Journal of Photochemistry and Photobiology B: Biology 119 (2013) 52–59

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Journal of Photochemistry and Photobiology B: Biology

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Near-infrared fluorescing IR820-chitosan conjugate for multifunctional cancertheranostic applications

Supriya Srinivasan a, Romila Manchanda a, Alicia Fernandez-Fernandez a,b, Tingjun Lei a,Anthony J. McGoron a,⇑a Florida International University, Biomedical Engineering Department, 10555 W. Flagler St., EC 2613, Miami, FL 33174, USAb Nova Southeastern University, Physical Therapy Department, 3200 S. University Dr., Fort Lauderdale, FL 33328, USA

a r t i c l e i n f o

Article history:Received 28 August 2012Received in revised form 17 December 2012Accepted 18 December 2012Available online 28 December 2012

Keywords:CancerChitosanHyperthermiaImagingIR820Theranostics

1011-1344/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jphotobiol.2012.12.008

⇑ Corresponding author. Tel.: +1 305 348 1352; faxE-mail address: mcgorona@fiu.edu (A.J. McGoron)

a b s t r a c t

This study reports the preparation and characterization of IR820-chitosan conjugates that have potentialmultifunctional imaging-hyperthermia applications in cancer. The conjugates were formulated by cova-lent coupling of chitosan to a carboxyl derivatized IR820, and studied for optical imaging and hyperther-mia applications. IR820-chitosan conjugates were able to generate heat upon exposure to 808 nm laserand produce hyperthermic cell growth inhibition in cancer cell lines MES-SA, SKOV-3 and Dx5. The levelof cell growth inhibition caused by hyperthermia was significantly higher for IR820-chitosan comparedto IR820 in MES-SA and Dx5 cells. Fluorescent microscope images of these cancer cell lines after 3-hexposure to 5 lM IR820-chitosan showed that the conjugates can be used for in vitro near-infrared imag-ing. In an in vivo rat model, the conjugates accumulated in the liver after i.v. injection and were excretedthrough the gastrointestinal tract, demonstrating a different biodistribution when compared to the freedye. The accumulation of these conjugates in bile with subsequent gastrointestinal excretion allows forpotential applications as gastrointestinal contrast agents and delivery vehicles. This formulation canpotentially be used in multifunctional cancer theranostics.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

Among imaging modalities, optical imaging is becoming one ofthe most versatile and informative methodologies, especially whencoupled with contrast agents. Near-infrared (NIR) dyes are impor-tant tools in biomedical and life science applications due to the en-hanced tissue penetration properties of NIR light compared tovisible light. They can be used as diagnostic and therapeutic toolsfor cancer, including imaging, photodynamic therapy and hyper-thermia. The NIR dye indocyanine green (ICG) has been widely ex-plored for clinical applications in cardiac output measurement,imaging, evaluation of liver and kidney function, and photody-namic therapy [1–4]. There are other potential candidates for theseapplications, such as the cyanine dye IR820.

Our group was the first to explore the hyperthermia generationof IR820 and characterize its optical and thermal properties com-pared to ICG [5]. At 5 lM, IR820 is sufficiently fluorescent to beused in cell imaging of healthy and cancer cells, and generates suf-ficient heat upon NIR laser exposure to create a cell growth inhibi-tion and/or cytotoxic effect. IR820 is also superior to ICG in terms

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of stability under different temperature and lighting conditions,with degradation half-times for IR820 which are approximatelydouble those of ICG. Our study also showed that IR820 allows forlonger image collection times for in vivo imaging in rats comparedto ICG. Abdominal fluorescent signals 24 h after tail vein injectionwere significantly stronger for IR820 than ICG. Recently, Prajapatiet al. showed that IR820 can be used as an inexpensive NIR contrastagent for in vivo imaging of injured tissue [6]. Moreover, Masottiet al. reported the formulation of an IR820-PEI (polyethyleneimine)polymer conjugate that can be used as a vector to bind to and de-liver DNA [12]. Meng and coworkers [7] used IR820 as a fluorescentprobe conjugated to MC-PEI to explore its in vivo distribution andbrain targeting ability. Our group has also reported ionic nanoplex-es of IR820 and PEG-diamine that can be used as a theranosticagent for imaging and hyperthermia treatment of cancer cells [8].

Chitosan, a partially acetylated glucosamine biopolymer poly-saccharide, has been extensively investigated for gene and drugdelivery applications due to its hydrophilicity, biodegradabilityand biocompatibility. Some of the advantages of chitosan includeits polycationic characteristics, good solubility at a pH value closeto the physiological range, and the presence of amino groups alongthe chitosan chain that can be used for further functionalizationwith specific components, such as drugs, peptides or other moie-ties [9].

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Recently, Qu et al. showed that Fe3O4–chitosan nanoparticleshad a remarkable heating effect and showed potential in hyper-thermia [10]. Also, chitosan degrades slowly and can be used fordelivery of genes to mucosal tissues in vivo [11]. Therefore, thereis potential for chitosan to act as the vehicle for multifunctionaldrug delivery and molecular imaging.

In the present study, IR820 was conjugated with chitosan to ob-tain a multifunctional vector and explore the imaging and hyper-thermia effects of this novel agent. We performed in vitro studiesto investigate optical properties, thermal generation properties,and toxicity effects of the conjugate on cellular systems. Further-more, the in vivo distribution of the IR820-chitosan was also mon-itored using a non-invasive optical imaging technique.

2. Materials and methodology

2.1. Chemicals and cell culture

Chitosan (85% deacetylated, 100–300 kDa), IR820, methanol,dimethylsulfoxide (DMSO >99.9% reagent grade), and 6-amino-hexanoic acid were purchased from Sigma–Aldrich (St. Louis, MO,USA). Dimethylformamide was purchased from Acros Organics(Fair Lawn, NJ, USA).

Human ovarian carcinoma SKOV-3 cells, human uterine sar-coma MES-SA cells and human uterine sarcoma MES-SA/Dx5(Dx5) cells, McCoy’s 5A medium, and fetal bovine serum (FBS)were purchased from ATCC (Manassas, VA, USA). D-poly coverslips,formalin and 24-well tissue culture plates were purchased fromFisher Scientific (Pittsburgh, PA, USA). Dulbecco’s Phosphate Buf-fered Saline (DPBS) and penicillin were purchased from SigmaAldrich.

2.2. Preparation of IR820-chitosan conjugate

The IR820-chitosan conjugate was formulated as illustrated inFig. 1 using a modification of the procedure reported by Masottiet al. [12]. The first step, preparation of IR-linker, involved substi-tution of the meso-chlorine group of IR820 with a linker 6-amino

Fig. 1. Scheme for synthesis of IR

hexanoic acid, to form a carboxyl-derivatized dye (IR-linker, Solu-tion 1). IR820 dye (30 mg, 0.283 mmol) was reacted with 6-amino-hexanoic acid (12.6 mg, 1.42 mmol) and triethylamine (19.8 ll,1.42 mmol) in 1 ml anhydrous DMF with magnetic stirring. Nitro-gen gas was purged into the solution for 20 min. The resultinggreen solution was stirred at 400 rpm for 3 h in a corked flask sub-merged in an oil bath to maintain a constant temperature of 85 �Cduring the procedure. The solution turned blue on completion ofthe reaction. The final solution was freeze-dried and lyophilized.The yield in this step was 60%. The IR-linker provided suitablefunctional groups as well as paved the way for less hindered sub-stitution of polymers. In the second step, the IR820-chitosan conju-gate was prepared by activating the carboxyl-derivatized dye (IR-linker) with a zero-length crosslinker N-(3-dimethylaminopro-pyl)-N-ethylcarbodiimide hydrochloride (EDC), in order to allowconjugation with chitosan’s amine functional groups. Briefly, anequimolar solution of EDC and IR-linker was allowed to react in9 ml anhydrous DMF. This step helped functionalize the IR-linkerto form an ester intermediate. Next, 2.2 ml of chitosan (preparedas 1% chitosan in 4% acetic acid) was added to the solution drop-wise. The reaction was allowed to proceed for one day. The esterintermediate of IR-linker reacted with the amine groups of chito-san to form an amide bond between the two.

The yield of the final product in this step was 55%. Purificationwas performed after step 2 (preparation of IR820-chitosan conju-gate) by dialysis, using a 100–300 kDa molecular weight cut-offmembrane against a mixture of 70%:30% methanol: water, and fi-nally with 100% water for 3 days before being recovered byfreeze-drying.

2.3. Chemical and optical characterization

2.3.1. Fourier Transform Infrared SpectroscopySample infrared spectra (IR-Linker, chitosan and IR820-Chito-

san) were obtained using a Perkin Elmer Spectrum 1000 spectrom-eter scanning from 4000 to 600 cm�1. Spectra were collected witha 4 cm�1 resolution and 4 scans were obtained and averaged foreach spectrum.

-linker and IR-820 chitosan.

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2.3.2. Dye content determinationThe IR820 content in the conjugate was determined by dissolv-

ing known amounts of the sample in deionized water and perform-ing serial-dilution absorbance measurements in the linear rangeusing a Cary UV spectrophotometer (Varian/Agilent Technologies,Switzerland). The dye content in the sample was determined froma previously obtained calibration curve of free IR820 in water.

2.3.3. Characterization of absorption propertiesStock samples of IR820, IR-linker and IR820-chitosan were pre-

pared in methanol at 1 mg/ml concentration of IR820 dye. Thesamples were diluted to the linear range. Sample absorption spec-trum was measured from 400–900 nm in 1-nm intervals with aCary UV spectrophotometer (Varian/Agilent Technologies, Switzer-land) and using methanol for blank subtraction.

2.3.4. Characterization of fluorescence propertiesSamples of IR820 and IR820-chitosan conjugate at 1 mg/ml dye

content were prepared as stock solutions in deionized water andserially diluted to the linear range. Sample emission spectra wererecorded from 690 nm to 850 nm after excitation at 688 nm usinga Fluorolog-3 spectrofluorometer (Jobin Yvon Horiba, Edison, NJ,USA). The fluorescent intensities were blank subtracted and plot-ted against their respective emission wavelengths.

2.4. Cellular imaging

Cells were grown in monolayers on poly-D-Lysine coated coverslips in McCoy’s 5A medium supplemented with 10% FBS and 1%penicillin in an atmosphere of 95% air and 5% CO2 at 37 �C. MES-SA, Dx5 and SKOV-3 cells were seeded at a density of 1 � 105 -cells/well for MES-SA and Dx5, and 5 � 104 cells/well for SKOV-3.After overnight attachment, the cell medium was replaced withmedium containing 5 lM IR820 or IR820-Chitosan with 5 lMdye content. After 3 h of incubation at 37 �C in the dark, wells werewashed 3� with Dulbecco’s Phosphate Buffered Saline (DPBS). Thecells were then fixed with 4% formaldehyde for 15 min at 37 �C.This was followed by 3�washing step with DPBS and then mount-ing of the coverslips on glass slides with antifade reagent/mount-ing medium mixture applied. The fixed cells were observedunder a fluorescence microscope (Olympus IX81, Japan) with a20� objective (for MES-SA and Dx5) and a 60� water mergedobjective (for SKOV-3), using kex = 785 nm and kem = 845 nm atan exposure time of 8000 ms (for MES-SA and Dx5) and 4000 ms(for SKOV-3). The fluorescence images were collected with a CCDcamera and pseudo-colored using IPLAB software (Olympus IX81,Japan). The settings of the microscope were kept constant.

2.5. Cytotoxicity assessment

The cytotoxicity of two different treatments (free IR820 versusIR820-chitosan) was investigated in three cancer cell lines (MES-SA, Dx5 and SKOV-3) using a Sulforhodamine B (SRB) colorimetricassay. Cells were seeded in 96-well plates at a density of20,000 cells/well and incubated overnight. After complete cellattachment, the wells were incubated with free IR820 or IR820-Chitosan for 24 h. The concentrations of IR820 tested ranged from0 to 5 lM (the wells labeled 0 lM were treated with DPBS). InIR820-chitosan wells, the amount of conjugate added was deter-mined from the dye content estimation to reach IR820 well con-centrations between 0 and 5 lM. After 24-h exposure, the SRBassay was performed to determine net cell growth.

In a similar experiment, the effect of hyperthermia was testedby seeding the cells, allowing for overnight attachment, and thenincubating with 5 lM IR820 or IR820-chitosan (normalized to5 lM IR820) for 1 h. After the incubation period, the cells were ex-

posed to an 808-nm laser for three minutes at a fluence rate of8.8 W/cm2. The laser system used for hyperthermia experimentshas been described in detail in our previous publications [13].The cells were then returned to the incubator and the SRB assaywas performed 24 h after the addition of IR820 or IR820 conjugateto the wells. Laser-only and dye-only groups were also included inthe experiment to evaluate the effect of the combination of laserand dye versus the two individual factors.

In all cytotoxicity experiments, four wells were used for eachtreatment in each cell line and were averaged to obtain the cellgrowth fraction. The cell growth fraction data for each treatmentwas normalized to control (cell growth fraction of cells with notreatment) and is represented as mean ± S.D. (n = 3). One-way AN-OVA was used to measure the statistical difference between treat-ment groups and between treatment and control groups.Significance was set at p < 0.05.

2.6. In vivo imaging studies

Fisher-344 rats (160–195 g) were purchased from Harlan (Indi-anapolis, IN), kept under standard housing conditions and fed adlibitum. All protocols followed the regulations of the InstitutionalAnimal Care and Use Committee. An imaging system consistingof a Sanyo DL 7140-201S laser (80 mW, 785 nm, 0.1–0.5 mWpower at the imaging plane) and a Retiga 1300 CCD camera wasused. IR820-chitosan conjugate samples (between 5.5 and5.7 mg) were dissolved in acetate buffer and injected in volumesof 0.25 ml to obtain animal dye doses of 0.13–0.14 mg/kg. On theday of the experiment, the animals were injected i.p. with a40 mg/kg dose of pentobarbital anesthesia. An image of the ratwas obtained under white light illumination to determine the po-sition of the target and to focus the camera before dye injection. Animage with focused laser illumination was also taken prior to dyeinjection to establish the background. Laser current was set to60 mA. The conjugate solution was injected through the tail vein.Camera recording was started immediately before the injection,and 10-s exposure images of the abdomen were recorded seriallyfor 45 min using QCapture Pro software. Twenty-four hours later,the rat was anesthetized and imaged again under the same operat-ing conditions. The protocol was completed in three rats. Organswere also carefully dissected and extracted after the 24-h imagingprotocol was completed. Organs were then placed in black-coatedPetri dishes and imaged using the same imaging setup. For organsthat had a fluorescent signal, images were processed in Matlab toselect a background-subtracted region of interest and calculatethe normalized intensity, R, as the ratio of total pixel intensity (pix-el index) in the region of interest to the area of the signal in lm2.To create the final figures, dye images were superimposed withtheir corresponding white image using Matlab.

3. Results and discussion

In the present study, we have conjugated IR820, a near-infrareddye, with chitosan to create a multifunctional probe for in vitro andin vivo imaging combined with therapeutic hyperthermia in can-cer. The covalent conjugation of IR820 to chitosan was performedin two steps as described in the experimental section and illus-trated in Fig. 1. Briefly, IR820 was first chemically modified tointroduce carboxyl groups and further conjugated to chitosan(polymer containing amine groups) by forming an amide bond be-tween IR820 and chitosan using carbodiimide chemistry. The con-jugate was characterized through FTIR spectra, absorption spectraand fluorescence spectra. The resulting conjugates were assessedfor their imaging, toxicity and hyperthermia-inducing capabilities.

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3.1. FTIR spectroscopy

IR820-chitosan samples were analyzed for the presence of anamide bond through FTIR. The IR-linker was found to possess acharacteristic absorption band at 1766 cm�1 (mc@o stretch) corre-sponding to the carbonyl stretch for carboxyl groups, and at3450 cm�1 (OH hydroxyl group) proving the presence of carboxylgroups in the compound. Chitosan (85% deacetylated) has charac-teristic absorption bands at 1649 cm�1 (mc@o stretch), 1584 cm�1

(ANH angular deformation), 3450 cm�1 (OH hydroxyl group), and1150–1040 cm�1 (CAOAC in glycosidic linkage. As a result of theconjugation, the absorbance of NH bands (1580–1490 cm�1) andCO deformation bands (1700–1630 cm�1) became gradually higherwith the increase of amide bonds in the compound. Observablepeak shifts with respect to pure chitosan (NH from 1584 to1542 cm�1, and C@O from 1650 to 1634 cm�1) further provedthe successful conjugation of IR-linker to chitosan.

3.2. Dye content determination

The dye content in IR820-chitosan conjugate was determinedfrom a calibration curve of IR820 in water. The dye content (mea-sured from A.U.688nm) was estimated to be 48 lg dye (5.65E-2 lmol dye) per mg conjugate. Percent substitution was calculatedto be approximately 0.96%. Based on an average MW of 200,000 Dafor chitosan polymer, and monomer MW 161 Da, then 1242 sitesare available for dye binding. Of those, approximately 12 wereoccupied by dye based on absorption dye content measurementsfor the final product, giving a substitution of 0.96%. This level ofsubstitution does not create significant modification of the poly-mer, ensuring that the physiological behavior of the conjugatewould remain very similar to that of chitosan. However, at thesame time, it provides us with enough content of IR820 for usein imaging applications.

3.3. Characterization of absorption properties

Representative UV–visible spectra of IR820, IR-Linker andIR820-chitosan are shown in Fig. 2. The IR-linker produced in thereaction scheme showed a characteristic peak located at 652 nmin methanol, whereas the free dye showed a peak at 820 nm inmethanol. A red shift (kmax = 669 nm) was observed in the absorp-tion spectra of the IR820-chitosan conjugate, which can be attrib-uted to an increase in conjugation length and formation ofextended p system. This red shift thus proves the successful conju-

Fig. 2. Absorption spectra of IR820 (dashed black), IR-linker (dotted gray) andIR820-chitosan (solid black) in methanol, in solutions prepared at 9.04 lM dyeconcentration. The peak absorption for IR820-chitosan occurs at 653 nm.

gation of IR820 to chitosan. This is consistent with the absorptionspectra reported by Masotti et al. for an IR820-PEI conjugate [12].

3.4. Characterization of fluorescence properties

A representative fluorescence emission spectrum of IR820-chitosan after 688-nm excitation is shown in Fig. 3. The Stokes shiftof IR820-chitosan is large, given that the peak absorption of IR820-chitosan occurs at 653 nm and the peak emission is located at745 nm. This indicates a good potential for the conjugate to beused as a fluorescent probe in the near-infrared region.

3.5. Cellular Imaging

Fig. 4 shows fluorescence microscopy images obtained fromIR820 and IR820-chitosan conjugate in SKOV-3 cell lines after 3 hincubation at 5 lM concentration of IR820. Fig. 5 shows the corre-sponding images for MES-SA and Dx5. The fluorescence observedfrom IR820 or IR820-chitosan after 3 h incubation with the threecell lines demonstrates the capability of the conjugate to be usedas an in vitro fluorescent probe. The uptake of this conjugate couldbe enhanced due to charge interactions between the positively-charged chitosan polymer and negatively-charged tumor cell sur-faces [14,15]. Such charge-targeting of polymers to tumor cell lineshas been widely investigated [16].

3.6. Cytotoxicity assessment

Drug resistance is an important factor governing the response ofcancer cells to different treatment modalities. Thus, MES-SA, Dx5and SKOV3 cells were chosen to investigate the cytotoxic potentialof IR820 and IR820-chitosan conjugate before and after laser expo-sure. MES-SA is a drug sensitive cell line, and Dx5 and SKOV-3 aredrug-resistant cell lines. As shown in Fig. 6, no significant differ-ence in net cell growth was observed between IR820 and IR820-chitosan for equivalent dye concentrations in any of the three celllines. MES-SA cells were found to exhibit up to 20% growth inhibi-tion compared to the control group (p < 0.05) when exposed to5 lM IR820 or IR820-chitosan with equivalent dye concentration.This drug-sensitive cell line seems to possess inherent sensitivityto environmental factors in comparison to the other two cell lines,which is consistent with our previous studies of IR820 and IR820-PEG-diamine conjugates [8]. Dx5 also showed reduction in cellgrowth to 5 lM IR820-chitosan, but the difference with controlsdid not reach statistical significance (p = 0.06). The enhanced toxic-ity of this conjugate could be attributed to the protonation of chito-

Fig. 3. Fluorescence spectrum of IR820-chitosan after 688-nm excitation in watersolution, 0.88 lM dye concentration.

Fig. 4. Fluorescent imaging of SKOV-3 after 3-h incubation with 5 lM dye content of IR820 (left) or IR820-chitosan (right); 60�, exposure time 4000 ms. kex = 775 nm andkem = 845 nm.

Fig. 5. Fluorescent imaging of MES-SA and Dx5 cells after 3-h incubation with 5 lM dye content of IR820-chitosan or IR820, 20�, exposure time 8000 ms. (A) MES-SA, IR820;(B) MES-SA, IR820-chitosan; (C) Dx5, IR820; (D) Dx5, IR820-chitosan. kex = 775 nm and kem = 845 nm.

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san residues, which increase destabilizing interactions betweenthis polycationic polymer and tumor cell membranes [17,18].

3.7. Hyperthermia studies

Hyperthermia-induced increases in temperature to 43 �C canselectively kill cancer cells and hence are widely used as an adju-vant therapeutic modality to chemotherapy and radiotherapy[19,20]. In our previous publication, we reported that 5 lM IR820resulted in temperature increases of 5 �C in vitro when exposedto an 808-nm laser for 3 min at a fluence rate of 8 W/cm2, and thisconcentration of IR820 induced cell growth inhibition upon lasertreatment in SKOV-3, Dx5, and MES-SA cells [5]. The current studyinvestigated the potential of an IR820-chitosan conjugate to inducehyperthermia-based cell killing in comparison to the free dye.IR820-chitosan (at equivalent concentration of 5 lM dye) was ableto increase the temperature of the wells from 37 �C to 42.5 �C after3 min of laser exposure, which is in the desired range for localizedhyperthermia to selectively kill tumor cells [21].

Cell proliferation of MES-SA, Dx5 and SKOV-3 after laser irradi-ation was investigated to test the response of these cells with dif-ferent drug sensitivity towards hyperthermia treatment. Laser

treatment without IR820 or IR820-chitosan (laser-only group)did not result in any changes in cell growth with respect to the truecontrol group that was not exposed to any treatment (neither lasernor dye). Fig. 7 shows cell net growth data obtained in the hyper-thermia experiments for the dye-only and dye plus laser groups.After laser exposure, both IR820 and IR820-chitosan significantlyreduced (p < 0.05) the net cell growth of all three cell lines com-pared to the corresponding dye-only group that had been exposedto only IR820 or IR820-chitosan but not laser. Thus, combinedexposure to laser plus dye resulted in significant reduction in netcell growth compared to exposure to dye without laser treatment.This toxicity profile suggests that the toxicity mediated by the dyeor the conjugate occurs only after exposure to laser and is notinherently due to the dye or the conjugate.

Additionally, when the two laser-plus-dye groups are comparedto each other, IR820-chitosan exposed to laser significantly inhib-ited the growth of drug-sensitive MES-SA and multidrug-resistantDX5 (p < 0.05) compared to IR820 after laser exposure, thus sug-gesting an enhancement in cell thermosensitivity. Thermal resis-tance or tolerance in cancer cells is often associated withexpression of heat shock proteins, which stabilize and refold cellu-lar proteins on exposure to heat [22]. Some studies report that

Fig. 6. Net cell growth after 24-h exposure to IR820-chitosan or IR820, n = 3experiments, four wells per treatment. � = significant difference (p < 0.05) versuscontrol (0 lM) group.

Fig. 7. Cytotoxicity of IR820-chitosan compared to free IR820 with and withoutlaser exposure, n = 3 experiments, four wells/experiment. � = significant difference(p < 0.05) versus non-laser group, �� = significant difference (p < 0.05) between lasergroups.

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nondrug-resistant tumor cells and tumor cells with acquired drugresistance do not differ in their thermal sensitivity [23–25]. Our re-port of similar enhanced thermal response to IR820-chitosan ofboth drug sensitive MES-SA and MDR+Dx5 is thus consistent withreports in the literature, indicating that mechanisms concerningheat response of tumor cells are independent of multi-drug resis-tance mechanisms like elevated P-gp activity. Other studies, how-ever, suggest that drug-resistant cell lines are more temperaturesensitive than their drug sensitive counterparts due to elevatedexpression of multi-drug resistance proteins such as P-glycopro-tein (or) P-gp [26,27]. Therefore, further research on the relation-ship between acquired drug resistance and thermosensitivity isstill needed.

After laser exposure, IR820-chitosan did not significantly inhibitthe growth of SKOV-3 cells in comparison to IR820 exposed to la-ser. These results are consistent with similar observations reportedby van de Broek et al. [28], who found that treating SKOV-3 cellswith branched gold nanoparticles exposed to laser (38 W/cm2,5 min, 11 �C temperature increase) was not sufficient to induce celldeath, even though gold nanoparticles have a higher thermal crosssection in comparison to organic dyes [29]. Thus, from the abovediscussion, it can be concluded that SKOV-3 has inherent thermo-tolerance, which may be related to the p53 mutation that confersdrug resistance to these cells. Cytotoxicity mediated by hyperther-mia can be influenced by both p53 dependent and independentpathways [30–32]. Since in our study SKOV-3 failed to show a sig-nificant increase in thermal response whereas Dx5 and MES-SA didshow enhanced cell growth inhibition, it seems that the particularmechanism involved in IR820 and IR820-chitosan hyperthermiamay be p53-dependent. However, based on available literature,resistance or sensitivity to thermal exposure seems to stem froma multitude of biological factors and chromosomal variations, sothat clear correlations to a specific root cause cannot be made.

3.8. In vivo imaging

Our group has previously reported the use of IR820 for in vivoimaging in a rat model [5]. In this study, we performed in vivoimaging experiments with an IR820-chitosan conjugate. Duringthe image collection period, the IR820-chitosan conjugate accumu-lated primarily in the liver starting within 5–10 min after injection.Subsequently, the signal was observed traveling through the intes-tines, indicating that the pathway of elimination is primarilythrough the digestive tract. Fig. 8 shows the IR820-chitosan conju-gate signal coming from the abdomen 45 min after injection (A–C)and 24 h after injection (D–F) for a 60 mA laser module current inthe three rats. In the images obtained 45 min after injection, theaverage R value ± standard deviation was 4.65 ± 0.15, indicatinggood reproducibility between subjects. In abdominal images takenafter 24 h, the signal had decreased significantly, with a ratio valuethat is between 10% and 20% of the value on the day of injection.Average R value ± standard deviation was 0.68 ± 0.19. Thus, theaverage signal at 24 h was 14.6% of the average value 45 min afterinjection. Imaging of the excised organs after 24 h showed a fluo-rescent signal in the intestines and/or the feces, confirming thepresence of intact dye in the gastrointestinal tract and fecal elimi-nation (Fig. 9). The proportion of signal present in feces versusintestines varied for each animal, which can be explained by vari-ability in intestinal motility and elimination patterns.

Our results demonstrate the feasibility of using the IR820-chito-san conjugate as an imaging agent. The biodistribution of the con-jugate is different from free IR820, which shows a strong liversignal even 24 h after injection and also localizes to lungs and kid-neys [5]. The IR820-chitosan conjugate accumulates only in the li-ver and is excreted in bile, subsequently traveling through thegastrointestinal tract and being retained in high amounts after24 h. The retention of fluorescence by the conjugates after biliaryexcretion indicates their potential application as gastrointestinal

Fig. 8. Abdominal images of rats after i.v. injection with IR820-chitosan. (A–C) were taken 45 min after injection, and (D–F) 24 h after injection. Image ratios R indicate signalintensity per unit area.

Fig. 9. Intestinal and fecal images 24 h after i.v. injection with IR820-chitosan.

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contrast agents in cases where oral administration is not medicallyappropriate. The conjugate has an advantage over the free dye inthis application because it is not taken up by other organs suchas the kidneys or the lungs, and it provides a wide window for col-lection of images of the gastrointestinal tract. Near-infrared opticalimaging of the lower gastrointestinal tract in humans can beaccomplished using techniques such as microendoscopy [33], andfurther advances in optical imaging instrumentation may expandits applications. Additionally, biliary excretion provides an oppor-tunity for absorption across the gastrointestinal barrier as the con-jugate travels through the intestines. Literature shows thatchitosan can potentially enhance transmucosal drug delivery byincreasing epithelial permeability [14]. Possible intestinal applica-tions of chitosan, and thus of our conjugate, could include genedelivery, mucosal vaccination, absorption enhancer for drugs withpoor GI bioavailability, and colonic delivery of therapeutic agentsfor colorectal cancer or ulcerative colitis [34]. The mucoadhesiveproperties of chitosan can result in local increases in drug concen-tration and enhance diffusion-mediated uptake into cells [35]. Theconjugate also provides an advantage over the free dye in thera-nostic applications based on the multifunctionality imparted bythe presence of chitosan, which opens the possibility of combiningthe inherent optical and heat generation abilities of IR820 with thecapability of delivering therapeutic agents such as drugs or genes.Delivery of these therapies would be co-localized with the hyper-thermia agent, which in the conjugate form also demonstrates an

enhanced ability to inhibit growth in some cancer cell lines uponlaser exposure, making it preferable to the free dye. A managementtriad of imaging, genetic or chemical therapy, and hyperthermiawith enhanced cell growth inhibition can thus be accomplishedby the use of IR820-chitosan. The localization and biodistributionof an IR820-chitosan delivery vehicle for drugs or genes could bemonitored and confirmed using non-invasive optical imaging,and could be then coupled with hyperthermia interventions, pro-viding a multifunctional theranostic capability to these agents.

4. Conclusion

In this study, we successfully conjugated IR820 to chitosan andtested its cytotoxicity and cellular uptake in cancer cell lines MES-SA, Dx5 and SKOV-3. We were able to demonstrate that IR820 isstill able to retain its imaging and hyperthermia-generating capa-bilities after conjugation to chitosan. Thus, these conjugates canbe used as multifunctional agents for localized image-guidedhyperthermia. The IR820-chitosan conjugate was eliminated viathe hepatobiliary system, and detected in the feces, different fromfree IR820. Because of their in vivo distribution properties and pref-erential elimination through the gastrointestinal tract, furtherdevelopment is possible to create multimodal drug or gene deliv-ery systems. Future studies will focus on developing nanoformula-tions of these conjugates using DNA for gene therapy.

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Acknowledgements

The authors gratefully acknowledge the help rendered by Den-ny Carvajal (MS, Biomedical Engineering, FIU) for phlebotomy pro-cedures during in vivo studies, and by Erika Esselan (Chemistrydepartment, FIU) for FTIR studies. This work was conducted usingthe facilities of the Biomedical Engineering Department at FloridaInternational University. This work was partly funded by the FIUBME Coulter Young Inventor Award to R.M.

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