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 NaYF
: 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.
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.
Nanoparticle surface engineering can change its chemical identity to enable surface coupling with functional biomolecules. However, common surface coupling methods such as physical adsorption or ...chemical conjugation often suffer from the low coupling yield, poorly controllable orientation of biomolecules, and steric hindrance during target binding. These issues limit the application scope of nanostructures for theranostics and personalized medicine. To address these shortfalls, we developed a rapid and versatile method of nanoparticle biomodification. The method is based on a SiO2-binding peptide that binds to the nanoparticle surface and a protein adaptor system, Barnase*Barstar protein pair, serving as a “molecular glue” between the peptide and the attached biomolecule. The biomodification procedure shortens to several minutes, preserves the orientation and functions of biomolecules, and enables control over the number and ratio of attached molecules. The capabilities of the proposed biomodification platform were demonstrated by coupling different types of nanoparticles with DARPin9.29 and 4D5scFvmolecules that recognize the human epidermal growth factor receptor 2 (HER2/neu) oncomarkerand by subsequent highly selective immunotargeting of the modified nanoparticles to different HER2/neu-overexpressing cancer cells in one-step or two-step (by pretargeting with HER2/neu-recognizing molecule) modes. The method preserved the biological activity of the DARPin9.29 molecules attached to a nanoparticle, whereas the state-of-the-art carbodiimide 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysulfosuccinimide method of conjugation led to a complete loss of the functional activity of the DARPin9.29 nanoparticle–protein complex. Moreover, the method allowed surface design of nanoparticles that selectively interacted with antigens in complex biological fluids, such as whole blood. The demonstrated capabilities show this method to be a promising alternative to commonly used chemical conjugation techniques in nanobiotechnology, theranostics, and clinical applications.
Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used ...in combination with a variety of genetic engineering manipulations allowing for precise spatio‐temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore‐assisted light inactivation (CALI) of proteins, as well as for photo‐induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini‐antibodies or selective scaffold proteins and photo‐induced cytotoxicity of miniSOG, which is particularly promising for selective non‐invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.
The review focuses on biological applications of a flavoprotein miniSOG, genetically encoded photosensitizer capable of generating singlet oxygen upon blue light irradiation. It describes applications of miniSOG in correlative light electron microscopy (CLEM) and chromophore‐assisted light inactivation (CALI) and for photo‐induced cell ablation in cell cultures and C. elegans. Recent approach of immunophotosensitizing, exploiting the specificity of mini‐antibodies and ROS generating capacity of miniSOG, is highlighted.
The targeted delivery of nanodrugs to malignant neoplasm is one of the most pressing challenges in the development of modern medicine. It was reported earlier that a bacteriorhodopsin-derived pH low ...insertion peptide (pHLIP) targets acidic tumors and has the ability to translocate low molecular weight cargoes across the cancer cell membrane. Here, to better understand the potential of pHLIP-related technologies, we used genetically engineered fluorescent protein (EGFP) as a model protein cargo and examined targeting efficiencies of EGFP-pHLIP hybrid constructs in vitro with the HeLa cell line at different pHs. By two independent monitoring methods we observed an increased binding affinity of EGFP-pHLIP fusions to HeLa cells at pH below 6.8. Confocal images of EGFP-pHLIP-treated cells showed bright fluorescence associated with the cell membrane and fluorescent dots localized inside the cell, that became brighter with time. To elucidate the pHLIP-mediated EGFP cell entry mechanisms, we performed a series of experiments with specific inhibitors of endocytosis. Our results imply that EGFP-pHLIP internalization is realized by endocytosis of various types.
•EGFP- pHLIP fusions effectively bind to HeLa cells at pHs below 6.8.•Confocal images demonstrate fluorescence associated with the cell membrane and cytoplasm.•Several types of endocytosis are responsible for the internalization of the EGFP-pHLIP constructs.
The upregulation of epithelial cell adhesion molecule (EpCAM) expression, found in a substantial fraction of renal cell carcinomas (RCCs), renders it a potential molecular target for the treatment of ...disseminated RCC. However, the heterogeneous expression of EpCAM necessitates first identifying the patients with sufficiently high expression of EpCAM in tumors. Using the specific radionuclide-based visualization of EpCAM might enable such identification. The designed ankyrin repeat protein, Ec1, is a small (molecular weight, 18 kDa) targeting protein with a subnanomolar affinity to EpCAM. Using a modified Ec1, a tracer was developed for the radionuclide-based visualization of EpCAM in vivo, i.e., an EpCAM-visualizing designed ankyrin repeat protein (EVD). EVD was labelled with either technetium-99m using technetium tricarbonyl or with iodine-125 (as a surrogate for iodine-123) by coupling it to para-125Iiodobenzoyl (125IPIB) groups. Both the 125I-labelled EVD (125I-EVD) and 99mTc-labelled EVD (99mTc-EVD) bound specifically to EpCAM-expressing SK-RC-52 renal carcinoma cells. The binding affinity (KD value) of 99mTc-EVD to SK-RC-52 cells was 400±28 pM. The tracers' uptake in SK-RC-52 ×enografts at 3 h after injection was 5.2±1.4%ID/g for 125I-EVD and 6.0±1.4%ID/g for 99mTc-EVD (no significant difference). These uptake values in SK-RC-52 ×enografts were significantly higher (P<0.001) than those in Ramos lymphoma xenografts (used as EpCAM-negative control). The tumor-to-blood uptake ratio was significantly higher for 99mTc-EVD (25±6) compared with that of 125I-EVD (14±3). However, 125I-EVD was associated with higher tumor-to-liver, tumor-to-salivary gland, tumor-to-spleen and tumor-to-intestinal wall ratios. This makes it the preferable tracer for visualizing EpCAM expression levels in the frequently occurring abdominal metastases of RCC.
Despite an enhanced permeability and retention effect typical of many solid tumors, drug penetration is not always sufficient. Possible strategies for the drug delivery improvement are a modification ...of the tumor cell-to-cell junctions and usage of cell membrane permeabilization proteins. In this review we discuss epithelial cell junctions as targets for a combined anticancer therapy and propose new possible sources of such agents. We suggest considering viral and bacterial pathogens disrupting epithelial layers as plentiful sources of new therapeutic agents for increasing tumor permeability for other effector agents. We also observe the application of pore forming proteins and peptides of different origin for cytoplasmic delivery of anti-cancer agents and consider the main obstacles of their use in vivo.
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Proton therapy is one of the promising radiotherapy modalities for the treatment of deep-seated and unresectable tumors, and its efficiency can further be enhanced by using boron-containing ...substances. Here, we explore the use of elemental boron (B) nanoparticles (NPs) as sensitizers for proton therapy enhancement. Prepared by methods of pulsed laser ablation in water, the used B NPs had a mean size of 50 nm, while a subsequent functionalization of the NPs by polyethylene glycol improved their colloidal stability in buffers. Laser-synthesized B NPs were efficiently absorbed by MNNG/Hos human osteosarcoma cells and did not demonstrate any remarkable toxicity effects up to concentrations of 100 ppm, as followed from the results of the MTT and clonogenic assay tests. Then, we assessed the efficiency of B NPs as sensitizers of cancer cell death under irradiation by a 160.5 MeV proton beam. The irradiation of MNNG/Hos cells at a dose of 3 Gy in the presence of 80 and 100 ppm of B NPs led to a 2- and 2.7-fold decrease in the number of formed cell colonies compared to control samples irradiated in the absence of NPs. The obtained data unambiguously evidenced the effect of a strong proton therapy enhancement mediated by B NPs. We also found that the proton beam irradiation of B NPs leads to the generation of reactive oxygen species (ROS), which evidences a possible involvement of the non-nuclear mechanism of cancer cell death related to oxidative stress. Offering a series of advantages, including a passive targeting option and the possibility of additional theranostic functionalities based on the intrinsic properties of B NPs (e.g., photothermal therapy or neutron boron capture therapy), the proposed concept promises a major advancement in proton beam-based cancer treatment.
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.
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.
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 (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. 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 (IC(50) = 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.
These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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