Magnus Bergkvist

Paradoxical glomerular filtration of carbon nanotubes

The molecular weight cutoff for glomerular filtration is thought to be 30–50 kDa. Here we report rapid and efficient filtration of molecules 10–20 times that mass and a model for the mechanism of this filtration. We conducted multimodal imaging studies in mice to investigate renal clearance of a single-walled carbon nanotube (SWCNT) construct covalently appended with ligands allowing simultaneous dynamic positron emission tomography, near-infrared fluorescence imaging, and microscopy. These SWCNTs have a length distribution ranging from 100 to 500 nm. The average length was determined to be 200–300 nm, which would yield a functionalized construct with a molecular weight of ∼350–500 kDa. The construct was rapidly (t1/2 ∼ 6 min) renally cleared intact by glomerular filtration, with partial tubular reabsorption and transient translocation into the proximal tubular cell nuclei. Directional absorption was confirmed in vitro using polarized renal cells. Active secretion via transporters was not involved. Mathematical modeling of the rotational diffusivity showed the tendency of flow to orient SWCNTs of this size to allow clearance via the glomerular pores. Surprisingly, these results raise questions about the rules for renal filtration, given that these large molecules (with aspect ratios ranging from 100:1 to 500:1) were cleared similarly to small molecules. SWCNTs and other novel nanomaterials are being actively investigated for potential biomedical applications, and these observations—that high aspect ratio as well as large molecular size have an impact on glomerular filtration—will allow the design of novel nanoscale-based therapeutics with unusual pharmacologic characteristics.

Cu–Cu diffusion bonding enhancement at low temperature by surface passivation using self-assembled monolayer of alkane-thiol

Self-assembled monolayer (SAM) of 1-hexanethiol is applied on copper (Cu) surface to retard surface oxidation during exposure in the ambient. This SAM layer can be desorbed effectively with an annealing step in inert N2 ambient to provide a clean Cu surface. Using this passivation method with SAM, wafers covered with thin Cu layer are passivated, stored, desorbed, and bonded at 250 °C. The bonded Cu layer presents clear evidence of substantial interdiffusion and grain growth despite prolonged exposure in the ambient. This method of passivation is proven to be effective and can be further optimized to enable high quality Cu–Cu direct bonding at low temperature for application in three-dimensional integration.

Synthesis and Biodistribution of Oligonucleotide-Functionalized, Tumor-Targetable Carbon Nanotubes

Single-wall carbon nanotubes (SWNT) show promise as nanoscale vehicles for targeted therapies. We have functionalized SWNT using regioselective chemistries to confer capabilities of selective targeting using RGD ligands, radiotracing using radiometal chelates, and self-assembly using oligonucleotides. The constructs contained approximately 2-7 phosphorothioate oligonucleotide chains and 50-75 amines per 100 nm length of SWNT, based on a loading of 0.01-0.05 mmol/g and 0.3-0.6 mmol/g, respectively. Dynamic light scattering suggested the functionalized SWNT were well dispersed, without formation of large aggregates in physiologic solutions. The SWNT-oligonucleotide conjugate annealed with a complementary oligonucleotide sequence had a melting temperature of 54 degrees C. Biodistribution in mice was quantified using radiolabeled SWNT-oligonucleotide conjugates. Appended RGD ligands allowed for specific binding to tumor cells in a flow cytometric assay. The techniques employed should enable the synthesis of multifunctional SWNT capable of self-assembly in biological settings.

Detecting Small Changes and Additional Peptides in the Canine Parvovirus Capsid Structure

ABSTRACT Parvovirus capsids are assembled from multiple forms of a single protein and are quite stable structurally. However, in order to infect cells, conformational plasticity of the capsid is required and this likely involves the exposure of structures that are buried within the structural models. The presence of functional asymmetry in the otherwise icosahedral capsid has also been proposed. Here we examined the protein composition of canine parvovirus capsids and evaluated their structural variation and permeability by protease sensitivity, spectrofluorometry, and negative staining electron microscopy. Additional protein forms identified included an apparent smaller variant of the virus protein 1 (VP1) and a small proportion of a cleaved form of VP2. Only a small percentage of the proteins in intact capsids were cleaved by any of the proteases tested. The capsid susceptibility to proteolysis varied with temperature but new cleavages were not revealed. No global change in the capsid structure was observed by analysis of Trp fluorescence when capsids were heated between 40°C and 60°C. However, increased polarity of empty capsids was indicated by bis-ANS binding, something not seen for DNA-containing capsids. Removal of calcium with EGTA or exposure to pHs as low as 5.0 had little effect on the structure, but at pH 4.0 changes were revealed by proteinase K digestion. Exposure of viral DNA to the external environment started above 50°C. Some negative stains showed increased permeability of empty capsids at higher temperatures, but no effects were seen after EGTA treatment.

Sequential Reactions of Surface- Tethered Glycolytic Enzymes

The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the flagellar principal piece by means of glycolytic enzymes, several of which are tethered to a cytoskeletal support via germ-cell-specific targeting domains. Inspired by this design, we have produced recombinant hexokinase type 1 and glucose-6-phosphate isomerase capable of oriented immobilization on a nickel-nitrilotriacetic acid modified surface. Specific activities of enzymes tethered via this strategy were substantially higher than when randomly adsorbed. Furthermore, these enzymes showed sequential activities when tethered onto the same surface. This is the first demonstration of surface-tethered pathway components showing sequential enzymatic activities, and it provides a first step toward reconstitution of glycolysis on engineered hybrid devices.

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.

Targeted in vitro photodynamic therapy via aptamer-labeled, porphyrin-loaded virus capsids

Virus capsids have emerged as multifunctional platform systems for development of bio-derived nanomaterials. In this work we investigate the use of aptamer decorated MS2 bacteriophage capsids, loaded with photosensitizer for targeted photodynamic therapy in vitro. MS2 capsids were loaded with approximately 250 cationic porphyrins through a novel assembly packaging mechanism, followed by exterior decoration of the capsid with a cancer-targeting nucleic acid aptamer via chemical conjugation. The ability of these aptamer-virus-porphyrin constructs to specifically target and eradicate MCF-7 human breast cancer cells upon photoactivation was assessed. Photoinduced cytotoxicity was evaluated via live/dead staining and a metabolic activity assay with MCF-10A cells as a control. Results show that MCF-7 cells incubated with targeted, porphyrin-loaded virus capsids exhibited cell death whereas the MCF-10A cells did not. Furthermore, MCF-7 cells incubated with porphyrin-loaded viruses decorated with a non-targeting aptamer exhibited no observable phototoxicity. Combined, the results presented in this work demonstrate our unique virus-based loading strategy offers a viable approach for efficient targeted delivery of photoactive compounds for site-specific photodynamic cancer therapy using bio-derived nanomaterials.

Recreating a human trabecular meshwork outflow system on microfabricated porous structures

Glaucoma is the leading cause of irreversible blindness, resulting from an increase in intraocular pressure (IOP). IOP is the only modifiable risk factor of glaucoma and is controlled by the outflow of the aqueous humor through the human trabecular meshwork (HTM). Currently, the lack of a proper in vitro HTM model impedes advances in understanding outflow physiology and discovering effective IOP‐lowering anti‐glaucoma therapeutics. Therefore, we designed and constructed an in vitro HTM model using micropatterned, porous SU‐8 scaffolds, which support cells to recapitulate functional HTM morphology and allow the study of outflow physiology. The pore size of SU‐8 scaffolds, surface coating, cell seeding density, and culture duration were evaluated for HTM cell growth. The bioengineered HTM was characterized by F‐actin staining and immunocytochemistry of HTM markers. A stand‐alone perfusion chamber with an integrated pressure sensing system was further constructed and used for the investigation of the outflow facility of the bioengineered HTM treated with latrunculin B—an IOP lowering agent. Cells in the in vitro model exhibited HTM‐like morphology, expression of α‐smooth muscle actin, myocilin, and αB‐crystallin, outflow characteristics and drug responsiveness. Altogether, we have developed an in vitro HTM model system for understanding HTM cell biology and screening of pharmacological or biological agents that affect trabecular outflow facility, expediting discovery of IOP‐lowering, anti‐glaucoma therapeutics. Biotechnol. Bioeng. 2013;110: 3205–3218.

Entropically Driven Self-Assembly of Lysinibacillus sphaericus S-Layer Proteins Analyzed Under Various Environmental Conditions

S-Layer proteins are an example of bionanostructures that can be exploited in nanofabrication. In addition to their ordered structure, the ability to self-assembly is a key feature that makes them a promising technological tool. Here, in vitro self-assembly kinetics of SpbA was investigated, and found that it occurs at a rate that is dependent on temperature, its concentration, and the concentration of calcium ions and sodium chloride. The activation enthalpy (120.81 kJ . mol(-1)) and entropy (129.34 J . mol(-1) . K(-1)) obtained infers that the incorporation of monomers incurs in a net loss of hydrophobic surface. By understanding how the protein monomers drive the self-assembly at different conditions, the rational optimization of this process was feasible.

Targeted fibrillar nanocarbon RNAi treatment of acute kidney injury

Fibrillar carbon nanotubes simultaneously deliver two small interfering RNAs, which safely prevent acute kidney injury and prolong survival in mice. Double trouble for kidney toxicity The kidneys can be damaged by drugs, such as antibiotics and chemotherapy, as well as by surgery, which robs the organs of oxygen. To prevent injury, Alidori et al. devised a nanomedicine treatment approach that delivers two small interfering RNAs (siRNAs) to the main cells of the kidney, the renal proximal tubule cells. siRNAs targeting Mep1b and Trp53 were attached to fibrillar carbon nanotubes and delivered simultaneously to mice before drug-induced kidney insult. With such RNA interference, the kidney cells could not produce meprin-1β and p53—two key proteins involved in kidney injury; the mice lived longer and remained injury-free, but only if given both siRNAs. The nanotube/siRNA complexes were also safe and had favorable pharmacokinetics in monkeys. The next steps will be testing the dual siRNAs in other animal models of kidney injury. RNA interference has tremendous yet unrealized potential to treat a wide range of illnesses. Innovative solutions are needed to protect and selectively deliver small interfering RNA (siRNA) cargo to and within a target cell to fully exploit siRNA as a therapeutic tool in vivo. Herein, we describe ammonium-functionalized carbon nanotube (fCNT)–mediated transport of siRNA selectively and with high efficiency to renal proximal tubule cells in animal models of acute kidney injury (AKI). fCNT enhanced siRNA delivery to tubule cells compared to siRNA alone and effectively knocked down the expression of several target genes, including Trp53, Mep1b, Ctr1, and EGFP. A clinically relevant cisplatin-induced murine model of AKI was used to evaluate the therapeutic potential of fCNT-targeted siRNA to effectively halt the pathogenesis of renal injury. Prophylactic treatment with a combination of fCNT/siMep1b and fCNT/siTrp53 significantly improved progression-free survival compared to controls via a mechanism that required concurrent reduction of meprin-1β and p53 expression. The fCNT/siRNA was well tolerated, and no toxicological consequences were observed in murine models. Toward clinical application of this platform, fCNTs were evaluated for the first time in nonhuman primates. The rapid and kidney-specific pharmacokinetic profile of fCNT in primates was comparable to what was observed in mice and suggests that this approach is amenable for use in humans. The nanocarbon-mediated delivery of siRNA provides a therapeutic means for the prevention of AKI to safely overcome the persistent barrier of nephrotoxicity during medical intervention.