Folate Receptor-β in Activated Macrophages: Ligand Binding and

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Folate Receptor‑β in Activated Macrophages: Ligand Binding and Receptor Recycling Kinetics Bindu Varghese,† Erina Vlashi,† Wei Xia,† Wilfredo Ayala Lopez,† Chrystal M. Paulos,† Joseph Reddy,‡ Le-Cun Xu,‡ and Philip S. Low*,† †

Department of Chemistry, Purdue University, West Lafayette, Indiana 49707, United States Endocyte, Incorporated, West Lafayette, Indiana 49707, United States

‡

S Supporting Information *

ABSTRACT: Activated macrophages overexpress a receptor for the vitamin folic acid termed the folate receptor β (FR-β). Because conjugation of folate to low molecular weight drugs, genes, liposomes, nanoparticles, and imaging agents has minor effects on FR binding, the vitamin can be exploited to target both therapeutic and imaging agents to activated macrophages without promoting their uptake by other healthy cells. In this paper, we characterize the binding, internalization, and recycling kinetics of FR-β on activated macrophages in inflamed tissues of rats with adjuvant-induced arthritis. Our results demonstrate that saturation of macrophage FR is achieved at injection doses of ∼150−300 nmol/kg, with more rapidly perfused tissues saturating at lower doses than inflamed appendages. After binding, FR-β internalizes and recycles back to the cell surface every ∼10−20 min, providing empty receptors for additional folate conjugate uptake. Because the half-life of low molecular weight folate conjugates in the vasculature is usually 94%. Analysis of Folate Receptor Saturation in Vivo. Arthritic animals were administered increasing doses (0, 2, 5, 16, 88, 264, and 881 nmol/kg) of 111In-DTPA-folate by intraperitoneal injection. To determine the specificity of the 111 In-DTPA-folate for the folate receptor, one group of animals was treated with 88 nmol/kg of 111In-DTPA-folate plus a 1000fold excess folic acid. Animals were sacrificed 4 h later and organs were collected, weighed, and counted for radioactivity. Values were decay corrected and relative accumulated radioactivity values in each tissue converted to nanomoles per kilogram of tissue. Analysis of the Rate of Folate Receptor Recycling in Macrophages in Vivo. Analysis of the rate of FR recycling in vivo was based on three assumptions: (1) only empty cell surface receptors can mediate endocytosis of 111In-DTPAfolate, (2) de novo synthesized folate receptors represent only a small fraction of empty FR that move to the cell surface during any recycling experiment, and (3) dissociation of bound folate from extracellular FR at neutral pH is slow.47 On the basis of these premises, after initial saturation of FR with 111In-DTPAfolate (500 nmol/kg), the rate of reappearance of empty FR on the cell surface can be used to estimate the rate of FR recycling through intracellular compartments. To measure this rate of FR reappearance, saturating doses of 111In-DTPA-folate were injected into different sets of arthritic rats at different regular intervals. If additional uptake of 111In-DTPA-folate was observed, the time between injections was assumed to be long enough to allow for return of empty FR to the cell surface. If little or no increase in cell radioactivity was observed, then insufficient time must have elapsed for FR to release its cargo inside the cell and return to the cell surface. Thus, by noting the shortest time interval between 111In-DTPA-folate injections that yielded an increase in 111In-DTPA-folate retention, the minimum time for FR recycling could be estimated. For this purpose, six different frequencies of injection (every 5, 10, 20, 30, 60, and 120 min) were examined in six different sets of arthritic rats, and all animals were sacrificed 4 h after the last injection to allow equal time for clearance of unbound 111InDTPA-folate from the tissues. Tissues were then weighed and levels of accumulated radioactivity quantitated. Analysis of the Rate of Clearance of Folate Conjugates from Various Tissues. Animals with adjuvantinduced arthritis were administered a single dose of 500 nmol/ kg 111In-DTPA-folate via intraperitoneal injection. Animals were sacrificed 1, 3, 6, 12, 24, 48, 96, or 120 h postinjection, and tissues were collected, weighed, and counted for 111InDTPA-folate.

macrophage by endocytosis, release the bound drug into the cytoplasm, and return empty to the cell surface (recycling time), and (3) the time involved in clearance of unbound folate conjugates from the vasculature.

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MATERIALS AND METHODS Materials. Diethylenetriamine-pentaacetic acid (DTPA)folate and folate-rhodamine were generously provided by Endocyte, Inc. Folate−Oregon Green was prepared by solidphase synthesis as previously reported.43 All three folate conjugates (see structures in Supporting Information Figure 1) were linked via their gamma carboxyl groups to their imaging agents. Lewis rats (75−99g) were purchased from Harlan (Indianapolis) and folate-deficient rodent chow was obtained from Harlan Teklad (Madison, WI). Heat-killed Mycoplasma butyricum was purchased from BD Biosciences (Sparks, MD). Anti-ED1 (CD68) antibody and FITC/PE conjugates (ED1 clone) were purchased from Biolegend Inc. (San Diego). Induction of Adjuvant Induced Arthritis. Because commercial diets contain excessive amounts of folic acid, all animals were placed on a folate deficient diet 2 weeks prior to the induction of arthritis and maintained on the diet throughout the study. This allowed serum folate concentrations to reach normal physiological levels. For induction of adjuvant induced arthritis (AIA), animals were first anesthetized with ketamine/xylazine and then injected in the right hind paw with 100 μL (0.5 mg) of a suspension of heat-killed Mycoplasma butyricum in oil at a concentration of 5.0 mg/mL. We chose to use AIA because it is a well-established animal model of human inflammatory disease, characterized by inflammation in the paws, spleen, and liver.34,36,44 Studies were performed on day 25 postinduction of arthritis. All animal studies were approved by the Purdue Animal Care and Use Committee in accordance with NIH guidelines. Induction of Activated Peritoneal Macrophages by Thioglycollate Treatment. Lewis rats were treated with a 3.0 mL of sterile thioglycollate medium via intraperitoneal injection. Injected rats were then euthanized on day 5, and elicited macrophages removed from the peritoneal cavity by lavage. Labeling of Peritoneal Macrophages in Vivo with Folate-Fluorophore Conjugates for Confocal Microscopy and Flow Cytometry. Lewis rats with adjuvant induced arthritis were injected intraperitoneally with 500 nmol/kg of either folate-rhodamine (for confocal microscopy) or folateOregon Green (for flow cytometry).45 At the indicated times, peritoneal macrophages were extracted and washed with cold PBS and then labeled with mouse anti-ED1 antibody followed by FITC- or phycoerythrin-conjugated antimouse-IgG (each for 30 min at 4 °C). After washing once in cold PBS, cells were again labeled with Hoescht dye and examined by confocal microcopy or flow cytometry. For competition studies, 1000fold excess free folic acid was administered with the folate-dye conjugate to block all unoccupied FR. Preparation of 111In-DTPA-Folate. 111In- DTPA-folate was prepared on the same day as the shipment of 111In arrived and used immediately in experiments. The specific activity was assumed to be the same in each case. 111In-DTPA-folate conjugate was prepared as described previously.46 Briefly, 0.6 mL of sodium citrate buffer (3.33 mg trisodium citrate dihydrate, 1.89 mg NaOH in 1.0 mL water) was added to a vial containing 5.0 mCi (185 Mbq) 111In-chloride in 0.05 N

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RESULTS Expression of Folate Receptors on Rat Macrophages. To ensure that activated rat macrophages express a functional folate receptor similar to activated human macrophages,17,21,33,36,37,48 cells were removed from the peritoneal cavities of rats stimulated to develop adjuvant-induced arthritis and analyzed for FR expression by incubation with folateconjugated fluorescent dyes. As shown in the flow cytometry data of Figure 1A, cells that stain with an antibody to ED1 (a subpopulation of rat macrophages that are thought to be more 3610

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activated than those lacking ED149,50) also bind folate-Oregon Green.

in the inflamed appendages was directly assessed by joint resection and gamma counting. As seen in Figure 1C, FR binding was found to be competitively blocked by coadministration of 1000-fold excess free folic acid, not only in the affected appendages but also in inflamed macrophage-rich organs. These data collectively demonstrate that activated rat macrophages internalize folate conjugates via a process that is FR-mediated. Saturation of Folate Receptors with 111In-DTPA-Folate in Vivo. To determine the dose at which 111In-DTPA-folate saturates FR in vivo, a dose escalation study was conducted where arthritic animals were administered 0, 2, 5, 16, 88, 264, or 881 nmol/kg 111In-DTPA-folate, and after allowing 4 h for unbound conjugates to clear, animals were sacrificed and inflamed tissues were resected and counted. As shown in Figure 2A, 111In-DTPA-folate binding to activated macrophages in

Figure 1. Folate conjugate binding can be competed off with excess folic acid. (A). Cells were isolated from the peritoneal cavities of thioglycollate-induced rats and stained with both anti-ED1 and folateOregon Green in the absence (left panel) and presence (right panel) of 1000-fold excess free folic acid (see Methods). Cells were then examined by flow cytometry for coexpression of FR and ED1. (B). Arthritic rats were injected intraperitoneally with folate-rhodamine, and peritoneal macrophages were harvested 30 min later and stained with both Hoechst dye (to label the nuclei blue) and anti-ED1 + fluorescein-conjugated antimouse IgG (to label macrophages). Cells were visualized using an Olympus confocal microscope. Scale bar =10 μm. (C) Rats with adjuvant-induced arthritis were injected intraperitoneally with either 88 nmol/kg 111In-DTPA-folate or 88 nmol/kg 111 In-DTPA-folate plus 1000x folic acid and sacrificed 4 h later. Relevant tissues were then resected, weighed, and counted for uptake of 111In-DTPA-folate. Radioactivity in the right foot of rats in this and subsequent studies was not counted because the right foot was the appendage that was always injected to induce development of the adjuvant-induced arthritis. Data are expressed as mean ± SE and were plotted using GraphPad Prism.

Figure 2. Saturation of 111In-DTPA-folate binding by inflamed tissues of AIA rats in vivo. Rats with adjuvant-induced arthritis were administered a single dose of 2, 5, 16, 88, 264, or 881 nmol/kg of 111 In-DTPA-folate by intraperitoneal injection. Rats were sacrificed and dissected 4h later, and radioactivity was analyzed in (A) the lefthand, right-hand, and left foot, and (B) the liver and spleen. Data are expressed as mean ± SE and were plotted using GraphPad Prism.

Not surprisingly, some peritoneal cells that do not express ED1 also take up folate-Oregon Green, which we presume constitute a subset of ED1 negative macrophages.51 That the observed folate-Oregon Green binding is FR specific can be demonstrated by the marked suppression of folate conjugate binding in the presence of excess folic acid (Figure 1A, right panel). Related fluorescence microscopy studies with folaterhodamine (Figure 1B) confirm that ED1+ macrophages take up folate conjugates and further demonstrate that the bound folate-dye conjugates are internalized into endosomes (see punctate cytoplasmic distribution). Finally, to establish that macrophages in the arthritic joints also take up folate conjugates in an FR-dependent manner, 111In-DTPA-folate was injected intraperitoneally into arthritic rats and radioactivity

inflamed joints saturates at injected doses >300 nmol/kg. The comparable saturating doses in macrophage-rich organs (i.e., liver and spleen due to systemic inflammation) were ∼150 nmol/kg (Figure 2B). Except for the kidneys (known to express very high levels of FR-α52), accumulation of folate conjugates in all other tissues was negligible (data not shown). Measurement of the ratio of 111In-DTPA-folate in inflamed tissues relative to blood can provide information on the injection dose that yields the most efficient targeting to diseased tissues (Figure 3). 3611

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Figure 3. Ratio of folate conjugate uptake in inflamed tissues to blood. Rats with adjuvant-induced arthritis were injected intraperitoneally with 2, 5, 16, 88, 264, or 881 nmol/kg of 111In-DTPA-folate and sacrificed 4 h later for analysis of tissue radioactivity. Ratios of left foot:blood, liver:blood, and spleen:blood radioactivity are shown as a function of the dose of 111In -DTPA-folate administered. Data were plotted using GraphPad Prism.

Although this ratio reaches a maximum in the inflamed foot somewhere near 88 nmol/kg, the more rapidly perfused tissues (i.e., liver and spleen) clearly capture a larger share of the conjugate at lower concentrations, whereas inflamed joints only accumulate significant conjugate at higher concentrations. At still higher doses, uptake of 111In -DTPA-folate by FR positive tissues becomes saturated, leading to a monotonous decline in tissue:blood ratio. Because increased unbound and circulating folate conjugate can only lead to off-target drug release (due to premature cleavage of the folate-drug bond in the vasculature) with its consequent uptake by normal cells, assessment of the injection dose that yields the highest inflamed to normal tissue ratio can improve both imaging and therapeutic applications. Rate of Folate Receptor Internalization and Recycling in Vivo. The rate of folate conjugate internalization by inflammatory macrophages was assessed in vivo by injecting arthritic rats intraperitoneally with folate-rhodamine and withdrawing the peritoneal cell suspension at different times postinjection. As seen in the confocal images of Figure 4A, folaterhodamine was already internalized by 1 min postinjection and the macrophage cytosol was nearly saturated with targeted dye by 10 min postinjection. No further accumulation of folate conjugate was noted at the 30 min time point, suggesting that internalization was essentially complete within 10 min of exposure. That this uptake was FR-mediated could be established by demonstrating that 1000-fold excess of free folic acid largely prevented conjugate endocytosis (Figure 4A, competition group). The frequency of folate receptor recycling in macrophages in vivo was then estimated by repeatedly administering 100 nmol/kg 111In-DTPA-folate at increasingly shorter time intervals and noting the shortest time interval where maximal conjugate accumulation still occurred (Table 1). Thus, receptor recycling was assumed to have occurred if unoccupied receptors were encountered on the cell surface when each subsequent dose was injected (i.e., as indicated by the increased net accumulation over the 120 min duration of the study). In contrast, an insufficient time interval for receptor recycling to occur was assumed to have elapsed if retention of additional 111In-DTPA-folate uptake was not observed upon more frequent dosing. As seen in Figure 4B, 111In-DTPA-folate accumulation in inflamed joints began to decline as the time intervals between doses were shortened to 90%) is released from receptor-negative tissues within 24 h, with the half-life of the conjugate in the blood only ∼30 min, whereas the conjugate persists in FR+ tissues considerably longer. Even after 24 h, approximately 40% of the conjugate remains in the arthritic paws. We presume that the 111 In-DTPA-folate that clears slowly from paws, liver, and spleen constitutes surface-bound conjugate that has not yet become entrapped within the macrophage cytoplasm and that the retained fraction of 111In-DTPA-folate represents conjugate that has already been deposited in the cell interior. Obviously, this contrasting distribution of tissue residence times between inflamed and healthy tissues is advantageous for the development of new targeted medicines or imaging agents with high therapeutic indices.

CONCLUSIONS Folate-targeted therapeutics constitute an attractive strategy for the treatment of inflammatory diseases, having already proven successful for the targeted therapies of several cancers.56−65 The data presented here provide a quantitative basis for selecting optimal dosing concentrations and frequencies in vivo, based on the kinetics of receptor binding and recycling. Additionally, they confirm our hypothesis that folate conjugates are capable of sustained accumulation in folate receptor positive tissues and rapid clearance from nontargeted tissues. ASSOCIATED CONTENT

S Supporting Information *

Chemical structures of folate-rhodamine, folate-Oregon Green, and111In-DTPA-folate. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (765) 494-5273. Fax: (765) 494-5272. Address: Department of Chemistry, Purdue University, 720 Clinic Drive, West Lafayette, Indiana 47907, United States. Author Contributions

B.V., E.V., W.X., W.A.L., and L.X. performed experiments; C.M.P. analyzed results. B.V. and E.V. performed experiments, analyzed results and prepared the figures; B.V., E.V., and P.S.L. designed the research and wrote the paper. Notes

The authors declare the following competing financial interest(s): P.S.L. is Founder and CSO of Endocyte Inc., which helped support this work.

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ACKNOWLEDGMENTS We thank Christopher P. Leamon for his advice. This work was supported by a grant from Endocyte, Inc. ABBREVIATIONS ADCC, antibody-dependent cellular cytotoxicity; Id, idiotype; BCR, B cell receptor; Ig V, immunoglobulin variable 3614

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dx.doi.org/10.1021/mp500348e | Mol. Pharmaceutics 2014, 11, 3609−3616