Bifunctional PAMAM Dendrimer Conjugates of Folic Acid and

Feb 24, 2012 - Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) ...
0 downloads 0 Views 475KB Size
Download PDF
Article pubs.acs.org/Biomac

Bifunctional PAMAM Dendrimer Conjugates of Folic Acid and Methotrexate with Defined Ratio Hong Zong,*,‡,† Thommey P. Thomas,‡,† Kyung-Hoon Lee,§ Ankur M. Desai,‡ Ming-hsin Li,‡ Alina Kotlyar,‡ Yuehua Zhang,‡ Pascale R. Leroueil,‡ Jeremy J. Gam,‡ Mark M. Banaszak Holl,§ and James R. Baker, Jr.*,‡ ‡

Michigan Nanotechnology Institute for Medicine and Biological Sciences and §Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) dendrimers. In those studies we conjugated the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) sequentially. This complex macromolecule was shown to selectively bind and kill KB tumor cells that overexpress folate receptor (FR) in vitro and in vivo. However, the multistep conjugation strategy employed in the synthesis of the molecule resulted in heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX. This led to inconsistent and sometimes biologically inactive batches of molecules, especially during large-scale synthesis. We here resolved this issue by using a novel triazine scaffold approach that reduces the number of dendrimer conjugation steps required and allows for the synthesis of G5 conjugates with defined ratios of FA and MTX. Although an unoccupied γ-glutamyl carboxylate of FA has been previously suggested to be nonessential for FR binding, the functional requirement of an open α-carboxylate still remains unclear. In an attempt to also address this question, we have synthesized isomeric FA dendrimer conjugates (α-carboxyl or γ-carboxyl linked). Competitive binding studies revealed that both linkages have virtually identical affinity toward FR on KB cells. Our studies show that a novel bifunctional triazine-based conjugate G5-Triazine-γMTX-αFA with identical numbers of FA and MTX binds to FR through a polyvalent interaction and induces cytotoxicity in KB cells through FR-mediated cellular internalization, inducing higher toxicity as compared to conjugates synthesized by the multistep strategy. This work serves as a proof of concept for the development of bifunctional dendrimer conjugates that require a defined ratio of two functional molecules.

■

INTRODUCTION Current cytotoxic cancer chemotherapeutics often have a low therapeutic index and, thus, are accompanied by deleterious side effects. Substantial effort has been focused on the development of drug delivery systems to enhance the therapeutic index of chemotherapeutic drugs.1,2 Conjugation of ligands to macromolecular carriers3 offers a mechanism to enhance the solubility of hydrophobic therapeutics, prolong blood circulation time, minimize nonspecific uptake, improve intracellular penetration and allow for site-specific targeting via both passive4−7 and active targeting methods.8,9 For over a decade, nanoparticle-based therapeutics have been studied as tumor-specific therapeutics and diagnostic agents.10,11 Despite a significant number of small-animal studies documenting the biological activity of a variety of cancer nanoparticle-based therapeutics, the number of devices tested at the clinical triallevel remains depressingly low.10 One reason for this is the inherent difficulty in producing consistent and homogeneous batches of multifunctional molecules. Although complex, multistep synthetic approaches can often be employed when © 2012 American Chemical Society

synthesizing small scale batches of material. However, any problems with consistency of these processes are exacerbated during scale-up to the point that the quantity of material needed to be clinically viable cannot be produced. Dendritic macromolecules have been extensively utilized as drug delivery platforms by virtue of their well-defined structure, size and shape, low toxicity and immunogenicity, and the possession of a large number of reactive surface functional groups.11−13 We and others have utilized the polyamidoamine (PAMAM) dendrimers as a platform for functionalizing different types of biological molecules for the targeted delivery in different types of cancer models.14−16 Importantly, our studies have shown in vitro and in vivo receptor-mediated cellular internalization and improved chemotherapeutic activity for a generation 5 (G5) PAMAM dendrimer onto which multiple folic acids (mFA) and antifolate drug methotrexates Received: November 20, 2011 Revised: February 21, 2012 Published: February 24, 2012 982

dx.doi.org/10.1021/bm201639c | Biomacromolecules 2012, 13, 982−991

Biomacromolecules

Article

Chart 1. Structures of PAMAM Dendrimer Conjugates 15−18

(nMTX) (G5-FAm-MTXn) were conjugated.17−19 The synthetic strategy we used for the production of the G5-FAm-MTXn included a multistep, sequential dendrimer surface modification that required: (1) partial acetylation of the surface amino groups, (2) coupling of FA through amide linkage, (3) glycidolation of the remaining free amino groups, and (4) conjugation of MTX through ester linkage.17 Despite this complex synthesis, we were successful at reproducing smallscale (mg to gram scale) batches of material that showed consistent in vitro and in vivo efficacy. However, when we attempted to scale up to hundreds of grams or kilogram scale batches necessary for clinical trial studies, we found that the material was significantly different from the small-scale batches. Indeed, it had markedly less cytotoxic activity in vitro and had insignificant antitumor activity in vivo in FRα-bearing mice tumor models when compared to the small-batch material. We recently showed that our sequential strategy for synthesizing multifunctional dendrimer conjugates resulted in polydispersity that could limit its use.19−21 For example, based on HPLC analytical data and by theoretical extrapolation of the distribution of a FA and MTX modified G5 dendrimer, it is possible that less than 5% of the material would contain the exact mean numbers of desired FA and MTX (4 and 5, respectively).21 Because both FA and MTX attached to the dendrimer in this manner can bind dihydrofolate reductase (DHFR),14 (and possibly other enzymes involved in folate metabolism), the presence of G5-FAm-MTXn populations with variability in the ratios of the two molecules greatly alters the consistency and biological activity of each batch of material. In addition, it is possible that other MTX-inhibiting enzymes, such as thymidylate synthase, could play a role in the therapeutic action of the conjugated FA and MTX. Thus, the synthesis of

dendrimers with defined and uniform ratios of FA and MTX is crucial in obtaining consistent and biological reproducible activity. A simple approach for coupling FA or MTX to a PAMAM dendrimer is through the nonselective activation of the carboxyl groups of the glutamic acid moiety in either small molecule by carbodiimide.17 Unfortunately, this method leads to the formation of two structural isomers because both FA and MTX can be linked through either the α- or γ-carboxyl group. Although it has been reported that FA linked via the γ-carboxyl group retains a stronger affinity toward its receptor, there is still controversy surrounding whether an unoccupied α-carboxyl group is essential for receptor binding.14,22−25 In the case of MTX, a free α-carboxyl group is essential for retaining MTX’s binding to target enzymes such as DHFR, whereas chemical modifications of the γ-carboxyl group have a lesser impact on functional activity.26,27 Also, the availability of both α- and γcarboxyl groups on MTX or FA could result in reactions with amino groups on the dendrimer surface that lead to dendrimer dimers or inactive conjugates. Finally, it is unclear whether the distribution of ligand regiochemistry on the dendrimer also affects the polydispersity of a conjugate, potentially changing activity due to less- or nonactive conjugates and contributing to its “functional” polydispersity. To synthetically define the ratios of FA to MTX and reduce polydispersity, we have chosen to use a trivalent triazine molecule as a linker scaffold. This small molecule has been previously utilized as a backbone for synthesizing dendrimer structures and conjugates28−31 and for conjugation of MTX to iron nanoparticles.32 The ease of displacement of chlorine atoms in 2,4,6-trichloro-1,3,5-triazine by various nucleophiles in a controlled manner makes this reagent useful for making 983

dx.doi.org/10.1021/bm201639c | Biomacromolecules 2012, 13, 982−991

Biomacromolecules

Article

3.22−3.65 (m, 21H), 4.62 (m, 1H), 5.40 (m, 1H), 5.88 (m, 1H), 6.43 (m, 1H); ESI-MS m/z, 485.2 (M + H+); calcd for C19H37N10O5, 485.3. Synthesis of 2-(4-(2-(2-Aminoethoxy)ethylamino)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethanol (Triazine-N3-OH-NH2; 4). Triazine-N3-OH-NHBoc 3 (485 mg, 1.0 mmol) was dissolved in CH2Cl2 (10 mL). TFA (2 mL) was added and the mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and the residue was dissolved in H2O (20 mL) and filtered off. NaOH (1 N) was added dropwise to the filtrate until the solution became basic. CH2Cl2 (20 mL) was added, and the organic layer was washed with brine and H2O, dried over Na2SO4, filtered, and evaporated under reduced pressure to yield 4 as a white solid (358 mg, 93%). The product was sufficiently pure for further reactions: ESI-MS m/z, 385.2 (M + H+); calcd for C14H29N10O3, 385.2. Synthesis of 2-(4-(2-(2-Aminoethoxy)ethylFA)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethanol [Triazine-N3-OH-αFA (5) and Triazine-N3-OH-γFA (6)]. FA (195 mg, 0.44 mmol) in DMSO (10 mL) was added to a solution of 4 (170 mg, 0.44 mmol) and N,N′dicyclohexylcarbodiimide (DCC; 182 mg, 0.88 mmol) in DMSO (5 mL). The reaction was stirred at room temperature for 24 h. The reaction mixture was filtered and H2O (30 mL) was added to the filtrate. The resulting precipitate was filtered, washed with H2O, methanol, and acetone, and dried under reduced pressure. The cruder product was further purified by semiprep HPLC (see Semiprep reverse phase high performance liquid chromatography (HPLC)). Two isomers 5 and 6 were isolated and purified as yellow solids, 5 (94 mg 26%) and 6 (53 mg, 15%): 1H NMR (500 MHz, DMSO-d6) 5 δ 1.23 (m, 1H), 1.77 (m, 2H), 1.85 (m, 1H), 1.96 (m, 1H), 2.26 (m, 2H), 3.49 (m, 25H), 4.35 (m, 1H), 4.50 (s, 1H), 6.63 (d, J = 8.5 Hz, 2H), 7.28 (m, 2H), 7.65 (d, J = 8.5 Hz, 2H), 7.87−7.98 (m, 2H), 8.10 (m, 1H), 8.27 (m, 1H), 8.66 (s, 1H); 6 δ 1.23 (m, 1H), 1.76 (m, 2H), 1.85 (m, 1H), 2.04 (m, 1H), 2.19 (m, 2H), 3.17 (t, J = 6.0 Hz, 1H), 3.35−3.61 (m, 24H), 4.28 (m, 1H), 4.50 (s, 1H), 6.64 (d, J = 8.5 Hz, 2H), 7.22 (m, 2H), 7.65 (d, J = 8.5 Hz, 2H), 7.89 (m, 1H), 8.11 (m, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.27 (m, 1H), 8.66 (s, 1H); ESI-MS m/z, 808.3 (M + H+); calcd for C33H46N17O8, 808.4. Synthesis of (R)-5-(2-(tert-Butoxycarbonylamino)ethyl) 1-tertButyl 2-(4-(((2,4-Diaminopteridin-6-yl)methyl)(methyl)amino)benzamido)pentanedioate (MTX-α-OtBu-γ-C2NHBoc; 10). MTX-αOtBu 9 (80 mg, 0.16 mmol) in DMF (2 mL) was added to a solution of tert-butyl 2-hydroxyethylcarbamate (38 mg, 0.24 mmol), 2-chloro-1methyl-pyridinium iodide (CMPI; 44 mg, 0.17 mmol), and 4(dimethylamino)pyridine (DMAP; 44 mg, 0.36 mmol) in DMF (3 mL). The reaction was stirred at room temperature for 6 h. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by column chromatography on silica gel (eluent CH3OH/CH2Cl2 = 6/94) to give 10 as a yellow solid (30 mg, 30%): 1H NMR (500 MHz, THF-d4) δ 1.38 (s, 9H), 1.45 (s, 9H), 1.88 (m, 1H), 2.21 (m, 1H), 2.39 (m, 2H), 2.54 (s, 2H), 3.19 (s, 3H), 3.25 (m, 2H), 3.93 (m, 1H), 4.12 (m, 1H), 4.66 (m, 1H), 4.75 (s, 2H), 6.43 (t, J = 5.5 Hz, 1H), 6.79 (d, J = 8.5 Hz, 2H), 7.08 (s, 2H), 7.38 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 9.0 Hz, 2H), 8.53 (s, 1H), 10.9 (s, 1H); ESI-MS m/z, 654.3 (M + H+); calcd for C31H44N9O7, 654.3. Synthesis of (R)-5-(2-Aminoethoxy)-2-(4-(((2,4-diaminopteridin6-yl)methyl)(methyl)amino)benzamido)-5-oxopentanoic acid (MTX-α-OtBu-γ-C2NH2; 11). Compound 10 (30 mg, 0.046 mmol) was dissolved in 4 mL of CH2Cl2/TFA (1:1) and the mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure. Ether (10 mL) was added to the residue. The solid was collected by centrifugation, washed with H2O and acetone, and dried under vacuum to yield 11 as a yellow solid (21 mg, 92%). The product is sufficiently pure for further reactions: ESI-MS m/z, 496.3 (M − H+); calcd for C22H26N9O5, 496.2. Synthesis of 2-(4-(2-(2-Aminoethoxy)ethylFA)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethyl (4-Nitrophenyl)carbonate (Triazine-N3-OC6H4NO2-αFA; 7). Compound 5 (20 mg, 0.025 mmol) was dissolved in DMSO (1 mL), and then triethylamine (10 μL) was added to the mixture, followed by bis(4-nitrophenyl)carbonate (15 mg, 0.049 mmol). The mixture was kept at room temperature for 48 h.

trisubstituted triazines. Our approach to preparing a welldefined dendrimer therapeutic is using triazine as a trifunctional molecule on which two sites are coupled to FA and MTX in fixed 1:1 ratio while an azido ligand is incorporated to its third functional site. These triazine derivatives are purified and characterized before being attached to an alkyne ligandmodified G5 PAMAM dendrimer using highly efficient click chemistry (Chart 1). This avoids producing populations containing only FA or MTX and reduces the extensive characterization and isolation procedures required between chemical conjugations.21,33 To the best of our knowledge, this is the first time triazine has been utilized for the simultaneous conjugation of two molecules to a dendrimer or other nanoparticle.

■

MATERIALS AND METHODS

General Information. 1H NMR spectra were obtained using a Varian Inova 500 MHz or an Inova 400 spectrometer. Matrix-assisted laser desorption ionization time-of-flight mass spectra (MALDI-TOFMS) were recorded on a PE Biosystems Voyager System 6050, using 2,5-dihydroxybenzoic acid (DHB) as the matrix. Electrospray ionization mass spectra (ESI-MS) was recorded using a Micromass Quattro II Electronic HPLC/MS/MS mass spectrometer. Materials. All solvents and chemicals were of reagent grade quality, purchased from Sigma-Aldrich (St. Louis, MO), and used without further purification unless otherwise noted. Thin-layer chromatography (TLC) and column chromatography were performed with 25 DC-Plastikfolien Kieselgel 60 F254 (Merck), and Baxter silica gel 60 Å (230−400 mesh), respectively. Synthesis. MTX-α-OtBu 9 was prepared as reported.34 Synthesis of N-(3-Azidopropyl)-4,6-dichloro-1,3,5-triazin-2-amine (Triazine-N3; 1). To a solution of cyanuric chloride (18.42 g, 0.10 mol) in acetone (180 mL) in an ice−water bath was added diisopropylethylamine (DIPEA; 12.92 g, 0.10 mol). 1-Azido-3-aminopropane (5.00 g, 0.050 mol) in acetone (50 mL) was added slowly over 2 h. The reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by column chromatography on silica gel (eluent CH2Cl2) to give 1 as a white solid (5.62 g, 45%): 1H NMR (400 MHz, CDCl3) δ 1.17 (br s, 1H), 1.85 (p, J = 6.4 Hz, 2H), 3.36 (t, J = 6.4 Hz, 2H), 3.55 (q, J = 6.4 Hz, 2H); ESI-MS m/z, 248.1 (M + H+); calcd for C6H8Cl2N7, 248.0. Synthesis of 2-(4-(3-Azidopropylamino)-6-chloro-1,3,5-triazin-2ylamino)ethanol (Triazine-N3-OH; 2). Ethanolamine (2.47 g, 40.4 mmol) in acetone (20 mL) was added to a solution of 1 (2.0 g, 8.1 mmol) in acetone (30 mL). The reaction was stirred at room temperature for 24 h. Acetone was removed by rotary evaporation and the residue was suspended in CH2Cl2. The mixture was filtered off. The solid collected was washed successively with CH2Cl2 and H2O and dried under reduced pressure to obtain 2 as a white solid (1.86 g, 85%). The product was sufficiently pure for further reactions: 1H NMR (500 MHz, acetone-d6) δ 1.89 (sept, J = 8.5 Hz, 2H), 3.42−3.50 (m, 4H), 3.50−3.57 (m, 2H), 3.66−3.73 (m, 2H), 3.93 (br s, 1H), 6.70−6.84 (m, 1H), 6.98 (m, 1H); ESI-MS m/z, 273.1 (M + H+); calcd for C8H14ClN8O, 273.1. Synthesis of tert-Butyl 2-(2-(4-(3-Azidopropylamino)-6-(2-hydroxyethylamino)-1,3,5-triazin-2-ylamino)ethoxy)ethylcarbamate (Triazine-N3-OH-NHBoc; 3). DIPEA (142 mg, 1.10 mmol), tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (273 mg, 1.10 mmol), and 2 (150 mg, 0.55 mmol) were dissolved in THF (5 mL). The mixture was stirred at 70 °C under N2 overnight. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by column chromatography on silica gel (eluent CH3OH/CH2Cl2 = 2/98) to give 3 as a white solid (124 mg, 47%): 1H NMR (500 MHz, CDCl3) δ 1.36 (s, 9H), 1.75 (m, 2H), 984

dx.doi.org/10.1021/bm201639c | Biomacromolecules 2012, 13, 982−991

Biomacromolecules

Article

b. Cu(I) Catalyzed Cycloaddition. G5-NHAc-alkyne 14 and G3FITC(FI)-alkyne 19 were synthesized according to the literature.21,35 G5-NHAc-alkyne 14 (white solid): MALDI-TOF mass 32774. The 1H NMR integration determined the mean number of acetyl groups per dendrimer is 80.1. The mean number of alkyne ligands per dendrimer is 12.3. G3-FI-alkyne 19 (orange solid): MALDI-TOF mass 14311. The 1H NMR integration determined the mean number of FIs per dendrimer is 3.2. The mean number of alkyne ligands per dendrimer is 3.5. G5-NHAc80-alkyne12 14 (14 mg, 0.43 μmol) was dissolved in Cu(II) sulfate (10 mol % per triazine-azide, 1 mg/mL H2O) and sodium ascorbate (60 mol % per triazine-azide, 1 mg/mL H2O solution) solution. The triazine-azide substrates (5, 6, 8, and 13; 7.5 azide mole ratio to G5-NHAc80-alkyne12, 10 mg/mL DMSO solution) were added. The reaction mixture was stirred at room temperature under N2 overnight. Samples were purified using 10000 MWCO centrifugal filtration devices. Purification consisted of ten cycles (20 min at 4800 rpm) using PBS (5 cycles) and DI water (five cycles). The purified dendrimer samples were lyophilized to yield 15−18 as brown solids. G5-Triazine-γMTX3.1-αFA3.1 15 (14.3 mg, 80%): MALDI-TOF mass 35094; the 1H NMR integration determined the mean number of triazine-γMTX-αFA attached is 3.1. G5-Triazine-OH-αFA4.2 16 (14.0 mg, 84%): MALDI-TOF mass 33059; the 1H NMR integration determined the mean number of triazine-OH-αFA is 4.2. G5-TriazineγMTX3.7-NHBoc 17 (11.7 mg, 68%): MALDI-TOF mass 34266; the 1 H NMR integration determined the mean number of triazine-γMTXNHBoc is 3.7. G5-Triazine-OH-γFA4.0 18 (13.6 mg, 82%): MALDITOF mass 33421; the 1H NMR integration determined the mean number of triazine-OH-αFA is 4.0. G3-Triazine-αFA-FI 20 and G3-Triazine-γFA-FI 21 were synthesized in a similar fashion as G5 conjugates by “clicking” triazine-azide substrates 5 or 6 onto G3-FI3.2-alkyne3.5 19. Samples were purified using 3500 MWCO centrifugal filtration devices and lyophilized to yield 20 (70% yield) and 21 (69% yield) as orange solids. The G3Triazine-αFA-FI 20: MALDI-TOF mass 16816; the 1H NMR integration indicated the mean number of triazine-OH-αFA is 2.8. G3-Triazine-γFA-FI 21: MALDI-TOF mass 16341; the 1H NMR integration determined the mean number of triazine-OH-γFA is 2.8. Ultra Performance Liquid Chromatography (UPLC). UPLC analysis was carried out on a Waters Acquity Peptide Mapping System controlled by Empower 2 software and equipped with a photodiode array detector. The G5 PAMAM dendrimer conjugates were run on an Acquity BEH C4 column (100 × 2.1 mm, 1.7 μm). The analysis was carried out using gradient elution ranging from 99:1 (v/v) water/ acetonitrile (ACN) to 20:80 water/ACN over 13.40 min. The gradient was then re-equilibrated to starting conditions in the next minute. Flow rate was maintained at 0.208 mL/min. Trifluoroacetic acid (TFA) at 0.14 wt % concentration in water as well as in ACN was used as a counterion. Sample was injected using a “partial loop with needle overfill”, an inbuilt 55 sample loop option within the software. The column temperature was maintained at 35 °C. The concentration of dendrimer conjugates were maintained at 1 mg/mL. Semiprep Reverse Phase High Performance Liquid Chromatography (HPLC). HPLC was utilized to separate and purify α and γ isomers of triazine-N3-OH-FA (5 and 6). A Waters Delta 600 HPLC system equipped with a 2996 photodiode array detector, auto sampler, controlled by Empower 2 software was used for this isolation, employing an Atlantis Prep T3 column (250 × 10 mm, 5 μL). The mobile phase for the elution with a two-step linear gradient was beginning with 90:10 (v/v) water/ACN and ending with 75:25 (v/v) water/ACN over 20 min at a flow rate of 4.02 mL/min. The gradient was gradually changed to 25:75 (v/v) water/ACN over the next 10 min, and TFA at 0.14 wt % concentration in water, as well as in ACN, was used as a counterion. Surface Plasmon Resonance (SPR) Spectroscopy. SPR experiments were performed in Biacore X (Pharmacia Biosensor AB, Uppsala, Sweden). Folate binding protein (FBP, bovine milk, SigmaAldrich), was immobilized on a CM5 sensor chip for SPR measurements using EDC/NHS chemistry.36 The immobilized FBP

Ether (10 mL) was added to the residue. The solid was collected by centrifugation, washed with CH2Cl2, methanol, and acetone, and dried under vacuum to give 7 as a yellow solid (crude product, 20 mg). The sample was used for the next step without further purification. 1H NMR (500 MHz, DMSO-d6) δ 1.09 (m, 1H), 2.00 (m, 1H), 2.13 (m, 1H), 2.68 (m, 2H), 3.17−3.50 (m, 24H), 4.01 (m, 1H), 4.33 (m, 1H), 4.49 (m, 2H), 6.38 (d, J = 8.5 Hz, 2H), 6.97 (m, 3H), 7.42 (d, J = 9.0 Hz, 2H), 7.61−7.69 (m, 3H), 7.99 (m, 1H), 8.07 (m, 1H), 8.28 (m, 2H), 8.64 (s, 1H). Synthesis of 2-(4-(2-(2-Aminoethoxy)ethylFA)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethylMTX (Triazine-N3-γMTX-αFA; 8). Compound 11 (25 mg, 0.050 mmol) in DMSO (0.5 mL) was added to a solution of 7 and DIPEA (32 mg, 0.25 mmol) in DMSO (0.5 mL). The reaction was stirred at room temperature for 24 h. Ether (10 mL) was added to the residue. The solid was collected by centrifugation, washed with methanol and acetone, and dried under vacuum. The crude product was further purified by semiprep (see Semiprep reverse phase high performance liquid chromatography (HPLC)) HPLC to yield 8 as yellow solids (12 mg, 36%): 1H NMR (500 MHz, DMSO-d6) δ 1.74 (m, 2H), 1.83 (m, 1H), 1.95 (m, 2H), 2.08 (m, 2H), 2.17 (m, 2H), 2.39 (m, 3H), 3.07−3.46 (m, 28H), 3.95−4.03 (m, 2H), 4.18−4.25 (m, 2H), 4.31 (m, 2H), 4.48 (m, 2H), 4.78 (m, 2H), 6.62 (m, 4H), 6.82 (m, 3H), 6.95 (m, 1H), 7.22 (m, 1H), 7.46 (m, 2H), 7.62−7.73 (m, 5H), 7.86 (m, 1H), 7.97 (m, 1H), 8.56 (s, 1H), 8.63 (s, 1H); ESI-MS m/z, 664.3 (M − 2H+)/2; calcd for (C56H70N26O14 − 2H+)/2, 664.3. Synthesis of tert-Butyl (2-(2-(2-((4-((3-Azidopropyl)amino)-6-((2(((4-nitrophenoxy)carbonyl)oxy)ethyl)amino)-1,3,5-triazin-2-yl)amino)ethoxy)ethoxy)ethyl)carbamate (Triazine-N3-OC6H4NO2NHBoc; 12). Triazine-N3-OH-NHBoc 3 (158 mg, 0.33 mmol) was dissolved in DMF (3 mL). Triethylamine (200 μL) was added to the mixture, followed by bis(4-nitrophenyl)carbonate (198 mg, 0.65 mmol). The mixture was kept at room temperature for 24 h. The solvent was removed under reduced pressure. The resulting residue was purified by column chromatography on silica gel (eluent: CH3OH/CH2Cl2 = 5/95) to give 12 as a white solid (182 mg, 86%): 1H NMR (500 MHz, CDCl3) δ 1.45 (s, 9H), 1.84 (m, 2H), 2.17 (s, 1H), 3.32−3.77 (m, 18H), 4.42 (m, 2H), 5.25 (m, 1H), 5.94 (m, 1H), 6.30−6.48 (m, 1H), 7.37 (d, J = 9.0 Hz, 2H), 8.27 (d, J = 8.5 Hz, 2H); ESI-MS m/z, 650.2 (M + H+); calcd for C26H40N11O9, 650.3. Synthesis of 2-(4-(2-(2-Aminoethoxy)ethoxy)ethyl)carbamate)-6(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethylMTX (TriazineN3-MTX-NHBoc; 13). Compound 11 (10 mg, 0.020 mmol) in DMF (0.5 mL) was added to a solution of 12 (26 mg, 0.040 mmol) and DIPEA (26 mg, 0.20 mmol) in DMF (0.5 mL). The reaction was stirred at room temperature for 24 h. CH2Cl2 (5 mL) was added to the residue. The solid was collected by centrifugation, washed with acetone and CH2Cl2, and dried under vacuum to give 13 as a yellow solid (7 mg, 35%). The sample was used for the next step without further purification: 1H NMR (500 MHz, DMF-d7) δ 1.41 (m, 2H), 1.45 (s, 9H), 2.10 (m, 1H), 2.25 (m, 1H), 2.53 (m, 2H), 3.29 (m, 10H), 3.48−3.65 (m, 24H), 4.13 (m, 1H), 4.60 (m, 1H), 4.86 (m, 2H), 6.92 (d, J = 9.0 Hz, 2H), 7.03 (m, 2H), 7.87 (m, 3H), 8.69 (m, 1H); ESI-MS m/z, 1006.5 (M − H+); calcd for C42H60N19O11, 1006.5. General Procedure for Syntheses of Dendrimer Conjugates. a. Characterization. All the conjugates and their intermediate reaction products were analyzed by MALDITOF, HPLC, and NMR, the methods of which have been previously described.21 The number of ligands that attached to the dendrimer was obtained from the integration of the methyl protons of the terminal acetyl groups to the aromatic protons on the conjugated ligands (e.g., FA and MTX). The number of acetyl groups per dendrimer was determined by first computing the total number of end groups from the number average molecular weight from gel permeation chromatography (GPC) and potentiometric titration data for G5-NH 2(100%) as previously described. 17 The total number of end groups was applied to the ratio of primary amines to acetyl groups, obtained from the 1H NMR of the partially acetylated dendrimer, to compute the average number of acetyl groups per dendrimer. 985

dx.doi.org/10.1021/bm201639c | Biomacromolecules 2012, 13, 982−991

Biomacromolecules

Article

Scheme 1. Synthesis of Triazine-N3-γMTX-αFA 8

gave 9500 response unit (RU), equivalent to 9.5 ng/mm2 FBP. Samples were run with HBS-EP buffer at a flow rate of 30 μL/min. After each measurement, the surface of the chip was regenerated by injection of 10 μL of 10 mM glycine−HCl (pH 2.5). The SPR sensorgrams collected for the conjugates run in channel 2 were corrected with the reference channel 1 (ΔRU = RU2 − RU1), to minimize the background due to nonspecific interactions. The kinetic binding parameters, the rate of association (kon), and the rate of dissociation (koff) were generated by individually fitting each curve using the Langmuir kinetic model (Biacore evaluation software). Dissociation constants (KD = koff/kon) for each conjugate were calculated by averaging the data obtained from at least three sets of measurements for each concentration; each measurement was required to have a chi square (χ2) value less than 5 for inclusion. Cell Culture. KB cells (tested to be mycoplasma free) were maintained in FA-free RPMI medium and the B16−F10 cells in DMEM medum, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin, under 5% CO2. Flow Cytometry Analysis. The cellular binding of G5-TriazineγMTX-αFA 15 was determined by a competition assay involving a fluorescently labeled G5-FI-FA as previously described.18 For this analysis, KB cells plated in 24-well plates were treated simultaneously

with a mixture of 50 nM of the G5-FI-FA and varying concentrations of the newly synthesized nonfluorescent triazine based conjugates. The cells were incubated with this mixture at 37 °C for 1 h, washed to remove unbound material and the fluorescence was determined by flow cytometry using a Beckman-Coulter EPICS-XL MCL. The data were analyzed using Expo32 software (Beckman-Coulter,Miami, FL), with the mean FL1-fluorescence of 10000 cells determine on a population gated for viable cells.18 XTT Cytotoxicity Assay. Cells were seeded in 96-well microtiter plates (3000 cells/well) in medium containing dialyzed serum. Two days after plating, the cells were treated with the dendrimer conjugates in the tissue culture medium. A colorimetric “XTT” (sodium 3-[1(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay (Roche Molecular Biochemicals, Indianapolis, IN) was performed, following the vendor’s protocol. After incubation with the XTT labeling mixture, the microtiter plates were read on an ELISA reader (Synergy HT, BioTek) at 492 nm with the reference wavelength at 690 nm.18 Stability Test. A 100 μM aliquot of the dendrimer conjugate 15 was incubated with RPMI medium containing 10% serum, and stirred continuously at 37 °C for varying time periods. The proteins were then precipitated with 10% DMSO in ACN and the supernatant obtained was subjected to UPLC analysis. UV absorbance peak area of 986

dx.doi.org/10.1021/bm201639c | Biomacromolecules 2012, 13, 982−991

Biomacromolecules

Article

MTX and FA was run separately to determine the concentration of released FA and MTX, as compared to standard curves made with free drugs.

■

RESULTS AND DISCUSSION Synthesis. The trifunctional triazine derivative 4 was synthesized from cyanuric chloride by consecutive aromatic nucleophilic substitution reactions in a stepwise manner, and these reactions were controlled by the temperature employed (Scheme 1). The monosubstituted triazine, triazine-N3 1, was synthesized by adding 1-azido-3-aminopropane to a solution of cyanuric chloride in acetone at 0 °C. A large excess of cyanuric chloride was used to minimize the bisubstituted byproduct. The second substitution was carried out in the same solvent at room temperature with 2-aminoethanol. The bisubstituted triazineN3-OH 2 was treated with tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate in the presence of DIPEA in THF at 70 °C to form triazine-N3-OH-NHBoc 3, which contained a protected primary amine that is used for conjugation with FA. The Boc-protected diamine compound was necessary because efforts to make trifunctional 4 using unprotected 2,2′-(ethane1,2-diylbis(oxy))diethanamine were unsuccessful due to the formation of a triazine dimer. After removal of the BOC protecting group, FA was coupled to triazine-N3-OH-NH2 4 by N,N′-dicyclohexylcarbodiimide (DCC), through an amide bond that should be stable under biological conditions. This sample was purified by reversed phase column chromatography, and characterized by NMR, mass spectroscopy, and UPLC (Figure S1). Two isomers, α-carboxyl 5 and γ-carboxyl 6 group linked products were separated; relatively more (60%) α-carboxyl linked product was obtained as compared to the γ isomer (40%) according to HPLC analysis. We took two different strategies to test the FR-binding efficiency and regioisomer selectivity of the triazine-linked FAs. First, we conjugated two triazine-N3-OH-FA isomers, 5 and 6 to a fluorescently labeled generation 3 (G3) PAMAM dendrimer G3-FI-alkyne 19,35 using click chemistry (Scheme 4). The G3-Triazine-αFA-FI 20 and G3-Triazine-γFA-FI 21 were then tested for binding with FR-expressing KB cells by flow cytometry. As shown in Figure 1, both G3-Triazine-αFAFI 20 and G3-Triazine-γFA-FI 21 bound to the KB cells in a dose-dependent manner with similar saturation kinetics, reaching maximum binding between 100 and 300 nM; this was similar to what we have previously observed for a sequentially synthesized G5-FI-FA conjugate.18 The binding was inhibited by preincubation with excess free FA (Figure 1), showing specificity for the folate receptor. Second, we conjugated compounds 5 and 6 to a G5-dendrimer to generate G5-Triazine-OH-αFA4.2 16 and G5-Triazine-OH-γFA4.0 18 (Scheme 4). The FR-binding capacity of 16 and 18 was examined by SPR, using FBP coupled to CM5 sensor chip as a FR surrogate. Both conjugates showed similar binding affinities to FBP (Table 1), consistent with the cell binding data (Figure 1). The dissociation constants (KD) obtained for the α- and γregioisomers were 19 and 9.8 nM, respectively; this suggested that conjugation to the dendrimer increased the avidity 1000fold higher than free FA.36 These studies demonstrated that (1) FA linked through a triazine linker retains its ability to bind to FR; (2) the α- and γ-isomers conjugates have similar ability to bind to the FR; and (3) the two conjugates bound to the KB cells through the FR, as demonstrated by the reversal of uptake by free FA. This finding is consistent with other results indicating that when utilizing folate as a macromolecular drug

Figure 1. Dose-dependent association of the G3-Triazine-αFA-FI 20 and G3-Triazine-γFA-FI 21 in KB cells. KB cells in serum- and FA-free RPMI medium were treated with different concentrations of 20 (diamond symbols) and 21 (triangle symbols) for 1 h at 37 °C; the cells were trypsinized and suspended in PBS containing 0.1% BSA and were subjected to flow cytometry analysis as given in the Materials and Methods (filled symbols). Some cells were pretreated with 50 μM FA prior to incubation with the conjugate also in the presence of FA (open symbols). The data shown represents the mean FL1fluorescence of 10000 cells, with similar data obtained in an independent experiment.

Table 1. Binding Constants of G5-Triazine-αFA 16 and G5Triazine-γFA 18 with FBP, Measured by SPR conjugates G5-αFA 16 G5-γFA 18

koff (s−1) −3

1.11 × 10 1.05 × 10−3

kon (M−1 s−1)

KD (M)

5.82 × 104 1.16 × 105

1.91 × 10−8 9.83 × 10−9

targeting ligand there is no preferred isomeric conjugation (αcarboxyl vs γ-carboxyl).24 The α-carboxyl group linked isomer 5 was used for further studies due to its relatively higher yield. The first approach for the synthesis of trifunctional triazineN3-γMTX-αFA 8 was the direct coupling MTX to triazine-N3OH-αFA 5 using DCC/4-(dimethylamino)pyridine (DMAP) or DCC/4-pyrrolidinopyridine.37 Both reactions were unsuccessful due to very low yield, and an alternate synthesis was devised. This involved the synthesis of γMTX-ethylamine 11 initially using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) activated 4-amino4-deoxy-N10-methyl pteroic acid (APA) in DMSO reacted with the potassium salt of glutamic acid α-tert-butyl ester to form the α-carboxyl protected MTX derivative (Scheme 2).34 A γcarboxyl linked ester bond was chosen for MTX conjugation with the hypothesis that inside the cell this could enhance release of free drug from the conjugate through cellular esterases, and enhance its ability to inhibit DHFR.26 The selectively protected MTX derivative was then coupled to tertbutyl (2-hydroxyethyl)carbamate by 2-chloro-1-methyl-pyridinium iodide (CMPI)/DMAP. The t-butyl and BOC protecting groups were then removed by TFA at the same time to afford the γMTX-ethylamine 11. The hydroxyl group of 5 was first activated with bis(4-nitrophenyl) carbonate to provide carbonate 7 and then treated with γMTX-ethylamine 11 to form the target compound triazine-N3-γMTX-αFA 8. In parallel, triazine-N3-γMTX-NHBoc 13 was synthesized as a control compound in a similar manner (Scheme 3). The bifunctional dendrimer conjugate G5-Triazine-γMTX-αFA 15 and the control conjugate G5-Triazine-γMTX-NHBoc 17 were 987

dx.doi.org/10.1021/bm201639c | Biomacromolecules 2012, 13, 982−991

Biomacromolecules

Article

Scheme 2. Synthesis of γMTX-ethylamine 11

Scheme 3. Synthesis of Triazine-N3-γMTX-NHBoc 13

Scheme 4. Synthesis of Dendrimer Conjugates G5-Triazine-γMTX-αFA 15, G5-Triazine-αFA 16, G5-Triazine-γMTX 17, G5Triazine-γFA 18, G3-Triazine-αFA-FI 20, and G3-Triazine-γFA-FI 21 by “Click” Reactions

988

dx.doi.org/10.1021/bm201639c | Biomacromolecules 2012, 13, 982−991

Biomacromolecules

Article

then synthesized by “click” reactions of G5-NHAc-alkyne 14 with corresponding triazine derivatives (Scheme 4). In Vitro Functional Studies. Because there is no fluorescence tag on the G5-Triazine-γMTX-αFA 15, we verified its cellular binding by a competition assay in KB cells with a traditionally synthesized FA conjugated FITC dendrimer.17,18 This conjugate contains 6 FA and 5 FI per dendrimer. As shown in Figure 2, the G5-Triazine-γMTX-αFA 15 competed

Figure 3. Stability test of G5-triazine-γMTX-αFA 15 in cell culture medium. Compound 15 (100 μM) was incubated with RPMI medium containing final 10% serum for different time periods at 37 °C, and the medium proteins were precipitated with 10% DMSO in acetonitrile. The supernatants obtained were subjected to UPLC analysis. The values given on the Y-axis are expressed as the percent mole of total MTX present per mole of the dendrimer. Inset: UPLC elution profile showing the peak areas of the released FA and MTX.

Figure 2. Competition of the triazine-based conjugates 15 and 17 with a sequentially synthesized G5-FI5-FA6 conjugate. The indicated concentrations of the triazine-based conjugates were premixed with 50 nM of the G5-FI5-FA6 and the competition for binding to KB cells was assessed by flow cytometry as given in Materials and Methods. The data represent the mean ± SE of triplicate cell samples, with similar data obtained in an independent experiment.

effectively with G5-FI5-FA6 in a dose-dependent manner, indicating receptor-mediated binding of the conjugate. The lack of competition by the control conjugate G5-TriazineγMTX-NHBoc 17 may be attributed to the known lower affinity (∼100-fold) of MTX for FR as compared to that of FA. These results confirmed that the G5-Triazine-γMTX-αFA 15 is bound by KB cells through the FR. To effectively target tumors, the synthesized conjugates must be stable until they reach the site of tumor before releasing drug or targeting ligand.38 To test this, we incubated the dendrimer conjugate in cell culture medium and tested its stability. For this analysis, the G5-Triazine-γMTX-αFA 15 was incubated in cell culture medium for 1, 4, 24, and 72 h. As shown in Figure 3, less than 5% of the total MTX present on the dendrimer was released into the serum-containing medium at 37 °C, over a period of 72 h. The amide-linked FA was stable, suggesting that the slow release of MTX was probably caused by serum esterase-mediated hydrolytic cleavage. The cytotoxicity of triazine-based conjugates was then determined in vitro in KB cells using XTT assay. The G5Triazine-OH-αFA 16 that lacks the MTX moiety actually promoted cell growth to ∼20%, due to the presence of FA, as observed with G5-FA conjugates made by sequential synthesis.18 Compared to the sequentially synthesized G5-FA5MTX7 conjugate, G5-Triazine-γMTX-αFA 15 inhibited KB cell growth in a dose-dependence fashion (Figure 4) with a lower IC50 (10 vs 75 nM), which is close to that of free MTX (5 nM). Examination of a time-course analysis showed that the conjugate also inhibited cell growth in a time-dependent fashion (Figure S2). Although the FA-lacking control conjugate G5-Triazine-γMTX-NHBoc 17 failed to compete with the G5FI5-FA6 (Figure 1), the conjugate is modestly cytotoxic. It is

Figure 4. Cytotoxicity of the triazine-based conjugates G5-TriazineγMTX-αFA 15, G5-Triazine-αFA 16, and G5-Triazine-γMTX 17. KB cells were incubated for 48 h with the indicated conjugates, the medium and drugs were then replaced with fresh medium and drugs and incubated for additional 48 h, and the cytotoxicity was determined by XTT assay, as given in Materials and Methods. Also shown is the cytotoxicity of a previous batch of G5-FA-MTX containing an average 5 FA and 7 MTX per dendrimer, synthesized by the conventional synthetic route. The data represents mean ± SE of six replicate cell samples, with similar data obtained in two independent experiments.

possible that the G5-Triazine-γMTX-NHBoc 17 conjugate is poorly taken up through the reduced folate carrier (RFC), similar to the previously observed reduced efflux of polyglutamated MTX versus free MTX.39 Therefore, the observed cytotoxicity could be due to slow FR binding and subsequently poorer internalization of the conjugate during the 48 h incubation period; this would also explain the lack of discernible binding during the 1 h competition assay. Alternatively, macropinocytic/endocytic mechanism of the conjugate also cannot be ruled out. Recent studies have shown that the B16−F10 cells have negligible uptake of G5-FI5-FA6 (1 h incubation with G5-FI5FA6 gave a background-subtracted mean fluorescence of 2.6 in B16−F10 cells vs 234.1 in KB cells under identical conditions). We, therefore, utilized these cells as a negative control for cytotoxicity measurements. In a 72 h incubation, B16−F10 cells had a dose-dependent inhibition by free MTX (>80% at 300 nM), where the G5-Triazine-γMTX-αFA 15 did not demonstrate any growth inhibition until 300 nM (95% of the MTX still remained on the dendrimer at the end of a 48 h incubation period (Figure 3). Specific uptake is also supported by the observation that a 4 h exposure of 100 nM conjugate followed by washing and incubation of cells for 2 days in drug-free medium resulted in significant cytotoxicity (Figure S2) and by the lack of cytotoxicity in the B16−F10 cells (Figure S3). In addition, the G5-Triazine-γMTX-NHBoc 17 demonstrated much less cytotoxic potential than compound 15, having folate, confirming extracellular released MTX was not the reason for cytotoxicity. Although the exact reason for the increased cytotoxicity of 15 versus the sequentially synthesized G5-FAMTX is not clear from this study, it is possible that more functionally active dendrimers were present having both FA and MTX. In addition, as MTX has ∼1000-fold more affinity toward DHFR versus FA, it is possible that the close molecular proximity of the FA and MTX on 15 may allow the preferential binding of MTX versus FA not observed in the sequential conjugate. This close proximity between MTX and FA is shown in 1 ns molecular dynamics simulations of the equilibrated structure in Figure S4.

■

ASSOCIATED CONTENT

S Supporting Information *

HPLC chromatogram of triazine-N3-OH-αFA 5 and triazineN3-OH-γFA 6, cytotoxicity of 15 with different incubation times, cytotoxicity of 15 in B16−F10 cells, a modified figure of Figure 4, including data for free FA, and plotting the data based on the MTX or FA concentrations on the dendrimer. NMR and UPLC profiles of dendrimer conjugates, and full experimental details of molecular dynamics (MD) simulations. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: (734) 615-0623 (H.Z.); (734) 647-2777 (J.R.B.). Fax: (734) 615-0621 (H.Z.); (734) 615-2506 (J.R.B.). E-mail: [email protected] (H.Z.); [email protected] (J.R.B.). Author Contributions †

These authors have contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Award 1 R01 CA119409. The authors thank Dr. Ying Song (MIT) for valuable discussion. We also thank Prof. Charles L. Brooks III for allowing us to use his dual Quad-core Intel Xeon cluster for the simulation work.

■

CONCLUSIONS Trifunctional triazine derivatives containing FA and the therapeutic drug MTX have been synthesized. FA and MTX have been coupled selectively through α-carboxyl or γ-carboxyl group by amide and ester bond, respectively. Bifunctional dendrimer therapeutics can be achieved by one-step “click reaction”, which does not introduce cross-linking byproduct as seen with typical coupling reactions. The procedure also allows the synthesis of bifunctional conjugates with an exact 1:1 ratio of the two ligands. The current study shows that the triazinelinked conjugate with a fixed ratio of FA and MTX (3.1 molecules per dendrimer) induces higher cytotoxicity in KB cells as compared to a sequentially synthesized conjugate with higher numbers of FA and MTX, 5 and 7, respectively. Our FR binding studies also show that α carboxyl-linked FA-conjugate is equally effective in FR-targeting as the γ carboxyl-linked conjugate. The triazine-based conjugate carrying more homogeneously distributed populations with a fixed ratio of FA and MTX. By minimizing the dendrimer modifications and reaction steps used in our sequential synthetic strategy, this approach may also result in the improvement of batch-to-batch reproducibility. The sequential reactivity of the three chlorine atoms in the triazine backbone provides these molecules synthetic flexibility, useful for the synthesis of different combinations of drugs and targeting agents. Although the currently synthesized molecule has a fixed 1:1 ratio of targeting molecule and drug, by using linkers with different numbers of terminal functional groups (e.g., hydroxyl or amino), it would be possible to tune the ratio between targeting molecule and

■

REFERENCES

(1) Riehemann, K.; Schneider, S. W.; Luger, T. A.; Godin, B.; Ferrari, M.; Fuchs, H. Angew. Chem., Int. Ed. 2009, 48, 872−897. (2) de Bono, J. S.; Ashworth, A. Nature 2010, 467, 543−549. (3) Haag, R.; Kratz, F. Angew. Chem., Int. Ed. 2006, 45, 1198−1215. (4) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Controlled Release 2000, 65, 271−284. (5) Torchilin, V. P. Nat. Rev. Drug Discovery 2005, 4, 145−160. (6) Duncan, R. Nat. Rev. Cancer 2006, 6, 688−701. (7) Maeda, H.; Bharate, G. Y.; Daruwalla, J. Eur. J. Pharm. Biopharm. 2009, 71, 409−419. (8) Khandare, J.; Minko, T. Prog. Polym. Sci. 2006, 31, 359−397. (9) Allen, T. M. Nat. Rev. Cancer 2002, 2, 750−763. (10) Davis, M. E.; Chen, Z.; Shin, D. M. Nat. Rev. Drug Discovery 2008, 7, 771−782. (11) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A. Angew. Chem., Int. Ed. 1990, 29, 138−175. (12) Lee, C. C.; MacKay, J. A.; Frechet, J. M. J.; Szoka, F. C. Nat. Biotechnol. 2005, 23, 1517−1526. (13) Soliman, G. M.; Sharma, A.; Maysinger, D.; Kakkar, A. Chem. Commun. 2011, 47, 9572−9587. (14) Thomas, T. P.; Shukla, R.; Majoros, I. J.; Myc, A.; Baker Jr., J. R. In Nanobiotechnology: Concepts, Methods and Perspectives; Mirkin, Ed.; Wiley-VCH: New York, 2007. (15) Nanjwade, B. K.; Bechra, H. M.; Derkar, G. K.; Manvi, F. V.; Nanjwade, V. K. Eur. J. Pharm. Sci. 2009, 38, 185−196. (16) Tomalia, D. A.; Reyna, L. A.; Svenson, S. Biochem. Soc. Trans. 2007, 35, 61−67. 990

dx.doi.org/10.1021/bm201639c | Biomacromolecules 2012, 13, 982−991

Biomacromolecules

Article

(17) Majoros, I. J.; Thomas, T. P.; Mehta, C. B.; Baker, J. R. J. Med. Chem. 2005, 48, 5892−5899. (18) Thomas, T. P.; Majoros, I. J.; Kotlyar, A.; Kukowska-Latallo, J. F.; Bielinska, A.; Myc, A.; Baker, J. R. Jr. J. Med. Chem. 2005, 48, 3729−3735. (19) Kukowska-Latallo, J. F.; Candido, K. A.; Cao, Z. Y.; Nigavekar, S. S.; Majoros, I. J.; Thomas, T. P.; Balogh, L. P.; Khan, M. K.; Baker, J. R. Cancer Res. 2005, 65, 5317−5324. (20) Gillies, E. R.; Frechet, J. M. J. Drug Discovery Today 2005, 10, 35−43. (21) Mullen, D. G.; Fang, M.; Desai, A.; Baker, J. R.; Orr, B. G.; Holl, M. M. B. ACS Nano 2010, 4, 657−670. (22) Rosowsky, A.; Forsch, R.; Uren, J.; Wick, M. J. Med. Chem. 1981, 24, 1450−1455. (23) Wang, S.; Lee, R. J.; Mathias, C. J.; Green, M. A.; Low, P. S. Bioconjugate Chem. 1996, 7, 56−62. (24) Leamon, C. P.; DePrince, R. B.; Hendren, R. W. J. Drug Targeting 1999, 7, 157−169. (25) Bettio, A.; Honer, M.; Muller, C.; Bruhlmeier, M.; Muller, U.; Schibli, R.; Groehn, V.; Schubiger, A. P.; Ametamey, S. M. J. Nucl. Med. 2006, 47, 1153−1160. (26) Wells, X. E.; Bender, V. J.; Francis, C. L.; He-Williams, H. M.; Manthey, M. K.; Moghaddam, M. J.; Reilly, W. G.; Whittaker, R. G. Drug Dev. Res. 1999, 46, 302−308. (27) Kaminskas, L. M.; Kelly, B. D.; McLeod, V. M.; Sberna, G.; Boyd, B. J.; Owen, D. J.; Porter, C. J. H. Mol. Pharm. 2011, 8, 338− 349. (28) Blotny, G. Tetrahedron 2006, 62, 9507−9522. (29) Simanek, E. E.; Abdou, H.; Lalwani, S.; Lim, J.; Mintzer, M.; Venditto, V. J.; Vittur, B. Proc. R. Soc. A 2010, 466, 1445−1648. (30) Steffensen, M. B.; Hollink, E.; Kuschel, F.; Bauer, M.; Simanek, E. E. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 3411−3433. (31) Lo, S. T.; Stern, S.; Clogston, J. D.; Zheng, J.; Adiseshaiah, P. P.; Dobrovolskaia, M.; Lim, J.; Patri, A. K.; Sun, X.; Simanek, E. E. Mol. Pharm. 2010, 7, 993−1006. (32) Young, K. L.; Xu, C.; Xie, J.; Sun, S. J. Mater. Chem. 2009, 19, 6400−6406. (33) Mullen, D. G.; Desai, A. M.; Waddell, J. N.; Cheng, X. M.; Kelly, C. V.; McNerny, D. Q.; Majoros, I. J.; Baker, J. R.; Sander, L. M.; Orr, B. G.; Holl, M. M. B. Bioconjugate Chem. 2008, 19, 1748−1752. (34) Nagy, A.; Szoke, B.; Schally, A. V. Proc. Natl. Acad. Sci. U.S.A 1993, 90, 6373−6376. (35) Zhang, Y.; Thomas, T. P.; Lee, K. H.; Li, M. H.; Zong, H.; Desai, A. M.; Kotlyar, A.; Huang, B. H.; Holl, M. M. B.; Baker, J. R. Bioorg. Med. Chem. 2011, 19, 2557−2564. (36) Hong, S.; Leroueil, P. R.; Majoros, I. J.; Orr, B. G.; Baker, J. R. Jr.; Banaszak Holl, M. M. Chem. Biol. 2007, 14, 107−115. (37) Devineni, D.; Blanton, C. D.; Gallo, J. M. Bioconjugate Chem. 1995, 6, 203−210. (38) Vicent, M. J.; Duncan, R. Trends Biotechnol. 2006, 24, 39−47. (39) Rosenblatt, D. S.; Whitehead, V. M.; Vera, N.; Pottier, A.; Dupont, M.; Vuchich, M. J. Mol. Pharmacol. 1978, 14, 1143−1147.

991

dx.doi.org/10.1021/bm201639c | Biomacromolecules 2012, 13, 982−991