Melanoma Treatment Via Non-Specific Adhesion of Cancer Cells Using
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www.nature.com/scientificreports OPEN Melanoma treatment via non‑specifc adhesion of cancer cells using charged nano‑clays in pre‑clinical studies Sahel N. Abduljauwad1, Habib‑ur‑Rehman Ahmed1* & Vincent T. Moy2 The incidence of malignant melanoma has rapidly increased in the last two decades. There are many challenges associated with the current conventional therapies, including tumour size and location, the specifcity of treatments, tumour resistance, non‑mutually exclusive mutations, drug resistance, and many adverse side efects. Due to conventional therapies having several limitations, we have explored an alternative therapy such as nano‑clays; nano‑sized natural materials originating from clay fraction of the soil. Recently, clay nanoparticles have increasingly been used as a drug carrier for cancer treatment due to their high absorption, ability to engulf microbes, and low toxicity. In this study, we evaluated the efects of a nano‑clays mix on melanoma cell proliferation and cell viability in vitro and melanoma growth in vivo xenograft animal model. The in vitro study revealed that nano‑clay treatments signifcantly reduced melanoma cell proliferation and cell viability in a dosage‑dependent manner. The in vivo tumour xenograft model demonstrated that nano‑clay mix treatment led to signifcantly reduced tumour size and weight, decreased tumour cell mitosis, and induced tumour necrosis. These processes owe to the most probable changes in the membrane potential of the cancer cells once nano‑clays bind with the former through the high non‑specifc adhesion characteristic of the cancer cells. As the data suggest an important role of nano‑clays as an inhibitor of melanoma cell proliferation and survival, these prove to be a natural and efective medicine for the treatment of melanoma. The proven compatibility of nano‑clays with the human cells with little side‑efects makes them a highly preferred choice for the treatment of melanoma and probably other types of cancers. Melanoma, although less common but due to being most deadly among skin cancers, accounts for the vast major- ity of skin cancer death1. Recently, an estimate shows that there would be 96,480 new cases of melanoma in the United States and 7230 deaths from the disease1. Unlike many forms of cancer, the incidence of melanoma has increased over the last two decades2. Current conventional therapies include excisional surgery, immune therapy, and targeted therapy3,4. However, there are multiple challenges associated with these treatment options, including tumour size and location, the specifcity of medicines, tumour resistance, and adverse side efects. Although there exist specifc drugs (such as vemurafenib and dabrafenib) that decrease tumour burden, these are efective only for a limited period5. Even as regards combination drug therapy, there are still several critical questions relevant to which combination(s) are most efective. Although, the discovery of processes underlying the traditional immune suppressive features of advanced melanomas has led to essential progress in using immune checkpoint therapies5, several immune checkpoint inhibitor drugs, available these days, have got several side efects. Te use of natural materials, such as nano-clays, could be one of the alternatives for overcoming several challenges associated with the several available options for the melanoma treatment. Nano-clays are nano-sized natural materials originating from minerals of the sedimentary rocks. Smectites and palygorskite are two main groups of the nano-clay minerals. Due to isomorphous substitution in their molecular structure, these nano-clays exhibit charge defciency on their surfaces. Tis charge defciency on their surfaces is neutralized by the water molecules and the dissolved cations (Fig. S1). Trough considerable research by the authors, we have developed basic characterization and behavior modelling of the charged clay minerals 6–8. In biomedical feld, nano-clays have been successfully used afer incorporating these into the cancer medicines 1Civil and Environmental Engineering Department, King Fahd University of Petroleum and Minerals (KFUPM), Dhahran, Saudi Arabia. 2Department of Physiology and Biophysics, University of Miami, Coral Gables, FL, USA. *email: [email protected] Scientifc Reports | (2021) 11:2737 | https://doi.org/10.1038/s41598-021-82441-8 1 Vol.:(0123456789) www.nature.com/scientificreports/ including 5-fuorouracil and Trastuzumab 9–15. Nano-clays can, therefore, be categorized as an alternate medicine for several ailments 16–20. Clay nanoparticles, being sticky in nature, have been a part of several sustained-release medicines21,22; researchers23 used anionic clays to be sandwiched with an anti-cancer drug, Methotrexate (MTX). Tese researchers used the methods based on co-precipitation followed by the hydrothermal action for the prepa- ration of a stable and sustained-release drug-nano clay hybrid. Another group of researchers24 bentonite clays could be incorporated in the drugs as delivery vehicles owing to their compatibility and high loading capacity. Resultantly, they prepared a sustained-release delivery system consisting of doxorubicin and nano-clay complex for the treatment of melanoma during chemotherapy. Furthermore, researchers 25 also studied Montmorillonite clay as drug carrier material in the controlled release of medicine. Some other researchers 26 also found that nano-clays are quite efective to be used in drug delivery systems and it helps by increasing the efciency of the drug delivery and hence reducing the toxicity of some of the medicines for the treatment of the thyroid cancer. Tere are other studies on Halloysite nano-clays that prove the compatibility of these natural materials with the healthy human tissues. Naumenko and Fakhrullin27 concluded in their studies that Halloysite nano-clay is biocompatible with the human healthy tissues. Tarasova et al., in their study28 also concluded that during their experimentation they found that the pristine Halloysite caused no changes in the morphology of the human cell nuclei. General confguration and the associated physicochemical properties of these nano-clays are provided in the baseline studies 29 and the supplementary material. In a previous research30, authors showed high afnity of the clay nanoparticles to the other charged particles and surfaces. A combined efect of high afnity of the nano-clays and the high non-specifc adhesion property of the cancer cells makes the former highly efective in controlling the tumour metastasis. In that study, we demon- strated that an optimum mix of two charged clay minerals (Na-montmorillonite and palygorskite) could be used for the control of cancer metastasis. Considering these fndings30, we further hypothesized that nano-clays could also have an inhibitory efect on the cancer cell proliferation and viability. To test this hypothesis, we conducted cell proliferation and cell viability analyses in melanoma cells in vitro and melanoma growth in vivo human melanoma xenograf animal models. Both the in vitro and in vivo studies were conducted at the laboratories of the School of Medicine at the University of Miami, Florida, USA. To substantiate the most probable reasons for the inhibition in the cell proliferation and necrosis, adhesion measurements among healthy and cancerous cells, without and with clay nanoparticles, was also carried out using Atomic Force Microscopy (AFM). Scanning Elec- tron Microscopy (SEM) was also used to visualize the physical interactions of the nano-clays with the cancer cells. Materials and methods In vitro study. In vitro studies were carried out on human malignant melanoma cells. Tese cells were exposed to various doses of clay nanoparticle formulations, and their growth and vitality were studied. Tese doses of nano-clay mix consisting of Na-montmorillonite and Palygorskite were studied and fnalized as a pro- portion of 75:25 in the authors’ previous research30. Materials. Cell culture. Human malignant melanoma cell line SK-Mel-28 (skin primary melanoma) were obtained from American Type Culture Collection (ATCC, Manassas, VA), while Human Epidermal Melano- cytes (HEMs) (adult primary normal cells) were purchased from Cell Applications, Inc. (San Diego). Te cells were grown in Dulbecco’s Modifed Eagles Medium (DMEM). DMEM was supplemented with 5% Fetal Bovine Serum (FBS) (HyClone Inc.) and antibiotic–antimycotic of 100 μg/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Mediatech, Manassas, VA) (DMEM growth media). Cells were maintained in a Termo culture incubator at 37 °C and 5% CO2. Clay nano-particles isolation and purifcation. Na-montmorillonite (SWy-3) and palygorskite (PFl-l) clay sam- ples were acquired from the Clay Minerals Society (CMS)29,31 (Tables S1 to S3). Nano-sized particles of pure clay minerals were obtained by a multi-step procedure involving the defocculation of the initial dry powdered state and the removal of non-clay minerals and impurities. Most of the steps followed the general guidelines provided in ASTM D792832. First, nanoparticle aggregates in each clay sample were broken down by mixing 5 g of sample with 12.5 ml defocculant solution (40 g sodium hexametaphosphate in 1000 ml of deionized water) and leaving for 16 h for uniform absorption. Next, the mixture was diluted to 100 ml using deionized water and centrifuged in a standard cup at a speed of 1000 rpm for 30 min. Te supernatant was poured into a glass cylinder and fur- ther diluted to 1000 ml. Te mixture then