Exosomes Secreted from Amniotic Membrane Contribute to Its Anti-Fibrotic Activity.

Amniotic membranes (AM) have anti-fibrotic activity. Exosomes (nano-sized vesicles) function as conduits for intercellular transfer and contain all the necessary components to induce the resolution of fibrosis. In this study, we tested the hypothesis that the anti-fibrotic activity of AM is mediated by exosomes. AM-derived exosomes or amniotic stromal cell-derived exosomes were isolated and characterized. Anti-fibrotic activity of exosomes was evaluated using human hepatic stellate cells (LX-2), an in vitro model of fibrosis. Exosomes isolated from AM tissue-conditioned media had an average size of 75 nm. Exosomes significantly inhibited the proliferation of TGFß1-activated LX-2 but had no effect on the proliferation of non-activated LX-2 cells. Exosomes also reduced the migration of LX-2 in a scratch wound assay. Furthermore, exosomes reduced the gene expression of pro-fibrotic markers such as COL1A1, ACTA, and TGFß1 in LX-2 cells. Interestingly, exosomes isolated from AM tissue under hypoxic conditions seemed to show a stronger anti-fibrotic activity than exosomes isolated from tissue under normoxic conditions. Exosomes released by in vitro cultured AM stromal cells were smaller in size compared with tissue exosomes and also showed anti-fibrotic activity on LX-2 cells. In conclusion, AM-tissue-released exosomes contribute to the anti-fibrotic activity of AM. This is the first report of isolation, characterization, and functional evaluation of exosomes derived from amniotic tissues with the direct comparison between tissue-derived exosomes and cultured cell-derived exosomes.

Disassembly of Nanospheres with a PEG Shell upon Adsorption onto PEGylated Substrates.

Polymeric nanospheres have the ability to encapsulate drugs and are therefore widely used in drug delivery applications. Structural transformations that affect drug release from nanospheres are governed by the surrounding environment. To understand these effects, we investigated the adsorption behavior of three types of nanospheres onto model surfaces using quartz crystal microbalance with dissipation (QCM-D) and by atomic force microscopy (AFM). Substrates were prepared from polymers with different degrees of PEGylation (0, 1, and 15%). Nanospheres were prepared via self-assembly of block copolymers. Tyrosine-derived nanospheres are A-B-A triblock copolymers with methoxy poly(ethylene glycol) (PEG) as the A-blocks and an alternating copolymer of desaminotyrosyl-tyrosine octyl ester and suberic acid oligo(DTO-SA) as the B-block. On non-PEGylated substrates, these nanospheres assembled into a close-packed structure; on PEGylated substrates, the adsorbed nanospheres formed a continuous film, thinner than the size of the nanospheres suggesting unraveling of the PEG corona and disassembly of the nanospheres. Also, the adsorption was concentration-dependent, the final thickness being attained at exponentially longer times at lower concentrations. Such substrate- and concentration-dependent behavior was not observed with Pluronic F-127 and PEG-poly(caprolactone) (PCL) nanospheres. Since the essential difference among the three nanospheres is the composition of the core, we conclude that the core influences the adsorption characteristics of the nanospheres as a consequence of their disassembly upon adsorption. These results are expected to be useful in designing nanospheres for their efficient transport across vascular barriers and for delivering drugs to their targets.

Polymerized Salicylic Acid Microparticles Reduce the Progression and Formation of Human Neutrophil Extracellular Traps (NET)s.

Neutrophils can contribute to inflammatory disease propagation via innate mechanisms intended for inflammation resolution. For example, neutrophil extracellular traps (NETs) are necessary for trapping pathogens but can contribute to clot formation and blood flow restriction, that is, ischemia. Currently, no therapeutics in the clinic directly target NETs despite the known involvement of NETs contributing to mortality and increased disease severity. Vascular-deployed particle-based therapeutics are a novel and robust alternative to traditional small-molecule drugs by enhancing drug delivery to cells of interest. This work designs a high-throughput assay to investigate the immunomodulatory behavior and functionality of salicylic acid-based polymer-based particle therapeutics against NETosis in human neutrophils. Briefly, this work finds that polymeric composition plays a role, and particle size can also influence rates of NETosis. Salicylate-based polymeric (Poly-SA) particles are found to functionally inhibit NETosis depending on the particle size and concentration exposed to neutrophils. This work demonstrates the high throughput method can help fast-track particle-based therapeutic optimization and design, more efficiently preparing this innovative therapeutics for the clinic.

Tyrosine-derived polymeric surfactant nanospheres insert cholesterol in cell membranes.

HYPOTHESIS: The design of biodegradable tyrosine-derived polymeric surfactants (TyPS) through the use of calculated thermodynamic parameters could lead to phospholipid membrane surface modifiers capable of controlling cellular properties such as viability. Delivery of cholesterol by TyPS nanospheres into membrane phospholipid domains could provide further controlled modulation of membrane physical and biological properties. EXPERIMENT: Calculated Hansen solubility parameters (∂T) and hydrophile:lipophile balances (HLB) were applied to design and synthesize a small family of diblock and triblock TyPS with different hydrophobic blocks and PEG hydrophilic blocks. Self-assembled TyPS/cholesterol nanospheres were prepared in aqueous media via co-precipitation. Cholesterol loading and Langmuir film balance surface pressures of phospholipid monolayers were obtained. TyPS and TyPS/cholesterol nanosphere effects on human dermal cell viability were evaluated by cell culture using poly(ethylene glycol) (PEG) and Poloxamer 188 as controls. FINDINGS: Stable TyPS nanospheres incorporated between 1% and 5% cholesterol. Triblock TyPS formed nanosphere with dimensions significantly smaller than diblock TyPS nanospheres. In accord calculated thermodynamic parameters, cholesterol binding increased with increasing TyPS hydrophobicity. TyPS inserted into phospholipid monolayer films in a manner consistent with their thermodynamic properties and TyPS/cholesterol nanospheres delivered cholesterol into the films. Triblock TyPS/cholesterol nanospheres increased human dermal cell viability, which was indicative of potentially beneficial TyPS effects on cell membrane surface properties.

Nanosphere size control by varying the ratio of poly(ester amide) block copolymer blends.

HYPOTHESIS: Blending amphiphilic triblock (A-B-A) and diblock (A-B) copolymers comprised of the same hydrophobic tyrosine-derived oligomeric B-block and hydrophilic poly(ethylene glycol) methyl ether (mPEG) A-block can provide highly tunable self-assembled nanosphere particle sizes suitable for biomedical applications. EXPERIMENT: Triblock and diblock copolymers were synthesized via carbodiimide chemistry and were characterized by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The amount of free PEG present in the purified copolymers was determined using a standard addition calibration curve and GPC peak deconvolution methods. Nanospheres were prepared by co-precipitation of each copolymer and of copolymer blends over a range of mole ratios. Nanospheres were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and % polymer recovery post-preparation. FINDING: Precise synthesis control produced triblock and diblock copolymers with narrow molecular weight distributions and minimal residual reactants. Self-assembled nanosphere particle sizes were 33 nm for the triblock and 129 nm for the diblock, and the size of their blends increased continuously as a function of mole ratio within that biomedically relevant range. Addition of unreacted PEG had minimal impact on either triblock or diblock nanosphere particle sizes whereas addition of unreacted oligomeric B-block increased nanosphere sizes.