BackgroundCurcumin is a polyphenolic compound isolated from the rhizomes of the plant Curcuma longa and shows intrinsic anti-cancer properties. Its medical use remains limited due to its extremely low water solubility and bioavailability. Addressing this problem, drug delivery systems accompanied by nanoparticle technology have emerged. The present study introduces a novel nanocarrier system, so-called CurcuEmulsomes, where curcumin is encapsulated inside the solid core of emulsomes.ResultsCurcuEmulsomes are spherical solid nanoparticles with an average size of 286 nm and a zeta potential of 37 mV. Encapsulation increases the bioavailability of curcumin by up to 10,000 fold corresponding to a concentration of 0.11 mg/mL. Uptaken by HepG2 human liver carcinoma cell line, CurcuEmulsomes show a significantly prolonged biological activity and demonstrated therapeutic efficacy comparable to free curcumin against HepG2 in vitro - with a delay in response, as assessed by cell viability, apoptosis and cell cycle studies. The delay is attributed to the solid character of the nanocarrier prolonging the release of curcumin inside the HepG2 cells.ConclusionsIncorporation of curcumin into emulsomes results in water-soluble and stable CurcuEmulsome nanoformulations. CurcuEmulsomes do not only successfully facilitate the delivery of curcumin into the cell in vitro, but also enable curcumin to reach its effective concentrations inside the cell. The enhanced solubility of curcumin and the promising in vitro efficacy of CurcuEmulsomes highlight the potential of the system for the delivery of lipophilic drugs. Moreover, high degree of compatibility, prolonged release profile and tailoring properties feature CurcuEmulsomes for further therapeutic applications in vivo.

The present study introduces a novel nanocarrier system comprising lipidic emulsomes and S-layer (fusion) proteins as functionalizing tools coating the surface. Emulsomes composed of a solid tripalmitin core and a phospholipid shell are created reproducibly with an average diameter of approximately 300 nm using temperature-controlled extrusion steps. Both wildtype (wt) and recombinant (r) S-layer protein SbsB of Geobacillus stearothermophilus PV72/p2 are capable of forming coherent crystalline envelope structures with oblique (p1) lattice symmetry, as evidenced by transmission electron microscopy. Upon coating with wtSbsB, positive charge of emulsomes shifts to a highly negative zeta potential, whereas those coated with rSbsB become charge neutral. This observation is attributed to the presence of a negatively charged glycan, the secondary cell wall polymer (SCWP), which is associated only with wtSbsB. The present study shows for the first time the ability of recombinant and wildtype S-layer proteins to cover the entire surface of emulsomes with its characteristic crystalline lattice. Furthermore, in vitro cell culture studies reveal that S-layer coated emulsomes can be uptaken by human liver carcinoma cells (HepG2) without showing any significant cytotoxicity over a wide range of concentrations. The utilization of S-layer fusion proteins equipped in a nanopatterned fashion by identical or diverse functions may lead to further development of emulsomes in nanomedicine, especially for drug delivery and targeting.

Isolated subunits of the crystalline cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were recrystallized on positively charged unilamellar liposomes. Liposomes were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and hexadecylamine (HDA) in a molar ratio of 10:5:4 and they were prepared by the dehydration-rehydration method followed by an extrusion procedure. The S-layer protein to DPPC ratio was 5.7 nmol/micromol which approximately corresponds to the theoretical value estimated by using the areas occupied by the S-layer lattice and the lipid membrane. Coating of the positively charged liposomes with S-layer protein resulted in inversion of the zeta-potential from +29.1 mV to -27.1 mV. Covalent crosslinking of the recrystallized S-layer protein was achieved with glutaraldehyde. Chemical analysis revealed that almost all amino groups (>95%) from HDA in the liposomal membrane were involved in the reaction. To study the influence of an S-layer lattice on the stability of the liposomes, the hydrophilic marker carboxyfluoresceine (CF) was encapsulated and its release was determined for plain and S-layer-coated liposomes in the course of mechanical and thermal challenges. In comparison to plain liposomes, S-layer-coated liposomes released only half the amount of enclosed CF upon exposure to shear forces or ultrasonication as mechanical stress factors. Furthermore, temperature shifts from 25 degrees C to 55 degrees C and vice versa induced considerably less CF release from S-layer-coated than from plain liposomes. A similar stabilizing effect of the S-layer lattice was observed after glutaraldehyde treatment of plain and S-layer-coated liposomes.

Polyelectrolyte multilayer (PE) deposition and S-layer technology have been combined to make novel robust biomimetic surfaces and membranes. Isolated subunits of the bacterial cell surface layer from Bacillus sphaericus CCM2177 SbpA was self-assembled on PE multilayer supports, with the composition of the multilayer playing a crucial role in determining the structure of the resulting supported protein layers. Flat substrates were studied using atomic force microscopy and neutron reflectometry; protein on suitable PE combinations showed a crystalline structure with lattice constants equal to those found in vivo on bacterial surfaces. The mechanical stability of the S-layer is higher when recrystallized on PEs than directly on silicon supports. The recrystallization process was subsequently used to coat colloidal particles, permitting the determination of zeta potentials before and after coating. Hollow capsules could also be coated in the same way, as proven by various techniques. Our results suggest that electrostatic interactions via divalent cations are important for the assembly process. The results also demonstrate that the versatility of the PE multilayer membranes can be successfully combined with the well-defined surface chemistry and structure of 2D protein crystals.

In the current work we have developed a lab-on-a-chip containing embedded amperometric sensors in four microreactors that can be addressed individually and that are coated with crystalline surface protein monolayers to provide a continuous, stable, reliable and accurate detection of blood glucose. It is envisioned that the microfluidic device will be used in a feedback loop mechanism to assess natural variations in blood glucose levels during hemodialysis to allow the individual adjustment of glucose. Reliable and accurate detection of blood glucose is accomplished by simultaneously performing (a) blood glucose measurements, (b) autocalibration routines, (c) mediator-interferences detection, and (d) background subtractions. The electrochemical detection of blood glucose variations in the absence of electrode fouling events is performed by integrating crystalline surface layer proteins (S-layer) that function as an efficient antifouling coating, a highly-oriented immobilization matrix for biomolecules and an effective molecular sieve with pore sizes of 4 to 5 nm. We demonstrate that the S-layer protein SbpA (from Lysinibacillus sphaericus CCM 2177) readily forms monomolecular lattice structures at the various microchip surfaces (e.g. glass, PDMS, platinum and gold) within 60 min, eliminating unspecific adsorption events in the presence of human serum albumin, human plasma and freshly-drawn blood samples. The highly isoporous SbpA-coating allows undisturbed diffusion of the mediator between the electrode surface, thus enabling bioelectrochemical measurements of glucose concentrations between 500 μM to 50 mM (calibration slope δI/δc of 8.7 nA mM(-1)). Final proof-of-concept implementing the four microfluidic microreactor design is demonstrated using freshly drawn blood. Accurate and drift-free assessment of blood glucose concentrations (6. 4 mM) is accomplished over 130 min at 37 °C using immobilized enzyme glucose oxidase by calculating the difference between autocalibration (10 mM glc) and background measurements. The novel combination of biologically-derived nanostructured surfaces with microchip technology constitutes a powerful new tool for multiplexed analysis of complex samples.