Elsevier

Chemical Engineering Journal

Volume 373, 1 October 2019, Pages 468-484
Chemical Engineering Journal

Dendrimer functionalized carbon quantum dot for selective detection of breast cancer and gene therapy

https://doi.org/10.1016/j.cej.2019.05.023Get rights and content

Highlights

  • Green synthesis of carbon quantum dot (CD) from sweet lemon peel waste.

  • Amine functionalization of CDs (CDP) through conjugation of PAMAM dendrimer.

  • Efficient targeted gene transfection efficiency in triple negative breast cancer.

  • Highly selective detection of Cu(II) ion with 93% quenching efficiency.

  • CDP may be promising theranostic tool for TNBC patient.

Abstract

Triple negative breast cancer (TNBC) is the most fatal subtype among all other types of breast cancer and there are no targeted chemotherapy for the treatment of TNBC due to the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2). To address this problem, we develop a targeted non-viral vector which could efficiently deliver the gene of interest and could also diagnose of TNBC. We have synthesized carbon quantum dots (CD) from sweet lemon peel, conjugated it with different generation of polyamidoamine (PAMAM) dendrimers to get CD-PAMAM conjugates (CDPs). RGDS peptide was further conjugated to CDP to target αvβ3 integrin which is over expressed in TNBC. DNA complexation assay, cellular cytotoxicity, hemolysis assay, DNase I assay, cellular uptake and in vitro transfection showed CDP3 as a promising gene carrier system TNBC gene therapy. Besides, CDP3 shows selective quenching of fluorescence in presences of Cu(II) with a quenching efficiency of about 93%. As the Cu(II) ion concentration remains upregulated in TNBC, CDP3 could be a promising theranostic tool for TNBC treatment.

Introduction

Breast cancer is the second leading cause of death in women worldwide [1]. Among the different types of breast cancers diagnosed, triple negative breast cancer (TNBC) accounts about nearly 10–15% in women having the average age within 53 years. Due to the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2), the diagnosis and treatment of TNBC patient are remain challenging which results high relapse rate, short progression-free survival (PFS) and overall survival (OS) [2]. TNBC is most aggressive type of cancer having relapse rate less than 5 years [3], [4]. However, metastases are the major issues of TNBC which makes this subtype of breast cancer more fatal. In a search for the proteins and the signaling pathways involved in tumor cell malignancy, MacDonald et al. [5] found that Memo is an essential enzyme for the breast cancer cell motility in association of various receptor tyrosine kinases. They also showed that Memo is a metal-binding enzyme which utilizes Cu(II) ion for its oxidase activity. They further noticed that Memo increases the local reactive oxygen species (ROS) production only in the presence of Cu(II) ion among the eight different cations and results cell migration and finally metastasis. Therefore, Cu(II) ion has an important role on tumor growth and angiogenesis. Apart from Memo, some other proteins such as MeK1 [6], LOX [7] and SPARC [8] were also identified which also have an important role in cancer metastases in presence of Cu(II) ions. Finney et al. also revealed that around 80–90% of total copper ion accumulates in the intracellular compartment during angiogenesis [9]. In a recent clinical study conducted by Pavithra group showed that the copper ion concentration in serum increased significantly from 110µg/dL to 202 µg/dL in breast cancer patients compared to healthy women [10]. Therefore, a highly selective and sensitive biosensor for copper ion detection may be an attractive diagnostic tool for TNBC patients.

Despite the tremendous progress in cancer research, chemotherapy still remains the most widely used clinical therapy of any type of cancer including TNBC. Due to severe side effect of chemotherapy, researchers are trying to find out alternate therapeutic approach for cancer treatment. Recently, theranostic approach has gained remarkable attention in biomedical field due its simultaneous diagnosis and treatment concept [11], [12]. Warner proposed a simplest definition of theranostic and it is “diagnostics plus therapy” [13]. It is assumed that the theranostic approach will not only improve the drug development and disease management but also will reduce the treatment risk and cost. During the past decade, various nanoparticles including iron oxide nanoparticles [14], [15], carbon nanotubes [16], quantum dots (QDs) [17], gold nanoparticles [18], [19], graphene [11], [20] and silica nanoparticles [21], [22] have been extensively studied for cancer theranostic. High surface energy and smaller particle size of nanoparticles result the aggregation and cellular toxicity, respectively which limit their clinical application [23], [24]. In this context, over the last few years, carbon quantum dots (CQDs) have gained marvelous attention towards the scientist due to its exceptional water solubility, low toxicity, ease of synthesis and functionalization, exceptional photoluminescence (PL) and optical properties and large scale production with low cost.

The previous research works directed us to hypothesis the present work that the development of CQD based non-viral vector system could efficiently transit the gene of interest with high transfection efficiency and no toxicity to TNBC. Along with the therapeutic benefit if the vector could detect the presence of Cu(II) which in turn detect the stage of TNBC then it might be effective theranostic tool for TNBC.

To implement the hypotheses, we have prepared CD from a renewable source like sweet lemon peel and different generation of PAMAM dendrimers were conjugated with it and the synthesis was confirmed by transmission electron microscopy (TEM), UV–vis spectroscopy, fourier-transform infrared (FTIR) spectroscopy, X-ray Diffraction (XRD) analysis and fluorescence spectroscopy. Plasmid DNA (pDNA) was used to mimic the gene binding efficiency of our carrier system. Agarose gel electrophoresis assay was carried out to determine the gene binding efficiency. Cellular uptake study and in vitro transfection were performed in TNBC cell such as MDA-MB-231 cell. Selective detection of Cu(II) was carried out and intracellular fluorescence quenching in presence of Cu(II) was also evaluated in MDA-MB-231 cell.

Section snippets

Materials and methods

Ethylenediamine (EDA), Methylacrylate (MA) were purchased from Merck, India. 1-(3- Dimethylaminopropyl)-3-ethyl carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4-morpholineethane sulphonic acid sodium salt buffer (MES), ethidium bromide, Triton X-100, thiazolyl blue tetrazolium bromide (MTT) were obtained from Sisco Research Laboratories Pvt. Ltd., India. Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate buffer saline (DPBS), trypsin, penicillin-streptomycin, fetal bovine serum

Synthesis of CD and CD-PAMAM conjugates

Here we developed CD based highly sensitive and selective biosensor for the detection of Cu(II) ion through conjugation of amine terminated full generation PAMAM dendrimer with CD. The synthesis of CD from sweet lemon peel waste followed by conjugation of PAMAM dendrimer of different generations (G1, G2 and G3) was confirmed by FTIR and XRD analysis as shown in Figs. S1 and S2. Fig. S1 shows the FTIR spectra of CD, CDP1, CDP2 and CDP3. The broad peak at 3400 cm−1 region and 2934 cm−1

Conclusion

In the present work, we have successfully synthesized CD from renewable source and conjugated it with PAMAM dendrimers with different generations. Among the CDP conjugates, CDP3 showed superior gene complexation capability and also gene protection capability against the enzymatic digestion. The low toxicity even up to 500 µg/ml concentration, blood compatibility and improved gene transfection efficiency show promising gene delivery system for TNBC gene therapy. In addition to this, CDP3 showed

Acknowledgement

This work is financially supported by DST-SERB funding agency through two sanctioned projects EEQ/2016/000712 and ECR/2016/002018. K.S. also acknowledge UGC for UGC-BSR Research Start-Up Grant (F.30-363/2017(BSR), Dt- 08/08/2017) and CU for UPE-II Nanofabrication project fund (UGC/166/UPE-II, Dt- 03/04/2017) for financial support. K.G also thanks to Pritiranjan Mondal for his help to prepare the manuscript.

Conflicts of interest

The authors declare no conflict of interest.

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