Hybrids of Polymer Multilayers, Lipids, and Nanoparticles: Mimicking the Cellular Microenvironment.

Here we address research directions and trends developed following novel concepts in 2D/3D self-assembled polymer structures established in the department led by Helmuth Möhwald. These functional structures made of hybrids of polymer multilayers, lipids, and nanoparticles stimulated research in the design of the cellular microenvironment. The composition of the extracellular matrix (ECM) and dynamics of biofactor presentation in the ECM can be recapitulated by the hybrids. Proteins serve as models for protein-based biofactors such as growth factors, cytokines, hormones, and so forth. A fundamental understanding of complex intermolecular interactions and approaches developed for the externally IR-light-triggered release offers a powerful tool for controlling the biofactor presentation. Pure protein beads made via a mild templating on vaterite CaCO3 crystals can mimic cellular organelles in terms of the compartmentalization of active proteins. We believe that an integration of the approaches developed and described here offers a strong tool for engineering and mimicking both extra- and intracellular microenvironments.

Structure of the complex of cytochrome c with cardiolipin in non-polar environment.

The complex of mitochondrial protein cytochrome c (CytC) with anionic phospholipid cardiolipin (CL) plays a crucial role in the initiation of apoptosis by catalyzing lipid peroxidation in mitochondrial membranes. In our previous papers, we found that CytC and CL mixed in millimolar concentrations form a sediment showing microcrystals composed of nanospheres (Cyt-CL) of 11-12 and 8 nm in diameter. The hypothesis was proposed that Cyt-CL, having hydrophobic shell, may appear inside the membrane lipid bilayer in mitochondria and peroxidase membrane phospholipids so initiating the apoptotic cascade. In this work, Cyt-CL complex dissolved in chloroform or hexane was investigated as a model of the complex in mitochondrial membranes. We used dynamic light scattering method to measure the size of the particles. The analysis of particles size distribution of Cyt-CL in chloroform allows to reveal three dominant diameters of 12.1 ±â€¯1.4, 7.8 ±â€¯1.0, and 4.7 ±â€¯0.7 nm. The first two values are closed to those, earlier obtained with small-angle X-ray scattering method in Cyt-CL microcrystals, 11.1 ±â€¯1.0 and 8.0 ±â€¯0.7 nm. CL extracted in chloroform-methanol forms a real solution of particles with diameter of 0.7 ±â€¯0.1 nm. In methanol-water phase, CL and CL + CytC mixture form particles of 83.7 ±â€¯9.8 and 71.3 ±â€¯11.6 nm, respectively. Apparently, cardiolipin in 50% methanol forms single-layer liposomes regardless of the presence of CytC in the medium. Partial unfolding of CytC in the complex was evidenced by (a) appearance of fluorescence of tyrosine and tryptophan residues and (b) disappearance of the absorption band at 699 nm due to breakdown of heme iron - methionine bond > F⋯S(Met80). In hydrophobic solvent Cyt-CL exhibited quasi-lipoperoxidase and lipoxygenase activity as was shown in kinetic measurements of chemiluminescence enhanced by coumarin C-525, a selective sensitizer of chemiluminescence, associated with reactions of lipid peroxyl radicals. Our data in this model system do not contradict the hypothesis (Vladimirov, Y.A. et al. Biochemistry (Mosc) 78, 1086-1097) that nanospheres of Cyt-CL complex, embedded into the lipid phase of mitochondrial membrane, catalyze lipid peroxidation, thereby initiating apoptosis.

The mechanism of catalase loading into porous vaterite CaCO3 crystals by co-synthesis.

Porous vaterite CaCO3 crystals are nowadays extensively used as high-capacity bio-friendly sacrificial templates for the fabrication of such protein-containing nano- and micro-particles as capsules and beads. The first step in the protein encapsulation is performed through loading of the protein molecules into the crystals. Co-synthesis is one of the most useful and simple methods proven to effectively load crystals with proteins; however, the loading mechanism is still unknown. To understand the mechanism, in this study, we focus on the loading of a model protein catalase into the crystals by means of adsorption into pre-formed crystals (ADS) and co-synthesis (COS). Analysis of the physico-chemical characteristics of the protein in solution and during the loading and simulation of the protein packing into the crystals are performed. COS provides more effective loading than ADS giving protein contents in the crystals of 20.3 and 3.5 w/w%, respectively. Extremely high loading for COS providing a local protein concentration of about 550 mg mL-1 is explained by intermolecular protein interactions, i.e. formation of protein aggregates induced by CaCl2 during the co-synthesis. This is supported by a lower equilibrium constant obtained for COS (5 × 105 M-1) than for ADS (23 × 105 M-1), indicating a higher affinity of single protein molecules rather than aggregates to the crystal surface. Fitting the adsorption isotherms by classical adsorption models has shown that the Langmuir and BET models describe the adsorption phenomenon better than the Freundlich model, proving the aggregation in solution followed by adsorption of the aggregates into the crystals. We believe that this study will be useful for protein encapsulation through CaCO3 crystals using the COS method.

Temperature-induced molecular transport through polymer multilayers coated with PNIPAM microgels.

Polyelectrolyte multilayers serve as effective reservoirs for bioactive molecules which are stored and released from the multilayers for cellular applications. However, control over the release without significantly affecting the multilayers and biomolecules is still a challenge. On the other hand, externally stimulated release would make the multilayers promising for the development of stimuli-sensitive planar carriers with release performance switched on demand. In this study soft composite films are designed by coating hyaluronic acid/poly-l-lysine (HA/PLL) multilayers with temperature responsive poly(N-isopropylacrylamide) (PNIPAM) microgels. Microgels are flattened and immersed into the multilayers to maximize the number of contacts with the surrounding polyelectrolytes (HA and PLL). The microgel coating serves as an efficient switchable barrier for the PLL transport into the multilayers. PLL diffusion into the film is significantly hindered at room temperature but is dramatically enhanced at 40 °C above the volume phase transition temperature (VPTT) of PNIPAM at 32 °C associated with microgel shrinkage. Scanning force microscopy micrographs show that the mechanism of volume phase transition on soft surfaces cannot be directly deduced from the processes taking place at solid substrates.

Protein loading into porous CaCO3 microspheres: adsorption equilibrium and bioactivity retention.

Formulation of proteins into particulate form is a principal strategy to achieve controlled and targeted delivery, as well as to protect fragile protein molecules. Control over size, mechanical properties, and surface area (porosity) of particulate proteins has been successfully achieved by hard templating under mild conditions using porous CaCO3 microspheres. A crucial step in this approach, which determines protein content, is the loading of proteins into the CaCO3 microspheres. In this study, the adsorption of different proteins into the microspheres has been investigated. Proteins differing in characteristics such as molecular weight and charge have been employed: catalase (Cat), insulin (Ins), aprotinin (Apr), and protamine (Pro). Thermodynamics of adsorption equilibria have been studied, together with quantitative and qualitative analysis of protein loading and distribution in the microspheres. Protein interaction with the CaCO3 microspheres is not limited by the diffusion of protein molecules (protein dimensions are significantly smaller than microsphere pores) but is determined by the protein affinity for the microsphere surface. Cat and Ins bind much more strongly to the microspheres than Apr and Pro, which can be explained by electrostatic attractive forces. Protein binding/release and protein biological activity have been investigated as a function of pH. It is shown that pH variation during the adsorption process plays a principal role and defines not only the amount of protein adsorbed/released but also protein biological activity. Protein adsorption and microsphere elimination (by EDTA) do not affect protein bioactivity. In addition to applications for protein particle/capsule formulations, the findings of this study might help in understanding protein interactions with carbonate minerals such as calcium carbonate, which is used as a natural material for multiple applications.

Stability and cell uptake of calcium carbonate templated insulin microparticles.

Therapeutic proteins are an integral part of today's pharmaceutical practice, but they still present challenges from the drug delivery point of view. In this work, a new approach is studied based on hard templating for fabrication of microparticles composed of pure insulin, which may enable effective delivery, for instance pulmonary delivery. The approach is both simple and versatile: the protein particles are prepared by selective precipitation into porous CaCO3 microtemplates, followed by full decomposition of the template at the isoelectric point of the protein (pH 5.2). Control over the main material parameters (mechanical properties, porosity, morphology and stability at physiological conditions) are critical for the envisioned application in drug delivery. It is demonstrated that these critical parameters can be significantly tuned by a slight final pH variation around the isoelectric point (pH range 4-6) and by the denaturation degree of insulin. Electrostatic interactions and inter-protein crosslinking in the protein particles as well as their internal structure are considered, to explain the variation in the particle properties. The particle property parameters are explored using atomic force microscopy, optical microscopy and circular dichroism spectra. Finally, phagocytic clearance of the protein particles in vitro was studied to explore possible enhancements in particle fabrication to improve the efficiency of insulin delivery by inhalation.

Patchiness of embedded particles and film stiffness control through concentration of gold nanoparticles.

Patchy particles are fabricated using a method of embedding-into and extracting-from thick, biocompatible, gel-like HA/PLL films. Control over the patchiness is achieved by adjusting the stiffness of films, which affects embedding and masking of particles. The stiffness is adjusted by the concentration of gold nanoparticles adsorbed onto the surface of the films.

Surface-supported multilayers decorated with bio-active material aimed at light-triggered drug delivery.

In this work, we report on the functionalization of layer-by-layer films with gold nanoparticles, microcapsules, and DNA molecules by spontaneous incorporation into the film. Exponentially growing films from biopolymers, namely, hyaluronic acid (HA) and poly-L-lysine (PLL), and linearly growing films from the synthetic polymers, namely, poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH), were examined for the embedding. The studied (PLL/HA)(24)/PLL and (PAH/PSS)(24)/PAH films are later named HA/PLL and PSS/PAH films, respectively. The HA/PLL film has been found to be more efficient for both particle and DNA embedding than PSS/PAH because of spontaneous PLL transport from the interior of the whole HA/PLL film to the surface in order to make additional contact with embedded particles or DNA. DNA and nanoparticles can be immobilized in HA/PLL films, reaching loading capacities of 1.5 and 100 microg/cm(2), respectively. The capacities of PSS/PAH films are 5 and 12 times lower than that for films made from biopolymers. Polyelectrolyte microcapsules adsorb irreversibly on the HA/PLL film surface as single particles whereas very poor interaction was observed for PSS/PAH. This intrinsic property of the HA/PLL film is due to the high mobility of PLL within the film whereas the structure of the PSS/PAH film is "frozen in". Gold nanoparticles and DNA form micrometer-sized aggregates or patches on the HA/PLL film surface. The diffusion of nanoparticles and DNA into the HA/PLL film is restricted at room temperature, but DNA diffusion is triggered by heating to 70 degrees C, leading to homogeneous filling of the film with DNA. The film has not only a high loading capacity but also can be activated by "biofriendly" near-infrared (IR) laser light, thanks to the gold nanoparticle aggregates on the film surface. Composite HA/PLL films with embedded gold nanoparticles and DNA can be activated by light, resulting in DNA release. We assume that the mechanism of the release is dependent on the disturbance in bonding between "doping" PLL and DNA, which is induced by local thermal decomposition of the HA/PLL network in the film when the film is exposed to IR light. Remote IR-light activation of dextran-filled microcapsules modified by gold nanoparticles and integrated into the HA/PLL film is also demonstrated, revealing an alternative release pathway using immobilized light-sensitive carriers (microcapsules).

Remote near-IR light activation of a hyaluronic acid/poly(l-lysine) multilayered film and film-entrapped microcapsules.

Spontaneous embedding of gold nanoparticle (NP) aggregates or polyelectrolyte microcapsules modified with NPs in biocompatible hyaluronic acid/poly(l-lysine) films is reported. The NPs were adsorbed in the aggregated state to induce near-IR light absorption. The films functionalized with gold NPs become active in response to a "biologically friendly" near-IR laser at a power of about 20 mW. The activation is characterized by a localized temperature increase in the film, allowing conversion of light energy to heat into confined volumes. Microcapsules adsorbed onto the film can release its cargo under stimulation with near-IR light because of localized permeability changes in their walls. This work is aimed at layer-by-layer film-based biomedical coatings and active surfaces with light-sensitive features wherein metal NPs and microcapsules are used as active centers or carriers with remote control of functionalities.

Inclusion of proteins into polyelectrolyte microparticles by alternative adsorption of polyelectrolytes on protein aggregates.

A new method of protein immobilization into polyelectrolyte microparticles by alternative adsorption of the oppositely charged polyelectrolytes on the aggregates obtained by salting out of protein is proposed. The model protein alpha-chymotrypsin (ChT) was included in the polyelectrolyte microparticles obtained by various number of polyelectrolyte adsorption steps (from 1 to 11). The main parameters of ChT inclusion into microparticles were calculated. Scanning electron and optical microscopy were used for characterization of morphology and determination of particle size which was from 1 to 10 micro m in most cases. It was shown that the size and shape of protein-containing particles and protein aggregates used as a matrix were similar. Change in ChT enzymatic activity during entrapment into polyelectrolyte particles and activity of released protein were studied. The effect of pH on release of incorporated proteins was investigated; it was shown that change in pH and the number of polyelectrolyte adsorption steps allows protein release to be manipulated.

Encapsulation of proteins by layer-by-layer adsorption of polyelectrolytes onto protein aggregates: factors regulating the protein release.

A novel method of protein encapsulation is proposed. Preformed protein aggregates are covered with polyelectrolyte layers by means of layer-by-layer adsorption. The polyelectrolyte membrane prevents protein leakage out of the capsule. Using chymotrypsin as a model enzyme the capsule wall selective permeability was demonstrated for substrates and inhibitors of different molecular weight and solubility.