Photodynamic therapy of melanoma skin cancer using carbon dot – chlorin e6 – hyaluronate conjugate

doi:10.1016/j.actbio.2015.08.027

Acta Biomaterialia

Volume 26, 15 October 2015, Pages 295-305

https://doi.org/10.1016/j.actbio.2015.08.027 Get rights and content

Abstract

Despite wide application of photodynamic therapy (PDT) for the treatment of melanoma skin cancers, there are strong biomedical unmet needs for the effective generation of singlet oxygen after targeted delivery of photosensitizers. Here, we investigated a facile PDT of melanoma skin cancer using transdermal carbon dot – chlorine e6 – hyaluronate (Cdot-Ce6-HA) conjugates. The Cdot-Ce6-HA conjugate was synthesized by the coupling reaction of diaminohexane modified HA (DAH-HA) with the carboxylic group of Ce6. The singlet oxygen generation of Cdot-Ce6-HA conjugates in aqueous solution was more significant than that of free Ce6. The enhanced transdermal and intracellular delivery of Cdot-Ce6-HA conjugates to B16F10 melanoma cells in tumor model mice were corroborated by confocal microscopy and two-photon microscopy. The laser irradiation after topical treatment with Cdot-Ce6-HA conjugates resulted in complete suppression of melanoma skin cancers. The antitumor effect was confirmed by histological analysis with H&E staining and TUNEL assay for tumor apoptosis. Taken together, we could confirm the feasibility of Cdot-Ce6-HA conjugate for transdermal PDT of melanoma skin cancers.

Statement of Significance

To our knowledge, this is the first report on a facile transdermal photodynamic therapy (PDT) of melanoma skin cancer using carbon dot – chlorine e6 – hyaluronate (Cdot-Ce6-HA) conjugates.

We found that the singlet oxygen generation of Cdot-Ce6-HA conjugates in aqueous solution was more significant than that of free Ce6.

Confocal microscopy and two-photon microscopy clearly confirmed the enhanced transdermal and intracellular delivery of Cdot-Ce6-HA conjugates to B16F10 melanoma cells in tumor model mice.

Taken together, we could confirm the feasibility of Cdot-Ce6-HA conjugate for transdermal PDT of melanoma skin cancers.

Graphical abstract

Introduction

Photodynamic therapy (PDT) is a promising therapeutic modality for metastatic melanoma skin cancers [1], [2], [3]. Three main requisites for an effective PDT include a photosensitizer (PS) delivered to tumor tissues, light with an appropriate wavelength, and oxygen in the tumor site [4], [5]. PS activated by light can transfer the photon energy to surrounding oxygen molecules, generating singlet oxygen (¹O₂) for the necrosis and/or apoptosis of cancer cells [6]. However, it is known to be difficult to transdermally deliver PS to cancerous tissues due to the limited penetration-depth [7]. Accordingly, target-specific transdermal delivery of PS is strongly needed for effective PDT, preventing normal cells from destruction. Meanwhile, Ce6 as a PS has several advantages for PDT, such as a high singlet oxygen quantum yield of ca. 0.75, bright fluorescence at wavelengths of 660–670 nm, and rapid clearance from the body [8]. The drawbacks of Ce6 are its poor water solubility and pharmacokinetics, and lack of tumor targeting. Huang et al. [9] reported a PDT using chlorin e6 (Ce6) conjugated to carbon dot (Cdot). They used Cdot to improve water solubility of Ce6 and extend half-life of Ce6 in blood. In addition, Cdot excited Ce6 indirectly by Förster fluorescence resonance energy transfer (FRET). Cdot has advantages of excitation dependent emission, non-cytotoxicity compared to quantum dots, and cheap synthetic process [9], [10], [11], [12], [13].

A naturally occurring linear polysaccharide of hyaluronate (HA) has been widely investigated for various drug delivery and tissue engineering applications [14], [15]. HA has been also used as a promising transdermal delivery carrier [16], [17], [18]. Although the transdermal delivery mechanism of HA is not quite clear yet, hygroscopic HA can hydrate stratum corneum, enhancing the permeability of HA with a hydrophobic patch domain through the skin. Especially, cancerous skin has over-expressed HA receptors such as cluster determinant 44 (CD44) and lymphatic vessel endothelial HA receptor-1 (LYVE-1) on the cellular surface [19]. We have previously reported the HA receptor-mediated transdermal delivery of human growth hormone (hGH) – HA conjugate and nanographene oxide (NGO) – HA conjugate [16], [17]. After transdermal delivery, the bioavailability of hGH was as high as 15% [16]. Furthermore, NGO-HA conjugate could be successfully exploited for transdermal photo-ablation therapy of melanoma skin cancer [17].

Here, we report a facile PDT of melanoma skin cancer using transdermal Cdot-Ce6-HA conjugates in the presence of laser irradiation at 660 nm. Melanoma skin cancer is one of the most aggressive and lethal skin cancers. Melanoma is known to grow rapidly into the skin and metastasize [20]. Although PDT has been applied for the treatment of melanoma [21], [22], to our knowledge, this is the first report to transdermally deliver Cdot-Ce6-HA conjugate for the treatment of melanoma skin cancers. Tumor tissues over-express HA receptors and have relatively leaky structures. The transdermal delivery of Cdot-Ce6 using HA can be safer and more effective than the systemic delivery, because it is locally accumulated in cancerous skin, making facile repeated administration possible and minimizing the side effect in the body. After ex vivo confocal and in vivo two-photon fluorescence imaging for the transdermal delivery of Cdot-Ce6-HA conjugates, we assessed and discussed the PDT of Cdot-Ce6-HA conjugates for the treatment of melanoma skin cancer in mice.

Section snippets

Materials

Sodium hyaluronate (HA) with a molecular weight (MW) of 230 kDa was purchased from Lifecore Co. (Chaska, MN). 1,6-Diaminohexane (DAH) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Sigma–Aldrich (St. Louis, MO). Chlorin e6 (Ce6) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride was purchased from Tokyo Chemical Industry (Tokyo, Japan) and N-hydroxysulfosuccinimide sodium

Preparation and characterization of Cdot-Ce6-HA conjugate

As previously reported elsewhere [9], Cdot-Ce6 was prepared by coupling reaction of Ce6 and Cdot in DMSO. Cdot-Ce6-HA conjugate was prepared by amide bond formation between Cdot-Ce6 and DAH-HA in PBS using the EDC chemistry (Fig. 1). Because three carboxyl groups of HA were reported to be the recognition site of HA receptors [26], HA with a DAH content of 10 mol% was prepared to facilitate receptor mediated transdermal delivery of Cdot-Ce6-HA conjugate into skin tissues. Cdot-Ce6-HA conjugates

Conclusion

Cdot-Ce6-HA conjugate was synthesized by amide bond formation between amine group of DAH-HA and carboxyl group of Cdot-Ce6 using the EDC chemistry. UV–Vis spectra, PL spectra, FT-IR, AFM, and TEM analyses confirmed the successful synthesis of Cdot-Ce6-HA conjugate. SOSG assay revealed the more effective generation of singlet oxygen by Cdot-Ce6-HA conjugate than Ce6 and Cdot-Ce6 due to enhanced water solubility. According to in vitro PDT in B16F10 cells, the photodynamic effect of Cdot-Ce6-HA

Acknowledgments

This study was supported by Mid-career Researcher Program through NRF Grant funded by the MEST (No. 2012R1A2A2A06045773). This research was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health and Welfare, Korea (HI14C1658). This research was supported by the MSIP (Ministry of Science, ICT and Future Planning), Korea, under the “IT Consilience Creative Program” (NIPA-2014-H0201-14-1001) supervised by the NIPA (National IT Industry Promotion Agency). We appreciate

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Photodynamic therapy of melanoma skin cancer using carbon dot – chlorin e6 – hyaluronate conjugate

Abstract

Statement of Significance

Graphical abstract

Introduction

Section snippets

Materials

Preparation and characterization of Cdot-Ce6-HA conjugate

Conclusion

Acknowledgments

Photodiagn. Photodyn. Ther.

Biomaterials

Biophys. J.

Exploitation of long-lived 3IL excited states for metal-organic photodynamic therapy: verification in a metastatic melanoma model

J. Am. Chem. Soc.

A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation

Nat. Commun.

Fluorescence-guided surgery of malignant gliomas based on 5-aminolevulinic acid: paradigm shifts but not a panacea

Nat. Rev. Cancer

Imaging and photodynamic therapy: mechanisms, monitoring, and optimization

Chem. Rev.

Ultrafine hydrogel nanoparticles: synthetic approach and therapeutic application in living cells

Angew. Chem. Int. Ed.

Porphyrin FRET acceptors for apoptosis induction and monitoring

J. Am. Chem. Soc.

The influence of oxygen depletion and photosensitizer triplet-state dynamics during photodynamic therapy on accurate singlet oxygen luminescence monitoring and analysis of treatment dose response

Photochem. Photobiol.

Light-triggered theranostics based on photosensitizer-conjugated carbon dots for simultaneous enhanced-fluorescence imaging and photodynamic therapy

Adv. Mater.

Graphene oxide-photosensitizer conjugate as a redox-responsive theranostic agent

Chem. Commun.

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