A nanoparticle-based molecular beacon for directly detecting attomolar small RNA from plasma without purification.

Molecular beacons (MBs) are DNA-based probes that detect DNA or RNA fragments and hold promise for monitoring diseases and studying protein-nucleic acid interactions. MBs usually use fluorescent molecules as fluorophores for reporting the target detection event. However, the fluorescence of the traditional fluorescent molecules can bleach and even be interfered with the background autofluorescence, reducing the detection performance. Hence, we propose to develop a nanoparticle-based MB (NPMB) that uses upconversion nanoparticles (UCNPs) as a fluorophore, which can be excited by near-infrared light to avoid background autofluorescence and thus enables us to detect small RNA from complicated clinical samples such as plasma. Specifically, we employ a DNA hairpin structure, with one segment complementary to the target RNA, to position a quencher (gold nanoparticles, Au NPs) and the UCNP fluorophore in close proximity, leading to the quenching of the fluorescence of UCNPs in the absence of a target nucleic acid. Only when the hairpin structure is complementary with the detection target, will the hairpin structure be destroyed to separate Au NPs and UCNPs, resulting in the instant recovery of the fluorescence signal of UCNPs and the consequent ultrasensitive detection of the target concentrations. The NPMB has an ultra-low background signal because UCNPs can be excited with NIR light with a wavelength longer than the emitted visible light. We demonstrate that the NPMB can successfully detect a small (22-nt) RNA (using a microRNA cancer biomarker, miR-21, as an example) and a small single-stranded DNA (complementing the cDNA of miR-21) in aqueous solutions from 1 aM to 1 pM, with the linear detection range being 10 aM to 1 pM for the former and 1 aM to 100 fM for the latter. We further show that the NPMB can be used to detect unpurified small RNA (miR-21) in clinical samples such as plasma with the same detection region. Our work suggests that the NPMB is a promising label-free and purification-free method for detecting small nucleic acid biomarkers in clinical samples with a detection limit as low as the aM level.

Selectively Suppressing Tumor Angiogenesis for Targeted Breast Cancer Therapy by Genetically Engineered Phage.

Antiangiogenesis is a promising approach to cancer therapy but is limited by the lack of tumor-homing capability of the current antiangiogenic agents. Angiogenin, a protein overexpressed and secreted by tumors to trigger angiogenesis for their growth, has never been explored as an antiangiogenic target in cancer therapy. Here it is shown that filamentous fd phage, as a biomolecular biocompatible nanofiber, can be engineered to become capable of first homing to orthotopic breast tumors and then capturing angiogenin to prevent tumor angiogenesis, resulting in targeted cancer therapy without side effects. The phage is genetically engineered to display many copies of an identified angiogenin-binding peptide on its side wall and multiple copies of a breast-tumor-homing peptide at its tip. Since the tumor-homing peptide can be discovered and customized virtually toward any specific cancer by phage display, the angiogenin-binding phages are thus universal "plug-and-play" tumor-homing cancer therapeutics.

Guiding nanomaterials to tumors for breast cancer precision medicine: from tumor-targeting small-molecule discovery to targeted nanodrug delivery.

Precision medicine emphasizes patient-specific formulation for treatment of diseases, especially cancer. However, in targeted cancer treatment, because the expression level of tumor receptors in each patient varies even for the same type of cancer, the ligand/receptor-mediated approach does not seem promising for precision medicine. In this work, we demonstrated our strategy of using a phage display technique for breast cancer precision medicine. Using in vivo biopanning, we first selected an MCF-7 breast tumor-targeting peptide, then tested the effectiveness of the as-selected peptide in tumor homing and finally conjugated the peptide to a model photothermal drug, namely, gold nanorods, to achieve enhanced cancer killing efficacy. The peptides identified by the phage display technique can guide the drug to the tumors without the need to know the exact receptors on the tumor. This approach requires significantly less effort to explore patient-specific targeting molecules for precision medicine.

Tuning photothermal properties of gold nanodendrites for in vivo cancer therapy within a wide near infrared range by simply controlling their degree of branching.

Although dendritic nanoparticles have been prepared by many different methods, control over their degree of branching (DB) is still impossible, preventing us from understanding the effect of the DB on the properties of the nanodendrites as cancer therapeutics. Herein, we developed a novel seed-mediated method to prepare gold nanodendrites (AuNDs) in an organic solvent using long chain amines as a structural directing agent. We discovered that the DB could be tuned facilely by simply adjusting synthetic parameters, such as the solvent type, the type and concentration of the long chain amines. We found that DB tuning resulted in dramatic tunability in the optical properties in the near infrared (NIR) range, which led to significantly different performance in the photothermal cancer therapy. Our in vitro and in vivo studies revealed that AuNDs with a higher DB were more efficient in photothermal tumor destruction under a lower wavelength NIR irradiation. In contrast, those with a lower DB performed better in tumor destruction under a higher wavelength NIR irradiation, indicating that AuNDs of even lower DB should have even better photothermal cancer therapy efficiency within the second NIR window. Thus, the tunable optical properties of AuNDs in the NIR range allow us to selectively determine a suitable laser wavelength for the best cancer therapeutic performance.

Stem cells loaded with nanoparticles as a drug carrier for in vivo breast cancer therapy.

A novel anti-cancer drug carrier, mesenchymal stem cells (MSCs) encapsulating drug-loaded hollow silica nanoparticles, is used to carry a photosensitizer drug and deliver it to breast tumors, due to the natural high tumor affinity of the MSCs, and inhibit tumor growth by photo dynamic therapy. This new strategy for delivering a photo sensitizer to tumors by using tumor-affinitive MSCs addresses the challenge of the accumulation of photosensitizer drugs in tumors in photodynamic therapy.

Silica-based branched hollow microfibers as a biomimetic extracellular matrix for promoting tumor cell growth in vitro and in vivo.

A novel scaffold composed of loosely branched hollow silica microfibers that has been proven to be highly biocompatible is proposed for the 3D culture of cancer cells. The MCF-7 cancer cells can grow and proliferate freely inside the scaffold in the form of multicellular spheroids. MCF-7 cancer cells cultured on the current 3D silica scaffold retained significantly more oncological characters than those cultured on the conventional 2D substrate and can serve as in vitro tumor model for studying cancer treatment.

Bacteriophage bionanowire as a carrier for both cancer-targeting peptides and photosensitizers and its use in selective cancer cell killing by photodynamic therapy.

A photosensitizer, pyropheophorbid-a (PPa), is conjugated to SKBR-3 breast cancer cell-specific biological nanowire phage, to form a novel PPa-phage complex, which is further successfully used in selectively killing SKBR-3 breast cancer cells by the mechanism of photodynamic therapy (PDT).

Morphology-controlled synthesis of silica nanotubes through pH- and sequence-responsive morphological change of bacterial flagellar biotemplates.

Bacterial flagella are naturally-occurring self-assembling protein nanofibers protruding from the bacterial surface to assist the swimming of bacteria. They are rigid and exhibit diverse morphologies depending on the ionic strength, the pH values, temperature, and subunit sequences. Here, the silica nanotubes (SNTs) with controllable morphologies were synthesized using flagella as biological templates in aqueous solution under mild conditions. The morphologies and surface features of flagella-templated SNTs can be simply tuned by adjusting the pH value or surface chemistry of flagella by peptide display. A variety of different morphologies (coiled, straight, and curly with different wavelengths) and surface features (smooth, rough, granular and pear-necklace-like) of SNTs were obtained. When pH varies from acidic to alkaline conditions, in general, SNTs varied from bundled coiled, to characteristic sinusoidal waves, helical, and straight morphology. Under genetic control, flagella displaying negatively-charged peptides exhibited thinner layer of silica condensation but rough surface. However, flagella with positively-charged peptide inserts induced the deposition of thicker silica shell with smooth surface. Incorporation of hydroxyl bearing amino acid residues such as Ser into the peptide displayed on flagella highly enhanced the biotemplated deposition of silica. This work suggests that bacterial flagella are promising biotemplates for developing an environmentally-benign and cost-efficient approach to morphology-controlled synthesis of nanotubes. Moreover, the dependency of the thickness of the silica shell on the peptides displayed on flagella helps us to further understand the mechanism of biomimetic nucleation of silica on biological templates.

Detection of serum anti-P53 antibodies from patients with colorectal cancer in China using a combination of P53- and phage-ELISA: correlation to clinical parameters.

BACKGROUND: Colorectal cancer is one of the most common malignant tumors in China. The aims of this research were to increase the sensitivity of anti-p53 antibody detection in the sera of patients with colorectal cancer and to assist in their diagnosis. METHODS: Sixty-seven non-selected Chinese with colorectal cancer were involved in this study. Anti-p53 antibodies in serum were detected by ELISA using recombinant human wild- type p53 protein and hybrid phage as the coating antigen. Correlations between the anti-p53 antibodies and clinicopathological parameters were also analyzed. RESULTS: The detection efficiency of anti-p53 antibodies in the patients with colorectal cancer was increased (46.3%, 31/67) through the combination of the two ELISA methods compared with each method alone. The titer of serum anti-p53 antibodies was not associated with clinicopathological parameters, but there was a significant correlation between their presence, the CEA level, and the stage of the patient's colorectal cancer. CONCLUSIONS: These results demonstrate that combination of the two ELISA methods increased the detection rate of anti-p53 antibodies in patients with colorectal cancer. This research may provide a useful method to complement conventional clinical diagnosis.

[Isolation and purification of flavones from Murraya exotica L. by high-speed counter-current chromatography].

High-speed counter-current chromatography (HSCCC) was used to isolate and purify flavones from Murraya exotica L. The optimum separation conditions were as follows: A two-phase solvent system was petroleum ether-ethyl acetate-methanol-water (5:5:4.8:5, v/v/ v/v). The lower phase as the mobile phase was operated at a flow rate of 2.0 mL/min, while the apparatus rotated at 800 r/min. Each time 200 mg of the sample was loaded. Under these conditions, 54.31 mg of recrystallized 5,7,3',4',5'-pentamethoxyflavone, 107.68 mg of 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 215.54 mg of 5-hydroxy-6,7, 8,3', 4'-pentamethoxyflavone, and 84.36 mg of 5-hydroxy-6,7,8,3',4',5 '-hexamethoxyflavone with their purities over 95% were successfully obtained from 4.0 g of the crude extract of Murraya exotica L. The four compounds were analyzed by high performance liquid chromatography (HPLC), and identified by mass spectrometry (MS), ' H-nuclear magnetic resonance (NMR) and 13C-NMR. The compound 5-hydroxy-6,7,3',4'-tetramethoxyflavone was for the first time isolated and purified from Murraya exotica L.