NEW MATERIALS FOR BIOMEDICAL APPLICATIONS OBTAINED BY THE SELF-ASSEMBLY OF DENDRIMER-SINGLE STRANDED DNA CONJUGATES

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Fine tuning of the pH-sensitivity of laponite–doxorubicin nanohybrids by polyelectrolyte multilayer coating

Publication . Xiao, Shili; Castro, Rita; Maciel, Dina; Gonçalves, Mara; Shi, Xiangyang; Rodrigues, João; Tomás, Helena

Despite the wide research done in the field, the development of advanced drug delivery systems with improved drug delivery properties and effective anticancer capability still remains a great challenge. Based on previous work that showed the potentialities of the nanoclay Laponite as a pH-sensitive doxorubicin (Dox) delivery vehicle, herein we report a simple method to modulate its extent of drug release at different pH values. This was achieved by alternate deposition of cationic poly(allylamine) hydrochloride and anionic poly(sodium styrene sulfonate) (PAH/PSS) polyelectrolytes over the surface of Dox-loaded Laponite nanoparticles using the electrostatic layer-by-layer (LbL) self-assembly approach. The successful formation of polyelectrolyte multilayer-coated Dox/Laponite systems was confirmed by Dynamic Light Scattering and zeta potential measurements. Systematic studies were performed to evaluate their drug release profiles and anticancer efficiency. Our results showed that the presence of the polyelectrolyte multilayers improved the sustained release properties of Laponite and allowed a fine tuning of the extension of drug release at neutral and acidic pH values. The cytotoxicity presented by polyelectrolyte multilayer-coated Dox/Laponite systems towards MCF-7 cells was in accordance with the drug delivery profiles. Furthermore, cellular uptake studies revealed that polyelectrolyte multilayer-coated Dox/Laponite nanoparticles can be effectively internalized by cells conducting to Dox accumulation in cell nucleus.

2016Journal article

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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

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Dendrimer-assisted formation of fluorescent nanogels for drug delivery and intracellular imaging

Publication . Gonçalves, Mara; Maciel, Dina; Capelo, Débora; Xiao, Shili; Sun, Wenjie; Shi, Xiangyang; Rodrigues, João; Tomás, Helena; Li, Yulin

Although, in general, nanogels present a good biocompatibility and are able to mimic biological tissues, their unstability and uncontrollable release properties still limit their biomedical applications. In this study, a simple approach was used to develop dual-cross-linked dendrimer/alginate nanogels (AG/G5), using CaCl2 as cross-linker and amine-terminated generation 5 dendrimer (G5) as a cocrosslinker, through an emulsion method. Via their strong electrostatic interactions with anionic AG, together with cross-linker Ca(2+), G5 dendrimers can be used to mediate the formation of more compact structural nanogels with smaller size (433 ± 17 nm) than that (873 ± 116 nm) of the Ca(2+)-cross-linked AG nanogels in the absence of G5. Under physiological (pH 7.4) and acidic (pH 5.5) conditions, the sizes of Ca(2+)-cross-linked AG nanogels gradually decrease probably because of their degradation, while dual-cross-linked AG/G5 nanogels maintain a relatively more stable structure. Furthermore, the AG/G5 nanogels effectively encapsulate the anticancer drug doxorubicin (Dox) with a loading capacity 3 times higher than that of AG nanogels. The AG/G5 nanogels were able to release Dox in a sustained way, avoiding the burst release observed for AG nanogels. In vitro studies show that the AG/G5-Dox NGs were effectively taken up by CAL-72 cells (a human osteosarcoma cell line) and maintain the anticancer cytotoxicity levels of free Dox. Interestingly, G5 labeled with a fluorescent marker can be integrated into the nanogels and be used to track the nanogels inside cells by fluorescence microscopy. These findings demonstrate that AG/G5 nanogels may serve as a general platform for therapeutic delivery and/or cell imaging.

2014Journal article

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PAMAM dendrimer/pDNA functionalized-magnetic iron oxide nanoparticles for gene delivery

Publication . Xiao, Shili; Castro, Rita; Rodrigues, João; Shi, Xiangyang; Tomás, Helena

Herein, we report an easy and ingenious method to functionalize magnetic iron oxide nanoparticles (MNPs) with plasmid DNA (pDNA) to obtain nanohybrid systems suitable for nucleic acid therapy. The nanohybrids were prepared by combining complexes of dendrimers and pDNA (dendriplexes) and poly(styrene) sulfonate-coated MNPs through electrostatic interactions. The effects of the dendrimer generation (generations 2, 4 and 6) and the amine to phosphate group (N/P) ratio on the hydrodynamic diameter, zeta potential, cell viability, cellular internalization and transfection efﬁciency of the nanohybridswere systematically investigated at different transfection conditions (including incubation time, pDNA concentration, presence or absence of an external magnetic ﬁeld, and presence or absence of fetal bovine serum). The results conﬁrmed that the nanohybrids were able to transfect NIH 3T3 cells, and that the level of gene expression (the luciferase protein reporter gene was used) was strongly dependent on the dendrimer generation, the N/P ratio, and the pDNA concentration. The best system was based on dendriplex-coated MNPs formed by generation 6 dendrimers at an N/P ratio of 10 that, at optimized conditions, led to a gene expression level which was not signiﬁcantly different from that obtained only using dendriplexes. In summary, a coherent set of results was reached indicating the potential of the developed nanohybrids as effective gene delivery nanomaterials.

2015Journal article

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

Publication . Ramalingam, Kirthiga; Castro, Rita; Pires, Pedro; Shi, Xiangyang; Rodrigues, João; Xiao, Shili; Tomás, Helena

A gene delivery platform for potential use in tissue engineering applications was developed by surface functionalization of biodegradable electrospun poly(lactic-co-glycolic acid) (PLGA) fibers with nanolayers of chitosan (cationic polymer) and alginate (anionic polymer) using the Layer-by-Layer (LbL) technique. The developed system not only supported the attachment and growth of human Mesenchymal Stem Cells (hMSCs), but also was capable of delivering pDNA/dendrimer complexes and inducing cell differentiation towards the osteogenic lineage when a pDNA codifying for human Bone Morphogenetic Protein-2 (BMP-2) was used. Beyond providing a means for pDNA/dendrimer complex immobilization, the polyelectrolyte coating conferred sustained release properties to the scaffold that resulted in pDNA protection from degradation. The polyelectrolyte coating, by itself, also contributed to enhance cell differentiation.

2016Journal article

Open access