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.

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.

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.

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.

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.

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.

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.

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.

Morphological differences in BMP-2-induced ectopic bone between solid and crushed hyaluronan hydrogel templates.

The possibility to affect bone formation by using crushed versus solid hydrogels as carriers for bone morphogenetic protein 2 (BMP-2) was studied. Hydrogels, based on chemical crosslinking between hyaluronic acid and poly(vinyl alcohol) derivatives, were loaded with BMP-2 and hydroxyapatite. Crushed and solid forms of the gels were analyzed both in vitro via a release study using ¹²5I radioactive labeling of BMP-2, and in vivo in a subcutaneous ectopic bone model in rats. Dramatically different morphologies were observed for the ectopic bone formed in vivo in the two types of gels, even though virtually identical release profiles were observed in vitro. Solid hydrogels induced formation of a dense bone shell around non-degraded hydrogel, while crushed hydrogels demonstrated a uniform bone formation throughout the entire sample. These results suggest that by crushing the hydrogel, the construct's three-dimensional network becomes disrupted. This could expose unreacted functional groups, making the fragment's surfaces reactive and enable limited chemical fusion between the crushed hydrogel fragments, leading to similar in vitro release profiles. However, in vivo these interactions could be broken by enzymatic activity, creating a macroporous structure that allows easier cell infiltration, thus, facilitating bone formation.

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.

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.

Generation and evaluation of bispecific affibody molecules for simultaneous targeting of EGFR and HER2.

Coexpression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here, we have generated six such bispecific targeting proteins, each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly(4)Ser)(3) or (Ser(4)Gly)(3), and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays, it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the inherent monomeric affibody molecules was favorable to promote the binding to the respective target. Interestingly, bispecific constructs containing the novel (Ser(4)Gly)(3) linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly(4)Ser)(3). It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimer formation and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.

Nuclisome--targeting the tumor cell nucleus.

The Nuclisome concept builds on a novel two-step targeting strategy with the aim to deliver short-range Auger-electron-emitting radionuclides to nuclear DNA of tumor cells. The concept is based on the use of Nuclisome-particles, i.e., tumor-targeted PEG-stabilized liposomes loaded with a unique DNA-intercalating compound that enables specific and effective delivery of radionuclides to DNA. The specific and potent two-step targeting leads to eradication of tumor cells while toxicity to normal organs is reduced to a minimum. Results of in vitro and in vivo studies point towards the Nuclisome concept as a promising strategy for the treatment of small tumor masses and, in particular, for the elimination of spread single cells and micrometastases.

Avoiding false negative results in specificity analysis of protein-protein interactions.

The competition measurement using simultaneous incubation of labeled and unlabeled Ligand is a common method to assess the specificity of a biomolecular interaction. In this paper we show that invalid assumptions about the interactions may lead to improper experimental setups which in turn can result in inaccurate conclusions about the specificity. To improve understanding of competition measurements, simulations in MATLAB as well as real-time interaction analysis using LigandTracer have been performed. We show that use of a concentration of unlabeled Ligand of at least 10 × K(D) is necessary for assay accuracy. Increasing the incubation time to assure equilibrium, adding a pre-incubation phase, and a general understanding of the reversibility of an interaction may also improve the reliability of the measurement and the conclusions drawn about specificity. These findings may lower the risk of false negative results as well as reducing the amount of reagent needed.

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.

Blocking EGFR in the liver improves the tumor-to-liver uptake ratio of radiolabeled EGF.

Overexpression of epidermal growth factor receptor (EGFR) in several types of malignant tumors correlates with disease progression. EGFR could, therefore, be an excellent candidate for targeted radionuclide diagnostics. However, the high natural expression of EGFR in the liver may be problematic. The aim of this study was to improve the tumor-to-liver ratio of radiolabeled epidermal growth factor (EGF) by blocking its uptake by the liver with a nonradiolabeled EGFR-targeting molecule in tumor-bearing mice. Intraperitoneally injected nonradiolabeled EGF was first evaluated as a blocking agent, preadministered at various time intervals before intravenous injection of (125)I-labeled EGF. The anti-EGFR Affibody molecule (Z(EGFR:955))(2) was then assessed as a blocking agent of (111)In-labeled EGF in a dual isotope study (50, 100, and 200 microg, preadministered 30 or 60 min before (111)In-EGF). The 30-min preadministration of nonradiolabeled EGF significantly decreased (125)I-EGF uptake in the liver, whereas uptake in the tumor remained unchanged. Furthermore, preadministration of only 50 microg (Z(EGFR:955))(2) as a blocking agent 30 min before the (111)In-EGF decreased the uptake of (111)In-EGF by the liver and increased its uptake by the tumor, thereby increasing the tumor-to-liver ratio sixfold. We conclude that the Affibody molecule (Z(EGFR:955))(2) shows promise as a blocking agent that could enhance the outcome of radionuclide-based EGFR-expressing tumor diagnostics and imaging.

Nuclisome: a novel concept for radionuclide therapy using targeting liposomes.

PURPOSE: For the treatment of cancer, the therapeutic potential of short-range, low-energy Auger-electron emitters, such as (125)I, is getting progressively wider recognition. The potency of Auger-electron emitters is strongly dependent on their location in close vicinity to DNA. We have developed a new two-step targeting strategy to transport (125)I into cancer-cell nuclei using PEG-stabilized tumour-cell targeting liposomes named "Nuclisome-particles". METHODS: In the present study, epidermal growth factor (EGF) was used as a tumour-cell-specific agent to target the EGF-receptor (EGFR) and the liposomes were loaded with (125)I-Comp1, a recently synthesized daunorubicin derivative. RESULTS: As analysed with cryo-TEM, the derivative precipitates inside liposomes at a drug-to-lipid molar ratio of 0.05:1. Receptor-specific uptake in cultured U-343MGaCl2:6 tumour cells of EGFR-targeting liposomes increased with time while non-specific and receptor-blocked uptake remained low. Nuclisome-particles were able to target single U-343MGaCl2:6 cells circulating in human blood during 4 h, with low uptake in white blood cells, as demonstrated in an ex vivo system using a Chandler loop. Autoradiography of targeted cells indicates that the grains from the radiolabelled drug are mainly co-localized with the cell nuclei. The successful targeting of the nucleus is shown to provide high-potency cell killing of cultured U-343MGaCl2:6 cells. At the concentration used, Nuclisome-particles were up to five orders of magnitude more effective in cell killing than EGFR-targeting liposomes loaded with doxorubicin. CONCLUSION: The results thus provide encouraging evidence that our two-step targeting strategy for tumour cell DNA has the potential to become an effective therapy against metastasizing cancer cells in the bloodstream.