br Corresponding authors at IICT RMIT Research Centre CSIR I
Corresponding authors at: IICT-RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad, India.
E-mail addresses: [email protected] (D. Pooja), [email protected] (S. Bhargava), [email protected] (S. Ramakrishna).
R. Radhakrishnan, et al.
used to incorporate both hydrophilic and hydrophobic drugs alike, and also can be administered through a variety of routes (Ramteke et al., 2012). These nanoparticles, therefore, worked as a protective as well as biodegradable vehicle for delivery.
One of the major advantages that EGCG holds is its pH-dependent degradation, with the molecule degrading as the pH moves from acidic pH towards physiological pH, with complete degradation at basic pH (Radhakrishnan et al., 2016). As tumour microenvironments have been shown to be slightly acidic in nature, only EGCG that is delivered in the cancer tissue will be active, while that delivered in normal cells will be degraded due to pH-dependent instability.
In the present study, we have developed a system that would act against cells marked with the targeted tumour marker. Non-specificity has to be circumvented to reduce non-specific organ damage which gives rise to serious side-eﬀects (Monsuez et al., 2010). The dissipation of the drug in other areas also results in the concomitant increase in drug dosages, as the intended organ may not get the total therapeutic dose required (Coates et al., 1983).
To provide this additional specificity we have grafted a ligand onto the nanoparticle vehicle specific for breast cancer. Throughout the years, targeting has been mediated via monoclonal Nifurtimox (Kulhari et al., 2015), biomolecules such as sugars, peptides and proteins (Pooja et al., 2015), and chemicals such as folates (Ruoslahti et al., 2010; Shohdy and Alfaar, 2013). Peptides provide an increasing advantage as they are small in size, thus allowing the conjugation of more peptide molecules per nanoparticle. They are also intrinsically biocompatible and non-toxic which leaves them undetected by the macrophages of the RES. GRPR is a glycoprotein receptor with 7 transmembrane domains consisting of 384 amino acids. It has been shown to overexpress in a number of cancers including breast cancer (Halmos et al., 1995; Zhou et al., 2019). In a recent study of 1432 primary breast tumours, GRPR overexpression was found in 75.8% of the cases (Morgat et al., 2017). Thus, the lower expression of GRPR in normal tissues and compara- Chemistry and Physics of Lipids xxx (xxxx) xxx–xxx
India). MDA-MB-231 human breast cancer cell lines and B16F10 mouse melanoma cells were obtained from American Type Culture Collection (ATCC, Manassas, USA). Dichloromethane was obtained from Merck Chemicals (Mumbai, India). Tween 80 was purchased from sd Fine Chemicals Limited (Hyderabad, India).
3.1. Preparation of nanoparticles
SLNs were prepared by the double emulsification-evaporation method as optimized in our previous work (Pooja et al., 2015). Briefly, GMS, stearic acid and lecithin soy were dissolved in the organic phase containing dichloromethane. The inner aqueous phase (1% w/v P-F68) containing dissolved EGCG was added to this organic phase under so-nication to form a primary water-in-oil (w/o) emulsion. This emulsion was added to 2% w/v P-F68 solution to give a water-in-oil-in-water (w/ o/w) emulsion. The nanoparticle suspension was stirred for 2 h for evaporation of the solvent and further centrifuged for 30 min at 11,200 g to obtain nanoparticle pellets. Nanoparticles were washed thrice with MilliQ water to remove the free drug present on the nano-particle surface. The supernatant was used for determination of the entrapment eﬃciency.
3.2. Entrapment eﬃciency
The drug entrapment eﬃciency (EE) was calculated by an indirect method. Briefly, the supernatant obtained after centrifugation of the nanoparticle suspension was used for estimation of the unloaded drug content by UV–vis spectroscopy on a Jasco UV650 spectrophotometer at the absorption maxima of EGCG at 274.5 nm. The percent EE was calculated by the following formula:
tively higher expression levels in tumour tissue, gives a greater mole-cular basis for choosing this receptor for targeting in chemotherapy
%EE = Total amopunt of EGCC added − Amount of EGCG insupernatant Total amount of EGCG added
approaches. These findings validate the use of GRPR as a promising indication for cancerous tissue, consequently aiding the meticulous delivery of nanoparticle cargo. Bombesin (BBN), a tetradecapeptide, is an amphibian analogue to the mammalian gastrin releasing peptide which is the natural ligand to GRPR (Begum et al., 2016). GRP and bombesin have a highly conserved 7 amino-acid terminal sequence that is essential for its high aﬃnity binding and immunogenicity towards GRPR (Sunday et al., 1988). It has shown a very strong aﬃnity towards GRPR and hence was used a ligand towards the tumour marker (Reubi et al., 2002).