Recent Studies of Pickering Emulsions: Particles Make the Difference.

In recent years, emulsions stabilized by micro- or nanoparticles (known as Pickering emulsions) have attracted much attention. Micro- or nanoparticles, as the main components of the emulsion, play a key role in the preparation and application of Pickering emulsions. The existence of particles at the interface between the oil and aqueous phases affects not only the preparation, but also the properties of Pickering emulsions, affording superior stability, low toxicity, and stimuli-responsiveness compared to classical emulsions stabilized by surfactants. These advantages of Pickering emulsions make them attractive, especially in biomedicine. In this review, the effects of the characteristics of micro- and nanoparticles on the preparation and properties of Pickering emulsions are introduced. In particular, the preparation methods of Pickering emulsions, especially uniform-sized emulsions, are listed. Uniform Pickering emulsions are convenient for both mechanistic research and applications. Furthermore, some biomedical applications of Pickering emulsions are discussed and the problems hindering their clinical application are identified.

Porous TiO2 materials through Pickering high-internal phase emulsion templating.

We report a facile method for preparing porous structured TiO2 materials by templating from Pickering high-internal phase emulsions (HIPEs). A Pickering HIPE with an internal phase of up to 80 vol %, stabilized by poly(N-isopropylacrylamide)-based microgels and TiO2 solid nanoparticles, was first formulated and employed as a template to prepare the porous TiO2 materials with an interconnected structure. The resultant materials were characterized by scanning electron microscopy, X-ray diffraction, and mercury intrusion. Our results showed that the parent emulsion droplets promoted the formation of macropores and interconnecting throats with sizes of ~50 and ~10 µm, respectively, while the interfacially adsorbed microgel stabilizers drove the formation of smaller pores (~100 nm) throughout the macroporous walls after drying and sintering. The interconnected structured network with the bimodal pores could be well preserved after calcinations at 800 °C. In addition, the photocatalytic activity of the fabricated TiO2 was evaluated by measuring the photodegradation of Rhodamine B in water. Our results revealed that the fabricated TiO2 materials are good photocatalysts, showing enhanced activity and stability in photodegrading organic molecules.

Harnessing the potential of halogenated natural product biosynthesis by mangrove-derived actinomycetes.

Mangrove-derived actinomycetes are promising sources of bioactive natural products. In this study, using homologous screening of the biosynthetic genes and anti-microorganism/tumor assaying, 163 strains of actinomycetes isolated from mangrove sediments were investigated for their potential to produce halogenated metabolites. The FADH2-dependent halogenase genes, identified in PCR-screening, were clustered in distinct clades in the phylogenetic analysis. The coexistence of either polyketide synthase (PKS) or nonribosomal peptide synthetase (NRPS) as the backbone synthetases in the strains harboring the halogenase indicated that these strains had the potential to produce structurally diversified antibiotics. As a validation, a new enduracidin producer, Streptomyces atrovirens MGR140, was identified and confirmed by gene disruption and HPLC analysis. Moreover, a putative ansamycin biosynthesis gene cluster was detected in Streptomyces albogriseolus MGR072. Our results highlight that combined genome mining is an efficient technique to tap promising sources of halogenated natural products synthesized by mangrove-derived actinomycetes.

Targeted delivery of insoluble cargo (paclitaxel) by PEGylated chitosan nanoparticles grafted with Arg-Gly-Asp (RGD).

Poor delivery of insoluble anticancer drugs has so far precluded their clinical application. In this study, we developed a tumor-targeting delivery system for insoluble drug (paclitaxel, PTX) by PEGylated O-carboxymethyl-chitosan (CMC) nanoparticles grafted with cyclic Arg-Gly-Asp (RGD) peptide. To improve the loading efficiency (LE), we combined O/W/O double emulsion method with temperature-programmed solidification technique and controlled PTX within the matrix network as in situ nanocrystallite form. Furthermore, these CMC nanoparticles were PEGylated, which could reduce recognition by the reticuloendothelial system (RES) and prolong the circulation time in blood. In addition, further graft of cyclic RGD peptide at the terminal of PEG chain endowed these nanoparticles with higher affinity to in vitro Lewis lung carcinoma (LLC) cells and in vivo tumor tissue. These outstanding properties enabled as-designed nanodevice to exhibit a greater tumor growth inhibition effect and much lower side effects over the commercial formulation Taxol.

Microcosmic mechanisms for protein incomplete release and stability of various amphiphilic mPEG-PLA microspheres.

The microcosmic mechanisms of protein (recombinant human growth hormone, rhGH) incomplete release and stability from amphiphilic poly(monomethoxypolyethylene glycol-co-D,L-lactide) (mPEG-PLA, PELA) microspheres were investigated. PELA with different hydrophilicities (PELA-1, PELA-2, and PELA-3) based on various ratios of mPEG to PLA were employed to prepare microspheres exhibiting a narrow size distribution using a combined double emulsion and premix membrane emulsification method. The morphology, rhGH encapsulation efficiency, in vitro release profile, and rhGH stability of PELA microspheres during the release were characterized and compared in detail. It was found that increasing amounts of PLA enhanced the encapsulation efficiency of PELA microspheres but reduced both the release rate of rhGH and its stability. Contact angle, atomic force microscope (AFM), and quartz crystal microbalance with dissipation (QCM-D) techniques were first combined to elucidate the microcosmic mechanism of incomplete release by measuring the hydrophilicity of the PELA film and its interaction with rhGH. In addition, the pH change within the microsphere microenvironment was monitored by confocal laser scanning microscopy (CLSM) employing a pH-sensitive dye, which clarified the stability of rhGH during the release. These results suggested that PELA hydrophilicity played an important role in rhGH incomplete release and stability. Thus, the selection of suitable hydrophilic polymers with adequate PEG lengths is critical in the preparation of optimum protein drug sustained release systems. This present work is a first report elucidating the microcosmic mechanisms responsible for rhGH stability and its interaction with the microspheres. Importantly, this research demonstrated the application of promising new experimental methods in investigating the interaction between biomaterials and biomacromolecules, thus opening up a range of exciting potential applications in the biomedical field including drug delivery and tissue regeneration.

Compatible and Incompatible Mycorrhizal Fungi With Seeds of Dendrobium Species: The Colonization Process and Effects of Coculture on Germination and Seedling Development.

Orchids highly rely on mycorrhizal fungi for seed germination, and compatible fungi could effectively promote germination up to seedlings, while incompatible fungi may stimulate germination but do not support subsequent seedling development. In this study, we compared the fungal colonization process among two compatible and two incompatible fungi during seed germination of Dendrobium officinale. The two compatible fungi, i.e., Tulasnella SSCDO-5 and Sebacinales LQ, originally from different habitats, could persistently colonize seeds and form a large number of pelotons continuously in the basal cells, and both fungi promoted seed germination up to seedling with relative effectiveness. In contrast, the two incompatible fungi, i.e., Tulasnella FDd1 and Tulasnella AgP-1, could not persistently colonize seeds. No pelotons in the FDd1 treatment and only a few pelotons in the AgP-1 treatment were observed; moreover, no seedlings were developed at 120 days after incubation in either incompatible fungal treatment. The pattern of fungal hyphae colonizing seeds was well-matched with the morphological differentiation of seed germination and seedling development. In the fungal cocultural experiments, for both orchids of D. officinale and Dendrobium devonianum, cocultures had slightly negative effects on seed germination, protocorm formation, and seedling formation compared with the monocultures with compatible fungus. These results provide us with a better understanding of orchid mycorrhizal interactions; therefore, for orchid conservation based on symbiotic seed germination, it is recommended that a single, compatible, and ecological/habitat-specific fungus can be utilized for seed germination.

Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles.

Chitosan-based nanoparticles (NPs) are widely used in drug delivery, device-based therapy, tissue engineering, and medical imaging. In this aspect, a clear understanding of how physicochemical properties of these NPs affect the cytological response is in high demand. The objective of this study is to evaluate the effect of surface charge on cellular uptake profiles (rate and amount) and intracellular trafficking. We fabricate three kinds of NPs (∼ 215 nm) with different surface charge via SPG membrane emulsification technique and deposition method. They possess uniform size as well as identical other physicochemical properties, minimizing any differences between the NPs except for surface charge. Moreover, we extend our research to eight cell lines, which could help to obtain a representative conclusion. Results show that the cellular uptake rate and amount are both positively correlated with the surface charge in all cell line. Subsequent intracellular trafficking indicates that some of positively charged NPs could escape from lysosome after being internalized and exhibit perinuclear localization, whereas the negatively and neutrally charged NPs prefer to colocalize with lysosome. These results are critical in building the knowledge base required to design chitosan-based NPs to be used efficiently and specifically.

Porous quaternized chitosan nanoparticles containing paclitaxel nanocrystals improved therapeutic efficacy in non-small-cell lung cancer after oral administration.

Clinical application of paclitaxel (PTX) is limited because of its poor solubility in aqueous media. To overcome this hurdle, we devised an oral delivery system by encapsulating PTX into N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride (HTCC) nanoparticles. These nanoparticles were small (~130 nm), had a narrow size distribution, and displayed high loading efficiency owing to the homogeneous distribution of PTX nanocrystals. The matrix hydrophilicity and porous structure of the obtained nanoparticles accelerated their degradation and improved drug release. In vitro and in vivo transport experiments had proved that the presence of positive charges enhanced the intestinal permeability of these nanoparticles. Further in vitro experiment of cytotoxicity showed that the PTX-loaded HTCC nanoparticle (HTCC-NP:PTX) was more effective than native PTX owing to enhanced cellular uptake. Drug distribution in tissues and in vivo imaging studies confirmed the preferred accumulation of HTCC-NP:PTX in subcutaneous tumor tissue. Subsequent tumor xenograft assays demonstrated the promising therapeutic effect of HTCC-NP:PTX on inhibition of tumor growth and induction of apoptosis in tumor cells. Additional investigation into side effects revealed that HTCC-NP:PTX caused lower Cremophor EL-associated toxicities compared with Taxol. These results strongly supported the notion that HTCC nanoparticle (HTCC-NP) is a promising candidate as an oral carrier of PTX for cancer therapy.

Prophylactic vaccine delivery systems against epidemic infectious diseases.

Prophylactic vaccines have evolved from traditional whole-cell vaccines to safer subunit vaccines. However, subunit vaccines still face problems, such as poor immunogenicity and low efficiency, while traditional adjuvants are usually unable to meet specific response needs. Advanced delivery vectors are important to overcome these barriers; they have favorable safety and effectiveness, tunable properties, precise location, and immunomodulatory capabilities. Nevertheless, there has been no systematic summary of the delivery systems to cover a wide range of infectious pathogens. We herein summarized and compared the delivery systems for major or epidemic infectious diseases caused by bacteria, viruses, fungi, and parasites. We also included the newly licensed vaccines (e.g., COVID-19 vaccines) and those close to licensure. Furthermore, we highlighted advanced delivery systems with high efficiency, cross-protection, or long-term protection against epidemic pathogens, and we put forward prospects and thoughts on the development of future prophylactic vaccines.

[Molecular imprinting-flow injection chemiluminescence method for determination of doxycycline hydrochloride].

Doxycycline hydrochloride can enhance the chemiluminescence of potassium ferricyanide and luminol in alkaline medium. So a molecular imprinting-flow-injection chemiluminescence method for the determination of doxycycline hydrochloride was established by using doxycycline hydrochloride-imprinted polymers as recognition material and potassium ferricyanide and luminol as detection system. Doxycycline hydrochloride-imprinted polymer was synthesized using methacrylic acid as functional monomer and ethylene glycol dimethacrylate as cross-linker. The linear range is 9.0 x 10(-7)-6.0 x 10(-5) g x mL(-1) and the detection limit is 3.2 x 10(-7) g x mL(-1). The relative standard deviation for 6.0 X 10(-6) g x mL(-1) of doxycycline hydrochloride was 3.5% (n = 9). This method has been successfully applied to the determination of doxycycline hydrochloride in tablets and in urine samples.

Transport of a graphene nanosheet sandwiched inside cell membranes.

The transport of nanoparticles at bio-nano interfaces is essential for many cellular responses and biomedical applications. How two-dimensional nanomaterials, such as graphene and transition-metal dichalcogenides, diffuse along the cell membrane is, however, unknown, posing an urgent and important issue to promote their applications in the biomedical area. Here, we show that the transport of graphene oxides (GOs) sandwiched inside cell membranes varies from Brownian to Lévy and even directional dynamics. Specifically, experiments evidence sandwiched graphene-cell membrane superstructures in different cells. Combined simulations and analysis identify a sandwiched GO-induced pore in cell membrane leaflets, spanning unstable, metastable, and stable states. An analytical model that rationalizes the regimes of these membrane-pore states fits simulations quantitatively, resulting in a mechanistic interpretation of the emergence of Lévy and directional dynamics. We finally demonstrate the applicability of sandwiched GOs in enhanced efficiency of membrane-specific drug delivery. Our findings inform approaches to programming intramembrane transport of two-dimensional nanomaterials toward advantageous biomedical applications.

Fabrication of rigid and macroporous agarose microspheres by pre-cross-linking and surfactant micelles swelling method.

A novel combined method of pre-cross-linking and surfactant micelles swelling was proposed in this study to fabricate highly cross-linked and macroporous agarose (HMA) microspheres. Agarose was chemically modified by allylglycidyl ether (AGE) as heterobifunctional cross-linker via its active glycidyl moieties before gel formation and pre-cross-linking was achieved. By this means, the effective concentration of cross-linker presented in agarose gel increased significantly, and thus cross-linking with a high-efficiency was achieved. Further to enhance the intraparticle mass transfer of agarose microspheres, the surfactant micelles swelling method was utilized to create interconnected macropores. Under the optimal condition, HMA microspheres with homogeneous reticular structure and pore size of hundreds nanometers were successfully prepared. They exhibited a low backpressure with a flow velocity as high as 1987 cm/h, which was much higher than that of commercial Sepharose 4 F F. HMA microspheres were then derivatized with carboxymethyl (CM) groups and applied in ion-exchange chromatography. As expected, CM-HMA column separated model proteins effectively even at a flow velocity three times higher than that of commercial CM-4 F F. Visualization of dynamic protein adsorption by confocal laser scanning microscope (CLSM) revealed that the intraparticle mass transfer of CM-HMA microspheres was intensified due to its macroporous structure. All of the results indicated the newly developed agarose microspheres were a promising medium for high-speed chromatography.

Preparation of hierarchical hollow CaCO3 particles and the application as anticancer drug carrier.

One-pot approach to couple the crystallization of CaCO(3) nanoparticles and the in situ symmetry-breaking assembly of these crystallites into hollow spherical shells was developed under the templating effect of a soluble starch. Further functional study using HP-a as an anticancer drug carrier (DOX) demonstrated its advantages for localizing drug release by the pH value-sensitive structure and enhancing cytotoxicity by increasing cellular uptake, perinuclear accumulation, and nuclear entry.

Bioprocess of uniform-sized crosslinked chitosan microspheres in rats following oral administration.

Chitosan microspheres have a great potential in pharmaceutical application. In this study, uniform-sized chitosan microspheres crosslinked with glutaraldehyde (CG microspheres) were prepared by Shirasu Porous Glass (SPG) membrane emulsification technique. Based on the characterizations of uniform size and autofluorescence, it was possible to develop a new detecting system for observing and quantifying the CG microspheres in rats with three different diameters (2.1, 7.2 and 12.5 microm) synchronously after oral administration. This system was a combination of scanning electron microscopy (SEM), laser scanning confocal microscope (LSCM) and flow cytometer technique, which showed the advantages of being simple, intuitionistic, repeatable and sensitive. After oral administration of three kinds of particles with different diameters, bioadhesion in gastrointestinal tract, absorption in gastrointestinal tract, distribution in systemic tissues, and biodegradation in reticuloendothelial system (RES) were studied firstly in detail. The CG microspheres showed different fates in bioadhesion, absorption and distribution according to their diameters, while the biodegradation also varied due to the different locations in RES. These original results would indicate a better way for the CG microspheres in the clinical application.

Preparation and characterization of thermo-responsive albumin nanospheres.

Thermo-responsive poly(N-isopropylacrylamide-co-acrylamide)-block-polyallylamine-conjugated albumin nanospheres (PAN), new thermal targeting anti-cancer drug carrier, was developed by conjugating poly(N-isopropylacrylamide-co-acrylamide)-block-polyallylamine (PNIPAM-AAm-b-PAA) on the surface of albumin nanospheres (AN). PAN may selectively accumulate onto solid tumors that are maintained above physiological temperature due to local hyperthermia. PNIPAM-AAm-b-PAA was synthesized by radical polymerization, and AN was prepared by ultrasonic emulsification. AN with diameter below 200 nm and narrow size distribution was obtained by optimizing the preparative conditions. Rose Bengal (RB) was used as model drug for entrapment into the AN and PAN during the particle preparation. The release rate of RB from PAN compared with AN in trypsin solution was slower, and decreased with the increase of PNIPAM-AAm-b-PAA molecular weight, which suggested that the existence of a steric hydrophilic barrier on AN made digestion of AN more difficult. Moreover, the release of RB from PAN above the cloud-point temperature (T(cp)) of PNIPAM-AAm-b-PAA became faster. This was because the density of temperature-responsive polymers on AN was not so high, so that the interspace between the polymer chains increased after they shrunk due to the high temperature. As a result, the biodegradable AN was attacked more easily by trypsin. The design of PAN overcame the disadvantages of temperature-responsive polymeric micelles.

Uniform-sized PLA nanoparticles: preparation by premix membrane emulsification.

Nanoparticle size is crucial to drug release behavior and biodistribution in vivo, but few studies have been performed on biodegradable nanoparticles with narrow size distribution. In this note, uniform-sized nanoparticles were prepared by a facile method combining emulsion-solvent removal and premix membrane emulsification for the first time. After preparation of coarse emulsions, additional premix membrane emulsification with very high pressure was occupied to achieve uniform-sized nanodroplets, and nanoparticles were formed by further solidification. Polylactide (PLA) was selected as a model polymer. Several factors played key roles to obtain uniform-sized PLA nanoparticles, including type of organic solvent, the volume ratio of oil phase and external water phase, pore size of the microporous membrane and transmembrane pressure. The coefficient of variation (CV) value of PLA nanoparticles could be controlled below 16.9% under an optimum condition. The novel method also has the advantages of high productivity, simplicity and easy scale-up. The uniform-sized nanoparticles prepared by this novel method have great potentials in drug delivery.

Preparation of uniform-sized pH-sensitive quaternized chitosan microsphere by combining membrane emulsification technique and thermal-gelation method.

In this study, uniform-sized pH-sensitive quaternized chitosan microsphere was prepared by combining Shirasu porous glass (SPG) membrane emulsification technique and a novel thermal-gelation method. In this preparation process, the mixture of quaternized chitosan solution and alpha-beta-glycerophosphate (alpha-beta-GP) was used as water phase and dispersed in oil phase to form uniform W/O emulsion by SPG membrane emulsification technique. The droplets solidified into microspheres at 37 degrees C by thermal-gelation method. The whole process was simple and mild. The influence of process conditions on the property of prepared microspheres was investigated and the optimized preparation condition was obtained. As a result, the coefficient of variation (C.V.) of obtained microspheres diameters was below 15%. The obtained microsphere had porous structure and showed apparent pH-sensitivity. It dissolved rapidly in acid solution (pH 5) and kept stable in neutral solution (pH 7.4). The pH-sensitivity of microspheres also affected its drug release behavior. Bovine serum albumin (BSA) as a model drug was encapsulated in microspheres, and it was released rapidly in acid solution and slowly in neutral medium. The novel quaternized chitosan microspheres with pH-sensitivity can be used as drug delivery system in the biomedical field, such as tumor-targeted drug carrier.

Porogen effects in synthesis of uniform micrometer-sized poly(divinylbenzene) microspheres with high surface areas.

A new method of synthesizing uniform poly(divinylbenzene) (polyDVB) microspheres with high specific surface areas was designed by combining Shirasu porous glass (SPG) membrane emulsification, suspension polymerization, and post-crosslinking techniques. It was shown that the physicochemical properties of porogens have a great influence on the size distribution and porous features of microspheres. The low aqueous solubility of porogen facilitated preparation of uniform emulsions and microspheres, and high aqueous solubility led to polydispersed emulsions and poor microsphere yields. Such aqueous solubility effects can be tailored by adding a low molecular weight polystyrene (LPST) as costabilizer in porogen, thus improving the uniformity of microspheres. Moreover, different affinities of porogens for copolymers demonstrate various contributions to specific surface areas of microspheres in suspension polymerization especially post-crosslinking. Solvating porogen requires a much higher addition than nonsolvating porogen to obtain equal specific surface areas in polymerization, but has more potential to enhance the specific surface area in post-crosslinking. Two kinds of uniform microspheres were obtained with high specific surface areas, up to 706.6 m2/g by heptane and 937.5 m2/g by toluene.

Preparation of uniform-sized PELA microspheres with high encapsulation efficiency of antigen by premix membrane emulsification.

Relatively uniform-sized poly(lactide-co-ethylene glycol) (PELA) microspheres with high encapsulation efficiency were prepared rapidly by a novel method combining emulsion-solvent extraction and premix membrane emulsification. Briefly, preparation of coarse double emulsions was followed by additional premix membrane emulsification, and antigen-loaded microspheres were obtained by further solidification. Under the optimum condition, the particle size was about 1 mum and the coefficient of variation (CV) value was 18.9%. Confocal laser scanning microscope and flow cytometer analysis showed that the inner droplets were small and evenly dispersed and the antigen was loaded uniformly in each microsphere when sonication technique was occupied to prepare primary emulsion. Distribution pattern of PEG segment played important role on the properties of microspheres. Compared with triblock copolymer PLA-PEG-PLA, the diblock copolymer PLA-mPEG yielded a more stable interfacial layer at the interface of oil and water phase, and thus was more suitable to stabilize primary emulsion and protect coalescence of inner droplets and external water phase, resulting in high encapsulation efficiency (90.4%). On the other hand, solidification rate determined the time for coalescence during microspheres fabrication, and thus affected encapsulation efficiency. Taken together, improving the polymer properties and solidification rate are considered as two effective strategies to yield high encapsulation.