TSPO Targeted Dendrimer Imaging Agent: Synthesis, Characterization, andCellular Internalization

Lynn E. Samuelson, Madeline J. Dukes, Colette R. Hunt, Jonathan D. Casey, and Darryl J. Bornhop*

Department of Chemistry, The Vanderbilt Institute for Chemical Biology and Vanderbilt-Ingram Cancer Center, VanderbiltUniversity, VU Station B 351822 Nashville, Tennessee 37235-1822. Received May 11, 2009; Revised Manuscript ReceivedSeptember 25, 2009

While it has become common practice for dendrimers to deliver imaging and therapeutic agents, there are fewreported examples of cellular internalization of dendrimers. Moreover, targeting of dendrimers to the mitochondriain cells has not yet been reported. Previously, we have delivered small molecule imaging agents into glioma andbreast cancer cells by targeting the translocator protein (TSPO; formerly known as the peripheral benzodiazepinereceptor or PBR) with a family of high-affinity conjugable ligands. The 18 kDa multimeric TSPO is expressedin steroid-producing cells, primarily on the outer mitochondrial membrane. This protein is a prime candidate formolecular targeting because tumors and other disease-related cells contain high densities of TSPO. Here, wepresent the synthesis, characterization, and cellular internalization into C6 rat glioma cells of a TSPO targeteddendrimer imaging agent.

INTRODUCTION

Developments of clinically relevant molecular probes toimage diseases or biological processes are essential for theadvancement of personalized medicine. A variety of biomarkersspecific to cancer and other diseases have been identified andused for therapeutic and/or imaging agent specific site delivery(1) (2, 3). For example, targeting proteins has been accomplishedusing antibodies (4), proteins (5), small molecules (6-8),nanoparticles (9-11), and dendrimers (1, 12, 13). Attachmentof multiple different species to a single dendrimer has produceda single molecule which targets, images, and/or delivers atherapeutic dose to the site of disease (14, 15). The advantagesof using dendrimers include (a) multiple moieties or multiplecopies of the targeting agent on a single particle, (b) biocom-patibility, (c) size solubility, and (d) tunability. However, thechallenges of using dendermers include (a) the difficulty incharacterization of functionalized molecule, (b) nonuniformdistribution of the adduct(s), (c) complexity in determining theoptimum size, and (d) ensuring that the targeting moiety retainsactivity after attachment to the dendrimer. It is clear thatmacromolecules will play an important role in personalizedmedicine; however, the current synthetic scaffolds are particu-larly ineffective in targeting intracellular receptors.

The translocator protein (TSPO, previously known as theperipheral-type benzodiazepine receptor or PBR) spans themitochondrial membrane and has become an attractive receptorfor therapeutics and targeted imaging (16). TSPO is associatedwith a number of biological processes including cell prolifera-tion, apoptosis, steroidogenesis, and immunomodulation; yet,its exact physiological role is still not fully defined (16-18).Overexpression of TSPO has been shown in numerous typesof cancer including brain, breast, colorectal, prostate, and ovariancancers, as well as astrocytomas, hepatocellular, and endometrialcarcinomas (17). Two well-characterized cell lines with highTSPO expression are C6 Rat Glioma cells (19) and MDA-MB-

231 human breast cancer cells (20). Several small, high binding,selective molecules have been developed for TSPO and madeinto positron emission tomography (PET), magnetic resonance(MR), and optical imaging agents (621-24). While attractive,prior to this report macromolecules targeting TSPO have notbeen reported, possibly due to difficulty in internalizing syntheticmolecules.

Dendrimers have been studied as potential delivery systemsto target, image, and/or deliver a therapeutic specifically todiseased tissue because of their globular shape, multiple surfaceattachment sites, biocompatibility, tunable size, monodispersity,and low production cost (25). The multiple surface end groupsavailable on a dendrimer allow for attachment of severalmoieties and or copies of each drug, ligand, or imaging agent.Furthermore, dendrimers can be adapted for advancements incoupling technology allowing them to be used with emergingtargets, therapeutics, and imaging strategies. Multiple attachmentpoints can provide increased therapeutic efficacy, reduced drugside effects, and increased signal-to-noise for improved imagingdetection limits. Methods of targeting include attaching severalcopies of a binding molecule (small molecules, peptides,proteins, or antibodies) to the surface of the dendrimer tocapitalize on polyvalency or the enhanced permeability andretention (EPR) effect where tumor accumulation occurs dueto the particle size and the increased vasculature of thetumor (26, 27). Imaging with dendrimers is possible throughthe attachment of small molecule fluorophores (for optical),metal chelates (for fluorescence, MRI, PET and SPECT), orwith a switch activated by a physiological process for enhancedtissue characterization (28, 29). Dendrimers can serve aseffective prodrug scaffolds with the drug molecules beingattached through a linkage which activates the drug in vivo oncethey reach the delivery site. Cleavage from the scaffold canoccur through a change in pH, enzyme reaction, or slowdegradation of the macromolecule (29). While internalizationof large nanoparticles through the cellular membrane has beenshown, targeting of specific organelles has scarcely beenreported (30, 31). In this paper, we present the synthesis,characterization, cellular internalization, and mitochondria label-ing of a dendrimer nanoparticle. Here, we demonstrate that a

* To whom correspondence should be addressed. Darryl Bornhop,Ph.D., Department of Chemistry, Vanderbilt University, VU Station B351822,Nashville,TN37235-1822,Tel:615.322.4226,Fax:615.343.1234,e-mail: darryl.j.bornhop@vanderbilt.edu.

Bioconjugate Chem. 2009, 20, 2082–20892082

10.1021/bc9002053 CCC: $40.75 2009 American Chemical SocietyPublished on Web 10/28/2009

functionalized fourth generation (G(4)-PAMAM) dendrimerparticle, which targets an internal cellular receptor and bindsthat target by passing through the cellular membrane undernormal physiological conditions and without the externalperturbation of the cellular membrane, can be prepared,characterized, and imaged.

EXPERIMENTAL PROCEDURES

Materials. G(4)-PAMAM dendrimer in a 10% w/w solutionof methanol was purchased from Fischer Scientific and pipettedinto a preweighed vial immediately before use. The methanolwas evaporated under a stream of Ar(g), and the resulting viscousoil was dissolved in water, frozen, and lyophilized to give afluffy white powder. The mass of the dendrimer was determinedby reweighing the vial and calculating the mass difference. 1-(2-Chlorophenyl)isoquinoline-3-carboxylic acid (ClPhIQ Acid) wassynthesized in our laboratory as previously reported (6, 32).All 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) and DOTA derivatives were purchased form Macro-cyclics and used as received. All other chemicals were purchasedfrom Fischer Scientific and used as is unless otherwise indicated.

MALDI-TOF MS were obtained on a PerSeptive BiosystemsVoyager-DE STR mass spectrometer. Freshly recrystallizedtrans-indole acrylic acid (IAA) was used as the matrix in a 10mg/mL solution of DMSO. The plate was spotted with 1 µL ofa 10:1 solution of matrix to analyte. NMR spectra were obtainedon a 400 MHz Bruker AV-400 instrument with a 5 mmZ-gradient broadband inverse probe. NMR spectra were obtainedin d6-dimethyl sulfoxide. UV-vis spectra were obtained on aShimadzu 1700 UV-vis spectrophotometer. Fluorescence spec-tra were obtained using a ISS PCI spectrofluorometer at roomtemperature.

ClPhIQ-G(4)-PAMAM (1). First, ClPhIQ Acid (152.9 mg,540 µmol) and benzotriazol-1-yloxytris(dimethylamino)-phos-phonium hexafluorophosphate (BOPCl, 288.1 mg, 651 µmol)were dissolved in 2 mL of DMSO each. Triethyl amine (Et3N,100 µL) was added to the ClPhIQ Acid solution. The BOPClsolution was added to the ClPhIQ Acid solution. The mixturewas stirred for 5-10 min (sometimes a change from clear tobright orange would occur) and was then added to a stirredsolution of G(4)-PAMAM dendrimer (252.2 mg, 18 nmol) inDMSO (5 mL). The reaction was stirred at room temperatureovernight. Unreacted ClPhIQ Acid was removed by dilutingthe solution 4-fold with water and placing the solution in anAmicon centrifugation molecular weight filter (molecular weightcutoff ) 5 kDa) and concentrating the solution to approximately500 µL via centrifuging at 3000 rpm (1419 × g) for 1 h. Thesolution was diluted and reconcentrated three times with waterto wash any unreacted materials away. Finally, the solution waslyophilized to give 441.2 mg (100% yield) of a slightly yellowpowder. The same procedure was followed for synthesizing the(ClPhIQ)12-PAMAM using fewer equivalents of ClPhIQ Acidand BOPCl. ClPhIQ30-G(4)-PAMAM: MALDI-TOF MS 19 500amu 1H NMR (300 MHz, CDCl3) 8.8 (1H, s), 8.6 (1H, s),8.2-8.5 (5H, m), 7.5-8.0 (9H, multi), 2.9 (3H, bs), and 3.1-3.5(28H, m) ppm.

ClPhIQ-G(4)-PAMAM-Lissamine (2). G(4)-PAMAM (9mg, 400 nmol) with or without ClPhIQ Acid was dissolved in2 mL of dimethylformamide (DMF). Next, 23 µL of a 17.3mM solution of Lissamine sulfonyl chloride was added to thestirred dendrimer solution. The reaction was stirred overnightfollowed by purification in Amicon centrifugation filters asdescribed above and lyophilized to give 5.6 mg (61% yield) ofa pink fluffy solid. The presence of the fluorophor was confirmedwith UV-vis (max absorbance at 551 nm) and fluorescence(ex 551 nm, em 586 nm) spectroscopies. Specific conditionsand yields provided above were for ClPhIQ23-G(4)-PAMAM,

but UV-vis and fluorescence spectra were identical forClPhIQ12-PAMAM and PAMAM-Liss.

Cell Internalization Experiments. with G(4)-PAMAM-Lissamine derivatives: Either 20 000 C6 rat glioma or 40 000MDA-MB-231 breast cancer cells were plated into collagencoated glass-bottomed microscopy (MatTeck) dishes and al-lowed to grow for two days in cell media. After two days, thecells had normal morphology and were attached, and ap-proximately 20% confluent. G(4)-PAMAM-Lissamine com-pounds (with or without other moieties) were diluted to 1 µM(in ClPhIQ) with media from a 10 mg/mL (12 µM in ClPhIQ)stock solution in DMSO. The media over the cells was replacedwith the media containing the fluorophor (either ClPhIQ-PAMAM-Liss or PAMAM-Liss) diluted as noted above andincubated for 6-12 h. The media was then poured off, and thecells were carefully rinsed 3-4 times with Dulbecco’s PhosphateBuffered Saline (DPBS). The cells were imaged live. Both whitelight and fluorescence pictures were obtained.

For fixed cells, the rinsed cells were incubated at roomtemperature for 30 min with a 4% para-formaldehyde solution.The cells were carefully washed 4 times with DPBS and storedunder DPBS at 4 °C until imaged (not longer than 1 week,typically overnight). Immediately before imaging, the fixed cellswere incubated with a 25 nM solution of mitotracker green(MTG) for 10 min. Excess MTG was removed by four washingswith DPBS and the cells were imaged. Cellular images wereobtained using a A Nikon Eclipse TE2000-U fluorescencemicroscope (Lewisville, TX) equipped with a Nikon FITC-TexasRed filter sets employed for the in vitro imaging. The TexasRed filter (ex, 560-580 nm; polychromatic mirror, 585-665nm; barrier filter, 600-650 nm) set was used to imageLissamine, and the FITC filter was used to image MTG (ex,450-505 nm; polychromatic mirror, 510-555 nm; barrier filter,515-545 nm). Fluorescence integration times ranged 100-5000ms, but were typically 100-500 ms. All controls were integratedfor at least 5000 ms. Sample homogeneity was maintained bykeeping the samples under DPBS the entire time of imaging.For live cells, images were taken within 20 min after replacingmedia with DPBS and were discontinued if the cellularmorphology changed in any way from the media containingmorphology.

RESULTS AND DISCUSSION

The TSPO targeted dendrimer was synthesized using 1-(2-chlorophenyl)isoquinoline-3-carboxylic acid (ClPhIQ Acid), aprecursor of the conjugable TSPO binding molecule previouslyreported (6). ClPhIQ Acid (Scheme 1) was activated withbenzotriazol-1-yloxytris(dimethylamino)-phosphonium hexaflu-orophosphate (BOPCl), in pyridine and dimethyl sulfoxide(DMSO), and added to a solution of G(4)-PAMAM dendrimerin DMSO. An average of 12 and 23 ClPhIQ Acids were attachedby reacting G(4)-PAMAM dendrimer with 15 and 30 mol equivof the activated TSPO ligand, respectively, to obtain twoClPhIQ-PAMAM compounds, 1a and 1b (Scheme 1). ExcessClPhIQ Acid and coupling agent were removed by diafiltrationwith a molecular weight cutoff (MWCO) of 5 kDa. Both

Scheme 1. Synthesis of ClPhIQ-PAMAM

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ClPhIQ-PAMAM molecules were characterized with nuclearmagnetic resonance (NMR) and matrix assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF-MS).

The average number of TSPO ligands per dendrimer wasestimated using NMR integrations. It was shown from protonNMR spectra of the PAMAM and ClPhIQ pieces individuallythat the aromatic proton signals are exclusively from the ClPhIQAcids attached (7.0-7.9 ppm) to the particle, while the interiorPAMAM methylenes are the only signal observed from 2 to 4ppm and the amides are found from 8.4 to 9 ppm (Figure 1).By calculation, there are approximately 930 methylene protonsin a G(4)-PAMAM dendrimer and 9 protons (all aromatic) perClPhIQ Acid molecule. To determine the average number ofClPhIQ Acids per dendrimer, the integral from 7.0 to 8.2 wascalibrated to 9 and the resulting integral from 2 to 4 ppm wasdivided into 930. As shown in Figure 1, we find 23 ClPhIQAcids attached to the dendrimer when the dendrimer was reactedwith 30 equiv of ClPhIQ Acid. NMR spectra indicate that 12ClPhIQ acids are attached when 15 equiv of ligand are reactedwith the dendrimer (data not shown). Interestingly, the amountof ClPhIQ that binds to the dendrimer nearly doubles (from 12to 23) when doubling the amount of ClPhIQ in the reaction(from 15 to 30).

MALDI-TOF MS was also utilized to confirm the averagenumber of ClPhIQ Acids attached to the dendrimer (Figure 2).First, a MALDI-TOF MS was acquired of the G(4)-PAMAMwith the average molecular weight (MW) found to be 13 244Da, significantly lower than the 14 215 Da of a G(4)-PAMAMwithout any of the widely reported structural defects (33). Thesedefects include incomplete reactions, making one arm of thedendrimer consistently a generation behind, and side reactionsthat stop the expansion of that branch of dendrimer altogether.The ClPhIQ-PAMAM (1a) has an average MW of 16 513, andthus, by calculation has 12 ClPhIQ Acid molecules perPAMAM. This result is consistent with the NMR data, as wasthe number of ClPhIQ Acids per PAMAM for ClPhIQ-PAMAM(1b) with 23 ClPhIQ ligands attached. Additionally, it wascalculated that ClPhIQ-PAMAM, 1b, had approximately 24ClPhIQ Acids per PAMAM, consistent with NMR data.

Following characterization of the ClPhIQ-PAMAM moleculeswith NMR and MALDI-TOF-MS, the dendrimer was furtherfunctionalized by reacting it with Lissamine rhodamine B

sulfonyl chloride (Lissamine) in dimethyl formamide (DMF)overnight to give 1 or 2 Lissamine dyes per dendrimer (Scheme2). The DMF was removed in vacuo and excess dye wasremoved by diafiltration (MWCO ) 5000) in water. UV-visand fluorescence were employed to characterize the opticalimaging agent. Attaching the dye to the dendrimer produced acompound with an absorbance maximum at 571 nm (versus 563nm for free dye) and a slightly red-shifted emission at 586 nm(versus 583 nm for free dye). A control compound (PAMAM-Liss), the agent absent of the targeting moiety, was synthesizedsimilarly to the targeting agent by reacting G(4)-PAMAMdendrimer with Lissamine (Scheme 2). The PAMAM-Lissconjugate was also purified by diafiltration in which the firstlow molecular weight fraction was slightly pink (from dye) andsubsequent washing resulted in lighter color until no color waspresent. A minimum of three colorless washings in the lowmolecular weight fraction were completed to provide pureClPhIQ-PAMAM-Liss with no unreacted Lissamine dye. Amolecular weight difference for the PAMAM and PAMAM-Liss of 500-600 amu was observed in the MALDI-TOF MSspectrum, consistent with an average of 1 dye per dendrimer.The small spectral shift in absorbance and emission was similarto the targeted moiety.

Finally, the solubility extraction coefficients between octanoland water were determined for both the PAMAM compoundsby using the shake method. Briefly, the shake method for findinglog P is determined by mixing an aqueous solution of thePAMAM-dye with a solution PAMAM-dye in octanol andallowing the two solvents to equilibrate. The absorbances ofeach layer (octanol and water) were then recorded and used todetermine the concentration of dye in each layer. The log P forClPhIQ-PAMAM-Liss is estimated to be 2.9, while that forPAMAM-Liss (no ClPhIQ) is 1.0. These log P values indicatethat the ClPhIQ-PAMAM-Liss is more lipophilic than thePAMAM-Liss and therefore is more likely to interact favorablywith the cellular membrane.

To evaluate the biological activity of the imaging agent, C6rat glioma cells, shown to express a high concentration ofTSPO (34, 35), were incubated with the dendrimer compounds2b and 3 for 12 h in collagen coated MatTek dishes andvisualized by fluorescence microscopy. Live cell imaging waspreformed immediately after rinsing the cells with PBS or salineto remove media and unbound or uninternalized imaging agent.A Nikon Eclipse TE2000-U fluorescence microscope (Lewis-ville, TX) equipped with Texas Red and FITC filter sets wasemployed for the in vitro imaging. As shown in Figure 3, theClPhIQ-PAMAM-Liss labels the cell, while the control (PAM-AM-Liss) does not produce fluorescence. As another control,cells were incubated with Lissamine rhodamine B sulfonylchloride and again no fluorescence was observed (data notshown). The imaging experiments were performed in triplicatewith observations being consistent with respect to absorbanceand fluorescence intensity and the absence of fluorescence inthe controls. We hypothesize that the dendrimer with the TSPObinding ligand (ClPhIQ-PAMAM-Liss, 2b) produced bright,localized cellular fluorescence due to its ability to pass throughthe cellular membrane and bind to the target protein TSPO(Figure 3). Since ClPhIQ Acid conjugated dye is known to bindTSPO, glioma cells overexpress TSPO, and it is an organellularprotein, we believe that the dendrimer labels the mitochondriaby binding to TSPO. In the labeling experiment where thedendrimer without the targeting ligand (compound 3) was used,no fluorescence was found in the cell (either intracellularly orat the membrane) at exposure times as long as 5 s. Takentogether, these results indicate that the ClPhIQ moiety isnecessary for cellular uptake of the dendrimer at labelingconcentrations of 1 µM and equilibration times of 6-12 h.

Figure 1. NMR of 23 ClPhIQ Acids attached to G(4)-PAMAM(ClPhIQ23-PAMAM).

2084 Bioconjugate Chem., Vol. 20, No. 11, 2009 Samuelson et al.

To further confirm that the agent was intracellular, a seriesof z-stacked images (pseudoconfocal) were obtained allowingdifferent regions of the cells to be viewed (Figure 4). Thesez-stacked images were accomplished by changing the lensposition incrementally, moving the focal planes of the micro-scope through cells, and collecting the images by viewingfluorescence. In the center focal planes, there is a dark hole,consistent with the nucleus as shown in the fluorescence-DICoverlay (Figure 4). This observation suggests that the agentundergoes internalization, passive or active, and does not justlabel the membrane due to the lipophilic nature of the ClPhIQmoiety. Since only a small amount of out-of-focus light isobserved in the membrane slices of the z stack (data not shown),labeling of the membrane appears to be negligible. Furthermore,fluorescence signal is observed through the cell to the entirenuclear membrane, but not in the nucleus, indicating uptake ofthe dendrimer by the cells but not the nucleus. Previouslyreported small molecule agents have not labeled the nucleus,although low concentrations of TSPO are present there (24).The reported macromolecular agent behaved as expected andtargeted the mitochondria and was not taken up into the nucleusas indicated in Figure 4.

To further explore whether ClPhIQ-PAMAM-Liss was tar-geting the mitochondria, a co-incubation experiment waspreformed. This was accomplished by first incubating C6 ratglioma cells with ClPhIQ-PAMAM-Liss as described above for

the live cellular imaging, followed by fixation using paraform-aldehyde, and then treating the cells with Mitotracker Green(MTG) according to the manufacturer’s guidelines. The cellswere then imaged using a Texas Red filter set to detect theLissamine (red) fluorescence and a FITC filter set to detect theMTG (green) fluorescence. Figure 5 shows C6 rat glioma cellsincubated with either ClPhIQ-PAMAM-Liss or the control,PAMAM-Liss. The DIC images in Figure 5A and G are presentto indicate the location of the cells relative to fluorescence signalof ClPhIQ-PAMAM-Liss (Figure 5A) and PAMAM-Liss dosedcells (Figure 5G). Fluorescence from the imaging agent isdisplayed in red (Figure 5B) and fluorescence from the MTGas green (Figure 5C). As demonstrated above with live cellimaging, the ClPhIQ-PAMAM-Liss agent labels the fixed cellsprincipally perinuclearly (Figure 5B). MTG was used in lowconcentration (25 nM) to ensure that it primarily labeledmitochondria in the cell. The MTG labels the mitochondria ofcells, as shown by numerous investigators (36, 37), particularlywhen used in low concentrations as those employed here(25nM). More careful investigation of the fluorescence signalsallow insight into where the species are within the cell. Figure5 panels D and E display co-registration of ClPhIQ-PAMAM-Liss and MTG with yellow indicating areas of overlap whilered and green indicate areas where ClPhIQ-PAMAM-Liss (red)and MTG (green) are not coincident. It is encouraging to seethat most of the red and green converge to yellow, indicating

Figure 2. Characterization and determination of the average number of ClPhIQ per dendrimer by MALDI-TOF MS. Left: MALDI-TOF spectrumof G(4)-PAMAM. Middle: MALDI-TOF spectrum of ClPhIQ(12)-PAMAM. Right: MALDI-TOF spectrum of ClPhIQ(23)-PAMAM.

Scheme 2. Attachment of Lissamine Dye to PAMAM

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that the majority of ClPhIQ-PAMAM-Liss is in the samelocation of the cell as the MTG. This good colocalization isfurther evidence that ClPhIQ-PAMAM-Liss is labeling themitochondria and not other parts of the cell. As demonstratedin Figure 5 panels FsI and in the live cell imaging (Figures 3and 4), fluorescence from the dendrimer is only detected ifClPhIQ Acid is incorporated into the dendrimer. In other words,images from cells inoculated with the control agent (PAMAM-Liss) exhibit no fluorescence (Figure 5I). MTG fluorescencewas not affected (Figure 5C,F,H) by the presence or absenceof dendrimer agent, and the cellular membrane was notperturbed by nonphysiological chemical or physical processesprior to or during the incubation with ClPhIQ-PAMAM-Liss.Of course, excess imaging agent was removed prior to fixationin all experiments. Taken together, the fluorescence images inFigure 5 indicate that the ClPhIQ-PAMAM-Liss is targetingthe mitochondria and that the ClPhIQ ligand is necessary forthis mitochondrial labeling to occur. The z-stacked images andcoincubation experimental results indicate that the imaging agentis internalized into the cell through normal cellular functions.

Although no quantitative binding studies were completed, theagent binds with high enough affinity to remain in the cell afterfixation, washing, and treatment with MTG. Studies beyond thescope of this report are necessary to determine the exactmechanism of cellular transport and binding; however, insightis shown into some initial experiments performed using shorterincubation times with the fluorescent agents (ClPhIQ-PAMAM-Liss and PAMAM-Liss). In these trials, no fluorescence wasobserved for either compound (with and without ClPhIQ ligand)after incubation for as long as 3 h (data not shown). It wasonly after a minimum of 6 h of incubation that fluorescence

was found in the dosed cells. One possible explanation for thisobservation is that the dendrimeric compound is slowly takenup into the cell and the ClPhIQ is absolutely necessary foruptake. Another possibility is that the molecule freely entersand exits the cell and eventually binds the target (TSPO), thatdye is completely removed (both inter- and intracellular) whenthe cells are rinsed immediately after incubating.

Imaging with IEC-6 cells was completed as an initial estimateas to whether the ClPhIQ-PAMAM-Liss would be taken up andbind mitochondria in cells that express low levels of TSPO.Since TSPO is necessary for cellular function, there are no celllines that do not express TSPO. No fluorescence was seen whenimaging the IEC-6 cells with ClPhIQ-PAMAM-Liss at dosingconcentrations as high as 1 µM in dye (data not shown). Theindication is that the ClPhIQ-PAMAM-Liss imaging agent isTSPO protein specific. Again, the mechanism of cellular uptakeinto the cell needs further investigation in order to pinpoint theexact mechanism of cellular uptake.

Further evidence of the utility of ClPhIQ-PAMAM-Liss wasdemonstrated by labeling MDA-MB-231 human breast cancercells, an aggressive human cancer cell line. Like the C6 ratglioma cells, the MDA-MB-231 human breast cancer cells havebeen shown to overexpress TSPO (20). The cells were incubatedwith ClPhIQ-PAMAM-Liss following the same procedures aswith the C6 rat glioma cells, fixed, and treated with MTG forcoregistration investigations. The individual red and greenfluorescence of ClPhIQ-PAMAM-Liss and MTG, respectively,in fixed MDA-MB-231 cells is demonstrated in panels A andC of Figure 6, while panels E and F show the lack of redfluorescence in the PAMAM-Liss treated cells. Associationbetween the red and green (shown in yellow) is evident in theoverlays of panels B and D indicating colocalization of theClPhIQ-PAMAM-Liss and MTG. These additional resultsrepresent further evidence that ClPhIQ-PAMAM-Liss is ableto label TSPO rich mitochondria in cancer cells.

The observations made in the cellular imaging experimentsshow that the ClPhIQ labeled dendrimers enter the cell and bind

Figure 4. Selected montage of red fluorescence images of ClPhIQ-PAMAM-Liss dosed cells from the z series. Panel A: slice 10, PanelB: slice 15. Panel C: slice 20, Panel D: slice 21. Panel E slice 22.Panel F: slice 23. Panel G: slice 24. Panel H: slice 325. Panel I: slice30. Panel J: slice 35. Panel K: DIC Fluorescence overlay.

Figure 3. Live cell images of C6 rat glioma cells. Panel A: Redfluorescence of ClPhIQ-PAMAM-Liss dosed cells. Panel B: DIC imageof ClPhIQ-PAMAM-Liss dosed cells. Panel C: DIC image of PAMAM-Liss dosed cells. Panel D: Red fluorescence image of PAMAM-Lissdosed cells.

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the mitochondria, but do not appear in the nucleus or cellularmembrane. The dendrimers without the ClPhIQ do not labelthe cell in any significant way. Although the molecules are smallenough to enter through the cellular and nuclear membranes,as shown previously the literature suggests that it is not as trivialas size (38). First, dendrimers are structurally different thanbiological molecules such as proteins and antibodies. Biologicalmolecules are typically well-defined, folded structures, whiledendrimers are floppier, not well-defined structures. Second, theend functional groups dictate the interactions with the mem-

branes, which may block or enhance the uptake of thecompound, depending on the chemical interactions. In amine-terminated dendrimers, the amines can be protonated and donot pass through the lipophilic cellular membrane. Our observa-tions in the cellular imaging experiments, along with the log Pdata, indicated that the lipophilicity of the ClPhIQ ligand allowsfor the dendrimeric molecule to pass into the cell.

In conclusion, a TSPO targeted imaging agent was synthe-sized using a G(4)-PAMAM backbone, ClPhIQ Acid, andLissamine (ClPhIQ-PAMAM-Liss). The imaging agent was

Figure 5. Fluorescence images of fixed C6 rat glioma cells. Panel A: DIC of ClPhIQ(23)-PAMAM-Liss and MTG dosed cells. Panel B: Redfluorescence of ClPhIQ(23)-PAMAM-Liss and MTG dosed cells. Panel C: Green fluorescence of ClPhIQ(23)-PAMAM-Liss and MTG dosed cells.Panel D: Red and Green fluorescence overlay of ClPhIQ(23)-PAMAM-Liss and MTG dosed cells. Panel E: DIC, Red, and Green fluorescenceoverlay of ClPhIQ(23)-PAMAM-Liss and MTG dosed cells. Panel F: DIC, Red and, Green Fluorescence overlay of PAMAM-Liss and MTG dosedcells. Panel G: DIC of PAMAM-Liss and MTG dosed cells. Panel H: Green fluorescence of PAMAM-Liss and MTG dosed cells. Panel I: Redfluorescence of PAMAM-Liss and MTG dosed cells.

Figure 6. Microscope images of MDA-MB-231 cells. Panel A: Red fluorescence of ClPhIQ(23)-PAMAM-Liss and MTG dosed cells. Panel B: Redand Green Fluorescence overlay of ClPhIQ(23)-PAMAM-Liss and MTG dosed cells. Panel C: Green fluorescence of ClPhIQ(23)-PAMAM-Lissand MTG dosed cells. Panel D: Red and Green Fluorescence and DIC overlay of ClPhIQ(23)-PAMAM-Liss and MTG dosed cells. Panel E: Redfluorescence of PAMAM-Liss dosed cells. Panel F: DIC image of PAMAM-Liss dosed cells.

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characterized with MALDI-TOF-MS and NMR and imaged inC6 rat glioma and MDA-MB-231 human breast cancer cells.To our knowledge, this is the first example of a TSPO-targeteddendrimer and among the few dendrimeric compounds that areinternalized into the cell under normal physiological conditions.Co-localization of the ClPhIQ-PAMAM-Liss and MTG indicatesthat the compound binds to the mitochondria. The ability forsynthetic dendrimers to be internalized into the cell and targeta specific protein is a step closer to understanding biologicalevents at the molecular level and ultimately enabling personal-ized medicine; however, further investigation is required todetermine the exact mechanism by which this agent enters thecell.

ACKNOWLEDGMENT

This work was supported by grants from the NationalScience Foundation (Bes-0323281), Department of Defense(W81XWH-04-1-0432), and the National Institute of Health(5P20 GM072048-4).

LITERATURE CITED

(1) Tomalia, D. A., Reyna, L. A., and Svenson, S. (2007)Dendrimers as multi-purpose nanodevices for oncology drugdelivery and diagnostic imaging. Biochem. Soc. Trans. 35, 61–67.

(2) Portney, N. G., and Ozkan, M. (2006) Nano-oncology: drugdelivery, imaging, and sensing. Anal. Bioanal. Chem. 384, 620–630.

(3) Bremer, C., Ntziachristos, V., and Weissleder, R. (2003)Optical-based molecular imaging: contrast agents and potentialmedical applications. Eur. Radiol. 13, 231–243.

(4) Sharkey, R. M., Cardillo, T. M., Rossi, E. A., Chang, C. H.,Karacay, H., McBride, W. J., Hansen, H. J., Horak, I. D., andGoldenberg, D. M. (2005) Signal amplification in molecularimaging by pretargeting a multivalent, bispecific antibody. Nat.Med. 11, 1250–1255.

(5) Lee, L. A., and Wang, Q. (2006) Adaptations of nanoscaleviruses and other protein cages for medical applications. Nano-medicine 2, 137–49.

(6) Manning, H. C., Goebel, T., Marx, J. N., and Bornhop, D. J.(2002) Facile, efficient conjugation of a trifunctional lanthanidechelate to a peripheral benzodiazepine receptor ligand. Org. Lett.4, 1075–1078.

(7) Bai, M., Wyatt, S. K., Han, Z., Papadopoulos, V., and Bornhop,D. J. (2007) A novel conjugable translocator protein ligandlabeled with a fluorescence dye for in vitro imaging. BioconjugateChem. 18, 1118–22.

(8) Bai, M. F., Sexton, M., Stella, N., and Bornhop, D. J. (2008)MBC94, a conjugable ligand for cannabinoid CB2 receptorimaging. Bioconjugate Chem. 19, 988–992.

(9) Mulder, W. J. M., Strijkers, G. J., van Tilborg, G. A. F.,Griffioen, A. W., and Nicolay, K. (2006) Lipid-based nanopar-ticles for contrast-enhanced MRI and molecular imaging. NMRBiomed. 19, 142–164.

(10) Morawski, A. M., Winter, P. M., Crowder, K. C., Caruthers,S. D., Fuhrhop, R. W., Scott, M. J., Robertson, J. D., Abend-schein, D. R., Lanza, G. M., and Wickline, S. A. (2004) Targetednanoparticles for quantitative imaging of sparse molecularepitopes with MRI. Magn. Reson. Med. 51, 480–486.

(11) Li, K. C. P., and Bednarski, M. D. (2002) Vascular-targetedmolecular imaging using functionalized polymerized vesicles.J. Magn. Reson. Imaging 16, 388–393.

(12) Gillies, E. R., and Frechet, J. M. J. (2005) Dendrimers anddendritic polymers in drug delivery. Drug DiscoVery Today 10,35–43.

(13) Lee, C. C., Yoshida, M., Frechet, J. M. J., Dy, E. E., andSzoka, F. C. (2005) In vitro and in vivo evaluation of hydrophilicdendronized linear polymers. Bioconjugate Chem. 16, 535–541.

(14) Wickline, S. A., Neubauer, A. M., Winter, P., Caruthers, S.,and Lanza, G. (2006) Applications of nanotechnology toatherosclerosis, thrombosis, and vascular biology. Arterioscler.Thromb. Vasc. Biol. 26, 435–441.

(15) Sinha, R., Kim, G. J., Nie, S. M., and Shin, D. M. (2006)Nanotechnology in cancer therapeutics: bioconjugated nanopar-ticles for drug delivery. Mol. Cancer Ther. 5, 1909–1917.

(16) Papadopoulos, V., Baraldi, M., Guilarte, T. R., Knudsen, T. B.,Lacapere, J. J., Lindemann, P., Norenberg, M. D., Nutt, D.,Weizman, A., Zhang, M. R., and Gavish, M. (2006) Translocatorprotein (18 kDa): new nomenclature for the peripheral-typebenzodiazepine receptor based on its structure and molecularfunction. Trends Pharmacol. Sci. 27, 402–409.

(17) Decaudin, D., Castedo, M., Nemati, F., Beurdeley-Thomas,A., De Pinieux, G., Caron, A., Pouillart, P., Wijdenes, J.,Rouillard, D., Kroemer, G., and Poupon, M. F. (2002) Peripheralbenzodiazepine receptor ligands reverse apoptosis resistance ofcancer cells in vitro and in vivo. Cancer Res. 62, 1388–1393.

(18) Casellas, P., Galiegue, S., and Basile, A. S. (2002) Peripheralbenzodiazepine receptors and mitochondrial function. Neuro-chem. Int. 40, 475–486.

(19) Hardwick, M., Fertikh, D., Culty, M., Li, H., Vidic, B., andPapadopoulos, V. (1999) Peripheral-type benzodiazepine receptor(PBR) in human breast cancer: Correlation of breast cancer cellaggressive phenotype with PBR expression, nuclear localization,and PBR-mediated cell proliferation and nuclear transport ofcholesterol. Cancer Res. 59, 831–842.

(20) Olson, J. M., Mcneel, W., Young, A. B., and Mancini, W. R.(1992) Localization of the peripheral-type benzodiazepine bind-ing-site to mitochondria of human glioma-cells. J. Neuro-Oncol.13, 35–42.

(21) Chen, J. W., Breckwoldt, M. O., Aikawa, E., Chiang, G., andWeissleder, R. (2008) Myeloperoxidase-targeted imaging ofactive inflammatory lesions in murine experimental autoimmuneencephalomyelitis. Brain 131, 1123–33.

(22) Nahrendorf, M., Sosnovik, D., Chen, J. W., Panizzi, P.,Figueiredo, J. L., Aikawa, E., Libby, P., Swirski, F. K., andWeissleder, R. (2008) Activatable magnetic resonance imagingagent reports myeloperoxidase activity in healing infarcts andnoninvasively detects the antiinflammatory effects of atorvastatinon ischemia-reperfusion injury. Circulation 117, 1153–60.

(23) Hammoud, D. A., Endres, C. J., Chander, A. R., Guilarte,T. R., Wong, D. F., Sacktor, N. C., McArthur, J. C., and Pomper,M. G. (2005) Imaging glial cell activation with [C-11]-R-PK11195 in patients with AIDS. J. NeuroVirol. 11, 346–355.

(24) Manning, H. C., Goebel, T., Thompson, R. C., Price, R. R.,Lee, H., and Bornhop, D. J. (2004) Targeted molecular imagingagents for cellular-scale bimodal imaging. Bioconjugate Chem.15, 1488–1495.

(25) Svenson, S., and Tomalia, D. A. (2005) Dendrimers inbiomedical applications--reflections on the field. AdV. DrugDeliVery ReV. 57, 2106–29.

(26) Khandare, J., and Minko, T. (2006) Polymer-drug conjugates:Progress in polymeric prodrugs. Prog. Polym. Sci. 31, 359–397.

(27) Satchi-Fainaro, R., Duncan, R., and Barnes, C. M. (2006)Polymer therapeutics for cancer: Current status and futurechallenges. Polymer Therapeutics II: Polymers as Drugs,Conjugates and Gene DeliVery Systems 193, 1–65.

(28) Winter, P. M., Cai, K. J., Chen, J., Adair, C. R., Kiefer, G. E.,Athey, P. S., Gaffney, P. J., Buff, C. E., Robertson, J. D.,Caruthers, S. D., Wickline, S. A., and Lanza, G. M. (2006)Targeted PARACEST nanoparticle contrast agent for the detec-tion of fibrin. Magn. Reson. Med. 56, 1384–1388.

(29) Pham, W., Choi, Y. D., Weissleder, R., and Tung, C. H. (2004)Developing a peptide-based near-infrared molecular probe forprotease sensing. Bioconjugate Chem. 15, 1403–1407.

(30) Kolonko, E. M., and Kiessling, L. L. (2008) A polymericdomain that promotes cellular internalization. J. Am. Chem. Soc.130, 5626+.

(31) Kitchens, K. M., Foraker, A. B., Kolhatkar, R. B., Swaan,P. W., and Ghandehari, H. (2007) Endocytosis and interaction

2088 Bioconjugate Chem., Vol. 20, No. 11, 2009 Samuelson et al.

of poly (amidoamine) dendrimers with Caco-2 cells. Pharm. Res.24, 2138–2145.

(32) Newman, A. H., Lueddens, H. W. M., Skolnick, P., and Rice,K. C. (1987) Novel irreversible ligands specific for peripheraltype benzodiazepine receptors - (() (+) and (-)-1-(2-chlo-rophenyl)-N-(1-methylpropyl)-N-(2-isothiocyanatoethyl)-3-isoquinolinecarboxamide and 1-(2-isothiocyanatoethyl)-7-chloro-1,3-dihydro-5-(4-chlorophenyl)-H-2-1,4-benzodiazepin-2-one. J. Med. Chem. 30, 1901–1905.

(33) Hummelen, J. C., vanDongen, J. L. J., and Meijer, E. W.(1997) Electrospray mass spectrometry of poly(propylene imine)dendrimers - The issue of dendritic purity or polydispersity.Chem.sEur. J. 3, 1489–1493.

(34) Kozikowski, A. P., Kotoula, M., Ma, D. W., Boujrad, N.,Tuckmantel, W., and Papadopoulos, V. (1997) Synthesis andbiology of a 7-nitro-2,1,3-benzoxadiazol-4-yl derivative of 2-phe-nylindole-3-acetamide: A fluorescent probe for the peripheral-type benzodiazepine receptor. J. Med. Chem. 40, 2435–2439.

(35) Starostarubinstein, S., Ciliax, B. J., Penney, J. B., Mckeever,P., and Young, A. B. (1987) Imaging of a glioma using peripheralbenzodiazepine receptor ligands. Proc. Natl. Acad. Sci. U.S.A.84, 891–895.

(36) Sehgal, I., Sibrian-Vazquez, M., and Vicente, M. G. H. (2008)Photoinduced cytotoxicity and biodistribution of prostate cancercell-targeted porphyrins. J. Med. Chem. 51, 6014–6020.

(37) Sacchi, S., Bernasconi, M., Martineau, M., Mothet, J. P.,Ruzzene, M., Pilone, M. S., Pollegioni, L., and Molla, G. (2008)pLG72 modulates intracellular D-serine levels through itsinteraction with D-amino acid oxidase - Effect on schizophreniasusceptibility. J. Biol. Chem. 283, 22244–22256.

(38) Najlah, M., Freeman, S., Attwood, D., and D’Emanuele, A.(2006) Synthesis, characterization and stability of dendrimerprodrugs. Int. J. Pharm. 308, 175–182.

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