Chemically Modified Dendritic Starch: A Novel Nanomaterial for siRNA Delivery

Nanostructured starches are naturally derived nanomaterials that can be chemically modified to allow for the introduction of functional groups, enhancing their potential for drug delivery and other biotechnology applications. In this proof of concept study, we investigate chemically modified, enzymatically synthesized glycogen (ESG) nanodendrites as a biodegradable, biocompatible, siRNA delivery system. Commercially available ESG was modified using glycidyltrimethylammonium chloride (GTMA), introducing quaternary ammonium groups via an epoxide ring opening reaction. This cationic ESG (cESG) electrostatically bound siRNA and successfully knocked down protein expression in an in vitro ovarian clear cell carcinoma model. The construct exhibited sustained siRNA delivery for up to 6 days while exhibiting less toxicity than a common liposome-based siRNA delivery reagent, Lipofectamine RNAiMAX. These promising results set the stage for the use of dendritic starch as a cost-effective, easily modifiable nanoscale delivery system for a diverse range of cargo including nucleic acids and therapeutic compounds

Immobilization Mechanisms of Deoxyribonucleic Acid (DNA) to Hafnium Dioxide (HfO₂) Surfaces for Biosensing Applications

Immobilization of biomolecular probes to the sensing substrate is a critical step for biosensor fabrication. In this work we investigated the phosphate-dependent, oriented immobilization of DNA to hafnium dioxide surfaces for biosensing applications. Phosphate-dependent immobilization was confirmed on a wide range of hafnium oxide surfaces; however, a second interaction mode was observed on monoclinic hafnium dioxide. On the basis of previous materials studies on these films, DNA immobilization studies, and density functional theory (DFT) modeling, we propose that this secondary interaction is between the exposed nucleobases of single stranded DNA and the surface. The lattice spacing of monoclinic hafnium dioxide matches the base-to-base pitch of DNA. Monoclinic hafnium dioxide is advantageous for nanoelectronic applications, yet because of this secondary DNA immobilization mechanism, it could impede DNA hybridization or cause nonspecific surface intereactions. Nonetheless, DNA immobilization on polycrystalline and amorphous hafnium dioxide is predominately mediated by the terminal phosphate in an oriented manner which is desirable for biosensing applications

Biomimicry Enhances Sequential Reactions of Tethered Glycolytic Enzymes, TPI and GAPDHS

<div><p>Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.</p></div

Design of recombinant proteins and verification of purified His-TPI and His-GAPDHS.

<p>a) A hexahistidine tag was introduced to modify the amino terminal, germ cell-specific domain (gcs) of sperm TPI. b) SDS-PAGE showing representative coomassie brilliant blue (CBB) protein staining and immunoblot analysis of purified His-TPI with antibodies against the His-tag (His) and the protein (TPI). c) A hexahistidine tag replaced the amino-terminal proline-rich domain (PRD) of GAPDHS. d) SDS-PAGE showing representative protein staining (CBB) and immunoblot analysis of purified His-GAPDHS with antibodies against the His-tag (His) and the protein (GAPDHS).</p

Properties of recombinant proteins.

<p>Enzyme activities in solution were determined as described. K_m values are for the substrates, glyceraldehyde 3-phosphate (GAP) for TPI and 3-phosphoglyceric acid (3-PGA) for GAPDHS.</p

Site-specific immobilization improved the coupled reaction of tethered enzymes.

<p>a) Forward TPI-GAPDHS coupled reaction. b) Reverse GAPDHS-TPI coupled reaction. Both enzymes were tethered via their His tags and site-specific immobilization (); both enzymes were tethered via carboxyl-amine binding (); control chips had no attached protein, but the complete reaction mixtures () [n = 9 (a), n = 12 (b); mean values are plotted with SE]. c) Comparison of forward TPI-GAPDHS activities at the 50 min timepoint calculated from (a). d) Comparison of reverse GAPDHS-TPI activities at the 60 min timepoint calculated from (b). Site-specific immobilization of His-NiNTA showed significantly higher activity (*p = 0.0001 ** p<0.001).</p

Site-specific immobilization improved specific activities of tethered enzymes.

<p>For both TPI and GAPDHS, site-specific immobilization using the His tag significantly improved enzyme specific activities versus carboxyl-amine binding. Although the total amounts of TPI (a) and GAPDHS (c) immobilized to carboxyl (C-A) or Ni-NTA activated surfaces were statistically identical, the specific activity of His-TPI bound to Ni-NTA was significantly higher than when bound via carboxyl-amine attachments (b; *p = 0.0143, n = 9). Similarly, the specific activity of His-GAPDHS was higher when bound to Ni-NTA versus carboxyl-amine binding (d; **p = 0.0234, n = 7).</p

Deploying RNA and DNA with Functionalized Carbon Nanotubes

Carbon nanotubes internalize into cells and are potential molecular platforms for siRNA and DNA delivery. A comprehensive understanding of the identity and stability of ammonium-functionalized carbon nanotube (f-CNT)-based nucleic acid constructs is critical to deploying them in vivo as gene delivery vehicles. This work explored the capability of f-CNT to bind single- and double-strand oligonucleotides by determining the thermodynamics and kinetics of assembly and the stoichiometric composition in aqueous solution. Surprisingly, the binding affinity of f-CNT and short oligonucleotide sequences was in the nanomolar range, kinetics of complexation were extremely rapid, and from one to five sequences were loaded per nanotube platform. Mechanistic evidence for an assembly process that involved electrostatic, hydrogen bonding, and π-stacking bonding interactions was obtained by varying nanotube functionalities, oligonucleotides, and reaction conditions. ³¹P NMR and spectrophotometric fluorescence emission data described the conditions required to assemble and stably bind a DNA or RNA cargo for delivery in vivo and the amount of oligonucleotide that could be transported. The soluble oligonucleic acid–f-CNT supramolecular assemblies were suitable for use in vivo. Importantly, key evidence in support of an elegant mechanism by which the bound nucleic acid material can be “off-loaded” from the f-CNT was discovered