Dual centrifugation as a novel and efficient method for the preparation of lipodisks.

Polyethylene glycol (PEG)-stabilized lipodisks have emerged as innovatiive, promising nanocarriers for several classes of drugs. Prior research underscores the important role of lipid composition and preparation method in determining the lipodisk size, uniformity, and drug loading capacity. In this study, we investigate dual centrifugation (DC) as a novel technique for the production of PEG-stabilized lipodisks. Moreover, we explore the potential use of DC for the encapsulation of two model drugs, curcumin and doxorubicin, within the disks. Our results show that by a considerate choice of experimental conditions, DC can be used as a fast and straightforward means to produce small and homogenous lipodisks with a hydrodynamic diameter of 20-30 nm. Noteworthy, the technique works well for the production of both cholesterol-free and cholesterol-containing disks and does not require pre-mixing of the lipids in organic solvent. Furthermore, our investigations confirm the efficacy of DC in formulating curcumin and doxorubicin within these lipodisks. For doxorubicin, careful control and optimization of the experimental conditions resulted in formulations displaying an encouraging encapsulation efficiency of 84 % and a favourable drug-to-lipid ratio of 0.13 in the disks.

Investigation of supramolecular structures in various aqueous solutions of an amyloid forming peptide using small-angle X-ray scattering.

Aggregation of peptide molecules into amyloid fibrils is a characteristic feature of several degenerative diseases. However, the details behind amyloid-formation, and other self-assembled peptide aggregates, remain poorly understood. In this study, we have used small-angle X-ray scattering (SAXS), static and dynamic light scattering (SLS and DLS) as well as cryogenic transmission electron microscopy (cryo-TEM) to determine the structural geometry of self-assembled peptide aggregates in various dilute aqueous solutions. Pramlintide was used as a model peptide to assess the aggregation behaviour of an amyloid-forming peptide. The effects of adding sodium chloride (NaCl), sodium thiocyanate (NaSCN), and sodium fluoride (NaF) and the co-solvent dimethyl sulfoxide (DMSO) on the aggregation behaviour were studied. Our scattering data analysis demonstrates that small oligomeric fibrils aggregate to form networks of supramolecular assemblies with fractal dimensions. The choice of anion in small amounts of added salt has a significant impact on the size of the fibrils as well as on the fractal dimensions of supramolecular clusters. In DMSO the fractal dimension decreased with increasing DMSO concentration, indicating the formation of a less compact structure of the supramolecular assemblies.

A closer look at calcium-induced interactions between phosphatidylserine-(PS) doped liposomes and the structural effects caused by inclusion of gangliosides or polyethylene glycol- (PEG) modified lipids.

The effects of polyethylene glycol- (PEG) modified lipids and gangliosides on the Ca2+ induced interaction between liposomes composed of palmitoyl-oleoyl phosphatidylethanolamine (POPE) and palmitoyl-oleoyl phosphatidylserine (POPS) was investigated at physiological ionic strength. Förster resonance energy transfer (FRET) studies complemented with dynamic light scattering (DLS) and cryo-transmission electron microscopy (Cryo-EM) show that naked liposomes tend to adhere, rupture, and collapse on each other's surfaces upon addition of Ca2+, eventually resulting in the formation of large multilamellar aggregates and bilayer sheets. Noteworthy, the presence of gangliosides or PEGylated lipids does not prevent the adhesion-rupture process, but leads to the formation of small, long-lived bilayer fragments/disks. PEGylated lipids seem to be more effective than gangliosides at stabilizing these structures. Attractive interactions arising from ion correlation are proposed to be a driving force for the liposome-liposome adhesion and rupture processes. The results suggest that, in contrast with the conclusions drawn from previous solely FRET-based studies, direct liposome-liposome fusion is not the dominating process triggered by Ca2+ in the systems studied.

Vesicle-enriched secretomes alter bacterial competitive abilities and are drivers of evolution in microbial communities.

Microbial membrane vesicles can carry compounds that inhibit bacterial growth, but how they impact the fitness of the vesicle-producing bacterial species and influence community dynamics remain unexplored questions. To address these questions, we examined the effect of vesicle-enriched secretomes (VESs) in different single-species and multi-species systems. Effects of VESs on single-species growth dynamics were determined for nine bacterial species belonging to four genera (Escherichia, Salmonella, Pseudomonas and Bacillus) in nutrient-rich and poor growth media. Results showed both species-specific and nutrient-dependent effects of the VESs on bacterial growth. The strongest antagonistic effects were observed for VES isolated from the natural isolates of E. coli, while those isolated from P. aeruginosa PA14 affected the highest number of species. We further demonstrated that these VESs altered the competitive abilities of the species involved in two-species (S. Typhimurium LT2 and S. arizonae) and three-species systems (E. coli, S. Typhimurium LT2 and B. subtilis). Finally, using experimental evolution we showed that different bacterial species could rapidly acquire mutations that abrogated the antagonistic effects of VESs. This study demonstrates how VESs can contribute in shaping microbial communities, both by increasing the competitive ability of a given bacterial species and as a driver of genetic adaptation.

Spontaneous Formation of Ultrasmall Unilamellar Vesicles in Mixtures of an Amphiphilic Drug and a Phospholipid.

We have observed ultrasmall unilamellar vesicles, with diameters of less than 20 nm, in mixtures of the tricyclic antidepressant drug amitriptyline hydrochloride (AMT) and the unsaturated zwitterionic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in physiological saline solution. The size and shape of spontaneously formed self-assembled aggregates have been characterized using complementary techniques, i.e., small-angle neutron and X-ray scattering (SANS and SAXS) and cryo-transmission electron microscopy (cryo-TEM). We observe rodlike mixed micelles in more concentrated samples that grow considerably in length upon dilution, and a transition from micelles to vesicles is observed as the concentration approaches the critical micelle concentration of AMT. Unlike the micelles, the spontaneously formed vesicles decrease in size with each step of dilution, and ultrasmall unilamellar vesicles, with diameters as small as about 15 nm, were observed at the lowest concentrations. The spontaneously formed ultrasmall unilamellar vesicles maintain their size for as long we have investigated them (i.e., several months). To the best of our knowledge, such small vesicles have never before been reported to form spontaneously in a biocompatible phospholipid-based system. Most interestingly, the size of the vesicles was observed to be strongly dependent on the chemical structure of the phospholipid, and in mixtures of AMT and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), the vesicles were observed to be considerably larger in size. The self-assembly behavior in the phospholipid-drug surfactant system in many ways resembles the formation of equilibrium micelles and vesicles in mixed anionic/cationic surfactant systems.

Tailoring the Lamellarity of Liposomes Prepared by Dual Centrifugation.

Dual centrifugation (DC) is a new and versatile technique for the preparation of liposomes by in-vial homogenization of lipid-water mixtures. Size, size distribution, and entrapping efficiencies are strongly dependent on the lipid concentration during DC-homogenization. In this study, we investigated the detailed structure of DC-made liposomes. To do so, an assay to determine the ratio of inner to total membrane surfaces of liposomes (inaccessible surface) was developed based on either time-resolved or steady-state fluorescence spectroscopy. In addition, cryogenic electron microscopy (cryo-EM) was used to confirm the lamellarity results and learn more about liposome morphology. One striking result leads to the possibility of producing a novel type of liposome-small multilamellar vesicles (SMVs) with low PDI, sizes of the order of 100 nm, and almost completely filled with bilayers. A second particularly important finding is that VPGs can be prepared to contain open bilayer structures that will close spontaneously when, after storage, more aqueous phase is added and liposomes are formed. Through this process, a drug can effectively be entrapped immediately before application. In addition, dual centrifugation at lower lipid concentrations is found to produce predominantly unilamellar vesicles.

Excited-state and charge-carrier dynamics in binary conjugated polymer dots towards efficient photocatalytic hydrogen evolution.

Aqueous dispersed conjugated polymer dots (Pdots) have shown promising application in photocatalytic hydrogen evolution. To efficiently extract photogenerated charges from type-II heterojunction Pdots for hydrogen evolution, the mechanistic study of photophysical processes is essential for Pdot optimization. Within this work, we use a PFODTBT donor (D) polymer and an ITIC small molecule acceptor (A) as a donor/acceptor (D/A) model system to study their excited states and charge/energy transfer dynamics via steady-state and time-resolved photoluminescence spectroscopy, respectively. Charge-carrier generation and the recombination dynamics of binary Pdots with different D/A ratios were followed using femtosecond transient absorption spectroscopy. A significant spectral relaxation of photoluminescence was observed for individual D Pdots, implying an energetic disorder by nature. However, this was not seen for charge carriers in binary Pdots, probably due to the ultrafast charge generation process at an early time (<200 fs). The results showed slower charge recombination upon increasing the ratio of ITIC in binary Pdots, which further resulted in an enhanced photocatalytic hydrogen evolution, twice that as compared to individual D Pdots. Although binary Pdots prepared via the nanoprecipitation method exhibit a large interfacial area that allows high charge generation efficiencies, it also provides a high possibility for charge recombination and limits the further utilization of free charges. Therefore, for the future design of type-II heterojunction Pdots, suppressing the charge carrier recombination via increasing the crystallinity and proper phase segregation is necessary for enhanced photocatalytic hydrogen evolution.

Polymer Dots as Photoactive Membrane Vesicles for [FeFe]-Hydrogenase Self-Assembly and Solar-Driven Hydrogen Evolution.

A semiartificial photosynthesis approach that utilizes enzymes for solar fuel production relies on efficient photosensitizers that should match the enzyme activity and enable long-term stability. Polymer dots (Pdots) are biocompatible photosensitizers that are stable at pH 7 and have a readily modifiable surface morphology. Therefore, Pdots can be considered potential photosensitizers to drive such enzyme-based systems for solar fuel formation. This work introduces and unveils in detail the interaction within the biohybrid assembly composed of binary Pdots and the HydA1 [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The direct attachment of hydrogenase on the surface of toroid-shaped Pdots was confirmed by agarose gel electrophoresis, cryogenic transmission electron microscopy (Cryo-TEM), and cryogenic electron tomography (Cryo-ET). Ultrafast transient spectroscopic techniques were used to characterize photoinduced excitation and dissociation into charges within Pdots. The study reveals that implementation of a donor-acceptor architecture for heterojunction Pdots leads to efficient subpicosecond charge separation and thus enhances hydrogen evolution (88 460 µmolH2·gH2ase-1·h-1). Adsorption of [FeFe]-hydrogenase onto Pdots resulted in a stable biohybrid assembly, where hydrogen production persisted for days, reaching a TON of 37 500 ± 1290 in the presence of a redox mediator. This work represents an example of a homogeneous biohybrid system combining polymer nanoparticles and an enzyme. Detailed spectroscopic studies provide a mechanistic understanding of light harvesting, charge separation, and transport studied, which is essential for building semiartificial photosynthetic systems with efficiencies beyond natural and artificial systems.

Influence of Chain Length of Gradient and Block Copoly(2-oxazoline)s on Self-Assembly and Drug Encapsulation.

Amphiphilic gradient copolymers represent a promising alternative to extensively used block copolymers due to their facile one-step synthesis by statistical copolymerization of monomers of different reactivity. Herein, an in-depth analysis is provided of micelles based on amphiphilic gradient poly(2-oxazoline)s with different chain lengths to evaluate their potential for micellar drug delivery systems and compare them to the analogous diblock copolymer micelles. Size, morphology, and stability of self-assembled nanoparticles, loading of hydrophobic drug curcumin, as well as cytotoxicities of the prepared nanoformulations are examined using copoly(2-oxazoline)s with varying chain lengths and comonomer ratios. In addition to several interesting differences between the two copolymer architecture classes, such as more compact self-assembled structures with faster exchange dynamics for the gradient copolymers, it is concluded that gradient copolymers provide stable curcumin nanoformulations with comparable drug loadings to block copolymer systems and benefit from more straightforward copolymer synthesis. The study demonstrates the potential of amphiphilic gradient copolymers as a versatile platform for the synthesis of new polymer therapeutics.

Structural Characterization Study of a Lipid Nanocapsule Formulation Intended for Drug Delivery Applications Using Small-Angle Scattering Techniques.

Lipid nanocapsules (LNCs) are increasingly being used for various drug delivery applications due to their versatile nature and ability to carry a wide variety of therapeutic drug molecules. In the present investigation, small-angle X-ray (SAXS) and neutron scattering (SANS) techniques were used to elucidate the structure of LNCs. Overall, size measurements obtained from SAXS and SANS techniques were complemented with dynamic light scattering, zeta potential, and cryogenic transmission electron microscopy measurements. The structural aspects of LNCs can be affected by drug loading and the properties of the drug. Here, the impact of drug loading on the overall structure was evaluated using DF003 as a model drug molecule. LNCs with varying compositions were prepared using a phase inversion method. Combined analysis of SAXS and SANS measurements indicated the presence of a core-shell structure in the LNCs. Further, the drug loading did not alter the overall core-shell structure of the LNCs. SANS data revealed that the core size remained unchanged with a radius of 20.0 ± 0.9 nm for unloaded LNCs and 20.2 ± 0.6 nm for drug-loaded LNCs. Furthermore, interestingly, the shell becomes thicker in an order of ∼1 nm in presence of the drug compared to the shell thickness of unloaded LNCs as demonstrated by SAXS data. This can be correlated with the strong association of hydrophilic DF003 with Kolliphor HS 15, a polyethylene glycol-based surfactant that predominantly makes up the shell, resulting in a drug-rich hydrated shell.

Investigation of the enhanced ability of bile salt surfactants to solubilize phospholipid bilayers and form mixed micelles.

The self-assembly in mixtures of the anionic bile salt surfactant sodium deoxycholate (NaDC) and the zwitterionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in physiological saline solution has been investigated using light scattering, small-angle X-ray scattering and cryo-transmission electron microscopy. Rather small tri-axial ellipsoidal NaDC-DMPC mixed micelles form at a high content of bile salt in the mixture, which increase in size as an increasing amount of DMPC is incorporated into the micelles. Eventually, the micelles begin to grow substantially in length to form long wormlike micelles. At higher mole fractions of DMPC, the samples become turbid and cryo-TEM measurements reveal the existence of large perforated vesicles (stomatosomes), coexisting with geometrically open disks. To our knowledge, stomatosomes have not been observed before for any bile salt-phospholipid system. Mixed micelles are found to be the sole aggregate structure in a very wide regime of bile salt-phospholipid compositions, i.e. up to about 77 mol% phospholipid in the micelles. This is much higher than the corresponding value of 25 mol% observed for the conventional surfactant hexadecyltrimethylammonium bromide (CTAB) mixed with DMPC in the same solvent. The enhanced ability of bile salt surfactants to solubilize phospholipid bilayers and form mixed micelles is rationalized using bending elasticity theory. From our theoretical analysis, we are able to conclude that amphiphilic molecules rank in the following order of increasing spontaneous curvature: phospholipids < conventional surfactants < bile salts. The bending rigidity of the different amphiphilic molecules increases according to the following sequence: bile salts < conventional surfactants < phospholipids.

Formulation development and upscaling of lipid nanocapsules as a drug delivery system for a novel cyclic GMP analogue intended for retinal drug delivery.

Lipid nanocapsules (LNCs) were prepared with a novel cyclic GMP analogue, DF003, intended for the treatment of neurodegenerative retinal degenerations. LNCs loaded with DF003 were prepared by a phase inversion method and characterized for particle size, polydispersity index, drug loading, entrapment efficiency, stability, and in vitro drug release. Particle size, PdI and zeta potential of selected optimized formulation were 76 ± 1.2 nm, 0.16 ± 0.02, and -11.6 ± 0.4 mV, respectively, with an entrapment efficiency of 69 ± 0.5%. The selected formulation showed a sustained drug release for up to 6 days in phosphate buffer as well as in vitreous components. Stability evaluation of LNCs in presence of vitreous components demonstrated structural stability and compatibility. Further, the nanoparticle preparation process was upscaled to 1000 times (10 L) of the typical lab scale (0.01 L). Product parameters were observed to be unaffected by the upscaling, demonstrating that the LNCs were of the same quality as those prepared at lab scale. Additionally, the manufacturing process was adapted and assessed for a continuous production of LNCs to leverage it for industrial viability. Overall, these findings reveal the remarkable potential of LNCs as drug delivery vehicles and their possibility for clinical translation.

Panchromatic Ternary Polymer Dots Involving Sub-Picosecond Energy and Charge Transfer for Efficient and Stable Photocatalytic Hydrogen Evolution.

Panchromatic ternary polymer dots (Pdots) consisting of two conjugated polymers (PFBT and PFODTBT) based on fluorene and benzothiadiazole groups, and one small molecular acceptor (ITIC) have been prepared and assessed for photocatalytic hydrogen production with the assistance of a Pt cocatalyst. Femtosecond transient absorption spectroscopic studies of the ternary Pdots have revealed both energy and charge transfer processes that occur on the time scale of sub-picosecond between the different components. They result in photogenerated electrons being located mainly at ITIC, which acts as both electron and energy acceptor. Results from cryo-transmission electron microscopy suggest that ITIC forms crystalline phases in the ternary Pdots, facilitating electron transfer from ITIC to the Pt cocatalyst and promoting the final photocatalytic reaction yield. Enhanced light absorption, efficient charge separation, and the ideal morphology of the ternary Pdots have rendered an external quantum efficiency up to 7% at 600 nm. Moreover, the system has shown a high stability over 120 h without obvious degradation of the photocatalysts.

Effect of gangliosides on structure and integrity of polyethylene glycol (PEG)-stabilized liposomes.

The in vivo efficacy and tolerance of polyethylene glycol (PEG)-decorated drug nanocarriers, such as liposomes, is compromised bytheir tendency to induce the generation of PEG-specific immunoglobulin M (IgM) antibodies. Recently, a number of independent studies have reported on an attenuated anti-PEG immune response upon incorporation of gangliosides in the membrane of PEGylated liposomes.In the present study we investigate the effect of gangliosides on the self-assembled structures found in lipid dispersions based on hydrogenated egg phosphatidylcholine (HEPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG(2000)). Results from cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS) investigations show that gangliosides promote structural transitions from liposomes to bilayer disks. In case of samples comprising 5 mol% PEG-conjugated lipids (PEG-lipid), inclusion of 2.5 mol% ganglioside (porcine ganglioside extract) results in the presence of a small but significant amount of disks. With increasing ganglioside content the population of disks grows at the expense of the liposomes. Comparative investigations using isolated ganglioside components reveal that disialoganglioside GD1a is more potent than monosialoganglioside GM1 in promoting disk formation. Experiments involving liposome encapsulated carboxyfluorescein confirm that the ganglioside-induced structural transformations have a detrimental effect on the total entrapped aqueous volume of the samples. The reported coexistence of liposomes and bilayer disks may if overlooked have important implications for the therapeutic efficacy and immunogenicity of ganglioside-supplemented liposomal formulations.

Tumor-Targeted Delivery of the p53-Activating Peptide VIP116 with PEG-Stabilized Lipodisks.

Stapled peptides targeting the interaction between p53 and its negative regulators MDM2 and MDM4 have exhibited great potential as anti-cancer drugs, albeit with room for improvement in formulation and tumor specificity. Lipid bilayer disks (lipodisks) have emerged as promising drug nanocarriers and can by attachment of targeting moieties be directed selectively towards tumor cells. Tumor-targeted delivery of stapled peptides by use of lipodisks may therefore increase the uptake in the tumors and limit toxicity in healthy tissue. Here, we utilized epidermal growth factor receptor (EGFR)-targeted lipodisks to deliver p53-activating stapled peptide VIP116 to EGFR-expressing tumor cells. We demonstrate that VIP116 can be stably formulated in lipodisks (maximum peptide/lipid molar ratio 0.11). In vitro cell studies verify specific binding of EGF-decorated lipodisks to tumor cells and confirm that targeted delivery of VIP116 significantly decreases tumor cell viability.

Osmoprotective effect of ubiquinone in lipid vesicles modelling the E. coli plasma membrane.

Bacteria need to be able to adapt to sudden changes in their environment, including drastic changes in the surrounding osmolarity. As part of this adaptation, the cells adjust the composition of their cytoplasmic membrane. Recent studies have shown that ubiquinones, lipid soluble molecules involved in cell respiration, are overproduced by bacteria grown in hyperosmotic conditions and it is thus believed that these molecules can provide with osmoprotection. Hereby we explore the mechanisms behind these observations. Liposomes with a lipid headgroup composition mimicking that of the cytoplasmic membrane of E. coli are used as suitable models. The effect of ubiquinone-10 (Q10) on water transport across the membranes is characterized using a custom developed fluorescence-based experimental approach to simultaneously determine the membrane permeability coefficient and estimate the elastic resistance of the membrane towards deformation. It is shown that both parameters are affected by the presence of ubiquinone-10. Solanesol, a molecule similar to Q10 but lacking the quinone headgroup, also provides with osmoprotection although it only improves the resistance of the membrane against deformation. The fluorescence experiments are complemented by cryogenic transmission electron microscopy studies showing that the E. coli membrane mimics tend to flatten into spheroid oblate structures when osmotically stressed, suggesting the possibility of lipid segregation. In agreement with its proposed osmoprotective role, the flattening process is hindered by the presence of Q10.

17AAG-induced internalisation of HER2-specific Affibody molecules.

The geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) is known to induce internalisation and degradation of the otherwise internalisation-resistant human epidermal growth factor receptor 2 (HER2) receptor. In the present study, 17-AAG was used to increase internalisation of the HER2-specific Affibody molecule ABY-025. The cellular redistribution of halogen-labelled 211At-ABY-025 and radiometal-labelled 111In-ABY-025 following treatment with 17-AAG was studied. 17-AAG treatment of SKOV-3 human ovarian carcinoma and SKBR-3 human breast carcinoma cells to some extent shifted the localisation of 111In-ABY-025 from the cell surface to intracellular compartments in the two cell lines. ABY-025 labelled with the high-linear energy transfer α emitter 211At was also internalised to a higher degree; however, due to its physiological properties, this nuclide was excreted faster. The results indicate that 17-AAG may be used to facilitate cell-specific intracellular localisation of a suitable cytotoxic or radioactive agent coupled to ABY-025 in HER2-overexpressing cells.

Pre-incubation of chemically crosslinked hyaluronan-based hydrogels, loaded with BMP-2 and hydroxyapatite, and its effect on ectopic bone formation.

The effects of pre-incubation of hyaluronan hydrogels, for different lengths of time after the initiation of chemical crosslinking and prior to injection, were explored both by investigating the in vitro BMP-2 release kinetics from the hydrogel and by studying the ectopic bone formation in rats. From the curing profile, obtained from rheological analysis, appropriate pre-incubation times (1 min, 5 h and 3 days) were selected, to prepare slightly, moderately and fully cured hydrogels. Comparable release profiles were observed for all three test groups in vitro. Furthermore, radiography, pQCT and histology of the explanted grafts showed cancellous bone formation in all groups after 5 weeks in vivo. However, longer pre-incubation times gave rise to an increase in bone volume, but a decrease in bone density. Moreover, the 5 h and the 3 days grafts appeared to be more ordered and resistant to deformation from the surrounding tissue than the 1 min grafts. The observed variations in mechanical and biological properties could potentially be used to adapt the treatment for a specific indication.

Characterization of recombinant human bone morphogenetic protein-2 delivery from injectable hyaluronan-based hydrogels by means of 125I-radiolabelling.

This study presents a thorough in vitro and in vivo characterization of the delivery of bone morphogenetic protein 2 (BMP-2) from a hyaluronan-based hydrogel system. The in vitro release of BMP-2 from similar hydrogels has previously been studied by enzyme-linked immunosorbent assay (ELISA), by which only a fraction of the loaded protein is detected. In the current study, (125) I radiolabelling was used instead to monitor BMP-2 in vitro and in vivo. To minimize protein loss during handling, (125) I-BMP-2 adsorption to different tubes was studied at different times and temperatures. The data showed that Protein LoBind tubes exhibited the lowest protein affinity. Furthermore, a biphasic release profile of biologically active BMP-2 was observed both in vitro and in vivo, with the initial fast phase during the first week, followed by a slower release during the remaining 3 weeks. The initial fast-release phase corresponded to the early bone formation observed after 8 days in an ectopic model in rats. Bone volume and mineral content increased until day 14, after which a decrease in bone volume was observed, possibly due to resorption in response to decreased amounts of released BMP-2. Overall, the results suggested that cautious protein handling and a reliable quantification technique are essential factors for successful design of a BMP-2 delivery system.

Evaluation of real-time immunohistochemistry and interaction map as an alternative objective assessment of HER2 expression in human breast cancer tissue.

Immunohistochemical study (IHC) is a critical tool in the clinical diagnosis of breast cancer. One common assessment is the expression level of the HER2 receptor in breast cancer tissue samples with the aim of stratifying patients for applicability of the therapeutic antibody Herceptin. In this study, we aimed to investigate whether a novel assay, real-time IHC combined with Interaction Map analysis, offers the possibility of objective assessment of HER2 expression. Interaction Map presents real-time interaction data as a collection of peaks on a surface, and it was performed on 20 patient tissue samples previously scored for HER2 expression. The result shows that the relative weight of the peaks in the maps contains novel information that could discriminate between high and low HER2 expression in an operator-independent manner (P<0.001). We conclude that the real-time IHC assay has a promising potential to complement conventional IHC and may improve the precision in the future clinical diagnostics of breast cancer.