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. Author manuscript; available in PMC: 2012 Sep 1.
Published in final edited form as: Arthritis Rheum. 2011 Sep;63(9):2671–2680. doi: 10.1002/art.30459

Folate-targeted nanoparticles show efficacy in the treatment of inflammatory arthritis

Thommey P Thomas 1, Sascha N Goonewardena 1, Istvan Majoros 1, Alina Kotlyar 1, Zhengyi Cao 1, Pascale R Leroueil 1, James R Baker Jr 1,*
PMCID: PMC3168725  NIHMSID: NIHMS296541  PMID: 21618461

Abstract

Objective

To investigate the uptake of a poly(amidoamine) dendrimer (generation 5 (G5)) nanoparticle covalently conjugated to polyvalent folic acid (FA) as the targeting ligand into macrophages, and the activity of a FA- and methotrexate-conjugated dendrimer (G5-FA-MTX) as a therapeutic for the inflammatory disease of arthritis.

Methods

In vitro studies were performed in macrophage cell lines and in isolated mouse macrophages to check the cellular uptake of fluorescently tagged G5-FA nanoparticles, using flow cytometry and confocal microscopy. In vivo studies were conducted in a rat model of collagen-induced arthritis to evaluate the therapeutic potential of G5-FA-MTX.

Results

Folate targeted dendrimer bound and internalized in a receptor-specific manner into both folate receptor β-expressing macrophage cell lines and primary mouse macrophages. The G5-FA-MTX acts as a potent anti-inflammatory agent and reduces arthritis-induced inflammatory parameters such as ankle swelling, paw volume, cartilage damage, bone resorption and body weight decrease.

Conclusion

The use of folate-targeted nanoparticles to specifically target MTX into macrophages may provide an effective clinical approach for anti-inflammatory therapy in rheumatoid arthritis.

Keywords: Dendrimer, nanotechnology, macrophages, folic acid, methotrexate, rheumatoid arthritis

Folate receptors (FR) comprise a family of glycosylphophatidylinositol (GPI)-anchored, high affinity receptors for folic acid (FA), and are the products of at least four different genes; FRα, β, γ, and δ (1, 2). Although much research has focused on the folate receptor-α (FRα) as a target for therapy and imaging in oncology, several recent studies have used the FRβ as a therapeutic target on macrophages in inflammatory diseases (3–5). In rheumatoid arthritis (RA), for example, FRβ has been shown to be specifically expressed on activated macrophages in inflamed joints, thus providing a target for the delivery of both therapeutics and imaging agents (6–9). Studies have demonstrated the specific internalization/accumulation of radionuclide-conjugated FA in inflamed joints of mice, rats, dogs, horses and even humans (3). The feasibility of using the FRβ to mediate the specific delivery of molecules is based on several unique biological characteristics of the FR. First, expression of the FRβ is specific to the myelomonocytic/macrophage lineages of hematopoietic cells, while there is negligible expression of this molecule on other blood cells such as lymphocytes, granulocytes or erythrocytes (9, 10). Second, elevated levels of functional glycosylated FRβ are expressed only on activated macrophages involved in inflammatory responses, but not on quiescent resident macrophages (5, 11). In addition, the FRα is known to be preferentially expressed at the apical (luminal) side (e.g., the urine side in kidney tubules and the air sac side of the lungs) of normal tissues; therefore it is possible that there will be minimal FRα binding of circulating folate-targeted drugs in normal tissues (12). Finally, although free FA and MTX can access normal cells through the ubiquitously expressed reduced folate carrier (RFC), uptake through this channel does not occur when FA or MTX are conjugated to a macromolecule like a nanoparticle. These findings together suggest that activated macrophages are the only predominant normal cells that could specifically bind and internalize folate targeted nanoparticle drugs from circulation through the FRβ.

Methotrexate (MTX) is an “antifolate” drug that competes with FA for binding to the dihydrofolate reductase enzyme and blocks the one-carbon transfer reaction used in the biosynthesis of nucleotides. This inhibits DNA formation and results in reduced cell proliferation and induction of apoptosis. For over two decades MTX has been employed as a disease-modifying anti-rheumatic drug (DMARD) for the treatment of RA, either as a single agent or in combination with biologics such as TNF inhibitors (13, 14). The mechanisms by which MTX suppresses arthritis are complex and may extend beyond the inhibition of DNA synthesis in inflammatory cells. It may include increased release of adenosine, altered production of cytokines, formation of reactive oxygen species, T cell apoptosis, and suppression of B cell responses (4, 15, 16). Several studies have suggested that at least one mechanism for MTX activity in RA is through anti-proliferative effects on activated macrophages following specific uptake into these cells via the FRβ (4, 5, 9, 10, 13, 15, 17–19). Unfortunately, about one-third of RA patients respond poorly to MTX while another third develop toxic side effects. Patients with increased side effects may have polymorphisms in proteins such as RFC and 5,10-methylenetetrahydrofolate reductase involved in the transport and metabolism of free MTX (20–22). Since the side effects of the MTX are the result of its action on normal cells, either preventing the uptake of MTX in normal cells or specifically targeting MTX to inflammatory cells independent of the RFC may prevent these problems.

Poly(amidoamine) (PAMAM) dendrimers have been extensively employed as a macromolecular delivery platform and have several attractive properties for this purpose (Figure 1). These properties include uniformity, biocompatibility, defined structure and the capability for chemically coupling multiple molecular entities to primary surface amino groups (23–26). Many biological functions of molecules coupled to the dendrimer’s surface are retained with a variety of molecules including hormones such as FA (27, 28), peptides (29), antibodies (30), chemotherapeutic drugs (28, 31), or an apoptosis-sensing fluorochrome (32). Importantly, prior studies from our laboratory have shown that dendrimers bearing FA can target FRα-expressing xenograft tumors in mice and increase the therapeutic index of carried MTX more than 10-fold (31). In another report, the anti-inflammatory drug indomethacin (IM) encapsulated in a generation 4 dendrimer-FA conjugate has been shown to preferentially target inflamed tissues in rats, presumably by targeting FRβ in macrophages (33). In the present study, we investigated using FA-bearing G5 dendrimers to target macrophages in vitro and deliver MTX as an anti-inflammatory agent in a collagen-induced arthritis model in rats.

Figure 1.

Figure 1

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Schematic representation of the structures and synthesis of different generations of PAMAM dendrimers, starting from the ethylene diamine (EDA) core, and that of the functionalized dendrimer conjugate G5-FA-MTX. The details of the synthesis steps and characterization of the conjugate have been previously described (24, 28). Briefly, Michael addition of methylacrylate to EDA followed by condensation reaction (amidation) gives the generation 0 (G0). These reaction steps are then repeated to obtain the higher generations as shown. The G5 is partially acetylated (60 – 70%), the FA is incorporated through amide linkage, followed by glycidolation (to fully neutralize the surface), and finally the MTX is conjugated through ester linkage.

MATERIALS AND METHODS

Materials

G5 PAMAM was synthesized and characterized at either the Michigan Nanotechnology Institute for Medicine and Biological Sciences (MNIMBS), University of Michigan, or at the Dendritech Inc., in Midland, MI. Methanol (HPLC grade), acetic anhydride (99%), triethylamine (99.5%), dimethyl sulfoxide (DMSO, 99.9%), dimethylformamide (DMF, 99.8%), glycidol, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide HCl (EDC, 98%), citric acid (99.5%), sodium azide (99.99%) and D2O were from Sigma. The fluorescein isothiocyanate (FITC, FI) and 5-Carboxytetra-methylrhodamine, succinimidyl ester [5-TAMRA, SE, (5T)] were purchased from Invitrogen (Carlsbad, California). The murine macrophage cell lines RAW264.7 and J774 were obtained from ATCC (Rockville, MD). The low FR-expressing MCA-207 mouse sarcoma cell line was kindly provided by Dr. Kevin McDonough at the University of Michigan. The RPMI cell culture medium, trypsin-EDTA, penicillin/streptomycin, Dulbecco’s phosphate buffered saline (PBS, pH 7.4), Hank’s balanced salt solution (HBSS), and Fetal Bovine Serum (FBS) were from Gibco/BRL (Gaithersburg, MD). Brewer Thioglycollate Medium (BTM; Sigma) was prepared by suspending 4.05 g of BTM in 100 ml de-ionized water, boiling to dissolve the medium completely and autoclaving at 15 lbs. pressure (121°C) for 15 minutes.

Synthesis and characterization of the dendrimer conjugates

The intermediates of the synthesis were extensively purified by dialysis and/or ultrafiltration before proceeding to the subsequent synthetic step. The final products and all intermediates have been characterized using 1H nuclear magnetic resonance (1H-NMR), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), mass spectrometry (MS), high-performance liquid chromatography (HPLC), gel-permeation chromatography (GPC) and UV spectroscopy, as we have described previously (28, 32, 34). A summary of the synthetic procedures used is given below, and the physical properties of the different conjugates are given in Table 1.

Table 1.

HDD (nM)1 PDI2 Zeta Potential3 Purity4 % MW (kDa)5 FA6 MTX6 5T FI
G5-FI 5.6 1.012 - >98 34.4 - - - 4.6
G5-FI-FA-1 5.9 1.058 - >98 40.5 4.3 - - 5.2
G5-FI-FA-2 - - - >95 38.3 4.0 - - 3.0
G5-5T 6.0 1.017 - >99 34.1 - - 6.2 -
G5-5T-FA 7.6 1.092 - >99 35.8 4.1 - 5.3 -
G5-FA-MTX-1 5.9 1.125 11.9 >99 40.7 2.5 8.0 - -
G5-FA-MTX-2 6.1 1.096 7.4 >99 38.2 5.0 10.5 - -
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The analytical data shown are performed at the MNIMBS (G5-FI, G5-FI-FA, G5-5T, G5-5T-FA), NCL, NCI (drug conjugates), and Cambrex Inc (the number of ligands for G5-FA-MTX-2).

1

Hydrodynamic diameter calculated as the volume-weighted average over a particular range of size populations corresponding to the most prominent peak in the % volume distribution obtained in dynamic light scattering (DLS).

2

Poly dispersity index (PDI).

3

Measured in PBS. The zeta potential of the amine-terminated starting dendrimer was 58.4±1.3. The slightly positive zeta potential for the conjugate may be attributed to residual secondary amines on the surface.

4

Based on HPLC analysis of percent conjugate peak area vs. total peak areas.

5

Average molecular weight determined by size-exclusion chromatography (SEC) coupled with a Multiple Angle Laser Light Scattering (MALLS) and by MALDI-TOF analyses.

6

Average numbers of FA and MTX per dendrimer, determined by UV-visible spectroscopy and GPC-MALLS.

FITC-Conjugates

(the number of each ligand present per conjugate molecule is given in parenthesis): G5-Ac(74)-FITC(5)-Gly(31) (G5-FI), G5-Ac(74)-FITC(5)-FA(4)-Gly(26) (G5-FI-FA-1) were synthesized and characterized using methods previously described (28). G5-FITC(3)-FA(4)-Gly(103) (G5-FI-FA-2) was synthesized as described (35). The molecular weight of the material was determined by GPC and the number of ligands (FA/FITC) on average attached to the dendrimer surface was calculated using this value in combination with 1H-NMR, and potentiometric titration of the starting G5-NH2 dendrimer (Table 1).

MTX-Conjugates

Two large scale batches of G5-Ac-FA-Gly-MTX (G5-FA-MTX-1 & G5-FA-MTX-2) were synthesized and characterized by Cambrex Corporation (East Rutherford, NJ) and by the National Characterization Laboratory, National Cancer Institute (Frederick, MD), using protocols similarly described (Figure 1 and Table 1) (28).

5TAMRA-Conjugates

G5-Ac(65)-5T(5)-Gly(48) (G5-5T) and G5-Ac(65)-5T(5)-FA(4)-Gly(40) (G5-5T-FA) conjugates were synthesized and characterized using procedures similar to that was described for the synthesis of 6TAMRA-based conjugates (36), as given in the Supplementary Data.

Cell culture and in vitro studies

RAW264.7 cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air, in RPMI medium containing 10% FBS supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml). The 10% FBS resulted in a final FA concentration equivalent to that present in human serum (~20 nM), and is denoted as ‘low FA medium’. The J774 and MCA207 cell lines were cultured in DMEM in similar conditions. In order to induce the expression of FA receptor, the medium was switched to FA-free RPMI with 10% FBS 5 to 7 days prior to performing experiments. Cells were plated in the low FA medium in 24-well plates (for flow cytometry), 35 mm dishes with glass-chambered dishes (for confocal microscopy).

Isolation of mouse peritoneal cavity macrophages (MPCM)

MPCM were isolated using the procedure described previously (37). Briefly, the mice were given intraperitoneal injection with ~2 ml of BTM 6 days prior to cell isolation. The abdominal wall was exposed, washed with 70% alcohol and injected ~5 ml of a solution of HBSS containing 5% fetal bovine serum (FBS) and 10 units/ml sodium heparin. The peritoneal fluid formed was aspirated gently. The cells were collected by centrifugation at 300 × g for 10 minutes at 4°C. The cell pellet obtained was suspended in 1 ml 10% HBSS, vortexed for 10–15 seconds to lyse any RBC, and diluted to isotonicity with HBSS. The cells were spun, counted and resuspended in a medium containing RPMI with 20% FBS, 15 mM HEPES, 20 mM L-glutamine, 50 μM 2-mercaptoethanol, and 50 μg/ml gentamicin, and transferred to 12-well plates, and the dishes were incubated for 1 hour at 37° in a humidified 5% CO2 incubator to allow the macrophages to attach to the dish. The non-adherent cells were removed and rinsed to remove any residual loosely adherent cells, and incubated with low folate medium overnight at 37°C in a humidified 5% CO2 incubator. The medium was changed to fresh medium prior to addition of the conjugates.

Flow cytometry studies

The binding of fluorescently labeled folate targeted dendrimers (G5-FI-FA) to macrophages (RAW264.7 and MPCM) was evaluated using a Beckman-Coulter EPICS-XL MCL flow cytometer and the data were analyzed using Expo32 software (Beckman-Coulter, Miami, FL). The viable cells were gated and the mean FL1-fluorescence of 10,000 cells was quantified.

Confocal microscopy studies

RAW 264.7 macrophage cell line was visualized using an IX81 inverted microscope (Olympus America, Center Valley, PA) equipped with an FV1000 confocal unit and a 60x/1.4 NA/0.15 mm oil emersion objective. For cell imaging, macrophages were plated in glass-chambered dishes (Nalge Nunc International, Rochester, NY) and allowed to adhere for 24 hour at 37°C, followed by washing two times with PBS to remove nonadherent cells. For in vitro staining, cells were incubated with G5-5T-FA (30 nM) at 37°C for 1 hour before washing 2 times to remove unbound dendrimer conjugate. A 543 nm HeNe laser was used to excite the dendrimer-5T and a 405-nm laser diode was used to excite the DAPI flourophore. Two-color imaging was performed with two spectral detectors (DAPI, excitation 405 nm, detector range 455 nm; 5T, excitation 543 nm, detector range 573–648 nm). Images were processed using FLUOVIEW software (Olympus America).

In vivo studies in a rat arthritic model

The in vivo studies were conducted at the Bolder BioPATH Inc., Boulder, CO. Female Lewis rats (Charles River Laboratories, Inc., Wilmington, MA) weighing an average of 150 g on day 0 of the study were acclimated to the laboratory conditions for ~7 days. The animals were anesthetized with isoflurane and given subcutaneous/intradermal (SC/ID) injections with 300 μl of Freund’s Incomplete Adjuvant (Difco, Detroit, MI) containing 2 mg/ml bovine type II collagen (Elastin Products, Owensville, Missouri) at the base of the tail and 2 sites on the back on days 0 and 6. Drug dosing was initiated on day 0 of the study and continued through day 16. Immunized rats (n=10/group) were treated intravenously three times per week with either 70 mg/Kg of G5-FA-MTX (two separate batches, equivalent to 5 mg/Kg MTX), 0.2 mg/kg free MTX, or normal saline. Control immunized rats (n=4) were also treated with normal saline using a similar regimen. The rats were weighed on days 0, 3, 6, and 9 to 17 of the study, and caliper measurements of the ankles were taken every day beginning on day 9. After a final body weight measurement on day 17, animals were euthanized, blood was drawn for serum, and tissues were collected. Hind paws were transected at the level of the medial and lateral malleolus, weighed, and placed in formalin, with knees, for microscopy. For histopathological evaluation, preserved and decalcified (5% formic acid) ankle and knee joints were cut in half longitudinally (ankles) or in the frontal plane (knees), processed through graded alcohols and a clearing agent, infiltrated and embedded in paraffin, sectioned, stained with Toluidine Blue, and analyzed by microscopy.

RESULTS

In vitro studies

We initially determined the time- and dose-dependent uptake of the fluorescent conjugate G5-FI-FA in the RAW264.7 cells by flow cytometric analysis. We have utilized two separate lots of G5-FI-FA in this study. Both these lots were initially tested in KB cells which express high FR, and was shown to bind in a dose-dependent and receptor-saturable fashion, and the binding was completely inhibited by 50-fold excess free FA (data not shown). An initial time-course analysis using the G5-FI-FA-1 showed that the conjugate bound to the RAW cells in a time-dependent manner with maximum binding at 3–4 h (300 nM the G5-FA-FI-1 showed a mean FL-1 fluorescence of 154 ± 7 at 4 h). As shown in Figure 2A, the G5-FI-FA-2 was taken up into the RAW264.7 cells in a dose-dependent fashion. The macrophage activating agent LPS failed to show any increase in the uptake of the conjugate up to 100 nM of the conjugate, but showed a modest increase at 300 nM. The control conjugate G5-FI (300 nM) failed to show any significant uptake into the RAW264.7 cells up to 4 h (incubation with 300 nM the G5-FI gave a net FL-1 fluorescence of 5.9±0.7). As the auto-fluorescence of RAW264.7 cells at the emission wavelength of FITC was significant for confocal analysis, we synthesized conjugates using the dye 5-TAMRA (5T) whose excitation/emission wavelength didn’t result in significant auto-fluorescence. As shown in Figure 2B, the G5-5T-FA-1 internalized into the RAW264.7 cells, showing localization in the cytosolic compartment. Consistent with the flow cytometric analysis, the control conjugate G5-5T failed to show any cellular accumulation. We also tested the conjugate uptake in the mouse macrophage cell line J774 and compared it with the low FR-expressing mouse sarcoma cell line MCA207 (38). In cells treated with 100 nM G5-FI-FA-1 for 1 hour, flow cytometric analysis gave the mean channel FL1 fluorescence values of 20.9±8.5 and 4.7±0.8 over PBS controls for the J774 and MCA207 cells, respectively. This is consistent with our previous studies showing lack of cytotoxicity of a G5-FA-Methotrexate drug conjugate in the MCA207 cells during a 3 day incubation period (38).

Figure 2.

Figure 2

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A. Dose dependent binding of G5-FI-FA-2 (3 h incubation) to RAW264.7 cells in the presence (red) or absence (blue) of 1 μg/ml LPS (15 h pre-incubation), analyzed by flow cytometry. B. Confocal images of RAW264.7 cells incubated with the indicated conjugates (orange) and the nuclei stained with DAPI (blue). C. Binding of G5-FI-FA on isolated mouse peritoneal cavity macrophages (MPCM). MPCM were incubated with 1 μM of G5-FI or G5-FI-FA-1 at 37°C for 2 h in the presence (red) or absence (blue) of 100-fold excess of free FA, and the mean fluorescence (given in parenthesis) was quantified. The mean fluorescence for the FR-negative MCA 207 cells, and the J774 macrophage cells treated with G5-FI-FA-1 were 4.7±0.8 and 20.9±8.5 (100 nM and 1 h incubation), respectively. The data shown are representative of treatments made in triplicate wells, with similar results obtained in an independent experiment.

We then isolated mouse peritoneal cavity macrophages (MPCM) and the uptake of the G5-FI-FA conjugate in these cells was tested. As had been observed for the cultured macrophage cell lines given above, the G5-FI-FA-1 bound to the MPCM, whereas the control conjugate G5-FI showed only low binding (Figure 2C). In order to test the FR-specificity of the conjugate, the effect of pre-incubation of cells with excess free FA was tested. A 100 X-fold molar excess of free FA partially blocked the binding of the G5-FI-FA in the MPCM (Figure 2C). Similar partial blocking of the uptake of G5-FI-FA by excess free FA was also observed in the RAW264.7 cells.

In vivo studies in the rat arthritis model

Our previous studies demonstrated that the dendrimer conjugate ‘G5-FA-MTX’ acts as a tumoricidal agent in vivo in the KB xenograft mouse tumor model with a higher therapeutic index than free MTX (31). G5-FA-MTX for these studies was synthesized using the same multistep procedure (28) and then tested in female Lewis rats using a 17-day type II collagen arthritis protocol. The collagen-induced arthritic rats (n = 10 for each group) were treated intravenously thrice a week with either vehicle (saline), two different batches of G5-FA-MTX (70 mg/kg for each conjugate, equivalent to 5 mg/kg of MTX), or free MTX (0.2 mg/kg), and the non-arthritic control rats (n = 4) were similarly treated with vehicle (saline). Efficacy evaluation was based on ankle caliper measurements and animal body weight measured from day 9–17, and also based on the histopathological analysis performed on day 17 following euthanasia of the animals. As shown in Figure 3, the increased ankle diameter and paw weight measurements caused by arthritis were decreased in rats treated with the conjugate. Significant inhibition of ankle diameter AUC was seen in rats treated with G5-FA-MTX-1 (88% inhibition), G5-FA-MTX-2 (101%), or MTX (54%), as compared to disease controls. Final paw weights were significantly inhibited by treatment with G5-FA-MTX-1 (89% inhibition), G5-FA-MTX-2 (105%), or MTX (46%), as compared to disease controls. Moreover, significant inhibition of body weight loss due to arthritis was seen in rats treated with G5-FA-MTX-1 (42% inhibition), G5-FA-MTX-1 (48%), or MTX (38%), as compared to disease controls (Figure 3). Histopathological evaluations at the end of the study analyzing ankle and knee inflammation and pannus infiltration, cartilage damage and bone resorption clearly indicated protection from arthritis-altered parameters by treatment with the conjugates and free MTX. As shown demonstrated in Figure 4C, normal rat treated with vehicle showed normal synovium (‘S’) and normal cartilage (large arrow) with no pannus or bone destruction (small arrow). Arthritic rat treated with vehicle had severe synovitis, moderate cartilage damage, and mild pannus and bone destruction. Arthritic rat treated with G5-FA-MTX-1 and G5-FA-MTX-2 had minimal synovitis, no cartilage damage, and no pannus or bone destruction. Arthritic rat treated with free MTX had moderate synovitis, minimal cartilage damage and minimal pannus and bone destruction. Photomicrographs with identical trends were observed for the knee histopathology from the respective groups (not shown). These parameters were scored on a scale of 0 to 5, with 0, 1, 2, 3, 4 and 5 scored as normal, minimal, mild, moderate, marked and severe respectively. The effects of the drug conjugates were, however, significantly greater than those of free drug.

Figure 3.

Figure 3

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Effect of G5-FA-MTX treatment on the ankle diameter (A, B), paw weight (C) and body weight (D) of arthritic rats. Normal and arthritic rats were treated with free MTX (0.6 mg/Kg/wk) or G5-FA-MTX (225 mg/Kg/wk = 15 mg/Kg/wk MTX equivalent), or their vehicle, and the ankle diameter (day 9 through 17, A), paw weight (C) and body weight (day 0 to 17, D) were measured. B shows the area under the curve (AUC) for the data given on A. * p < 0.05 vs. Arthritis Vehicle.

Figure 4.

Figure 4

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Histopathological evaluation of the ankles (A) and knees (B) of arthritic rats treated for 17 days with G5-FA-MTX or free MTX, obtained for the data shown in Figure 3. * p < 0.05 vs. the Arthritis Vehicle controls. For each of the indicated treatment conditions, the bars represent (from left to right) inflammation, pannus, cartilage damage, bone resorption, and the average of these four parameters. These parameters for control non-arthritic rats were all scored as zero (not shown). C. Representative photomicrographs of stained ankle from rats from normal rats (1), and arthritic rats treated with vehicle (2), G5-FA-MTX-1 (3), G5-FA-MTX-2 (4), and MTX (5). (S= synovium; large and small arrows indicate cartilage and bone/pannus, respectively).

The effects of the conjugates and the free MTX on organ weights of the animals were also examined (Figure 5). Relative liver and thymus weights for treatment groups did not differ significantly from disease controls. Relative spleen weights were significantly increased (above normal and disease controls) following treatment with G5-FA-MTX-1 (18% increase), or G5-FA-MTX-2 (12%), and were significantly reduced (below normal and disease controls) following treatment with MTX (9% decrease).

Figure 5.

Figure 5

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Effect of G5-FA-MTX on the relative weights of liver (A), spleen (B) and thymus (C). Collagen-induced arthritic rats (n=10) were treated with free MTX or two different batches of G5-FA-MTX, or their vehicle, and the organ weights were taken on the 17th day of treatment, as described in the Methods. The values are expressed as the percent of the body weight of the individual animals. * p < 0.05 vs. the respective Arthritis Vehicle control.

An independent study was conducted to test the dose responsive efficacy of the conjugate vs. free MTX (Figure S1). At the maximum dose used for the conjugate (225 mg/Kg/wk) and at the known MTD for MTX (2 mg/Kg/wk), the ankle diameter was reversed to the normal control values.

DISCUSSION

Several lines of evidence have indicated that macrophage activation plays a significant role in the inflammatory pathogenesis of RA (39–42). The concept of targeting macrophages through the FRβ to treat RA is supported by the expression of this target in activated macrophages (39–42), the effectiveness of MTX as a drug for RA (13, 14, 42, 43), and our previously documented utility of G5-FA-MTX as a therapeutic agent in FRα-expressing tumor models. We therefore sought to examine G5-FA-MTX as a DMARD in vivo in a collagen-induced arthritis model in rats. The results of our studies provide clear evidence G5-FA-MTX acts as a targeted therapeutic for macrophages through the FRβ effectively suppressing inflammation and arthritis in the rat model.

Our initial in vitro studies documented that a FITC-labeled, folate targeted nanoparticle (G5-FI-FA) bound to both FRβ-expressing macrophage cell lines and activated peritoneal macrophages in a receptor-specific manner and subsequent confocal microscopic analysis demonstrated the internalization and cytosolic localization of 5-TAMRA labeled folate-targeted nanoparticles in RAW264.7 cells. The receptor specificity of this binding was apparent given that the FA conjugate bound to the FR-expressing macrophages and cell lines while control dendrimer conjugates lacking folate, G5-FI and G5-5TAMRA, failed to bind to these cells. In contrast to our previous studies in KB cells (28), however, the binding of G5-FI-FA could only be partially inhibited by excess free FA. The reasons for this incomplete inhibition are not clear, but given that the control conjugates G5-FITC and G5-5TAMRA were not taken up by macrophages it is unlikely that a portion of the uptake of the targeted conjugate was not dependent on the FRβ. Our findings are similar to a previous study that showed that the uptake of FA-targeted liposomes was completely inhibited by free FA in FRα-expressing IGROV cells but was only ~50% inhibited in FRβ-expressing macrophages FA (44). While it is tempting to speculate that polyvalent binding of the FA-targeted nanoparticle to FRβ-expressing macrophages is more avid and therefore more difficult to compete than with the FRα-expressing KB cells, this finding requires more extensive examination.

Our in vivo studies in the collagen-induced rat arthritis model clearly documented that administration of G5-FA-MTX yields significant and beneficial protection from the inflammatory characteristics of collagen-induced arthritis (Figures 3). In addition, the time-dependent increase in the ankle diameter and paw weight associated with arthritis was almost completely prevented by administration of the G5-FA-MTX conjugate. Moreover, weight loss due to arthritis was prevented in rats treated with the conjugate (Figure 3) and necropsy histopathology substantiated the prevention of the inflammatory pathology of the arthritis (Figure 4). Thus, essentially all arthritis-induced effects, including infiltration of inflammatory cells, edema of the knee and ankle, pannus infiltration in cartilage and bone, cartilage damage, and bone resorption were all significantly prevented by the targeted conjugates (G5-FA-MTX-1 and G5-FA-MTX-2) (Figure 4). Importantly, as compared to animals treated with free MTX, the G5-FA-MTX conjugate treated animals showed no abnormalities in their spleen tissue architecture (Figure 5B). This suggest high efficacy for this conjugate in treating joint inflammation, with the potential to have fewer side effects.

Although the inflammatory symptoms were almost completely reversed by the conjugates and MTX toward the normal control, the body weight loss was only partially reversed by the drugs (Figure 3). The reason for this is not clear, and may be related to handling of the animals. In this regard, a similar discrepancy has been observed with Lewis rats treated with MTX and cyclosporine A (45). In addition, we used a cumulative dose 0.6 mg/Kg/week of free MTX and 15 mg/Kg/week of conjugate MTX equivalent. Although the MTD of free MTX in Lewis rats is ~2 mg/Kg/week (46), the observed lack of toxicity of the conjugates at 7.5-fold excess of the MTD predicts the increased therapeutic index of the targeted drug as compared to the free MTX. A dose-responsive efficacy study (Figure S1) efficacy study showed that the complete reversal of the symptoms of the arthritis required free MTX levels at its MTD (~2 mg/Kg/wk). The conjugate showed similar maximum efficacy at ~225 mg/Kg/wk (equivalent to 15 mg/Kg/wk MTX), which is ~60% its MTD. The apparent increased therapeutic index of the conjugate vs. MTX may be attributed to the fact that the conjugate is primarily targeted to the macrophages only through the FRβ, whereas the free MTX is taken up non-specifically through the RFC, which is ubiquitously present in cells. Single and multiple dose studies of the targeted drug in rats and dogs revealed no dose limiting toxicity at the concentrations used in this study, also suggesting a wide therapeutic index for this compound (James G. Laveglia, PhD, MPI Research, Mattawan, MI, personal communication).

Together our studies provide evidence that the in vivo effect of G5-FA-MTX is a consequence of targeted drug delivery into macrophages. This parallels our prior work that showed the specific targeting of G5-FA-MTX into FR-expressing tumor cells and the current studies documenting the inability of this conjugate to be taken up by FR-negative cells. This was reinforced by previous studies suggesting the in vitro and in vivo macrophage uptake of other FA-conjugated molecules (6–9) and the in vitro documentation of targeted uptake of the G5-FI-FA into macrophages in this work (Figure 2). Together these findings support the utility of macrophage-targeted therapy for this disease. Despite this, there are significant differences in activity of G5-FA-MTX in this arthritis model and what was previously observed in tumor models. The two G5-FA-MTX lots used in this study were effective in suppressing arthritis, but the G5-FA-MTX-1 lot was ineffective as a tumoricidal agent (unpublished observation). The two lots of G5-FA-MTX differed primarily in the average number of FAs conjugated to the surface of the G5 dendrimer (~2.5 in the first lot vs. ~5 in the second). This suggested that polyvalent interactions that occur with the FRβ may require less folate for targeting, or may be augmented by MTX interactions with this receptor to yield a sufficiently high binding affinity to obtain targeting. Further dose ranging studies will clarify these effects. Unfortunately the only prior previous report using a folate-conjugated G4-PAMAM to treat arthritis does not clarify these issues because the drug involved (indomethacin) was complexed but not covalently linked to amine-surfaced dendrimers (33). Thus, the observed increase in indomethacin in the joint may be attributed to non-specific internalization of the positively charged dendrimer (47). Further confirmation of overall pharmacokinetics of folate targeting could come from MRI imaging of animals given folate and gadolinium conjugated dendrimers (48).

In conclusion, we have demonstrated the FRβ-specific targeting of MTX into macrophages via dendrimers beneficially suppresses inflammatory changes associated with type II collagen induced arthritis in rats. This approach may also serve as a useful targeted therapeutic strategy for treating other macrophage inflammatory diseases such as the inflammatory bowel disease, granulomatous diseases and atherosclerosis. Alternatively, the current FA-based dendrimer could be utilized as a platform to improve the delivery of other DMARDS such as the TNF blockers, gold salts, chloroquine (42, 49), or NFκB inhibitors (50).

Supplementary Data

Synthesis of G5-5TAMRA and G5-5TAMRA-FA

G5-NH2 dendrimer 1 (Scheme 1; with 94 surface primary amine groups, as determined by potentiometric titration) dissolved in methanol was partially acetylated by reacting with 65-fold molar excess of acetic anhydride in presence of triethylamine to generate G5-NH2(29)-Ac(65) 2 (with 65 molecules of acetyl groups per dendrimer, as quantified by 1H-NMR analysis). For conjugation of FA, an active ester of FA was initially prepared by reacting FA with 14-fold excess EDC in presence of DMF and DMSO. A 7-fold molar excess of the FA-active ester was then reacted with 2 to generate G5-NH2(25)-Ac(65)-FA(4.1) 5. HPLC analysis showed that the purified 5 was completely free of any free FA. 5 dissolved in 0.1 M NaHCO3 was reacted with 6-fold excess 5TAMRA to generate the final product G5-NH2(25)-Ac(65)-FA(4.1)-NH2(20)-5T(5) 6. The excess amine groups in 6 was finally neutralized by glycidolation by reacting with 62-fold molar excess of glycidol to obtain the final product G5-NH2(25)-Ac(65)-FA(4.1)-OH(20)-5T(5) 7 (G5-5T-FA). For the synthesis of the control conjugate G5-5T, 2 was directly reacted with 5TAMRA to obtain 3, followed by glycidolation as described above, to obtain G5-NH2(25)-Ac(65)-OH(24)-5T(5) 4.

Dose Responsive Efficacy Study in Rats using G5-FA-MTX

A dose responsive efficacy study was performed using G5-FA-MTX-1 and free MTX. In this independent study, G5-FA-MTX-1 at 5, 20 and 65 mg/Kg/injection (administered IV, twice a week), or free MTX at 0.25 and 1mg/Kg (administered PO, twice a week) (MTX equivalent of 0.67, 2.7 and 8.7 mg/Kg/wk for the conjugate vs. 0.5 and 2 mg/Kg/wk of free MTX doses), under the other conditions of the experiment described in Figure 3 and in ‘Methods’. As shown in Figure S1, The G5-FA-MTX-1 induced a dose-dependent reversal of ankle diameter. Other arthritic symptoms, as presented in Figure 3, were also reversed in a dose-dependent fashion (data not shown). Similar efficacy was seen at the MTD of free MTX (2 mg/Kg/wk) and at 225 mg/Kg/wk (equivalent of 15 mg/kg MTX).

Supplementary Material

Scheme S1

Scheme S1: Synthesis of G5-5T and G5-5T-FA

Figure S1. Comparative dose responsive efficacy of G5-FA-MTX-1 vs. free MTX. G5-FA-MTX-1 at 5, 20 and 65 mg/Kg/injection (administered via IV, twice a week), or free MTX at 0.25 and 1 mg/Kg (administered PO, twice a week), under the other conditions of the experiment described in Figure 3 and in ‘Methods’. The respective MTX equivalent administered per week are given in the boxes. For comparison, included also is the data from the independent trial described in Figure 3 (dashed lines; both the conjugate and the free MTX administered IV three times a week, to reach the cumulative weekly doses shown). Statistical t-test analysis showed that all the drug-administered values given were significantly different from the respective Vehicle values, except for the 0.67 mg/Kg/wk G5-FA-MTX-1 set (solid triangle symbols) which showed significantly different values only at days 11, 14 and 15 (p < 0.05).

Acknowledgments

Grants and Financial Supporters: This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under award 1 R01 CA119409, and from the National Institute of Biomedical Imaging and Bio-Engineering, National Institutes of Health, under award RO1 EB005028.

This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under award 1 R01 CA119409, and from the National Institute of Biomedical Imaging and Bio-Engineering, National Institutes of Health, under award RO1 EB005028. We thank Alison M. Bendele, DVM, Ph.D., DACVP, Bolder BioPATH Inc., Boulder, CO, for directing the in vivo studies. We thank Dr. Anil K. Patri, NCL, NCI, Frederick, MD for conducting the characterization of some of the dendrimer conjugates. We thank Steve Lundy Ph.D., Internal Medicine/Rheumatology, University of Michigan, for a critical review of the manuscript.

References

  • 1.Antony AC. Folate receptors. Annu Rev Nutr. 1996;16:501–21. doi: 10.1146/annurev.nu.16.070196.002441. [DOI] [PubMed] [Google Scholar]
  • 2.Spiegelstein O, Eudy JD, Finnell RH. Identification of two putative novel folate receptor genes in humans and mouse. Gene. 2000;258(1–2):117–25. doi: 10.1016/s0378-1119(00)00418-2. [DOI] [PubMed] [Google Scholar]
  • 3.Low PS, Henne WA, Doorneweerd DD. Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc Chem Res. 2008;41(1):120–9. doi: 10.1021/ar7000815. [DOI] [PubMed] [Google Scholar]
  • 4.Cutolo M, Sulli A, Pizzorni C, Seriolo B, Straub RH. Anti-inflammatory mechanisms of methotrexate in rheumatoid arthritis. Ann Rheum Dis. 2001;60(8):729–35. doi: 10.1136/ard.60.8.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Nakashima-Matsushita N, Homma T, Yu S, Matsuda T, Sunahara N, Nakamura T, et al. Selective expression of folate receptor beta and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis. Arthritis Rheum. 1999;42(8):1609–16. doi: 10.1002/1529-0131(199908)42:8<1609::AID-ANR7>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  • 6.Nagayoshi R, Nagai T, Matsushita K, Sato K, Sunahara N, Matsuda T, et al. Effectiveness of anti-folate receptor beta antibody conjugated with truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages. Arthritis Rheum. 2005;52(9):2666–75. doi: 10.1002/art.21228. [DOI] [PubMed] [Google Scholar]
  • 7.Paulos CM, Turk MJ, Breur GJ, Low PS. Folate receptor-mediated targeting of therapeutic and imaging agents to activated macrophages in rheumatoid arthritis. Adv Drug Deliv Rev. 2004;56(8):1205–17. doi: 10.1016/j.addr.2004.01.012. [DOI] [PubMed] [Google Scholar]
  • 8.Paulos CM, Varghese B, Widmer WR, Breur GJ, Vlashi E, Low PS. Folate-targeted immunotherapy effectively treats established adjuvant and collagen-induced arthritis. Arthritis Res Ther. 2006;8(3):R77. doi: 10.1186/ar1944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.van der Heijden JW, Oerlemans R, Dijkmans BA, Qi H, van der Laken CJ, Lems WF, et al. Folate receptor beta as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patients. Arthritis Rheum. 2009;60(1):12–21. doi: 10.1002/art.24219. [DOI] [PubMed] [Google Scholar]
  • 10.Xia W, Hilgenbrink AR, Matteson EL, Lockwood MB, Cheng JX, Low PS. A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages. Blood. 2009;113(2):438–46. doi: 10.1182/blood-2008-04-150789. [DOI] [PubMed] [Google Scholar]
  • 11.Turk MJ, Breur GJ, Widmer WR, Paulos CM, Xu LC, Grote LA, et al. Folate-targeted imaging of activated macrophages in rats with adjuvant-induced arthritis. Arthritis Rheum. 2002;46(7):1947–55. doi: 10.1002/art.10405. [DOI] [PubMed] [Google Scholar]
  • 12.Hilgenbrink AR, Low PS. Folate receptor-mediated drug targeting: from therapeutics to diagnostics. J Pharm Sci. 2005;94(10):2135–46. doi: 10.1002/jps.20457. [DOI] [PubMed] [Google Scholar]
  • 13.Fiehn C. The future of folic acid antagonist therapy in rheumatoid arthritis. Arthritis Rheum. 2009;60(1):1–4. doi: 10.1002/art.24216. [DOI] [PubMed] [Google Scholar]
  • 14.Pincus T, Ferraccioli G, Sokka T, Larsen A, Rau R, Kushner I, et al. Evidence from clinical trials and long-term observational studies that disease-modifying anti-rheumatic drugs slow radiographic progression in rheumatoid arthritis: updating a 1983 review. Rheumatology (Oxford) 2002;41(12):1346–56. doi: 10.1093/rheumatology/41.12.1346. [DOI] [PubMed] [Google Scholar]
  • 15.Wessels JA, Huizinga TW, Guchelaar HJ. Recent insights in the pharmacological actions of methotrexate in the treatment of rheumatoid arthritis. Rheumatology (Oxford) 2008;47(3):249–55. doi: 10.1093/rheumatology/kem279. [DOI] [PubMed] [Google Scholar]
  • 16.Cronstein BN. Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis. Pharmacol Rev. 2005;57(2):163–72. doi: 10.1124/pr.57.2.3. [DOI] [PubMed] [Google Scholar]
  • 17.Hu SK, Mitcho YL, Oronsky AL, Kerwar SS. Studies on the effect of methotrexate on macrophage function. J Rheumatol. 1988;15(2):206–9. [PubMed] [Google Scholar]
  • 18.Johnson WJ, DiMartino MJ, Meunier PC, Muirhead KA, Hanna N. Methotrexate inhibits macrophage activation as well as vascular and cellular inflammatory events in rat adjuvant induced arthritis. J Rheumatol. 1988;15(5):745–9. [PubMed] [Google Scholar]
  • 19.Puig-Kroger A, Sierra-Filardi E, Dominguez-Soto A, Samaniego R, Corcuera MT, Gomez-Aguado F, et al. Folate Receptor beta Is Expressed by Tumor-Associated Macrophages and Constitutes a Marker for M2 Anti-inflammatory/Regulatory Macrophages. Cancer Research. 2009;69(24):9395–403. doi: 10.1158/0008-5472.CAN-09-2050. [DOI] [PubMed] [Google Scholar]
  • 20.Aletaha D, Smolen JS. The rheumatoid arthritis patient in the clinic: comparing more than 1,300 consecutive DMARD courses. Rheumatology (Oxford) 2002;41(12):1367–74. doi: 10.1093/rheumatology/41.12.1367. [DOI] [PubMed] [Google Scholar]
  • 21.Maetzel A, Wong A, Strand V, Tugwell P, Wells G, Bombardier C. Meta-analysis of treatment termination rates among rheumatoid arthritis patients receiving disease-modifying anti-rheumatic drugs. Rheumatology (Oxford) 2000;39(9):975–81. doi: 10.1093/rheumatology/39.9.975. [DOI] [PubMed] [Google Scholar]
  • 22.Bohanec Grabar P, Logar D, Lestan B, Dolzan V. Genetic determinants of methotrexate toxicity in rheumatoid arthritis patients: a study of polymorphisms affecting methotrexate transport and folate metabolism. Eur J Clin Pharmacol. 2008;64(11):1057–68. doi: 10.1007/s00228-008-0521-7. [DOI] [PubMed] [Google Scholar]
  • 23.Vicent MJ, Duncan R. Polymer conjugates: nanosized medicines for treating cancer. Trends in Biotechnology. 2006;24(1):39–47. doi: 10.1016/j.tibtech.2005.11.006. [DOI] [PubMed] [Google Scholar]
  • 24.Thomas TP, Shukla R, Majoros IJ, Myc A, Baker JR., Jr . Poly (amidoamine) Dendrimer-based Multifunctional Nanoparticles. In: Mirkin, editor. Nanobiotechnology: Concepts, Methods and Perspectives. Wiley-VCH; 2007. [Google Scholar]
  • 25.Svenson S, Tomalia DA. Dendrimers in biomedical applications--reflections on the field. Advanced Drug Delivery Reviews. 2005;57(15):2106–29. doi: 10.1016/j.addr.2005.09.018. [DOI] [PubMed] [Google Scholar]
  • 26.Nanjwade BK, Bechra HM, Derkar GK, Manvi FV, Nanjwade VK. Dendrimers: emerging polymers for drug-delivery systems. Eur J Pharm Sci. 2009;38(3):185–96. doi: 10.1016/j.ejps.2009.07.008. [DOI] [PubMed] [Google Scholar]
  • 27.Konda SD, Aref M, Wang S, Brechbiel M, Wiener EC. Specific targeting of folate-dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenografts. Magma. 2001;12(2–3):104–13. doi: 10.1007/BF02668091. [DOI] [PubMed] [Google Scholar]
  • 28.Thomas TP, Majoros IJ, Kotlyar A, Kukowska-Latallo JF, Bielinska A, Myc A, et al. Targeting and inhibition of cell growth by an engineered dendritic nanodevice. Journal of Medicinal Chemistry. 2005;48(11):3729–35. doi: 10.1021/jm040187v. [DOI] [PubMed] [Google Scholar]
  • 29.Shukla R, Thomas TP, Peters J, Kotlyar A, Myc A, Baker JR., Jr Tumor angiogenic vasculature targeting with PAMAM dendrimer-RGD conjugates. Chem Commun (Camb) 2005;46:5739–41. doi: 10.1039/b507350b. [DOI] [PubMed] [Google Scholar]
  • 30.Shukla R, Thomas TP, Peters JL, Desai AM, Kukowska-Latallo J, Patri AK, et al. HER2 specific tumor targeting with dendrimer conjugated anti-HER2 mAb. Bioconjugate Chemistry. 2006;17(5):1109–15. doi: 10.1021/bc050348p. [DOI] [PubMed] [Google Scholar]
  • 31.Kukowska-Latallo JF, Candido KA, Cao Z, Nigavekar SS, Majoros IJ, Thomas TP, et al. Nanoparticle targeting of anticancer drug improves therapeutic response in animal model of human epithelial cancer. Cancer Research. 2005;65(12):5317–24. doi: 10.1158/0008-5472.CAN-04-3921. [DOI] [PubMed] [Google Scholar]
  • 32.Myc A, Majoros IJ, Thomas TP, Barker JR., Jr Dendrimer-based targeted delivery of an apoptotic sensor in cancer cells. Biomacromolecules. 2007;8(1):13–8. doi: 10.1021/bm060815l. [DOI] [PubMed] [Google Scholar]
  • 33.Chandrasekar D, Sistla R, Ahmad FJ, Khar RK, Diwan PV. The development of folate-PAMAM dendrimer conjugates for targeted delivery of anti-arthritic drugs and their pharmacokinetics and biodistribution in arthritic rats. Biomaterials. 2007;28(3):504–12. doi: 10.1016/j.biomaterials.2006.07.046. [DOI] [PubMed] [Google Scholar]
  • 34.Majoros IJ, Keszler B, Woehler S, Bull T, Baker JR., Jr Acetylation of Poly (amidoamine) Dendrimers. Macromolecules. 2003;36:5526–9. [Google Scholar]
  • 35.Zhang Y, Thomas TP, Desai A, Zong H, Leroueil PR, Majoros IJ, et al. Targeted Dendrimeric Anticancer Prodrug: A Methotrexate-Folic Acid-Poly(amidoamine) Conjugate and a Novel, Rapid, “One Pot” Synthetic Approach. Bioconjug Chem. 2010 doi: 10.1021/bc9003958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Thomas TP, Ye JY, Chang YC, Kotlyar A, Cao Z, Majoros IJ, et al. Investigation of tumor cell targeting of a dendrimer nanoparticle using a double-clad optical fiber probe. J Biomed Opt. 2008;13(1):014024. doi: 10.1117/1.2870105. [DOI] [PubMed] [Google Scholar]
  • 37.McCarron RM, Goroff DK, Luhr JE, Murphy MA, Herscowitz HB. Methods for the collection of peritoneal and alveolar macrophages. Methods Enzymol. 1984;108:274–84. doi: 10.1016/s0076-6879(84)08092-7. [DOI] [PubMed] [Google Scholar]
  • 38.Myc A, Douce TB, Ahuja N, Kotlyar A, Kukowska-Latallo J, Thomas TP, et al. Preclinical antitumor efficacy evaluation of dendrimer-based methotrexate conjugates. Anticancer Drugs. 2008;19(2):143–9. doi: 10.1097/CAD.0b013e3282f28842. [DOI] [PubMed] [Google Scholar]
  • 39.Kinne RW, Brauer R, Stuhlmuller B, Palombo-Kinne E, Burmester GR. Macrophages in rheumatoid arthritis. Arthritis Res. 2000;2(3):189–202. doi: 10.1186/ar86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Yanni G, Whelan A, Feighery C, Bresnihan B. Synovial tissue macrophages and joint erosion in rheumatoid arthritis. Ann Rheum Dis. 1994;53(1):39–44. doi: 10.1136/ard.53.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Kinne RW, Stuhlmuller B, Burmester GR. Cells of the synovium in rheumatoid arthritis. Macrophages. Arthritis Res Ther. 2007;9(6):224. doi: 10.1186/ar2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Hamilton JA, Tak PP. The dynamics of macrophage lineage populations in inflammatory and autoimmune diseases. Arthritis Rheum. 2009;60(5):1210–21. doi: 10.1002/art.24505. [DOI] [PubMed] [Google Scholar]
  • 43.Bookbinder SA, Espinoza LR, Fenske NA, Germain BF, Vasey FB. Methotrexate: its use in the rheumatic diseases. Clin Exp Rheumatol. 1984;2(2):185–93. [PubMed] [Google Scholar]
  • 44.Turk MJ, Waters DJ, Low PS. Folate-conjugated liposomes preferentially target macrophages associated with ovarian carcinoma. Cancer Lett. 2004;213(2):165–72. doi: 10.1016/j.canlet.2003.12.028. [DOI] [PubMed] [Google Scholar]
  • 45.Jaffee BD, Kerr JS, Jones EA, Giannaras JV, McGowan M, Ackerman NR. The effect of immunomodulating drugs on adjuvant-induced arthritis in Lewis rats. Agents Actions. 1989;27(3–4):344–6. doi: 10.1007/BF01972818. [DOI] [PubMed] [Google Scholar]
  • 46.Morgan SL, Baggott JE, Bernreuter WK, Gay RE, Arani R, Alarcon GS. MTX affects inflammation and tissue destruction differently in the rat AA model. J Rheumatol. 2001;28(7):1476–81. [PubMed] [Google Scholar]
  • 47.Thomas TP, Majoros I, Kotlyar A, Mullen D, Holl MM, Baker JR., Jr Cationic poly(amidoamine) dendrimer induces lysosomal apoptotic pathway at therapeutically relevant concentrations. Biomacromolecules. 2009;10(12):3207–14. doi: 10.1021/bm900683r. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Swanson SD, Kukowska-Latallo JF, Patri AK, Chen C, Ge S, Cao Z, et al. Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement. Int J Nanomedicine. 2008;3(2):201–10. [PMC free article] [PubMed] [Google Scholar]
  • 49.Sokka T, Envalds M, Pincus T. Treatment of rheumatoid arthritis: a global perspective on the use of antirheumatic drugs. Mod Rheumatol. 2008;18(3):228–39. doi: 10.1007/s10165-008-0056-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Hattori Y, Sakaguchi M, Maitani Y. Folate-linked lipid-based nanoparticles deliver a NFkappaB decoy into activated murine macrophage-like RAW264.7 cells. Biol Pharm Bull. 2006;29(7):1516–20. doi: 10.1248/bpb.29.1516. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Scheme S1

Scheme S1: Synthesis of G5-5T and G5-5T-FA

Figure S1. Comparative dose responsive efficacy of G5-FA-MTX-1 vs. free MTX. G5-FA-MTX-1 at 5, 20 and 65 mg/Kg/injection (administered via IV, twice a week), or free MTX at 0.25 and 1 mg/Kg (administered PO, twice a week), under the other conditions of the experiment described in Figure 3 and in ‘Methods’. The respective MTX equivalent administered per week are given in the boxes. For comparison, included also is the data from the independent trial described in Figure 3 (dashed lines; both the conjugate and the free MTX administered IV three times a week, to reach the cumulative weekly doses shown). Statistical t-test analysis showed that all the drug-administered values given were significantly different from the respective Vehicle values, except for the 0.67 mg/Kg/wk G5-FA-MTX-1 set (solid triangle symbols) which showed significantly different values only at days 11, 14 and 15 (p < 0.05).

NIHMS296541-supplement-Scheme_S1.doc (90.5KB, doc)

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