Tumor Imaging
`
`Tumor Fibroblast Specifi c Activation of a Hybrid
`Ferritin Nanocage-Based Optical Probe for Tumor
`Microenvironment Imaging
`
` Tianjiao Ji , Ying Zhao , * Jing Wang , Xin Zheng , Yanhua Tian , Yuliang Zhao ,
` and Guangjun Nie *
`
` During various phases of tumor progression, such as deg-
`radation of the extracellular matrix (ECM), angiogenesis,
`tumor invasion, and metastasis, secretion of specifi c enzymes
`plays crucial roles during these disease processes in tumor
`microenvironment. [ 1 , 2 ] These specifi c enzymes are therefore
`attractive targets for both tumor therapy and imaging. [ 3 ] Our
`previous studies have shown that inhibition of both expres-
`sion and activity of matrix metalloproteinases (MMPs) can
`signifi cantly prevent malignant tumor invasion and metas-
`tasis via the prison the tumor cell strategy, in which deg-
`radation of ECM was dramatically decreased after the
`metallofullerene nanoparticles treatment. [ 4–6 ] For clinical
`diagnostics perspective, to examine the exact site of tumors
`and the expression degree of these enzymes are particularly
`valuable. To meet the urgent demand for bioimaging in live
`systems, continuous efforts are required for developing intel-
`ligent and sensitive imaging probes, and the applications of
`functional nanomaterials may provide enormous opportunity.
`Some novel enzyme-responsive nanoprobes have been devel-
`oped to image and monitor the abnormal expression of these
`enzymes in tumor tissue, where the activity of an enzyme
`elicited a specifi c response via the nanoparticle assembly-dis-
`assembly process to produce an activity-depending signal. [ 7–9 ]
`Enzymes that have attracted much recent attention as candi-
`date targets for nanoprobes involve caspase-3, [ 10 , 11 ] urokinase
`plasminogen activator (uPA), [ 12 ] and MMPs. [ 13 , 14 ] Despite
`progress achieved, further improvements are still required
`regarding design of novel nanoprobes with enhanced effi -
`ciency and specifi city, as the enzymes currently used may not
`be the most abundant and potent enzymes with lack of tumor
`type specifi city. Among various cells present in tumor tissues,
`it is likely that enzymes expressed by the greatly abundant
`stromal cells will be increasingly noticed in the near future as
`
` T. Ji, Prof. Y. Zhao, Prof. J. Wang, X. Zheng, Y. Tian,
`Y. Zhao, Prof. G. Nie
`Chinese Academy of Sciences Key Laboratory
`for Biomedical Effects of Nanomaterials
`and Nanosafety, National Center for Nanoscience
`and Technology, China, Beijing 100190, China
`E-mail:zhaoying@nanoctr.cn; niegj@nanoctr.cn
`
` DOI: 10.1002/smll.201300600
`
`effi cient and specifi c new targets and may provide important
`insights into cancer biology. [ 15 ]
` Fibroblast activation protein- α (FAP- α ), a membrane-
`bound serine protease, is highly and specifi cally expressed by
`cancer-associated fi broblasts (CAFs) and pericytes in epithe-
`lial tumors, without any notable expression in normal adult
`tissues. [ 16–18 ] FAP- α exhibits both dipeptidyl peptidase and
`collagenase proteolytic activity, being able to cleave N-ter-
`minal dipeptides from polypeptides with L-proline or L-ala-
`nine in the penultimate position, as well as degrade gelatin
`and native type I collagen. [ 16 , 19–21 ] Thus, FAP- α plays a crit-
`ical role in shaping the microenvironment to promote tumor
`growth and invasion through degrading ECM. [ 22–24 ] Although
`the biological function of FAP- α still needs further study, the
`specifi c expression and the unique enzymatic activity of FAP-
` α make it a potentially attractive therapeutic and diagnostic
`target in the tumor microenvironment. [ 25 ] The tumor growth
`and invasion can be inhibited by suppressing the activity of
`FAP- α . [ 25 , 26 ] However, the diagnosis value of FAP- α has not
`been exploited as a specifi c biomarker to precisely diagnose
`FAP- α positive tumors and follow the prognosis. Moreover,
`FAP- α -positive stromal cells compose the most abundant
`tumor stromal components. [ 20 , 27 ] In the tumor microenviron-
`ment, FAP- α -positive stromal cells dominantly surround the
`tumor-derived vascular endothelial cells, [ 28 ] further facili-
`tating the effi cient and specifi c accumulation and activation
`of the FAP- α -responsive nanoprobes.
` In this study, we designed a ferritin-based FAP- α -
`responsive fl uorescence probe for CAFs positive carcinoma
`imaging. Unlike tumor cell-targeting probes, which need to
`reach a certain depth into tumor tissue to interact with tumor
`cells, the ferritin-based probe could be activated imme-
`diately (about 0.5 h after injection) as they penetrate the
`tumor blood vessels and come across the FAP- α molecules
`on the membrane of CAFs in the tumor microenvironment
`( Scheme 1 ). Hybrid ferritin has well been developed as an
`approach to build multi-functional nanoparticles for protein-
`based nanoprobe construction. [ 29–31 ] Ferritin is a family of
`proteins consisting of 24 subunits that exist in many types of
`organisms. As a type of stable cage-like biomolecules circu-
`lated in blood, ferritin-based nanomaterials are supposed to
`possess good biocompatibility and suffi cient stability. Further-
`more, its interior cavity for biomineralization and exterior
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`small 2013, 9, No. 14, 2427–2431
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`© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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`wileyonlinelibrary.com
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`
`

`

`communications
`
`T. Ji et al.
`
` Scheme 1 . The schematic illustration of the ferritin-based CAFs-responsive fl uorescence nanoprobe (FPB-Ft). A) Design, synthesis and B) assembly
`of hybrid ferritins and the FPB-Ft; C) the CAFs-dependent activation mechanism of the FPB-Ft fl uorescence probe in the tumor microenvironment.
`
`surface for further functionalization endeavor ferritins as the
`ideal molecules to form multifunctional nanostructures. [ 29 , 30 ]
`Moreover, the formation and dissociation of the cage-like
`nano-sized structure of single ferritin molecule is subject to
`be regulated by the environmental pH. It has been observed
`that in strong acidic environments (pH = 2.0) the ferritin cage
`would disassemble into discrete subunits, while at pH = 7.4
`these subunits once again self-assemble through non-cova-
`lent interactions and restore the cage-like structure. [ 29–31 ] This
`unique pH-responsive architecture has rendered it especially
`convenient to build multi-functional hybrid ferritins by con-
`jugating different small molecules onto the subunits. Here,
`we constructed a series of hybrid ferritin fl uorescence probes
`by coupling a fl uorescence-tagged peptide that can be spe-
`cifi cally cleaved by FAP- α on ferritin subunits, followed by
`reassembly them under physiological conditions. Considering
`these favorable properties, our design is making full use of
`the good biocompatibility and longer half-life of ferritins
`in the blood circulation; we fi nally achieved fl uorescence
`imaging in vivo by intravenously injecting this nanoprobe
`into mice. [ 32 , 33 ] This strategy exhibited high specifi city and
`stimuli-responsive sensitivity in the tumor microenvironment.
`Based on the results, this novel nanoprobe could be further
`developed as a promising nanomaterial for tumor microenvi-
`ronment imaging and tumor diagnosis.
` Construction of the hybrid ferritin was carried out
`according to the procedure shown in Scheme 1 A,B. A
`designed carboxyfl uorescein-tagged (FAM-tagged) peptide
`(FAM-DRGETGPAC, MW 1262) that can be specifi cally
`cleaved by FAP- α at the amide bond between Pro and Ala
`(resulting a fl uorescent fragment FAM-DRGETGP, MW
`1088) was coupled onto horse spleen ferritins to create the
`fi rst set of functionalized ferritin, FAM-peptide-ferritin (FP-
`Ft); meanwhile a second set of functionalized ferritin, black
`hole quencher (BHQ) linked ferritin, BHQ-ferritin (B-Ft),
`was prepared by modifying the protein with a fl uorescence
`
`quencher, BHQ-1. Both sets of ferritins maintained the
`cage structure after modifi cation, which was confi rmed by
`their morphologies under transmission electron micros-
`copy (TEM), as shown in Figure 1 A,B. The two sets of fer-
`ritins were then mixed and broke down into subunits at pH
`2.0, followed by retuning the pH back to neutral (pH 7.4) to
`allow the reassembly of ferritin particles. [ 29–31 ] During the
`reassembling process, the two sets of subunits hybridized
`to form the optimal hybrid ferritin (FPB-Ft, FP/B = 1:2),
`
` Figure 1 . The morphology of ferritins during the process of synthesis.
`TEM images of the assemblies of A) FP-Ft and B) B-Ft at pH 7.4;
`C) the disassembled subunits of FP-Ft and B-Ft at pH 2.0, and D) the
`reassemblies of the hybrid ferritin (FPB-Ft) formed by adjusting the
`acidic solution back to pH 7.4.
`
`2428 www.small-journal.com
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`© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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`small 2013, 9, No. 14, 2427–2431
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` 16136829, 2013, 14, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.201300600 by Jeff Kushan - <Shibboleth>-member@gwu.edu , Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Petitioner GE Healthcare – Ex. 1045, p. 2428
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`

`

`Tumor Fibroblast Specifi c Activation of Nanocage-Based Optical Probe
`
`i.e., the designed fl uorescence quenched
`nanoprobe, as showed in Figure 1 C,D. As
`a fl uorescence resonance energy transfer
`(FRET) quencher, [ 34 , 35 ] BHQ-1 quenched
`the fl uorescence of nearby fl uorescence-
`tagged peptide, hence the nanoprobe was
`at a quiescent stage until activated by
`encountering with FAP- α . This enzyme
`triggered activation would signifi cantly
`improve both the sensitivity and speci-
`fi city for optical imaging. The fl uorescent
`peptide, FAM-DRGETGPAC, has been
`demonstrated as being selective for FAP-
` α even after the conjugation with ferritins,
`which was validated by mass spectrometric
`analysis (Figure S1 in the Supporting
`Information). When FAP- α was added
`into free FAM-tagged peptide, the pep-
`tide peak at m/z = 1263 disappeared and
`the peak m/z = 1089 corresponding to the
`fl uorescence fragment FAM-DRGETGP
`emerged, 5 h after the treatment; and in
`the case of FP-Ft, the peak of m/z = 1263
`was not present in mass spectrum due to
`chemical binding between the peptide
`and ferritin, while the peak m/z = 1089 was observed after
`the addition of FAP- α , indicating the releasing of the same
`fragment. It could thus be concluded that the FAM-tagged
`peptide was successfully linked to ferritins and the peptide
`could be cleaved as desired from ferritins by FAP- α . Details
`of peptide and BHQ-1 coupling, purifi cation procedures and
`HPLC analysis can be found in the Supporting Information
`(Supporting Information Figure S2).
` To evaluate
`the effi ciency of such quench/restore
`approach and to determine the optimal composition of nan-
`oprobe, we prepared hybrid ferritins (1 mg/mL) solutions
`with different FP/B subunits ratios (1:0, 1:1, 1:2, and 1:3) as
`substrates, which were then mixed with 0.2 μ
` g/mL FAP- α ,
`respectively. For each ferritin hybrid, stability was tested in
`the same condition as mentioned above, but with no FAP-
` α added. Fluorescence intensity of each solution was meas-
`ured by a fl uorescence spectrometer (excitation at 494 nm,
`emission at 520 nm) every 0.5 h. None of the controls has
`demonstrated to have any signifi cant change in fl uorescence
`intensity in the 12 h interval ( Figure 2 A), indicating that
`the nanoprobes could not self-activate spontaneously and
`remained in stable stage. However, for all the four enzyme-
`substrate mixtures, the fl uorescence intensity increased grad-
`ually after the addition of FAP- α and subsequently reached
`maximum in 5 h, suggesting that the activation of nanoprobes
`by FAP- α and the activation started as long as FAP- α was
`added (Figure 2 B). When analyzed by mass spectrometry, the
`peak m/z = 1089 was present in the spectra of all the mixtures
`after reaction. Among the four ratios, the hybrid ferritins with
`the ratio of FP/B 1:2 exhibited both the highest quenching
`effi ciency and the optimal fl uorescence emission, with a 6-fold
`increase in fl uorescence between the activation state and the
`quenched state (Figure 2 B,C). Increase in FB-Ft proportion
`(1:1) decreased quenching effi ciency (3-fold), while increase
`
` Figure 2 . Kinetic changes of the fl uorescence intensity of hybrid ferritin with different FP/B
`ferritin ratios. A) In the absence of FAP- α , the probes could not be activated. B) In the presence
`of FAP- α , the probes were activated and the fl uorescence intensity increased gradually with
`time. C) Activation capacities of the different hybrid ferritin formulas (with different FP/B
`ferritin ratios) derived from (A) and (B).
`
`in B-Ft proportion (1:3) led to a weaker fl uorescence activa-
`tion (Figure 2 C). Therefore, the hybrid ferritin with FP/B 1:2
`was used as the optimal nanoprobe during further in vitro
`experiment, whose fl uorescence intensity was measured both
`in cells and culture media. Prostate CAF, hTERT immortal-
`ized, cell line was analyzed with PCR and agarose gel elec-
`trophoresis to confi rm that the CAFs indeed express FAP- α .
`Two pairs of primers (183 bp and 165 bp, primer sequences in
`the Supporting Information) were designed to investigate the
`existence of the corresponding mRNA, with positive results
`showing the expression of FAP- α (Supporting Information
`Figure S3). The CAFs were then monitored by laser-scanning
`confocal microscopy (LSCM) at various time points during
`incubation with the nanoprobes (0.05 mg/mL). The imaging
`results showed that the nanoprobe was activated in an hour
`and the fl uorescence intensity continuously increased with
`time in the fi rst 5 h time interval ( Figure 3 A). The intensity
`at 12 h was approximately the same as at 5 h. Considering
`that most of fl uorescent fragments would widely distribute
`in the culture medium after being cut off from FPB-Ft, the
`fl uorescent emission of the culture media was also analyzed
`by fl uorescence spectroscopy (Figure 3 B,C). The results were
`consistent with Figure 3 A, and the fl uorescence intensity of
`the cell culture medium at 12 h was slightly lower than that
`at 5 h, possibly because more fragments were taken up by the
`surrounding CAFs. However, no fl uorescence was detected
`in tumor cells, human prostate cancer cell line (PC-3) in the
`same condition because the tumor cells do not express FAP- α
`as reported. [ 20 ] These in vitro imaging data provided positive
`indications of the nanoprobe's capability to target FAP- α -
`positive stromal cells in the tumor microenvironment in vitro.
` The potential application of the FAP- α activatable probes
`for tumor imaging was further investigated in vivo for FAM
`signal detection (Maestro in vivo imaging system, excitation
`
`small 2013, 9, No. 14, 2427–2431
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`© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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`www.small-journal.com
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` 16136829, 2013, 14, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.201300600 by Jeff Kushan - <Shibboleth>-member@gwu.edu , Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Petitioner GE Healthcare – Ex. 1045, p. 2429
`
`

`

`communications
`
` Figure 3 . In vitro imaging of the FPB-Ft. The confocal microscopy images of PC-3 cells and CAFs
`incubated with FPB-Ft probe at various time points. Green and blue fl uorescence are FAM and
`DAPI fl uorescence, respectively. The enhanced fl uorescence intensity of the culture media at
`the corresponding time points with (A). B,C) Fluorescence intensity of the CAFs group (B) and
`the PC-3 group (C).
`
`T. Ji et al.
`
`investigated. As
`quenched), were also
`shown in Supporting Information Figure S5,
`the positive control group showed a
`whole body distribution of fl uorescence
`at 1.5 h after injection and the intensity
`decreased quickly. Only a weak signal
`could be detected in the tumor after 5 h.
`No fl uorescence signal was observed in
`the negative control group. These studies
`suggested that the FPB-Ft reached the
`tumor through blood circulation and was
`activated by FAP- α in the co-inoculated
`group (Scheme 1 C), but mainly remained
`quenched in the PC-3 only group. Ex
`vivo measurements also exhibited con-
`sistent results. The main visceral organs
`and tumors of both groups were imaged
`1.5 h after intravenous injection. In the
`co-inoculated group, the tumor showed
`much stronger fl uorescence compared
`to the rather low signal of other organs
`(except for liver, which showed a detect-
`able signal) (Figure 4 G); yet in the PC-3
`group, no obvious signal was detected in
`either the major organs or tumor. Such
`results implied that the FPB-Ft nano-
`probes were only specifi cally activated
`by FAP- α in tumors. The possible reason
`that the PC-3 group had not depicted
`any targeted signal could be that without
`CAFs inoculation, and one week tumor
`inoculation was still not suffi cient for the
`
`and emission wavelengths were 490 and 520 nm, respec-
`tively). All experiments with mice followed the guidelines for
`experimental animals and were approved by the local animal
`welfare committee. One group of BALB/c nude mice (male,
`16–18 g body weight) were co-inoculated with 1:1 mixture of
`PC-3 cells and CAFs (1 × 10 6 each), [ 36 , 37 ] and another group
`(PC-3 group) was inoculated with PC-3 cells (2 × 10 6 each)
`only. A week later, the diameter of tumors in co-inoculated
`group grew to 0.5 cm on average, while tumors in the PC-3
`group were smaller (0.3 cm) in diameters, which was in
`accordance with the known promotion effect of CAFs on
`tumor growth. [ 22–24 ] FPB-Ft (150 μ L, 0.5 mg/mL in PBS) was
`intravenously injected into both groups of mice. As expected,
`the observed FAM signal was largely localized in the co-
`inoculated group ( Figure 4 A–C), with the intensity reaching
`its maximum at 1.5 h after injection (Figure 4 B). Afterwards
`the signal decreased gradually, and only a weak signal could
`be detected in the tumor tissue 5 h after injection, because
`most fl uorescent fragments were metabolized and only a few
`of them still localized in the tumor site (Figure 4 C). Inter-
`estingly, no localized signal was observed in the PC-3 group
`(Figure 4 D–F). Two control groups of mice co-inoculated
`with PC-3 cells and CAFs (1 × 10 6 each), where the positive
`control group was treated with FAM-ferritin (F-Ft, with no
`peptide linked) and the negative control group with FAM-
`BHQ-ferritin (FB-Ft, no peptide
`linked, fl uorescence
`
` Figure 4 . A–F) In vivo and G) ex vivo fl uorescence images of the mice
`injected with FPB-Ft probe. In the co-inoculated group (A–C), the FPB-Ft
`probe was activated and the FAM signal reached the maximum 1.5 h
`after FPB-Ft probe injection (B), and the signal could still be observed
`5 h after the probe injection (C). In the PC-3 group, no specifi c signal
`was detected with time (D–F), showing the FAP- α specifi c activation of
`CAFs, but not the tumor cells.
`
`2430 www.small-journal.com
`
`© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`small 2013, 9, No. 14, 2427–2431
`
` 16136829, 2013, 14, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.201300600 by Jeff Kushan - <Shibboleth>-member@gwu.edu , Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Petitioner GE Healthcare – Ex. 1045, p. 2430
`
`

`

`Tumor Fibroblast Specifi c Activation of Nanocage-Based Optical Probe
`
`tumor cells (PC-3) to recruit or activate the endogenous
`fi broblasts to become cancer associated fi broblasts in the
`mice, i.e., not suffi cient for the formation of a FAP- α -positive
`tumor microenvironment. [ 37 , 38 ] Furthermore, although FAP-
` α is highly specifi cally expressed in various tumor tissues, it
`also expressed in granulation tissue and some pathological
`lesions, such as tissue repair. [ 39 ] Special care should be taken
`to ensure not to include these false-positive observations
`under such conditions.
` In summary, we have reported a ferritin-based fl uores-
`cence nanoprobe that was specifi cally activated by FAP- α
`expressed in the tumor microenvironment, by which specifi c
`fl uorescence imaging in vivo was realized through tail vein
`injection. Owing to abundant and stable FAP- α expression
`in tumor microenvironment and dominantly surrounding the
`tumor-derived vascular endothelial cells, there was no need
`to make the probes reach tumor cells to be activated, likely
`to result in rapid, specifi c and sensitive tumor imaging. Our
`work suggests a promising nanomaterial for tumor imaging
`through responding to the enzymes of stromal cells in the
`tumor microenvironment, which may be a new strategy for
`highly specifi c and sensitive tumor imaging and diagnosis.
`
` Supporting Information
`
` Supporting Information is available from the Wiley Online Library
`or from the author.
`
` Acknowledgements
`
`from MOST 973
` This work was supported by the grants
`(2012CB934004, 2010CB933601 and 2012CB932601) and the
`NSFC (31170962, 51203032, and 21277037).
`
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` Received: February 25, 2013
` Revised: April 11, 2013
`Published online: July 22, 2013
`
`small 2013, 9, No. 14, 2427–2431
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`Petitioner GE Healthcare – Ex. 1045, p. 2431
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