Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

available in PMC 2011 September 1Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8+ T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.Ragon Institute of MGH, MIT and HarvardBill & Melinda Gates FoundationUnited States. Dept. of Defense (contract W911NF-07-D-0004)National Institutes of Health (U.S.) (P41RR002250)National Institutes of Health (U.S.) (RC2GM092599

Bioactive Electrospun Scaffolds Delivering Growth Factors and Genes for Tissue Engineering Applications

A biomaterial scaffold is one of the key factors for successful tissue engineering. In recent years, an increasing tendency has been observed toward the combination of scaffolds and biomolecules, e.g. growth factors and therapeutic genes, to achieve bioactive scaffolds, which not only provide physical support but also express biological signals to modulate tissue regeneration. Huge efforts have been made on the exploration of strategies to prepare bioactive scaffolds. Within the past five years, electrospun scaffolds have gained an exponentially increasing popularity in this area because of their ultrathin fiber diameter and large surface-volume ratio, which is favored for biomolecule delivery. This paper reviews current techniques that can be used to prepare bioactive electrospun scaffolds, including physical adsorption, blend electrospinning, coaxial electrospinning, and covalent immobilization. In addition, this paper also analyzes the existing challenges (i.e., protein instability, low gene transfection efficiency, and difficulties in accurate kinetics prediction) to achieve biomolecule release from electrospun scaffolds, which necessitate further research to fully exploit the biomedical applications of these bioactive scaffolds

Barriers to Non-Viral Vector-Mediated Gene Delivery in the Nervous System

Efficient methods for cell line transfection are well described, but, for primary neurons, a high-yield method different from those relying on viral vectors is lacking. Viral transfection has several drawbacks, such as the complexity of vector preparation, safety concerns, and the generation of immune and inflammatory responses when used in vivo. However, one of the main problems for the use of non-viral gene vectors for neuronal transfection is their low efficiency when compared with viral vectors. Transgene expression, or siRNA delivery mediated by non-viral vectors, is the result of multiple processes related to cellular membrane crossing, intracellular traffic, and/or nuclear delivery of the genetic material cargo. This review will deal with the barriers that different nanoparticles (cationic lipids, polyethyleneimine, dendrimers and carbon nanotubes) must overcome to efficiently deliver their cargo to central nervous system cells, including internalization into the neurons, interaction with intracellular organelles such as lysosomes, and transport across the nuclear membrane of the neuron in the case of DNA transfection. Furthermore, when used in vivo, the nanoparticles should efficiently cross the blood-brain barrier to reach the target cells in the brain

The multiple faces of self-assembled lipidic systems

Lipids, the building blocks of cells, common to every living organisms, have the propensity to self-assemble into well-defined structures over short and long-range spatial scales. The driving forces have their roots mainly in the hydrophobic effect and electrostatic interactions. Membranes in lamellar phase are ubiquitous in cellular compartments and can phase-separate upon mixing lipids in different liquid-crystalline states. Hexagonal phases and especially cubic phases can be synthesized and observed in vivo as well. Membrane often closes up into a vesicle whose shape is determined by the interplay of curvature, area difference elasticity and line tension energies, and can adopt the form of a sphere, a tube, a prolate, a starfish and many more. Complexes made of lipids and polyelectrolytes or inorganic materials exhibit a rich diversity of structural morphologies due to additional interactions which become increasingly hard to track without the aid of suitable computer models. From the plasma membrane of archaebacteria to gene delivery, self-assembled lipidic systems have left their mark in cell biology and nanobiotechnology; however, the underlying physics is yet to be fully unraveled

Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts

BackgroundPoly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce.MethodsWe conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells’ proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits.ResultsCell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells’ proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts.ConclusionsThese data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies

Influence of Rheology of Dispersion Media in the Preparation of Polymeric Microspheres through Emulsification Method

Chitosan microspheres as drug delivery system have attained importance and attracted the attention of researchers in last few years. This study was aimed toward the elucidation of the effect of viscosity of external oil phase on the properties of microspheres prepared by emulsification method. Chitosan microspheres were prepared utilizing oil phase of different viscosity viz. castor oil, heavy liquid paraffin, light liquid paraffin and mixture of light paraffin, and petroleum ether (1:1 v/v ratio). Microspheres prepared in highly viscous castor oil exhibited an average size of 11.52 ± 0.57 µm with a percentage drug entrapment of 43.12 ± 2.14. On the other hand, very small microspheres of 3.15 ± 0.04 µm and 68.87 ± 1.03% drug entrapment were obtained when mixture of liquid paraffin and petroleum ether was utilized as oil phase. Effect of viscosity on percent mucoadhesion, percent drug entrapment, zeta potential, percent process yield, etc. of microspheres has been observed. In vitro drug release in phosphate buffer pH 7.4 was determined for different batch of microspheres. The results revealed a difference in the drug release pattern of the different microspheres prepared as a function of viscosity of different oil phase. Use of low viscose oil resulted in the formulation of spherical and small size microspheres. This work was a part of our ongoing thrust and project to develop microparticulate drug delivery system