Two-Step Synthesis of Multivalent Cancer-Targeting Constructs

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Biomacromolecules 2010, 11, 160–167

Two-Step Synthesis of Multivalent Cancer-Targeting Constructs Jill M. Stukel,† Ronald C. Li,‡ Heather D. Maynard,‡ and Michael R. Caplan*,† School of Biological and Health Systems Engineering, Center for Interventional Biomaterials, Arizona State University, Tempe, Arizona, and Department of Chemistry and Biochemistry, University of California, Los Angeles, California Received September 9, 2009; Revised Manuscript Received October 30, 2009

Selective targeting of constructs to pathological cells by conjugating one or more ligands for an overexpressed receptor has been proposed to enhance the delivery of therapeutics to and imaging of specific cells of interest. Previous work in our lab has demonstrated the efficacy of targeting glioblastoma cells with a multivalent, biomacromolecular construct targeted to the R6β1-integrin. However, solid-phase synthesis of this construct was inefficient in terms of cost and number of steps. Here we show proof-of-concept of a two-step synthesis that can be used to create similar constructs targeted to glioblastoma cells. Specifically, a well-defined aldehyde side chain polymer was synthesized and oxime chemistry was employed to conjugate ligands specific for the R6β1integrin. These constructs were then tested in competitive binding, fluorescence binding, and toxicity assays, through which we demonstrate that constructs are multivalent, preferentially target glioblastoma cells, and are nontoxic. Rapid, potentially low-cost synthesis of targeting constructs will enable their use in the clinic and for personalized medicine.

Introduction Glioblastoma multiforme is a common form of brain cancer that affects approximately 3 out of 100 000 people each year.1 The prognosis for survival is typically less than 12 months. This is due to cells that migrate away from the primary tumor to establish secondary tumors so that even if the primary tumor is resected these secondary tumors remain. Systemic chemotherapy is not completely effective in treating these because most drugs have limited permeability through the blood-brain barrier; therefore, some of these secondary tumors survive, spread migrant cells of their own, and eventually result in death. We and others2-7 have proposed injecting targeting constructs directly into brain tissue to address this problem. In particular, our laboratory has studied biomacromolecular, multivalent constructs, which can perfuse freely in brain tissue and selectively target glioblastoma cells.6,8 Previously, our laboratory synthesized these constructs using traditional peptide synthesis chemistry, which is time-consuming and expensive. Here we seek to create similar constructs in a two-step process that is both quick and potentially inexpensive enough to be a viable treatment strategy. Recent research in targeting drugs or imaging contrast agents to cancer seeks to exploit receptor overexpression by tumor cells relative to healthy cells.9,10 Potential advantages include minimizing adverse side effects by achieving the same concentration at the cancer cell while decreasing total dose, achieving earlier diagnosis of cancer by increasing imaging contrast between tumor and normal tissue, and increasing treatment efficacy by keeping the overall dose the same, resulting in increased concentration in the tumor tissue. In the brain, where systemic drug delivery is much less effective because of the blood-brain barrier, the use of these targeting constructs may allow effective treatment of glioblastoma where current treatments have failed. * To whom correspondence should be addressed. Tel: (480) 965-5144. Fax: (480) 727-7624. E-mail: [email protected]. † Arizona State University. ‡ University of California.

Current technologies that have accomplished varying degrees of success in targeting cancer include antibody-conjugated drugs or radioisotopes,11-13 quantum dots (QDs),14-17 and biomacromolecular constructs.6,18-20 For the case of glioblastoma specifically, antibody-conjugated and interleukin-conjugated drugs are currently in clinical trials.4,5,21 We and others have used biophysical models to predict that the specificity that targeting constructs demonstrate for the tumor cells can be substantially increased by making such targeting constructs multivalent.8,22-24 Previously, Rosca et al.6 synthesized trivalent, biomacromolecular constructs targeted to R6β1-integrin via a dodecapeptide ligand derived from laminin (TWYKIAFQRNRK).25,26 This synthesis was performed using sequential solid-phase synthesis steps, as is typically done in peptide synthesis; however, this is both time-consuming and expensive. Here we propose to create similar multivalent constructs through atom transfer radical polymerization (ATRP) to form poly(3,3-diethoxypropyl methacrylate) (PDEPMA), which can then be conjugated to targeting ligands using an oxime ligation scheme.27-29 The polymer can be created, stored, and then mixed with aminooxy ligands to form the desired multivalent biomacromolecular constructs. Aminooxy-functionalized drug or imaging agent can be mixed with the peptide in this first step to incorporate these into the construct. A second step is necessary to react aminooxytetra(ethylene glycol) (TEG) on the remaining side groups both to quench these sites and to increase the solubility of the final construct. Alternatively, all components can be mixed together, as we have found that the composition in the final polymer mirrors the feed ratios.28 In this study we create aminooxy-functionalized TWYKIAFQRNRK peptides that are then reacted with PDEPMA that has been presubjected to acidic conditions to reveal aldehyde moieties. Three different ratios of peptide to polymer are used in an attempt to achieve different valence constructs. The products, after subsequent reaction with TEG, are characterized with NMR. We then perform three biological assays to characterize the function of these constructs: a competition assay

10.1021/bm9010276  2010 American Chemical Society Published on Web 11/19/2009

Multivalent Cancer-Targeting Constructs

Figure 1. Schematic of synthesis scheme: (A) acetal protected polymer backbone, represented by straight lines; (B) deprotected polymer side chains shown with stars; (C) partial side chains reacted with peptide (wavy lines); (D) remainder of side chains conjugated with Alexa Fluor 488 molecule (closed circle) and TEG (open circles).

to determine construct avidity, a fluorescence assay to determine specificity of binding between glioma and astrocyte (normal) cells, and a cell viability assay to determine toxicity. Through this synthesis and characterization, we established the proofof-principle for a generalizable method of creating economical constructs. This method can be utilized to target any pathology in which a cell surface receptor is overexpressed.

Experimental Details Construct Synthesis. A protocol similar to Li et al.28 was followed. In brief, to deprotect the side groups, ∼40 mg of polymer (10 200 MW) was dissolved in 2 mL of dichloromethane (DCM) for the low, medium, and highly substituted unlabeled polymer. For the fluorescently labeled highly substituted polymer, 14.5 mg of polymer stock was mixed with 2 mL of DCM. To the polymer solution, trifluoroacetic acid (TFA) and water were added in a 2:1:1 volumetric ratio of DCM/TFA/H2O and mixed at room temperature for ∼30 min (Figure 1A,B). Prior to the substitution reactions, sodium bicarbonate salt (NaHCO3) was added to 10 mL of saturated sodium acetate (NaCH3CO2) in methanol and mixed into the reaction solution until a pH of 5.5 to 6.0 was achieved (Figure 1B,C). Aminooxy-peptide (1683 g/mol) was added and reacted for 4 h at room temperature in molar ratios of 1:10, 2:10, or 3:10 (peptide/PDEPMA side group) to synthesize low, medium, and highly substituted polymer constructs (Figure 1B,C), respectively. In the case that a fluorescently labeled construct was desired for imaging, TEG conjugation was preceded by a 16 h reaction with AlexaFluor 488 hydroxylamine (Invitrogen) using a molar ratio of one PDEPMA polymer chain per fluorophore molecule (Figure 1C,D). To maximize water solubility, the remaining polymer side chains were reacted with aminooxy tetra(ethylene glycol) (TEG, 223.27 g/mol) in a molar ratio of 1:1 (TEG/number of PDEPMA side chains remaining unconjugated) (Figure 1C,D). After allowing the final reaction with TEG to occur for 16 h, the solution was removed in vacuo, and the product was subsequently dissolved in water. The sample was purified by centrifuge filtration with a Millipore Amicon 5000 MWCO filter, five times for 15 min each (1800g, Beckman Coulter Allegra X-22), followed by redissolving in water and centrifuging for 10 min (46 100g, Beckman

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Coulter Optima MAX-E ultracentrifuge) to ensure that aggregates and insoluble species were excluded. The filtered product and supernatant were collected for filtered centrifugation and ultracentrifugation respectively, and the resulting product was subsequently lyophilized and stored at -20 °C until used. Characterization. 1H NMR and UV-vis absorbance spectroscopy were employed to determine the relative amounts of peptide conjugated to polymer. For NMR analysis, spectra of the final conjugated products and peptide alone, were compared with results of polymer alone,27,28 100% polymer-TEG conjugate, and Alexa Fluor 488 reacted polymer. Each conjugate and peptide were dissolved in D2O (0.75 mL) and placed in an NMR tube, and the spectra were recorded. NMR spectra were acquired on a Varian Inova500 spectrometer using “wet” analysis to suppress residual water peaks. Absorbance spectroscopy at 280 nm was used to quantify the concentration of tyrosine and tryptophan in the conjugated polymer to determine peptide concentration. The concentration of fluorescent label was determined by the intensity of Alexa Fluor 488 measured using UV-vis absorbance spectroscopy at 495 nm, corresponding to the excitation wavelength per manufacturer instructions. For labeled construct, the absorbance of peptide was correspondingly measured at 280 nm. From absorbance measurements, a 20× concentration of peptide relative to fluorophore was calculated for the highly substituted fluorescent construct (on average 1 fluorophore per 20 peptide side-chain substitutions). Concentrations for fluorescence imaging were based on the measured fluorophore concentration. Europium Binding Assays. Normal human astrocytes (NHAs) and glioma cells (SF767) were generously supplied by Dr. Michael Berens (Translational Genomic Institute, Phoenix, Arizona). Cells were maintained under standard culture conditions (37 °C and 5% CO2) and were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% bovine growth serum (BGS). The procedure of Rosca et al.6 was followed by binding of the Europium (Eu)-labeled dodecapeptide competed off by the peptide alone or the low- and medium-substituted polymer-peptide conjugate. In brief, the day prior to the experiment, cells were passaged and plated in DMEM at a density of 60 000 cells per well in a black, clear-bottomed 96-well plate coated with 0.1% gelatin. After plating (24 h), the cells were washed with phosphate-buffered saline (PBS) and incubated for 30 min at 4 °C with 100 µL of binding media (DMEM, 1 mM 1,10-phenanthroline, 200 mg/L bacitracin, 0.5 mg/L leupeptin, and 3% bovine serum albumin (BSA)). Unlabeled construct in binding media spanning a range of peptide concentrations (100 µM-2 pM) was added simultaneously to 10nM Eu-labeled peptide as the competitor for 1.5 h at 4 °C. Following incubation, cells were washed four times with wash buffer (50 mM Tris-HCl, 30 mM NaCl, and 0.5% BSA). Enhancement solution (PerkinElmer) was added (100 µL/well) and incubated at room temperature for 1 h. Using the standard time-resolved fluorescence (TRF) protocol (emission 340 nm, excitation 615 nm), the fluorescence of each well was read on the Perkin-Elmer Wallac VictorV instrument. For each concentration of each construct, four replicates were performed, with the error bars in data analysis representing plus and minus the standard error of the normalized mean of intensity. Construct concentration is determined by dividing the overall concentration of peptide in the low and medium conjugate sample (determined by spectrophotometry) by the number of peptides determined as conjugated to each construct via NMR. For these calculations, the average percent substitution values of 6 and 21% for the low- and medium-substituted constructs were used to estimate the number of peptides per construct. Additionally, avidity (KD) values were calculated for each of the four replicates of each substitution, and four values for KD were obtained on the basis of replicate runs. Using these four KD values for each construct type, an average KD for each construct (low, medium, or peptide alone) was calculated, and t tests (p < 0.05) were used to determine the statistical significance between various substitutions. Imaging of Binding. Visual inspection of binding was performed on glioblastoma SF767 and NHA cells using the highly substituted construct conjugated with Alexa Fluor 488. NHA and SF767 cells were

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maintained under standard conditions (37 °C and 5% CO2) and were grown in DMEM supplemented with 10% BGS. The day prior to the experiment, cells were passaged and plated in DMEM at a density of 30 000 cells per well of a black, clear-bottomed 96-well plate coated with 0.1% gelatin. On the day of the experiment, the cells were washed twice with PBS and incubated for 30 min at 4 °C with 50 µL of binding media to block for nonspecific binding prior to incubation with fluorophore-labeled construct. To minimize construct internalization by the cells, experiments were conducted at 4 °C. After this initial incubation period, 50 µL of either fluorophore-labeled highly substituted construct or fluorophore-labeled peptide was added to each cell type for 1 h at 4 °C on a rotating plate at fluorophore concentrations of 0.5 and 5 µM. After 1 h of incubation, cells were washed four times with wash buffer and resuspended in PBS supplemented with Ca2+ and Mg2+ (100 mg/L). Binding was visualized using the 488/512 nm filter for fluorophore detection at 120s exposure time for all Figures except Figure 4C,D, which was captured at 21s exposure because of saturation of the camera at 120s. Random fields (three to four per well) were selected for pictures, and two replicate wells were conducted for each construct concentration and each cell type. Images were acquired using a Leica DM IRB inverted microscope with a Diagnostic Instruments SPOT RT3 camera (Leica Microsystems and Diagnostic Instruments, respectively). Contrast levels for each image were adjusted identically across all samples in Adobe Photoshop to enhance visualization of the cells. All image analysis and quantification were performed in Photoshop. Phase contrast images were overlaid with their respective fluorescent image. Each individual cell in each picture was outlined in phase contrast. After outlining in phase contrast and taking all cells within the image into account (so as to estimate results conservatively), each picture was adjusted to display the fluorescence overlay, and a histogram of the green fluorescence pixel intensity was taken for each cell. The mean fluorescence intensity value per cell was calculated using the histogram tool in Photoshop for each individual cell. At least 30-40 cells were analyzed per image, and five images were analyzed for each treatment group. These values are plotted in the histogram in Figure 6. We calculated specificity values cited in the text by dividing the average fluorescence intensity of SF767 cells by the average fluorescence intensity per cell of the corresponding NHA treatment. Toxicity Studies. Toxicity of the various constructs was determined using the LIVE/DEAD viability/cytotoxicity kit (Molecular Probes). Cells were plated in a 96-well plate at a density of 1000 cells per well and incubated with various conjugate types at concentrations 1nM, 1 µM, and 10 µM for 5 days. Cells were labeled according to the instructions provided by the manufacturer. Three fields were randomly selected per well for imaging. The number of dead cells per field was counted manually, and the number of live cells was calculated by dividing the number of green pixels by the average number of pixels per cell. Standard error of the mean was used to configure the error bars, and ANOVA (p < 0.05), in combination with posthoc t test with media alone, was used to determine statistical significance.

Results Construct Synthesis. Multivalent polymer constructs derived from a poly(3,3-diethoxypropyl methacrylate) (PDEPMA) backbone were synthesized. An aminooxy-functionalized 12 amino acid peptide (TWYKIAFQRNRK) was conjugated, and the remaining free side chains of the polymer were then reacted with aminooxy-functionalized TEG to ensure solubility in water. Solid-phase peptide synthesis was employed to synthesize the aminooxy-terminated dodecapeptide, with the aminooxy group attached to the N-terminus threonine. Subsequent to peptide synthesis, MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectroscopy) was used to confirm the molecular weight of the dodecapeptide. Prior to

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Figure 2. NMR spectra of (A) low, (B) medium, and (C) high percent substitutions of peptide to polymer backbone and (D) peptide alone. Peaks within each spectrum correspond to (a) syn and anti bond peaks of the protons in the oxime bond, (b) polymer side chain peaks, and (c) ester methylene proton peaks.

conjugation, we deprotected the PDEPMA polymer side chains by hydrolyzing the acetal groups (Figure 1A) to form aldehydes (Figure 1B). NMR spectra of the three conjugated polymer-peptide conjugates, peptide alone, and polymer alone were compared to determine the relative substitution of peptide on the polymer. Figure 2A-C shows the spectra corresponding to low, medium, and high relative peptide substitution percents (therefore high, medium, and low TEG substitution), respectively, relative to the spectra for peptide alone (Figure 2D). From the NMR spectra of the low and medium peptide-conjugated polymers (Figures 2A,B), the formation of the oxime bond (peaks denoted by (a)), which is indicative of both peptide and TEG binding, shows that oxime linkages have been formed between the polymer and the substituted groups. The aldehyde peaks characteristic of unreacted side chains are no longer observed (∼9.7 ppm, see the Supporting Information for extended NMR spectra), and instead, the syn and anti hydrogen of the oxime bond are shown at ∼7.42 and 6.82 ppm for both spectra (a). Comparison of the integral sum of these peaks (a) relative to the characteristic polymer peaks at 2.5 and 2.7 ppm (b) of the anti and syn


methylene protons β to the ester indicated that conjugation was efficient. In both cases, the syn to anti ratio was 1.3:1, which was consistent with that of the PEG substitution reported by Li et al.28 More specifically, the percent peptide substitution to the polymer backbone was calculated by comparing the integral values of the protons in the aromatic peptide peaks relative to the protons involved in the oxime, polymer side chain, and ester bonds. The peaks in the ∼6.6 to 7.7 ppm region correspond to the presence of aromatic amino acids in the side chain, namely, tyrosine, tryptophan, and phenylalanine. For the low peptide substitution, the integration of the aromatic region not overlapping with oxime peaks (7.07 to 7.37 ppm) corresponding to eight peptide protons (C6H5 of F, NHCCHC and NHCCHCH of W) was compared with the syn and anti oxime peak integrations (subtracting one peptide H integration for NHCCHCHCHCHC of W and two CHC(OH)CH integrations for Y, respectively). This gave a percent substitution of 8.4%. The same peptide region was compared with the integration of the syn and anti peaks of the polymer side chain at 2.40 to 2.75 (OCH2CH2) subtracting the underlying peptide peak integrations, thereby resulting in a percent substitution of 6.4%. Comparing the peptide integrations to ester methylene protons at ∼4.1 ppm (c) and subtracting the underlying peptide peaks gave a substitution of 3.9%. The average of these three substitution results for the low conjugation reaction was calculated to be 6.2%, and this value was used to determine the number of peptides as 2.82 on the polymer (approximately trivalent construct). Similarly, the percent peptide conjugated to the polymer for the medium substitution reaction was calculated as described above. The integration of the aromatic region not overlapping with oxime peaks was taken into account such that nine peptide peaks were considered including NHCCHCHCHCHC of W. This was then compared with the syn oxime peak, the side chain methylene syn and anti peak, and the ester methylene protons minus the peptide peaks. The percent peptide substitution was calculated to be 19% from the oxime, 32% from the side chain, and 12.4% from the ester protons, respectively, resulting in an average peptide substitution value of 21% for the medium substitution reaction, corresponding to 9.90 peptides per polymer (approximately decavalent construct). For the highest peptide substitution reaction (Figure 2C), the peptide peaks are large relative to the oxime bond peaks; therefore, some peak broadening is experienced, confounding the analysis. Whereas the highly conjugated spectrum was integrated similarly as the low- and medium-substituted spectra, the increased peak presence due to the aromatic amino acids confounds a clean integration and therefore quantitative assessment of integration values for oxime bond peaks. When compared with the peptide spectrum alone, as shown in Figure 2D, it is expected that both aromatic amino acid-amino acid and amino acid-oxime peaks in the 6.6 to 7.7 ppm range make deciphering the exact substitution in this region difficult because integration values are indistinguishable because of the multiple peaks present. For the highly substituted sample, this makes additional evaluation of the ratio of peptide substitution relative to polymer side chain peaks (b) difficult as well. However, it is noted that whereas the oxime peaks are not as easily quantified in Figure 2C, the relative amount of peptide in the 6.6 to 7.7 ppm region (a) increases relative to the polymer backbone peaks between 0.5 and 2 ppm as the amount of peptide used in the conjugation step is increased in all spectra (Figure 2A-C). Furthermore, observation of the peptide peaks in the other


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Figure 3. Competitive binding of labeled dodecapeptide versus (A) peptide, (B) low-, and (C) medium-substituted polymer backbone. Plot shows normalized Eu intensity of the various constructs as a function of construct concentration. Four replicate runs of data (points) and affinity fit (lines) were used to calculate an avidity (open circle) with standard error.

regions of the spectra (for example between 2 and 3 ppm) indicates a relative increase in peptide presence relative to polymer. Again, because the presence of free aldehydes (9.5 to 10.5 ppm) is not detected (extended region not shown), this signifies that all deprotected side groups reacted. Whereas the exact amount of TEG is not as significant as peptide substitution, because it is used for purposes of solubility, the presence of TEG conjugation is denoted by the presence of CH2 and OCH3 peaks in the 3.2 to 3.8 ppm region. Therefore, although the exact substitution was not obtainable for the highly substituted construct, it can be considered to be greater than that of the medium construct (i.e., greater than 21% peptide substitution). Binding Activity. Construct function was investigated with a competitive binding assay using either dodecapeptide alone or a polymer-peptide construct to compete off a Eu-labeled dodecapeptide. Binding assays were performed using adherent glioma (SF767) cells. Figure 3 shows the relative amount of labeled peptide binding to glioma cells in the presence of each unlabeled construct type at varying concentrations. First, it can be observed that the Eu-labeled dodecapeptide can compete off the construct, indicating that the construct is bound to the R6β1integrin rather than being nonspecifically bound to the cell

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Figure 4. Binding of highly substituted construct (5 µM) to (A) SF767 and (B) NHA cells. Binding of monovalent dodecapeptide (5 µM) to (C) SF767 and (D) NHA cells.

membrane or glycocalyx. It was expected that the highly conjugated construct would exhibit stronger avidity in comparison with the peptide alone and low-and medium-substituted constructs. However, because of the difficulty of characterizing the exact number of peptides on the high substitution reaction, the competitive binding analysis results are shown only for the peptide (Figure 3A), low- (Figure 3B), and medium-substituted (Figure 3C) constructs. Results from the competitive binding assay plot the normalized binding intensity of the labeled dodecapeptide versus the construct concentration for the low and medium conjugates and peptide concentration for the sample of peptide alone. The low substitution construct displays strong binding affinity to the cells compared with the dodecapeptide alone (KD ) 5.4 ( 2.0 vs 17.5 ( 3.1 µM for the low vs peptide respectively, p < 0.001). In addition, the medium-substituted construct is more avid than the monovalent dodecapeptide (KD ) 2.6 ( 0.6 µM, p < 0.001). The medium-substituted construct is slightly more avid than the low-substituted construct (2.6 ( 0.6 vs 5.4 ( 2.0 µM, p < 0.05). Imaging of Binding. Binding of the highly substituted polymer versus peptide was investigated with cells. Peptide and construct were labeled with Alexa Fluor 488 for imaging analysis. Experiments were conducted at 4 °C to minimize internalization. SF767 and NHA cells were imaged after incubation with 0.5 and 5 µM (fluorophore concentration) polymer-peptide construct and dodecapeptide. Figure 4A,B shows binding of the polymer-peptide construct (5 µM) to the SF767 (glioma) and NHA cells, respectively. Qualitatively, it is noted that the SF767 cells have greater peripheral binding of construct than do the NHAs, which exhibit punctate binding. Figure 4C,D shows images of the SF767 and NHA cells after incubation with monovalent dodecapeptide alone. In comparison with binding of multivalent constructs to both cell types (Figure 4A,B), little binding is observed when monovalent dodecapeptide is added (Figure 4C,D). It should be noted that these images are at a shorter exposure time because of saturation of the camera at the exposure time used for Figure 4A,B; however, this is likely due to high background fluorescence in the media above the cells. As can be seen in Figure 4C,D, there is still high background, indicating that dodecapeptide may have been bound to cells when media was exchanged but that any bound peptide quickly dissociated before imaging. Whereas competitive binding assays could not demonstrate multivalency for the highly substituted construct because of the inexact determination of

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Figure 5. Binding of highly substituted construct (0.5 µM) to (A) SF767 and (B) NHA cells or binding of monovalent dodecapeptide (0.5 µM) binding to (C) SF767 and (D) NHA cells.

Figure 6. Histogram of cell fluorescence intensity for (A) 5 and (B) 0.5 µM construct concentrations during fluorescence binding assay for SF767 tumor cells (black bars) and NHA cells (gray bars).

peptide substitution, the fluorescence binding assay suggests that their avidity is certainly stronger than peptide alone, thereby demonstrating multivalency, which would slow the effective dissociation rate. This is corroborated by the comparison between Figure 5A and 5C or 5B and 5D, which were able to be taken using the same exposure settings. On the basis of this determination, we expected to observe a concentration-dependent increase in specificity for SF767s versus NHAs as the concentration is decreased.8 The intensity of binding is expected to decrease somewhat on the SF767s when construct concentration is decreased from 5.0 (Figure 4A) to 0.5 µM (Figure 5A), and binding is expected to decrease even more significantly on NHAs (Figure 4B vs 5B). Instead, we note that SF767s and NHAs exhibit similar decreases in intensity, thus maintaining specificity unchanged (Figure 6). Two likely possible reasons for this include (1) that the experiment may not have decreased concentration below the threshold for enhanced specificity, which is expected to be near the receptor-ligand bond affinity (17 ( 3 µM) but could be less or (2) that high nonspecific binding of the relatively hydrophobic polymer may have prevented NHAs from decreasing their intensity significantly. Fluorescence intensities of SF767 and NHA cells were quantitativelycomparedforboththe5.0and0.5µMpolymer-peptide conjugate cases in Figure 6. The mean fluorescence intensities were 76.23 and 52.10 for the 5 µM SF767 and NHA cell populations, and 43.39 and 30.28 for the SF767 and NHA cells incubated with 0.5 µM construct concentration. For each concentration, the mean intensity difference of the SF767 and NHA cell populations is statistically significant (p < 0.05). This is demonstrated in Figure 6 by the rightmost shift in the fluorescence cell intensity trend of the SF767 cells relative to



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Table 1. Results from Toxicity Assay for Each Cell and Construct Type at Varying Concentrations SF767

5 nM

low percent substitution medium percent substitution high percent substitution peptide alone 100% TEG media alone NHA

97.49 97.19 99.37 99.30 97.09 97.88

( ( ( ( ( (

0.78 0.87 0.29 0.17 1.32 0.68

5 nM

low percent substitution medium percent substitution high percent substitution peptide alone 100% TEG media alone

99.32 98.89 99.00 97.80 96.07 96.56

( ( ( ( ( (

0.57 0.74 0.50 0.89 1.17 2.24

the NHA trends for each concentration. Furthermore, the corresponding specificities of binding between cell types were 1.46 and 1.43 for 5 and 0.5 µM, respectively. This is in comparison with the ratio of receptor expression of 2.84 ( 0.07 for SF767/NHA expression (SF767: 37 600 ( 270; NHA: 13 210 ( 340).8 Toxicity Studies. Cells were cultured in the presence of all construct substitutions and peptide alone at varying concentration for 5 days. After 5 days, the live/dead assay was performed, and the percent of live and dead cells was determined. Table 1 depicts the results of these assays, demonstrating that varying concentrations of these constructs are not toxic to SF767 or NHA cells. For both cell types, at least 96% of cells are viable after 5 days. Comparison of constructs to controls using peptide alone, 100% TEG conjugations, and media alone showed no statistical difference. Experiments were conducted for 5 days to avoid cell confluence, which would result in cell death at longer times because of contact inhibition.

Discussion Cancer-targeting biomacromolecules have been made by linking peptides via oligomeric linkers;6,10,23,28-30 however, their synthesis is both expensive and time-consuming. Previous research by Rosca et al. used solid-phase peptide synthesis techniques to create such multivalent constructs.6 That synthesis requires one reaction step per amino acid in each ligand (12 amino acids per ligand times 3 ligands) and one reaction step per oligomer (3 oligomers per linker times 2 linkers) for a total of 42 reaction steps. This reaction scheme cannot use presynthesized and purified peptides because incorporating large molecules such as premade peptides greatly reduces the synthesis yield. Here we have demonstrated proof-of-principle for what we consider to be a two-step synthesis (assuming polymer and peptide are available) of multivalent biomacromolecules, which is potentially less expensive and faster. Assuming an off-the-shelf polymer and a previously synthesized peptide, the synthesis scheme shown here requires only the addition of aminooxy-peptide ligand and then aminooxy-TEG. In this work, the polymer was synthesized de novo; however, the polymer was dried, shipped, and stored prior to conjugation with the peptide ligand, so the synthesis scheme should be compatible with bulk synthesis of the polymer followed later by conjugation of peptide ligands (also purchased or synthesized prior to conjugation). The possibility of rapid synthesis of multivalent constructs in response to patient-specific pathologies may allow this strategy to be used in personalized medicine. For example, if a

0.5 µM 97.77 96.55 99.52 99.23 98.37

( ( ( ( (

0.68 0.89 0.17 0.28 1.03

0.5 µM 98.95 98.31 98.21 97.69 98.15

( ( ( ( (

0.37 0.73 0.96 0.98 0.48

5 µM 99.25 96.66 98.29 98.20 93.59

( ( ( ( (

0.26 0.94 0.17 0.34 1.94

5 µM 98.32 96.40 98.29 97.00 96.28

( ( ( ( (

0.73 1.19 0.64 0.99 0.96

patient’s tumor was biopsied and found to have a particular receptor overexpressed, then aminooxy-peptide ligands for that pathology could be synthesized (or taken out of storage), conjugated to the off-the-shelf polymer, and purified, and the resulting multivalent construct could be ready for use within 1 week. In this way, this synthesis scheme could potentially decrease the financial and time costs of personalized medicine by an order of magnitude or more into a range that is feasible. The incorporation of aminooxy-fluorophores in the peptide conjugation steps in this work also indicates the possibility of mixing several different ligands for several different receptors to achieve a heterovalent construct without increasing the difficulty or time required for the synthesis. In fact, this is one of the advantages of the oxime linkage where resulting composition mirrors the relative amounts of different ligands placed in the reaction flask.28 Heterovalent constructs have been proposed by us and others as a way to target cancer cells for which no single overexpressed receptor would result in specific targeting. Recent work by our collaborators23 indicates that no single receptor ligand is likely to target all cancers of a particular type. Therefore, it is likely that a cocktail of several constructs or a single heterovalent construct will be necessary to achieve targeting to all possible phenotypes of a particular cancer. Using the synthesis scheme validated here, it may be possible to biopsy the cells first to determine their phenotype and then to tailor the targeting construct to the particular phenotype present using either the cocktail or heterovalent approach. On the basis of NMR characterization as well as functional assays of multivalent construct binding, the synthesis scheme does create multivalent constructs with enhanced avidity and specificity for cells overexpressing the R6β1-integrin. NMR results show that conjugation of the peptide ligand to the polymer backbone does occur and that, as the ratio of peptide to polymer increases, there are more peptides conjugated to the polymer. This is exemplified by the increased peptide substitution (6 versus 21%) for the low and medium constructs, respectively. Although NMR could not derive an exact peptide substitution for the highly substituted construct, the imaging data (Figures 4 and 5) demonstrate that the highly substituted construct behaves as a multivalent construct exhibiting enhanced avidity. Functional assays demonstrate that these constructs behave as biomacromolecular, multivalent targeting constructs. Comparison between the highly substituted construct and dodecapeptide at 5 and 0.5 µM shows that binding is intense for the construct but not for the dodecapeptide; this indicates that the construct exhibits substantially increased avidity, as would be

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expected for multivalency. Furthermore, through the quantification of fluorescent cell intensity, SF767 tumor cells demonstrate increased binding relative to NHA (astrocyte) cells by the statistically significant shift in fluorescence intensity, as seen for both concentrations when comparing each cell type. Competitive binding assays results corroborate these conclusions in that there is a significant difference in binding demonstrated between the low- and medium-substituted constructs and peptide alone. Whereas the exact peptide content of the highly substituted construct is unknown, it has equal to, if not stronger avidity than, the medium-substituted construct (data not shown). The specific purpose in conjugating these polymers with peptide ligands for the R6β1-integrin is to target glioblastoma cells.25,26 Compared with previous work in our lab by Rosca et al.6,8 in which trivalent constructs show peripheral binding to glioblastoma cells and less intense punctuate staining for astrocytes, the multivalent constructs synthesized here perform similarly. One detail that should be noted is that the imaging experiments are performed at 4 °C to prevent internalization of the peptides or constructs. The performance of biomacromolecular constructs at 37 °C has been characterized and shows that astrocytes internalize constructs more rapidly than do glioblastoma cells.6,8 Ongoing work in our lab seeks to address this issue. This aside, the polymer-based constructs studied here seem to duplicate the ability to target glioblastoma cells using cooperative binding. There are two reasons why using biomacromolecular constructs may be preferable to using nanoparticles for glioblastoma targeting.6 The first is the enhanced perfusion of biomacromolecular constructs through tissue. Localized delivery to the brain is necessary because all multivalent constructs are too large to traverse the blood-brain barrier. The distances over which a construct can diffuse or perfuse within tissue is limited;31 however, nanoparticles exacerbate this problem by being large enough for their movement to be inhibited by the tissue. In our previous work, comparing perfusion of biomacromolecular peptide constructs with QDs approximately 30 nm in diameter, the best-fit filtration coefficient is 0.25 for QDs but 1.0 for biomacromolecular constructs, indicating that biomacromolecular constructs are not inhibited but that nanoparticles are 75% inhibited.6 The second reason why biomacromolecular constructs may be preferable involves biocompatibility. There is concern about the biocompatibility of nanoparticles in general and, specifically, about QDs containing heavy metals. Toxicity studies of the polymeric constructs characterized in this work show that >96% viability is maintained after 5 days of culture for all constructs. No significant difference was found between cells (either NHAs or SF767s) treated with these polymers with varying substitution percentages. Additionally, in comparison with the controls of media and/or peptide alone, no significant difference was seen in viability, thereby demonstrating in vitro that these constructs are nontoxic. Although this does not guarantee biocompatibility for in vivo applications, an added indication that biocompatibility is likely is that these molecules are small enough to be cleared from blood rapidly by excretion in the kidney.32

Conclusions We have demonstrated proof-of-principle for synthesizing multivalent constructs using a rapid and potentially economical method. Aminooxy-terminated dodecapeptides were conjugated to a polymethacrylate polymer via oxime bond formation, resulting in a construct with enhanced avidity and specificity

Stukel et al.

for SF767 over NHA cells compared with the monovalent ligand. Competitive binding assay results show enhanced avidity of the low- and medium-substituted constructs; fluorescence binding assays with the highly substituted construct corroborate these results in terms of enhanced binding and visualization. In addition to their multivalent properties and enhanced binding, these polymeric constructs are nontoxic to cells in vitro. Acknowledgment. We thank the Arizona Biomedical Research Commission for funding through a Category III grant (no. 06-06) and the National Institute for Neurologic Diseases and Stroke for funding through R21 NS051310. H.D.M. thanks the Alfred P. Sloan Foundation for additional funding. Supporting Information Available. Extended NMR spectra for each of the “high”-, “medium”-, and “low”-substituted constructs including peaks to 10 ppm. This material is available free of charge via the Internet at http://pubs.acs.org.

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BM9010276

Two-Step Synthesis of Multivalent Cancer-Targeting Constructs

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