A success targeted nano delivery to lung cancer cells with multi-walled carbon nanotubes conjugated to bromocriptine

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Abstract​

In this research, a new nano drug-based multi-walled carbon nanotubes (MWCNTs) was prepared and evaluated qualitatively. Bromocriptine (BRC) was conjugated to functionalized carbon nanotubes. Then, the CHNS, FT-IR, SEM, and RAMAN tests for characterization of the conjugated drug were done. The nanofluid-containing nano-drug was evaluated on lung cancer cells (A549 & QU-DB) and MRC5 by MTT and flow cytometry tests. Then, the gene expression studies of dopamine receptor genes were done before and after nano-drug treatment. After that, a western blotting test was carried out for further investigation of dopamine receptors protein production. Finally, Bax and Bcl-2 secretion were measured by the ELISA method in cells affected by MWCNTs-BRC Nf compared to untreated cells. The results showed that the nano-drug had a significant lethal effect on cancer cells, while it had no toxicity on MRC5. Also, the nano-drug could significantly induce apoptosis in lung cancer cells at a lower dose compared to the drug alone. In this study, a targeted nano-drug delivery system was designed, and its performance was evaluated based on neurotransmitter pathways, and the results showed that it may be useful in the treatment of lung cancer. However, additional studies on animal models are underway.

 

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Introduction​

Lung cancer is one of the most prevalent cancers and the leading cause of death worldwide. Recently, the relationship between the nervous and immune systems for identifying risk factors for lung cancer has been considered. In the past, neurotransmitters were known exclusively as transmitters of messages between cells within the nervous system. Still, today it is believed that these mediators are involved in regulating the immune and the nervous systems. However, the mechanism of this regulation is not fully understood. New evidence sheds more light on the role of neurotransmitters in immune function and regulating the migration of lymphocytes and tumor cells. Evidence of growing researches toughly supports the notion that dopamine receptors (DRDs) are associated with the regulation of tumor activities, such as tumor apoptosis, proliferation, invasion, and migration, which can limit tumor progress through triggering tumor immunity<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Wang, X. et al. The prospective value of dopamine receptors on bio-behavior of tumor. J. Cancer 10, 1622 (2019)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR1">1</a>.

DRD2 subtype is the main target for most anti-neurological drugs<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Noble, E. P. The DRD2 gene in psychiatric and neurological disorders and its phenotypes. Pharmacogenomics 1, 309–333 (2000)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR2">2</a>. The role of DRD2 signaling in several malignant tumors has been investigated; however, the results are contradictory. Increased levels of DRD2 expression are associated with the development of several tumors, and some DRD2 antagonists have been used to reduce the progress of these malignancies<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Grossrubatscher, E. et al. High expression of dopamine receptor subtype 2 in a large series of neuroendocrine tumors. Cancer Biol. Ther. 7, 1970–1978 (2008)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR3">3</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Sachlos, E. et al. Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells. Cell 149, 1284–1297 (2012)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR4">4</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Shin, J. H. et al. Sertindole, a potent antagonist at dopamine D2 receptors, induces autophagy by increasing reactive oxygen species in SH-SY5Y neuroblastoma cells. Biol. Pharm. Bull. 35, 1069–1075 (2012)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR5">5</a>. Conversely, it has been revealed that DRD2 agonist has inhibitory properties in several cancer types, such as lung cancer, gastric cancer, prostate cancer, and leukemia<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Yang, Y. et al. Repositioning dopamine D2 receptor agonist bromocriptine to enhance docetaxel chemotherapy and treat bone metastatic prostate cancer. Mol. Cancer Ther. 17, 1859–1870 (2018)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR6">6</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Qun, H. &amp; Yuan, L.-B. Dopamine inhibits proliferation, induces differentiation and apoptosis of K562 leukaemia cells. Chin. Med. J. 120, 970–974 (2007)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR7">7</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Ganguly, S. et al. Dopamine, by acting through its D2 receptor, inhibits insulin-like growth factor-I (IGF-I)-induced gastric cancer cell proliferation via up-regulation of Krüppel-like factor 4 through down-regulation of IGF-IR and AKT phosphorylation. Am. J. Pathol. 177, 2701–2707 (2010)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR8">8</a>.

DRDs have five subgroups, including D1, D2, D3, D4, and D5, which have inhibitory or stimulatory effects depending on the type of receptors. Bromocriptine (BRC) is a dopamine-specific agonist and was accepted as the first therapeutic agent for adenomas. BRC binds to receptor D2 and inhibits prolactin secretion, and is used to treat Parkinson’s disease, type 2 diabetes, hyperprolactinemia, cocaine addiction, and neuroleptic malignant syndrome<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="De Leeuw Van Weenen, J. et al. The dopamine receptor D2 agonist bromocriptine inhibits glucose-stimulated insulin secretion by direct activation of the alpha2-adrenergic receptors in beta cells. Biochem. Pharmacol. 20, 1827–1836 (2010)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR9">9</a>.

These conditions are due to a decrease in dopamine secretion from a specific area of the brain. BRC is an accepted therapeutic agent to reduce the secretion of the growth hormone prolactin and reduce cell proliferation and tumor cell size<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Liu, X. et al. The mechanism and pathways of dopamine and dopamine agonists in prolactinomas. Front. Endocrinol. 9, 768 (2019)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR10">10</a>. Previous studies have shown that the expression profile of dopamine receptor genes in people with lung cancer is different from that in healthy people. This profile is changed after the patient recovers from the disease<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Shaikhpoor, M., Ahangari, G., Sadeghizadeh, M., Khosravi, A. &amp; Derakhshani Deilami, G. Significant changes in D2-like dopamine gene receptors expression associated with non-small-cell lung cancer: could it be of potential use in the design of future therapeutic strategies?. Curr. Cancer Ther. Rev. 8, 304–310 (2012)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR11">11</a>. BRC induced significant cell apoptosis in a cell line of lung adenocarcinoma. It inhibited the proliferation of lung cancer cells in a dose-dependent manner. At the effective dose of the drug, the expression of D2 receptor genes increased appropriately after treatment<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Sheikhpour, M., Ahangari, G., Sadeghizadeh, M. &amp; Deezagi, A. A novel report of apoptosis in human lung carcinoma cells using selective agonist of D2-like dopamine receptors: a new approach for the treatment of human non-small cell lung cancer. Int. J. Immunopathol. Pharmacol. 26, 393–402 (2013)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR12">12</a>. In another study, BRC-based liposomal nanosystems were designed and fabricated to act purposefully through a specific receptor and increase drug penetration. The nano liposome-containing BRC caused about 50% of apoptosis in lung cancer cells<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Sheikhpour, M. et al. Co-administration of curcumin and bromocriptine nano-liposomes for induction of apoptosis in lung cancer cells. Iran. Biomed. J. 24, 24 (2020)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR13">13</a>. Common treatments of lung cancer include drug treatment, radiotherapy, chemotherapy, surgery and targeted therapy, and immunotherapy. Recently, new methods, such as drug delivery systems and the use of nano drugs, have been widely considered.

Various nanomaterials, such as carbon nanotubes (CNTs), have been used to treat and diagnose cancers<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Sheikhpour, M., Barani, L. &amp; Kasaeian, A. Biomimetics in drug delivery systems: A critical review. J. Control. Release 253, 97–109 (2017)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR14">14</a>. CNTs are allotropes of carbon that, in addition to their ability as carriers for a wide range of therapeutic molecules, their high surface area and the ability to manipulate physical surfaces and dimensions make them suitable for thermal conductivity to kill cancer cells. CNTs are divided into two main categories: single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs), and can play a key role in biomimetic nanomedicine toward personalized medicine for different diseases<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Sheikhpour, M., Golbabaie, A. &amp; Kasaeian, A. Carbon nanotubes: a review of novel strategies for cancer diagnosis and treatment. Mater. Sci. Eng. C 76, 1289–1304 (2017)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR15">15</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Hao, H., Chen, Y. &amp; Wu, M. Biomimetic nanomedicine toward personalized disease theranostics. Nano Res. 14, 2491–2511. https://doi.org/10.1007/s12274-020-3265-z (2021)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR16">16</a>.

Many studies have been conducted on different biomedical effects of MWCNTs in several diseases and cell signaling pathways such as apoptosis and autophagy. The results of extensive investigations show that MWCNTs have high efficient drug loading capacity. They could be used as proper drug nanocarriers and promising nanoplatforms for cancer therapy. Besides, to improve their biocompatibility and conjugation capacity, and reduce their toxicity, they are functionalized with chemical groups such as hydroxyl or carboxyl<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Li, Z. et al. The toxicity of hydroxylated and carboxylated multi-walled carbon nanotubes to human endothelial cells was not exacerbated by ER stress inducer. Chin. Chem. Lett. 30, 582–586. https://doi.org/10.1016/j.cclet.2018.12.011 (2019)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR17">17</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Yang, Q. et al. Pre-incubated with BSA-complexed free fatty acids alters ER stress/autophagic gene expression by carboxylated multi-walled carbon nanotube exposure in THP-1 macrophages. Chin. Chem. Lett. 30, 1224–1228 (2019)." href="https://www.nature.com/articles/s41598-021-03031-2#ref-CR18">18</a>.

This study aimed to prepare a nano-drug with a high and targeted anti-cancer effect and also to deliver it to cancer cells with higher efficiency compared to the free drug. Also in this research by functionalization of MWCNTs, conjugation of drug, and preparation of nanofluid containing this nano-drug, it was tried to increase the stability of the drug in nanofluid and reduce its toxicity on normal cells compared to cancer cells.

 

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