Synthetic PAMAM–RGD Conjugates Target and Bind To Odontoblast

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Synthetic PAMAM–RGD Conjugates Target and Bind To Odontoblast-like MDPC 23 cells and the Predentin in Tooth Organ Cultures Elliott Hill,*,† Rameshwer Shukla,‡ Steve S. Park,‡ and James R. Baker, Jr.‡ Departments of Biologic and Materials Sciences and Internal Medicine, University of Michigan, Ann Arbor, Michigan. Received January 22, 2007; Revised Manuscript Received July 30, 2007

Screening techniques now allow for the identification of small peptides that bind specifically to molecules like cells. However, despite the enthusiasm for this approach, single peptides often lack the binding affinity to target in vivo and regulate cell function. We took peptides containing the Arg-Gly Asp(RGD) motif that bind to the RVβ3 integrin and have shown potential as therapeutics. To improve their binding affinity, we synthesized polyamidoamine (PAMAM) dendrimer–RGD conjugates that that contain 12–13 copies of the peptide. When cultured with human dermal microvessel endothelial cells (HDMEC), human vascular endothelial cells (HUVEC), or odontoblast-like MDPC-23 cells, the PAMAM dendrimer conjugate targets this receptor in a manner that is both time- and dose-dependent. Finally, this conjugate selectively targets RGD binding sites in the predentin of human tooth organ cultures. Taken together, these studies provide proof of principle that synthetic PAMAM–RGD conjugates could prove useful as carriers for the tissue-specific delivery of integrin-targeted therapeutics or imaging agents and could be used to engineer tissue regeneration.

INTRODUCTION Biologically inductive materials have revolutionized our concept of restorative medicine and reconstructive surgery (1). This comes from the recognition of the potential of restoring the form and function of lost tissues by engineering approaches using the identical type of functional tissue that was lost. Though relatively few of these materials have been approved by the Food and Drug Administration (FDA), more than 70 companies are spending a total of $600 million per year to develop new products (2–5). In the context of oral facial tissues and dentition, a primary effort involves developing materials that do not elicit an immune response or will camouflage structural deficiencies. While some advances in this area have been made with dental filling materials that release fluoride, or wound dressings that release regenerative growth factors, the molecular cues that enhance and maintain a healthy dental pulp and periodontal ligament are largely unknown. Extensive numbers of studies have introduced the concept that, in general, tissue health and homeostasis are regulated though the interplay of a variety of signals including insoluble signaling molecules, insoluble ligands, mechanical cues, and cell to cell interaction (6–8). Integrin adhesion receptors play a crucial role in regulating interactions between cells and the extracellular matrix (ECM) (9). Integrin activation initiates multiple intracellular signaling pathways and results in regulation of cell functions such as motility, proliferation, and differentiation (10). One such integrin-binding peptide contains the ArgGly Asp (RGD) motif that commonly binds to extracellular matrix proteins. Previous studies have demonstrated that proteins modified with RGD peptides bind with high affinity to Rvβ3 receptors (11–13). The Rvβ3 integrin is found on the luminal surface of endothelial cells only during angiogenesis (14). RGD peptides have been used to deliver doxorubicin (15) and * Corresponding author. Elliott Hill, Division of Biologic and Materials Sciences, 1011 N. University, University of Michigan, Ann Arbor, MI 48109, Tel: 734-647-2110, e-mail: [email protected]. † Department of Biologic and Materials Sciences. ‡ Department of Internal Medicine.

proapoptotic peptides (16) to the tumor vasculature in targeted chemotherapy. Taken together, these data illustrate the important role RGD plays in regulation of normal cell function. In addition, this important ligand can be potentially utilized to target therapeutic molecules to tissues that express the Rvβ3 receptor. In this regard, antibody and peptide inhibitors of Rvβ3 integrins are currently in phase I/II clinical trials for the inhibition of angiogenesis in cancer (17). There also has been growing interest in the use of polymers as nonviral vectors for the delivery of targeting and or therapeutic molecules (18–22). One such polymer, polyamidoamine (PAMAM) dendrimers, have been synthesized as building blocks for conjugation to biological molecules, including RGD (23). PAMAM dendrimers are water-soluble and biocompatible macromolecules that have been coupled to many biological molecules such as proteins, synthetic drugs, and small molecules. These dendrimers can be coupled to multiple RVβ3selective ligands to target tumor-associated capillary beds and allow the delivery of cytotoxic agents to kill the new vessels (6, 24, 25). Also, these dendrimer–RGD conjugates may provide useful building blocks for the creation of new biomaterials in dental applications such as adhesive agents for dental implants or delivery vectors for bone morphogenic proteins and transforming growth factors. In the present work, we describe the synthesis and characterization of a generation 5 dendrimer conjugated to c(RGDyK) labeled with fluorescein. In addition, we examine the binding properties and cellular uptake of this conjugate in endothelial cells, and we report unique binding of the dendrimer–RGD conjugate to the predentin of human tooth organs and dental pulp-like MDPC-23 cells.

EXPERIMENTAL PROCEDURES General. G5-PAMAM dendrimer was prepared at the Michigan Nanotechnology Institute for Medicine and Biological sciences, University of Michigan, and was analyzed extensively by 1H and 13C NMR, matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry, high-performance liquid chromatography (HPLC), gel-permeation chromatography (GPC), and polyacrylamide gel electrophoresis

10.1021/bc0700234 CCC: $37.00  2007 American Chemical Society Published on Web 10/31/2007

Synthetic PAMAM–RGD Bind to MDPC 23 Cells

(PAGE) (26). Prepacked Sephadex G-25 PD-10 columns were purchased from Amersham Pharmacia Biotech (Piscataway, NJ) and equilibrated with degassed eluting buffer before sample introduction. Synthesis of G5-Ac (1). G5 amine dendrimer (0.265 g, 0.0099 mmol) and triethyl amine (0.088 g, 0.8635 mmol) were dissolved in 30 mL anhydrous MeOH and allowed to stir for 30 min. A solution of acetic anhydride (0.076 g, 0.744 mmol) in anhydrous MeOH (15 mL) was added dropwise while stirring. The reaction mixture was allowed to stir overnight at room temperature. After evaporation of the solvent, the residue was dissolved in H2O and dialyzed in 10 000 MWCO regenerated cellulose dialysis bags initially against PBS buffer, followed by water. The partially acetylated dendrimer was lyophilized to give a colorless powder (0.270 g, 91.2%). The molecular weight of synthesized G5-PAMAM dendrimer was found to be 26 530 g/mol by GPC, and the average number of primary amino groups was estimated to be 108 by potentiometric titration. The average number of acetyl groups (80) has been determined on the basis of a 1H NMR calibration curve drawn by plotting a ratio of acetyl protons and sum of all methylene protons vs degree of actetylation (27). 1H NMR (300 MHz, CDCl3): δ ) 1.8 (s, 240H), 2.32–2.38 (m, 499H), 2.51–2.52 (m, 260H), 2.65–2.80 (m, 500H), 3.15 (br m, 62H), 3.4 (s,br, 596H), 3.45 (s, 62H). MS (MALDI): 300 050. G5-Ac-Fl (2). To G5-Ac (102.05 mg, 3.4 mmol) in DMSO (10 mL) was added FITC (5.8 mg, 15.0 mmol) in DMSO (2 mL) dropwise. The reaction was allowed to stir overnight. The reaction mixture was diluted 1:1 in PBS, and free dye was separated from conjugate by gel filtration on a G-25 Sephadex column. The eluted conjugate was concentrated using a centriprep device (MWCO 10 000) and dialyzed against PBS and H2O before lyophilization. 1H NMR (300 MHz, CDCl3): δ ) 1.8 (s, 240H), 2.32–2.38 (m, 499H), 2.51–2.52 (m, 260H), 2.65–2.80 (m, 500H), 3.15 (br m, 62H), 3.4 (s,br, 596H), 3.45 (s, 62H), 6.25 (br s, 16H), 7.02 (br s, 8H), 7.5 (br s, 8H), 7.71 (br s, 8H). MS (MALDI): 34 470. G5-Ac-Fl-glutarate (3). Glutaric anhydride (0.0029 g, 0.0257 mmol) dissolved in anhydrous MeOH (2 mL) was added dropwise to a solution of G5-Ac80-Fl4 (0.0203 g, 0.0006 mmol) and TEA (0.0026 g, 0.0257 mmol) in anhydrous MeOH (18 mL) while stirring, and the reaction mixture was allowed to stir for another 24 h at room temperature. The solvent was evaporated in vacuo, and the residual material was dissolved in H2O, purified by extensive ultrafiltration against PBS and H2O using a centricon device (10k MWCO), then lyophilized. 1 H NMR (300 MHz, CDCl3): δ ) 1.8 (s, 240H), 1.7 (m, 56h), 2.32–2.38 (m, 499H), 2.2 (m, 112H), 2.51–2.52 (m, 260H), 2.65–2.80 (m, 500H), 3.15 (br m, 62H), 3.4 (s,br, 596H), 3.45 (s, 62H), 6.25 (br s, 16H), 7.02 (br s, 8H), 7.5 (br s, 8H), 7.71 (br s, 8H). MS (MALDI): 38 370. G5-Ac-Fl-glutarate-c(RGD) (4). An active ester was prepared by reacting G5-Ac80-Fl4-glutarate (12.26 mg, 0.00036 mmol) in H2O (2.0 mL) with EDC (1.02 mg, 0.00533 mmol) for 3 h. RGD (0.005 g, 0.0036 mmol) in DMSO (0.5 mL) was added dropwise to the above solution and allowed to stir overnight. The product was purified by extensive ultrafiltration against PBS buffer (pH 7.4) and H2O using a centricon device (10k MWCO), then lyophilized. 1H NMR (300 MHz, CDCl3): δ ) 1.8 (s, 240H), 2.32–2.38 (m, 499H), 2.51–2.52 (m, 260H), 2.65–2.80 (m, 500H), 3.15 (br m, 62H), 3.4 (s,br, 596H), 3.45 (s, 62H), 6.25 (br s, 16H), 7.02 (br s, 8H), 7.27 (br, s, 40H), 7.5 (br s, 8H), 7.71 (br s, 8H). MS (MALDI): 42 416. Cell Cultures. L1210 cell lines were grown in RPMI medium (with folate), supplemented with 10% FBS, 1% penicillin/ streptomycin. and 50 nM L-glutamine. These cells were grown in suspension, and the media were changed every other day.

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MDPC-23 cell cultures were grown in high-glucose DMEM (Gibco) supplemented with 1% P/S and 10% FBS. HDMEC and HUVEC were grown in EGMV-2 media supplemented with 5% FBS, 1% gentamycin, 1% VEGF, 1% ascorbic acid, 0.5% hydrocortisone, 3% hFGF, 1% IGF, and 1% hEGF. HUVEC, HDMEC, and MDPC-23 cells were incubated as a monolayer at 37 °C and 5% CO2 and grown in 75 cm3 flasks until 85% confluent (2 × 106 cells/flask). Approximately 0.5 × 106 cells were seeded onto 6 well culture dishes (MatTek Corp., Ashland, MA) and cultured in 3 mL of medium at least 24 h before the initiation of experimental conditions. Samples were examined quantitatively for G5-conjugate uptake via flow cytometry (Coulter-Epiflow) at time points ranging from 1 h to 7 days normalized to controls that were prepared for each time point. Cell Targeting and Binding Assay. The cellular uptake of G5-Ac-Fl-RGD conjugate was measured in human dermal microvessel endothelial cells (HDMEC), human vascular endothelial cells (HUVEC), fetal mouse molar papillae derived (MDPC-23) cells, and mouse lymphocytic leukemia cell line (L1210) cells. Endothelial cells were cultured in ECM-2 media supplemented with fetal bovine serum, EGF, VEGF, IGF, and IL-8. All other cell lines were cultured in RPMI media (GIBCO) supplemented with 10% FBS/1% P/S. A time course for G5 (100 nM) binding was done, and results were analyzed via flow cytometric analysis (BectonDickinson FACScan analyzer, Fullerton, CA). The mean fluorescence of 10 000 cells was quantified. Confocal microscopic analysis was performed on HDMEC cells cultured in 35 mm glass-bottomed culture dishes (MatTek Corp., Ashland, MA) using a Carl Ziess confocal microscope. Fluorescence and differential interference contrast (DIC) images were collected simultaneously using an argon laser and FluoView software. Targeting of Tooth Organ Cultures. Adult human molars were extracted, soft tissue was carefully removed, and teeth were stored in 70% ethanol. One millimeter cross sections of medial and distal roots were made with a diamond wheel saw (South Bay Technology Inc., Temple City, CA) at a point 1 mm apical to the cement–enamel junction. Pulp contents were carefully removed, and the sections were placed in 35 mm glass-bottomed culture dishes (MatTek Corp., Ashland, MA). The targeted binding of G5-Ac-Fl-RGD conjugates was examined by coculturing the conjugate with human molar tooth slice sections under three different conditions: (1) tooth slices cocultured with G5-Ac-Fl-glutarate (with out the targeting RGD moiety), (2) tooth slices cocultured with G5-Ac-Fl-RGD, (3) tooth slices preincubated for 1 h with free RGD peptide and then cocultured with G5-Ac-Fl-RGD conjugate. All cocultures were incubated for 3 h; cultures were rinsed with sterile water (3×) and analyzed via confocal microscopic analysis using a Carl Ziess confocal microscope. Fluorescence and differential interference contrast (DIC) images were collected simultaneously using an argon laser and FluoView software.

RESULTS The conjugate synthesis and characterization was performed as follows: (1) The synthesis and partial surface modification of G5 dendrimer with acetic anhydride (75 mol %) in the presence of triethylamine (Figure 1) was initiated via the use of 75 molar excess of acetic anhydride. This reaction leaves approximately 25% of the primary amines on the surface for further conjugation. In addition, it prevents aggregation, intermolecular interaction, and problems arising from decreased solubility. The degree of acetylation and the purity of the G5 dendrimer conjugate were analyzed using 1H NMR spectroscopy, which shows a distinct signal for the terminal NHCOCH3 protons of the dendrimer. The degree of acetylation was measured by comparing the ratio of NHCOCH3 protons with

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Figure 2. HPLC analysis used to compare G5-FITC to G5-FITC-RGD dendrimer showed a peak typical of a homogeneous peptide conjugate λ max 285 nm. In addition, the difference in retention time between the final conjugate the precursor material gives indirect evidence that RGD peptide conjugation was successful.

Figure 1. Schematic of PAMAM dendrimer generation 5 (G5)-FLRGD synthesis.

the sum of all methylene protons in the dendrimer to a calibration curve as described previously (27). (2) To detect the conjugate by flow cytometry or for visual imaging via confocal microscopy, a detectable fluorescent probe is required. We labeled the partially acetylated dendrimer with fluorescein isothiocyanate (FITC). The partially acetylated dendrimer was reacted with 5 mol equiv of FITC dissolved in dimethyl sulfoxide (DMSO). After purification, the conjugate was characterized using 1H NMR spectroscopy and high-performance liquid chromatography (HPLC) (Figure 2). The 1H NMR spectrum of the conjugate shows broad signals in the aromatic region for the FITC (data not shown). The number of dye molecules attached to the dendrimer was calculated to be ∼4 based on UV/vis spectroscopy and 1H NMR. (3) Remaining surface amines on the dendrimer were converted to carboxylic acid by reacting with excess glutaric anhydride in MeOH, in the presence of TEA as base. This conversion provides a carboxyl terminal linker while eliminating primary amines that could cause nonspecific charge interactions. The loss of the methylene peaks (–CH2CH2NH2) at δ 2.97 and 3.32 ppm and the emergence of the new methylene peaks (-NHCO(CH2)3COOH) at δ 1.65 and 2.05 ppm indicate complete conversion of the remaining terminal amines to give G5-Ac-Fl-glutarate. (4) Finally, the carboxyl groups were activated by reacting with EDC followed by the addition of 10 equiv of c(RGDyK) to give the dendrimer–RGD conjugate. The

Figure 3. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) analysis of G5-FL conjugate before and after RGD conjugation revealed a difference in mass consistent with the successful conjugation of 13 RGD peptides per G5-FL conjugate.

dendrimer–RGD conjugate was purified by ultrafiltration and lyophilized. The 1H NMR spectrum of the conjugate shows overlapping signals in the aromatic region for both the FITC and phenyl ring of the peptide apart from the expected aliphatic signals for the dendrimer overlapping with some aliphatic signals from the peptide. The number of peptides was calculated to be 12–13 peptides per dendrimer based on matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (Figure 3) and 1H NMR. The cellular uptake of G5Ac-Fl-RGD examined in MDPC23 odontoblast-like cells, via flow cytometric analysis, revealed time-dependent uptake of G5 conjugate (Figure 4). Measurable binding was observed at 30 min with an increased uptake beginning at 3 h that seemed to become constant at 6 h. The binding was also dose-dependent with a 10-fold increase seen at 300 nM G5RGD as compared to that seen at 30 nM (Figure 4b). To examine specific binding of G5-Ac-Fl-RGD to HDMEC, HUVEC, and MDPC-23 cells, these cells were cultured with and without preincubation with free RGD peptide and examined via flow cytometry (Figure 5). This approach also demonstrated that when incubated for 6 h at 37 °C the G5 conjugate exhibited RGD-dependent binding to HDMEC, HUVEC, and MDPC-23 cells. The binding effect was not enhanced by longer incubation times, and binding could be completely inhibited when the cells

Synthetic PAMAM–RGD Bind to MDPC 23 Cells

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Figure 4. Flow cytometry data of time dependent binding of G5-Ac-Fl-RGD to MDPC-23 (odontoblast like) cells, shows targeted binding after 3 h incubation that increased and stabilized at 6 h (a). The binding demonstrated at 6 h was dose dependent (b).

Figure 5. In vitro binding of G5-FL-RGD conjugate to HUVEC (solid block), HDMEC (striped block), or odontoblast-like MDPC-23 (shaded block) cells for 6 h was eliminated by preincubation with free RGD peptide. Negative control L1210 cells exhibited no binding (white block).

were preincubated with free RGD peptide. Finally, no binding was observed when the conjugate was incubated with L1210 cells, cells known to lack RGD binding affinity. To confirm the flow cytometry data, we repeated the assessment of time-dependent uptake of G5-FL-RGD and its target specific binding to MDPC-23 cells using confocal microscopy. MDPC-23 cells were cultured in 35 cm culture dishes and exposed to 300 nM G5-Fl-RGD conjugate for 30 min, 3 h, or 6 h. The cells demonstrated time-dependent uptake of the conjugate where, at 30 min, the confocal images revealed

no apparent internalization of the conjugate (Figure 6a), while after 3 h with the conjugate, the cells showed modest uptake of the G5-FL-RGD (Figure 6b). After 6 h in coculture, the conjugate appears throughout the cytoplasm in a characteristic punctuate pattern (Figure 6c). When HDMEC, HUVEC, and MDPC-23 cells were cultured and exposed to 300 nM G5-FLRGD with and without preincubation with free RGD peptide, confocal examination confirmed conjugate binding and inhibition by free RGD peptide preincubation (Figure 7). Finally, when the G5-FL-RGD conjugate was incubated for 3 h in an acellular tooth organ culture, binding to the predentin was clear and robust (Figure 8a–c). This binding was completely eliminated when cultures were preincubated with 300 nM free RGD peptide for 30 min prior to G5 conjugate incubation (Figure 8d–f). Taken together, these data suggest that the G5FL-RGD conjugate selectively binds to the RVβ3 integrin present in the predentin.

DISCUSSION Tissue engineering is an interdisciplinary field that applies the principles of engineering and the life sciences to the development of biological substitutes that repair, maintain, or improve tissue function that has been lost, impaired, or damaged due to injury, trauma, disease, or cancer (28). Currently, in dental medicine, restoration of impaired tooth function depends on materials that share few of the chemical characteristics of natural tooth (29–31). About 90% of the organic matrix of tooth structure is type I collagen (32, 33). Much work has been done investigating the role of noncollagenous proteins in tooth development (29, 31, 34, 35) and pulp biology (9, 36, 37). To

Figure 6. Confocal images of cultured MDPC-23 cells cocultured with G5-Ac-Fl-RGD for 30 min (a), 3 h (b), and 6 h (c) show uptake of this targeted dendrimer in a time-dependent manner.

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Figure 7. Confocal photomicrographs of odontoblast-like MDPC-23 cells (a,d), human umbilical vein endothelial cells HUVEC (b,e), and human dermal microvascular cells HDMEC (c,f) (60×) show no binding or uptake of G5-Fl-RGD conjugate when cells are preincubated with free RGD peptide (a–c), while binding and uptake of this conjugate are clearly seen in cells not preincubated with free RGD peptide (d–f).

Figure 8. Confocal photomicrographs (20×) of human adult tooth slices (a), when cocultured with RGD targeted G5-Ac-Fl-RGD conjugate (b,c), demonstrate targeted binding of G5-Fl-RGD conjugate to predentin. This effect is not seen in G5-Fl conjugate without the RGD targeting moiety (d,e), and eliminated when coculture of tooth slice and G5-Ac-Fl-RGD was preincubated with free RGD (f) peptide prior to coculture.

build on this work, the present studies examine possible roles for ligands that bind to the organic matrix, specifically the RVβ3 integrin. Integrins constitute a 24-member family of cell adhesion molecules. Each of these molecules consist of two subunits, R and β, which are noncovalently associated (38). These subunits bind with specificity to integrin receptors either on extracellular matrix molecules or on the cell surface, thus mediating both cell–cell and cell–matrix interactions (38). Strategies focusing on targeting integrin adhesion receptors with short bioadhesive oligopeptides have demonstrated in vitro control of cell adhesion and differentiation. More importantly, these peptides have also demonstrated enhancement of tissue healing responses in vivo including bone formation (39), nerve regeneration (40, 41) and corneal tissue repair (42, 43). Here, we extend this work by showing that synthetic PAMAM–RGD conjugates can be designed to present the RGD motif to cells known to express the RVβ3 integrin. By characterizing these conjugates via HPLC, MALDI-TOF, and 1H NMR analysis, we present direct and

indirect evidence that RGD peptides were successfully conjugated to a PAMAM-FITC dendrimer platform. In addition, when cultured in vitro with human dermal microvessel endothelial cells (HDMEC), human vascular endothelial cells (HUVEC), and odontoblast-like MDPC-23 cells, we demonstrate both timeand dose-dependent uptake of these conjugates into all three cell types that can be measured quantitatively and visualized via confocal microscopy. We also presented compelling data on the specificity of this binding, which was completely prevented by preincubation with free RGD peptide. Taken together, these data indicate that the G5-FL-RGD conjugate described here selectively binds to cells expressing the RVβ3 receptor. The RGD motif has been widely studied in targeting molecule cancer therapeutics (11), and more specifically in targeting the neovasculature of tumor-enhanced angiogenesis (12, 23, 44). And, while the role of RGD peptides in angiogenesis is established, the present work is the first study to our knowledge to demonstrate selective binding of a synthetic polymer con-

Synthetic PAMAM–RGD Bind to MDPC 23 Cells

jugate to the RVβ3 integrin binding sites of the predentin matrix of human teeth. The binding, seen via flow cytometry and confirmed by confocal microscopy, suggests robust expression of the RVβ3 integrin and uptake of the RGD conjugate in odontoblast-like cells. We further demonstrated that in both the odontoblast-like cells and the predentin of adult tooth organs, this binding is eliminated by preincubation with free RGD peptide. Taken together, these data substantiate a role for this receptor in endothelial cells as well as suggest a possible role in tissue associated with the dental pulp. These data suggest that targeted binding of RGD to tooth predentin may be an important event in pulp biology. The induction of bone and dentin formation by molecules found in the dental matrix of adult teeth was established several decades ago (45, 46). However, the molecular signals responsible for this regenerative response and their signaling pathways are still being studied (29, 34, 47–50). In addition, the role of one of the most abundant molecules in the dentin matrix of adult teeth (RGD) is unknown. The current study argues that this often overlooked molecule may play an important role in odontoblast regeneration and/or homeostasis. In addition, we show that synthetic polymers can be used to deliver RGD to both pulplike cells and their mineralized matrix in a way that specifically engages the RVβ3 receptor. Since current therapies include mechanical debridment of tooth dentin, diseased via tooth decay or periodontal infection, and this process exposes dentinal tubules and in some cases predentin, the development of functional polymers that can exploit native binding molecules (integrins) for the nonviral delivery of therapeutic drugs makes this therapy a realistic goal for the future.

ACKNOWLEDGMENT The authors gratefully acknowledge financial support of the National Cancer Institute through contract N01-CM-97065-32.

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