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Dendrimers Bind Human Serum Albumin E. Froehlich,† J. S. Mandeville,† C. J. Jennings,‡ R. Sedaghat-Herati,‡ and H. A. Tajmir-Riahi*,† De´partement de Chimie-Biologie, UniVersite´ du Que´bec a` Trois-RiVie`res, C. P. 500, Trois-RiVie`res (Que´bec), G9A 5H7, Canada, and Department of Chemistry, Missouri State UniVersity, Springfield, Missouri 65897 ReceiVed: February 6, 2009; ReVised Manuscript ReceiVed: March 24, 2009
Dendrimers are synthetic, highly branched, spherical macromolecules with nanometer dimensions and potential applications in DNA and drug delivery systems. Human serum albumin (HSA) is a major transporter for delivering several endogenous compounds and drugs in vivo. The aim of this study was to examine the interaction of human serum albumin with several dendrimers such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions, using constant protein concentration and various dendrimer compositions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods were used to analyze macromolecule binding mode, the binding constant and the effects of dendrimers complexation on HSA stability and conformation. Structural analysis showed that dendrimers bind HSA via polypeptide polar groups (hydrophilic) with number of bound polymer (n) 1.08 (mPEG-PAMAM-G3), 1.50 (mPEG-PAMAM-G4), and 0.96 (PAMAM-G4). The overall binding constants estimated were of KmPEG-G3 ) 1.3 ((0.2) × 104 M-1, KmPEG-G4 ) 2.2 ((0.4) × 104 M-1, and KPAMAM-G4 ) 2.6 ((0.5) × 104 M-1. HSA conformation was altered by dendrimers with a major reduction of R-helix and increase in random coil and turn structures suggesting a partial protein unfolding. Introduction Dendrimers are highly branched, monodispersed synthetic macromolecules with a large number of reactive terminal groups all emanating from a central core.1-3 Among dendrimers, poly(amidoamine) (PAMAM) (Scheme 1) have received most attention as potential transfection agents for gene and drug delivery, as these macromolecules can bind DNA and drug at physiological pH.4-6 However, PAMAM dendrimers are toxic in cells and animals due to their polycationic character.7 It has been demonstrated that modification of the amino groups on the periphery of the dendrimer with poly(ethylene glycol) chains reduces the toxicity and increases the biocompatibility of the resulting polymer.8 This is because poly(ethylene glycol) is nontoxic, nonimmunogenic, and water-soluble, and its conjugation with other substrates produces conjugates that combine the properties of both the substrate and the polymer. However, conjugate formation can alter the binding affinity of PAMAM to DNA, drug, and protein in general. Human serum albumin (Scheme 2) is the most abundant serum protein, which carries several endogenous compounds including fatty acids.9 HSA has long been the center of attention of pharmaceutical industry due to its ability to bind various drug molecules and alters their pharmacokinetic properties.10 HSA is a globular protein composed of three structurally similar domains (I, II, and III), each containing two subdomains (A and B) and stabilized by 17 disulfide bridges.10-17 Aromatic and heterocyclic ligands were found to bind within two hydrophobic pockets in subdomains IIA and IIIA, namely site I and site II.10-17 Seven binding sites for fatty acids are localized in subdomains IB, IIIA, IIIB, and on the subdomain interfaces.10 * To whom correspondence should be addressed. Fax: 819-376-5084. Tel: 819-376-5011 (ext. 3310). E-mail:
[email protected]. † Universite´ du Que´bec a` Trois-Rivie`res. ‡ Missouri State University.
HSA has also a high affinity metal binding site at the N-terminus.11 These multiple binding sites underline the exceptional ability of HSA to interact with many organic and inorganic molecules and make this protein an important regulator of intercellular fluxes, as well as the pharmacokinetic behavior of many drugs.10-18 Therefore, it was of interest to study the binding of dendrimers with HSA in aqueous solution in order to examine the effects of dendrimers on protein secondary structure, conformation, and stability. In this report, we present a spectroscopic analysis of the interaction of HSA with dendrimers PAMAM (G4), m-PEGPAMAM (G3), and m-PEG-PAMAM (G4) in aqueous solution at physiological conditions, using constant protein concentration and various dendrimer compositions. Structural information regarding dendrimers binding mode and the effects of polymerHSA complexation on the protein stability and secondary structure is reported here. Experimental Section Materials. HSA fraction V was purchased from Sigma Chemical Company and used as supplied. PAMAM-G4 (MW 14214 g/mol) was purchased form Aldrich Chemical Co and used as supplied. mPEG-PAMAM-G3 (MW 5697 g/mol) and mPEG-PAMAM-G4 (MW 8423 g/mol) were synthesized according to published methods.19,20 Other chemicals were of reagent grade and used without further purification. Preparation of Stock Solutions. Human serum albumin was dissolved in aqueous solution (40 mg/ml or 0.5 mM) containing 10 mM Tris-HCl buffer (pH 7.4). The protein concentration was determined spectrophotometrically using the extinction coefficient of 36 500 M-1 cm-1 at 280 nm.21 In this study, HSA did not have its fatty acids removed in a way to reproduce normal physiological conditions where between 0.1 to 2 fatty acid molecules are bound to albumin.11,17 Dendrimers 1 mM
10.1021/jp9011119 CCC: $40.75 2009 American Chemical Society Published on Web 04/21/2009
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SCHEME 1: Chemical Structure of PAMAM-G4
were prepared in distilled water and diluted to various concentrations in Tris-HCl. FTIR Spectroscopic Measurements. Infrared spectra were recorded on a FTIR spectrometer (Impact 420 model), equipped with deuterated triglycine sulfate (DTGS) detector and KBr beam splitter, using AgBr windows. A solution of dendrimers was added dropwise to the protein solution with constant stirring to ensure the formation of homogeneous solution and to reach the target dendrimers concentrations of 0.125, 0.25, and 0.5 with a final protein concentration of 0.25 mM (20 mg/ml). Spectra were collected after 2 h incubation of HSA with polymer solution at room temperature, using hydrated films. Interferograms were accumulated over the spectral range 4000-600 cm-1 with a nominal resolution of 4 cm-1 and 100 scans. The difference spectra [(protein solution + dendrimers solution) (protein solution)] were generated using the polypeptide antisymmetric and symmetric C-H stretching bands,22 located at 2900-2800 cm-1, as internal standard. These bands, which are due to protein C-H stretching vibrations, do not undergo any spectral changes (shifting or intensity variation) upon polymer complexation and therefore, they are commonly used as internal standard. When producing difference spectra these bands were adjusted to the baseline level in order to normalize difference spectra. Details regarding infrared spectral treatment are given in our recent publication.23 Analysis of Protein Conformation. Analysis of the secondary structure of HSA and its dendrimers complexes was carried
out on the basis of the procedure previously reported.24 The protein secondary structure is determined from the shape of the amide I band, located around 1650-1660 cm-1. The FT-IR spectra were smoothed and their baselines were corrected automatically using Grams AI software. Thus the root-mean square (rms) noise of every spectrum was calculated. By means of the second derivative in the spectral region 1600-1700 cm-1 seven major peaks for HSA and the complexes were resolved. The above spectral region was deconvoluted by the curve-fitting method with the Levenberg-Marquadt algorithm, and the peaks corresponding to R-helix (1656-1658 cm-1), β-sheet (1614-1638 cm-1), turn (1660-1677 cm-1), random coil (1640-1648 cm-1) and β-antiparallel (1680-1692 cm-1) were adjusted and the area was measured with the Gaussian function. The area of all the component bands assigned to a given conformation were then summed up and divided by the total area.25 The curve-fitting analysis was performed using the GRAMS/AI Version 7.01 software of the Galactic Industries Corporation. Circular Dichroism. CD Spectra of HSA and its dendrimers’ complexes were recorded with a Jasco J-720 spectropolarimeter. For measurements in the far-UV region (178-260 nm), a quartz cell with a path length of 0.01 cm was used in nitrogen atmosphere. HSA concentration was kept constant (12.5 µM), while varying each polymer concentration (0.125, 0.25, and 0.5 mM). An accumulation of three scans with a scan speed of 50 nm per minute was performed and data were collected for each nm from 260 to 180 nm. Sample temperature was maintained
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SCHEME 2: Human Serum Albumin
at 25 °C using a Neslab RTE-111 circulating water bath connected to the water-jacketed quartz cuvettes. Spectra were corrected for buffer signal and conversion to the Mol CD (∆ε) was performed with the Jasco Standard Analysis software. The protein secondary structure was calculated using CDSSTR, which calculates the different assignments of secondary structures by comparison with CD spectra, measured from different proteins for which high quality X-ray diffraction data are available.26,27 The program CDSSTR is provided in CDPro software package, which is available at the Web site: http:// lamar.colostate.edu/∼sreeram/CDPro. Absorption Spectroscopy. The absorption spectra were recorded on a Perkin-Elmer Lambda 40 Spectrophotometer. Quartz cuvettes of 1 cm were used for determination of protein concentration. Fluorescence Spectroscopy. Fluorometric experiments were carried out on a Varian Cary Eclipse. Stock solutions of dendrimers 1 mM in buffer (pH ) 7.4) were prepared at room temperature (24 ( 1 °C). Various solutions of polymers (10 to 400 µM) were prepared from the above stock solutions by successive dilutions also at 24 ( 1 °C. A solution of HSA (15 µΜ) in 10 mM Tris-HCl (pH. 7.4) was also prepared at 24 ( 1 °C. The above solutions were kept in the dark and used soon after. Samples containing 0.4 mL of the above HSA solution and 0.4 mL of various polymer solutions were mixed to obtain final polymer concentration of 5 to 200 µΜ with constant HSA content 7.5 µΜ. The fluorescence spectra were recorded at λexc ) 280 nm and λem from 287 to 500 nm. The intensity at 337 nm (tryptophane) was used to calculate the binding constant (K) according to previous literature reports.28-31
Results and Discussion FTIR and CD Spectra of Dendrimers-HSA Complexes. The dendrimers-HSA complexation was characterized by infrared spectroscopy and its derivative methods. Since there was no major spectral shifting for the protein amide I band at 1656 cm-1 (mainly CdO stretch) and amide II band at 1544 cm-1 (C-N stretching coupled with N-H bending modes)22-24 upon polymer interaction, the difference spectra [(protein solution + dendrimer solution) - (protein solution)] were obtained in order to monitor the intensity variations of these vibrations, and the results are shown in Figures 1 and 2. Similarly, the infrared self-deconvolution with second derivative resolution enhancement and curve-fitting procedures24 were used to determine the protein secondary structures in the presence of dendrimers (Figure 3 and Table 1). CD spectroscopy was also used to analyze the protein conformation in the polymer-HSA complexes and the results are shown in Figure 5 and Table 2. At low polymer concentration (0.125 mM), an increase in intensity was observed for the protein amide I at 1656 and amide II at 1545 cm-1 in the difference spectra of the mPEG-PAMAMG3-, mPEG-PAMAM-G4-, and PAMAM-G4-HSA complexes (Figures 1 and 2, diff. 0.125 mM). Positive features are located in the difference spectra for amide I and II bands at 1652, 1529 cm-1 (mPEG-PAMAM-G3), 1653, 1544 cm-1 (mPEG-PAMAMG4), and 1653, 1545 cm-1 (PAMAM-G4 (Figure 1 and 2, diff., 0.125 mM). These positive features are related to an increase in the intensity of the amide I and amide II bands upon dendrimers complexation The increase in intensity of the amide I and amide II bands is due to polymer binding to protein CdO, C–N and N-H groups. Additional evidence to support the polymer interactions with C-N and NH groups comes from
Dendrimers Bind Human Serum Albumin
Figure 1. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.4) for free HSA (0.25 mm), free mPEG-PAMAM-G3 (0.5 mM), mPEG-AMAM-G3-HSA (0.5 mM), and difference spectra (diff.) of polymer-HSA complexes (bottom two curves) obtained at different polymer concentrations (indicated on the figure).
the shifting of the protein amide A band at 3296 cm-1 (N-H stretching mode) in the free HSA to 3298 (mPEG-PAMAMG3), 3293 (mPEG-PAMAM-G4), and 3294 cm-1 (PAMAMG4) upon polymer complexation. As polymer concentration increased to 0.5 mM, strong negative feature for amide I was observed at 1656 cm-1 (mPEGPAMAM-G3), 1653 cm-1 (mPEG-PAMAM-G4), and 1658 cm-1 (PAMAM-G4) in the difference spectra of polymer-HSA complexes (Figures 1 and 2, diff, 0.5 mM). The observed decrease in intensity of the amide I band at 1656 in the spectra of the polymer-protein complexes suggests a major reduction of protein R-helical structure at high dendrimers concentrations. Similar infrared spectral changes observed for protein amide I band in several ligand-HSA complexes, where major protein conformational changes occurred.32 A quantitative analysis of the protein secondary structure for the free HSA and its polymer adducts in hydrated films has been carried out and the results are shown in Figure 3 and Table 1. The free protein has 55% R-helix (1657 cm-1), β-sheet 17% (1616, 1625, and 1634 cm-1), turn structure 14% (1677 cm-1), β-antiparallel 7% (1688 cm-1), and random coil 7% (1643 cm-1) (Figure 4A and Table 1). The β-sheet structure is composed of three components at 1616 (inter β-strand), 1625 (intra β-strand), and 1634 cm-1 (hydrated) that are consistent with the spectroscopic studies of human serum albumin.33-35 Upon polymer interaction, a major decrease of R-helix from 55% (free HSA) to 49% (mPEG-PAMAM-G3-HSA), 51% (mPEG-PAMAMG4-HSA), and 43% (PAMAM-G4) with an increase in random coil from 8% (free HSA) to 19% (mPEG-PAMAM-G3-HSA), 19% (mPEG-PAMAM-G4-HSA), and 22% (PAMAM-HSA) with reduction of β-sheet structure from 17% (free HSA) to 9% (mPEG-PAMA-G3-HSA), 10% (mPEG-PAMAM-G4-
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Figure 2. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.4) for (A) free HSA (0.25 mm), free mPEG-PAMAM-G4 (0.5 mM), mPEG-PAMAM-G4-HSA (0.5 mM), and difference spectra (diff.) of polymer-HSA complexes (bottom two curves) and (B) for free HSA (0.25 mm), free PAMAM-G4 (0.5 mM), PAMAM-G4-HSA (0.5 mM), and difference spectra (diff.) of polymer-HSA complexes (bottom two curves) obtained at different polymer concentrations (indicated on the figure).
Figure 3. Second derivative resolution enhancement and curve-fitted amide I region (1700-1600 cm-1) for free HSA and its polymer adducts with 0.5 mM dendrimers and 0.25 mM protein concentrations at pH 7.4.
HSA), and 12% (PAMAM-G4-HSA) (Figure 3 and Table 1). These results are consistent with the decrease in the intensity of the protein amide I band discussed above. The major decrease in R-helix structure and increase in random coil suggest a partial protein unfolding at high polymer concentration. Hydrophobic Interactions. The effect of dendrimers-HSA complexation on polymer antisymmetric and symmetric CH2 stretching vibration in the region of 3000-2800 cm-1 was
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TABLE 1: Secondary Structure Analysis (Infrared Spectra) from the Free HSA and Its Dendrimers Complexes in Hydrated Film at pH 7.4 amide I components (cm-1) 1692-1680 1680-1660 1660-1650 1648-1641 1640-1610
β-anti turn R-helix random coil β-sheet
free HSA (%) 0.25 mM
mPEG G3-HSA (%) 0.5 mM
mPEG G4-HSA (%) 0.5 mM
PAMAM-G4-HSA (%) 0.5 mM
7 14 55 7 17
9 13 49 19 9
9 16 46 19 10
6 17 43 22 12
Figure 5. Circular dichroism of free HSA and its polymer complexes in aqueous solution with protein concentration of 12.5 µM and polymer concentrations of 0.125, 0.25, and 0.5 mM in 10 mM Tris-HCl buffer pH 7.4 at 25 °C.
Figure 4. Spectral changes of dendrimers CH2 symmetric and antisymmetric stretching vibrations upon HSA complexation (the contribution from free protein vibrations has been subtracted in this region).
TABLE 2: Secondary Structure of HSA Complexes (CD Spectra) with Dendrimers at pH 7.4 Calculated by CDSSTR Software dendrimer concentration
R-helix (%)
β-sheet (%)
turn (%)
random (%)
free HSA (12.5 µM) mPEG 3-HSA (0.5 mM) mPEG G4-HSA (0.5 mM) PAMAM G4-HSA (0.5 mM)
58 48 51 38
9 14 10 19
11 14 16 19
22 24 23 24
investigated by infrared spectroscopy. The CH2 bands of the free mPEG-PAMAM-G3 located at 2942 and 2884 cm-1 shifted to 2956, 2938, and 2873 cm-1 (mPEG-PAMAM-G3-HSA); free mPEG-PAMAM-G4 with CH2 bands at 2942, and 2876 cm-1 shifted to 2956, 2940, and 2874 cm-1 (mPEG-PAMAM-G4HSA) and free PAMAM-G4 with CH2 bands at 2965, 2945, and 2835 cm-1 shifted to 2957, 2938, and 2872 cm-1 (PAMAMG4-HSA) in the polymer-protein complexes (Figure 4). Major shifting of the polymer antisymmetric and symmetric CH2
stretching vibrations in the region 3000-2800 cm-1 of the infrared spectra suggest the presence of hydrophobic interactions via polymer aliphatic chain and hydrophobic pockets in HSA. CD Spectra and Protein Conformation. The CD spectroscopic results shown in Figure 5 and Table 2 exhibit marked similarities with those of the infrared data. Secondary structures calculations based on CD data suggests that free HSA has a high R-helix content 58%, β-sheet 9% turn, 11% and random coil 22% (Figure 5 and Table 2), which is consistent with the literature report.36 Upon dendrimers complexation, major reduction of R helix was observed from 58% free HSA to 38% in mPEG-PAMAM-G3, 51% mPEG-G4 and 48% PAMAM (Figure 4 and Table 2). The major decrease in R-helix was accompanied by an increase in the random coil and turn structure (Table 2). The major reduction of the R-helix with an increase in the random coil and turn structure are consistent with the infrared results that showed reduction of R-helix and increase of random coil structure (Tables 1 and 2). It should be noted that there are minor differences between IR versus CD results regarding free HSA conformation (Tables 1 and 2). The reason for the differences may be due to sample preparation since IR measurements were performed in hydrated films, whereas CD experiments were conducted in aqueous solutions.37
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Fluorescence Spectra and Binding Constants of Dendrimers-HSA Complexes. HSA contains a single polypeptide of 585 amino acids with only one tryptophan (Trp 214) located in subdomain II A. Tryptophan 214 emission dominates HSA fluorescence spectra in the UV region. When other molecules interact with HSA, tryptophan fluorescence may change depending on the impact of such interaction on the protein conformation, or via direct quenching effect. On the assumption that there are (n) substantive binding sites for quencher (Q) on protein (B), the quenching reaction can be shown as following
nQ + B S Qn B
(1)
The binding constant (KA) can be calculated as
KA ) [QnB] ⁄ [Q]n[B]
(2)
where [Q] and [B] are the quencher and protein concentration, respectively, [QnB] is the concentration of non fluorescent fluorophore-quencher complex and [B0] gives total protein concentration.
[QnB] ) [B0] - [B]
(3)
KA ) [B0] - [B] ⁄ [Q]n[B]
(4)
The fluorescence intensity is proportional to the protein concentration as described
[B] ⁄ [B0]∝F ⁄ F0
(5)
Results from fluorescence measurements can be used to estimate the binding constant of polymer-protein complex. From eq 4
log[(F0 - F) ⁄ F] ) log KA + n log[Q]
(6)
The accessible fluorophore fraction (f) can be calculated by modified Stern-Volmer equation.
F0 ⁄ (F0 - F) ) (1 ⁄ f)K[Q] + 1 ⁄ f
(7)
where F0 is initial fluorescence intensity and F is fluorescence intensities in the presence of quenching agent (or interacting molecule). K is the Stern-Volmer quenching constant, [Q] is the molar concentration of quencher, and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional fluorescence contribution of the total emission for an interaction with a hydrophobic quencher.38 The plot of F0/(F0 - F) versus 1/[Q] yields f -1 as the intercept on y axis and (f K)-1 as the slope. Thus, the ratio of the ordinate and the slope gives K. The decrease of fluorescence intensity of HSA has been monitored at 330 nm for HSA-polymer systems (Figures 6 shows representative results for each system). The plot of F0/(F0 - F) versus 1/[dendrimers] (Figure 6 A-C show representative plots). Assuming that the observed changes in fluorescence come from the interaction between polymer and HSA, the quenching constant can be taken as the binding constant of the complex formation. The K values given here are averages of four-replicate and six-replicate runs for HSA/polymer systems, each run involving several different concentrations of dendrimers (Figure 6). The binding constants obtained were KmPEG-G3 ) 1.3 × 104 M-1 and KmPEG-G4 ) 2.2 × 104 M-1 and KPAMAM) 2.6 × 104 M-1 (Figure 6A′–C′). The association constants calculated for the polymer-HSA suggest low affinity dendrimers-HSA binding, compared to the other strong ligand-protein complexes.10,39 However, lower binding constants (104 to 105 M-1) were also reported for several other ligand-protein complexes using fluorescence spectroscopic methods.40-42
Figure 6. Fluorescence emission spectra of dendrimers-HSA systems in 10 mM Tris-HCl buffer pH 7.4 at 25 °C for (A) mPEGPAMAM-G3-HSA (a) free HSA (7.5 µM), (b-l) HSA 7.5 µM with mPEG-PAMAM-G3 at 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, and 200 µM; (B) mPEG-PAMAM-G4-HSA (a) free HSA (7.5 µM), (b-h) HSA 7.5 µM with mPEG-PAMAM-G4 at 20, 30, 40, 80, 100, 150, and 200 µM; (C) PAMAM-G4-HSA (a) free HSA (30 µM), (b-g) with PAMAM-G4 at 8, 20, 30, 40, 65, and 80 µM. The plot of F0/(F0 - F) as a function of 1/dendrimers concentration. The binding constant K being the ratio of the intercept and the slope for (A′) mPEG-PAMAM-G3-HSA, (B′) mPEG-PAMAM-G4-HSA, and (C′) PAMAM-G4-HSA complexes.
Similar polymer-protein interaction was observed for several dendrimers and bovin serum albumin.43,44 The binding constant for PAMAM-G4/HSA is expected to be the highest as there are 64 amino groups on the periphery of the dendrimer as compared with 16 on mPEG-PAMAM-G4 and 8 on mPEG-PAMAM-G3 dendrimers. Additionally, the intramolecular hydrogen bonding between mPEG and the PAMAM dendron should result in a weaker interaction in mPEG-PAMAM/HSA complexes. The f values shown in Figure 6 suggest that polymer interact with fluorophore via hydrophobic interactions. As a result, we predict that polymer binds mainly with the only fluorophore Trp 214, which is buried inside the HSA. This argument is based on the fact that the emission λmax of Trp 214 was 330 nm (Figure 6A-C), which is the emission region of hidden tryptophan molecules known to be usually around 340 nm,44,45 while fluorescence emission of exposed tryptophan molecule is around 340 nm due to solvent relaxation. The tightening of protein structure through intramolecular interactions, such as hydrogen bonds seem to bury Trp 214 in a more hydrophobic environment. The change in fluorescence intensity of Trp 214 in the presence of dendrimers may arise as a direct quenching or as a result of protein conformational changes induced by polymer-HSA complexation. The results indicate that polymer interaction do not
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Froehlich et al. periphery of the dendrimer increases the stability of polymerprotein complexation. The interaction of dendrimers leads to major HSA conformational changes indicating a partial protein unfolding at high polymer concentration. Abbreviations HSA, human serum albumin; PEG, poly(ethylene glycol); mPEG, methoxypoly(ethylene glycol); PAMAM, poly(amidoamine); FTIR, Fourier transform infrared spectroscopy; CD, circular dichroism. Acknowledgment. This work is supported by a grant from Natural Sciences and Engineering Research Council of Canada (NSERC). R.S.-H. acknowledges Missouri State University for a faculty grant. References and Notes
Figure 7. The plot of log (F0 - F)/F as a function of log[dendrimers] for calculation of number of bound polymer (n) in dendrimersHSA complexes.
change the emission λmax at 330 nm. No spectral shift was observed for the emission spectra upon polymer-HSA complexation, indicating that Trp 214 was not exposed to any change in polarity. The emission λmax of quenched tryptophan remains at 330 nm suggesting that dendrimers interact with HSA via hydrophobic region located inside the protein. This argument is consistent with the infrared analysis of dendrimers CH2 antisymmetric and symmetric stretching vibrations discussed earlier (Figure 4). The number of polymer bound (n) is calculated from log[(F0 - F)/F] ) log KS + n log[dendrimers] for the static quenching.46-50 The linear plot of log[(F0 - F]/F] as a function of log[dendrimers] shown in Figure 7. The n values from the slope of the straight line are 1.08 (mPEG-PAMAMG3), 1.50 (mPEG-PAMAM-G4), and 0.96 (PAMAM-G4) (Figure 7A-C). It seems about one molecule of the bulkier PAMAM-G4 can bind HSA tightly in comparison with smaller mPEG-PAMAM-G3 and mPEG-PAMAM-G4 dendrimers. Conclusions On the basis of our spectroscopic results dendrimers bind HSA via hydrophilic and hydrophobic interactions with the order of binding PAMAM-G4 > mPEG-PAMAM-G4 > mPEG-PAMAM-G3. Addition of amino groups on the
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