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. liposomesscaffoldsdelivery systemsbioactive agentsstem cellsThe authors thank the Portuguese Foundation for Science and Technology for the PhD grant to N.S.M. (SFRH/BD/62465/2009), the post-doctoral grants of A.M. (SFRH/BPD/73663/2010). This study was also partly supported by POLARIS (FP7-REGPOT-2012-2013-1), RL3-TECT-NORTE-01-0124-FEDER-000020, co-financed by the North Portugal Regional Operational Programme (ON.2-O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF), the OsteoGraphy (PTDC/EME-MFE/2008) and MaxBone (PTDC/SAU-ENB/115179/2009) projects

Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

BACKGROUND:To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA). METHODOLOGY:Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated. RESULTS:The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations--DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold. CONCLUSIONS:Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections

Mechanism of calcium ion induced multilamellar vesicle DNA interaction

The effect of Ca2+ on the DNA interaction with anionic and neutral multilamellar vesicles (MLV) has been investigated. DNA from wheat (Triticum aestivum L. Gerek) was introduced to a suspension of MLV, composed of phosphatidylcholine (PC): dicetylphosphate (DCP):cholesterol (CHOL) at different molar ratios, to which Ca2+ (5-75 mM) was subsequently added. Indication of aggregation and/or fusion was obtained via light-scattering examination following the addition of Ca2+ and DNA to the MLV medium. Using a UV spectrophotometric assay, it was observed that although DNA alone has no effect on negatively charged MLV, it enhances liposomal interaction in the presence of calcium ions. The minimal Ca2+ concentration required to promote the interaction was detected to be 10 mM, and the highest level of interaction was observed at 75 mM. The aggregation/fusion of vesicles was detected for uncharged MLV (with no DCP in their structure), as well as for the anionic ones containing c. 10% CHOL, but not for anionic MLV containing 40% CHOL. This is explained in terms of cholesterol decreasing the membrane fluidity (above the Tc of components) as a result of which more rigid vesicles become less prone to aggregation/fusion interactions

Formation Of Supramolecular Structures By Negatively Charged Liposomes In The Presence Of Nucleic Acids And Divalent Cations

Cationic liposomes are being increasingly studied as delivery vehicles for bioactive agents such as DNA and other polynucleotides, The mechanism of interaction of DNA with liposomes and the organization of these interacting structures during and after the interaction are still poorly understood. Nucleic acids are known to induce aggregation and size enlargement of liposomes, In the case of phosphatidylcholine (PC) vesicles, these processes depend on the presence and concentration of divalent metal cations and the amount of cholesterol in the liposomes. In this study, anionic small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) composed of dicetylphosphate (DCP):PC:cholesterol at 2:7:1 molar ratios were prepared and incubated with the DNA (from wheat) and Ca2+ (50 mM) at 25 degrees C with the aim of transferring the genetic material into the liposomes by inducing fusion of liposome-liposome aggregates created in the presence, and with the help, of DNA, The organization and the nature of the resultant liposome-DNA-Ca2+ complexes were investigated by scanning tunneling microscopy (STM) and fluorescence microscopy, Observations of complexes with similar appearances with both SUV and MLV, as shown by two quite different microscopic approaches, prove that the resultant forms are real and not artifacts of the methodology used. At this stage it is not clear whether the detected complexes represent an intermediate state before fusion of liposomes which will lead to engulfing of the genomic material by the fused liposomes, or the final form. In either case the structures consisting of some adhered or semifused liposomes bearing the nucleic acid seem to be candidates as vehicles for in-vitro and in-vivo transfection.WoSScopu

A review of scanning probe microscopy investigations of liposome-DNA complexes

Liposome-DNA complexes are one of the most promising systems for the protection and delivery of nucleic acids to combat neoplastic, viral, and genetic diseases. In addition, they are being used as models in the elucidation of many biological phenomena such as viral infection and transduction. In order to understand these phenomena and to realize the mechanism of nucleic acid transfer by liposome-DNA complexes, studies at the molecular level are required. To this end, scanning probe microscopy (SPM) is increasingly being used in the characterization of lipid layers, lipid aggregates, liposomes, and their complexes with nucleic acid molecules. The most attractive attributes of SPM are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. This review describes the application of scanning tunneling microscopy and atomic force microscopy, the two most commonly applied SPM techniques, in the characterisation of liposome-DNA complexes

Histomorphometric Analysis of Periodontal Tissue Regeneration by the Use of High Density Polytetrafluoroethylen Membrane in Grade II Furcation Defects of Dogs

Statement of Problem: There are limited histomorphometric studies on biologic efficacy of high density tetrafluoroethylen (d-PTFE) membrane. Objectives: To investigate the healing of surgically induced grade II furcation defects in dogs following the use of dense polytetrafluoroethylene as the barrier membrane and to compare the results with the contra lateral control teeth without the application of any membrane. Materials andMethods: Mandibular and maxillary 3rd premolar teeth of 18 young adult male mongrel dogs were used for the experiment. The furcation defects were created during the surgery. 5 weeks later, regenerative surgery was performed. The third premolar teeth were assigned randomly to control and test groups. In the test group, after a full thickness flap reflection, the d-PTFE membrane was placed over furcation defects. In the control group, no membrane was placed over the defect. 37 tissue blocks containing the teeth and surrounding hard and soft tissues were obtained three months post-regenerative surgery. The specimens were demineralized, serially sectioned, mounted and stained with Hematoxylin and Eosin staining technique. From each tissue block, 35-45 sections of 10 μm thickness within 60μm interval captured the entire surgically created defect. The histological images were transferred to computer and then the linear measurement ranges of the defects area, interadicular alveolar bone, epithelial attachment and coronal extension of the new cementum were done. Then, the volume and area of aforementioned parameters were calculated considering the thickness and interval of the sections. To compare the parameters between the control and test teeth, we calculated the amount of each one proportionally to the original amount of defects. Results: The mean interradicular root surface areas of original defects covered with new cementum was 74.46% and 29.59% for the membrane and control defects, respectively (p < 0.0001). Corresponding values for epithelial attachment were 16.03% and 48.93% for the membrane and control defects, respectively (p < 0.005). The mean volume of the new bone formed in the inter-radicular defects was 61.80% and 35.93% for the membrane and control defects, respectively (p < 0.02). Conclusions: The present study provided a biological rationale for high density polytetrafluoroethylen (d-PTFE, n-PTFE) as a barrier membrane for enhancement of the bone and cementum regeneration in grade II furcation defects subjected to regeneration therapy

Selective cytotoxicity of green synthesized silver nanoparticles against the MCF-7 tumor cell line and their enhanced antioxidant and antimicrobial properties

Sadegh Khorrami,1 Ali Zarrabi,1 Moj Khaleghi,2 Marziyeh Danaei,3 MR Mozafari3 1Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran; 2Department of Biology, Faculty of Sciences, Shahid Bahonar University, Kerman, Iran; 3Australasian Nanoscience and Nanotechnology Initiative, Monash University, Clayton, VIC, Australia Introduction: Silver nanoparticles (AgNPs) are of great interest due to their unique and controllable characteristics. Different synthesis methods have been proposed to produce these nanoparticles, which often require elevated temperatures/pressures or toxic solvents. Thus, green synthesis could be a replacement option as a simple, economically viable and environmentally friendly alternative approach for the synthesis of silver nanoparticles.Methods: Here, the potential of the walnut green husk was investigated in the production of silver nanoparticles. An aqueous solution extracted from walnut green husk was used as a reducing agent as well as a stabilizing agent. Then, the synthesized nanoparticles were characterized with respect of their anticancer, antioxidant, and antimicrobial properties. Results: Results showed that the synthesized nanoparticles possessed an average size of 31.4 nm with a Zeta potential of -33.8 mV, indicating high stability. A significant improvement in the cytotoxicity and antioxidant characteristics of the green synthesized Ag nanoparticles against a cancerous cell line was observed in comparison with the walnut green husk extract and a commercial silver nanoparticle (CSN). This could be due to a synergistic effect of the synthesized silver nanoparticles and their biological coating. AgNPs and the extract exhibited 70% and 40% cytotoxicity against MCF-7 cancerous cells, respectively, while CSN caused 56% cell death (at the concentration of 60 &micro;g/mL). It was observed that AgNPs were much less cytotoxic when tested against a noncancerous cell line (L-929) in comparison with the control material (CSN). The free radical scavenging analysis demonstrated profound anti-oxidant activity for the synthesized nanoparticles in comparison with the extract and CSN. It was also detected that the synthesized AgNPs possess antibacterial activity against nosocomial and standard strains of both Gram-positive and Gram-negative bacteria (minimum inhibitory concentration =5&ndash;30 &micro;g/mL). Conclusion: These findings imply that the synthesized nanoparticles using green nanotechnology could be an ideal strategy to combat cancer and infectious diseases. Keywords: green synthesis, silver nanoparticles, antimicrobial agent, antioxidant agent, anticancer agent, selective cytotoxicit