Poly-L-Lysine and Human Plasmatic Fibronectin Films as Proactive Coatings to Improve Implant Biointegration

The success of stable and long-term implant integration implies the promotion, control, and respect of the cell microenvironment at the site of implantation. The key is to enhance the implant–host tissue cross talk by developing interfacial strategies that guarantee an optimal and stable seal of soft tissue onto the implant, while preventing potential early and late infection. Indeed, implant rejection is often jeopardized by lack of stable tissue surrounding the biomaterial combined with infections which reduce the lifespan and increase the failure rate of implants and morbidity and account for high medical costs. Thin films formed by the layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes are particularly versatile and attractive for applications involving cell–material contact. With the combination of the extracellular matrix protein fibronectin (Fn, purified from human plasma) and poly-L-lysine (PLL, exhibiting specific chain lengths), we proposed proactive and biomimetic coatings able to guarantee enhanced cell attachment and exhibiting antimicrobial properties. Fn, able to create a biomimetic interface that could enhance cell attachment and promote extracellular cell matrix remodeling, is incorporated as the anionic polymer during film construction by the LbL technic whereas PLL is used as the cationic polymer for its capacity to confer remarkable antibacterial properties.

Schistosome immunomodulators

Schistosomes are long lived, intravascular parasitic platyhelminths that infect >200 million people globally. The molecular mechanisms used by these blood flukes to dampen host immune responses are described in this review. Adult worms express a collection of host-interactive tegumental ectoenzymes that can cleave host signaling molecules such as the “alarmin” ATP (cleaved by SmATPDase1), the platelet activator ADP (SmATPDase1, SmNPP5), and can convert AMP into the anti-inflammatory mediator adenosine (SmAP). SmAP can additionally cleave the lipid immunomodulator sphingosine-1-phosphate and the proinflammatory anionic polymer, polyP. In addition, the worms release a barrage of proteins (e.g., SmCB1, SjHSP70, cyclophilin A) that can impinge on immune cell function. Parasite eggs also release their own immunoregulatory proteins (e.g., IPSE/α1, omega1, SmCKBP) as do invasive cercariae (e.g., Sm16, Sj16). Some schistosome glycans (e.g., LNFPIII, LNnT) and lipids (e.g., Lyso-PS, LPC), produced by several life stages, likewise affect immune cell responses. The parasites not only produce eicosanoids (e.g., PGE2, PGD2—that can be anti-inflammatory) but can also induce host cells to release these metabolites. Finally, the worms release extracellular vesicles (EVs) containing microRNAs, and these too have been shown to skew host cell metabolism. Thus, schistosomes employ an array of biomolecules—protein, lipid, glycan, nucleic acid, and more, to bend host biochemistry to their liking. Many of the listed molecules have been individually shown capable of inducing aspects of the polarized Th2 response seen following infection (with the generation of regulatory T cells (Tregs), regulatory B cells (Bregs) and anti-inflammatory, alternatively activated (M2) macrophages). Precisely how host cells integrate the impact of these myriad parasite products following natural infection is not known. Several of the schistosome immunomodulators described here are in development as novel therapeutics against autoimmune, inflammatory, and other, nonparasitic, diseases.

Elucidating morphological effects in membrane mineral fouling using real-time particle imaging and impedance spectroscopy

Mineral fouling is a major hindrance to high recovery effluent nanofiltration, with calcium phosphate (Ca-P) and calcium carbonate (CaCO3) the most prevalent mineral foulants. In this study, we used a novel combination of real-time in-line microscopy, electrical impedance spectroscopy (EIS), post SEM analysis, and filtration metrics (water flux and rejection) to study mineral fouling mechanisms of Ca-P and CaCO3 salts in synthetic effluent nanofiltration. We used nanofiltration (NF) polyelectrolyte multilayer (PEM) membranes, prepared by static layer-by-layer (LbL) coating of a cationic polymer - polydiallyl dimethylammonium chloride, and anionic polymer - poly styrenesulfonate (six bi-layer) on a polyethersulfone (PES) ultrafiltration (UF) membrane. Increasing permeate recovery over filtration time was simulated through additions of CaCl2 with NaHCO3 or NaH2PO4/Na2HPO4. Using the novel combination of methods, we delineated the mechanisms governing fouling development with time for both CaCO3 and Ca-P. For CaCO3, a transition from heterogeneous precipitation on the membrane surface (scaling) to particulate fouling due to bulk precipitation was identified. For Ca-P, a transition from fouling by amorphous particles to fouling by crystalline particles was identified; and this phase-change was captured in real-time images using an in-line microscope. We also found that for similar precipitation potentials measured by weight, Ca-P fouling was more detrimental to water flux (86% decrease) compared to CaCO3 (20% decrease) due to the voluminous amorphous phase. We established in-line microscopy as a new useful method to study mineral fouling, as it gives invaluable information on the suspended particles in real-time. Combining it with EIS gives complementary information on mineral accumulation on the membrane surface. Insight from this study and further use of these methods can guide future strategies towards higher effluent recovery by membrane filtration.

Gene-Activated Matrix with Self-Assembly Anionic Nano-Device Containing Plasmid DNAs for Rat Cranial Bone Augmentation

We have developed nanoballs, a biocompatible self-assembly nano-vector based on electrostatic interactions that arrange anionic macromolecules to polymeric nanomaterials to create nucleic acid carriers. Nanoballs exhibit low cytotoxicity and high transfection efficiently in vivo. This study investigated whether a gene-activated matrix (GAM) composed of nanoballs containing plasmid (p) DNAs encoding bone morphogenetic protein 4 (pBMP4) could promote bone augmentation with a small amount of DNA compared to that composed of naked pDNAs. We prepared nanoballs (BMP4-nanoballs) constructed with pBMP4 and dendrigraft poly-L-lysine (DGL, a cationic polymer) coated by γ-polyglutamic acid (γ-PGA; an anionic polymer), and determined their biological functions in vitro and in vivo. Next, GAMs were manufactured by mixing nanoballs with 2% atelocollagen and β-tricalcium phosphate (β-TCP) granules and lyophilizing them for bone augmentation. The GAMs were then transplanted to rat cranial bone surfaces under the periosteum. From the initial stage, infiltrated macrophages and mesenchymal progenitor cells took up the nanoballs, and their anti-inflammatory and osteoblastic differentiations were promoted over time. Subsequently, bone augmentation was clearly recognized for up to 8 weeks in transplanted GAMs containing BMP4-nanoballs. Notably, only 1 μg of BMP4-nanoballs induced a sufficient volume of new bone, while 1000 μg of naked pDNAs were required to induce the same level of bone augmentation. These data suggest that applying this anionic vector to the appropriate matrices can facilitate GAM-based bone engineering.

Interaction of Linear Polyelectrolytes with Proteins: Role of Specific Charge–Charge Interaction and Ionic Strength

We present a thermodynamic study of the interaction of synthetic, linear polyelectrolytes with bovine serum albumin (BSA). All polyelectrolytes are based on poly(allyl glycidyl ether) which has been modified by polymer-analogous reaction with anionic (-SO3Na), cationic (-NH3Cl or -NHMe2Cl) or zwitterionic groups (-NMe2(CH2)3SO3). While the anionic polymer shows a very weak interaction, the zwitterionic polymer exhibits no interaction with BSA (pI = 4.7) under the applied pH = 7.4, ionic strength (I = 23–80 mM) and temperature conditions (T = 20–37 °C). A strong binding, however, was observed for the polycations bearing primary amino or tertiary dimethyl amino groups, which could be analysed in detail by isothermal titration calorimetry (ITC). The analysis was done using an expression which describes the free energy of binding, DGb, as the function of the two decisive variables, temperature, T, and salt concentration, cs. The underlying model splits DGb into a term related to counterion release and a term related to water release. While the number of released counter ions is similar for both systems, the release of bound water is more important for the primary amine compared to the tertiary N,N-dimethyl amine presenting polymer. This finding is further traced back to a closer contact of the polymers’ protonated primary amino groups in the complex with oppositely charged moieties of BSA as compared to the bulkier protonated tertiary amine groups. We thus present an investigation that quantifies both driving forces for electrostatic binding, namely counterion release and change of hydration, which contribute to a deeper understanding with direct impact on future advancements in the biomedical field.