed by FACS for cellular association of Tf-fluor.

and normalized by the A465/A280 ratio for holo-transferrin (Figure 4A). The Tf-PEG-AD conjugate had only 40% iron-loading efficiency. The transferrin iron-loading efficiency was then determined at each step in the synthesis process: in the pH 5 reaction buffer, after oxidation, after reaction with AD-PEG-hydrazide, and after hydrazone reduction with sodium cyanoborohydride. Each product was isolated by gel filtration before ironloading. Efficient iron-loading was obtained at pH 5 (98%), but was substantially reduced after oxidation with sodium periodate (30%). The loss in iron-loading efficiency of the oxidized Tf was maintained upon conjugation to the AD-PEG and was not reversible; the addition of a reducing agent only slightly increased iron-loading efficiency of the conjugated Tf (54%). The Tf-PEG-AD conjugates were tested for transferrin receptor-binding affinity. PC-3, a human prostate carcinoma cell line that expresses elevated levels of transferrin receptor (Tf-R) (36), were exposed to 250 nM of fluorescein-labeled transferrin (Tf-fluor) and 100 nM of hTf (holo-transferrin), Tf-PEG-AD, or Tf-(PEGAD)2, and analyzed for Tf-fluor cellular association (Figure 4B). Transferrin conjugates with high receptor binding affinities should compete efficiently against Tffluor for receptor binding sites, thus reducing average


cell fluorescence. The fraction of cells in each sample with Tf-fluor/Tf-R binding above a threshold value was determined by flow cytometry and normalized by the fraction of fluorescein positive cells in samples exposed to 250 nM Tf-fluor alone. The native holo-Tf ligand has the highest binding affinity for the Tf-R and prevents Tffluor/Tf-R association (0.6% fluorescein positive cells), even at 40% the concentration of Tf-fluor. Oxidized transferrin has significantly reduced Tf-R binding affinity (72%), as do the purified Tf-PEG-AD (76%) and Tf(PEG-AD)2 (107%) conjugates. Because the synthesis of Tf-PEG-AD via the reductive amination scheme causes a significant reduction in the Tf/Tf-R binding, another conjugation method was pursued. Tf-PEG-AD Synthesized by Lysine Conjugation. A second approach was used to prepare Tf-PEG-AD conjugates (Figure 2). Tf was modified at surface-available lysines via reaction with adamantane-PEG-vinyl sulfone. The Tf-PEG-AD conjugates were separated and purified by hydrophobic interaction column (Figure 3A). Each conjugate (Tf-PEG-AD, Tf-(PEG-AD)2, and Tf(PEG-AD)3), could therefore be isolated. Clean separation of the mono-, di-, and trisubstituted transferrins was demonstrated by HPLC analysis with products confirmed by Maldi-ToF mass spectroscopy (Figure 3B). Transferrin is an 80 kDa protein, and the molecular weight of PEGAD is approximately 5300. As expected, mass spectroscopy of the transferrin yields a sharp signal at 79 kDa. The isolated fractions for Tf-PEG-AD, Tf-(PEG-AD)2, and Tf-(PEG-AD)3 reveal peaks at 84.6 kDa, 89.8 kDa, and 95.1 kDa, respectively. The purified transferrin conjugates were then iron-loaded as described above. Unlike the conjugates synthesized by transferrin oxidation and Schiff base formation, Tf-PEG-AD’s prepared by reaction with surface lysines retain high iron-loading efficiencies (all around 100% iron loading, Figure 5A). The Tf-(PEG-AD), Tf-(PEG-AD)2, and Tf-(PEGAD)3 compounds were analyzed for receptor-binding to transferrin receptors on both PC-3 and K562 cells as described above (Figure 5B and 5C). The monosubstituted Tf-PEG-AD showed the highest binding affinity to Tf-R of the three conjugates and was only slightly reduced from the native holo-transferrin protein (for PC3, 6% cells with Tf-fluor association compared with 0.6% in the presence of holo-transferrin and for K562, 6% cells with Tf-fluor association compared with 2% in the presence of holo-transferrin). The ability of Tf-(PEGAD)2 to bind to Tf-R was affected by PEG-AD conjugation (34% of PC-3 and 25% of K562 cells fluorescein positive), and Tf-(PEG-AD)3 revealed the lowest receptor-binding of the three conjugates (36% cells fluorescein positive for both PC-3 and K562 cells). Therefore, the monosubstituted Tf-PEG-AD synthesized by lysine conjugation was used for all further experiments described. Formulation of Tf-Modified Particles. Transferrinmodified particles were prepared by mixing the following components (prepared in water solutions) in equal volumes: (1) CDP-Imid, a linear, cyclodextrin-based polycation that condenses and protects plasmid DNA, (2) adamantane-PEG (AD-PEG), a particle modifier that provides for PEGylation to give stabilization against nonspecific interactions including self-aggregation, (3) AD-PEG-Tf (synthesized by lysine conjugation), another modifier that introduces transferrin for targeting to Tf-R-expressing cells (for controls, holo-transferrin was used instead of AD-PEG-Tf), and (4) plasmid DNA. The first three components are premixed and added to the fourth solution. Particles were prepared with various


Bellocq et al.

Figure 6. Zeta potential of transferrin modified particles. Transferrin-modified particles were prepared at 3 +/- (CDPImid to pGL3-CV) with 100% adamantane modification (total of AD-PEG and Tf-PEG-AD). Particles were analyzed using a ZetaPals dynamic light scattering detector.

Figure 5. (A) Comparison of iron-loading efficiency of transferrin and Tf-PEG-AD conjugates synthesized by lysine reaction. Iron-loading efficiency was measured by the ratio of UV absorption at 465 nm to 280 nm and normalized by the A465/ A280 ratio for holo-transferrin. Relative binding of Tf-PEGAD conjugates (synthesized by lysine conjugation) to Tf-R to PC3 (5B) and K562 (5C) cells. Cells were exposed to 250 nM of Tffluor in the presence of transferrin or various transferrin-based conjugates for 15 min. Cells were collected, washed, and analyzed by FACS for celllar association of Tf-fluor.

amounts of Tf or Tf-PEG-AD (0%, 0.5%, 1%, and 2% of total cyclodextrins by mole). The particle size and surface charge were measured by dynamic light scattering and zeta potential, respectively. Average particle diameter of all formulations was ∼100 to 150 nm. Transferrin modification was verified by monitoring the average zeta potential of the particles

(Figure 6). Transferrin is an anionic protein; therefore, transferrin association with the positively charged particles should decrease the surface charge. Addition of free transferrin decreases average particle zeta potential from +30 mV to +25 mV (at 1% and 2% transferrin to CD). The effect of transferrin on surface charge levels off at 1% (∼+25 mV for both 1% and 2% transferrin). The zeta potential of particle formulations with Tf-PEG-AD also decreased with higher concentration of Tf-PEG-AD, and indicated successful modification of particles with TfPEG-AD. At 1% and 2% Tf-PEG-AD, average particle zeta potentials are +20 mV and +17 mV, respectively. Transferrin-modified particles (Tf-PEG-particle) should also have higher association to transferrin receptors on cell surfaces than Tf-PEG-AD because the Tf-PEGparticle has the ability to bind to several Tf-R while TfPEG-AD/Tf-R binding is a single interaction event. PC-3 cells were exposed for 15 min to 250 nM of Tf-fluor and 75 nM of Tf or Tf-PEG-AD in the presence and absence of CDP-Imid-based particles (at a 2 Tf per 100 cyclodextrin mole ratio). The cells were prepared and analyzed by flow cytometry as described previously for the receptor binding assay. Particle addition alone did not affect Tffluor association to PC-3 cells. However, formulation of Tf-PEG-AD with particles reduces the percentage of fluorescein positive cells by 15% from the percentage when exposed directly to Tf-PEG-AD and Tf-fluor. Similarly, exposure of cells to Tf in the presence of particles only reduces the percentage by 6%. The salt stability of various Tf-PEG-particle formulations was determined by monitoring particle size in physiological salt solutions by dynamic light scattering. Tf-PEG-particles were formulated with 0%, 0.05%, 0.1%, 0.5%, 1.0%, 2%, and 5% Tf-PEG-AD (of total cyclodextrins by mole with the remaining balance to 100% comprised of AD-PEG) in a small volume (40 µL) of water. PBS, pH 7.2, was then added (1.2 mL) and average particle diameter recorded every minute for 5 min (Figure 7). Unmodified particles aggregated rapidly in salt (final diameter of 300 nm), but PEGylated particles remained stable with diameters of ∼70 nm. The stability of Tfmodified particles depended on the amount of Tf-PEGAD present during formulation. Formulations with 0.05%, 0.1%, and 0.5% Tf-PEG-AD were stable in salt (respective diameters of 75, 80, and 83 nm after 5 min in salt solutions) while formulations with 1%, 2%, and 5% TfPEG-AD aggregated slowly upon salt addition (107, 129, and 168 nm after 5 min, respectively).



equiv of holo-Tf. These samples did not reveal much increase in luciferase gene delivery (4.7 × 104 RLU/mg) over the PEGylated particle. The transfection by particles formulated with holo-Tf was likewise reduced in the presence of free transferrin (3 × 104 RLU/mg). In addition, no toxicity was observed in any of the samples. DISCUSSION

Figure 7. Salt stability of transferrin-modified particles. Transferrin-modified particles were prepared at 3 +/- (CDPImid to pGL3-CV) with 100% adamantane modification (total of AD-PEG and Tf-PEG-AD). PBS (1.2 mL) was added to the particle solution and average particle diameter every minute for 5 min by dynamic light scattering.

Figure 8. Luciferase transfection of transferrin-particles to K562 cells. K562 cells were plated in 24-well plates and exposed to 1 µg of pGL3-CV in four formulations: particles modified with 100% AD-PEG, particles modified with 100% AD-PEG, and mixed with 0.05% holo-Tf (by mole to cyclodextrins), particles modified with 100% AD-PEG and 0.05% Tf-PEG-AD (TfPEG-part.), and Tf-PEG-part. mixed with 10 equiv of holo-Tf (with respect to Tf-PEG-AD). Experiments were conducted with five replicates with average values shown.

In Vitro Transfection of Tf-Modified Particles to K562 Cells. Tf-PEG-particles containing pGL3-CV were formulated at 0.05% Tf-PEG-AD (and 99% AD-PEG) and 3 +/- charge ratio (ratio of polymer to plasmid charge by mole) and used to transfect K562 cells. Control samples included PEGylated particles (PEG-particles, 1 AD-PEG to 1 cyclodextrin by mole), PEG-particles formulated with holo-Tf (at 0.05% Tf by mole cyclodextrin), PEG-particles formulated with holo-Tf (at 0.05% Tf by mole cyclodextrin) mixed with 10 equiv of free holoTf, and Tf-PEG-particles mixed with 10 equiv of free holo-Tf. Transfections were carried out in serum-free media, and cells were lysed and analyzed for luciferase activity 48 h after transfection with results reported in relative light units per mg of protein (RLU/mg protein, protein levels determined by Lowry protein assay, Figure 8). The positively charged, PEGylated particles transfected K562 cells (4.6 × 104 RLU/mg). Formulation of these particles with holo-Tf increased delivery efficiency by 1.7-fold (7.7 × 104 RLU/mg). The Tf-PEG-particles, with Tf attachment to the particles via inclusion complex formation, demonstrated the highest luciferase expression (1.8 × 105 RLU/mg) with a 4-fold enhancement over the PEG-particles. To confirm specific transfection enhancement by Tf-mediated delivery, Tf-PEG-particle transfections were also conducted in the presence of 10

The goal of this work is to develop a transferrinmediated, tumor-targeting gene delivery system that is chemically well-defined. Although targeting to tumors via mixtures of tranferrin and liposomal or polymeric delivery systems has been reported (15, 16, 18, 37), the characteristics and efficacy of these constructs are highly dependent on formulation methods. We strived to prepare stable formulations without significantly diminished interaction affinity between the transferrin targeting ligand and Tf-R on the surfaces of carcinoma cells. Here, Tf-PEG-AD conjugates were synthesized for assembly with cyclodextrin-containing particles by inclusion complex formation between the adamantanes and particle surface displayed cyclodextrins. The Tf-PEG-AD interacts with the particles through charge (anionic Tf-cationic particles) and inclusion complexation formation (ADCD). A Tf-PEG-AD ligand was first synthesized by sodium periodate oxidation of the apo-transferrin carbohydrate moieties followed by a reductive amination reaction with AD-PEG-hydrazide (Figure 1). The synthesis protocol was adapted from published procedures for preparing Tf-poly(L)lyine and Tf-poly(ethylenimine) conjugates (12, 13, 23). The various transferrin products were purified by hydrophobic interaction chromatography, an established method for isolation of PEGylated proteins (38, 39). The mono-, di-, and tri-PEGylated transferrins were successfully isolated and converted to the ironloaded form by iron citrate treatment in bicarbonate. A distinct difference in color was noted between holo-Tf and holo-Tf-PEG-AD solutions at the same concentrations. This discrepancy was quantified by measuring the UV absorbance at 465 nm (for the oxidized iron) and 280 nm (for total protein concentration). The results confirmed that the Tf-PEG-AD conjugates suffered from low ironloading efficiency. The amount of iron-loading into transferrin was therefore measured at each reaction step, and the cause of reduced iron-loading was determined to be the oxidation of transferrin with sodium periodate (Figure 4A). The oxidized transferrin was separated from excess sodium periodate by chromatography before iron citrate treatment. Iron-loading of oxidized transferrin was only 30%, and efficiency was not recovered by reaction with AD-PEG-hydrazide or by subsequent reduction with sodium cyanoborohydride. The iron-loading protocol conditions (concentration of iron citrate and iron-loading time) were also varied in an attempt to increase efficiencies, but did not result in any substantial improvements. The binding affinity of the transferrin conjugates for Tf-R on surfaces of cultured cells was assessed by monitoring the competitive binding against fluorescently labeled transferrin (Figure 4B). Holo-transferrin completely displaces the fluorescently labeled transferrin, even at a reduced concentration (the Tf-fluor was also synthesized by reductive amination with fluorescein hydrazide and has lower receptor binding affinity than the natural ligand). The transferrin iron-loading efficiency was found to be a good indicator of receptor binding. Transferrin with low iron-loading efficiencies


(oxidized transferrin and Tf-PEG-AD conjugates) also had low receptor binding. Indeed, apo-transferrin is reported to have lower binding affinity for the transferrin receptor at physiologic pH to unloading after recycling to cell surfaces (40). After finding that the affinity for the Tf-R was greatly reduced when conjugating AD-PEG to Tf via carbohydrate oxidation, an alternative synthetic approach was pursued. Tf-PEG-AD was synthesized by reaction of lysine amines with a vinyl sulfone that was prepared as AD-PEG-vinyl sulfone (Figure 2). The vinyl sulfone group is known to be highly selective at pH 7 for reaction with sulfhydryl groups (relative to reaction with amino groups). However, it has been shown that the reaction with lysine sites occurs slowly at pH 9.4 and is essentially complete (41). A reaction conducted at pH 7 revealed that the vinyl sulfone did not react with cysteine thiols groups of transferrin because no free thiols are available on the protein surface (as confirmed by Ellman’s titration (34)). The Tf-PEG-AD conjugates were thus synthesized at pH 9.4 by reaction between the vinyl sulfone and the lysine groups. The resulting product was a physical mixture of conjugates with variable number of attachments of AD-PEG chains. These conjugates were purified and Tf-PEG-AD, Tf-(PEG-AD)2, and Tf-(PEGAD)3 were isolated by hydrophobic interaction chromatography (Figure 3A) with product confirmation by MaldiToF mass spectroscopy (Figure 3B). Iron-loading was efficient for all transferrin conjugates synthesized by lysine conjugation (Figure 5A). During this process, it was found that it was important to freeze the protein conjugates slowly in dry ice before lyophilization. Rapid freezing in liquid nitrogen impaired the iron-loading efficiency. The transferrin conjugates were tested for transferrin receptor binding to both PC-3 and K562 cell lines. PC-3, an adherent cell line, and K-562, a suspension cell line, have upregulated transferrin receptors on their surfaces; K-562 cells are reported to have 1-5 × 105 Tf-R per cell (40, 42). Unlike transferrin conjugates synthesized by carbohydrate reaction, all lysine-reacted Tf-PEG-AD were able to compete effectively with fluorescein-labeled transferrin for receptor binding in both cell lines (6-36% fluorescein positive cells for lysine-reacted conjugates compared with 76-100% fluorescein positive cells for carbohydrate-reacted conjugates, Figure 5B and 5C). The transferrin receptor binding affinity was decreased by modification with multiple PEGs. While Tf-PEG-AD demonstrated slightly lower binding constants from the natural ligand, the difference was larger for the di- and tri-PEGylated conjugates. With higher PEGylation events, it becomes more likely for the reaction site to interfere with the ligand/receptor binding site. In addition, a multiPEGylated protein may be sterically inhibited from receptor interactions. On the basis of these receptor binding studies, the mono-Tf-PEG-AD conjugate was used for all subsequent experiments. Transferrin-modified particles were formulated using the Tf-PEG-AD conjugate. The Tf-modified particles are composed of four components: the nucleic acid of interest, a polycation for condensation of nucleic acid, AD-PEG for particle stabilization, and AD-PEG-Tf for tumor targeting. CDP-Imid, a linear, cyclodextrin-based polycation end-modified with imidazoles, was used as the nucleic acid condensing agent. The parent cyclodextrinbased polymer has been optimized structurally for in vitro transfection and the imidazoles further boost transfection efficiency and act as buffers in endosomes (24). The increased gene expression may be occurring by

Bellocq et al.

mechanisms similar to those proposed for histidylated polymers and poly(ethylenimine) (43, 44). AD-PEG5000 was used because previous experiments demonstrated that a molecular weight of 5000 was necessary for stabilizing cyclodextrin-based particles in salt-containing media (27). The particles were formulated by mixing the three polymer solutions together and then adding the polymer solution to the nucleic acid solution. The polymers and DNA self-assembled quickly (in seconds) to form particles with diameters ∼100-150 nm (as measured by dynamic light scattering). The amount of Tf-PEG-AD (by mole % to cyclodextrin) included in the formulations ranged from 0% to 2% with the balance (to 100% CD) consisting of AD-PEG. This range was chosen by estimating the amount of Tf that can pack around a 100 nm sphere, subject to space constraints. The zeta potential for each of the formulations was also measured (Figure 6). As expected, the zeta potentials decrease with increasing concentration of Tf or Tf-PEGAD in the formulations. Transferrin, an anionic protein, decreases zeta potential as it associates with the positively charged particle surface via electrostatic interactions. Formulations with Tf-PEG-AD have a larger impact on zeta potential, likely due to the higher surface affinity resulting from both inclusion complex formation and electrostatic interactions. With both ligands, the decrease in zeta potential levels off in the formulations with 1-2% transferrin and likely indicates surface saturation. Incorporation of the Tf-PEG-AD ligand in the particle was further confirmed by competitive receptor-binding studies. Fluorescein-labeled transferrin and either transferrin or Tf-PEG-AD were incubated with PC-3 cells, both as free ligands in solution and as a component in a particle formulation. The percentage of fluorescein-positive cells, indicating Tf-fluor binding to receptors, was determined by flow cytometry. The fluorescence profile of the cells was the same for cells exposed to 250 nM Tffluor alone and to 250 nM Tf-fluor/PEGylated particles (no Tf on the particles); therefore, the presence of particles alone does not affect Tf-fluor association with PC-3 cells. Formulation of 250 nM Tf-fluor/75 nM TfPEG-AD with the particles reduced the number of fluorescein positive cells by 15% as compared with 250 nM Tf-fluor/75 nM Tf-PEG-AD alone, while formulation of 250 nM Tf-fluor/75 nM Tf with the particles reduced the number by 6% as compared with 250 nM Tffluor/75 nM Tf alone. The difference in receptor binding between free ligand and ligand formulated with particles (keeping the concentration of transferrin constant) can therefore be attributed to an increase in binding affinity resulting from multidentate interactions between the ligand-modified particles and cell surface receptors. The stronger interaction between Tf-PEG-AD and the CDPImid particles (as opposed to Tf and particle) is reflected in the larger difference in receptor binding affinity between free ligand and ligand/particle formulations. It has been demonstrated previously that AD-PEG imparts salt stability to cyclodextrin-based particles by forming a hydrophilic layer on the particle surface (27). It is expected that, above certain proportions, incorporation of the large and bulky Tf-PEG-AD ligand will interfere with the PEG layer and eliminate its stabilization effect. The particle sizes of several Tf-particle formulations were monitored by dynamic light scattering to determine their salt stability in 150 mM PBS (Figure 7). Formulations with 0%, 0.05%, 0.1%, and 0.5% TfPEG-AD were stable, whereas average particle diam-


eters of formulations with 1%, 2%, and 5% Tf-PEG-AD increased steadily after salt addition (although not to the extent of the unmodified particles). The Tf-particle formulation was then tested for gene delivery efficiency to K562 cells (Figure 8). K562 cells are suspension cells that are notoriously difficult to transfect. The PEGylated particles are positively charged and can bind via electrostatic interaction with proteoglycans on the cell surface with eventual uptake by endocytosis (45). Formulations with 0.05% transferrin result in a 1.7-fold increase in delivery efficiency. The transferrin associates with the particles by electrostatic interactions and may facilitate cell association and uptake. The TfPEG-AD particle preparations (with 0.05% Tf-PEGAD) demonstrate the highest transfection, a 4-fold increase over AD-PEG-particles. The enhanced transfection is due to transferrin-mediated uptake; the addition of excess transferrin (10 equiv) as a competitor for the Tf-R eliminates the transfection enhancement. In summary, we describe the synthesis, purification, and in vitro use of a transferrin conjugate for targeting to tumor cells that are known to have upregulated transferrin receptors. While the potential of transferrinmediated targeting has been clearly demonstrated by others in the field (4, 7, 8, 14, 23, 46-49), we focused on preparing delivery systems with high receptor binding affinities. To this end, monofunctionalized Tf-PEG-AD conjugates were synthesized and purified by hydrophobic interaction chromatography. Isolation of the monofunctionalized transferrin is important for maintaining high receptor binding affinity. Sato et al. also isolated Tf-DNA conjugates by selecting conjugates that bind strongly to Tf-R affinity columns (19). However, most other conjugation chemistries for transferrin impair receptor binding or result in polymeric products. Glutaraldehyde crosslinking of transferrin with drug molecules forms polymeric products (4) with receptor binding affinities half that of transferrin (21), SPDP cross-linking of Tf with polycations yields multiple populations (including polymeric products) (50), and reaction by carbohydrate oxidation adversely affects receptor binding (as described previously). The transferrin conjugates described here are monofunctionalized and retain high receptor binding affinity. Physiochemical characterization and in vitro studies demonstrate that these conjugates should be useful in providing transferrin receptor-targeted drug delivery systems. The synthesis and purification protocols discussed here can also be extended to prepare transferrin conjugates for small molecule delivery. Applications of the transferrin-modified nucleic acid delivery system described herein are currently being investigated for the in vivo delivery of both oligonucleotide and plasmids in mouse xenograph models (29, 31). ACKNOWLEDGMENT

We thank Prof. Ernest Beutler (Scripps Institute) for his scientific advice and Mona Shahgholi (California Institute of Technology) for her assistance with MaldiToF analysis. Mark E. Davis is a consultant to and has financial interest in Insert Therapeutics, Inc. LITERATURE CITED (1) Green, N., and Seymour, L. (2002) Adenoviral vectors: systemic delivery and tumor targeting. Cancer Gene Ther. 9, 1036-42. (2) Nielsen, L., and Maneval, D. (1998) P53 tumor suppressor gene therapy for cancer. Cancer Gene Ther. 5, 52-63.

Bioconjugate Chem., Vol. 14, No. 6, 2003 1131 (3) Singh, M. (1999) Transferrin as a targeting ligand for liposomes and anti-cancer drugs. Curr. Pharm. Des. 5, 44351. (4) Qian, Z. M., Li, H., Sun, H., and Ho, K. (2002) Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pharm. Rev. 54, 561-587. (5) Thorstensen, K., and Romslo, I. (1993) The transferrin receptor: its diagnostic value and its potential as therapeutic target. Scand. J. Clin. Lab Invest. 53, 113-120. (6) Li, H., Sun, H., and Qian, Z. M. (2002) The role of the transferrin-transferrin-receptor system in drug delivery and targeting. Trends in Pharm. Sci. 23, 206-209. (7) Kratz, F., Beyer, U., Roth, T., Tarasova, N., Collery, P., Lechenault, F., Cazabat, A., Schumacher, P., Unger, C., and Falken, U. (1998) Transferrin conjugates of doxorubicin: synthesis, characterization, cellular uptake, and in vitro efficacy. J. Pharm. Sci. 87, 338-346. (8) Kratz, F., Beyer, U., Schumacher, P., Kruger, M., Zahn, H., Roth, T., Fiebig, H., and Unger, C. (1997) Synthesis of new maleimide derivatives of daunorubicin and biological activity of acid labile transferrin conjugates. Bioorg. Med. Chem. Lett. 7, 617-622. (9) Cotten, M., La¨ngle-Rouault, F., Kirlappos, H., Wagner, E., Mechtler, K., Zenke, M., Beug, H., and Birnstiel, M. (1990) Transferrinspolycation-mediated introduction of DNA into human leukemic cells: Stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels. Proc. Natl. Acad. Sci. U.S.A. 87, 4033-4037. (10) Kircheis, R., Blessing, T., Brunner, S., Wightman, L., and Wagner, E. (2001) Tumor targeting with surface-shielded ligand-polycation DNA complexes. J. Controlled Release 72, 165-170. (11) Kircheis, R., Wightman, L., Schreiber, A., Robitza, B., Rossler, V., Kursa, M., and Wagner, E. (2001) Polyethylenimine/DNA complexes shielded by transferrin target gene expression to tumors after systemic application. Gene Ther. 8, 28-40. (12) Kircheis, R., Kichler, A., Wallner, G., Kursa, M., Ogris, M., Felzmann, T., Buchberger, M., and Wagner, E. (1997) Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther. 4, 409-418. (13) Ogris, M., Steinlein, P., Kursa, M., Mechtler, K., Kircheis, R., and Wagner, E. (1998) The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther. 5, 1425-1433. (14) Ogris, M., Brunner, S., Schu¨ller, S., Kircheis, R., and Wagner, E. (1999) PEGylated DNA/transferrinsPEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 6, 595-605. (15) Simoes, S., Slepushkin, V., Gaspar, R., Pedroso de Lima, M., and Du¨zgu¨nes, N. (1998) Gene delivery by negatively charged ternary complexes of DNA, cationic liposomes and transferrin or fusigenic peptides. Gene Ther. 5, 955-964. (16) Xu, L., Frederik, P., Pirollo, K. F., Tang, W.-H., Rait, A., Xiang, L.-M., Huang, W., Cruz, I., Yin, Y., and Chang, E. (2002) Self-assembly of a virus-mimicking nanostructure system for efficient tumor-targeted gene delivery. Hum. Gene Ther. 13, 469-481. (17) Fisher, K., Ulbrich, K., Subr, V., Ward, C., Mautner, V., Blakey, D., and Seymour, L. (2000) A versatile system for receptor-mediated gene delivery permits increased entry of DNA into target cells, enhanced delivery to the nucleus and elevated rates of transgene expression. Gene Ther. 7, 13371343. (18) Seki, M., Iwakawa, J., Cheng, H., and Cheng, P.-W. (2002) p53 and PTEN/MMAC1/TEP1 gene therapy of human prostate PC-3 carcinoma xenograft, using transferrin-facilitated lipofection gene delivery strategy. Human Gene Therapy. 13, 761-773. (19) Sato, Y., Yamauchi, N., Takahashi, M., Sasaki, K., Fukaura, J., Neda, H., Fujii, S., Hirayama, M., Itoh, Y., Koshita, Y., Kogawa, K., Kato, J., Sakamaki, S., and Niitsu, Y. (2000) In Vivo gene delivery to tumor cells by transferrin-streptavidinDNA conjugate. FASEB J. 14, 2108-2118.

1132 Bioconjugate Chem., Vol. 14, No. 6, 2003 (20) Yeh, C., Taylor, C., and Faulk, W. (1984) Transferrin binding by peripheral blood mononuclear cells in human lymphomas, myelomas and leukemias. Vox Sang. 46, 217223. (21) Berczi, A., Ruthner, M., Szuts, V., Fritzer, M., Schweinzer, E., and Goldenberg, H. (1993) Influence of conjugation of doxorubicin to transferrin on the iron uptake by K562 cells via receptor-mediated endocytosis. Eur. J. Biochem. 213, 427-436. (22) Faulk, W., Taylor, C., Yeh, C., and McIntyre, J. (1990) Preliminary clinical study of transferrin-adriamycin conjugate for drug deliveyr to acute leukemia patients. Mol. Biother. 2, 57-60. (23) Wagner, E., Cotten, M., Mechtler, K., Kirlappos, H., and Birnstiel, M. L. (1991) DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety, Bioconjugate Chem. 2, 226-231. (24) Davis, M., Pun, S., Bellocq, N., Reineke, T., Popielarski, S., Mishra, S., and Heidel, J. (2003) Self-assemblying nucleic acid delivery vehicles via linear, water-soluble, CD-containing polymers. Curr. Med. Chem., in press. (25) Gonzalez, H., Hwang, S., and Davis, M. (1999) New class of polymers for the delivery of macromolecular therapeutics. Bioconjugate Chem. 10, 1068-74. (26) Hwang, S., Bellocq, N., and Davis, M. (2001) Effects of Structure of Beta-Cyclodextrin-Containing Polymers on Gene Delivery. Bioconjugate Chem. 12, 280-290. (27) Pun, S. H., and Davis, M. (2002) Development of a NonViral Gene Delivery Vehicle for Systemic Application. Bioconjugate Chem. 13, 630-639. (28) Bellocq, N., Pun, S., Grubbs, B., Jensen, G., Liu, A., Cheng, J., Mavis, M., Bakker, A., Janssens, B., Floren, W., Janicot, M., Peeters, J., and Brewster, M. (2002). Development of transferrin-modified, cyclodextrin-based particles for the delivery of RNA-cleaving DNA enzyme (DNAzyme) molecules. Paper presented at the 2nd International Symposium on Tumor Targeted Delivery Systems, Rockville, MD. (29) Pun, S., Bakker, A., Bellocq, N., Grubbs, B., Jensen, G., Liu, A., Cheng, J., Janssens, B., Floren, W., Peeters, J., Janicot, M., Mavis, M., and Brewster, M. Transferrintargeted, cyclodextrin polymer-based gene delivery systems. II. Applications in the delivery of RNA-cleaving DNA enzyme (DNAzyme) molecules. Manuscript in preparation. (30) Bellocq, N. C., Davis, M. E., Engler, H., Jensen, G. S., Liu, A., Machemer, T., Maneval, D. C., Quijano, E., Pun, S. H., Schleup, T., and Wen, S. (2003). Transferrin-targeted, cyclodextrin polycation-based gene vector for systemic delivery. Presented at the American Society of Gene Therapy Annual Conference, Washington, D. C. (31) Schleup, T., Bellocq, N., Davis, M., Engler, H., Liu, A., Machemer, T., Maneval, D., Philopena, J., Quijano, E., Pun, S. H., and Wen, S. Tumor-targeted nonviral gene delivery vehicle for systemic delivery. Manuscript in preparation. (32) Larson, R., Menard, V., Jasobs, H., and Kim, S. (2001) Physicochemical characterization of poly(ethylene glycol)modified anti-GAD antibodies. Bioconjugate Chem. 12, 8619. (33) Sehgal, D., and Vijay, I. (1994) A method for the high efficiency of water-soluble carbodiimide-mediated amidation. Anal. Biochem. 218, 87-91. (34) Hermanson, G. T. (1996) Bioconjugate Techniques, p 132, Academic Press, Rockford, IL. (35) Hermanson, G. T. (1996) Bioconjugate Techniques, pp 313315, Academic Press, Rockford, IL.

Bellocq et al. (36) Keer, H., Kozlowski, J., Tsai, Y., Lee, C., McEwan, R., and Grayhack, J. (1990) Elevated transferrin receptor content in human prostate cancer cell lines assessed in vitro and in vivo. J. Urol. 143, 381-385. (37) Xu, L., Pirollo, K. F., Tang, W.-H., Rait, A., and Chang, E. (1999) Transferrin-liposome-mediated systemic p53 gene therapy in combination wtih radiation results in regression of human head and neck cancer xenografts. Hum. Gene Ther. 10, 2941-2952. (38) Snider, J., Neville, C., Yuan, L.-C., and Bullock, J. (1992) Characterization of the heterogeneity of poly(ethylene glycol)modified superoxide dismutase by chromatographic and electrophoretic techniques. J. Chromatogr. 599, 141-155. (39) Seely, J. E., and Richey, C. W. (2001) Use of ion-exchange chromatography and hydrophobic interaction chromatography in the preparation and recovery of poly(ethylene glycol)linked proteins. J. Chromatogr. A 908, 235-241. (40) Klausner, R., VanRenswoude, J., Ashwell, G., Kempf, C., Schechter, A., Dean, A., and Bridges, K. (1983) Receptormediated Endocytosis of Transferrin in K562 Cells. J. Biol. Chem. 258, 4715-4724. (41) Morpurgo, M., Veronese, F., Kachensky, D., and Harris, J. (1996) Preparation and characterization of poly(ethylene glycol) vinyl sulfone. Bioconjugate Chem. 7, 363-8. (42) Barabas, K., and Faulk, W. (1993) Transferrin Receptors Associate with Drug Resistance in Cancer Cells. Biochem. Biophys. Res. Commun. 197, 702-708. (43) Pack, D., Putnam, D., and Langer, R. (2000) Design of imidazole-containing endosomolytic biopolymers for gene delivery. Biotechnol. Bioeng. 67, 217-23. (44) Boussif, O., Lezoualcl’h, F., Zanta, M., Mergny, M., Scherman, D., Demeneix, B., and Behr, J.-P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301. (45) Mislick, K., and Baldeschwieler, J. (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 93, 12349-12354. (46) Dash, P., Read, M., Fisher, K., Howard, K., Wolfert, M., Oupicky, D., Subr, V., Strohalm, J., Ulbrich, K., and Seymour, L. (2000) Decreased Binding to Proteins and Cells of Polymeric Gene Delivery Vectors Surface Modified with a Multivalent Hydrophilic Polymer and Retargeting through Attachment of Transferrin. J. Biol. Chem. 275, 3793-3802. (47) Fritzer, M., Szekeres, T., Szuts, V., Jarayam, H., and Goldenberg, H. (1996) Cytotoxic Effects of a DoxorubicinTransferrin Conjugate in Multidrug-Resistant KB Cells. Biochem. Pharm. 51, 489-493. (48) Wagner, E., Zenke, M., Cotten, M., Beug, H., and Birnstiel, M. L. (1990) Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. U.S.A. 87, 34103414. (49) Liang, K. W., Hoffman, E. P., and Huang, L. (2000) Targeted delivery of plasmid DNA to myogenic cells via transferrin-conjugated peptide nucleic acid. Mol. Ther. 1, 236-243. (50) Wagner, E., Cotten, M., Foisner, R., and Birnstiel, M. (1991) Transferrin-polycation-DNA complexes: The effect of polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. U.S.A. 88, 4255-4259.

BC034125F

Transferrin-Containing, Cyclodextrin Polymer-Based Particles for

Recommend Documents