Folate-Targeted Dendrimers Selectively Accumulate at Sites of

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Folate-targeted dendrimers selectively accumulate at sites of inflammation in mouse models of ulcerative colitis and atherosclerosis Scott Poh, Karson S. Putt, and Philip S. Low Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00728 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 2, 2017

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Folate-targeted dendrimers selectively accumulate at sites of inflammation in mouse models of ulcerative colitis and atherosclerosis

Scott Poha, Karson S. Puttb, Philip S. Lowb,c,*

a

College of Engineering and Science - Chemistry, Louisiana Tech University, Ruston LA 71272 b

Institute for Drug Discovery, Purdue University, West Lafayette IN 47907 c

Department of Chemistry, Purdue University, West Lafayette IN 47907

*Author to whom all correspondence should be addressed:

KEYWORDS: Dendrimer, Folate receptor, Folate targeting, Activated macrophages, Inflammatory disease

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ABSTRACT

Folate receptor positive activated macrophages are critical for the development and maintenance of many chronic inflammatory and autoimmune diseases. Previously, small molecule folatetargeted conjugates were found to specifically bind to these activated macrophages in vitro and selectively accumulate at sites of inflammation in vivo. While these small molecule conjugates have shown promise, the use of a folate-targeted, higher cargo capacity nanovehicle may prove superior in delivering imaging and/or therapeutic agents in vivo. This nanoparticle strategy has been demonstrated in oncology where targeted dendrimers have shown superior delivery capabilities, however, little research has been pursued in the area of folate-targeted dendrimers for inflammation and autoimmune diseases.

Therefore, we endeavored to create a folate-

decorated dendrimer to explore its uptake in mouse models of ulcerative colitis and atherosclerosis. Herein, we demonstrate that our final PEG-coated, acetic anhydride-capped, folate-targeted PAMAM dendrimer exhibits no discernable cytotoxicity in vitro, specifically binds to a folate receptor-expressing macrophage cell line in vitro, and selectively accumulates in areas of inflammation in vivo.

Introduction Activated macrophages are a primary driver of chronic inflammatory and autoimmune diseases1. During the progression of inflammatory disease, these activated macrophages recruit other immune cells by releasing cytokines2,3, chemokines4, and prostaglandins5 while directly damaging normal tissue via release of digestive enzymes6 and reactive oxygen species7. Currently, the primary clinical response to modulate these detrimental macrophage activities is

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the administration of corticosteroids8-10. While steroid treatment is highly efficacious, prolonged systemic steroid treatment often results in severe side effects, most commonly liver toxicity11. There is a clear need to identify better delivery systems to reduce these unwanted and harmful side effects to treat the myriad of inflammatory diseases. One such delivery strategy that reduces side effects brought on by systemic administration of a toxic drug is targeted delivery via cellular receptors that are upregulated at the site of disease. This targeting strategy concentrates the therapeutic at its intended site of action thus allowing for lower systemic exposure. Fortunately at sites of inflammation, activated macrophages have been shown to greatly overexpress folate receptor beta (FR-β), while resting or quiescent macrophages express this receptor at very low levels12. Subsequently, FR-β has been exploited for the folatetargeted delivery of small molecule imaging and drug conjugates in a variety of inflammatory diseases including atherosclerosis13, Crohn’s disease, osteoarthritis14, rheumatoid arthritis15,16, and systemic lupus erythematosus17. These studies show that folate-targeted small molecule conjugates are able to successfully accumulate in areas of inflammation, specifically deliver therapeutics, and ameliorate disease13,15-17. While small molecule folate conjugates show promise as diagnostics and therapeutics for inflammatory diseases, the use of higher cargo capacity nanoparticle delivery systems may provide an even greater benefit. One such nanoparticle delivery system is dendrimers. Folate targeted dendrimers have been extensively studied for anti-cancer applications such as the delivery of imaging agents18, siRNA19, radiotherapy18, and various drugs20-22. However, there has been less effort in exploring folate-targeted dendrimers for anti-inflammatory applications with the few studies performed focusing exclusively on arthritis23-27. Therefore, in an effort to determine if folate-targeted dendrimers could be used as a general delivery strategy for

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inflammatory and autoimmune diseases, we created a poly(ethylene glycol) (PEG)-coated, acetic anhydride-capped, folate-targeted poly(amidoamine) (PAMAM) dendrimer to study the uptake in inflamed tissues in mouse models of ulcerative colitis and atherosclerosis. Herein, we show that these dendrimers are not cytotoxic in vitro, specifically bind to a FR positive macrophage cell line in vitro and selectively accumulate in areas of inflammation in both disease models.

Materials and Methods Materials. PEG chemicals were purchased from Laysan Bio (Arab, AL). Cyanine 5.5 ester was purchased from Lumiprobe (Hallandale Beach, FL).

Cell culture media and dialysis

membranes were purchased from Thermo Scientific (Waltham, MA). RAW 264.7 cells were a kind gift from Endocyte (West Lafayette, IN). CellTiter-Glo was purchased from Promega (Madison, WI).

Mice, normal diet and folate deficient diet were purchased from Harlan

(Indianapolis, IN). PAMAM (G3) and all other chemicals were purchased from Sigma Aldrich (St. Louis, MO). All other consumables were purchased from VWR (Chicago, IL). Dendrimer preparation. Dendrimers were synthesized as previously described20,28-30. Briefly, 8 eq. of mPEG2000-succinimidyl amido succinate were dissolved in chloroform and reacted overnight with PAMAM in the presence of N,N-diisopropylethylamine (DIPEA) under argon. The product was purified via filtration on a Sephadex G-25 column equilibrated with 0.15M NaHCO3 and freeze-dried to create non-targeted dendrimers (NT-Dend). To create a folate targeted dendrimer (Fol-Dend), first, folate was conjugated to PEG3500-NHS as previously described20,30. The PEG-PAMAM product then was dissolved in DMSO and 4 eq. of NHS activated folate conjugated PEG3500 was added. DIPEA then was added and the solution was left to react overnight. The resulting folate-PEG-dendrimers were dialyzed and freeze-dried. To

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create fluorescent dendrimers, 2.5 eq. cyanine 5.5-NHS or FITC-NHS was added to NT-Dend or Fol-Dend. Unreacted free amines on all of the dendrimers were capped by adding excess acetic anhydride (Scheme 1). In vitro imaging and flow cytometry. RAW 264.7 cells were seeded into 8-well plates and maintained at 37 oC. After 1 day, media was replaced with medium containing 0.5 mg/mL fluorescent dendrimers in the presence or absence of 1 µM free folate. Cells were incubated for 2 hours at 37 oC then washed 3X in PBS. Cells were imaged using an Olympus BH-2 confocal microscope or removed from the plate using trypsin and analyzed via flow cytometry. For FITC dendrimers, a 488 nm excitation and 520 nm emission was used.

For Cy 5.5, a 670 nm

excitation and 700 nm emission was used. Cytotoxicity. RAW 264.7 cells were seeded into 8-well plates and maintained at 37 oC. After 1 day, media was replaced with medium containing 0.5 mg/mL dendrimers.

Cells were

incubated for 24 hours at 37 oC then washed with PBS. For Trypan blue staining, Trypan blue was added and the total number of cells was counted. The number of cells staining blue (nonviable) also was counted and the percentage of viable cells then was calculated. For CellTiterGlo, luminescent substrate was added and luminescence was recorded as per the manufacturer’s instructions.

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Scheme 1.

Decoration of PAMAM dendrimers.

Reagents and conditions:

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a) mPEG2000-

succinimidyl amido succinate, CHCl3, DIPEA, overnight; b) DMSO, folate-PEG3500-NHS, DIPEA, overnight; c) cyanine 5.5-NHS or FITC-NHS, DIPEA, overnight; d) (CH3CO2)O, DIPEA, overnight. Animal Husbandry. All animal procedures were approved by the Purdue Animal Care and Use Committee in accordance with guidelines from the National Institutes of Health. Mice were housed in a humidity and temperature controlled room on a standard 12 hour light/dark cycle. Food and water were provided ad libitum. Mice were maintained on a folate-deficient diet for at

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least 7 days prior to experimentation to lower the concentration of folate to a normal human physiological level15,31. Induction and imaging of colitis mouse model.

Six week old male C57BL6 mice were

maintained on a folate deficient diet. To induce colitis, drinking water was supplemented with 3% dextran sodium sulfate for 6 days26. Mice were randomly divided into treatment groups (n = 4) where they received either 1 mg/kg Fol-Dend-Cy5.5 or NT-Dend-Cy5.5 via tail vein injection. A group of healthy mice were also administered 1 mg/kg Fol-Dend-Cy5.5. Mice were sacrificed via CO2 asphyxiation 12 hours post-injection and images were acquired with a Kodak imaging station using the parameters detailed below. After in vivo imaging, colons were removed and imaged separately. Induction and imaging of atherosclerosis mouse model.

Female offspring of ApoE (-/-)

breeding trios were weaned at 3 weeks of age. These mice then were maintained on normal rodent chow. At 5 weeks, a group of mice were transferred to a high fat “western diet” consisting of 2% cholesterol and 21.2% fat13. After 10 weeks on a high fat diet, mice were randomly assigned to groups (n = 4) and administered either 1 mg/kg Fol-Dend-Cy5.5 or NTDend-Cy5.5. A group of mice maintained on normal rodent chow were administered 1 mg/kg Fol-Dend-Cy5.5. Mice were sacrificed via CO2 asphyxiation 12 hours post-injection and images were acquired with a Kodak imaging station using the parameters detailed below. After in vivo imaging, aortas were removed and imaged separately. In vivo Imaging parameter. In vivo images were acquired with a Kodak imaging station coupled to a charge coupled device camera and operated with Kodak molecular imaging software (version 4.0). For white light imaging, acquisition time = 0.05s, f-stop = 11, focal plane = 7, field of view = 200, and no binning was used. For fluorescent imaging, acquisition time =

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30s, excitation filter = 625 nm, emission filter = 700 nm, f-stop = 4, focal plane = 7, field of view = 200, and binning = 4.2 was used. Statistical Analysis. Data was analyzed via a t-test or 1-way ANOVA followed by Tukey ad hoc post-analyses where appropriate using GraphPad prism (GraphPad Software, San Diego CA). p-values < 0.05 were considered significant.

Results and Discussion Dendrimer preparation.

Dendrimers were prepared based upon the polyamidoamine

(PAMAM) backbone. PAMAMs were chosen as they allow for precise control of size, shape and functionalization32. Specifically, generation 3 (G3) PAMAM dendrimers were chosen as they have shown reduced toxicity32,33 and are able to load drugs with good entrapment efficiencies (D) as compared to higher generation dendrimers.

Additionally, their lower

molecule weight aids in their cellular uptake as compared to G4 and G4 dendrimers34 and should also improve their diffusion into the extravascular space, especially in areas of inflammation where partially damaged vascular is present.

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Figure 1. Dendrimer cytotoxicity and binding to RAW 264.7 mouse macrophage cells.

A)

Cells were incubated with NT-Dend or FolDend for 24 hours and viability was assessed via trypan blue staining. Error bars represent standard deviation.

B) Cells were incubated

with unlabeled 0.5 mg/mL (i) Fol-Dend, (ii) Fol-Dend-Cy5.5, (iii) NT-Dend-Cy5.5 or (iv) Fol-Dend-Cy5.5 in the presence of 1 µM folate for 2 hr at 37 oC. Cells then were washed with PBS and the remaining fluorescence was visualized via confocal microscopy (λex = 670 PAMAM dendrimers were synthesized as nm, λem = 700 nm). previously described20,29,30 and separated by gel filtration chromatography.

As summarized in Scheme 1, polyethylene glycol with a

molecular weight of ~2000 (PEG2000) was coupled to the surface to reduce toxicity and increase circulation time in vivo32,35. These non-targeted dendrimers are further referred to as NT-Dend. To create a targeted dendrimer, a PEG3500-folate conjugate was incorporated into the pegylatedPAMAM (Fol-Dend). The longer PEG3500 was utilized to ensure the attached folate could access the folate receptor (FR), as shorter PEG linkers are known to interfere with binding36. In an effort to create a fluorescent dendrimer for in vitro binding studies, FITC was incorporated into Fol-Dend. Additionally, far-red fluorescent dendrimers were created for in vivo imaging by

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Figure 2. FITC dendrimer binding and uptake to RAW 264.7 mouse macrophage cells. Cells were incubated with 0.5 mg/mL Fol-Dend-FITC in the presence or absence of 1 µM folate for 2 hr at 37 oC. Cells then were washed with PBS and the remaining fluorescence was either visualized via confocal microscopy (λex = 488 nm, λem = 520 nm) or analyzed by flow cytometry. incorporating cyanine 5.5 (Cy5.5) into both NT-Dend and Fol-Dend. Lastly, in an effort to further reduce toxicity, all free amines on all dendrimers were capped with acetic anhydride (37). Dendrimer cytotoxicity to RAW 264.7 macrophage cell line. As cytotoxicity of non- or littledecorated PAMAM dendrimers has been previously reported32,35,37, we first determined if our PEG2000-decorated and acetic anhydride-capped PAMAM dendrimers reduced this known cytotoxicity. To accomplish this, dendrimers were incubated with RAW 264.7 cells, a mouse macrophage cell line for 24 hours and the percentage of viable cells was determined via trypan blue staining and CellTiter Glo luminescence assays. As shown in Figure 1A, no significant differences were observed between non-treated and Fol-Dend or NT Dend treated cells (p = 0.629 and 0.808, respectively) when analyzed by Trypan blue staining.

Results using the

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CellTiter Glo viability assay were essentially identical with no significant differences observed (data not shown). Dendrimer

binding

macrophage cell line.

to

RAW

264.7

To ensure that the

targeted dendrimers would selectively bind to FR-expressing

macrophages,

FITC-

and

Cy5.5-conjugated NT-Dend, Fol-Dend, and Fol-Dend in the presence of 1 µM folate were incubated with RAW 264.7 cells, which are known to express FR23. As shown in Figure Figure 3. In vivo and ex vivo imaging of Cy5.5labeled dendrimers in an ulcerative colitis model.

Drinking

water

of

mice

was

supplemented with 3% dextran sodium sulfate (DSS) for 6 days to induce colitis. Healthy or DSS induced mice were administered 1 mg/kg Fol-Dend-Cy5.5 or NT-Dend-Cy5.5 via tail vein injection (n=4).

After 12 hours, mice were

sacrificed and whole body images were acquired (excitation = 625 nm, emission = 700 nm).

Colons then were excised and imaged

separately.

1B,

Fol-Dend-Cy5.5

internalized

into

was

RAW

bound

264.7

cells

and as

visualized via confocal microscopy (panel ii). Importantly, when in the presence of free folate, binding and internalization of FolDend-Cy5.5 was completely abrogated (panel iv). Additionally, the non-targeted NT-DendCy5.5 exhibited very weak binding/uptake (panel ii).

Similarly, Fol-Dend-FITC was

bound and internalized as determined by both confocal microscopy and flow cytometry, but binding was blocked by the addition of free

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folate (Figure 2). Taken together, these data show that Fol-Dend binding is indeed folate receptor mediated. In vivo imaging of a mouse colitis model. As the folate-targeted dendrimer was shown to bind FR expressing cells in vitro and activated macrophages are known to express FR-β12, we then

Figure 4. In vivo and ex vivo fluorescence quantitation of Cy5.5-labeled dendrimer uptake in an ulcerative colitis model.

Fluorescence

intensity was determined in the whole animal imaging (A) or excised colons (B) from Figure 3. Error bars represent SEM. * denotes p-value < 0.05, ** denotes p-value < 0.005.

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explored the ability of this dendrimer to accumulate in areas of inflammation in vivo. First, a mouse model of colitis was employed where the drinking water was supplemented with 3% dextran sodium sulfate (DSS) for 6 days prior to injection of the dendrimers. Once colitis was induced, 1 mg/kg Fol-Dend-Cy5.5 or NT-Dend-Cy5.5 was administered. After 12 hours, whole animal fluorescence imaging was performed. As shown in Figure 3, very little Fol-Dend-Cy5.5 signal was detected in healthy mice while abundant signal was observed in the DSS-induced colitis mice. Importantly, when the non-targeted dendrimer was imaged in the colitis mice, very little signal was observed. When the colons were removed and imaged, a similar pattern of fluorescence was identified with high dendrimer uptake present in the colitis mice administered Fol-Dend-Cy5.5 and little uptake in the two other groups.

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the

fluorescent

signal

was

quantitated, differences between the groups were readily apparent in both whole animals (Figure 4A) and excised colons (Figure 4B, 1way ANOVA p-value = 0.049 and 0.018, respectively). Figure 5. In vivo and ex vivo imaging of Cy5.5labeled dendrimers in an atherosclerosis model (ApoE knockout).

ApoE (-/-) mice were

maintained on a high fat “western” diet for 10 weeks to induce atherosclerosis.

Mice fed a

normal or high fat diet were administered 1 mg/kg Fol-Dend-Cy5.5 or NT-Dend-Cy5.5 via tail vein injection (n=4). After 12 hours, mice were sacrificed and whole body images were acquired (excitation = 625 nm, emission = 700 nm).

Aortas then were excised and imaged

separately.

In both image analyses, the

relative fluorescence intensity of Fol-DendCy5.5 in colitis mice was found to be significantly different than both Fol-DendCy5.5 fluorescence in healthy mice (p-value = 0.007 and 0.022 for whole animal and excised colons, respectively) and NT-Dend-Cy5.5 fluorescence in colitis mice (p-value = 0.0134 and 0.048 for whole animal and excised colons, respectively). No statistical difference was observed between the healthy mouse group and NT-Dend-Cy5.5 treated colitis group in either whole animals or excised colons

(p-value

=

0.940

and

0.898,

respectively). Taken together, these data show that the non-targeted dendrimers do not passively accumulate to a great extent in the site of inflammation due to any enhanced permeability retention effects. Therefore, the 3- to 4-fold higher levels of fluorescence observed in the

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inflamed colons by the folate-targeted dendrimers is indicative of FR-mediated accumulation and retention. In vivo imaging of a mouse atherosclerosis model. To ensure that the uptake of Fol-Dend is a general process in inflammatory diseases and not specific to colitis, a second mouse model of disease, atherosclerosis was employed. To induce atherosclerosis, ApoE (-/-) knockout mice were fed a high fat “western” diet for 10 weeks. Exactly as the colitis model, 1 mg/kg of FolDend-Cy5.5 and NT-Dend-Cy5.5 was administered to ApoE (-/-) mice on either a normal or high fat (atherosclerotic-inducing) diet.

After 12 hours, animals were imaged followed by the

excision and imaging of the aorta. As shown in Figure 5, the pattern of dendrimer uptake was similar to that in the colitis model where Fol-Dend-Cy5.5 signal was observed in the area of inflammation in whole-body imaging or in the excised aorta, but not in ApoE (-/-) mice fed a normal diet. As expected, very little NT-Dend-Cy5.5 accumulation was observed in the aorta of the atherosclerotic mice.

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Figure 6.

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In vivo and ex vivo fluorescence

quantitation of Cy5.5-labeled dendrimer uptake in an atherosclerosis model.

Fluorescence

intensity was determined in the (A) whole animal imaging or (B) excised aortas from Figure 5. Error bars represent SEM. * denotes p-value < 0.05, ** denotes p-value < 0.005.

Upon quantitation of Cy5.5 fluorescence (Figure 6A and B), the same pattern of dendrimer uptake was again observed where Fol-Dend-Cy5.5 in the atherosclerotic mice was statistically different than in mice fed a normal diet (p-value = 0.001 and 0.001 for whole animal and aorta imaging, respectively) and in atherosclerotic mice administered NT-Dend-Cy5.5 (p-value = 0.004 and 0.003 for whole animal and aorta imaging, respectively). As before, no statistical difference was observed between the normal diet Fol-Dend-Cy5.5 group and the atherosclerotic NT-Dend-Cy5.5 group (p-value = 0.106 and 0.8426, respectively). These results again show that the accumulation of targeted dendrimers is folate-dependent and uptake is similar in different inflammatory diseases.

Conclusions As the majority of folate-targeted dendrimers have been tested for their selective uptake in tumors, we endeavored to create a non-toxic, folate-targeted dendrimer that would selectively

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accumulate at sites of inflammation. To accomplish this, we coupled folate to a PEG-coated, acetic anhydride-capped, PAMAM dendrimer that has the ability to transport imaging or therapeutic cargo. These folate-targeted dendrimers were shown to bind macrophages in a folate receptor dependent fashion in vitro. To explore the effectiveness of folate-targeted dendrimers in inflammatory diseases in vivo, animal models of arthritis have been used nearly exclusively23-27. In an effort to ensure that folate-targeted dendrimers also would selectively accumulate in other inflammatory diseases, mouse models of colitis and atherosclerosis were utilized. Importantly, these dendrimers were able to selectively accumulate in these inflammatory sites in vivo, showing that the folate targeting of dendrimers to areas of inflammation is a general process and would likely show efficacy in other inflammatory disease models as well. Interestingly, the fluorescence intensity of Fol-Dend-Cy5.5 was ~3 to 4 times greater in inflamed mice than in healthy animals whereas small molecule folate-targeted conjugates generally only exhibit a ~2-fold increase in intensity between inflamed and healthy mice (2). This result implies that the strategy of using a higher capacity cargo dendrimer does indeed appear to deliver more agent to the sites of inflammation than a small molecule conjugate. Additionally, in both disease models tested, the accumulation of these folate-targeted dendrimers was similar to that of other higher cargo capacity folate-targeted nanoparticles, such as liposomes38.

However, the uptake of non-targeted dendrimers was less than non-targeted

liposome nanoparticles38 in the same two mouse models of disease. This may be due to a more rapid clearance of non-targeted dendrimers than other types of nanoparticles and this increased clearance rate may be beneficial in reducing the unintentional release of imaging/therapeutic cargo in non-targeted tissues.

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In conclusion, as these folate-targeted dendrimers did not exhibit cytotoxicity, were able to successfully accumulate in areas of inflammation, and appeared to deliver more cargo than small molecule conjugates, we believe these dendrimers warrant further study as a targeted delivery system for inflammatory and autoimmune diseases.

Corresponding Author Philip S. Low Email: [email protected] Phone: 765-494-5272 Department of Chemistry Purdue University 560 Oval Drive West Lafayette IN 47907

Acknowledgements We wish to thank Johnnie Shen, Michael Hansen, Charity Wayua, Nimalka Bandara and Lindsay Kelderhouse for their valuable assistance. June Lu and Kristin Wollak (Endocyte, Inc) for their assistance and providing cell samples. This work was supported by a grant from Endocyte, Inc. The authors gratefully acknowledge the campus-wide mass spectroscopy facility and support from the Purdue University Center for Cancer Research, P30CA023168.

References

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1. Jang, J. C.; Nair, M. G. Alternatively activated macrophages revisited: new insights into the regulation of immunity, inflammation and metabolic function following parasite infection. Curr Immunol Rev 2013, 9(3), 147-156. 2. Arango Duque G, Descoteaux A. Macrophage cytokines: involvement in immunity and infectious diseases. Front Immunol Arango Duque G, Descoteaux 2014, 5, 491. 3. Billiau, A. D.; Roskams, T.; Van Damme-Lombaerts, R.; Matthys, P.; Wouters, C. Macrophage activation syndrome: characteristic findings on liver biopsy illustrating the key role of activated, IFN-gamma-producing lymphocytes and IL-6- and TNF-alphaproducing macrophages. Blood 2005, 105(4), 1648-1651. 4. Ylä-Herttuala, S.; Lipton, B. A.; Rosenfeld, M. E.; Särkioja, T.; Yoshimura, T.; Leonard, E. J.; Witztum, J. L.; Steinberg, D. Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci 1991, 88(12), 5252-5256. 5. Humes, J.; Bonney, R.; Pelus, L.; Dahlgren, M.; Sadowski, S. J.; Kuehl, F. A. Jr.; Davies, P. Macrophage synthesis and release prostaglandins in response to inflammatory stimuli. Nature 1977, 269(5624), 149-151. 6. Werb, Z.; Gordon, S. Secretion of a specific collagenase by stimulated macrophages. J Exp Med 1975, 142(2), 346-360. 7. Cruz, C. M.; Rinna, A.; Forman, H. J.; Ventura, A. L.; Persechini, P. M.; Ojcius, D. M. ATP activates a reactive oxygen species-dependent oxidative stress response and

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secretion of proinflammatory cytokines in macrophages. J Biol Chem 2007, 282(5), 2871-2879. 8. Barnes, P. J. How corticosteroids control inflammation: Quintiles Prize Lecture 2005. Br J Pharmacol 2006, 148(3), 245-254. 9. Tsianakas, A.; Varga, G.; Barczyk, K.; Bode, G.; Nippe, N.; Kran, N.; Roth, J.; Luger, T. A.; Ehrchen, J.; Sunderkoetter, C. Induction of an anti-inflammatory human monocyte subtype is a unique property of glucocorticoids, but can be modified by IL-6 and IL-10. Immunobiology 2012, 217(3), 329-335. 10. Varga, G.; Ehrchen, J.; Tsianakas, A.; Tenbrock, K.; Rattenholl, A.; Seeliger, S.; Mack, M.; Roth, J.; Sunderkoetter, C. Glucocorticoids induce an activated, anti-inflammatory monocyte subset in mice that resembles myeloid-derived suppressor cells. J Leukoc Biol 2008, 84(3), 644-650. 11. Gerdes, H. Z. Glucocorticoid and mineralocorticoid hormones in chronic liver diseases. Z Gastroenterol 1979, 17(7), 439-446. 12. Reddy, J. A.; Haneline, L. S.; Srour, E. F.; Antony, A. C.; Clapp, D. W.; Low, P. S. Expression and functional characterization of the beta-isoform of the folate receptor on CD34(+) cells. Blood, 1999, 93(11), 3940-3948. 13. Ayala-Lopez, W.; Xia, W.; Varghese, B.; Low, P. S. Imaging of atherosclerosis in apoliprotein e knockout mice: targeting of a folate-conjugated radiopharmaceutical to activated macrophages. J Nucl Med 2010, 51(5), 768-774.

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14. Tsuneyoshi, Y.; Tanaka, M.; Nagai, T.; Sunahara, N.; Matsuda, T.; Sonoda, T.; Ijiri, K.; Komiya, S.; Matsuyama, T. Functional folate receptor beta-expressing macrophages in osteoarthritis synovium and their M1/M2 expression profiles. Scand J Rheumatol 2012, 41(2), 132-140. 15. Xia, W.; Hilgenbrink, A. R.; Matteson, E. L.; Lockwood, M. B.; Cheng, J. X.; Low, P. S. A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages. Blood 2009, 113(2), 438-446. 16. Matteson, E. L.; Lowe, V. J.; Prendergast, F. G.; Crowson, C. S.; Moder, K. G.; Morgenstern, D. E.; Messmann, R. A.; Low, P. S. Assessment of disease activity in rheumatoid arthritis using a novel folate targeted radiopharmaceutical Folatescane. Clin Exp Rheumatol 2009, 27(2), 253-259. 17. Varghese, B.; Haase, N.; Low, P. S. Depletion of folate-receptor-positive macrophages leads to alleviation of symptoms and prolonged survival in two murine models of systemic lupus erythematosus. Mol Pharm 2007, 4(5), 679-685. 18. Zhu, J.; Zhao, L.; Cheng, Y.; Xiong, Z.; Tang, Y.; Shen, M.; Zhao, J.; Shi, X. Radionuclide (131)I-labeled multifunctional dendrimers for targeted SPECT imaging and radiotherapy of tumors. Nanoscale 2015, 7(43), 18169-18178. 19. Ohyama, A.; Higashi, T.; Motoyama, K.; Arima, H. In vitro and in vivo tumor-targeting siRNA delivery using folate-PEG-appended dendrimer (G4)/α-cyclodextrin conjugates. Bioconjug Chem 2016, 27(3), 521-532.

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20. Singh, P.; Gupta, U.; Asthana, A.; Jain, N. K.

Folate and folate-PEG-PAMAM

dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice. Bioconjug Chem 2008, 19(11), 2239-2252. 21. Xu, X.; Li, J.; Han, S.; Tao, C.; Fang, L.; Sun, Y.; Zhu, J.; Liang, Z.; Li, F. A novel doxorubicin loaded folic acid conjugated PAMAM modified borneol, a nature dualfunctional product of reducing PAMAM toxicity and boosting BBB penetration. Eur J Pharm Sci 2016, 88, 178-190. 22. Thomas, T. P.; Joice, M.; Sumit, M.; Silpe, J. E.; Kotlyar, A.; Bharathi, S.; KukowskaLatallo, J.; Baker, J. R.; Choi, S. K. Design and in vitro validation of multivalent dendrimer methotrexates as a folate-targeting anticancer therapeutic. Curr Pharm Des 2013, 19(37), 6594-6605. 23. Thomas, T. P.; Goonewardena, S. N.; Majoros, I. J.; Kotlyar, A.; Cao, Z.; Leroueil, P. R.; Baker, J. R. Jr.

Folate-targeted nanoparticles show efficacy in the treatment of

inflammatory arthritis. Arthritis Rheum 2011, 63(9), 2671-2680. 24. Chandrasekar, D.; Sistla, R.; Ahmad, F. J.; Khar, R. K.; Diwan, P. V. 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), 504-512. 25. Chandrasekar, D.; Sistla, R.; Ahmad, F. J.; Khar, R. K.; Diwan, P. V. Folate coupled poly(ethyleneglycol)

conjugates

of

anionic

poly(amidoamine)

dendrimer

for

inflammatory tissue specific drug delivery. J Biomed Mater Res A 2007, 82(1), 92-103.

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Biomacromolecules

26. Qi, R.; Majoros, I.; Misra, A. C.; Koch, A. E.; Campbell, P.; Marotte, H.; Bergin, I. L.; Cao, Z.; Goonewardena, S.; Morry, J.; Zhang, S.; Beer, M.; Makidon, P.; Kotlyar, A.; Thomas, T. P.; Baker J. R. Jr. Folate receptor-targeted dendrimer-methotrexate conjugate for inflammatory arthritis. J Biomed Nanotechnol 2015, 11(8), 1431-1441. 27. Benchaala I, Mishra MK, Wykes SM, Hali M, Kannan RM, Whittum-Hudson JA. Folate-functionalized dendrimers for targeting Chlamydia-infected tissues in a mouse model of reactive arthritis. Int J Pharm 2014, 466(1-2), 258-265. 28. Singh P, Gupta U, Asthana A, Jain NK. Folate and folate-PEG-PAMAM dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice. Bioconjug Chem 19(11), 2239-2252. 29. Chandrasekar, D.; Sistla, R.; Ahmad, F. J.; Khar, R.; K.; Diwan, P. V. 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), 504-512. 30. Zhang, Y.; Sun, Y.; Xu, X.; Zhang, X.; Zhu, H.; Huang, L.; Qi, Y.; Shen, Y. M. Synthesis, biodistribution, and microsingle photon emission computed tomography (SPECT)

imaging

study

of

technetium-99m

labeled

PEGylated

dendrimer

poly(amidoamine) (PAMAM)-folic acid conjugates. J Med Chem 2010, 53(8), 32623272. 31. Lu, Y.; Stinnette, T. W.; Westrick, E.; Klein, P. J.; Gehrke, M. A.; Cross, V. A.; Vlahov, I. R.; Low, P. S.; Leamon, C. P. Treatment of experimental adjuvant arthritis with a

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novel folate receptor-targeted folic acid-aminopterin conjugate. Arthritis Res Ther 2011, 13(2), R56. 32. Jevprasesphant, R.; Penny, J.; Jalal, R.; Attwood, D.; McKeown, N. B.; D’Emanuele, A. The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int J Pharm 2003, 252(1-2), 263-266. 33. Shao N, Su Y, Hu J, Zhang J, Zhang H, Cheng Y.

Comparison of generation 3

polyamidoamine dendrimer and generation 4 polypropylenimine dendrimer on drug loading, complex structure, release behavior, and cytotoxicity.

Int J Nanomedicine

2011, 6, 3361-3372. 34. Lv T, Yu T, Fang Y, Zhang S, Jiang M, Zhang H, Zhang Y, Li Z, Chen H, Gao Y. Role of generation on folic acid-modified poly(amidoamine) dendrimers for targeted delivery of baicalin to cancer cells. Mater Sci Eng C Mater Biol Appl 2017, 75, 182-190. 35. Kim, Y.; Klutz, A. M.; Jackobson, K. A. Systematic investigation of polyamidoamine dendrimers surface-modified with poly(ethylene glycol) for drug delivery applications: synthesis, characterization, and evaluation of cytotoxicity. Bioconjug Chem 2008; 19(8), 1660-1672. 36. Gabizon, A.; Horowitz, A. T.; Goren, D.; Tzemach, D.; Mandelbaum-Shavit, F.; Qazen, M. M.; Zalipsky, S.

Targeting folate receptor with folate linked to extremities of

poly(ethylene glycol)-grafted liposomes: in vitro studies. Bioconjug Chem 1999, 10(2), 289-298.

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37. Jain, K.; Kesharwani, P.; Gupta, U.; Jain, N. K. Dendrimer toxicity: Let’s meet the challenge. Int J Pharm 2010, 394(1-2), 122-142. 38. Poh, S.; Chelvam, V.; Ayala- López, W.; Putt, K. S.; Low, P. S. Selective liposome targeting of folate receptor positive immune cells in inflammatory diseases. Nanomedicine (Lond) 2017, accepted.

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Scheme 1. Decoration of PAMAM dendrimers. Reagents and conditions: a) mPEG2000-succinimidyl amido succinate, CHCl3, DIPEA, overnight; b) DMSO, folate-PEG3500-NHS, DIPEA, overnight; c) cyanine 5.5-NHS or FITC-NHS, DIPEA, overnight; d) (CH3CO2)O, DIPEA, overnight. 170x148mm (300 x 300 DPI)

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Figure 1. Dendrimer cytotoxicity and binding to RAW 264.7 mouse macrophage cells. A) Cells were incubated with NT-Dend or Fol-Dend for 24 hours and viability was assessed via trypan blue staining. Error bars represent standard deviation. B) Cells were incubated with unlabeled 0.5 mg/mL (i) Fol-Dend, (ii) FolDend-Cy5.5, (iii) NT-Dend-Cy5.5 or (iv) Fol-Dend-Cy5.5 in the presence of 1 µM folate for 2 hr at 37 oC. Cells then were washed with PBS and the remaining fluorescence was visualized via confocal microscopy (λex = 670 nm, λem = 700 nm). 82x114mm (300 x 300 DPI)

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Figure 2. FITC dendrimer binding and uptake to RAW 264.7 mouse macrophage cells. Cells were incubated with 0.5 mg/mL Fol-Dend-FITC in the presence or absence of 1 µM folate for 2 hr at 37 oC. Cells then were washed with PBS and the remaining fluorescence was either visualized via confocal microscopy (λex = 488 nm, λem = 520 nm) or analyzed by flow cytometry. 170x88mm (300 x 300 DPI)

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Figure 3. In vivo and ex vivo imaging of Cy5.5-labeled dendrimers in an ulcerative colitis model. Drinking water of mice was supplemented with 3% dextran sodium sulfate (DSS) for 6 days to induce colitis. Healthy or DSS induced mice were administered 1 mg/kg Fol-Dend-Cy5.5 or NT-Dend-Cy5.5 via tail vein injection (n=4). After 12 hours, mice were sacrificed and whole body images were acquired (excitation = 625 nm, emission = 700 nm). Colons then were excised and imaged separately. 82x98mm (300 x 300 DPI)

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Figure 4. In vivo and ex vivo fluorescence quantitation of Cy5.5-labeled dendrimer uptake in an ulcerative colitis model. Fluorescence intensity was determined in the whole animal imaging (A) or excised colons (B) from Figure 3. Error bars represent SEM. * denotes p-value < 0.05, ** denotes p-value < 0.005. 82x95mm (300 x 300 DPI)

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Figure 5. In vivo and ex vivo imaging of Cy5.5-labeled dendrimers in an atherosclerosis model (ApoE knockout). ApoE (-/-) mice were maintained on a high fat “western” diet for 10 weeks to induce atherosclerosis. Mice fed a normal or high fat diet were administered 1 mg/kg Fol-Dend-Cy5.5 or NT-DendCy5.5 via tail vein injection (n=4). After 12 hours, mice were sacrificed and whole body images were acquired (excitation = 625 nm, emission = 700 nm). Aortas then were excised and imaged separately. 82x76mm (300 x 300 DPI)

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Figure 6. In vivo and ex vivo fluorescence quantitation of Cy5.5-labeled dendrimer uptake in an atherosclerosis model. Fluorescence intensity was determined in the (A) whole animal imaging or (B) excised aortas from Figure 5. Error bars represent SEM. * denotes p-value < 0.05, ** denotes p-value < 0.005. 82x97mm (300 x 300 DPI)

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88x35mm (300 x 300 DPI)

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