Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers ...have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.
MicroRNAs (miRNAs) are noncoding RNAs that provide an endogenous negative feedback mechanism for translation of messenger RNA (mRNA) into protein. Single miRNAs can regulate hundreds of mRNAs, ...enabling miRNAs to orchestrate robust biological responses by simultaneously impacting multiple gene networks. MiRNAs can act as master regulators of normal and pathological tissue development, homeostasis, and repair, which has motivated expanding efforts toward the development of technologies for therapeutically modulating miRNA activity for regenerative medicine and tissue engineering applications. This review highlights the tools currently available for miRNA inhibition and their recent therapeutic applications for improving tissue repair.
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This article presents a critical evaluation of silver nanorod arrays as substrates for assaying nucleic acid hybridization by surface enhanced Raman scattering (SERS). SERS spectra acquired on ...complementary oligos, alone or in combination, contain the known spectral signatures of the nucleotides that comprise the oligo; however, no signature bands characteristic of the hybrid were observed. Spectra acquired on an oligo with a 5′- or 3′-thiol were distinctly different from that acquired on the identical oligo without a thiol pendant group suggesting a degree of control over the orientation of the oligo on the nanorod surface. A set of oligos consisting of adenine tracts in a polycytosine chain served as molecular rulers to probe the distance dependence of the SERS enhancement. Using these, we have identified the point at which the characteristic bands for the nucleotides that comprise the oligo disappear from the spectrum. These findings suggest that the applicability of SERS for label-free detection of nucleic acid hybridization is limited to short oligos of less than nine nucleotides.
Self‐assembled polymer/porous silicon nanocomposites overcome intracellular and systemic barriers for in vivo application of peptide nucleic acid (PNA) anti‐microRNA therapeutics. Porous silicon ...(PSi) is leveraged as a biodegradable scaffold with high drug‐cargo‐loading capacity. Functionalization with a diblock polymer improves PSi nanoparticle colloidal stability, in vivo pharmacokinetics, and intracellular bioavailability through endosomal escape, enabling PNA to inhibit miR‐122 in vivo.
This work examines the influence of charge density and surface passivation on the DNA-induced corrosion of porous silicon (PSi) waveguides in order to improve PSi biosensor sensitivity, reliability, ...and reproducibility when exposed to negatively charged DNA molecules. Increasing the concentration of either DNA probes or targets enhances the corrosion process and masks binding events. While passivation of the PSi surface by oxidation and silanization is shown to diminish the corrosion rate and lead to a saturation in the changes by corrosion after about 2 h, complete mitigation can be achieved by replacing the DNA probe molecules with charge-neutral PNA probe molecules. A model to explain the DNA-induced corrosion behavior, consistent with experimental characterization of the PSi through Fourier transform infrared spectroscopy and prism coupling optical measurements, is also introduced.
Peptide nucleic acids (PNA) are a unique class of synthetic molecules that have a peptide backbone and can hybridize with nucleic acids. Here, a versatile method has been developed for the automated, ...in situ synthesis of PNA from a porous silicon (PSi) substrate for applications in gene therapy and biosensing. Nondestructive optical measurements were performed to monitor single base additions of PNA initiated from (3-aminopropyl)triethoxysilane attached to the surface of PSi films, and mass spectrometry was conducted to verify synthesis of the desired sequence. Comparison of in situ synthesis to postsynthesis surface conjugation of the full PNA molecules showed that surface mediated, in situ PNA synthesis increased loading 8-fold. For therapeutic proof-of-concept, controlled PNA release from PSi films was characterized in phosphate buffered saline, and PSi nanoparticles fabricated from PSi films containing in situ grown PNA complementary to micro-RNA (miR) 122 generated significant anti-miR activity in a Huh7 psiCHECK-miR122 cell line. The applicability of this platform for biosensing was also demonstrated using optical measurements that indicated selective hybridization of complementary DNA target molecules to PNA synthesized in situ on PSi films. These collective data confirm that we have established a novel PNA–PSi platform with broad utility in drug delivery and biosensing.
Current imaging techniques capable of tracking nanoparticles in vivo supply either a large field of view or cellular resolution, but not both. Here, we demonstrate a multimodality imaging platform of ...optical coherence tomography (OCT) techniques for high resolution, wide field of view in vivo imaging of nanoparticles. This platform includes the first in vivo images of nanoparticle pharmacokinetics acquired with photothermal OCT (PTOCT), along with overlaying images of microvascular and tissue morphology. Gold nanorods (51.8 ± 8.1 nm by 15.2 ± 3.3 nm) were intravenously injected into mice, and their accumulation into mammary tumors was non-invasively imaged in vivo in three dimensions over 24 hours using PTOCT. Spatial frequency analysis of PTOCT images indicated that gold nanorods reached peak distribution throughout the tumors by 16 hours, and remained well-dispersed up to 24 hours post-injection. In contrast, the overall accumulation of gold nanorods within the tumors peaked around 16 hours post-injection. The accumulation of gold nanorods within the tumors was validated post-mortem with multiphoton microscopy. This shows the utility of PTOCT as part of a powerful multimodality imaging platform for the development of nanomedicines and drug delivery technologies.
A versatile and scalable method for fabricating shape-engineered nano- and micrometer scale particles from mesoporous silicon (PSi) thin films is presented. This approach, based on the direct ...imprinting of porous substrates (DIPS) technique, facilitates the generation of particles with arbitrary shape, ranging in minimum dimension from approximately 100 nm to several micrometers, by carrying out high-pressure (>200 MPa) direct imprintation, followed by electrochemical etching of a sub-surface perforation layer and ultrasonication. PSi particles (PSPs) with a variety of geometries have been produced in quantities sufficient for biomedical applications (≫10 μg). Because the stamps can be reused over 150 times, this process is substantially more economical and efficient than the use of electron beam lithography and reactive ion etching for the fabrication of nanometer-scale PSPs directly. The versatility of this fabrication method is demonstrated by loading the DIPS-imprinted PSPs with a therapeutic peptide nucleic acid drug molecule, and by vapor deposition of an Au coating to facilitate the use of PSPs as a photothermal contrast agent.
Thermal Stability of Silver Nanorod Arrays Beavers, Kelsey R; Marotta, Nicole E; Bottomley, Lawrence A
Chemistry of materials,
04/2010, Volume:
22, Issue:
7
Journal Article
Peer reviewed
Silver nanorod arrays fabricated by glancing angle deposition have found application as substrates for surface enhanced Raman scattering. A critical issue in the continued development of these ...substrates is their stability over time. The thermal stability of the arrays was systematically investigated. Thermally induced changes in surface morphology were evaluated using scanning electron microscopy and X-ray diffraction and correlated with changes in SERS enhancement. The findings presented herein suggest that the shelf life of silver nanorod arrays is limited by migration of silver on the surface. This rate of migration is enhanced by the presence of a surface layer of chemisorbed oxygen.
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