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Fundação para a Ciência e a Tecnologia

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Publications

Synthesis of FITC-PAMAM conjugates for in vitro cell studies

Publication . Fernandes, João Tiago Moniz; Rodrigues, João Manuel Cunha; Tomás, Helena Maria Pires Gaspar

2014-12-29Master thesis

Open access

RGD-Modified dendrimers for drug encapsulation and targeted inhibition of tumor cells

Publication . Xuedan, He; Xiangyang, Shi; Tomás, Helena Maria Pires Gaspar

In this study, cyclic arginine-glycine-aspartic acid (RGD) peptide-modified amine-terminated generation 5 poly(amidoamine) (G5.NH2 PAMAM) dendrimers were prepared for the encapsulation of the anticancer drug doxorubicin (DOX) for targeted delivery to cancer cells overexpressing αvβ3 integrin cell surface receptors. First, the thiolated RGD peptide was linked to polyethylene glycol (PEG) via the bifunctional cross-linking reagent 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS). Then a dendrimer modification process was performed in which the PEGylated RGD peptide and fluorescein isothiocyanate (FI) were covalently attached to the G5 dendrimers. This process was finally followed by acetylation of the remaining dendrimer terminal amines. The experimental results show that each G5.NHAc-FI-PEG-RGD dendrimer approximately encapsulated six DOX molecules. This formed complex is water soluble and stable. In vitro release studies proved that the multifunctional dendrimers facilitate a sustained release of DOX. More interesting, one-dimensional NMR and two-dimensional NMR were applied to investigate the interactions between dendrimers and DOX. Here, the impact of the environmental pH on the release rate of DOX from G5.NHAc-FI-PEG-RGD/DOX was fully studied. Furthermore, cell biological studies demonstrated that G5.NHAc-FI-PEG-RGD dendrimers have no cytotoxicity towards U87-MG cancer cells but that G5.NHAc-FI-PEG-RGD/DOX complexes have almost the same cytotoxicity as DOX alone. Moreover, due to the targeting ability of RGD, this dendrimer/drug system can also specifically target and display therapeutic efficacy to cancer cells overexpressing αvβ3 integrins. The cellular internalization of the multifunctionalized dendrimer was shown to be receptor mediated to an important extent. According to this study, we can say that G5.NHAc-FI-PEG-RGD is a promising system for the targeted therapy of different types of cancer.

2014-07-30Master thesis

Open access

Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the layer-by-layer technique

Publication . Ramalingam, Kirthiga; Tomás, Helena Maria Pires Gaspar; Xiao, Shii

Tissue engineering is an important branch of regenerative medicine that uses cells, materials (scaffolds), and suitable biochemical and physicochemical factors to improve or replace specific biological functions. In particular, the control of cell behavior (namely, of cell adhesion, proliferation and differentiation) is a key aspect for the design of successful therapeutical approaches. In this study, poly(lactic-co-glycolic acid) (PLGA) fiber mats were prepared using the electrospinning technology (the fiber diameters were in the micrometer range). Furthermore, the electrospun fiber mats thus formed were functionalized using the layer-by- layer (LbL) technique with chitosan and alginate (natural and biodegradable polyelectrolytes having opposite charges) as a mean for the immobilization of pDNA/dendrimer complexes. The polyelectrolyte multilayer deposition was confirmed by fluorescence spectroscopy using fluorescent-labeled polyelectrolytes. The electrospun fiber mats coated with chitosan and alginate were successfully loaded with complexes of pDNA and poly(amidoamine) (PAMAM) dendrimers (generation 5) and were able of releasing them in a controlled manner along time. In addition, these mats supported the adhesion and proliferation of NIH 3T3 cells and of human mesenchymal stem cells (hMSCs) in their surface. Transfection experiments using a pDNA encoding for luciferase showed the ability of the electrospun fiber mats to efficiently serve as gene delivery systems. When a pDNA encoding for bone morphogenetic protein-2 (BMP-2) was used, the osteoblastic differentiation of hMSCs cultured on the surface of the mats was promoted. Taken together, the results revealed that merging the electrospinning technique with the LbL technique, can be a suitable methodology for the creation of biological active matrices for bone tissue engineering.

2014-09Master thesis

Open access

Antitumor efficacy of doxorubicin-loaded laponite/alginate hybrid hydrogels

Publication . Gonçalves, Mara; Figueira, Priscilla; Maciel, Dina; Rodrigues, João; Shi, Xiangyang; Tomás, Helena; Li, Yulin

Degradable hybrid hydrogels with improved stability are prepared by incorporating nanodisks of biocompatible laponite (LP) in alginate (AG) hydrogels using Ca2+ as a crosslinker. The Dox‐loaded hybrid hydrogels give a controlled Dox release at physiological environment in a sustained manner. Under conditions that mimic the tumor environment, both the sustainability in the Dox release (up to 17 d) and the release efficiency from LP/AG‐Dox hydrogels are improved. The in situ degradation of these hybrid hydrogels gives rise to nanohybrids that might serve as vehicles for carrying Dox through the cell membrane and diminish the effect of Dox ion‐trapping in the acidic extracellular environment of the tumor and/or in the endo‐lysosomal cell compartments.

2014-01Journal article

Restricted access

Redox-responsive alginate nanogels with enhanced anticancer cytotoxicity

Publication . Maciel, Dina; Figueira, Priscilla; Xiao, Shili; Hu, Dengmai; Shi, Xiangyang; Rodrigues, João; Tomás, Helena; Li, Yulin

Although doxorubicin (Dox) has been widely used in the treatment of different types of cancer, its insufficient cellular uptake and intracellular release is still a limitation. Herein, we report an easy process for the preparation of redox-sensitive nanogels that were shown to be highly efficient in the intracellular delivery of Dox. The nanogels (AG/Cys) were obtained through in situ cross-linking of alginate (AG) using cystamine (Cys) as a cross-linker via a miniemulsion method. Dox was loaded into the AG/Cys nanogels by simply mixing it in aqueous solution with the nanogels, that is, by the establishment of electrostatic interactions between the anionic AG and the cationic Dox. The results demonstrated that the AG/Cys nanogels are cytocompatible, have a high drug encapsulation efficiency (95.2 ± 4.7%), show an in vitro accelerated release of Dox in conditions that mimic the intracellular reductive conditions, and can quickly be taken up by CAL-72 cells (an osteosarcoma cell line), resulting in higher Dox intracellular accumulation and a remarkable cell death extension when compared with free Dox. The developed nanogels can be used as a tool to overcome the problem of Dox resistance in anticancer treatments and possibly be used for the delivery of other cationic drugs in applications beyond cancer.

2013Journal article

Restricted access