Oxidized Carbon Black: Preparation, Characterization and Application in Antibody Delivery across Cell 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.

Hydrophilic and Cell-Penetrable Pyrrolidinyl Peptide Nucleic Acid via Post-synthetic Modification with Hydrophilic Side Chains.

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose-phosphate was replaced by a peptide-like backbone. The absence of negative charge in the PNA backbone leads to several unique behaviors including a stronger binding and salt independency of the PNA-DNA duplex stability. However, PNA possesses poor aqueous solubility and cannot directly penetrate cell membranes. These are major obstacles that limit in vivo applications of PNA. In previous strategies, the PNA can be conjugated to macromolecular carriers or modified with positively charged side chains such as guanidinium groups to improve the aqueous solubility and cell permeability. In general, a preformed modified PNA monomer was required. In this study, a new approach for post-synthetic modification of PNA backbone with one or more hydrophilic groups was proposed. The PNA used in this study was the conformationally constrained pyrrolidinyl PNA with prolyl-2-aminocyclopentanecarboxylic acid dipeptide backbone (acpcPNA) that shows several advantages over the conventional PNA. The aldehyde modifiers carrying different linkers (alkylene and oligo(ethylene glycol)) and end groups (-OH, -NH2, and guanidinium) were synthesized and attached to the backbone of modified acpcPNA by reductive alkylation. The hybrids between the modified acpcPNAs and DNA exhibited comparable or superior thermal stability with base-pairing specificity similar to those of unmodified acpcPNA. Moreover, the modified apcPNAs also showed the improvement of aqueous solubility (10-20 folds compared to unmodified PNA) and readily penetrate cell membranes without requiring any special delivery agents. This study not only demonstrates the practicality of the proposed post-synthetic modification approach for PNA modification, which could be readily applied to other systems, but also opens up opportunities for using pyrrolidinyl PNA in various applications such as intracellular RNA sensing, specific gene detection, and antisense and antigene therapy.

Proretinal nanoparticles: stability, release, efficacy, and irritation.

Despite many potent biological activities, retinoids such as retinoic acid (RA) and retinal possess dose-related broad side effects. In this study, we show that this problem, which has been unsolvable for a long time, can be tackled through a controlled release strategy in which retinal is continuously delivered to the skin via sustained release from proretinal nanoparticles. The water dispersible proretinal nanoparticles are stable when kept in water at neutral pH and at room temperature for 8 months under light-proof conditions, and show sustained release of retinal into human synthetic sebum at a pH of 5. In the daily topical application tests performed for 4 weeks on rats' skin, the nanoparticles showed superior ability to increase epidermal thickness compared to RA and retinal, with no skin irritation observed for the proretinal particles, but severe skin irritation observed for RA and free retinal. When tested under occlusion conditions in human volunteers, insignificant skin irritation was observed for the proretinal nanoparticles. The 12-week, double-blind, split-face study on human volunteers indicates better antiaging efficacy of the particles as compared to the free RA.

Depositing α-mangostin nanoparticles to sebaceous gland area for acne treatment.

Although entrapment of nanoparticles of appropriate sizes at hair follicles has been clarified, there is no report on specific clinical application of this finding. Since sebaceous gland is associated with hair follicle, we hypothesize that effective acne vulgaris treatment/prevention can be achieved by depositing anti-acne agent in nanoparticle form at the hair follicles. Challenge of this strategy, however, lies at the finding of effective anti-acne particles with minimal skin irritation. Here using cellulose-based nanoparticles as nano-reservoir and α-mangostin (an active component isolated from the edible Garcinia mangostana Linn. fruit) as anti-acne agent, we prepare nanoparticles highly loaded with α-mangostin. Ability of the obtained particles to sustained release α-mangostin into synthetic sebum is demonstrated. The obtained mangostin particles are verified for their insignificant skin irritation through the two-week, twice-daily open application test in 20 healthy human volunteers. Excellent entrapment and sustainment of the mangostin nanoparticles at the hair follicles are elucidated in six human volunteers by detecting the presence of α-mangostin at the roots of hairs pulled from the treated skin area. The 4-week-randomized, double-blind, placebo-controlled, split-face study in 10 acne patients indicates significant improvement in acne vulgaris condition on the side twice daily applied with mangostin nanoparticles.

Shape Effect on Particle-Lipid Bilayer Membrane Association, Cellular Uptake, and Cytotoxicity.

Although computer simulation and cell culture experiments have shown that elongated spherical particles can be taken up into cells more efficiently than spherical particles, experimental investigation on effects of these different shapes over the particle-membrane association has never been reported. Therefore, whether the higher cellular uptake of an elongated spherical particles is a result of a better particle-membrane association as suggested by some calculation works or a consequence of its influence on other cellular trans-membrane components involved in particle translocation process, cannot be concluded. Here, we study the effect of particle shape on the particle-membrane interaction by monitoring the association between particles of various shapes and lipid bilayer membrane of artificial cell-sized liposomes. Among the three shaped lanthanide-doped NaYF4 particles, all with high shape purity and uniformity, similar crystal phase, and surface chemistry, the elongated spherical particle shows the highest level of membrane association, followed by the spherical particle with a similar radius, and the hexagonal prism-shaped particle, respectively. The free energy of membrane curvature calculated based on a membrane indentation induced by a particle association indicates that among the three particle shapes, the elongated spherical particle give the most stable membrane curvature. The elongated spherical particles show the highest cellular uptake into cytosol of human melanoma (A-375) and human liver carcinoma (HepG2) cells when observed through a confocal laser scanning fluorescence microscope. Quantitative study using flow cytometry also gives the same result. The elongated spherical particles also possess the highest cytotoxicity in A-375 and normal skin (WI-38) cell lines, comparing to the other two shaped particles.

Bringing macromolecules into cells and evading endosomes by oxidized carbon nanoparticles.

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.

Acrylate-tethering drug carrier: covalently linking carrier to biological surface and application in the treatment of Helicobacter pylori infection.

The development of carriers to sustain drugs at stomach surface is an attractive strategy to increase drug bioavailability locally and systematically. So far, the only reported carrier that can form a covalent bond with mucus, the thiolated carrier, relies on a reversible disulfide exchange reaction between thiols on the carrier and disulfide bridges on the mucus. Here we show the design and fabrication of a cellulose carrier with tethering acrylate groups (denoted here as clickable carrier) that, under a nontoxic condition, can efficiently react with thiols on biomaterials in situ through the thermodynamically driven and kinetically probable Michael thiol-ene click reaction. Here we show the attachments of the clickable carriers to a mucin protein, a surface of human laryngeal carcinoma cells, and a surface of a fresh porcine stomach. We also show that the required thiol moieties can be generated in situ by reducing existing cystine disulfide bridges with either the edible vitamin C or the relatively nontoxic tris(2-carboxyethyl) phosphine. Comparing to a control carrier, the clickable carrier can increase some drug concentrations in an ex vivo stomach tissue, and improve the Helicobacter pylori treatment in infected C57BL/6 mice.

Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles.

Garcinia mangostana Linn extract (GME) is a natural product that has received considerable attention in cancer therapy, and has the potential to reduce side effects of chemotherapeutics and improve efficacy. We formulated GME-encapsulated ethyl cellulose (GME-EC) and a polymer blend of ethyl cellulose and methyl cellulose (GME-EC/MC) nanoparticles. We achieved high drug-loading and encapsulation efficiency using a solvent-displacement method with particle sizes around 250 nm. Cellular uptake and accumulation of GME was higher for GME-encapsulated nanoparticles compared to free GME. In vitro cytotoxicity analysis showed effective anticancer activity of GME-EC and GME-EC/MC nanoparticles in HeLa cells in a dose-dependent manner. GME-EC/MC nanoparticles showed approximately twofold-higher anticancer activity compared to GME-EC nanoparticles, likely due to their enhanced bioavailability. GME-encapsulated nanoparticles primarily entered HeLa cells by clathrin-mediated endocytosis and trafficked through the endolysosomal pathway. As far as we know, this is the first report on the cellular uptake and intracellular trafficking mechanism of drug-loaded cellulose-based nanoparticles. In summary, encapsulation of GME using cellulose-derivative nanoparticles - GME-EC and GME-EC/MC nanoparticles - successfully improved the bioavailability of GME in aqueous solution, enhanced cellular uptake, and displayed effective anticancer activity.

Ethyl cellulose nanoparticles: clarithomycin encapsulation and eradication of H. pylori.

The extreme acidic environment of the stomach, its regular voidance of contents and the restricted access to the mucus covered habitat combined with the antibiotic resistance of the bacteria, all contribute to the poor success in the treatment of Helicobacter pylori gastric infections. Here, we demonstrate that by encapsulating clarithromycin into ethyl cellulose (EC) nanoparticles, the efficiency of H. pylori clearance in C57BL/6 mice infected with these bacteria was significantly improved. Clarithomycin-loaded EC nanoparticles were prepared via a simple yet effective anti-solvent particle induction method, to yield sub-micron sized particles with 22.3 ± 0.17% (w/w) clarithromycin loading at 86 ± 0.5% (w/w) encapsulation efficiency. The particles dispersed well in water and simulated gastric fluid and gave a minimum inhibitory concentration of 0.09-0.18 µg/ml against four strains of H. pylori. Encapsulation into EC particles not only enhanced the anti-adhesion activity of clarithromycin when tested with H. pylori and Hep-2 cells, but also gave significant enhancement of H. pylori clearance in the stomach of C57BL/6 mice infected with the bacteria.

Comparison of the skin penetration of Garcinia mangostana extract in particulate and non-particulate form.

The aim of the present study was to solve the water insolubility limitation of the medically and cosmetically interesting substance Garcinia mangostana Linn (GML) extract by encapsulation, and to evaluate and investigate the penetration efficacy of free and encapsulated GML in two different vehicles (water and cream) in porcine ear skin. The follicular penetration depth was determined in 50 hair follicles for each of the four formulations by means of fluorescence microscopy. Tape stripping was used to compare the distribution properties of GML with all formulations on the stratum corneum. The results showed that encapsulated and free GML in the cream base penetrated deeper into hair follicles than if applied in an aqueous base. In addition, encapsulated GML could be distributed more homogeneously on the stratum corneum than the free GML. In conclusion, it was found that encapsulated GML in a cream base had the most effective penetration level in porcine ear skin.

Combating Helicobacter pylori infections with mucoadhesive nanoparticles loaded with Garcinia mangostana extract.

AIM: To combat the resistance of Helicobacter pylori to antibiotics through the use of Garcinia mangostana extract (GME) in the form that can be localized at stomach mucosa. MATERIALS & METHODS: GME and its major active component, α-mangostin, are encapsulated into the moderately acid stable mucoadhesive nanocarriers, and tested for anti-H. pylori and antiadhesion activities in vitro and their ability to eradicate H. pylori in infected mice. RESULTS: The two in vitro activities are observed and are enhanced when the materials are encapsulated into nanocarriers. Preliminary in vivo tests revealed the ability to combat H. pylori in mice following oral administration of the encapsulated GME, but not the unencapsulated GME. CONCLUSION: Nanoencapsulated GME is a potential anti-H. pylori agent.

Mucosa-plate for direct evaluation of mucoadhesion of drug carriers.

The method to prepare mucosa-plates, glass slides covalently coated with mucin, is demonstrated. The use of the plate to evaluate mucoadhesion of nanocarriers made from different four polymeric materials, N-succinylchitosan (NS-chitosan), alginate (ALG), ethylcellulose (EC), and a blend of EC and methylcellulose (EC/MC), was demonstrated. While different mucoadhesion of the four carriers could be detected using mucosa-plate, the conventional viscosity measurement could not differentiate their mucin-binding ability. ALG and NS-chitosan nanospheres showed the best attachment to the mucosa-plate compared to the EC/MC and EC spheres. Capsaicin, a model hydrophobic drug, was loaded into the carriers and the ability of the different polymeric carriers to retain capsaicin at the stomach tissue was compared using an ex vivo fresh porcine stomach assay. Ability to retain capsaicin at the stomach tissue correlated well with binding affinity toward the mucosa-plate and the loading capacity of the carriers.