Surface Engineered Poly(lactide-co-glycolide) Nanoparticles for Intracellular Delivery: Uptake and CytotoxicitysA Confocal Raman Microscopic Study Gabriela Romero, † Irina Estrela-Lopis, ‡ Jie Zhou, †,§ Elena Rojas, † Ana Franco, | Christian Sanchez Espinel, | Africa Gonza ´ lez Ferna ´ ndez, | Changyou Gao, § Edwin Donath, ‡ and Sergio E. Moya* ,† CIC BiomaGUNE, Paseo Miramo ´n 182 Ed. Emp. C, San Sebastia ´ n, Spain, Institute of Biophysics and Medical Physics, University of Leipzig, Leipzig, Germany, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China, and Laboratorio de Inmunologı ´a, Edificio Ciencias Experimentales, Campus Lagoas Marcosende, Universidad de Vigo, CP 36310, Vigo, Pontevedra, Spain Received July 13, 2010; Revised Manuscript Received August 31, 2010 Confocal Raman Microscopy (CRM) is used to study the cell internalization of poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) fabricated by emulsion techniques with either poly(ethylene imine) (PEI) or bovine serum albumin (BSA) as surface stabilizers. HepG2 cells were exposed to PEI and BSA stabilized PLGA NPs. Spontaneous Confocal Raman Spectra taken in one and the same spot of exposed cells showed bands arising from the cellular environment as well as bands characteristic for PLGA, proving that the PLGA NPs have been internalized. It was found that PLGA NPs preferentially colocalize with lipid bodies. The results from Raman spectroscopy are compared with flow cytometry and confocal scanning laser microscopy (CLSM) data. The advantages of CRM as a label-free technique over flow cytometry and CLSM are discussed. Additionally, cell viability studies by means of quick cell counting solution and MTT tests in several cell lines show a generally low toxicity for both PEI and BSA stabilized PLGA NPs, with BSA stabilized PLGA NPs having an even lower toxicity than PEI stabilized. Introduction Polymeric nanoparticles (NPs) of submicrometer size are appealing systems for drug delivery. 1–7 They can be easily synthesized and in most of the cases the protocols for drug encapsulation are performed in parallel with fabrication. Fur- thermore, polymeric NPs are normally suitable to be function- alized for targeted delivery by multiple procedures. There is a wide variety of biocompatible and biodegradable polymers from natural sources like BSA, collagen, gelatin, chitosan, and alginate, which can be employed for the fabrication of devices for controlled drug delivery. 8 During the last three decades, synthetic biodegradable polymers, for example, poly(amino acids), poly(alkyl-2-cyanoacrylates), polyesters, polyorthoesters, polyanhydrides, and polyurethanes, have been synthesized and employed in drug loading devices. Among them, the homopoly- mer of lactic acid (PLA) 9,10 and its copolymer, poly(lactide- co-glycolide) (PLGA), 6,11–16 are commonly used to fabricate NPs. PLA and PLGA NPs are easy to produce and display excellent biocompatibility and biodegradability. Several tech- niques have been developed for the preparation of PLA and PLGA NPs such as simple or double emulsions-solvent evapo- ration, 17 nanoprecipitation, 18,19 spray drying, 20 and so on. The simple or double emulsions-solvent evaporation method is most often employed. The size of the NPs can be easily controlled with this method, and it can also be applied for the encapsulation of both hydrophobic and hydrophilic drugs. 21–24 We have previously shown that PLGA NPs can be prepared employing PEI or BSA as surface stabilizers. 25–27 The charge of PEI or BSA allows for the application of the layer by layer technique (LBL) for further noncovalent modification of the NPs. The LBL coating, which can be performed by the stepwise adsorption of the biocompatible chitosan and alginate, 26 for example, has then been used for the attachment of folic acid to increase NP uptake in cancer cell lines. 26,27 In our previous work, cellular uptake of the PLGA NPs has been quantified by means of the combination of flow cytometry and confocal laser scanning microscopy (CLSM). For labeling, either fluorescein or rhodamine was encapsulated in the PLGA NPs during preparation. It was shown that the uptake of NPs is influenced by the nature of the surface of the PLGA NPs. Large uptake was observed for PLGA NPs coated with PEI and BSA. A coating with chitosan/alginate or PEGylation 27 reduced the uptake significantly. Subsequent modification with folic acid facilitated again uptake by specific targeting, because it is known that tumor cells overexpress on their membranes folate receptors (FRs). 25–27 It is nevertheless difficult to differentiate whether the NPs have been internalized in the cells or remained attached to the cell membrane. Flow cytometry measures the fluorescence per cell and does not provide information about localization of labeled NPs, whether attached to or indeed taken up by the cells under study. Visualization of the NPs by CLSM is not a straightforward task because it is not always possible to distinguish between NPs that have been internalized in the cell and NPs attached to the cell membrane, which in many cases * To whom correspondence should be addressed. Fax: +34 943005311. E-mail: smoya@cicbiomagune.es. † CIC biomaGUNE. ‡ University of Leipzig. § Zhejiang University. | Universidad de Vigo. Biomacromolecules 2010, 11, 2993–2999 2993 10.1021/bm1007822  2010 American Chemical Society Published on Web 09/30/2010