Design of Dendritic Macromolecules Containing Folate or

addition, a similar conjugation of the antitumor drug methotrexate to the dendrimer was also investigated. Once optimized, these molecules may form th...
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Bioconjugate Chem. 1999, 10, 1115−1121

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Design of Dendritic Macromolecules Containing Folate or Methotrexate Residues Kenji Kono,‡ Mingjun Liu, and Jean M. J. Fre´chet* Department of Chemistry, University of California, Berkeley, California 94720-1460. Received June 18, 1999; Revised Manuscript Received September 20, 1999

Polyether dendritic compounds bearing folate residues on their surface were prepared as model drug carriers with potential tumor cell specificity. Starting from ester-terminated polyether dendrimers, hydrazide groups were easily introduced to the surface of the dendrimers by reaction with hydrazine. Folate residues were then conjugated to the hydrazide chain ends of the dendrimers by direct condensation with folic acid in the presence of a condensing agent or by reaction with an active ester derivative of folic acid. Essentially complete functionalization of the terminal hydrazide groups was achieved for both the first and the second generation dendrimers with four and eight hydrazide groups. For the G-2 dendrimer with 16 hydrazide groups, an average number of only 12.6 folate residues were attached to each dendrimer. The conjugates are soluble in aqueous medium above pH 7.4. In addition, a similar conjugation of the antitumor drug methotrexate to the dendrimer was also investigated. Once optimized, these molecules may form the basis for a novel family of multivalent drug carriers.

Dendrimers are unique synthetic macromolecules that have attracted much interest due to their unique structures and properties since their introduction in the mid1980s. Their highly branched and well-defined structure, globular shape, and controlled surface functionality are some of the important characteristics of dendrimers (13). While applications of dendrimers have been explored in a very broad range of fields, their use in drug delivery systems has remained largely unexplored. The functional groups present at the surface of dendrimers have been utilized for the conjugation of various biologically active molecules. For example, antibody (46) and sugar moieties (7, 8) have been conjugated to the chain ends of poly(amidoamine) dendrimers, and these modifications have afforded dendrimers that may possess site-specific properties via antibody-antigen binding or via interactions between sugars and their receptors on cell surfaces. The interior of dendrimers has also been shown to be capable of encapsulating guest molecules. Meijer and coworkers have reported that guest molecules, such as Rose Bengal, may be physically entrapped into the internal “cavity” of high generation poly(propylene imine) dendrimers when an amino acid derivative was used to cap each end group of the dendrimer (9). Because the amino acid derivative used was bulky, sterically crowded, and subject to hydrogen bonding, the entrapped guest molecules could not diffuse out of the dense packed shell of the dendrimer into the surrounding solution. Yet another strategy for the encapsulation of guest molecules in dendrimers makes use of hydrophobic interactions. Thus, Newkome et al. have prepared dendritic macromolecules * To whom correspondence should be addressed. Phone: (510) 643-3077. Fax: (510) 643-3079. E-mail: Frechet@ CChem.Berkeley.edu. ‡ Present address: Department of Applied Materials Science, College of Engineering, and Department of Applied Bioscience, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.

with hydrophobic interior and hydrophilic chain ends (10), and these molecules were shown to behave as unimolecular micelles capable of solubilizing various hydrophobic compounds in aqueous solution (11). In early work, we have also reported that simple and well-characterized poly(aryl ether) dendrimers bearing carboxylic groups as chain ends were able to solubilize pyrene in water (12). More recently, we have explored the possibility of using more appropriately designed dendritic unimolecular micelles as drug delivery agents, and the result showed that ca. 11 wt % of a hydrophobic drug such as indomethacin could be easily entrapped inside the hydrophobic interior of dendritic micelles and released slowly into buffer solution (13, 14). Another, yet less explored (15, 16), approach to the use of dendrimers in drug delivery is to attach drug molecules to the chain ends of the dendrimers; therefore, a high concentration of drug may be carried by a simple well-defined molecule. If an appropriate ligand is conjugated to one of the terminal groups of the dendrimers, a targeting feature may eventually be added to these dendrimer-drug conjugates. It is known that a membrane-associated folate receptor, folate binding protein, is overexpressed on the surface of a variety of human tumor cells (17-19). When folic acid molecules are covalently linked to proteins, the folate-protein conjugates are internalized into cells via receptor-mediated endocytosis (20). Therefore, folate has been used as a ligand for the targeting of proteins (21), liposomes (22, 23), and other molecules (24, 25) to tumor cells that express the folate-binding proteins. The folic acid molecule (see Figure 1) possesses two carboxyl groups, termed R- and γ-, and the latter exhibits a much higher reactivity in a carbodiimide-mediated coupling to amino groups (26). In addition, folate linked via its γcarboxyl group retains a strong affinity toward its receptor (26). In this study, we have designed and synthesized dendritic molecules containing folate residues as models

10.1021/bc990082k CCC: $18.00 © 1999 American Chemical Society Published on Web 10/21/1999

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Figure 1. Structure of folic acid and MTX.

for drug carriers with tumor cell specificity. The synthesis is started from readily accessed ester-terminated dendrimers. After conversion of the ester groups to hydrazide groups, conjugation of folic acid to the dendrimers was accomplished by coupling the carboxylic groups of folic acid to the hydrazide groups of the dendrimers. The conjugation of methotrexate (MTX) (see Figure 1), an anti-cancer drug which has a structure similar to that of folic acid, to the dendrimer was also investigated. EXPERIMENTAL PROCEDURES

General Methods. Folic acid and methotrexate (MTX) were purchased from Sigma. Poly(ethylene glycol) (PEG) monomethyl ether (Mn ) 2000) was obtained from Shearwater Polymers, Inc. Other materials were obtained from Aldrich and used without further purification unless otherwise stated. DMSO was distilled under vacuum. 1H NMR spectra were recorded on a Bruker AMX-300 (300 Hz) spectrometer and the solvent signals were used as the standard. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were recorded on a Voyager-DE PerSeptive Biosystems in a positive ion mode using 2,5-dihydroxybenzoic acid as the matrix. Synthesis of Hydrazide-Terminated Dendritic Compounds. Diethyl isophthalate-terminated polyether dendrimers were synthesized according to the published procedure of Leon et al. (27). Hydrazide-terminated polyether dendrimers were prepared by reaction of esterterminated dendritic precursors with hydrazine. (H2NHNCO)4-[G-1]-OH (1). To a solution of (EtO2C)4[G-1]-OH (0.50 g, 0.90 mmol) in DMF (10 mL) was added hydrazine (anhydrous, 1.5 mL), and the solution was stirred at 60 °C for 2 days under N2 atmosphere. Methanol (50 mL) was added to the reaction mixture, and the white precipitate was collected by filtration. The crude product was redissolved in DMF and precipitated by addition of methanol. The precipitate was collected and dried under vacuum. Yield: 0.38 g, 84%. 1H NMR δ 4.44 (s, 2 H, benzylic H), 4.53 (s, 8 H, NH2), 5.15 (s, 5 H, benzylic H + OH), 6.59 (s, 1 H, aromatic H), 6.63 (s, 2 H, aromatic H), 8.00 (s, 4 H, aromatic H), 8.19 (s, 2 H, aromatic H), 9.84 (s, 4 H, CONH). (H2NHNCO)8-[G-2]-OH (2). This was prepared via the same procedure from (EtO2C)8-[G-2]-OH and hydrazine. Yield: 66%. 1H NMR δ 4.30-4.70 (m, 18 H, benzylic H and NH2), 4.90-5.30 (m, 13 H, benzylic H + OH), 6.506.80 (m, 9 H, aromatic H), 8.01 (s, 8 H, aromatic H), 8.20 (s, 4 H, aromatic H), 9.84 (s, 8 H, CONH). 1 Abbreviation: MTX, methotrexate; MALDI-TOF, matrixassisted laser desorption ionization time-of-flight; DCC, N,N′dicyclohexylcarbodiimide; EDC, 1-[3-(dimethylamino)propyl]-3ethylcarbodiimide, PEG, poly(ethylene glycol).

Kono et al.

{(H2NHNCO)8-[G-2]}2-[C] (3). This was prepared via the same procedure (except DMSO used as solvent instead of DMF) from {(EtO2C)8-[G-2]}2-[C] and hydrazine. Yield: 94%. 1H NMR δ 4.51 (s, 32 H, NH2), 5.01 (s, 12 H, benzylic H), 5.14 (s, 16 H, benzylic H), 6.60-6.80 (m, 18 H, aromatic H), 7.00 (d, 4 H, J ) 8.7 Hz, aromatic H), 7.46 (d, 4 H, J ) 8.7 Hz, aromatic H), 7.98 (s, 16 H, aromatic H), 8.16 (s, 8 H, aromatic H), 9.79 (s, 16 H, CONH). Conjugation of Folic Acid to Hydrazide-Terminated Dendritic Macromolecules. Direct Condensation. To a solution of folic acid (0.28 g, 0.63 mmol) and triethylamine (0.27 mL) dissolved in DMSO (10 mL) were added 1-hydroxybenzotriazole (0.19 g, 1.41 mmol) and EDC (0.27 g, 1.41 mmol), and the solution was stirred for 1 h at room temperature. Then, (H2NHNCO)4-[G-1]OH 1 (0.040 g, 0.072 mmol) in DMSO (2 mL) was added and the mixture was stirred for 2 days at room temperature. Acetone was added to the reaction mixture, and a yellow precipitate formed. The precipitate was collected by filtration and dried under vacuum. The crude product was dissolved in water, and the pH was adjusted to ca. 9 using 1 N NaOH(aq) solution. The solution was loaded to a Sephadex G-25 column (40 cm × 2.5 cm) using water as effluent. Fractions containing the conjugate were collected, and the pH of the solution was adjusted to ca. 3 using 1 N HCl. The product precipitated out and was collected by filtration. The product was washed with water and dried under vacuum. Yield: 0.14 g. Active Ester Method. The active ester of folic acid was prepared via the method reported by Lee and Low (22). To a solution of folic acid (0.30 g, 0.68 mmol) and triethylamine (0.10 g, 1.0 mmol) dissolved in DMSO (10 mL) was added DCC (0.14 g, 0.68 mmol), and the solution was stirred for 1 h at room temperature. Then, Nhydroxysuccinimide (0.12 g, 1.0 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was filtered and the filtrate was precipitated into ether. The active ester of folic acid was collected by filtration, washed with dry THF, and dried under vacuum. The active ester of folic acid was dissolved in DMSO (25 mg/mL), and the hydrazide-terminated dendrimers dissolved in DMSO (10 mg/mL) was added. The solution was stirred for 5 days (for 1 and 2) and 10 days (for 3) at room temperature. Acetone was added to the reaction mixture, and a yellow precipitate formed. The precipitate was collected by filtration and dried under vacuum. The product was purified using a Sephadex G-25 column as described above. Conjugation of MTX to Hydrazide-Terminated Dendritic Macromolecule. To a solution of MTX (0.072 g) and triethylamine (0.050 mL) dissolved in DMSO (6 mL) was added DCC (0.066 g), and the mixture was stirred for 1 h at room temperature. Then, (H2NHNCO)8[G-2]-OH (0.020 g) in DMSO (1 mL) was added, and the resulting mixture was stirred for 2 days. Ether was added to the reaction mixture, and a yellow precipitate formed. The precipitate was collected by filtration and dried under vacuum. The product was purified using a Sephadex G-25 column as described above. Yield: 0.028 g. Synthesis of (EtO2C)8-[G-2]-PEG. To a dispersion of NaH (0.105 g) in dry THF (5 mL) was added PEG monomethyl ether (1.46 g) dissolved in THF (15 mL) dropwise at room temperature, and the solution was stirred for 2 h. Then, (EtO2C)8-[G-2]-Br (1.12 g) dissolved in THF (15 mL) was added and the reaction mixture was stirred for 2 h. The reaction mixture was filtered and the

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Figure 2. Structure of the hydrazide-terminated dendrimers.

crude product was precipitated by addition of ether to the filtrate. The crude product was loaded to silica gel column and washed with 15% THF/CH2Cl2. The eluent was changed to CH2Cl2, then 5% methanol/CH2Cl2. The product (EtO2C)8-[G-2]-PEG was eluted using 5% methanol/CH2Cl2. Yield: 0.58 g. Synthesis of (H2NHNCO)8-[G-2]-PEG. (EtO2C)8-[G2]-PEG (0.13 g) and hydrazine (2 mL) in DMSO (3 mL) were heated at 40 °C for 3 days. The product was precipitated by adding ether to the reaction mixture. The precipitate was dissolved in methanol, reprecipitated into ether, and dried under vacuum. Yield: 0.10 g. Synthesis of Folate-[G-2]-PEG Conjugate. (H2NHNCO)8-[G-2]-PEG (0.040 g) was allowed to react with folic acid (0.132 g) in the presence of EDC (0.058 g) and triethylamine (0.092 mL) in DMSO (10 mL) for 4 days as described above. The conjugate was precipitated by addition of ether, collected by filtration, and purified using a Sephadex G-25 column eluted with water. Yield: 0.080 g.

Water-Solubility Measurement. The water solubility of the folate-dendrimer conjugates was examined by measuring the turbidity of their aqueous solutions. The conjugate sample (1.2 mg) was dissolved in 10 mM phosphate buffer solution containing 140 mM NaCl (3 mL) under an alkaline condition (pH 9-11). The pH of the solution was adjusted to a given value by the addition of 1 N HCl, and the optical density of the solution was measured using a spectrophotometer (JASCO V-560). RESULTS AND DISCUSSION

1. Synthesis of Hydrazide-Terminated Dendritic Compounds. The selection of the functional groups used for conjugation of folic acid onto the dendrimer surface is important. In this work, we chose hydrazide groups because of their easy synthesis and high reactivity toward coupling with folic acid. Small dendritic polyvalent hydrazides have previously been used as cross-linkers for the preparation of hydrogels of hyaluronic acid (28). Starting from ester-terminated dendrimers, hydrazide

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Figure 4. 1H NMR spectrum for the folate-[G-1]-OH conjugate in DMSO-d6.

Figure 3. 1H NMR spectra for the ester-terminated (A) and the hydrazide-terminated (B) [G-2]2-[C] dendrimers.

groups could easily be introduced onto dendrimers by reaction with hydrazine. We prepared three different hydrazide-terminated dendritic compounds with 4, 8, and 16 hydrazide groups. Figure 2 shows the structure of the hydrazide-terminated dendritic compounds used in this work for the construction of conjugates. Characterization by 1H NMR confirmed the complete transformation of ester groups to hydrazide groups. Figure 3 shows the 1H NMR spectra for the precursor dendrimer {(EtO2C)8-[G2]}2-[C] and the corresponding hydrazide derivative {(H2NHNCO)8-[G-2]}2-[C]. Signals for ethyl groups appear at 1.42 and 4.42 ppm in the spectrum for the esterterminated dendrimer. In contrast, these signals disappeared completely after reaction with hydrazine, and a new signal corresponding to the protons from the hydrazide groups appeared at 4.50 and 9.78 ppm. The same changes in 1H NMR spectra were also observed in the transformation of the precursor dendrimers, (EtO2C)4[G-1]-OH and (EtO2C)8-[G-2]-OH, into the corresponding hydrazides, (H2NHNCO)4-[G-1]-OH and (H2NHNCO)8-[G2]-OH. 2. Conjugation of Folic Acid to Dendrimers. Conjugation of folic acid to (H2NHNCO)4-[G-1]-OH was carried out via condensation between the γ carboxyl group of folic acid and the hydrazide groups of the Scheme 1

dendrimer. Two methods were tested for this condensation (Scheme 1): direct condensation in the presence of the carbodiimide condensing agent EDC and reaction of the hydrazide groups with the active ester of folic acid as previously reported (21-23, 26). The conjugates were characterized by 1H NMR spectroscopy as shown in Figure 4. It is apparent that the spectrum of the conjugate contains signals originating from both folic acid and the dendrimer. From the integral ratio of the signal at 8.64 ppm, which corresponds to the proton at the 7-position of the pterin ring, to the signal at 5.15 ppm, which corresponds to the benzylic proton from the dendritic moiety, the average number of folate residues in the conjugate can be evaluated. By using excess of active ester of folic acid with the G-1 dendrimer, an average number of 3.6 folate molecules became attached to the dendrimer. However, when the conjugate was prepared via direct condensation, the average number of folate molecules per conjugate was 3.0. The lower number of folate per conjugate may be the result of reaction between the two carboxyl groups of one folic acid molecule and two hydrazide groups. Such coupling is possible since each of the two carboxyl groups of folic acid may be activated by EDC. After attachment of folic acid to the dendrimer by coupling between one carboxyl group and one hydrazide group, the other carboxyl group may react with second hydrazide groups on the same dendrimer, leading to a macrocyclic structure. Thus, the possibility of attachment of a folate residue via two hydrazide groups may be higher for the conjugation method involving carbodiimide-mediated direct conden-

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Figure 5. MALDI-TOF mass spectrum for the folate-[G-1]-OH conjugate. Table 1. Syntheses of Folate-[G-2]-OH and Folate-[G-2]2-[C] Conjugates

conjugate folate-[G-2]-OH folate-[G-2]2-[C]

the ratio of folic acid/ dendrimer in reaction mixture

folate residues per conjugate

8.0 15.2 24.0 48.0

3.2 7.3 8.2 12.6

sation. Therefore, the active ester method was used in the construction of all of the conjugates described in this work. The folate-[G-1]-OH conjugate was further characterized using MALDI-TOF mass spectrometry. Figure 5 depicts the MALDI-TOF mass spectrum for the conjugate prepared via the active ester method. The spectrum shows strong peaks at 2268 and 2247, which correspond to the [M + Na]+ ion and the [M + H]+ ion of the conjugate with four folate residues, respectively. In addition, relatively small peaks were observed at 1850 and 1826 corresponding to the [M + Na]+ ion and the [M + H]+ ion of the conjugate with three folate residues. Conjugation of folic acid to (H2NHNCO)8-[G-2]-OH and {(H2NHNCO)8-[G-2]}2-[C] using the active ester method was also examined. Preparation conditions and the average number of folate residues per conjugate are listed in Table 1. For folate-[G-2]-OH conjugate, the average number of folates per conjugate increases as the folic acid/ dendrimer ratio in the reaction mixture increases. Essentially complete functionalization of the hydrazide groups was achieved when 3 equiv of folic acid active ester were used for each hydrazide group. However, in the case of {(H2NHNCO)8-[G-2]}2-[C] dendrimer with 16 hydrazide groups, only an average of 12.6 folate residues were found to be attached to the dendrimer even after 10 days of reaction. When the same reaction was carried out for 5 days, only 10.0 folate residues could be attached to the dendrimer. These findings suggest that the terminal hydrazide groups of the {(H2NHNCO)8-[G-2]}2-[C] dendrimer are somewhat less accessible than those of the (H2NHNCO)8-[G-2]-OH, perhaps as a result of the increased steric congestion on the dendrimer surface, or

Figure 6. MALDI-TOF mass spectra for the folate-[G-2]-OH conjugate (A) and the folate-[G-2]2-[C] conjugate (B).

due to reason intrinsic to this demanding carbodiimidemediated coupling. The molecular weight of the conjugates was investigated by MALDI-TOF mass spectrometry. For the folate[G-2]-OH conjugate (Figure 6A), strong peaks are observed at a mass of 4614, which corresponds to the [M + Na]+ ion of the conjugate with eight folate residues. Also, several peaks were observed around 4177 that correspond to the conjugate with seven folate residues. The existence of peaks between those for conjugates with seven and eight folate residues suggests the occurrence of fragmentation of the conjugate residues during measurement. It has been reported that folate residues fragment readily with formation of pteridinyl radicals with a molecular mass of 176 Da (26). Therefore, peaks observed around 4417 are likely due to the molecular ions and Na adducts of the fragments of conjugates with eight folate residues that have lost a single pteridinyl radical. It is not unlikely that this kind of fragmentation could occur during the MALDI-TOF measurement since higher power is frequently used for such high MW compounds. Small peaks around 5033 correspond to the conjugate containing eight

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Figure 7. Turbidity of solutions of the folate-[G-2]-OH conjugate (2); the folate-[G-2]2-[C] conjugate (1); and pure folic acid (b) in 10 mM phosphate-buffered solutions containing 140 mM NaCl at varying pH values. The ordinate shows the optical density of the solution at 550 nm.

folate residue at the chain ends plus one folate residue at the focal point. The MALDI-TOF spectrum for the folate-[G-2]2-[C] conjugate with 12.6 folate residues as measured by NMR analysis is shown in Figure 6B. The conjugate gave a broad peak centered at a mass of 7100 that corresponds to a conjugate with an average of 10.7 folate residues. 3. Water Solubility of the Folate-Dendrimer Conjugates. Since the folate-dendrimer conjugates have carboxyl groups on their surface, they are expected to be soluble in water under conditions where these groups are charged. Thus, pH dependence of the water solubility of the conjugates was examined. Figure 7 shows the turbidity of the solutions of the conjugates and folic acid at varying pH values. The solution of folic acid was not turbid under either neutral or alkaline conditions. However, its turbidity increased drastically near pH 5.4, indicating precipitation of the folic acid. Potentiometric titration of an aqueous solution of folic acid at pH 8.3 using 10 mM HCl showed that 2 equiv of HCl was consumed to neutralize the folate anion near pH 5.4, indicating that both the R- and the γ-carboxyl groups of folic acid were neutralized at this pH value where precipitation is expected to take place. For the folate-[G2]-OH conjugate, the solution was completely clear under alkaline conditions, showing that the conjugate is soluble in water under such conditions. However, an increase in turbidity was observed near pH 7.4. Similarly, an increase in turbidity was also seen for the solution of folate[G-2]2-[C] conjugate at the same pH. Agarose gel electrophoresis analysis of the folate-[G-2]2-[C] conjugate showed that the conjugate was negatively charged at pH 8.0. Therefore, it is considered that these conjugates indeed acquired water solubility as a result of the presence of a negative charge on the remaining carboxylate group of the folate residues. Protonation of these carboxylate groups results in the precipitation of the conjugates. Precipitation of these conjugates took place at higher pH than observed for free folic acid, probably due to the hydrophobic nature of the dendritic moieties. 4. Conjugation of MTX. Because methotrexate (MTX), a widely used antitumor drug, is structurally similar to folic acid, coupling of MTX to the dendritic macromolecules is expected to occur through the same process. Conjugation of MTX to synthetic polymers has already been attempted and the MTX-polymer conjugates have shown antitumor activity or cell growth inhibitory activity (29, 30). The conjugation of MTX to hydrazide terminated dendrimer (H2NHNCO)8-[G-2]-OH was examined briefly to demonstrate its feasibility. Using an

Figure 8. MALDI-TOF spectra of the ester-terminated [G-2]PEG (A) and the folate-[G-2]-PEG conjugate (B).

MTX/dendrimer (mole/mole) ratio of 10.4, the 1H NMR of the product showed that an average of 4.7 MTX residues/conjugate was bound. 5. Folate-dendron-PEG Conjugates. The G-2 dendron (EtO2C)8-[G-2]-OH possesses one hydroxyl group at the focal point, and this group may be used for the attachment of another molecule. This might be a targeting moiety or a solubilizing group. In a first exploration, we examined the introduction of a PEG chain at the focal point of the dendrimer since this PEG chain is expected to modify the behavior of the conjugate in the body and increase the overall water solubility of the conjugate (31, 32). The hybrid molecule (EtO2C)8-[G-2]-PEG was prepared via a Williamson ether synthesis involving (EtO2C)8[G-2]-Br and a PEG monomethyl ether with Mn 2000 and a polydispersity of 1.01. Figure 8A shows the MALDITOF spectrum of the resulting (EtO2C)8-[G-2]-PEG. A series of peaks centered at 3294 were observed. These correspond to the hybrid molecule with a PEG chain of MW 1968. The mass difference between two adjacent peaks in the MALDI spectrum is 44, which is the mass of one oxyethylene repeating unit. Unfortunately, the PEG sample is not monodisperse and its effect on broadening the dispersity of the dendrimer is quite significant. The hydrazide derivative of the hybrid and its subsequent folate conjugate were prepared via the same procedure as described above without optimization for complete coupling. The MALDI-TOF spectrum of the folate-[G-2]-PEG conjugate shown in Figure 8B consists of a broad peak centered at 4000, which corresponds to the conjugate with two folate residues only. This result demonstrates that the hydrazide groups of the dendrimer-PEG hybrid are still reactive and available for conjugation, although their reactivity appears to be reduced by the introduction of PEG chain. Further studies with optimized dendritic structure will focus on increased functionalization of such hybrid structures. CONCLUSION

Our work has shown that folate residues could easily be conjugated to the chain ends of a hydrazide-terminated polyether dendrimer. The folate-dendrimer conju-

Design of Dendritic Macromolecules

gates were soluble in phosphate buffer solution at physiological pH. The folate-dendrimer conjugates possess hydrophobic interior and hence might be useful to entrap hydrophobic drugs. Because these molecules have folate residues on their surface, they are expected to exhibit a strong affinity for tumor cells that overexpress folate receptor on the cell surface. Thus, these types of conjugates may potentially be useful as drug carriers with targeting specificity. In addition, the anticancer drug MTX has been successfully attached to a dendrimer. Although the toxicology of the polyether dendrimers remains to be clarified, these molecules or analogues thereof based on more biocompatible dendritic building blocks may be used in the design of a sophisticated type of multivalent drug carriers. Interactions of the conjugates with cells are currently under investigation. ACKNOWLEDGMENT

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