Micro/nano-scale strategies for engineering in vitro the celular microenvironment using biodegradable biomaterials

Programa doutoral em BioengenhariaBiological tissues result of a specific spatial organization of cells, extracellular matrix (ECM) molecules, and soluble factors. These micro and nanoscaled biological entities organize into regional tissue architectures, creating highly complex and heterogeneous cellular microenvironments. To generate functional tissue equivalents in vitro, engineered biomaterials should mimic the structural, chemical and cellular complexity by recapitulating the unique native microenvironments. Thus, the main goal of this thesis was to engineer biodegradable polymers using various micro and nanofabrication techniques, with specific structural, biochemical and cellular cues for improved performance. The main governing hypotheses of this thesis were: 1) substrates with improved structural properties can be engineered using biodegradable polymers that have previously shown good results in in vivo studies, 2) biochemical cues can be incorporated into biodegradable polymers, yielding biomaterials with integrated chemical cues for improved cellular performance, and 3) these structural and biochemical cues can be incorporated into a single system. To develop biomaterials with structural cues, micromolding of poly(butylene succinate) (PBS) was performed to engineer surfaces with features at a microscale that induced the alignment of human adipose stem cells. Although this polymeric material has been processed at a macroscale into scaffolds, this was the first report on the engineering of this material at a microscale, demonstrated by the development of twenty features with different dimensions. Improved substrates with structural cues were also engineered using the polysaccharide gellan gum (GG), which has been extensively studied at 3B’s Research Group. Microcapsules of GG, aimed at being used as drug or cell carriers and/or delivery agents, were engineered using a two-phase system. The principle of hydrophobic-hydrophilic repulsion forces was combined with a microfabrication process by means of a needle/syringe pump system. Microcapsules with different diameters were produced by varying the system parameters. As an original proof-of-concept, fluorescent beads, cell suspensions and cell aggregates were encapsulated within this microfabrication system. To develop biomaterials with enhanced biochemical cues, GG was chemically modified with ester bonds, yielding novel hydrogels crosslinkable by ultraviolet (UV) light. Methacrylated GG (MeGG) hydrogels were formed using physical and chemical mechanisms resulting in hydrogels with tunable mechanical properties, matching those of natural tissues from soft to hard, as the brain or collagenous bone. In a subsequent step, this material was combined with chitosan (CHT), a natural polysaccharide, resulting in a polyelectrolyte complex (PEC) hydrogel that combined the most advantageous properties of CHT and MeGG. PEC hydrogels are commonly formed by the interaction between the chains of oppositely charged polymers and are thus held together by ionic forces, which can be disrupted by changes in physiological conditions. However, in our new system, the biochemical cues earlier introduced in GG, allowed to crosslink the MeGG-CHT hydrogel using UV light, stabilizing the structure of the hydrogel. This rather important property also enabled for the development of microgels by photolithography. The encapsulation of rat cardiac fibroblasts within MeGG before PEC hydrogel production, led to the fabrication of microgels with combined biochemical, structural and cellular cues. The developed MeGG-CHT hydrogel was further engineered into a multi-hierarchical fibrous hydrogel by means of combining fluidics technology and chemistry principles of the interaction of two oppositely charged polymers. Two converging fluidic channels were used to extrude the MeGG-CHT hydrogel, formed by the assembly of the polymeric chains at the location where the channels converged. The resulting hydrogel closely mimicked the architecture of natural collagen fibers not only at a micro but also at a nanoscale. The developed hydrogel with relevant biological structural properties was enhanced by incorporating cell adhesive motifs (RGD peptides) into the MeGG backbone before processing. The research work described in this thesis addresses strategies to mimic several parameters of the native microenvironment of tissues. Biochemical and cellular cues were incorporated into biomaterials that were microprocessed with relevant biological micro and nanoscale features. In summary, the works reported in this thesis show the importance of combining different areas of knowledge into the development of improved systems for biomedical engineering applications. Undoubtfully, chemistry and micro and nanofabrication technologies are two areas of knowledge that allow the fabrication of micro and nanostructured materials. Herein, this synergy was achieved with a top-down approach (by micromolding, photolithography or fluidics technologies) and/or with a bottom-up approach (by the assembly of polymer chains). The last work of this thesis is the result of the original combination of both approaches for the development of enhanced micro and nanostructured biomaterials, thus presenting significant improved features compared to currently developed systems to be successfully used in several regenerative medicine approaches.A funcionalidade dos tecidos biológicos está associada à organização espacial de células, à composição e distribuição de moléculas da matriz extracelular e a outros componentes solúveis. Estas entidades biológicas à escala micro/nanométrica organizam-se em arquitecturas locais específicas, criando micro-ambientes celulares complexos e heterogéneos. Existe portanto um grande interesse no desenvolvimento de equivalentes funcionais dos tecidos humanos usando biomateriais de modo a mimetizar a complexidade química, estrutural e celular. Acredita-se que estes biomateriais poderão recapitular as características únicas dos micro-ambientes dos tecidos, favorecendo a sua regeneração funcional. O objectivo principal desta tese consistiu em produzir e desenvolver polímeros biodegradáveis com estímulos químicos, estruturais e celulares de modo a obter uma elevada funcionalidade, usando para isso diferentes técnicas de micro/nano-fabricação. As hipóteses científicas que estão na base do trabalho descrito nesta tese são: 1) é possível desenvolver substratos com estímulos estruturais usando polímeros biodegradáveis que já tenham demonstrado resultados promissores in vivo, 2) é possível incorporar estímulos bioquímicos em sistemas baseados em polímeros biodegradáveis, produzindo biomateriais com sinais bioquímicos integrados para o melhor desempenho biológico dos materiais, e 3) é possível combinar estes sinais estruturais e bioquímicos num único sistema. O polímero polibutileno succinato foi micro-moldado de modo a desenvolver superfícies com topografias à escala micrométrica, visando o desenvolvimento de biomateriais com sinais estruturais, capazes de induzir o alinhamento de células do tecido adiposo humano. Embora este material tenha sido processado anteriormente sob a forma de estrutura 3D porosa, esta foi a primeira vez que foi descrito o processamento deste material à escala micrométrica, demonstrado pelo desenvolvimento de vinte padrões com diferentes dimensões. O polissacarídeo goma gelana (GG), extensivamente estudado no Grupo de Investigação 3B’s, foi usado para desenvolver substratos com sinais estruturais. Micro-cápsulas de GG foram fabricadas usando um sistema de duas fases, com o intuito de serem usadas para o transporte ou libertação de drogas ou células. O princípio de repulsão entre soluções hidrofóbicas e hidrófilas foi combinado com um processo de micro-fabricação, usando uma bomba de injecção. De modo a demonstrar o conceito, partículas fluorescentes, suspensões celulares e agregados celulares foram encapsulados usando este sistema. Para desenvolver biomateriais com sinais bioquímicos, a GG foi modificada quimicamente com ligações éster, produzindo hidrogéis reticuláveis por radiação ultravioleta (UV). Os hidrogéis de GG metacrilada (MeGG) são formados com mecanismos físicos e químicos, resultando em géis com propriedades mecânicas ajustáveis numa gama que se situa próximo da dos tecidos humanos moles e duros, como o cérebro e o osso. Este material foi posteriormente combinado com quitosano, um polissacarídeo de origem natural, resultando num complexo polieletrolítico (PEC) que combina as melhores propriedades do quitosano e da MeGG. A formação de hidrogéis de PECs resulta da interacção entre cadeias de polímeros com cargas opostas, sendo o mecanismo de ligação dependente de forças iónicas, as quais podem ser perturbadas por mudanças na composição da solução. Os sinais bioquímicos introduzidos anteriormente permitiram reticular o hidrogel MeGG-CHT com a radiação UV, estabilizando a estrutura do hidrogel. Este material permitiu também o desenvolvimento de micro-géis por fotolitografia. O encapsulamento de fibroblastos do coração de ratos na MeGG previamente à produção dos hidrogéis conduziu à fabricação de micro-géis com sinais bioquímicos, estruturais e celulares integrados num mesmo sistema. O sistema de hidrogel MeGG-CHT foi usado para obter um hidrogel fibroso hierárquico, através da combinação de microfluídica e complexação polieletrolitica. Extrudiu-se o MeGGCHT em dois canais convergentes com o objectivo de obter a complexação das cadeias poliméricas na forma de fibra. O hidrogel desenvolvido mimetiza a arquitectura das fibras de colagénio existentes no corpo humano, não só ao nível micrométrico mas também à escala nanométrica. O hidrogel desenvolvido foi funcionalizado através da incorporação de moléculas adesivas (péptidos RGD) na MeGG antes do seu processamento. O trabalho de investigação descrito nesta tese demonstra o potencial de diferentes estratégias para mimetizar várias características do micro-ambiente existente nos tecidos. Sinais bioquímicos e celulares foram incorporados em biomateriais que foram posteriormente processados para obter estruturas biológicas relevantes à escala micro/nanométrica. Esta tese demonstra a importância de combinar diferentes áreas do conhecimento para o desenvolvimento de sistemas funcionais para aplicações biomédicas. É inquestionável que a química e as tecnologias de micro e nano-fabricação são duas áreas de conhecimento que se complementam e permitem a fabricação de materiais micro e nanoestruturados. Esta sinergia foi alcançada usando para o efeito uma abordagem top-down (através de fotolitografia, micro-moldação ou microfluídica) e/ou uma abordagem bottom-up (através da complexação de cadeias poliméricas). No último trabalho da tese estas duas abordagens convergem para o desenvolvimento de biomateriais micro e nano-estruturados. Este tipo de sistemas permitem a funcionalização de biomateriais até níveis de aproximação dos tecidos biológicos não tem paralelo nos sistemas convencionais, o que se traduz no desenvolvimento de sistemas de elevado desempenho para diferentes abordagens em engenharia de tecidos

Development of micro-patterned surfaces of poly(butylene succinate) by micromolding for guided tissue engineering

Native tissues present complex architectures at the micro- and nanoscale that dictate their biological function. Several microfabrication techniques have been employed for engineering polymeric surfaces that could replicate in vitro these micro- and nanofeatures. In this study, biomimetic surfaces of poly(butylene succinate) (PBS) were engineered by a micromolding technique. After the optimization of the system parameters, 20 surfaces with different combinations of groove and ridge sizes were developed and characterized by scanning electron microscopy (SEM). The influence of the engineered microfeatures over the viability and attachment of human adipose derived adult stem cells (hASCs) was evaluated. hASCs cultured onto the engineered surfaces were demonstrated to remain viable for all tested patterns. SEM and immunostaining showed adequate attachment and spreading of the stem cells for all the patterned groove/ridge combinations. This study indicated that it is possible to engineer micropatterned surfaces of PBS and that the developed structures could have great potential for tissue engineering where cell alignment is an essential requisite.Fundação para a Ciência e a Tecnologia (FCT)MIT-Portugal ProgramNoE EXPERTISSUES (NMP3-CT-2004-500283

Magnetoliposomes based on NixCu1-xFe2O4 or NiFe2-yAlyO4 nanocrystals for applications in magnetic separation and classification

Ferrites are a broad class of compounds with general formula MFe2O4, where M stands for a divalent metallic cation. Among these, copper ferrite (CuFe2O4) presents a moderate saturation magnetization, while nickel ferrite (NiFe2O4) holds a large one. Mixed ferrites with composition NixCu1-xFe2O4 were then prepared by coprecipitation method, in order to control the saturation magnetization with the fraction of Ni. Another possibility is the partial substitution of iron atoms with aluminium, as NiAl2O4 nanocrystals show very low saturation magnetization. Thus, ferrites of NiFe2-yAlyO4 composition were also obtained. XRD spectra of the obtained nanoparticles show the spinel-type crystalline phase. Solid magnetoliposomes of the prepared mixed ferrites were obtained, and their bilayer structure was proven by the use lipophilic fluorescence probes.info:eu-repo/semantics/publishedVersio

The effect of chitosan on the in vitro biological performance of chitosan-poly(butylene succinate) blends

Chitosan blends with synthetic biodegradable polymers have been proposed for various biomedical applications due to their versatile mechanical properties and easier processing. However, details regarding the main surface characteristics that may benefit from the blending of these two types of materials are still missing. Hence, this work aims at investigating the surface properties of chitosan-based blends, illustrating the way these properties determine the material-proteins interactions and ultimately the behavior of osteoblast-like cells. The surface characteristics of modified and nonmodified blends were assessed using complimentary techniques such as optical microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR-ATR), X-ray photoelectron spectroscopy (XPS), contact angle measurements and surface energy calculations. The adsorption of human serum albumin (HSA) and human plasma fibronectin (HFN) onto the different surfaces was quantified by association of an indirect method with a colorimetric assay. It was found that the presence of chitosan on the surface promoted the adsorption of proteins. Moreover, a preferential adsorption of albumin over fibronectin was registered. The in vitro biological performance of the studied materials was further investigated by a direct contact assay with an osteoblastic-like cell line (SaOs-2). A synergistic effect of the two components of the blend was observed. While the synthetic polyester promoted the adhesion of SaOs-2, the presence of chitosan significantly enhanced the osteoblastic activity of these cells. This work further confirmed the interest in designing polymeric blends with natural polymers as a successful strategy to enhance the biological performance of a biomaterial

Cryopreservation of cell laden natural origin hydrogels for cartilage regeneration strategies

Statement of Purpose: An increase of scientific published literature and clinical experience supports the requirement of providing products like cultured cells and tissues to the market. One of the main prospects of cartilage tissue engineering is the possibility of developing custom-made regenerative medicine solutions on an individual patient basis. The efficient preservation and storage procedure will provide products available as needed which could be adapted to an autologous immediate solution. Thus, the aim of this study was to examine the effects of the cryopreservation on the chondrogenic differentiation characteristics of human mesenchymal stem cells isolated from adipose tissue (hASCs). Furthermore, we also propose to determine hASCs-hydrogel stability and confirm the potential of these bioengineered constructs to be applied in cartilage regeneration. The results obtained show that the hydrogels withstand the cryopreservation process maintaining their structural integrity, with good cell content after cryopreservation. Thus, cell encapsulation systems of natural based hydrogels may be an interesting approach for the long term preservation of cartilage tissue engineered products. Methods: The κ-carrageenan (κCR) hydrogels were produced using an ionotropic gelation method. Then, stem cells, namely human adipose derived stem cells (hASCs), were encapsulated in κCR discs (5 mm dia. x 3 mm height) at a density of 5x106 cell/cm3 and cultured for 21 days in standard basal (BM) or chondrogenic media (ChM). The cell hydrogels were cryopreserved in liquid nitrogen for up to 30 days. The overall morphology of κCR discs with encapsulated hASCs was observed under light microscope. hASCs viability and proliferation rate was determined by double stand DNA quantification. Additionally, chondrogenic differentiation of hASCs encapsulated in the hydrogels is being characterized by histological staining for selective cartilage staining and real time PCR analysis (Sox9, aggrecan, and collagen: type I, type II and type X). DMA analysis allowed determining the mechanical properties of κ-carrageenan hydrogels, namely storage (elastic) and loss (viscous) while immersed in wet state at 24 °C and throughout a physiological relevant range of frequencies. The described characterization assays were performed both before (BC) and after cryopreservation/freezing (AC) time points. Results: The cell morphology, distribution and appearance of the hydrogels are clearly observed from the microscopic light images (Figure 1A). It is possible to observe the smooth and homogeneous surface, the well defined and stable shape before and after the freezing process. Encapsulated hASCs were able to maintain cellular content, despite an expected decrease observed upon cryopreservation (Figure 1B), which is associated to a recovery time after thawing. The microscopic images and biological evaluation of κCR hydrogel revealed that the cryopreservation process did not change the cellular morphology; the surface and integrity of the hydrogel disc and enables maintenance of hASCs after exposure to low temperatures environments. Figure 1. (A) Representative optical micrographs of hASCs encapsulated in κCR hydrogels and cultured in ChM and BM before and after cryopreservation and (B) cell proliferation results, based on the quantification of dsDNA content. Scale bar represent 100 μm. Conclusions: The results obtained so far indicated the feasibility of hASCs-κCR system in cartilage tissue engineering regeneration strategies due to its ability to support hASCs viability before and after cryopreservation. Ongoing studies on the assessment of chondrogenic features of these cryopreserved systems will provide information on the effect of cryopreservation indicative of a stable chondrocyte phenotype. In summary, this study provided information on the potential of ASCs-hydrogel constructs for a long term storage and ready to use bioengineered tissue substitutes for cartilage regeneration strategies. References: (Popa EG. Biomacromolecules 2011;12:3952-3961

Self-assembled hydrogel fiber bundles from oppositely charged polyelectrolytes mimic micro-/nanoscale hierarchy of collagen

Fiber bundles are present in many tissues throughout the body. In most cases, collagen subunits spontaneously self-assemble into a fibrilar structure that provides ductility to bone and constitutes the basis of muscle contraction. Translating these natural architectural features into a biomimetic scaffold still remains a great challenge. Here, a simple strategy is proposed to engineer biomimetic fiber bundles that replicate the self-assembly and hierarchy of natural collagen fibers. The electrostatic interaction of methacrylated gellan gum with a countercharged chitosan polymer leads to the complexation of the polyelectrolytes. When directed through a polydimethylsiloxane channel, the polyelectrolytes form a hierarchical fibrous hydrogel demonstrating nanoscale periodic light/dark bands similar to D-periodic bands in native collagen and align parallel fibrils at microscale. Importantly, collagen-mimicking hydrogel fibers exhibit robust mechanical properties (MPa scale) at a single fiber bundle level and enable encapsulation of cells inside the fibers under cell-friendly mild conditions. Presence of carboxyl- (in gellan gum) or amino- (in chitosan) functionalities further enables controlled peptide functionalization such as Arginylglycylaspartic acid (RGD) for biochemical mimicry (cell adhesion sites) of native collagen. This biomimetic-aligned fibrous hydrogel system can potentially be used as a scaffold for tissue engineering as well as a drug/gene delivery vehicle.S.S. and D.F.C. contributed equally to the work. This research was funded by the US Army Engineer Research and Development Center, the Institute for Soldier Nanotechnology, the NIH (HL092836, EB007249), and the National Science Foundation CAREER award (A.K.). This work was in part supported by FCT through funds from the POCTI and/or FEDER programs and from the European Union under the project NoE EXPERTISSUES (NMP3-CT-2004-500283). D.F.C. acknowledges the Foundation for Science and Technology (FCT), Portugal and the MIT-Portugal Program for personal grant SFRH/BD/37156/2007. S.S. acknowledges the postdoctoral fellowship awarded by Le Fonds Quebecois de la Recherche sur la Nature et les Technologies (FQRNT), Quebec, Canada and interdisciplinary training fellowship (NIH NRSA T32) awarded by System-based Consortium for Organ Design and Engineering (SysCODE). The authors would like to thank Dr. Iva Pashkuleva and Dr. Maria Ericsson for scientific discussions and technical assistance with TEM, respectivelyinfo:eu-repo/semantics/publishedVersio

Fluorescence studies on new potential antitumoral 1,3-diarylurea derivatives in the thieno[3,2-b]pyridine series encapsulated in magnetoliposomes

Magnetic nanoparticles of magnetite and of nickel core with silica shell were prepared and either covered with a lipid bilayer or entrapped in liposomes, forming magnetoliposomes. New potential antitumoral 1,3-diarylurea derivatives of thieno[3,2-b]pyridines were then encapsulated in liposomes and magnetoliposomes and their photophysical behavior was investigated.Fundação para a Ciência e a Tecnologia (FCT), FEDER, COMPETE/QREN/EU for financial support to CFUM (PEst-C/FIS/UI0607/2011) and CQ/UM (PEst-C/QUI/UI0686/2011) and to research projects PTDC/QUI/81238/2006 (FCOMP-01-0124-FEDER-007467), PTDC/QUIQUI/111060/2009 (FCOMP-01-0124-FEDER-015603)

New 1,3-diarylureas linked by C-C Suzuki coupling to the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate moiety: synthesis and fluorescence studies in solution and in lipid membranes

New six fluorescent 1,3-diarylureas linked by C-C Suzuki coupling to the 6-position of the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate moiety were prepared by reaction of the amino groups on the ortho or meta positions relative to the C-C bond of the Suzuki coupling products, with different para-substituted arylisocyanates (H, OMe, CN), in high to excellent yields. The fluorescence properties of the 1,3-diarylureas in solution and in lipid membranes of egg-yolk phosphatidylcholine (Egg-PC), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG) or dioctadecyldimethylammonium bromide (DODAB), with or without cholesterol (Ch), were studied. The six 1,3-diarylureas have reasonable fluorescence quantum yields in several solvents (between 0.02 and 0.69) and present a moderately solvent sensitive emission, but are not fluorescent in alcohols and water. The compounds bearing the arylurea moiety in the meta position relative to the C-C bond, especially with the OMe and CN substituents, present the better solvatochromic properties. Incorporation of the six compounds in lipid membranes indicates that all the compounds are deeply located in the hydrophobic region of the lipid bilayers, feeling the transition between the rigid gel phase and fluid phases.To the Foundation for the Science and Technology (FCT, Portugal) for inancial support to the NMR portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho). To the FCT and FEDER (European Fund for Regional Development)-COMPETE-QREN-EU for financial support to the Research Centres, CQ/UM [PEst-C/QUI/UI0686/2011 (FCOMP-01-0124-FEDER-022716)] and CFUM [PEst-C/FIS/UI0607/2011 (F-COMP-01-0124-FEDER-022711)], and to the research projects PTDC/QUI/81238/2006 (FCOMP-01-0124-FEDER-007467) (photophysical studies) and PTDC/QUI-QUI/111060/2009 (F-COMP-01-0124-FEDER-015603) (organic synthesis)

Genetic ablation of inositol 1,4,5-Trisphosphate receptor type 2 (IP3R2) fails to modify disease progression in a mouse model of Spinocerebellar Ataxia type 3

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by an abnormal polyglutamine expansion within the ataxin-3 protein (ATXN3). This leads to neurodegeneration of specific brain and spinal cord regions, resulting in a progressive loss of motor function. Despite neuronal death, non-neuronal cells, including astrocytes, are also involved in SCA3 pathogenesis. Astrogliosis is a common pathological feature in SCA3 patients and animal models of the disease. However, the contribution of astrocytes to SCA3 is not clearly defined. Inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is the predominant IP3R in mediating astrocyte somatic calcium signals, and genetically ablation of IP3R2 has been widely used to study astrocyte function. Here, we aimed to investigate the relevance of IP3R2 in the onset and progression of SCA3. For this, we tested whether IP3R2 depletion and the consecutive suppression of global astrocytic calcium signalling would lead to marked changes in the behavioral phenotype of a SCA3 mouse model, the CMVMJD135 transgenic line. This was achieved by crossing IP3R2 null mice with the CMVMJD135 mouse model and performing a longitudinal behavioral characterization of these mice using well-established motor-related function tests. Our results demonstrate that IP3R2 deletion in astrocytes does not modify SCA3 progression.This work has been funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020, PTDC/NEUNMC/3648/2014 and COMPETE-FEDER (POCI-01-0145-FEDER-016818); fellowships to DCG (2021.08121.BD), DMF (SFRH/BD/147947/2019), JSC (SFRH/BD/140624/2018), ANC (SFRH/BPD/118779/2016), AVF (UMINHO/BIL-CNCG/2022/11), SGG (SFRH/BD/101298/2014), and JFV (2020.05109.BD); FCT Scientific Employment Stimulus (CEEC)—Individual Call position to SDS (CEECIND/00685/2020); grants from the Bial Foundation (037/18) and “the la Caixa” Foundation (LCF/PR/HR21/52410024) to JFO; and by the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). It was also supported by grants from the ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122 and national funds through the Foundation for Science and Technology (FCT)

Magnetoliposomes containing calcium ferrite nanoparticles for applications in breast cancer therapy

Magnetoliposomes containing calcium ferrite (CaFe2O4) nanoparticles were developed and characterized for the first time. CaFe2O4 nanoparticles were covered by a lipid bilayer or entrapped in liposomes forming, respectively, solid or aqueous magnetoliposomes as nanocarriers for new antitumor drugs. The magnetic nanoparticles were characterized by UV/Visible absorption, XRD, HR-TEM, and SQUID, exhibiting sizes of 5.2 ± 1.2 nm (from TEM) and a superparamagnetic behavior. The magnetoliposomes were characterized by DLS and TEM. The incorporation of two new potential antitumor drugs (thienopyridine derivatives) specifically active against breast cancer in these nanosystems was investigated by fluorescence emission and anisotropy. Aqueous magnetoliposomes, with hydrodynamic diameters around 130 nm, and solid magnetoliposomes with sizes of ca. 170 nm, interact with biomembranes by fusion and are able to transport the antitumor drugs with generally high encapsulation efficiencies (70%). These fully biocompatible drug-loaded magnetoliposomes can be promising as therapeutic agents in future applications of combined breast cancer therapy.This research was funded by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CF-UM-UP (UID/FIS/04650/2013; UID/FIS/04650/2019), CQUM (UID/QUI/00686/2016; UID/QUI/00686/2019) and LA-26 (PEst-C/SAU/LA0026/2013), and through the research project PTDC/QUI-QFI/28020/2017 (POCI-01-0145-FEDER-028020), financed by FCT, European Fund of Regional Development (FEDER), COMPETE2020 and Portugal2020. The magnetic measurements were supported by projects UTAP-EXPL/NTec/0046/2017, NORTE-01-0145-FEDER-028538 e PTDC/FIS-MAC/29454/2017. The APC was also funded by FCT. B.D.C. acknowledges FCT for a PhD grant (SFRH/BD/141936/2018)