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Biomacromolecules 2005, 6, 2305-2311

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PEG-g-poly(GdDTPA-co-L-cystine): Effect of PEG Chain Length on in Vivo Contrast Enhancement in MRI Aaron M. Mohs,† Yuda Zong,† Junyu Guo,‡ Dennis L. Parker,‡ and Zheng-Rong Lu*,† Department of Pharmaceutics and Pharmaceutical Chemistry and Department of Radiology, University of Utah, Salt Lake City, Utah Received March 12, 2005; Revised Manuscript Received May 2, 2005

Biodegradable macromolecular Gd(III) complexes, Gd-DTPA cystine copolymers (GDCP), were grafted with PEG of different sizes to modify the physicochemical properties and in vivo MRI contrast enhancement of the agents and to study the effect of PEG chain length on these properties. Three new PEG-grafted biodegradable macromolecular gadolinium(III) complexes were synthesized and characterized as blood pool MRI contrast agents. One of three different lengths of MPEG-NH2 (MW ) 550, 1000, and 2000) was grafted to the backbone of GDCP to yield PEGn-g-poly(GdDTPA-co-L-cystine), PEGn-GDCP. The PEG chain length did not dramatically alter the T1 relaxivity, r1, of the modified agents. The MRI enhancement profile of PEGn-GDCP with different PEG sizes was significantly different in mice with respect to both signal intensity and clearance profiles. PEG2000-GDCP showed more prominent enhancement in the blood pool for a longer period of time than either PEG1000-GDCP or PEG550-GDCP. In the kidney, PEG2000GDCP had less enhancement at 2 min than PEG1000-GDCP, but both PEG550-GDCP and PEG1000-GDCP showed a more pronounced signal decay thereafter. The three agents behaved similarly in the liver, as compared to that in the heart. All three agents showed little enhancement in the muscle. Chemical grafting with PEG of different chain lengths is an effective approach to modify the physiochemistry and in vivo contrast enhancement dynamics of the biodegradable macromolecular contrast agents. The novel agents are promising for further clinical development for cardiovascular and cancer MR imaging. Introduction Low molecular weight gadolinium (III) chelates are routinely used in contrast enhanced magnetic resonance imaging exams. Gd-DTPA, Gd-DOTA, and their derivatives are used to aid the diagnosis of a wide range of pathologies by enhancing the morphology and functionality of the tissue.1-4 These are agents that have little inherent toxicity because they are rapidly eliminated as intact chelates. This results in undesirable pharmacokinetic performance, however, including transient retention time. The implication is that these agents are not ideal for imaging where blood pool retention is desired, such as cardiovascular and tumor imaging.5 Not only do these agents have transient blood pool retention, they are also extravascular, meaning they extravasate from the vasculature, decreasing the spatial resolution.6 Finally, these agents have a relatively low relaxivity, necessitating a higher dose to achieve the same signal intensity as an agent with higher relaxivity. Macromolecular Gd(III) complexes are a useful alternative to extravascular agents because the large size of macromolecules results in a long circulation half-life, confinement to the blood pool, and increased signal intensity in some cases, all of which allow for a more complete data acquisition from * Corresponding author. Address: 421 Wakara Way, Suite 318, Salt Lake City, UT 84108. Phone: 801 587-9450. Fax: 801 585-3614. E-mail: [email protected]. † Department of Pharmaceutics and Pharmaceutical Chemistry. ‡ Department of Radiology.

the MR exam.7 Macromolecular contrast agents have been developed from the conjugation of Gd-DOTA, Gd-DTPA, or their derivatives to synthetic polymers, such as polylysine and dendrimers,8,9 or to biological macromolecules.10 Unfortunately, the clinical development of these agents is limited because their slow clearance may result in a metabolic release of toxic Gd3+ ions. To alleviate this safety concern, we have designed novel biodegradable macromolecular agents based on polydisulfides.11 The disulfide bonds in the macromolecules can be readily reduced by the thiol-disulfide exchange reaction with endogenous or exogenous thiols. More prominent contrast enhancement was observed in the vasculature with the agents than a low molecular weight control, Gd(DTPABMA). The biodegradable macromolecular agents excrete as quickly as the control agent via renal filtration after MR imaging.12 Low molecular weight in vivo degradation products were identified in the mass spectra of the urine samples collected from the rats injected with the agents.11,13 Structural modification of the agents resulted in a variety of physiochemical and pharmacokinetic properties.13 Poly(ethylene glycol) is a nontoxic, nonantigenic, and biocompatible polymer that has been used in the modification of proteins and biomedical polymers to alter the in vivo behavior of these materials. Previously, we modified (Gd-DTPA) cystine copolymers (GDCP) with MPEG-NH2, yielding PEG-g-(GdDTPA-co-L-cystine).14 MPEG-NH2 (MW ) 2000) was added in a low (PEGa-GDCP) and a high PEG/

10.1021/bm050194g CCC: $30.25 © 2005 American Chemical Society Published on Web 06/16/2005

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Gd ratio (PEGb-GDCP). Both PEGylated contrast agents resulted in superior and more prolonged in vivo contrast enhancement compared to unPEGylated GDCP and a lowmolecular-weight control agent. Although PEGa-GDCP with a lower degree of PEG grafting had a higher in vitro relaxivity and larger hydrodynamic volume, PEGb-GDCP with a higher grafting degree provided superior contrast enhancement in mice as a blood pool agent. Most likely, higher-degree PEG grafting resulted in an agent less susceptible to degradation mechanisms via the disulfide bond in turn decreasing the elimination of the copolymers. Here we report further modification to PEG-GDCP by altering the length of the PEG chain to better understand the effect that PEG has on the physicochemical properties and in vivo contrast enhancement of PEG-GDCP. Three PEG-GDCP agents with different PEG sizes but similar grafting density were prepared. The size of PEG did not dramatically alter the T1 relaxivity of the agents, but significantly modified their in vitro degradation rate and in vivo contrast enhancement. Experimental Proceedures DTPA was purchased from J. T. Baker (Philipsburg, NJ). was purchased from Sigma (St. Louis, MO). Gd(OAc)3 was purchased from Alfa Aesar (Ward Hill, MA). Monomethoxy-poly(ethylene glycol)-amine (MPEG-NH2, MW ) 2000, 1000, 550) was purchased from Nektar Therapeutics (Huntsville, AL). DTPA-dianhydride was synthesized according to the literature.15 Poly(GdDTPA-co-Lcystine) was similarly prepared as previously described12,13 and fractionated by size-exclusion chromatography (SEC) on an AKTA FPLC with a HiPrep 26/60 column loaded with Sephacryl S-300 beads (Pharmacia, Piscataway, NJ) and eluted with 150 mM NaCl at a rate of 80 mL/h. Synthesis of PEGn-g-poly(GdDTPA-co-L-cystine). PEG2000-g-poly(GdDTPA-co-L-cystine) (PEG2000-GDCP) was synthesized from the 1:1 stoichiometric ratio of GDCP (100 mg, 0.13 mmol cystine) to mPEG-NH2 (MW ) 2000 g/mol, 266 mg, 0.13 mmol) via NHS (152.8 mg, 1.3 mmol) and EDC (381.9 mg, 2.0 mmol). GDCP was first dissolved in 2 mL of DI H2O with stirring. NHS and EDC were then added to the solution, and the solution was stirred for 20 min. MPEG-NH2-2000 was added slowly to the mixture. The reaction was stirred overnight at room temperature. Excess PEG was then removed by ultrafiltration using a Centricon-10 concentrator (membrane MWCO ) 10 000 Da). The purified PEG2000-GDCP graft copolymers were concentrated to dryness. PEG1000-g-poly(GdDTPA-co-Lcystine) (PEG1000-GDCP) and PEG550-g-poly(GdDTPA-coL-cystine) (PEG550-GDCP) were prepared and purified by using the same procedure with the same PEG/cystine molar ratio. The purified PEGylated GDCP copolymers were characterized by SEC and gadolinium contents were determined by ICP-OES (Perkin-Elmer, Norwalk, CT, Optima 3100 XL). PEG content on the graft copolymers was determined from the difference between the weight of the sample and the amount of GDCP in the sample based on Gd content given by ICP-OES and the molecular weight L-Cystine

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of the PEG chain in the graft copolymers. The size of the grafted copolymeric contrast agents was characterized by the number-averaged molecular weight, Mn, and the weightaveraged molecular weight, Mw. PEG2000-GDCP: Mn ) 28.5 kDa, Mw ) 37.7 kDa, Gd content ) 320.84 µmol Gd/g polymer, PEG/Gd ) 1.2. PEG1000-GDCP: Mn ) 31.1 kDa, Mw ) 37.8 kDa, Gd content ) 564.52 µmol Gd/g polymer, PEG/Gd ) 1.3. PEG550-GDCP: Mn ) 30.8 kDa, Mw ) 33.7 kDa, Gd content ) 614.89 µmol Gd/g polymer, PEG/ Gd ) 1.3. Relaxivity of PEGn-GDCP. The T1 relaxivity for the three PEGylated grafted GDCP contrast agents were determined on a Siemens Trio 3T MR scanner. T1 relaxation times for three concentrations of each contrast agent were determined by the sequential application of a standard inversionrecovery (IR) pulse sequence, with TR ) 5000 ms, TE ) 17 ms for inversion times TI ) 22, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, and 3500 ms in a water bath at room temperature, 21 °C. Net magnitization amplitude data for each sample were determined from the appropriate region of interest (ROI) using Osirix software and fit to the following multiparametric nonlinear regression (eq 1): MTI ) M0(1-2e-(1/T1×TI))

(1)

where TI is the inversion time, MTI is the net amplitude of a selected ROI at time, TI. The maximum amplitude at TI ) 0, M0, and T1, the concentration-dependent relaxation time of the agent, are fitted parameters. M0 and T1 were determined simultaneously for each concentration using Matlab software. Plotting 1/T1 versus [Gd(III)] for each polymer then gave the relaxivity, r1. In Vitro Degradation of PEGn-GDCP. PEG2000-GDCP, PEG1000-GDCP, and PEG550-GDCP (0.64 mM cystine based on Gd3+ content of the copolymers) were separately incubated in an aqueous cysteine solution at varying concentrations of cysteine (10, 100, 1,000, 10,000 µM) for 1 h at 37°C. The incubation mixture was analyzed by SEC to examine the effect of thiol concentration and PEG chain length on the degradation of the polymeric contrast agent. In Vivo MR Imaging. In vivo contrast enhanced MR imaging using PEG2000-GDCP, PEG1000-GDCP, and PEG550GDCP was investigated in female nu/nu athymic mice (Charles River Laboratories). The animals were cared for under an approved protocol and the guidelines of the University of Utah Institutional Animal Care and Use Committee. The mice were anesthetized by an i.p. injection of ketamine (80 mg/kg) and xylazine (12 mg/kg). Contrast enhanced images of the mice were obtained on a Siemens Trio 3T MR scanner with a wrist coil using a 3D FLASH pulse sequence. The imaging parameters were TE ) 2.4 ms, TR ) 7.4 ms, 25° flip angle, 3D acquisition with 64 slices/ slab, 120 mm FOV, and 0.5 mm coronal slice thickness. Each 3D data set had an acquisition time of 1 min 40 s. The PEGylated polymeric contrast agents were injected at a dose of 0.05 mmol/kg via a tail vein into an anesthetized mouse. Images were acquired before and 2, 5, 10, 15, 30, and 60 min postinjection of the contrast agents. The temperature of

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In Vivo Contrast Enhancement of PEG-GDCP Scheme 1. Synthesis of PEGn-GDCP

each mouse was maintained between image acquisitions by using a warming pad. Each PEGylated contrast agent was investigated in a group of three mice. Contrast-enhanced MR data was analyzed using the Osirix software package. Signal intensity values were calculated as follows (eq 2): Signal Intensity Ratio(t) )

Signaltissue(t) Signalmuscle(0)

(2)

where the signal intensity ratio at time t is the ratio of the signal in the selected ROI at time t, Signaltissue(t), relative to the signal in a thigh muscle at time t ) 0, Signalmuscle(0). Statistical Analysis. MR signal intensity at each data point is the average from three different mice. Error bars are ( the standard deviation. Statistical differences between the three polymeric contrast agents at each data point for each tissue were calculated using a one-way ANOVA with a Tukey post-test to determine which pair of data points accounted for the statistical difference of the grouping of three if their was one. A statistical difference was considered to be p < 0.05. Results Synthesis of PEGn-g-poly(GdDTPA-co-L-cystine). The synthetic procedure for PEG2000-GDCP, PEG1000-GDCP and PEG550-GDCP is shown in Scheme 1. GDCP was first fractionated, and a fraction with narrow molecular weight distribution (Mn ) 23.2 KDa; Mw ) 28.1 KDa) was used for modification with PEG. Monomethoxy-PEG amine with three different molecular weights, 2000, 1000, and 550 Da, was grafted onto the GDCP backbone. Each PEG graft was synthesized in a ratio of one PEG molecule to one L-cystine molecule. For each PEG grafting, the molecular weight distribution of the copolymers shifted to a higher molecular weight after the reaction, and unreacted PEG was removed by ultrafiltration, as verified by SEC. The apparent molecular weight of PEG2000-GDCP (MW ) 37.7 kDa) and

PEG1000-GDCP (MW ) 37.8 kDa) were similar, and it was slightly smaller for PEG550-GDCP (MW ) 33.7 kDa). These molecular weights are comparable to globular proteins of MW ) 92.2, 92.1, and 84.7 kDa for PEG2000-GDCP, PEG1000GDCP, and PEG550-GDCP, respectively. All three polymers had PEG/Gd molar ratios greater than one (PEG2000-GDCP, 1.2; PEG1000-GDCP, 1.3; PEG550-GDCP, 1.3). On the basis of the number-averaged molecular weight of GDCP, there are approximately 30 Gd3+ chelate units per polymeric contrast agent and 35-40 PEG chains grafted to GDCP. It is likely that salt remained in GDCP after dialysis, which affected the stoichiometry of the reactants. The physicochemical parameters of the copolymers are summarized in Table 1. Relaxivity of PEGn-GDCP. The relaxivity of the PEGng-poly(GdDTPA-co-L-cystine) did not change significantly as compared to that of its precursor, GDCP, which has a T1 relaxivity of 8.31 mM-1 s-1 (based on Gd concentration) at 3 T. When PEG2000 was grafted the relaxivity increased marginally to 8.73 mM-1 s-1. For PEG1000-GDCP and PEG550-GDCP, the relaxivity decreased slightly to 7.79 and 7.83 mM-1 s-1, respectively. The T1 relaxivity of PEG2000GDCP was higher than that of PEG1000-GDCP and PEG550GDCP. Degradation of PEGn-GDCP. PEG2000-GDCP, PEG1000GDCP, and PEG550-GDCP were incubated with L-cysteine of various concentrations to verify the degradability of the copolymers via the disulfide-thiol exchange reaction and to study the effect of PEG chain length on the degradation. Figure 1 shows that for all three polymeric contrast agents, a low concentration of cysteine (10 µM) had little effect on the molecular weight distribution of each polymer after 1 h. As the concentration of L-cystine increased the fraction of high-molecular-weight polymers decreased while the fraction of low molecular weight PEGylated degradation units increased. PEG2000-GDCP had the least reduction in the highmolecular-weight fraction. PEG550-GDCP on the other hand had a more rapid decrease from the high-molecular-weight fractions and a more dramatic increase in the low-molecularweight fractions. PEG1000-GDCP also had a significant increase in the low-molecular-weight fraction, but not as substantial as PEG550-GDCP. It appears that the length of PEG affects the degradation rate though the disulfide-thiol exchange reaction. In Vivo MR Imaging. Figure 2 shows the 3D maximum intensity projection (MIP) MR images of mice contrast enhanced with PEG2000-GDCP, PEG1000-GDCP, and PEG550GDCP at various time points. In these images the heart, kidneys, and vessels of the head and neck are all clearly shown by the contrast enhancement through the first five minutes postinjection. The signal intensity then gradually faded away based on the chain length of PEG in the graft copolymers. The contrast enhancement in the heart from PEG2000-GDCP remained strong throughout the first fifteen minutes and was still visible at 30 and 60 min. PEG1000GDCP gave enhancement profiles in which the signal intensity in the heart visibly decreased at 15 min and became background level at 60 min. The enhancement from PEG550-

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Table 1. Physicochemical Parameters of PEGn-GDCP molecular weight (kDa)

ICP characterization

polymeric contrast agent

Mw

Mn

Mw/Mn

GDCP PEG2000-GDCP PEG1000-GDCP PEG550-GDCP

28.1 37.7 37.8 33.7

23.2 28.5 31.1 30.8

1.2 1.3 1.2 1.1

Gd3+

(µmol/g agent) 846.52 320.84 564.52 614.89

relaxivity

PEG/Gd3+

mM-1 s-1

N/A 1.2 1.3 1.3

8.31 8.73 7.79 7.83

Figure 2. 3-D maximum intensity projection (3D-MIP) MR images of contrast enhancement of mice by PEG2000-GDCP (A), PEG1000GDCP (B), and PEG550-GDCP (C) at pre-injection (a) and 2 (b), 5 (c), 10 (d), 15 (e), 30 (f), and 60 (g) min after injection macromolecular contrast agent. Contrast agents were injected at a dose of 0.05 mmol/kg.

Figure 1. The molecular-weight distribution of PEGn-GDCP in the incubation with L-cysteine of various concentrations.

GDCP faded away more rapidly than that of PEG1000-GDCP, and it disappeared to background level after 30 min. A similar enhancement pattern was also observed in the blood vessels for the three agents. Figure 3 shows coronal slice images through the mouse abdomen to show the resolution of the descending aorta and common iliac arteries contrast enhanced by the agents. PEG2000-GDCP shows prominent enhancement of the vessels through 1 h, while PEG1000-GDCP gives sufficient enhancement until 30 min and PEG550-GDCP through 15 min. The ratios of the signal intensities of the heart, liver, and kidney to muscle are shown in Figure 4. The signal intensity in the heart with PEG2000-GDCP at 2 min was statistically different from the signal intensity in the same tissue compared to PEG550-GDCP (p < 0.05) but not to PEG1000GDCP (Figure 4A). PEG2000-GDCP gave statistically dif-

Figure 3. MR coronal slices through mouse blood vessels before (a) and 2 (b), 5 (c), 10 (d), 15 (e), 30 (f), and 60 (g) min after contrast enhancement by PEG2000-GDCP (A), PEG1000-GDCP (B), and PEG550GDCP (C).

ferent (p < 0.05) signal intensities in the heart compared to PEG550-GDCP at all time points. This difference was most pronounced (p < 0.001) at 10, 15, and 30 min. The difference between PEG1000-GDCP and PEG2000-GDCP in the heart enhancement was statistically significant at 5 min after injection and thereafter. There was no statistical difference in the signal intensity between PEG1000-GDCP and PEG550GDCP in the heart. In the kidneys, the contrast enhancement profile was different compared to that in the heart (Figure 4B). At 2 min after injection of contrast agent, PEG1000-GDCP showed the strongest signal intensity in the kidneys, but this

In Vivo Contrast Enhancement of PEG-GDCP

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Figure 4. Pharmacokinetic profile (mean ( SD, N ) 3) of PEGn-GDCP contrast agents in the heart (A), kidney (B), liver (C), and muscle (D).

difference was not significant. After 2 min, however, the signal intensity of both PEG1000-GDCP and PEG550-GDCP decreased more quickly than PEG2000-GDCP, whose signal decayed much slower. Two minutes and thereafter, PEG2000GDCP had a significantly different signal intensity than PEG550-GDCP. At 5 and 15 min after injection, PEG1000GDCP is significantly different from PEG550-GDCP as well. PEG2000-GDCP did not become significantly different than PEG1000-GDCP, most likely due to the relatively larger standard deviation in the enhancement of PEG1000-GDCP. The signal enhancement patterns in the kidneys were also clearly visible in Figure 2. In the 2 min column, the kidneys were visible with each polymeric contrast agent. For PEG2000GDCP this signal intensity was maintained out through 30 min. The kidneys for mice given either PEG1000-GDCP or PEG550-GDCP were visible in the 3D-MIP images at 10 min, but not as intensely as those with PEG2000-GDCP, and they were slightly visible for PEG1000-GDCP thereafter, but not for PEG550-GDCP. In the liver the contrast agents behaved similar to in the heart (Figure 4C). PEG2000-GDCP always had the highest signal intensity and was significantly different from PEG550GDCP at all time points. At 10 and 15 min, PEG1000-GDCP showed a significantly different enhancement pattern from PEG2000-GDCP. Overall, the enhancement from the three polymeric contrast agents in the liver was lower than that in either the heart or the kidneys. There was little contrast enhancement in the muscle and the difference between the agents was not significant (Figure 4D). Discussion Previously, we modified the biodegradable macromolecular contrast agent GDCP with PEG-2000 at different grafting ratios.14 The grafting ratios significantly affected the

physicochemical properties and in vivo blood pool contrast enhancement of the contrast agents. PEGb-GDCP with a high PEG grafting ratio (PEG/Gd ) 0.76) provided more significant and prolonged contrast enhancement than either GDCP or PEGa-GDCP (PEG/Gd ) 0.33) in mice. It is also interesting to know how the size of PEG affects the physicochemical properties and in vivo contrast enhancement of the modified agents. To minimize the potential variations due to the wide molecular distribution of GDCP copolymers, the copolymers were fractionated, and the same narrow fraction (Mw ) 28.2 KDa, Mw/Mn ) 1.2) was used for grafting with PEG of different molecular weights. PEG modified GDCP has approximately one PEG molecule per repeat unit, regardless of the size of PEG. The apparent molecular weight of the copolymers increased after the modification. The impact of PEG modification with different sizes on the T1 relaxivity of the contrast agents was moderate. The modified agents had similar T1 relaxivity as their precursor, GDCP (8.31 mM-1s-1). PEG2000-GDCP (8.73 mM-1s-1) has slightly higher relaxivity than PEG1000-GDCP (7.79 mM-1 s-1) or PEG550-GDCP (7.83 mM-1 s-1). The relaxivity of these agents is lower than that of the agents with lower grafting degrees that we previously reported.14 The T1 relaxivity of PEG2000-GDCP with grafting ratios of 0.33, 0.76, and 1.2 is 16.3, 12.7, and 8.73 mM-1 s-1, respectively. It appears that the increase of PEG density in the copolymer chains decreases the T1 relaxivity of the agent. A plausible explanation is that the high density of PEG on the polymer chains may interfere with the interaction of water molecules with the Gd(III) complexes because PEG can associate with water molecules via hydrogen bonding.16 The size of PEG in PEGn-GDCP significantly affects the contrast enhancement in the blood pool (Figures 2, 3, and 4A). Although MR signal intensity in the tissue of interest

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does not linearly correlate to the concentration of the contrast agents, its dynamic changes implicate the impact of PEG size on the in vivo properties of the modified agents. PEG2000GDCP displayed more significant and persistent contrast enhancement in the blood pool than either PEG1000-GDCP or PEG550-GDCP. PEG550-GDCP clears from the blood pool more rapidly than PEG2000-GDCP and PEG1000-GDCP. The phenomenon correlates well with the degradability of modified agents (Figure 1). The modification with PEG of high molecular weight results in contrast agents with a slow degradation rate and prolonged contrast enhancement in the blood pool. PEG increases the biocompatibility of biomedical polymers, sterically preventing interaction with other biomolecules.17 In this case, PEG-2000 most likely creates a larger hydrodynamic volume than either PEG-1000 or PEG550 around GDCP. Therefore, PEG2000-GDCP has a larger PEG volume protecting the biodegradable GDCP backbone by decreasing the probability of a low molecular thiol or enzyme in the blood stream interacting with the disulfide bond. Similar to the heart, the signal decay of the contrast agents modified with high molecular weight PEG in the kidney is slower than those with a lower molecular weight PEG (Figure 4B). PEG2000-GDCP resulted in more persistent contrast enhancement in the kidney than PEG1000-GDCP and PEG550GDCP. Two major factors, slow degradation rate and relatively large size of PEG-2000, may contribute to the slow clearance of PEG2000-GDCP in the kidneys. MPEG (MW ) 2000) is less readily cleared than PEG chains of shorter length via glomerular filtration.17 Therefore, the prolonged blood pool and kidney enhancement of PEG2000-GDCP may be attributed to higher T1 relaxivity, slow degradation rate of the macromolecules, and slow renal excretion of the degradation products due to the longer PEG chains. The contrast enhancement in the liver of the three PEGylated agents had similar uptake and clearance profiles. The initial signal intensity in the liver of each agent was lower than its signal intensities in the heart and kidney (Figure 4). The agent modified with high molecular weight PEG had relatively high signal intensity. PEG is often used to modify proteins and polymers to alter their pharmacokinetics and biodistribution. It has been shown that the presence of PEG on proteins decreases their uptake by reticuloendothelial cells while obtaining a longer half-life.18 PEG grafting has also been used on dendrimeric contrast agents, resulting in decreased liver uptake and more prolonged plasma enhancement.19 A similar effect was seen with the grafting of PEG to Gd(DTPA)-polylysine.20 We expect that the modification of the biodegradable macromolecular MRI contrast agents with PEG improves their pharmacokinetic properties and minimizes nonspecific tissue uptake, particularly the uptake in the liver. In this study we investigated the effect of PEG of different sizes on the physicochemical properties and in vivo contrast enhancement of PEGylated biodegradable macromolecular contrast agents. It was demonstrated that these properties can be significantly altered by the size of the PEG. Further detailed studies, including pharmacokinetics and safety of

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the agents, are needed to evaluate the potential of the modified agents in preclinical and clinical development. Conclusion The chain length of PEG in PEG-g-(GdDTPA-co-Lcystine) was altered to examine the effect that PEG has on the physicochemical and blood pool contrast enhancement profiles of the agent. PEG2000-GDCP showed the most prominent and prolonged contrast enhancement in the blood pool, while PEG550-GDCP and PEG1000-GDCP were cleared more quickly. The longer PEG grafts resulted in more persistent contrast enhancement in the kidney, indicating that the length of PEG may affect renal clearance of the agents. Acknowledgment. This research was supported in part by NIH grant R01 EB00489 and R33 CA095873. Aaron Mohs is also supported by the Predoctoral Fellowship from the PhRMA Foundation. We thank Dr. Yong-en Sun and Mr. Harvey Y. Feng for their technical assistance with MR imaging. References and Notes (1) Schneider, G.; Ahlhelm, F.; Seidel, R.; Fries, P.; Kramann, B.; Bo¨hm, M.; Kindermann, I. Contrast-Enhanced Cardiovascular Magnetic Resonance Imaging. Top. Magn. Reson. Imaging 2003, 14, 386402. (2) Semelka, R. C.; Helmberger, T. K. G. Contrast Agents for MR Imaging of the Liver. Radiology 2001, 218, 27-38. (3) Raghunand, N.; Howison, C.; Sherry, A. D.; Zhang, S.; Gillies, R. J. Renal and Systemic pH Imaging by Contrast-Enhanced MRI. Magn. Reson. Med. 2003, 49, 249-257. (4) Su, M.-Y.; Cheung, Y.-C.; Fruehauf, J. P.; Yu, H.; Nalcioglu, O.; Mechetner, E.; Kyshtoobayeva, A.; Chen, S.-C.; Hsueh, S.; McLaren, C. E.; Wan; Y.-L. Correlation of Dynamic Contrast Enhanced MRI Parameters with Microvessel Density and VEGF for Assessment of Angiogenesis in Breast Cancer. J. Magn. Reson. Imaging 2003, 18, 467-477. (5) Li, D.; Deshpande, V. Magnetic Resonance Imaging of the Coronary Arteries. Top. Magn. Reson. Imaging 2001, 12, 337-348. (6) Marchand, V.; Douek, P. C.; Benderbous, S.; Corot, C.; Canet, E. D. V. M. Pilot MR Evaluation of Pharmacokinetics and Relaxivity of Specific Blood Pool Agents for MR Angiography. InVest. Radiol. 2000, 35, 41-49. (7) Corot, C.; Violas, X.; Robert, P.; Gagneur, G.; Port., M. Comparison of Different Types of Blood Pool Agents (P792, MS325, USPIO) in a Rabbit MR Angiography-like Protocol. InVest. Radiol. 2003, 38, 311-319. (8) Schuhmann-Giampieri, G.; Schmitt-Willich, H.; Franzel, T.; Press, W.-R.; Weinmann, H.-J. In Vivo and In Vitro Evaluation of GdDTPA-Polylysine as a Macromolecular Contrast Agent for Magnetic Resonance Imaging. InVest. Radiol. 1991, 26, 969, 974. (9) Kobayashi, H.; Kawamoto, S.; Jo, S.-K.; Bryant, H. L.; Brechbiel, M. W.; Star, R. A. Macromolecular MRI Contrast Agents with Small Dendrimers: Pharmacokinetic Differences between Sizes and Cores. Bioconjugate Chem. 2003, 14, 388-394. (10) Schmiedl, U.; Ogan, M.; Paajanen, H.; Marotti, M.; Crooks, L. E.; Brito, A. C.; Brasch, R. C. Albumin Labeled with Gd-DTPA as an Intravascular, Blood Pool-Enhancing Agent for MR Imaging: Biodistrubution and Imaging Studies. Radiology 1987, 162, 205-210. (11) Lu, Z.-R.; Parker, D. L.; Goodrich, K. C.; Wang, X.; Dalle, J. G.; Buswell, H. R. Extracellular Biodegradable Macromolecular Gadolinium (III) Complexes for MRI. Magn. Reson. Med. 2004, 51, 2734. (12) Wang, X.; Feng, Y.; Ke, T.; Schabel, M.; Lu, Z.-R. Pharmacokinetics and Tissue Retention of (GD-DTPA)-Cystamine Copolymers, a Biodegradable macromolecular magnetic resonance Imaging Contrast Agent. Pharm. Res. 2005, 22, 596-602. (13) Zong, Y.; Wang, X.; Goodrich, K. G.; Mohs, A. M.; Parker, D. L.; Lu, Z.-R. Contrast Enhanced MRI with New Biodegradable Macromolecular Gd(III) Complexes in Tumor-Bearing Mice. Magn. Reson. Med. 2005, 53, 835-42.

In Vivo Contrast Enhancement of PEG-GDCP (14) Mohs, A. M.; Wang, X.; Goodrich, K. C.; Zong, Y.; Parker, D. L.; Lu Z.-R. PEG-g-poly(GdDTPA-co-L-cystine): A Biodegradable Macromolecular Blood Pool Contrast Agent for MR Imaging. Bioconjugate Chem. 2004, 15, 1424-1430. (15) Hantowich, D. J.; Friedman, B.; Clancy, B.; Novac, M. Labeling of preformed liposomes with Ga-67 and Tc-99m by chelation. J. Nucl. Med. 1981, 22, 810-814. (16) To´th, E.; van Uffelen, I.; Helm, L.; Merbach, A. E.; Ladd, D.; BrileySæbø, K.; Kellar., K. E. Gadolinium-based linear polymer with temperature-independent proton relaxivities: A unique interplay between the water exchange and rotational contributions. Magn. Reson. Chem. 36, 1998, S125-S134. (17) Harris, J. M.; Martin, N. E.; Modi, M. Pegylation: A Novel Process for Modifying Pharmacokinetics. Clin. Pharmacokinet. 2001, 40, 539-551.

Biomacromolecules, Vol. 6, No. 4, 2005 2311 (18) Veronese, F. M.; Monfardini, C.; Caliceti, P.; Schiavon, O.; Scrawen, M. D.; Beer, D. Improvement of pharmacokinetic, immunological and stability properties of asparaginase by conjugation to linear and branched monomethoxy poly(ethylene glycol). J. Controlled Relat. 1996, 40, 199-209. (19) Kobayashi, H.; Kawamoto, S.; Saga, T.; Sato, N.; Hiraga, A.; Ishimori, T.; Konishi, J.; Togashi, K.; Brechbeil, M. W. Positive effects of poly ethylene glycol conjugation to generation-4 polyamidoamine dendrimers as macromolecular MR contrast agents. Magn. Reson. Med. 2001, 46, 781-788. (20) Weissleder, R.; Bogdanov, A.; Tung, C. H.; Weinmann, H.-J. Size optimization of synthetic graft copolymers for in vivo angiogenesis imaging. Bioconjugate Chem. 2001, 12, 213-219.

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