High-Efficiency Excitation Energy Transfer in Biohybrid Quantum Dot-Bacterial Reaction Center Nanoconjugates.

Reaction centers (RCs) are the pivotal component of natural photosystems, converting solar energy into the potential difference between separated electrons and holes that is used to power much of biology. RCs from anoxygenic purple photosynthetic bacteria such as Rhodobacter sphaeroides only weakly absorb much of the visible region of the solar spectrum, which limits their overall light-harvesting capacity. For in vitro applications such as biohybrid photodevices, this deficiency can be addressed by effectively coupling RCs with synthetic light-harvesting materials. Here, we studied the time scale and efficiency of Förster resonance energy transfer (FRET) in a nanoconjugate assembled from a synthetic quantum dot (QD) antenna and a tailored RC engineered to be fluorescent. Time-correlated single-photon counting spectroscopy of biohybrid conjugates enabled the direct determination of FRET from QDs to attached RCs on a time scale of 26.6 ± 0.1 ns and with a high efficiency of 0.75 ± 0.01.

Genetically Encoded Stealth Nanoparticles of a Zwitterionic Polypeptide-Paclitaxel Conjugate Have a Wider Therapeutic Window than Abraxane in Multiple Tumor Models.

Small-molecule therapeutics demonstrate suboptimal pharmacokinetics and bioavailability due to their hydrophobicity and size. One way to overcome these limitations-and improve their efficacy-is to use "stealth" macromolecular carriers that evade uptake by the reticuloendothelial system. Although unstructured polypeptides are of increasing interest as macromolecular drug carriers, current recombinant polypeptides in the clinical pipeline typically lack stealth properties. We address this challenge by developing new unstructured polypeptides, called zwitterionic polypeptides (ZIPPs), that exhibit "stealth" behavior in vivo. We show that conjugating paclitaxel to a ZIPP imparts amphiphilicity to the polypeptide chain that is sufficient to drive its self-assembly into micelles. This in turn increases the half-life of paclitaxel by 17-fold compared to free paclitaxel, and by 1.6-fold compared to the nonstealth control, i.e., ELP-paclitaxel. Treatment of mice bearing highly aggressive prostate or colon cancer with a single dose of ZIPP-paclitaxel nanoparticles leads to near-complete eradication of the tumor, and these nanoparticles have a wider therapeutic window than Abraxane, an FDA-approved taxane nanoformulation.

Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy.

Splice-switching antisense oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood-brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141-150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.

DNA-gadolinium-gold nanoparticles for in vivo T1 MR imaging of transplanted human neural stem cells.

The unambiguous imaging of transplanted cells remains a major challenge to understand their biological function and therapeutic efficacy. In vivo imaging of implanted cells is reliant on tagging these to differentiate them from host tissue, such as the brain. We here characterize a gold nanoparticle conjugate that is functionalized with modified deoxythymidine oligonucleotides bearing Gd(III) chelates and a red fluorescent Cy3 moiety to visualize in vivo transplanted human neural stem cells. This DNA-Gd@Au nanoparticle (DNA-Gd@AuNP) exhibits an improved T1 relaxivity and excellent cell uptake. No significant effects of cell uptake have been found on essential cell functions. Although T1 relaxivity is attenuated within cells, it is sufficiently preserved to afford the in vivo detection of transplanted cells using an optimized voxel size. In vivo MR images were corroborated by a post-mortem histological verification of DNA-Gd@AuNPs in transplanted cells. With 70% of cells being correctly identified using the DNA-Gd-AuNPs indicates an overall reliable detection. Less than 1% of cells were false positive for DNA-Gd@AuNPs, but a significant number of 30% false negatives reveals a dramatic underestimation of transplanted cells using this approach. DNA-Gd@AuNPs therefore offer new opportunities to visualize transplanted cells unequivocally using T1 contrast and use cellular MRI as a tool to derive biologically relevant information that allows us to understand how the survival and location of implanted cells determines therapeutic efficacy.

Quantification of nanomaterial bioconjugation based on electrophoretic mobility shift.

Electrophoretic mobility shift assay (EMSA) is a well-established technique to monitor interactions between biomolecules particularly DNA and proteins. Even though numerous variations of this method have been presented, challenges in the form of detection sensitivity and/or variations in the stability of the formed complex still remain. With advances in the area of nanomaterials improvements in EMSA have been also suggested. Recently, Zhang and Wang (Electrophoresis 2015, 36, 1011-1015) presented electrophoretic mobility shift method for determination of number of DNA molecules conjugated to quantum dots (QDs), which was further utilized for calculation of enzymatic activity, sequence specific DNA detection, and neutral molecule quantification.

Development of spectrofluorimetric method for determination of certain aminoglycoside drugs in dosage forms and human plasma through condensation with ninhydrin and phenyl acetaldehyde.

A simple and sensitive spectrofluorimetric method has been developed and validated for determination of amikacin sulfate, neomycin sulfate and tobramycin in pure forms, pharmaceutical formulations and human plasma. The method was based on condensation reaction of cited drugs with ninhydrin and phenylacetaldehyde in buffered medium (pH 6) resulting in formation of fluorescent products which exhibit excitation and emission maxima at 395 and 470nm, respectively. The different experimental parameters affecting the development and stability of the reaction products were carefully studied and optimized. The calibration plots were constructed with good correlation coefficients (0.9993 for tobramycin and 0.9996 for both neomycin and amikacin). The proposed method was successfully applied for the analysis of cited drugs in dosage forms with high accuracy (98.33-101.7)±(0.80-1.26)%. The results show an excellent agreement with the reference method, indicating no significant difference in accuracy and precision. Due to its high sensitivity, the proposed method was applied successfully for determination of amikacin in real human plasma.

Size exclusion chromatography as a universal method for the purification of quantum dots bioconjugates.

The bioconjugation of fluorescent quantum dots (QDs) and purification of QDs bioconjugates are of vital importance in bioapplications. In this paper, we systematically investigated the purification efficiency of QDs bioconjugates and their stability during separation process by using size exclusion chromatography (SEC) technique, fluorescence spectroscopy, and fluorescence correlation spectroscopy (FCS). In this study, commercial QDs and fluorescein isothiocyanate (FITC) were used as labeling probes, and bovine serum albumin (BSA) and antibody (Erbitux) were used as mode samples. The covalently linkage and the electrostatic interaction were used in bioconjugation procedures. We systematically studied the effects of certain factors such as the scales of column, loading volume, elution buffer, and separation media on the purification efficiency of QDs bioconjugates by a home-built SEC device. And highly pure and monodisperse QDs bioconjugates could be obtained by SEC purification twice under the optimized conditions. Furthermore, we investigated the stability of QDs bioconjugates in different conjugation ways and purification conditions by FCS, and found that the stability of bioconjugates were poor when electrostatic binding mode was used. We also observed that Sephacryl S300 HR (separation range for globular proteins: 1 × 10(4) -1.5 × 10(6) Da) was the best choice for purifying the vast majority of QDs-bioconjugates. Our work described here will be constructive to popularization and applications of QDs in life science.

Targeted RGD nanoparticles for highly sensitive in vivo integrin receptor imaging.

A new magnetic resonance imaging (MRI) contrast bearing RGD peptide is reported. In this study, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles with various sizes were prepared. Particles sizes between 6 and 13 nm were tuned by varying the stirring rate. Remarkable negative contrast was observed because USPIO nanoparticles (13.1 ± 2.1 nm) generated high transversal relaxivity r2 (188 ± 3 m m(-1) s(-1) ) and saturation magnetization (94 emu g(-1) Fe). The USPIO nanoparticles were coated with PDA [2-(pyridyldithio)-ethylamine; PDA nanoparticles] containing functional polymer, which can be readily synthesized by Michael addition. The PDA nanoparticles were conjugated with RGD peptide (RGD nanoparticles) for targeting the specific site. The target specificity and high relaxivity allowed RGD nanoparticles to differentiate the expression level of integrin receptor on several cell lines and tumors (MCF-7, A-549, HT-29 and HT-1080) by in vitro and in vivo MR imaging. Importantly, a remarkable negative contrast (-51.3 ± 6.7%) was observed for in vivo MR imaging of MCF-7 tumor. This result implies that the RGD nanoparticles that greatly enhance the MR imaging are highly sensitive for early stage tumor detection.

Chemically induced self-assembly of enzyme nanorings.

Continued exploration into the field of chemically induced dimerization (CID) has revealed a number of applications for its use in a broader context as a method of structural assembly (1-4). In particular, the use of CID technology to generate self-assembled (and selectively disassembled) protein toroids serves as a key advancement toward developing stable and controllable protein-based platforms. Such structures have broad application to the development of novel therapeutics, lab-on-a-chip technologies, and multi-enzyme assemblies (5, 6). This chapter describes a method of developing an enzymatically active protein nanostructure incorporating both a CID-based assembly region containing dihydrofolate reductase (DHFR) and an enzymatic region consisting of histidine triad nucleotide binding protein 1 (Hint1). Details of both the production and the characterization of this structure are provided.