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Journal of Controlled Release 115 (2006) 175 182 www.elsevier.

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A thermally responsive biopolymer for intra-articular drug delivery


Helawe Betre a , Wenge Liu a , Michael R. Zalutsky a,b , Ashutosh Chilkoti a , Virginia B. Kraus c , Lori A. Setton a,d,
a

Department of Biomedical Engineering, 136 Hudson Hall, Box 90821, Duke University, Durham, NC 27708, USA b Department of Radiology, Duke University Medical Center, Durham, NC, USA c Department of Medicine, Duke University Medical Center, Durham, NC, USA d Department of Surgery, Duke University Medical Center, Durham, NC, USA Received 9 April 2006; accepted 21 July 2006 Available online 26 July 2006

Abstract Intra-articular drug delivery is the preferred standard for targeting pharmacologic treatment directly to joints to reduce undesirable side effects associated with systemic drug delivery. In this study, a biologically based drug delivery vehicle was designed for intra-articular drug delivery using elastin-like polypeptides (ELPs), a biopolymer composed of repeating pentapeptides that undergo a phase transition to form aggregates above their transition temperature. The ELP drug delivery vehicle was designed to aggregate upon intra-articular injection at 37 C, and form a drug depot that could slowly disaggregate and be cleared from the joint space over time. We evaluated the in vivo biodistribution and joint half-life of radiolabeled ELPs, with and without the ability to aggregate, at physiological temperatures encountered after intra-articular injection in a rat knee. Biodistribution studies revealed that the aggregating ELP had a 25-fold longer half-life in the injected joint than a similar molecular weight protein that remained soluble and did not aggregate. These results suggest that the intra-articular joint delivery of ELP-based fusion proteins may be a viable strategy for the prolonged release of disease-modifying protein drugs for osteoarthritis and other arthritides. 2006 Elsevier B.V. All rights reserved.
Keywords: Intra-articular; Osteoarthritis; Drug delivery; Sustained release; Drug carriers; Thermogelling; Thermally responsive; Elastin-like polypeptide

1. Introduction Osteoarthritis (OA) is a degenerative joint disease that affects more patients than any other musculoskeletal disorder [1,2]. For symptomatic relief of osteoarthritis, the mainstay of therapy is chronic and oral administration of non-steroidal anti-inflammatories (NSAIDs), although they can cause gastrointestinal bleeding and ulceration [3] in a sub-population of patients. Joint symptoms are also treated with intra-articular administration of either corticosteroids or viscosupplements (e.g., hyaluronan based products) [4] although these therapies vary in their ability to control patient symptoms and have no disease-modifying effects. Numerous strategies have been developed to promote

Corresponding author. Department of Biomedical Engineering, 136 Hudson Hall, Box 90821, Duke University, Durham, NC 27708, USA. Tel.: +1 919 660 5131; fax: +1 919 681 8490. E-mail address: setton@duke.edu (L.A. Setton). 0168-3659/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.07.022

sustained intra-articular drug release in order to minimize the required frequency of intra-articular injection for osteoarthritis. For example, liposomes have been developed to encapsulate drugs for sustained local delivery and are believed to have promise for intra-articular drug therapy [57], although they generally exhibit a burst release due to instability in physiologic environments. Microspheres have also been used to encapsulate drugs for arthritis (e.g., methotrexate, paclitaxel), using degradable polymers such as poly(lactic) or poly(lactic-coglycolic acid) [814], albumin [12,15], N-N-dicarboxymethyl chitosan [16,17] and gelatin/chondroitin sulfate [18]. These approaches have been found to improve the residence time of synthetic drugs or active compounds within the joint space by as much as 10-fold. Current methodologies for synthesizing microspheres utilize high temperatures, organic solvents and/ or harsh non-selective cross-linkers; however, these conditions are not suitable for most bioactive proteins. This may preclude use of microsphere technologies with newer classes of antiarthritis drugs such as the TNF- or IL-1 blockers, that make

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use of soluble receptors, receptor antagonists or antibody fragments to inhibit pro-inflammatory activities associated with these cytokines [19]. Strategies that could significantly increase the intra-articular half-life of anti-arthritic protein drugs would have the potential to favorably impact the progression of arthritis in a targeted joint. One strategy of interest for intra-articular drug delivery is the concept of thermogelling polymers, such as polyethylene glycol (PEG) copolymers or chitosans, that have been evaluated as drug depot-forming materials for sustained release in systemic drug delivery [2022]. Similar to these thermogelling polymers, elastin-like polypeptides (ELPs) are biopolymers that consist of a repeating pentapeptide sequence present in native elastin (VPGX-G, where X is any amino acid except proline) [23]. ELPs are soluble in aqueous solution and exhibit an inverse temperature phase transition above a given transition temperature (Tt) characterized by the formation of micron and sub-micron size aggregates. ELPs have been studied for their ability to spontaneously entrap or encapsulate drugs at temperatures above their Tt [24] or to serve as a targeted release to solid tumors following systemic drug delivery [25]. ELPs have some unique advantages for biomedical applications in that they are biocompatible, non-immunogenic [26,27], and they can be conjugated to protein drugs at the gene level for expression as fusion proteins [2831]. The objective of this study was to evaluate ELPs as intraarticular drug delivery vehicles that could aggregate upon injection into a joint. ELPs were evaluated for their ability to form an aggregating drug depot at the injection site from which protein would disaggregate into the joint space over time, thus providing a mechanism for sustained delivery of the co-administered drug. The in vivo biodistribution and joint half-life of aggregating ELPs (Tt b 32 C), versus and non-aggregating or soluble ELPs (Tt N 50 C), were evaluated after intra-articular injection in a rat model. Results indicate that the soluble polypeptides have a halflife of less than 4 h within the joint space, while aggregating ELPs have a half-life of more than 85 h, an approximate 25-fold increase in intra-articular half-life. The kinetics of joint, blood and tissue levels of ELPs reveal that the aggregating ELPs concentrate in the joint upon injection, and slowly disaggregate promoting protein release, distribution and clearance over time. These results are promising for the use of ELPs for the biocompatible and sustained release of protein drugs to the intra-articular joint space as a treatment modality for osteoarthritis. 2. Methods 2.1. ELP synthesis Two genes encoding two different ELP biopolymers were used for this study. One was selected as a soluble or nonaggregating ELP with guest residues Val/Gly/Ala at a ratio of 1:7:8 (Tt N 50 C, MW 61 kDa) [32]. A second ELP was selected as the aggregating ELP drug carrier with guest residues Val only (Tt b 32 C, MW 47 kDa). These ELPs were selected from pre-existing gene libraries in order to encode proteins of similar molecular weights. The thermally-induced phase transition

profile has been characterized for both ELPs using turbidity measurements [32] and the rheological properties of the aggregating ELP [VPGVG] have also been reported above and below its transition temperature [33]. Both ELPs were expressed in E. coli and purified using the inverse transition cycling (ITC) technique [28]. 2.2. ELP radiolabeling The purified proteins were reconstituted at 1 mM and labeled by a reductive methylation reaction [34]. In a typical labeling reaction, the purified ELP was mixed with [14C]formaldehyde with specific activity 53 mCi/mmol (PerkinElmer, Boston, MA) and sodium cyanoborohydride (Pierce, Rockford, IL) at a molar ratio of 1:5:20 (ELP/HCOH/NaCNBH3) in PBS. The reaction mixture was incubated overnight at room temperature, and then dialyzed against 4L PBS (72 h at 4 C) until radioactivity in the dialysate was less than 200 cpm/ml. The dialyzed ELP was further purified by two rounds of inverse temperature cycling, reconcentrated to 1 mM and sterile filtered through a 0.22 m filter. The purity of the labeled protein was examined by SDS PAGE and autoradiography. The amount of 14C incorporated into the ELP was determined in two 10 l aliquots of the dialyzed ELP by spectrophotometrically measuring concentration followed by quantifying the radioactivity of the same aliquot on a liquid scintillation counter (15 ml of ACS-II scintillation cocktail, Amersham; RackBeta, PerkinElmer, Shelton, CT). The specific activity of each labeled protein (nCi/nmol) was calculated and the labeling ratio of the protein determined as the moles of 14C incorporated per mol of ELP. 2.3. Stability of
14

C radiolabel on ELP

Instability of the radiolabel by removal or cleavage of the radiolabel from [14C]ELP is a possibility for proteins exposed to bodily fluids and could interfere with accurate interpretation of the in vivo biodistribution data. For this reason, studies were undertaken to evaluate radiolabel stability in body fluids in vitro. [14C]ELP (VPGVG-containing) was diluted in either rat serum or 1% BSA solution in PBS to a final ELP concentration of 10 M. The mixtures (1 ml, n = 4 for each group) were then incubated at 37 C for 0, 9, 24 and 48 h, with 200 l aliquots removed at each time point and stored at 80 C. The 48 h time point was chosen to encompass a time greater than the maximum half-life of the non-aggregating ELP in the joint, and thus was the maximum time point of interest for stability of the radiolabel in the control population. At the end of the experiment, the small and large molecular weight fractions were separated by a low binding cellulose ultrafiltration membrane (UltraCel PL-10 10,000 MW cutoff, Millipore, Nepean, ON, Canada). Briefly, the [14C]ELP incubated with serum or PBS was transferred to the insert and centrifuged at 2000g for 30 min. Radioactivity in the filtrate and retentate was then measured on a scintillation counter. From the activities of both filtrate and proteins on the insert, the fraction of flow-through to total protein was calculated as a measure of 14C retention on

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ELP in serum. Data were normalized to time zero, and the effect of time and treatment (serum or PBS) on the stability of [14C]ELP was analyzed using two-factor ANOVA with StudentNewman Keuls post hoc test, at the significance level of = 0.05. 2.4. Biodistribution of non-aggregating and aggregating ELPs in vivo 2.4.1. Experimental protocol The biodistribution of the radiolabeled ELPs (non-aggregating and aggregating) were evaluated after intra-articular injection in a rat joint. Female Wistar rats (180200 g, Charles River Laboratories, Wilmington, MA) were sedated by i.m. administration of ketamine (40 mg/kg) and xylazine (5 mg/kg, Lloyd Labs, Shenandoah, IA), and the right knee prepared for aseptic injection. Animals then received one intra-articular injection of 30 l [14C]ELP at a concentration of 650 M (maximum dose 1 Ci/injection) in the right knee with a 30gauge needle (n = 5 animals per group for each time point radioactive dose of 0.005 Ci/g body weight). After injection, animals were housed in an approved animal facility with free access to food and water. Animals injected with soluble ELP were sacrificed at 10 min, 1.5, 3, 6, 15 or 48 h post injection. Animals injected with aggregating ELP were sacrificed at 10 min, 6 h, 1, 2, 4, 7, 14 or 28 days to track biodistribution of protein over longer periods of time. All procedures were approved by the IACUC and were performed in accordance with University guidelines. At each time point, the following body fluids and tissues were harvested: right and left knee synovial fluid, meniscus, cartilage, synovium, as well as blood, heart, lung, liver, kidney and bladder. Tissues and bodily fluids were weighed immediately after harvesting and then incubated in toluene based Tissue Solubilizer solution (Amersham) for 36 h at 50 C to ensure complete digestion. Synovial fluid was harvested by absorption onto pre-weighed filter paper [35]. Harvesting of all other fluids and tissues was routine. Radioactivity in tissue digests and body fluids was measured by liquid scintillation counting. Total counts per tissue weight were measured for each animal and for each tissue or body fluid. Values for all animals were evaluated to test for differences between soluble ELP and aggregating ELP groups at each time point (n = 5 per group and per time point). In addition, values for joint tissues and fluids were summed into a total joint value (synovial fluid, meniscus, joint cartilages, synovium) to test for comparisons between left (uninjected control) and right (injected) joints. A radioactive half-life (t1/2) was determined for the injected knee joint compartment by summing the total radioactivity count for the joint as described, and fitting to an exponential solution describing first-order decay kinetics. The half-life was obtained as t1/2 = ln(2), where is the time constant obtained from fitting to the exponential function. 2.4.2. Data analyses One-factor ANOVA with StudentNewmanKeuls post hoc tests at the significance level of = 0.05 were used to detect an effect of time on [14C]ELP/g tissue within each ELP formu-

lation, and to test for differences between right and left knee compartments within each ELP formulation. ANOVA was also used to test for differences between [14C]ELP in total blood volume and the injected (right) knee synovial fluid volume at a given time, within each ELP formulation. Differences in [14C] ELP/g between joint tissues (synovium, cartilage, meniscus, synovial fluid) of animals injected with aggregating ELP and soluble ELP, were also tested by ANOVA at matched time points (10 min, 6 h and 2 days). Finally, ANOVA was used to detect differences in the joint half-life between aggregated and soluble ELP formulations. 3. Results and discussion 3.1. ELP synthesis and radiolabeling After the reductive methylation reaction, [14C]ELP was measured to have a specific activity of 36.8 and 32.3 mCi/mmol for soluble and aggregating ELP, respectively (equivalent to 0.84 and 0.77 14C atoms per respective ELP). Thus, the reaction resulted in an approximate 80% conversion of ELP lysine residues to the methyl derivative, which is comparable to the 70% to 90% conversion efficiency previously reported for various globular proteins, including albumin [34,36]. 3.2. Stability of
14

C radiolabel on ELP

[14C]ELP incubated in PBS exhibited an average 14C cleavage of 0.57 0.19% from the labeled peptide during the 48 h observation period. [14C]ELP simply mixed with serum showed activity of 2.80 0.47% total labeled peptide at time zero, indicating a relatively high level of baseline activity for the animal sera. Therefore, data for free 14C after incubation in sera or PBS at all time points until 48 h are normalized to time zero values

Fig. 1. Stability of 14C conjugated to ELP shown after incubation in serum (hatched bars) or physiological saline (open bars) at 37 C as a function of time. Percent of free 14C released into incubation medium was measured by scintillation counting of fractions following ultra-filtration, normalized to time zero values, and plotted against incubation time for the [14C]ELP conjugate. Data normalized to time zero and expressed as mean S.E.M. (n = 4, p N 0.05, ANOVA, no difference with time in either saline or serum).

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and are shown in Fig. 1. [14C]ELP incubated in either solution did not show any significant increase in free label levels with time ( p N 0.05, ANOVA). These findings suggest that the cleavage of 14C from ELP was negligible throughout the experimental time period. 3.3. Biodistribution of soluble and aggregating ELPs in vivo 3.3.1. Non-specific ELP distribution The total radioactivity recovered from all tissues and fluids in the joint (average weight collected 0.73 0.07 g) was as-

Fig. 3. Profile of (A) soluble ELP (Tt N 50 C) and (B) aggregating ELP (Tt b 35 C) in the total volume of synovial fluid (injected knee) and blood with time. Data normalized to the total 14C amount recovered in injected knee at time zero (10 min post injection) and expressed as mean S.E.M. (n =5). ,# Statistical differences ( p b 0.05) from time zero within each tissue and from blood at a given time, respectively. (A) Soluble ELP rapidly clears from synovial fluid of the injected joint reaching below 1% of the injected dose with the first 24 h. It appears that a large amount of the cleared protein ends up in blood levels that peak at 30% of the injected dose at 15 h after injection, and then slowly clears from the body. (B) The synovial fluid concentrations of aggregating ELP drops rapidly upon injection as protein is cleared into the blood volume, and then appears to stabilize for nearly 7 days at 1235% injected dose. The amount of cleared protein in the blood remains at less than 12% of the injected dose throughout the experiment.

Fig. 2. Biodistribution of soluble [14C]ELP (Tt N 50 C) after intra-articular injection in a rat knee. The injected dose (ID) is amount of 14C recovered from the injected knee compartment (per gram of recovered tissues and fluids) at time zero (10 min post injection). The 14C-dose per tissue or fluid is normalized by the ID on a weight basis, to determine a normalized %ID/g for each tissue or fluid. All data expressed as mean S.E.M. (n = 5); dashed line at 1% ID/g represents background levels for most tissues. (A) p b 0.05, statistically different from time zero; +p b 0.05, statistically different from uninjected (left) knee compartment. (B) p b 0.05, statistically different from time zero; +p b 0.05, statistically different from blood. (C) The concentration of [14C]ELP was found to be below 1% ID/g for all organs, and these tissues were not found to be statistically different from the uninjected (left) knee at all time points.

sessed at 10 min after injection of [14C]ELP and found to be 74.1 6.2% and 71.0 7.1% of the administered dose for soluble and aggregating ELP, respectively. This suggests that the ELPs were largely absorbed and distributed in the joint cavity, including articular cartilage, synovium, synovial fluid and meniscus. The amount not recovered (2530%) was regarded as having been non-specifically lost during injection. To correct for non-specific distribution across all tissues and fluids, the value for the total injected dose for each animal was corrected to be equal to the amount in the injected knee joint cavity at time zero (10 min post-injection). In this manner, the percent of injected dose per gram (%ID/g) of tissue relative to the recovered amount at time zero was determined for all recovered tissues and fluids as reported below. 3.3.2. Soluble ELP distribution The distribution of soluble [14C]ELP in tissues and body fluids of Wistar rats is shown in Fig. 2. In general, values of %

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ID/g were found to differ amongst time points and between [14C]ELP injected and uninjected knees, blood and kidney (Fig. 2A and B, p b 0.05; ANOVA). The amount of radiolabeled protein in the injected knee decreased with time reaching background levels (b 1% ID/g) within the first 48 h after injection (Fig. 2A). With the exception of blood and kidney, the amount of soluble ELP in the other organs was below

background at all times (Fig. 2C). Thus, the soluble ELP does not appear to preferentially accumulate in any of the organs studied. Doses in the blood and kidney increased for the first 15 h to maxima of 4.3 0.8% and 1.7 0.4% ID/g, respectively. By 48 h, the level of radioactivity in the kidney was below background levels, and radioactivity in blood decreased to 1.30 0.37% ID/g. The amount of soluble [14C]ELP in the total synovial fluid volume, and the amount in total blood volume was estimated from animal weight (60 ml/kg of body weight [37]), and normalized to respective amounts at time zero (Fig. 3A). In general, values for %ID were found to differ amongst time points and between blood and synovial fluid compartments over time ( p b 0.05, ANOVA). By the first 15 h, 60.0 11.5% of the initial dose of nonaggregating ELP was distributed in the blood. In contrast, the amount of soluble ELP in synovial fluid was just above 1% at the same time point. These data clearly indicate that the soluble protein rapidly cleared from synovial fluid into blood. By 48 h, the amount of [14C]ELP in the total blood volume decreased to 18.3 5.2% of the injected dose, illustrating slow clearance of the polypeptide from the animal body. The moderate increase of ELP in the kidneys within the first 15 h and decrease by 48 h parallels the trends for blood, which suggests that ELPs may be cleared from the animal body through glomerular filtration renal clearance [38]. Urine was not collected in this study, however, so that this observation must be further studied to confirm the path of excretion. 3.3.3. Aggregating ELP distribution The distribution of aggregating [14C]ELP in the rat tissues and body fluids after a single intra-articular injection is shown in Fig. 4. As in the case of soluble ELP, the aggregating ELP did not appear to accumulate in the peripheral organs. With the exception of blood and the injected knee (right knee), all tissues including the uninjected (left) knee were near or below 1% ID/g. The right knee retained high levels of aggregating [14C]ELP for an extended period of time (Fig. 4A). The amount of ELP in the injected knee dropped from over 80% ID/g at 48 h to less than 10% ID/g at 14 days; however, ELP in the injected knee did not drop to 1% ID/g until 28 days post-injection. In comparison, soluble ELP in the injected joint dropped below 1% ID/g within the first 48 h after injection as described above (Fig. 2A). Aggregating ELP amounts in blood increased from background levels at time zero and 24 h to reach 1.02 0.75% ID/g at 48 h; values decreased back to background levels by day 7 (Fig. 4B). These findings illustrate the dramatic differences between the soluble and aggregating forms of ELP and suggest that the thermally-driven ELP aggregation process occurs spontaneously upon injection, as proposed. The amount of aggregating ELP in the total blood and synovial fluid volumes is shown in Fig. 3B. Similar to soluble ELP, the amount of aggregating ELP in the synovial fluid rapidly decreases in the first 24 h after injection. Unlike the soluble protein, however, the synovial fluid concentrations of aggregating ELP appear to stabilize and stay between 10% and 35% ID out to 7 days. Nevertheless, blood levels of the aggregating ELP at all times (between 6% and 9% ID) were lower than blood

Fig. 4. Biodistribution of the aggregating [14C]ELP (Tt b 35 C) after intraarticular injection in a rat knee. The injected dose (ID) is amount of 14C recovered from the injected knee compartment (per gram of recovered tissues and fluids) at time zero (10 min post injection). The 14C-dose per tissue or fluid is normalized by the ID on a weight basis, to determine a normalized %ID/g for each tissue or fluid. All data expressed as mean S.E.M. (n = 5); dashed line at 1% ID/g represents background levels for most tissues. (A) p b 0.05, statistically different from time zero; +p b 0.05, statistically different from uninjected (left) knee compartment. (B) p b 0.05, statistically different from time zero; +p b 0.05, statistically different from blood. (C) The concentration of [14C]ELP was found to be below 1% ID/g for all organs, and these tissues were not found to be statistically different from the uninjected (left) knee at all time points.

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Table 1 Biodistribution of [14C]ELP in tissues of the injected (right) and uninjected (left) knee compartments for both soluble and aggregating ELP formulations 10 min post injection Soluble ELP Right knee Condyle Meniscus Synovial fluid Synovium Tibia plateau Left knee a Condyle Meniscus Synovial fluid Synovium Tibia plateau
a

6 h post injection Aggregating ELP 25.65 5.41 5.23 0.88 42.52 7.01 , 6.98 1.40 12.45 2.99 0.08 0.02 0.01 0.00 0.01 0.00 0.05 0.04 0.06 0.02 Soluble ELP 7.24 0.60 , , 3.07 0.88 2.03 0.39 , 5.11 1.31 4.70 0.83 , 0.06 0.01 0.01 0.00 0.01 0.00 0.02 0.00 0.06 0.01 Aggregating ELP 16.46 5.14 10.39 3.14 34.50 9.31 4.95 2.38 7.15 2.22 0.06 0.01 0.04 0.03 0.03 0.02 0.03 0.01 0.08 0.02

2 days post injection Soluble ELP 0.24 0.05 , 0.09 0.02 , 0.02 0.00 0.13 0.03 , 0.13 0.02 , 0.04 0.00 0.01 0.00 0.01 0.00 0.01 0.00 0.03 0.00 Aggregating ELP 18.56 5.57 14.98 7.27 12.95 4.54 8.41 2.99 13.90 4.34 0.03 0.01 0.01 0.00 0.01 0.00 0.01 0.00 0.05 0.02

15.24 4.14 4.92 1.66 47.41 13.61 4.57 1.44 9.11 2.24 0.07 0.03 0.01 0.00 0.01 0.00 0.02 0.01 0.08 0.03

Bolded text denotes statistical difference ( p b 0.05) from amount of soluble ELP for matched tissue and matched time point. a Data for 14C in each tissue first normalized to total 14C injected into the right knee at time zero (%ID). All data expressed as mean S.E.M., n = 5. p b 0.05, statistically different from matched tissue in uninjected (left) knee at a given time point. p b 0.05, statistically different from time zero within the same knee for a matched tissue. p b 0.05, statistically different from day two within the same knee for a matched tissue.

levels for the soluble protein that reached as high as 60% ID (Fig. 3A), suggesting that the aggregating ELP also acts to reduce systemic exposure. The initial drop in fluid ELP levels might be associated with the distribution of the aggregating ELP formulation into other joint tissues, such as articular cartilage, synovium and meniscus. In addition, any disaggregated or free ELP was continuously removed from the joint space through the synovium, thus driving towards a more rapid disaggregation process than would be observed in a static system. This continuous clearance of ELP from the joint space promotes the formation of free ELP from ELP aggregates, and likely contributed to the rapid drop in ELP joint fluid levels at short time periods postinjection. 3.3.4. Comparison of soluble and aggregating ELP biodistribution within the joints Data for soluble and aggregating ELP levels in the tissues of the injected and control knees are shown in Table 1 at matched experimental points (10 min, 6 h and 2 days). At all time points, the amount of both ELPs in the control knee (left knee) was less than 1% ID/g, which is within background activity levels. This suggests that ELP does not re-enter the joint space, once it is cleared from the injected joint. This observation further suggests that intra-venous or other systemic delivery of protein drugs for the treatment of localized joint disease may not be effective in attaining therapeutic drug levels at the affected joint. This concern has motivated many investigators to explore novel intra-articular drug delivery methods, as well as gene therapy, for delivering disease-modifying protein drugs to the joint [39,40]. When soluble and aggregating ELP levels within joint tissues were directly compared with each other, statistically significant differences were observed across time points and between ELP formulations (Table 1). Differences were not observed between ELP formulations for the first 6 h after injection for all joint tissues except for synovial fluid, for which a 16-fold higher %ID/g was seen for the aggregating ELP

compared to soluble ELP. By day 2, aggregating ELP levels were significantly higher than soluble ELP levels in all joint tissues except meniscus. In general, the data showed that both

Fig. 5. Biodistribution of [14C]ELP in the injected joint compartment over time. The injected dose (ID) is amount of 14C recovered from the injected knee compartment (per gram of recovered tissues and fluids) at time zero (10 min post injection); all data for subsequent times are normalized to the time zero value. (A) Soluble ELP (Tt N 50 C) and (B) aggregating ELP (Tt b 35 C). Data expressed as mean S.E.M. (n = 5) were fit to a first-order exponential decay function (solid line) and plotted on a semi-log axis. Confidence intervals (95%, dashed lines) are also shown. The time constant () for each fit was used to calculate the joint half-life of the two ELP types t1/2 = ln(2)).

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the aggregating and non-aggregating ELPs distributed across the joint tissues shortly after injection, and that the aggregating ELP remained in the joint tissues at much higher levels (65- to 650-fold) than the non-aggregating ELPs over the 2-day timeframe. 3.3.5. Intra-articular ELP half-life The profiles of both soluble and aggregating ELP in the injected knee joint compartment are shown in Fig. 5, along with numerical fit of an exponential function to determine joint halflife of each ELP. The joint half-life of the soluble ELP was found to be 3.37 0.22 h, while that of the aggregating ELP was 87.6 4.6 h (3.7 0.2 days). These data illustrate that the aggregation property of ELP significantly prolonged the half-life of the peptide in articular joints by over 25-fold ( p b 0.05, significantly different from soluble ELP). Taken together, the data for intra-articular joint tissue distribution, synovial fluid levels, and joint half-life provide evidence that the proposed continuous and sustained release mechanism of the thermally-driven aggregation process could serve as an effective drug delivery system. 4. Conclusion In this study, an intra-articular drug delivery system was proposed based upon a thermally responsive elastin-like polypeptide (ELP) that can spontaneously aggregate upon injection into the knee joint, forming a drug depot from which polymer will disaggregate or re-solubilize over time. Data for the biodistribution of ELPs after intra-articular injection in vivo demonstrate that aggregating ELPs have a joint half-life that is over 25-fold longer compared to soluble, non-aggregating ELPs. Synovial fluid levels of the aggregating ELPs were sustained, between 10% and 35% of the injected dose from 6 h to 7 days after injection, suggesting not only extended joint halflife but also sustained release of the free peptide in the joint fluid. This proof-of-concept study provides support for a drug delivery mechanism whereby sustained drug release is achieved when co-administered with a spontaneously aggregating ELP formulation. This mechanism is similar to that proposed for ELP formulations that form small and stable particles upon thermally-induced phase transitioning [24], with the principal difference that the ELPs studied here appear to convert to a free form and become gradually cleared from the joint space over time. ELPs have several distinct advantages in serving as the thermally responsive biopolymer for this application. As a genetically engineered protein, ELP can be coupled to a protein drug as a fusion protein, as demonstrated for numerous drugs previously investigated for drug delivery to solid tumors [25]. In an ongoing work, ELP has been synthesized as a fusion to the anti-inflammatory protein drug, IL-1 receptor antagonist (IL-1Ra) [30]. IL1Ra has been shown to inhibit progression of osteoarthritic lesions in animal models [39,41,42], and to reduce pain and swelling of inflammatory arthritis in humans [43]. In order to overcome problems with rapid clearance of the drug from the body in treating osteoarthritis, investigators have focused attention on delivery of the IL-1Ra gene directly to the affected joint

space [39], or delivery of high IL-1Ra doses (150 mg/injection) via intra-articular injections [40] . For purposes of protein drug delivery, it is apparent that a well-conceived intra-articular drug delivery strategy could benefit the treatment of localized joint disease of a variety of etiologies. Furthermore, ELPs are biocompatible with all tissues, tissue fluids and bloods according to ASTM recommendations for materials and devices [26]. ELPs are biodegradable [44], can be easily purified with endotoxin levels below suggested guidelines [45], and elicit no known immune response [26]. ELPs can also be produced and purified with high yield at a cost comparable to or less than that of other recombinant proteins [31,46]. Ongoing work in the design and synthesis of effective ELPdrug fusion proteins will be important for further evaluating the efficacy of the drug delivery system in treating localized joint disease. Acknowledgements This work was supported by the NIH (AR047442 and EB002263) and UNCF-Merck graduate dissertation fellowship (HB). The authors would like to thank Ms. Katia Peixoto for assistance with animal studies and Ms. Charlene Flahiff for assistance with manuscript preparation. References
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