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Photodynamic Detection of Oral Cancers with High Performance Chitosan-Based Nanoparticles Shu-Jyuan Yang, Cha-Fu Lin, Min-Liang Kuo, and Ching-Ting Tan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm400820s • Publication Date (Web): 02 Aug 2013 Downloaded from http://pubs.acs.org on August 4, 2013
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Photodynamic Detection of Oral Cancers with High Performance Chitosan-Based Nanoparticles Shu-Jyuan Yang,1 Cha-Fu Lin,1 Min-Liang Kuo,1,2 and Ching-Ting Tan3,* 1. Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan 2. Institute of Biochemical Sciences, College of Live Science, National Taiwan University, Taipei, Taiwan 3. Department of Otolaryngology, National Taiwan University Hospital, and National Taiwan University College of Medicine, Taipei, Taiwan *Corresponding author: Tan, C.-T. Tel: 886-2-23123456 ext; 88600 Fax: 886-2-23217522
E-mail: [email protected]; [email protected]
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ABSTRACT Oral cancer, a subtype of head and neck cancer, is one of the leading causes of cancer death and is difficult to detect in the early stages. Improved methods of detecting primary oral lesions during endoscopy would significantly improve cancer survival rates. Here we report a high-performance nanoparticle for photodynamic detection of oral cancer. Succinate-modified chitosan (SCHI) is physically complexed with folic acid-modified chitosan to form nanoparticles with a high drug loading efficiency and to improve drug release in the cellular lysosome. The z-average diameter and zeta potential of the prepared nanoparticles (fSCN) was 110.0 nm and 18.6 mV, respectively, enough to keep the nanoparticles stable in aqueous suspension without aggregating. When loaded with 5-aminolaevulinic acid (5-ALA; 72.8% loading efficiency) in the prepared fSCNA, there were no significant differences between the fSCN and fSCNA in particle size or zeta potential. Moreover, the fSCNA nanoparticles were readily engulfed by oral cancer cells via folate receptor-mediated endocytosis. The release of loaded 5-ALA in the lysosome was promoted by the reduced attraction intensity between chitosan and 5-ALA via the deprotonated SCHI molecules, which resulted in a higher accumulation of intracellular protoporphyrin IX (PpIX) for photodynamic detection. These results demonstrate that N-succinyl-chitosan-incorporated and folic acid-conjugated chitosan nanoparticles are an excellent vector for oral-specific delivery of 5-ALA for fluorescent endoscopic detection. Keywords: oral cancer, chitosan, N-succinyl chitosan, 5-aminolevulinic acid, nanoparticle, fluorescence
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INTRODUCTION Photo-detection, employing an exogenous chromophore excited by optima light to generate fluorescence, is one of the most promising non-invasive methods used recently to detect malignant or premalignant tissue.1, 2 A photosensitive fluorophore, protoporphyrin IX (PpIX) can be used as a source of fluorescence, because it can be converted from 5-aminolevulinic acid (5-ALA) during the heme group synthesis process in the cell mitochondria and decomposed at different rates in cancer and normal cells.3, 4 5-ALA is a hydrophilic and zwitterionic drug that has a poor affinity toward the cell membrane in a physiological environment and is very easily taken up by bacteria to produce misleading results in endoscopic observation.5 Therefore, a suitable carrier is needed that can prevent bacterial uptake of 5-ALA and help it pass through the lipophilic barrier for photo-detection.6
Oral cancer, a subtype of head and neck cancer, is the sixth most common cancer among men and women worldwide. It is one of the leading causes of mortality rate and is commonly difficult to detect at an early and treatable stage.7 Current clinical diagnostic procedures for oral cancer typically involve performing invasive needle biopsies followed by histological examination of the excised tissue, a process that may cause patients psychological trauma and create risk of infection. Moreover, precancerous lesions appear harmless or occur in hidden sites, with the result that they are often overlooked even with white-light endoscopy. Therefore, finding a powerful and highly sensitive tool for the detection of precancerous lesions is very important.8 Several methods using nano-engineered materials have been developed that might prove effective for detecting the disease at an early stage.9-11 ACS Paragon Plus Environment 3
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Chitosan is a linear polymer composed of 2-amino-2-deoxy-β-D-glucan by glycosidic linkages. It is biocompatible, biodegradable and possesses antibacterial properties that can prevent it from bacterial uptake in the GI track.12 A number of free amino groups on the molecular chain give chitosan several special characteristics, such as its ability to easily approach the cell membrane and muco-adhere to locations of interest with a long retention time, making it useful for pharmaceutical applications.13-15 Furthermore, its cationic nature also allows for ionic cross-linking with multivalent anions, such as sodium tripolyphosphate (STPP), to prepare nanoparticles in aqueous solution without using toxic organic solvents.16 In order to enhance the nanoparticle targeting and engulfment by the targeted cells, several targeting ligands have been conjugated onto the particle surface.9,
11, 17
Folic acid, a stable,
inexpensive, and poorly immunogenic chemical with a high affinity for the folate receptor is useful for enhancing nanoparticle endocytosis via folate receptor-mediated endocytosis.18, 19 Because the folate receptor is overexpressed on many human epithelial cancer cells, the conjugation of folic acid with drugs and macromolecules via folate’s γ-carboxyl moiety can improve their uptake and targeting ability. In previous studies, it has been shown that the incorporation of negatively charged polymers, such as alginate, can improve the drug releasing or transfection efficiency of chitosan-based nanoparticles by reducing the intensity of interactions between chitosan and the drug or DNA.20, 21 Therefore, the N-succinyl chitosan (SCHI) can be exploited as a negatively charged polymer to enhance the 5-ALA releasing in oral cancer cells. The synthesis of SCHI can be proceeded by the introduction of succinyl groups into chitosan at the N-position of the glucosamine units, ACS Paragon Plus Environment 4
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transforming chitosan molecules from acetic acid aqueous solution-soluble into water-soluble.22 Furthermore, it has low toxicity and is less biodegradable in the body, therefore can be expected to be useful as a drug carrier that allows long-term retention in the body. The purpose of this study was to modify chitosan molecules with folic acid and succinate at the N-position of the glucosamine units, respectively, and then physically incorporate N-succinyl chitosan into the folic acid-modified chitosan nanoparticles as a 5-ALA carrier (fSCNA). With these prepared fSCNA nanoparticles, 5-ALA could be easily taken up into oral cancer cells via folate receptor-mediated endocytosis. In addition, the release of 5-ALA in cancer cells could be meliorated by the anionic N-succinyl residue competing with the 5-ALA for the cationic residues of chitosan, enhancing the accumulation of PpIX for fluorescent endoscopic detection (Figure 1).
EXPERIMENTAL SECTION Materials. Chitosan (85% deacetylation and 15 kDa MW) was purchased from Polysciences, Inc. (Pennsylvania, USA); 5-aminolevulinic acid (5-ALA), succinic anhydride and sodium tripolyphosphate (STPP) were purchased from Sigma-Aldrich Co. (Missouri, USA). Folic acid came from TCI (Tokyo, Japan). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from Acros Organics (New Jersey, USA); anhydrous dimethyl sulfoxide (DMSO) was purchased from Riedel-de Haën (Germany). All were reagent grade and used without further purification.
Conjugation of folic acid or succinic anhydride with chitosan. The process of conjugation of folic acid to chitosan molecules was performed as previously described.23 First, folic acid and EDC ACS Paragon Plus Environment 5
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were thoroughly dissolved and mixed in 20 ml anhydrous DMSO (molar ratio 1:1) and then added slowly to 0.5 % (w/v) chitosan in acetic acid aqueous solution (0.1 M, pH 4.7). Folic acid-chitosan conjugate was formed at the molar ratio of folic acid to amino groups of 0.1. Stirring at room temperature in the dark for 16 hours, the solution was brought to pH 9.0 with 1M NaOH aqueous solution and centrifuged at 2,500 rpm to spin down the folic acid-chitosan conjugate. The precipitate was dialyzed against phosphate buffered saline (PBS) and water for seven days, and then isolated as a sponge by lyophilization and kept for follow-up study. SCHI was synthesized by referring the procedure described by Yang et al.24 1g chitosan powder was added into 2% (w/v) of succinic anhydride pre-dissolved in 100 ml anhydrous DMSO solution, and reacted at 70 oC for 24 hours. The solution was dialyzed first against 0.01M NaOH aqueous solution for two days and then against water for five days to remove the remanent succinic anhydride and DMSO. Finally, the SCHI was isolated as a sponge by lyophilization and kept for follow-up study.
Characterization of the N-succinyl chitosan and folic acid-chitosan conjugate. The 1H-NMR spectra of folic acid-chitosan conjugate and SCHI were recorded on a Bruker Avance-400MHz NMR spectrometer (Bruker BioSpin, MA, USA) using deuterium acetic acid-d4/ deuterium oxide solution (1/4 v/v) and 1 N Sodium deuteroxide solution, respectively, as solvents.
Synthesis of nanoparticles. The chitosan nanoparticles loaded with 5-ALA were prepared based on the ionic gelation interaction between positively charged chitosan and negatively charged 5-ALA ACS Paragon Plus Environment 6
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and STPP at room temperature. As described in previous studies,20, 23, 25 0.05 % chitosan and folic acid-chitosan conjugate solutions were prepared by dissolving 0.5 mg chitosan powder and folic acid-chitosan conjugate sponge in 1 ml 0.01 M acetic acid solution at pH 4.0, respectively; 0.15% 5-ALA solution was prepared by dissolving 1.5 mg 5-ALA powder in 1 ml 0.05% STPP-PBS solution. Either 2 ml of STPP-PBS solution or 2 ml of 5-ALA solution were added to 5 ml of chitosan or folic acid-chitosan conjugate solution to prepare the CN, CNA, fCN and fCNA nanoparticle suspensions. The preparation of fSCN and fSCNA was similar to that of fCN and fCNA, but the STPP and 5-ALA solutions were exchanged for 0.015% SCHI and 5-ALA/SCHI (10/1 w/w) solutions, respectively, which were pre-dissolved in 0.05% STPP-PBS solution. The prepared suspended nanoparticle solutions were later used directly without further treatment for the characteristic determination.
Characteristics of the prepared nanoparticles. Measurements of particle size and zeta potential of various nanoparticles were performed using the Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK) by dynamic light scattering measurements and laser Doppler electrophoresis, respectively. Particle size was measured at 25 °C with a 90° scattering angle based on the Zetasizer Nano-ZS90 internal setting. Measurements of zeta potential were made using the aqueous flow cell in automatic mode at 25 °C.26 Carbon-coated 200 mesh copper grids pre-immersed in CNA, fCNA and fSCNA solutions were placed on filter paper to absorb excess liquid and then dried under a lamp for 30 minutes. The dried copper grids with CNA, fCNA, and fSCNA were examined under the Hitachi TEM H-7650 ACS Paragon Plus Environment 7
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(Tokyo, Japan). The loading efficiency of 5-ALA in the prepared nanoparticles was analyzed by centrifuging the particle solution at a 20,000 g force to spin down the particles. The suspension was collected and then reacted with 2,4,6-trinitrobenzene sulfonic acid (TNBS) as an assay reagent to detect 5-ALA. The assay was performed using a TNBS kit (Pierce Chemical Company; Rockford, IL) according to the manufacturer’s instructions, and a serial of gradient 5-ALA standard solutions were used to prepare the calibration curve. The loading efficiency of 5-ALA in the nanoparticles was calculated with the following equation:
Loading efficiency (%) =
Ct − C Ct
f
×100 %
where Ct and Cf were the total amount of 5-ALA and the free amount of 5-ALA in the suspension, respectively.
Determination of folate receptor expression among oral cancer cell lines. Western blotting analysis was done to determine the expression of folate receptor on cell membranes as previously described.23 Two oral cancer cell lysate samples (TW1.5 and TW2.6) were collected by scraping the cell cultures into cold RIPA buffer (Millipore, Temecula, CA) containing protease inhibitors. The protein concentrations were assayed using the Pierce® BCA protein assay (Thermo scientific, IL, USA) with bovine serum albumin as the standard. Total protein (30 µg) was separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transblotted. Anti-human folate receptor antibody was purchased from Santa Cruz Biotechnology, Inc. (CA, USA); anti-α-tubulin was purchased from Sigma-Aldrich. The signals of anti-human folate receptor in the blots were ACS Paragon Plus Environment 8
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quantified with ImageQuant 5.1 software, and the values were normalized to α-tubulin expression.
Measurement of accumulated PpIX and cytotoxicity of the prepared nanoparticles. Cell lines TW1.5 and TW2.6 were cultured in 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture (GIBCOTM; Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum, at 37 °C in an atmosphere of 5% CO2, respectively. Cultured medium was changed on alternate days until confluent. TW1.5 and TW2.6 oral cancer cell lines were seeded onto 24-well culture plates, respectively, at the cell density of 1×104 per well. After cell lines were cultured for 24 hours, the medium was replaced with fresh medium containing CNA, fCNA and fSCNA, where the CNA, fCNA and fSCNA solutions were concentrated to 10 times by Amicon Ultra centrifugal filter devices (Millipore, Billerica, MA, USA) to remove the unloaded 5-ALA beforehand. Cells were further cultured for 18 hours to allow engulfment of nanoparticles and to convert 5-ALA to PpIX. In order to determine the association of folic acid-folate receptor mediated endocytosis with the endogenously synthesized PpIX, fresh culture media, both with and without free folic acid (5mM), were tested at the same time. After removal of the cultured medium and PBS wash, the PpIX were extracted with DMSO. The fluorescent intensity of PpIX was determined by spectrofluorometer (Molecular Device SPECTRA MAX GEMINE XS; Sunnyvale, CA) with an excitation wavelength of 405 nm and the emission wavelength at 635 nm. Fluorescent intensity was expressed as an accumulative percentage. For determination of the effect of the prepared nanoparticles on cell toxicity, TW1.5 or TW2.6 cells seeded onto 24-well platesACS wereParagon treated Plus withEnvironment fresh medium containing CNA, and fCNA, 9
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fSCNA. After 18 hours of incubation, the particle-containing medium was replaced with 500 µl of fresh medium that contained 0.5 mg/ml MTT assay reagent (Alfa Aesar, San Francisco, CA), and then incubated at 37°C for 3 hours. Then, the MTT-containing medium was replaced with 200 µl of acid-isopropanol solution (0.04 M HCl) to dissolve formazan, the MTT product. Finally, 150 µl samples were transferred to 96-well plates and the absorbance was measured at 570 nm with the Thermo Scientific Multiskan® FC microplate photometer (Thermo Fisher Scientific Inc., USA). The viability of untreated control cells was defined as 100%. To evaluate the feasibility of using the prepared nanoparticles for detection of oral cancer cells, the glass plate was placed in a Petri dish and seeded with either TW1.5 or TW2.6 cells. After cells were cultured for 24 hours, medium was replaced with fresh medium containing CNA, fCNA, fSCNA and 5-ALA. To confirm the nanoparticle uptake content, based on a correlation between the folate receptor expression and the folic acid modification, a fluorescent compound, calcein, was incorporated simultaneously into CNA, fCNA and fSCNA as a fluorescent marker. After 18 hours, cells were washed three times with PBS, fixed with 10% formalin and then examined under a spectral confocal and multiphoton system (Leica TCS SP5, Wetzlar, Germany). The average fluorescent intensity of calcein in cells was quantified with the MetaMorph® imaging software (Version 7.5, Molecular Devices, USA) to demonstrate the content of nanoparticle uptake. The data represented an average of five regions obtained from randomly chosen areas of each sample, and the brightness of the green emission light of the CNA group was designated as 100% for the individual oral cancer cell lines.
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Competition binding assay. The competition binding assay was done as previously described:20 10 ml of calcein-loaded fCN nanoparticle solution was concentrated to 10 times to remove the unloaded calcein, then mixed with 0.14 ml of PBS solution or 0.14 ml of 0.3% SCHI solution in a CelluSep dialysis membrane (Membrane Filtration Products, Seguin, TX, USA) with the molecular weight cut-off of 3.5 kD. In order to simulate the pH value in the cytoplasm and lysosomal environments, the dialysis membrane was soaked in 45 ml of buffer at pH 7.4 or pH 5.0 at 37 °C, respectively. After shaking for a period of time, 0.1 ml of the buffer was removed, and the optical density (OD) of calcein was recorded at a wavelength of 500 nm using the Thermo Scientific Multiskan® FC microplate photometer. The release profiles are represented as the correlation between the OD value of calcein and the incubation period.
Generation of subcutaneous tumor models and in vivo photodynamic detection. Because it is difficult to develop and treat oral cancers in mouse’s mouth, the subcutaneous tumor models were generated for in vivo photodynamic detection. The in vivo experimental protocols were approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC). As with our previous study,27 female C.B-17/Icr-scid-bg mice (4 weeks old, weighing 18–20 g) were subcutaneously inoculated in their flanks with 1×106 TW1.5 cells per mouse to generate a subcutaneous (SC) tumor model. Tumor sizes were measured using calipers, and tumor volume was calculated using the following formula: volume=width2×length×0.52. When the volume of SC tumors reached about 100 mm3, the mice were randomly divided into four treatment groups (n = 6 for each group): (a) injected intratumorally ACS Paragon Plus Environment 11
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(i.t.) with 50 µl of PBS solution, (b) injected i.t. with 50 µl of the prepared CNA solution in a 5-ALA concentration of 5 mM, (c) injected i.t. with 50 µl of the prepared fSCNA solution in a 5-ALA concentration of 5 mM, and (d) injected i.t. with 50 µl of 5 mM free 5-ALA solution. Eighteen hours after injection, the tumor tissues were resected and imbedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA), and then snap-frozen on dry ice. Cryosections of 5 µm thickness were fixed with acetone at 4 oC for 5 min and examined directly using a Zeiss Axiovert 200 M inverted fluorescence microscope (Carl Zeiss, Göttingen, Germany).
Statistical Analysis. Mean±standard deviation (SD) and graphs were used to describe the data. One-way analysis of variance (ANOVA) with post hoc muti comparison methods, such as Fisher’s least significant difference test and Tamhane’s T2 test, was used to assess the differences in the fluorescent intensity of PpIX and calcein in oral cancer cells fed with CNA, fCNA and fSCNA. All p values were two-sided and their significance level was 0.05. The statistical package for social
sciences 11.0 (SPSS 11.0) software was used to conduct all statistical analysis.
RESULTS AND DISCUSSION Chitosan was chosen as a carrier for ionized drugs because of its many free amino groups and quick micro/nanoparticle-forming ability once in contact with polyanions.28, 29 When covalently conjugated with folic acid, chitosan molecules had a high affinity for folate receptors and cellular uptake ability via folate receptor-mediated endocytosis.23 Moreover, the incorporation of an anionic polymer into the chitosan-based nanoparticles would enhance the release of loading drug.20, 21
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Characterization of the N-succinyl chitosan and folic acid-chitosan conjugate. Successful synthesis of folic acid-chitosan conjugate can be confirmed by 1H NMR spectroscopy; the 1H NMR spectra of chitosan and folic acid-chitosan conjugate are shown in Figure 2A and B. The signals at δ 2.00, 3.12 and 3.67-3.85 ppm were assigned to the resonance of the monosaccharide residue
protons, -COCH3, -CH-NH- and –CH2-O-, respectively.30, 31 Compared with the 1H NMR spectra of chitosan, the signals appearing at δ 6.78-8.70 ppm in the 1H NMR spectra of folic acid-chitosan conjugate were attributed to the resonance of the folate aromatic protons. They revealed that the coupling of the folate residue to the chitosan could be achieved via EDC-mediated reaction.23, 30 Because of the reaction conditions chosen and the steric constraints, folic acid could be successfully conjugated onto chitosan molecules via its γ-carboxyl moiety mostly.23 Moreover, the 1H NMR spectra of SCHI is shown in Figure 2C. The multiplets at δ 2.28–2.57 ppm were assigned to the methylene groups, -CH2-, of succinate, revealing that the modification of the succinyl residue onto
the chitosan could also be achieved by ring-opening reaction.24, 32
Characteristics of the prepared nanoparticles. The z-average diameter, zeta potential, and loading efficiency of the chitosan-based nanoparticles prepared under different conditions are shown in Table 1. The z-average diameter of the prepared nanoparticles did not vary significantly with 5-ALA loading, but increased remarkably from 77 to 110 nm when folic acid was conjugated and SCHI was incorporated. The zeta potential analysis showed that the nanoparticles prepared under different conditions all bore a positive charge, but the charge decreased with the simultaneous folate-conjugation (fCN) and SCHI-incorporation (fSCN). When the CN, fCN, or fSCN were ACS Paragon Plus Environment 13
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loaded with 5-ALA (CNA, fCNA, and fSCNA, respectively), the zeta potential exhibited no significant decreases. These results revealed that the z-average diameter and zeta potential of the prepared nanoparticles depended on the folate-conjugation and SCHI-incorporation but not on the 5-ALA-load Moreover, zeta potential greatly influences particle stability through electrostatic repulsion between particles and may result in a narrower size distribution.33 As shown in Table 1 and Figure 3A, the zeta potential of the CN and fCNA was about 20-29 mV, providing enough surface charge to stabilize the particles from aggregation and showing a narrower size distribution (in the range of 20-200 nm). When the protoned amino groups of chitosan were displaced with the negative succinyl derivatives, the zeta potential of the fSCN decreased as far as 18 mV and the particle size distribution fell into the range of 30-500 nm. Figure 3B shows the particle morphology of CNA, fCNA and fSCNA as determined by transmission electron microscopic examination (TEM): all formed as solid spheres and were as small as the z-average diameter determined by Zetasizer Nano-ZS90. The loading efficiency of 5-ALA in CNA was 37.7% and decreased to 34.0% with folic acid conjugation; however, when simultaneously incorporating SCHI into fCNA to form fSCNA, the loading efficiency increased to 72.8%. It was suggested that in fCNA particles, the replacement of electro-neutral folate derivatives reduced the numbers of protonated amino groups on chitosan molecules to attract 5-ALA molecules,29 and the spatial hindering of folate derivatives resisted the interaction between chitosan and 5-ALA molecules, resulting in a lower loading efficiency than the prepared CNA. Moreover, because of the zwitterionic property of 5-ALA,25 partial 5-ALA molecules would bear positive charges and interact with the negatively charged SCHI molecules in ACS Paragon Plus Environment 14
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the prepared conditions, resulting in a high 5-ALA loading efficiency in the prepared fSCNA. Based on these evidences, the loading efficiency also appeared strongly dependent on the folic acid conjugation and SCHI incorporation.
Correlation between folate receptor expression and PpX accumulation in oral cancer cell lines. It is well known that folate receptor is over-expressed on various types of cancer cell membrane; these cells can actively regulate the internalization of folic acid-conjugated molecules through folate receptor-mediated endocytosis.34 Therefore, the western blotting method was used to determine the expression of folate receptor on TW1.5 and TW2.6 oral cancer cell lines with results shown in Figure 4. It was apparent that expression of folate receptor depended on the cell line: TW1.5 cells exhibited 1.3-fold higher expression of folate receptors than did TW2.6 cells. Figure 5A shows the relative fluorescent intensity of PpIX in TW1.5 and TW2.6 cells cultured in a medium with or without free folic acid addition, and the brightness of the emission light of the CNA group was designated as 100% for the individual oral cancer cell lines. When the cells were co-cultured with fCNA for 18 hours, the intensity of emission light in TW1.5 and TW2.6 cells increased to 136.3% and 119.5%, respectively. This enhancement of PpIX accumulation might be due to the high folate receptor expression in TW1.5 cells, which resulted in the stronger affinity of TW1.5 cells to the folic acid-conjugated nanoparticles and the higher efficacy of internalization via folate receptor-mediated endocytosis, compared to TW2.6 cells. However, when the prepared fSCNA nanoparticles were fed to cells, the increase in intensity of brightness was 45.3% for TW2.6 cells and event up to 95.5% for TW1.5 cells, suggesting that the interaction between 5-ALA molecules and chitosan could be reduced by the negatively charged SCHI molecules, resulting in an ACS Paragon Plus Environment 15
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increased release of 5-ALA and accumulation of PpIX in cells. Moreover, the role of folic acid in the cellular engulfment of the prepared nanoparticles was evaluated by co-incubating 5mM free folic acid and the prepared nanoparticles with the TW1.5 and TW2.6 cells. As shown, there were no significant differences of brightness intensity between CNA and fCNA groups, revealing that free folic acid molecules could compete with fCNA for folate receptors and suppress the receptor-mediated endocytosis of the prepared particles. Additionally, the addition of free folic acid when fed with the fSCNA could only decrease the emission light brightness to 120% and 109% in TW1.5 and TW2.6 cells, respectively, suggesting that free folic acid did not affect the competition between 5-ALA and SCHI with chitosan and the 5-ALA release in the cells. Figure 5B shows the red fluorescence of PpIX in TW1.5 and TW2.6 cells excited by a green laser and observed under a spectral confocal and multiphoton system, after being fed with CNA, fCNA, fSCNA or 5-ALA for 18 hours. Pure 5-ALA or 5-ALA in the prepared nanoparticles both could be released in oral cancer cell lysosomes and then converted into PpIX, which accumulated in cells and then was excited by a green laser and emitted red light. Moreover, the fSCNA group exhibited a stronger red fluorescence than either the fCNA or CNA groups. Herein, we used the MTT assay to assess the cytotoxicity of the prepared nanoparticles on the TW1.5 and TW2.6 cells. The viability of untreated control cells was set as 100%. As shown in Figure 5C, the cell viability was as high as the untreated control group while TW1.5 cells were co-cultured with the CNA, fCNA or fSCNA. There was only a modest decrease in cell viability (about 97%) in TW2.6 cells for the CNA, fCNA, or fSCNA group. These results revealed that the prepared nanoparticles at this experimental condition would not cause the immediate cytotoxicity. ACS Paragon Plus Environment 16
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Competition binding of N-succinyl chitosan induces drug release in cells. The prepared nanoparticles, CNA, fCNA, or fSCNA, were simultaneously loaded with a membrane-impermeant fluorescent dye, calcein, by the electrostatic interaction between the negatively charged calcein and positively charged chitosan, as demonstrated in a previous study.23 Therefore, after incubating WT1.5 and WT2.6 cells with the calcein-incorporated CNA, fCNA, or fSCNA nanoparticles for 18 hours, the emission from calcein released from the prepared nanoparticles and excited by UV light was shown to increase with the modification of folic acid, and was further intensified following incorporation of SCHI (Figure 6A). Compared with TW2.6 cells, TW1.5 cells incubated with the folic acid-conjugated nanoparticles (fCNA) exhibited a substantial calcein fluorescent intensity, which might be attributed to the high expression of folate receptor on the TW1.5 cells and highly active folate receptor-mediated endocytosis. Moreover, when SCHI was incorporated into the nanoparticles and then incubated with either TW1.5 or TW2.6 cells, it exhibited two times greater fluorescent intensity than that of fCNA, suggesting that the calcein encapsulated in the fSCNA could be liberated in the lysosome by the competitive displacement reaction for binding sites on chitosan in the presence of the negatively charged SCHI molecules. In order to confirm that the competition of SCHI with 5-ALA occurred in the lysosomes of the targeted cells, we analyzed the in vitro release profiles of calcein at pH 7.4 and pH 5.0 in the presence or absence of SCHI molecules. As shown in Figure 6B, in a pH 7.4 environment, no significant difference was exhibited between the calcein release profiles of CN, fCN and fCN in the presence of SCHI, suggesting that the deprotonation of amino groups on chitosan resulted in weak ACS Paragon Plus Environment 17
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electrostatic interaction between chitosan and calcein or SCHI, and that the calcein release from the particles was mainly governed by diffusion. However, in the lysosome-mimicking buffer with a pH of 5.0, the release rate of calcein in the presence of SCHI was higher than in the absence of SCHI for fCN groups. We compared the absolute values of the slopes of the curves generated over the first five hours of the experiment. When SCHI was added into the fCN solution, the increase in the slope of the calcein release curve was 45%. This suggested that the intensity of the electrostatic interaction between the negatively charged SCHI and the positively charged chitosan might be higher than that between calcein and chitosan, thereby allowing release of calcein under these conditions (Figure 6C). These results also revealed that the release of 5-ALA in the lysosome could be improved by the incorporation of SCHI, and subsequent accumulation of PpIX might be sufficiently enhanced for fluorescent endoscopic detection.
In vivo photodynamic detection. The histology of subcutaneous tumors in C.B-17/Icr-scid-bg strain mice injected intratumorally with PBS, the prepared CNA, fSCNA, and free 5-ALA was evaluated and observed under an inverted fluorescence microscope. There was no visible red fluorescence in the mouse subcutaneous tumors when they were injected with only PBS (Figure 7); the red fluorescence of PpIX could be detected in tumor tissues in mice injected with the CNA. However, the subcutaneous tumors injected intratumorally with the prepared fSCNA exhibited clearly increased red fluorescence when compared with mice injected with free 5-ALA and CNA, suggesting that fSCNA could be substantially internalized into tumor tissues via folate receptor-mediated endocytosis, and the 5-ALA release and PpIX accumulation could actually be ACS Paragon Plus Environment 18
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improved by the SCHI incorporation. These results indicate that 5-ALA may be an optimal precursor chromophore for detecting cancer lesions and that the incorporated N-succinyl chitosan in folic acid-modified chitosan nanoparticles should be an excellent vector for this molecule.
CONCLUSIONS In this study, a folic acid-conjugated and succinate-modified chitosan nanoparticle loaded with 5-ALA was taken up by oral cancer cells via receptor-mediated endocytosis. After uptake of the nanoparticles, the 5-ALA molecules were released in the lysosomes by the reduced intensity of attraction between chitosan and 5-ALA molecules due to the presence of deprotonated succinyl residue in N-succinyl chitosan, resulting in increased 5-ALA release and PpIX accumulation for photodynamic detection. This study demonstrates that chitosan-based nanoparticles may be an excellent vector for oral-specific delivery of 5-ALA for fluorescent endoscopic detection.
REFERENCES (1) Allison, R. R.; Mota, H. C.; Sibata, C. H. Photodiagnosis and Photodynamic Therapy, 2004, 1, 263–277. (2) Allison, R. R.; Cuenca, R.; Downie, G. H.; Randall, M. E.; Bagnato, V. S.; Sibata, C. H., Photodiagnosis and Photodynamic Therapy, 2005, 2, 51–63.
(3) Pottier, R. H.; Chow, Y. F. A.; Laplante, J. P.; Truscott, T. G.; Kennedy, J. C.; Beiner, L. A. Photochem. Photobiol. 1986, 44, 679–687.
(4) Kennedy, J. C.; Pottier. R. H.; Ross. D. C. J. Photochem. Photobiol. B: Biol. 1990, 6, 143–148. ACS Paragon Plus Environment 19
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(5) Gederaas, O. A.; Rash, M. H.; Berg, K.; Lagerberg, J. W. M.; Dubbleman, T. M. A. R. J. Photochem. Photobiol. B: Biol. 1999, 49, 162–170.
(6) Berscht, P. C.; Nies, B.; Liebendorfer, A.; Kreuter, J. Biomaterials 1994, 15, 593–600. (7) Virupakshappa, B. Oral Health Dent. Manag. 2012, 11, 62–68. (8) Kah, J. C.; Kho, K. W.; Lee, C. G.; James, C.; Sheppard, R.; Shen, Z. X.; Soo, K. C.; Olivo, M. C. Int. J. Nanomedicine 2007, 2, 785–798. (9) Yang, K.; Cao, Y. A.; Shi, C.; Li, Z. G.; Zhang, F. J.; Yang, J.; Zhao, C. Oral Oncol. 2010, 46, 864–868., (10) Wu, Y. N.; Yang, L. X.; Shi, X. Y.; Li, I. C.; Biazik, J. M.; Ratinac, K. R.; Chen, D. H.; Thordarson, P.; Shieh, D. B.; Braet, F. Biomaterials 2011, 32, 4565–4573. (11) Melancon, M. P.; Lu, W.; Zhong, M.; Zhou, M.; Liang, G.; Elliott, A. M.; Hazle, J. D.; Myers, J. N.; Li, C.; Stafford, R. J. Biomaterials 2011, 32, 7600–7608. (12) Du, J.; Zhang, S.; Sun, R.; Zhang, L. F.; Xiong, C. D.; Peng, Y. X. J. Biomed. Mater. Res. Part B 2005, 72B, 299–304.
(13) Thanou, M.; Verhoef, J. C.; Junginger, H. E. Adv. Drug Deliv. Rev. 2001, 52, 117–126. (14) Agnihotri, S. A.; Mallikarjuna, N. N.; Aminabhavi, T. M. J. Control. Release 2004, 100, 5–28. (15) Qi, L.; Xu, Z.; Jiang, X.; Hu, C.; Zou, X. Carbohydr. Res. 2004, 339, 2693–2700. (16) Xu, Y.; Du, Y.; Huang, R.; Gao, L. Biomaterials 2003, 24, 5015–5022. (17) Austin, L. A.; Kang, B.; Yen, C. W.; El-Sayed, M. A. J. Am. Chem. Soc. 2011, 133, 17594–17597. (18) Park, E. K.; Lee, S. B.; Lee, Y. M. Biomaterials 2005, 26, 1053–1061. ACS Paragon Plus Environment 20
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(19) Chan, P.; Kurisawa, M.; Chung, J. E.; Yang, Y. Y. Biomaterials 2007, 28, 540–549. (20) Yang, S. J.; Lin, F. H.; Tsai, H. M.; Lin, C. F.; Chin, H. C.; Wong, J. M.; Shieh, M. J. Biomaterials 2011, 32, 2174–2182.
(21) Douglas, K. L.; Piccirillo, C. A.; Tabrizian, M. J. Control. Release 2006, 115, 354–361. (22) Kato, Y.; Onishi, H.; Machida, Y. Biomaterials 2000, 21, 1579–1585. (23) Yang, S. J.; Lin, F. H.; Tsai, K. C.; Wei, M. F.; Tsai, H. M.; Wong, J. M.; Shieh, M. J. Bioconjug. Chem. 2010, 21, 679–689.
(24) Yan, C.; Chen, D.; Gu, J.; Hu, H.; Zhao, X.; Qiao, M. Yakugaku Zasshi 2006, 126, 789–793. (25) Yang, S. J.; Shieh, M. J.; Lin, F. H.; Lou, P. J.; Peng, C. L; Wei, M. F.; Yao, C. J.; Lai, P. S.; Young T. H. Cancer Lett. 2009, 273, 210–220. (26) Gan, Q.; Wang. T.; Cochrane, C.; McCarron, P. Colloid Surf. B-Biointerface 2005, 44, 65–73. (27) Yang, S. J.; Chang, S. M.; Tsai, K. C.; Tsai, H. M.; Chen, W. S.; Shieh, M. J. J. Biomed. Mater. Res. B Appl. Biomater. 2012, 100, 1746–1754.
(28) Qi, L.; Xu, Z.; Jiang, X.; Li, Y.; Wang, M. Bioorg. Med. Chem. Lett. 2005, 15, 1397–1399. (29) Sanyakamdhorn, S.; Agudelo, D.; Tajmir-Riahi, H. A. Biomacromolecules 2013, 14, 557–563. (30) Dubé, D.; Francis, M.; Leroux, J. C.; Winnik, F. M. Bioconjug. Chem. 2002, 13, 685–692. (31) Chan, P.; Kurisawa, M.; Chung, J. E.; Yang, Y. Y. Biomaterials 2007, 28, 540–549. (32) Lu, B.; Sun, Y. X.; Li, Y. Q.; Zhang, X. Z.; Zhuo, R. X. Mol. Biosyst. 2009, 5, 629–637. (33) Hu, Y.; Jiang, X.; Ding, Y.; Ge, H.; Yuan, Y.; Yang, C. Biomaterials 2002, 23, 3193–3201. (34) Wang, S.; Low, P. S. J. Control. Release 1998, 53, 39–48.
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Table 1. The z-average diameter, zeta potential, and 5-ALA loading efficiency in the prepared nanoparticles. Sample
Z-average diameter (nm)
PDI
Zeta potential 5-ALA loading efficiency (mV) (%)
CN CNA fCN
89±1.2 77±1.1 86±7.4
0.278 0.244 0.276
29.7±2.33 24.3±2.95 19.1±0.89
― 37.7±4.65 ―
fCNA fSCN fSCNA
86±6.1 110±0.7 108±2.2
0.285 0.282 0.256
17.4±0.73 18.6±0.29 17.0±1.06
34.0±2.51 ― 72.8±8.00
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Figure 1. Schematic illustration of the preparation of folic acid-conjugated and SCHI-incorporated nanoparticles (fSCNA) for PpIX accumulation and photodynamic detection of oral cancer cells.
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-CH2-O
-COCH3 -CH-NH-
-CH2-O -COCH3 -CH-NH-
Folate aromatic protons
-CH2-
-CH2-O -COCH3
Figure 2. 1H NMR spectrum of chitosan (A), folic acid-chitosan conjugate (B) in an acetic acid-d4/D2O solution (1/4 v/v), and SCHI (C) in a sodium deuteroxide/D2O solution (4 wt. %).
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(A)
(B) CNA
fCNA
200 nm
200 nm
fSCNA
200 nm
Figure 3. Size distribution (A) and TEM photos (B) of the prepared nanoparticles.
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*
Figure 4. (A) Western blot analysis of folate receptor expression and (B) the ratio of folate receptor to α-Tubilin in TW1.5 and TW2.6 oral cancer cells, quantified with the software ImageQuant 5.1. *: p
Article
Photodynamic Detection of Oral Cancers with High Performance Chitosan-Based Nanoparticles Shu-Jyuan Yang, Cha-Fu Lin, Min-Liang Kuo, and Ching-Ting Tan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm400820s • Publication Date (Web): 02 Aug 2013 Downloaded from http://pubs.acs.org on August 4, 2013
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Photodynamic Detection of Oral Cancers with High Performance Chitosan-Based Nanoparticles Shu-Jyuan Yang,1 Cha-Fu Lin,1 Min-Liang Kuo,1,2 and Ching-Ting Tan3,* 1. Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan 2. Institute of Biochemical Sciences, College of Live Science, National Taiwan University, Taipei, Taiwan 3. Department of Otolaryngology, National Taiwan University Hospital, and National Taiwan University College of Medicine, Taipei, Taiwan *Corresponding author: Tan, C.-T. Tel: 886-2-23123456 ext; 88600 Fax: 886-2-23217522
E-mail: [email protected]; [email protected]
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ABSTRACT Oral cancer, a subtype of head and neck cancer, is one of the leading causes of cancer death and is difficult to detect in the early stages. Improved methods of detecting primary oral lesions during endoscopy would significantly improve cancer survival rates. Here we report a high-performance nanoparticle for photodynamic detection of oral cancer. Succinate-modified chitosan (SCHI) is physically complexed with folic acid-modified chitosan to form nanoparticles with a high drug loading efficiency and to improve drug release in the cellular lysosome. The z-average diameter and zeta potential of the prepared nanoparticles (fSCN) was 110.0 nm and 18.6 mV, respectively, enough to keep the nanoparticles stable in aqueous suspension without aggregating. When loaded with 5-aminolaevulinic acid (5-ALA; 72.8% loading efficiency) in the prepared fSCNA, there were no significant differences between the fSCN and fSCNA in particle size or zeta potential. Moreover, the fSCNA nanoparticles were readily engulfed by oral cancer cells via folate receptor-mediated endocytosis. The release of loaded 5-ALA in the lysosome was promoted by the reduced attraction intensity between chitosan and 5-ALA via the deprotonated SCHI molecules, which resulted in a higher accumulation of intracellular protoporphyrin IX (PpIX) for photodynamic detection. These results demonstrate that N-succinyl-chitosan-incorporated and folic acid-conjugated chitosan nanoparticles are an excellent vector for oral-specific delivery of 5-ALA for fluorescent endoscopic detection. Keywords: oral cancer, chitosan, N-succinyl chitosan, 5-aminolevulinic acid, nanoparticle, fluorescence
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INTRODUCTION Photo-detection, employing an exogenous chromophore excited by optima light to generate fluorescence, is one of the most promising non-invasive methods used recently to detect malignant or premalignant tissue.1, 2 A photosensitive fluorophore, protoporphyrin IX (PpIX) can be used as a source of fluorescence, because it can be converted from 5-aminolevulinic acid (5-ALA) during the heme group synthesis process in the cell mitochondria and decomposed at different rates in cancer and normal cells.3, 4 5-ALA is a hydrophilic and zwitterionic drug that has a poor affinity toward the cell membrane in a physiological environment and is very easily taken up by bacteria to produce misleading results in endoscopic observation.5 Therefore, a suitable carrier is needed that can prevent bacterial uptake of 5-ALA and help it pass through the lipophilic barrier for photo-detection.6
Oral cancer, a subtype of head and neck cancer, is the sixth most common cancer among men and women worldwide. It is one of the leading causes of mortality rate and is commonly difficult to detect at an early and treatable stage.7 Current clinical diagnostic procedures for oral cancer typically involve performing invasive needle biopsies followed by histological examination of the excised tissue, a process that may cause patients psychological trauma and create risk of infection. Moreover, precancerous lesions appear harmless or occur in hidden sites, with the result that they are often overlooked even with white-light endoscopy. Therefore, finding a powerful and highly sensitive tool for the detection of precancerous lesions is very important.8 Several methods using nano-engineered materials have been developed that might prove effective for detecting the disease at an early stage.9-11 ACS Paragon Plus Environment 3
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Chitosan is a linear polymer composed of 2-amino-2-deoxy-β-D-glucan by glycosidic linkages. It is biocompatible, biodegradable and possesses antibacterial properties that can prevent it from bacterial uptake in the GI track.12 A number of free amino groups on the molecular chain give chitosan several special characteristics, such as its ability to easily approach the cell membrane and muco-adhere to locations of interest with a long retention time, making it useful for pharmaceutical applications.13-15 Furthermore, its cationic nature also allows for ionic cross-linking with multivalent anions, such as sodium tripolyphosphate (STPP), to prepare nanoparticles in aqueous solution without using toxic organic solvents.16 In order to enhance the nanoparticle targeting and engulfment by the targeted cells, several targeting ligands have been conjugated onto the particle surface.9,
11, 17
Folic acid, a stable,
inexpensive, and poorly immunogenic chemical with a high affinity for the folate receptor is useful for enhancing nanoparticle endocytosis via folate receptor-mediated endocytosis.18, 19 Because the folate receptor is overexpressed on many human epithelial cancer cells, the conjugation of folic acid with drugs and macromolecules via folate’s γ-carboxyl moiety can improve their uptake and targeting ability. In previous studies, it has been shown that the incorporation of negatively charged polymers, such as alginate, can improve the drug releasing or transfection efficiency of chitosan-based nanoparticles by reducing the intensity of interactions between chitosan and the drug or DNA.20, 21 Therefore, the N-succinyl chitosan (SCHI) can be exploited as a negatively charged polymer to enhance the 5-ALA releasing in oral cancer cells. The synthesis of SCHI can be proceeded by the introduction of succinyl groups into chitosan at the N-position of the glucosamine units, ACS Paragon Plus Environment 4
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transforming chitosan molecules from acetic acid aqueous solution-soluble into water-soluble.22 Furthermore, it has low toxicity and is less biodegradable in the body, therefore can be expected to be useful as a drug carrier that allows long-term retention in the body. The purpose of this study was to modify chitosan molecules with folic acid and succinate at the N-position of the glucosamine units, respectively, and then physically incorporate N-succinyl chitosan into the folic acid-modified chitosan nanoparticles as a 5-ALA carrier (fSCNA). With these prepared fSCNA nanoparticles, 5-ALA could be easily taken up into oral cancer cells via folate receptor-mediated endocytosis. In addition, the release of 5-ALA in cancer cells could be meliorated by the anionic N-succinyl residue competing with the 5-ALA for the cationic residues of chitosan, enhancing the accumulation of PpIX for fluorescent endoscopic detection (Figure 1).
EXPERIMENTAL SECTION Materials. Chitosan (85% deacetylation and 15 kDa MW) was purchased from Polysciences, Inc. (Pennsylvania, USA); 5-aminolevulinic acid (5-ALA), succinic anhydride and sodium tripolyphosphate (STPP) were purchased from Sigma-Aldrich Co. (Missouri, USA). Folic acid came from TCI (Tokyo, Japan). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from Acros Organics (New Jersey, USA); anhydrous dimethyl sulfoxide (DMSO) was purchased from Riedel-de Haën (Germany). All were reagent grade and used without further purification.
Conjugation of folic acid or succinic anhydride with chitosan. The process of conjugation of folic acid to chitosan molecules was performed as previously described.23 First, folic acid and EDC ACS Paragon Plus Environment 5
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were thoroughly dissolved and mixed in 20 ml anhydrous DMSO (molar ratio 1:1) and then added slowly to 0.5 % (w/v) chitosan in acetic acid aqueous solution (0.1 M, pH 4.7). Folic acid-chitosan conjugate was formed at the molar ratio of folic acid to amino groups of 0.1. Stirring at room temperature in the dark for 16 hours, the solution was brought to pH 9.0 with 1M NaOH aqueous solution and centrifuged at 2,500 rpm to spin down the folic acid-chitosan conjugate. The precipitate was dialyzed against phosphate buffered saline (PBS) and water for seven days, and then isolated as a sponge by lyophilization and kept for follow-up study. SCHI was synthesized by referring the procedure described by Yang et al.24 1g chitosan powder was added into 2% (w/v) of succinic anhydride pre-dissolved in 100 ml anhydrous DMSO solution, and reacted at 70 oC for 24 hours. The solution was dialyzed first against 0.01M NaOH aqueous solution for two days and then against water for five days to remove the remanent succinic anhydride and DMSO. Finally, the SCHI was isolated as a sponge by lyophilization and kept for follow-up study.
Characterization of the N-succinyl chitosan and folic acid-chitosan conjugate. The 1H-NMR spectra of folic acid-chitosan conjugate and SCHI were recorded on a Bruker Avance-400MHz NMR spectrometer (Bruker BioSpin, MA, USA) using deuterium acetic acid-d4/ deuterium oxide solution (1/4 v/v) and 1 N Sodium deuteroxide solution, respectively, as solvents.
Synthesis of nanoparticles. The chitosan nanoparticles loaded with 5-ALA were prepared based on the ionic gelation interaction between positively charged chitosan and negatively charged 5-ALA ACS Paragon Plus Environment 6
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and STPP at room temperature. As described in previous studies,20, 23, 25 0.05 % chitosan and folic acid-chitosan conjugate solutions were prepared by dissolving 0.5 mg chitosan powder and folic acid-chitosan conjugate sponge in 1 ml 0.01 M acetic acid solution at pH 4.0, respectively; 0.15% 5-ALA solution was prepared by dissolving 1.5 mg 5-ALA powder in 1 ml 0.05% STPP-PBS solution. Either 2 ml of STPP-PBS solution or 2 ml of 5-ALA solution were added to 5 ml of chitosan or folic acid-chitosan conjugate solution to prepare the CN, CNA, fCN and fCNA nanoparticle suspensions. The preparation of fSCN and fSCNA was similar to that of fCN and fCNA, but the STPP and 5-ALA solutions were exchanged for 0.015% SCHI and 5-ALA/SCHI (10/1 w/w) solutions, respectively, which were pre-dissolved in 0.05% STPP-PBS solution. The prepared suspended nanoparticle solutions were later used directly without further treatment for the characteristic determination.
Characteristics of the prepared nanoparticles. Measurements of particle size and zeta potential of various nanoparticles were performed using the Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK) by dynamic light scattering measurements and laser Doppler electrophoresis, respectively. Particle size was measured at 25 °C with a 90° scattering angle based on the Zetasizer Nano-ZS90 internal setting. Measurements of zeta potential were made using the aqueous flow cell in automatic mode at 25 °C.26 Carbon-coated 200 mesh copper grids pre-immersed in CNA, fCNA and fSCNA solutions were placed on filter paper to absorb excess liquid and then dried under a lamp for 30 minutes. The dried copper grids with CNA, fCNA, and fSCNA were examined under the Hitachi TEM H-7650 ACS Paragon Plus Environment 7
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(Tokyo, Japan). The loading efficiency of 5-ALA in the prepared nanoparticles was analyzed by centrifuging the particle solution at a 20,000 g force to spin down the particles. The suspension was collected and then reacted with 2,4,6-trinitrobenzene sulfonic acid (TNBS) as an assay reagent to detect 5-ALA. The assay was performed using a TNBS kit (Pierce Chemical Company; Rockford, IL) according to the manufacturer’s instructions, and a serial of gradient 5-ALA standard solutions were used to prepare the calibration curve. The loading efficiency of 5-ALA in the nanoparticles was calculated with the following equation:
Loading efficiency (%) =
Ct − C Ct
f
×100 %
where Ct and Cf were the total amount of 5-ALA and the free amount of 5-ALA in the suspension, respectively.
Determination of folate receptor expression among oral cancer cell lines. Western blotting analysis was done to determine the expression of folate receptor on cell membranes as previously described.23 Two oral cancer cell lysate samples (TW1.5 and TW2.6) were collected by scraping the cell cultures into cold RIPA buffer (Millipore, Temecula, CA) containing protease inhibitors. The protein concentrations were assayed using the Pierce® BCA protein assay (Thermo scientific, IL, USA) with bovine serum albumin as the standard. Total protein (30 µg) was separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transblotted. Anti-human folate receptor antibody was purchased from Santa Cruz Biotechnology, Inc. (CA, USA); anti-α-tubulin was purchased from Sigma-Aldrich. The signals of anti-human folate receptor in the blots were ACS Paragon Plus Environment 8
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quantified with ImageQuant 5.1 software, and the values were normalized to α-tubulin expression.
Measurement of accumulated PpIX and cytotoxicity of the prepared nanoparticles. Cell lines TW1.5 and TW2.6 were cultured in 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture (GIBCOTM; Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum, at 37 °C in an atmosphere of 5% CO2, respectively. Cultured medium was changed on alternate days until confluent. TW1.5 and TW2.6 oral cancer cell lines were seeded onto 24-well culture plates, respectively, at the cell density of 1×104 per well. After cell lines were cultured for 24 hours, the medium was replaced with fresh medium containing CNA, fCNA and fSCNA, where the CNA, fCNA and fSCNA solutions were concentrated to 10 times by Amicon Ultra centrifugal filter devices (Millipore, Billerica, MA, USA) to remove the unloaded 5-ALA beforehand. Cells were further cultured for 18 hours to allow engulfment of nanoparticles and to convert 5-ALA to PpIX. In order to determine the association of folic acid-folate receptor mediated endocytosis with the endogenously synthesized PpIX, fresh culture media, both with and without free folic acid (5mM), were tested at the same time. After removal of the cultured medium and PBS wash, the PpIX were extracted with DMSO. The fluorescent intensity of PpIX was determined by spectrofluorometer (Molecular Device SPECTRA MAX GEMINE XS; Sunnyvale, CA) with an excitation wavelength of 405 nm and the emission wavelength at 635 nm. Fluorescent intensity was expressed as an accumulative percentage. For determination of the effect of the prepared nanoparticles on cell toxicity, TW1.5 or TW2.6 cells seeded onto 24-well platesACS wereParagon treated Plus withEnvironment fresh medium containing CNA, and fCNA, 9
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fSCNA. After 18 hours of incubation, the particle-containing medium was replaced with 500 µl of fresh medium that contained 0.5 mg/ml MTT assay reagent (Alfa Aesar, San Francisco, CA), and then incubated at 37°C for 3 hours. Then, the MTT-containing medium was replaced with 200 µl of acid-isopropanol solution (0.04 M HCl) to dissolve formazan, the MTT product. Finally, 150 µl samples were transferred to 96-well plates and the absorbance was measured at 570 nm with the Thermo Scientific Multiskan® FC microplate photometer (Thermo Fisher Scientific Inc., USA). The viability of untreated control cells was defined as 100%. To evaluate the feasibility of using the prepared nanoparticles for detection of oral cancer cells, the glass plate was placed in a Petri dish and seeded with either TW1.5 or TW2.6 cells. After cells were cultured for 24 hours, medium was replaced with fresh medium containing CNA, fCNA, fSCNA and 5-ALA. To confirm the nanoparticle uptake content, based on a correlation between the folate receptor expression and the folic acid modification, a fluorescent compound, calcein, was incorporated simultaneously into CNA, fCNA and fSCNA as a fluorescent marker. After 18 hours, cells were washed three times with PBS, fixed with 10% formalin and then examined under a spectral confocal and multiphoton system (Leica TCS SP5, Wetzlar, Germany). The average fluorescent intensity of calcein in cells was quantified with the MetaMorph® imaging software (Version 7.5, Molecular Devices, USA) to demonstrate the content of nanoparticle uptake. The data represented an average of five regions obtained from randomly chosen areas of each sample, and the brightness of the green emission light of the CNA group was designated as 100% for the individual oral cancer cell lines.
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Competition binding assay. The competition binding assay was done as previously described:20 10 ml of calcein-loaded fCN nanoparticle solution was concentrated to 10 times to remove the unloaded calcein, then mixed with 0.14 ml of PBS solution or 0.14 ml of 0.3% SCHI solution in a CelluSep dialysis membrane (Membrane Filtration Products, Seguin, TX, USA) with the molecular weight cut-off of 3.5 kD. In order to simulate the pH value in the cytoplasm and lysosomal environments, the dialysis membrane was soaked in 45 ml of buffer at pH 7.4 or pH 5.0 at 37 °C, respectively. After shaking for a period of time, 0.1 ml of the buffer was removed, and the optical density (OD) of calcein was recorded at a wavelength of 500 nm using the Thermo Scientific Multiskan® FC microplate photometer. The release profiles are represented as the correlation between the OD value of calcein and the incubation period.
Generation of subcutaneous tumor models and in vivo photodynamic detection. Because it is difficult to develop and treat oral cancers in mouse’s mouth, the subcutaneous tumor models were generated for in vivo photodynamic detection. The in vivo experimental protocols were approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC). As with our previous study,27 female C.B-17/Icr-scid-bg mice (4 weeks old, weighing 18–20 g) were subcutaneously inoculated in their flanks with 1×106 TW1.5 cells per mouse to generate a subcutaneous (SC) tumor model. Tumor sizes were measured using calipers, and tumor volume was calculated using the following formula: volume=width2×length×0.52. When the volume of SC tumors reached about 100 mm3, the mice were randomly divided into four treatment groups (n = 6 for each group): (a) injected intratumorally ACS Paragon Plus Environment 11
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(i.t.) with 50 µl of PBS solution, (b) injected i.t. with 50 µl of the prepared CNA solution in a 5-ALA concentration of 5 mM, (c) injected i.t. with 50 µl of the prepared fSCNA solution in a 5-ALA concentration of 5 mM, and (d) injected i.t. with 50 µl of 5 mM free 5-ALA solution. Eighteen hours after injection, the tumor tissues were resected and imbedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA), and then snap-frozen on dry ice. Cryosections of 5 µm thickness were fixed with acetone at 4 oC for 5 min and examined directly using a Zeiss Axiovert 200 M inverted fluorescence microscope (Carl Zeiss, Göttingen, Germany).
Statistical Analysis. Mean±standard deviation (SD) and graphs were used to describe the data. One-way analysis of variance (ANOVA) with post hoc muti comparison methods, such as Fisher’s least significant difference test and Tamhane’s T2 test, was used to assess the differences in the fluorescent intensity of PpIX and calcein in oral cancer cells fed with CNA, fCNA and fSCNA. All p values were two-sided and their significance level was 0.05. The statistical package for social
sciences 11.0 (SPSS 11.0) software was used to conduct all statistical analysis.
RESULTS AND DISCUSSION Chitosan was chosen as a carrier for ionized drugs because of its many free amino groups and quick micro/nanoparticle-forming ability once in contact with polyanions.28, 29 When covalently conjugated with folic acid, chitosan molecules had a high affinity for folate receptors and cellular uptake ability via folate receptor-mediated endocytosis.23 Moreover, the incorporation of an anionic polymer into the chitosan-based nanoparticles would enhance the release of loading drug.20, 21
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Characterization of the N-succinyl chitosan and folic acid-chitosan conjugate. Successful synthesis of folic acid-chitosan conjugate can be confirmed by 1H NMR spectroscopy; the 1H NMR spectra of chitosan and folic acid-chitosan conjugate are shown in Figure 2A and B. The signals at δ 2.00, 3.12 and 3.67-3.85 ppm were assigned to the resonance of the monosaccharide residue
protons, -COCH3, -CH-NH- and –CH2-O-, respectively.30, 31 Compared with the 1H NMR spectra of chitosan, the signals appearing at δ 6.78-8.70 ppm in the 1H NMR spectra of folic acid-chitosan conjugate were attributed to the resonance of the folate aromatic protons. They revealed that the coupling of the folate residue to the chitosan could be achieved via EDC-mediated reaction.23, 30 Because of the reaction conditions chosen and the steric constraints, folic acid could be successfully conjugated onto chitosan molecules via its γ-carboxyl moiety mostly.23 Moreover, the 1H NMR spectra of SCHI is shown in Figure 2C. The multiplets at δ 2.28–2.57 ppm were assigned to the methylene groups, -CH2-, of succinate, revealing that the modification of the succinyl residue onto
the chitosan could also be achieved by ring-opening reaction.24, 32
Characteristics of the prepared nanoparticles. The z-average diameter, zeta potential, and loading efficiency of the chitosan-based nanoparticles prepared under different conditions are shown in Table 1. The z-average diameter of the prepared nanoparticles did not vary significantly with 5-ALA loading, but increased remarkably from 77 to 110 nm when folic acid was conjugated and SCHI was incorporated. The zeta potential analysis showed that the nanoparticles prepared under different conditions all bore a positive charge, but the charge decreased with the simultaneous folate-conjugation (fCN) and SCHI-incorporation (fSCN). When the CN, fCN, or fSCN were ACS Paragon Plus Environment 13
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loaded with 5-ALA (CNA, fCNA, and fSCNA, respectively), the zeta potential exhibited no significant decreases. These results revealed that the z-average diameter and zeta potential of the prepared nanoparticles depended on the folate-conjugation and SCHI-incorporation but not on the 5-ALA-load Moreover, zeta potential greatly influences particle stability through electrostatic repulsion between particles and may result in a narrower size distribution.33 As shown in Table 1 and Figure 3A, the zeta potential of the CN and fCNA was about 20-29 mV, providing enough surface charge to stabilize the particles from aggregation and showing a narrower size distribution (in the range of 20-200 nm). When the protoned amino groups of chitosan were displaced with the negative succinyl derivatives, the zeta potential of the fSCN decreased as far as 18 mV and the particle size distribution fell into the range of 30-500 nm. Figure 3B shows the particle morphology of CNA, fCNA and fSCNA as determined by transmission electron microscopic examination (TEM): all formed as solid spheres and were as small as the z-average diameter determined by Zetasizer Nano-ZS90. The loading efficiency of 5-ALA in CNA was 37.7% and decreased to 34.0% with folic acid conjugation; however, when simultaneously incorporating SCHI into fCNA to form fSCNA, the loading efficiency increased to 72.8%. It was suggested that in fCNA particles, the replacement of electro-neutral folate derivatives reduced the numbers of protonated amino groups on chitosan molecules to attract 5-ALA molecules,29 and the spatial hindering of folate derivatives resisted the interaction between chitosan and 5-ALA molecules, resulting in a lower loading efficiency than the prepared CNA. Moreover, because of the zwitterionic property of 5-ALA,25 partial 5-ALA molecules would bear positive charges and interact with the negatively charged SCHI molecules in ACS Paragon Plus Environment 14
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the prepared conditions, resulting in a high 5-ALA loading efficiency in the prepared fSCNA. Based on these evidences, the loading efficiency also appeared strongly dependent on the folic acid conjugation and SCHI incorporation.
Correlation between folate receptor expression and PpX accumulation in oral cancer cell lines. It is well known that folate receptor is over-expressed on various types of cancer cell membrane; these cells can actively regulate the internalization of folic acid-conjugated molecules through folate receptor-mediated endocytosis.34 Therefore, the western blotting method was used to determine the expression of folate receptor on TW1.5 and TW2.6 oral cancer cell lines with results shown in Figure 4. It was apparent that expression of folate receptor depended on the cell line: TW1.5 cells exhibited 1.3-fold higher expression of folate receptors than did TW2.6 cells. Figure 5A shows the relative fluorescent intensity of PpIX in TW1.5 and TW2.6 cells cultured in a medium with or without free folic acid addition, and the brightness of the emission light of the CNA group was designated as 100% for the individual oral cancer cell lines. When the cells were co-cultured with fCNA for 18 hours, the intensity of emission light in TW1.5 and TW2.6 cells increased to 136.3% and 119.5%, respectively. This enhancement of PpIX accumulation might be due to the high folate receptor expression in TW1.5 cells, which resulted in the stronger affinity of TW1.5 cells to the folic acid-conjugated nanoparticles and the higher efficacy of internalization via folate receptor-mediated endocytosis, compared to TW2.6 cells. However, when the prepared fSCNA nanoparticles were fed to cells, the increase in intensity of brightness was 45.3% for TW2.6 cells and event up to 95.5% for TW1.5 cells, suggesting that the interaction between 5-ALA molecules and chitosan could be reduced by the negatively charged SCHI molecules, resulting in an ACS Paragon Plus Environment 15
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increased release of 5-ALA and accumulation of PpIX in cells. Moreover, the role of folic acid in the cellular engulfment of the prepared nanoparticles was evaluated by co-incubating 5mM free folic acid and the prepared nanoparticles with the TW1.5 and TW2.6 cells. As shown, there were no significant differences of brightness intensity between CNA and fCNA groups, revealing that free folic acid molecules could compete with fCNA for folate receptors and suppress the receptor-mediated endocytosis of the prepared particles. Additionally, the addition of free folic acid when fed with the fSCNA could only decrease the emission light brightness to 120% and 109% in TW1.5 and TW2.6 cells, respectively, suggesting that free folic acid did not affect the competition between 5-ALA and SCHI with chitosan and the 5-ALA release in the cells. Figure 5B shows the red fluorescence of PpIX in TW1.5 and TW2.6 cells excited by a green laser and observed under a spectral confocal and multiphoton system, after being fed with CNA, fCNA, fSCNA or 5-ALA for 18 hours. Pure 5-ALA or 5-ALA in the prepared nanoparticles both could be released in oral cancer cell lysosomes and then converted into PpIX, which accumulated in cells and then was excited by a green laser and emitted red light. Moreover, the fSCNA group exhibited a stronger red fluorescence than either the fCNA or CNA groups. Herein, we used the MTT assay to assess the cytotoxicity of the prepared nanoparticles on the TW1.5 and TW2.6 cells. The viability of untreated control cells was set as 100%. As shown in Figure 5C, the cell viability was as high as the untreated control group while TW1.5 cells were co-cultured with the CNA, fCNA or fSCNA. There was only a modest decrease in cell viability (about 97%) in TW2.6 cells for the CNA, fCNA, or fSCNA group. These results revealed that the prepared nanoparticles at this experimental condition would not cause the immediate cytotoxicity. ACS Paragon Plus Environment 16
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Competition binding of N-succinyl chitosan induces drug release in cells. The prepared nanoparticles, CNA, fCNA, or fSCNA, were simultaneously loaded with a membrane-impermeant fluorescent dye, calcein, by the electrostatic interaction between the negatively charged calcein and positively charged chitosan, as demonstrated in a previous study.23 Therefore, after incubating WT1.5 and WT2.6 cells with the calcein-incorporated CNA, fCNA, or fSCNA nanoparticles for 18 hours, the emission from calcein released from the prepared nanoparticles and excited by UV light was shown to increase with the modification of folic acid, and was further intensified following incorporation of SCHI (Figure 6A). Compared with TW2.6 cells, TW1.5 cells incubated with the folic acid-conjugated nanoparticles (fCNA) exhibited a substantial calcein fluorescent intensity, which might be attributed to the high expression of folate receptor on the TW1.5 cells and highly active folate receptor-mediated endocytosis. Moreover, when SCHI was incorporated into the nanoparticles and then incubated with either TW1.5 or TW2.6 cells, it exhibited two times greater fluorescent intensity than that of fCNA, suggesting that the calcein encapsulated in the fSCNA could be liberated in the lysosome by the competitive displacement reaction for binding sites on chitosan in the presence of the negatively charged SCHI molecules. In order to confirm that the competition of SCHI with 5-ALA occurred in the lysosomes of the targeted cells, we analyzed the in vitro release profiles of calcein at pH 7.4 and pH 5.0 in the presence or absence of SCHI molecules. As shown in Figure 6B, in a pH 7.4 environment, no significant difference was exhibited between the calcein release profiles of CN, fCN and fCN in the presence of SCHI, suggesting that the deprotonation of amino groups on chitosan resulted in weak ACS Paragon Plus Environment 17
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electrostatic interaction between chitosan and calcein or SCHI, and that the calcein release from the particles was mainly governed by diffusion. However, in the lysosome-mimicking buffer with a pH of 5.0, the release rate of calcein in the presence of SCHI was higher than in the absence of SCHI for fCN groups. We compared the absolute values of the slopes of the curves generated over the first five hours of the experiment. When SCHI was added into the fCN solution, the increase in the slope of the calcein release curve was 45%. This suggested that the intensity of the electrostatic interaction between the negatively charged SCHI and the positively charged chitosan might be higher than that between calcein and chitosan, thereby allowing release of calcein under these conditions (Figure 6C). These results also revealed that the release of 5-ALA in the lysosome could be improved by the incorporation of SCHI, and subsequent accumulation of PpIX might be sufficiently enhanced for fluorescent endoscopic detection.
In vivo photodynamic detection. The histology of subcutaneous tumors in C.B-17/Icr-scid-bg strain mice injected intratumorally with PBS, the prepared CNA, fSCNA, and free 5-ALA was evaluated and observed under an inverted fluorescence microscope. There was no visible red fluorescence in the mouse subcutaneous tumors when they were injected with only PBS (Figure 7); the red fluorescence of PpIX could be detected in tumor tissues in mice injected with the CNA. However, the subcutaneous tumors injected intratumorally with the prepared fSCNA exhibited clearly increased red fluorescence when compared with mice injected with free 5-ALA and CNA, suggesting that fSCNA could be substantially internalized into tumor tissues via folate receptor-mediated endocytosis, and the 5-ALA release and PpIX accumulation could actually be ACS Paragon Plus Environment 18
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improved by the SCHI incorporation. These results indicate that 5-ALA may be an optimal precursor chromophore for detecting cancer lesions and that the incorporated N-succinyl chitosan in folic acid-modified chitosan nanoparticles should be an excellent vector for this molecule.
CONCLUSIONS In this study, a folic acid-conjugated and succinate-modified chitosan nanoparticle loaded with 5-ALA was taken up by oral cancer cells via receptor-mediated endocytosis. After uptake of the nanoparticles, the 5-ALA molecules were released in the lysosomes by the reduced intensity of attraction between chitosan and 5-ALA molecules due to the presence of deprotonated succinyl residue in N-succinyl chitosan, resulting in increased 5-ALA release and PpIX accumulation for photodynamic detection. This study demonstrates that chitosan-based nanoparticles may be an excellent vector for oral-specific delivery of 5-ALA for fluorescent endoscopic detection.
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Table 1. The z-average diameter, zeta potential, and 5-ALA loading efficiency in the prepared nanoparticles. Sample
Z-average diameter (nm)
PDI
Zeta potential 5-ALA loading efficiency (mV) (%)
CN CNA fCN
89±1.2 77±1.1 86±7.4
0.278 0.244 0.276
29.7±2.33 24.3±2.95 19.1±0.89
― 37.7±4.65 ―
fCNA fSCN fSCNA
86±6.1 110±0.7 108±2.2
0.285 0.282 0.256
17.4±0.73 18.6±0.29 17.0±1.06
34.0±2.51 ― 72.8±8.00
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Figure 1. Schematic illustration of the preparation of folic acid-conjugated and SCHI-incorporated nanoparticles (fSCNA) for PpIX accumulation and photodynamic detection of oral cancer cells.
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-CH2-O
-COCH3 -CH-NH-
-CH2-O -COCH3 -CH-NH-
Folate aromatic protons
-CH2-
-CH2-O -COCH3
Figure 2. 1H NMR spectrum of chitosan (A), folic acid-chitosan conjugate (B) in an acetic acid-d4/D2O solution (1/4 v/v), and SCHI (C) in a sodium deuteroxide/D2O solution (4 wt. %).
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(A)
(B) CNA
fCNA
200 nm
200 nm
fSCNA
200 nm
Figure 3. Size distribution (A) and TEM photos (B) of the prepared nanoparticles.
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*
Figure 4. (A) Western blot analysis of folate receptor expression and (B) the ratio of folate receptor to α-Tubilin in TW1.5 and TW2.6 oral cancer cells, quantified with the software ImageQuant 5.1. *: p
