432 Current Nanoscience, 2012, 8, 432-440 1875-6786/12 $58.00+.00 © 2012 Bentham Science Publishers Evaluation of Effect of Ligand on Cellular Internalization: A Comparative Study of Nanoparticles and Multifunctional Nanoparticles on MDA-MB-231 Cells Anand Mahalwar a , Arvind Gulbake a , Ashish Jain a , Satish Shilpi a , Bhawna Sharma b , Beenu Joshi b and Sanjay K. Jain a * a Pharmaceutics Research Projects Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar (M.P.) 470003 India; b Department of Immunology, National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra (U.P.), India Abstract: The present work was aimed to develop, explore & compare the use of multifuctional nanoparticles (MNPs) (drug loaded) ma- de of the conjugate poly (lactide-co-glycolide) (PLGA) - polyethylene glycol (PEG) & – folic acid with PLGA nanoparticles (NPs) (drug loaded) for targeting solid tumor. For that first optimum cytotoxic concentration of PLGA (polymer) and cisplatin (drug) were optimized through MTT assay. The optimum size and percent entrapment efficiency were found to be 170±6.5 nm and 74.9±2.3% for PLGA NPs and 186±4.2 nm and 76.9±3.1% for MNPs. The in vitro cytotoxic activity of MNPs and PLGA NPs were investigated & compared with drug solution (cisplatin) on MDA-MB-231 breast cancer cells, which revealed that MNPs are more cytotoxic in a time dependent man- ner. The rhodamine B isothiocyanate loaded NPs and MNPs were prepared & compared for cell uptake studies which conformed that tar- geted NPs (MNPs) were more taken up by the MDA-MB-231 cells. To determine the effect of ligand (folic acid) on internalization, cells were incubated with MNPs, NPs and 10 fold excess folic acid with MNPs. Results confirmed that the presence of ligand gradually in- creases internalization of carriers and exhibited maximum uptake of MNPs whereas, little difference was observed on uptake between NPs and excess folate treated cells. Results suggesting that MNPs are promising approach for targeting solid tumor & to achieve deeper cellular internalization. Keywords: PLGA poly (lactide-co-glycolide), nanoparticles, multifunctional nanoparticles, cytotoxicity, cell uptake. INTRODUCTION Cisplatin (cis-diaminedichloroplatinum) is one of the most fre- quently used antineoplastic agent [1], as it has proved its superior antitumor activity against variety of solid tumors and used in most of first-line chemotherapeutic [1-4]. Platinum has a strong affinity to bind at N7 of guanine on DNA. The binding to DNA results in an altered protein conformation and changes in biological activity, especially when enzymatic reactions are affected [5-7]. It shows high toxicity i.e. nephrotoxicity, gastrointestinal toxicity, ototoxic- ity, cardiotoxicity and neurotoxicity [8, 9] when it is given in non- site specific manner, thus it limits its therapeutic potential. To make it potent, past research was aimed to reduce its toxicity by making either its complex with different polymers like polycar- boxylates [10], poly(amidoamines) [11], polyamidoamine dendrim- ers [12] and the complexes with N-(2-hydroxypropyl) methacryla- mide [13], or encapsulating it in the form of vesicular systems, such as liposomes [14,15], PEGylated liposomes [16], poly(aspartic) acid–poly (ethylene glycol) micelles [17] and poly(caprolactone)– poly(ethylene glycol) or poly(caprolactone)–poly[2-(N,N-dimethyl- amino)ethyl methacrylate] micelles [18]. Some other approaches also utilized by the researchers to reduce its side effects such as encapsulation of it in the polymeric matrix, such as PLGA micro- sphers, PLGA nanoparticles [19]. As cisplatin with long-circulating carriers (PEGylated) alters drug pharmacokinetics and results in increased drug accumulation in tumors, based on the ‘‘enhanced permeability and retention’’ (EPR) effect [20, 21] the PEGylated PLGA nanoparticles [21], and PEGylated PCL nanoparticles was formulated [2]. To the best of our knowledge, no studies so far have been re- ported on, ligand anchored targeted delivery of encapsulated *Address correspondence to this author at the Pharmaceutics Research Projects Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya, Sagar (Madhya Pradesh), India; Tel:/Fax: 07582-264236; E-mail: drskjainin@yahoo.com cisplatin in nanocarriers for cancer cells hence, a target specific, stealth, polymeric nanocarrier (MNPs) where designed & compared with another polymeric nanocarriers (PLGA NPs) having also abil- ity to target (passive targeting due to enhanced permeability and retention (EPR) effect [22]. Researchers reported earlier that, folic acid internalizes via caveolae assisted receptor mediated endocyto- sis, and thus it makes it efficient to release on the nucleus of the cytosolic environment [23-30] with bypass mechanism of harsh environment of lysosomal degradation, where the nucleus resides [31-35]. So that folic acid is used as ligand for present investiga- tion. Multifunctional nanoparticles (MNPs) as the name indicate that these were designed to do multiple works instead of single work. In the present work term MNPs is utilized because we have used spe- cific constituents for the making of stable nanoparticles, these con- stituents bear special characteristics in each i.e. 1. Folic acid- (i) makes carrier target potential [23,36], (ii) provides deeper cellular internalization & nucleus directed release due to caveolin assisted receptor mediated endocytosis [34], (iii) due to strong uptake of folic acid at cellular nucleus [23] can manage efflux mechanism of P-glycoprotein (P-gp) causes multidrug resistance (MDR) [37], 2. PEG- (i) makes carrier long circulatory by preventing opsonization [38,39] (ii) coats hydrophilic layer over the surface of carrier thus MDR receptors (hydrophobic vacuum cleaner) will unable to efflux them out [37], (iii) can improve encapsulation efficiency of hydro- philic drug and also increases sustainability in drug release [40-42], 3. PLGA- (i) biocompatible and biodegradable polymer [43, 44], (ii) improves encapsulation efficiency of hydrophilic drug [45, 46], (iii) sustained as well as controlled release can be possible [43, 45], so that the composed form of all the functionality in a single stable construct termed as multifunctional, and as it is in nano-size range, we said it multifunctional nanoparticles. In spite the use and advan- tages of monofunctional nanoparticles, MNPs consist different functionalities in it. So, in this present study the targeting potential as well as importance of ligand (folic acid) on cellular internaliza- tion were evaluated & compared with PLGA nanoparticles.