Thermodynamic insights into interaction of protein coated gold nanoclusters with DNA and influence of coating on drug binding

Highlights

• HSA coated gold nanoclusters have been synthesized and characterized.

• Binding of anticancer drugs with HSA_AuNCs compared with drug-HSA binding

• HSA_AuNCs showed better drug binding efficiency than free HSA.

• Binding of HSA_AuNCs does not affect the conformation of DNA.

• Binding of drug-nanocluster complex with DNA compared with drug-DNA binding

Abstract

Biocompatibility and ease of surface modification make gold nanoclusters potential to be used in nanomedicine and revolutionizing cancer therapy. Low solubility and bioavailability of drug limits the use of conventional drug administrative methods and hence the need for drug delivery vehicles arises. Different drug delivery vehicles have been used in the past but usually, the information is limited to qualitative in nature. Here, we have reported quantitative insights into interaction of human serum albumin coated gold nanoclusters with anticancer drugs cyclophosphamide, 5-fluorouracil and hydroxyurea, and further interaction of drug-nanoclusters complexes with deoxyribonucleic acid (DNA). Calorimetry, in combination with spectroscopy has been used to characterize HSA coated gold nanoclusters and to obtain thermodynamic parameters associated with interaction of drug with nanoclusters and drug-nanoclusters complex with DNA. Binding of drugs with HSA coated gold nanoclusters has been compared with binding of drugs with free HSA. We found, HSA coated gold nanocluster show better binding efficiency than free HSA due to the minor conformational changes in HSA upon coating. Binding of HSA coated gold nanoclusters has also been studied with DNA to understand the effect of nanocluster binding upon conformation of DNA. Thermodynamic signatures for interaction of drug-nanoclusters complex with DNA imparted the significant role of hydrogen bond and electrostatic interactions in the binding of the charged drug to DNA where as a neutral drug with aromatic ring favour the entropically driven binding. Energetics of interaction correlated with structural feature of drugs provides the structure-energetics relationship and assist to get guidelines for the knowledge-based development of efficient drug delivery vehicles.

Introduction

Development of suitable drug delivery vehicles to achieve maximum therapeutic effect of drug is of utmost importance. New developments in medicine indicate the emergence of drugs with low solubility and low permeability thus limits the use of conventional oral administrative method for these drugs [1,2]. Drug delivery vehicles are of great interest for delivering these drugs due to their ability to accommodate hydrophobic drugs [3,4]. Micelles [5,6], liposomes [7], nanoparticles [8], protein drug conjugates [9] and dendrimers [10] are being used as nanocarrier for different drugs. Nanotechnology has further assisted to improve the formulation of delivery vehicles by incorporating different feature to the vehicles such as image-guidance, external trigger response, magnetic resonance-guidance, target oriented and sustained release of drug [11]. This improves the therapeutic effect of drugs as well as reduces the toxicity associated with drug and therapy.

Nanostructured materials have gained attention in the area of drug delivery because of their tuneable physico-chemical properties [12]. Nanoparticles of noble metal functionalised with biocompatible groups have been formulated and can be used in the treatment of vulnerable diseases like cancer, chronic lung disease, diabetes etc. [[13], [14], [15], [16]]. Controlling colloidal properties of nanoparticles is difficult but has been achieved for the small size regime of 1–3 nm and particles of this small regime have been called nanoclusters [17]. Core size of nanoclusters is comparable to Fermi energy of conduction electron and they exhibit different optical and electronic properties. Gold nanoclusters stand out among the other noble metals due to their easy synthesis, intrinsic fluorescence, water solubility, biocompatibility and low toxicity [18]. Functionalisation of gold nanoclusters with biomolecules not only enhances the biocompatibility but also provide stability. Additionally coating of protein is advantageous over other biomolecule due to its capacity to reduce gold from +3 oxidation state to 0 oxidation state which also makes synthesis of nanoclusters green as no external reducing agent is required to reduce gold.

Human serum albumin (HSA) is the main component of blood serum protein and responsible to transport drugs to target sites. HSA provides multiple sites for drug binding and drug-protein complex serves a as reservoir of drugs in body after renal excretion of free drug [19]. Drug binding property and biocompatibility of HSA makes it suitable as coating material to nanoclusters. Albumin has already been used in the preparation of albumin based nanoparticles for drug delivery [20]. HSA is a globular protein with three domains, each divided into sub-domains A and B. Helical structure of HSA is maintained by different intermolecular forces such as hydrophobic interactions, hydrogen bonding, disulfide bonds and Vander Waals forces. Three main sits are identified as major drug/metabolite binding sites and help in transporting the same in body.

Tunable binding of drug with delivery vehicle is required for effective transportation and release of drug at target site. For this interaction between drug and delivery vehicle are crucial and plays an important role in the development of efficient delivery vehicles. Further, the interactions of these vehicles with target biomolecule like DNA are also important for biological applications. Gold nanoclusters with or without protein coating are highly fluorescent and facilitate bio-imaging of cells [21], drug delivery for hydrophobic drugs [22], clinical diagnosis and sensing of heavy metals, small molecules, and toxic compounds [23]. In recent time various drug delivery systems have been identified for drug delivery applications; nanoclusters of noble metal coated with protein are important due to their green synthesis, low toxicity, biocompatibility and drug loading capacity. Additionally, they also permit continuous monitoring of target regime.

In the present study, HSA coated gold nanoclusters have been synthesized by green method reported earlier by Xie, J. et al. [18] and their interaction with anticancer drugs such as cyclophosphamide (CPA), 5-fluorouracil (5-FU) and hydroxyurea (HU) has been studied to use them as delivery vehicles. Cyclophosphamide is an alkylating agent containing nitrogen mustard which was first recognized as clinical agent for human cancer [24]. Cyclophosphamide is a widely used anticancer and immunosuppressive agent [25] 5-fluorouracil is a pyrimidine antagonist used to inhibit the activity of thymidylate synthetase [26] and affects primidine synthesis. DNA/RNA of tumour cells can take up 5-fluorouracil which leads to base pair mismatch and ultimately terminates the chain. It has been used in the treatment colon, esophageal, stomach, pancreatic, breast, and cervical cancer [27]. Hydroxyurea also known as hydroxycarbamide inhibits the synthesis of DNA by inhibition of ribonucleotide reductase [28]. Hydroxyurea improves the clinical results in sickle-cell disease, chronic myelogenous leukaemia and cervical cancer [29]. Synthesized nanoclusters were characterized by different spectroscopic techniques including fluorescence, circular dichroism, dynamic light scattering, mass spectrometry and microscopy. Quantitative insights of interaction of drugs with these nanoclusters have been studied by means of isothermal titration calorimetry and fluorescence quenching experiments. Detailed comparison of thermodynamic parameters for interaction of anticancer drugs with HSA coated gold nanoclusters and free HSA has been done in order to understand the effect of HSA coating on gold nanoclusters upon drug-protein binding. Further the interaction of these drug loaded nanoclusters with DNA has been studied by means of calorimetry and competitive dye displacement method to illustrate the binding mechanism of drug-nanoclusters complex with DNA.