A cyanobacterial polysaccharide, sacran, which has a high molecular length over 30 μm, forms in-plane oriented film by casting. The film creates uniaxially-swelling hydrogels with a micrometer thickness.

A great challenge exists in finding safe, simple, and effective delivery strategies to bring matters across cell membrane. Popular methods such as viral vectors, positively charged particles and cell penetrating peptides possess some of the following drawbacks: safety issues, lysosome trapping, limited loading capacity, and toxicity, whereas electroporation produces severe damages on both cargoes and cells. Here, we show that a serendipitously discovered, relatively nontoxic, water dispersible, stable, negatively charged, oxidized carbon nanoparticle, prepared from graphite, could deliver macromolecules into cells, without getting trapped in a lysosome. The ability of the particles to induce transient pores on lipid bilayer membranes of cell-sized liposomes was demonstrated. Delivering 12-base-long pyrrolidinyl peptide nucleic acids with d-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) complementary to the antisense strand of the NF-κB binding site in the promoter region of the Il6 gene into the macrophage cell line, RAW 264.7, by our particles resulted in an obvious accumulation of the acpcPNAs in the nucleus and decreased Il6 mRNA and IL-6 protein levels upon stimulation. We anticipate this work to be a starting point in a new drug delivery strategy, which involves the nanoparticle that can induce a transient pore on the lipid bilayer membrane.

Modulating biomolecular networks in cells with peptides and proteins has become a promising therapeutic strategy and effective biological tools. A simple and effective reagent that can bring functional proteins into cells can increase efficacy and allow more investigations. Here we show that the relatively non-toxic and non-immunogenic oxidized carbon black particles (OCBs) prepared from commercially available carbon black can deliver a 300 kDa protein directly into cells, without an involvement of a cellular endocytosis. Experiments with cell-sized liposomes indicate that OCBs directly interact with phospholipids and induce membrane leakages. Delivery of human monoclonal antibodies (HuMAbs, 150 kDa) with specific affinity towards dengue viruses (DENV) into DENV-infected Vero cells by OCBs results in HuMAbs distribution all over cells’ interior and effective viral neutralization. An ability of OCBs to deliver big functional/therapeutic proteins into cells should open doors for more protein drug investigations and new levels of antibody therapies and biological studies.

No abstract

We propose a self-similar assembly to generate planar orientation of megamolecular polysaccharides on the nanometer scale and submicron scale. Evaporating the aqueous liquid crystalline (LC) solution on a planar air-LC interface induces polymer layering by self-assembly and rational action of macroscopic capillary forces between the layers. To clarify the mechanisms of nanometer- and submicron-scale layering, the polymer films are investigated by electron microscopy.

Molecularly oriented hydrogels of sacran, which is a supergiant liquid crystalline polysaccharide extracted from Aphanothece sacrum biomaterials, showing ultrahigh anisotropy of swelling is successfully prepared by two-step chemical crosslinking. Divinyl sulfone (DVS) works as a chemical cross-linker of sacran chains in a dilute aqueous solution to form hydrogels, but some of the added DVS remains in the hydrogel without cross-linking. The remaining DVS cross-links further with the preformed networks of sacran chains in liquid crystalline state during slow drying to produce in-plane oriented xerogels. The xerogels show heterogeneous anisotropy in the successive swellings steps; the linear swelling ratio in the thickness direction is 10000−40000-fold higher than that in the width direction due to the molecular orientation of the sacran hydrogels. X-ray diffraction imaging of the hydrogels reveal not only the orientation of the xerogel films but also the unusual orientation of water molecules binding to sacran networks in the hydrogel state.

We have detected a second-order nonlinear optical response from aggregates of the ampholytic megamolecular polysaccharide sacran extracted from cyanobacterial biomaterials, by using optical second harmonic generation (SHG) microscopy. The SHG images of sacran cotton-like lump, fibers, and cast films showed SHG intensity microspots of several tens of micrometers in size. The dependence of the SHG spot intensity on an excitation light polarization angle was observed to illustrate sacran molecular orientation in these microdomains. We also observed SHG signals around a special region of the cast film edges of sacran. These results show that sacran megamolecules aggregate in several different ways.

Uses of micro-/nano-sized particles to deliver biologically active entities into cells are common for medical therapeutics and prophylactics and also for cellular experiments. Enhancing cellular uptake and avoiding destruction by lysosomes are desirable for general particulate drug delivery systems. Here, we show that the relatively nontoxic, negatively charged oxidized carbon black particles (OCBs) can enhance cellular penetration of micro- and nano-particles. Experiments with retinal-grafted chitosan particles (PRPs) with hydrodynamic sizes of 1200 ± 51.5, 540 ± 29.0, and 430 ± 11.0 nm (three-sized model particles) indicate that only the sub-micron-sized particles can penetrate the first layer of multilayered liposomes. However, in the presence of OCBs, the micron-sized PRPs and the two submicron-sized PRPs can rapidly enter the interiors of all layers of the multilayered liposomes. Very low cellular uptakes of micro- and submicron-sized PRPs into keratinocytes cells are usually observed. However, in the presence of OCBs, faster and higher cellular uptakes of all of the three-sized PRPs are clearly noticed. Intracellular traffic monitoring of PRP uptake into HepG2 cells in the presence of OCBs revealed that the PRPs did not co-localize with endosomes, suggesting a nonendocytic uptake process. This demonstration of OCB’s ability to enhance cellular uptake of micro- and submicron-particles should open up an easy strategy to effectively send various carriers into cells.