Nelson Monteiro

Liposomes in tissue engineering and regenerative medicine

Liposomes are vesicular structures made of lipids that are formed in aqueous solutions. Structurally, they resemble the lipid membrane of living cells. Therefore, they have been widely investigated, since the 1960s, as models to study the cell membrane, and as carriers for protection and/or delivery of bioactive agents. They have been used in different areas of research including vaccines, imaging, applications in cosmetics and tissue engineering. Tissue engineering is defined as a strategy for promoting the regeneration of tissues for the human body. This strategy may involve the coordinated application of defined cell types with structured biomaterial scaffolds to produce living structures. To create a new tissue, based on this strategy, a controlled stimulation of cultured cells is needed, through a systematic combination of bioactive agents and mechanical signals. In this review, we highlight the potential role of liposomes as a platform for the sustained and local delivery of bioactive agents for tissue engineering and regenerative medicine approaches.

Antibacterial activity of chitosan nanofiber meshes with liposomes immobilized releasing gentamicin.

Chitsan (Ch) nanofiber mesh (NFM) is a material with natural characteristics favoring its use in human wound dressing. The present work proposes a gentamicin-loaded liposome immobilized at the surface of Ch NFMs to promote its antibacterial activity. To achieve this purpose, Ch NFMs were functionalized with thiol groups, and gentamicin-loaded liposomes were covalently immobilized by the reaction of the SH groups with maleimide. The maximum concentration of SH groups (55.52±11.19nmolcm(-2)) was obtained at pH 7. A fluorescent dye was covalently bound to the SH groups present at the surface of electrospun Ch NFMs. Their spatial distribution was uniform throughout the NFMs when analyzed by fluorescence microscopy. Gentamicin was successfully encapsulated into the liposomes with an efficiency of 17%. Gentamicin-loaded liposomes were uniformly distributed at the surface of the Ch NFMs and the drug release kinetic showed a sustained release of gentamicin during 16h, achieving a steady state at 24h. The in vitro susceptibility tests confirmed that the gentamicin released from the liposomes immobilized at the surface of electrospun Ch NFM has bactericidal activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The results show that the developed system has promising performance for wound dressing applications, avoiding infections caused by these common pathogens.

Immobilization of bioactive factor-loaded liposomes on the surface of electrospun nanofibers targeting tissue engineering

Electrospun nanofiber meshes (NFM), due to their morphology and fibrous structure, are extensively proposed as biomedical devices, for tissue engineering on scaffolds and also as drug delivery systems. Liposomes are nanoparticles prepared from a biologically derived material (phospholipid), which are already in clinical use as a drug release device. Liposomes may be combined with biomaterial scaffolds to promote a local and sustained delivery of loaded bioactive agents. The main objective of the present study is to evaluate the efficacy of dexamethasone (Dex)-loaded liposomes immobilized on the surface of electrospun polycaprolactone (PCL) NFM for promoting the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). The in vitro release profile demonstrates a sustained release of Dex over 21 days, after an initial burst release over 12 h. Biological assays show that Dex-loaded liposomes immobilized on the surface of electrospun PCL NFMs do not exhibit any cytotoxic effect, being able to successfully promote the osteogenic differentiation of hBMSCs. We herein validate the concept of using liposomes immobilized on the surface of a nanostructured fibrous system to be used as an advanced cell carrier device with autonomous release of growth/differentiation factors relevant for tissue engineering and regenerative medicine strategies.

Nanoparticle-based bioactive agent release systems for bone and cartilage tissue engineering

The inability to deliver bioactive agents locally in a transient but sustained manner is one of the challenges on the development of bio-functionalized scaffolds for tissue engineering (TE) and regenerative medicine. The mode of release is especially relevant when the bioactive agent is a growth factor (GF), because the dose and the spatiotemporal release of such agents at the site of injury are crucial to achieve a successful outcome. Strategies that combine scaffolds and drug delivery systems have the potential to provide more effective tissue regeneration relative to current therapies. Nanoparticles (NPs) can protect the bioactive agents, control its profile, decrease the occurrence and severity of side effects and deliver the bioactive agent to the target cells maximizing its effect. Scaffolds containing NPs loaded with bioactive agents can be used for their local delivery, enabling site-specific pharmacological effects such as the induction of cell proliferation and differentiation, and, consequently, neo-tissue formation. This review aims to describe the concept of combining NPs with scaffolds, and the current efforts aiming to develop highly multi-functional bioactive agent release systems, with the emphasis on their application in TE of connective tissues.

On the use of dexamethasone-loaded liposomes to induce the osteogenic differentiation of human mesenchymal stem cells

Stem cells have received considerable attention by the scientific community because of their potential for tissue engineering and regenerative medicine. The most frequently used method to promote their differentiation is supplementation of the in vitro culture medium with growth/differentiation factors (GDFs). The limitations of that strategy caused by the short half‐life of GDFs limit its efficacy in vivo and consequently its clinical use. Thus, the development of new concepts that enable the bioactivity and bioavailability of GDFs to be protected, both in vitro and in vivo, is very relevant. Nanoparticle‐based drug delivery systems can be injected, protect the GDFs and enable spatiotemporal release kinetics to be controlled. Liposomes are well‐established nanodelivery devices presenting significant advantages, viz. a high load‐carrying capacity, relative safety and easy production, and a versatile nature in terms of possible formulations and surface functionalization. The main objective of the present study was to optimize the formulation of liposomes to encapsulate dexamethasone (Dex). Our results showed that the optimized Dex‐loaded liposomes do not have any cytotoxic effect on human bone marrow‐derived mesenchymal stem cells (hBMSCs). More importantly, they were able to promote an earlier induction of differentiation of hBMSCs into the osteogenic lineage, as demonstrated by the expression of osteoblastic markers, both phenotypically and genotypically. We concluded that Dex‐loaded liposomes represent a viable nanoparticle strategy with enhanced safety and efficacy for tissue engineering and regenerative medicine. Copyright

Dual release of a hydrophilic and a hydrophobic osteogenic factor from a single liposome

Delivery systems may be designed to protect and control the release kinetics of growth/differentiation factors in a spatiotemporal manner. Liposomes are examples of biological-based bioactive agent delivery systems. In this work, ascorbic acid (AscA) was encapsulated in the inner compartment of the liposome and dexamethasone (Dex) was encapsulated within the lipid bilayer in order to develop a dual release system of these bioactive agents involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). The particle size (∼150 nm) of the prepared liposomes showed a monodisperse distribution. The bioactive agent release study showed that Dex was released more rapidly from the liposomes than AscA. The Dex release profile showed an initial burst release within 12 h; afterwards, a slower and sustained release was observed until 21 days. The release of AscA from the liposomes was not detected until 6 h; afterwards, a linear release was observed from 24 h until 21 days. The effect of Dex–AscA-loaded liposomes on the viability, proliferation and osteogenic differentiation of human bone marrow-derived MSCs (hBMSCs) were assessed. The cell culture results showed that the Dex–AscA-loaded liposomes (in a single dose or in repeated doses) do not have any cytotoxic effect. Dex–AscA-loaded liposomes given once did not promote induction of hBMSCs differentiation into the osteogenic lineage. However, Dex–AscA-loaded liposomes given repeatedly promoted the hBMSCs differentiation into the osteogenic lineage, both in basal medium and complete osteogenic medium. These results were genotypically demonstrated by the expression of osteoblastic markers. In conclusion, Dex–AscA-loaded liposomes represent a biological nanoparticle strategy with potential safety and efficacy for bone tissue engineering approaches.