Galactose residues could be specifically recognized by the asialoglycoprotein receptor (ASGPR) which is highly exhibited on liver tissues. However, ASGPR has not been widely investigated on different tumor cell lines except for hepatoma carcinoma cells, which motivates us to investigate the possibility of galactose serving as a board tumor ligand. In this study, a galactose (Gal)-based probe conjugated with fluorescence dye MPA (Gal-MPA) was constructed for the evaluation of tumor affinities/targeted ability on different tumor cell lines. In the vitro cell study, it was indicated that the fluorescence probe Gal-MPA displayed higher cell affinity to tumor cells (HepG2, MCF-7 and A549) than that of the normal liver cells l02. In the vivo dynamic study of Gal-MPA in tumor-bearing mice (HepG2, MCF-7, A549, HCT116, U87, MDA-MB-231 and S180), it was shown that its high tumor targeted ability with the maximal tumor/normal tissue ratio reached up to 6.8. Meanwhile, the fast tumor-targeted ability within 2 hours and long retention on tumor site up to 120 hours were observed. Our results demonstrated that galactose should be a promising broad ligand for multiple tumor imaging and targeted therapy. Subsequently, Gal was covalently conjugated to doxorubicin (DOX) to form prodrug Gal-DOX for tumor targeted therapy. The therapeutic results of Gal-DOX than DOX being better suggested that galactosylated prodrugs might have the prospective potential in tumor targeted therapy.

Targeting tumors using miniature antibodies is a novel and attractive therapeutic approach, as these biomolecules exhibit low immunogenicity, rapid clearance, and high targeting specificity. However, most of the small-sized antibodies in existence do not exhibit marked anti-tumor effects, which limit their use in targeted cancer immunotherapy. To overcome this difficulty in targeting multiple biomarkers by combination therapies, we designed a new bifunctional antibody, named MaAbNA (multivalent antibody comprised of nanobody and affibody moieties), capable of targeting EGFR1 and HER2, which are widely overexpressed in a variety of tumor types. The small-sized (29 kDa) MaAbNA, which was expressed in E.coli, consists of one anti-EGFR1 nanobody and two anti-HER2 affibodies, and possesses high affinity (KD) for EGFR1 (~4.1 nM) and HER2 (~4.7 nM). In order to enhance its anti-tumor activity, MaAbNA was conjugated with adriamycin (ADM) using a PEG2000 linker, forming a new complex anticancer drug, MaAbNA-PEG2000-ADM. MaAbNA exhibited high inhibitory effects on tumor cells over-expressing both EGFR1 and HER2, but displayed minimal cytotoxicity in cells expressing low levels of EGFR1 and HER2. Moreover, MaAbNA-PEG2000-ADM displayed increased tumoricidal effects than ADM or MaAbNA alone, as well exhibited greater antitumor efficacy than EGFR1 (Cetuximab) and HER2 (Herceptin) antibody drugs. The ability of MaAbNA to regulate expression of downstream oncogenes c-jun, c-fos, c-myc, as well as AEG-1 for therapeutic potential was evaluated by qPCR and western-blot analyses. The antitumor efficacy of MaAbNA and its derivative MaAbNA-PEG2000-ADM were validated in vivo, highlighting the potential for use of MaAbNA as a highly tumor-specific dual molecular imaging probe and targeted cancer therapeutic.

Potato late blight, caused by Phytophthora infestans (Mont.) de Bary, represents a great food security threat worldwide and is difficult to control. Recently, Bacillus spp. have been considered biocontrol agents to control many plant diseases. Here, Bacillus subtilis WL-2 was selected as a potent strain against P. infestans mycelium growth, and its functional metabolite was identified as Iturin A via electrospray ionization mass spectrometry (ESI-MS). Analyses using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that Iturin A caused cell membrane disruption and an irregular internal cell structure. In addition, Iturin A triggered oxidative stress reactions similarly to reactive oxygen species (ROS) in P. infestans cells and caused mitochondrial damage, including mitochondrial membrane potential (MMP), mitochondrial respiratory chain complex activity (MRCCA), and ATP production decline. These results highlight that the cell structure disruption, oxidative stress, and energy supply dysfunction induced by Iturin A play an important role in inhibiting P. infestans. Additionally, B. subtilis WL-2 and Iturin A have great potential for inhibiting P. infestans mycelium growth and controlling potato late blight in the future.

Oomycete Phytophthora infestans [(Mont.) de Bary] is the cause of potato late blight, a plant disease which poses a serious threat to our global food security and is responsible for huge economic losses worldwide. Lipopeptides produced by Bacillus species are known to be potent antibacterial compounds against many plant pathogens. In this study, we show that Bacillus megaterium WL-3 has an antagonistic effect against potato late blight. Electrospray ionization mass spectrometry (ESI-MS) revealed that lipopeptides derived from the WL-3 strain contained three subfamilies, surfactin (C13 - C15), Iturin A (C14 - C16), and Fengycin A (C15 - C19). The Iturin A and Fengycin A lipopeptide families were each confirmed to have anti-oomycete effects against P. infestans mycelium growth as well as obvious controlling effects against potato late blight in greenhouse experiments and field assays. Furthermore, Iturin A and Fengycin A were able to promote plant photosynthetic efficiency, plant growth, and potato yield. Most importantly, the combination of Iturin A and Fengycin A (I + F) was superior to individual lipopeptides in controlling potato late blight and in the promotion of plant growth. The results of this study indicate that B. megaterium WL-3 and its lipopeptides are potential candidates for the control of late blight and the promotion of potato plant growth.

Development of alternative linear peptides for targeting αvβ3 integrin has attracted much attention, as the traditional peptide ligand, cyclic RGD, is limited by inferior water-solubility and complex synthesis. Using pharmacophore-based virtual screening and high-throughput molecular docking, we identified two novel linear small peptides RWr and RWrNM with high affinity and specificity to αvβ3 integrin. The competitive binding with cyclic RGD (c(RGDyK)) and cellular uptake related to the integrin expression levels verified their affinity to αvβ3 integrin. The intermolecular interaction measurement and dynamics simulation demonstrated the high binding affinity and stability, especially for RWrNM. In vivo peptide-guided tumor imaging and targeted therapy further confirmed their specificity. Results indicated that the newly identified small linear peptide RWrNM, with high affinity and specificity to αvβ3 integrin, better water-solubility, and simplified synthetic process, could overcome limitations of the current cyclic RGD peptides, paving the way for diverse use in diagnosis and therapy.