CC BY
6DOI 10.24412/cl-37136-2023-1-156-159
ASSOCIATION OF PHOTOTHERMAL THERAPY WITH OPTICAL CLEARING AGENTS FOR THE TREATMENT OF CUTANEOUS MELANOMA, USING AN INDOCYANINE GREEN
NANOEMULTION
LETÍCIA MARTINELLI1 , GABRIEL JASINEVICIUS1 , LILIAN MORIYAMA1 , HILDE BUZZÁ2 , JUAN CHEN3 , GANG ZHENG3 AND CRISTINA KURACHI1
1Sao Carlos Institute of Physics, University of Sao Paulo, Brazil 2 Institute of Physics, Pontificia Universidad Católica de Chile, Chile 3Department of Medical Biophysics, University of Toronto, Canada
cristina@,ifsc.usp .br
ABSTRACT
Melanoma is a pigmented tumor that originates from melanin-producing cells called melanocytes. According to the National Cancer Data Base (NCDB-US), approximately 91.2% of melanomas originate in the skin [1]. Its main cause is prolonged exposure to the sun, especially in childhood and adolescence, due to ultraviolet rays (UV) [2]. Even representing only 5% of skin cancers, it is the most aggressive and invasive type, due to its high possibility of causing metastasis, being responsible for the highest rate of deaths from skin cancer, with approximately 80% to 85% [3]. First-line diagnostic procedures include clinical analysis of the lesions, mainly their ABCDEs macroscopic characteristics, which are evaluated in terms of asymmetry, borders, diameter, color and evolving [4]. Dermoscopy is also performed, which is a noninvasive means of evaluating suspicious lesions using magnification and light to visualize structures in the epidermis and superficial dermis that are not visible in routine exams with the naked eye [5]. When malignancy is suspected, an excisional biopsy is performed, where the melanoma is confirmed by histopathological examination. In the initial stages, the most used treatment today is surgery [6,7]. In more advanced cases, palliative treatments such as radio, chemo and immunotherapy are used. The difficulty in finding an effective treatment is mainly due to the presence of melanin: a highly absorbing and light-scattering agent, in addition to having an antioxidant and photoprotective action [8-10]. Knowing the limitations of melanoma treatment, new therapies such as photothermal therapy have been studied. It needs a molecule that is sensitive to light (photosensitizer) and to light in the NIR region (700-2000nm). By absorbing the energy from light, the photosensitizer goes to an excited state of higher energy level and undergoes a process called vibrational relaxation, where it returns to its fundamental state of energy, releasing heat [11,12]. This increase in temperature causes protein aggregation and denaturation, cytosol evaporation and cell lysis, leading to cell and tissue death. In biological systems, the range of 42 to 60°C is known as hyperthermia, and this is where conformational changes occur in molecules, accompanied by destruction of bonds and membrane changes and cell immobility. At 60°C, protein and collagen denaturation occurs, which leads to tissue coagulation and cell necrosis [13]. The photosensitizers with the best response to photothermal therapy are nanomolecules with strong absorbance in the infrared region [14]. Indocyanine green, used as a fluorescence imaging probe, is an FDA-approved photothermal agent with low dark toxicity and rapid clearance by the liver [15,16]. Because it has these characteristics, it has been studied encapsulated in nanoparticles or associated with drugs [17,18], for example. However, no study showed melanoma eradication in vivo. Therefore, we have been investigating the use of an ICG nanoemulsion (NanoICG) developed by Prof. Gang Zheng (University Health Network, Toronto, Canada), differing from other photothermal nanoparticles, since the ICG is not located inside the nanoparticle, but outside, and its interior is composed of glycerol trioctanoate (Figure 1). Its physical-chemical characterization demonstrated the stable formation of dimers and, therefore, greater applicability for photothermal and photoacoustic applications.
A B
p. ICG
jLr-»' Oil
/Jg^s \ )
• m m'i Lo oj
il 1 S-0 o-?
la ■ i ......^ ICG 0 0 Na'
Figure 1: (A) Estructure of NanoICG; (B) Molecule ICG.
The animals used are immunosuppressants, balbC/nude and B16F10 melanoma cells injected intradermally. Treatment was performed when the tumor reached 3mm in thickness. Two concentrations of NanoICG (100 and 200^M) and two irradiances (1 and 0.5W/cm2 ) were tested in an in vivo model for intratumoral administration. The nanoemulsions were also administered intraperitoneally and its kinetics and treatment responses were measured by intradermal needle and thermographic camera. This group received 9.6 mg/kg of NanoICG and were irradiated with 0.5 W/cm2 for 10 minutes. So far, the group that presented the best results was with 200 ^M of NanoICG intratumorally. The control group had a temperature increase of 12°C, which is expected since melanin is a highly light absorbing component and the treatment one of 33°C. With the thermographic camera, it was possible to visualize a localized temperature increase, meaning that the molecule did not spread to other tissues (Figure 2).
Figure 2: (A) Cinical image of melanoma; (B) Thermographic image while the tumor was being
irradiated.
The Kepler-Meier curve was performed for the best experimental group, where within 25 days there was no tumor regrowth (Figure 3-A). Qualitatively, the tumors right after irradiation showed a whitish aspect due to the damage caused by the treatment and there was growth of a scab, showing a therapy-induced tumor necrosis (Figure 3-C). On the fourteenth day of treatment, there was skin regeneration in 67% of the animals and the absence of melanoma cells Immunohistochemical analyzes are being carried out to confirm with certainty that there were no remnants of tumor cells, and studies are also being carried out to determine whether there was any type of metastasis. Intravenous administration will also be performed and finally associate photothermal therapy with optical clearing agents (OCAs).
Figure 3: (A) Kepler-Meier curve for intratumoral administration of200 pM and systemic administration of 3.1mM; (B) Cinical image of melanoma before treatment; (C) Melanoma 3 days after treatment; (D)
Melanoma after 14 days of treatment with regrowth.
OCAs are non-toxic hyperosmic compounds with a refractive index ~ 1.4. They promote local osmotic dehydration, and tissue refractive index matching, reducing light scattering and improving the light penetration into the tumor [19-22]. Our group has previously demonstrated its potential to enhance light-based therapies in cutaneous pigmented melanoma, enabling the eradication of lesions with up to 1mm in thickness, using photodynamic therapy [23,24]. When the treatment is associated with clearings, it has already been verified that the light reaches deeper into the melanoma, making it more optically homogeneous [25]. Therefore, associating photothermia with OCAs leads us to believe that the response to treatment will be even more effective and the best protocol tested so far.
REFERENCES
[1] Chang AE, Kamell LH, Menck HR. The National Cancer Data Base Report on Cutaneous and Noncutaneous Melanoma. Cancer. 1998;83(8):1664-78.
[2] Gandini S, Sera F, Cattaruzza MS, Pasquini P, Zanetti R, Masini C, et al. Meta-analysis of risk factors for cutaneous melanoma: III. Family history, actinic damage and phenotypic factors. Eur J Cancer. 2005 Sep;41(14):2040-59.
[3] Aladowicz E, Ferro L, Vitali GC, Venditti E, Fornasari L, Lanfrancone L. Molecular networks in melanoma invasion and metastasis. Vol. 9, Future Oncology. 2013. p. 713-26.
[4] Tsao H, Olazagasti JM, Cordoro KM, Brewer JD, Taylor SC, Bordeaux JS, et al. Early detection of melanoma: Reviewing the ABCDEs American Academy of Dermatology Ad Hoc Task Force for the ABCDEs of Melanoma. J Am Acad Dermatol. 2015 Apr 1;72(4):717-23.
[5] Zager JS, Sondak VK, Kudchadkar R. Melanoma. 2016.
[6] Zalaudek I, Ferrara G, Argenziano G, Ruocco V, Soyer HP. Diagnosis and treatment of cutaneous melanoma: a practical guide. Vol. 2, Skinmed. 2003.
[7] Eggermont AMM, Spatz A, Robert C. Cutaneous melanoma. In: The Lancet. Lancet Publishing Group; 2014.p.816-27.
[8] Mroz P, Huang YY, Szokalska A, Zhiyentayev T, Janjua S, Nifli AP, et al. Stable synthetic bacteriochlorins overcome the resistance of melanoma to photodynamic therapy. FASEB J. 2010 Sep;24(9):3160-70.
[9] Huang Y-Y, Vecchio D, Avci P, Yin R, Garcia-Diaz M, Hamblin MR. Melanoma resistance to photodynamic therapy: new insights. Biol Chem [Internet]. 2013 Feb 1;394(2):239-50. Available from: http://www.degruyter.com/view/j/bchm.2013.394.issue-2/hsz-2012-0228/hsz-2012-0228.xml
[10] Wang Z, Dillon J, Gaillard ER. Antioxidant Properties of Melanin in Retinal Pigment Epithelial Cells. Photochem Photobiol. 2006;82(2):474.
[11] Cortezon-Tamarit F, Ge H, Mirabello V, Theobald MBM, Calatayud DG, Pascu SI. Carbon Nanotubes and Related Nanohybrids Incorporating Inorganic Transition Metal Compounds and Radioactive Species as Synthetic Scaffolds for Nanomedicine Design. In: Inorganic and Organometallic Transition Metal Complexes with Biological Molecules and Living Cells. Elsevier Inc.; 2017. p. 245-327.
[12] Eskiizmir G, Baskin Y, Yapici K. Graphene-based nanomaterials in cancer treatment and diagnosis. In: Fullerens, Graphenes and Nanotubes [Internet]. Elsevier; 2018. p. 331-74. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780128136911000099
[13] Niemz MH. Laser-Tissue Interactions [Internet]. Third. Elias Greenbaum, editor. Berlin, Heidelberg: Springer Berlin Heidelberg; 2002. Available from: http://link.springer.com/10.1007/978-3-662-04717-0
[14] Doughty ACV, Hoover AR, Layton E, Murray CK, Howard EW, Chen WR. Nanomaterial applications in photothermal therapy for cancer. Materials (Basel). 2019;12(5).
[15] Shirata C, Kaneko J, Inagaki Y, Kokudo T, Sato M, Kiritani S, et al. Near-infrared photothermal/photodynamic therapy with indocyanine green induces apoptosis of hepatocellular carcinoma cells through oxidative stress. Sci Rep. 2017 Dec 1;7(1).
[16] Boni L, David G, Mangano A, Dionigi G, Rausei S, Spampatti S, et al. Clinical applications of indocyanine green (ICG) enhanced fluorescence in laparoscopic surgery. Surg Endosc. 2015 Jul 19;29(7):2046-55.
[17] Hwang J, Jin J-O. Attachable Hydrogel Containing Indocyanine Green for Selective Photothermal Therapy against Melanoma. Biomolecules [Internet]. 2020 Jul 29; 10(8): 1124. Available from: https://www.mdpi.com/2218-273X/10/8/1124
[18] Ledezma DK, Balakrishnan PB, Cano-Mejia J, Sweeney EE, Hadley M, Bollard CM, et al. Indocyanine Green-Nexturastat A-PLGA Nanoparticles Combine Photothermal and Epigenetic Therapy for Melanoma. Nanomaterials [Internet]. 2020 Jan 17;10(1): 161. Available from: https://www.mdpi.com/2079-4991/10/1/161
[19] Millon SR, Roldan-Perez KM, Riching KM, Palmer GM, Ramanujam N. Effect of optical clearing agents on the in vivo optical properties of squamous epithelial tissue. Lasers Surg Med. 2006 Dec;38(10):920-7.
[20] Zhu D, Larin K V., Luo Q, Tuchin V V. Recent progress in tissue optical clearing. Laser Photonics Rev. 2013 Sep;7(5):732-57.
[21] Sdobnov AY, Darvin ME, Genina EA, Bashkatov AN, Lademann J, Tuchin V V. Recent progress in tissue optical clearing for spectroscopic application. Spectrochim Acta - Part A Mol Biomol Spectrosc. 2018 May 15;197:216-29.
[22] Sdobnov AY, Darvin ME, Schleusener J, Lademann J, Tuchin V V. Hydrogen bound water profiles in the skin influenced by optical clearing molecular agents—Quantitative analysis using confocal Raman microscopy. J Biophotonics [Internet]. 2019 May 4; 12(5). Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/jbio.201800283
[23] Pires L, Demidov V, Vitkin IA, Bagnato V, Kurachi C, Wilson BC. Optical clearing of melanoma in vivo: characterization by diffuse reflectance spectroscopy and optical coherence tomography "Optical clearing of melanoma in vivo: characterization by diffuse reflectance spectroscopy and optical coherence tomography Optical clearing of melanoma in vivo: characterization by diffuse reflectance spectroscopy and optical coherence tomography. J Biomed Opt [Internet]. 2016;21(8):81210. Available from: https://www. spiedigitallibrary.org/terms -of-use
[24] Pires L, Demidov V, Wilson BC, Salvio AG, Moriyama L, Bagnato VS, et al. Dual-agent photodynamic therapy with optical clearing eradicates pigmented melanoma in preclinical tumor models. Cancers (Basel). 2020 Jul 1;12(7): 1-17.
[25] Martinelli LP, Iermak I, Moriyama LT, Requena MB, Pires L, Kurachi C. Optical clearing agent increases effectiveness of photodynamic therapy in a mouse model of cutaneous melanoma: an analysis by Raman microspectroscopy. Biomed Opt Express [Internet]. 2020 Nov 1; 11(11):6516. Available from: https://www.osapublishing.org/abstract.cfm?URI=boe-11-11-6516
