RESEARCH PAPER Folate-Targeted Multifunctional Amino Acid-Chitosan Nanoparticles for Improved Cancer Therapy Vítor M. Gaspar & Elisabete C. Costa & João A. Queiroz & Chantal Pichon & Fani Sousa & Ilídio J. Correia Received: 21 May 2014 /Accepted: 15 August 2014 /Published online: 4 September 2014 # Springer Science+Business Media New York 2014 ABSTRACT Purpose Tumor targeting nanomaterials have potential for im- proving the efficiency of anti-tumoral therapeutics. However, the evaluation of their biological performance remains highly challeng- ing. In this study we describe the synthesis of multifunctional nanoparticles decorated with folic acid-PEG and dual amino acid-modified chitosan (CM-PFA) complexed with DNA and their evaluation in organotypic 2D co-cultures of cancer-normal cells and also on 3D multicellular tumor spheroids models. Methods The physicochemical characterization of CM-PFA mul- tifunctional carriers was performed by FTIR, 1 H NMR and DLS. 2D co-culture models were established by using a 1:2 cancer-to- normal cell ratio. 3D organotypic tumor spheroids were assem- bled using micromolding technology for high throughput screen- ing. Nanoparticle efficiency was evaluated by flow cytometry and confocal microscopy. Results The CM-PFA nanocarriers (126–176 nm) showed hemocompatibility and were internalized by target cells, achieving a 3.7 fold increase in gene expression. In vivo-mimicking 2D co- cultures confirmed a real affinity towards cancer cells and a negligible uptake in normal cells. The targeted nanoparticles pen- etrated into 3D spheroids to a higher extent than non-targeted nanocarriers. Also, CM-PFA-mediated delivery of p53 tumor suppressor promoted a decrease in tumor-spheroids volume. Conclusion These findings corroborate the improved efficiency of this delivery system and demonstrate its potential for application in cancer therapy. KEY WORDS cancer therapy . gene delivery . targeted nanoparticles . 2D co-cultures . 3D tumor spheroids INTRODUCTION Targeted delivery through specialized nanocarriers cur- rently attracts remarkable attention since bioactive mol- ecules can be selectively transported into malignant tis- sues with improved efficiency and limited systemic tox- icity (1). In line with this focus, nanosized systems are currently being developed for the delivery of a broad range of anti-tumoral therapeutics that span from plas- mid DNA (pDNA (2)), small interfering RNA (siRNA (3)), polyphenolic compounds (Resveratrol, tea catechins (4)), up to general pharmaceuticals (Doxorubicin, Pacli- taxel and Cisplatin (5)). Nevertheless, despite these advances, the majority of the nanocarriers engineered so far present limited in vivo targeting capacity, generally taking advantage of a probabilistic accu- mulation in cancer cells through the so-termed enhanced permeability and retention effect (EPR) (6). The EPR effect is a consequence of an uncontrolled angiogenesis which orig- inates disorganized and leaky vascular networks that have characteristic fenestrations with sufficiently large sizes for pas- sive nanocarrier extravasation into diseased tissues (100 - 300 nm) (7,8). However, this range is highly variable, depends on tumor type, location, and vascular density, both at the periphery and tumor core (9). To further hinder accumula- tion, solid tumors have a high interstitial fluid pressure, an additional barrier that affects nanoparticle convective diffu- sion and tissue penetration (10). Consequently, these proper- ties significantly impair the pharmacokinetic/ pharmacodynamic profile and therapeutic effectiveness of non-targeted delivery systems (9,11). In order to overcome these issues various classes of targeting ligands such as anti- bodies (Herceptin ® (12)), aptamer bioconjugates (13), homing Electronic supplementary material The online version of this article (doi:10.1007/s11095-014-1486-0) contains supplementary material, which is available to authorized users. V. M. Gaspar : E. C. Costa : J. A. Queiroz : F. Sousa : I. J. Correia (*) CICS-UBI – Health Sciences Research Centre, Universidade da Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal e-mail: icorreia@ubi.pt C. Pichon Centre de Biophysique Moléculaire, CNRS UPR4301, Inserm and University of Orléans, 45071 cedex 02 Orléans, France Pharm Res (2015) 32:562–577 DOI 10.1007/s11095-014-1486-0