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Singlet Oxygen Production and Tunable Optical Properties of Deacetylated Chitin-Porphyrin Cross-Linked Films Kai Li, Paula Berton, Steven P. Kelley, and Robin D. Rogers Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00605 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 18, 2018
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Biomacromolecules
Singlet Oxygen Production and Tunable Optical Properties of Deacetylated Chitin-Porphyrin Cross-Linked Films Kai Li,a,b Paula Berton,a,† Steven P. Kelley,a and Robin D. Rogersa,b,c,* a
Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montreal, QC H3A 0B8, Canada b College of Arts & Sciences, The University of Alabama, Tuscaloosa, AL 35487, USA c 525 Solutions, Inc., P.O. Box 2206, Tuscaloosa, AL 35403, USA *
Corresponding author:
[email protected] Abstract The increasing need for biocompatible materials as supports to immobilize photosensitizer molecules for photodynamic therapy (PDT), led us to investigate the use of chitin as a support for 4,4′,4′′,4′′′-(porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) (mTCPP) for singlet oxygen production. Chitin was first extracted from shrimp shells using the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), coagulated as a floc into water, and then deacetylated to varying degrees of deacetylation using 4 M NaOH. The deacetylated chitin (DA-chitin) was dissolved in [C2mim][OAc] and mTCPP was covalently attached by reaction between the amino groups of DA-chitin and the carboxyl groups of mTCPP using N-(3-dimethylaminopropyl)-Nˊethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as activators. The resulting composite polymers were cast as a film and coagulated with water to remove IL and excess reagents, resulting in homogenous DA-chitin/mTCPP films. Attempts to prepare films by coagulation from a solution containing chitin and mTCPP to physically entrap the porphyrin, resulted in aggregation of mTCPP in the film. The DA-chitin/mTCPP films had strong optical absorbance and their absorbance intensity could be tuned by changing the mTCPP content and degrees of deacetylation of DA-chitin in a predictive manner. In addition, metal ions (Cu2+, Zn2+, Gd3+, and Fe3+) could be easily chelated into the DA-chitin/mTCPP films through mixing metal salt solutions with the films and heating. After chelating metal ions, optical properties, such as absorption region and intensities, of the films changed, suggesting chelating metal ions could tune their optical properties. Moreover, the DA-chitin/mTCPP films could generate singlet oxygen under light irradiation, and hence might serve as a photosensitizer in PDT. The methodology used in this study is also applicable for developing other functional biomaterial devices. 1 ACS Paragon Plus Environment
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Introduction The production of reactive oxygen species (ROS) using immobilized photosensitizers is attracting increased attention for photodynamic therapy (PDT).1 In PDT, the photosensitizer, e.g., a porphyrin, phthalocyanine, chlorin, or porphycene, is activated by light irradiation and generates ROS, 3-,4,5,6 mainly singlet oxygen (1O2), to elicit cell death. For several types of cancers, the use of solid supports for PDT allows the recovery and removal of the device after treatment.
7 , 8
Although solid supports, such as polyacrylamide resins,
9
silica,
10
gold
nanoparticles, 11 and quantum dots 12 have been developed, biocompatibility is one the most important properties when choosing supports for this application. 13 , 14 Therefore, there is an increasing need for more biocompatible materials to immobilize photosensitizers. Biopolymers have been proposed in the last decade for materials with medical applications due to their sustainability and biocompatibility. 15 , 16 Among biopolymers, chitin, which is composed of β(1-4) linked N-acetylglucosamine units (Fig. 1),17 possesses attractive properties, such as biocompatibility, biodegradability, nontoxicity, and the ability to promote tissue regrowth. 18-20 This biopolymer and its derivatives have been applied in the medical area for wound dressings,19 tissue engineering,20 drug delivery,21 and cancer treatment.22 However, the insolubility of chitin in almost any non-reactive solvent and the need for harsh solvent systems to isolate and dissolve it greatly complicate the production of chitin-based medical devices.23,24
Figure 1. The structure of chitin, deacetylated chitin, and 4,4′,4′′,4′′′-(porphine-5,10,15,20tetrayl)tetrakis(benzoic acid) (mTCPP). Ionic liquids (IL), defined as salts with melting points below 100 °C,25 have shown excellent capabilities to dissolve chitin and other biopolymers.23,26,27 In particular, the IL 1-ethyl-3-methyl-
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Biomacromolecules
imidazolium acetate ([C2mim][OAc]) allows the direct extraction of chitin from shrimp shells, without the need of acid and base treatments. 28 Since the IL dissolves pure chitin, IL-based solution processes can also be used to manipulate the chitin into materials with different shapes, functionalities, and applications.19,26,29-33 Because of the unique N-acetylglucosamine groups on chitin, its chemical functionalization is relatively simple. Most frequently, it is accomplished by deacetylation of chitin to give chitosan, a polymer of glucosamine that typically has better solubility and lower strength than chitin 19,32 Chitosan and its derivatives have been used in a vast range of applications, including as a support for Rose Bengal as a photosensitizer to generate singlet oxygen for the photoxidation of organic molecules in water treatment. 34-,35,36 We envisioned that the use of chitin as a support would provide stronger, insoluble materials with similar photoactivity which would be suitable for applications where dissolution of the photosensitizer is not desired, such as a medical device that can be implanted and removed from the body. Our group took advantage of the good solubility of chitin in ILs and prepared hydrogels based on chemically cross-linked chitin using epichlorohydrin as cross-linker and suggested IL-based process could aid in fabricating materials from biopolymers.37 In another example, using the IL-based process, we generated chitin fibers which could be surface deacetylated and functionalized with an amidoxime extractant for the extraction of uranium from seawater.32 Using this approach, only the surface of the material was modified, thus combining the strength and low solubility of the core chitin and the functionalization of the surface for that particular application. Here, we explored the use of deacetylated chitin (extracted from shrimp shells) as a solid support
for
the
photosensitizer
porphyrin
4,4′,4′′,4′′′-(porphine-5,10,15,20-
tetrayl)tetrakis(benzoic acid) (mTCPP, Fig. 1) for PDT applications. We hypothesized that, after partial deacetylation, the amino groups of the deacetylated chitin would react with the carboxylic acid groups of mTCPP in an IL solution, and a composite film could be directly cast without additional steps.33 We also believed that a partially deacetylated chitin prepared from our higher MW shrimp-shell extracted chitin could be used to form stable films, where commercial chitosan is often unsuitable for such purposes. It was anticipated that the incorporated porphyrin would endow the DA-chitin/mTCPP film the ability to generate singlet oxygen under light irradiation and afford tunable optical properties.
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Experimental Chemicals and Materials All chemicals were used as received except as noted. The IL [C2mim][OAc] (> 95% purity) was purchased from IoLiTec, Inc. (Tuscaloosa, AL). N-(3-dimethylaminopropyl)-Nˊethylcarbodiimide hydrochloride (EDC bioxtra), N-hydroxysuccinimide (NHS, > 98% purity), mTCPP, zinc(II) acetate ([Zn(OAc)2]), copper(II) acetate ([Cu(OAc)2], > 98% purity), iron (II) chloride (FeCl2, > 98% purity), gadolinium acetate ([Gd(OAc)3]), 9,10-dimethylanthracene (DMA,
99%
purity),
meso-tetraphenylporphyrin
(TPP,
99%
purity),
and
N,
N-
dimethylmethanamide (DMF) were purchased from Sigma-Aldrich (Milwaukee, WI). Sodium hydroxide (NaOH, > 97% purity) was bought from ACP Chemicals Inc. (Montreal, QC). Raw Black Tiger shrimp shells (Penaeus monodon) were kindly donated by a local restaurant (Montreal, QC). Commercial chitosan was bought from Bonding Chemistry (Medical grade, 100% degree of deacetylation (DD), Katy, TX).
Extraction and Deacetylation of Chitin Chitin was first extracted from the Raw Black Tiger shrimp shells using [C2mim][OAc] according to our reported method with small modifications.19 In general, 1 g shrimp shells powder (
