Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 1

http://dx.doi.org/10.10014-4827/& 2014 E

nCorresponding autE-mail address: g

Research Article

Synthesis and functional characterization of afluorescent peptide probe for non invasive imaging ofcollagen in live tissues

Ushasri Chilakamarthia, Jaipal Kandhadib, Srinivas Gundaa, AvinashRaj Thatipallia, Mahesh Kumar Jeralda, Giribabu Lingamallub, Rakesh C. Reddyb,Arabinda Chaudhurib, Gopal Pandea,n

aCSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, IndiabCSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India

a r t i c l e i n f o r m a t i o n

Article Chronology:

Received 18 January 2014Received in revised form5 May 2014Accepted 10 May 2014Available online 5 June 2014

Keywords:

Liver fibrosisDiagnostic probeType I collagenPorphyrin

016/j.yexcr.2014.05.005lsevier Inc. All rights reser

hor. Fax: þ91 40 27160591pande@ccmb.res.in (G. Pa

a b s t r a c t

Targeted molecular imaging to detect changes in the structural and functional organization oftissues, at the molecular level, is a promising approach for effective and early diagnosis ofdiseases. Quantitative and qualitative changes in type I collagen, which is a major component inthe extra cellular matrix (ECM) of skin and other vital organs like lung, liver, heart and kidneys,are often associated with the pathophysiology of these organs. We have synthesized a fluorescentprobe that comprises collagelin, a specific collagen binding peptide, coupled to fluorescentporphyrin that can effectively detect abnormal deposition of collagen in live tissues by emittingfluorescence in the near infra red (NIR) region. In this report we have presented the methodologyfor coupling of 5-(4-carboxy phenyl)-10, 15, 20-triphenyl porphyrin (C-TPP) to the N-terminal ofcollagelin or to another mutant peptide (used as a control). We have evaluated the efficacy of

these fluorescent peptides to detect collagen deposition in live normal and abnormal tissues. Ourresults strongly suggest that porphyrin-tagged collagelin can be used as an effective probe for thenon invasive in vivo detection of tissue fibrosis, especially in the liver.

& 2014 Elsevier Inc. All rights reserved.

Introduction

Non invasive imaging of live tissues in experimental animals,for doing either basic studies or pre-clinical trials for early diseasediagnosis, has been increasingly practiced in recent past. Design-ing of novel and effective imaging probes for targeting biomarkersin live tissues is a major challenge. The protocol for designing an

ved.

.nde).

imaging probe generally includes selection/synthesis of an appro-priate probe molecule (antibody, ligand or peptide) thatis specifically targeted to a in vivo biomarker, conjugation ofthe probe to a biocompatible contrast/detection reagent and amethod for using the labeled probe, in combination with anappropriate imaging methodology, to specifically and sensitivelyvisualize the targeted biomarker in live tissues. It has been

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 192

suggested that an ideal molecular probe for non-invasive imagingshould be safe for use in vivo; should be stable and readilypermeable into the target tissue; should have high bindingaffinity for the targeted biomarker and hence should be specifi-cally retained in the target tissue and rapidly cleared from non-target tissues [1].Non-invasive fluorescence imaging of live tissues has emerged

as a safe, inexpensive and rapid tool in this field as compared toother techniques. Fluorochromes that emit signals in the nearinfrared (NIR) region serve as good contrast agents becauseminimum auto-fluorescence of tissues is observed in the NIRregion [1]. A NIR dye Indo-Cyanine Green (ICG) has beenextensively used by cardiologists and ophthalmologists since1960s [2], but its wider use in other tissues has been restricteddue to rapid clearance of the dye from liver [3] and also somereports of adverse reactions [4] Porphyrins (another group ofreagents emitting in NIR) have been used widely for the photo-dynamic therapy of cancer [5], but their use as diagnostic probeshas not been explored extensively. Recently platinum conjugatedtetra-phenyl-tetra-naphtho-porphyrin [Pt (TPNP)] was used forimaging of tumors in nude mice [6] and 5-aminolevulinic acidinduced porphyrin fluorescence was used for the detection ofCervical Intraepithelial Neoplasia (CIN) in humans [7].Biomarker targeted peptides coupled with various contrast

agents have proven to be efficient for live tissue imaging [8,9].Collagen, the major protein in ECM, has been targeted as abiomarker for this purpose [10,11]. In our study we have used aNIR contrast reagent 5-(4-carboxy phenyl)-10,15,20-triphenylporphyrin (C-TPP), coupled to collagelin—a specific collagenbinding peptide whose properties have been reported earlier[11] as a targeted probe for visualization of collagen in live tissues.First we covalently tethered the C-terminus of C-TPP to the N-terminus of collagelin and evaluated its collagen binding proper-ties in solution assays. We found that porphyrin coupling did notsignificantly alter the collagen binding properties of collagelin,however if mutations were introduced in the sequence ofcollagelin its binding ability was reduced significantly. For study-ing in vivo tissue binding the probe was injected into normal andfibrotic Balb/c and nude mice—fibrosis of liver in these mice wasinduced by carbon tetra chloride (CCl4) injections as per theprotocols described earlier [12,13]. Fluorescence of C-TPP taggedcollagelin and mutant peptide, either from live animals or fromexcised organs of injected animals, was estimated in NIR regionusing a multi-spectral animal imager. Our results showed that theprobe binds strongly and specifically to collagen I (Col I) of fibroticliver tissue and either weakly, or not at all, to normal liver tissue.This was also confirmed by co-visualization of normal and fibrotic

Fig. 1 – Scheme showing the ste

liver tissues labeled with fluorescent anti-Col I antibody and theC-TTP tagged collagelin. In summary, we have reported a novelin vivo collagen-specific NIR probe that holds diagnostic promisefor non invasive early detection of liver fibrosis.

Materials and methods

Preparation of the fluorescent probe

Synthesis of C-TPPThe methodology for synthesis of C-TPP is shown in Fig. 1. 4-carboxy benzaldehyde (1 eq.) (Sigma Chemicals) and benzalde-hyde (3 eq.) (Sigma Chemicals) were taken in 400 ml of propionicacid. The reaction mixture heated up to 120 1C and to this 4 eq.pyrrole (Sigma Chemicals) was added drop wise. Then thereaction mixture was brought to reflux for 1 h. The reactionmixture cooled to room temperature and kept in refrigerator forovernight to precipitate. The precipitate was filtered, washed withwater and dried under vacuum. The obtained solid material wassubjected to silica gel column chromatography (100–200 mesh)and eluted with CHCl3/CH3OH (95:5% v/v). The second purplecolor band was collected and re-crystallized from CHCl3/CH3OH toget the desired compound.

Coupling of C-TPP with collagelinResin bound collagelin and control mutant peptides (both with freeN-terminus) were commercially obtained from USV Ltd. Mumbai.The sequence of collagelin was 2HN-SGSGCPGRVMHGLHLGDDEGPC-COOH and the mutant peptide sequence was 2HN-SGSGLG-GLVMHGLHLGNNQGPC-COOH (the changed residues are shown inbold). Both peptides were coupled with C-TPP using solid phasepeptide chemistry as shown in Fig. 2. The peptide resin, C-TPP andall coupling reagents were desiccated for 4 h and 50 mg of the resinbound collagelin or mutant peptide was swollen in dimethylformamide (DMF) for 1 h at room temperature. Then 10 eq. ofC-TPP, 10 eq. of hexafluoro-benzotriazole-tetramethyl-uronium-phosphate (HBTU) Novabiochem (San Diego, CA) and 10 eq. ofhydroxybenzotriazole (HOBT) Novabiochem (San Diego, CA) weredissolved in DMF separately and mixed in a round bottom flask.The mixture was left for 5 min at room temperature to activate thecarboxy functional group of C-TPP. This mixture was added to thepre swollen resin and 10 eq. of di-isopropyl-ethyl-amine (DIPEA)(Sigma-Aldrich) was added to start the coupling reaction. Thereaction was allowed to proceed for 12 h in a round bottom flask.The resin was then washed several times with DMF to remove theun-reacted C-TPP and other reagents. A fresh reaction mixture was

ps in the synthesis of C-TPP.

Fig. 2 – Characterization of C-TPP.(A) MALDI-TOF massspectrometry of C-TPP and (B) absorption and fluorescencespectra of C-TPP. Red colored spectra is absorption spectrashowing absorption maxima Ex. λ¼420 nm and the black oneis emission spectra showing Em. λ¼650 nm.

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 1 93

prepared as above and added to the same resin and reaction wasallowed to continue further for 12 h. Resin was washed with DMF toremove unreacted reagents until the filtrate was colorless. The resinwas dried and de-protection of side groups and cleavage of peptidefrom the resin was done by treatment with a solution containingtrifluoro acetic acid: thioanisole: phenol: water: ethane dithiol at avolume to volume ratio of 82.5:5:5:5:2.5 at room temperature for12 h. The C-TPP coupled peptides were precipitated several timeswith cold diethyl ether to further remove the unreacted reagents.Finally the precipitate was dissolved in autoclaved distilled waterand further purified by HPLC.

Peptide purification

The diethyl ether precipitated conjugates were dissolved in waterand analyzed for coupling by reverse phase HPLC using a 5–100%acetonitrile step gradient in water containing 0.1% TFA for115 min. at a 0.5 ml/min flow rate. The parameters were set forUV detection at 214 nm (specific for peptide bond absorption) and420 nm (specific for porphyrin absorption) and fluorescent detec-tion at 650 nm (porphyrin emission wavelength). HPLC analysisand separations were performed using a semi-preparative Waters

Prep NovaPak C18 column (7.8�300 mm2, pore size 6 mm). Thepeptide conjugate containing fractions were collected and lyo-philized to get pure coupled products. The mass of coupledproducts was verified by MALDI-TOF mass spectrometry (AppliedBiosystems, USA) and Vayoger software. The absorption proper-ties of the coupled products were analyzed in a UV–vis spectro-meter (UV3600 Shimadzu) and fluorescence was studied inFluorolog 3 (Horiba Jobin NJ.USA) fluorimeter. The purifiedlabeled peptides were used further for in vitro, ex vivo andin vivo experiments.

In vitro collagen binding assays

In vitro binding studies were performed in solution phase withisolated rat tail collagen. 25 mg of C-TPP tagged collagelin ormutant peptide were added to 100 mg of rat tail collagen in0.25 mM acetic acid solution and volume was made up to 250 mlwith PBS (pH-7) and the reaction was kept for 1 h at roomtemperature. In the other tubes 100 mg of collagen or 25 mg eachof C-TPP labeled peptides, suspended in the same buffer, wereincubated alone. After 1 h all the solutions were precipitated byadding NaCl to a final concentration of 2.4 M and left overnight at4 1C. The precipitates were centrifuged and supernatants werecollected in other tubes. The pellets and supernatants from all thetubes were observed in multispectral FXPRO fluorescence imager(Rochester, NY USA) at porphyrin specific wavelengths 420 nmexcitation and 650–690 nm band pass filter set. In order to getcollagen binding profiles of fluorescent collagelin and fluorescentmutant collagelin a more quantitative experiment was designedand performed.25 mg (3.3 mM) of collagen dissolved in 0.25 mM acetic acid

solution was taken in different tubes and coupled peptide ormutant peptide were added ranging from 0.02 mM to 5.3 mM.After 1 h of incubation at room temperature the solutions in allthe tubes were precipitated by adding NaCl to final concentrationof 2.4 M (the concentration at which collagen I gets precipitated)and left overnight at 4 1C. The precipitates were centrifuged, thepellets were washed twice with cold PBS, dissolved in 0.25 mMacetic acid and collagen bound fluorescence was imaged in theMultispectral FX-PRO fluorescence imager (Rochester, NY USA) atporphyrin specific wavelengths of 420 nm excitation and 650 nmemission. The fluorescence intensity of the solutions in the sametubes was also measured using FL� 800 microplate fluorescencereader (Bio-Tek instruments, INC, UK) at excitation and emissionwavelengths of 420 nm and 650 nm respectively. The amount ofcollagen bound peptide(s) in the precipitates was calculated fromporphyrin- tagged-peptide(s) standard curves (SupplementaryFig. SF1). The quantities of collagen-bound fluorescent peptides(in mM) were plotted as a function of increasing amounts ofcollagelin or mutant collagelin used in the reaction.To examine whether coupling of porphyrin had altered the

affinity of collagelin to collagen, we performed a similar assayas described above, however here we measured the quantity ofthe residual peptide (in mM) in the supernatants using a BCA(Bicinchoninic acid) assay kit (Thermo scientific, USA) (seeSupplementary Fig. SF2). The collagen bound amounts of eachpeptide were estimated from these values and the same wereplotted as a function of increasing total peptide concentrations ineach assay.

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 194

Induction of fibrosis in Balb/c and nude mice

All animal experiments were done as per the protocol approvedby the Institutional Animal Ethics Committee of CCMB (ProtocolNo. IAEC 35/2012). Inbred, 8–12 weeks old Balb/c mice of eithergender were injected with 0.5 ml/kg body weight CCl4 twice aweek for 4 weeks. Fibrotic model of nu�/� nude mice (8–12weeks age) were prepared by administering 0.25 ml of CCl4 per kgbody weight twice a week for 4 weeks. In control animals 200 mlPBS was injected at the same time points. 6 animals were used infibrosis group and 4 animals in the control group. After injectionsboth sets of animals were left under normal maintenance for 4weeks at which time imaging and histochemistry experimentswere done. Tissues from fibrotic and normal Balb/c mice wereused for ex vivo fluorescence imaging and histochemistry. Anaes-thetized normal and fibrotic nude mice were used for non-invasive fluorescence imaging studies.

Peptide injections

6mg/kg body weight of porphyrin tagged collagelin or mutantpeptide was intravenously administered to normal or CCl4 treatedBalb/c and nu-/- nude mice. After 4 h Balb/c mice were sacrificed andintact heart, lung, kidney and liver were removed from the animaland observed for porphyrin fluorescence. Nude mice 4 h after peptideinjection were anaesthetized with 25mg/kg body weight ketamineand 5mg/kg body weight xylazine and placed in the fluorescenceimager (see details below) for localization of collagen in live mice.

Collagen I immunofluorescence in liver

Liver tissues, from normal and CCl4 treated Balb/c mice, werefrozen using OCT and isobutane and kept at �80 1C until furtheruse. 7 mm thick sections of the frozen tissues were cut in aCryomicrotome (Leica). The sections were fixed in 4% paraformal-dehyde solution, dried and kept at �80 1C. Before staining thesections were taken from �80 1C and washed with PBS. Then 1%BSA was added for blocking and left for 20 min. Again the sectionswere washed twice with PBS. Collagen I anti body (Abcam, USA)1:100 dilution was added to the sections and kept for 2 h at roomtemperature. The sections were again washed with PBS and 21antibody FITC conjugated goat anti rabbit 1:1000 dilution wasadded and kept in dark for 2 h at room temperature. Then thesections were washed with PBS, counter stained with DAPI andobserved in Axioplan-2 fluorescence microscope (Carl ZeissGermany). 455–485 nm band pass filter was used for excitationand 505–555 nm band pass filter for emission detection. Axiovi-sion (Version 3.1) software was used to analyze the images.

Collagelin binding to frozen liver sections

The sections of normal and fibrotic livers were fixed in 10%buffered formaldehyde for 30 min. Then sections were washedwith PBS and then 1% BSA was added for blocking and left for20 min. Again the sections were washed twice with PBS. 25 mg ofcollagelin or mutant peptide, prepared in 200 ml of PBS, wasapplied to the sections and kept in dark for 2 h at roomtemperature. The sections were washed twice with PBS at roomtemperature, counter stained with DAPI and observed inAxioplan-2 fluorescence microscope (Carl Zeiss, Germany). Band

pass 395–440 nm filter set was used for excitation and 550 nmlong pass filter set for emission detection. Axiovision (Version 3.1)software was used to analyze the images.

Collagelin fluorescence in excised organs

Normal and fibrotic Balb/c mice injected with 6 mg/kg bodyweight of porphyrin tagged collagelin or mutant peptide weresacrificed 4 h after peptide injection and internal organs heart,lung, kidney and liver of each mouse were carefully excised. Allthe organs, immediately after excision, were examined in KODAKCarestream FXPRO imager under the conditions described above.After live imaging the liver lobes were frozen to prepare 7 mmthick sections. Further analysis of collagelin fluorescence in tissuesections was done in the Zeiss Axioplan-2 fluorescence micro-scope using specific filters as described above. One set of normaland fibrotic livers were harvested and washed in cold saline.500 mL of lysis buffer (0.1 M Tris–HCl, 2 mM EDTA and 0.2% TritonX-100, pH7.8) was added to each liver and homogenized using amechanical homogenizer. The homogenates were centrifuged at14,000 rpm for 10 min. at 4 1C and fluorescence of 100 mL of thesupernatant was measured at excitation and emission wavelengthof 420 nm and 650 nm at FLx 800 microplate fluorescence reader(Bio-Tek instruments, INC, UK). For comparison similar estima-tions were also done on extracts of other tissues such as lung,kidney and heart. For each sample, the background fluorescencewas subtracted and the fluorescence peptide intensities obtainedfrom each tissue sample was plotted as a histogram display.

Collagelin fluorescence in intact mice

Liver fibrosis in nude mice was induced as indicated previously.6 mg/kg body weight of peptide or mutant peptide was intrave-nously injected to the normal as well as 8th week fibrotic animals.The fluorescent imaging was done after 4 h giving mild anesthesia(by using lower dose of anesthetics) under the same conditions asdescribed for Balb/c mice. Immediately after imaging, the micewere sacrificed and tissue specific fluorescence was analyzed inthe FXPRO multispectral imager and liver sections were studied inAxioplan 2 microscope as described above.

Results

Chronic liver disease is characterized by the accumulation of ECMproteins especially collagen [14]. Accumulation of ECM proteinsdestroys the hepatic architecture by forming fibrous scar becauseof which pseudo lobules are formed [15]. The formation of pseudolobules by the accumulation of type I collagen was depicted inFig. SF3.

Preparation and characterization of the fluorescent probe

C-TPP was synthesized as per reactions shown in Fig. 1. It waspurified by silica gel column chromatography and characterizedby mass spectrometry, and absorption and fluorescence spectro-scopy. The calculated mass of synthetic C-TPP (C45H30N4O2) was658 and in our analysis we observed a mass of 659.28 (Fig. 2A).The absorption and emission properties of C-TPP were also in the

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 1 95

expected value ranges: Emax absorption at 420 nm and Emax

emission at 650 nm (Fig. 2B).This C-TPP was coupled to the N-terminus of collagelin and its

mutant peptide as per the reactions shown in Fig. 3. The HPLC profiles

Fig. 4 – HPLC profiles of (A) C-TPP coupled collagelin (B) C-TPP couwavelengths as indicated in the panels. Mass spectrometry of (C) c

Fig. 3 – Scheme showing the steps in the coupling of porphyrinto collagelin and mutant collagelin.

of the peptide conjugates are shown in Figs. 4A and 4B. The C-TPPcoupled collagelin and mutant peptide eluted at 57min (Fig. 4A)and 55min (Fig. 4B) respectively, whereas uncoupled collagelinand mutant peptide eluted much before at 34min and uncoupledC-TPP eluted much later at 67 min. This separation in elution wasbased on different hydrophobicities of these three molecular species.Uncoupled C-TPP is more hydrophobic and hence elutes last whereaspeptide coupled C-TPP has increased aqueous solubility due to whichit elutes early. HPLC was done several times and each time fractionscontaining the conjugated molecules (at 57 min and 55min) werecollected, pooled, dried and quantified.After de-protection, cleavage from the resin and HPLC purifica-

tion we obtained 2 mg of coupled product from 50 mg of0.25 mmole substituted resin. There was no change in theabsorption and fluorescence properties of the coupled products(data not shown) indicating that coupling of peptide did not alterthe photophysical properties of C-TPP. The masses of the con-jugates were confirmed by mass spectrometry. The analysisshowed calculated and observed masses of the collagelin

pled mutant collagelin at various excitation and emissionoupled collagelin (D) coupled mutant collagelin.

Fig. 5 – In vitro studies to evaluate the collagen binding and reporting capacity of the probe (A) X-ray and fluorescent images of thetubes containing the precipitates of 1—collagen 2—collagenþfluorescent collagelin, 3—collagenþfluorescent mutant collagelin,4—Fluorescent collagelin, 5—Fluorescent mutant collagelin, 6–10 are the supernatants from the above respective tubes. (B) X-ray(upper panel) fluorescence (lower panel) images of tubes containing the reaction of fluorescent collagelin (concentrations rangingfrom 0, 0.02 to 5.3 mM with constant amount of collagen (3.3 uM). (C) Same as A, but with fluorescent mutant collagelin. (D) Graphshowing the binding profiles of fluorescent collagelin and fluorescent mutant collagelin with collagen (E) Graph showing theaffect of porphyrin coupling on collagen binding profile of collagelin.

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 196

conjugate at 2819 and 2819.66 respectively (Fig. 4C) and that ofmutant peptide conjugate at 2745 and 2744.5 respectively(Fig. 4D).

In vitro studies

First we assayed collagen binding to C-TPP collagelin in solutionphase under conditions described in Materials and Methods.The X-ray and fluorescence images of all the precipitates andsupernatants are shown in Fig. 5A. Precipitates of collagen,collagelin and mutant peptide alone did not exhibit muchfluorescence (tubes 1, 4 and 5) but intense fluorescence wasobserved in the corresponding supernatants of collagelin andmutant peptide (tubes 9 and 10) and no fluorescence was seenin the supernatant of collagen 1 (tube 6). These results indicatedthat fluorescent peptides or unbound non-fluorescent collagenalone did not contribute to the fluorescence of the precipitates

in the collagen binding reactions (seen in tube 2 and 3). Strongfluorescence was seen in precipitates obtained from collagenand fluorescent collagelin incubation (tube 2), but lesser fluor-escence was observed when collagen was incubated withfluorescent mutant peptide (tube 3), indicating a reducedbinding of the mutant peptide to collagen. The correspondingsupernatants of both reactions did not show any fluorescence(tubes 7 and 8).

In order to compare the collagen binding profiles of fluorescentnative and mutant collagelin a quantitative binding assay wasperformed as described in Materials and Methods. The images ofnative and mutant peptides, bound to collagen, are shown inFig. 5B and 5C and their respective bound quantities, as a functionof total peptide used, are shown in Fig. 5D. The inset graph showsbinding profiles of these peptides specifically at initial concentra-tions i.e. before they reached the saturation point. These resultsclearly demonstrate that the mutant collagelin exhibited

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 1 97

significantly lower binding to collagen as compared to nativecollagelin.

In the experiment conducted to evaluate the alteration of collagelinbinding to collagen, before and after coupling with C-TPP, althoughwe observed a marginal reduction in the maximum amount of boundcollagelin after C-TPP coupling (�20%) but the overall binding profileremained unaltered (Fig. 5E) suggesting that the C-TPP coupledcollagelin was still efficient in detection of collagen.

Fig. 6 – Fluorescence images of normal and fibro-tic liver tissue secas indicated in the panels. The white arrowheads show the bindin

Fig. 7 – Ex vivo studies to evaluate the efficacy of the probe to detecfibrotic livers excised from Balb/c mice injected with fluorescent colivers injected with mutant collagelin.

Further confirmation of collagelin binding to collagen from fibroticliver was done by incubation of the fluorescent peptides on sectionsof liver tissue sections from normal and fibrotic mice. Fluorescentcollagelin showed good binding to fibrotic liver sections (Fig. 6B), butdid not show any fluorescence on normal liver sections (Fig. 6A);mutant peptide did not show fluorescence on normal liver tissuesection (Fig. 6C), but showed some fluorescent spots due to nonspecific binding (Fig. 6D) on fibrotic liver sections.

tions stained with the fluorescent collagelin, mutant collageling sites of native and mutant collagelin to fibrotic liver section.

t fibrotic-liver. (A) X-ray and fluorescent images of normal andllagelin (B) X-ray and fluorescent images of normal and fibrotic

Fig. 8 – Non invasive in vivo imaging of normal and fibrotic livers to detect collagen deposition. A and B are normal nude miceinjected with fluorescent collagelin and mutant coUegeltn probes respectively.(A–i and B–i) In vivo fluorescence images. (A–ii &B–ii) Visual images of excised vital organs (A–iii and B–iii) X-ray image overlayed with fluorecence of excised vital organs. C and Dare fibrotic nude mice injected with fluorescent collagelin and mutant collagelin probes respectively. lC–i and D–i) In vivofluorescence images. (C–ii and D–ii) Visual images of excised vital organs (C–iii and D–iii) X-ray image overlayed with fluorecenceof excised vital organs. Li—Liver, K—Kidney, Lu—Lung. H—heart.

Fig. 9 – Quantification of the C-TPP fluorescence in the variousorgans by bio-distribution analyses of representative animals.Cntrl- without any injections, FC- fluorescent collagelin.FMC-fluorescent mutant collagelin.

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 198

Ex vivo studies

C-TPP-tagged collagelin or mutant peptides were intravenouslyinjected into normal (n¼4) and fibrotic (n¼6) Balb/c mice, foreach peptide, as described in Materials and Methods. X-ray andfluorescence images of excised normal and fibrotic livers fromeach group of mice are shown in Fig. 7. Panel A shows images ofnormal and fibrotic livers of mice injected with collagelin andpanel B shows images of isolated livers from mice injected withmutant peptide. TPP specific fluorescence was observed only infibrotic livers of mice injected with C-TPP tagged collagelin. Noor very low levels of fluorescence respectively could be seen innormal livers injected with C-TPP tagged collagelin and infibrotic livers injected C-TPP tagged mutant peptide. This wasconfirmed by the comparative bio-distribution analysis ofC-TPP fluorescence where the highest presence of C-TPP wasseen in fibrotic livers injected with native C-TPP taggedcollagelin (Fig. 9).

In vivo studies

Imaging of intact animals, after injecting collagelin or mutantpeptides into fibrotic (n¼3) and normal (n¼2) nude mice foreach peptide, was done as described in Materials and Methods.Intense fluorescence was observed in the livers of fibrotic micebut very low fluorescence was visible in the livers of normal mice(Fig. 8A panel i and 8C panel i). Weak fluorescence could also beseen in the lungs of both types of animals.The fluorescence imaging of excised organs also confirmed

the above observations in the various groups (Fig. 8A–D). Wefurther quantified the C-TPP fluorescence in the various organs

by doing bio-distribution analyses of representative animalsas described in Materials and Methods and the data areshown in Fig. 9. The maximum C-TPP fluorescence could beseen in the fibrotic liver tissue. The residual fluorescence inthe lungs of both fibrotic and normal animals injected withmutant or native collagelin could be due to the passive trappingof the probe molecules that possibly could penetrate non-specifically through the fenestrated capillaries of the lungepithelium.

Fig. 10 – Co-localization of fluorecent collagelin probe with coltagen-l antibody. Collagen -l antibody staining of liver sections fromnude mice injected with fluorescent collagelin probe. Staining with OAPI, Col I, collagelin fluorescence and merged images areshown as indicated in the panels. CV—Central vein, PT—Portai triads.

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 1 99

Co-localization of peptide and anti-collagen1 antibodybinding regions

The fibrotic liver sections taken from mice injected with collagelinwere stained with FITC tagged anti-ColI antibody to check for theco-localization of collagelin (NIR) with the antibody (FITC). Theseresults are shown in Fig. 10, where it can be clearly seen thatcollagelin specific fluorescence was exactly co-localized with theFITC signal from the anti-ColI anti body in the interlobular spacesand the peri-portal tracks. It has been earlier reported that CCl4treated rat liver tissues exhibit extensive accumulation of con-nective tissue that results in the formation of pseudo-lobules dueto continuous interlobular septa [16]. In our study also, collagelinand collagen antibody presence was detected in similar regions.

Discussion

Collagen has been used as a useful biomarker for diagnosticpurposes in earlier studies. Collagen binding domains from thebacterial adhesion protein CNA-35 and integrin α1 labeled withOregon Green 488 were used to visualize collagen in cell cultureand on tissues [17]. CNA-35/OG488 was also used to visualizeatherosclerotic plaques [18]. Affinity of CNA-35 to collagen couldbe improved by combining multiple CNA-35 moieties in a micelle[19]. Muzard et al. reported the use of Technetium (Tc) coupledcollagelin for tissue imaging purposes for detection of myocardialinfarction and lung fibrosis in rat [20] and they have patented thecyclic peptide [21].This peptide is peptidomimetic of the glyco-protein (GPVI), the major platelet collagen receptor. PreviouslyGPVI was also implicated for imaging purposes. The endothelialdamage in the rabbit carotid and femoral arteries were imagedex vivo using a fluorescently labeled GPVI Fc [22]. In another studythe same probe was used to detect atherosclerotic arteries [23]and the radioiodinated GPVI was used to image injured arteriallesions in vivo in ApoE(�/�) mice [24].

We have for the first time successfully used C-TPP coupledcollagelin as a safe and efficient fluorescent probe to detect liverfibrosis in mice. Porphyrins have high extinction coefficients(4-5�105 cm�1 M�1) and hence can be detected from within thetissues in intact animals in a non invasive manner. In addition, theunique chelating property of porphyrin with metals like manganese(Mn) makes them attractive as MRI contrast agents also.Along with collagelin we also used a mutant peptide by

replacing acidic with basic amino acids (E with Q and D withN); polar with non-polar amino acids (R with L and C with L) andbulky side chain to small side chain containing amino acid (P withG) in the collagelin peptide sequence. These changes were madewith the aim to reduce or abolish the binding of collagelin tocollagen. As expected, much weaker, reduced or non specificbinding with the collagen was observed when tissue sectionswere stained with the fluorescent mutant peptide (Fig. 6D),However, the fluorescent collagelin was able to strongly bind tocollagen in fibrotic liver (Fig. 7A and Fig. 8C) which the mutantsequence could not; and interestingly the regions of collagelinbinding overlapped with that of collagen I antibody (Fig. 10). Thestaining of fluorescent collagelin and mutant peptide were carriedout on frozen sections and these results are consistent with thein vivo staining of liver sections with fluorescent collagelin andmutant peptide (Fig. 6 and Fig.10).Generally porphyrins are hydrophobic and dissolve only in non

polar organic solvents. Coupling them with peptides makes themsoluble in water and usable for biomedical and cell biologicalapplications [25,26] On this principle, porphyrin coupled humanimmunodeficiency virus I transcriptional activator (HIV-TAT)peptide was used for its specific localization in the nucleus andcytoplasm of hepatocytes [26].There are many advantages as well as limitations in the non-

invasive fluorescence imaging of live tissues. The advantagesinclude ease of operation, high sensitivity lower cost portableequipment and non-usage of radioactive isotopes, in contrast toX-ray and PET imaging methods. The limitations of this

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 1100

methodology include low penetration of signal and lack ofquantification of the signal intensity [27].In the earlier study related to the fibrosis of the lung and

myocardial infarction Tc-99 m tagged collagelin was used todetect myocardial scars and fibrosis levels[11], however theapplication of this method have been limited due to the generalrisks associated with radioactive isotopes and their low stability.Using NIR fluorescence tagged collagelin in our study we coulddifferentiate the localization of collagen in fibrotic vs. normal liver.The mutant collagelin, which contained replaced amino acids in 6positions in the sequence of collagelin, showed very low level ornon specific binding to fibrotic liver sections suggested that theseamino acids could be involved in the preferential binding ofcollagelin to collagen. Further studies on specific replacements ofamino acids of collagelin, or similar peptides, are required tomake better probes for more specific identification of fibroticcollagen. On the positive side, our study clearly demonstrates thepotential of peptides as targeted molecular probes and utility ofporphyrins in non invasive NIR imaging of live tissues. Fibrosis ifuntreated may lead to the formation of nodules of regeneratinghepatocytes and finally to cirrhosis. Previously fibrosis is consid-ered as irreversible process, but clinical reports in 1970s sug-gested that it can be potentially reversible [28]. Hence any Probethat can detect fibrosis at early stage would be helpful in themanagement of chronic liver disease.

Conclusions

In summary collagelin (a specific collagen binding peptide) wastagged to tetra phenyl porphyrin to develop a fluorescentmolecular probe targeted for diagnostic imaging of early liverfibrosis. This probe demonstrated specific binding to collagenunder in vitro, ex vivo and in vivo conditions. The probe coulddifferentiate fibrotic and normal livers.

Acknowledgments

This work is supported by Department of Science and Technology(DST), Ministry of Science and Technology, Govt. of India andIndian Council of Medical Research (ICMR, New Delhi) Grants no.GAP 0301 & GAP 0383 to GP. Authors greatly acknowledge thecontributions of Mrs. Subbalakshmi and Mrs. Krishna Kumari forHPLC; Mr. Bhikshapathi, Dr. Anoop Rawat and Ms. Sugata forpeptide coupling and Ms. Reeta for porphyrin synthesis and Dr. C.Tripura for collagen antibody staining.

Appendix A. Supporting information

Supplementary data associated with this article can be foundin the online version at http://dx.doi.org/10.1016/j.yexcr.2014.05.005.

r e f e r e n c e s

[1] R. Weissleder, V. Ntziachristos, Shedding light onto live mole-cular targets, Nat. Med. 9 (2003) 123–128.

[2] J.S. Slakter, L.A. Yannuzzi, D.R. Guyer, J.A. Sorenson, D.A. Orlock,Indocyanine-green angiography, Curr. Opin. Ophthalmol. 6(1995) 25–32.

[3] J. Caesar, S. Shaldon, L. Chiandussi, L. Guevara, S. Sherlock, Theuse of indocyanine green in the measurement of hepaticblood flow and as a test of hepatic function, Clin. Sci. 21 (1961)43.

[4] M. Hope-Ross, L.A. Yannuzzi, E.S. Gragoudas, D.R. Guyer, J.S.Slakter, J.A. Sorenson, S. Krupsky, D.A. Orlock, C.A. Puliafito,Adverse reactions due to indocyanine green, Ophthalmology 101(1994) 529–533.

[5] K. Sugisaki, T. Usui, N. Nishiyama, W.D. Jang, Y. Yanagi, S.Yamagami, S. Amano, K. Kataoka, Photodynamic therapy forcorneal neovascularization using polymeric micelles encapsu-lating dendrimer porphyrins, Invest. Ophthalmol. Vis. Sci. 49(2008) 894–899.

[6] R. Kumar, T.Y. Ohulchanskyy, I. Roy, S.K. Gupta, C. Borek,M.E. Thompson, P.N. Prasad, Near-infrared phosphorescentpolymeric nanomicelles: efficient optical probes for tumorimaging and detection, ACS Appl. Mater. Interfaces 1 (2009)1474–1481.

[7] P. Hillemanns, H. Weingandt, R. Baumgartner, J. Diebold,W. Xiang, H. Stepp, Photodetection of cervical intraepithelialneoplasia using 5‐aminolevulinic acid–induced porphyrin fluor-escence, Cancer 88 (2000) 2275–2282.

[8] J.E. Bugaj, S. Achilefu, R.B. Dorshow, R. Rajagopalan, Novelfluorescent contrast agents for optical imaging of in vivo tumorsbased on a receptor-targeted dye-peptide conjugate platform,J. Biomed. Opt. 6 (2001) 122–133.

[9] W.K. Moon, Y. Lin, T. O'Loughlin, Y. Tang, D.-E. Kim, R. Weissleder,C.H. Tung, Enhanced tumor detection using a folate receptor-targeted near-infrared fluorochrome conjugate, BioconjugateChem. 14 (2003) 539–545.

[10] Y. Ye, S. Bloch, B. Xu, S. Achilefu, Design, synthesis, and evaluationof near infrared fluorescent multimeric RGD peptides for tar-geting tumors, J. Med. Chem. 49 (2006) 2268–2275.

[11] J. Muzard, L. Sarda-Mantel, S. Loyau, A. Meulemans, L. Louedec,C. Bantsimba-Malanda, F. Hervatin, J. Marchal-Somme, J.B.Michel, D. Le Guludec, P. Billiald, M. Jandrot-Perrus, Non-invasivemolecular imaging of fibrosis using a collagen-targeted pepti-domimetic of the platelet collagen receptor glycoprotein VI, PloSOne 4 (2009) e5585.

[12] S. Wasser, C.E. Tan, , Experimental Models of Hepatic Fibrosis inthe Rat, 28, Annals of the Academy of Medicine, Singapore109–111.

[13] M.N. Chobert, D. Couchie, A. Fourcot, E.S. Zafrani, Y. Laperche,P. Mavier, A. Brouillet, Liver precursor cells increase hepaticfibrosis induced by chronic carbon tetrachloride intoxication inrats, Lab. Investigation; J. Tech. Methods Pathol. 92 (2012) 135–150.

[14] S.L. Friedman, Liver fibrosis–from bench to bedside, J. Hepatol. 38(2003) 38–53.

[15] H. Popper, S. Udenfriend, Hepatic fibrosis: correlation of bio-chemical and morphologic investigations, Am. J. Med. 49 (1970)707–721.

[16] H. Ebaid, S. Bashandy, I.M. Alhazza, A. Rady, S. El-Shehry, Folicacid and melatonin ameliorate carbon tetrachloride-inducedhepatic injury, oxidative stress and inflammation in rats, Nutr.Metab. 10 (2013) 20.

[17] K.N. Krahn, C.V. Bouten, S. van Tuijl, M.A. van Zandvoort,M. Merkx, Fluorescently labeled collagen binding proteins allowspecific visualization of collagen in tissues and live cell culture,Anal. Biochem. 350 (2006) 177–185.

[18] R.T. Megens, M.G. oude Egbrink, J.P. Cleutjens, M.J. Kuijpers,P.H. Schiffers, M. Merkx, D.W. Slaaf, M.A. van Zandvoort, Imagingcollagen in intact viable healthy and atherosclerotic arteriesusing fluorescently labeled CNA35 and two-photon laser scan-ning microscopy, Mol. Imag. 6 (2007) 247–260.

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 9 1 – 1 0 1 101

[19] M. Breurken, E.H. Lempens, R.P. Temming, B.A. Helms, E. Meijer,M. Merkx, Collagen targeting using multivalent protein-functionalized dendrimers, Bioorg. Med. Chem. 19 (2011) 1062–1071.

[20] J. Muzard, L. Sarda-Mantel, S. Loyau, A. Meulemans, L. Louedec,C. Bantsimba-Malanda, F. Hervatin, J. Marchal-Somme, J.B.Michel, D. Le Guludec, Non-invasive molecular imaging offibrosis using a collagen-targeted peptidomimetic of the plateletcollagen receptor glycoprotein VI, PloS One 4 (2009) e5585.

[21] M. Jandrot-Perrus, J. Muzard, P. Billiald, G.D. Le, L. Sarda, A.Meulemans, Polypeptides, Cyclic Polypeptides and Pharmaceu-tical Comprising Thereof for Non Invasive Specific Imaging ofFibrosis, Google Patents, 2009.

[22] B. Bigalke, S. Lindemann, T. Schönberger, I. Pohlmeyer, A.Chiribiri, A. Schuster, R.M. Botnar, C.M. Griessinger, B.J. Pichler,M. Gawaz, Ex vivo imaging of injured arteries in rabbits usingfluorescence-labelled glycoprotein VI–Fc, Platelets 23 (2012) 1–6.

[23] B. Bigalke, I. Pohlmeyer, T. Schönberger, C.M. Griessinger, M.Ungerer, R.M. Botnar, B.J. Pichler, M. Gawaz, Imaging of injured

and atherosclerotic arteries in mice using fluorescence-labeledglycoprotein VI–Fc, Eur. J. Radiol. 79 (2011) e63–e69.

[24] M. Gawaz, I. Konrad, A.I. Hauser, S. Sauer, L. Zhongyan, H.J.Wester, F.M. Bengel, M. Schwaiger, A. Schomig, S. Massberg, Non-invasive imaging of glycoprotein VI binding to injured arteriallesions, Thromb. Haemost. 93 (2005) 910–913.

[25] S. Flynn, S. George, L. White, W. Devonish, G. Takle, Water-soluble, meso-substituted cationic porphyrins-a family of com-pounds for cellular delivery of oligonucleotides, Biotechniques 26(1999) 736–747.

[26] M. Sibrian-Vazquez, T.J. Jensen, R.P. Hammer, M.G.H. Vicente,Peptide-mediated cell transport of water soluble porphyrinconjugates, J. Med. Chem. 49 (2006) 1364–1372.

[27] V. Ntziachristos, C. Bremer, R. Weissleder, Fluorescence imagingwith near-infrared light: new technological advances that enablein vivo molecular imaging, Eur. Radiol. 13 (2003) 195–208.

[28] E. Albanis, S.L. Friedman, Hepatic fibrosis: pathogenesis andprinciples of therapy, Clin. Liver Dis. 5 (2001) 315–334.