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- Lipid Status of A2780 Ovarian Cancer Cells after Treatment with Ruthenium Complex Modified with Carbon Dot Nanocarriers: A Multimodal SR-FTIR Spectroscopy and MALDI TOF Mass Spectrometry StudyPublication . Nesic, Maja D.; Ducic, Tanja; Algarra, Manuel; Popović, Iva; Stepić, Milutin; Gonçalves, Mara; Petkovic, MarijanaIn the last decade, targeting membrane lipids in cancer cells has been a promising approach that deserves attention in the field of anticancer drug development. To get a comprehensive un derstanding of the effect of the drug [Ru(η 5 -Cp)(PPh3 )2CN] (RuCN) on cell lipidic components, we combine complementary analytical approaches, matrix-assisted laser desorption and ionization time of-flight mass spectrometry (MALDI TOF MS) and synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectroscopy. Techniques are used for screening the effect of potential metallodrug, RuCN, without and with drug carriers (carbon dots (CDs) and nitrogen-doped carbon dots (N-CDs)) on the lipids of the human ovarian cancer cell line A2780. MALDI TOF MS results revealed that the lysis of ovarian cancer membrane lipids is promoted by RuCN and not by drug carriers (CDs and N-CDs). Furthermore, SR-FTIR results strongly suggested that the phospholipids of cancer cells undergo oxidative stress after the treatment with RuCN that was accompanied by the disordering of the fatty acid chains. On the other hand, using (N-)CDs as RuCN nanocarriers prevented the oxidative stress caused by RuCN but did not prevent the disordering of the fatty acid chain packing. Finally, we demonstrated that RuCN and RuCN/(N-)CDs alter the hydration of the membrane surface in the membrane–water interface region.
- Antitumor efficacy of doxorubicin-loaded laponite/alginate hybrid hydrogelsPublication . Gonçalves, Mara; Figueira, Priscilla; Maciel, Dina; Rodrigues, João; Shi, Xiangyang; Tomás, Helena; Li, YulinDegradable 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.
- Fine tuning of the pH-sensitivity of laponite–doxorubicin nanohybrids by polyelectrolyte multilayer coatingPublication . Xiao, Shili; Castro, Rita; Maciel, Dina; Gonçalves, Mara; Shi, Xiangyang; Rodrigues, João; Tomás, HelenaDespite 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.
- The effect of PAMAM dendrimers on mesenchymal stem cell viability and differentiationPublication . Gonçalves, M.; Castro, R.; Rodrigues, J.; Tomás, H.Stem cells and nanomaterials are two new and exciting fields of science that are evolving very fast and that are starting to establish ties. Nanomaterials should, however, be designed to interact with stem cells without compromising their biological characteristics, in other words, without affecting their viability and differentiation potential. In the present report and for the first time, the effects of poly(amidoamine) (PAMAM) dendrimers on the viability and differentiation ability towards the osteogenic and adipogenic lineages of human mesenchymal stem cells (hMSCs) are systematically evaluated. Studies were done as a function of the cell culture media composition and PAMAM dendrimer surface functionalization, generation, and concentration. hMSCs were exposed to amino and hydroxyl (generations 2, 4 and 6), and carboxylate (generations 1.5, 3.5 and 5.5) functionalized dendrimers, at two different concentrations (10 μg/mL and 0.5 μg/mL), for a period of 21 days. Overall, the results revealed that amino functionalized dendrimers can be severely cytotoxic, the extension of cell death being dependent on the concentration of amino groups in solution. However, in all cases, the differentiation of hMSCs towards the osteogenic and adipogenic phenotypes seems not to be affected as demonstrated by staining in in vitro cultures.
- Biochemical changes in cancer cells induced by photoactive nanosystem based on carbon dots loaded with Ru-complexPublication . Nesic, Maja D.; Ducic, Tanja; Gonçalves, Mara; Stepić, Milutin; Algarra, Manuel; Soto, Juan; Gemović, Branislava; Bandosz, Teresa J.; Petkovic, MarijanaCarbon dots (CDs) and N-carbon dots (N-CDs) loaded with Ru-complex (CDs@RuCN, N-CDs@RuCN, respec tively) were investigated as media imposing biochemical changes induced by UV illumination of ovarian cancer, A2780, and osteosarcoma, CAL72, cells. Synchrotron radiation-based Fourier Transform Infrared Spectroscopy was performed, and the spectra were subjected to a Principal Component Analysis. The CDs@RuCN and N CDs@RuCN effects on cancer cells were analyzed by the theoretical modelling of the stability of the composite systems and a protein database search. Moreover, a detailed evaluation of surface and optical properties of CDs@RuCN and N-CDs@RuCN was carried out. Results demonstrated selective action of the CDs@RuCN and N CDs@RuCN-based photodynamic therapy, with N-CDs@RuCN being the most active in inducing changes in A2780 and CDs@RuCN in CAL72 cells. We assume that different surface charges of nanoparticles led to direct interactions of N-CDs@RuCN with a Wnt signalling pathway in A2780 and those of CDs@RuCN with PI3–K/Akt in CAL72 cells and that further biochemical changes occurred upon light illumination.
- Thermo/redox/pH-triple sensitive poly(N-isopropylacrylamide-co-acrylic acid) nanogels for anticancer drug deliveryPublication . Zhan, Yuan; Gonçalves, Mara; Yi, Panpan; Capelo, Débora; Zhang, Yuhong; Rodrigues, João; Liu, Changsheng; Tomás, Helena; Li, Yulin; He, PeixinThe clinical application of doxorubicin (DOX), like other anticancer drugs, is limited by insufficient cellular uptake and the numerous drug resistance mechanisms existing in cells. The development of smart nanomaterials capable of carrying the drugs into the cells and of releasing them under the control of the microenvironment is an interesting approach that may increase the success of the anticancer drugs currently in use. Herein, we report an easy process to prepare biocompatible nanogels (NGs) with thermo/ redox/pH-triple sensitivity, which are highly effective in the intracellular delivery of DOX. Redox-sensitive/ degradable NGs (PNA-BAC) and nondegradable NGs (PNA-MBA) were prepared through in situ polymerization of N-isopropylacrylamide (NIPAM) and acrylic acid (AA) in the presence of sodium dodecyl sulfate (SDS) as a surfactant, using N,N0-bis(acryloyl)cystamine (BAC) as a biodegradable crosslinker or N,N0-methylene bisacrylamide (MBA) as a nondegradable crosslinker, respectively. After that, the cationic DOX drug was loaded into the NGs through electrostatic interactions, by simply mixing them in aqueous solution. Compared to nondegradable PNA-MBA NGs, PNA-BAC NGs not only presented a higher DOX drug loading capacity, but also allowed a more sustainable drug release behavior under physiological conditions. More importantly, PNA-BAC NGs displayed thermo-induced drug release properties and an in vitro accelerated release of DOX under conditions that mimic intracellular reductive conditions and acidic tumor microenvironments. The thermo/redox/pH multi-sensitive NGs can quickly be taken up by CAL-72 cells (an osteosarcoma cell line), resulting in a high DOX intracellular accumulation and an improved cytotoxicity when compared with free DOX and DOX-loaded nondegradable PNA-MBA NGs. The developed NGs can be possibly used as an effective platform for the delivery of cationic therapeutic agents for biomedical applications.
- pH-sensitive Laponite®/doxorubicin/alginate nanohybrids with improved anticancer efficacyPublication . Gonçalves, Mara; Figueira, Priscilla; Maciel, Dina; Rodrigues, João; Qu, Xue; Liu, Changsheng; Tomás, Helena; Li, YulinThe efficacy of the anticancer drug doxorubicin (Dox) is limited by an insufficient cellular uptake and drug resistance, which is partially due to ion trapping in acidic environments such as the extracellular environment of solid tumors and the interior of endolysosome vesicles. Herein, we describe the preparation and in vitro evaluation of a new type of nanohybrid for anticancer drug delivery which is capable of carrying a high load of the cationic Dox through the cell membrane. In addition, the nanohybrids use the acidic environment of the endolysosomes to release the drug, simultaneously helping to disrupt the endolysosomes and diminishing endolysosome Dox trapping. Furthermore, as the nanohybrid carriers are capable of sustained drug delivery, those that remain in the cytoplasm and still contain Dox are expected to exert a prolonged anticancer activity. Briefly, Dox is loaded onto biocompatible anionic Laponite(®) (LP) nanodisks with a high aspect ratio (25 nm in diameter and 0.92 nm in thickness) through strong electrostatic interactions to get Dox-loaded LP disks. Alginate (AG), a biocompatible natural polymer, is then coated onto the Dox-loaded LP disks (LP/Dox/AG nanohybrids) to prevent the burst release of the drug. The results demonstrate that the nanohybrids have a high encapsulation efficiency (80.8 ± 10.6%), are sensitive to pH and display a sustained drug release behavior. Cell culture experiments indicate that the LP/Dox/AG nanohybrids can be effectively internalized by CAL-72 cells (an osteosarcoma cell line), and exhibit a remarkable higher cytotoxicity to cancer cells than the free Dox. The merits of Laponite(®)/alginate nanohybrids, such as biocompatibility, high loading capacity and stimulus responsive release of cationic chemotherapeutic drugs, render them as excellent platforms for drug delivery.
- Nanotechnology approaches for the delivery of antitumor drugs: the case of doxorubicinPublication . Gonçalves, Mara Isabel Jesus; Tomás, Helena Maria Pires Gaspar; Rodrigues, João Manuel Cunha; Lin, YulinOver the years, nanotechnology had a huge evolution and gathered the attention of many scientists, including those involved in medical sciences. Nanomedicine thus appeared, trying to overcome obstacles that still exist in conventional medicine, by providing innovative approaches for the diagnosis and treatment of diseases. Nowadays, cancer is considered one of the major causes of worldwide death. Doxorubicin (DOX) is a chemotherapeutic drug which is routinely used for cancer treatment. Due to its broad spectrum of activity, DOX is used as a first-line treatment combined with other drugs and procedures. However, this drug has several associated side effects, being the injury of the cardiac muscle tissue and myelosuppression the most reported. Cancer nanomedicine stands up as an alternative to conventional cancer therapy by using nanomaterials as drug carriers which, potentially, make the treatment more efficient and safe. Polymer-based nanomaterials are very promising vehicles for drug delivery, due to the easiness in modelling their properties. Over the years, polymers have proven to be capable of encapsulating and releasing drugs in a sustained manner, improving their biodistribution and accumulation in tumours. The main goal of this thesis was to find new drug delivery systems that could be able to encapsulate DOX and successfully deliver it inside cancer cells. Hopefully, using nanomaterials for DOX delivery, it will be possible to overcome the side effects which are frequently associated to this antitumor drug.
- Polyester Dendrimers Based on Bis-MPA for Doxorubicin DeliveryPublication . Gonçalves, Mara; Kairys, Visvaldas; Rodrigues, João; Tomás, HelenaAlthough doxorubicin (DOX) is one of the most used chemotherapeutic drugs due to its efficacy against a wide group of cancer types, it presents severe side effects. As such, intensive research is being carried out to find new nanoscale systems that can help to overcome this problem. Polyester dendrimers based on the monomer 2,2-bis- (hydroxymethyl)propionic acid (bis-MPA) are very promising systems for biomedical applications due to their biodegradability properties. In this study, bis-MPA-based dendrimers were, for the first time, evaluated as DOX delivery vehicles. Generations 4 and 5 of bis-MPA-based dendrimers with hydroxyl groups at the surface were used (B-G4-OH and B-G5-OH), together with dendrimers partially functionalized with amine groups (B-G4-NH2/OH and B-G5-NH2/OH). Partial functionalization was chosen because the main purpose was to compare the effect of different functional groups on dendrimers’ drug delivery behavior without compromising cell viability, which is often affected by dendrimers’ cationic charge. Results revealed that bis-MPA-based dendrimers were cytocompatible, independently of the chemical groups that were present at their surface. The B-G4-NH2/OH and B-G5-NH2/OH dendrimers were able to retain a higher number of DOX molecules, but the in vitro release of the drug was faster. On the contrary, the hydroxyl-terminated dendrimers exhibited a lower loading capacity but were able to deliver the drug in a more sustained manner. These results were in accordance with the cytotoxicity studies performed in several models of cancer cell lines and human mesenchymal stem cells. Overall, the results confirmed that it is possible to tune the drug delivery properties of bis-MPA-based dendrimers by modifying surface functionalization. Moreover, molecular modeling studies provided insights into the nature of the interactions established between the drug and the bis-MPA based dendrimersDOX molecules attach to their surface rather than being physically encapsulated.
- Dendrimer-assisted formation of fluorescent nanogels for drug delivery and intracellular imagingPublication . Gonçalves, Mara; Maciel, Dina; Capelo, Débora; Xiao, Shili; Sun, Wenjie; Shi, Xiangyang; Rodrigues, João; Tomás, Helena; Li, YulinAlthough, 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.