An Efficient Method for Vault Nanoparticle Conjugation with Finely Adjustable Amounts of Antibodies and Small Molecules.

Vaults are eukaryotic ribonucleoproteins consisting of 78 copies of the major vault protein (MVP), which assemble into a nanoparticle with an about 60 nm volume-based size, enclosing other proteins and RNAs. Regardless of their physiological role(s), vaults represent ideal, natural hollow nanoparticles, which are produced by the assembly of the sole MVP. Here, we have expressed in Komagataella phaffi and purified an MVP variant carrying a C-terminal Z peptide (vault-Z), which can tightly bind an antibody's Fc portion, in view of targeted delivery. Via surface plasmon resonance analysis, we could determine a 2.5 nM affinity to the monoclonal antibody Trastuzumab (Tz)/vault-Z 1:1 interaction. Then, we characterized the in-solution interaction via co-incubation, ultracentrifugation, and analysis of the pelleted proteins. This showed virtually irreversible binding up to an at least 10:1 Tz/vault-Z ratio. As a proof of concept, we labeled the Fc portion of Tz with a fluorophore and conjugated it with the nanoparticle, along with either Tz or Cetuximab, another monoclonal antibody. Thus, we could demonstrate antibody-dependent, selective uptake by the SKBR3 and MDA-MB 231 breast cancer cell lines. These investigations provide a novel, flexible technological platform that significantly extends vault-Z's applications, in that it can be stably conjugated with finely adjusted amounts of antibodies as well as of other molecules, such as fluorophores, cell-targeting peptides, or drugs, using the Fc portion as a scaffold.

Natural Inhibitors of Amyloid Aggregation.

Amyloid aggregates are diverse proteinaceous assemblies, including one or more protein species, wherein the molecules interact according to characteristic patterns [...].

Addressing Critical Issues Related to Storage and Stability of the Vault Nanoparticle Expressed and Purified from Komagataella phaffi.

The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We also found that the protein displayed a significant propensity to aggregate. We thus investigated this phenomenon and the related structural changes by Fourier-transform spectroscopy and dynamic light scattering, which led us to determine the most suitable storage conditions. In particular, the addition of either trehalose or Tween-20 ensured the best preservation of the protein in native, soluble form.

Pathological ATX3 Expression Induces Cell Perturbations in E. coli as Revealed by Biochemical and Biophysical Investigations.

Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.

Epigallocatechin-3-gallate and related phenol compounds redirect the amyloidogenic aggregation pathway of ataxin-3 towards non-toxic aggregates and prevent toxicity in neural cells and Caenorhabditis elegans animal model.

The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.

A fast and straightforward procedure for vault nanoparticle purification and the characterization of its endocytic uptake.

BACKGROUND: Vaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97 kDa major vault protein that assembles into a barrel-like, "nanocapsule" enclosing poly(ADP-ribose) polymerase, telomerase-associated protein-1 and small untranslated RNAs. Overall, the molecular mass of vault particles amounts to about 13 MDa. Although it has been implicated in several cellular functions, its physiological roles remain poorly understood. Also, the possibility to exploit it as a nanovector for drug delivery is currently being explored in several laboratories. METHODS: Using the baculovirus expression system, vaults were expressed and purified by a dialysis step using a 1 MDa molecular weight cutoff membrane and a subsequent size exclusion chromatography. Purity was assessed by SDS-PAGE, transmission electron microscopy and dynamic light scattering. Particle's endocytic uptake was monitored by flow cytometry and confocal microscopy. RESULTS: The purification protocol here reported is far simpler and faster than those currently available and lead to the production of authentic vault. We then demonstrated its clathrin-mediated endocytic uptake by normal fibroblast and glioblastoma, but not carcinoma cell lines. In contrast, no significant caveolin-mediated endocytosis was detected. CONCLUSIONS: These results provide the first evidence for an intrinsic propensity of the vault complex to undergo endocytic uptake cultured eukaryotic cells. GENERAL SIGNIFICANCE: The newly developed purification procedure will greatly facilitate any investigation based on the use of the vault particle as a natural nanocarrier. Its clathrin-mediated endocytic uptake observed in normal and in some tumor cell lines sheds light on its physiological role.

Targeting Amyloid Aggregation: An Overview of Strategies and Mechanisms.

Amyloids result from the aggregation of a set of diverse proteins, due to either specific mutations or promoting intra- or extra-cellular conditions. Structurally, they are rich in intermolecular ß-sheets and are the causative agents of several diseases, both neurodegenerative and systemic. It is believed that the most toxic species are small aggregates, referred to as oligomers, rather than the final fibrillar assemblies. Their mechanisms of toxicity are mostly mediated by aberrant interactions with the cell membranes, with resulting derangement of membrane-related functions. Much effort is being exerted in the search for natural antiamyloid agents, and/or in the development of synthetic molecules. Actually, it is well documented that the prevention of amyloid aggregation results in several cytoprotective effects. Here, we portray the state of the art in the field. Several natural compounds are effective antiamyloid agents, notably tetracyclines and polyphenols. They are generally non-specific, as documented by their partially overlapping mechanisms and the capability to interfere with the aggregation of several unrelated proteins. Among rationally designed molecules, we mention the prominent examples of ß-breakers peptides, whole antibodies and fragments thereof, and the special case of drugs with contrasting transthyretin aggregation. In this framework, we stress the pivotal role of the computational approaches. When combined with biophysical methods, in several cases they have helped clarify in detail the protein/drug modes of interaction, which makes it plausible that more effective drugs will be developed in the future.

Protein Environment: A Crucial Triggering Factor in Josephin Domain Aggregation: The Role of 2,2,2-Trifluoroethanol.

The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially α-helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain.

Inhibition of α-Synuclein Fibril Elongation by Hsp70 Is Governed by a Kinetic Binding Competition between α-Synuclein Species.

The Hsp70 family of chaperones plays an essential role in suppressing protein aggregation in the cell. Here we investigate the factors controlling the intrinsic ability of human Hsp70 to inhibit the elongation of amyloid fibrils formed by the Parkinson's disease-related protein α-synuclein. Using kinetic analysis, we show that Hsp70 binds preferentially to α-synuclein fibrils as a consequence of variations in the association and dissociation rate constants of binding to the different aggregated states of the protein. Our findings illustrate the importance of the kinetics of binding of molecular chaperones, and also of potential therapeutic molecules, in the efficient suppression of specific pathogenic events linked to neurodegeneration.

Epigallocatechin-3-gallate and tetracycline differently affect ataxin-3 fibrillogenesis and reduce toxicity in spinocerebellar ataxia type 3 model.

The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) when its polyQ tract is expanded beyond a critical length. This results in protein aggregation and generation of toxic oligomers and fibrils. Currently, no effective treatment is available for such and other polyQ diseases. Therefore, plenty of investigations are being carried on to assess the mechanism of action and the therapeutic potential of anti-amyloid agents. The polyphenol compound epigallocatechin-3-gallate (EGCG) and tetracycline have been shown to exert some effect in preventing fibrillogenesis of amyloidogenic proteins. Here, we have incubated an expanded AT3 variant with either compound to assess their effects on the aggregation pattern. The process was monitored by atomic force microscopy and Fourier transform infrared spectroscopy. Whereas in the absence of any treatment, AT3 gives rise to amyloid ß-rich fibrils, whose hallmark is the typical glutamine side-chain hydrogen bonding, when incubated in the presence of EGCG it generated soluble, SDS-resistant aggregates, much poorer in ß-sheets and devoid of any ordered side-chain hydrogen bonding. These are off-pathway species that persist until the latest incubation time and are virtually absent in the control sample. In contrast, tetracycline did not produce major alterations in the structural features of the aggregated species compared with the control, but substantially increased their solubility. Both compounds significantly reduced toxicity, as shown by the MTT assay in COS-7 cell line and in a transgenic Caenorhabditis elegans strain expressing in the nervous system an AT3 expanded variant in fusion with GFP.

Peptide-nanoparticle ligation mediated by cutinase fusion for the development of cancer cell-targeted nanoconjugates.

The relationship between the positioning of ligands on the surface of nanoparticles and the structural features of nanoconjugates has been underestimated for a long time, albeit of primary importance to promote specific biological recognition at the nanoscale. In particular, it has been formerly observed that a proper molecular orientation can play a crucial role, first optimizing ligand immobilization onto the nanoparticles and, second, improving the targeting efficiency of the nanoconjugates. In this work, we present a novel strategy to afford peptide-oriented ligation using genetically modified cutinase fusion proteins, which combines the presence of a site-directed "capture" module based on an enzymatic unit and a "targeting" moiety consisting of the ligand terminal end of a genetically encoded polypeptide chain. As an example, the oriented presentation of U11 peptide, a sequence specific for the recognition of urokinase plasminogen activator receptor (uPAR), was achieved by enzyme-mediated conjugation with an irreversible inhibitor of cutinase, an alkylphosphonate p-nitrophenol ester linker, covalently bound to the surface of iron oxide nanoparticles. The targeting efficiency of the resulting protein-nanoparticle conjugates was assessed using uPAR-positive breast cancer cells exploiting confocal laser scanning microscopy and quantitative fluorescence analysis of confocal images. Ultrastructural analysis of transmission electron micrographs provided evidence of a receptor-mediated pathway of endocytosis. Our results showed that, despite the small average number of targeting peptides presented on the nanoparticles, our ligand-oriented nanoconjugates proved to be very effective in selectively binding to uPAR and in promoting the uptake in uPAR-positive cancer cells.

How Epigallocatechin-3-gallate and Tetracycline Interact with the Josephin Domain of Ataxin-3 and Alter Its Aggregation Mode.

Epigallocatechin-3-gallate (EGCG) and tetracycline are two known inhibitors of amyloid aggregation able to counteract the fibrillation of most of the proteins involved in neurodegenerative diseases. We have recently investigated their effect on ataxin-3 (AT3), the polyglutamine-containing protein responsible for spinocerebellar ataxia type 3. We previously showed that EGCG and tetracycline can contrast the aggregation process and toxicity of expanded AT3, although by different mechanisms. Here, we have performed further experiments by using the sole Josephin domain (JD) to further elucidate the mechanism of action of the two compounds. By protein solubility assays and FTIR spectroscopy we have first observed that EGCG and tetracycline affect the JD aggregation essentially in the same way displayed when acting on the full-length expanded AT3. Then, by saturation transfer difference (STD) NMR experiments, we have shown that EGCG binds both the monomeric and the oligomeric JD form, whereas tetracycline can only interact with the oligomeric one. Surface plasmon resonance (SPR) analysis has confirmed the capability of the sole EGCG to bind monomeric JD, although with a KD value suggestive for a non-specific interaction. Our investigations provide new details on the JD interaction with EGCG and tetracycline, which could explain the different mechanisms by which the two compounds reduce the toxicity of AT3.

The conformational ensemble of the disordered and aggregation-protective 182-291 region of ataxin-3.

BACKGROUND: Intrinsically disordered proteins (IDPs) are an emerging part of structural biology that has challenged the classic paradigm of structure-function relationship. Indeed, IDPs have been associated with different physiological functions and associated with several pathologies, such as polyglutamine (polyQ) related diseases. Ataxin-3 (AT3) is the smallest polyQ protein, composed by the N-terminal folded Josephin domain (JD), which is amyloidogenic on its own, and a C-terminal unstructured part. The disordered region between the polyQ and the JD, AT3182-291 plays a key role in the development of the disease. METHODS: We integrated different biophysical experimental techniques, atomistic explicit-solvent molecular dynamics (MD) simulations and graph theory to study AT3182-291 structure. RESULTS: AT3182-291 is a monomeric intrinsically disordered (ID) domain in solution and it is characterized by different conformational states, ascribable to pre-molten globule populations with different degrees of compactness. If isolated, it decreases the aggregation of the entire AT3. CONCLUSIONS: We provided the first structural description of an ID domain associated to a polyQ protein and we also showed that it exerts protective effects against AT3 aggregation. This effect is likely to be induced by intermolecular interactions between AT3 and the ubiquitin-interacting motifs of AT3182-291. Electrostatic interactions play a pivotal role in regulating the topology and tertiary propensity of the fragment and hub residues have been identified. GENERAL SIGNIFICANCE: Synergistic use of atomistic simulations and biophysical techniques should be more generally applied to the study of IDPs. Since ID domains and polyQ-proteins are intimately connected, the study here provided can be of interest for other members of the group.

Different ataxin-3 amyloid aggregates induce intracellular Ca(2+) deregulation by different mechanisms in cerebellar granule cells.

This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca(2+) levels and the abnormal Ca(2+) signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca(2+) responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca(2+) response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.

Immobilization of carboxypeptidase from Sulfolobus solfataricus on magnetic nanoparticles improves enzyme stability and functionality in organic media.

BACKGROUND: Superparamagnetic iron oxide nanoparticles (MNP) offer several advantages for applications in biomedical and biotechnological research. In particular, MNP-based immobilization of enzymes allows high surface-to-volume ratio, good dispersibility, easy separation of enzymes from the reaction mixture, and reuse by applying an external magnetic field. In a biotechnological perspective, extremophilic enzymes hold great promise as they often can be used under non-conventional harsh conditions, which may result in substrate transformations that are not achievable with normal enzymes. This prompted us to investigate the effect of MNP bioconjugation on the catalytic properties of a thermostable carboxypeptidase from the hyperthermophilic archaeon Sulfolobus solfataricus (CPSso), which exhibits catalytic properties that are useful in synthetic processes. RESULTS: CPSso was immobilized onto silica-coated iron oxide nanoparticles via NiNTA-His tag site-directed conjugation. Following the immobilization, CPSso acquired distinctly higher long-term stability at room temperature compared to the free native enzyme, which, in contrast, underwent extensive inactivation after 72 h incubation, thus suggesting a potential utilization of this enzyme under low energy consumption. Moreover, CPSso conjugation also resulted in a significantly higher stability in organic solvents at 40°C, which made it possible to synthesize N-blocked amino acids in remarkably higher yields compared to those of free enzyme. CONCLUSIONS: The nanobioconjugate of CPSso immobilized on silica-coated magnetic nanoparticles exhibited enhanced stability in aqueous media at room temperature as well as in different organic solvents. The improved stability in ethanol paves the way to possible applications of immobilized CPSso, in particular as a biocatalyst for the synthesis of N-blocked amino acids. Another potential application might be amino acid racemate resolution, a critical and expensive step in chemical synthesis.

Surface properties of the seas of Titan as revealed by Cassini mission bistatic radar experiments.

Saturn's moon Titan was explored by the Cassini spacecraft from 2004 to 2017. While Cassini revealed a lot about this Earth-like world, its radar observations could only provide limited information about Titan's liquid hydrocarbons seas Kraken, Ligeia and Punga Mare. Here, we show the results of the analysis of the Cassini mission bistatic radar experiments data of Titan's polar seas. The dual-polarized nature of bistatic radar observations allow independent estimates of effective relative dielectric constant and small-scale roughness of sea surface, which were not possible via monostatic radar data. We find statistically significant variations in effective dielectric constant (i.e., liquid composition), consistent with a latitudinal dependence in the methane-ethane mixing-ratio. The results on estuaries suggest lower values than the open seas, compatible with methane-rich rivers entering seas with higher ethane content. We estimate small-scale roughness of a few millimeters from the almost purely coherent scattering from the sea surface, hinting at the presence of capillary waves. This roughness is concentrated near estuaries and inter-basin straits, perhaps indicating active tidal currents.

Site-specific conjugation of ScFvs antibodies to nanoparticles by bioorthogonal strain-promoted alkyne-nitrone cycloaddition.

Particularly suitable: An N-terminal serine mutant of anti-HER2 scFv antibody was conjugated to polymer-coated magnetofluorescent nanoparticles by strain-promoted alkyne-nitrone cycloaddition. The resulting nanoparticles (see scheme) proved effective in targeting and labeling HER2-positive breast cancer cells.

Saporin Toxin Delivered by Engineered Colloidal Nanoparticles Is Strongly Effective against Cancer Cells.

Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin's main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.

Site-specific mutation of Staphylococcus aureus VraS reveals a crucial role for the VraR-VraS sensor in the emergence of glycopeptide resistance.

An initial response of Staphylococcus aureus to encounter with cell wall-active antibiotics occurs by transmembrane signaling systems that orchestrate changes in gene expression to promote survival. Histidine kinase two-component sensor-response regulators such as VraRS contribute to this response. In this study, we examined VraS membrane sensor phosphotransfer signal transduction and explored the genetic consequences of disrupting signaling by engineering a site-specific vraS chromosomal mutation. We have used in vitro autophosphorylation assay with purified VraS[64-347] lacking its transmembrane anchor region and tested site-specific kinase domain histidine mutants. We identified VraS H156 as the probable site of autophosphorylation and show phosphotransfer in vitro using purified VraR. Genetic studies show that the vraS(H156A) mutation in three strain backgrounds (ISP794, Newman, and COL) fails to generate detectable first-step reduced susceptibility teicoplanin mutants and severely reduces first-step vancomycin mutants. The emergence of low-level glycopeptide resistance in strain ISP794, derived from strain 8325 (ΔrsbU), did not require a functional σ(B), but rsbU restoration could enhance the emergence frequency supporting a role for this alternative sigma factor in promoting glycopeptide resistance. Transcriptional analysis of vraS(H156A) strains revealed a pronounced reduction but not complete abrogation of the vraRS operon after exposure to cell wall-active antibiotics, suggesting that additional factors independent of VraS-driven phosphotransfer, or σ(B), exist for this promoter. Collectively, our results reveal important details of the VraRS signaling system and predict that pharmacologic blockade of the VraS sensor kinase will have profound effects on blocking emergence of cell wall-active antibiotic resistance in S. aureus.

Multiple presentation of Scfv800E6 on silica nanospheres enhances targeting efficiency toward HER-2 receptor in breast cancer cells.

Spherical silica nanoparticles (SNP) have been synthesized and functionalized with anti-HER-2 scFv800E6 antibody by both localized histidine-tag recognition, leading to an oriented protein ligation, and glutaraldehyde cross-linking, exploiting a statistical reactivity of lysine amine groups in the primary sequence of the molecule. The targeting efficiency of nanocomplexes in comparison with free scFv was evaluated by flow cytometry using a HER-2 antigen-positive MCF-7 breast cancer cell line, exhibiting a 4-fold increase in scFv binding efficacy, close to the affinity of intact anti-HER-2 monoclonal antibody, which suggests the effectiveness of presenting multiple scFv molecules on nanoparticles in improving antigen recognition. Unexpectedly, the conjugation method did not affect the binding efficacy of scFv, suggesting a structural role of lysines in the scFv molecule. Confocal laser scanning microscopy confirmed the binding of nanocomplexes to HER-2 and also provided evidence of their localization at the cell surface.