PEGylated Dendrimers with Core Functionality for Biological

Jan 4, 2008 - of a Waters Alliance separation module 2695 and a Waters 410 differential RI detector. SEC was performed in a G2500 PWXL. (6 μm) and a ...
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Bioconjugate Chem. 2008, 19, 461–469

461

PEGylated Dendrimers with Core Functionality for Biological Applications Steven J. Guillaudeu,† Megan E. Fox,† Yarah M. Haidar,† Edward E. Dy,‡ Francis C. Szoka,‡ and Jean M. J. Fréchet*,† College of Chemistry, University of California, Berkeley, California 94720-1460, and Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446. Received July 14, 2007; Revised Manuscript Received November 29, 2007

The synthesis of a variety of core functionalized PEGylated polyester dendrimers and their in vitro and in vivo properties are described in this report. These water-soluble dendrimers have been designed to carry eight functional groups on their dendritic core for a variety of biological applications such as drug delivery and in vivo imaging as well as eight solubilizing groups. Using a common symmetrical aliphatic ester dendritic core and trifunctional amino acid moieties, a library of dendrimers with phenols, alkyl alcohols, alkynes, ketones, and carboxylic acid functionalities has been synthesized without the need for column chromatography. The amines were PEGylated, leaving the other functionality of the amino acid available for further manipulation such as the attachment of drugs and/or labels. Radiolabeling experiments with the PEGylated dendrimers showed that they had a long circulation half-life in mice, confirming the potential of this class of dendrimers for therapeutic and/or diagnostic applications. A carboxylic acid functionalized dendrimer was elaborated to carry doxorubicin bound via a hydrazone bond. The drug-loaded carrier accumulated more in tumors and less in healthy organs than the clinically used PEGylated liposomal formulation Doxil. The efficient synthesis, high versatility, and favorable biological properties make these PEGylated polyester dendrimers promising structures for therapeutic and/or imaging applications.

INTRODUCTION Dendritic polymers are currently being evaluated for many in vivo biological applications including drug delivery, tissue growth scaffolding, imaging (magnetic resonance, near-infrared, positron emission), and boron-neutron capture therapy (1–4). PEGylated dendrimers, in which a multifunctional dendritic core is conjugated to polyethylene glycol (PEG, or polyethylene oxide) chains, are an interesting subclass of dendritic polymers for these applications (5). Various PEG-dendrimer hybrids have shown lower toxicity and hemolytic properties, long blood circulation times, low organ accumulation, and high accumulation in tumor tissue due to the enhanced permeation and retention (EPR) effect (6–14). PEG-dendrimer hybrids can be constructed by two basic architectures: PEG (linear or branched) can be attached to the apex (10) (Figure 1a) or the peripheral groups (15) (Figure 1b and c) of dendrimers. In the former, the dendrimer periphery (if compatible with the solvent) is more exposed to the surrounding environment, while in the latter, a PEG shell is the most solvent accessible. From the reported works cited above, it appears that both show great promise for biotechnological applications, but the latter has shown the best performance with respect to long blood circulation half-lives and low organ accumulation (7–9, 12, 16). In our previous work exploring PEG-dendrimer hybrids (10, 12, 15–21), we have found that a nontoxic, biodegradable, asymmetric polyester dendrimer with eight PEG chains of 5000 Da molecular weight and multiple sites for drug attachment, a bowtie dendrimer, is efficient at delivering the chemotherapeutic agent doxorubicin (Dox) to C26 tumors in mice; this led to complete tumor regression in all of the treated mice with only a single dose (16). The effective tumor therapy with the bow* Corresponding author. Tel: (510)-643-3077. Fax: (510)-643-3079. E-mail: [email protected]. † University of California, Berkeley. ‡ University of California, San Francisco.

tie is believed to stem from the highly branched dendritic architecture that lowers the rate of filtration through renal membranes and the PEG-shell/drug-core structure that shields the therapeutic from recognition by the reticuloendothelial system (12, 16). Such a system, with a PEG shell and functional core, may prove useful in many of the above applications where a long circulation time, accumulation at leaky blood vessel sites such as in tumors, inflammation sites, or arterial injuries, and biocompatibility and biodegradability are desired. Dendrimers with a PEGylated periphery that have accessible functionality on the dendrimer have been produced by substoichiometrically PEGylating amine-terminated dendrimers such as PAMAM or polylysine (7–9, 22–28), by using elegant control of the reactivity on melamine dendrimers (6), through ether formation on bifunctional polyether dendrimers (18, 19), and by PEGylating an asymmetrically protected ester dendrimer (15). We now describe the high-yielding column-free synthesis of a family of PEGylated ester dendrimers bearing some analogy in terms of functionality and architecture to the bow-tie dendrimer. However, these new dendrimers are symmetric about the core, with different multifunctional handles easily incorporated at their periphery, thereby increasing their ease of preparation and versatility for potential biomedical applications. One generation of bis-2,2-hydroxymethylpropanoic acid (bisHMPA) building block was attached to a pentaerythritol core, followed by eight copies of a trifunctional amino acid molecule via EDCI-mediated coupling of their carboxylic acid functionality. Following deprotection, the terminal amines were PEGylated using a carbamate linkage, leaving the third functionality of the amino acid available for further elaboration. Because the amino acid-terminated dendrimers do not require column chromatography for purification, these compounds are more readily accessed than the functionally analogous bow-tie dendrimers we have successfully used in cancer chemotherapy. In this article, we demonstrate the versatility of this approach by presenting structures with ketones, alkynes, carboxylic acids, alkyl alcohols, and phenols present at the core of the macro-

10.1021/bc700264g CCC: $40.75  2008 American Chemical Society Published on Web 01/04/2008

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molecule. Because of our interest in the potential of these compounds for drug delivery to tumors, the stability of these PEGylated dendrimers in buffer and plasma was studied, and biodistributions of the radiolabeled phenolic dendrimer in nontumored mice and of the carboxylate dendrimer conjugated to Dox in tumored mice were determined.

MATERIAL AND METHODS Materials. Materials were used as obtained from commercial sources unless otherwise noted. Dimethylformamide (DMF), pyridine, and CH2Cl2 for syntheses were purged for 1 h with nitrogen and further dried by passing them through commercially available push stills (Glass Contour). Benzene was distilled from CaH2. Monomethoxy polyethyleneglycol p-nitrophenyl carbonate (PEO-PNP) (29) and 1 (30) were synthesized according to published procedures. Solvents were removed under reduced pressure using a rotary evaporator or by vacuum pump evacuation. Characterization. NMR spectra were recorded on Bruker AV 300, AVB 400, AVQ 400, or DRX 500 MHz instruments. Fourier transform infrared (FTIR) spectroscopic analyses were performed using a thin film cast from an appropriate solvent on a NaCl plate. Elemental analyses were performed at the UC Berkeley Mass Spectrometry Facility. MALDI-TOF MS was performed on a PerSeptive Biosystems Voyager-DE, generally using the following matrices: trans-3-indoleacrylic acid (IAA) for tert-butyloxycarbonyl (Boc)-protected dendrimers or 2,5dihyroxybenzoic acid (DHB) for amine-terminated dendrimers. Samples were prepared by diluting dendrimer solutions (∼1 M) 40-fold in 100 mM matrix solutions in tetrahydrofuran and spotting 0.5 µL on the sample plate. Size exclusion chromatography (SEC) system A consisted of a Waters 515 pump, a Waters 717 autosampler, a Waters 996 Photodiode Array detector (210–600 nm), and a Waters 2414 differential refractive index (RI) detector. SEC was performed at 1.0 mL/min in a PLgel Mixed B (10 µm) and a PLgel Mixed C (5 µm) column (Polymer Laboratories, both 300 × 7.5 mm), in that order, using DMF with 0.2% LiBr as the mobile phase and linear PEO (4,200–478,000 MW) as the calibration standards. The columns were kept at 70 °C. SEC system B consisted of the same equipment as that in system A and was performed at 1.0 mL/ min in two SDV Linear S (5 µm) columns (Polymer Standards Service, 300 × 8 mm) using DMF with 0.2% LiBr as the mobile phase. The columns were kept at 70 °C. SEC system C consisted of a Waters Alliance separation module 2695 and a Waters 410 differential RI detector. SEC was performed in a G2500 PWXL (6 µm) and a G3000 PWXL (6 µm) column (TosoHaas, both 300 × 7.8 mm), in that order, at ambient temperature using phosphate buffered saline (pH 7.4) as the mobile phase. Radioactivity from 125I was quantified with 1480 Wallac Wizard 3 Automatic Gamma Counter. Counts emitted from samples contained in screw-cap scintillation vials were measured over the course of 1 min and were recorded as counts per minute (cpm). Animal Experiments. All animal experiments were performed in compliance with the National Institutes of Health guidelines for animal research under a protocol approved by the Committee on Animal Research at University of California (San Francisco, CA) (UCSF). C-26 colon carcinoma cells obtained from the UCSF cell culture facility were cultured in RPMI medium 1640 containing 10% FBS. Nomenclature. A pentaerythritol core (PE) with one generation of bis-2,2-hydroxymethylpropanoic acid (G1) and serine protected as a benzyl ether for the side chain and with a Boc on the amine (Ser(Bn)Boc) is abbreviated PE-G1-(Ser(Bn)Boc)8 because there are eight serines. Standard nomenclature is used for other amino acids Xaa and their protecting groups (PG).

Guillaudeu et al.

The PEGylated versions are abbreviated as PE-G1-(Xaa(PG)PEO)8. In the title of this article, we term the final polymers core functionalized because the reactive functionalities used to attach the drugs and or imaging labels are located near the core of the macromolecules, which is surrounded and engulfed by long PEG linear chains. General Procedure for the Acylation of 1 with Amino Acids. Synthesis of PE-G1-(Ser(Bn)Boc)8 (3b). Compound 1 (201 mg, 0.334 mmol), L-Boc-Ser(Bn)-OH (946 mg, 3.20 mmol), and 4-dimethylaminopyridine (DMAP) (195 mg, 1.60 mmol) were dissolved in DMF (2.7 mL), and then 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDCI) (613.3 mg, 3.20 mmol) was added. After 4 h, the reaction was complete as determined by MALDI. The solution was diluted with diethyl ether (75 mL) and washed with 1 M NaHSO4 (3 × 50 mL), 1 M NaOH (3 × 50 mL), and water (2 × 50 mL). The organic layer was dried with MgSO4, and filtered, and the solvent was removed to yield a white solid (903 mg, quant). 1 H NMR (400 MHz, CDCl3): δ 1.16 (s, 12), 1.41 (s, 72), 3.64 (m, 8), 3.81 (m, 8), 4.01 (d, 4, J ) 11.1), 4.10 (d, 4, J ) 11.1), 4.19 (dd, 8, J ) 10.6, 11.2), 4.44 (m, 32), 5.47 (s, 8), 7.28 (m, 40). 13C NMR (100 MHz, CDCl3): δ 17.52, 28.36, 46.78, 53.97, 61.73, 65.30, 65.46, 69.91, 73.25, 79.87, 127.72, 127.80, 128.44, 137.59, 155.39, 170.14, 171.59. MALDI-TOF MS. [M + Na]+ (C145H196N8O48Na) Calcd: m/z ) 2840.3. Found: m/z ) 2839.9. Anal. (C145H196N8O48) C, H, N. PE-G1-(Tyr(Bn)Boc)8 (3a). Yield 92%. 1H NMR (400 MHz, CDCl3): δ 1.12 (s, 12), 1.35 (s, 72), 2.87 (m, 8), 3.04 (m, 8), 4.18–4.37 (m, 24), 4.51 (m, 8), 4.93 (s, 16), 5.16 (s, 8), 6.84 (d, 16, J ) 7.0), 7.04 (d, 16, J ) 7.0), 7.28–7.38 (m, 40). 13C NMR (100 MHz, CDCl3): δ 17.70, 28.36, 37.15, 46.68, 54.59, 65.61, 69.97, 70.65, 79.79, 115.00, 127.51, 127.92, 128.41, 128.56, 130.36, 137.12, 155.20, 157.93, 171.76. MALDI-TOF MS. [M + Na]+ (C193H228N8O48Na) Calcd: m/z ) 3449.6. Found: m/z ) 3446.3. Anal. (C193H228N8O48) C, H, N. PE-G1-(Asp(Bn)Boc)8 (3c). The synthesis was performed as described in the general procedure. Following the extractions, a small amount of benzyl alcohol was present; it was removed by two precipitations of the dendrimer from ether into excess hexanes. Because the product stuck to the glass surfaces as a white gummy material, the hexanes were decanted, and the residual solvent was removed under vacuum to yield a white solid (95%). 1H NMR (400 MHz, CDCl3): δ 1.19 (s, 12), 1.40 (s, 72), 2.88 (m, 16), 4.13–4.38 (m, 24), 4.61 (br s, 8), 5.09 (m, 16), 5.61 (s, 8), 7.31 (br s, 40). 13C NMR (100 MHz, CDCl3): δ 17.74, 28.32, 36.62, 43.01, 46.48, 50.02, 62.25, 65.71, 66.85, 79.98, 128.32, 128.57, 135.54, 155.31, 170.55, 170.78, 171.74. MALDI-TOF MS. [M + Na]+ (C153H196N8O56Na) Calcd: m/z ) 3064.3. Found: m/z ) 3065.1. Anal. (C153H196N8O56) C, H, N. PE-G1-(PropargylGlyBoc)8 (3d). 1H NMR (300 MHz, CDCl3): δ 1.31 (s, 12), 1.45 (s, 72), 2.14 (d, 8, J ) 2.5), 2.72 (s, 16), 4.13–4.48 (m, 28), 5.45 (d, 8, J ) 7.9). 13C NMR (100 MHz, CDCl3): δ 17.19, 22.58, 28.30, 46.62, 52.02, 62.10, 65.74, 72.15, 78.70, 80.20, 155.01, 170.25, 171.62. MALDI-TOF MS. [M + Na]+ (C105H148N8O40Na) Calcd: m/z ) 2185.4. Found: m/z ) 2183.6. Anal. (C105H148N8O40) C, H, N. PE-G1-((4-AcetylPhe)Boc)8 (3e). 1H NMR (300 MHz, CDCl3): δ 1.19 (s, 12), 1.35 (s, 72), 2.55 (d, 24, J ) 1.8), 3.01 (br s, 8), 3.19 (m, 8), 4.16–4.35 (m, 24), 4.59 (br s, 8), 5.30 (br s, 8), 7.25 (d unresolved from solvent, 16, J ) 7.5), 7.86 (d, 16, J ) 8.1). 13C NMR (100 MHz, CDCl3): δ 17.69, 28.28, 37.85, 46.55, 54.17, 61.93, 65.66, 77.26, 80.08, 128.65, 129.53, 135.97, 141.91, 155.15171.42, 171.76, 197.78. MALDI-TOF MS. [M + Na]+ (C153H196N8O48Na) Calcd: m/z ) 2936.3. Found: m/z ) 2934.3. Anal. (C153H196N8O48) C, H, N.

Core-Functional PEGylated Dendrimers

General Deprotection by Anhydrous HCl. PE-G1(Ser(Bn)NH3Cl)8 (b). Compound 3b (98.8 mg, 0.35 mmol) was dissolved in ethanol (0.5 mL) and cooled to 0 °C. Acetyl chloride (0.4 mL) was added dropwise over 2 min. After 30 min, the reaction was complete as determined by MALDI (IAA). The solvents and remaining acetyl chloride were removed by rotary evaporation, and the white solid product was dried under high vacuum. General Deprotection by Trifluoroacetic Acid. PE-G1(Asp(Bn)NH3TFA)8 (c). Compound 3c (251.2 mg, 82.6 µmol) was dissolved in a 1:1 mixture of trifluoroacetic acid (TFA) and CH2Cl2. After 30 min, deprotection was complete as determined by MALDI (DHB). The solvents were removed under vacuum. General PEGylation Reaction. PE-G1-(Asp(Bn)PEO)8 (4c). PEO-PNP (3.678 g, 700 µmol), DMAP (38 mg, 310 µmol), N,N-diisopropylethylamine (520 µL, 3.0 mmol), pyridine (2.5 mL), and benzene (5 mL) were added to deprotected 3b (described above, 82.6 µmol). After 3 days, piperidine (600 µL, 6.1 mmol) was added to quench excess PEO-PNP, and the reaction was stirred for an additional 16 h. The solution was diluted with benzene (25 mL) and the product was precipitated into ether (600 mL). The white solid was collected by filtration, rinsed with ether, and dialyzed against water in 3500 molecular weight cutoff (MWCO) regenerated cellulose membranes (Spectra-Por) for 18 h. The retained water solution was lyophilized to yield a white powder (3.15 g). 1H NMR (400 MHz, CDCl3): δ 1.13 (br s, 12), 2.9 (m, 16), 3.38 (s, 24), 3.65 (br m, ∼4300), 4.2–4.4 (br m, 42), 4.62 (br s, 8), 5.07 (m, 16), 5.9 (br s, ∼6), 7.31 (m, unresolved from solvent, ∼40). PE-G1-(Tyr(Bn)PEO)8 (4a). 1H NMR (500 MHz, CDCl3): δ 1.16 (s, 12), 2.88 (br s, 8), 3.03 (br s, 8), 3.38 (s, 24), 3.65 (br m, ∼4380), 4.0–4.4 (br m, 42), 4.53 (br s, 8), 4.93 (s, 16), 5.45 (br s, ∼6), 6.84 (d, 16, J ) 6.5), 7.02 (d, 16, J ) 7.6), 7.2–7.4 (m, unresolved from solvent, ∼40). PE-G1-(Ser(Bn)PEO)8 (4b). 1H NMR (500 MHz, CDCl3): δ 1.13 (s, 12), 3.38 (s, 24), 3.65 (br m, ∼3900), 4.1–4.5 (br m, 55), 5.74 (br s, ∼5), 7.20–7.35 (br m, unresolved from solvent, ∼40). PE-G1-(PropargylGlyPEO)8 (4d). 1H NMR (500 MHz, CDCl3): δ 1.30 (s, 12), 2.15 (s, unresolved from residual water, ∼8), 2.74 (s, 16), 3.38 (s, 24), 3.65 (br m, ∼4500), 4.1–4.6 (br m, 38), 5.78 (br s, ∼4). PE-G1-((4-AcetylPhe)PEO)8 (4e). 1H NMR (500 MHz, CDCl3): δ 1.14 (s, 12), 2.54 (s, 24), 3.02 (br s, 8), 3.17 (br s, 8), 3.38 (s, 24), 3.65 (br m, ∼4100), 4.0–4.3 (br m, 42), 4.6 (br s, 8), 5.66 (br s, ∼5), 7.28 (m, unresolved from solvent), 7.86 (br s, 16). PE-G1-(AspPEO)8 (5). Compound 4c (96.4 mg) was deprotected in CH3OH (5 mL) by hydrogenolysis using Pd/C (10 wt%, 10 mg) at 1 atm for 16 h. The solution was filtered through celite, and the solvent was removed under reduced pressure. 1 H NMR (500 MHz, CDCl3): δ 1.27 (s, 12), 2.80 (br s, 8), 2.97 (br s, 8), 3.38 (s, 24), 3.64 (br m, ∼3600), 4.0–4.4 (br m, 42), 4.62 (br s, 8), 6.22 (br s, ∼4). N′-(4-Hydroxymethyl-benzyl)-hydrazinecarboxylic Acid tert-Butyl Ester (6). 4-(Hydroxymethyl) benzoic acid (0.760 g, 5 mmol), HOBt (0.341 g, 2.5 mmol, 0.5 equiv), and tertbutyl carbazate (0.925 g, 7 mmol, 1.4 equiv) were suspended in CH2Cl2 (25 mL) at 0 °C. EDCI (1.06 g, 5.5 mmol, 1.1 equiv) was added, and the mixture was stirred for 24 h. The mixture became almost homogeneous after a few hours, and the product later precipitated. The white solid was filtered, rinsed with CH2Cl2 (3 × 5 mL), and dried (790 mg, 59%). 1H NMR (MeOD-d3): δ 1.49 (s, 9), 4.66 (s, 2), 7.45 (d, 2, J ) 8), 7.83 (d, 2, J ) 8). 13C NMR (MeOD-d3): δ 27.34, 63.19, 80.63, 126.40, 127.37, 131.00, 146.02, 156.63, 168.34. IR (cm-1):

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3726.8, 1720.2, 1664.1, 860.3. mp ) 159–160 °C (dec.). Calcd: [M + H]+ (C13H19N2O4) m/z ) 267.1345. Found: FABHR MS: [M + H]+ m/z ) 267.1350. Anal. (C13H18N2O4) C, H, N. PE-G1-(Asp(BnCONNBoc)PEO)8 (7). Coumpound 5 (200 mg, 38 µmol COOH) was dissolved in DMF (0.75 mL). Coumpound 6 (300 mg, 1.13 mmol), N,N′-dicyclohexylcarbodiimide (245 mg, 1.19 mmol), and DMAP p-toluenesulfonate salt (13 mg, 44 µmol) were added. After 24 h, the solution was diluted with water (10 mL), filtered through celite, and dialyzed against water in 3500 MWCO membranes for 18 h. The retained water solution was lyophilized to yield a white powder (172 mg). By 1H NMR, comparing the OMe peak at δ 3.38 to the Boc peak at δ 1.48, it was determined that ca. 50% of the COOH groups reacted with the linker. 1H NMR (400 MHz, CDCl3): δ 1.11 (s, 12), 1.49 (s, 37), 2.34 (unresolved from residual water in solvent), 2.95 (br m, ∼12), 3.38 (s, 24), 3.65 (br m, ∼3100), 4.0–4.4 (br m, 25), 4.63 (br s, 3), 4.75 (s, 1), 5.11 (br s, ∼5), 6.1 (br s, ∼2), 7.81 (br m, ∼8). PE-G1-(Asp(BnCONNDox)PEO)8 (8). Coumpound 7 (15.2 mg, 1.5 µmol NNBoc) was dissolved in 1:1 TFA/CH2Cl2 for 2 h. The solvent was removed under a stream of N2. Residual TFA was removed by dissolving the solid in CH3OH and evaporating it under a stream of N2. The polymer was then dissolved in CH3OH (1 mL) and Dox (4.5 mg, 7.8 µmol) was added. The reaction was stirred at 60 °C in the dark for 16 h. The polymer was eluted on an LH-20 column with CH3OH. The first dark red band (high MW material) was collected, the solvent was removed by evaporation under a stream of N2, and the product was further purified on a PD-10 column using water as the eluent. The collected dark red fraction was lyophilized to yield a red powder (10 mg). The loading of Dox was determined by the absorbance at 486 nm (ε ) 11,500) (31) to be 2.9 wt %, or ∼50% of the maximum theoretical loading. PE-G1-TyrPEO8 (9a). Compound 4a (195 mg) was dissolved in a mixture of TFA (2 mL) and thioanisole (0.25 mL). After 16 h, TFA was removed under reduced pressure, and NEt3 (0.5 mL) was added. The solution was diluted with CH3OH (6 mL) and dialyzed 24 h against CH3OH in a 3500 MWCO regenerated cellulose membrane. After removing the retentate solvent under reduced pressure, the product was purified on a PD-10 column and lyophilized (yield: 135 mg, 69%). 1H NMR (400 MHz, CDCl3): δ 1.03 (s, 12), 2.95 (br s, 16), 3.38 (s, 24), 3.65 (br m, ∼4350), 3.9–4.3 (br m, 34), 4.49 (br s, 8), 5.55 (br s, ∼6), 6.76 (br s, 16), 6.94 (br s, 16), 7.52 (br s, ∼6). Polymer Aqueous Buffer Hydrolysis Study. Compound 4b was dissolved at a concentration of 1 mg/mL in a solution of PBS, pH 7.4, with 30 mM NaNO3. The polymer solutions were incubated at 37 °C, and samples were taken periodically over a period of 20 days. Samples were frozen at -80 °C until they could be prepared for molecular weight determination by SEC. Samples were prepared for SEC (system B) by running solutions through PD-10 desalting columns in distilled water, followed by lyophilization. Polymer Plasma Degradation Study. A stock polymer solution of 4a in PBS with 30 mM NaNO3, pH 7.4, was made at a concentration of 20 mg polymer/mL. Lyophilized human plasma (Sigma) was reconstituted in PBS, and the pH was adjusted to 7.4 using 1 M HEPES containing 9 g/L NaCl and 13.5 g/L NaOH. The choice of buffer is important to prevent a change in plasma pH over the course of the experiment. For each time point, 40 µL of polymer solution was added to 160 µL of preheated human plasma in a 1.5 mL centrifuge vial and vortexed briefly. Samples were incubated at 37 °C. At the desired time point, 200 µL of a 1:1 methanol/water with 2 wt % ZnSO4 solution was added to the vial to precipitate the proteins. The sample was briefly vortexed, centrifuged at 14,100 rcf for 5 min, then frozen at -80 °C until it could be prepared

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Figure 1. Schematic representations of the PEG-dendrimer hybrid structures. PEG can be attached (a) at the dendrimer apex or the periphery of (b) symmetric or (c) asymmetric bow-tie dendrimers.

for molecular weight determination by SEC. Individual samples were prepared for SEC (system C) by filtering the supernatant through 0.2 µm pore size PTFE syringe filters. Radioiodination of the Free Phenols on Tyrosine (Tyr)Functionalized PE Dendrimers (9b). For tracing of the dendrimer in biodistribution studies, the free phenols of 9a were radiolabeled with 125I using a published procedure (32). Briefly, 9a (3 mg, 75 pmol) was dissolved in 0.49 mL of boric acid buffer (0.1 M, pH 8.5). A solution of potassium iodide in water (1 µM) was added (136 µL), followed by chloramine T in boric acid buffer (1 µM, 272 µL). The mixture was allowed to react for 15 min, then sodium bisulfite solution (50 µM, 6.8 µL) was added to stop the reaction. The material was purified on a PD10 column and lyophilized overnight to yield 3 mg of iodinated polymer. The polymer was iodinated at 1 µCi per PEGylated dendrimer. A solution of iodinated polymer was combined with a solution of non-iodinated polymer, resulting in a final solution of 5 mg polymer/mL and with an activity of 14.2 µCi/mL. Biodistribution Studies in Nontumored Mice. Radioiodinated polymer solutions (200 µL, ∼6.5 million cpm/mouse) were injected intravenously in the tail vein of 6–8 week old female CD-1 mice. The mice were sacrificed at three different times following injection for biodistribution analyses: 30, 540, and 2880 min postinjection (three mice per experimental group). The blood (collected by heart puncture), heart, lungs, liver, stomach, spleen, intestines, kidneys, and carcass (divided into three portions) were collected for analyses. Each were weighed, and the amount of radioactivity present in each organ was quantified. For the 2880 min time point, the mice were housed in metabolic cages and the urine and feces collected. Additionally, retro-orbital (RO) bleeds were taken at four different time points to construct a blood concentration curve. A 10 min RO bleed was taken from the 30 min sacrifice mice, 60 and 1440 min RO were taken from the 2880 min sacrifice mice, and a 180 min RO bleed was taken from the 540 min sacrifice mice. All samples were weighed and the amount of radioactivity present quantified. Urine was analyzed by running 1 mL of urine from each polymer biodistribution through a G-50 Sephadex column (column volume ∼10 mL). Fractions were collected and analyzed for radioactivity to determine whether radioactivity in the urine was polymer-associated or low MW-associated. A set of PEO polymers with known MW polymers was also run on the G-50 column for comparison. The % injected dose per gram (%ID/g) of blood versus time curve was analyzed using a two compartment model described by eq 1. The two compartment model (33) was chosen as the blood concentration curve showed two distinct rates of decay. The pharmacokinetic parameters A, R, B, and β for the twocompartment model were estimated using the residuals model. All pharmacokinetic quantities were calculated from these parameters.

Cp ) Ae-Rt + Be-βt

(1)

Biodistribution Studies in Tumored Mice. Six to eight week old female Balb/C mice were injected in the right rear flank with C26 colon carcinoma cells by subcutaneous administration (3 × 105 cells in 50 µL media). Tumors were allowed to grow 13 days. Mice were injected intravenously in the tail vein with one of the following solutions in PBS: 8 (8 mg Dox equiv/kg), DOXIL (8 mg Dox equiv/kg), or free Dox (8 mg/kg) (three mice per experimental group). Two control mice were also injected with 200 µL of PBS. The mice were sacrificed 2880 min postinjection. The blood (collected by heart puncture), heart, lungs, liver, spleen, kidney, muscle, and tumor were collected for analyses. Each organ was weighed, and 200–300 mg of the collected organs were combined with ∼0.6 mL zirconia beads and 1 mL of acidified isopropyl alcohol (0.075 M HCl, 90% IPA) in 2.0 mL centrifuge tubes. The samples were homogenized for 30 s and incubated at 4 °C for 24 h. Samples were then frozen at -80 °C in a freezer until quantification of Dox content by fluorescence measurements could be made. At measurement time, samples were thawed and centrifuged for 5 min at 14,000 rpm (15,400g). Then, 80 µL of supernatant was combined with 920 µL of acidified IPA for fluorescence measurements. Dox fluorescence (ex 485 nm, em 595 nm) was measured on a Spex Fluorolog fluorimeter. Calibration curves were made from organ samples collected from the control mice.

RESULTS AND DISCUSSION Synthesis of the PEGylated Dendrimers. The synthesis of the core functional PEGylated dendrimers is straightforward and nearly quantitative at all dendronization steps (Scheme 1). The first generation pentaerythritol bisHMPA dendrimer 1 (PE-G1OH8) was synthesized as previously described (30). This molecule was chosen as a core because a large number of radially emanating functional groups could be attained in a minimum number of steps on a large scale, and the biodegradable ester backbone is desirable for biological applications. Attachment of the amino acids 2a-e using DMAP catalyzed EDCI coupling provided analytically pure dendrimers (3), requiring only an extractive workup for most versions. None of the dendrimers required column chromatography for analytically pure material, thus facilitating scale up. The reaction was monitored by MALDI-TOF MS to detect incomplete couplings (Figure 2a for 3c). In all cases, the reaction went to completion, normally within 4 h. Occasionally, loss of either a single tertbutyl group or a number of Boc groups was seen in the MALDITOF spectrum, but the peaks associated with a loss of 8 Boc groups, however, were higher than those associated with 7 or 6, indicating that this was an artifact of the ionization process because of the use of an acidic matrix. In the workup of 3c, some benzyl alcohol was produced, likely due to the ester

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Scheme 1. Synthesis of PEGylated Ester Dendrimers with Core Functionalitya

a

(i) EDC, DMAP, DMF; (ii) TFA/DCM or AcCl/EtOH; (iii) PEO-PNP, DMAP, Pyr, PhH, DIPEA.

Figure 3. Size exclusion chromatogram (SEC) of 4c and residual linear PEG in DMF solvent.

Figure 2. MALDI-TOF spectra of (a) 3c and (b) 3c after Boc deprotection. Note that the deprotected product was isolated as the octa(trifluoroacetate) salt.

hydrolysis of some of the water-soluble starting amino acid. This was removed by precipitation of the material into hexanes. These dendrimers are soluble in a variety of common organic solvents (CH2Cl2, CHCl3, CH3OH, EtOAc, etc.). Dialysis in 3500 molecular weight cutoff regenerated cellulose membranes against methanol is an alternative purification procedure to extractive workups, generally producing yields greater than 80%. All of the amino acids attached are commercially available, although the unnatural amino acids are more expensive and may not be readily available on a large scale. By using this variety of amino acids, PEGylated dendrimers ready for radiolabeling, esterification, amidation (with limitations, vida infra), click

reactions, Pd coupling, and hydrazone/oxime formation are easily accessible. Deprotection of the amine-protecting Boc groups could be monitored by MALDI-TOF MS (Figure 2b for 3c). Products were isolated by the removal of the solvent and used in the next step without further purification. In the MALDI-TOF spectrum of 3a, the loss of a single benzyl group was seen, but this was a minor event that did not interfere with the PEGylation reaction. Monomethoxy-PEO-p-nitrophenyl carbonate (PEOPNP, 5000 MW) (29) was used in ∼5% excess to fully cover the amine periphery of the dendritic core. After 3 days, size exclusion chromatography in DMF showed a peak around 38,000 Da, and another at 6,000 Da corresponding to the excess PEO-PNP (Figure 3 for 4c). A ninhydrin test indicated that all primary amines were reacted in each case. For applications where excess PEO-PNP could be problematic, the addition of a slight excess of piperidine to the crude reaction mixture, followed by 12 h of stirring, effectively capped the remaining activated 5000 Da PEO. At this stage, the products could be purified from small molecules by dialysis in water. We have shown previously (15) that excess linear PEG can be removed from the PEGylated dendrimer by careful dialysis, but in this work, no such purification was attempted as it leads to a significant lowering of the yield of the PEGylated dendrimer, and the small amount of lower molecular weight linear PEG impurity should not alter the in vivo characteristics of the PEGylated dendrimers.

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Guillaudeu et al.

Scheme 2. Linker Synthesis, Attachment, and Dox Loadinga

a

(i) H2, Pd/C, ME OH; (ii) EDCl, HOBt, DCM; (iii) DMF, DCC, DPTS; (iv) TFA, DCM, then MeOH, Dox, 60 °C.

Dox Loading of the PEGylated Dendrimers. To demonstrate that the dendritic cores are accessible for further chemical manipulation, 4c was elaborated to contain Dox conjugated through a hydrazone bond (Scheme 2). This linkage shows promise for releasing Dox in its biologically active form after endocytosis but is stable at physiological pH 7.4 (34–37). Although the most straightforward route to a hydrazidecontaining polymer would be attachment of tert-butylcarbazate directly to the aspartate moiety, we found that the R-amido ester bonds were not completely stable in the presence of excess amine and some degradation of the dendrimer occurred. It should be noted that piperidine, the secondary amine used to deactivate excess PEO-PNP, does not cause ester degradation.

Similar results were seen when deprotecting the ε-Cbz of a lysine version of the dendrimer (data not shown). No further attempts were made to optimize the conditions to amidate with primary amines. To circumvent this degradation by a nucleophilic amine, hydroxyl-hydrazide linker 6 was synthesized and, following hydrogenolysis of the benzyl ester-protecting group of 4c to afford 5, was attached to the dendrimer. A comparison of the Boc peak at 1.48 ppm against the OMe of the PEG chains at 3.38 ppm in the 1H NMR spectrum indicated that roughly 50% coverage (4 molecules of 6 attached to 7) is achieved under these conditions. Such coverage is sufficient for the intended application.

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Figure 4. SEC of 8 in DMF. The delay between the UV (abs 584 nm) and RI traces is due to the sequential placement of the detectors.

Deprotection of the hydrazide Boc group followed by hydrazone formation with Dox in methanol afforded polymer 8 containing 2.9 wt % Dox. This value is 50% of the theoretical loading, consistent with previous work in our laboratories (16). Such partial loading may result from the presence of residual trifluoroacetic acid in the reaction mixture from the deprotection step, which lowers the pH of the solution below the ideal level for hydrazone formation. Optimization of the reaction conditions for linker attachment and drug conjugation should result in significantly higher (∼4-fold) loadings. Although some linear PEG was still present in the reaction mixture, the absorbance at 584 nm (in 0.2% LiBr in DMF) in the size exclusion chromatogram shows that, as expected, Dox was only attached to the PEGylated dendrimer and not to free PEG (Figure 4). The refractive index (RI) trace of the drug-loaded carrier (Figure 4) appeared different from that of the initial PEGylated dendrimer (Figure 3) and all the intermediates in terms of the amount of excess linear PEG. Initially, when the carboxylate groups were protected, ca. 5% excess PEG was seen, as expected. As the core was deprotected, linker added, and drug added, the ratio of the PEGylated dendrimer peak area to the linear PEG area decreased. The peaks were eluted at the same retention time for each, indicating that the molecular weight did not change and that degradation had not occurred. Therefore, we believe this was an artifact related to the changing refractive index of the material and that the amount of residual free PEG did not actually change. The drug-loaded material 8 was highly water soluble. In Vitro Biodegradability of the PEGylated Dendrimers. While prior work has shown that PEGylated ester dendrimers can be hydrolyzed in aqueous solutions (12), we carried out experiments to determine if these PEGylated dendrimers could be degraded by enzymes since it has been suggested that PEGylation can inhibit access of enzymes and physiological fluids to a dendritic core (38). The properties of 4a in biologically relevant situations was evaluated by tracking its stability in pH 7.4 buffer and in human plasma at 37 °C. As expected, over the course of 20 days in buffer, the PEG chains were hydrolyzed leading to a mixture of molecules with lower overall molecular weight and higher polydispersity than that of the starting 4a. However, a significant portion of the material was still of higher molecular weight, suggesting that it had remained intact. Initial studies in plasma reconstituted using distilled water, as instructed by the manufacturer, showed that complete degradation of the ester dendrimer took place within only 48 h, leading to a product in which the largest molecules appeared to be 5 kDa PEG chains. However, it was found that the reconstituted plasma had a pH of ∼9, and thus, the high level of ester degradation seen was likely a result of the basic

Figure 5. (a) Blood circulation profile of radiolabeled 9b given as the average percent ID of polymer in the blood over time (three mice per time point). The line represents the fit curve resulting from the calculated pharmacokinetic parameters detailed in Table 1. (b) Biodistribution of radiolabeled 9b in nontumored mice taken at three time points: 30, 540, and 2880 min postinjection (three mice per time point). (c) Fortyeight hour biodistribution, given as the average %ID Dox/g tissue, of 8 in mice tumored subcutaneously in the right flank with C26 colon carcinoma. DOXIL and Dox HCl were run as controls (three mice per treatment group).

pH and not enzymatic degradation. After careful adjustment of the pH to 7.4 using a nonvolatile buffer such as HEPES buffer, the plasma samples showed degradation profiles essentially identical to those in PBS, pH 7.4. Therefore, this preliminary work does not shed light on the accessibility of the polyester dendritic core itself to enzymes. Biodistribution Studies of Radiolabeled Polymer. To follow the fate of the PEGylated dendrimer in vivo, the phenolic groups of 4a were deprotected and radiolabeled with 125I (compound 9b, not shown). Six to eight week old female Balb/C mice were injected intravenously with polymer solutions, and the distribution of the radiolabel in tissues was followed over time. The blood concentration profile (Figure 5a), given as the

468 Bioconjugate Chem., Vol. 19, No. 2, 2008 Table 1. Pharmacokinetic Data for Radiolabeled 9b t1/2,R (h) t1/2,β (h) V1 (g blood) AUC0f∞ (%ID · h/g) k21 (L/min) k12 (L/min) kel (L/min)

0.32 ( 0.11 23.8 ( 2.5 1.94 ( 0.07 1,200 ( 100 0.024 ( 0.012 0.01 ( 0.02 0.0007 ( 0.0004

average %ID of radioactivity, shows >16%ID remained in the blood after 48 h. A two-compartment pharmacokinetic model (33) was fitted to the data, and from this the pharmacokinetic parameters were determined. In particular, the blood circulation half-life t1/2β was found to be 23.8 ( 2.5 h (Table 1), and the area under the curve, AUC0f∞, was determined to be 1,200 ( 100%ID · h/g blood. The long circulation half-life and large AUC0f∞ are indicative of high levels of polymer in the blood and increased opportunity for accumulation in a tumor due to the EPR effect. This is consistent with previously reported results for PEGylated bow-tie dendrimers of similar molecular weight (12) and is more favorable than results obtained with linear 50 kDa PEO (39). The biodistribution study of 9b (Figure 5b) showed low levels of polymer in all vital organs after 48 h, less than 5%ID/g tissue. However, despite the long blood circulation half-life, 28.5%ID and 5.0%ID were found in the urine and feces, respectively, after 48 h. To determine whether this radioactivity was polymerassociated (>5,000 Da) or low MW-associated (