HER2-specific liposomes loaded with proteinaceous BRET pair as a promising tool for targeted self-excited photodynamic therapy.

Photodynamic therapy (PDT) for deep-seated tumors is still challenging due to the limited penetration of visible light through tissues. To resolve this limitation, systems based on bioluminescence resonance energy transfer (BRET), that do not require an external light source are proposed. Herein, for BRET-activated PDT we developed proteinaceous BRET-pair consisting of luciferase NanoLuc, which acts as energy donor upon addition of luciferase specific substrate furimazine, and phototoxic protein SOPP3 as a photosensitizer. We have shown that hybrid protein NanoLuc-SOPP3 is an excellent BRET pair with BRET ratio of 1.12. Targeted delivery of NanoLuc-SOPP3 BRET pair via tumor-specific small liposomes (∼100 nm) to tumors overexpressing the HER2-receptor (human epidermal growth factor receptor 2) was demonstrated in vitro and in vivo. The proposed BRET-activated system has been shown to significantly suppress tumor growth in a model of subcutaneous and, more importantly, deep-seated tumor model. Taking into account the in vivo efficiency of proposed BRET-activated system, we believe that it has great potential for depth-independent PDT and can significantly broaden the application of PDT in the clinic.

Genetically encoded BRET-activated photodynamic therapy for the treatment of deep-seated tumors.

Photodynamic therapy (PDT) is one of the most appealing photonic modalities for cancer treatment based on anticancer activity of light-induced photosensitizer-mediated reactive oxygen species (ROS), but a limited depth of light penetration into tissues does not make possible the treatment of deep-seated neoplasms and thus complicates its widespread clinical adoption. Here, we introduce the concept of genetically encoded bioluminescence resonance energy transfer (BRET)-activated PDT, which combines an internal light source and a photosensitizer (PS) in a single-genetic construct, which can be delivered to tumors seated at virtually unlimited depth and then triggered by the injection of a substrate to initiate their treatment. To illustrate the concept, we engineered genetic NanoLuc-miniSOG BRET pair, combining NanoLuc luciferase flashlight and phototoxic flavoprotein miniSOG, which generates ROS under luciferase-substrate injection. We prove the concept feasibility in mice bearing NanoLuc-miniSOG expressing tumor, followed by its elimination under the luciferase-substrate administration. Then, we demonstrate a targeted delivery of NanoLuc-miniSOG gene, via tumor-specific lentiviral particles, into a tumor, followed by its successful elimination, with tumor-growth inhibition (TGI) coefficient exceeding 67%, which confirms a great therapeutic potential of the proposed concept. In conclusion, this study provides proof-of-concept for deep-tissue "photodynamic" therapy without external light source that can be considered as an alternative for traditional PDT.

DARPin_9-29-Targeted Gold Nanorods Selectively Suppress HER2-Positive Tumor Growth in Mice.

Near-infrared phototherapy has great therapeutic potential for cancer treatment. However, for efficient application, in vivo photothermal agents should demonstrate excellent stability in blood and targeted delivery to pathological tissue. Here, we demonstrated that stable bovine serum albumin-coated gold mini nanorods conjugated to a HER2-specific designed ankyrin repeat protein, DARPin_9-29, selectively accumulate in HER2-positive xenograft tumors in mice and lead to a strong reduction in the tumor size when being illuminated with near-infrared light. The results pave the way for the development of novel DARPin-based targeted photothermal therapy of cancer.

Comparative Evaluation of Engineered Polypeptide Scaffolds in HER2-Targeting Magnetic Nanocarrier Delivery.

Targeted drug delivery is one of the most intriguing and challenging issues in modern biomedicine. For active targeting, full-size IgG molecules (150 kDa) are usually used. Recent studies have revealed that small artificial polypeptide scaffolds such as DARPins (14 kDa) and affibodies (8 kDa) are much more promising tools for drug delivery due to their small size, artificial nature, low immunogenicity, and many other properties. However, there is no comparative information on the targeting abilities of scaffold polypeptides, which should be taken into account when developing drug delivery systems (DDSs). The present work is the first comprehensive study on the comparison of the effectiveness of different HER2-targeting proteins within the architecture of nanoparticles. Namely, we synthesized trimodal nanoparticles: magnetic, fluorescent, and directed toward HER2 oncomarker on cancer cells. The magnetic particles (MPs) were covalently modified with (i) full-size IgG, 150 kDa, (ii) DARPin_G3, 14 kDa, and (iii) affibody ZHER2:342, 8 kDa. We showed that the number of DARPin_G3 and affibody ZHER2:342 molecules conjugated to the nanoparticle surface are 10 and 40 times higher, respectively, than the corresponding value for trastuzumab. Using the methods of magnetic particle quantification (MPQ)-cytometry and confocal microscopy, we showed that all types of the obtained magnetic conjugates specifically labeled HER2-overexpressing cells. Namely, we demonstrated that particle binding to HER2-positive cells is 1113 ± 39 fg/cell for MP*trastuzumab, 1431 ± 186 fg/cell for MP*ZHER2:342, and 625±21 fg/cell for MP*DARPin_G3, which are 2.77, 2.75, and 2.30 times higher than the corresponding values for control HER2-negative cells. Thus, we showed that the smallest HER2-recognizing polypeptide affibody ZHER2:342 is more effective in terms of specificity and selectivity in nanoparticle-mediated cell labeling.

Dual Regioselective Targeting the Same Receptor in Nanoparticle-Mediated Combination Immuno/Chemotherapy for Enhanced Image-Guided Cancer Treatment.

When combined with immunotherapy, image-guided targeted delivery of chemotherapeutic agents is a promising direction for combination cancer theranostics, but this approach has so far produced only limited success due to a lack of molecular targets on the cell surface and low therapeutic index of conventional chemotherapy drugs. Here, we demonstrate a synergistic strategy of combination immuno/chemotherapy in conditions of dual regioselective targeting, implying vectoring of two distinct binding sites of a single oncomarker (here, HER2) with theranostic compounds having a different mechanism of action. We use: (i) PLGA nanoformulation, loaded with an imaging diagnostic fluorescent dye (Nile Red) and a chemotherapeutic drug (doxorubicin), and functionalized with affibody ZHER2:342 (8 kDa); (ii) bifunctional genetically engineered DARP-LoPE (42 kDa) immunotoxin comprising of a low-immunogenic modification of therapeutic Pseudomonas exotoxin A (LoPE) and a scaffold targeting protein, DARPin9.29 (14 kDa). According to the proposed strategy, the first chemotherapeutic nanoagent is targeted by the affibody to subdomain III and IV of HER2 with 60-fold specificity compared with nontargeted particles, while the second immunotoxin is effectively targeted by DARPin molecule to subdomain I of HER2. We demonstrate that this dual targeting strategy can enhance anticancer therapy of HER2-positive cells with a very strong synergy, which made possible 1000-fold decrease of effective drug concentration in vitro and a significant enhancement of HER2 cancer therapy compared to monotherapy in vivo. Moreover, this therapeutic combination prevented the appearance of secondary tumor nodes. Thus, the suggested synergistic strategy utilizing dual targeting of the same oncomarker could give rise to efficient methods for aggressive tumors treatment.

Genetically encoded immunophotosensitizer 4D5scFv-miniSOG is a highly selective agent for targeted photokilling of tumor cells in vitro.

Tumor-targeted delivery of cytotoxins presents considerable advantages over their passive transport. Chemical conjugation of cytotoxic module to antibody is limited due to insufficient reproducibility of synthesis, and recombinant immunotoxins are aimed to overcome this disadvantage. We obtained genetically encoded immunophotosensitizer 4D5scFv-miniSOG and evaluated its photocytotoxic effect in vitro. A single-chain variable fragment (scFv) of humanized 4D5 antibody was used as a targeting vehicle for selective recognition of the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) overexpressed in many human carcinomas. As a phototoxic module we used a recently described photoactivated fluorescent flavoprotein miniSOG. We found that recombinant protein 4D5scFv-miniSOG exerts a highly specific photo-induced cytotoxic effect on HER2/neu-positive human breast adenocarcinoma SK-BR-3 cells (IC50= 160 nM). We demonstrated that the 4D5scFv-miniSOG specifically binds to HER2-positive cells and internalizes via receptor-mediated endocytosis. Co-treatment of breast cancer cells with 4D5scFv-miniSOG and Taxol or junction opener protein JO-1 produced remarkable additive effects.

Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III.

RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner.