br a Guangdong Provincial Key Laboratory of Malignant
a Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Department of Thoracic Surgery, Sun yat-sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China b Department of Thoracic Surgery, Cancer Center of Guangzhou Medical University, Guangzhou 510000, China
c Department of Operating Theatre, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
d Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
Copper oxide nanoparticles (CuO NPs) are fabricated using Coleus aromaticus leaf extract with an environmental friendly method and studied using various microscopic and spectroscopic techniques. Also, a new aptamer-conjugated hybrid delivery system using green synthesized CuO NPs is developed to deliver miRNA-29b to A549 cells. This delivery system can eﬀectively deliver miRNAs to cancer cells, with superior performance compared to traditionally available transfection agents, thus acting as an eﬃcient platform for intracellular miRNA de-livery and improving therapeutic outcomes for lung cancer.
Copper oxide nanoparticles (CuO NPs) are p-type semiconductor nanomaterials with a band gap of 1.7 eV, and have attracted significant scientific interest because of their wide range of applications, such as giant magnetoresistance materials, superconductors, gas sensors, na-nocomposites, and lubricants, as well as in the field of lithography [1–4]. CuO NPs have also been used in clothes, plastic materials, paints for killing bacteria, and anti-fouling agents [5,6], and in pesticide production .
Various synthetic approaches have been taken for the production of CuO NPs, including chemical ABT 263 , microwave irradiation  and electrochemical processes ; however, CuO NPs synthesized by chemical methods suﬀer from the adsorption of toxic chemicals onto their surface, rendering them unsuitable for biological applications. Therefore, there is an increasing requirement for novel, environmental friendly methods of CuO NP synthesis.
Green synthesis methods for metal NPs, such as gold and silver, using plant polyphenols and microbes have already been developed [11,12], for example, plant extracts of gum karaya , Albizia lebbeck  and Aloe vera  have been used for the synthesis of CuO NPs.
However, the production of CuO NPs using Coleus aromaticus leaf ex-tract has not yet been reported.
mRNA 29b is considered as a highly expressed class among all the families of mRNA 29 . Earlier reports have established that mRNA-29b plays a key role in cancer therapy by controlling cell diﬀerentia-tion, apoptosis and proliferation [17–20]. mRNA 29b can be down-regulated or silenced in lung and other cancer and hence considered as an attractive candidate for cancer therapy because of its ability to suppress tumor. It is reported in literature that aptamer functionalized nanoparticles can eﬀectively deliver the mRNA29b to cancer cells. For instance, Yu et al. have reported that MUC1 aptamer-conjugated na-noparticles eﬀectively improved the delivery of mRNA to breast cancer cells .
Here, Coleus aromaticus plant extract is used for the biofabrication of CuO NPs. The particle size and morphology is investigated using var-ious characterization techniques and the in-vitro cytotoxicity of the biofabricated CuO NPs against A549 cells derived from lung carcinoma is investigated. Also, we have fabricated a new aptamer-conjugated hybrid delivery system using synthesized CuO NPs and studied for its delivery of miRNA-29b to A549 cells.
☆ The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www. textcheck.com/certificate/j5Iwcq.
E-mail address: [email protected] (M. Wang).
2. Experimental section
CuSO4, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide (MTT) reagent, dimethyl sulfoxide (DMSO) and other chemicals and solvents used were obtained from Sigma-Aldrich Chemicals Ltd. (St. Louis, MO, USA) Aptamer (5′-GCA GTT GAT CCT TTG GAT ACC CTG G-3′) modified with 3-NH2, AlexaFluor-555-labeled wheat germ agglutinin (WGA-AF-555), Lysotracker-Red, and MiRIDIAN mimic with miR-29b were purchased from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ, USA). All experiments were performed using double distilled water.
Preparation of Coleus aromaticus extract.
For preparation of the plant extract, 1.0 g of dried powder of Coleus aromaticus leaf was mixed with 100 mL of double distilled water in a round bottomed flask and heated at 90 °C for 1 h in a water bath. Then, the obtained plant extract was passed through a 0.22 μm cellulose ni-trate filter paper to obtain a clear extract, which was stored in a re-frigerator for later use in CuO NPs synthesis.