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Ultrasmall Chitosan-genipin Nanocarriers Fabricated from Reverse Microemulsion Process for Tumor Photothermal Therapy in Mice Xiaojie Song, Hao Wu, Shen Li, Yanfang wang, Xiaojun Ma, and Mingqian Tan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00511 • Publication Date (Web): 15 Jun 2015 Downloaded from http://pubs.acs.org on June 21, 2015

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Ultrasmall Chitosan-genipin Nanocarriers Fabricated from Reverse Microemulsion Process for Tumor Photothermal Therapy in Mice

Xiaojie Song a,b, Hao Wu a,b, Shen Li a,b,Yanfang Wang a,b, Xiaojun Ma a,*, and Mingqian Tan a,*

a

Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences,

457 Zhongshan Road, Dalian 116023, China; Fax: +86-411-84379139 b

University of the Chinese Academy of Sciences, Beijing 100049, China

*To whom correspondence should be addressed. E-mail: [email protected]; [email protected]; Tel: 86-411-84379139; Fax: 86-411-84379139 Running headline: Ultrasmall Nanocarriers for Tumor Photothermal Therapy

Table of contents (TOC)

ACS Paragon Plus Environment

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Abstract: Nanocarriers play an important role in improving the photo- and thermal-stability of photosensitizers to gain better pharmacokinetics behavior in tumor photothermal therapy. Herein, PEGylated chitosan (CG-PEG) nanoparticles with ultrasmall size (~ 5 nm) were prepared through a W/O reverse microemulsion method using genipin as a crosslinker. Particle size and zeta-potential can be tuned by varying the molar ratio between chitosan amino groups and genipin. CG-PEG-ICG nanoparticles were fabricated by adding ICG to CG-PEG aqueous solution through a self-assembly method via electrostatic interaction. The resulted CG-PEG-ICG nanoparticles exhibited improved photo- and thermal-stability, good biocompatibility and low toxicity. When irradiated with a laser, the cells incubated with CG-PEG-ICG nanoparticles showed very low cell viability (15%), indicating the CG-PEG-ICG nanoparticles possess high in vitro photothermal toxicity. Moreover, the CG-PEG nanocarriers can significantly alter the biodistribution and prolong the retention time of ICG in the mice body after intravenous injection. In vivo photothermal study of tumors injected with CG-PEG-ICG nanoparticles containing ICG concentration more than 100 μg·mL-1 (100 μL) induced irreversible tissue damage. The growth of U87 tumors was dramatically inhibited by CG-PEG-ICG nanoparticles, demonstrating that the CG-PEG nanoparticles may act as potential ICG nanocarriers for effective in vivo tumor photothermal therapy.

Keywords: Nanocarriers, indocyanine green, photothermal therapy, chitosan, genipin


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1. Introduction Photothermal therapy (PTT), an effective noninvasive treatment for many diseases, has been extensively investigated in cancer treatment due to its unique minimal invasion, relatively simple execution, and particular direct heating in tumor1, 2. Photosensitizers play an important role in PTT treatment that strongly absorb near infrared (NIR) light and convert luminous energy to thermal energy, producing cytotoxic heat for tumor treatment. One particular organic NIR dye, indocyanine green (ICG), has been approved by the United States Federal Drug Administration (FDA) for NIR clinical imaging agents due to its unique photochemical, photobiological, and pharmacokinetics properties3. ICG absorbs around 780 nm and emits around 800 nm that make it highly suitable for bio-imaging applications with high signal to-background ratio. Moreover, it can convert the absorbed light energy to produce heat for PTT treatment4-7. However, the use of ICG for imaging and PTT is limited by its several drawbacks: 1) ICG is photo-degraded in aqueous solution which followed first-order kinetics, and the degradation was accelerated by light exposure. 2) ICG is markedly thermal-degraded in higher temperatures of aqueous solutions. 3) ICG undergoes degradation in aqueous media resulting in a simultaneous loss of the ability of absorption and fluorescence. 4) It is easy to bind nonspecifically to human serum albumin which results in its rapid clearance by the liver with a short circulation time 2-4 min8. All these drawbacks restrict its utility in effective and prolonged PTT applications. To overcome these limitations, great effort has been made recently in improving ICG photoand thermal-stability, pharmacokinetics and biodistribution in tumor tissue through versatile nanocarriers, considering multiple factors such as shape, size, surface charge, water solubility and cytotoxicity9. For example, nanocarriers such as perfluorocarbon nanoemulsions (~164.2 nm) 10, phospholipid-polyethylene glycol (PL-PEG) nanostructures5, hybrid polypeptide micelles11, poly


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(lactic-co-glycolic acid) (PLGA) nanoparticles (>300 nm) 8, folate-amphiphilic chitosan micelles (~136 nm)

12

, silica/calcium phosphate nanoparticles13,

14

, have been developed as delivery

vehicles for ICG in tumor imaging and photothermal effect study. These nanocarriers exhibited relatively good biocompatibility and low toxicity, improved stability and prolonged plasma half-life. In comparison with various nanocarriers mentioned above, the polymer-based with ultrasmall size (< 10 nm) have been rarely reported, probably due to the difficulty in controlling the polymerization degree of the polymers to give a uniform size distribution. Recently, we have developed a chitosan-based ICG-containing nanostructure for effective molecular tumor imaging 15

. This has motivated us to search for a new strategy for the controllable synthesis of

chitosan-based nanocarriers for effective PTT study in mice models. Chitosan is a partially deacetylated glycosaminoglycan derived from chitin, possessing great potential in a wide range of areas such as food, cosmetics, wastewater treating, biotechnology and medicine because of its good biodegradability, nontoxicity and biocompatibility16-19. It is an ideal candidate for the preparation of nanocomposites with good biocompatibility for drug delivery 20

.Chitosan-based nanocarriers have been prepared by a number of methods, such as ionotropic

gelation, polyelectrolyte complex, emulsification-cross linking, complex coacervation, solvent evaporation and co-precipitation methods21. Alternatively, reverse microemulsion method can provide water-in-oil (W/O) “nano-reactor” for controlled particle growth, which offers the advantages in giving uniform sub-100 nm nanocarriers with a narrow size distribution. Chitosan nanoparticles are usually cross-linked by the reagents such as gluteraldehyde, diisocyanate, diepoxy compounds and dialdehyde to acquire quite stable matrixes

22, 23

. However, these toxic

reagents have attracted attention because they are more or less cytotoxic and may impair the biocompatibility of a chitosan delivery system

24

. Recently, genipin has been used as


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cross-linking reagent for the preparation of chitosan nanoparticles through the reverse microemulsion method25. The W/O droplets not only act as nano-reactors for the formation of chitosan nanoparticles but also prevent the forming particles from aggregation

26

. The products

cross-linked by genipin were previously reported 10,000 times less cytotoxic than glutaraldehyde25. Therefore, the chitosan nanoparticles prepared using genipin as the cross-linking reagent may act as good nanocarriers for ICG delivery in tumor photothermal therapy. In this study, we reported a method for the preparation of polyethylene glycol (PEG) modified chitosan nanoparticles cross-linked by genipin through a W/O reverse microemulsion strategy as ICG nanocarriers (CG-PEG-ICG) for tumor photothermal therapy in mice. Scheme 1 shows the ultra-small chitosan-PEG nanoparticles prepared via a modified reverse microemulsion method by the PEG modification on chitosan-genipin precursor. PEG modification were used to improve the particle stability and prolong blood circulation time27. The physicochemical properties

were

characterized

by

Fourier

transform

infrared

spectroscopy

(FTIR),

ultraviolet-visible (UV-Vis) spectroscopy, dynamic laser scattering (DLS), transmission electron microscopy (TEM), and fluorescent measurements. The energy conversion ability from luminous light to heat was investigated by using free ICG as a control. Cytotoxicity, PTT of CG-PEG-ICG was evaluated using glioma U87 cells and tumor-bearing mice model. The results demonstrated that the CG-PEG-ICG nanostructures were water-soluble ( ≥ 50 g/L), highly stable against the light quenching and effective in producing heat for tumor treatment in photothermal therapy. All of above indicated PEG decorated chitosan-genipin nanoparticles may be used as nanocarriers in more drug delivery applications.


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OH

NH2 HO O

Chi

n-Octanol Triton X-100

O

SCM-PEG

N

OH

chitosan

O

①

Micro 2

O

OH O

O HO

O

OCH 3 HO O

O

NH 2

OH

OH

NH2

O

O HO

②

OH

SCM-PEG

N

OH O OS O

O

N

NH

HO O O

N

OH

OH OH

genipin

Micro 1

O

O HO

HO O OONa S O

NH

O

OH O

O HO

NH OH

ICG

n O

CG-PEG

O

O

CG-PEG-ICG

Scheme 1. Schematic illustration of ICG loaded chitosan-genipin nanocarriers synthesized from a W/O reverse microemulsion method.

2. Materials and methods 2.1 Materials

Indocyanine green (ICG) was purchased from Yichuang Co. Ltd. (Dandong, China). Chitosan (CS) (Mw=5000 Da, DD=90%) was purchased from Golden-shell pharmaceutical Co. Ltd(Zhejiang, China). Genipin (HPLC, 98%) was purchased from Linchuanzhixin Co. Ltd (Fuchuan, China). SCM-PEG (2090 Da, ≥98%) was purchased from Shanghai Yare Bitech, inc (Shanghai, China). N, N-diisopropylethylamine (DIPEA) was purchased from Aladdin Industrial Co. Ltd. (Shanghai, China). Triton X-100 and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma Aldrich (USA). Cyclohexane was purchased from Fuchen chemical reagent Co. Ltd (Tianjin, China). Acetone, ethanol and i-octanol were supplied by Sinopharm


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Chemical Reagent Co. Ltd. and Damao chemical reagent Co. Ltd. (Tianjin, China). Glioma U87 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA). BD matrigel was purchased from Shanghai QCBIO Science & Technologies Co. Ltd. (Shanghai, China). All reagents for synthesis were analytical grade.

2.2 Instruments Transmission electron microscopy (TEM) images were collected on a JEM-2000 electron microscope operating at 200 kV. Nano ZS90 Zetasizer (Malvern Instruments, Malvern, UK) was used to determine the Zeta potential. The fluorescence and UV-vis absorption spectra were measured on Perkin Elmer LS 55 and Shimadzu UV-2550 spectrometers, respectively. The FTIR spectra were measured on a VECTOR 22 spectrometer using the KBr pellet method. In vivo imaging was carried out with a CRi Meastro Ex in vivo imaging system (Caliper Life Sciences Inc. USA). Temperatures and infrared thermographic maps were recorded with a FLIR-i3 infrared thermal imaging camera. Cells and tumors for photothermal therapy were irradiated under 808 nm laser (Shanxi Alaxy Technologies Photonics Co., Ltd). The morphology of cells was observed and recorded by phase contrast microscopy (Nikon Co., Japan). The cell viability after laser was measured at 570 and 630 nm on an automated microtiter plate reader (Wellscan MK3, Labsystems Dragon, Finland). 2.3 Preparation of CG-PEG and CG-PEG-ICG nanoparticles CG-PEG nanoparticles were fabricated in a W/O reverse microemulsion. Briefly, cyclohexane (18.15 mL), i-octanol (4.41 mL), Triton X-100 (4.74 g) were mixed and stirred for 30 min to form transparent microemulsion. Chitosan (33 mg) in 1.1 mL of deionized water was added to the microemulsion and the mixture was treated with ultrasound to form homogeneous


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solution. The polymerization reaction was initiated by adding 200 μL of genipin in ethyl alcohol and allowed to continue for 12 h at room temperature. The SCM-PEG (50 mg) in 1.1 mL deionized water was added to the microemulsion containing 18.15 mL of cyclohexane, 4.41 mL of i-octanol and 4.74 g Triton X-100. The resulted mixture was dropped into above-mentioned chitosan microemulsion for another 12 h reaction. The chitosan nanoparticles were isolated by adding acetone, centrifuging, and washing with ethanol for 3 times to remove surfactant and impurities. The resulted nanoparticles were throughly washed with deionized water by centrifugation using molecular weight cutoff centrifugal filters (10 kDa cutoff, Millipore). The dried precipitate was dissolved in deionized water and further dried by lyophilizer. The CG-PEG-ICG nanoparticles were prepared via a self-assembly method by adding 1 mL of ICG (5 mg·mL-1) to 4 mL of CG-PEG nanoparticles solution (1 mg·mL-1) under stirring for 24 h at room temperature. The CG-PEG-ICG nanoparticles were further purified by centrifugation using a centrifuge tube of 10kDa at room temperature to remove the extra free ICG (Millipore, 3,500 r/min, 5min). 2.4 Physiochemical characterization of CG-PEG and CG-PEG-ICG nanoparticles Size, surface charge, polydispersity index (PDI), size distribution of CG-PEG and CG-PEG-ICG nanoparticles were acquired using a Nano ZS90 Zetasizer (Malvern Instruments, Malvern, UK) at 25oC. The TEM samples were prepared by placing a drop of nanoparticles solution onto a 200-300 mesh copper grid (Zhongjingkeyi Technology Co. Ltd., Beijing). The adsorption efficiency and loading efficiency of ICG in CG-PEG nanoparticles were detected by isolating free ICG from aqueous suspension medium by ultracentrifuge (3,500 r/min, 5min) (TL-16R Table-top & high-speed refrigerated centrifuge, Shanghai) and the concentration of ICG loaded in nanoparticles was determined by Shimadzu UV-2550 spectrometer. The adsorption


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efficiency and loading efficiency were acquired through the following formula: adsorption efficiency (%) = (weight of loaded drug) / (weight of initially added drug) ×100; Loading efficiency (%) = (weight of loaded drug) / (total weight of nanoparticles) ×100. 2.5 Fluorescent stability and temperature measurement during light irradiation. To evaluate the fluorescence stability under illumination, the free ICG and CG-PEG-ICG with same ICG concentration (50 μg·mL-1) were irradiated under lamp for 0~60 min. Fluorescent intensities of free ICG and CG-PEG-ICG nanoparticles at 820 nm were measured by fluorescence spectroscopy (Perkin Elmer LS 55). Moreover, the temperature increase of PBS, free ICG and CG-PEG-ICG with different ICG concentration was measured with an aquarium thermometer under the irradiation with a 0.17 w/cm2 808 nm laser for 15 min. 2.6 In vitro photothermal toxicity of CG-PEG-ICG nanoparticles Photothermal toxicity of CG-PEG-ICG nanoparticles were evaluated by adding100 μL of medium, free ICG, CG-PEG-ICG nanoparticles at different concentrations of ICG (4.68, 9.38, 18.75, 37.50, 75.00 μg·mL-1) into a 96-well plate seeded with U87 cells (1×104 cells·well-1). After incubation for 4 h, the plate was irradiated with a 0.17 w/cm2 808nm laser for 10 minutes in 37oC digital incubator. Cell viability was evaluated using MTT assay. 20 μL of MTT (5 mg·mL-1) reagent was added to each well. The cells were washed with PBS three times after further incubation for 4 h and 100 μL of dimethylsulfoxide was added. Optical density (OD) at 570 nm of each well was measured using a Microplate Reader (Wellscan MK3, Labsystems). The cell viability was estimated according to the following equation: Cell Viability [%] = (ODtreated /ODcontrol) × 100% (ODcontrol is measured in the absence of reagent, and ODtreated donates the intensity obtained in the presence of ICG or CG-PEG-ICG nanoparticles. Cell viability incubated with CG-PEG-ICG nanoparticles at different concentrations of ICG (4.68, 9.38, 18.75, 37.50,


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75.00 μg·mL-1) with and without 808 nm laser irradiation was also evaluated using the MTT assay. Cell morphology was tested by phase contrast microscopy (Nikon Co., Japan). 2.7 Animal tumor model BALB/c-nu mice (5-6 weeks) were purchased from the Animal Center of Dalian Medical University. All mice received care incompliance with the guidelines outlined in the Guide approved by the Animal Care Committees of the Dalian Medical University. U87 cells (3 ×106) in a mixture of 50 μL of culture medium and 50 μL of Matrigel were subcutaneously injected into the flank region of mice. The tumor volume was calculated as (tumor length) × (tumor width)2/228. 2.8 In vivo imaging and biodistribution studies When the tumor volume reached to 100~200 mm3, 200 μL of free ICG, CG-PEG-ICG nanoparticles containing 50 μg·mL-1 of ICG were injected into the mice bearing xenograft tumors via tail vein. In vivo tumor imaging was performed with a CRi Meastro Ex in vivo imaging system. Spectra fluorescent images were obtained using the appropriate filter for ICG (excitation: 735 nm; maximum emission: 805 nm; acquisition settings: 780~900 nm in 10 nm steps, long-pass filter). The mice were sacrificed after 24 h and various organs and tumors were collected and imaged on the Maestro EX in vivo imaging system according to the protocol. Biodistribution of free ICG, CG-PEG-ICG nanoparticles was studied by the comparison of the fluorescence intensity of the major organs and tumor tissue. To investigate the retention effect of the CG-PEG-ICG nanoparticles in tumor, 100 μL of CG-PEG-ICG nanoparticles containing 50 μg·mL-1 ICG was injected into the tumors by using same amount free ICG as a control. The mice bearing xenograft tumors were imaged at different time points with the CRi Meastro Ex in vivo imaging system. The ICG signal was quantified by


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spectrally extracting from the multispectral fluorescent images with Maestro software after subtracting the background auto-fluorescence and regions of interest (ROIs) were selected over the tumors. Average signal values in tumor ROIs were normalized to the non-tumor ROI. 2.9 In vivo tumor photothermal treatment To investigate the efficiency for tumor therapy, CG-PEG-ICG nanoparticles (100 μL) with different concentration ICG (0, 50, 100, 200, 500 μg·mL-1) were intratumorally injected into the mice. The tumors of mice were irradiated with 808 nm laser at 0.3 W/cm2 up to 15 min. Temperatures at different time points were recorded every minute with a FLIR-i3 infrared thermal imaging camera and the morphology of the tumor was also recoded after 24 h irradiation. When the tumor volume reached to 100~200 mm3, the tumor-bearing mice were divided into three groups (three per group). 100 μL of PBS, free ICG and CG-PEG-ICG nanoparticles containing 75 μg·mL-1 of ICG were intratumorally injected into the mice. For the laser treatment groups, the tumors of mice were irradiated with 808 nm laser at 0.3 W/cm2 for 15 min. After 3 days, the mice were secondly injected with 100 μL of PBS, free ICG and CG-PEG-ICG nanoparticles containing 200 μg·mL-1 of ICG. The tumors were irradiated once again with the same procedure. The tumor volumes and body weight of each mouse were recorded every day. To further detect the effect of photothermal therapy in vivo, the tumors were collected at 48 h after irradiation and stained by hematoxylin and eosin (H&E). Sections were examined by biological inverted microscope. 3. Results and discussions 3.1 Preparation and characterization of chitosan nanoparticles The chitosan-genipin (CG) nanoparticles were prepared using a water-in-oil reverse microemulsion method. The crosslinking reaction of chitosan and genipin took place when mixed


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chitosan and genipin microemulsion together as described above which involves reaction between primary amine of chitosan and different sites of genipin, carbon-3 and ester group to form an amide29. We noted that the microemulsion medium became dark blue, which might come from the oxygen radical-induced polymerization of genipin as well as its reaction with amino groups

24

. The reaction can be monitored by UV-Visible absorption spectra because dark blue

genipocyanin generated30. As shown in Fig. 1A, the prepared chitosan nanoparticles showed an obvious absorption feature centered at 605 nm and the long reaction time made the absorption peak more evidently. After reaction for 7 days, the intensity of UV absorbance at 605 nm remained unchanged, indicating the reaction reached to the end. The reverse microemulsion template can provide a “nano-reactor” for controlled growth of the chitosan nanoparticle with uniform sub-100 nm particles and a narrow size distribution31. Herein, water soluble oligomeric chitosan (Mw= 5000 Da) was used to solve the precipitate problems of the normal chitosan as the pH increased to physiological conditions25. Genipin amount is crucial for the formation of stabile nanoparticles, because excessive genipin may consume a large number of primary amine and caused aggregation, otherwise insufficient to generate nanoparticles. The molar ratios 1:1, 1.5:1, 2:1, 4:1 and 8:1 between -NH2(chitosan) and genipin were investigated to the effect on primary amine consumption, nanoparticle hydrodynamic size, PDI and zeta-potential. The FTIR spectra (Fig. 1B) showed the peak of chitosan at 1514 cm-1 decreased gradually along with the increase of genipin amount, indicating the consumption of primary amino groups and the formation of the nanoparticles. The DLS measurement (Fig. 1C) showed that the particle size was in the range of 60~150 nm, with a PDI around 0.2~1.0. The dynamic diameters of the particles are around 60 nm when the molar ratio between -NH2(chitosan) and genipin less than 2:1, while they increase significantly for those with -NH2/genipin molar ratio of 4:1 and 8:1. The Zeta-potential analysis


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in Fig. 1D demonstrated that all the nanoparticles had positive charge (~40 mV) in deionized water, which can be used for loading ICG through the electrostatic self-assembly32. There is hardly Zeta-potential change of the chitosan nanoparticles, probably due to the presence of sufficient amino groups on the chitosan nanoparticles surface, which exhibit the similar Zeta-potential. The nanoparticles prepared with molar ratio of –NH2 (chitosan) to genipin 1.5:1 were selected in the following experiment because of their relatively small size.

2

B

2d 3d 4d 5d 6d 7d 8d

Transmittance

0h 2h 4h 8h 12h 1d

3

1

0

C

200

400

500 600 Wavelength(nm)

* * 1514 * 1620 1678

2000 1500 1000 Wavenumber (cm-1)

500

D

Size PDI

2

100

1

50

1:1 1.5:1 2:1 4:1 8:1 (-NH2/Genipin) Molar Ratio

0

PDI

3

150

0

CG 1/1 CG 1.5/1 CG 2/1 CG 4/1 CS 2500

700

Zeta-potential (mv)

Absorbance

A

Size (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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40

20

0

1:1 1.5:1 2:1 4:1 8:1 (-NH2/Genipin) Molar ratio

Fig.1. Study of the crosslinking reaction of the chitosan-genipin nanoparticles.(A) Evolution of the UV-visible absorption spectra of chitosan microemulsion during the preparation of chitosan nanoparticles trigged with genipin(-NH2/genipin molar ratio=1.5:1). (B) FTIR spectra, (C) Particle size and PDI and (D) Z-potential of chitosan nanoparticles as a function of molar ratio between -NH2(chitosan) and genipin. CG1/1,CG1.5/1, CG2/1, CG4/1, CG8/1 represent that the molar ratio between -NH2(chitosan) and genipin of CG nanoparticles are 1:1, 1.5:1, 2:1, 4:1 and 8:1, respectively.


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Moreover, PEG modified nanoparticles (CG-PEG) were prepared due to the unique properties of PEG in protein resistance, thus improving biocompatibility27. Fig. 2A showed the FTIR spectra of CG and CG-PEG nanoparticles. For CG-PEG nanoparticles, we can observe absorption band caused by deformation vibrations of -CH2 at 1409 cm-1 and the intensity increase of peak at 1584 cm-1 came from the amide bond generated by succinimidyl carboxymethyl and primary amine 33. Significant increase in the intensity of absorption band at 1089 cm-1 (circled) belonging to the C-O-C bond was also observed. The CG nanoparticles absorption bands at 1089 cm-1 is much less intense than CG-PEG nanoparticles. The intensity of the absorption bands in the range between 3000 - 2900 cm-1 of CG-PEG corresponding to stretching vibrations of C-H bands in PEG was also changed after modification of chitosan nanoparticles with PEG. All of the above results revealed that PEG had been successfully conjugated on the surface of CG nanoparticles. The content of PEG in nanoparticles was determined to be 8.89% by the analysis of the intensity of the characteristic absorption band of C-O-C bond in PEG chains at 1089 cm-1 in FTIR spectra of CG-PEG nanoparticles, calculated by the calibration cure as displayed in Fig. S2. The size of all CG-PEG nanoparticles was in the range of 40~65 nm after PEG modification when pH values varied from 2.8 to 7.3 (Fig. 2B). Diameter decreased slightly when pH changed from 7.3 to 2.8 because of the protonation of residual primary amine, while the PDI did not change. PDI was less than 0.3 for all samples indicating the CG-PEG nanoparticles were rather uniform at different pH buffer. In addition, no aggregation was observed when stored at room temperature over a month. So the improved stability of CG-PEG nanoparticles may benefit from the hypothesis that PEG provided a steric shielding of the chitosan-genipin core from the crosslink reaction34. The morphology and size distribution of the CG-PEG nanoparticles studied by TEM were illustrated


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in Fig. 2 C, D. The nanoparticles showed an ultra-small diameter around 5 nm with uniform size distribution within the range of 1-10 nm. The size of CG-PEG measured with TEM was smaller than that of DLS results, probably due to the aggregation of partial nanoparticles, which contribute great to total particle size. It is necessary to mention that the differing electron densities between the polymer forming the core and the PEG on the surface of the particles, respectively, can result in different contrasts of the respective domains, thus giving a relatively smaller size in TEM analysis (Table S1). These results demonstrated that the CG-PEG nanoparticles with relative narrow size distribution, spherical morphology and good stability were fabricated.

A

B Size

80

0.8

CG NPs

CG-PEG NPs

60

PDI

Size (d.nm)

PDI

Transmittance

40

0.4

20 800

1600

2400

3200

0

4000

2.8

-1

Wavenumber (cm )

C

D Percentage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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3.8

4.7 5.8 pH

6.8

7.3

0

36 27 18 9 0

1

2

3

4 5 6 7 8 Particle size (nm)

9 10

Fig. 2.Characterization of the CG-PEG nanoparticles. (A) FTIR spectra of CG and CG-PEG nanoparticles. Circle points the absorption band at 1089 cm-1 belonging to the C-O-C bond. (B) Particle size and PDI of CG-PEG nanoparticles at different pH values. (C) Morphology and (D)


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particle

size

distribution

of

CG-PEG

nanoparticles

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obtained

by

TEM

(-NH2

of

chitosan/genipin=1.5:1). Scale bar= 100 nm. 3.2 Fabrication and characterization of CG-PEG-ICG nanoparticles CG-PEG-ICG nanoparticles were simply fabricated via a self-assembly method by adding ICG to CG-PEG nanoparticle aqueous solution under stirring through electrostatic interaction. The extra free ICG can be removed by the centrifugation with a centrifuge tube. Fig. 3A showed the nanoparticle size and PDI before (CG-PEG) and after (CG-PEG-ICG) adsorption of NIR organic dye ICG. The nanoparticle size increased from 62 to 73 nm, while the PDI decreased from 0.27 to 0.17 after adsorption of ICG. The zeta potential changed from 40 to 24 mV because of the negative charge of the ICG, which dramatically neutralized the positive charge of the CG-PEG nanoparticles through the electrostatic interaction. The adsorption efficiency and loading efficiency of ICG was calculated to be 19.3 wt % and 1.93 wt %, respectively, calculated by the calibration cure as displayed in Fig. S1. The absorption and emission spectra of free ICG, CG-PEG and CG-PEG-ICG nanoparticles in aqueous solution were measured, respectively (Fig. 3C). In this condition, a small absorption peak centered at 605 nm of CG-PEG nanoparticles was observed coming from the above mentioned dark blue genipocyanin, while no emission peak was found. After adsorption of ICG, the absorption peak at 778 nm of CG-PEG-ICG nanoparticles decreased as compared to that of free ICG. This revealed that the ICG local environment was changed after forming the CG-PEG-ICG nanoparticles, indicating the occurrence of self-assembling between ICG and CG-PEG. Meanwhile, the emission peak at 805 nm for CG-PEG-ICG did not shift after adsorption of ICG with CG-PEG carrier (Fig. 3D).


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B

A

0.8

0.4

40

0.2

20 0

Zeta potential (mv)

0.6

60

PDI

Size (d.nm)

80

CG-PEG

CG-PEG-ICG

0.0

45

30

15

0 CG-PEG

CG-PEG-ICG

2.0

1.5

D

ICG CG-PEG CG-PEG-ICG

Fluorescence intensity (a.u.)

C Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.0

0.5

0.0 300

400

500 600 700 Wavelengh (nm)

800

900

ICG CG-PEG-ICG

4000 3000 2000 1000 0 750

800

850

900

950

Wavelength (nm)

Fig. 3. (A) Particle size and PDI of CG-PEG and CG-PEG-ICG nanoparticles. (B) Z-potential of chitosan nanoparticles as a function of molar ratio between –NH2(chitosan) and genipin. (C) Absorbance and (D) emission fluorescence spectra of free ICG, CG-PEG and CG-PEG-ICG nanoparticles in aqueous solution.

The stability was studied by comparing the fluorescent intensity of free ICG and CG-PEG-ICG samples containing 50 μg·mL-1 of ICG irradiated under lamp for 0~60 min. The fluorescence signal intensity of free ICG and CG-PEG-ICG nanoparticles decreased 70.4% and 27.8%, respectively, after irradiation for 60 min (Fig. 4A). The max fluorescence emission wavelength did not shift when ICG degraded over time (Fig. S3). This indicated the CG-PEG nanocarriers can protect the ICG from photo-quenching very well. Fluorescence quenching may be due to light exposure and degradation of the ICG molecules, the improved fluorescent stability of CG-PEG-ICG could be explained by the isolation of ICG from surroundings by the CG-PEG


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nanocarriers and reducing the chance to interact with other molecules35. Thus the CG-PEG nanocarriers can effectively shield the ICG from degradation. To evaluate the photothermal efficiency, the temperature changes of different concentration CG-PEG-ICG nanoparticles under laser irradiation was investigated as displayed in Fig. 4 B. With the laser irradiation at 808 nm, 0.17 W/cm2 for 15 min, the temperature of all samples increased rapidly, but there was little temperature variation for PBS. The temperature increase showed a concentration-dependent behavior and the maximum temperature of the CG-PEG-ICG containing 75 μg·mL-1 ICG could achieve 44 oC with 20 oC increase over the thermal ablation threshold

36

. As for free ICG, the

temperature increase is relatively small in the same concentration as compared with CG-PEG-ICG. The photothermal study above demonstrated that the CG-PEG-ICG nanocarriers can effectively shield the ICG from degradation over time and result in greater temperature increase. The higher temperature produced by the CG-PEG-ICG is essential for tumor photothermal therapy.

CG-PEG-ICG ICG

100 80

B

50

PBS ICG 1 CG-PEG-ICG 1 ICG 2 CG-PEG-ICG 2

45

O

Temperature ( C)

A

Normalized intensity (a. u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60 40

40 35

20

30 0

0

2 5 10 15 30 Irradiation time (min)

60

0

2

4 6 8 10 12 14 Irradiation time (min)

Fig. 4.(A) Fluorescence intensity changes of free ICG and CG-PEG-ICG samples containing 50 μg·mL-1 of ICG irradiated under lamp for up to 60 min. (B) Temperature increase of ICG and CG-PEG-ICG solutions under laser irradiation (808 nm, 0.17 W/cm2) with different ICG concentration: ICG1: 5 μg·mL-1, ICG2: 75 μg·mL-1, CG-PEG-ICG1: 5 μg·mL-1, CG-PEG-ICG2:


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75 μg·mL-1.

3.3 In vitro photothermal therapy To investigate the photothermal therapy efficiency of CG-PEG-ICG nanoparticles in cancer cells, U87 cells were incubated with different concentration nanoparticles for 4 h and then exposed to an 808 nm laser at a power density of 0.17 W/cm2 for 10 min. Cell viability was evaluated after photothermal therapy treatment using a standard MTT assay. As shown in Fig. 5 A, the cancer cells with CG-PEG-ICG nanoparticles irradiated by a laser exhibited a concentration-dependent cytotoxicity of all groups. When the concentration of ICG was 75 μg·mL-1, the cancer cells exhibited a very low cell viability (15%), suggesting that the CG-PEG-ICG nanoparticles possessed high photothermal therapy efficiency. An interesting phenomenon was that free ICG led to lower cell viability than CG-PEG-ICG in condition of low concentration (ICG 18.75 μg·mL-1). The phenomenon may be due to the self-quenching of free ICG at low concentration was not significant and high toxicity was caused37. At high concentration, the ICG molecules were well protected by the CG-PEG-ICG nanocarriers, leading the death of more cells under the laser irradiation. U87 cells irradiated with laser showed no obvious toxic effect, suggesting that the laser had hardly effect on cell viability. The cell viability of U87 cells incubated with CG-PEG-ICG nanoparticles without laser irradiation were not affected significantly by the CG-PEG-ICG nanoparticles, revealing that the nanoparticles were biocompatible and had low toxicity. Moreover, the cell viability of U87 cells without adding CG-PEG-ICG nanoparticles remained more than 95% irradiated by the 808 nm laser for 10 min (data not shown). This indicated that there was hardly any effect for the laser


Biomacromolecules

irradiation to cell viability. To further study the in vitro photothermal toxicity of CG-PEG-ICG nanoparticles to the U87 cells, the cell morphology was studied by phase contrast microscopy. Round, fuzzy and volume reduction of the cells was observed after 10 min laser irradiation incubated with CG-PEG-ICG nanoparticles (Fig. 5B). However, the control group without adding CG-PEG-ICG nanoparticles showed no change, revealing that the laser irradiation were non-toxic and only led to the cell death with CG-PEG-ICG nanoparticles. These results demonstrated that CG-PEG-ICG nanoparticle is a promising photodynamic agent for cancer therapy. A 150

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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CG-PEG-ICG - laser CG-PEG-ICG + laser ICG + laser

100

50

0

4.68 9.38 18.75 37.5 75.00 Concentrition of ICG (µg/mL)

Fig. 5. (A) Relative viabilities of U87 cells by MTT assays after incubation with free ICG and CG-PEG-ICG nanoparticles irradiated by the 808 nm laser for 15 min or without laser as a control. (B) Microscopy photographs of U87 cells incubated with CG-PEG-ICG nanoparticles (75 μg·mL-1) before and after irradiation for 15 min. U87 cells without adding of CG-PEG-ICG nanoparticles were used as a control. Means ± SD (n=3). Scale bar=47μm. 3.4 In vivo imaging and biodistribution studies In order to check whether the PEGylation of CG-PEG-ICG nanoparticles can increase the circulation time, the tumor-bearing mice were intravenously injected with free ICG and CG-PEG-ICG nanoparticles. Fig. 6 showed the fluorescent imaging of the whole body of the mice at different time points, and major organs and tumor tissue 24 h post-injection, as well as


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their relative fluorescence intensity. The mice injected with CG-PEG-ICG nanoparticles exhibited intensive and strong fluorescence signal in contrast with those with free ICG (Fig. 6 A). This indicated that the PEGylated CG nanoparticles could prolong the retention time of ICG in the body. When the mice were sacrificed and various organs were imaged 24 h post-injection, the comparative major organs and tumors distribution of free ICG and CG-PEG-ICG nanoparticles were shown in Fig. 6 B and C. Most of ICG accumulated in the liver, kidney and tumor after 24 h intravenous injection of the agents and the CG-PEG nanocarriers dramatically increased the accumulation of ICG in the tumor compared with free ICG. The results demonstrated that the PEGylated chitosan-genipin nanoparticles could significantly alter the biodistribution and elimination patterns of the ICG, which might be a benefit for the tumor photothermal therapy.

C Fluorescence intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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liver

CG-PEG-ICG ICG

8

6

kidney 4

lung

tumor

spleen

2

muscle

heart

Fig. 6. (A) Fluorescence image of nude mice bearing U87 xenograft tumors after intravenous injection of free ICG or CG-PEG-ICG nanoparticles at different time. (B) Fluorescence image of major

organs

and

tumor

tissue

24

h

post-injection.

(C)

Quantitative analysis

of

fluorescence intensity of major organs and tumor tissue 24 h post-injection.

During the PTT process, one of the important concerns after intratumoral injection is whether the CG-PEG-ICG nanoparticles would accumulate in xenograft tumor or organs or clear


Biomacromolecules

out of the body. By fluorescent imaging of the tumor-bearing mice, the retention effect of CG-PEG-ICG nanoparticles accumulated in tumor was investigated at different time points. Fig. 7 showed the ICG fluorescence signal and intensity distribution around the tumor after intratumoral injections of free ICG and CG-PEG-ICG nanoparticles. Except the tumor area where the ICG was injected, the fluorescent signal in other areas of the mice body was relatively weak because of a relatively low dose of ICG (Fig. 7A). The fluorescence intensity increased at the first 2 hours because of the photosensitizer diffusion (Fig. 7B). The signal intensity around the tumor at 4, 8, 12 and 24 h post-injection for CG-PEG-ICG nanoparticles were relatively 18.1 %, 26.6 %, 31.2 % and 21.3 % higher than those of tumors injected with free ICG. The relatively faster signal intensity decrease of free ICG was attributed to the quenching effect in physiological environments, where the free ICG was rapidly cleared out from the body. In contrast, the CG-PEG-ICG nanoparticles were significantly retained in the tumor area even after 24 h. The improved retention of CG-PEG-ICG nanoparticles was possibly due to the tumor passive targeting of EPR effects. The retention of photosensitizer in the tumor is important for photothermal therapy if the nanocarrier containing ICG could be retained there and the ICG could be released into the tumor cells. The prolonged retention of CG-PEG-ICG nanoparticles could improve thermodynamic stability of free ICG, and finally enhance photothermal effect of the photosensitizer in tumor photothermal therapy. B 2000 Fluorescence intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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CG-PEG-ICG ICG

1500

1000

500

0

0


2 4 8 12 Injection time (h)

24

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Fig. 7. (A) In vivo imaging and (B) quantitative intensity of nude mice bearing U87 xenograft tumors after intratumoral injection of free ICG or CG-PEG-ICG nanoparticles (n=3, ICG= 50 μg·mL-1).

3.5 Photothermal treatments in vivo To confirm a lethal dose of CG-PEG-ICG nanoparticles to tumors with little damage to surrounding normal tissue as possible, temperatures and infrared thermographic maps were recorded with a FLIR-i3 infrared thermal imaging camera after intratumoral injection of CG-PEG-ICG nanoparticles (Fig. 8A). We could see that the temperature increase showed a concentration-dependent manner. Temperature of all samples increased rapidly at the first 5 min then gradually in accordance with the results in vitro(Fig. 8B). The maximum temperatures of tumors treated with PBS and CG-PEG-ICG nanoparticles containing different concentration ICG were 36.8, 41.2, 43.3, 45.3 and 50.8 oC, respectively, after irradiation for 15 min. It had been reported that even below the plasma formation threshold energy, laser pulses can also induce irreversible damage to the biological sample by reduction of enzyme activity, protein denaturation and increase in cell membrane stress38. In this case, CG-PEG-ICG nanoparticles with ICG concentration of 100, 200 and 500 μg·mL-1 induced irreversible tissue damage which was confirmed in Fig. 8A “tumor” column, while those loaded with a low dose of ICG were non-effective for in vivoU87 tumor photothermal therapy.


Biomacromolecules

B 55 5

PBS 1 2 3 4

50 5

O

Temperature ( C)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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45 4 40 4 35 3 30 3 0

2

4 6 8 10 12 14 Irrradiation tim me (min)

16

Fig. 8. (A A) Infraredd thermo-graphic imaages and morphology m of tumors at 24 h after a laser irradiationn (808 nm, 0.3 W/cm2, 15 min). (B) Tempeerature increase of tum mor under continuous c laser irraddiation injected with CG-PEG-IC C CG nanopartticles contaaining ICG, 1-50 μg·mL-1, 2-100 -1 μg·mL-1, 3-200 μg·m mL-1, 4-500 μg·mL μ .

In vivo v mice experimentts were peerformed to o evaluate the efficaccy of CG--PEG-ICG nanoparticles PTT effect onn the desstruction of o U87 xeenograft tuumors. To o achieve better phootothermal therapeutic t effect, the CG-PEG-IC C CG nanoparrticles were directly injjected into the tumorr site in the mice. As shhown in Figg. 9A, tumors volume exhibited e litttle change at the first 4 days, suuggesting 75 μg·mL-1 of o ICG was not sufficieent to inhibbit tumor groowth as we expected. After injeection of thhe second dose containning 200 μg g·mL-1 of IC CG at day ffive, the miice treated with CG--PEG-ICG nanoparticle n es show thee smallest tumor t size (523 mm3 oof tumor size) at day eleven, when w compaared with thhe mice treaated with PB BS (5472 m mm3 of tumoor size) and d free ICG (1437 mm m3 of tumorr size). The growth of U87 tumorrs inhibited by CG-PEG G-ICG nano oparticles, free ICG and PBS shhowed significant diffeerences. Thee possible reason r was that ICG ad dsorbed in CG-PEG nanoparticlles had highher condenssed concenttration thann free ICG, aand the nan noparticles could be retained thhere and diiffuse ICG into the tu umor cells, resulting iin the high her energy efficiencyy and lowerr heat dissiipation in thhe CG-PEG G-ICG nanooparticles aafter laser irrradiation.


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Moreover, the body weights of the mice in all groups were measured during the treatment period and did not induce any noticeable changes for the CG-PEG-ICG group, whereas slight increases of the bodyweight were observed for PBS and free ICG groups because of the tumor growth (Fig. 9B). During the course of PTT treatment, the tumor-bearing mice treated with CG-PEG-ICG nanoparticles resulted in a scar formation (Fig. 9A, inset), and later on the skin at their radiation site got peeled off. The therapeutic efficacy and toxicity was also studied by the histological tissue images through H&E staining. We could see that the H&E staining tumor tissue in PBS group showed vigorous growth, a tight arrangement and intact shape, while cell necrosis, lysis and fragmentation to a certain extent in CG-PEG-ICG nanoparticles groups (Fig. 9C) was observed, which was consistent with the therapeutic efficacy. All of these results indicate that the CG-PEG may act as a good nanocarrier for effective tumor phototherapy.

Tumor volume(mm3)

A 8000 6000

PBS ICG CG-PEG-ICG

4000

* *

* *

First injection Second injection

2000

0 2

B

40

Body weight (g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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35

4

6 8 Time(day)

10

12

PBS ICG CG-PEG-ICG

30

25

20 1

2

3

4

5 6 7 8 Time(day)

9 10 11

Fig. 9. (A) Growth curve of U87 xenograft tumors within 11 days in different groups (n=4). Inset shows the picture of tumor-bearing mice treated with CG-PEG-ICG nanoparticles at day 11. (B)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Body weight of U87 tumor bearing mice treated with different formulations. (C) Morphology of tumor at 24 h after laser irradiation and histological staining of the excised tumors 48 h after irradiation (808 nm, 0.3 W/cm2, 15 min). (Scale bar=47 μm) 4. Conclusions In summary, we have successfully fabricated a new type of ultra-small chitosan nanoparticles cross-linked by genipin through a W/O reverse microemulsion method. The chitosan-genipin

nanoparticles

after

PEG

modification

showed

good

dispersibility,

monodispersity and stability at different pH value, which could be used as ICG nanocarriers for tumor photothermal therapy. Our results demonstrated that the CG-PEG-ICG nanoparticles exhibited quite low cytotoxicity, which not only induced in vitro U87 cells death under irradiation of laser 808 nm, but also suppressed tumor growth in tumor-bearing mice modal. Hence, the CG-PEG nanoparticles might have potential as nanocarriers for photosensitizer in tumor photothermal therapy.

Supporting Information: This information is available free of charge via the Internet at http://pubs.acs.org/.

Acknowledgements This work was supported by the National Nature Science Foundation of China (91227126, 81441052), National Special Fund for Key Scientific Instrument and Equipment Development (2013YQ17046307) and the Nature Science Foundation of Liaoning Province, China (2013020177). We are thankful for the support by the Animal Experiment Combined Workstation


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of Dalian Institute of Chemical Physics and Dalian Medical University.

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