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Nanoparticle-based chemotherapy is considered to be an effective approach to cancer diagnostics and therapy in modern biomedicine. However, efficient tumor targeting remains a great challenge due to the lack of specificity, selectivity, and high dosage of chemotherapeutic drugs required. A two-step targeted drug delivery strategy (DDS), involving cancer cell pre-targeting, first with a first nontoxic module and subsequent targeting with a second complementary toxic module, is a solution for decreasing doses for administration and lowering systemic toxicity. To prove two-step DDS efficiency, we performed a direct comparison of one-step and two-step DDS based on chemotherapy loaded PLGA nanoparticles and barnase*barstar interface. Namely, we developed and thoroughly characterized the two-step targeting strategy of HER2-overexpressing cancer cells. The first targeting block consists of anti-HER2 scaffold polypeptide DARPin9_29 fused with barstar. Barstar exhibits an extremely effective binding to ribonuclease barnase with Kaff = 1014 M−1, thus making the barnase*barstar protein pair one of the strongest known protein*protein complexes. A therapeutic PLGA-based nanocarrier coupled to barnase was used as a second targeting block. The PLGA nanoparticles were loaded with diagnostic dye, Nile Blue, and a chemotherapeutic drug, doxorubicin. We showed that the two-step DDS increases the performance of chemotherapy-loaded nanocarriers: IC50 of doxorubicin delivered via two-step DDS was more than 100 times lower than that for one-step DDS: IC50 = 43 ± 3 nM for two-step DDS vs. IC50 = 4972 ± 1965 nM for one-step DDS. The obtained results demonstrate the significant efficiency of two-step DDS over the classical one-step one. We believe that the obtained data will significantly change the direction of research in developing targeted anti-cancer drugs and promote the creation of new generation cancer treatment strategies. [ABSTRACT FROM AUTHOR]

Innovative luminescent nanomaterials, termed upconversion nanoparticles (UCNPs), have demonstrated considerable promise as molecular probes for high-contrast optical imaging in cells and small animals. The feasibility study of optical diagnostics in humans is reported here based on experimental and theoretical modeling of optical imaging of an UCNP-labeled breast cancer lesion. UCNPs synthesized in-house were surface-capped with an amphiphilic polymer to achieve good colloidal stability in aqueous buffer solutions. The scFv4D5 mini-antibodies were grafted onto the UCNPs via a high-affinity molecular linker barstar:barnase (Bs:Bn) to allow their specific binding to the human epidermal growth factor receptor HER2/neu, which is overexpressed in human breast adenocarcinoma cells SK-BR-3. UCNP-Bs:Bn-scFv4D5 biocomplexes exhibited high-specific immobilization on the SK-BR-3 cells with the optical contrast as high as 10:1 benchmarked against a negative control cell line. Breast cancer optical diagnostics was experimentally modeled by means of epi-luminescence imaging of a monolayer of the UCNP-labeled SK-BR-3 cells buried under a breast tissue mimicking optical phantom. The experimental results were analyzed theoretically and projected to in vivo detection of early-stage breast cancer. The model predicts that the UCNP-assisted cancer detection is feasible up to 4 mm in tissue depth, showing considerable potential for diagnostic and image-guided surgery applications. [ABSTRACT FROM AUTHOR]

Semiconductor quantum dots represent a novel class of fluorophores with unique physical and chemical properties which could enable a remarkable broadening of the current applications of fluorescent imaging and optical diagnostics. Complexes of quantum dots and antibodies are promising visualising agents for fluorescent detection of selective biomarkers overexpressed in tumor tissues. Here we describe the construction of self-assembling fluorescent complexes of quantum dots and anti-HER1 or anti-HER2/neu scFv antibodies and their interactions with cultured tumor cells. A binding strategy based on a very specific non-covalent interaction between two proteins, barnase and barstar, was used to connect quantum dots and the targeting antibodies. Such a strategy allows combining the targeting and visualization functions simply by varying the corresponding modules of the fluorescent complex. [ABSTRACT FROM AUTHOR]

A bioengineering method for self-assembly of multifunctional superstructures with in-advance programmable properties has been proposed. The method employs two unique proteins, barnase and barstar, to rapidly join the structural components together directly in water solutions. The properties of the superstructures can be designed on demand by linking different agents of various sizes and chemical nature, designated for specific goals. As a proof of concept, colloidally stable trifunctional structures have been assembled by binding together magnetic particles, quantum dots, and antibodies using barnase and barstar. The assembly has demonstrated that the bonds between these proteins are strong enough to hold macroscopic (5 nm-3 μm) particles together. Specific interaction of such superstructures with cancer cells resulted in fluorescent labeling of the cells and their responsiveness to magnetic field. The method can be used to join inorganic moieties, organic particles, and single biomolecules for synergistic use in different applications such as biosensors, photonics, and nanomedicine. [ABSTRACT FROM AUTHOR]

Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185HER-2-ECD antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p1852HER-2-ECD-expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach. [ABSTRACT FROM AUTHOR]

Background: RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells. Methodology/Principal Findings: In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC50) ranging from 0.2 to 13 μM and to leukemia cell lines with IC50 values ranging from 2.4 to 82 μM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC50 = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3. Conclusions/Significance: These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells. [ABSTRACT FROM AUTHOR]

Theranostic approach is currently among the fastest growing trends in cancer treatment. It implies the creation of multifunctional agents for simultaneous precise diagnosis and targeted impact on tumor cells. A new type of theranostic complexes was created based on NaYF4: Yb,Tm upconversion nanoparticles coated with polyethylene glycol and functionalized with the HER2-specific recombinant targeted toxin DARPin-LoPE. The obtained agents bind to HER2-overexpressing human breast adenocarcinoma cells and demonstrate selective cytotoxicity against this type of cancer cells. Using fluorescent human breast adenocarcinoma xenograft models, the possibility of intravital visualization of the UCNP-based complexes biodistribution and accumulation in tumor was demonstrated. [ABSTRACT FROM AUTHOR]

Highlights • Method of magnetite particles stabilization and functionalization was developed. • Barnase ∗ Barstar interface makes the method universal in terms of self-assembly. • Fusion protein of C-terminal part of Mms6 and Barstar stabilized particles in PBS. • Targeted delivery of modified particles to HER2/neu+ cancer cells was realized. Abstract Surface modification of nanoparticles with various biologically active molecules makes it possible to realize the huge biomedical potential of various nanoobjects. Functionally active particles are usually obtained through stabilization by polymers and subsequent chemical conjugation with biomolecules, such as, antibodies or aptamers. This entails a number of problems, such as non-oriented conjugation, low coupling yield and the inability to easily vary the attached components on demand. To solve this problem, we developed a novel method of magnetite nanoparticles stabilization with simultaneous modification by functionally active protein – Barstar, for subsequent self-assembly of the necessary components with these particles through the interaction of high-affinity protein pair Barnase ∗ Barstar (K aff = 1014 M−1). Namely, we developed a biocompatible Bs-C-Mms6 fusion protein containing the C-terminal part of the Mms6 (magnetite-binding protein of magnetotactic bacteria) and Barstar (an inhibitor of bacterial ribonuclease Barnase). We obtained stable in PBS magnetite nanoparticles modified with Bs-C-Mms6. These particles can be used for self-assembly with any type of Barnase-containing molecules. To demonstrate the effectiveness of this approach for the development of targeted nanoparticles, we performed a self-assembly of these particles with a fusion protein of Barnase and DARPin9.29, namely DARPin9.29-Bn. DARPin9.29 recognizes the extracellular domain of clinically important HER2/neu oncomarker. We have shown that such particles selectively bind this oncomarker and can be used to detect HER2/neu-positive cancer cells for diagnostic purposes. [ABSTRACT FROM AUTHOR]

Abstract: Overexpression of the P185HER2 protein determines the malignancy and unfavorable prognosis of ovarian and breast tumors. In this work, the distribution of P185HER2 in human cancer cells was studied by electron microscopy, using a novel approach. It is based on the interaction between barnase (a ribonuclease from Bacillus amyloliquefaciens) and its specific inhibitor barstar. The monoclonal antibody 4D5 scFv to extracellular P185HER2 domain fused with two molecules of barnase was used as a recognizing agent, and the conjugate of colloidal gold with barstar, as an electron dense label for electron microscopic visualization. For labeling, we used supramolecular complexes 4D5 scFv-dibarnase:barstar-Au. The distribution of P185HER2 in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was studied at 4 °C and 37 °C. It was shown that at 4 °C the protein P185HER2 occurs exclusively on the cell surface, mainly on protrusions or close to their bases. At 37 °C, the internalization of P185HER2 caused by its interaction with 4D5 scFv-dibarnase was observed. Inside the cells, P185HER2 was located in the coated pits and vesicles, endosomes and multivesicular bodies. The data obtained indicate that the supramolecular 4D5 scFv-dibarnase:barstar–gold complex can be used as a new immunodetection system for exploring the P185HER2 distribution. [Copyright &y& Elsevier]