Weien Yuan

Porous microsphere and its applications

Porous microspheres have drawn great attention in the last two decades for their potential applications in many fields, such as carriers for drugs, absorption and desorption of substances, pulmonary drug delivery, and tissue regeneration. The application of porous microspheres has become a feasible way to address existing problems. In this essay, we give a brief introduction of the porous microsphere, its characteristics, preparation methods, applications, and a brief summary of existing problems and research tendencies.

Formulating erythropoietin-loaded sustained-release PLGA microspheres without protein aggregation

We report a simple method to microencapsulate erythropoietin (EPO, a protein easily denatured and antigenized by contact with water-organic solvent interfaces) into poly(lactic-co-glycolic acid) (PLGA) microspheres with minimal aggregation. This formulation process involved an aqueous-aqueous emulsion formed at reduced temperature. EPO was first dissolved in water together with dextran (MW=70,000) and polyethylene glycol (MW=8000), followed by a freezing process during which dextran separated out as the dispersed phase with EPO partitioned in preferentially. The frozen sample was then lyophilized to powder and washed with dichloromethane to remove the PEG continuous phase. Once loaded in the polysaccharide particles, 1-4 microm in diameter, EPO gained resistance to organic solvents and was encapsulated into PLGA microspheres without significant aggregation (<2%). EPO released from the composite PLGA microspheres in a sustained-release manner with minimal burst (<20% at the first day) and incomplete (<20%) release. Single injection of these EPO-loaded PLGA microspheres to mice resulted in a red blood cell elevation equivalent to twelve injections of the solution formulation. An ELISA assay suggested that the mice injected with the EPO-loaded PLGA microspheres did not develop anti-EPO IgG more than those given solution form EPO.

Development of protein delivery microsphere system by a novel S/O/O/W multi-emulsion.

A novel method has been developed to protect protein drugs in poly (lactic-co-glycolic acid) (PLGA) microspheres using S/O/O/W multi-emulsion method. This method develops a novel protein drug sustained-release system, which is based on the combination of protein-loaded dextran glassy microparticles (protein-loaded AqueSpheres) and PLGA microspheres. The protein molecules are encapsulated in the dextran glassy particles and the drug-containing dextran glassy particles are further dispersed in the PLGA microspheres. The protein-loaded AqueSpheres based PLGA composite microspheres have spherical shape and a smooth surface. They possess a normal size distribution and a mean diameter of 67.08 microm. The method may decrease protein aggregations and incomplete release due to avoiding protein contacting with oil/water interfaces and hydrophobic PLGA. The dextran glassy particles can stabilize proteins in the PLGA matrix, which is the major advantage of this novel protein sustained-release system over preparation for the PLGA microspheres using W/O/W double-emulsion method.

Dissolving and biodegradable microneedle technologies for transdermal sustained delivery of drug and vaccine

Microneedles were first conceptualized for drug delivery many decades ago, overcoming the shortages and preserving the advantages of hypodermic needle and conventional transdermal drug-delivery systems to some extent. Dissolving and biodegradable microneedle technologies have been used for transdermal sustained deliveries of different drugs and vaccines. This review describes microneedle geometry and the representative dissolving and biodegradable microneedle delivery methods via the skin, followed by the fabricating methods. Finally, this review puts forward some perspectives that require further investigation.

Polyspermine Imidazole‐4,5‐imine, a Chemically Dynamic and Biologically Responsive Carrier System for Intracellular Delivery of siRNA

SUMMARY We report a structurally simple yet highly functional cationic polymer, polyspermine imidazole-4,5-imine, which accomplishes four of the total five essential tasks to achieve therapeutically feasible delivery of siRNA. INTRODUCTION The five essential tasks for a therapeutically feasible synthetic carrier of siRNA to achieve include: (A) packing siRNA tightly into nanoparticulate forms to avoid pre-phagocytosis degradation; (B) adsorbing onto diseased cells selectively; (C) facilitating endosomal escape of siRNA after phagocytosis; (D) releasing siRNA in the cytoplasm of the target cell in time; and (E) metabolizing itself into nontoxic and eliminable species. Among these five tasks, A, C, D and E may be achieved concurrently with a rationally designed polycationic carrier system, as they are universal for almost all the cell types. In addition, a chemical design to address these multiple biologic issues is best to be structurally simple in order to avoid metabolic complex and synthetic complication. With all these criteria taken into consideration, we designed, synthesized and examined polyspermine imidazole-4,5-imine (PSI) as an ideal polycationic carrier to accompolish the four universal tasks (A, C, D, and E). Figure 1. Synthesis and chemical structure of polyspermine imidazole-4, 5-imine (PSI). As schematically demonstrated in Figure 1, this polymer is formed by condensing spermine, an endogenous monomer condensing genes professionally in sperms, with bisformaldehyde imidazole through a pH-responsive linkage, a bis-imine bond conjugated with the imidazole ring. The beauty of the chemistry is that the imidazole-conjugated π linkage possesses a pKa in the range of 5~6, which causes it to be stable under neutral or basic pH, but turn to be unstable and dissociate to spermine and imidazole bisformate, a safety-known drug metabolite, by absorbing a proton under the endosomal pH. Moreover, the spermine generated by decomposition of each repeating units of the polymer possesses two free primary amino groups which may absorb two additional protons, further enhancing the proton-sponging effect, and in turn facilitating endosomal rupture. While the polymer degradation occurs, the packed siRNA is released simultanously. Since the endosome ruptured by proton-sponging may be recovered before escaping of the engulfed particle, if it is still in a particulate form, the concurrent dissociation of PSI-siRNA polyplex upon endosome ruptureing may offer an additional advantage in terms of avoiding reencapsulation by the recovered endosome. EXPERIMENTAL METHODS The synthetic process of PSI was as simple as a single condensation reaction: dropping approximately an equivalent molar amount of imidazole-4,5-dialdehyde into spermine (both dissolved in DMF), in the presence of catalytic amount of p-toluenesulfonic acid. The polymerization was allowed to further proceed at 80°C for 24 hours under stirring, followed by filtration, evaporation, as well as dialysis in ultrapure water through a cellulose membrane, 10 KDa in cut off molecular weight. The final product was lyophilized to a yellowish powder, prior to storage at -80°C. The polymer was subject to a number of assays as pH-responsive degradation rate, cytotoxicity assay and siRNA transfection. RESULTS AND DISCUSSION The degradability of PSI was examined by incubating the polymer in formic acid buffers, 7.4, 5.8 and 5.0 in pH for simulating the environment of body fluid, endosomes and lysosomes, at 37°C, followed by monitoring average molecular weight (<Mw>) changes using SEC (with PEI 25 KDa as a standard). The average molecular weight of PSI declined over time at remarkably different rates in the different buffers (with pH 7.4 << pH 5.8 << pH 5.0). The degradation rates were numerically summarized in the form of the time required for <Mw> to reach half of its original. This time period was 48.8 h, 22.6 h, and 0.6 h for PSI incubated in pH 7.4, 5.8 and 5.0, respectively (Figure 2). Figure 2. pH-responsive degradability of PSI. Cytotoxicity of PSI was examined by culturing the polymer with COS-7 and SMMC7721 cells stably transfected luciferase gene, followed by monitoring viability changes. As displayed in Figure 3, the viability of both the cell lines remains unchanged as the polymer concentration was raised up to 100 μg/mL. For the same cell lines treated with PEI 25 KDa, however, viability dropped dramatically to 20 % of its original when the polymer concentration reached 20 μg/mL. Gene silencing assays was carried out by using PSI and PEI-25KDa to pack and deliver anti pGL3-luciferase siRNA (at various N/P ratios) to COS-7 cells pre-transfected with pGL3-Lipofactmine-2000 lipoplexes. As result, the cells treated with PSI delivered antisense siRNA showed significantly lower luciferase expression but higher viability than those treated with PEI-25KDa delivered antisense siRNA at each corresponding N/P ratio (Figure 4). The rate of pGL3 silencing for the cells treated with the former, was up to 95.5 % but only 30.4% for the later for N/P ratio of 50/1, (defined by the difference in luciferase expression between the cells treated with antisense and nonsense siRNA). Clearly, the suppressed pGL3 gene expression by PSI polyplexes was not due to cell death but superior siRNA delivery efficiency of PSI. Figure 3. Cytoxicity profile of PSI. Figure 4. Gene silencing efficiency of PSI. CONCLUSION Polyspermine imidazole-4,5-imine (PSI) represents a type of cationic polymers capable of siRNA condensing, endosomal escaping, incytoplasm releasing and carrier self metabolizing (task A, C, D, E mentioned above). REFERENCES 1. S. Duan, W. Yuan, F. Wu, T. Jin, Angew. Chem. 2012, 124, 8062. ACKNOWLEDGMENTS This study was financially supported by National Science Foundation of China (30472096), and BioPharm Solutions, Inc.

Biscarbamate cross-linked polyethylenimine derivative with low molecular weight, low cytotoxicity, and high efficiency for gene delivery

Polyethylenimine (PEI), especially PEI 25 kDa, has been widely studied for delivery of nucleic acid drugs both in vitro and in vivo. However, it lacks degradable linkages and is too toxic for therapeutic applications. Hence, low-molecular-weight PEI has been explored as an alternative to PEI 25 kDa. To reduce cytotoxicity and increase transfection efficiency, we designed and synthesized a novel small-molecular-weight PEI derivative (PEI-Et, Mn: 1220, Mw: 2895) with ethylene biscarbamate linkages. PEI-Et carried the ability to condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed complete condensation of pDNA at w/w ratios that exceeded three. The particle size of polymer/pDNA complexes was between 130 nm and 180 nm and zeta potential was 5–10 mV, which were appropriate for cell endocytosis. The morphology of PEI-Et/pDNA complexes observed by atomic force microscopy (AFM) was spherically shaped with diameters of 110–190 nm. The transfection efficiency of polymer/pDNA complexes as determined with the luciferase activity assay as well as fluorescence-activated cell-sorting analysis (FACS) was higher than commercially available PEI 25 kDa and Lipofectamine 2000 in various cell lines. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration in three cell lines. Therefore, our results indicated that the PEI-Et would be a promising candidate for safe and efficient gene delivery in gene therapy.

A scalable fabrication process of polymer microneedles.

While polymer microneedles may easily be fabricated by casting a solution in a mold, either centrifugation or vacuumizing is needed to pull the viscous polymer solution into the microholes of the mold. We report a novel process to fabricate polymer microneedles with a one-sided vacuum using a ceramic mold that is breathable but water impermeable. A polymer solution containing polyvinyl alcohol and polysaccharide was cast in a ceramic mold and then pulled into the microholes by a vacuum applied to the opposite side of the mold. After cross-linking and solidification through freeze-thawing, the microneedle patch was detached from the mold and transferred with a specially designed instrument for the drying process, during which the patch shrank evenly to form an array of regular and uniform needles without deformation. Moreover, the shrinkage of the patches helped to reduce the needles’ size to ease microfabrication of the male mold. The dried microneedle patches were finally punched to the desired sizes to achieve various properties, including sufficient strength to penetrate skin, microneedles-absorbed water-swelling ratios, and drug-release kinetics. The results showed that the microneedles were strong enough to penetrate pigskin and that their performance was satisfactory in terms of swelling and drug release.

Effect of bases with different solubility on the release behavior of risperidone loaded PLGA microspheres.

Poly (D, L-lactide-co-glycolide) (PLGA) microspheres are attractive delivery vehicles due to their excellent sustained release capabilities. One major problem with PLGA microspheres is that the hydrophobic properties of PLGA generally cause a lag period in the process of drug release, leading to fluctuation of drug concentration in the blood and various resulting adverse reactions. Herein, Mg(OH)₂, an inorganic base, and arginine, an organic base, were separately co-encapsulated into risperidone-loaded PLGA microspheres at varying concentration using the solvent evaporation method to improve release profiles from the microspheres. High encapsulation efficiencies were obtained in all formulations. The surface of base-free microspheres was smooth, whereas a few pores formed in base co-encapsulated microspheres. After 7-days degradation, many inter-connecting pores were formed in the interior of the microspheres containing 10 mg Mg(OH)₂. The final pH in the microspheres with Mg(OH)₂ was higher than in those with arginine after 28-days degradation. The initial release of risperidone from microspheres containing Mg(OH)₂ was higher than from those containing arginine, and the latter release exhibited a more uniform pattern. Microspheres with 5mg and 10mg arginine exhibited zero-order release kinetics. However, both bases eliminated the lag phase of release. These results indicate that the incorporation of bases has potential in addressing the problem of the lag period in drug release from PLGA microspheres, and improving release behavior toward an ideal model.

Advances with microRNAs in Parkinson’s disease research

Parkinson’s disease (PD) is the second-most common age-dependent neurodegenerative disorder and is caused by severe degeneration of dopaminergic neurons in the substantia nigra pars compacta. Unfortunately, current treatment only targets symptoms and involves dopamine replacement therapy, which does not counteract progressive degeneration. MicroRNAs (miRNAs) are a class of small RNA molecules implicated in post-transcriptional regulation of gene expression during development. Recent studies show that miRNAs are playing an important role in the pathophysiology of PD. miRNA-based therapy is a powerful tool with which to study gene function, investigate the mechanism of the disease, and validate drug targets. In this review, we focus on the recent advances of the use of miRNAs in the pathogenesis of PD.

Preparation of polysaccharide glassy microparticles with stabilization of proteins.

This study investigates a method of preparing hazard-resistant protein-loaded polysaccharide glassy microparticles using freezing-induced phase separation method without exposure to water/oil, water/air interface and cross-linking reagents. Model protein (such as bovine serum albumin, myoglobin and beta-galactosidase (beta-Gal)) was dissolved in water together with dextran and polyethylene glycol (PEG), followed by a freezing process to form a temperature-stabilized aqueous-aqueous emulsion wherein dextran separated out as the dispersed phase with protein partitioned in preferentially. The frozen sample was freeze-dried and washed with dichloromethane (DCM) to remove the PEG continuous phase, after which protein-loaded polysaccharide particles, 1-4 microm in diameter, were harvested. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) patterns showed that the particles were in glassy state. These glassy polysaccharide microparticles can well protect the delicate structure of proteins and preserve their bioactivities under deleterious environment interacting with organic solvents, vortex and centrifugation processes that often involve during the formulation processes leading to polymer-based sustained-release systems. Therefore, this freezing-induced phase separation method is a mild and effective way to encapsulate protein into hazard-resistant polysaccharide glassy particles, which ensure its stability in subsequent formulating processes that leads to polymer-based sustained-release system.